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Current Trends in Biotechnology and Pharmacy 346
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0973-8916 (Print), 2230-7303 (Online)

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Current Trends in Biotechnology and Pharmacy i
Vol. 14 (5), ISSN No. 0973-8916 (Print), 2230-7303 (Online)

Current Trends in Biotechnology and Pharmacy

Volume 14 (5) CONTENTS December, 2020

In Silico Investigation on the Probable Macromolecular Drug Targets Involved in the Anti-Schizophrenia
Activity of Ocimum sanctum 1-10
Goh Yen Joe, Anand Gaurav, Mayasah Al-Nema
DOI : 10.5530/ctbp.2020.4s.1

Comparative Study on Antioxidant and Anti-Inflammatory Activities of Red and Brown Species of
Areca catechu L. Nut Extracts 11-18
Parthasarathi Perumal, Suresh Rathnasamy, Balaji Tirupathi, Purushoth Prabhu Thiraviam,
Ashok Kumar Balaraman, Vinothkumar S P, Senthil Kumar G P
DOI : 10.5530/ctbp.2020.4s.2

Antioxidant and Antihyperlipidemic Activity of Methanolic Fraction of Maytenus heyneana Root on STZ
Induced Diabetic Wistar Rats 19-31
G Sumithira, G P SenthilKumar, Maya Sharma, B Krisnamoorthy, R Suresh, Ashok Kumar Balaraman
DOI : 10.5530/ctbp.2020.4s.3

Development and Assessment of Modified Glover Nilsson Vaping Behavioural Questionnaire Among
Malaysian Electronic Cigarettes Users 32-37
Aziz-ur-Rahman, Mohamad Haniki Nik Mohamed, Syed Mahmood, Ashok Kumar Balaraman,
Muhammad Ahsan Iftikhar Baig
DOI : 10.5530/ctbp.2020.4s.4

ATR-FTIR Spectroscopy Methods for Determination of Aminoglycoside Antibiotics in Ophthalmic and

Parenteral Preparations with Full Partial Least Squares Algorithm 38-54
Yau Xin Yi, Bontha Venkata Subrahmanya Lokesh, Gabriel Akyirem Akowuah
DOI : 10.5530/ctbp.2020.4s.5

Assessment of Knowledge, Attitude and Practice of Malaysian Women Towards Osteoporosis 55-63
Yee Thong Cheng, Fazlollah Keshavarzi, Muhammad Junaid Farrukh,
Safia Sabry Lotfy Aly Mahmoud
DOI : 10.5530/ctbp.2020.4s.6

Barriers and Enhancers of Medication Error Reporting Among Hospital Pharmacists,

a Qualitative Exploration 64-71
Keat Jiu Yoo, Fazlollah Keshavarzi
DOI : 10.5530/ctbp.2020.4s.7

Malaysian Community Pharmacists' Knowledge, Attitudes and Practice

Toward Vaccination and Immunization; A Cross-Sectional Study 72-81
Ali Saleh Noori Istehkam, Fazlollah Keshavarzi, Aziz Ur Rahman
DOI : 10.5530/ctbp.2020.4s.8

Knowledge, Attitude and Practice of General Public Towards Counterfeit and Adulterated Medicines :
a Cross-sectional Study in Malaysia 82-91
Choo Shiuan Por, Fazlollah Keshavarzi, Chuan Sheng Yap, Yee Chang Soh
DOI : 10.5530/ctbp.2020.4s.9

Evaluation of Self-Medication Practice Among University Students 92-100

Tan Puay Luan, Khaled M. Alakhali, Fazlollah Keshavarzi, Omotayo Oladuntoye Fatokun
DOI : 10.5530/ctbp.2020.4s.10
Current Trends in Biotechnology and Pharmacy ii
Vol. 14 (5), ISSN No. 0973-8916 (Print), 2230-7303 (Online)

Course Satisfaction and Perception of Malaysian Provisionally Registered Pharmacists toward

Their Training : A Qualitative Study 101-111
Mei Qi Hee, Fazlollah Keshavarzi, R. Mogana
DOI : 10.5530/ctbp.2020.4s.11

Evaluation of Antibacterial Activity against Multidrug Resistance (MDR)

Bacteria by the Bark Fractions of Canarium patentinervium Miq. 112-118
Sook Shuan T, R. Mogana, Sasikala Chinnappan, Ashok Kumar Balaraman, S Chandramathi, K Geethanjali
DOI : 10.5530/ctbp.2020.4s.12

Enzymatic and Non-enzymatic Antioxidant Potential of Methanolic Fractions of Artabotrys suaveolens 119-127
R. Mogana, Jubair Najwan, WL Koh, LM Foh, Theresa WT Lee.
JH Foo, Sasikala Chinnappan, Ashok Kumar Balaraman, C. Wiart
DOI : 10.5530/ctbp.2020.4s.13

In silico Screening of Selected Flavanones for HMG CoA Reductase Inhibitory Activity 128-139
Tan Ker Ying, Mohamed Saleem Abdul Shukkoor, Shaik Ibrahim Khalivulla
DOI : 10.5530/ctbp.2020.4s.14

Gamification Technique to Estimate Mini Mental State Examination Scores : A Validation Study 140-146
Muhammad Junaid Farrukh, Mohd Makmor Bakry, Ernieda Hatah, Tan Hui Jan
DOI : 10.5530/ctbp.2020.4s.15

Risk Assessment of Sleep Apnoea and Quality Of Sleep Among General Public in Klang Valley 147-155
Muhammad Qamar, Leong Mun Yee, Muhammad Ahsan Iftikhar Baig,
Muhammad Haseeb Tariq, Muhammad Junaid Farrukh
DOI : 10.5530/ctbp.2020.4s.16

Anti-Angiogenic Effect of Ethanolic Extract and its Phenolic Rich Fraction of Acacia auriculiformis
Bark in the Chick Embryo Chorioallantoic Membrane Model 156-161
Chong Wei Chean, Sasikala Chinnappan, R. Mogana, Ashok Kumar B
DOI : 10.5530/ctbp.2020.4s.17

Anti-Angiogenic Effect of Ethanolic Extract and its Phenolic Rich Fraction of Filicium decipiens in the
Chick Embryo Chorioallantoic Membrane Model 162-167
Looi Kah Xin, Sasikala Chinnappan, R. Mogana, Ashok Kumar B
DOI : 10.5530/ctbp.2020.4s.18

Knowledge and Awareness About Blood Pressure, Stroke and Prevalence of Hypertension :
A Cross-Sectional Study in a Private University, Kuala Lumpur 168-175
Chuan Sheng Yap, Zi Xuan Khor, Peng Nam Yeoh, R. Mogana, Chia Yook Chin
DOI : 10.5530/ctbp.2020.4s.19

Investigation on Antibacterial Activity of Pyrogallol in Methicillin Resistant Staphylococcus aureus 176-180

Yik-Ling Chew, Joo-Kheng Goh, Chairunnisa Arasi
DOI : 10.5530/ctbp.2020.4s.20

Analysis of the Effectiveness of Drug Awareness Campaigns Using Google Trends 181-187
Deng Ruolan, Muhammad Shahzad Aslam
DOI : 10.5530/ctbp.2020.4s.21

Sun Protection Effect of 2-Hydroxy-4-(Octyloxy) Benzophenone in Sunscreen Creams Formulations

by a Combination of Inorganic UV Filters 188-193
Asmiyenti Djaliasrin Djalil, Anisa Tri Susanti, Bella Apriani, Muhammad Arba, Ika Yuni Astuti
DOI : 10.5530/ctbp.2020.4s.22
Current Trends in Biotechnology and Pharmacy iii
Vol. 14 (5), ISSN No. 0973-8916 (Print), 2230-7303 (Online)

In Silico Studies of Green Tea Catechins Against HER-2 Receptor in Breast Cancer 194-199
Fitriyani, Taufik M. Fakih, Daryono H. Tjahjono
DOI : 10.5530/ctbp.2020.4s.23

Revisiting the Intractable Barriers Affecting Medication Adherence Among Outpatients

with Schizophrenia 200-205
Julaeha Julaeha, Umi Athiyah, Verra Yuliana, J.P Ayuningtyas, Andi Hermansyah
DOI : 10.5530/ctbp.2020.4s.24

A Production and Activity Test of Anti-bacterial Compounds of Endophytic fungi BR-S1

(a) Isolate extract in Different General Growth Media 206-212
Kurniawan and Mustiah Yulistiani
DOI : 10.5530/ctbp.2020.4s.25

Phytochemical Investigation, Cytotoxicity and Anti-Diabetic Activity of Whole Fresh and Dry Ethanolic
Extracts of Sudanese Portulaca quadrifida 213-217
Layla Fathi Yassin, Ayat Ahmed Alrasheid, Khalid Abdallah Enan,
Ali Abdalla Adam, MazinYousif Babiker
DOI : 10.5530/ctbp.2020.4s.26

Analysis of Prednisone in Indonesian Uric Acid Herbs Using High Performance Liquid Chromatography 218-221
Pri Iswati Utami, Elza Sundhani, Deka Maulyani
DOI : 10.5530/ctbp.2020.4s.27

An Analysis of Rat Meat with FTIR and GC/MS for Halal Authentication 222-225
Wiranti Sri Rahayu, Pri Iswati Utami, Irfan Nugraha, Rati Janah
DOI : 10.5530/ctbp.2020.4s.28

Epidemiological Studies of Schistomiasis in Bauchi Central Senatorial Zone, Nigeria 226-232

Usman, A.M
DOI : 10.5530/ctbp.2020.4s.29

Anti-Osteoporotic Effects of Alendronate and Sitagliptin in Streptozotocin Induced Type 2 Diabetes

Mellitus in Ovariectomized Rats 233-240
Vadivelan Ramachandran, Gautam Adhikari, Manogaran Elumalai
DOI : 10.5530/ctbp.2020.4s.30

Perception and Satisfaction Among Single and Dual Users Malaysian Vapers Towards Electronic
Cigarettes. A One Year Observational Study 241-248
Aziz-ur-Rahman, Mohamad Haniki Nik Mohamed, Syed Mahmood, Ashok Kumar Balaraman
DOI : 10.5530/ctbp.2020.4s.31

Investigation of Process Variables in the Development of Nateglinide Nanocrystals 249-257

Ng Chia Huey, Ashok Kumar Janakiraman, Shiek Abdul Kadhar Mohamed Ebrahim Habibur Rahman
DOI : 10.5530/ctbp.2020.4s.32

Evaluation of Antibacterial Activity Against Multidrug-Resistance (MDR)

Bacteria by the Fractions of Artabotrys suaveolens (Blume) 258-265
Jian-You C, R. Mogana, Chandramathi SR, Ashok Kumar B, Sasikala C and Geethanjali K
DOI : 10.5530/ctbp.2020.4s.33

In-Vitro Cytotoxic Activities of Brassica oleracea Var capitata by Using Brine Shrimp Lethality Assay 266-271
Ashok Kumar Balaraman, Sasikala Chinnappan, R. Mogana, Aziz Ur Rahman and Tan Zhe Way
DOI : 10.5530/ctbp.2020.4s.34
Current Trends in Biotechnology and Pharmacy 1
Vol. 14 (5) 1-10, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.1

In Silico Investigation on the Probable Macromolecular Drug Targets Involved

in the Anti-Schizophrenia Activity of Ocimum sanctum
Goh Yen Joe1, Anand Gaurav1*, Mayasah Al-Nema1
1Faculty of Pharmaceutical Sciences, UCSI University, Jalan Menara Gading, Taman Connaught,
Cheras, 56000 Kuala Lumpur, Malaysia
*Corresponding author:anand.pharma@gmail.com

Abstract observed in schizophrenia is attributed to the

Despite the low prevalence of schizophrenia, it hyperactivation of mesolimbic pathway and dysfunction
negatively impacts the quality of life for patients and their of the mesocortical pathway leading to an imbalance in
families. The present antipsychotics improve only the the dopaminergic, serotonergic, GABAergic as well as
positive symptoms of the illness, but they do not affect glutamatergic neurotransmission in certain regions of the
the negative symptoms or cognitive impairment. Thus, brain. In addition, other factors can also contribute to the
there is no satisfactory and effective remedy available to development of psychosis, i.e. heredity, oxidative stress,
prevent, manage, and cure schizophrenia. Ocimum N-methyl-D-aspartate (NMDA) receptor antagonists, drug
sanctum is an Ayurvedic medicinal plant, the active abuse and traumatic injury. The positive symptoms are
constituents of the plant are known to have certain considered the most common symptoms of schizophrenia.
activities which might be useful in alleviating the These symptoms are effectively treated with the currently
symptoms of schizophrenia. Therefore, the objective of available antipsychotics. Whereas the negative symptoms
this study is to identify the active constituents of Ocimum and cognition dysfunction do not respond to the available
sanctum with the highest affinity for the probable medications. Thus, there is no satisfactory and effective
macromolecular drug targets involved in the aetiology of remedy available to prevent, manage and cure
schizophrenia and, thereby determine the structural schizophrenia (1, 2).
features of the ligands that involve in the interactions with Recently, the major attention has been directed toward
the drug targets and propose some modifications for exploring and studying the therapeutic potential of natural
designing of new drugs. The phytochemicals present in herbs in the management of schizophrenia. Ocimum
Ocimum sanctum were filtered first based on their anti- sanctum (also known as tulsi or holy basil) is one of the
schizophrenia activity and Lipinski's rule of 5. Then, the plants that is renowned for its therapeutic uses. It is an
selected active constituents were subjected to molecular Ayurvedic medicinal plant which is believed to act as
docking against the dopamine receptor, N-methyl-D- potent adaptogenic herb that protect the body from the
aspartate receptor, Gamma-aminobutyric acid receptor effects of various stressors (3). Despite the numerous
and phosphodiesterase 10A. Apigenin exhibited a therapeutic values of Ocimum sanctum, the attention was
particularly high binding affinity towards the four targets. drawn mainly to certain activities which might be useful
Thus, apigenin can serve as a starting point for the in alleviating the symptoms of schizophrenia. These
designing of new compounds with the highest affinity activities include antioxidant and anti-stress activities,
for the target receptors. memory and cognition-enhancing activities and
Key words : Schizophrenia, Antipsychotics, Ocimum neuroprotection activities. Therefore, recent research has
sanctum, Molecular Docking, Apigenin been driven to investigate the therapeutic potential of
Ocimum sanctum in treating schizophrenia (4-6).
1. Introduction
Computational (in silico) methods have been applied
Schizophrenia is a mental disorder that affects the widely and frequently in the process of drug discovery
ability of an individual to think, feel, and behave. It is and development to help in the selection of potent lead
characterised by three symptomatic domains : positive molecule, thereby minimise the failures during the late
symptoms, negative symptoms and cognitive stage of clinical development (7). The aim of the present
dysfunction.The pathophysiology of schizophrenia is study was to identify the potential anti-schizophrenia
unclear but it has been postulated that the psychosis phytochemical compounds present in the Ocimum

In silico study of the active constituents of Ocimum sanctum

Current Trends in Biotechnology and Pharmacy 2
Vol. 14 (5) 1-10, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.1

sanctum. Molecular docking has been used to study the were saved as separate files to be used as standards for
protein-ligand interactions by predicting the preferred the docking studies. The protein structures were imported
orientation and free energies of binding between the active into AutoDock where AutoDock Tools (ADT) 1.5.6 was
constituents of Ocimum sanctum (ligands) and the target used to prepare each protein by removing the water
receptors (macromolecules) which are known to be molecules, adding the hydrogen atoms, and Gasteiger
related to the pathophysiology of schizophrenia. charges (12).
2. Materials and Methods Preparation of ligands
Selection of phytochemicals from Ocimum sanctum The two dimensional (2D) structures of the ligands
(the standard compounds and the selected active
A Literature review has been conducted to identify a
constituents) were drawn using ACD/ChemSketch
number of phytochemicals present in various parts of
software (13). Followed by conversion the ligands into
Ocimum sanctum. All these phytochemicals were selected
their 3D structures using Open Babel (14). The 3D
from reported scientific literature based on their
structures of the ligands were imported into AutoDock
pharmacological activities. Only the active constituents
where they were subjected to the addition of charges.
were further filtered according to the Lipinski's rule of 5
Then the rotatable bonds were set by ADT and all the
and polar surface area (PSA). The inclusion criteria are
torsions were allowed to rotate for the ligands (12).
presented in Table 1 (8, 9). The violation of either one of
these properties will result in the exclusion of the active Identification of the proteins' binding sites
constituent. The amino acid residues of each prepared protein
Table 1: Lipinski's rule of 5 and polar surface area which interact with its co-crystallised ligand were
S.No. Inclusion Criteria determined by using Biovia DS (11). The binding site of
1 Molecular weight (MW) ≤ 500 Dalton (g/mol) the protein was identified based on the amino acid
residues involved in the interactions. ADT was used to
2 Hydrophobicity (logP) ≤ 5
determine the Grid Box that covers the entire identified
3 Number of hydrogen bond donor (nOHNH) ≤ 5
binding site of the protein (12).
4 Number of hydrogen bond acceptor (nON) ≤ 10
Molecular docking
5 Number of rotatable bonds (nrotb) < 8
AutoDockVina 1.1.2 was used to perform molecular
6 Polar surface area (PSA) of 20 – 90 Å2
docking of the prepared ligands against the binding sites
of the prepared macromolecular drug targets (12). During
Preparation of proteins
the docking process (Figure 1), flexible ligands were
The three dimensional (3D) structures of dopamine docked into a rigid binding site of the protein structure.
(D2) receptor, N-methyl-D-aspartate (NMDA) receptor, The ligands were ranked based on their affinity (binding
gamma-aminobutyric acid GABAA receptor, and energy) to the target receptor in which ligands with lower
phosphodiesterase (PDE) 10A were downloaded from the binding energies were ranked higher. Additionally, the
Protein Data Bank (PDB) and used as macromolecular orientation of the top ligands and the mode of interactions
targets for the docking study (Table 2) (10). with each drug target were studied using Biovia DS (11).
Table 2 : Macromolecular targets with their respective
PDB ID, co-crystallised ligands, and standard
compounds.
Macromolecular Targets PDB ID Co-crystallized Standard
Ligands Compounds
Dopamine (D2) 6CM4 Risperidone Risperidone
N-Methyl-D-Aspartate (NMDA) 5U8C PEAQX PEAQX
Gamma-Aminobutyric Acid (GABAA) 6D6T Flumazenil Alprazolam
Phosphodiesterase(PDE) 10A 2WEY Papaverine Mardepodect

The protein structures were prepared for docking by

removing the co-crystallised ligands first using Biovia
Discovery Studio (DS) (11). Those co-crystallised ligands Figure 1 Flow chart of the molecular docking study.

Goh et al
Current Trends in Biotechnology and Pharmacy 3
Vol. 14 (5) 1-10, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.1

3. Results and Discussion

Selection of phytochemicals from Ocimum Sanctum
The phytochemicals were selected based on inclusion criteria mentioned previously. The selected compounds
along with their lipinski’s rule of 5 and PSA values are presented in Table 3.
Table 3 : The active constituents of Ocimum sanctum with their lipinski’s rule of 5 and PSA values.

No. Ligands MW Log P nOHNH nON nrotb PSA

1 4-Hydroxybenzaldehyde 122.12 1.25 1 2 1 37.30

OH

2 Apigenin 270.24 2.46 3 5 1 90.89

O OH

O OH

HO

3 α-Bisabolol 222.37 4.68 1 1 4 20.23

OH

4 Borneol 154.25 2.35 1 1 0 20.23

HO

5 Isoborneol 154.25 2.35 1 1 0 20.23

HO

In silico study of the active constituents of Ocimum sanctum

Current Trends in Biotechnology and Pharmacy 4
Vol. 14 (5) 1-10, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.1

6 Bornyl acetate 196.29 3.05 0 2 2 26.3

7 Carvacrol 150.22 3.81 1 1 1 20.23

OH

8 Cirsimaritin 314.29 2.79 2 6 3 89.14

OH O

O O

OH

9 Eugenol 164.20 2.10 1 2 3 29.46

OH

10 Myrtenol 152.24 2.30 1 1 1 20.23

HO

Molecular Docking As can be seen in Table 4, all the active constituents

186 phytochemicals are presented in Ocimum sanctum displayed low to intermediate affinities toward the four
(Supplementary Materials). These phytochemicals were receptors except apigenin which exhibited the highest
screened based on their pharmacological properties and affinity (binding energy < -9.5 Kcal/mol) to the target
Lipinski's rule of 5. As a consequence, only ten active receptors followed by cirsimaritin (binding energy <
-8.2 Kcal/mol) then -bisabolol (binding energy < -7.0
constituents have been selected to be docked against the
Kcal/mol). However, the binding energies that displayed
probable macromolecular drug targets of schizophrenia,
by the four standards were the lowest in comparison to
D2 receptor, NMDA receptor, GABAA receptor, and
the active constituents of Ocimum sanctum, thus these
PDE10A receptor. The docking results are presented in standards had the highest affinities toward the target
Table 4. receptors.

Goh et al
Current Trends in Biotechnology and Pharmacy 5
Vol. 14 (5) 1-10, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.1

Table 4 : Binding energy of the standard compounds and the active constituents of Ocimum sanctum with the probable
macromolecular drug targets of schizophrenia.
No. Ligands Binding Energy (kcal/mol)
D2Receptor NMDA GABAA PDE10A
Standard compound for each Receptor
receptor Receptor Receptor
1 Risperidone -12.0 - - -
2 PEAQX - -10.6 - -
3 Alprazolam - - -11.2 -
4 Mardepodect - - - -9.8
Active constituents of Ocimum sanctum
5 4-Hydroxybenzaldehyde -5.7 -5.7 -5.2-5.2 -6.2-6.2 -5.6 -5.6
6 Apigenin -10.3 -9.5 -9.7 -9.6
7 α-Bisabolol -8.6 -7.3 -7.8 -7.0
8 Borneol -6.3 -5.9 -6.4 -5.2
9 Isoborneol -6.6 -6.0 -6.9 -5.4
10 Bornyl acetate -7.2 -6.5 -6.9 -6.0
11 Carvacrol -7.0 -6.2 -7.0 -6.8
12 Cirsimaritin -9.5 -8.2 -9.3 -8.9
13 Eugenol -6.5 -6.0 -6.9 -6.3
14 Myrtenol -6.4 -6.1 -5.9 -5.3

Apigenin
in the interactions and the bonded and non-bonded
Apigenin is a plant-derived flavonoid that belongs to interactions that contribute to the strength of binding.
the flavone class. It can be found abundantly in herbs, Furthermore, the mode of interactions of the standard
fruits and vegetables. The structure of apigenin contains compounds has been studied as well to illustrate the
a fused benzene ring (Ring A) and oxygen-containing important amino acids that are essential for the binding
heterocycle (Ring C) that form a benzopyrone moiety, affinity and selectivity.
besides the phenyl ring (Ring B) at position 2. The
benzopyrone moiety and the phenyl ring constitute the Dopamine (D2) receptor
phenylchromane nucleus of apigenin (15). It is substituted Risperidone is a benzisoxazole derivative that belongs
with three hydroxyl groups at positions 4', 5, and 7 to the atypical antipsychotic medications. It is widely
forming a trihydroxyflavone with the molecular formula prescribed as a treatment for the positive symptoms of
of C15H10O5 (Figure 2). schizophrenia due to its potent D2 receptor antagonism
activity (16, 17). Thus, risperidone has been used as a
standard compound for assessing the docking results of
the active constituents of Ocimum sanctum with D2
receptor. Based on the docking results, risperidone has
shown the highest binding affinity (binding energy -12.3
kcal/mol) to the D2 receptor. The high affinity might be
due to the strong interactions between the ligand and the
Figure 2 : a) General structure of flavones. b) Structure of active site's residues of D2 receptors. These interactions
apigenin (4',5,7-trihydroxy-flavone)
involve two hydrogen bonds between the oxygen atom
Based on the docking results, apigenin displayed high in benzisoxazole and the residues Thr119 and Ser197 and
affinity to all macromolecular drug targets involved in n-n T-shaped interactions between the pyrimidone ring
schizophrenia. Therefore, the mode of interactions of and phenyl ring of the ligand and the aromatic rings of
apigenin with the target receptors has been studied Trp100 and Trp386, respectively. Additionally, the
thoroughly to identify the amino acids that are involved fluorine group of risperidone displays a unique interaction

In silico study of the active constituents of Ocimum sanctum

Current Trends in Biotechnology and Pharmacy 6
Vol. 14 (5) 1-10, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
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(halogen interaction) with the residue Cys118 of the and position 2 to lengthen the molecule hence strengthen
receptor. This carbon-fluorine interaction has similar the binding. In this case, a salt bridge will be formed
structural significance as the week hydrogen bond. The between the basic nitrogen atom (positively charged) and
benzisoxazole moiety of risperidone is important for the acidic Asp114 residue (negatively charged). Besides,
tethering the ligand in the active site of the D2 receptor. the basic nitrogen will contribute to the reduction of the
This moiety extends into a deep binding pocket, that form logP value, thus enhancing brain penetration. This
a sub-pocket underneath the orthosteric site of the D2 structural modification will add some advantage to
receptor, thereby contributing to the significant binding apigenin (9).
affinity and the strength of binding (Figure 3) (18).
N-Methyl-D-Aspartate (NMDA) receptor
The NMDA receptor hypofunction is strongly
implicated in the aetiology of schizophrenia. Thus,
NMDA receptor agonist is required to reverse this effect.
PEAQX is a competitive NMDA receptor antagonist. This
compound has been used in the docking study for the
comparison of the docking results due to unavailability
of NMDA receptor agonist (1, 21, 22). The binding mode
of PEAQX within the active site of the NMDA receptor
Figure 3. Molecular docking of risperidone with D2 receptor.
a) ligand-binding site of D2 receptor. b) 2D representation of
showed that the ligand interacts with the receptor via
D2-risperidone complex interactions. seven hydrogen bonds. These hydrogen bonds form
between the nitrogen atom and two carbonyl groups of
Apigenin showed the highest binding affinity (-10.3
quinoxaline-dione moiety and the residues Ser114,
kcal/mol) to D2 receptor in comparison to the rest of the
Thr116, Arg121 and Ser173 from one side and the three
active constituents that have subjected to molecular
oxygen atoms of the phosphoryl group and the residues
docking. The ligand has involved in three hydrogen bonds
Ser173, Thr174 and Tyr214 from the other side. All the
with the active site residues that contribute to the strong
amino acid residues act as hydrogen bond donors.
affinity to the D2 receptor. These hydrogen bonds form
Moreover, the phenyl ring of quinoxaline-dione moiety
between the two hydroxyl groups and the carbonyl group
interacts with the aromatic ring of His88 and the
of apigenin and the residues Cys118, Tyr416 and Ser193,
bromophenyl interacts with the Thr116 (Figure 5).
respectively. The residues Phe198, Trp386, and Phe390
stacked on the phenyl ring of apigenin vertically to form
three n-n T-shaped interactions indicating the significance
of the phenyl ring (ring B) in the deep pocket-binding of
D2 receptor (Figure 4).

Figure 5. Molecular docking of PEAQX with NMDA receptor.

a) ligand-binding site of NMDA receptor. b) 2D representation
of NMDA-PEAQX complex interactions.

On the other hand, apigenin interacts with the NMDA

Figure 4. Binding interactions of D2-apigenin complex. receptor through six hydrogen bonds. The hydrogen
a) 3D representation. b) 2D representation. bonds form between the carbonyl group and the three
Previous studies have illustrated the important role hydroxyl groups of the ligand and the residues Ser173,
of Asp114, Ser193, and Ser197 residues in the binding Thr174, Tyr214, Tyr245, Glu16 and Ala241, respectively
of D2 receptor agonists and antagonists (17, 19, 20). (Figure 6). Besides, the phenol ring of apigenin interacts
Therefore, tertiary nitrogen (eg. ethyl piperidine) has been with the residue Thr116, similar interaction was observed
suggested to be added as a bridge between position 1' with PEAQX.

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Current Trends in Biotechnology and Pharmacy 7
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DOI : 10.5530/ctbp.2020.4s.1

atom from Thr. Additionally, the phenyl ring of the ligand

interacts with the aromatic ring of Phe77 and Tyr58 and
the triazole ring interacts with the Lys156. Furthermore,
the chloride group displays an interaction (halogen
interaction) with the residue Gln204 (Figure 7). All these
interactions contribute to the strength of binding and the
high affinity of alprazolam towards the benzodiazepine
binding sites.
Figure 6. Binding interactions of apigenin with NMDA
receptor. a) 3D representation. b) 2D representation.
Both PEAQX and apigenin exhibited similar affinity
to NMDA receptor (binding energy -10.60 Kcal/mol for
PEAQX and -9.5 Kcal/mol for apigenin). The high
binding affinity is attributed to the strong interactions
with the binding site residues in which both ligands
interact with the residues Ser173, Thr174, and Tyr214
through hydrogen bonds. These hydrogen bonds are
important for stabilising the ligands in the active site of
Figure 7. Molecular docking of alprazolam with GABAA
the NMDA receptor. receptor. a) ligand-binding site of GABAA receptor. b) 2D
Two studies have studied the binding mode of NMDA representation of GABAA-alprazolam complex interactions.
receptor antagonists including PEAQX as glutamate site
The binding mode of apigenin within the
ligand. They found that the bromophenly group interacts
benzodiazepine binding site of GABAA receptor showed
with the carboxylate group of Glu781 from GluN1
that the two hydroxyl groups and the carbonyl group of
(different residue number is due to the differences
the ligand involve in four hydrogen bonds with the
between human and rat NMDA receptor). This interaction
residues His102, Ser159, and Ala161, respectively. Four
displaces three water molecules, results in water-mediated
aromatic interactions can be observed between the
polar interaction at Glu781 of GluN1 and Ser689 and
chromene moiety of the ligand and the aromatic rings of
Thr690 of GluN2A (23, 24). Based on this finding, the
the residues Tyr160 and Tyr210 (Figure 8). Besides, the
replacement of the hydroxyl group attached to the phenyl
dihydropyranone ring of the chromene moiety involves
ring (ring B) of apigenin with bromine group could
in an interaction with the sulfur atom of Met130 (Pi-Sulfur
increase the water-mediated polar interaction upon the
interaction).
displacement of water molecules by interaction of
bromophenyl.
Gamma-aminobutyric acid GABAA receptor
Alprazolam is a short-acting benzodiazepine that binds
to specific sites on the GABAA receptor. The binding of
alprazolam to these sites (known as benzodiazepine
binding sites) modulates the effect of GABAA receptors
and, thus, of GABAergic neurons resulting in sedative,
hypnotic and anxiolytic properties. In this study,
alprazolam has been used as a standard compound where
it displayed the highest binding affinity towards the Figure 8. Binding interactions of apigenin with GABAA
GABAA receptor (binding energy -11.2 Kcal/mol). The receptor. a) 3D representation. b) 2D representation.
ligand involved in several interactions with the In order to increase the binding affinity of apigenin to
benzodiazepine binding site of the GABAA receptor in the benzodiazepine binding site, an amino group can be
which the nitrogen atom of the triazole ring forms a added at position 3. This group will interact via a
hydrogen bond with residues Thr142. The ligand acts as hydrogen bond thus increasing the ligand's affinity and
hydrogen bond acceptor where it accepted the hydrogen strengthening the binding.

In silico study of the active constituents of Ocimum sanctum

Current Trends in Biotechnology and Pharmacy 8
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Phosphodiesterase (PDE) 10A

Mardepodect, also known as MP-10, is a selective
inhibitor of PDE10A that has been developed by Pfizer
for the treatment of schizophrenia. This compound
showed the lowest binding energy to PDE10A (-9.8 kcal/
mol). The low binding energy is attributed to the strong
interactions between the protein, PDE10A, and the ligand.
Mardepodect forms a hydrogen bond with Tyr524 where
the Tyr donated the hydrogen atom to the ligand. Two
aromatic interactions have been observed between the
quinoline group of the ligand and the aromatic rings of Figure 10. Binding interactions of apigenin with PDE10A. a)
Phe696 and Phe729. Moreover, the quinoline group 3D representation. b) 2D representation.
occupies the hydrophobic clamp (P-clamp) of PDE10A An in silico study conducted by Wu Q. et al. to
in which the Phe696 and Phe729 are on one side and investigate the various quinoline derivatives as potent
Ile692 on the other side (Figure 9). Occupation the P- and selective PDE10A inhibitors using 3D-QSAR,
clamp is very important for stabilizing the ligand in the molecular docking and molecular dynamics simulations
active site. methods has been reported. The authors have found that
the bulky substituents at position 8 and around the
pyridine ring (ring D) increase the biological activity of
the inhibitor (Figure 11). The replacement of the hydroxyl
group at position 8 with a bulky group declines the
inhibitory activity. The introduction of a heterocyclic ring
containing a hydrogen bond acceptor atom at position 18
on the ring D is desirable to enhance the binding affinity
and the inhibitory potency. And finally, the hydrophobic
interactions with Ile692 and Met713, as well as the
aromatic interactions with Phe696 and Phe729 are
Figure 9. Molecular docking of mardepotect with PDE10A. essential factors to increase the binding affinity (25).
a) ligand-binding site of PDE10A. b) 2D representation of
PDE10A-mardepotect complex interactions.
In contrast to mardepodect, apigenin interacts with
the active site residues of PDE10A through four hydrogen
bonds. These hydrogen bonds form between the carbonyl
group and the three hydroxyl groups of the ligand and
the residues Tyr542, Gln726, Asp674 and Leu635,
respectively. Apigenin also occupies the P-clamp and
Figure 11. The structural features of quinoline derivatives
interacts with the residues Phe696, Phe729 and Ile692. indicated by in Wu Q et al.
The phenol ring of the ligand shows an interaction with
the aromatic ring of Phe729. Furthermore, apigenin shows Apigenin fulfilled the key interactions including
unique interactions with the metal ions Zn+2 and Mg+2 at hydrogen bonding with invariant Gln726, and
the active site of PDE10A (Figure 10). These interactions, hydrophobic contacts with Ile692, Phe696 and Phe729
hydrogen bonding with the invariant glutamine, Gln726, and the aromatic interaction with Phe729. Modification
hydrophobic interactions with the three residues that form can be performed to increase the binding affinity of
the P-clamp and the interactions with the metal ions apigenin by replacing the hydroxyl group at position 7
contribute to the high affinity of the ligand to PDE10A. on ring A with a heterobenzene ring containing a hydrogen
bond acceptor atom.

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Current Trends in Biotechnology and Pharmacy 9
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DOI : 10.5530/ctbp.2020.4s.1

4. Conclusion 8. Lipinski, C. A., Lombardo, F., Dominy, B. W.,

The docking results of the ten active constituents of Feeney, P. J. (1997). Experimental and
Ocimum sanctum with the four macromolecular drug computational approaches to estimate solubility and
targets involved in the pathophysiology of schizophrenia permeability in drug discovery and development
have shown that apigenin possess the highest affinities settings. Advanced drug delivery reviews, 23:3-25.
towards the four targets. The high affinities are attributed 9. Pajouhesh, H., Lenz, G. R. (2005). Medicinal
to the hydrogen bonding interactions, aromatic chemical properties of successful central nervous
interactions and the hydrophobic interactions between system drugs. NeuroRx, 2:541-53.
the ligand and the receptors. All these interactions are
10. Burley, S. K., Berman, H. M., Bhikadiya, C., Bi,
important for stabilising the ligand in the active site of
C., Chen, L., Di Costanzo, L., et al. (2019). RCSB
the target receptor. This study has highlighted the binding
mode and the binding interactions between apigenin and Protein Data Bank: biological macromolecular
structures enabling research and education in
the four receptors, thus, may serve as a starting point for
fundamental biology, biomedicine, biotechnology
the identification of a potential treatment for
schizophrenia from natural sources. Further structure and energy. Nucleic acids research, 47:D464-D74.
modifications can be performed to improve the key 11. BIOVIA, D. S. discovery studio visualizer. San
interactions with each receptor to enhance the binding Diego: Dassault Systèmes,2016.
affinity and design a new compound that can act as an
12. Morris, G. M., Huey, R., Lindstrom, W., Sanner,
anti-schizophrenic drug.
M. F., Belew, R. K., Goodsell, D. S., et al. (2009).
5. References AutoDock4 and AutoDockTools4: Automated
docking with selective receptor flexibility. Journal
1. Kumar, A., Yadav, M., Parle, M., Dhingra, S., Dhull,
of computational chemistry, 30:2785-91.
D. K. (2017). Potential drug targets and treatment
of schizophrenia. Inflammopharmacology, 25:277- 13. Hunter, A. D. ACD/ChemSketch 1.0 (freeware);
92. ACD/ChemSketch 2.0 and its tautomers, dictionary,
and 3D plug-ins; ACD/HNMR 2.0; ACD/CNMR
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2.0. ACS Publications; 1997.
Pharmacy and Therapeutics (5th edition), Churchill
Livingstone Elsever. 14. O'Boyle, N. M., Banck, M., James, C. A., Morley,
3. Cohen, M. M. (2014). Tulsi-Ocimum sanctum: A C., Vandermeersch, T., Hutchison, G. R. (2011).
herb for all reasons. Journal of Ayurveda and Open Babel: An open chemical toolbox. Journal of
integrative medicine, 5:251. cheminformatics, 3:33.

4. Sharma, K., Parle, M., Yadav, M. (2016). Evaluation 15. Salehi, B., Venditti, A., Sharifi-Rad, M., Kr?giel,
of antipsychotic effect of methanolic extract of D., Sharifi-Rad, J., Durazzo, A., et al. (2019). The
Ocimum sanctum leaves on laboratory animals. therapeutic potential of apigenin. International
Journal of Applied Pharmaceutical Science, 6:171- journal of molecular sciences, 20:1305.
7. 16. Gupta, S., Black, D. W., Smith, D. A. (1994).
5. Kadian, R., Parle, M. (2015). Antipsychotic Risperidone: review of its pharmacology and
potentials of Ocimum sanctum leaves. International therapeutic use in schizophrenia. Annals of Clinical
journal of pharmaceutical sciences and drug Psychiatry, 6:173-80.
research 7:46-51. 17. Kalani, M. Y. S., Vaidehi, N., Hall, S. E., Trabanino,
6. Kadian, R., Parle, M. (2012). Therapeutic potential R. J., Freddolino, P. L., Kalani, M. A., et al. (2004).
and phytopharmacology of tulsi. International The predicted 3D structure of the human D2
Journal of Pharmacy & Life Sciences, 3. dopamine receptor and the binding site and binding
7. Wadood, A., Ahmed, N., Shah, L., Ahmad, A., affinities for agonists and antagonists. Proceedings
Hassan, H., Shams, S. (2013). In-silico drug design: of the National Academy of Sciences, 101:3815-
An approach which revolutionarised the drug 20.
discovery process. OA drug design & delivery, 1:3- 18. Wang, S., Che, T., Levit, A., Shoichet, B. K.,
7. Wacker, D., Roth, B. L. (2018). Structure of the D2

In silico study of the active constituents of Ocimum sanctum

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DOI : 10.5530/ctbp.2020.4s.1

dopamine receptor bound to the atypical schizophrenia. Annals of the New York Academy
antipsychotic drug risperidone. Nature, 555:269- of Sciences, 1003:318-27.
73. 23. Romero-Hernandez, A., Furukawa, H. (2017).
19. Duan, X., Zhang, M., Zhang, X., Wang, F., Lei, M. Novel mode of antagonist binding in NMDA
(2015). Molecular modeling and docking study on receptors revealed by the crystal structure of the
dopamine D2-like and serotonin 5-HT2A receptors. GluN1-GluN2A ligand-binding domain complexed
Journal of Molecular Graphics and Modelling, to NVP-AAM077. Molecular pharmacology, 92:22-
57:143-55. 9.
20. Ostopovici-Halip, L., Rad-Curpan, R. (2014). 24. Lind, G. E., Mou, T.-C., Tamborini, L., Pomper, M.
Modeling of ligand binding to dopamine D2 G., De Micheli, C., Conti, P., et al. (2017). Structural
receptor. Journal of the Serbian Chemical Society, basis of subunit selectivity for competitive NMDA
79:175-83. receptor antagonists with preference for GluN2A
21. Javitt, D. C. (2007). Glutamate and schizophrenia: over GluN2B subunits. Proceedings of the National
phencyclidine, N?methyl?d?aspartate receptors, Academy of Sciences, 114:E6942-E51.
and dopamine-glutamate interactions. International 25. Wu, Q., Gao, Q., Guo, H., Li, D., Wang, J., Gao,
review of neurobiology, 78:69-108. W., et al. (2013). Inhibition mechanism exploration
22. COYLE, J. T., TSAI, G., GOFF, D. (2003). of quinoline derivatives as PDE10A inhibitors by
Converging evidence of NMDA receptor in silico analysis. Molecular BioSystems, 9:386-
hypofunction in the pathophysiology of 97.

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Current Trends in Biotechnology and Pharmacy 11
Vol. 14 (5) 11-18, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.2

Comparative Study on Antioxidant and Anti-Inflammatory Activities of Red

and Brown Species of Areca catechu L. Nut Extracts
Parthasarathi Perumal1, Suresh Rathnasamy2, Balaji Tirupathi2, Purushoth Prabhu Thiraviam3,
Ashok Kumar Balaraman 4,5*, Vinothkumar S P 6, Senthil Kumar G P7
1Department of Plant Biology and Plant Biotechnology, Presidency College (Autonomous), Chennai-05, Tamil Nadu, India
2Department of Pharmacology, Greensmed Labs, Thoraipakkam, Chennai, Tamil Nadu, India
3Department of Pharmacognosy, CL Baid Metha College of Pharmacy, Thoraipakkam, Chennai, Tamil Nadu, India
4Faculty of Pharmaceutical Sciences, Block G, UCSI University, Kuala Lumpur, Malaysia
5School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia
6Department of Pharmaceutical chemistry, Erode college of pharmacy, Erode, Tamilnadu,
7Department of pharmaceutical chemistry, Bharathi college of pharmacy, Mandya, Karnataka

*Corresponding author: drashokbalaraman@gmail.com

Abstract LPS-induced nitric oxide production assay in RAW 264.7

The bioactive products from medicinal plants have rich cell line revealed that the brown areca nut extract reduces
sources of secondary metabolites helpful to the treatment the nitric oxide level than red areca nut. The maximum
of several biological applications. Traditionally, areca nuts inhibitory activity of brown areca nut extract from GC-
are the medicinal source that grows in India, Bangladesh, MS analysis shows the following active constituents i.e.
Sri Lanka, Myanmar, Taiwan, the tropical Pacific region, (2S,3S)-(-)-3-Propyloxiranemethanol, Propanedioic acid,
and the parts of East Africa. Several investigations propyl, 1,1-Dodecanediol, diacetate, 2-Nonenoic acid,
identified the secondary metabolites of Areca catechu Carbromal, 3-Nonenoic acid, Pentanoic acid, 2-
species inhibits anti-cancer activity, anti-inflammatory (Aminooxy)-, 2R,3S-9-[[1,3-Dihydroxy-4-fluoro-3-
activity, anti-diabetic activity, and anti-microbial activity, butoxy]methyl]guanine, respectively. The results suggest
respectively. The present study was aimed to investigating the brown areca nut extract inhibits the anti-inflammatory
the phytoconstituents along with in vitro antioxidant activity of nitric oxide LPS-stimulated RAW 264.7 cell
activity and potential anti-inflammatory activity of red line. Thus, the regular consumption of areca nut reduces
and brown varieties of ethanol extracts of areca nut for the inflammatory diseases.
the inhibition of nitric oxide production in LPS-stimulated Key words : Areca catechu L. nut; Phytoconstituents;
RAW 264.7 cells. The qualitative phytochemical analysis Antioxidant activity; RAW 264.7 cell line; Anti-
and thin layer chromatography differentiate the bioactive inflammatory activity; MTT assay; Lipopolysaccharide
components. The ethanol extract of both the species areca
1. Introduction
nut red and brown varieties have identified the
phytoconstituents such as tannins, cardiac glycosides, Areca nut or betel nut is the seed of the fruit areca
flavonoids, steroids, terpenoids, proteins, and phenolic palm (Areca catechu Linn. family Palmaceae), which
compounds, respectively. The TLC fingerprinting grows in Asia (India, Bangladesh, Sri Lanka, Myanmar,
observed six bands in the red areca nut and five bands Taiwan), tropical Pacific, and in some parts of East Africa.
were identified in brown areca nut. The antioxidant It is a common ingredient for many products and
activity of areca nut extracts was performed by DPPH approximately 600 million people are widely using it for
and ABTS radical scavenging assay. The IC50 values of chew products (13,22). Various parts of the tree (A.
DPPH assay from brown and red areca nut was found to catechu) have been used to prepare lubricants, dye-
be 24.54 ± 0.14 µg/ml, and 28.26 ± 0.11 µg/ml followed making, preparing pickles, weaves, and fuel wood etc.
by the standard ascorbic acid shows 12.14 ± 0.17 µg/ml, Traditionally the parts from A. catechu's roots, shoots,
respectively. The effective brown areca nut and the leaves, buds, and nuts are appropriately used for healing
moderate effects of red nut were found to be 26.55 ± 0.08 burn wounds and skin ulcers (6); the users of A. catechu's
µg/ml, 32.24 ± 0.01 µg/ml, and the standard ascorbic acid believe that the plant is also helpful for the supporting
showed 6.10 ± 0.01 µg/ml, respectively. Significantly, the the digestive system (7).
MTT assay shows the maximum concentrations of viable The major phytoconstituents of the areca nut are
cells from both the extracts on RAW 264.7 cells. The alkaloids, polyphenols, flavonoids, carbohydrates,

Comparative study on antioxidant and anti-inflammatory activities of Areca catechu

Current Trends in Biotechnology and Pharmacy 12
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minerals, fats, proteins, and crude fiber (27). Several Test for alkaloids
studies have reported that the secondary metabolites from To the crude extract, 2 ml of Mayer's reagent was
A. catechu has various biological applications like added, a dully white precipitate revealed the presence of
antioxidant, anti-inflammatory, anti-nematodal, anti- alkaloids.
diabetic, hypolipidemic, anti-hypertensive, anti-bacterial,
anti-fungal, anti-aging, anti-malarial, anti-viral, anti-HIV, Test for saponins
anti-aging, anti-depressant, anti-convulsant, treatment for 1 ml of the crude extract, 5 ml of water was added,
Alzheimer's, wound healing, anti-ulcer, anti-migraine, and the tube was shaken vigorously. The formation of
anti-hypertensive, anti-depressant, anti-allergic, stable foam indicated the presence of saponins.
anthelmintic, aphrodisiac, anti-venom, hepatoprotective,
and cytoprotective (16). Test for tannins
2 ml of 2% ferric chloride solution was added to the
Inflammation occurs to eliminate the preliminary
crude extract. Greenish black color indicates the presence
cause of cell injury due to the response of the body's
of tannins.
protection. Though, sometimes, inflammation it can also
become self-perpetuating and cause further inflammation. Test for cardiac glycosides
Chronic inflammation, involved in many diseases, for 1 ml of crude extract, 2 ml of glacial acetic acid
example, Alzheimer's, dermatitis, ulcerative colitis, containing a drop of ferric chloride. An equal volume of
inflammatory bowel disease, systemic lupus concentrated sulphuric acid was added from the sides of
erythematous, cancer, osteoarthritis, rheumatoid arthritis, the test tube. A brown color ring indicates the presence
and cardiovascular diseases (25). of cardiac glycosides.
In the previous study, the hydroalcoholic extract of
Areca catechu was reported to possess significant anti- Test for flavonoids
inflammatory activity (5). In this context, the present The crude extract was treated with a 10% sodium
study was planned to evaluate the in vitro anti- hydroxide solution; the formation of intense yellow color
inflammatory potency of red and brown Areca catechu indicates the presence of flavonoid.
nuts in murine macrophages raw 264.7 cell lines.
Test for phenols
2. Materials and Methods The crude extract was mixed with 3 ml of 10% lead
Collection and extraction of plant material acetate solution was added. A bulky white precipitate
indicates the presence of phenolic compounds.
The Areca catechu red and the brown nut were
collected from Dindigul, Tamil Nadu, India, and the Test for steroids
collected plant materials were taxonomically 1 ml of crude extract was dissolved in 10 ml of
authenticated. The red and brown areca nuts were washed chloroform and an equal volume of concentrated the
under the running tap water to remove dirt and then shade sulphuric acid was added from sides of the test tube. The
dried. The dried nuts were grinded to obtain coarse upper layer turns red and the sulphuric acid layer showed
powder by using the electrical blender. The powdered yellow with green fluorescence indicates the presence of
materials were stored in an airtight container for further steroids.
use. 100 g of each plant materials were subjected to
Soxhlet extraction using 95 % ethanol for 48 h. The Test for terpenoids
extracts were subsequently filtered through Whatman 1 ml of crude extract was mixed in 2 ml of chloroform,
No.1 filter paper and concentrated to dryness in a vacuum and concentrated sulphuric acid was carefully added to
under reduced pressure using a rotary evaporator (20). the sides of the test tube. A reddish-brown coloration of
Preliminary qualitative phytochemical analysis the interface indicates the presence of terpenoids.
Preliminary phytochemical analysis was carried out Test for quinones
to identify the phytoconstituents like alkaloids, saponins, The crude extract was treated with an alcoholic
tannins, cardiac glycosides, flavonoids, phenols, steroids, potassium hydroxide solution. The appearance of color
terpenoids, quinones, and proteins present in the ethanol ranging from red to blue indicates the presence of
extracts of areca nuts red and brown (10). quinones.

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Current Trends in Biotechnology and Pharmacy 13
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Test for Proteins The reaction mixture was kept at room temperature in
1 ml of crude extract was taken, and few drops of the dark for 30 min, then the absorbance was measured
freshly prepared Ninhydrin reagent were added and at 734 nm and the percentage of inhibition was calculated
heated. The appearance of purple color indicates the as follows (21) :
presence of proteins. control-test sample
% of free radical scavenging = ------------------------ x 100
Thin Layer Chromatography (TLC) analysis control
A 10 cm long TLC plate silica gel 60 F254 (Merk,
In vitro cell culture and maintenance
Darmstadt, Germany) was cut and marked carefully. 10
mg/ml of stock solution of the samples areca nut red and The murine macrophage RAW 264.7 cell line was
brown were spotted at about 0.5 cm from the bottom of purchased from National Centre for Cell Science (NCCS,
the plate with the help of a capillary tube. Butanol: acetic Pune, India) and maintained in DMEM supplemented
acid: water (4 ml: 1 ml: 2 ml) was used as a mobile phase. with 10% FBS, 100 µg/l streptomycin, and 100 IU/ml
The TLC plate was then placed in the chamber containing penicillin at 37°C in a 5% CO2 atmosphere.
the respective solvent system. After that, the plate was MTT cell viability assay
removed from the chamber and air-dried. The plates were
RAW 264.7 cells were seeded in 96-well plates at the
then observed under UV light at 254 and 366 nm. The
density of 5 x 104 cells/well. After 24 h of incubation,
iodine and potassium permanganate reagents were
the adhered cells were treated with various concentrations
sprayed on the TLC plate and were heated continuously
(3.125 - 100 µg/ml) of the extracts. 24 h later, after
to 100 ºC to visualize the spots. The visible colored spots
changing the medium, MTT was added to a final
were marked and the retention factor (Rf) was calculated
concentration of 5 mg/ml, and the cells were incubated
as follow (4) :
for 4 h at 37°C and 5% CO2. The medium was then
Distance traveled by substance
removed, and the formazan crystal was solubilized in
Rf = -----------------------------------------
Distance traveled by the solvent DMSO. The absorbance was measured at 570 nm using
a microplate reader (14).
Determination of in vitro antioxidant assay
Inhibition of nitric oxide (NO) production in LPS-
Free radical scavenging assay on DPPH
induced Raw 264.7 cell line
The DPPH? radical scavenging activity of the crude
The RAW 264.7 cells were seeded at a density of 5 x
extracts (Areca nut red and brown) was determined
104 cells/well in 96-well plate and incubated for 24 h at
according to the method described by some modification
37°C and 5% CO2. Then media of each well were removed
(21). Samples at different concentrations (3.125 - 100
and fresh FBS-free DMEM media were replaced.
µg/ml) were added to 2 ml solution of DPPH (0.4 mM)
Different concentrations of samples (3.125 - 100 µg/ml)
in methanol. Ascorbic acid was used as a reference
were prepared in FBS-free DMEM to give a total volume
standard. The tubes were shaken and allowed to stand
of 100 µl in each well of a microtiter plate. After 1 h
for 30 min at 37°C under dark conditions. The absorbance
treatment, cells were stimulated with 1 µg/ml of LPS for
was measured at 517 nm. % of inhibition was calculated
24 h. After, 100 µl of cell culture medium with an equal
as follow :
volume of Griess reagent in a new 96-well plate was
control - sample
% of inhibition = ----------------------- x 100 incubated at room temperature for 10 min. Then the
control absorbance was measured at 540 nm in a microplate
reader. The amount of nitrite in the media was calculated
ABTS+ scavenging assay from sodium nitrite (NaNO2) standard curve (14).
Radical scavenging assay of areca nut red and brown
Gas Chromatography-Mass Spectroscopy (GC-MS)
extracts were described by ABTS+ cation decolorization
analysis
assay (21). ABTS+ was developed by reacting 7 mM
ABTS + aqueous solution with 2.4 mM potassium The GC-MS analysis of the potent brown variety of
persulfate in the dark for 16 h at room temperature. areca nut extract during in vitro anti-inflammatory studies
Ascorbic acid was used as a reference standard. Samples on murine macrophages RAW 264.7 cell line was carried
at different concentrations (3.125 - 100 µg/ml) were added out using a Clarus 500 Perkin - Elmer (Auto system XL)
to 1 ml of diluted ABTS solution and mixed thoroughly. Gas Chromatograph equipped and coupled to a mass

Comparative study on antioxidant and anti-inflammatory activities of Areca catechu

Current Trends in Biotechnology and Pharmacy 14
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DOI : 10.5530/ctbp.2020.4s.2

detector Turbo mass gold - Perkin Elmer Turbomass 5.2 and potassium permanganate spraying reagents. The
spectrometer with an Elite - 5MS (95% Dimethyl ethanol extract of areca nut red showed six spots with Rf
polysiloxane), 30m x 250 m DF of the capillary column. values 0.26, 0.53, 0.66, 0.8, 0.86, and 0.93 respectively
The instrument was set to an initial temperature of 60 °C while the ethanol extract of areca nut brown showed five
and maintained at this temperature for 2 min. At the end spots with Rf values 0.26, 0.53, 0.66, 0.8, and 0.93,
of this period, the oven temperature was rose up 300°C, respectively (Figure 1).
at the rate of an increase of 5°C /min, and maintained for
6 min. Injection port temperature was ensured as 240°C
and Helium flow rate as one ml/min. The ionization
voltage was 70eV. The sample was injected in split mode
as 10:1. The mass spectral scan range was set at 40-600
(m/z). Using computer searches on a NIST Version Year
2011 were used GC - MS data library and comparing the
spectrum obtained through GC - MS compounds present
in the plant sample was identified (15).
Statistical analysis
All the data were expressed as mean ± standard
deviation. The data were evaluated with GraphPad Prism
5.01, GraphPad Software Inc. P value less than or equal
to 0.05 were considered significant. Figure 1 : TLC fingerprint of red and brown varieties of
3. Results and Discussion areca nut extracts (a) UV 254 nm; (b) Iodine spraying
reagent; (c) Potassium permanganate spraying reagent
Qualitative phytochemical screening
In vitro antioxidant activity
The present study revealed that ethanol extract of both
red and brown varieties of Areca catechu nut that contains DPPH assay
tannins, cardiac glycosides, flavonoids, phenols, steroids, The antioxidant properties of crude ethanol extract
terpenoids, and proteins. However, the phytoconstituents from Areca catechu nuts (red and brown) were determined
of alkaloids, saponins, and quinones were not found in using DPPH radical scavenging assay. The results shown
both the red and brown areca nut (Table 1). in Table 2 were found to be that the extract of brown
Table 1 : Qualitative phytochemical screening of ethanol areca nut showed better DPPH radical scavenging activity
extracts of red and brown varieties of Areca catechu compared with the red areca nut. The mean IC50 values
L. nut of brown and red areca nut were found in the order to be
Phytoconstituents Ethanol extract 24.54 ± 0.14 µg/ml, and 28.26 ± 0.11 µg/ml followed by
Red areca nut Brown areca nut the standard (ascorbic acid) which showed at 12.14 ±
Alkaloids - - 0.17 µg/ml, respectively.
Saponins - -
Tannins + + Table 2 : In vitro antioxidant activity of ethanol extracts
Cardiac glycosides + + of red and brown varieties of Areca catechu L.
Flavonoids + +
Antioxidant IC50 values of samples (µg/ml)
Phenols + +
activity Red Brown Ascorbic
Steroids + +
areca nut areca nut acid
Terpenoids + +
DPPH 28.26 ± 0.21 24.54 ± 0.14 12.14 ± 0.07
Quinones - -
ABTS 32.24 ± 0.01 26.55 ± 0.08 6.10 ± 0.01
Proteins + +
*Data represented as mean ± Standard deviation (n=3);
TLC analysis
statistically significant level at (p<0.05).
Thin-layer chromatography on silica gel revealed that
ABTS+ assay
to identify the bioactive molecules from medicinal plants.
The secondary metabolites of areca nut (red and brown) The IC50 values of ABTS+ scavenging assay of red
extracts were observed under UV light at 254 nm, iodine, and brown areca nut extracts are shown in Table 2. The

Parthasarathi Perumal et al
Current Trends in Biotechnology and Pharmacy 15
Vol. 14 (5) 11-18, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.2

most effective of brown areca nut and least activity of 84.52, 73.74, 65.26, 38.5, 16.92 and 3.41nitric oxide/ml),
red nut were found to be 26.55 ± 0.08 µg/ml, 32.24 ± respectively.
0.01 µg/ml, and the standard ascorbic acid showed 6.10
GC-MS analysis
± 0.01 µg/ml, respectively.
The Gas Chromatography-Mass Spectroscopy (GC-
In vitro cell line assay MS) of the brown areca nut extract of chromatogram
Ethanol extracts of red and brown areca nut on Raw shows in Figure 4. The identified secondary metabolites
264.7 cell viability assay follow (2S,3S) - (-) -3-Propyloxiranemethanol,
The ethanol extracts of Areca catechu nut (red and Propanedioic acid, propyl, 1,1-Dodecanediol, diacetate,
brown) against Raw 264.7 cell line was screened by MTT 2-Nonenoic acid, Carbromal, 3-Nonenoic acid, Pentanoic
assay. The IC50 values of both extracts red and brown acid, 2-(Aminooxy)-, 2R,3S-9-[[1,3-Dihydroxy-4-fluoro-
areca nut were found to be >100 µg/ml, respectively 3-butoxy]methyl]guanine, respectively (Table 3).
(Figure 2). The secondary metabolites of plant-derived chemical
compounds are important for drug development from
pharmaceutical industries (2, 23, 24). Tannins, alkaloids,
and flavonoids compounds were found to be present from
the ethanol extract of areca nut (23). Acetone extract of
both the leaves and root showed the presence of saponins
and tannins. Root extract also showed the alkaloids,
carbohydrates, terpenoids, and sterols. The
phytoconstituents has antimicrobial activity which
developed the zone of inhibition against bacteria viz.,
Klebsiella pneumonia, Staphylococcus epidermidis,
Bacillus subtilis, Enterococcus faecalis and Enterobacter
aerogenes, and fungus viz., Aspergillus fumigatus,
Aspergillus niger, Candida albicans, Candida glabrata,
and Candida tropicalis (1). The methanol extract of areca
nut shows tannins, glycosides, steroids, phenols, resins,
carbohydrates, and quinones. The presence of bioactive
components was reported strongly active against protein
denaturation assay (11). The phenolic compounds were
identified from water, methanol, and DMSO extract from
areca nut shows the Rf value 0.73, respectively (17).
Figure 2 : Different concentration of areca nut (red and Antioxidant activity plays an important role in
brown) extracts on RAW 264.7 cell line. (a) Represents therapeutic applications. It prevents oxidative stress and
red areca nut extract treated on RAW 264.7 cell line. control the various diseases from humans (26). Anthikat
(b) Represents brown areca nut extract treated on RAW and Michael, (2012) investigated the aqueous extract of
264.7 cell line areca nut shows the DPPH and ABTS radical scavenging
Inhibition of nitric oxide production in LPS-stimulated assay of IC50 value found to be 95 µg/ml, and ABTS
Raw 264.7 cell line assay found to be 9.5 µg/ml, respectively (3). The author
investigated methanol extract from the areca nut showed
In figure 3 represents there is no nitrite could be
the IC50 value of 0.021 µg/ml, respectively (9).
detected in the untreated Raw 264.7 cells. Once the cells
were stimulated with LPS, the increased level of nitrite During inflammation, macrophages play an important
was produced. The brown areca nut inhibited nitric oxide role in the body against pathogens (8). The secondary
formation more than red areca nut. Various concentrations metabolites of Areca catechu are pharmacologically
(3.125 - 100 µg/ml) of brown and red areca nut extracts important for several therapeutic purposes especially in
shows the percentage of nitrite level as follow (81.22, treating inflammatory conditions. In the present study,
69.26, 60.53, 33.59, 13.72 and1.95 nitric oxide/ml, and the MTT assay showed the cell viability of areca nut (red

Comparative study on antioxidant and anti-inflammatory activities of Areca catechu

Current Trends in Biotechnology and Pharmacy 16
Vol. 14 (5) 11-18, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.2

and brown) extracts have no toxicity against RAW 264.7

cells. The maximum concentration (100 µg/ml) of
samples showed the IC50 value ranging from > 100 µg/
ml, respectively. In this LPS-induced nitric oxide
production assay in RAW 264.7 cell line, the various
concentrations of brown areca nut significantly decreased
the nitric oxide content than red areca nut extract.
Similarly, it has been reported that ethanol extract from
areca leaf inhibits the NO levels against RAW 264.7 cell
line (18). The natural compound linalool shows anti-
inflammatory activity against the RAW 264.7 cells (12). Figure 3 : Inhibitory effect of red and brown varieties of
The extraction of hawthorn fruit aqueous fraction ethanol extract of areca nut on nitric oxide production in a
significantly decreases the NO production in RAW 264.7 culture medium of LPS-stimulated RAW 264.7 cell lin
cells (19). *Data represented as mean±Standard deviation (n=3);
statistical significant level at (p<0.05).

Figure 4 : GC-MS chromatogram of ethanol extract of brown areca nut

Table 3 : Compounds identified in the ethanol extract of brown areca nut

RT Name of the compound Molecular formula Molecular weight
27.073 [(2S,3S)-3-propyloxiran-2-yl] C6H12O2 116.16
methanol
27.368 Propanedioic acid, propyl C6H10O4 146.14
27.483 1,1-Dodecanediol, diacetate C16H30O4 286.41
27.603 2-Nonenoic acid C9H16O2 156.22
28.074 Carbromal C7H13BrN2O2 237.09
28.314 3-Nonenoic acid C9H16O2 156.22
28.424 Pentanoic acid, C6H13NO3 147.17
2-Aminooxy)-
28.674 2R,3S-9-[[1,3-Dihydroxy-4- C10H14FN5O4 287.25
fluoro-3-butoxy] methyl]
guanine

Parthasarathi Perumal et al
Current Trends in Biotechnology and Pharmacy 17
Vol. 14 (5) 11-18, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.2

4. Conclusion 2004. Inflammatory resolution: new opportunities

The conclusion of this present study indicates that the for drug discovery. Nat Rev Drug Discov., 3, pp.401-
brown areca nut from ethanol extract has more effective 16.
in reducing the level of nitric oxide in lipopolysaccharide 9. Hamsar, M.N., Ismail, S., Mordi, M.N., Ramanathan,
(LPS)-stimulated RAW 264.7 cells than red areca nut S. and Mansor, S.M., 2011. Antioxidant activity and
extract. The given evidence from GC-MS shows the the effect of different parts of Areca catechu extracts
brown areca nut extract comprises the compound on Glutathione-S-Transferase activity in vitro. Free
guanidine that supports the treatment of inflammatory Radicals and Antioxidants, 1(1), pp.28-33.
diseases. 10. Harborne, A.J., 1998. Phytochemical methods a
Acknowledgment guide to modern techniques of plant analysis.
springer science & business media.
The authors are thankful to Greensmed Labs,
Thoraipakkam, Chennai-97, for providing support to 11. Hemand, A, Krishnaja, M, and Anisree, P.A., 2015.
carry out the research work in their laboratory. Proximate analysis, preliminary phytochemical
screening and in vitro anti-inflammatory activity of
5. References premature areca nuts (Areca catechu). IJIPSR, 3(7),
1. Ambika, K, and Rajagopal, B., 2017. Antimicrobial pp.804-811.
and phytochemical properties of Areca catechu L. 12. Huo, M., Cui, X., Xue, J., Chi, G., Gao, R., Deng,
leaf and root extracts. Int. J. Curr. Res. Biosci. Plant X., Guan, S., Wei, J., Soromou, L.W., Feng, H. and
Biol., 4(4), pp.107-112. Wang, D., 2013. Anti-inflammatory effects of
2. Amudhan, M.S., Begum, V.H. and Hebbar, K.B., linalool in RAW 264.7 macrophages and
2012. A review on phytochemical and lipopolysaccharide-induced lung injury model.
pharmacological potential of Areca catechu L. seed. Journal of surgical research, 180(1), pp.e47-e54.
IJPSR, 3(11), pp.4151-4157. 13. Johnson, N.W. and Amarasinghe, H.K., 2016.
3. Anthikat, R.R.N. and Michael, A., 2012. Anti- Epidemiology and aetiology of head and neck
inflammatory and antioxidant effect of Areca cancers. In Head and neck cancer (pp. 1-57).
catechu. IJPSR, 3(7), pp.2031. Springer, Cham.
4. Barupal, T., Meena, M. and Sharma, K., 2019. 14. Joo, T., Sowndhararajan, K., Hong, S., Lee, J., Park,
Inhibitory effects of leaf extract of Lawsonia inermis S.Y., Kim, S. and Jhoo, J.W., 2014. Inhibition of nitric
on Curvularia lunata and characterization of novel oxide production in LPS-stimulated RAW 264.7 cells
inhibitory compounds by GC-MS analysis. by stem bark of Ulmus pumila L. Saudi journal of
Biotechnology Reports, 23, pp.e00335. biological sciences, 21(5), pp.427-435.
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Hadambar, A.A. and Thorve, V.S., 2010. Potential Phytochemical screening and GC-MS studies on the
analgesic, anti-inflammatory and antioxidant ethanolic extract of Turnera ulmifolia L. Int. J.
activities of hydroalcoholic extract of Areca catechu Pharm. Phytopharmacol. Res., 4, pp.179-181.
L. nut. Food and Chemical Toxicology, 48(12), 16. Keshava, B.S, Ashwin, D, Mythri, and S, Sukesh,
pp.3412-3417. B., 2018. Areca nut (Areca catechu L.) is not
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N.M, 2017. Efficacy and effect of Areca catechu J. Med. Health Res., 4(1), pp.35-40.
nuts. J. Terr. Mar. Res.,1, pp.50-53. 17. Koontongkaew, S., Thaweboon, B. and
7. Garg, A., Chaturvedi, P. and Gupta, P.C., 2014. A Tungtrongchitr, R., 1986. The toxicity of betel nut
review of the systemic adverse effects of areca nut extract determined by inhibition of microbial
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oncology: official journal of Indian Society of pp.419-429.
Medical & Paediatric Oncology, 35(1), pp.3. 18. Lee, K.P., Sudjarwo, G.W., Kim, J.S., Dirgantara,
8. Gilroy, D.W, Lawrence, T, Perretti, M, Rossi, A.G, S., Maeng, W.J. and Hong, H., 2014. The anti-

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inflammatory effect of Indonesian Areca catechu leaf immunomodulatory activity of areca nut extract from
extract in vitro and in vivo. Nutrition research and Aceh, Indonesia, against Staphylococcus aureus
practice, 8(3), pp.267-271. infection in Sprague-Dawley rats, Veterinary World,
19. Li, C. and Wang, M.H., 2011. Anti-inflammatory 13(1), pp.134-140.
effect of the water fraction from hawthorn fruit on 24. Silva, A.P., Nascimento da Silva, L.C., Martins da
LPS-stimulated RAW 264.7 cells. Nutrition research Fonseca, C.S., de Araújo, J.M., Correia, M.T.,
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20. Murthuza, S. and Manjunatha, B.K., 2018. In vitro Antimicrobial Activity and Phytochemical Analysis
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of Mesua ferrea, Saraca asoca, Viscum album & Frontiers in microbiology, 7, pp.963.
Anthocephalus cadamba in murine macrophages raw 25. Soonthornsit, N., Pitaksutheepong, C., Hemstapat,
264.7 cell lines and Wistar albino rats. Beni-Suef W., Utaisincharoen, P. and Pitaksuteepong, T., 2017.
University journal of basic and applied sciences, In vitro anti-inflammatory activity of Morus alba L.
7(4), pp.719-723. Stem extract in LPS-stimulated RAW 264.7 cells.
21. Perumal, P. and Saravanabhavan, K., 2018. Evidence-Based Complementary and Alternative
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extract of Piper betle L. leaves in catfish, Clarias 26. Taepongsorat, L. and Konsue, A., 2019. Biological
gariepinus. Asian J Pharm Clin Res, 11(3), pp.194- screening of tri-jannarose as a recipe from Thai
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R.A., 2015. Environmental toxins and the heart. In 27. Vanimakhal, R.R. and Ezhilarasi, B.S., 2016.
Heart and toxins (pp. 75-132). Academic Press. Phytochemical qualitative analysis and total tannin
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Current Trends in Biotechnology and Pharmacy 19
Vol. 14 (5) 19-31, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.3

Antioxidant and Antihyperlipidemic Activity of Methanolic Fraction of

Maytenus heyneana Root on STZ Induced Diabetic Wistar Rats
G Sumithira1, G P Senthil Kumar2, Maya Sharma3, B Krisnamoorthy4,
R Suresh5, Ashok Kumar Balaraman 6,7*
1Department of Pharmacology, The Erode College of Pharmacy, Erode, Tamilnadu, India.
2Department of Pharmaceutical Chemistry, Bharathi College of Pharmacy, Mandya, Karnataka, India.
3Department of Pharmaceutical Chemistry, Pacific college of pharmacy, Udaipur, Rajasthan,India.
4Department of Pharmaceutics, Sanjivani college of pharmaceutical sciences, Jhunjhunu, Rajasthan,India.
5Department of Pharmacology, Greensmed Labs, Thoraipakkam, Chennai, Tamil Nadu, India
6Faculty of Pharmaceutical Sciences, Block G, UCSI University, Kuala Lumpur, Malaysia
7School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia

*Corresponding author : drashokbalaraman@gmail.com

Abstract 1. Introduction
In earlier in-vitro anti diabetic and anti oxidant study At least 68 % of individual's age of 65 or more with
with serial fractionation of Maytenus heyneana root, diabetes dies from some type of coronary illness; and 16%
indicated methanol fraction as the most effective.In the dies of stroke. Grown-ups with diabetes are two to
present investigation, methanol fraction wasfurther tested multiple times more likely to die from coronary illness
for in vivo antioxidant and hyperlipidemic activity on STZ than Grown-ups without diabetes (1). The American Heart
induced diabetic rats. Secondary metabolites of MFMH Association considers diabetes to be one of the seven
wereidentified by phytochemical screening. Single dose significant controllable risk factors for cardiovascular
interperitoneal injection of STZ (45 mg/kg) was used to disease. There are afew factors that play important role
induce the hyperglycemia. To confirm the hyperglycemia, in pathogenesis of diabetes namelyoxidative stress and
blood glucose was measured,where as hyperglycemia hyperlipidemia leading to higher risk of complications
induced oxidation was the determined by using enzymatic (2). Oxidative stress ( ROS) owe to the prolonged
(SOD & CAT), non enzymatic (GSH) antioxidants and hyperglycemia is possibly the onset and development of
oxidative stress parameter was evaluated by LPO type 1 & II diabetes and its complications such as stroke,
(TBARS). To access the antihyperlipidemic activity, the retinopathy, nephropathy and neuropathy and also
TC, TG, HDL-C and LDL-C were measured. Histology itdevelop the vascular complications in diabetes
of liver was screened. Phytochemical studies revealed the particularly type 2 diabetes. In diabetes, oxidative stress
presence of alkaloids, saponins, flavonoids, phenols, is brought about by advanced glycation end products
cardiac glycosides and terpenoids. Treatment with the (AGEs) and free radical development via autoxidation of
methanolic fraction of Maytenus heyneana was effective unsaturated lipids in plasma and membrane proteins. It
in reducing the blood glucose level and also found to be might be amplified & propagated by an autocatalytic cycle
of metabolic stress, tissue damage, and cell death leading
potent antioxidant by significantly increase SOD, CAT
to a concurrent increment in free radical production and
& GSH and significant decrease in oxidative stress LPO.
compromised inhibitory and scavenger mechanisms,
The dose dependent MFMHon antihyperlipidemicactivity
which further exacerbate the oxidative stress (3).
was found, by ameliorating the increasedlevel of TC, TG,
LDL-C and increased the level of HDL-C. Degenerated Oxidative stress in diabetes is coincides with a
hepatocytes of STZ diabetic rats were restored to normal decrease in the antioxidant power. Diabetes tends to
morphological features as like reference standard decrease "good" cholesterol levels (HDL-c) and increase
glibenclamide. Hence from observation, the MFMH triglyceride and "bad" cholesterol levels (LDL-c), which
possesses antioxidant as well as antihyperlidimic activity builds the risk for heart disease and stroke, a condition
on STZ induced diabetic rats. called Diabetic dyslipidemia (4). Hyperlipidemia
associated with diabetes is a medical as well as social
Key words : Maytenus heyneana root, Antioxidant,
problem, leading to increasing both morbidity and
Antihyperlipidemic activity, Methanolic fraction, STZ
mortality. Hyperglycemia & hypercholesterolemia were
diabetic rats
related with oxidative change of LDL-C, protein

Antioxidant and antihyperlipidemic activity of Maytenus heyneana

Current Trends in Biotechnology and Pharmacy 20
Vol. 14 (5) 19-31, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.3

glycation, glucose -autoxidation.The significant objective induced oxidative stress and hyperlipidemia in diabetic
of diabetes treatment is to keep blood glucose, lipid wistar rats.
protein and lipids levels near to normal and scavenge
2. Materials and Methods
ordetoxify the free radicals. As of late, drug formulation
Chemicals and reagents
from natural plants, for treatment of diabetes mellitus
and other diseases, fascinated the attention of many All the chemicals and reagents used were of analytical
scientists (5). Since various studies have demonstrated grade. Streptozotocin (STZ) was purchased from Sigma
that hyperglycaemia in diabetes mellitus commits to (Sigma-Aldrich Ltd., Mumbai, India). Glibenclamide
oxidative stress, it has been suggested that the constant (GLA) was gifted from Hetro Pharm, Vishakpatnam.
supply of natural products as an antioxidants might
Preparation of samples
reduce the oxidative stress, and hence protect harmful
effects of oxidation stress on tissues (6, 7, 8). The selected plant fraction was evaporated to remove
the solvent and concentrated powder was used for in-
On account of strong antioxidant properties; during vivo evaluation. The plant fractions were designated as
recent years many species of plants have been studied MFMH (methanolic fraction Maytenus heyneana). The
for the management and treatment of various diseases. dried fraction powder were dissolved in saline solution
For this reason, research work is being conducted to and givento animals by orally.
suggest an approach that involves certain agents tending
to alleviate the hyperglycemia induced oxidative stress Toxicity studies and selection of the dose
and hyperlipidemia. Genus of Maytenus, aflowering and The acute toxicity studies was performed for the
fruiting plants consists of about 300 species and it is MFMHaccording to OECD-423 (Organization of
widely distributed in dry deciduous forests in tropical Economic and Cooperation Development), fixed dose
and subtropical areas and it has a wide range of guidelines. All the experiments on animals were designed
pharmacological actions such as antibacterial, and conducted in accordance to ethical norms approved
antileukemia, gastroprotective, anti-inflammatory, by (Institutional animal ethical committee) IAEC (NCP/
antiulcer. analgesic, antinociceptive, antidiarrheal (9), IAEC/2016-17-17). Generally, healthy adult albino
anti-malarial (10), anti amobic dysentery, anti- female rats of Wistar strain (n=3) were used. The animals
tuberculosis (11). Maytenus heyneana (Fig 1) armed were starved for 3 hours with free access to water only.
shrubs, synonym of Gymnosporia heyneana belongs to Single oral doses of MFMH were administered with a
Celastraceae family distributed mainly southern parts preliminary dose of 200mg/kg body weight (b.w). The
(Tamil Nadu, Kerala, Andhra Pradesh and Karnataka) of animals were monitored for mortality for 3 days. If
India. It has been used as antiarthritic, antidiabetic, anti- mortality was not ascertained the procedure is repeated
snake venom, immunity boosters, cancerous wound with higher dose (2000 mg/kg) of the selected plant
healing, and antidysentery (12). Research shows that bark fractions. General behaviors (13) was also observed
of Maytenus senegalensis, leaf of Maytenus emarginatus during the acute toxicity study such as Hypnotics,
and root of Maytenus putterkloides found to possess Sedative, Convulsion, Motor activity Ptosis, Analgesia,
antidiabetic activity. Maytenus senegalensis and Stupar reaction, Muscle relaxant, Pilo erection, Changes
Maytenus royleanus have reported to possess an in skin color, Stool Consistency and Lachrymal secretion.
antioxidant effects.
Animals
In vitro antidiabetic and antioxidant activities of
differentsolvent fractions of M. heyneana root have been Healthy, adult male albino wistar rats were selected
recorded in our previous studies (12). Furthermore, we for the study. They were housed in clean polypropylene
have observed significant increase in free radical cages under standard laboratory conditions with
scavenging activity against DPPH and ABTS and temperature around 26 °C. The rats were acclimatized to
inhibitory action against -amylase and -glucosidase the laboratory conditions prior to any treatment for 10
with the methanol fraction of M. heyneana root (12). days prior to any treatment and had ad libitum access the
Based on the in vitro anti diabetic and antioxidant food and water on a 12:12 day/night cycle. Throughout
properties of M. heyneana, the current experiment was the experiment cage bedding of animals was changed
designed to evaluate the protective effects of methanol frequently following the STZ induction of diabetes due
fraction of M. heyneana (MFMH) root against the STZ to polyuria

Sumithira et al
Current Trends in Biotechnology and Pharmacy 21
Vol. 14 (5) 19-31, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.3

Induction of Diabetes Serumpreparation

Diabetes was provoked in male rats by intraperitoneal The collected blood without EDTA was allowed to
injection (i.p) of 45 mg/kg of STZ (Sigma Aldrich clot by keeping undisturbed in a room temperature for
Chemical Co, St. Louis, MO, United states of America 30 min. The clot was removed by centrifuging for 10
(USA) reconstituted in 0.1 M cold citrate buffer (pH 4.5) min at 4000 rotation per minute (rpm) in a refrigerated
after an overnight fast. The glucose levels were centrifuge (REMI, INDIA). The supernatant serum was
determined in blood collected by tail vein puncture, 72 collected and used for serum lipid profile analysis.
hrs after STZ administration, utilizing Accu-check
Preparation of liverhomogenate
Advantage II clinical glucometer (Roche Diagnostics
Co.,). Fasting blood Rats with glucose 250 mg/dl were The liver was rapidly removed and perfuse
considered diabetic and were included in the study immediately with ice-cold 0.9% sodium chloride (NaCl).
A portion of the liver was homogenized in chilled Tris-
Study Design for in vivo Studies
HCl buffer (0.025 M, pH 7.5) using a homogenizer
To assess the antioxidant and antihyperlipidemic (REMI, Bombay, India) with a Teflon plunger. The
activity of the MFMH following 5 groups were made homogenate thus obtained was centrifuged at 5000 rpm
and six animals for each group (Table 1). for 10 min. The supernatant was collected and used for
Table 1. Depicts Study Design for in vivo studies oxidative stress parameter (Lipid peroxidation-TBARS
method).
Group 1 Normal control rats were given normal
saline (5 ml/kg/b.w) orally Hemolysate preparation for antioxidant activities
Group 2 Diabetic rats were given normal saline (5 Hemolysate was prepared by McCord and Fridovich
ml/kg/b.w)orally et al., proposed method (14). 5 ml of blood sample was
Group 3 Diabetic rats were given MFMH collected in an EDTA bulb, 2 ml was separated for
(Maytenus heyneana methanol fraction) estimation of GSH and the remaining sample is
200mg/kg dissolved in normal centrifuged at 2000 rpm for 10 min. The plasma was then
salineorally removed. The Red blood cell (RBCs) was washed twice
with normal saline. Nearly 3.5 ml of purified water was
Group 4 Diabetic rats were given MFMH added to the RBCs. This hemolysate was utilized for
(Maytenus heyneana methanol fraction)
catalase estimation. Then the HB (hemoglobin)
400mg/kg dissolved in normal
concentration of the remaining hemolysate was measured.
salineorally The concentration of HB was adjusted to 10gm/dl. To
Group 5 Diabetic rats given Glibenclamide (GLB) 0.5ml of Hb adjusted hemolysate, 3.5 ml of ice-cold water
at 10 mg/kg dissolved in 0.5% Carboxy (distilled water) was added and mixed well on a
methyl cellulose (CMC)orally cyclomixer until all the particulate matter disappears. To
this solution, 1 ml of ethanol was added and again mixed
The MFMH, Glibenclamide were administered by
on cyclomixer. Finally, added 0.6ml of chloroform and
orally to their respective group of rats up to 14 days.
mixed well. Then this hemolysate was centrifuged for
During the study the rats were fed with 10% w/v glucose
10 min at 3000 rpm. The clear supernatant was used to
solution, in their cages in order to avoid the diabetic
estimate the superoxide dismutaseactivity.
shock. The blood was collected from tail of the rats. The
glucose level was measured by glucometer (Accu Chek). Estimation of blood glucose and antioxidant parameters
At the 14th day (end of the study period), all the animals The blood was collected from tail vein at 0, 7th and
were anaesthetized with diethyl ether and the blood 14th day and the level of blood glucosewere monitoring
samples were obtained by puncture of retro-orbital plexus using Accu-chek advantage glucometer and Roche
using glass capillary (20mm) and stored with or without glucostrip (15).
ethylenediaminetetraacetic acid (EDTA) (2mg/ml) to
Enzymatic antioxidant reduced glutathione (GSH) in
evaluate the lipid profile and antioxidant parameters.
blood was estimatedspectrophotometrically by Beutler
Then, they were sacrificed and theliver was collected
et al 1983 (16). Non enzymatic antioxidant such as
immediately after dissection and stored at -20 oC for
Superoxide dismutase (SOD) in hemolysate was
further histopathological studies and small portion of liver
estimated by the method of Marklund and marklund
was dissected for tissue homogenate preparation.
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Current Trends in Biotechnology and Pharmacy 22
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1974 (17) and the catalase (CAT) in hemolysate assay of phytoconstituents such as saponins, phenols,
was estimated by the method of Aebi 1983 (18). The terpenoids, flavonoids and tanninsin the MFMH root may
oxidative stress parameter Lipid peroxidation (LPO) in contribute to its hypoglycemic activity (12), and these
liver homogenate was estimated colorimetrically by compounds have been shown to beresponsible for
TBARS (thiobarbituric acid reactive substance) by the hypoglycemic activity in Momordica charantia (27).
method of Niehaus and Samuelsson 1968 (19). Presence of flavonoids and phenols in MFMH could be
a reason for scavenge active oxygen species and
Estimation of lipid profile parameters
effectively prevent from oxidative cell damage (12). Our
The total serum concentration of cholesterol results are confirmed with those announced by
wasdetermined by enzymatic CHOD-PAP method Ramachandran V and Saravanan R 2013 (28).
(Allainet al.,1974) (20) using commercially available
biochemical kits (Recon Diagnostics). The total serum Acute toxicity studies
triglyceride was estimated by GPO-PAP (Bucolo G and WHO recommends that medicinal plants would be
Harold D 1973) (21) using commercially available dominant source to obtain a wide range of drugs.
biochemical kits (Recon Diagnostics).The serum HDL- Therefore, medicinal herbs must be investigated for its
C was estimated by precipitation method and followed better understanding of their pharmacological activity,
by CHOD-PAP method described by Burstein et al., effectiveness and safety and well involves the recognition
1970(22). The serum LDL-Cand VLDL-C was estimated of a dose level that causes mortality (29).Thus, acute
by calculation using the empirical relationships according toxicity studies of MFMH was performed on adult albino
to Friedewald's formula (Friedwald et al., 1972) (23). female rats of wistar strain were used because of minor
Preparation of liver for histopathologicalstudies difference in sensitivity between sexes; therefore females
rats were commonly used for its more sensitiveness (30)
The whole isolated liver was washed in saline
to finding out the quantitative aspect of lethal dose (LD50)
solution and 10% formalin was used for fixing and
and observing the behavioral studies. From the results
paraffin for embedding and sections were cut of 3-5 m
(Table 2) it was observed that MFMH did not shown any
thickness and stained with haematoxylin & eosin are
sign of toxicity, mortality or a dying status in a female
basic dye and acidic dye respectively. The sections were
rat. The LD50 of MFMH roots was observed to be greater
microscopical studied at 10× and 40× magnifications for
than the test dose (2000mg/kg). Therefore, low dose of
the islet cell characteristics using a binocular compound
200mg/kg b.w and high dose of 400 mg/kg b.w were used
microscope.
for our studies.
Statisticalanalysis
Hyperglycemic activity
In animal study, data were denoted as mean ± Standard
Error Mean (SEM). The statistical analysis was done by Hyperglycemia was induced in male rats, because
using Analysis of Variance (ANOVA), followed by female rats are less susceptible to streptozotocin at high
Dunnett's test for multiple comparisons using Graph Pad doses, this decreased susceptibility of females may be
Prism (version 5) software and values of Probability Value presence of estradiol, which able to protect the oxidative
(P)< 0.05 were considered as statistically significant. stress induced pancreatic cells apoptosis (31) the single
dose of IP injection of 45 mg/kg of STZ reconstituted in
3. Results and Discussion the 0.1 M cold citrate buffer at pH, in order to prevent
Diabetes mellitus is a chronicmetabolic disorder that the degradation and maintain the stability of STZ (32).
has arguably achieved epidemic proportions. It affects After administration STZ, triphasic response of blood
more than 371 million people globally, and itis projected glucose was produced. Initially raise of blood glucose
to affect 522 millionpersons by the year of 2030 (24) due to breakdown of liver glycogen, secondly persistent
(25) (26). For some decades, phyto-therapy has played hypoglycemia and it was more pronounced in fasted rats,
asignificantrole in the treatment of the disease particularly which may cause early mortalities, 10% glucose solution
in poor resource countries. Clearly, theidentification of has given to experimental rats to prevent from mortality.
plant materials that can treat the diabetes mellitusand its Finally, permanent hyperglycemia was observed (33).
complications would seven million of people life, This STZ hyperglycemia was might be due to cytotoxic
particularly in developing countries, from untimely death. effect of on GLUT2 receptor present in cell membrane,
From our earlier reported studies showed the presence liver and kidney, this cytotoxic action is more pronounced

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on cell membranes, which in turn to triggers the multiple The oxidatative stress LPO in diabetic group shows
pathways such as advanced glycation end (AGE) product, significant increases (P<0.001) from 11.61 to 24.06 nM/
hexosamine pathway, polyol pathway, protein Kinase C mg. Whereas, the diabetic groups with MFMH treatment
pathway, and poly adipose ribose polymerase pathway at different doses of 200 and 400 mg/kg b.w decreases
all pathways contribute towards oxidative stress by the LPO to 19.60 and 13.34 nM/mg respectively, which
generation of ROS in mitochondria. The results disclosed are statistical significance of p<0.001, compared with the
that single dose STZ is highly effective cytotoxic agent diabetic group.
for pancreatic cells and complications in liver, kidney Diabetic group shows enzymatic antioxidant
and other vital organs such as heart, brain and muscles superoxide dismutase was significantly declined
(34). STZ - diabetes has been described as a useful (P<0.001) to 9.89 IU/mg compared to that of the normal
experimental model to study the antidiabetic activity of control group 20.30 nM/mg. However, the SOD in
several agents (35).Glibenclamide the reference standard hemolysate was significantly raised (P<0.001) to 12.89
is like other sulphonylureas, is effective against in mild &15.15 nM/mg in groups with MFMH treatment at the
diabetic condition, but ineffective against severe diabetic concentrations of 200, 400 mg/kg of body weight
condition, where the cells were destroyed completely respectively. Whereas, catalase (CAT) is pronounceable
(34). dropped (P<0.001) to 14.86 nM/mg, when it is compared
The change in level of blood glucose of control and to the normal group 27.87 nM/mg. The levels of CAT in
experimental groups of diabetic rats were observed are hemosylate is observed significantly increased (P<0.001)
represented graphically in Fig 2. On the day 0, the level to 21.47 & 24.36 nM/mg in the diabetic animal groups
of blood glucose was significantly raised (P<0.001), treats with MFMH at the dose of 200, 400 mg/kg
compared to control group. Remarkably (P<0.001) respectively. In case of non-enzymatic level of antioxidant
increase in blood glucose level on 7th day was noticed as glutathione (GSH) was significantly (P<0.001) decreased
288.83 mg/dl on diabetic control group, when compared to 31.45 nM/mg, compared to normal group (52.07 nM /
with normal control group. However, treatment given to mg). The glutathione in hemolysate was significantly
diabetic rat with MFMH at the dose 200 & 400 mg/kg, increased (P<0.001) to 37.89 and 46.41 nM /mg in the
the level of blood glucose was significantly (P<0.001) diabetic groups treatment with MFMH at the
decreased to 234.50 & 184.67 mg/dl respectively. In concentrations of 200, 400 mg/kg respectively. Also
diabetic group, the level of blood glucose was further noticed from the results that effect of MFMH (400mg)
significantly increased (P<0.001) to 308.67 mg/dl at the on LPO,CAT,SOD & GSH exhibits identical to standard
last of 14th day. When treated STZ diabetic rats with glibenclamide and normal group.
MFMH at the dose 200 mg/kg, the level of blood glucose Chronic hyperglycemia can generates oxidative stress,
was significantly (P<0.001)declinedto179.33mg/dl and which gives rise to cellular tissue damage through
it was further significantly turned down to 147.50 mg/dl important five mechanisms (37). The highly complexed
with the case of treated STZ diabetic rats with MFMH at antioxidant systems in the human body (enzymatic and
a dose 400 mg/kg. Thus the antidiabetic action of MFMH non-enzymatic), which work together synergistically to
may act directly by stimulating the insulin secretion in defend the cells and organs against free radical
existing active cells. Further, the fundamental damage.STZ acts selectively on -cells of pancreas lead
antihyperglycemic activity of MFMH was associated with to increase ROS in pancreas & other tissues are resulted
increase in plasma insulin level and or insulin like extra in tissue damage & increases LPO i.e., membrane lipid
pancreatic action of reduction of hepatic gluconeogenesis oxidation. Increase LPO is an indication of decreasing
& glycogenolysis and with increase utilization of glucose defense mechanisms of both enzymatic and non-
by peripheral tissue. Our results are in confirmed with enzymatic antioxidants (38). MDA has reported as a
those announced by Raju Patil et al., (36). primary biomarker of the free radical mediated lipid
Antioxidant activity damage and oxidative stress. The elevated level of MDA
in an erythrocytes ofthe diabetic rats was observed, this
Table 3. Illustrates that the oxidatative stress LPO
could be a reason of ROS mediated chain propagation
level in liver homogenate, enzymatic antioxidant
reaction might be lost activity of defense antioxidant
superoxide dismutase, catalasein hemolysateand non-
system to adequately scavenge the free radicals produced
enzymatic antioxidant Glutathione inwhole blood.
by STZ induced diabetes. Conversely, decreased in the

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serum LPO level and MDA was monitored in MFMH glibenclamide. A similar finding has been reported on
and glibenclamide treated diabetic animals, this perhaps the study with Mangiferin (41).
by the inhibition of the propagation chain reaction of LPO
Lipid profile assessment
(39). Additionally, possible way of oxidative stress in
hyperglycemia also may by the account of an autoxidation Fig 3. Represents effect of leaves of EAFOG and
of glucose, redox imbalances, reduced concentration of MFMH root on total cholesterol, triglycerides, HDL-c
LMW antioxidants, such as GSH (reduced glutathione) &LDL-C. The level of cholesterol was significantly high
and reduced activities of antioxidant defense enzymes (P<0.001) from 91.14 to 145.28 mg/dl while in diabetic
such as SOD (superoxide dismutase) and CAT (catalase) animals, the cholesterol have significantly (P<0.05)
(40). The enzymatic antioxidant (GSH) and the non- reduced to 130.58 by the treatment EAFOG at the dose
enzymatic antioxidants (SOD, CAT) are the cellular of 200mg/kg. Similarly significant declined (P<0.001)
defense mechanism that acts on the free radical by in cholesterol (110.28 mg/dl) in Streptozotocin induced
removal of hydroxyl, hydrogen peroxide and superoxide diabetic group treated with extracts MFMH at the dose
radicals. SOD is the key enzyme in detoxifying the level of 400 mg/kg.
superoxide radical and converts into hydrogen peroxide Whereas, the serum triglyceride levels was
and water. H2O2 is highly reactive small molecule which significantly (P<0.001) increased to 146.11 mg/dl in
formed as results of the energy metabolism of natural diabetic animals. However, there is significantly
product which in an excessive level may cause (P<0.001) reduced to 111.42 and 105.19 mg/dl while
appreciableamount damages to RNA, DNA, lipids, and in treatment with MFMH at different doses of 200 and
proteins (41). CAT is the main regulator of H 2 O 2 400 g/kg respectively. In case of serum HDL-c level is
metabolism and which enzymatically neutralizes it, thus seen significantly declined (P<0.001) to 26.56 mg/dl in
protects the pancreatic cells from hydroxyl radicals' the diabetic group. In the case of diabetic control animals,
attack (42) (43). The diabetic groups shown decrease in which were fraction of EAFOG at the dose of 200, 400
erythrosylate SOD and CAT concentration. This might mg/kg are observed in increases upto 37.24 & 38.31 mg/
be the cause of an excess production of superoxide dl respectively. The results of HDL-c showed,
radicals which inhibit the activity of the SOD and CAT significantly increased (P<0.001) to 96.90 mg/L, when
activities (44), which consequently enhanced after it is compared with normal group. The increased in LDL-
treatment with MFMH and glibenclamide, be a sign of c level is significantly lowered (P<0.001) to 86.61 and
strife against ROS generation. GSH act as reducing agent, 66.01 mg/dl by MFMH at the dese level of 200 and 400
which detoxify the H2O2 in the existence of glutathione mg/kg respectively.
peroxidase (GPx) enzyme (44).The current study shows Dyslipidemia is a well known complication of diabetes
decreased in concentration of erythrocytes GSH in (47) and accompany with hyperglycemia characterized
diabetic control, this could be because of increased by increased LDL-c, increased triglycerides (TGs) and
scavengingactivity in the repair of free radicals caused decreased cholesterol. In present study, the elevated serum
biological damage. Reduction in antioxidant capacity with cholesterol, triglycerides and LDL-c while decreased
increased oxidative stress could be related to the levels of HDL-c was observed in Streptozotocin induced
complications in patients with diabetes like oxidative diabetic rats, this observation also conforms with reports
DNA damage and insulin resistance (45) due to reduced of earlier studies (48) In diabetic induced rats, the glucose
antioxidant potential of blood, diabetes complications level is increased together with the lipids level shows
increase which include nephropathy, cardiovascular that the insulin dependent tissue plays main role in
disease, nerve damage and blindness. Thus, the increasing glucose and lipid homeostasis (49). Insulin resistance
the occurrence of diabetes is a remarkable health concern initiates the intracellular hormone-sensitive lipase, which
beyond the disease itself (46). Whereas, MFMH and in turn to increases the release of NEFA (non-esterified
glibenclamide treatments restore the GSH concentration, fatty acid) from triglycerides in adipose tissue (50); the
maybe by reason of the up regulation of GSH redox raised NEFA consecutively increases the formation of
system in liver to counteract oxidative stress. The hepatictriglyceride. The NEFA is converted to cholesterol
treatment and MFMH also observed declines in the level and phospholipids when combining hepatic triglyceride
of LPO and elevated concentrations of SOD and CAT in and released as in form of lipoproteins into blood stream.
diabetic animals as equal as reference standard Hyperlipidaemia was produced in Streptozotocin diabetic

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DOI : 10.5530/ctbp.2020.4s.3

group might be regarded as the result of uninhibited scattered inflammatory cells amidst these hepatocytes.
actions of lipolytic action of hormones in the fat storage The veins in the central are appears and few sinusoids
(51). LDL-c oxidation positively and HDL-C dysfunction are seen dilated (Fig 4). The GLB treated liver section
negatively contributes to risk of cardiovascular disease shows, recovery of liver structure and normal granule of
(52). The increased TG in the diabetic groups was glycogen contents present in hepatocytes. Liver
observed that may be chance as to the increased the parenchyma shows intact architecture. The perivenular
absorption & production of TG in form of chylomicrons hepatocytes and mid zonal hepatocytesappears.
and decreased uptake of TG by peripheral tissues. The As the sensitive organ liver have a great capability to
elevated total cholesterol may be because of increased detoxify the toxic substances, excrete the xenobiotic and
small intestione absorption of cholesterol (53). Whereas, its metabolitesand also plays vital roles in carbohydrate
treatment with MFMH at different doses of 200 and 400 metabolism (55). Consequently, the pathological damages
mg/kg of b.w have not just decrease the total cholesterol, that are impose on the liver by hepatotoxic agents show
TG and LDL-c but also increases the HDL-c, HDL-c plays to be fatal in diabetic conditions. The liver is most
a key role in transport of peripheral tissue cholesterol frequently damaged organ during diabetes, as a
back to liver by the pathway termed as "reverse consequence of increased oxidative stress, which is
cholesterol transport' which consider to be a cardio generated by free radicals and dysregulation of immune
protective lipid. A highly negative relationship between function (56) (57). Additionally long term insulin
HDL-c and the occurrence of atherosclerosis are reported resistance and impaired insulin secretion contribute the
and our findings are in an agreement with earlier studies deterioration of diabetes by impairment in mitochondria
of Poonam S et al., (54). Interestingly, MFMH treated of islet cells (58) along with mitochondrial dysfunction
groups shown significant development in the lipid profile of insulin-sensitive tissues such as muscle,liver and heart
comparable to glibenclamide in the control of (59). Hepatocytes degeneration, cytoplasm vacuolization,
hyperglycemia (diabetic dyslipidaemia) this might be due congested central vein and hypertrophied Kupffer cells
to presence of active metabolites. This hypolipidemic were noticed in diabetic group.Interestingly these severely
effect could represents the protective mechanism against damaged pathological conditions which were seen in
atherosclerosis development. diabeticrats are restored almost to its normal liver
Assessmentof histopathology of liver morphological features by MFMH and glibenclamide,
which depicts evidence of cellular regeneration. These
Histopathology of liver revealed the existence of
results are echoed by the earlier published reports (60)
normal histological structure, regular distinct hepatocytes
(61).
with sinusoidal spaces arranged radically around the
central vein, normal hepatic parenchyma with normal 4. Conclusion
portal triads and sinusoids in normal control group. In Overall, the administration of MFMH led to lower
diabetic group, the section liver shows that hepatocytes the ameliorated hyperglycaemia and promotedthe
degeneration, cytoplasm vacuolization, cloudy swelling, correction of dyslipidemic activity in the treatment
loss of glycogen granule and congested central vein. A groups. In addition, MFMH increases the level of SOD,
hemorrhagic focus has replacing necrotic hepatocytes. CAT and GSH- activity and reduced level of LPO activity
Infiltration of leucocytes in portal triads is observed. Also and consequently could alleviate complications of liver
visualization of Kupffer cells are hypertrophied along this might be due to the improvement of glycaemia
with hepatic sinusoids, hepatocytes with various degree promoted by MFMH. The imbalance between the
of degeneration. Treatment with MFMH at the low dose generation of ROS and enzyme activity be controlled and
of 200 mg/kg shows Leucocytic infiltration in portal triad also protect against the development of atherosclerosis
and thickening of the portal artery wall. Kupffer cells on diabetic rats.Hence, it may be concluded that MFMH
are Hypertrophied also seen along with the sinusoids and root at 400 mg/kg b.w exhibited promising antioxidant
partially distorted architecture, leucocytic infiltration and and hyperlipdimic activity in STZ-induced diabetic rats.
proliferation of bile canaliculi is also visualizes. In case However, Further studies is needed to isolate the active
of high dose of MFMH 400 mg/kg shows, shows mild components of MFMH.
inflammatory infiltration in periportal region. There are

Antioxidant and antihyperlipidemic activity of Maytenus heyneana

Current Trends in Biotechnology and Pharmacy 26
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Fig 1. (a) Moderate-sized armed shrub with terminating short shoots

(b) Exhibits large dominant taproot.

B lo od g lucose
400
0 day

300 7 t h day
1 4 t h day
200

100

Gro up s
Fig 2. Effect of leaves of MFMH root on the level of Blood Glucose

Fig 3. Represents effect of MFMH root on total cholesterol, triglycerides, HDL-c &LDL-C

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Fig 4. (a) : Normal control rats, (b) STZ induced diabetic control rats,
(c) Diabetic rats treated with MFMH 200mg/kg (d) Diabetic rats treated with MFMH 400mg/kg.
(e) Diabetic rats treated with reference standard glibenclamide 10mg/kg

Table 2. Specifies the observation of behavioral studies of EAFOG and MFMH on rats

General Behaviors Time (hrs)

1 2 3 12 24 72
Hypnotics - - - - - -
Sedative - - - - - -
Convulsion - - - - - -
Motor activity - - - - - -
Ptosis - - - - - -
Analgesia - - - - - -

Stupar reaction - - - - - -
Muscle relaxant - - - - - -
Pilo erection - - - - - -

Change in skin - - - - - -
color
Stool Consistency - - - - - -
Lacrimal secretion - - - - - -

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Table 3. Effect of EAFOG and MFMH on Antioxidant parameters SOD, CAT, GSH & Oxidatative stress parameter
LPO

Oxidative
Antioxidant parameters
stress
Groups parameter
SOD CAT GSH LPO
(IU/mg of (nM of H2O2 (nM /mg of (nM of
protein) decomposed/ protein) MDA/mg of
min/mg of protein) protein)
Group I Normal 20.30±0.41 27.87±0.24 52.07±0.99 11.61±0.99
control Normal saline
10ml/kg

Group II Diabetic 9.89±0.34 14.86±0.48 a*** 31.45±0.69 24.06±0.69

control a*** a*** a***
STZ 45mg/kg

Group III 12.89±0.24 21.47±0.66 b*** 37.89±0.25 19.60±0.25

STZ +MFMH- b*** b*** b***
200mg/kg

Group IV 15.15±0.39 24.36±0.25 b*** 46.41±0.50 13.34±0.50

STZ +MFMH- b*** b*** b***
400mg/kg

GroupV 19.59±0.75 26.68±0.75 b*** 50.41±0.89 11.85±0.89

STZ +GLB- b*** b*** b***
10mg/kg

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Current Trends in Biotechnology and Pharmacy 32
Vol. 14 (5) 32-37, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.4

Development and Assessment of Modified Glover Nilsson Vaping Behavioural

Questionnaire Among Malaysian Electronic Cigarettes Users
Aziz-ur-Rahman1*, Mohamad Haniki Nik Mohamed2, Syed Mahmood3, Ashok Kumar Balaraman4,
Muhammad Ahsan Iftikhar Baig1
1Department of Clinical Pharmacy, Faculty of pharmaceutical sciences
UCSI University, Kuala Lumpur, Malaysia
2Department of Pharmacy Practice, Kulliyyah of pharmacy, International Islamic University of Malaysia (IIUM),
Kuantan Campus, 25200, Pahang, Malaysia
3Department of Pharmaceutical Engineering, Faculty of engineering technology
University Malaysia Pahang, Gambang, 26300
4Department of Pharmaceutical Biology, Faculty of pharmaceutical sciences
UCSI University Kuala Lumpur, Malaysia

Corresponding author email: aziz@ucsiuniversity.edu.my

Abstract scale needs to be validated further with a large sample

The Glover Nilsson smoking Behavioural size for further robust authentication.
Questionnaire (GNSBQ) is the commonly used scale to Key words : Electronic cigarette, Nicotine, Vaping,
assess the behavioural nicotine dependence through Behaviour, Dependency, Scale
conventional tobacco cigarettes (TCG). But the GNSBQ 1. Introduction
does not evaluate the subject's behavioural dependence Nicotine dependence is a complicated experience that
to nicotine that administered via electronic cigarette (EC). encompasses both physiological and behavioural
The study aim was to develop and assess an equivalent components [1]. The diagnostic and statistical manual
modified Glover Nilsson vaping behavioural of mental disorders edition 5 (DSM-5) stated that tobacco
questionnaire (GNVBQ) which measures the nicotine use disorder is assigned to individuals who are dependent
behavioural dependency via EC. The modified developed on the drug nicotine. According to DSM-5, there are 11
GNVBQ scale is identical to the original GNSBQ. The possible criteria for nicotine dependence of which at least
scale scores indicate the EC nicotine behavioural 2 must exist in the last 12 months among smokers:
dependency ranking as slight (1-6), mild (7-11), moderate 1)Tolerance, indicated by increased amounts of tobacco
(12-22), strong (23-33) and very strong (> 33). The scale to achieve the desired effect or a markedly diminished
piloted among 15 EC single users i.e. used only EC. The effect with continued use of the same amount of tobacco
assessment of the scale did among 69 EC single users 2) Withdrawal symptoms, shown by either the
and observed their nicotine behavioural dependency status characteristic withdrawal syndrome or the use of tobacco
for a one-year period. The modified scale revealed a to relieve the withdrawal symptoms. 3) Craving or urge
satisfactory Cronbach's alpha value of 0.74. Further test- to use tobacco 4) Tobacco has taken in larger amounts or
retest reliability of the scale showed an acceptable over longer periods of time 5) Persistent desire or
spearman's rank correlation coefficient value of 0.75 (p unsuccessful efforts to cut down nicotine 6) Spending
> 0.05). A one-year observation showed that out of 69 much time to obtain tobacco 7) Recurrent tobacco use
EC single users, 11 single users completely stopped resulting in a failure to fulfil the major role obligations at
nicotine intake. The EC users who completely stopped work, school, or home 8) Continued tobacco uses despite
nicotine intake after one year had a low nicotine having persistent or interpersonal problems caused e.g.,
behavioural dependency scores between 7-11 measured disputes with others for tobacco use 9) Important social,
by the modified GNVBQ scale. The modified GNVBQ occupational, or recreational activities are given up or
scale has accurately identified the behavioural dependence reduced because of tobacco use 10) Recurrent of tobacco
of nicotine that administered via EC. Therefore, as per use in situations in which it is physically hazardous 11)
the current study results, the modified GNVBQ scale may Tobacco use is continued despite knowledge of having a
useful to predict the nicotine behavioural dependency that persistent or psychological problem that likely to cause
administered through various EC products. However, the by tobacco use.
Development and assessment of modified glover nilsson vaping
Current Trends in Biotechnology and Pharmacy 33
Vol. 14 (5) 32-37, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.4

The criteria from 1 to 3 such as tolerance, withdrawal If a smoker shows, the high score on both the scales
and craving show the physiological dependence to requires both behavioural counselling and
nicotine. Whereas other characteristics signify the pharmacological treatment.
behavioural dependence to nicotine. The physical Literature findings revealed lacking the scale that
dependence on nicotine is measured by various scales. measures the behavioural dependence to nicotine that
The most applied scales are Fagerstrom test for nicotine administered through electronic cigarette (EC). The
dependence (FTND) [2], nicotine dependence syndrome GNSBQ measured the nicotine dependence via tobacco
scale (NDSS) [3] and Wisconsin inventory of smoking cigarettes (TCG) but do not point out the subject's
dependence motives (WISDM-68)[4]. The world health behavioural dependency to nicotine by using EC. There
organisation (WHO) international classification of are various nicotine EC products in the markets with
diseases-10 also assess the physical dependence on distinct concentration [12-13]. Therefore, it is a necessity
nicotine [5]. However, none of the earlier mention scales to assess the behavioural dependency of nicotine by
addresses the nicotine behavioural element of dependence means of EC for consumer's safety and public awareness
[6]. The behavioural component of nicotine addiction is purposes. In order to treat effectively the nicotine
demonstrated through the smoker's patterns of tobacco dependency among vapers, it is suitable to recognize both
use such as smoking style, opening the cigarette pack, behavioural as well as physical dependence to nicotine
pulling a cigarette out of the pack, hand to mouth cigarette via EC. Thus, the current study developed a scale by
action, smoking along with some daily activities like modifying the existing GNSBQ scale which predicts the
drinking coffee or driving a car. The behavioural pattern behavioural dependence of nicotine that administered
of smoking includes the cognitive, social, and behavioural through the various EC products.
effects correlated with nicotine dependence. Smoking
cessation products can fulfil the physical dependence to 2. Materials and Methods
nicotine but not yet replaced the behaviour involved with Scale development
nicotine dependency [7].
The scale was modified from GNSBQ [10]. The
It has well documented that smokers are physically modified scale scores like original GNSBQ which
and behaviourally dependent on nicotine. Many earlier consists of 11 questions and measures the nicotine
studies have shown that smoking cessations medications behavioural dependence using EC. The scale has a total
like nicotine replacement therapy, varenicline, and score of 44. Each question is ranked on a 0-4 scale. The
bupropion along with behavioural and motivational values in scale described as 0=, not at all, 1=somewhat,
counselling showed a good quitting rate as compared to 2=moderately, 3=very much and 4=extremely
medication alone [8-9]. The FTND scale used extensively respectively. The modified Glover-Nilsson vaping
in the smoking-related research to assess the physical behavioural questionnaire (GNVBQ) explained about
dependence to nicotine, whereas behavioural dependence vapers relationship related to feeling, perception and any
on smoking measured by the Glover-Nilsson smoking rituals associated with EC. Higher the score, more
behavioural questionnaire (GNSBQ) [10]. The GNSBQ behavioural nicotine dependence to EC, whereas lower
scale exchange smoker and cigarette relationship related case shows low nicotine behavioural dependence via EC.
to feeling any rituals associated with smoking. The Scoring for behavioural dependence follows ranking as
GNSBQ consists of 11 questions and responses to the slight (1-6), mild (7-11), moderate (12-22), strong (23-
questions on a 0-4 scale, where 0=not at all, 1=somewhat, 33) and very strong (> 33). The modified scale has a
2=moderately, 3=very much and 4=extremely. GNSBQ variation as compared to the original GNSBQ scale. The
upper score indicates higher behavioural dependence on principal change is replacing the word tobacco cigarette
smoking, whereas lower numerical shows low with an electronic cigarette.
behavioural dependence. Scoring for GNSBQ follows
Validity and Reliability of the scale
ranking as mild (1-11), moderate (12-22), strong (23-33)
and very strong (> 33). The smokers who score more on The developed scale was sent to 5 experts in associated
GNSBQ scale may treat suitable through behavioural disciplines to evaluate the face and content validity. The
counselling[11]. However, a smoker revealed higher score reviewer individually ranked each item of the scale by
on FTND display high physical dependence to nicotine, using a four-point grading system i.e. 1=not relevant, 2=
which can manage well by pharmacological interventions. somewhat relevant, 3=relevant, 4=highly relevant. The

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Current Trends in Biotechnology and Pharmacy 34
Vol. 14 (5) 32-37, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.4

Item Content Validity Index (I-CVI) was applied to assess language. The face validity was determined on a 1-4 scale
the validity of each item. The items on the scale with which denoted as 4=strongly agree, 3=agree, 2=disagree,
CVIs ranged from 0.80 to 1.00 had retained. The and 1 strongly disagree. All experts were graded three or
calculated average I-CVI scale value was 0.95 with four on the scale and indicated the questionnaire is
average content validity ratio (CVR) of 0.99 [14]. The feasible and appeared understandable to the desired
Face validity of the scale was also assessed in terms of population. The items and scoring guide of the modified
feasibility, readability, clarity and uniformity of the developed scale has shown in table 1.
Table 1: Modified Glover Nilsson Vaping Behavioural Questionnaire

Scale: 0=Not at all, 1= somewhat; 2=moderately ; 3=Very much; 4=Extremely

How much do you value the following (Specific to Questions 1-2).

1. My vaping habit is very important to me 0 1 2 3 4

2. I handle and manipulate my electronic cigarette (EC) as part of the 0 1 2 3 4
ritual of vaping
Please indicate your choice by circling the number that best reflects your
choice. (Specific to Questions 3-11).
Scale: 0=never; 1=seldom; 2=sometimes; 3=often; 4=Always
3. Do you place something in your mouth to distract you from vaping? 0 1 2 3 4
4. Do you reward yourself with vaping after accomplishing a task 0 1 2 3 4
5. If you find yourself without vaping, will you have difficulties in 0 1 2 3 4
concentrating before attempting a task?
6. If you are not allowed to vape in certain places, do you then play with 0 1 2 3 4
your EC?
7. Do certain environmental cues trigger your vaping, e.g., favourite 0 1 2 3 4
chair, sofa, room, car, or drinking alcohol
8. Do you find yourself vape routinely without craving? 0 1 2 3 4
9. Do you find yourself placing an EC in off mode in your mouth and 0 1 2 3 4
sucking to get relief from stress, tension or frustration, etc
10. Does part of your enjoyment of vaping come from the steps while 0 1 2 3 4
switching on your EC?
11. When you are alone in a restaurant, bus terminal, party, etc., do you 0 1 2 3 4
feel safe, secure, or more confident if you are holding an EC?
Total
Scoring
1-6 : Slight ; 7-11 : Mild ; 12-22 : Moderate; 23-33 : Strong ; > 33 : Very Strong

Pilot study periods and the scale is stable over time. Finally, the
The modified developed scale was piloted among 15 approved piloted scale was tested among 69 EC single
EC single users i.e. who use only EC verified by exhaled users and observed their behavioural dependence of
carbon monoxide (CO) of < 8 ppm. The internal nicotine that administered through EC.
consistency of the scale was determined by using
Assessment of the developed scale
Cronbach's alpha which revealed a satisfactory value of
0.74. Moreover, the reliability of the scale was further The study participants were enrolled from EC sales
accomplished by the test-retest method by followed up points, vaping stations surrounding at the Kuantan and
among all 15 users of the pilot study after a period of two Pekan districts, province Pahang, Malaysia by distributing
weeks interval. The reliability of the scale after two-week flyer related to the study. The study partakers have
intervals showed a spearman's rank correlation coefficient contacted the researcher and clarified any queries related
value of 0.75 with p > 0.05. The P-value indicated that to the study before the enrolment process. The
there was no significant variation at two different interval information sheet and consent forms were given to the

Development and assessment of modified glover nilsson vaping

Current Trends in Biotechnology and Pharmacy 35
Vol. 14 (5) 32-37, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.4

committed participants. The participants who met the via EC. The developed scale has good internal consistency
eligibility criteria were selected for the enrolment process. shown by Cronbach alfa value of 0.74 and the reliability
The socio-demographic details, history of smoking packs value of 0.75 respectively. The developed scale revealed
per years, and EC were reported. In the previous study, that the participants who had low behavioural dependence
the investigator assesses the modified FTND scale to stopped completely the nicotine intake. The GNVBQ can
determine the physical dependence to nicotine that be considered as a predictive scale of behavioural nicotine
administered via EC. In the current study, the researcher dependence. The previous studies indicated that nicotine
among the same cohort assessed the behavioural users who had high behavioural dependence have more
dependency to nicotine by modified GNVBQ scale. The desire for nicotine as compared to low nicotine users,
participants were observed for a one-year period. At week irrespective of their physical dependence. The modified
52, all the subjects were verified through the biochemical GNVBQ scale accurately assess the desire for nicotine
validation by measuring the CO test irrespective of any via EC by the behavioural gesture. It is believed that the
smoking status. At week 52, self-reported complete desire for nicotine is the most challenging symptoms of
nicotine quit participants additionally evaluated by the nicotine withdrawal that may hinder vapers to completely
saliva NicAlert® strips to check their full nicotine-free stop nicotine intake via various EC products.
status. At week 52, subjects without biochemical The current study findings also suggested that the use
validation were documented as nicotine users for analysis of nicotine via EC induced behavioural nicotine
purpose. Intention-to-Treat (ITT) analysis was applied dependency. The current study results revealed that at
to evaluate the final outcomes of the study. That means the end of week 52 most of the participants were still
those users who lost to follow-up were categorised as using nicotine. The study supports the notion that nicotine
nicotine users. Also, participants who withdrew from the plays a vital role in the popularity of EC use [13]. The
study were omitted for analysis. current study also showed that the use of ECs even for
Ethical Committee Approval an extended period was unable to crack the nicotine
addiction. It is also possible that most of the study
The study was approved by the research ethics
participants were using ECs as an alternative device for
committee (IREC) of Kulliyyah of Medicine,
nicotine. The current study findings are comparable with
International Islamic University of Malaysia (IIUM)
previous literature studies which too indicated that
Kuantan on 9th October 2014, with IREC registration
administration of nicotine via vaping will not induce
number 302. The study was also registered in the National
complete nicotine cessation [15-16]. Therefore, there is
Medical Research Registration with NMRR.number:15-
a possibility that vaping may uphold nicotine addiction
180-24825.
due to availability of e-liquids in various flavours and
might renormalize smoking behaviour among vapers.
3. Results and Discussion
Therefore, tobacco control policy makers should restrict
After a one-year observation period out of 69 EC the sale of nicotine-containing EC to non-smokers and
single users, 11 EC single users were completely stopped youngsters to prevent its abuse. Most of the study
nicotine intake. The other 24 remained as EC single users, participants believed that EC is a worthy choice to curb
15 shifted to dual use i.e. using both EC and TCG and 19 smoking because it does not induce physical or
relapsed to TCG. All study participants nicotine status behavioural dependency. Though, the current study
validated by CO level and saliva cotinine tests results failed to establish the spotless capability of EC in
respectively. Those EC single users who completely complete nicotine cessation as compared to other FDA-
stopped nicotine intake after the one-year period had a approved smoking managements medications trials [17-
low nicotine behavioural dependency score of 7-11 18].
measured by modified GNVBQ scale. Figure 1 shown
participants behavioural nicotine dependency score The current study results indicated that it is necessary
measured by modified GNVBQ scale at baseline and their to identify nicotine behavioural dependence among
nicotine status at week 52. vapers in addition to physical nicotine dependence. Since
medication therapy along with behavioural and
The current study results demonstrated that the motivational counselling revealed a satisfactory nicotine
modified GNVBQ is a reliable and valid scale to assess cessation results in previous literature studies [8-9].
the nicotine behavioural dependence that administered Therefore, more customized nicotine cessation treatment

Aziz et al
Current Trends in Biotechnology and Pharmacy 36
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DOI : 10.5530/ctbp.2020.4s.4

is needed in vapers based on their nicotine physical vs quit. In addition, more clinical trials are needed to verify
behavioural dependency. the concept of nicotine behaviourally dependent. This is
The current study is not without limitations. The required to determine the exact treatment needed for
current study was conducted among small vapers nicotine-dependent users either by behavioural
population in just two locations of Malaysia i.e. Pekan intervention or pharmacological or a combination of both.
and Kuantan. Therefore, the scale needs to verify on a 4. Conclusion
large sample size before applying in any EC related
The modified GNVBQ scale may precisely identify
studies. Further, factor analysis and construct validities
the behavioural dependence to nicotine which
are needed to achieve for robust authentication of scale.
administered via EC. Therefore, the modified GNVBQ
Moreover, the existing study population mainly consists
scale can apply to predict the behavioural dependence
of male even though our enrolment was not aimed at male
on nicotine that administered through various EC
vapers. Therefore, it would be difficult to generalize these
products. However, the scale needs to be validated further
results to female vapers regardless of their intention to
with a large sample size for further authentication.

Figure 1 : participants EC behavioural nicotine dependence measured by modified Glover-Nilsson Vaping behavioural
questionnaire (MGNVBQ) scale at baseline and their nicotine status at week 52.

5. References 4. Piper, M. E., Piasecki, T. M., Federman, E. B., Bolt,

1. American Psychiatric Association (2013). Diagnostic D. M., Smith, S. S., Fiore, M. C., & Baker, T. B.
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7. Nakamura, M., Oshima, A., Fujimoto, Y., Maruyama, 13. Royal College of Physicians of London. (2016).
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acetylcholine receptor partial agonist, in a 12-week, 14. Lawshe, C. H. (1975). A quantitative approach to
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R. E. (2000). Treating tobacco use and dependence: and Medicine Division, National Academies of
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Therapeutic advances in the treatment of nicotine National Academies Press.
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Reliability and validity of the Glover-Nilsson in Kuantan and Pekan, Malaysia: a six-month
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11. Roberts, N. J., Kerr, S. M., & Smith, S. M. (2013). K. S., & Mayo, M. S. (2002). Sustained-release
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Current Trends in Biotechnology and Pharmacy 38
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DOI : 10.5530/ctbp.2020.4s.5

ATR-FTIR Spectroscopy Methods for Determination of Aminoglycoside

Antibiotics in Ophthalmic and Parenteral Preparations
with Full Partial Least Squares Algorithm
Yau Xin Yi1, Bontha Venkata Subrahmanya Lokesh1*, Gabriel Akyirem Akowuah1
1Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences
UCSI University, 56000 Kuala Lumpur, Malaysia
*Corresponding author : bvslk71@yahoo.com

Abstract 1. Introduction
Attenuate Total Reflectance-Fourier Transform AGAs are potent broad-spectrum bactericidal agent
Infrared (ATR-FTIR) Spectroscopy methods were that are particularly active against aerobic, gram-negative
developed and validated for the analysis of selected bacteria and act synergistically against certain gram-
aminoglycoside antibiotics (AGAs) as per ICH guidelines. positive bacteria well. Other than being antibacterial agent
This non-destructive technique was utilized as a direct for human health, the AGAs are widely used in veterinary
measurement of sample after grinding to prepare the medicine and agriculture as well (1-3). They are
powder required priorto read on diamond reader of ATR- chemically characterized by two or more amino sugars
FTIR spectrophotometer. Dilutions were made using linked by glycosidic bonds to an aminocyclitol
component. The cyclitol is 2-deoxystreptamine in most
spectroscopic grade potassium bromide (KBr) on %w/w
of the AGAs, one exception being streptomycin, which
basis.Linear concentration ranges from 0.25 - 15.0 (%w/
has streptidin moiety. Figure 1 portrays the main chemical
w)were observed with high regression value (r2> 0.995)on
moiety and addition of different functional groups at active
their unique peak bands using full spectrum partial least
sites in different types of AGAs.
squares (PLS)algorithm. These methods weredisplayed
low limit of detection (LOD) of 0.20-0.25 (%w/w) and
low limit of quantification (LOQ) of 0.60-0.80 (%w/w)
for three selected AGAs (Gentamicin, Tobramycin and
Kanamycin). The intra-day and inter-day precision values
were found with %RSD less than 4.00.Assay studies were
shown withtheir mean recovery estimated at 100.727 ±
2.65% for gentamicin, 101.04±1.076% for tobramycin,
and 100.67% ±1.08%at 95% confidence intervals for the
quantification of gentamicin and tobramycin in
ophthalmic preparations and kanamycin in injections.
These methods were found to be simple, faster, non-
destructive with full sample recovery and eco-friendly.
Hence, proposed ATR-FTIR spectroscopy methods can
be used for routine analysis and samples approval for
regulatory and therapeutic drug monitoring studies during
treatment with AGAs with good precision and accuracy
comparatively with conventional LC-MS methods, which
are time consuming and complex method parameters and
also needs dedicated space, funding, manpower and time Figure 1. Chemical Structures of AGAs
consuming for immediate results. The selected AGAs that will be tested in this study
Keywords : ATR-FTIR Spectroscopy, Aminoglycoside are gentamicin, tobramycin and kanamycin. Gentamicin
antibiotics, estimation, validation, non-destructive consists of three different closely related aminoglycoside
technique, injections and ophthalmic preparations sulphates, Gentamicin C1, C2, and C1a, obtained from

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Current Trends in Biotechnology and Pharmacy 39
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Micromonosporapurpurea and related species, with with OMNIC software to assist users in easy calibration
molecular formula C21H43N5O7 and average 477.60g/ and manipulation of spectra(17). It is widely applied in
mol molecular weight(4).According to United State ingredient identification, grade verification, content
Pharmacopoeia (USP) 39, the content of gentamicin C1 uniformity and concentration prediction.
is between 25-50%; the content of gentamicin C1a is The direct ATR-FTIR spectroscopic study was
between 10-35%; and the sum of content of gentamicin conducted for standard gentamicin and tobramycin with
C2a and C2 is between 25-55%. Slight variation in their respective pharmaceutical formulation available in
percentage was claimed by European Pharmacopeia(5- local pharmacy, which is ophthalmic solution. In the
7). On the other hand, tobramycin is a broad-spectrum
proposed method, the specific functional groupsof
antibiotic produced by Streptomyces temerarious which
gentamicin, tobramycin and kanamycinthat represent for
is effective against gram-negative bacteria, especially the
its identification and quantification were explored with
pseudomonas species. Its molecular formula is
PLS algorithms of good linear regression coefficient.
C18H37N5O9 with molecular weight of 467.51g/mol(8).
Drying process of the ophthalmic preparation and
For the determination of gentamicin and its impurities, injection sampleswas optimized to ensure the samples in
USP 39 and European Pharmacopoeia 6.8. (Ph. Eur. 6.8.) solid form after drying process. All method parameters
recommended ion pairing HPLC with pulsed were set and followed by ICH guidelines of analytical
amperometry detection (HPLC-PAD), whilemicro method development(18).ATR-FTIR spectroscopy
biological assay is described for quantitative analysis. methods were statistically compared with independent t-
The USP monograph of kanamycin and quantification in
test to observe the sensitivity among AGAs. Direct ATR-
formulation also utilize HPLC-PAD for assay (5,9-
FTIR methods were simple, fast, non-destructive and
11).The method of pre-column derivatization with 2,4-
accurate. determination of selected AGAs.
dinitrofluorobenzene followed by reversed-phase HPLC
with UV detection is described in USP for tobramycin 2. Materials and Methods
analysis (12).The reported methods for analysis for the Materials and reagents
selected AGAs cannot be used for routine analysis due
Gentamicin, tobramycin and kanamycin sulphate
to its complex method parameters and lack of UV
standard were purchased from Sigma-Aldrich company
chromophore. Also, expensive pH-stable column with the
(USA); IR spectroscopic grade potassium bromide
need of frequent maintenance for good column efficiency
(KBr)99.999%, and HPLC grade ethanol 95% were
is a factor to be considered as well (10,13,14). Microbial
purchased from Merck company (Darmstadt, Germany).
assay requires 24-72 hours incubation time, and is subject
to variability (agar thickness, inoculums concentration, Ophthalmic solution (Beagenta eye/ear drops 0.3% w/v
incubation temperature, exposure-time duration) and formulation in 5mL bottle, and Tobrex solution 0.3% w/
biological error (15). Therefore, development and v formulation in 5mL) were procured from local
validation of a good, fast, sensitive and economical pharmacies in Kuala Lumpur, while Meiji kanamycin IM
analytical method is crucial for the routine quality injection formulation in 3mL was procured
assessment. fromSomedico Pharma companyto be used for
quantification determination in compliance with the
The non-destructive attenuated total reflectance
claimed amount in the formulation.
Fourier transform infrared (ATR-FTIR) spectroscopy is
rapidlygaining popularity in the development of Instrumentation
alternative methodology for the quantification of Infrared spectra were obtained with NicoletTM iS5
pharmaceutical drug for its suitability in FTIR spectrometer (Thermo Scientific, Madison,
commercialization. The environmental-friendly method Wisconsin, USA) with iD5 ATR accessory featuring a
also reduces the usage of hazardous chemical reagent diamond crystal. The spectra were collected against the
(16). Therefore it is considered as green analytical diamond window background controlled by OMNIC
chemistry. Diamond sample reader facilitates the analysis software for spectra collection and TQ Analyst software
of all sample state and directly provide refined spectra for data processing (Thermo Scientific, USA). The
without sample destruction. PLS regression algorithm is instrument is equipped with iD5 ATR accessory featuring
a commonly used multivariate calibration method in
a top plate diamond crystal with a fixed angle of incidence
baseline correction which utilizes TQ software integrated
of 42o.

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Current Trends in Biotechnology and Pharmacy 40
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DOI : 10.5530/ctbp.2020.4s.5

ATR-FTIR method development the concentration range and analysis of each

All spectra were recorded at 4cm-1 resolution with concentration was repeated three time at different time
average of 20 scans in the range of 4000-600cm-1. In point. Intra-day repeatability and inter-day precision were
addition, a background scan of air was applied prior each measured and recorded in terms of relative standard
sample scan. An analytical balance with 0.01 mg deviation (%RSD). Quantification of AGA formulation
readability and minimum weight of 2mg in fine range was also done based on calibration curve. The samples
was used. All measurements were taken at ambient were dried into powder form in Binder RE53 Gravity
temperature and samples were stored in a desiccator when Convection Oven at 60 oc to remove water in the sample
not in use. and vortexed equally before direct analysis of the powder.
The powder was spiked with 0.4, 1 and 5% w/w of AGA
AGA standards were diluted with IR gradeKBr to
standards respectively. The FTIR spectrum of each spiked
prepare a series of concentration of drug standard ranging
sample was collected three times (n=3) and the accuracy
0.25% to 15% w/w. Different amounts of selected AGAs
was expressed as the mean percentage recovery of three
were weighted and mixed with proportionate amount of
replicates for each spiked sample.
pure KBr, to get a total weight of 0.1 g of each working
standard. The mixture of KBr and gentamicin standard Quantification of AGA based on FTIR method
was done in a dry mortar till homogenization reached. After validation process, quantification of selected
The mixture was then transferred to Eppendorf tube to AGAs in theirformulations (ophthalmic preparations and
be mixed using vortex for 5 minutes. Each working injection) was carried out after evaporating the liquid
standard was applied on the ATR-FTIR instrument to scan sample under thermostatically controlled oven and the
for its spectrum within the range of 4000 to 650cm-1. A dried powder was diluted with pure KBr as per calibration
background scan of air was done prior to each scan of curve and the content was determined by directly place
working standard. All spectra were recorded at 4cm-1 the grinded powder on the diamond reader accessory to
resolution with average of 20 scans per spectrum. The get the corresponding FTIR spectrum. The quantification
diamond crystal of the ATR accessory was cleaned by was conducted in triplicate and results were expressed
Ethanol 95% intermittently between each scan. The as mean ± standard deviation.
working standards were used to observe the standard
calibration curve with points ranging from 0.25-15% w/ 3. Results and Discussion
w. From OMNIC and TQ Analyst software, two types of Gentamicin
region including peak area and fixed height location were Method development
recorded for identification of suitable peak to generate During the development of ATR-FTIR method, the
calibration curve with R2 value higher than 0.995. The spectra of gentamicin standard were collected initially
measured data of standards and calculated standards were as a step in the qualification and quantification
used for linear regression analysis. process.Full spectrum of pure gentamicin sulphatewas
Validation of ATR-FTIR methods scanned from 4000-650 cm-1 and overlaidwith external
spectrum obtained from Spectrabase (CAS Registry
According to ICH guidelines, validation criteria
Number of 1405-41-0) (19). The overlaid spectrum was
included are inter-day and intra-day precision, accuracy,
displayed in Figure 2. A set of 9 gentamicin working
linearity and sensitivity. All samples spectra were tested
standards with wide range from 0.25% to 15% w/w was
following the above stated criteria. For linearity, nine
used to generate a calibration curve. Full spectra of pure
serial dilutions of AGA working standards with low
standard of concentrations ranging 0.25-15% w/w and
concentration ranging 0.25-15% w/w were used to
its calibration curve were shown in Figure 3 and Figure
generate a calibration curve in PLS algorithm by scanning
from 4000-650cm-1. The sensitivity of method was 4.
determined from calibration curve with respect to limit The FTIR spectra showed both N-H group, including
of detection (LOD) and limit of quantification (LOQ). secondary anime (3450-3350 cm-1) and primary amine
For the determination of LOD and LOQ, selected single (1650-1550cm-1), C-H stretch group ranging 3040-2940
spectrum band was measured at lowest concentration of cm-1, methyl group ranging 1540-1490 cm-1with bending
AGA standards, and repeated three times to obtain vibration, tertiary alcohol group ranging 1150-1085
substantial signal. Precision criteria was evaluated by cm-1, di-alkyl ether group ranging 1070-1030cm-1, and
preparing three different concentrations of standard across out-of-plane amine bend at 900-850cm -1 . The

ATR-FTIR methods for determination of aminoglycoside antibiotics

Current Trends in Biotechnology and Pharmacy 41
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DOI : 10.5530/ctbp.2020.4s.5

Figure 2. Spectrum of gentamicin standard overlaid with

external standard spectrum in full infrared region (4000-
650 cm-1). Figure 4. The calibration curve of gentamicin standard
full spectrum ranging 0.25% to 15% w/w with correla-
tion coefficient r2 of 0.9999.

interpretation of major peaks and their correspondence

to the chemical groups in gentamicin chemical structure
were shown in Table 1.
Band 1 (secondary amine) and band 4 (methyl
group)were the unique functional groups to ease
differentiation process of gentamicin sulphate from other
aminoglycosides. Band 1 is a secondary amine (R'R''CH-
NH-CH3) stretching vibration and band 4is amethyl group
(CH3C-R) with bending vibration. Both peaks were
Figure 3. Full spectrum of gentamicin standard with identified with strong intensity and met the selection
increasing concentration in full infrared region (4000- criteria.The absorbance and calibration curve of standard
650cm-1). 1% w/w at 3381cm-1.Since band 1 has higher correlation

Table 1: List of IR Band Assignments of predominant chemical groups present in Gentamicin Sulphate
Bonds Wavenumber (cm-1) Possible
Vibration r2value
Name Intensity* Theoretical Experimental Functional Group

N-H M 3350-3310 3450-3350 Secondary amine stretch 1.0000

C-H M-S 3000-2850 3040-2940 Alkane stretch 0.9938

N-H W-M 1650-1580 1650-1550 Amine bend 0.9998

C-H M-S 1470-1450 1540-1490 Methyl group bend 0.9975

C-O-H S 1205-1125 1150-1085 Tertiary alcohol stretch 0.9994

C-O-C S 1150-1085 1070-1030 Di-alkyl ether stretch 0.9690

out of plane
N-H - 800 900-850 Amine 0.9997
bending
*Intensity abbreviations: S- Strong; M- Medium; W- Weak;

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DOI : 10.5530/ctbp.2020.4s.5

coefficient value, secondary amine was selected as main from 4000cm-1 to 650cm-1 to find specific bands that
functional group. could provide highest correlation coefficient (r 2) with
value greater than 0.995. The correlation coefficient (r2)
FTIR Method Validation
values for calibration curve in full infrared region
Average height locations of peaks were used for the
corresponding to each band was listed in Table 2 below.
generation of calibration curve by scanning major peaks

Table 2: R2 values for calibration curve at each band based on PLS method for full ATR-FTIR spectrum
(4000-650cm-1) of gentamicin standard.
Correlation Root Mean Squared Error
Band Wavenumber (cm-1)
Coefficient (r2) Calibration (RMSEC)
1 3450-3350 1.0000 0.0254
2 3040-2940 0.9938 0.3840
3 1650-1550 0.9998 0.0021
4 1540-1490 0.9975 0.2420
5 1150-1085 0.9980 0.2180
6 1070-1030 0.9690 0.8530
7 900-850 0.9997 0.0861
Table 3: Linearity, LOD and LOQ values of gentamicin standards at band 1 single spectrum
Linear range Correlation RMSEC LOD LOQ
Spectrum
(%w/w) coefficient (r2)
(%w/w) (%w/w)

Band 1 0.25-15 1.0000 0.0254 0.2006 0.6080

Table 4: Intra-day precision of gentamicin standard using simple Beer's Law at band 1and band 4 single spectra
Analyte Selected band Intra-day precision
Concentration Intra-day
Wavelength (cm-1) Reading (n=3) Mean ± SD
(%) RSD (%)
1: 0.54
Band 1 2: 0.55 0.54 ± 0.0100 1.852
3: 0.53
0.5
1: 0.52
Band 4 2: 0.50 0.52 ± 0.0153 2.957
3: 0.53
1: 4.02
Band 1 2: 3.92 3.99 ± 0.0681 1.703
3: 4.05
4.0
1: 4.02
Band 4 2: 3.90 4.00 ± 0.0916 2.291
3: 4.08
1: 9.84
Band 1 2: 10.06 9.99 ± 0.1365 1.365
3: 10.09
10.0
1: 10.04
Band 4 2: 9.98 9.99± 0.0458 0.459
3: 9.95

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The calibration curve of band 1 was indicated with The gentamicin concentration was calculated using
correlation coefficient of 1.000 with good linearity. The regression equation from calibration curve using TQ
method was considered sensitive with LOD and LOQ Analyst software. The mean of labelled amount was found
values of 0.2006% w/w and 0.6080% w/w respectively. to be 104± 2.9143mg with (%RSD 2.799).
The linearity and sensitivity results of this method for Commercial gentamicin ophthalmic preparation
gentamicin standards at band 1 (3450-3350cm-1) single (Beagenta eye/ear drops(ED) 0.3% w/v, 5mL
spectrum was shown in Table 3. Linearity was indicated capacity)was procured from local pharmacy and dried in
by range value (%w/w) with its r2 and RMSEC values, Binder RE53 Gravity Convection Ovenr.at 50oCto
while sensitivity of the test was shown with LOD and evaporate liquid to obtain a white colored powder. Drying
LOQ values. The %RSD values of intra-day precision conditions were optimized by drying three samples of
were around 2%, and inter-day precision was achieved the same batch at 25oC, 40oC and 50oC temperature and
less than 4% with low deviation among triplicates. The dried in desiccator as well. Gentamicin is well noted for
calculated %RSD values for intra-day and inter-day being a heat stable antibiotic, retaining its activity even
precision were stated in Table 4 and Table 5. after autoclaving (20-22). The full spectrum of dried
Quantification of gentamicin in ophthalmic gentamicin 0.3% w/v ophthalmic formulation sample with
preparations excipient benzalkonium chloride 0.1% w/v.
The validated proposed ATR-FTIR method was used Peak of tertiary alcohol group (band 5) is shifted
for quantification of gentamicin eye drop formulation. towards1191cm-1, however secondary amine (band 1) is
Table 5: Inter-day precision of gentamicin standard using simple Beer's Law at band 1and band 4 single spectra
Analyte Selected band Inter-day precision
Concentration Intra-day
Wavelength (cm-1) Reading (n=3) Mean ± SD
(%) RSD (%)
1: 0.46
0.5 Band 1 2: 0.48 0.46 ± 0.0152 3.297
3: 0.45
1: 0.53
Band 4 2: 0.54 0.547 ± 0.0208 3.808
3: 0.57
1: 3.84
4.0 Band 1 2: 3.98 3.990 ± 0.1552 3.891
3: 4.15
1: 3.81
Band 4 2: 4.12 3.987 ± 0.1595 4.000
3: 4.03
1: 9.71
10.0 Band 1 2: 9.75 9.930 ± 0.3470 3.494
3: 10.33
1: 9.80
Band 4 2: 9.85 10.017 ± 0.3329 3.324
3:10.40
Table 6: Result of quantification of three gentamicin dried ED formulation in powder form
Empty beaker weight Beaker + Sample weight
Bottle Sample weight (mg)
(mg) (mg)
1 33259.2 33359.8 100.8
2 32710.8 32817.0 106.2
3 26057.0 26162.4 105.4
Mean ± SD of sample weight (mg) 104.1 ± 2.9143
RSD (%) 2.7987

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totally masked by broad OH spectrumranging R (%) = [(C-B)/A] x 100%

wavenumber 3400-3000cm-1. Since secondary amine where,
could not be detected in this case, tertiary alcohol group R = Percentage recovered,
will be the focus of the study to quantify gentamicin in A = Amount of known standard (exogenous addition),
selected preparation.Another three bottles of same batch B = Known sample concentration before addition, and
were dried at the same time optimized conditions. Average
C = Total concentration measured after addition.
weight of powder was obtained and tabulated in Table 6.
The statistics of recovery test were revealed with high
Samples with 5 different concentrations (0.25%, 0.5%,
recovery performance (98.75 -103.2%)with mean
1%, 2% and 4%) were prepared from dried sample
recovery percentage of 100.727% ± 2.3597 as shown in
powder. Sample linearity curve was plotted in Figure 5.
in Table 7. The sampling mean was followed a normal
Sample spectrum was shown in (Figure 5)
distribution. In this case, the standard error of the mean
(SEM) was calculated using the equation below .
S
s = = 1.3624

The mean recovery was significant with margin of

error(100 ±2.65% at 95% confidence interval). The
recovery results were clearly indicated that there was no
significant interferenceobserved from any excipients
present in the matrix and hence this method was proven
to be feasible without any solvent extraction. According
to USP, gentamicin sulphate ophthalmic preparation
contains equivalent of not less than 90% and not more
Figure 5. 0.3% w/v Gentamicin ophthalmic solution sam- than 135% of the labelled amount of gentamicin(5).
ple linearity curve with concentration ranging 0.25% -
4%. Method development
The primary step in qualification process of ATR-
Recovery studies FTIR method is to obtain the spectra The FTIR spectra
The accuracy was evaluated by calculating the of tobramycin standard.at full infrared region (4000-650
percentage recovery of gentamicin sample. The cm-1) was shown in Figure 6(a).The IR spectra was
driedgentamicin sample powder (1% w/w) was accurately showed peaks corresponding to the functional groups in
weighed and spiked with different concentrations (0.4%, the chemical structure that could be used for qualitative
1%, and 5% w/w of gentamicin) of pure standards using and quantitative analysis of tobramycin. The external
KBr as diluent andtopped up to 100mg. The FTIR standard spectrum of Tobramycin with KBr discwas
spectrum of each spiked sample was collected three times obtained from SDBS with CAS Registry Number of
and the accuracy was expressed as the mean percentage 32986-56-4 is shown in Figure 6(b)(25).
recovery of three replicates for each spiked sample. The Different concentrations of tobramycin standards
calculation was carried out by the given equation ranging from 0.25% to 15% were prepared and FTIR
below(23,24).

Table 7. Recovery test of gentamicin from samples after exogenous addition of known standards concentration.

Amount of Known sample

Total
known concentration Recovery
concentration %RSD
standard in before addition in Efficiency (R)
measured (C)
% (A) % (B)
0.4 1 1.395 ± 0.0458 3.273 98.75%
1 1 2.032 ± 0.0436 2.179 103.20%
5 1 6.024 ± 0.1015 1.691 100.48%

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(a) (b)
-1
Figure 6. (a) Pure tobramycin spectrum scanned at full infrared region (4000-650 cm ) with ATR-FTIR instrument;
(b) External standard spectrum of tobramycin with KBr disk from AIST SDBS with list of displayed peak wavenumber.

graded KBr was used as background spectrum. Figure 7

presents the tobramycin standard spectra with calibration
curve in full infrared region. Full spectrum of tobramycin
standard showed excellent calibration curve with
correlation coefficient r2 value of 0.9999, which is shown
in Figure 8.
The major two primary amine stretching at
wavenumber 3470-3420cm -1 and 3370-3320cm -1 ,
bending at 1620-1550cm-1, alkane stretching at 2915-
2875cm-1, methylCH3 -CHbending at a range of 1400-
1370cm-1,substituted alkane group at 845-835cm-1 and
790-755cm-1. As for O-H functional group,alcohol stretch
group is ranged within wavenumber 3305-3255cm-1, in- Figure 8. The calibration curve of tobramycin standard
plane OH bending is found at 1360-1340 cm-1, while full spectrum ranging 0.25% to 15% with r2 value of
primary OHgroup was noticed at 1050-1000cm-1. Ether 0.9999.
group is also found at 1145-1125cm-1. The interpretation
of major peaks and their correspondence to the chemical Peaks corresponding to hydroxide, alkane and ether
groups in tobramycin chemical structure are shown in functional groups were not considered for quantification
Table 8. purpose. In this study, band 2 (3375-3325cm-1) single
spectrum with r2 value of 0.9998 and band 10 (1050-
1000cm-1) single spectrum with r2 value of 0.9992 are
selected for the qualitative analysis of since they are the
unique functional group of tobramycin as compared to
gentamicin. The peak height location of band 2 is
displayed at 3347.3cm-1.
Method validation
Average height locations of peaks were used for the
generation of calibration curve by scanning major peaks
from 4000cm-1 to 650cm-1 to find specific bands that
could provide highest correlation coefficient (r 2) with
Figure 7. Spectra of tobramycin standards with increas- value more than 0.995. Table 9 showed the r2 values of
ing concentration in full infrared region (4000-650cm-1). calibration curve corresponding to each band.

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Current Trends in Biotechnology and Pharmacy 46
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DOI : 10.5530/ctbp.2020.4s.5

Table 8: List of major IR band assignments of predominant chemical groups present in tobramycin.
Bonds Wavenumber (cm-1) Possible
Functional Vibration rvalue
Name Intensity* Theoretical Experimental
Group
N-H M ~3500 3470-3420 Primary amine Stretch 0.9803
Aliphatic
N-H M 3400-3300 3370-3320 Stretch 0.9999
primary amine
O-H M, broad 3400-3300 3305-3255 Alcohol Stretch 1.0000
C-H M 3000-2840 2915-2875 Alkane Stretch 0.9978
N-H M 1650-1580 1620-1550 Primary amine Bend 0.9939
C-H W 1470-1450 1475-1450 Alkane Bend 0.9940

CH3 -CH M 1470-1450 1400-1370 Methyl group Bend 0.9929

In-plane
O-H M 1390-1310 1360-1340 Alcohol 0.9986
bending
R-O-R M 1150-1085 1145-1125 Aliphatic ether Stretch 0.9992
CH2OH M-S 1085-1050 1050-1000 Primary alcohol Stretch 0.9996
1,4-disubstituted
C-H M 810±20 845-835 Bend 0.9919
alkane
1,2,3-
C-H M 780±20 790-755 trisubstituted Bend 0.9940
alkane
*Intensity abbreviations: S- Strong; M- Medium; W- Weak;

Table 9: Correlation coefficient values for calibration curve at each band based on PLS method.
Root Mean Squared
Correction
Band Wavenumber (cm-1) Error Calibration
Coefficient (r2)
(RMSEC)
1 3470-3420 0.9610 0.939
2 3375-3325 0.9998 0.063
3 3305-3255 1.0000 0.016
4 2915-2875 0.9956 0.315
5 1620-1550 0.9878 0.526
6 1475-1450 0.9880 0.521
7 1400-1370 0.9859 0.564
8 1360-1340 0.9972 0.247
9 1145-1125 0.9984 0.195
10 1050-1000 0.9992 0.448
11 845-835 0.9839 0.606
12 790-750 0.9912 0.140

The calibration curve of Band 2 was indicated with Quantification of tobramycin in drug formulation
good linearity with r2 value of 0.9998. The method was The quantification of tobramycin eye drop was tested
considered sensitive with calculated LOD and LOQ using the validated ATR-FTIR method. The tobramycin
values of 0.2296% w/w and 0.7654% w/w respectively. concentration was calculated using regression equation
Table 10 shows the linearity and sensitivity results of from calibration curve using TQ Analyst software. The
this method for tobramycin standards at band 2(3375 - mean of labelled amount was found to be 84.57mg ±
3325cm-1) single spectrum. The %RSD of intra-day 2.658, with %RSD of 3.15.
readings were around 2, while %RSD were lower than 4
Tobrexophthalmic solution 0.3% w/v formulation in
among triplicate inter-day readings, with data shown in
5mL bottle was procured from local pharmacy and dried
Table 11 and 12.

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Table 10: Linearity, LOD and LOQ values of tobramycin standards at single spectrum
Linear range Correlation RMSEC LOD LOQ
Spectrum
(%w/w) coefficient (r2) (% w/w) (%w/w)
Band 2 0.25-15 0.9998 0.0626 0.2296 0.7654

Table 11: Intra-day precision of tobramycin standard using simple Beer's Law at single spectra
Analyte Selected band Intra-day precision
Concentration Wavelength Intra-day
Reading (n=3) Mean ± SD
(%) (cm-1) RSD (%)
1: 0.51
Band 2 2: 0.49 0.503 ± 0.0115 2.294
3: 0.51
0.5
1: 0.52
Band 10 2: 0.49 0.503 ± 0.0153 3.035
3: 0.50
1: 3.94
Band 2 2: 4.04 4.000 ± 0.0529 1.323
3: 4.02
4.0
1: 4.01
Band 10 2: 4.01 3.997 ± 0.0231 0.578
3: 3.97
1: 10.01
Band 2 2: 9.99 9.997 ± 0.0115 0.116
3: 9.99
10.0
1: 10.03
Band 10 2: 10.10 10.000 ± 0.1179 1.179
3: 9.87

Table 12: Inter-day precision of tobramycin standard using simple Beer's Law at single spectra

Analyte Selected band Inter-day precision

Concentration Inter-day
Wavelength (cm-1) Reading (n=3) Mean ± SD
(%) RSD (%)
1: 0.49
Band 2 2: 0.50 0.500 ± 0.0100 2.000
3: 0.51
0.5
1: 0.48
Band 10 2: 0.52 0.503 ± 0.0208 4.136
3: 0.51
1: 4.10
Band 2 2: 4.11 4.027 ± 0.1357 3.372
3: 3.87
4.0
1: 3.96
Band 10 2: 4.05 4.000 ± 0.0458 1.146
3: 3.99
1: 10.05
Band 2 2: 10.05 9.997 ± 0.0924 0.924
3: 9.89
10.0
1: 9.81
Band 10 2: 10.10 10.000 ± 0.1646 1.646
3: 10.09

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Table 13: Average weight of three dried ophthalmic formulation sample

Empty beaker weight Beaker + Sample weight
Bottle Sample weight (mg)
(mg) (mg)
1 26047.8 26134.0 86.2
2 32728.2 32809.7 81.5
3 33003.4 33089.4 86.0
Mean ± SD of sample weight (mg) 84.57 ± 2.658
RSD (%) 3.15

at similar condition as gentamicin to obtain white colored Kanamycin

powder. As mentioned in several reports, Method Development
tobramycinretains stability in high temperature, retaining
Full spectrum of kanamycin standard was scanned
its activity even after autoclaving (20,26). The full
from 4000 to 650cm-1 wavenumber range and the
spectrum of tobramycin 0.3% w/v ophthalmic solution spectrum was overlaid with an external spectrum obtained
with excipient benzalkonium chloride 0.1% w/v. from Spectrabase (CAS Registry Number of 25389-94-
Peak of primary amine (band 2) is being masked by 0), as shown in Figure 9. The calibration curve was
OH group at 3203.22cm-1, while band 10 representing obtained and shown in Figure 10with good calibration
primary OH group is still visible at wavenumber coefficient greater than 0.995.
1025.07cm-1. Since primary amine could not be detected
in this case, band 10 will be the focus of the study to
quantify tobramycin in selected formulation. Another
three bottles of same batch (Batch no: 18K25AG) were
dried at the same time in same condition. Average weight
of powder is obtained and tabulated in Table 13.
Sample with concentrations 0.25%, 0.5%, 1%, 2%
and 4%were prepared to plot linearity curve. Sample
linearity curve and spectrum could be seenrespectively.

Recovery studies
Figure 9. Spectrum of kanamycin standard overlaid with
The statistics of recovery studieswere revealed high external standard spectrum in full infrared region.
recovery performance (99.29 -103.00 %), with mean
recovery percentage of 101.04% ± 1.864 as shown in
Table 14.Since sampling mean was followed a normal
distribution, the standard error of the mean (SEM) was
1.076. The mean was significant (with margin of error of
±2.09%) at 95% confidence interval. The recovery results
revealed that there is no significant interference from any
excipients present in the matrix and hence proven that
this method is feasible without any solvent extraction.
According to USP 39, tobramycin ophthalmic solution
contains equivalent of not less than 90% and not more
than 120% of the labelled amount of tobramycin.(5)These
results clearly prove the validity of proposed direct
method using ATR-FTIR with PLS method for
Figure 10. Full spectrum of kanamycin standard with
quantitative analysis of tobramycin from its ophthalmic increasing concentration in full infrared region (4000-
formulation. 650cm-1).

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Table 14. Recovery studies of tobramycin from samples after exogenous addition of known standards concentration.

Amount of Known sample

Total
known concentration Recovery
concentration RSD (%)
standard in before addition in Efficiency (R)
measured (C)
% (A) % (B)
0.4 1 1.390 ± 0.0400 2.878 99.29%
1 1 2.060 ± 0.0265 1.284 103.00%
5 1 6.050 ± 0.0265 0.437 100.83%

Table 15: List of major IR band assignments of predominant chemical groups present in kanamycin sulphate.
Bonds Wavenumber (cm-1) Possible
Vibration rvalue
Name Intensity* Theoretical Experimental Functional Group
N-H M ~3500 3520-3460 Primary amine Stretch 0.9880
Aliphatic primary
N-H M 3400-3300 3355-3325 Stretch 0.9956
amine

C-H M 3000-2840 2895-2870 Alkane Stretch 0.9958

S 1610-1575 0.9997
N-H 1650-1580 Primary Amine Bend
S 1540-1500 0.9989

C-H W 1450 1465-1445 Methyl group Bend 0.9915

In-plane
O-H W 1390-1310 1365-1355 Alcohol 0.9992
bending
C-N W 1250-1020 1230-1215 Amine Stretch 0.9981

R-O-R M 1150-1085 1145-1135 Aliphatic ether Stretch 0.9913

O-H S 1124-1087 1125-1115 Secondary alcohol Stretch 0.9734

C-O S 1085-1050 1070-1055 Primary alcohol Stretch 0.9998

S=O S 1070-1030 1040-1010 Sulphoxide Stretch 0.9994

1,2,4-trisubstituted
C-H M 880±20 875-860 or 1,3-disubstituted Bend 0.9966
alkane
845-835 0.9895
1,4-disubstituted
C-H M 810±20 Bend
alkane
815-800 0.9880
1,2,3-trisubstituted
C-H M 780±20 785-775 Bend 0.9865
alkane
Monosubstituted
C-H M 750±20 765-755 Bend 1.0000
alkane
*Intensity abbreviations: S- Strong; M- Medium; W- Weak;

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The spectra displayed several important functional Table 15, band 4 (1610-1575cm-1) single spectrum with
groups in kanamycin structure, including primary amine r2 value of 0.9994 and band 5 (1540-1500cm-1) single
stretching group at wavenumber 3520-3460 cm-1 , spectrum with r 2 value of 0.9978 were selected for
aliphatic primary amine ranging 3355-3325 cm-1, primary analysis. The selected peaks were sharp and non-
amine bending groups (1610-1575 cm-1, 1540-1500cm- overlapping unlike tobramycin.
1 ), methyl group at 1465-1445cm -1 , and several
Method validation
substituted alkanes groups (875-860cm-1, 845-835cm-1,
Average height locations of peaks were used for the
785-775cm-1, 765-755cm-1 etc). Due to similarity in
generation of calibration curve by scanning major peaks
structure with tobramycin, primary alcohol group could
from 4000cm-1 to 650cm-1 to find specific bands that
be found in kanamycin spectra, ranging at wavenumber
could provide highest correlation coefficient (r2) with
1070-1055cm-1, and aliphatic ether peak of kanamycin
value more than 0.995 and the r2 values of calibration
could be found at 1145-1135cm-1 wavenumber as well.
curve corresponding to each functional group in
Since kanamycin has more secondary alcohol group and
kanamycin as shown in Table 16.
lesser primary amine group than tobramycin, the spectra
of both AGAs showed some significant difference The calibration curve of Band 4 was indicated with
especially in wavenumber ranging 3500-3300cm-1 and good linearity with r2 value of 0.9994. The method was
1650-1580cm-1 which usually depict amine functional considered sensitive with calculated LOD and LOQ
group. The difference in spectra could be noticed in both values of 0.2393% w/w and 0.7977% w/w respectively.
spectra. The linearity and sensitivity results for kanamycin
standards at band 4 (1610-1575cm-1) single spectrum
Tobramycin peak corresponding to hydroxide, alkane
were shown in Table 17. The %RSD among triplicate,
and ether functional groups were not considered for
the intra-day and inter-day readings were less than 2.00
quantification purpose , but certain bands were shown in
and 4.00 as shown in Table 18 and 19.

Table 16: Correlation coefficient values for calibration curve at each band based on PLS method

Correction
Band Wavenumber (cm-1) RMSEC
Coefficient (r2)
1 3520-3460 0.9761 0.733
2 3355-3325 0.9912 0.446
3 2895-2870 0.9914 0.434
4 1610-1575 0.9994 0.118
5 1540-1500 0.9978 0.222
6 1465-1445 0.9831 0.618
7 1365-1355 0.9984 0.193
8 1230-1215 0.9962 0.296
9 1145-1135 0.9827 0.627
10 1125-1115 0.9475 1.090
11 1070-1055 0.9996 0.092
12 1040-1010 0.9988 0.160
13 875-860 0.9932 0.389
14 845-835 0.9791 0.688
15 815-800 0.9761 0.734
16 785-775 0.9732 0.779
17 765-755 1.0000 0.033

Table 17: Linearity, LOD and LOQ values of kanamycin standards at single spectrum
Linear range Correlation RMSEC LOD LOQ
Spectrum
(%w/w) coefficient (r2) (% w/w) (%w/w)
Band 4 0.25-15 0.9994 0.118 0.2393 0.7977

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Table 18: Intra-day precision of kanamycin standard using simple Beer's Law at single spectra
Analyte Selected band Intra-day precision
Concentration Wavelength Intra-day
Reading (n=3) Mean ± SD
(%) (cm-1) RSD (%)
1: 0.51
Band 4 2: 0.49 0.50 ± 0.010 2.000
3: 0.50
0.5
1: 0.50
Band 5 2: 0.48 0.49 ± 0.020 4.000
3: 0.52
1: 4.00
Band 4 2: 4.01 3.99 ± 0.015 0.382
3: 3.98
4.0
1: 4.00
Band 5 2: 4.01 4.00 ± 0.010 0.250
3: 3.99
1: 9.81
Band 4 2: 10.03 10.0 ± 0.177 1.769
3: 10.16
10.0
1: 9.76
Band 5 2: 10.01 9.97 ± 0.189 1.894
3: 10.13

Table 19: Inter-day precision of kanamycin standard using simple Beer's Law at single spectra

Analyte Selected band Inter-day precision

Concentration Wavelength Intra-day
Reading (n=3) Mean ± SD
(%) (cm-1) RSD (%)
1: 0.51
Band 4 2: 0.52 0.52 ± 0.015 2.919
3: 0.54
0.5
1: 0.53
Band 5 2: 0.51 0.53 ± 0.020 3.774
3: 0.55
1: 4.03
Band 4 2: 4.10 4.0 ± 0.118 2.947
3: 3.87
4.0
1: 4.00
Band 5 2: 4.12 4.0 ± 0.120 3.000
3: 3.88
1: 9.71
Band 4 2: 10.03 10.0 ± 0.276 2.762
3: 10.26
10.0
1: 9.68
Band 5 2: 10.03 10.0 ± 0.306 3.061
3: 10.29

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Quantification of kanamycin injections significant interference from any excipients present in

Similar drying method was used on kanamycin the matrix. According to USP, Kanamycininjection must
formulation, however due to high concentration as meet the approval criteria of an amount of Kanamycin
compared to gentamicin and tobramycin, gel state was Sulfate equivalent to not less than 90.0 percent and not
formed. The drying was done with stirring at every one- more than 115.0 percent of the labeled amount of
hour point. Kanamycin white crystalline powder was kanamycin.(5).The results were proved the validity of
grinded in big mortar and pestle to ensure uniform mixing proposed ATR-FTIR spectroscopy method for
with KBr powder. quantitative analysis of kanamycin injection.
Full spectrum of kanamycin IM injection sample was LC-MS Reference Method
shown as a peak of primary amine bending group (band
Gentamicin
4) is visible within wavenumber 1650-1600cm-1, which
was the focus for the quantification study in selected Gentamicin is usually found in mixture of five
formulation. Another three vials of kanamycin IM components (C1, C1a, C2, C2a, and C2b). ESI mass
injection were dried at the same time under the same spectrum was recorded from Gentamicin standard and it
condition. Average weight of powder was obtained and was identified as mass spectral peaks that represented
tabulated in Table 20. The mean of labelled amount was the protonated species of all Gentamicin subtypes (m/z
found to be 390.63mg ± 5.472, with %RSD of 1.40. 478, 450, and 464), The molecule with the greatest m/z
value indicates the parent ion.
Sample with concentrations from 0.25% to 15% were
prepared to plot linearity curve. Sample calibration curve Good linearity of Gentamicin standard was shown
was shown with good linearity. withgood correlation coefficient (r2 0.9986). For recovery
studies, the percent purity was calculated according to
Recovery studies
the formula below.
The statistics of recovery studieswere revealed high
recovery performance (99.92 -101.75 %), with mean % Purity = x 100
recovery percentage of 100.67% ± 0.960 as shown in
Table 21. The sampling mean was followed a normal Where Cun is concentration of sample,
distribution, the standard error of the mean (SEM) Cstd is concentration of standard.
wasestimated to be 0.554. The mean was significant (with
margin of error of ±1.08%) at 95% confidence interval. The recovered concentration was 1.457µg/mL,
The recovery results were revealed that there was no therefore the calculated recovery percentage was 112%.

Table 20: Average weight of each of the three dried injection vial sample
Beaker + Sample weight
Bottle Empty beaker weight (mg) Sample weight (mg)
(mg)
1 27463.4 27859.2 395.8
2 32935.1 33320.0 384.9
3 32618.8 33010.0 391.2
Mean ± SD of sample weight (mg) 390.63 ± 5.472
RSD (%) 1.40

Table 21. Recovery test of kanamycin from samples after exogenous addition of known standards concentration.
Amount of Known sample
Total
known concentration Recovery
concentration RSD (%)
standard in before addition in Efficiency (R)
measured (C)
% (A) % (B)
0.4 1 1.405 ± 0.0134 0.956 100.33%
1 1 2.035 ± 0.0567 2.785 101.75%
5 1 5.995 ± 0.0353 0.589 99.92%

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In the reported reference method by Freneiletc, the rapid analysis of different AGAs in commercial
sensitivity was established, and the results was found to preparations. The relationship between IR spectra of the
be 1ng/mL for LOD. The percent RSD of intra-day (n=5) compound and its chemical structure were fully exploited
and inter-day precision (n=15) were 6.1 and 8 respectively before establishing analytical method validation
(27). parameters for detection and quantification of the selected
AGAs separately. ATR-FTIR spectroscopy with PLS
Tobramycin
algorithm for quick quality control analysis as a direct
ESI operated in positive ion mode was used to study method which does not require any complex
the fragmentation behavior of tobramycin and its known derivatization, chemical modification or solvent
related substances. Tobramycin was yielded a [M+H]+ intervention for the sample preparation except drying.
base peak at m/z 468. Another major spectral peak was Validation results of the present study were shown precise,
collected at m/z 288, which was a major product ion in accurate and reproducible. These methods were faster,
the fragmentation pathway. It was revealed from the simple and eco-friendly for sensitive detection and
literature about the fragmentation pathway of tobramycin accurate measurement of purity of active ingredient from
to its major product ion with their m/z value. The LC- its pharmaceutical preparations.
MS spectrum of reference method was optimized and
5. References
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1. Vidaver, A.K.(2002). Uses of Antimicrobials in Plant
The good linearity was established with LC-MS
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ATR-FTIR instrument has the potential of carrying

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Current Trends in Biotechnology and Pharmacy 54
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DOI : 10.5530/ctbp.2020.4s.5

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Assessment of Knowledge, Attitude and Practice of Malaysian Women

Towards Osteoporosis
Yee Thong Cheng1, Fazlollah Keshavarzi1*, Muhammad Junaid Farrukh1,
Safia Sabry Lotfy Aly Mahmoud1
1Faculty of Pharmaceutical Sciences, UCSI University, JalanMenaraGading, Taman Connaught,
Cheras, 56000 Kuala Lumpur, Malaysia

*Corresponding author : fazlollahk@yahoo.com

Abstract Key words : Osteoporosis, knowledge, attitude, practice,

Due to the increasing proportions of aging populations Malaysian women
in the Asian region, osteoporosis has become more
1. Introduction
prevalent and increases the health care expenditure in this
region. The majority of osteoporotic fractures occur in Osteoporosis is a common disease characterized by a
postmenopausal women. It is important to identify women systemic impairment of bone mass and microarchitecture
at the highest risk and to prevent further fractures. We that results in increase bone fragility and susceptibility
aimed to assess knowledge, attitude and practice towards to fracture (1). Due to the increasing proportions of aging
osteoporosis among Malaysian women in Klang Valley.A populations in the Asian region, osteoporosis has become
cross-sectional study was conducted in 384 Malaysian more prevalent and increases the health care expenditure
women aged above 18 years. A researcher-administered in this region (2). In Malaysia, 5.3 million of the
questionnaire was used to collect data. The participants population was aged 50 and above in 2013 and the number
were selected conveniently from obstetrics and is expected to increase rapidly to 13.9 million in 2050
gynecology (O&G) or orthopedic clinics from 6 districts (3). In 2000, WHO estimated that osteoporosis causes
of Klang Valley. Data analysis was done by SPSS version more than 8.9 million fractures worldwide every year (4).
22, using ANOVA, t-test, Chi-square test and Pearson There is limited data documented on the prevalence of
correlation. The findings show participants had a poor osteoporosis in Malaysia. However, a study in 2005
score of knowledge towards osteoporosis. There was a reported the prevalence of osteoporosis was 24.1%,
significant association between the level of osteoporosis predominantly at the hip (5).
knowledge and education level, employment status and The majority of osteoporotic fractures occur in
occupation of participants (P<0.05). The participants had postmenopausal women. Therefore, to manage
a moderate attitude towards osteoporosis. Age, race and postmenopausal osteoporosis, it is important to identify
education level of participants was significantly women at the highest risk and to prevent further fractures
associated with the attitude towards osteoporosis. Most (6). There were a few studies that determine the
participants had poor preventive practices against prevalence (3) and knowledge, attitude and practice (7,
osteoporosis. The level of practice to prevent osteoporosis 8) of osteoporosis among Malaysians. However, there is
was significantly associated with races, education level, limited study regarding osteoporosis among Malaysian
occupation and monthly income of participants. Both women.
knowledge and attitude towards osteoporosis were
correlated with the practices to prevent osteoporosis.The Postmenopausal osteoporosis is the main consequence
participants had inadequate knowledge, moderate attitude of bone loss. It is caused by estrogen deficiency and
and poor practice towards osteoporosis. This could serve therefore affects mainly women (9). The incidence of
as a stimulant for policymakers to increase the education osteoporosis increases markedly with age. Osteoporotic
of osteoporosis among younger women. Furthermore, the fractures are increasingly prevalent in women after 55
practice against osteoporosis among high-income years of age and in men after 65 years of age (10).
participants was higher than low-income. This indicates Ethnicity and race are also important factors to determine
that poverty should be addressed in Malaysia. the risk of osteoporosis. A survey on hip fracture incidence

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among Malaysian populations showed that 63% were determine the knowledge and attitude of Malaysian
Chinese(11). women towards osteoporosis. How Malaysian women
Older people with a history of fractures, family history practice preventing osteoporosis and the determining
of osteoporosis and history of rheumatoid arthritis are at factors of this practice also have been concerned in the
a particularly higher risk of osteoporotic fractures (9). current study.
Modifiable risk factors such as the history of smoking, 2. Materials and Methods
alcohol abuse, intake of caffeine and history of
The cross-sectional study was conducted from April
glucocorticoid use also increase the incidence of
to July 2018 and July to August 2019 in Klang Valley,
osteoporosis (9, 12).
Malaysia. A total of 384 Malaysian women aged 18 years
A study shows that self-efficacy for calcium intake and above were recruited in the study. Malaysian women
and physical exercise directly affect the corresponding aged below 18, foreigners and those who refused to take
practices among young adults (13). Another study in the part were excluded from the study. 111 samples were
US suggests that understanding the barriers to calcium recruited in 2018 while the other 273 samples were
supplements is essential to increase calcium intake and obtained in 2019.
reduce the risk of osteoporosis (14). Exercise is a safe
A two-stage cluster convenient sampling was used to
and effective way to prevent bone loss in postmenopausal
women. However, most studies showed females had an select the studied population. In the first stage, Klang
Valley is clustered into six districts. In the second stage,
inadequate physical exercise that could protect them from
from each district, 3 or more obstetrics & gynecology
osteoporosis such as Saudi Arabia (15).
(O&G) or orthopedic clinics were selected based on the
The National Osteoporosis Foundation recommends convenience of accessibility of researchers and patient
a total calcium intake of 1200 mg per day and a total flow in the clinics. From each district, 64 samples were
vitamin D intake of 800 to 1000 IU per day for people recruited.
aged over 50 to prevent osteoporosis(16). Increased
consumption of vegetables and fruits and low To estimate the sample size, a margin of error of less
than 5% and a confidence interval of 95% were used in
consumption of meat has shown to support bone status
Raosoft sample size calculator(22). The target population
(17). Hormone replacement therapy, selective estrogen
receptor modulators, bisphosphonates, calcitonin, size of 1 million and the response rate of 50% were
entered. A sample size of 384 women was needed to be
calcium and vitamin D can be used to treat osteoporosis
recruited in the study.
(18).
Ethical consideration : Faculty Research and Scholarly
An existing study revealed that there was a lack of
awareness of the diagnosis of osteoporosis and Activities Committee at UCSI University approved the
research project in February 2018. Ethical approval was
osteoporotic fracture risk among postmenopausal women
obtained from the Medical Research & Ethics Committee
in the US(19).The attitude towards osteoporosis is
dependent on one's beliefs related to the disease(20). of the Ministry of Health Malaysia. An informed consent
letter was signed by all respondents before they answer
Increasing knowledge, correcting health beliefs and
the questions.
promoting osteoprotective practices are effective
measures for building and maintaining strong bone Data collection tool : To develop the questionnaire,
throughout ones' lifespan(21). previous studies carried out on women of the same age
group in Sri Lanka (2), Saudi Arabia (15) eight European
In Malaysia, the aging population is alarming and
countries (17) and Canada (23)were utilized. The
urgent action is required to deal with the projected burden
questionnaire items were modified to meet local needs.
of osteoporosis. However, studies show that the calcium
The language used in the questionnaire was English and
and vitamin D intake remains low in Malaysian women
the data was collected by face to face interviews. The
who are at higher risk of getting osteoporosis. Most of
interviewer was able to communicate in Mandarin and
the studies emphasized to determine the prevalence of
BahasaMelayu, in addition to English. In case the
osteoporosis among Malaysian women but so far there is
respondent was not proficient in English, the interviewer
not any study that determined the knowledge, attitude
assisted the respondent to interpret the questions. The
and various methods of practice to prevent osteoporosis
questionnaire included four parts: demographic
among Malaysian women. Therefore, we decided to

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characteristics, knowledge, attitude towards osteoporosis 3. Results and Discussion

and practice/preventive behaviors against osteoporosis. There was a total of 384 completed questionnaires
Assessment of knowledge : The responses to knowledge collected. All the participants were women. Most of the
items include 'yes', 'no' and 'don't know'. Correct answers participants (49.2%) in the study aged 30-49. There were
were scored as 1, while wrong answers and don't know 179 Chinese, 46 Indian, 153 Malay and 6 other races
were scored as 0. The total knowledge score ranges from women enrolled in the study. Most women in the study
0 to 14. (61.7%) were married while the other 37.2% of women
Assessment of attitude : Likert scale items (strongly were single. There were 45.8% of women university-
agree, agree, disagree, strongly disagree) were used for educated, 31.5% secondary school educated, and 6% with
the assessment of attitude regarding osteoporosis. no formal education. More than half of the women were
Strongly agree responses were scored as 4, agree as 3, employed while 16.7% were housewives. There were
disagree as 2 and strongly disagree as 1. The total attitude only 26% of women worked in healthcare-related
score ranges from 8 to 32. industries while the rest had non-healthcare related jobs.
Most women (43.2%) had a monthly income of RM 1000
Assessment of practice : The items of practice/prevention
- RM 3000.
behaviors against osteoporosis include frequency of
intake of calcium-rich food, frequency of physical activity The classifications of knowledge, attitude and practice
and frequency of exposure to sun. A five-point scale was towards osteoporosis among participants are based on
used to rate the frequencies of each activity: never scored Bloom's cut off point (24) according to the mean scores
as 1, seldom scored as 2, occasionally scored as 3, often in each dimension. Table 1 presents the mean score and
scored as 4 and always scored as 5. Furthermore, the overall level of classification of KAP among the
history of the BMD test and history of calcium participants. The overall mean scores ± standard deviation
supplements were included in the assessment of practice for all participants were 6.52 ± 2.609 for knowledge,
against osteoporosis. If there is a history of these 22.72 ± 2.674 for attitude and 10.78 ± 2.963 for practice.
preventive measures, 1 point was given for each. The The participants had a poor level of knowledge and
total practice score ranges from 3 to 17. practice, and a moderate level of attitude towards
osteoporosis.
Data analysis : SPSS version 22 was used for data
analysis. Demographic characteristics and KAP scores Table 1 : Mean ± SD of knowledge, attitude and practice
were summarized using descriptive statistics and score towards osteoporosis among participants.
expressed as mean ± standard deviation and percentage.
Dimension Total (384) Level of
Bloom's cut off scale was used to assess the level of
classification
knowledge, attitude and practice towards osteoporosis Mean ± SD (% of the
of participants. The mean KAP total scores of 80-100% total score)
were classified as good knowledge, a positive attitude,
Knowledge 6.52 ± 2.609 (46.6%) Poor
and good practice, 60-79% as moderate, and <60%
Attitude 22.72 ± 2.674 (71%) Moderate
indicated a poor level of KAP.
Practice 10.78 ± 2.963 (63.4%) Poor
The differences in KAP scores by demographic
characteristics were tested using the one-way analysis of
Table 2 presents the result of the association between
variance (ANOVA). T-test was used to compare the means
of KAP scores between healthcare-related occupations demographic characteristics and KAP scores using
and non-healthcare related occupations. The P-value of ANOVA and t-test. ANOVA test showed knowledge score
less than 0.05 was considered statistically significant. Chi- was statistically associated with education level and
square analysis with Fisher's exact test was used to test employment status (P<0.05). Attitude score was
the relationship between race, marital status and associated with age, race and education level. While
employment status with KAP categories (good, moderate practice score was significantly associated with race,
and poor). Pearson's correlation was used to identify the education level and monthly income. T-test showed that
relationship between knowledge and practice as well as women with healthcare-related occupation had
attitude and practice towards osteoporosis among significantly higher mean knowledge and practice scores
participants. than non-health-care related jobs (both P<0.001).

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Table 2 : The association between demographic variables and KAP scores (n=384), based on ANOVA and
t test of significance.

Variables Categories N Knowledge Attitude Practice

Mean ± SD Mean ± SD Mean ± SD

Age 18-29 139 6.29 ± 2.728 23.12 ± 2.607 10.34 ± 2.645

30-49 189 6.60 ± 2.362 22.76 ± 2.636 10.89 ± 3.141

50-69 52 6.79 ± 3.108 21.75 ± 2.700 11.35 ± 3.016

>70 4 7.00 ± 2.944 20.00 ± 2.708 13.25 ± 1.708

P value 0.593 0.003* 0.045

Race Chinese 179 6.68 ± 2.576 22.09 ± 2.550 10.37 ± 3.173

Indian 46 6.57 ± 3.270 23.04 ± 3.076 11.13 ± 3.131

Malay 153 6.29 ± 2.403 23.38 ± 2.518 11.05 ± 2.535

Other 6 7.50 ± 3.146 22.17 ± 3.125 13.17 ± 3.817

P value 0.439 <0.001* 0.027*

Marital status Single 143 6.50 ± 2.737 23.04 ± 2.575 10.50 ± 2.656

Married 237 6.52 ± 2.545 22.51 ± 2.719 10.93 ± 3.123

Other 4 7.50 ± 1.915 23.50 ± 2.887 11.75 ± 3.594

P value 0.751 0.149 0.313

Education Secondary 121 6.47 ± 2.422 22.19 ± 2.498 10.07 ± 3.301

level school

College 64 6.25 ± 2.570 22.53 ± 2.588 10.80 ± 2.890

University 176 6.89 ± 2.659 23.43 ± 2.560 11.18 ± 2.627

Other 23 4.70 ± 2.566 20.65 ± 3.009 11.39 ± 3.187

P value 0.001* <0.001* 0.010*

*P < 0.05 is significant - t-test used for occupation; ANOVA used for age, race, marital status, education level,
employment status and monthly income.

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Based on the results of post-hoc Tukey tests, secondary attitude scores than secondary school educated women
school and university-educated women had significantly (P<0.001).
higher mean knowledge scores than women with no Regarding practice towards osteoporosis, the
formal education. Self-employed women had a secondary school-educated women had significantly
significantly higher mean knowledge score than lower mean practice scores than university-educated
housewives. women (P=0.008). Women with monthly income less than
RM 1000 had significantly lower practice scores than
The mean attitude score of women aged 18-29 were those with monthly income more than RM 5000
significantly higher than women aged 50-69 (P=0.008). (P=0.019).
Post-hoc study shows no significant difference between Tables 3, 4 and 5 reveal the results of Chi-square
races and practice scores although the overall P-value is analysis with Fisher's exact test to test the relationship
0.027. The mean attitude scores of women with no formal between race, marital status, employment status and KAP
education were significantly lower than women with categories. Knowledge and practice categories were
secondary school, college and university education (P- shown to be no relationship with race, marital status and
value of 0.044, 0.015 and <0.001 respectively). Women employment status. However, there is an association
with university education also had significantly higher between races and attitude categories.

Table 3 : Chi square analysis with Fisher's exact test to test the relationship between race, marital status,
employment status and knowledge categories.
Knowledge
Characteristics Frequency Fisher exact
Good Moderate Poor
Race Chinese 3 42 134 0.094
Indian 2 12 32
Malay 2 25 126
Other 1 1 4
Marital status Single 5 31 107 0.249
Married 3 47 187
Other 0 2 2
Employment Employed 4 44 167 0.055
status Self-employed 2 17 34
Unemployed 0 5 23
Housewife 0 8 56
Other 2 6 16

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Table 4 : Chi square analysis with Fisher's exact test to test the relationship between race, marital
status, employment status and attitude categories.
Attitude
Characteristics Frequency Fisher exact
Positive Moderate Negative
Race Chinese 15 136 28 0.003*
Indian 9 32 5
Malay 27 119 7
Other 1 4 1
Marital status Single 24 109 10 0.159
Married 27 179 31
Other 1 3 0
Employment Employed 26 168 21 0.802
status Self-employed 9 38 6
Unemployed 6 20 2
Housewife 9 46 9
Other 2 19 3

*P value<0.05, significant.

Table 5 : Chi square analysis with Fisher's exact test to test the relationship between race, marital
status, employment status and practice categories.
Practice
Characteristics Frequency Fisher exact
Good Moderate Poor
Race Chinese 33 54 92 0.076
Indian 11 18 17
Malay 27 58 68
Other 4 1 1
Marital status Single 21 51 71
Married 52 79 106
0.210
Other 2 1 1
Employment Employed 43 66 106 0.369
status Self-employed 15 21 17
Unemployed 5 11 12
Housewife 9 23 32
Other 3 10 11

Knowledge and attitude towards osteoporosis were (r=0.241, p<0.001) among the participants. The
correlated with practice using Pearson's correlation test. participants' attitude towards osteoporosis was weakly
Table 6 reveals that knowledge of osteoporosis was positively correlated with their practice towards the
weakly positively correlated with osteoporosis practice prevention of osteoporosis (r=0.189, p<0.001).

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Table 6 : Pearson correlation between knowledge and scored higher knowledge scores than women with no
practice as well as attitude and practice towards formal education. It is known that education can affect
osteoporosis among Malaysian women. one's ability and practice to access and interpret health
Variables r p knowledge and information(29, 30). This result is in line
with previous studies in the USA(31)and Saudi
Knowledge vs practice 0.241* <0.001 Arabia(15). Subjects with healthcare-related occupations
Attitude vs practice 0.189* <0.001 had better osteoporosis knowledge than others with non-
healthcare related jobs. This result is in agreement with
*P value<0.05, significant. a study in Vietnam, with nurses scored higher knowledge
This study reveals that Malaysian women had low than others(25). It may be due to their exposure to more
knowledge, moderate attitude and low practice regarding healthcare-related problems. The monthly income of
osteoporosis. Knowledge of osteoporosis was shown to subjects shows no relationship with osteoporosis
be associated with education level, employment status knowledge.
and occupation. Attitude towards osteoporosis was The mean osteoporosis attitude scores were associated
associated with age, race and education level. Practice with age, races and education level. Women subjects aged
against osteoporosis was associated with education level, 18-29, Malay and had higher education were shown to
occupation and monthly income of the participants. The have more positive attitudes towards osteoporosis.
study also found that both osteoporosis knowledge and Furthermore, the classifications of attitude are associated
attitude were significantly positively correlated with the with races of subjects. More Chinese women had a
practice of Malaysian women. People with good negative attitude regarding osteoporosis compared to
knowledge and a positive attitude towards health will be Indian women and Malay women. This may contribute
more likely to engage in health-related practice and to the prevalence of hip fractures in Malaysia in which
preventive measures(7). 63% were Chinese according to a survey in 2007(11).
The study shows a low level of knowledge towards In the study, results indicate that Malaysian women
osteoporosis among participants. This result is in line with had a poor level of practice towards the prevention of
a previous study which resulted in poor osteoporosis osteoporosis. This finding is in line with similar previous
knowledge among perimenopausal women in Vietnam studies in Malaysia and Saudi Arabia which showed a
(25) and Turkey (26). This contrasts with a recent study low frequency of preventive activity among females(7,
among middle-aged Chinese in Malaysia which shows 15). In our study, 32.0% of women exposed to the sun
moderate osteoporosis knowledge(7). The difference every day compared to only 6.8% of women had no
could be explained by the different study population, exposure to the sun. This may be due to the tropical
cultural difference and sample size of the studies. weather all year round in Malaysia and the participants'
In the current study, only 11.5% of women were aware job characteristics that expose them to the sun. Exposure
that osteoporosis is a silent disease. Another recent study to the sun at an appropriate time is essential to obtain
in Malaysia showed that 40.9% of the subjects were aware vitamin D which is good for bone health. Besides, only
that osteoporosis does not cause symptoms such as knee 18.5% of the participants had a history of BMD tests.
pain. This may be due to the participants were confused However, a lower percentage was found in another study
between osteoporosis and osteoarthritis (7). The which is 14% in Saudi Arabia(15). 41.1% of women
misconception should be addressed to the public. Besides, subjects had a history of taking calcium supplements.
54.4% knew that not getting active in regular exercise This is lower compared to a study in Saudi Arabia which
increases the risk of osteoporosis. 42.2% knew that early showed 55% of females had a history of calcium
menopause is a risk factor for osteoporosis. Higher supplement intake.
percentages were found in other studies: 90% of women In the study, there is no significant association between
in New Zealand (27) knew that regular exercise is a the level of practice towards osteoporosis and age.
preventive measure; 88% of women in America(28) knew Another study also found out that practice did not differ
to be menopausal increases the risk of getting by age(15). There is a significant relationship between
osteoporosis. preventive practice and level of education. University
In the current study, knowledge was not associated educated women had higher mean osteoporosis practice
with the age of the participants. This is different from scores than high school educated women. Women with
previous studies in Vietnam (25) and Saudi Arabia (29) healthcare-related occupations also had higher practice
which reported level of knowledge decreases with age. scores than those with non-healthcare related jobs.
Women subjects who finished high school and university Subjects with monthly income less than RM 1000 had

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significantly lower mean osteoporosis practice scores than the Chinese Population in Klang Valley, Malaysia.
those with monthly income more than RM 5000. It is Applied Sciences 9 (9), 1820. doi: 10.3390/
perhaps because people with higher income have more app9091820
access to calcium-rich foods, physical exercise, BMD 4. WHO scientific group on the assessment of
testing and calcium supplements. osteoporosis at primary health care level: Summary
Pearson's correlation shows both osteoporosis Meeting Report, Brussels, Belgium, 5-7 May 2004.
knowledge and attitude are correlated with practice to Geneva: World Health Organization (2007) Available
prevent osteoporosis. Subjects with inadequate healthcare from: www.who.int/chp/topics/Osteoporosis.pdf
knowledge and negative attitude tend to have a lower [cited 27 July 2020].
level of practice to prevent diseases. 5. Lim, P.S., Ong, F.B., Adeeb, N., Seri, S.S., Noor-
Limitations : This study was conducted only in Klang Aini, M.Y., Shamsuddin, K., Hapizah, N., Mohamed,
Valley which includes the capital city, Kuala Lumpur and A.L., Mokhtar, A. and Wan, H.W.H. (2005) Bone
the surrounding area. The results may be different in other health in urban midlife Malaysian women: risk
regions of the country that are not covered. The factors and prevention. Osteoporosis international
convenient method of sampling may increase the risk of 16 (12), 2069-2079.doi: 10.1007/s00198-005-2003-
selection bias. 4.
4. Conclusion 6. Rosen, C.J. (2005) Postmenopausal osteoporosis.
The study showed that Malaysian women had New England Journal of Medicine 353 (6), 595-603.
inadequate knowledge, moderate attitude and low level doi: 10.1056/NEJMcp043801.
of practice towards osteoporosis. This study could serve 7. Chan, C.Y., Subramaniam, S., Chin, K.-Y., Ima-
as a stimulant for policymakers to increase the education Nirwana, S., Muhammad, N., Fairus, A., Manap, A.,
and awareness of osteoporosis among the public Ng, P.Y., Nor Aini, J. and Aziz, N.A. (2019)
especially younger women. Further researchers can also Knowledge, Beliefs, Dietary, and Lifestyle Practices
assess the causes of low levels of osteoporosis knowledge Related to Bone Health among Middle-Aged and
and practice among Malaysians. Furthermore, the practice Elderly Chinese in Klang Valley, Malaysia.
against osteoporosis among high-income participants was International journal of environmental research and
higher than low-income. This indicates that poverty public health 16 (10), 1787.doi: 10.3390/
should be addressed in Malaysia in order to improve bone ijerph16101787.
health and to prevent osteoporosis. 8. Yeap, S.S., Goh, E.M.L. and Das Gupta, E. (2010)
Acknowledgment : This work was sponsored partially Knowledge about osteoporosis in a Malaysian
by the Faculty of Pharmaceutical Sciences, UCSI population. Asia Pacific Journal of Public Health 22
University, Malaysia [grant number UCSI/Pharmacy/ (2), 233-241. doi: 10.1177/1010539509343948.
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Assessment of knowledge, attitude and practice towards osteoporosis
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DOI : 10.5530/ctbp.2020.4s.7

Barriers and Enhancers of Medication Error Reporting Among

Hospital Pharmacists, a Qualitative Exploration
Keat Jiu Yoo1, Fazlollah Keshavarzi1*
1Faculty of Pharmaceutical Sciences, UCSI University, No 1, JalanMenara Gading,
Taman Connaught, Cheras, 56000 Kuala Lumpur, Malaysia

Corresponding author: fazlollahk@yahoo.com

Abstract In a study from the UK, the prevalence of prescribing

Reporting the medication errors (MEs) will help avoid errors was around 9%, wherein the 19 hospital's incident
the recurrence of the errors. The purpose of this study is reporting system, less than 0.2% of the detected
to explore the enhancers and barriers of ME reporting prescribing errors were voluntarily reported . In the USA,
among Malaysian hospital pharmacists. A qualitative direct observation of medication administration in 36
study using in-depth interviews of 18 hospital pharmacists hospitals revealed an 11.7% error rate compared with just
was conducted. Six themes and 29 codes were identified. 0.04% for errors detected through the incident reporting
Most of the participants agreed that medication errors scheme (5). The problem was probably even more serious,
were underreported in Malaysia. The main determinant with estimates of underreporting of events ranging from
of medication error reporting is perceived to be the 50% to 96% annually (7237).
severity of the error. Other themes include reporting According to a study in Malaysia, 17357 MEs (16%)
system, organizational factors, reporter's burden, provider- were reported and reviewed from January 2009 to
related factors and benefit from reporting. The December 2012, of which 92.1% was reported by
confidentiality of the reporting system and reporting pharmacists, primarily from public-funded hospitals (12).
culture in the hospital are other factors that affect MER. According to a study that included 12 Ministry Of
Huge paperwork increases reporter's burden thus hinders Health (MOH) primary care clinics in four states of
ME reporting. Simplifying the process of reporting, Malaysia, the most common clinical management errors
protecting the confidentiality of the process and separating (41.3%) were medication errors (1). From 2014 to 2015,
MER from staff performance evaluation are the most there were 3,526 MEs and 248,307 near misses reported
important interventions to improve the MER rate. to the Patient Safety Unite of the Ministry of Health .
Keywords : Hospital pharmacist. Medication error The attitudes and perceptions of doctors, nurses,
reporting. Medication incidents. Patient safety midwives and other healthcare professionals towards
1. Introduction medication errors in healthcare have been extensively
studied. However, despite their key role, there were only
Medical errors have been estimated to be the third
limited publications about the pharmacists' attitude,
leading cause of death in the USA in 2013 with over perception, and practice of ME reporting (1).
400,000 deaths hospitalized patients. Medication errors
(MEs) were among the most common types of medical A study investigated the perceptions and attitudes of
errors, harming about 1.5 million people every year and Malaysian healthcare professionals towards medication
were the main contributors to adverse events to error reporting in primary care clinics, but no study has
hospitalized patients (21). It imposed substantial costs been conducted to address the same issue among
between US$ 6 billion to US$ 29 billion per year . pharmacists, specifically (12).There is a small qualitative
study with four participants in a single hospital in Miri
The primary purpose of medical error reporting is to
Hospital, Sarawak that focuses on the causes of
maintain healthcare providers' responsibility for
medication errors from the viewpoint of hospital
performance and to produce or generate new knowledge
pharmacists.
and improve patient safety. MER allows the errors to be
corrected before serious harms occur (2). Considering that medication errors are among the most
common medical errors with a high global burden, and at

Barriers and enhancers of medication error in hospital

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the same time, ME reporting rate is low, investigating familiarization stage, the audio recording of each
the barriers of ME reporting matters. ME reporting is interview was listened to whilst the transcription was
essential and important to minimize the reoccurrence of being read. The researcher then examined a few
such errors and improve patient safety. Therefore, the transcripts line by line and assigned codes to denote
barriers should be identified by understanding hospital particular meaningful segments. This coding combined
pharmacists' perceptions and attitudes toward ME both deductive and inductive approaches. The codes were
reporting. grouped into categories or themes. These formed a
working analytical framework that was based on the data
2. Materials and Methods and a prior understanding of the literature.
This qualitative study uses semi-structured in-depth The framework was refined further to improve clarity,
individual interviews. Each interview planned to last reduce ambiguity and to produce the final thematic
about one hour. More interviewees were invited until framework as the process went further.
saturation was achieved. The term 'saturation' meant the
Subsequently, the data was charted into the framework
last few interviews contain no new point to improve the
matrix which involved summarizing data by category for
targeted exploration. A thematic framework was drafted
each transcript. The matrix enabled the researchers to
based on prior understanding. However, it was amended
make final conclusions regarding the rich data (1).
after data collection from participants. Purposive
sampling was conducted. The participants were recruited Ethics approval was obtained from Medical Research
from different departments of a variety of public and Ethics Approval (MREC), Ministry of Health, Malaysia.
private, large and small hospitals. To improve the validity 3. Results and Discussion
and reliability of the study, the recruited hospital
Table 1 shows the characteristics of the participants
pharmacists were selected purposely with diverse
(n = 18) comprised of hospital pharmacists from different
working experience. Recruitment of participants stopped
departments such as drug information service (DIS), in-
after saturation reached, which meant further interviews
patient pharmacy, out-patient pharmacy, as well as clinical
did not change the current themes and codes.
pharmacists from public and private sectors. Two invited
All practicing Malaysian hospital pharmacists with persons refused to participate, one of them because the
at least one year of working experience were eligible for supervisor asked not to take part in any interview and the
this study. The participants were invited through social other one insisted on no voice recording. The number of
media, personal and professional connections. Some of years of experience ranged from 22 months to twelve
the participants were in the researchers' social networks, years.
in advance and were invited to take part, as long as they Table 1: Characteristics of Participants
fulfilled the eligibility criteria. They were invited by
Practice Setting No of participants No of participants
electronic mails or messages through the online means (Private sector) (Public sector)
of communication.
In-patient Pharmacist 2 3
The interviews were carried out by the first author Out-patient 1 3
Pharmacist
who attended a workshop to learn the qualitative research Clinical Pharmacist 1 7
techniques, at convenient places such as café, home or Drug Information - 2
workplace, based upon the agreement between service
Duration of practice
interviewer and interviewee. 1 to 3 years 8
3 to 5 years 8
A semi-structured interview guide was adopted from
5 to 7 years 1
previous studies with some modifications to address the years 2
local requirements (Appendix I) (12)(1). The duration of
the interview was around 45 minutes to one hour, Six themes were developed by the 2 authors of this
depending on the situation. The interviews were all in article, primarily based on the frequency of the relevant
English. The field notes were taken in real-time during words, after 18 interviews with hospital pharmacists were
the interview. conducted. Table 2 outlines the themes with associated
The procedure of data analysis was started with the codes. The number of pharmacists who responded to each
verbatim transcription of the interviews. Next, in the code was recorded.

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Table 2 : Thematic Framework

No. of participant
Theme/Category Codes responded
Nature of error Severity of outcome 13
Type of drug 3
Frequency of errors 2
unimportant errors 1
Repetition of errors 2
Reporting Time
Reporting mode
13
MERS (online / paper-based) 2
QAP-1 form 1
Incident reporting 5
verbal
Confidentiality 16
Reporting form 10
Targeted reporting 1
Organizational Push factor 4
factors
Feedback 18
Maintain the professional relationship 9
Maintain a reputation 4
High turn-over rate 2
Reporting culture
Blaming culture 7
The concern of superiors' reaction 1
The concern of repercussion from patient 4
The concern of the colleague's reaction 1
The concern of confidentiality 3
The concern of getting a low mark in annual
performance appraisal 7
Reporter's burden Overlapping reporting 3
Workload pressure 6
Shortage of staff 1
Huge paperwork 7
Provider related Unclear 4
factors
Role in reporting 3
Responsibility 7
Routine task 3
Personal fear 5
Laziness 2
benefit from Change in practice 16
reporting
Prevent reoccurrence 17
Self-protection 1
Vigilance 14

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In general, the majority of the hospital pharmacists the MERS system." (In-patient pharmacist, Interviewee
understood the definitions of medication error and the 14)
existence of the medication error reporting system. Although confidentiality was respected in most of the
All of the participants agreed that medication error hospitals, in some of the settings the identity of the
reporting brought benefits to patients and the healthcare reporter was not protected. This might affect the number
system in the hospital. However, the majority of the of ME reports, according to some of the interviewees:
participants agreed that the underreporting issue occurred "People can recognize your handwriting, people see
in the hospital. 13 out of 18 participants agreed that the you writing. If you have an online system, maybe after
severity of the error outcome was the deciding factor for work, you go back to your house, you can at least just
medication error reporting. According to the participants, submit it. Yes, so if the reporting is more invisible." (Out-
multiple reporting systems were available at different patient pharmacist, Interviewee 8)
settings, Medication Error Reporting System (MERS)
10 out of 18 participants said that the reporting form
was the most widely used.
was tedious to fill out and too lengthy. Majority of them
Nature of error said a simplified reporting form or using an application
Nature of error was the first theme, identified from would encourage them to do more reporting:
these interviews. 13 out of 18 participants agreed that
"Just that maybe we can simplify the process of
the nature of error affecting their decision to do the
reporting. So the people will actually report it." (In-
medication error reporting. The majority of the
patient pharmacist, Interviewee 14)
participants agreed that the severity of outcomes was the
main factor to decide if the medication error should be Organizational factors
reported. Besides that, the type of drugs, frequency of There were a few organizational factors that affected
errors, the importance of errors and repetition of errors the behavior of medication error reporting. Pushing
also affected reporting decisions. factors from other professionals or superior and useful
In case the medication error brings no or mild harm feedback encourage medication error reporting. In
to the patient, less likely it to be reported: contrast, factors like blaming culture, concerns about
maintaining the relationship with other professionals,
"If no harm to the patient, I will not report. Just inform
maintaining the reputation and annual performance
the person that… they did errors, so that next time they
appraisal hinder medication error reporting.
will not do the error again." (DIS pharmacist, Interviewee
11) Practitioners learn from the feedback in order to
prevent the recurrence of the same medication error and
Reporting system to gain some new knowledge.
The majority of the participants were aware of the "We have email and in this time, we have all those
methods to do the ME reporting, however, some of them (like) WhatsApp, we have a lot of groups, normally they
were confused due to the multiple reporting modes will notify us by those kinds of channels." (Clinical
available in their practice setting. Confidentiality issues, pharmacist, Interviewee 16)
reporting form and targeted reporting in their setting were
Some participants said they tried to maintain a good
among other concerns of the interviewees.
relationship with other healthcare professionals and their
MERS, either online or manual, QAP-1 form, CP3 colleagues, therefore they didn't report some of the MEs
form, Incident Reporting and finally verbal report to the as long as the errors didn't bring any harm to the patient:
superior or person-in-charge are of those different ME
"They will feel like we are reporting them, then is
reporting systems.
something like not good, like complaint themsometimes
Most of the participants stated that MERS was the they will feel like… like revenge… revenge or what." (Out-
most commonly used reporting mode in the hospital patient, Interviewee 6)
setting:
The reporting culture in the practice setting is of
"We will fill up a medication error form. After that, importance and blaming culture, followed by the concern
we will send it to our drug information center and then of getting a low mark in annual performance appraisal
they will have… they will help us to key in the data into were participants' concerns. Some of the participants

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DOI : 10.5530/ctbp.2020.4s.7

mentioned considerations about their superior, patients, "But there are also staff who just don't care." (Out-
colleagues and others, too: patient pharmacist, Interviewee 6)
"I would say like staff, they are quite… sometimes Personal fear of own committed error reporting was
they quite resist of reporting any errors to us, which is one of the factors with a negative impact on medication
because … they afraid they will be punished." (Out- error reporting.
patient pharmacist, Interviewee 6) "let's say if you do something wrong, there will be
"... whenever somebody did an error, no matter is sure you will feel afraid yeah, you know boss is going to
actual error or near-miss error… even near-miss error scold me this and that…" (Out-patient pharmacist,
that is detected before… before actually dispensing Interviewee 9)
prescription, they want to like so-called minus marks."
Benefit from reporting
(Out-patient pharmacist, Interviewee 4)
All participants believed that medication errors
Reporter's burden reporting brings benefits to the patients as well as the
For some medication errors, participants said they healthcare system. The majority of them stated that
were required to fill out more than one reporting form. reporting had successfully prevented the reoccurrence of
This increased the burden for the reporters. similar errors. They strongly believed that medication
error reporting can increase the alertness and awareness
Most of the participants said they had a heavy
of practitioners. One of the participants considered
workload in the practice setting, so it was difficult for
reporting as self-protection.
them to do the reporting. If they wanted to report the
medication errors, it actually increased their workload: According to the participants, root-cause analysis of
occurred errors had been done with corrective measures
"We are busy from 8 until… until lunchtime. After
such as tall-man lettering, changing of the arrangement
lunchtime, the staff nurse will non-stop calling us, so I
of the medications in their practice setting to prevent the
think is a harsher to… actually to increase our workload."
same errors from occurring again:
(In-patient pharmacist, Interviewee 18)
"We will make sure the two different medications, we
Many participants stated that they needed to do a lot
put in a far place, farther place, or else we, we do tall
of paperwork to report the medication errors, they needed
man lettering..." (Out-patient pharmacist, Interviewee 2)
to provide lots of information for root-cause analysis,
investigation and so on: "For medication error, I feel the reporting is important
because it will alert the staff, okay, to… to be more
"I would say first, sometimes working in the
cautious, to prevent the same error from happening
government hospital, the pharmacist can be very busy,
again." (Out-patient pharmacist, Interviewee 6)
and the workload can be very high, so er… reporting all
these requires a lot of paperwork and sometimes we even This study revealed that Malaysian hospital
need to key into the computer system. So erm… it actually pharmacists are well aware of the existence of medication
encourages more underreporting because of lacking errors reporting in their settings. They viewed this system
time." (Out-patient pharmacist, Interviewee 8) positively, with benefits to the patients and the healthcare
system by improving the patient's safety. This finding, as
Provider-related factors well as the underreported medication error reporting, are
Referring to the attitude of the hospital pharmacists in accordance with other study findings, especially those
towards MER, this study came to know that most of the studies from Malaysia that estimated MER as low as 16%
participants took medication error reporting as their (12) or 9% in the UK . However, these statistics may be
responsibility. They perceived that medication error argued, as direct observation of medication administration
reporting can improve the patient's safety. However, some in 36 hospitals in the US revealed an 11.7% error rate
said they knew some coworkers who did not care and compared with just 0.04% for errors detected through
refused to do the reporting: the incident reporting scheme (5).
"Happy or not, it's a responsibility of reporting it. The severity of the outcome of the error was the main
Because ultimately is about patient safety so no matter factor to influence their decision to report. 13 out of 18
how, I think it's very important to report the error." (Out- participants emphasized on this point. It seems the barriers
patient pharmacist, Interviewee 4) would not hinder reporting if there is an actual severe

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medication error. This is in line with other studies consuming, which is in line with previous studies (12)(1).
(12)(8)(6)(1). This was because they strongly believed This was considered a major barrier to medication error
that medication error can be prevented by reporting reporting. The reporting form was complained to be very
medication errors. tedious, the reporting process was very lengthy, and a lot
In contrast, the hospital pharmacists are reluctant to of information was required to fill out in the medication
report the actual medication errors if there is no harm or error form. It was troublesome and needed to spend a lot
the harm to the patient is mild. This finding was parallel of time.
with previous studies which were published earlier Of all organizational factors, perhaps the feedback on
(1)(4)(12). their reported medication errors is the main factor that
Regarding the near miss medication errors and affects the decision of pharmacists to actively report
medication errors that impose mild or no harm to the medication errors . Positive feedback enhanced or
patient, our finding is also the same as previous studies encouraged pharmacists to do more reporting in the
(12)(1); the error might be reported only if it is being future. Lack of feedback on the reported medication errors
occurred repetitively in their practice setting. demotivated the pharmacists to do the reporting, on the
other hand. This finding is consistent with the other
There are multiple types of medication error reporting
studies which reported the demotivation after lack of
platforms available in Malaysia. Different hospitals use
getting feedback (1)(12). Demotivation was due to the
different reporting platforms. The examples in
perception that no actions were taken, or no changes
government hospitals include MERS (either online or
happened in their practice to prevent similar medication
physical manual form), QAP-1 form, incident reporting
errors from happening. Conversely, feedback on
and verbal reporting to the superior. Hospital pharmacists
medication errors motivated the pharmacist to do the
do not feel comfortable with the existence of multiple
reporting. This was because they knew their work was
reporting platforms, as it is reported to be confusing (1).
appreciated and they did something to improve patient
MERS was the most commonly used platform among
safety and healthcare system in their practice settings.
the participants of this study. This finding is different
from the previous publication where the participants use Another barrier to MER, based on our findings, is the
QAP and PF, majorly and perceived MERS as a duplicate concern about maintaining a good relationship with the
task. This different finding may be primarily due to the other healthcare professionals and maintaining a
difference in the participants' profession; ours is hospital harmonious working atmosphere. They had specific
pharmacists, whereas the previous study recruited other anxieties about the effects of reporting on
health-care providers in primary care clinics (12). The interprofessional working relationships with other
other reason for the observed difference could be the healthcare providers. The Same concerns have been
dominance of hospital pharmacists from the government reported by others (1). This problem can be relieved by a
sector (14 out of 18) in this study that warrants the usage continuous convincing education on the importance of
of the national medication error reporting system. In some MER in one hand and correcting the blaming culture in
situations, two different reporting forms needed to be case of the occurrence of medication errors, on the other
filled for a case which obviously increased the reporter's hand. Around half of the participants agreed that there
burden and hinders ME reporting. was a blaming culture in their practice setting and this
was a barrier to MER. This finding is consistent with
According to our participants, although there is no
other studies that stated the same findings (2)(1). They
intentional plan to disclose the identity of the reporters
were afraid to do the reporting especially if they were
by the hospital management, the existing procedure and the ones who involved in medication errors. This is
system of MER cannot completely protect the
probably because they believed that the culture of blaming
confidentiality of the report. It was believed that the
individuals was still present dominantly in the system.
number of medication errors reporting would have been They were afraid they will be punished on their committed
increased If the confidentiality of the reporting could be
medication error or disciplinary action will be taken
maintained. The previous Malaysian study also concluded
against the person who committed an error (3). The
the same point (12). culture of 'blaming on the system' was more successful
10 out of 18 of the participants stated that the reporting in motivating the people to do the reporting (12).
forms for ME reporting were too cumbersome and time-

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The concern of getting a low mark in annual medication error will be raised by providing some
performance appraisal was a major barrier to medication feedbacks such as briefing, memo or bulletin in their
error reporting. This finding was in line with other studies practice setting, this makes the colleagues more alert in
that stated they feared that these reports somehow would the future. However, the job will be accomplished when
affect their annual performance appraisal (12). Annual a root-cause analysis is conducted, and the causes of the
performance appraisal, reflected in Key Performance error are identified. Appropriateness of any solution
Index (KPI), normally affects the staff promotion, salary would be dependent on a proper root cause analysis.
increment, and bonus. In some of the settings, it was stated
4. Conclusion
that the reported medication error does not affect their
performance. However, in most of the hospital settings, Despite the availability of several different methods
they had the target and key performance index (KPI) that of medication error reporting, the rate of reporting is still
could be affected by MER. This hindered the pharmacists low. The diversity of reporting systems may not enhance
or co-workers from reporting. The impact of medication the MER if multiple methods are being deployed in one
error on their KPI in the government sector is apparently single center. At the same time, the procedure of MER
not as big as in the private sector. should as simple and quick as possible. The recent
attempts of using software and applications can be
MER perceived as a burden if the reporting process
evaluated to assess the impact of digitalization and online
was not simple and required too much extra work or time
services on the rate of MER, not only in severe cases but
. This tedious process increased paper workload and it
also in low risk and near-miss cases. Whatever the
hindered medication error reporting (12). They needed
method is, the confidentiality of the process should be
to spend a lot of time to do these procedures in order for
guaranteed by the healthcare managers. The managers
them to complete the form. The overlapping reporting
should also avoid blaming the reporters and separate the
requires more time and effort from the reporter to
performance evaluation from MER, where they put
complete all the reporting. (192)(1).
maximum effort into correcting the system and addressing
The responsibility of a pharmacist to improve the error by proper root cause analysis. Finally, it is
medication safety is an enhancer for medication error necessary to educate the healthcare practitioners about
reporting. In this context, the pharmacist takes the MER and conduct continuous reminding plans for them
responsibility of dispensing the medication properly, and to keep the patients' safety as the priority and practice
to make sure that the patient receives a proper medication MER with no connivance.
treatment. Medication error reporting prevents the
Acknowledgments : We would like to thank the Director-
reoccurrence of the mistake, thus improves patient safety.
General of Health, Malaysia for permission to publish
In some studies, it is stated that the healthcare providers'
this article.
responsibility cannot be ignored and not shirking from
their responsibility (1). Conflict of Interest : The authors of this article claim
no conflict of interest.
Personal fear of the consequences of reporting has
been proposed by other researchers. People may feel 5. References
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Keat and Fazlollah

Current Trends in Biotechnology and Pharmacy 72
Vol. 14 (5) 72-81, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.8

Malaysian Community Pharmacists' Knowledge, Attitudes and Practice

Toward Vaccination and Immunization; A Cross-Sectional Study
Ali Saleh Noori Istehkam1, Fazlollah Keshavarzi1*, Aziz Ur Rahman1
1Faculty of Pharmaceutical Sciences, UCSI University, No 1, JalanMenaraGading,
Taman Connaught, Cheras, 56000 Kuala Lumpur, Malaysia

Corresponding Author : fazlollahk@yahoo.com

Abstract Key words : Vaccination.Community Pharmacy.

Vaccine hesitancy is a serious problem that has been Knowledge, attitudes and practice. Health policy
increasing in the past few decades. The contribution of
1. Introduction
community pharmacists in vaccination was found to have
In line with the necessity of increased contribution in
a good impact on immunization rates in some countries.
public health, the Malaysian Pharmaceutical Society
This study aims to investigate the Malaysian community
(MPS) has recently started a campaign for the involvement
pharmacists' knowledge and attitude toward vaccine-
of community pharmacists in government vaccination and
related services, as well as the enhancers and barriers of
immunization programs (1). This is after prior MPS efforts
the contribution of the community pharmacists in public
in 2014 (2) and initial agreements between MPS and
immunization services. A cross-sectional study was
Malaysian Ministry of Health in 2015 and 2016, but later
conducted among Malaysian community pharmacists. A
the Ministry did not execute the plan (1). Malaysian
pre-validated questionnaire was collected from 273
Medical Association (MMA) afterwards reflected and
community pharmacists by using online and face to face
described it as 'counterproductive' (3). This particular
approaches, from August to October 2019. Overall, 80.2%
extended pharmacy service is now in the center of some
of the participants' level of knowledge scores were
controversies and conflicts.
moderate. The knowledge score on the influenza vaccine
was higher than the other vaccine types. 61.2% of the Vaccine hesitancy has been increasing in the past few
participants were willing to contribute to vaccine decades, probably due to the anti-vaccine propaganda via
administration services by giving priority to administering the internet and social media in many countries. Several
emergency (influenza pandemic) (61.9%), travel (43.6%) reports from western countries (4-6) shown the problem
and influenza (30.8%) vaccines. 35.9% of the pharmacists is serious enough to be addressed by health authorities.
counseled adults for vaccines and 35.6% promoted routine In Malaysia also, it is reported that the number of parents
immunizations of any type for adults. The barriers to with children aged below two years refusing vaccination
pharmacists-led immunization were the need for formal increased from 470 cases in 2013 to 1292 cases in 2014
certification (93.8%), extra education/training to (7). Re-emergence of diphtheria and measles with death
administer vaccines safely (89.7%), reimbursement tolls since 2013 (8) is now a major concern for the
concerns (69.6%), patients' perspective of receiving Ministry of Health. This situation warrants maximum
vaccines by pharmacist (63.4%), the average waiting time utilization of healthcare providers to enhance the process
for immunization services (52.8%), time needed for of immunization in the whole society.
professional development and training (50.5%) and costs Community pharmacists are in the frontline of the
needed for professional development and training healthcare system and most readily accessible healthcare
(49.5%). Despite the legal and administrative limitations providers. Compared with other healthcare providers,
of practicing vaccination by community pharmacists, the community pharmacies offer extended opening hours,
overall vaccination-related knowledge of the participants more convenient locations and access to professional
is moderate. The majority of the participants were willing knowledge and expertise without requiring an
to contribute to vaccine administration services. Further appointment (9). The long history of the contribution of
education and training campaigns along with the financial community pharmacists in vaccination and immunization
support are needed to overcome the barriers which hinder programs in the western countries strongly supports the
their contribution to the immunization programs. idea of the involvement of community pharmacists in

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Current Trends in Biotechnology and Pharmacy 73
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dispensing and administration of vaccines. The question immunization programs would increase public access and
is how prepared the Malaysian community pharmacists improve rates (84 %). 68 % agreed that pharmacists
are for such an extended pharmaceutical care service. should be permitted to immunize. Pharmacists indicated
In line with the policy of increasing accessibility to education, reimbursement, and negative interactions with
immunization services which is considered as a key other providers as main barriers to contribution to
element in the prevention of many serious infectious administering vaccines (12).
diseases, community pharmacists have been actively Another Canadian survey reports that 52% of
involved in immunization services in many developed community pharmacists show interest to administer
countries such as the USA (10), the United Kingdom vaccines, pending a legislative change. These pharmacists
(UK)(11), Canada (12), and New Zealand (13). It is were more interested in administering travel (92%), flu
evident that the involvement of pharmacists in (88%) and pandemic (85%) vaccines than regularly
immunization, whether as educators, facilitators, or scheduled vaccines for adults (65%) or children (18%).
administrators of vaccines, results in increased Leading barriers to pharmacist-led immunization were
immunization rates (14). lack of time (90%) and training (92%). The main positive
In the USA, a study was conducted to assess the determinants were identified as immunization training
involvement of immunization-certified community (95%) and adequate remuneration (92%)(18).
pharmacists as educators, facilitators, and direct In a study from Saudi Arabia, 55% of the respondents
immunizers. It demonstrated the willingness of the expressed their willingness to administer vaccines and
pharmacists to be certified immunization service establish an immunization service. The remaining (45%)
providers were more than 50% of the participants (15). respondents mentioned the lack of training (75.4%) and
Another American study was conducted to assess the concerns in maintaining patient safety (67.4%) as barriers
community pharmacists' opinions on providing to the delivery of immunization services (19).
immunizations. The main documented barriers to This study will enable Malaysian healthcare
pharmacists' contribution was reimbursement concerns policymakers and pharmaceutical professional bodies to
(66%), insufficient staffing (42%), lack of space for reevaluate the contribution of Malaysian community
administration (23%), lack of physicians' support and pharmacists in public vaccination programs. We are
record-keeping (22%), time for certification (10%), coast hopeful the results of this study highlight the knowledge
of certification (7%) and managing adverse events and gap and challenges of effective CP's involvement in
liability concerns (18%). Most respondents felt fully vaccination and immunization activities in general and
accepted as immunization providers by patients (97%), in a particular manner, in addition to the assessment of
the frequency of patients' requests for vaccine information CPs preparedness and willingness.
was 23% monthly, 19% weekly and 18% daily requests. The study results might be stimulation for extra
The majority of respondents (>80%) were comfortable educational and training sessions for the Malaysian
with providing information about influenza, herpes zoster, community pharmacists in the future that might have a
hepatitis A or hepatitis B vaccines. In contrast, they were positive impact on the immunization rates in Malaysia.
less comfortable with discussing pneumococcal, tetanus It may also be a starting point for Malaysian pharmacy
and meningococcal vaccines due to the lack of schools to help achieve conformity with national
knowledge, reimbursement concerns and being unsure immunization objectives by training future generations
about safety in different patient populations (16). of pharmacists, as they can improve immunization
A national survey among American community education and enhance the practical skills for
pharmacists reported that the main three barriers to the undergraduate students of pharmacy by incorporating
provision of immunization services were lack of time, vaccines into their curriculum.
concern for legal liability and reimbursement concerns
(17). 2. Materials and Methods

A survey from Canada shows a high willingness Study design

among Canadian community pharmacists towards This is a cross-sectional survey among Malaysian
involvement in vaccination programs. The study found community pharmacists, using a validated questionnaire
that 88 % agreed that the involvement of pharmacists in that is adapted from a Canadian study (12). The study

Ali et al
Current Trends in Biotechnology and Pharmacy 74
Vol. 14 (5) 72-81, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.8

aims were to assess the beliefs, attitudes, knowledge, and vaccines' adverse reactions, and other vaccine
practice towards vaccination and immunization among administration issues.
Malaysian community pharmacists. The community pharmacists' overall knowledge was
Study settings categorized by partitioning the total knowledge score into
Community pharmacists from different states and three equal ranges(20); high level of knowledge if the
cities (Terengganu, Johor, Kedah, Kelantan, Melaka, score was between 17 to 25, moderate if the score was
Pahang, Perak, Perlis, Penang, Sabah, Sarawak, Selangor, between 9 to 16 and low level of knowledge if the score
Negeri Sembilan, Putrajaya and Kuala Lumpur) was less than or equal to 8.
participated in this study. Sample Size. According to Raosoft online sample size
calculator (http://www.raosoft.com/samplesize.html)
Study participants assuming a 5% margin of error, 95% confidence level,
All community pharmacists who practice the 50% response rate and 3000 community pharmacies with
community pharmacy profession in Malaysia, including response distribution of 50%, the required sample size
provisionally registered pharmacists (PRPs), were for this study will be 341 respondents.
eligible. Hospital and industrial pharmacists were
Data collection
excluded from this study.
An online survey form was created to share the study
Study instrument questionnaire with different networks that are linked to
The validated questionnaire, previously published by the Malaysian community pharmacists. At the same time,
Canadian researchers was adopted and face validation to ensure that the minimum required sample size is
was performed by three expert academicians, followed achievable within a preplanned time frame, manual
by a pilot study, to ensure its compatibility with the local distribution of the questionnaire was performed, on a
conditions. Further corrections and modifications were convenience basis in Kuala Lumpur and the surrounding
made, based on the feedback from experts and area in the state of Selangor, such as Puchong, Petaling
respondents. The questionnaire (APPENDIX II) consisted Jaya, Cheras, Serdang, Gombak, Shah Alam, Klang,
of 88 questions; 5 demographic questions, 21 questions Subang Jaya, Sunway, Sri Kembangan, and Ampang Jaya.
to assess the immunization knowledge, 61 questions about The data was collected from mid-August to mid-October
the attitude toward immunization and one question about with a total of 273 responses, both manually and online.
vaccine-related services (Appendix II). The first 61 Data analysis
questions were partitioned into six sections. The first Data analysis was performed using SPSS version 22.
section (general attitude) consisted of three questions
Descriptive analysis and frequency assessment were
regarding community pharmacists' opinions about
applied to the demographic characteristics of the
expanding their scope of practice and adults' vaccination. participants. To assess the correlation and association of
The second section (specific attitude) consisted of 22
the variables, Spearman's correlation was performed. To
questions regarding participants' awareness about
compare the means and scores of the knowledge and
vaccines and their importance in addition to some of the attitude between the groups of the participants; Mann-
possible barriers to pharmacists' involvement in
Whitney U and Kruskal-Wallis tests were conducted.
immunization services. The third section (practical
implications) consisted of three questions regarding Study approvals
participants' willingness to administer vaccines and the The project approval was received from Faculty
barriers to their contribution to vaccine administration. Research and Scholarly Activities (FRSA), Faculty of
The fourth section consisted of 13 questions about Pharmaceutical Sciences, UCSI University. The study
pharmacist-related immunization practice. The 8 protocol was registered on the National Medical Research
questions in the fifth section were about patient-related Register (NMRR) website and ethical approval was
factors that impact immunization practice. The 9 obtained via the Medical Research Ethics Committee
questions of the sixth section were about the information (MREC), the Ministry of Health, Malaysia.
and requirements in vaccine administration.
3. Results and Discussion
The 21 knowledge-related questions were about A total of 273 community pharmacists participated in
vaccine types, vaccines indications/contraindications, the study. There was no significant difference between

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Current Trends in Biotechnology and Pharmacy 75
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DOI : 10.5530/ctbp.2020.4s.8

male and female participants. Since the questionnaire link Table 2 : Pharmacy Practice Variables
was shared with community pharmacists via different Variables N %
platforms the authors could not stipulate how many
pharmacists had reached the link. Therefore, the response State of primary
rate could not be estimated due to the missing
denominator. practice n=273
Selangor 121 44.3
The education level of 185 (67.8%) of the participants
was undergraduate whereas 88 (32.2%) were Kuala Lumpur 77 28.2
postgraduate as shown in Table 1. Sarawak 24 8.8
Table 1 : Demographic Data Variables Sabah 20 7.3
Variables N % Johor 12 4.4
Gender n=273 Putrajaya 8 2.9
Male 138 50.5 Perak 8 2.9
Female 136 49.5 Labuan 3 1.2
Qualification n=273 Years of working
Undergraduate 185 67.8
experience n=273
Postgraduate 88 32.2
1 to 5 yeas 104 38.1
Position n=273
6 to 10 years 85 31.1
Staff pharmacists 158 57.9
11 to 20 years 47 17.2
Manager 44 16.1
Less than one year 26 9.5
Relief pharmacist 31 11.4
21 to 30 years 11 4.1
Owner 22 8.1
Clinical pharmacist 18 6.6 Working hours per
week n=273
Regarding the participants' position, 158 (57.9%) of
the participants were staff pharmacists, 44 (16.1%) More than 40 hours 172 63
managers, 31 (11.4%) relief pharmacists, 22 (8.1%)
owners, and 18 (6.6%) clinical pharmacists. In terms of 25 to 40 hours 75 27.5
the primary state of practice, 121 (44.3%) of the 11 to 24 hours 23 8.4
participants were practicing in Selangor, 77 (28.2%) in
Kuala Lumpur, 24 (8.8%) in Sarawak, 20 (7.3%) in Sabah. Less than 10 hours 3 1.1
The primary practice state of the remaining (11.4%) was
The specific participants' knowledge about the
Johor (4.4 %), Putrajaya (2.9 %), Perak (2.9%) and
influenza vaccine is reflected in Table 3.
Labuan (1.2%).
For example, the necessity of immunization of
Regarding working experience, 104 (38.1%) of the
pregnant women with influenza vaccine if delivery during
participants reported 1 to 5 years, 85 (31.1%) stated 6 to
the influenza season is expected was questioned. The table
10 years, 47 (17.2%) had 11 to 20, 26 (9.5%) declared
demonstrates a high level of knowledge, compared to
less than one year and only 11 (4.1%) declared 21 to 30
other components of knowledge in the questionnaire.
years.
The findings of the knowledge of the participants
In response to a question about their weekly working
about the other types of vaccines are summarized in Table
hours, 172 (63%) were working more than 40 hours per
4. The questions of this section were about the
week, 75 (27.5%) reported 25 to 40 hours per week, 23
pneumococcal vaccine, tetanus-diphtheria vaccine, polio
(8.4%) replied 11 to 24 hours per week and only 3 (1.1%)
vaccine, and so on. The percentage of given correct
reported that they work less than 10 hours per week. Table
answers is low compared to the influenza vaccination
2 summarizes the participants' information about their
section.
pharmacy practice.

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DOI : 10.5530/ctbp.2020.4s.8

Table 3 : Influenza Vaccine Correct Responses approximately 1/10,000 vaccine doses administered",
Items (N= 273) N % 30.4% "The development of an urticarial skin rash (hives)
at the site of vaccine administration may occur as an early
Unvaccinated people with mild
214 78.4 sign of anaphylaxis and should be managed as
symptoms of influenza can spread the
disease to others. anaphylaxis" and only 6.6% "Epinephrine should be
administered in the same limb as the vaccine".
Influenza vaccine should not be given 114 41.8
during the first trimester of pregnancy. The community pharmacists were asked whether their
opinions should be sought when the scope-of-practice is
Annual influenza immunization is
recommended for all health care 246 90.1 expanded to include the administration of vaccines to
professionals in contact with individuals adults. The majority of the pharmacist (82.5%) reported
in high-risk groups. that their opinion should be sought to include the
What it the reported vaccine-associated administration of vaccines among the adults and only
adverse event after immunization with 246 90.1 while 13.9% neither agree nor disagree and the remaining
influenza vaccine that contraindicates to 9.9 % disagreed agree for the above query. In response
subsequent immunization. to the question of whether the provision of immunizations
Influenza vaccination is considered safe 139 50.9
to adults is adequate, 40.3% of the participants disagreed,
in pregnancy.
31.9% agreed and 27.8% neither agreed nor disagreed.
Table 4 : Other Vaccine Types Correct Responses Regarding the importance of receiving all adult vaccines
recommended by Malaysian guidelines, 83.5% chose
Items (N= 273) N %
"Agree", 9.2% chose "Neither agree nor disagree" and
Pneumococcal vaccination is 59 21.6 only 7.3% chose "Disagree". Referring to the importance
contraindicated for a splenic (without
of increasing the proportion of adults who receive
a spleen) patients.
recommended immunizations, 92.6% agree, 5.1% did not
One tetanus-diphtheria booster in
92 33.7 agree or disagree and only 2.2% disagree.
adults should be replaced with one
dose of tetanus, diphtheria, acellular The participants were asked about the importance of
pertussis (Tdap) vaccine. getting annual influenza vaccine and tetanus-diphtheria
47 17.2 toxoid vaccine every 10 years; 82.4% agree that the
polysaccharide or conjugate annual influenza vaccine is important, 11.8% didn't agree
pneumococcal vaccine. or disagree and only 5.9% reported that they disagree.
Which of these are Live Vaccines? Most of the pharmacists (75.1%) reported that they agree
Oral polio 54 19.8 with the importance of receiving the tetanus-diphtheria
Measles-Mumps-Rubella 179 65.6 vaccine every 10 years, 20.5% didn't agree or disagree
Varicella 108 39.6 and only 4.4% disagree.
In terms of risks and benefits of vaccines, 89.4% agree
The pharmacists were asked if there is any evidence
that vaccines produce more health benefits than health
to support an association between vaccines and multiple
risks, 7.3% did not agree or disagree and only 3.3%
sclerosis; 58.5% chose "Don't know", 26.5% "False" and
disagree. 72.9% of the participants agreed that vaccines
only 15% reported that this statement is true. The
are adequately tested for safety, 25.3% didn't agree or
participants were questioned regarding the vaccines that
disagree and only 1.8% disagreed. Most of the
could be given to individuals with an anaphylactic
respondents (85%) agree that the serious adverse
reaction to eggs, 58.2% of the pharmacists selected
reactions to vaccines are rare.
"Influenza", 32.6% "Measles-Mumps-Rubella", 21.7%
"Meningococcal", 20.9% "Hepatitis B" and 11.7% Regarding the frequency of the vaccine-related
"Pneumococcal". questions, asked by patients 28.2% disagree that they are
frequently asked by the patients for advice about vaccines,
Regarding anaphylaxis following vaccination, 56%
46.5% agree and 25.3% did not agree or disagree. When
of the community pharmacists chose "Prompt
the question was about their comfortability to answer the
administration of subcutaneous or intramuscular
patients' inquiries, 77.7% agree.
epinephrine is the most important step in the management
of anaphylaxis", 45.1% "Anaphylaxis occurs following The impact of the pharmacists' contribution to the

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DOI : 10.5530/ctbp.2020.4s.8

vaccination, was assessed by respondents positively of the respondents agreed that they had received an
(85.4%). The participants were also asked about vaccine adequate education or training. It is not surprising that
administration issues. 83.6% of the pharmacists agreed 89.7% stated that they need additional education and
that pharmacists should be permitted to expand their training to be able to administer vaccines safely. Some
practice to include administration of recommended adult Canadian studies (12, 18) along with a study from Saudi
vaccines. Arabia (19) reported a good level of vaccine-related
The incorporation of immunization services in knowledge among community pharmacists and at the
community pharmacy practice was also questioned, same time an adequate education and training courses
where 61.2% responded positively, 28.6% "Unsure" and about vaccines and immunization in the taught pharmacy
only 10.3% responded "No". programs. The same stipulation can be extrapolated to
the findings from the US, as well (15). The involvement
When the pharmacists were asked if an immunization
of American community pharmacists in vaccine
training or certification program was available for them
administration and services undoubtedly provides a good
which one of the immunization services they feel they
opportunity to expand their knowledge in this field. The
apply to them, 61.9% reported "I would vaccinate in
correlation between practicing a specific task and
emergency situations (e.g. influenza pandemic)", 39.2%
attributed knowledge level would be more obvious when
reported "I would vaccinate in a collaborative framework
the influenza-specific knowledge score is compared with
where a physician recommends a vaccine first and then I
the general vaccination knowledge in our respondents.
administer the vaccine", 41% reported "I would vaccinate
Influenza-specific knowledge is higher, simply because
in a collaborative framework where a physician
the community pharmacists are being queried by clients
vaccinated the first time and I could provide all
more. The fact that 92.7% of the respondents reflect
subsequent vaccinations of the same vaccine", 30.8%
comfortability and willingness to administer the influenza
reported "I would be comfortable administering influenza
vaccine is another aspect of the same concept. This is in
vaccine only", 43.6% reported "I would administer travel
line with a study results (16) that demonstrated American
vaccines" and only 8.8% reported, "I would not administer
community pharmacists were more knowledgeable on
vaccines".
influenza, herpes zoster, hepatitis A and hepatitis B
Currently, the Malaysian community pharmacists do vaccines as compared to their knowledge in
not administer vaccines, although they may provide some pneumococcal, tetanus and meningococcal vaccines.
vaccine-related services. 35.9% of the participants stated
Regarding the safety of vaccines, their relationship
that they actively counsel adults for vaccines and 35.5%
with multiple sclerosis and specifically the link between
reported that they don't provide any vaccine-related
egg allergy and influenza vaccine contraindication, the
services.
respondents' feedback shows that most of the community
The moderate level of knowledge in the Malaysian pharmacists are not well updated. While doubts about
community pharmacists can be attributed to low the link between vaccines and multiple sclerosis were
contribution to immunization programs in Malaysia (1- expressed in the older literature (24, 25), recent pieces
3). This may hinder a direct exposure to the practice and of evidence deny such a linkage (26, 27). Moreover, it
subsequently affect their experience and skills. The was believed that the influenza vaccine is contraindicated
outcome of the lack of exposure would result in or should be given with supervision and caution for
suboptimal knowledge and motivation to learn about patients with egg allergy (28)(29). However, recent
vaccines or to improve their current knowledge about studies show that the influenza vaccine can be given to
different aspects of immunization (21, 22). Almost 70% patients with egg allergy safely (30, 31). 73.5% and
0f the respondents failed to answer the question about 58.2% of our respondents failed to demonstrate the
the management of anaphylactic reactions to the vaccines updated knowledge regarding the linkage between
correctly. This problem can also be attributed to curricular vaccines and multiple sclerosis and egg allergy,
issues in the pharmacy program in Malaysia where most respectively.
of the private and public schools and colleges do not
Our findings show that the male participants have a
provide adequate training and education courses about higher level of knowledge than the female participants
vaccines and immunization programs (23). This is
(mean rank 147.11 vs. 126.67, p = 0.032). This is
supported by the current study finding where only 16.9%
correlated and perhaps explained by longer working

Ali et al
Current Trends in Biotechnology and Pharmacy 78
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experience of male participants than the female Table 5 : Frequent Barriers to Vaccine Administration
participants. Where the number of female participants in Item N %
the category of 'below one year working experience' was
significantly higher than the male participants (18 vs. 8,
Formal certification needed 256 93.8
p<0.05), the number of male participants in the category
of '11 to 20 years working experience' was significantly for vaccine administration
higher than the number of female participants (31 vs. 16, Reimbursement concerns 190 69.6
p<0.05). (time and staff support)
Despite all limitations and obstacles of community Time needed for professional 138 50.5
pharmacy practice in Malaysia, this study shows that development and training
Malaysian community pharmacists are willing to step up Costs needed for professional 135 49.5
their area of practice beyond the existing situation. Only development and training
10.3% of the participants were not keen to get involved
The availability of staff support 206 75.5
in vaccine administration and immunization programs.
The fact that 83.6% of the participants agreed that The average wait time for 144 52.8
community pharmacists should be permitted to administer immunization services
adult vaccination shows that community pharmacists are Patients’ perspective of receive 173 63.4
well aware of the necessity of establishing extended vaccines by pharmacist
pharmacy services, including vaccination and Patients’ confidence in
immunization services. 158 57.9
pharmacist ability to administer
The participants strongly believe (85.4%) that the vaccines
proportion of adults who receive recommended The need for more education/
208 76.2
immunizations would increase if community pharmacists training for vaccines
were permitted to administer vaccines to adults, due to administration
improved access to immunization services. Inadequate education/training 166 60.8
The current study results are comparable with the other about immunization
studies from Canada (12), the USA (15), and Saudi Arabia
Of all those potential barriers, lack of physician
(19) in terms of the attitude of the participants. Similar
support should be considered as a specific matter in the
to the American study, the majority of participants of
context of current pharmacy practice in Malaysia.
current study mentioned improving public healthcare as
Malaysian community pharmacists struggle for separating
their main driver of willingness toward being a certified
dispensing and prescribing has still been fruitless after
vaccine administrator. Regarding the frequency of
decades. Seeking for other areas of extended pharmacy
vaccine-related queries, 23% reported monthly, 19%
services, in the atmosphere of tension between community
reported weekly requests and 18% reported daily requests.
pharmacies and dispensing physicians, probably would
This load of queries may justify the high level of
not be welcomed by physicians. Malaysian Medical
willingness for practicing vaccine administration in
Association (MMA) reflection (3) to the Malaysian
community pharmacists.
Pharmaceutical Society (MPS) attempts toward
Despite the positive attitude of the participants vaccination and immunization services, then was not
towards practicing vaccination, the knowledge gap may surprising. Others have also reported the physicians'
play a negative role in such an achievement. However, opinion that the community pharmacists should focus on
the obstacles are far beyond the sole knowledge gap, their main role as medication experts to oppose their
according to the participants. The study participants contribution in extended services such as vaccination
pointed out the time needed for professional development (32).
training, the cost associated for professional development
Higher attitude scores among the participants from
training, reimbursement concerns (including the
Selangor and Kuala Lumpur states as compared to
availability of staff and their time to provide vaccines),
Sarawak, Sabah, Johor, Perak, and Labuan, is probably
physician support, people's confidence and safety about
due to the type of queries in metropolitan areas, including
receiving vaccines from pharmacists and lack of formal
higher public attention to the adult vaccination issues (33,
certification as potential obstacles (Table 5).
Study on malaysian community vaccination and immunization
Current Trends in Biotechnology and Pharmacy 79
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DOI : 10.5530/ctbp.2020.4s.8

34). Meanwhile, the current study findings show that the Ministry of Health Director-General for issuing the
willingness of pharmacists to practice vaccination is required approvals.
reversely correlated with working hours per week (rs = -
Conflict of interest
0.124, p= 0.041).
The authors of this article declare no conflict of
In response to the questions regarding the barriers to
interest.
vaccination practicing, the pharmacy owners obtain a
higher attitude score compared to the manager or clinical 5. References
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JB. The Star Online 2019 3 March 2019.
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Knowledge, Attitude and Practice of General Public Towards Counterfeit and

Adulterated Medicines : a Cross-sectional Study in Malaysia
Choo Shiuan Por1, Fazlollah Keshavarzi1*, Chuan Sheng Yap1, Yee Chang Soh1
1Faculty of Pharmaceutical Sciences, UCSI University, Kuala Lumpur, Malaysia

* Corresponding email : fazlollah@yahoo.com

Abstract the level of KAP of respondents, followed by the highest

Counterfeit and adulterated medicines have become a level of education and employment status. Health care
global threat. No documented benchmark and framework providers and health workers are encouraged to deliver
that can be used to evaluate knowledge and practice information of CFAM to the public to narrow the gap of
among Malaysian. The main objective of the research is knowledge of CFAM.
to assess the public's knowledge, attitude and practice Key words : Counterfeit and Adulterated Medicines,
(KAP) towards counterfeit and adulterated medicines Knowledge, Attitude, Practice, Kuala Lumpur
(CFAM) in Kuala Lumpur, Malaysia. The research is also
1. Introduction
conducted to evaluate the relationship between
demographic variables and the level of KAP of Malaysian According to the U.S. Food and Drug Administration,
general public on CFAM and assess the opinion of public counterfeit medicine is defined as medicine which may
on the use of education to combat the CFAM issue. A contain wrong or no active ingredient (1). Counterfeit
descriptive cross-sectional study using a validated, self- medicine includes those with right active ingredient but
administered questionnaire was conducted among public wrong dose or those medical products which are
using a convenient sampling technique. This study was contaminated. Adulterated medicines are medicines that
approved by the Medical Research Ethics Committee. A contain or are mixed with substances which may diminish
total of 387 volunteers participated in the study. Data the effect of medicines or jeopardise the health of
including respondents' demographic characteristics, KAP consumers (2).
regarding CFAM and opinion on education to combat Before a medicinal product can be marketed in a
CFAM were collected. IBM SPSS Statistic® version 20 certain country, it must be registered with local drug
was used to analyse the collected data.43.9% of regulatory authority. All medicinal products must be
respondents had a moderate level of knowledge towards compliant to the specifications of approval for registration
CFAM. 54.5% of respondents showed a positive attitude of medicinal products. Responsibility of the authority is
towards CFAM. However, 53% of respondents to ensure that all medicinal products conform to
demonstrated negative practice against CFAM. established criteria of quality, safety and efficacy (3).
Occupation of respondents had a significant association However, Counterfeit and adulterated medicine (CFAM)
with knowledge on CFAM. Highest level of education have become a growing threat worldwide. A systematic
and occupation of respondents were significantly review reported that the prevalence of CFAM in lower-
associated with attitude against CFAM. Significant and lower-middle-income countries throughout Africa and
associations between the highest level of education, Asia is high (4).
employment status and occupations with the practice of
Developing countries are main targets for distribution
respondents against CFAM were revealed. Overall, the
of CFAM due to unaffordability of costly medications by
general Malaysian public in Kuala Lumpur had a
the population (5). High demand in the market drives the
moderate level of knowledge, positive attitudes but
infiltration of CFAM into supply chain easily (6).
negative practice towards CFAM. Although knowledge
Therefore, most commonly counterfeited medicines are
of respondents is positively correlated with attitude and
those costly medicines used for the treatment of life-
practice on CFAM, the practice of respondents towards
threatening conditions such as cancers and infectious
CFAM is unsatisfactory despite positive attitude on
diseases (7). In developed countries, expensive lifestyle
CFAM. Occupation is the most important predictors for
medicines are most commonly counterfeited, such as
Knowledge, attitude and practice of public towards counterfeit and adulterated medicines
Current Trends in Biotechnology and Pharmacy 83
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hormones and steroids pills (7). CFAM has developed for each item in the knowledge domain was computed.
differently between developing and developed country. The higher the mean score (closer to 1), the better the
Consequently, countermeasures to overcome CFAM knowledge of respondents on the respective questionnaire
should be catered for such differences between regions. item.
From a survey conducted in 2013, almost 5% of C. Attitudes towards Counterfeit and Adulterated
medicines sold in Malaysia was fake (8). Serious concern Medicines. This section contained 10 statements to
has been raised in the country on the emerging issue of evaluate the attitudes of respondents towards CFAM.Each
CFAM. However, there are limited local studies to assess positive sentence was graded as follows: ''strongly
the knowledge, attitude and practice (KAP) of the general disagree", 1 point; ''disagree", 2 points; ''neutral", 3 points;
public on CFAM. Association between demographic ''agree", 4 points and "strongly agree", 5 points. In
characteristics of Malaysian with knowledge, attitude and contrast, the negative sentences (statement 1, 4 and 7)
practice towards CFAM are yet to be identified. Hence, were graded in reverse order. A sum score (from a
we conducted a study in the federal territory of Kuala minimum of '10' to maximum of "50') for each respondent
Lumpur, the capital of Malaysia to assess the level of was then computed. The respondent's attitude was
KAP of Malaysian general public on CFAM and evaluate stratified into negative (10-23), neutral (24-37) and
the relationship between demographic variables and the positive (38-50), respectively. Mean score was calculated
level of KAP of Malaysian general public on CFAM. for each item in the attitude domain. Respondents
demonstrate better attitude to the respective questionnaire
2. Materials and Methods
item with a higher mean score (closer to 5).
Study design : A descriptive cross-sectional study was
D. Practice against Counterfeit and Adulterated
conducted between May and August 2018 to assess the
Medicines. This section contained 5 statements to assess
KAP of the general public towards CFAM in Kuala
the practice of respondents about the genuine medicine
Lumpur, Malaysia. The study design and questionnaire
utilisation.Responses to the positive sentences
were approved by the Medical Research Ethics
(statements 1, 2, 3 and 4) were graded as follows: ''almost
Committee (MREC), Malaysia. (reference no:
never", 1 point; ''sometimes", 2 points; ''often", 3 points;
KKM.NIHSEC.P18-1673 (5)).Thiscross-sectional study
and ''always", 4 points. The negative sentence (statement
followed the Strengthening the Reporting of 5), was graded in reverse order. A sum score (from a
Observational Studies in Epidemiology (STROBE)
minimum of '5' to maximum of "20') for each respondent
guideline (9). Given that these are anonymous surveys,
was then computed. The respondent's practice was
the identity of the participants or their affiliated institution stratified into negative (5-10), neutral (11-15) and positive
was not collected. Responding to the surveys by
(16-20), respectively.Respondents practice positively
participants were considered as implied consent.
towards items with a higher mean score (closer to 4).
Study instrument : A self-administered questionnaire was E. Public advice on measures to reduce counterfeit
developed by adaptation and modification from a previous and adulterated medicines. This section contained two
study on counterfeit medicines to suit with local questions to seek public opinion on the use of education
population (10). to combat the CFAM issue.
The questionnaire was constructed in English and Reliability and validity of study instrument: Reliability
consisted of five sections : and validity testing of the questionnaire were performed.
A. Demographics. This section sought details of eight Content and construct validity of the questionnaire was
demographic data. conducted by pharmacy lecturers and subject matter
B. Knowledge about Counterfeit and Adulterated experts. The questionnaire was pilot tested in two phases
Medicines. This section contained 8 statements that the with a convenience sample of 60 Malaysian general
respondents used to describe the CFAM (Yes, No or public for face validity and reliability of the questionnaire.
Unsure).Each correct answer was given '1' point while Cronbach's alphas of the final questionnaire were 0.887
the unsure or wrong answer was given '0' point. A sum (knowledge domain), 0.838 (attitude domain) and 0.781
score (from a minimum of '0' to a maximum of '8') for (practice domain), respectively.
each respondent was then computed. The level of Sampling method and sample size: A convenience
respondent's knowledge was stratified into poor (0-2), sampling method was adopted in this study. The sampling
moderate (3-5) and good (6-8), respectively.Mean score frame is the Malaysian general public who reside in the

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DOI : 10.5530/ctbp.2020.4s.9

federal territory of Kuala Lumpur. Verbal verification of Table 1: Socio-demographic characteristics of

Malaysian citizenship of respondents was done before respondents (N=387)
distributing the questionnaire. By using Raosoft® sample Demographic Parameters Frequency (%)
size calculator, the minimum sample size required Age (years)
(confidence interval = 95%; margin of error = 5%) was 18-24 180 (46.5)
384 for this study (11). 25-39 159 (41.1)
Study participants and data collection: All Malaysian 40-54 29 (7.5)
citizens who reside in Kuala Lumpur, aged 18 or above More than 55 19 (4.9)
with English literacy were eligible to participate in the Race
study. The exclusion criterion was one who refuses to Malay
105 (27.1)
participate in the survey. The validated self-administered Chinese
237 (61.2)
questionnaires were distributed to the crowds in public Indian
35 (9.0)
areas. An informed consent form was attached along with Others
10 (2.6)
each questionnaire,participants were aware that their
Highest level of education
willingness to participate were fully respected andthe
Primary School
result of data analysis will be published in journal for 2 (0.5)
Secondary school
academic purpose. Their anonymity and confidentiality 34 (8.8)
Pre-university
were preserved. 69 (17.8)
Degree
252 (65.1)
Data analysis: The data were analysed using Statistical Master
16 (4.1)
Package for the Social Sciences version 22.0 (12). PhD
14 (3.6)
Descriptive analysis was used to tabulate the respondents'
demographics and level of KAP in frequencies and Employment status
percentages. Chi-square test was employed to assess the Employed
222 (57.4)
Self-employed
association between the demographic variables and the 13 (3.4)
Unemployed
KAP outcomes. Logistic regression analysis was used to 17 (4.4)
Housewife
determine the predictors of KAP. Multinomial logistic 9 (2.3)
Student
regression was employed for knowledge and practice 123 (31.8)
Retired
2 (0.5)
whereas binary logistic regression was utilised for attitude Others
1 (0.3)
domain. A p-value of less than 0.05 was regarded as
statistically significant.Spearman's rho correlationtest was Occupation
conducted to determine the possible relationship between Healthcare related
98 (25.3)
respondents' knowledge-attitude, knowledge-practice and Non healthcare related
188 (48.6)
Not applicable
attitude-practice on CFAM. 101 (26.1)
3. Results and Discussion Monthly income
387 questionnaires were completed and collected from <1,000
36 (9.3)
the respondents. 1,000-3,000
85 (22.0)
3,000-5,000
Socio-demographic characteristics of respondents: 118 (30.5)
>5,000
The socio-demographic characteristics of 387 study 34 (8.8)
Not applicable
participants are summarised in Table 1. Majority of the 114 (29.5)
respondents aged between 18 and 24 years (N= 180, Table 1 shows socio-demographic characteristics of
46.5%), Chinese (N=237, 61.2%) ethnic, and with degree 387 respondents participated into this survey.
qualification (N=252, 65.1%). The mean age of Knowledge ofcounterfeit and adulterated medicines:
respondents participated in this study is 28.13±10.11 Majority of respondents had a moderate level of
years (range = 18-68 years old).About 50% of the knowledge on CFAM (N=170, 43.9%) with a median
respondents are employed in non-healthcare related score of 4.37% (N=143) and 19.1% (74) of respondents
sector, while nearly one-third have monthly income have poor and good knowledge on CFAM respectively.
ranged from RM3,000 to RM5,000. It is also noteworthy Breakdown of respondents' knowledge score on CFAM
that approximately one-third of the respondents refused for the eight questionnaire itemsis shown at table 2. The
to disclose their occupation and monthly income. results revealed that most respondents were aware that
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Table 2 : Breakdown of respondents' rnowledge, attitude and practice score on CFAM per questionnaire item

No. Questions Score

Knowlegde
If there is no MeditagTM Hologram security sticker on the label/outer
1. packaging of a pharmaceutical product, then it’s a “counterfeit 0.421 ± 0.494
pharmaceutical product”.
If there is no registration number starting with the alphabets “MAL”
2. printed on the label of a pharmaceutical product, then it’s a “counterfeit 0.302 ± 0.460
pharmaceutical product”.
Brand and generic medicines are found counterfeited or adulterated and
3. 0.522 ± 0.500
sold for profit.
4. Quality, efficacy and safety of CFAM are not guaranteed. 0.455 ± 0.499
5. The problem of CFAM affects not only developed countries. 0.553 ± 0.498
Medicines for treating chronic and serious diseases (‘lifesaving
6. medicines’) such as heart disease or cancer can be counterfeited/ 0.279 ± 0.449
adulterated.
7. The Internet has become a major platform for circulating CFAM. 0.587 ± 0.493
CFAM can be discovered in the legal medicine supply chain, that is,
8. 0.248 ± 0.432
through licensed wholesalers and traders.
Attitude
1. Counterfeit drugs are not as good as authentic drugs. 3.698 ± 1.023
2. There is high probability that the counterfeit drug doesn’t work. 3.602 ± 1.041
3. Authentic drugs are worth the money they cost. 3.452 ± 0.984
I do not prefer to buy pharmaceutical products from online
4. 3.501 ± 1.184
pharmacy/drug retailer.
I agree that it is important for consumers to consult pharmacist before
5. 4.080 ± 1.026
buying any pharmaceutical products.
I agree that it is important for a pharmaceutical product to display
6. MeditagTM Hologram security sticker and registration number on the 4.010 ± 0.979
label/packaging.
I do not prefer to buy cheap pharmaceutical products regardless the
7. 3.897 ± 0.968
source of the products.
I should immediately inform my physician and pharmacist if I suspect
8. 3.858 ± 1.025
that the pharmaceutical products are counterfeits.
9. I agree that using CFAM will put my health at risk. 3.778 ± 0.991
I agree that purchasing CFAM will bring harm to economy of the country
10. 3.631 ± 0.947
through loss of taxation revenue.
Practice
I check if there is MeditagTM Hologram security sticker on
1. 1.995 ± 0.939
pharmaceutical product before purchasing any pharmaceutical product.
I check if there is registration number on the pharmaceutical product
2. 1.889 ± 0.908
before purchasing any pharmaceutical product.
I inform physician /pharmacist /drug retailer if there is no MeditagTM
3. 1.744 ± 0.919
Hologram security sticker on a pharmaceutical product.
I inform physician /pharmacist /drug retailer if there is no registration
4. 1.713 ± 0.895
number on a pharmaceutical product.
I do not buy the pharmaceutical product that is cheaper than other similar
5. 3.279 ± 0.775
product regardless of the source.

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the internet is a major platform for circulating. More than half of respondents (53%, N = 205) practice
Unsurprisingly, respondents demonstrated the poorest negatively in issues related to CFAM with a median score
knowledge on the discovery of CFAM in the legal of 10. Only 8% of respondents (N = 31) demonstrated
medicine supply chain. positive practice towards CFAM. According to Table 2,
question 5 under the domain of practice ("I buy the
Attitude on counterfeit and adulterated medicines:
pharmaceutical product as long as it is cheaper than other
Around 54.5% (N=211) of the respondents showed
similar product regardless of the source of the
positive attitude (score = 38-50 points) on CFAM with
pharmaceutical product.") was given the highest score
median score of 38. Respondents with a negative attitude
among all the question in practice domain (mean score =
(3.4%, N = 13) may feel that CFAMhave no harm on
3.279 ± 0.775) by the respondents.
their health, whereas respondents with neutral attitude
(42.1%, N = 163) have mixed opinion on harmful effects Association between level of knowledge, attitude and
practice with demographic characteristics of
of CFAM. Among the 10 questions assessing respondents'
respondents:Occupation of respondents is the only
attitude on CFAM (Table 2), most respondents agreed
parameter with a significant association with knowledge,
that consulting pharmacist before buying any
attitude and practice of respondents towards CFAM, as
pharmaceutical productis important. Interestingly,
shown in table 3. The other two characteristics found to
majority of respondents disagreed about authentic drugs
be significantly associated with attitude and practice
worth the money they cost.
arethe highest level of education and employment status
Practice on counterfeit and adulterated medicines: of respondents.

Table 3 : Association between knowledge, attitude and practice with demographic characteristics of respondents

Demographic Knowledge Attitude Practice

characteristics 2
p-value 2
p-value 2
p-value

Age 6.63 0.352 3.37 0.337 1.58 0.960

Race 8.65 0.059 3.44 0.323 10.72 0.080

Highest level of 11.79 0.063 17.28 0.001* 12.11 0.050*

education

Employment status 11.52 0.061 4.23 0.238 12.00 0.046*

Occupation 53.22 0.000* 53.22 0.003* 17.46 0.000*

Monthly income 11.91 0.186 4.84 0.307 3.09 0.935

x2: chi square

*: p value value less than 0.05

Predictors of level of knowledge, attitude and practice As shown in Table 6, employment status, the highest
on CFAM: Table 4 shows that the occupation of level of education and occupation were found to be the
respondents is the only significant predictor for the significant predictors for the level of practice on CFAM
respondents' level of knowledge on CFAM. Respondents of respondents. Unemployed respondents were less likely
with healthcare-related occupation will likely to have a to practice neutrally compared to the student.
better level of knowledge on CFAM than that of non- Respondents attained postgraduate level of education
healthcare related occupation.According to table 5, the were more likely to have neutral practice than the student.
highest level of education and occupation significantly When comparing positive practice to negative practice
predict one's attainment of positive attitude on CFAM. on CFAM, respondents working in the healthcare-related
Healthcare related workers are more likely to have a field were more likely to practice positively on CFAM
positive attitude to non-healthcareelated counterpart. compared to non-healthcarecounterpart.

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Table 4 : Forward stepwise multinomial logistic regression analysis of factors associated with poor knowledge on
CFAM
SE df OR 95%CI p-value
Reference: Poor level of knowledgeModerate level of knowledge vs. poor level of knowledge
Occupation
Healthcare related 0.358 1 4.626 2.294:9.329 0.000*
Non healthcare related (control group)
Reference: Poor level of knowledgeGood level of knowledge vs. poor level of knowledge
Occupation
Healthcare related 0.394 1 12.571 5.845:27.034 0.000*
Non healthcare related (control group)
R2 = 0.128 (Cox and Snell), 0.146 (Nagelkerke)
2
(2) = 52.785 *p<0.05
SE : standard error, df: degree of freedom, OR: odd ratio, CI: confidence interval, x2 : chi-square

Table 5 : Binary logistic regression analysis of factors associated with attitude on CFAM
SE df OR 95%CI p-value
Reference: Neutral attitude
Positive attitude vs. neutral attitude
Highest level of education
Pre-University 0.434 1 0.597 0.255:1.397 0.234
Degree 0.383 1 0.862 0.407:1.825 0.698
Postgraduate 0.561 1 0.166 0.055:0.499 0.001*
Secondary school (control group)
Occupation
Healthcare related 0.280 1 2.285 1.356:3.849 0.002*
Non healthcare related (control
group)
2
R = 0.072 (Cox and Snell), 0.096 (Nagelkerke)
2
(4) = 27.557 *p < 0.05
SE: standard error, df: degree of freedom, OR: odd ratio, CI: confidence interval, x2: chi-square

Correlation of knowledge, attitude and practice on education as part of the solution to combating/stopping
CFAM: There was a statistically significant positive CFAM. Public educationshould be part of the
correlation between knowledge with attitude and practice comprehensive approach to counteract the CFAM issue.
as shown in table 7.The higher the knowledge of
Demographic characteristics: Kuala Lumpur is the
respondents towards CFAM, the more positive the attitude
capital of Malaysia with a population of 1.8 million as of
and practice of respondents towards CFAM.
2018 (13). This city is the financial and economic centre
Education on counterfeit and adulterated medicines: of Malaysia, attracting citizens all around the country to
Majority of respondents(48.8%, N = 189) had never heard pursue their careers in Kuala Lumpur. Age distribution
about the term "Counterfeit and/or Adulterated of the respondents is skewed towards younger age with a
Medicines" before study participation. 10.3% (N = 40) mean age of28.13 ± 10.110years due to language barrier.
of respondents were unsure about the meaning of Older generation preferred to answer the questionnaire
counterfeit medicine whereas 40.8% (N = 158) of them only if it is written in their native languages, such as
heard about counterfeit medicine before.Interestingly, Malay, Chinese and Tamil. According to the population
around72.1% (N = 279) of respondents had never census conducted in 2010, the percentage of Malay and
suspected about the authenticity of purchased medicines. Chinese population in Kuala Lumpur are 45.9% and
As high as 75.5% of respondents (N = 292) agreed that 43.2%, respectively (14).

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Table 6 : Forward stepwise multinomial logistic regression analysis of factors associated with negative practice on
CFAM
SE df OR 95%CI p-value
Reference: Negative practiceneutral practice vs. negative practice
Employment status
Employed 0.255 1 0.641 0.389:1.058 0.082
Unemployed 0.804 1 0.112 0.023:0.540 0.006*
Housewife 0.890 1 0.394 0.069:2.257 0.296
Student (control group)
Highest level of education
Pre-University 0.472 1 0.923 0.366:2.329 0.865
Degree 0.413 1 0.880 0.391:1.977 0.756
Postgraduate 0.560 1 3.166 1.056:9.491 0.040*
Secondary school (control group)
Occupation
Healthcare related 0.270 1 1.535 0.904:2.606 0.113
Non healthcare related (control
group)
Reference: Negative practicepositive practice vs. negative practice
Employment status
Employed 0.502 1 0.599 0.224:1.601 0.307
Unemployed 0.913 1 0.500 0.083:2.992 0.447
Housewife 1.011 1 4.181 0.576:30.329 0.157
Student
Highest level of education
Pre-University 1.032 1 0.336 0.044:2.540 0.290
Degree 0.727 1 1.278 0.307:5.319 0.736
Postgraduate 1.285 1 0.652 0.053:8.089 0.739
Secondary school
Occupation
Healthcare related 0.454 1 7.324 3.010:17.819 0.000*
Non healthcare related
R2 = 0.141 (Cox and Snell), 0.169 (Nagelkerke) 2
(20) = 58.485
*p<0.05
SE: standard error, df: degree of freedom, OR: odd ratio, CI: confidence interval, x2: chi-square

Table 7 : Correlation respondents' level of KAP on CFAM Knowledge on CFAM : In general, the knowledge of the
general public in Kuala Lumpur on CFAM is classified
Knowledge Attitude Practice
as moderate (median score = 4). According to a study
Knowledge 1.000 0.376* 0.159* comparing knowledge of the population in urbanised and
Attitude 0.376* 1.000 0.013 remote settlement, respondents from urbanised area have
statistically higher knowledge than remote area. The study
Practice 0.159* 0.013 1.000 states that majority of respondents in urbanised area have
a moderate level of knowledge, which is consistent with
*p-value is less than 0.1 current finding.
However, the percentage of Chinese respondents The present study showed that there was a significant
(61.2%) recruited in the study is higher than Malay association between the occupation of respondent and
respondents (27.1%). Malay language is the national level of knowledge on CFAM. Generally, respondents
language in Malaysia whereas English is widely used in working in the healthcare-related field have better
speaking and communication. Preferenceof national knowledge on differentiating the counterfeit medicines
language by Malay population may attribute to a low from their genuine counterpart. Similarly, Linus et al.
percentage of Malay participants in the study (15, 16). reported that respondents in healthcare-related field can

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differentiate authentic antimalarial drugs from their hospital are covered by the budget allocated to the hospital
counterfeit counterparts compared to those without from the central treasury (23). Malaysians are eligible
healthcare-related background in Tanzania (17). A study for unrealistically cheap health care services with minimal
conducted in Haryana, India revealed that more than half payment in Malaysia. Hence, drivers of negative practice
of the doctors working in SHKM Government Medical towards CFAM such as affordability and accessibility of
College (tertiary care hospital) demonstrated correct medicines may not present in the public sector. Most
knowledge on CFAM (18). Although health care health care costs of the patients admitted in private health
practitionersare equipped with better knowledge, platform care facilities are covered by the health insurance scheme
for sharing of knowledge on CFAM with the public is (23). Therefore, they are less exposed to the
not established. Gap of knowledge between health care unaffordability of medicine, which may drive them to
practitioner and the public can be narrowed by educating seek cheaper alternatives for medicine.
the public on CFAM. Highest level of education, employment status and
Attitude towards CFAM : Respondents hada positive occupation were found associated with the level of
attitude towards CFAM. One of the effective ways to practice of recruited respondents towards CFAM.
combat CFAM is public education about the harmful Respondents with postgraduate qualification are
consequences of CFAM on their health (19). Although lesslikely to have negative practice on CFAM than
CFAM is sold at a low price, its harmful effect may incur secondary school. Respondents with postgraduate degree
higher health care cost to the patient in the future. capable of makingextensive decision making which
Education on economic consequences of CFAM such as requires extensive gathering and evaluation of
discouragement of the pharmaceutical company to invest information of products and several alternatives compared
in research and development of new medicine is also to respondents attained secondary school qualification
warranted (19). (24). Such practices have a positive impact on purchasing
The study has identified that the highest level of intention of CFAM by respondents. Respondents are more
education and occupation of respondents are predictors likely to have positive practice with a comprehensive
for the level of attitude towards CFAM. Overall, health evaluation of latest information relating to the CFAM.
care practitioners have more positive attitude towards Measures to combat CFAM : Definition of CFAM
CFAM than of non-health care employees. The finding remains unclear in Malaysia. Many terms are used
is consistent with a previous study conducted among interchangeably with CFAM, such as fake, illegal,
Iranian pharmacists, although their knowledge and unregistered and substandard medicine, which may create
practice level on CFAM is poor, they have a positive unnecessary confusion among consumers. Different terms
attitude on CFAM (20). AnupNagaraj et.al also indicate are used during the discussion in public health and in
that medical practitioners, dental practitioners and case of violation of intellectual property right (IPR) (19).
medical wholesaler distributors have a comparable WHO has replaced the old "substandard/spurious/falsely-
positive attitude towards the use of CFAM in India (21). labelled/falsified/counterfeit (SSFFC)" terminology with
Practice against CFAM : One of the main drivers for "Substandard and Falsified (SF)" due to lack of global
the rapid growth of CFAM is a high demand for CFAM consensus and understanding of the previous term on
in the market. The willingness of consumers to purchase 2017 (25). Standardisation of the term is the fundamental
CFAM further aggravate the distribution of CFAM in the approach for educating consumers and health care
country (22). Good attitude of consumers towards CFAM providers on CFAM.
does not necessarily result in positive purchasing practice Majority of the respondents agreed that education is
of the consumers. one of several effective measures to combat CFAM.
Most respondents refused to purchase the Public awareness program incorporating multifaceted
pharmaceutical product with an unknown source. The approaches such as comprehensive data supported by
positive practice of respondents may attribute to the health evidence, innovative technology, communication and
care system in Malaysia. Malaysia's health care system political commitment is effective in improving public
comprised of dichotomous public and private services. awareness on health-related issues (26). Public must be
Public sector provides healthcare services to cover around made aware of the presence of CFAM in the market even
65% of the population (23). Health care costs for from the legal supply chain. Imparting public with the
outpatient services and admission into government threatening effect of CFAM can be a good focus of the

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campaign to discourage them from purchasing CFAM towards CFAM, taking into account the entire population
(27). Mass media is commonly used to influence the from different states in Malaysia
opinion of public towards particular issues. It may be
4. Conclusion
useful to raise awareness of the public towards CFAM
due to huge coverage in the country as compared to the Overall, the present study revealed that the general
campaign. One of the disadvantages of utilisation of mass Malaysian public in Kuala Lumpur had a moderate level
media is short-lived of the awareness towards CFAM of knowledge, positive attitudes but negative practice
because issues focused by media may change over time towards CFAM. Occupation is the most important
(28). It may also decrease the confidence of the public predictors for the level of KAP of respondents, followed
towards conventional medicine and directs them towards by the highest level of education and employment status.
alternative medicines (27). Healthcare-related workers generally have a higher level
of KAP regarding CFAM as compared to those in
The public is the end consumer of the pharmaceutical
nonhealthcare related field. Although knowledge of
product. They have rights to ensure that the products are
respondents is positively correlated with attitude and
safe and quality. Lack of knowledge towards CFAM
practice on CFAM, the practice of respondents towards
hinders them from being able to identify whether which
CFAM is unsatisfactory despite positive attitude on
medicines are authentic. Healthcare providers could be
CFAM. Education on CFAM is recommended as one of
the main source of CFAM related information forthe
the measures to combat the CFAM issue in Malaysia since
general public. Nonetheless, the study respondents
the majority of respondents have moderately poor
demonstrateda lack of confidence on the competences of
knowledge towards CFAM. Health care providers are
healthcare providers in providing CFAM related
encouraged to deliver information of CFAM to the public
information. This is evident that almost half of the
to narrow the gap of knowledge of CFAM.
respondents agreed that education must be provided to
healthcare providers. Acknowledgements
Limitations of this study : Selection and participation We want to express our deep gratitude to the all the
biases maybe present in the recruitment of participants participants recruited in this study for their willingness
due to the convenient sampling was used. Therefore,the to complete the questionnaire.This research did not
sample population may not fully representative of the receive any specific grant from funding agencies in the
actual population in Kuala Lumpur. This research works public, commercial, or not-for-profit sectors.
well as a pilot study.It may not be possible to utilise
probability sampling to recruit participants due to huge Conflict of interest
sampling frame of the population. However, location- There are no financial and non-financial competing
based sampling is a feasible approach to reduce bias in interests to be reported.
the selection of participants (29).
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Evaluation of Self-Medication Practice Among University Students

Tan Puay Luan1, Khaled M. Alakhali1*, Fazlollah Keshavarzi1, Omotayo Oladuntoye Fatokun1
1Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, UCSI University, Kuala Lumpur, Malaysia

Corresponding Author : khaled@ucsiuniversity.edu.my; alakhalikhaled@gmail.com

Abstract university. It was indicated in conditions of mild illness

The World Health Organization refers the practice of such as headache, fever, cough, etc. Effective use of
self-medication to 'use over-the-counter medicines to treat medications as self-medication has been of benefit to
self-diagnosed symptoms or diseases or to continue and humans. Yet there are other factors that have made self-
reuse chronic medicines. Self-medication is a common medication the main cause of drug abuse.
and important part of the behavior of patients to cope Key words : self-medication; university; students.
with disease. The aim of present study is determine which
1. Introduction
classes of drugs the students used most often as self-
medication. Also, to analyze the nature and views of self- Self-medication was practiced by every human being
medication among UCSI students, and to assess the daily for their own health which was now increasingly
practice of self-medication among students. A cross- considered as a part of self-care (1). In addition, some
sectional study was carried out for three months. A pre- governments widely encouraged the practice of self-
validated questionnaire was distributed to the students at medication in the management of minor ailments (2,3).
private University in Kuala Lumpur, Malaysia. The study Self-medication was mostly implied when an individual
was participated by 367 students. 239 (65.1%) of the encounter a common health problem that he/she thinks
respondents practiced self-medication in the past one year, that a visit to doctor was not needed. Self-medication is
among which 101(42.3%) were males and 138 (57.7%) defined as the use of any medication for self-treatment
were females. 166 (45.2%) of the respondents think self- without consulting a healthcare professional (4). The term
medication is harmful. 209 (56.9%) of the respondents 'responsible' self-medication is frequently used in which
think that the medication used for self-medication gives the appropriate drugs including over the counter (OTC)
symptomatic relief but does not treat the main causes of drugs were indicated only when they are necessary (5).
the disease. 280 (76.3%) of the respondents read the leaflet The WHO (1995) emphasized that the prevention and
of the medication before using it. Pharmacy was the main treatment of minor health problem can be achieved
source of self-medication. The most common indication through rational self-medication at an affordable cost (6).
for self-medication was fever, followed by cough, However, this practice may cause some unwanted and
headache, common cold and pain. The most common drug serious drawbacks. There are chance of getting serious
classes for self-medication were antipyretics followed by adverse outcomes, drug interactions, polypharmacy and
cough syrups, vitamins, analgesics/anti-inflammatory and drug abuse and dependence (6). The reasons of self-
cold preparations. The three main reasons of self- medication include high medical cost, prolonged waiting
medication were "health problem is not serious", "seeking hours at clinics, time-wasting, social or family support,
quick relief" and "illness is minor". The main reasons previous experience of similar illness and its management,
against self-medication were "risk of using wrong lack of nearby health facilities and health professionals
medication"(80.9%), "risk of adverse effects" (64.9%)and (3). The practice of self-medication in Malaysia was
"risk of misdiagnosis of illness"(53.4%). 279 (76.0%) of widely studied among the adult's urban population instead
the respondents agreed that all medications including of students (7-9). There was only one study conducted in
herbal have adverse effects. 354 (96.5%) was aware that International Islamic University Malaysia (IIUM),
increasing drug dose can be dangerous, however, only Kuantan about the perceptions, knowledge, and practice
153 (41.7%) aware that decreasing drug dose can be of self-medication among pharmacy students (10).
dangerous.The study concludes that self-medication However, UCSI University's students may varied from
practice was widespread among students at the UCSI the general population due to demographic variability and

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Vol. 14 (5) 92-100, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.10

their knowledge regarding various diseases and drugs affecting the practice of self-medication. Part 4 evaluated
(11). The present study was carried out to evaluate the the students' view on some aspects of self-medication.
practice of self-medication among university students in The data collected was analyzed using SPSS version
UCSI University, Kuala Lumpur. 20. The descriptive data and categorical variables were
expressed as counts/frequency and percentages. In
2. Materials and Methods
addition, continuous variables were summarized as mean
A cross-sectional questionnaire-based study was (standard deviation). Independent sample t-test was
conducted at UCSI University, Kuala Lumpur. The target utilized to compare between male and female groups
students were undergraduate and postgraduate university where p<0.05 was considered statistically significant.
students. Students are recruited from 6 different faculties.
The sample size was assumed to have a confidence level 3. Results and Discussion
of 95%, 5% margin of error, 50% recruitment rate and a Only 367 out of 381 total students completely filled
maximal sample size of 8000 students. According to the the survey with response rate of 96.3%. Incomplete filling
Rao soft sample size calculator (http://www.raosoft.com/ of the questionnaires and the questionnaires with
samplesize.html), the minimum required sample size, was unsigned consent forms were not included in this study.
367 respondents. In addition, some of the respondents did not submit their
questionnaires for data analysis. Among 367 respondents,
The study subjects were informed that participation 149 (40.6%) were males, whereas 218 (59.4%) were
will be completely voluntary, and the information females. The mean age ± standard deviation was 20.88 ±
collected would be anonymous. The questionnaire was 1.81 years. This study recruited 142(38.7%) first-year
printed in English language and consists of four parts undergraduate students; 81(22.1%) second-year
which was modified from the other studies (3,4,11,12). undergraduate students; 95(25.9%) third-year
The preliminary letter explained the term 'self-medication' undergraduate students; 38 (10.4%) fourth-year
and require the participants to report on the use of self- undergraduate students and 11(3.0%) postgraduate
medication during past one year. Part 1 focused on the students. Among 367 respondents, 94 (25.6%) were
practice of self-medication in the past one year. Part 2 medical students, whereas, 273 (74.4%) were from non-
was used to assess the common drugs and indications for medical courses. The characteristics of participating
self-medication. Part 3 was used to examine the factors students are described in Table 1.

Table 1. Demographic characteristics of respondents (N=367)

Age (Mean ± SD) 20.88 ± 1.81
Gender Frequency Percentages (%)
Male 149 40.6
Female 218 59.4
Faculty
a) Medical
Faculty of Pharmaceutical Sciences 79 21.5
Faculty of Medicine & Health Sciences 15 4.1
Total 94 25.6
b) Non-medical
Faculty of Business & Information Sciences 116 31.6
Faculty of Engineering, Technology & Built Environment 87 23.7
Faculty of Applied Sciences 44 12.0
Faculty of Social Sciences & Liberal Arts 26 7.1
Total 273 74.4
Level of study
First year undergraduate 142 38.7
Second year undergraduate 81 22.1
Third year undergraduate 95 25.9
Fourth year undergraduate 38 10.4
Postgraduate 11 3.0

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DOI : 10.5530/ctbp.2020.4s.10

Self-medication practice by the students in the past one medication prior consumption. According to Table 2,
year majority (91.8%) of the respondents practiced self-
Self-medication was practiced by 239 (65.1%) of the medication at least once in the past year, which was
respondents in the past one year, among which 101 contradictory to the first question in Table 2 (65.1%).
(42.3%) were males and 138 (57.7%) were females. 166 The pharmacy was the main source of self-medication
(45.2%) of the respondents think self-medication is for 273 (74.4%) of the respondents followed by home
harmful. Majority (91.8%) of the respondents practice stock (16.3%), herbal store (7.1%), street market (1.1%)
self-medication not less than once in the past year. 244 and friends (1.1%). 214 (58.3%) of the respondents speak
(66.5%) of the respondents had an idea about rational to a pharmacist before taking a drug as self-medication.
drug use. 209 (56.9%) of the respondents think that the 100 (27.2%) of the respondents had been consumed
medication used for self-medication gives symptomatic antibiotics as a part of self-medication during the past
relief but does not treat the main causes of the disease. one year. More females than male respondents were
247 (67.3%) of the respondents realized about the having an idea about rational drug use and read the leaflet
potential adverse effects of self-medicated drugs. 280 of the medicine before consumption (p=0.041, 0.030) as
(76.3%) of the respondents read the leaflet of the shown in Table 2.
Table 2. Shown self-medication practice by the students in the past one year.
Did you have any self-medication in the past one year? Male (%) Female (%) Total (%) p (t-test)
Yes 101(67.8) 138 (63.3) 23965.1) 0.378
Have you ever treated yourself with medication without it to be prescribed by a doctor?
Yes 116(77.9) 172 (78.9) 288 78.5) 0.811
Do you think self-medication is harmful?
Yes 65 (43.6) 101 (46.3) 166 45.2) 0.610
How many times have you practiced self-medication and used over the counter drugs in the past year?
No 12 (8.1) 18 (8.3) 30 (8.2) 0.740
1 Time 39 (26.2) 49 (22.5) 88 (24.0)
2 Times 39 (26.2) 60 (27.5) 99 (27.0)
3 Times 21 (14.1) 37 (17.0) 58 (15.8)
4 Times 6 (4.0) 5 (2.3) 11 (3.0)
5 Times 3 (2.0) 4 (1.8) 7 (1.9)
>5 Times 29 (19.5) 45 (20.6) 74 (20.2)
How long was the average duration of self-medication?
No 5 (3.4) 9 (4.1) 14 (3.8) 0.869
1-2 days 73 (49.0) 99 (45.4) 172(46.9)
2-3 days 28 (18.8) 57 (26.1) 85 (23.2)
3-4 days 19 (12.8) 21 (9.6) 40 (10.9)
4-5 days 7 (4.7) 5 (2.3) 12 (3.3)
5-6 days 1 (0.7) 2 (0.9) 3 (0.8)
1 week 5 (3.4) 11 (5.0) 16 (4.4)
More than a week 11 (7.4) 14 (6.4) 25 (6.8)
Do you have any idea about rational drug use?
Yes 90 (60.4) 154 (70.6) 244(66.5) 0.041
Do you think that the medication you use to treat yourself gives symptomatic relief but does not treat the main causes
of the disease?
Yes 76 (51.0) 133 (61.0) 209(56.9) 0.058
Did you know the potential adverse effects of the drug by which you self-medicated?
Yes 94 (63.1) 153 (70.2) 247(67.3) 0.156
When you treat yourself with a medication, do you read the leaflet of the medication before using it?
Yes 105(70.5) 175 (80.3) 280(76.3) 0.030
What sources of self-medication do you use?
Pharmacy 110(73.8) 163 (74.8) 273(74.4) 0.706

Home stock 24 (16.1) 36 (16.5) 60 (16.3)

Street market 2 (1.3) 2 (0.9) 4 (1.1)

Herbal store 11 (7.4) 15 (6.9) 26 (7.1)

Friends 2 (1.3) 2 (0.9) 4 (1.1)

Do you speak to pharmacist before taking a drug as self-medication?
Yes 87 (58.4) 127 (58.3) 214(58.3) 0.980
Have you ever taken antibiotic as self-medication?
Yes 45 (30.2) 55 (25.2) 100 27.2) 0.295

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DOI : 10.5530/ctbp.2020.4s.10

Indications and drug classes for self-medication The most common drug classes for self-medication
The most common health condition for self- was antipyretics (59.7%), cough syrups (59.1%), vitamins
medication was fever (72.8%), cough (67.6%), headache (55.3%), analgesics/anti-inflammatory (45.8%) and cold
(67.0%), common cold (65.7%) and pain (30.5%). Other preparations (36.0%). Other than that, nutritional
indications include diarrhea (28.6%), mouth ulcers supplements (34.3%), nasal/ear/eye drops (34.1%), herbs
(26.4%), gastric pain (22.6%), allergy (15.8%), (21.8%), anti-gastritis (21.3%), antihistamines (19.6%),
constipation (9.5%), fungal/microbial infection (4.9%), topical agents (17.4%) and anti-diarrhea (17.2%) were
insomnia (3.8%), sex-related problem (2.2%), some other examples of drug classes indicated for self-
contraception (1.6%) and the least was sore throat (0.5%). medication. Nasal/ear/eye drops, topical agents and oral
The use of self-medication for headache was more contraceptives were more commonly used by females
common in males than females (78.5% vs. 59.2%, than males (p=0.002, 0.025, 0.027).
p=0.000).

Table3. Shown indications and drug classes for self-medication.

Indications Male (%) Female (%) Total (%) p (t-test)

Fever 114(76.5) 153(70.2) 267(72.8) 0.182
Cough 106(71.1) 142(65.1) 248(67.6) 0.229
Headache 117(78.5) 129(59.2) 246(67.0) 0.000
Common cold 96(64.4) 145(66.5) 241(65.7) 0.681
Pain 46(30.9) 66(30.3) 112(30.5) 0.903
Diarrhea 46(30.9) 59(27.1) 105(28.6) 0.429
Mouth ulcers 41(27.5) 56(25.7) 97(26.4) 0.697
Gastric pain 32(21.5) 51(23.4) 83(22.6) 0.667
Allergy 19(12.8) 39(17.9) 58(15.8) 0.186
Constipation 16(10.7) 19(8.7) 35(9.5) 0.518
Fungal/Microbial infection 6(4.0) 12(5.5) 18(4.9) 0.521
Insomnia 3(2.0) 11(5.0) 14(3.8) 0.137
Sex-related problem 4(2.7) 4(1.8) 8(2.2) 0.585
Contraception 2(1.3) 4(1.8) 6(1.6) 0.716
Sore throat 1(0.7) 1(0.5) 2(0.5) 0.787

Factors affecting the practice of self-medication The major causes against self-medication were "risk
The three main causes of self-medication were "health of using wrong medication" (80.9%), "risk of adverse
problem is not serious" (65.1%), "seeking quick relief" effects" (64.9%), "risk of misdiagnosis of illness"
(62.1%), "illness is minor" (53.7%) and the least was "I (53.4%), "risk of drug interaction" (35.7%) and "risk of
do not trust my physician" (0.8%). Among all the reasons, drug abuse and dependence" (32.2%). The risk of
avoidance of long waiting at clinics, suggestion of a misdiagnosis of illness was the most commonly reported
relative/friend and embarrassed of discussing own reason against self-medication in females than males
symptoms were more common in males than females in (p=0.041) as shown in Table 4.
order to acquire self-medication (p<0.05). More females Student's views on some aspects of self-medication
go for self-medication due to "illness is minor" than males
279 (76.0%) of the respondents agreed that all drugs
(p = 0.006).
including herbal cause adverse effects. 354 (96.5%) of
The three main reasons for seeking professional help the respondents were aware of the danger of increasing
were "symptoms are worsening" (71.1%), "symptoms last drug dose, however, only 153 (41.7%) aware of the danger
for more than one week" (64.9%), "thinking the problem of decreasing drug dose. Most of the respondents were
is serious" (64.6%) and the least was "in case of the mental aware that concurrent use of drugs can be dangerous
problem" (6.8%). The reasons for seeking professional (94.0%), physician help must be sought in case of adverse
help was not statistically significant difference between effects (95.9%) and using medications with unknown
male and female group (p > 0.05). substances in patients with liver and kidney disease is

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Vol. 14 (5) 92-100, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.10

Table 4. Shown factors affecting the practice of self-medication. p< 0.05 was considered significant when compared
between male and female groups (N=367)
Reasons in favor of self-medication Male (%) Female (%) Total (%) p (t-test)
Health problem is not serious 89(59.7) 150(68.8) 239(65.1) 0.074
Seeking quick relief 100(67.1) 128(58.7) 228(62.1) 0.104
Illness is minor 67(45.0) 130(59.6) 197(53.7) 0.006
High cost of medical consultation 72(48.3) 86(39.4) 158(43.1) 0.092
Avoidance of long waiting at clinics 46(30.9) 43(19.7) 89(24.3) 0.014
Suggestion of a relative/friend 25(16.8) 21(9.6) 46(12.5) 0.042
Physician's advice of self-management 13(8.7) 9(4.1) 22(6.0) 0.069
Embarrassed of discussing own symptoms 8(5.4) 1(0.5) 9(2.5) 0.003
I do not trust my physician 2(1.3) 1(0.5) 3(0.8) 0.357
Reasons for seeking professional help
Symptoms are worsening 109(73.2) 152(69.7) 261(71.1) 0.478
Symptoms last for more than one week 101(67.8) 137(62.8) 238(64.9) 0.332
Thinking the problem is serious 95(63.8) 142(65.1) 237(64.6) 0.787
Presence of severe pain 97(65.1) 131(60.1) 228(62.1) 0.333
Usual treatment is not effective 76(51.0) 109(50.0) 185(50.4) 0.850
Side effects of usual treatment 21(14.1) 23(10.6) 44(12.0) 0.306
In case of mental problem 10(6.7) 15(6.9) 25(6.8) 0.950
Reasons against self-medication
Risk of using wrong medication 123(82.6) 174(79.8) 297(80.9) 0.514
Risk of adverse effects 96(64.4) 142(65.1) 238(64.9) 0.889
Risk of misdiagnosis of illness 70(47.0) 126(57.8) 196(53.4) 0.041
Risk of drug interaction 45(30.2) 86(39.4) 131(35.7) 0.070
Risk of drug abuse and dependence 49(32.9) 69(31.7) 118(32.2) 0.804

dangerous (95.6%). 195 (53.1%) of the respondents More females than males were aware of the aspects
agreed that no drugs can be used during pregnancy. 62.9% of self-medication that "physician help must be sought
approved that mild illnesses do not require drug treatment in case of adverse effects" (p=0.036) and "mild medical
whereas 69.2% approved that self-medication can mask problems do not require drug treatment" (p=0.025) as
clinical presentations of the illness so that the physician shown in Table 6.
can overlook them easily as shown in Table 5.

Table 5. Shown student's views on some aspects of self-medication (N=367)

Student's views on self-medication Approve (%) Disapprove (%)
All medications (prescription, OTC and herbal) have adverse 279(76.0) 88(24.0)
effects.
Concomitant use of drugs can be dangerous. 345(94.0) 22(6.0)
Increasing drug dose can be dangerous. 354(96.5) 13(3.5)
Decreasing drug dose can be dangerous. 153(41.7) 214(58.3)
Physician help must be sought in case of adverse effects. 352(95.9) 15(4.1)
Using medications with unknown substances in patients with liver 351(95.6) 16(4.4)
and kidney disease is dangerous.
No drug can be used during pregnancy. 195(53.1) 172(46.9)
Mild medical problems do not require drug treatment. 231(62.9) 136(37.1)
Self-medication can mask signs and symptoms of disease so the 254(69.2) 113(30.8)
physician can overlook them easily.

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Table 6. Shown comparison of student's views on self-medication between male and female group (N=367)
Student's views on self-medication Approve (%) Disapprove (%) p value
(t-test)
Male Female Male Female
All medications (prescription, OTC 115(77.2) 164(75.2) 34(22.8) 54(24.8) 0.668
and herbal) have adverse effects.
Concomitant use of drugs can be 140(94.0) 205(94.0) 9(6.0) 13(6.0) 0.976
dangerous.
Increasing drug dose can be 143(96.0) 211(96.8) 6(4.0) 7(3.2) 0.679
dangerous.
Decreasing drug dose can be 61(41.0) 92(42.2) 88(59.1) 126(57.8) 0.810
dangerous.
Physician help must be soughtin case 139(93.3) 213(97.7) 10(6.7) 5(2.3) 0.036
of adverse effects.
Using medications with unknown 143(96.0) 208(95.4) 6(4.0) 10(4.6) 0.797
substances in patients with liver and
kidney disease is dangerous.
No drug can be used during 83(55.7) 112(51.4) 66(44.3) 106(48.6) 0.416
pregnancy.
Mild medical problems do not require 104(69.8) 127(58.3) 45(30.2) 91(41.7) 0.025
drug treatment.
Self-medication can mask signs and 102(68.5) 152(69.7) 47(31.5) 66(30.3) 0.797
symptoms of disease so the physician
can overlook them easily.

The study showed that UCSI university students to the involvement of pharmacy or health science students
commonly practiced self-medication for mild medical only in the studies. Health science students are familiar
illnesses to achieve quick relief. This cross-sectional with the signs and symptoms of diseases in order to
survey has shown that the mean age of the respondents practice self-medication and are well equipped the
was similar to the previous studies conducted in Egypt knowledge of self-medication in mild medical problems.
and Jordan reported (11,13). Apart from that, the study conducted in Karachi and
The response rate of the current study (96.3%) was Nigeria had a higher prevalence of self-care than our study
higher than the study conducted in the United Arab (18,19). This may be due to the higher number of
Emirates and Jordan (6,11). This may be due to the healthcare students, family education and the easy
method of data collection by using both web-based and availability of the drugs. It was clear that the pattern of
paper-based questionnaires in this study. In addition, the self-medication varies among the countries because of
respondents in this study were highly cooperative and geographical, demographic, and economical variation.
well understand the purpose of the study conducted. In In this current study, there was no significant
contrast, the study conducted in the United Arab Emirates difference in the practice of self-medication among
and Jordan utilized paper-based questionnaires only genders, similarly with the study conducted in Jordan,
(6,11). Iran and Bangladesh (11,16,17). However, the study
This study has shown that the university students conducted in Egypt and Nigeria reported that self-
practiced self-medication similarly to the study conducted medication was significantly associated with gender. This
in Egypt and Turkey (13,15). In contrast, the prevalence may due to the unequal distribution of male and female
of self-medication in Slovenia (92.3%) was greater than respondents in the study conducted in Egypt and Nigeria
the current study (4). This may be due to the larger sample (13,19).
size of 1294 students in Slovenia compared to our study Based on the current study, more than half of the
(4). Furthermore, the practice of self-medication was university students were aware of rational drug use
greater in Iran, Bangladesh, Jordan, United Arab consistent with the study conducted in the United Arab
Emirates, and Ethiopia (11,12,14,16,17). This may be due Emirates and Turkey (12,15). However, in Jordan, a

Tan et al
Current Trends in Biotechnology and Pharmacy 98
Vol. 14 (5) 92-100, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.10

higher percentage of the students were aware of rational education and sexual behavior of the university students
drug use than our study, this may be due to the inclusion involved in the studies.
of pharmacy students only in their study (11). Awareness In the current study, more males than females had been
towards rational drug use is vital to reduce healthcare self-medicated for headaches (p=0.000). However,
burden, the occurrence of adverse effects and the chance studies showed that headache was more prevalent in
of antibiotics resistance (20). females than males due to hormonal differences especially
In addition, the current study reported that university during menstruation and less tolerance towards the
students think that the medication used to treat them give sensation of pain (22). Hence, the difference between this
symptomatic relief but does not treat the main causes of study and the other studies may be due to the higher
the disease. Similarly, in Jordan, the pharmacy students number of male respondents that cause bias in the result.
think that the medication used to treat them gives According to this study, the most common drug classes
symptomatic relief but does not treat the main cause of for self-medication were antipyretics, cough syrups,
the disease (11). vitamins,analgesics/anti-inflammatory, cold preparations,
In this study, majority of the respondents read the nutritional supplements, nasal/ear/eye drops and herbs.
leaflet of the medication before using it which was In contrast with the United Arab Emirates, the most
consistent with the study conducted in Bangladesh, Egypt common drug classes were analgesics, antipyretics,
and Jordan (11,13,17). The habit of reading medication's vitamins and minerals and herbal teas. The more frequent
leaflet is important to avoid the misuse of self-medicated use of vitamins and herbs in the United Arab Emirates
drugs. than our study may be due to the knowledge of students
According to the current study, pharmacy was the main towards the use of vitamins and the cultural beliefs of
source of self-medication, similarly in Jordan, Egypt and the students. Generally, the use of vitamins and minerals
the United Arab Emirates (11-13). This is because are becoming more popular among university students
pharmacies are easily available and provide a wide range that are widely exposed to the health-related information
of over the counter (OTC) medication and prescription- (23). In addition, the students in the United Arab Emirates
only drugs. are more knowledgeable about the use of traditional
Moreover, 27.2% of the university students in this medicine and the perception that the use of herbals was
study had taken antibiotics as self-medication. Similarly, cheap, effective and without side effects. However, a
36.9% of university students in Turkey and 32.0% of recent study concluded that the use of herbs may cause
university students in the United Arab Emirates had taken liver toxicity and kidney toxicity due to overdose, drug-
antibiotics as self-medication (12,15). In contrast with drug interaction and the lack of control from the Drug
Nigeria, only 10.5% of the university students had taken Control Agency (22). On the other hand, our study was
antibiotics as self-medication (19). The relatively lower inconsistent with the study conducted in Bangladesh and
percentage of antibiotic use in Nigeria than our study Iran (16,17).
may be due to the high cost of antibiotics and the high According to this study, the fundamental cause of self-
awareness of the students towards the threat of antibiotics medication were health problem is not serious, seeking
resistance (20,21). Hence, pharmacist plays a crucial role quick relief, illness is minor, high cost of medical
in dispensing antibiotics only if the prescription is consultation and avoidance of long waiting at clinics,
presented. In our study, there was no significant while the least was "I do not trust my physician" similarly
difference in the use of antibiotics as self-medication with the study carried out in the United Arab Emirates
between genders, similarly in Turkey (15). However, in (12). However, in Karachi, the major reasons were the
Jordan, more males had taken antibiotics as self- problem is not serious, previous experience, lack of time,
medication compared with females (11). cost of the consultation, the urgency of the problem,
The main indications for self-medication among advice from friends and unavailability of transport (18).
university students in this study were fever, cough, Also, a study conducted in Egypt reported that minor
headache, common cold, pain, diarrhea, mouth ulcers, disease, knowledge from previous experience, same drugs
gastric pain, allergy, constipation and fungal/microbial prescribed by a doctor, save money and time, fast relief
infection, similarly in Bangladesh (17). The outcomes of and the least was the unavailability of health service were
other studies conducted in Egypt, Jordan, Ethiopia and the main causes of self-medication (13). In Iran, the main
Iran were inconsistent with our findings (11,13,14,16). causes of self-medication among health science students
The differences among the studies are attributed to the were previous experience about the similar illness, mild
demographic variation, lifestyles, diet consumption, disease, drug availability and history of drug use (16).
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DOI : 10.5530/ctbp.2020.4s.10
Furthermore, a study conducted in Jordan reported that (12,17). Hence, university students should be educated
the main reasons of self-care were time saving, health on the effects of drug dose, drug use during pregnancy,
problem was not serious and ease of drug availability the rationale of appropriate drug treatment in the
(11). In Bangladesh, the main reasons for self-medication maintenance of good health. In our study, more females
were illness is minor, easy availability of medicine and than males approved that "physician help must be sought
emergency (17). in case of adverse effects" (p=0.036) and "mild medical
In this study, the two major reasons for seeking problems do not require drug treatment" (p=0.025).
professional help were symptoms are worsening (71.1%) However, the other studies did not examine the significant
and symptoms last for more than one week (64.9%) differences between student's perceptions on self-
similarly in Slovenia (4). In contrast with the studies in medication and genders (4,11-19). The limitations of this
Jordan, and the United Arab Emirates. In addition, our current study were attributed to the evaluation of self-
study concluded that the reasons for seeking medical medication practice in the past year that may cause recall
advice were not statistically significant between genders, bias among respondents. This is because the respondents
whereas, the other studies did not investigate the tend to forget their previous use of drugs as self-care in
relationship between the reasons for seeking professional the past one year. In addition, self-reported basis of the
help and genders (4,11-19). questionnaires may cause over-reporting or under-
Based on our study, the reasons against self- reporting of the responses. The inclusion of more centers
medication were the risk of using the wrong medication, and larger sample size would enable us to better
risk of adverse effects, risk of misdiagnosis of illness, characterize the practice and perception of self-
risk of drug interaction and risk of drug abuse and medication among students.
dependence, consistent with United Arab Emirates (12). 4. Conclusion
According to our study, more females than males assumed Self-medication was commonly practiced among
that the risk of misdiagnosis of illness was the reason university students. The most common indications for
against self-medication (p= 0.041) while the other reasons self-medication among university students were fever,
were not significantly different between genders. On the cough, headache, common cold, pain, diarrhea and etc.
other hand, the other studies did not investigate the The knowledge of students regarding the reasons for and
significant difference between the reasons against self- against self-medication seems appropriate. The current
medication and genders (4,11-19). study recommends that proper education should be done
According to this study, majority of the university among university students to prevent and treat some
students approved that "increasing drug dose can be common indications for self-medication by incorporating
dangerous" similarly in United Arab Emirates, Jordan and health-related knowledge as a part of the curricula in all
Bangladesh (11,12,17). Besides that, our study showed university programs.
that majority of the students were aware that "physician Ethical approval
help must be sought in case of adverse effects" similarly The study has received the ethics initial approval:
in Jordan and Bangladesh (11,17). In our study, most of NMRR-19-1469-48803 (IIR), Reference: KKM/
the students agreed that "using medications with unknown NIHSEC/P19-1406(6), Date: 13-August-2019.
substances in patients with liver and kidney disease is
Acknowledgement
dangerous" and "the concomitant use of drugs can be
This work is supported by UCSI University, Faculty
dangerous", consistent with a study conducted in Jordan
of Pharmaceutical Sciences, Kuala Lumpur, Malaysia.
(11). However, our findings showed that more students
(76.0%) were aware that "all medications (prescription, Conflict of interest
OTC and herbal) have adverse effects" in contrast with The authors declare no conflict of interest.
United Arab Emirates (41%) and Jordan (49%) (11,12). 5. References
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Current Trends in Biotechnology and Pharmacy 101
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Course Satisfaction and Perception of Malaysian Provisionally Registered

Pharmacists Towards their Training : A Qualitative Study
Mei Qi Hee1*, Fazlollah Keshavarzi1, Mogana Rajagopal2
1Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, UCSI University Kuala Lumpur Campus, Malaysia,
2Department of Pharmaceutical Biology, Faculty of Pharmaceutical Sciences, UCSI University Kuala Lumpur Campus, Malaysia

*Corresponding Author : email : heemq@ucsiuniversity.edu.my

Abstract Pre-registration Training; Malaysia; Pharmacy;

In Malaysia, it is compulsory for pharmacy graduates Pharmacist
to undergo a one year provisionally registered pharmacist 1. Introduction
(PRP) training. The liberalisation of PRP training
In Malaysia, pharmacists are required to have
following the saturation of governmental institutions has
successfully completed an undergraduate pharmacy
raised many concerns about the transformation of the
degree program for a minimum of 4 years duration from
training into a scheme for exploiting the fresh pharmacy
an accredited public or private higher learning institution
graduates without a systematic training as its initial
(1) and passed the qualifying examination for practicing
purpose. The objective of this study is to explore the
as a pharmacist (2), prior to enrolling in a one year
experiences and perceptions of Malaysian pharmacists
compulsory training. The compulsory training known as
about PRP training at different settings, after the
provisionally registered pharmacist (PRP) training can
liberalisation of PRP training.A qualitative study was
be done in any training premises approved by Pharmacy
conducted in West Malaysia, mainly in Klang Valley.
Board of Malaysia (PBM), either in public or private
Through maximum variation purposeful sampling, data
sector such as government hospital, public institution,
were gathered from 33 participants from different settings
health clinic, community pharmacy, private hospital,
included government hospital, private hospital, health
research and development (academia), manufacturing and
clinic, community pharmacy, manufacturing non-manufacturing pharmaceutical industry (3). The aim
pharmaceutical industry, non-manufacturing of the PRP training is to provide pharmacists with
pharmaceutical industry and research and development sufficient in-depth clarity in the understanding of
(academia) using semi-structured in-depth interview pharmacy practice and to equip the pharmacists with
method. All the interviews were audio-recorded and relevant knowledge and skills by exposing them to the
conducted in English. The data were analyzed according real world setting through hands-on training modules (4-
to framework approach.A total of 4 themes and 24 codes 6).
were identified in this study, the themes included
The upsurge in the number of pharmacy graduates in
placement, payment, working condition and training. The
Malaysia in the past few years has been driven in part by
findings indicated a balance of positive and negative
the expansion of higher learning institutions worldwide
perceptions towards the PRP training in various settings.
and nationwide (7). Before 1996, only one public
The participated pharmacists believed that PRP training
institution offered pharmacy degree program (7-8),
was a necessary exposure to gain required experiences,
presently there are 19 institutions in Malaysia and 71
despite all difficulties and challenges. The focus of
institutions from other countries offering pharmacy degree
government in the past few years was to resolve the
program that recognized by PBM (1). The rate of
saturation of PRP placement in government hospitals
pharmacy graduates has been increased up to 1,400
through the liberalisation of PRP training. Emphasis
annually (9-10). These rising numbers have recently
should be in improving the inconsistency of quality in
caused a point of saturation in the PRP placement for
PRP training program in different practice settings in order
pharmacy graduates to undergo PRP training (10). The
to improve the experiences and perceptions of future PRPs
liberalisation of PRP training into the private sectors, as
in their training.
the alternative training premises to government hospitals,
Key words : Provisionally Registered Pharmacist (PRP); has been introduced by the PBM since October 2012. The

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primary aim of liberalisation is to resolve the saturation changed to government hospital setting. The data from
of PRP placement in government hospitals and in both settings was collected.
furtherance to provide more training opportunities for
Data collection
the pharmacy graduates (10-12). In 2017, the Ministry
of Health has introduced a new policy to offer a one year The data collection started from June to September
service contract to pharmacy graduates, with a maximum 2018. A semi-structured face to face interview was carried
contract of two years. This is to reduce the waiting time out with prior arrangement with the participants at a place
for the training placement at government sector due to and time of their convenience. The place of interview
the constraints in permanent posts (13-14). was mainly in Klang Valley (an area comprises of Kuala
Lumpur and conjoining cities and towns of Selangor state)
Previous studies have investigated the perception of
(18), except two interviews conducted at Melaka and one
PRP towards their training at government hospitals in
interview conducted at Ipoh. The verbal informed consent
Malaysia (4, 15, 16). These studies pointed out the
was obtained from all participants before the interview
positive and negative perceptions of the pharmacists
began and the study's objectives were stated and explained
toward PRP training, however suffer from major
to them. A pre-determined set of open-ended questions
drawbacks in terms of scope, sample size and
were asked during the interview and each interview lasted
methodology (4, 15, 16). The lack of reliable research on
from 30 minutes to 50 minutes. All the interviews were
the liberalisation of PRP training following the saturation
audio-recorded using a voice recorder and conducted in
of governmental institutions has raised many concerns
about the transformation of the training into a scheme English. The interview was continued until no new data,
for exploiting the fresh pharmacy graduates without a themes and coding were captured in the interviews (19).
systematic training as its initial purpose, requisite an in- Data analysis
depth qualitative investigation.The primary objective of As a preliminary step, the plausible themes and codes
this study is to explore the pharmacists' experiences and were developed, based on already-agreed-on professional
perceptions about PRP training at different settings. This
definitions found in literature reviews; from local and
study also provides an opportunity to improve the current
commonsense constructs; from researchers' values,
PRP training program based on the suggestions from
theoretical orientations and personal experiences. The
interviewees.
data analysis started with familiarizing with the data at
2. Materials and Methods the same time as the interviews were being transcribed
verbatim. The audio-recorded files were listened
Study design
repetitively and the transcriptions were reread several
The present qualitative study was conducted using in- times to provide leads for further data gathering and
depth interview method to develop a comprehensive
provoke insights. Once all of the interviews had been
understanding of the topic (17).
transcribed and checked, the transcriptions were
Recruitment of participants examined line by line to assign codes and sub codes that
All Malaysian pharmacists who had completed PRP denote particular meaningful segments as used in a
within the past 2 years or those who were currently doing grounded theory approach (20). This coding combines
PRP for at least 6 months had been eligible to participate both deductive and inductive approaches. The sub codes
in this study. Maximum variation purposeful sampling were compared in terms of similarities and differences,
was performed to make sure that they were the and those that implied the same meaning were assigned
representatives of different practices settings in this study. to one code which were further grouped into themes that
Selected pharmacists were invited through social media, reflected their central content. New sub codes were placed
email and personal connections. The recruitment of in previous code after assessment and code were formed
participants began in June until August 2018. A total of as the data analysis continued. To further improve the
33 participants from different PRP training sectors were clarity and reduce the ambiguity of the framework, sub
participated in the study. There were 5 participants from codes were compared and if possible merged, relabelled,
each sector except manufacturing pharmaceutical split as necessary and placed in a common code. By the
industry and research and development that had 4 end of this stage, all of the themes were generated to
participants each. One of the participants underwent PRP indicate the general content of the codes and sub codes.
training in community pharmacy for 11 months and later Upon completion of analysis, data saturation was attained

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at 21st interviews. Additional interviews were conducted Ethical consideration

to ensure no new or relevant information were emerging. This study was approved by the Medical Research
Subsequently, the data was tabulated into a thematic and Ethics Committee, Ministry of Health Malaysia
framework (Table 1). A total of 4 themes and 24 codes (NMRR-18-2009-42980) and Faculty Research and
were identified. At last the final conclusion was drawn Scholar Activities (FRSA), Faculty of Pharmaceutical
from the rich data. Science, UCSI University.Confidentiality and anonymity

Table 1 : Thematic Framework

Themes Codes Total number Consideration
of respondent about PRP
(n=34) pharmacist
Placement Waiting time 32 All settings
Selecting the PRP training centre 34 All settings
Emotional status of graduates 31 All settings
during PRP waiting period
Pros of PRP training centre 34 All settings
Cons of PRP training centre 34 All settings
Payment Agreement on allowance 34 All settings
Minimum allowance 34 All settings
Supplementary allowances 34 All settings
Working Status of PRP 34 All settings
Comparison between 34 All settings
condition PRP and employee
Working hour 34 All settings
Leave 33 All settings
Workload 34 All settings
Comparison of workload of PRP and 34 All settings
registered pharmacist
Comparison of responsibility between PRP 27 G, P, H, C, M,
and registered pharmacist
NM
PRP training in line with pharmacist’s job 34 All settings
scope
Accessibility to computerized system 23 G, P, H, M, NM
Training Regular and systematic training for PRP 34 All settings
Difference between PRP training and 34 All settings
academic attachment
Sufficiency of undergraduate program 34 All settings
Incorporation of PRP training into university 32 All settings
curriculum
Preceptor’s qualification 34 All settings
Relationship with preceptor 33 All settings
Experiences of PRP 34 All settings

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were informed and assured to all participants during the not been well structured and organized. This was probably
invitation stage that any type of disseminating of the data due to the newly established of PRP training module in
will be done anonymously and both PRP training centre the individual premise with poor guidance which affected
and pharmacist will be unidentified. The verbal informed their learning experience.
consent was obtained and audio recorded from all the Limitation is because we are still in new module so
participants at the beginning of the interview. sometimes our preceptor don't know how to handle us
3. Results and Discussion they don't know what should we do for in order to fulfil
the logbook all that. For example, for unit inspection
Characteristics of participants normally our pharmacist they will go outside for unit
Out of 33 participants, 23 of them were PRP and the inspection but for PRP she doesn't know how to conduct
remaining 10 participants were FRP. Most of the for the unit inspection but we just follow the pharmacist
participants (n =19) from government hospital, private to go for unit inspection.
hospital, health clinic and research and development were (Health clinic, Interview 4, Line 42, 46)
female. In pharmaceutical industry, most of the
participants were male. Majority of participants (n = 20) Payment
stayed near to the workplace and travelled by car (n = Minimum allowance
26).
The payment of PRPs were in line with the minimum
Main themes allowance policy except those under research and
The representative quote for each group of responses development setting. Some received monthly research
is shown to illustrate themes and codes. grant lower than the minimum allowance and some were
not receiving any allowances or research grant throughout
Placement the training. Participants from government sector had the
Waiting time same basic allowance, while for other settings, allowance
varied from one training premises to another.
Most of the participants waited less than 6 months
for the PRP placement from their graduation. During the I actually being paid by the grant, there is this fix
waiting period, they were working as a pharmacist amount that everybody get. The grant is for the research
assistant or intern to gain experiences in the relevant project, my supervisor will allocate some for the
pharmacy field or went for vacation. Participants who computer, some for the software, some for my allowance.
waited more than 6 months for the PRP placements were Actually very low because we are still student and I
those who applied for PRP training in government employed here as research assistant.
hospital. Those who failed to obtain a placement in (Research and development (academia), Interview 2, Line
government hospital,opted for private hospital or health 64, 66, 69)
clinic to do their PRP training. The acceptable waiting
Working condition
period for majority for PRP placement was 6 months.
Around 5 to 6 months. I feel compared to senior is not Work load
a very long duration. 5 to 6 months is enough for me to The workload of PRPs in government hospitals was
have some rest at the same time to work part-time to gain different from one department to another. Outpatient
some experiences before going to government hospital. pharmacy had the highest workload due to high volume
(Government hospital, Interview 4, Line 11) of patients and large amount of prescriptions. Overall,
their workload was reported to be manageable, likewise
Cons of PRP training centre in health clinic setting. In private hospitals, their workload
The main short coming of the PRP training centre was was mostly operation and pharmacy management. Heavy
limited learning experience. Participants in private workload was reported in manufacturing sector as some
hospital had limited exposure on clinical, TDM and TPN of them were placed in a specific department for an
due to the short training duration or unavailability of the extended period and they were required to handle multiple
services. Participants under health clinic, non- work tasks concurrently. The workload of PRP in non-
manufacturing pharmaceutical industry and research and manufacturing pharmaceutical industry was depend on
development reported that the PRP training program had the projects available at that time. Only selected work

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tasks can be delegated to PRPs because one project from the job and attended workshops occasionally. A
required a few months to be completed which minority of these training premises provided systematic
consequently limit their job scope and learning training module to the PRPs, such as monthly presentation
experience. In community pharmacy, the amount of and case studies, departmental training and monthly
workload was determined by the PRPs' job scope. They medication review. For non-manufacturing sector, the
have more workload if they are involved in branch PRP training was based on the projects and department's
opening, warehouse sale, management work and others. need such as presentation and workshop. Training
For research and development, the 9 months research schedule and checklist were prepared in certain
training was heavy but manageable and the 3 months departments but not all. In manufacturing sector, the
hospital training was hectic due to the high requirements standard operating procedure (SOP) training was usually
of logbook. given to the PRPs before on-the-job training. They needed
Workload for PRP definitely very tough because to do presentation and handle different projects. PRPs
besides operation work we do have a lot of management from research and development started with the briefing
work to do and have to fill in the logbook and spend for laboratory instruments and they were encouraged to
extra time to study. participate in research related workshops and
conferences.
(Private hospital, Interview 1, Line 203)
Depend if you are in department such as
PRP training in line with pharmacist's job scope pharmacovigilance and regulatory affairs where
Except the participants from pharmaceutical industry, everything is capture and they want thing to be done in
all other participants agreed that the PRP training was in particular way then yes a lot of training. If is something
line with pharmacist's job scope. In manufacturing more hey this is a question that we have do some research
pharmaceutical industry, they were involved in technical, then no training will be provided.
management and administrative work in addition to (Non-manufacturing pharmaceutical industry, Interview
pharmacist related work. Likewise, in non-manufacturing 4, Line 168)
sector, their job scope was wide-ranging across depart-
ments, for instance regulatory affair and pharma- Incorporation of PRP training into university
covigilance was more towards a typical pharmacist's role curriculum
compared to sales and marketing department. Participants who agreed and disagreed to incorporate
I'm not too sure as a pharmacist, but it is not the same PRP training into undergraduate program were
as a clinical pharmacist. If deal with the regulatory side, comparable. The benefit of incorporation was to provide
yes, will be in line with a pharmacist, if sale and pharmacy students with the pre-exposure of working
marketing it is more to selling, promoting and marketing environment and gain more practical experiences. Those
the products, so I wouldn't say is in line but we can use with partial agreement or disagreement was mainly
some pharmacy knowledge in sale and marketing. concerned with the students' capability, responsibility,
knowledge and working attitude.
(Non-manufacturing pharmaceutical industry, Interview
1, Line 118) Incorporated is quite hard I would say because during
university is still your learning time and I feel you should
Training learn the most from the lecturer but during PRP time you
Regular and systematic training for PRP are considered a working adult it comes with
responsibilities with whatever you do so is not just about
A vast majority of participants were uncertain whether
getting knowledge it comes with correct attitude.
regular and systematic PRP training was provided to them,
especially those under private sector. Half of them agreed (Government hospital, Interview 3, Line 133)
the PRP training was regular and systematic. Due to the Preceptor's qualification
nature of each training sector, the training method was
All the participants agreed the qualification of their
different from each other. The government training
preceptors was adequate to train a PRP. The preceptors
centres complied to the logbook requirements to provide
were knowledgeable and are experienced in the relevant
PRP training. Near to two third of the participants under
pharmacy field. However, there was one PRP from private
private hospital and community pharmacy reported there
hospital uncertain about the preceptor's qualification due
was no specific PRP training given to them, they learnt

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to the lack of interaction between PRP and the preceptor. in government sector has been largely reduced from
For my preceptor I don't really deal much with her average 18 months to 6 months or less after the service
and so I'm not sure about her qualification but for my contract commenced. Participants prefer to receive
direct superior he is a very good teacher I would say, he notification from government regarding the intake
actually taught me quite a lot of things. schedule, so they will know how long they should be
(Private hospital, Interview 3, Line 214) waited for the placement. The waiting period in private
sector was comparatively shorter, the most was 6 months
The present study examined qualitatively the
experience, satisfaction and perception of Malaysian and the fastest was less than a month. Participants who
preferred to do their PRP training in hospital settings
pharmacists towards their training with a total of 4 themes
predilected towards government hospital.The differences
and 24 codes. The first theme was placement. According
of PRP training module between government and private
to the findings, the waiting time for the PRP placement
hospital are as shown in Table 2.

Table 2 : The comparison PRP training module between government hospital and private hospital (39-40)

Government Hospital Differences Private Hospital

8 weeks Duration of ward pharmacy 4 weeks

Clinical Pharmacokinetics Services,

Compulsory Therapeutic Drug Monitoring Services (TDM) Optional
and Parental Nutrition Services (TPN)

In general, the clinical exposure and learning qualified graduates into the private sector who competed
opportunity in private hospital are lesser compared to for a limited number of training opportunities available.
government hospital. Besides that, the pharmacists in A more variable allowance was observed from one
private hospital are more focused on operational and training premise to another and/or one training setting to
pharmacy management. Participants also mentioned most another due to lack of standardized allowance scheme in
of the doctors in private hospital are consultants which private sector. According to our findings, all the
do not rely much on the pharmacist(21).They believed participants under private sector, except research and
that clinical knowledge is the most valuable asset for development, received minimum allowance of RM 2,600
pharmacists, much of gained are very useful even if the per month (22). However, not all the private training
pharmacists decide to venture into different fields. premises provided supplementary allowance to the PRPs,
Participants preferred a similar training duration for the it is hugely depending on the company. There were
clinical module as the government hospital in order to participants who raised the concern that minimum
get more exposures and learning experiences. Further allowance was not sufficient to cover their living costs
investigation is warranted to determine whether these and they suggested to provide supplementary allowance
optional training modules should be made compulsory. based on their needs. Those participants who practiced
The health clinic PRP training module was not a necessity under research and development shared the same concern
as reported by the participants because the knowledge aforementioned. As this training module is relatively new
and skill gained from hospital will be sufficient for them compared to others, hitherto there is no any guideline on
to manage the work tasks in health clinic.The training in providing allowance to the PRPs. Further studies are
health clinic was not sufficient for PRPs to handle work required to determine if all the private training premises
tasks in hospital. followed the minimum allowance as set in the government
The second theme was payment. In general, all the sector. Standardized allowance scheme should be
government training premises complied with the implemented in all sectors with the consideration of the
minimum allowance policy which provided at least current living costs and to prevent the possibilities of
RM2,600 per month to PRPs(22). The saturation in PRPs being exploited in the process of securing any PRP
government sector consequently drove a large influx of placements.

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The third theme was working condition. The status of settings. The pharmacy course content should be more
PRPs can affect their perception towards the PRP training. balance between clinical pharmacy practice and
Inequality between PRPs and other employees were pharmaceutical sciences in order to provide students with
noticed in government hospitals. PRPs are required to the necessary knowledge and skills for the pharmaceutical
do more basic work tasks and run errands that are industry. There were participants suggested the provision
irrelevant to their job scope, for example order food of pharmacy placement in non-manufacturing sector
delivery for lunch talk. Besides that, PRPs are would be beneficial for pharmacy graduates during the
circumscribed from mingling with registered pharmacists undergraduate program.
due to the hierarchical structure as reported in government The inconsistency in the quality of PRP training was
hospital. However, there was no issue of inequality in noticed across the practice settings. In addition, the
health clinic even if they are under the hierarchical discrepancies between preceptors in terms of knowledge,
structure. On the contrary, the status of PRPs in assessment, logbook requirement, and teaching style were
community pharmacy had a significant impact on their mentioned by the participants from government hospitals.
learning opportunities and job scope. If the PRPs are Similar findings was found in the previous study (15). It
treated as employee, they will be working closely with has recently been reported by local media that PRPs
the FRP sand able to involve more in management works. complained of lack of transparency in PRP evaluation
However, if the PRPs are treated as trainee, their job scope and unavailability of feedback loop (24). PRPs were not
and learning opportunities are limited because they are given permission to review their marks and they couldn't
not allowed to handle certain work tasks. The status of learn from their mistakes and improve on weakness (24).
PRPs under research and development was different from The provision of PRP training is highly dependent on the
all other settings, participants perceived themselves as a preceptors and training centre. Although the criteria for
student during the 9 months research training as their job pharmacists to become a preceptor was specified by PBM
scope is totally different from other employees within (25), there is no requirement to demonstrate their
the same organization. PRPs in pharmaceutical industry expertise in workplace assessment. Malaysia should
and private hospitals were treated as employee under implement a quality management system in PRP training
training. likewise the Pre-Registration Pharmacist Scheme (PRPS)
The last theme was training. The pharmacy curriculum launched in 2006 by Scottish Government with the
in Malaysia is a combination of 3 aspects included purpose of ensuring every PRP receive a high quality
pharmaceutical sciences, clinical pharmacy practice and training opportunity and experience in all practice settings
research and development, the emphasis on one aspect (26). According to Mills et al, a quality management
or other varied from one pharmacy school to another. system should encompass survey of PRPs and preceptors
Based on the findings, the pharmacy curriculum provided and visits to training sites (27-28). In Malaysia, the
sufficient basic knowledge for PRP training in different appraisal of preceptors by PRPs is optional and they are
settings except for pharmaceutical industry. Majority of required to send the form separately from PRP logbook
pharmacy schools emphasized more on clinical pharmacy to PBM (29). The direct feedback of PRPs on their
practice than the pharmaceutical sciences. The modules training is essential and should be made compulsory as
covered under pharmaceutical sciences were mostly to disclose problems and areas of PRP training needed
manufacturing related, in conjunction with non-avail- for improvement (27-28). The mechanism for PRPs
ability pre-registration placements in non-manufacturing feedback can be implemented either through a national
sector causing PRPs can only apply limited knowledge survey or locally implemented, or both (28). Another
into their training. According to Kirby-Smith et al, this important element is regular site visits by the responsible
may affect the pharmacy graduate's perception towards bodies in order to ensure the quality of PRP training
pharmaceutical industry as they might have provided by the respective training facilities (27-28). At
incomprehensive understanding of the wide range of present, there are no studies to evaluate the current
career opportunities available and caused a substantial training workshop for preceptors, further studies are
influence on loss of graduate pharmacists to the industrial warranted in order to determine the needs of developing
sector (23). Furthermore, they have a common perception a structured training program (16) and performance
that their job scope will be clinical related as the management system for preceptor (27) as proposed by
undergraduate program is structured towards clinical the previous studies. Otherwise the training would

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Current Trends in Biotechnology and Pharmacy 108
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continue to be offered variously resulting in serious however they may not immediately get the posts until
disparity (30) and negative learning experience (15, 16, there are vacancies (14). The first batch of contract
27, 30). The possibility to incorporate part of the PRP pharmacists had completed the 2 years contract service
training into pharmacy curriculum was agreed and in December 2018. Out of 500 pharmacists, 180 of them
disagreed by the same number of participants. Some have been selected to receive the permanent post, however
universities in United Kingdom offer a 5 year integrated there are no any updates from the government regarding
pharmacy degree program which incorporates the pre- their postings (24). The local media also reported that
registration training into a single program of curriculum PRPs urged for the transparency on the selection criteria
and training (31). The incorporation of PRP training into of PRPs to receive the permanent post in government
pharmacy curriculum guaranteed the pre-registration hospitals (24). Although PRPs are given briefing during
placements for all pharmacy students(32-34). the Mindset Transformation Programme (Program
Additionally, the employment rate and average starting Transformasi Minda), but they need more detailed
salary for 5 years pharmacy graduates was higher than briefing on the second and third year of the contract post.
those 4 years pharmacy graduates (35). The incorporation
of PRP training into Malaysia pharmacy education and 4. Conclusion
extending the duration of undergraduate program to 5 The participated pharmacists believed that PRP
years is possible, but from the student's point of view it training was a necessary exposure to gain required
could be challenging for them because they will need to experiences, despite all difficulties and challenges. The
pay tuition fees for one more year, instead of receiving focus of government in the past few years was to resolve
allowance (32-36). the saturation of PRP placement in government hospitals
Even though the PRP training was stressful and through the liberalisation of PRP training. However, more
challenging, the overall experiences of pharmacists focus should be put on improving the inconsistency of
toward their training were satisfactory and beneficial. quality in PRP training program in different practice
However, there were areas of concern addressed by the settings in order to improve the experiences and
participants from different settings. The 3 months hospital perceptions of future PRPs toward their training.
training should be reconsidered whether it should be put
Limitations
under research and development. The hospital training
is more relevant to PRPs who study clinical postgraduate This study was mainly conducted in Klang Valley area
programs or those doing clinical research, by contrary, it and the findings may not be generalized in other regions
is less relevant to those who study nonclinical courses. A of Malaysia. The majority of participants under
number of participants urged for revision of PRP training government hospital setting were from larger hospitals,
module, including the logbook requirements, duration of their experiences and perception might be different from
training module and learning outcomes.The purpose of those undergoing PRP training in smaller government
logbook is to serve as a guidance for PRPs to record their hospitals. The suggestion of participants for the PRP
training and experiences (37), however the requirements training can be subjective, since this is a novel study the
of logbook is too overwhelming and repetitive, they tend findings can be utilized as an indicator for further
to focus more on fulfilling the requirements than the assessment on PRP training in different settings.
objectives of PRP training. One of the interesting findings
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Current Trends in Biotechnology and Pharmacy 112
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DOI : 10.5530/ctbp.2020.4s.12

Evaluation of Antibacterial Activity Against Multidrug Resistance (MDR)

Bacteria by the Bark Fractions of Canarium patentinervium Miq.
Sook Shuan T1, R Mogana1, Sasikala Chinnappan1,
Asok Kumar Balaraman1, S Chandramathi2, K Geethanjali2
1Faculty of Pharmaceutical Sciences, UCSI University, Jalan Puncak Menara Gading, 56000, Kuala Lumpur, Malaysia.
2Dept of Microbiology, Faculty of Medicine, University of Malaya, Jalan University, 50603, Kuala Lumpur, Malaysia.

*Corresponding author : mogana@ucsiuniversity.edu.my

Abstract fractions of Canarium patentinervium Miq. bark supports

Rapid emergence of antimicrobial resistance has the evidence of its traditional use and can be explored for
become a concern worldwide. This is due to bioactive compounds as antibiotic alternatives.
indiscriminate increase in bacterial adaptation towards Key words: antibacterial, Canarium patentinervium
conventional antibiotics. This has led to exploration of Miq., MDR
bioactive compounds from plants. Canarium 1. Introduction
patentinervium Miq belongs to the family of Burseraceae
Canarium patentinervium Miq belongs to the family
Kunth and genus Canarium L. This plant has been used
of Burseraceae Kunth (torchwood family)and genus
traditionally in wound healing by indigenous people in
Canarium L. The genus Canarium L (derived from Malay
Malaysia. This study aimed to search for an alternative
name "kanari") comprises approximately 18 genera and
antibiotic from medicinal plant and to provide
700 species.1 The trees are mainly distributed in tropical
ethnopharmacological evidence for its traditional use. The
Asia and the Pacific regions. The genus Canarium found
study aims to fractionate the ethanol extract of the barks
in Asia Pacific region previously recorded for its usage
of Canarium patentinervium Miq by using three solvents
in wound healing by the indigenous people of Malaysia.
(petroleum ether, chloroform and water) and investigate
In previous studies, a number of chemical constituents
its antibacterial activity against multidrug resistance
such as tannins, flavonoids and sterols were presented in
(MDR) bacteria. Qualitative phytochemical analysis of
the ethanolic extract of leaves and barks of Canarium
the fractions of Canarium patentinervium Miq was
patentinervium Miq. 2 The extracts and pure chemical
examined for the presence of chemical constituents. The
constituents derived from Canarium species have been
antibacterial activity of the fractions against reference
revealed for antioxidant, antibacterial, anti-inflammatory,
bacteria, Methicillin sensitive Staphylococcus aureus
and antiacetylcholinerase properties.2,3,4,5
(MSSA) ATCC 29213, Klebsiella Pneumoniae
(K.Pneumoniae) ATCC 13883, Escherichia coli (E.coli) Study of plant secondary metabolites began the modern
ATCC 35218 and clinical isolates, Methicillin resistant medicinal plants research in the early 19th century. 6
Staphylococcus aureus (MRSA), K.Pneumoniae, Bioactive compound has been reported to be
Acinetobacter Baumannii (A.Baumannii) were screened therapeutically useful in treating disease. 7 Rapid
using disc diffusion method, minimum inhibitory emergence of multi drug resistance has become a concern
concentration (MIC) assay and minimum bactericidal issues in healthcare. Antibiotic has become less effective
concentration (MBC) assays. Petroleum ether fraction in treating infectious diseases due to increase of bacterial
exhibited bactericidal activity against MDR bacteria adaptation towards conventional antibiotics. Therefore,
MRSA, MIC=0.125 mg/ml, MBC= 0.5 mg/ml (MBC/ screening approach of a new effective medicinal leads
MIC ratio= 4) and A. Baumannii, MIC= 1.0 mg/ml, from medicinal plants is essential to combat pathogens
MBC= 2.0 mg/ml (MBC/MIC ratio= 2). Water fraction and to avoid the emergence of untreatable bacterial
displayed potent antibacterial activity against MDR strain infections.8,9
of MRSA (MIC= 0.125 mg/ml) and A.Baumannii (MIC= Screening the properties of fractions of ethanol extract
2.0 mg/ml) as compared to positive control respectively of the barks of Canarium patentinervium Miq., this study
(vancomycin, MIC= 0.78 µg/ml and gentamycin, MIC= aims to investigate their phytochemical constituents and
>25 µg/ml). The antibacterial activity of ethanol extract antibacterial activity to support the ethnopharmacological

Evaluation of antibacterial activity of Canarium patentinervium

Current Trends in Biotechnology and Pharmacy 113
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DOI : 10.5530/ctbp.2020.4s.12

evidence in its traditional use and correlate between water bath with 2 moles of hydrochloric acid, HCl (5ml).
antibacterial activity to investigate their antibacterial After cooling, the mixture was filtered and the filtrate
activity against 3 reference strains (MSSA ATCC 29213, was divided into two equal portions. One portion was
K.Pneumoniae ATCC 13883, E.coli ATCC 35218) and treated with 1ml of Dragendorff's reagent. A prominent
clinical isolates (MRSA, K.Pneumoniae, A.Baumannii). orange red precipitate indicates positive result.

2. Materials and Methods Flavonoids test (Shinoda test)

Plant material collection and authentication 4 mg fractions were dissolved in 0.2 ml ethanol and
The barks of Canarium patentinervium Miq (CP).were filtered. The filtrate was treated with a few drops of
previously collected from one individual tree from Bukit concentrated HCl and magnesium turnings (0.5g). The
Putih, Selangor, Malaysia (3º5'24'' N 101º46'0''E). The presence of flavonoids is indicative if pink or magenta-
plant was identified with a herbarium sample (PID red color developed within 3 minutes.
251210-12) has been deposited in the Forest Research Saponins test
Institute of Malaysia (FRIM). The fraction was shaken vigorously to froth and then
allowed to stand for 15-20minutes and classified for
saponins content as follows: (no froth= negative; froth
less than 1cm= weakly positive; froth 1-2cm high =
positive; and froth greater than 2cm high = strongly
positive).
Tannins test
About 1mg of fractions were dissolved in 1ml of hot
distilled water and filtered. The solution was divided into
two test tubes. To the first 0.9% sodium chloride (NaCl)
solution was added, to the second 0.9% NaCl and 1%
Fig 1. Canarium patentinervium Miq.
gelatin solution were added. Formation of a precipitate
Fractionation of the ethanol extract of the barks of in the second treatment suggests the presence of tannins,
Canarium patentinervium Miq. which result in white precipitate supports this inference.
The extraction of this plant was previously done by Sterols test (Salkowski Reaction)
supplier. 12.27 g ethanol extract of the bark of Canarium 4mg of fractions were dissolved in 0.2ml of
patentinervium Miq was dissolved in water. Then, it was chloroform and filtered. The filtrate was then added to
sonicated by using asonicator for about 20 minutes to 0.1ml of concentrated sulfuric acid, H2SO4. The presence
dissolve the extract completely. It was then subjected to of sterols is indicated by the 2 phase formation with a
fractionation by liquid-liquid partition using petroleum red color in the chloroform phase.
ether, chloroform and water solvents to yield the
respective solvent fractions by using separating funnel.4 Cardiac glycosides test (Keller-Kiliani test)
It was concentrated with a rotary evaporator (Buchi, R- A solution of glacial acetic acid (4.0ml) with 1 drop
200 Switzerland). Water fraction was placed in the fridge of 2.0% FeCl3 mixture was mixed with 10ml aqueous
at -80ºC for one day before undergoing freeze dryingusing plant extract and 1ml of concentrated H2SO4. A brown
a freeze dryer. ring formed between the layers which showed the entity
of cardiac steroidal glycosides.
Phytochemicals screening
Qualitative phytochemical analysis was carried out Evaluation of antibacterial activity
to identify the presence of secondary metabolites in the Bacterial strains
plant fractions. Qualitative phytochemical analysis of the For all the experiments, three different microbial
fractions to identify presence of secondary metabolites (ATCC reference) cultures and three clinical strains were
was determined as follows : 10,11,12 used. The clinical isolates of bacteria were obtained from
Alkaloids test University Malaya Medical Centre (UMMC). All bacteria
Dragendorff's reagent : 200mg of the fraction was strains were isolated from clinical specimens of
dissolved in 10ml of methanol and heated on a boiling hospitalized patients identified according to the Centers

Tan et al
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DOI : 10.5530/ctbp.2020.4s.12

for Disease Control and Prevention / National Healthcare 3. Results and Discussion
Safety Network (CDC/NHSN) criteria:13 Fractionation yield
Reference strains : MSSA ATCC 29213, K.Pneumoniae Based on Table 1, H2O solvent yielded the highest
ATCC 13883, E.coliATCC 35218 amount of fraction (77.0 %), followed by PE (18.1%)
Clinical isolates : MRSA, K.Pneumoniae and and CHCl3 (1.5%).
A.Baumannii Phytochemicals screening assay
Disc diffusion test Determination of the presence of secondary metabolite
In vitro antimicrobial activity of fractions of ethanol on the fractions was investigated by conducting
extract of the barks of Canarium patentinervium Miq was phytochemicals analysis (alkaloids, flavonoids, tannins,
studied against bacterial strains by using disc diffusion saponins, sterols, and cardiac glycosides).
method, also known as Kirby-Bauer test following Investigation of secondary metabolites on
guidelines provided by the Clinical and Laboratory phytochemicals analysis was described on Table 2.
Standards Institute (CLSI). 14 In the present study, Presence of secondary metabolites varies between each
antibiotic was used as positive control (vancomycin used fraction. Chloroform (CHCl3) fraction only contains
for gram positive bacteria, while gentamycin used for sterols. While petroleum ether (PE) fraction has all tested
gram negative bacteria) and dimethylsulfoxide (DMSO) phytochemicals except for alkaloid and water (H 2O)
as negative control. Each standardized inoculum was fraction contains alkaloid, flavonoids, tannins, and
adjusted at 0.5 Mcfarland standard / 625nm to yield 1 x cardiac glycosides. Previous study done by Mogana R et
108cfu/ml by using Mueller Hinton broth (MHB). The al. (2011) showed that ethanol extract of the barks of
inoculum was streaked onthe surface of agar plate. Paper Canarium patentinervium Miq consists of flavonoids,
discs were impregnated with the fractions already tannins and sterols.
dissolved in pure DMSO and placed on the surface of Antimicrobial susceptibility tests
inoculated agar with bacterial strain. Inhibition zones
Antibacterial activity of fractions of ethanol extract
around each disc after an incubation of 37°C for 24 hours
of bark of Canarium patentinervium Miq against
was measured and described as antibacterial activity.
reference bacteria strains (MSSA ATCC 29218,
Minimum inhibitory concentration (MIC) assay K.Pneumoniae ATCC 13883, E.coli ATCC 35218) and
MIC assay was described by Eloff15 and performed clinical isolates bacteria (MRSA, K.Pneumoniae,
in 96 wells plate by 2-folds serial dilutionfollowing the A.Baumannii) were evaluated by antibacterial
Clinical and Laboratory Standards Institute (CLSI) susceptibility tests such as disc diffusion method, MIC
guidelines.16 A serial dilution from the stock solution were and MBC assays.
ranging from 32mg/ml to 0.25 mg/ml using MHB. It For disc diffusion method, the results showed that H2O
aimed to evaluate antibacterial effects of the fractions. and PE fractions have significant antibacterial activity
with p < 0.05 against almost all the tested bacteria strains
Minimum bactericidal concentration (MBC) assay
except for K.pneumoniae and E.coli (ATCC 35218) which
MBC assay was performed using the method of
shown in Table 3. H2O and PE fractions displayed
Oztuk&Ercisli only for the susceptible bacteria from the
significant antibacterial activity against MDR bacteria,
MIC assay.17 Only samples that have MIC values of lower
MRSA with zone of inhibition of 19.7 mm and 18.3 mm
or equivalent to 0.5mg/ml (strong inhibitors: PE and H2O
respectively and A.Baumanii with zone of inhibition of
fractions) were tested for the MBC values 18 . Ten
10 mm and 9.7 mm respectively as compared to positive
microliters were taken from the well obtained from MIC
control. While, CHCl3 fraction has least antibacterial
value and two wells above the MIC well and spread on
activity among three fractions with sensitivity towards
MHA plates. The number of colonies was counted after
gram positive bacteria MSSA ATCC 29218 (inhibition
18-24 hours of incubation at 37ºC.
zone= 6.83 mm) and MRSA (inhibition zone= 10.3 mm).
Statistical analysis Only fractions (H2O, PE) with higher activity were
All the results were expressed as mean ± SD. One carried out MIC and MBC assay to determine the
way ANOVA and Tukey's test (Prism) were employed specificity of antibacterial activity. PE revealed
for the data analysis, when p < 0.05, the difference was bactericidal activity against MSSA ATCC 29213 (MBC/
considered significant. MIC=2), MRSA (MBC/MIC= 4), K.Pneumoniae ATCC

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Current Trends in Biotechnology and Pharmacy 115
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13883 (MBC/MIC=1) and A.Baumannii (MBC/MIC= 2). Table 2. Preliminary phytochemical screening of the
PE displayed high sensitivity towards MDR bacteria such fractions of ethanolic extract of the barks of CP.
as MRSA (MIC= 0.125 mg/ml, MBC= 0.5 mg/ml, MBC/
Tests CHCl3 PE H2O
MIC= 4) and A.Baumannii (MIC= 1.0 mg/ml, MBC= 2.0
mg/ml, MBC/MIC= 2).
Fraction Fraction Fraction
A.Baumannii was considerable resistant to gentamycin
Alkaloids - + -
(MIC= >25µg/ml), however it showed sensitivity towards
H2O (MIC=2.0 mg/ml) and PE fractions (MIC=1.0 mg/ Flavonoids - ++ +
ml, MBC= 2.0 mg/ml, MBC/MIC = 2). This may be Saponins - ++ +
implied as a clear finding for a novel therapeutic choice
to treat MDR A.Baumanii infection.
Tannins - - +
Table 1. Percentage of fraction yield of fractions of Sterols + ++ -
ethanolic extract of the bark of CP. Cardiac - ++ +
Solvents Fraction Glycosides
Petroleum ether (PE) 18.1 Keys: (+) present, (-) absent
Chloroform (CHCl3) 1.5
Water (H2O) 77.0

Table 3. Antimicrobial susceptibility tests of the fractions of ethanolic extract of the barks of CP Miq.

Bacteria strains Zone of inhibition (mm)

Plant fractions MBC,MIC values (mg/ml) Control antimicrobial agents
MBC,MIC values (µg/ml)
MBC/MIC ratio
CHCl3 PE H 2O Vancomycin Gentamycin
Gram positive 6.83±0.29 15.3±1.53A 16.0±1.0 A
11.0±0.0
MSSA NA 1.0/0.5 -/0.25 0.78/0.78 NA
(ATCC 29213) 2(+) - 1(+)
MRSA (MDR) 10.3±0.58B 18.3±1.15C 19.7±1.15C 10.3±1.15B
NA 0.5/0.125 -/0.125 0.78/0.78 NA
4(+) - 1(+)
Gram negative - 13.7±1.53D 14.0±1.0D 21.7±1.15
K.pneumoniae NA 1.0/1.0 -/1.0 NA 0.2/0.2H
(ATCC 13883) 1(+) - 1(+)
K.pneumoniae - 17.0±1.0
(MDR) NA - - NA 0.39/0.2
2(+)
E.coli (ATCC - 15.0±1.0
35218) NA - - NA 6.25/1.56
4(+)
A.baumannii - 9.7±0.58E 10.0±0.0E Resistant
(MDR) NA 2.0/1.0 -/2.0 NA -/>25I
2(+) - -

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Current Trends in Biotechnology and Pharmacy 116
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DOI : 10.5530/ctbp.2020.4s.12

Notes : CHCl3: Chloroform fraction at the concentration cereus, MRSA and gram negative bacteria Pseudomonas
of 0.6 mg/disc in DD assay, H2O: water and PE: petroleum aeruginosa.2
ether fractions at the concentration of 1 mg/disc in DD Phytochemical investigation of three fractions
assay, vancomycin and gentamycin at 1 µg/disc in DD revealed the presence of bioactive compounds that serve
assay, vancomycin used for gram positive bacteria, as defense mechanism against microbes such as
gentamycin used for gram negative bacteria, -: no activity flavonoids (Xie YX et al., 2014)19, saponins (Tagousop
noted, that is, inhibition zone of 6 mm, NA: not applicable CN et al., 2018)20, tannins (Akiyama H et al., 2001)21,
and MBC/MIC ratio ?4 = bactericidal (+) , >4 = sterols (Kavita K et al.,2014 and Dogan A et al.,2017)22,23
bacteriostatic (-). and alkaloids (Cushnie TPT et al., 2014)24. For flavonoid,
Data were obtained from triplicates experiments for evidences have shown that flavonoids exhibit inhibitory
disc diffusion method (n=9) and duplicate experiment effects against the efflux pump of MRSA and against -
for MIC assay (n=6) and represented as mean ± SD. lactamases producing bacteria due to the structure of C6-
Values with the same capital letter are considered as non- C3-C6 skeleton possess antibacterial activity to defense
significant different (p>0.05) followed by One way wide range of pathogenic microorganisms.19 According
ANOVA and Tukey multiple comparison test. to Tagousop CN et al, saponins have synergistic effect in
the combination of antibiotic which possibly associated
with sugar moiety. This study revealed saponins possess
highest inhibitory activity against S.aureus with less than
3 sugar moiety. Moreover, tannins owing antibacterial
activity could be due to the existence of tannic acids which
affect membranous structure of bacteria. According to
Dogan A et al., the study reported that sterols have
antibacterial activity can be explained based on peroxide
and vinyl bonds in their structure.23 Its mechanism may
be correlated to the similarity of sterols in the bacterial
cells. On the other hand, alkaloids such as indole alkaloids
undergo dimerization to reveal antibacterial activity
which possibly due to larger molecules of indole that are
less prone to bacterial efflux. Hence, it can be concluded
that fractions contain potent bioactive compounds with
antibacterial activity.
Since the qualitative antibacterial activity of fractions
Fig 2. Antibacterial activity of fractions (1 mg/disc) of
of CP was determined by disc diffusion assay, therefore
ethanol extract of bark of CP and positive control (1 µg/ MIC and MBC assays were performed to evaluate the
ml) tested by disc diffusion method mode of antibacterial actions either bactericidal or
In the present study, it was noted that the highest bacteriostatic. According to Fabry et al., it was indicated
percentage of fraction yield was with H2O, followed by that all plant extracts with MIC values < 8 mg/ml as active
PE and the lowest with CHCl3. The percentage of fraction inhibitory agents. On the other hand, Van Vuuren was
yield may be associated with the polarity of solvents and suggested medicinal plants with MIC < 2 mg/ml were
characteristic of chemical constituents in the fractions. considered as active.25 Hence, it can be considered that
The antibacterial activities of fractions of ethanolic H2O and PE fractions displayed high antibacterial activity
against references bacteria and clinical isolated bacteria
extract of the CP barkswere evaluated by using disc
with MIC values ranged from 0.125 mg/ml to 2.0 mg/ml.
diffusion method, MIC and MBC assays. The
antibacterial activity of the fraction could be due to the Generally, all fractions displayed antibacterial activity
presence of bioactive compounds that analyzed through more pronounced to gram positive bacteria than gram
phytochemical screening assay (Table 2). According to negative bacteria. This is most probably due to the
the preliminary study by Mogana R et al., the ethanol morphology of bacteria. Differences between gram
extract of leaves and barks and hexane extract of barks positive and gram negative bacteria were associated with
of CP revealed significant antimicrobial activity against the composition of cell wall (Munyendo WLL et al.,
gram positive bacteria Staphylococcus aureus, Bacillus 2011).26

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Current Trends in Biotechnology and Pharmacy 117
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DOI : 10.5530/ctbp.2020.4s.12

Moreover, a clear indication was implied that biotechnology, 2013, 986361. https://doi.org/
A.Baumannii was susceptible to H2O (MIC= 2.0 mg/ml) 10.5402/2013/986361
and PE (MIC= 1.0 mg/ml) fractions. In the present study, 4. Mogana, R., Teng-Jin, K., & Wiart, C. (2013). Anti-
there was considerable resistance to gentamycin with MIC Inflammatory, Anticholinesterase, and Antioxidant
value of >25 µg/ml. Fraction contains large amount of Potential of Scopoletin Isolated from Canarium
chemical constituents which may role in inhibiting the patentinervium Miq. (Burseraceae Kunth). Evidence-
growth of A.Baumannii and as a valuable source with based complementary and alternative medicine :
potent antimicrobial activity to reverse antibiotic eCAM, 2013, 734824. https://doi.org/10.1155/2013/
resistance (Khameneh B et al.,2019).27 734824
4. Conclusion 5. Mogana, R., Adhikari, A., Debnath, S., Hazra, S.,
Fractions of ethanolic extract of the barks of Canarium Hazra, B., Teng-Jin, K., & Wiart, C. (2014). The
patentinervium Miq. showed potent antibacterial activity antiacetylcholinesterase and antileishmanial
possibly due to the presence of various bioactive activities of Canarium patentinervium Miq. BioMed
compounds (tannins, flavonoids, saponins, sterols and research international, 2014, 903529. https://doi.org/
alkaloids). This study revealed to support the evidence 10.1155/2014/903529
of its traditional use and explored for bioactive
6. Saxena, M., Saxena, J., Nema, R., Singh, D., &
compounds as antibiotic alternatives. For future studies,
Gupta, A. (2013) Phytochemistry of Medicinal
it was recommended to carry out for bioassay guided
Plants. Journal of pharmacognosy and
isolation and identification of bioactive secondary
phytochemistry, 1(6). https://www.researchgate.net/
metabolites as well as to study the mechanism of bioactive
p u b lica t ion / 2 8 4 4 25 7 3 4 _ P hyt o chemis t r y_
compounds action on MDR bacteria.
of_Medicinal_Plants
Acknowledgement
7. A. Hussein, R., & A. El-Anssary, A. (2019). Plants
The authors would like to thank everyone who Secondary Metabolites: The Key Drivers of the
provided help in this project and UCSI University and Pharmacological Actions of Medicinal Plants. In
University of Malaya provide facilities to conduct this Herbal Medicine. https://doi.org/10.5772/
study. intechopen.76139
Conflict of interest 8. Podschun, R., & Ullmann, U. (1998). Klebsiella spp.
The authors declare that they have noconflict of as nosocomial pathogens: epidemiology, taxonomy,
interests. typing methods, and pathogenicity factors. Clinical
microbiology reviews, 11(4), 589-603.
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Current Trends in Biotechnology and Pharmacy 119
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DOI : 10.5530/ctbp.2020.4s.13

Enzymatic and Non-enzymatic Antioxidant Potential of

Methanolic Fractions of Artabotrys suaveolens
R. Mogana1*, Jubair Najwan1*, WL Koh1, LM Foh1, Theresa WT Lee1.
JH Foo1, Sasikala Chinnappan1, Ashok Kumar Balaraman1, C. Wiart2
1Faculty of Pharmaceutical Sciences, UCSI University, Kuala Lumpur 56000, Malaysia
2School of Pharmacy, University of Nottingham, JlnBroga, Semenyih, Malaysia

*Corresponding authors : mogana@ucsiuniversity.edu.my, najwanjubair@yahoo.com.

Abstract process but at high concentration, they contribute to

Artabotrys suaveolens is a tropical plant traditionally serious cellular damages (2).
used for treatment of inflammation. The stem and leaves In contrast, antioxidants are compounds that capable
extract of Artabotrys suaveolens plant were investigated of donating their electron to stabilize these reactive species
for 5-lipooxygenase inhibition (LOX) in both enzymatic and reduce their harmful effects to human body (3). They
and non-enzymatic invitro assays. The non-enzymatic are classified into two types; a) enzymatic which are
antioxidant potential was determined using 1,1'-diphenyl- endogenous antioxidants that present naturally in the body
2-picrylhydrazyl (DPPH) assay and beta-carotene such as superoxide dismutase (SOD), catalase (CAT),
bleaching assay while the enzymatic antioxidant potential glutathione peroxidase (GPX) and glutathione reductase
was measured by superoxide dismutase (SOD) assay. (GR) (4), and b) non-enzymatic which are further split
Nordihydroguaiaretic acid (NDGA) was used as positive into metabolic and nutrient antioxidants. Metabolic
standard. Phytochemical constituents of different fractions antioxidants are endogenous antioxidants produced from
were determined. The chloroform fraction of the stem metabolism such as transferrin, glutathione, coenzymeQ-
confronted antioxidant activity using DPPH assay (EC50: 10, L-arginine etc. while nutrient antioxidants are
7.89±0.50 µg/ml), beta-carotene bleaching assay (EC50: exogeneous antioxidants obtained from diet or
8.04±0.65 µg/ml) and SOD assay (IC50: 13.83±0.35 µg/ supplements such as vitamin C and E, trace metals
ml) with significant inhibition (IC50 value of 16.00±0.50 (selenium, manganese, zinc), carotenoids, flavonoids,
µg/ml) compared to NGDA (IC50 value of 55.80±1.00 omega-3 and omega-6 fatty acids etc (3).
µg/ml). The phytochemical analysis suggested the Both enzymatic and non-enzymatic antioxidants work
presence of alkaloids, cardiac glycosides and flavonoids together to ensure cell protection against oxidative
in the stem of this plant. The significance of results damages resulted from reactive oxygen species (ROS)
supports the role of chloroform fraction from the stem of and reactive nitrogen species (RNS) [5,6].
Artabotrys suaveolens as a lead compound with
5-lipooxygenase (5-LOX) is an important enzyme
therapeutic usefulnessin treatment of inflammatory
from arachidonic acid (AA) cascade [7], catalyses the
diseases.
synthesis of leukotrienes (LTs) [8]that are involved in the
Key words : Artabotrys suaveolens, 5-LOX inhibition, pathogenesis of various inflammatory diseases such as
SOD assay, DPPH assay, beta carotene bleaching assay allergic rhinitis, asthma, cardiovascular diseases and
1. Introduction certain types of cancer [9].
"Oxidative stress" is a situation when there is transient Currently, the only 5-LOX inhibitor approved for
or chronic imbalance between reactive oxygen species clinical uses is zileuton which is prescribed for treatment
(ROS) production and the ability of biological system to of asthma symptoms however; long term use of zileuton
detoxify them through antioxidants (1). The modulation is associated with liver toxicity [10]. Hence, there is a
of redox state is crucial for cell viability, activation, need to search for new 5-LOX inhibitors with fewer side
proliferation, and organ function.At low concentration, effects. Natural 5-LOX inhibitors from medicinal plants
ROS play vital role in body regulation and signaling become increasingly important.

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Current Trends in Biotechnology and Pharmacy 120
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DOI : 10.5530/ctbp.2020.4s.13

In continuation of our natural and medicinal research respective solvent fractions of different polarity using
programme on tropical rainforest plants [11,12], this study liquid-liquid partitioning technique. The same were
aims to investigate the inhibition of 5-LOX, the enzymatic repeated with SM and the fractions were labelled as LPE
and non-enzymatic antioxidant capacity of Artabotrys (petroleum ether fraction of leaves), LCL (chloroform
suaveolens plant. fraction of leaves), LW (water fraction of leaves), SPE
A. suaveolens (from Greek, artao = supports and botrys (petroleum ether fraction of stems), SCL (chloroform
= bunch of grapes and suavis = sweet) [13], is a plant fraction of stems), and SW (water fraction of stems).
belongs to the genus Artabotrys and family of Phytochemical analysis : was performed as follows [18-
Annonaceae. It is known by its local names, akarchenana 20]
and akarlarak in Malaysia [14] which is widely distributed Alkaloids : 6 drops of Dragendorff's reagent were added
in India (Nicobar Island), Malaysia, Philippines and Java, into 1 ml of filtrate containing sample fractions with HCl.
Indonesia [13]. A. suaveolens is traditionally used orally The presence of alkaloid was indicated by production of
as emmenagogue. In addition, it is used to relieve fatigue orange /brown precipitate.
after childbirth [15], to treat cholera [15] and to treat
Flavonoids : Shinoda test was done to test the presence
inflammation associated with enlarged spleen [16]. The
of flavonoids. Few drops of concentrated HCl was added
above study is the first reported study on this plant and is
intofiltrate of sample fraction and magnesium ribbon. The
documented by our team.
presence of flavonoids was indicated by appearance of
2. Materials and Methods pink-tomato red colour.
Materials : 1,1'-diphenyl-2-picrylhydrazyl (DPPH) from Saponins : Frothing test was used. The presence of
Calbiochem, beta-carotene, trolox, quercetin, dimethyl saponins was indicated by frothing of mixture containing
sulfoxide (DMSO) were purchased from R&M, tween sample fraction and distilled water which classified as
20, linoleic acid, superoxide dismutase (SOD) kit were follows: Negative results = no froth; Positive results =
bought from Cayman Chemical Company (Item number: froth height (<1 cm: weak; 1-2cm: medium; >2 cm:
706002; batch number: 0526463), while strong).
nordihydroguairetic acid (NDGA) was purchased from Tannins : 5% w/v FeCl3was added to filtrate containing
Sigma Aldrich, enzyme 5-lipoxygenase enzyme (human sample fraction. The presence of tannins was indicated
recombinant) was purchased from Cayman Chemical by the production of blue-black precipitate.
Company and potassium phosphate buffer was from
Sterols : Salkowski reaction was used. 1 ml of
Sigma Life Science.
concentrated H2SO4 was added into the solution. The
Plant materials : The leaves and stems of Artabotrys presence of sterols was shown by two phase formation
suaveolens were collected from a forest in Perak, with a red colour appearance.
Malaysia (4°46'N, 100°56'E). Plant identification was
Cardiac glycosides : In 2 ml plant fraction, glacial acetic
done by Forest Research Institute of Malaysia (FRIM).
acid, one drop of 5% ferric chloride (FeCl3) and
An herbarium sample has been placed at FRIM. The
concentrated sulphuric acid (H2SO4) were added.
leaves and barks were air-dried and grinded into small
Reddish brown colour appears at junction of the two
particles using industrial grinder.
liquid layers and upper layer appearedwith bluish green,
Extraction and fractionation : maceration was used for confirming the presence of glycosides.
extraction through which the plant was soaked in a closed
Antioxidant capacity tests : Extract and fractions of
conical flask at room temperature [17]. Samples from
leaves and stems were dissolved into DMSO prior to
leaves (2.7 kg) and stems (1.7 kg) were immersed in
DPPH and beta-carotene assays at a stock concentration
methanol (one part of the plant sample soaked in 3 parts
of 400 µg/ml and 2000 µg/ml respectively. Sample buffer
of methanol) for 2 hours at 60oC water bath. Then, leaves
supplied by SOD assay kit (Cayman Chemical) was used
extract was concentrated in a rotary evaporator (Eyela
to dissolve plant samples for SOD assay to yield stock
NVC-2200). The methanolic extracts of the leaves was
concentration of 1.25 mg/ml. All samples were then plated
indicated as LM (leaf methanolic crude extract) and the
in a 96-well microtiter plate in different concentrations
methanolic extracts of the stem was indicated as SM (stem
with serial dilution starting from 100 g/ml to 3.125 g/
methanolic crude extract). LM was partitioned with
ml. Trolox, quercetin and standard SOD enzyme were
petroleum ether, chloroform and water to produce

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used as positive controls. All three assays were performed the positive standard. The antioxidant activity of the tested
using BMG LABTECH FLUO star Omega microtiter extract and fractions were evaluated in terms of bleaching
plate reader, connected to a computer equipped with of -carotene using the following formula: antioxidant
(MARS Data Analysis Software 5.10 R2). GraphPad activity AA (%) = [1- (A0 - At)/(A'0 - A't)] × 100, where
Prism version 7.04 were used to generate the EC50 and A0 and A'0were absorbances measured at zero time of
IC50 value. Three independent tests were carried out in incubation for the test sample and control, respectively;
triplicates for each sample in DPPH and beta-carotene At and A'twere the absorbances measured in the test
assays whereas duplicates for each sample were done in sample and control, respectively, after incubation for 3
SOD assay. hours.
2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay : The Superoxide dismutase (SOD) assay : Cayman superoxide
DPPH assay was carried out according to Juan- dismutase (SOD) assay kit [24] was used in the present
Badaturuge method [21]. Aliquots of methanolic plant study. The kit worked by mimicking the action of xanthine
extract and fractions were dissolved in dimethyl sulfoxide oxidase in the body which generated ROS during
(DMSO) at a stock solution of 0.4 mg/ml. Samplesat conversionof hypoxanthine to xanthine and then to uric
different concentrations were plated out in triplicates acid. If the sample to be tested contained SOD enzyme,
using a 96-well microtiter plate, prepared as serial superoxide can be neutralized to produce hydrogen
dilutions from 100 g/ml to 3.125 g/ml. 1.183 mg of peroxide and oxygen. Rate of oxygen reduction had a
DPPH was added into 30 ml of methanol to obtain 0.1 linear relationship to xanthine oxidase activity and can
mM of DPPH solution. The plate was covered by be inhibited by SOD enzyme [25].
aluminium foil after adding the prepared DPPH solution, Failure of inhibition resulted in two simultaneous
gently shacked for 2 min and kept in the dark for 30 reactions : (1) oxidation of two superoxide anions to two
minutes. Spectrophotometric measurements were done molecular oxygen and (2) reduction of tetrazolium salt
at 550 nm to obtain the percentage of decolourisation (colourless) to formazan dye (yellow). The IC50 (50 %
(colour change from deep violet to light yellow) and EC50 inhibition activity of SOD or SOD-like samples) can be
values were determined. Standard antioxidants (ascorbic obtained by a colorimetric method. To perform the assay,
acid, trolox and quercetin) were selected as positive 20 L sample stock solution (2.5 mg/mL) was diluted in
control. The radical scavenging percentage for each 20 L of sample buffer to yield concentration of 1.25
sample was calculated using the equation: mg/mL. The samples were plated in a 96-well microtiter
DPPH radical scavenging activity (%) plate in various concentrations ranging from 100 g/mL
Abs control - Abs sample to 3.125 g/mL in which half of the concentration was
= ----------------------------------- x 100 reduced during each serial dilution. Standard SOD
Abs control
enzyme was also plated in different concentrations. 210
Where Abs control represents absorbance of DPPH L of radical detector and 20 L of enzyme were added
radical + methanol; Abs sample represents absorbance and incubated for 20 minutes at 37 ºC. All plant samples
of DPPH radical + sample extract /standard [21]. including standard SOD enzyme were diluted with sample
buffer provided by the kit except petroleum ether and
-Carotene bleaching assay : This assay was carried out
chloroform fraction of stems diluted with ethanol as both
based on the method described by Habtemariam and
fractions are insoluble in sample buffer.
Jackson [22]. Samples were plated out at different
concentration in a 96-well microtiter plate. -carotene Absorbance of formazan dye was measured at the
solution was prepared based on the previous study wavelength 450 nm using the BMG LABTECH
described by R. Mogana et al. [23]. Additionally, sample FLUOstar Omega microtiter plate reader, linked to a
plate for assay was prepared based on the previous study computer equipped with MARS Data Analysis Software
elaborated by R. Mogana et al [23]. In brief, 180 µL of 5.10 R2. Recorded absorbance was proportional to
the emulsion was pipetted into 20 µL of samples at concentration of superoxide hence absorbance has an
different concentrations in the 96-well microtiter plate. inverse relationship with SOD activity.SOD enzyme
The absorbance was obtained at 470 nm immediately and provided in the kit was used as standard control. Sample
after 3h incubation at 50ºC against a blank consisting of well with buffer served as sample blank control to correct
emulsion without -carotene by using a colour absorbance of samples. The rate of SOD inhibition
spectrophotometer. Trolox and quercetin were used as and IC50 were determined. The IC50 was obtained from

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Current Trends in Biotechnology and Pharmacy 122
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graph of SOD activity (%) against concentration of each E = enzyme's activity without test sample
plant sample. S = enzyme's activity with test sample
5-Lipoxygenase(5-LOX) inhibition assay : R. Mogana Similar with previous study, positive control
method for 5-LOX assay was used [25]. This method Nordihydroguaiaretic acid (NDGA) was used.
adapted the procedure outlined by Baylac and Racine [24] Statistical analysis : Data from three independent
and Kamatouet.al. [26] with certain modifications. experiments were performed in triplicates (n=9), except
Human recombinant 5-LOX enzyme (from Calbiochem) for SOD assay which performed in duplicates (n=6). All
was used. 100 U of enzyme was reconstituted using 4°C results were expressed as mean ± SD and nonlinear best
ice-cold buffer (potassium phosphate). DMSO was used fit was plotted. Concentration-response curves were
to dissolve 20 µL of sample. Samples were then plated calculated using the Prism software package 7.04 for
out in triplicates at various concentrations in a 96-well Windows, GraphPad Software, San Diego, California,
microtiter plate. Wells were added with 160 µL of 0.1M USA, http://www.graphpad.com/ (GraphPad, San Diego,
potassium phosphate buffer (pH 6.3) at room temperature. USA). One-way ANOVA with Tukey's multiple
Then 20 µL of enzyme solution were added to all wells comparison tests was performed. Statistical significance
and mixture was agitated. 10 µL of linoleic acid was is considered as p < 0.05.
added at room temperature and incubated for 10 mins. 3. Results and Discussion
Absorbance at 234nm was recorded. At this wave length,
Phytochemical analysis test
linoleic acid transformation (from 1-4-diene into 1-3-
diene) can be detected. 5-LOX catalyses oxidation of Alkaloids, cardiac glycosides, flavonoids, saponins,
unsaturated fatty acids containing 1-4 diene. Rates of sterols/steroids and tannins were detected in the
reaction of samples was compared to blank using the methanolic fractions of both leaves and stems of A.
formula below, percentage inhibition of enzyme was suaveolens(Table 1).
determined : Antioxidant capacity tests
E–S Antioxidant capacity of extract and fractions of A.
Percentage inhibation of enzyme = ------- x 100
E suaveolenswere performed using both enzymatic (SOD)
Table 1 : Phytochemical analysis of fractions of leaves and stems A. suaveolens
Phytochemicals LPE LCL LW SPE SCL SW
test
Alkaloids - ++ + + ++ -
(Dragendroff’s
test)
Cardiac + + - + ++ +
glycosides
(Keller-Killani
test)
Flavonoids - ++ ++ - +++ ++
(Shinoda test)
Saponins (Froth - ++ ++ - + +
test)
Sterols/Steroids + - - +++ + -
(Salkowski’s
test)
Tannins (Ferric - - ++ - - ++
Chloride test)

Key: +: low colour intensity, ++: moderate colour intensity, +++: high colour intensity. LPE: petroleum ether fraction of
leaves, LCL: chloroform fraction of leaves, LW: water extract of leaves, SPE: petroleum ether fraction of stems, SCL:
chloroform fraction of stems, SW: water fraction of stems

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DOI : 10.5530/ctbp.2020.4s.13

and non-enzymatic (DPPH, carotene) assays. Non- yellow solution. Competition reaction occurs with the
enzymatic method involves two principle; hydrogen atom presence of another antioxidant (sample) to react with
transfer (HAT) method by measuring the potential LOO* which leads to slower bleaching of the solution
antioxidant activity to convert unstable free radicals to detected at 470 nm spectrophotometrically [30].
stable form through the mechanism of hydrogen atom Results of DPPH assay and the -carotene bleaching
donation [26] and single electron transfer (SET) method assay are shown in Table 2. Fractions of both leaves and
by measuring the capacity of the antioxidant to transfer stems showed antioxidant activity.
one electron to reduce compound including metals,
DPPH assay results indicates that the LM confronted
carbonyls and radicals [27]. -carotene bleaching assay
stronger antioxidant activity (EC50 values of 26.62±0.26
includes HAT method while DPPH uses both methods
g/ml) compared to SM (EC50> 100 g/ml). The antioxidant
predominantly via SET method.
capacity for the leaf's fractions was as follows; LPE >
Stable free radical diphenyl-picryl-hydrazyl (DPPH) LW > LCL. However, for stem fractions, the antioxidant
[28]and its specific absorbance properties can be used to capacity was as: SCL > SPE > SW. The beta-carotene
estimate antioxidant activity [29]. The DPPH molecule bleaching assay also showed strong antioxidant activity
is characterized as stable free radical by virtue of spare possessed by the plant (Table 2). LM and SM showed
electron delocalisation over the molecule as a whole, so antioxidant activity in beta-carotene bleaching assay with
that the molecule does not dimerize [29]. The EC50 values of 2.37±0.50 g/ml and 6.11±0.45 g/ml
delocalisation give rise to deep violet colour. DPPH assay respectively compared to positive. controls trolox (EC50
is based on the ability of antioxidant to reduce stable = 3.59 ± 0.61 g/ml) and quercetin (EC50 = 4.85±0.50 g/
DPPH radical to form yellow coloured , -diphenyl- - ml). The lipid peroxidation capacity of the leaves fraction
picryl hydrazine thus decolourising the deep purple DPPH was as follows: LW> LPE > LCL while the stems
methanol solution. In the -carotene bleaching assay, the demonstrated activity of SCL> SPE & SW (EC50>100
linoleic acid, which is a lipid, undergo reaction in the g/ml). In both assays SCL demonstrated strong activity
presence of reactive oxygen species (ROS) and oxygen compared to all other fractions.
(O2) to produce an unstable peroxyl radical (LOO*). The
Enzymatic antioxidant potential is commonly
peroxyl radical then reacts with -carotene to produce a
determined by measuring the SOD-like activity of a
stable -carotene radical which causes the bleaching of

Table 2 : Antioxidant and anti-inflammatory activitiesof leaves and stems methanolic extracts of A. suaveolens
DPPH assay, EC50 -carotene assay, SOD assay, 5-LOX inhibition,
Sample
(µg/ml) EC50 (µg/ml) IC50 (µg/ml) IC50 (µg/ml)
LM 26.62 ± 0.26 2.37 ± 0.50 b 53.50 ± 0.30 13.00 ± 0.70
LPE 20.60 ± 0.85 22.50 ± 1.31d 55.12 ± 0.42 22.70 ± 0.50
LCL > 100 23.37 ± 0.61d 29.27 ± 0.81 26.90 ± 0.60
LW 32.90 ± 0.20 16.64 ± 0.6 19.67 ± 0.71 30.80 ± 0.60
SM > 100 6.11 ± 0.45c 49.80 ± 0.90 11.20 ± 0.60
SPE 10.30 ± 0.21 > 100 22.40 ± 0.36 28.70 ± 0.70
SCL 7.89 ± 0.50 a 8.04 ± 0.65 13.83 ± 0.35 16.00 ± 0.50
SW 11.80 ± 0.40 > 100 3.25 ± 0.08 > 100
Trolox 7.33 ± 0.28 a 3.59 ± 0.61b NA NA
Quercetin 5.56 ± 0.61 4.85 ± 0.50c NA NA
SOD NA NA 0.20± 0.02 NA
NDGA NA NA NA 55.80± 1.00

Key: LM: methanolic extract of leaves, LPE: petroleum ether fraction of leaves, LCL: chloroform fraction of leaves, LW:
water extract of leaves, SM: methanolic extract of stems, SPE: petroleum ether fraction of stems, SCL: chloroform fraction
of stems, SW: water fraction of stems, SOD: standard superoxide dismutase enzyme, NDGA: nordihydroguaiaretic acid,
NA: not applicable. Data were express as mean ± SD, each perform performed as triplicates (n=9) in 3 independent
experiments. Values with similar alphabet in the same column are not significantly different (p < 0.05) based on Tukey
multiple comparison test.

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Current Trends in Biotechnology and Pharmacy 124
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DOI : 10.5530/ctbp.2020.4s.13

sample compared to SOD enzyme. SOD enzyme is a first

line defence antioxidant that reduces and prevents
generation of free radicals by catalysing the dismutation
of superoxide anion to hydrogen peroxide [1]. In this
study, xanthine oxidase is used to generate superoxide
radical from oxygen. If the sample to be tested has SOD-
like activity, superoxide can be neutralized to produce
hydrogen peroxide and oxygen. If SOD-like activity is
absent in the sample, superoxide will be reduced by
tetrazolium salt (colourless) and colour of formazan dye
(yellow) would be observed.
Methanolic extracts exhibited SOD-like activity with
SM (IC50 = 49.80 ± 0.90 µg/ml) having higher activity Fig. 2 : Mechanism of flavonoid as antioxidant via
than LM (IC50 = 53.50 ± 0.30 µg/ml), versus the positive hydrogen atom transfer
standard SOD enzyme (IC50 = 0.20 ± 0.02 µg/ml). The In this study, SOD-like activity of SW and SCL was
leaves fraction enzymatic antioxidant activity was as respectively superior than the others. High superoxide
follows; LW> LCL> LPE while the stem fractions activity scavenging activity of water fraction could be contributed
was as follows; SW> SCL> SPE. The water and by polar compounds such as flavonoids [38-40] and
chloroform fractions of stem had the highest SOD-like tannins [41-43] where else the SOD-like activity of SCL
activity (SW = IC50 3.25 ± 0.08 µg/ml and SCL = IC50 could be due to the presence of cardiac glycosides,
13.83± 0.35 µg/ml). SCL consistently had relatively flavonoids and alkaloids [37] (Table 1). Various
higher antioxidant activity via all three assays. This effect flavonoids which possess high SOD-like activity are
is attributed to the presence of flavonoids, alkaloids and highly polar (Figure 3) and it was suggested that high
cardiac glycosides in these fractions [31-35]. superoxide scavenging activity is contributed by hydroxyl
group at C-3' in ring B and C-3 [44]. SOD-like activity
of tannins is due to its polyphenolic structure with
hydrophobic core which surrounded by polar compounds
that form hydrophilic shell (Figure 4) [43], making tannin
a water-soluble compound that remains in water fractions.
However, because there is no recent structural activity
relationship study of tannins on its superoxide scavenging
activity, the specific structural features of tannin that
contributes to its SOD-like activity is still not known.
Fig. 1 : General structural requirement of flavonoid as
antioxidant
Flavonoids have a general structure (Figure 1) consists
of three 6-membered rings with one of them is a pyran
ring having a carbonyl group [36]. Flavonoid works in
various types of mechanism of action such as direct Fig. 3 : Chemical structures of flavonoids.
scavenging of ROS/RNS, chelating of trace metal ions
5-Lipoxygenase inhibition assay : The presence of 5-
involved in free radical production, inhibiting enzymes LOX inhibitor decreases the breakdown of linoleic acid
involved in production of free radicals, and regeneration into leukotrienes by 5-LOX enzyme. The change in the
of membrane-bound antioxidants. However, the concentration of linoleic acid is detected using a
consideration of primary antioxidant mechanism of spectrophotometer at 234 nm. Both LM (IC 50 =
flavonoid is hydrogen atom transfer [37]. Thus, o- 13.00±0.70 µg/ml) and SM (IC50 = 11.20±0.60 µg/ml)
dihydroxy substitution in B ring, C2-C3 double-bond, showed significantly higher 5-LOX inhibition as
and C-4 carbonyl group in C ring are the structural activity compared to positive standard NDGA (IC50 = 55.80±1.00
requirements for hydrogen atom transfer (Figure 2). µg/ml).All fractions reported significantly higher
inhibitory activity compared to NDGA except the water
Mogana et al
Current Trends in Biotechnology and Pharmacy 125
Vol. 14 (5) 119-127, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
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Fig. 4 : Chemical structure of tannin. Shaded area indicates

the core structure of tannic acid - pentagalloylglucose. The Fig. 6 : Graphical abstract of A. suaveolens 5-LOX
hydrophobic core and hydrophilic shell are features inhibition activity
responsible for antioxidant action of tannin.
4. Conclusion
fraction of stems as shown in table 2. The 5-LOX
Artabotrys suaveolens is a tropical plant grows
inhibition activity of the leaves were; LPE> LCL> LW
naturally in India, Myanmar, Thailand, Malaysia,
while those of stems fractions were as follows; SCL>
Indonesia and Philippines. It istraditionally used for
SPE> SW.This study focused on various antioxidant and
treatment of inflammation. The extractand fractions of
anti-inflammatory assays include DPPH assay, beta-
Artabotrys suaveolens plant were investigated for 5-
carotene bleaching assay, superoxide dismutase (SOD)
lipooxygenase inhibition (LOX) in both enzymatic and
assay and 5-LOX assay because of their close relationship
non-enzymatic invitro assays. The findings of this study
in the anti-oxidative processes as the development of free
support the antioxidant and anti-inflammatory potential
radicals could be initiated by inflammation reaction [45]
of A. suaveolens as promising agents in treatment of
(Figure 5). linoleic acid (LA) is used in this study as it is
oxidative stress-related diseases. Further isolation,
structurally similar to arachidonic acid and is more stable
characterization of the bioactive components and in vivo
[46]. A graphical abstract of this study is illustrated in
antioxidant research need to be carried out to further
Figure 6.
support the in vitro data.
Acknowledgements
This project has no fund.
Conflict of interest
Authors have no conflict of interest.

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Enzymatic and non-enzymatic antioxidant potential of Artabotrys suaveolens

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DOI : 10.5530/ctbp.2020.4s.14

In silico Screening of Selected Flavanones for HMG CoA Reductase

Inhibitory Activity
Tan Ker Ying1, Mohamed Saleem Abdul Shukkoor1*, Shaik Ibrahim Khalivulla1
1Faculty of Pharmaceutical Sciences, UCSI University, No. 1, JalanMenaraGading, UCSI Heights,
Taman Connaught, Cheras 56000, Kuala Lumpur, Malaysia

*Corresponding author : E-mail: saleemskma@yahoo.com

Abstract binding energy than atorvastatin. However, they have total

Hypercholesterolemia is one of the potential polar surface area (TPSA) lower than 140 Å2 and do not
modifiable risk factors for cardiovascular diseases, the violate the Lipinski's Rule of Five. Eriocitrin and
main leading causes of death globally. Statins (HMG CoA hesperidin showed the estimated inhibition constant (Ki)
reductase inhibitors) are widely prescribed to keep serum in nanomolar range. Further in vitro and in vivo studies
levels of total cholesterol and LDL within the normal limit. are required to analyze the correlation of these in silico
Statins are generally well tolerated. However use of statins findings.
could lead to adverse effects such as elevated hepatic Key words : Insilico docking, flavanones, atorvastatin,
transaminases level, myalgia and increased risk of HMG Co-A reductase inhibitory activity, binding energy?
diabetes. These adverse effects could reduce patient
1. Introduction
compliance and results in poor therapeutic outcomes.
Various flavanones are shown to possess anti- Cardiovascular diseases are a group of disorders
hypercholesterolemic effect in vitro, in silico and in vivo. associated with the heart and blood vessels (1-4).
In this present study, the binding energies of the selected According to the World Health Organization (WHO), the
flavanone compounds against HMG CoA reductase were proportional mortality rate of cardiovascular diseases in
determined through in-silico screening. The selected Malaysia is 35% (5), which is the highest among the
flavanones are eriocitrin, eriodictyol, hesperitin, non-communicable diseases. As compared to the
hesperidin, neohesperidin, naringin, naringenin and neighbouring countries, Malaysians also develop
narirutin. Atorvastatin was used as a positive control to cardiovascular diseases at a younger age (58.5 years) than
validate the binding and to compare the binding energies in Thailand (63.5 years) and in Singapore (68 years) (6).
of the selected flavanones. The structure of the human Apart from that, it is reported that the drug expenditures
HMG CoA reductase (PDB ID: 1DQA) was downloaded in Malaysia continue to rise in the recent years, with the
from Protein Data Bank, whereas the structures of the highest increase in lipid modifying agents, antithrombotic
flavanones were downloaded from ZINC database. All agents and anti-diabetic agents for both public and private
the compounds were prepared using AutoDock Tools sectors (6). Atherosclerosis is the underlying pathology
1.5.6. Then, they were docked against the human HMG of cardiovascular diseases. It is often asymptomatic in
CoA reductase using AutoDockVina 1.1.2 and Accelrys the early phase and manifests as heart attacks and stroke
Discovery Studio 4.5. The interactions between in later years (2).
flavanones and the protein were analyzed and their drug The most important risk factor for atherosclerosis is
likeness was also determined. The binding energy of hypercholesterolemia (7), which is also one of the
atorvastatin was found to be -8.0 kcal/mol. The flavanone potential modifiable risk factors for cardiovascular
glycosides, eriocitrin (-10.0 kcal/mol), hesperidin (-9.7 diseases. The goal of current pharmacotherapy is to keep
kcal/mol), neohesperidin (-9.5 kcal/mol), narirutin (-9.5 the serum levels of total cholesterol and LDL cholesterol
kcal/mol) and naringin (-9.1 kcal/mol) exhibited greater within the normal limit. Statins are competitive inhibitors
binding affinity towards HMG CoA reductase, as of HMG Co-A reductase enzymes which are widely
compared to atorvastatin. The flavanon glycone prescribed in various countries for hypercholesterolemia
compounds, eriodictyol (-7.4 kcal/mol), hesperitin (-7.6 (8). Generally, statins are well tolerated (9). However,
kcal/mol) and naringenin (-7.4 kcal/mol) exhibited lower recent studies reported that the use of statins could lead

In silico screening of flavanones

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DOI : 10.5530/ctbp.2020.4s.14

to an increased risk of diabetes, possibly due to decline torsions followed by pdbqt file generation. The grid box
in insulin synthesis (10). Other side effects of statins was prepared with grid spacing at 1.0Å and grid points
include elevated hepatic transaminases and myalgia (9, at center_x = -26.052; center_y = 7.737; center_z =
11). The presence of these adverse effects could reduce 28.764; size_x = 40; size_y = 40 and size_z = 40 so that
patient compliance which lead to poor therapeutic it covers the ligand and all the binding site residues in
outcomes. Hence, new drug development is necessary to chain A of HMG Co-A reductase (Glu559, Cys561,
minimize the adverse effects and to increase patient Leu562, Ser565, Arg568, Arg590, Val683, Ser684,
compliance. Flavonoids are naturally occurring low- Asn686, Cys688, Asp690, Lys691, Lys692, Lys735,
molecular-weight polyphenolic compounds which are His752, Asn755, Asp767, Ser852, Leu853, Ala856 and
usually found in fruits and vegetables. Numerous studies Leu857) that are identified in a previous study (20). Chain
had reported that flavonoids have broad spectrum of A of HMG-CoA reductase (PDB ID: 1DQA) was selected
biological activities, including anti-inflammatory, anti- for docking as all other chains are identical. The selected
bacterial, anti-diabetic, anti-cholinesterase, antioxidant, flavanone compounds were then docked against the
human HMG CoA reductase by using AutoDockVina
hepatoprotective, anti-mutagenic, anti-carcinogenic and
1.1.2 by using all the default values (21). The results were
anti-hypercholesterolemia (12-15). Some studies showed
analyzed by binding energies and root mean square
that flavonoids such as epigallocatechin-3-gallate
deviation (RMSD) values. The estimated inhibition
(EGCG) and curcumin exhibited HMG CoA reductase
constant (Ki) was calculated for all the ligands by using
inhibitory activity in silico (16). Furthermore, some
a previously reported method by using the python script
flavanones like hesperidin, hesperitin, naringin and
available at https://github.com/virtualscreenlab/Virtual-
naringenin are shown to improve the metabolism of
Screen-Lab/blob/master/DelG_to_Kd_converter.py (22).
cholesterol in vivo. The total cholesterol level was
The resultant docking poses of the ligands and their
reduced upon administration of bergamot food extract interactions with the protein were analyzed using BIOVIA
which is rich in neoeriocitrin, naringin, neohesperidin, Discovery Studio Visualizer 4.5 (23). The drug likeliness
melitidin and brutieridin (13). Eriocitrin is also proved of all the compounds was also analyzed and the best HMG
to potentially reduce the total cholesterol level by Co-A reductase inhibitory compound was identified. The
minimizing the accumulation of lipid in liver (17). molecular properties of atorvastatin and the 9 ligands
In this study, selected flavanones (eriocitrin, were calculated online by SwissADME (24).
eriodictyol, hesperidin, hesperitin, neohesperidin, 3. Results and Discussion
naringin, naringenin and narirutin) were tested against Atorvastatin, as a positive control in this study, showed
HMG Co-A inhibitory activity in silico and their structure- binding affinity of -8.0 kcal/mol towards HMG Co-A
activity relationship was analysed. reductase (Table 2). Eriocitrin, hesperidin, neohesperidin,
2. Materials and Methods narirutin and naringin exhibited greater binding affinity
The selected flavanones (Table 1) to be tested for the compared to atorvastatin (Table 2). Among all the selected
flavanones, eriocitrin showed the highest binding energy
HMG CoA reductase inhibitory activity were eriocitrin,
which was -10.0 kcal/mol, followed by hesperidin with -
eriodictyol, hesperetin, hesperidin, naringenin, naringin,
9.7 kcal/mol (Table 2). Neohesperidin and narirutin
narirutin and neohesperidin. Atorvastatin was used as a
showed same binding energy, which was -9.5 kcal/mol,
positive control to compare the binding pose and energy
whereas the binding energy of naringin was -9.1 kcal/
of the selected flavanones. The structure of atorvastatin
mol (Table 2). Hesperitin, naringenin and eriodictyol
and the selected flavanones were downloaded from ZINC
showed lower but comparable binding energies as
database(18).The structure of human HMG CoA
compared to atorvastatin (Table 2). The binding energy
reductase with a complex with atorvastatin (PDB ID: of hesperitin was -7.6 kcal/mol, whereas naringenin and
1DQA)was downloaded from protein data bank (19). eriodictyol showed similar binding energy, which was -
AutoDockTools 1.5.6 was used for preparation of protein 7.4 kcal/mol (Table 2). The inhibition constant (Ki) of
molecule. Briefly, water molecules were removed, all the ligands in M range is given in Table 2. The
Kollman charges were added, polar hydrogens were binding interactions of atorvastatin and selected ligands
added, non-polar hydrogens were merged and pdbqt file are shown in 2D and 3D diagrams in Figures 1-6.Various
was generated. Similarly, the selected flavanones and molecular properties of all ligands are given in Table 2
atorvastatin were prepared by using AutoDock Tools 1.5.6 and the drug likeness of all flavanone compounds were
by adding polar hydrogens, accepting the proposed analyzed.

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Table 1. List of ligands with their respective ZINC ID, name, IUPAC name and chemical structures

ZINC
No. Name IUPAC Name Ligands
ID
1. 8234294 S-Eriocitrin (2S)-2-(3,4-dihydroxy-
phenyl)-5-hydroxy-7-
[(2S,3R,4S,5S,6R)-3,4,5-
trihydroxy-6-
[[(2R,3R,4R,5R,6S)-3,4,5-
trihydroxy-6-methyloxan-2-
yl]oxy-methyl]oxan-2-
yl]oxy-2,3-dihydrochromen-
4-one
2. 58117 S-Eriodictyol (2S)-2-(3,4-dihydroxy-
phenyl)-5,7-dihydroxy-2,3-
dihydrochromen-4-one

3. 39092 S-Hesperitin (2S)-5,7-dihydroxy-2-(3-

hydroxy-4-methoxyphenyl)-
2,3-dihydrochromen-4-one

4. 8143568 S-Hesperidin (2S)-5-hydroxy-2-(3-

hydroxy-4-methoxyphenyl)-
7-[(2S,3R,4S,5S,6R)-3,4,5-
trihydroxy-6-
[[(2R,3R,4R,5R,6S)-3,4,5-
trihydroxy-6-methyloxan-2-
yl]oxy-methyl]oxan-2-
yl]oxy-2,3-dihydrochromen-
4-one

In silico screening of flavanones

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5. 8234302 S- (2S)-7-[(2S,3R,4S,5S,6R)-
Neohesperidin 4,5-dihydroxy-6-(hydroxy-
methyl)-3-
[(2S,3R,4R,5R,6S)-3,4,5-
trihydroxy-6-methyloxan-2-
yl]oxyoxan-2-yl]oxy-5-
hydroxy-2-(3-hydroxy-4-
methoxyphenyl)-2,3-
dihydrochromen-4-one
6. 8234300 S-Narirutin (2S)-5-hydroxy-2-(4-
hydroxyphenyl)-7-
[(2S,3R,4S,5S,6R)-3,4,5-
trihydroxy-6-
[[(2R,3R,4R,5R,6S)-3,4,5-
trihydroxy-6-methyloxan-2-
yl]oxy-methyl]oxan-2-
yl]oxy-2,3-dihydrochromen-
4-one
7. 8143604 S-Naringin (2S)-7-[(2S,3R,4S,5S,6R)-
4,5-dihydroxy-6-(hydroxy-
methyl)-3-
[(2S,3R,4R,5R,6S)-3,4,5-
trihydroxy-6-methyloxan-2-
yl]oxyoxan-2-yl]oxy-5-
hydroxy-2-(4-
hydroxyphenyl)-2,3-
dihydrochromen-4-one

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8. 156701 S-Naringenin (2S)-5,7-dihydroxy-2-(4-

hydroxyphenyl)-2,3-
dihydrochromen-4-one

9 3920719 Atorvastatin (3R,5R)-7-[2-(4-

Fluorophenyl)-5-isopropyl-
3-phenyl-4-(phenyl-
carbamoyl)-1H-pyrrol-1-yl]-
3,5-dihydroxyheptanoic acid

Table 2. Comparison of molecular properties and affinity of the ligands

Ki
Binding
Structure Analysis using TPSA % (Inhibition
natoms Affinity
S. Lipinski’s rule of five (Ų) ABS constant,
Ligands (kcal/mol)
No.
MW
HBD HBA log P
(Da)
1. Atorvastatin 558.64 4 6 4.94 111.79 41 -8.0 70.4 1.302

2. Eriocitrin 596.53 9 15 -1.28 245.29 42 -10.0 24.4 0.043

3. Eriodictyol 288.25 4 6 1.45 107.22 21 -7.4 72.0 3.597

4. Hesperitin 302.28 3 6 1.91 96.22 22 -7.6 75.8 2.563

5. Hesperidin 610.56 8 15 -1.06 234.30 43 -9.7 28.2 0.073

6. Neohesperidin 610.56 8 15 -1.02 234.30 43 -9.5 28.2 0.102

7. Naringin 580.53 8 14 -0.87 225.06 41 -9.1 31.4 0.202

8. Naringenin 272.25 3 5 1.84 86.99 20 -7.4 80.0 3.597

9. Narirutin 580.53 8 14 -1.15 225.06 41 -9.5 31.4 0.102

Log P: octanol/water partition coefficient; TPSA: Molecular Polar Surface Area; natoms: Number of atoms;
MW: Molecular weight; nrotb: Number of rotatable bonds; HBA: Hydrogen bond acceptor; HBD: Hydrogen bond
donor; %ABS: Absorption

In silico screening of flavanones

Current Trends in Biotechnology and Pharmacy 133
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Docking of selected flavanones towards HMG Co-A

reductase Atorvastatin served as the reference standard
compound in this study to validate and compare the
binding energies of test compounds. According to the
literature, the catalytic portions of HMG Co-A reductase
are made up of amino acid residues 426-888(20, 25).
Based on the 2D diagram in Figure 1, atorvastatin forms
strong hydrogen bonds with Asn567, Arg568, Lys722,
Ser865 and Cys561, with bond lengths of <3.0Å. It also
forms weak hydrogen bonds with Ser565 and His866,
with bond lengths of >3.0Å. Based on the previous
studies, the weak van der Waals force interaction occurs
between the atorvastatin and Leu562, His752, Ser852,
Leu853, Ala856, Leu862, His869 and Tyr479 (26, 27).
Figure 2. Interaction of HMG Co-A reductase with
The amino acid residues around the binding pocket of atorvastatin on 3D diagram
atorvastatin found in this study were Asn567, Arg568,
Lys722, Ser865, Cys561, Ser565, His866, Leu562,
His752, Ser852, Leu853, Ala856, Leu862, His869 and
Tyr479 (Fig. 1) and found to be similar to the previously
reported studies (20, 26, 27). In general, the selected
flavanones showed binding interactions with all these
amino acid residues (Fig 3-6), except His752. Therefore,
the selected flavanones may share a common binding
method methodology with that of atorvastatin in HMG
Co-A reductase.

Figure 3. Interaction of HMG Co-A reductase with

eriocitrin on 2D diagram
Atorvastatin, as a control in this study showed a
binding affinity of -8.0 kcal/mol towards HMG Co-A
reductase (Table 1). The flavanone-O-glycosides
exhibited greater binding affinities than atorvastatin. They
were eriocitrin (-10.0 kcal/mol), hesperidin (-9.7 kcal/
mol), neohesperidin (-9.5 kcal/mol), narirutin (-9.5 kcal/
mol) and naringin (-9.1 kcal/mol) (Table 1). In contrast,
the aglycone flavanones exhibited lower but comparable
binding affinities with atorvastatin. They are hesperitin
(-7.6 kcal/mol), naringenin (-7.4 kcal/mol) and eriodictyol
(-7.4 kcal/mol) (Table 1). The binding affinities of each
Figure 1. Interaction of HMG Co-A reductase with
flavanone differ from one another due to chemical
atorvastatin on 2D diagram
structure differences.

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The binding energies of eriocitrin, hesperidin, naringin

and eriodictyol have been reported previously. According
to Radhakrishnan et al., eriocitrin exhibits the highest
binding energy towards HMG Co-A reductase, followed
by hesperidin, naringin and eriodictyol, which are
consistent with the results obtained in this study (28).
As in atorvastatin, all flavanones formed hydrogen
bonds with Asn567 at the oxygen atoms in heterocyclic
rings or in sugar moieties, except naringenin and
eriodictyol (Fig 2-6). Naringenin formed weak van der
Waals force with Asn567. Flavanones which interacted
with Asn567, showed greater binding affinities. Hence,
Figure 4. Interaction of HMG Co-A reductase with it is deduced that Asn567 plays an important role in the
eriocitrin on 3D diagram binding mechanism of flavanones towards HMG-CoA
reductase. Eriocitrin (Fig. 3) and hesperidin (Fig. 5) which
exhibited the highest binding affinities among all
flavanones, also formed moderate to strong hydrogen
bonds with Cys561 as in atorvastatin. It is proposed that
the formation of these hydrogen bonds further stabilizes
and enhances their bindings towards HMG Co-A
reductase.
Arg568 plays an important role in the binding of statins
towards HMG Co-A reductase (29, 30). All flavanone-
O-glycosides formed weak hydrogen bonds with Arg568
(hydrogen bond lengths > 3.0 Å), similar to a previous
study(31), except neohesperidin and naringin. The
hydrogen bond interaction between O43 of neohesperidin
Figure 5. Interaction of HMG Co-A reductase with and H21 of Arg568 is strong and mostly covalent, with a
hesperidin on 2D diagram bond length of 2.37 Å(31). In naringin, the hydrogen bond
is formed between Arg568 and the oxygen atom in
heterocyclic ring. Naringenin is the only flavanone which
formed hydrogen bond with Lys722, at a bond length of
2.66 Å. The hydrogen bond formation occurred between
O1 of naringenin and NH of Lys722.
Eriocitrin, the disaccharide derivative of eriodictyol,
consists of a dihydroxyphenyl ring substituted at position
2 of benzopyran-4-one ring. The hydroxyl groups in the
dihydroxyphenyl ring of eriocitrin might have acted as
hydrogen bond donors, which were responsible for
hydrogen bond formation with Ser852 and Cys561 in the
binding pocket of HMG Co-A reductase (Fig. 3). Besides,
the hydrogen bond formation also occurred between
Figure 6. Interaction of HMG Co-A reductase with His869 with the ketone group in the benzopyran-4-one
hesperidin on 3D diagram

In silico screening of flavanones

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ring, as well as between Asn567 and Gly542 with oxygen together through -1,2 glycosidic linkage. The hydrogen
atoms in its sugar moiety (Fig. 3). Hence, the results bonds formed between Arg568 with naringin and
suggest that the amino acid residues Asn567, Cys561, neohesperidin occurred at the oxygen atoms in their
Ser852, His869 and Gly542 are important for hydrogen phenyl rings attached to benzopyran-4-one. Hence,
bond formation between eriocitrin and HMG Co-A Arg568, Asn567 and Tyr479 are important for hydrogen
reductase (Fig. 3 and Fig. 4). bond formation between naringin and HMG Co-A
reductase.
Hesperidin differs from eriocitrin, as the hydroxyl
group (-OH) at position 4' is replaced with a methoxy Narirutin is the conformational isomer of naringin(33).
group (-OCH3), which provides steric hindrance to the The monosaccharides in narirutin are joined together
oxygen atoms and prevent hydrogen bond formation from through -1,6 glycosidic linkage. Narirutin formed
taking place. Hesperidin formed hydrogen bonds with hydrogen bonds with Asn567 at the benzopyran-4-one
Asn567 and Cys561 as observed in eriocitrin, at ring, and also with Lys474 and Gln552 at the oxygen
benzopyran-4-one ring and its sugar moiety respectively atoms in its sugar moiety. The results indicate that
(Fig. 5). Ser865 acted as hydrogen bond donor to oxygen Asn567, Lys474 and Gln552 are the amino acid residues
atom in the sugar moiety of hesperidin, in forming a in the binding pocket of narirutin in HMG Co-A reductase.
hydrogen bond. In contrast, Ser865 formed weak van der
For aglycone flavanones, hesperitin, with methoxy
Waals force with eriocitrin (Fig. 3). There is no hydrogen
group (-OCH3) substituted at C4' of the phenol ring only
bond formation with His869 and Gly542 in hesperidin,
interacted with Asn567 to form hydrogen bond. The
but weak van der Waals forces formed instead (Fig. 5).
presence of the -OCH3 group is thought to provide steric
Hence, the results suggest that the amino acid residues
hindrance to the oxygen atoms and prevent binding
Asn567, Cys561 and Ser865 are important for the
interactions. As in atorvastatin, hesperitin interacted with
hydrogen bond formation between hesperidin and HMG
Tyr479, Ser852, Leu853 and Leu862 through weak van
Co-A reductase.
der Waals force. Eriodictyol formed hydrogen bond with
Neohesperidin is a conformational isomer of Glu730, Glu782 and Asn734 at the hydroxyl groups in
hesperidin (32). The monosaccharides in neohesperidin dihydroxybenzene rings. Glu730 and Glu782 served as
are joined together through -1,2 glycosidic linkage, hydrogen bond acceptors, while Asn734 served as
whereas the monosaccharides in hesperidin are joined hydrogen bond donor. Naringenin has a phenol ring
together through -1,6 glycosidic linkage. Neohesperidin attached to the benzopyran-4-one. As in atorvastatin, it
exhibited interactions which are quite similar to that of formed a hydrogen bond with Lys722 at the ketone group
eriocitrin. Both the ligands formed hydrogen bonds with in benzopyran-4-one ring, and interacted with Asn567
Asn567, Ser852, His869 and Gly542. As in atorvastatin, through weak Van der Waals force. It also formed
Arg568 acted as hydrogen bond donor in neohesperidin, hydrogen bond with Glu719 at the hydroxyl group in
forming strong and most likely a covalent hydrogen bond benzopyran-4-one ring.
with its 3'-hydroxyl group. All other flavanone-O- Drug likeness analysis
glycosides formed weak hydrogen bonds with Arg568,
Lipinski's rules of 5 (Lo5) is used to evaluate the drug
except naringin. Hence, the results suggest that the amino
likeliness and pharmacokinetics (ADME - absorption,
acid residues Asn567, Ser852, Arg568, His869 and
distribution, metabolism and excretion) of drug
Gly542 are important for the formation of hydrogen bonds
substances, as well as to determine whether the drug is
between neohesperidin and HMG Co-A reductase.
biologically active. The components of Lo5 include (a)
Naringin, which has a phenol group attached to the molecule with molecular weight less than 500 Dalton,
benzopyran-4-one ring, formed hydrogen bonds with (b) no more than 5 hydrogen bond donors, (c) no more
Asn567, Arg568 and Tyr479. Both naringin and than 10 hydrogen bond acceptors and (d) octanol-water
neohesperidin have disaccharides which are joined partition coefficient log P is not greater than 5(34-36).

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Any drug substance which violates more than one of the 225.06 Å2), whereas the TPSA of all aglycone flavanones
Lipinski's rules is said to possess poor solubility, are less than 140 Å2 (eriodictyol = 107.22 Å2, hesperitin
absorption and permeability (34-36). = 96.22 Å 2 , naringenin = 86.99 Å 2 ). Therefore,
eriodictyol, hesperitin and naringenin are expected to
Based on the results obtained in Table 2, the molecular
have good permeability across the intestinal membrane.
weight of atorvastatin is more than 500 Da. It also has 4
However, these theoretical predictions need to be
hydrogen bond donors, 6 hydrogen bond acceptors and a
correlated with actual data as contrasting data has been
log P value of <5. Since it does not violate more than one
reported on atorvastatin's intestinal absorption and
Lo5, it is assumed to have good solubility and
bioavailability (43).
permeability. In contrast, all flavanone-O-glycosides
(eriocitrin, hesperidin, neohesperidin, naringin and TPSA can also be used to calculate the percentage of
narirutin) which exhibited greater binding affinities than intestinal absorption (%ABS) by %ABS = 109 - [0.345
atorvastatin have violated more than one of the Lipinski's x topological polar surface area (TPSA)], according to
rules. Their log P values were in accordance with Lo5, the method of Zhao et al (37, 44). Compounds, which
but they have molecular weights of >500 Da, more than have high TPSA values are expected to have low
5 hydrogen donors and more than 10 hydrogen acceptors. absorption (%ABS). Hence, based on the results obtained,
However, Lo5 is not applicable to substrates transported all flavanone-O-glycosides are expected to have poor
through active transporters (36). Hence, further in vitro intestinal absorption, whereas all aglycone flavanonesare
studies should be carried out in the future to investigate expected to have great intestinal absorption (eriodictyol
the drug-likeliness of flavanone-O-glycosides and their = 72.0%, hesperitin = 75.8%, naringenin = 80.0%), owing
transport mechanisms in vivo. to their good permeability across the intestinal membrane.
On the other hand, aglycone flavanones which 4. Conclusion
exhibited lower but comparable binding affinities have
Based on the findings of this study, all flavanone
molecular weights of <500Da, which are 288.25Da,
glycosides (eriocitrin, hesperidin, neohesperidin, narirutin
302.28Da, 272.25Da for eriocitrin, hesperitin and
and naringin) exhibited higher binding affinities towards
naringenin respectively. Furthermore, they have < 5
HMG Co-A reductase when compared to atorvastatin with
hydrogen bond donors, < 10 hydrogen bond acceptors
eriocitrin having the highest binding affinity. Eriocitrin
and octanol-water partition coefficient log P <5. Hence,
and hesperidin showed the estimated inhibition constant
the aglycone flavanonesobey the Lo5, which indicates
(Ki) in nanomolar range while other compounds showed
that they are drug-like substances, possess desirable
in micromolar range. Drug likeness analysis indicated
pharmacokinetic properties and most likely biologically
that all the flavanone aglycones have favorable absorption
active.
property when compared with flavanone glycosides.
The topological polar surface area (TPSA) refers to Further in vitro and in vivo studies are required to analyze
the surface of polar atoms, which correlates with the the correlation of these in silico findings.
passive molecular transport across membranes. TPSA is
Conflict of interest
often used to predict intestinal absorption and penetration
through blood brain barrier(37, 38). A TPSA of <60Å2 The authors declare that they have no conflict of interest.
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Gamification Technique to Estimate Mini Mental State Examination Scores :

A Validation Study
Muhammad Junaid Farrukh1,2, Mohd Makmor Bakry1*, Ernieda Hatah1, Tan Hui Jan3
1Faculty of Pharmacy, University Kebangsaan Malaysia, Kuala Lumpur, Malaysia.
2Faculty of Pharmaceutical Sciences, UCSI University, Kuala Lumpur, Malaysia.
3Faculty of Medicine, Pusat Perubatan Universiti Kebangsaan Malaysia (PPUKM).

*Corresponding author : mohdclinpharm@ukm.edu.my, junaid@ucsiuniversity.edu.my

Abstract suppressing neuronal excitability or enhancing inhibitory

Current cognitive screening methods are less neurotransmission (2). Thus, the main cognitive effects
interactive, costly, time consuming and require trained of AEDs are impaired attention, vigilance, and
psychomotor speed. Standard cognitive assessment tools
staff to perform the task. The goal of this study was to
such as the Montreal Cognitive Assessment (MoCA), and
demonstrate the validity of a freely available game-based
the Mini-Mental State Examination (MMSE) are used in
instrument for the self-assessment of cognitive function.
healthcare settings (3,4). The Bahasa Malaysia version
We conducted a cross-sectional observational clinical
of the MoCA (MoCA-BM), a validated translated version
study on 47 participants who were 18 years or older,
of the MoCA, is also available for cognitive assessment
diagnosed with neuromedical illnesses, and without
among Malaysian communities (5). These screening tools
physical and psychiatric illness. The result showed that a
are paper-and-pencil based and require administration by
total, 25 females and 22 males between the ages of 18 a trained healthcare professional. However, difficulties
and 78 years were included. Our assessment tools included in completing the MoCA have been reported due to the
the MMSE conducted and scored by physicians and the inability of some patients to hold a pencil (6). A similar
Holey Moley freely available game. Participants received issue was also reported for the MMSE, particularly for
instructions and brief practice prior to the assessment. the writing and drawing tasks (7). The Early Dementia
The actual assessment was conducted after the hands-on Questionnaire (EDQ) is a promising alternative to the
practice, and MMSE and game scores were recorded. MMSE for screening of cognitive assessment in primary
MMSE scores ranged from 9-30 with 12 participants care (8).
classified as having impaired cognition. The Holey Moley
Current cognitive screening methods are minimally
game scores ranged from 7-113. Our experiment results
interactive, providing patients with less motivation to
showed a normalised root mean square error of 8.1%
complete the assessment (9). Software suites such as
between the actual and estimated MMSE scores. There CogTest, the Cambridge Neuropsychological Test
was a significant positive correlation between MMSE and Automated Battery, Oxford's Cancellation Tools, Cognifit,
game score (r= 0.92, P <0.01). The feely available Holey and Lumosity offer computerised versions of traditional
Moley game is a promising instrument for cognitive cognitive tests (10-14). These tools can also be utilised
screening in clinical settings. This work demonstrates the for brain training and to improve cognitive function.
feasibility of utilising games for cognitive screening in a Additionally, WESIHAT 2.0 is a suitable tool for
health care environment. educating elderly people regarding lifestyle modification
Key words : Cognitive screening, Mobile games, approaches to aid in slowing the progression of cognitive
Cognitive screening tools, Feasibility decline (15). Validation issues may arise when
transitioning the test to a computer medium, and there is
1. Introduction a potential for lack of motivation while executing
Cognition refers to high-order processes, primarily moderately boring tasks on a computer. Accurate
involving the cortical structures of the brain, that program screening of cognitive function can aid in differentiating
adaptive behaviour, solve problems, memorise between age-related and abnormal cognitive decline (16).
information, and focus attention (1). Antiepileptic drugs Moreover, conventional screening methods are costly,
(AEDs) are known to affect cognitive function by time consuming and require highly skilled staff (17).

A validation study using gamification technique

Current Trends in Biotechnology and Pharmacy 141
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DOI : 10.5530/ctbp.2020.4s.15

To make cognitive assessment more enjoyable and Sample size

entertaining, gamification can be used to improve user A total of 47 participants were included, which was
experience and engagement (18). Games can be higher than the estimated sample size. The minimum
entertaining, helping the user to relax cognitively and estimated sample size, calculated based on a moderate
mentally (19). Additionally, these games can offer valid correlation at r = 0.5 with of 0.05 and study power of
cognitive assessment without loss of predictive validity 80%, was 29 participants (29).
in a cost-effective manner (20). Several games have been
Assessment tools
specifically developed for use in health care, such as to
manage juvenile diabetes, asthma, and depression (21- Our assessment tools included the MMSE, Holey
24). Another study reported the use of a technique Moley freely available game, and a 10-inch mobile tablet.
involving a game that focuses on improving cognitive Game selection
function (25). Games allow more effective monitoring Holey Moley, a mole whacking game offered by
of cognitive status and allow changes in cognitive status Refresh Creations and developed by Ryan Carso (30) was
to be detected more quickly (26). A Canadian study selected because it is free to use on Android and iOS
developed a game intended for cognitive assessment, operating systems, and comparable to the Whack-a-Mole
"Whack-A-Mole", that showed significant correlation game used by Tong et al. for cognitive assessment (26).
with the MMSE (27). These results cannot be generalised
The Whack-a-Mole and Holey Moley game differ in
as the study included only elderly patients (70 years or
target and distractor characters. The comparison between
older). Similarly, a study by Manera et al. using a cooking
the game variants are summarised in Table 1.
game was also performed on a very focused population,
including only patients with dementia (28). Thus, Table 1: Summary of comparison between game variants.
Parameters Games
validated game-like screening tools that can be completed
Whack-a-Mole Holey Moley
rapidly and independently by a broad range of adults with
Target Mole and Squirrel Mole and Mole with Hat
varying cognitive abilities are warranted. The goal of this Distractor Rabbit, Butterfly, Blue Mole Bomb
study was to validate a game-based cognitive assessment Game 60 sec 60 sec
delivered on tablet technology to a clinical sample of duration

patients with neuromedical illnesses.

Based on a literature review (20,26-27) , a checklist
2. Materials and Methods was created to compare the Holey Moley game with
different assessment methods such as the MMSE, the
Study population and sampling method
MoCa, the Whack-a-Mole game, Lumosity, and Cognifit.
The patients were recruited from the neurology clinic The parameters compared were focus, speed, memory,
at the tertiary care hospital in Malaysia. Data was problem solving, coordination, and language. Time
collected using simple random sampling. List of patients needed to complete the task and cost were also compared.
was obtained from the clinical appointment record and A panel of 5 experts, consisting of lecturers and healthcare
the patients were randomly called in the physicians room. practitioners, reviewed the checklist and compared
Patients were recruited based on the following criteria. assessments. Their evaluations were similar. Based on
Inclusion criteria their scoring, the Holey Moley game was chosen as it is
free, time efficient, and covers the majority of parameters
Patients with epilepsy, Parkinson's Disease, stroke and
(focus, speed, memory, coordination) in cognitive
Alzheimer's disease, who were 18 years or older and
assessment. The expert panel's scoring of the checklist is
taking neurological related medications for at-least 6
summarised in Table 2.
months,
Ethical considerations
Exclusion criteria
Ethical approval was obtained from the ethical
Patients with physical or psychiatric illness such as
committee of Universiti Kebangsaan Malaysia Medical
schizophrenia and major depression, critically ill patients
Centre, reference no. UKM PPI/111/8/JEP-2017-138.
and those who refused to participate in this study and
Written informed consent was obtained from all patients
patients not fit to be interviewed determined by the
before participation in the study. Patients who refused to
physicians were excluded.
participate in this study were excluded.

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DOI : 10.5530/ctbp.2020.4s.15

Table 2 : Checklist for the comparison of cognitive assessment methods.

Items Parameters
Assessment Time Cost Focus Speed Memory Problem Coordination Language
Needed Solving
Methods
MMSE 10 min $1.6/test 5/5 0/5 5/5 5/5 0/5 5/5
MoCA 10-15 min Free 5/5 0/5 5/5 5/5 0/5 5/5
Whack-A- 60 sec N/A 5/5 5/5 5/5 0/5 5/5 0/5
Mole game
Holey Moley 60 sec Free 5/5 5/5 5/5 0/5 5/5 0/5
Lumosity >10 min $15/ 5/5 5/5 5/5 0/5 5/5 0/5
month
Cognifit >10 min $19.95/ 5/5 5/5 5/5 5/5 5/5 0/5
month
*Value in table is number of people in agreement/total no of people in panel
Data collection
scores ranged from 9 to 30, with 12 participants classified
Data collection included age, sex, race, education,
as having impaired cognitive function. The Holey Moley
comorbidities, MMSE score, and game score. The MMSE game scores ranged from 7 to 113. The socio-
was conducted and scored by physicians who were demographic characteristics of participants and game
blinded to game score. After completion of the MMSE, scores are listed in Table 3.
researchers explained the graphical interface of the game Table 3 : Socio-demographic characteristics and game scores
and gave instructions to the participants on how to play
Item Descriptive Game Score Stat
the game. Participants were given time to interact with
Value Mean (SD) (p-value)
the tablet, involving hands-on practice for up to 5 minutes
to verify if they had any difficulty in understanding the
Age, year (SD) 47.1 (15.2) (<0.01)a
graphical and/or textual information provided on the
screen. The actual assessment was conducted for 1 minute
after the hands-on practice. Participant scores on the Sex, n (%)

MMSE, and the Holey Moley game were recorded for Male 22 (46.8) 78.3 (25.7) (0.493)b
correlation analyses and validation. Female 25 (53.2) 72.2 (33.)
Race, n (%)
Statistical analyses
Malay 19 (40.4) 67.4 (31.4) (0.170)c
Statistical analyses were performed using the IBM
Chinese 19 (40.4) 75.4 (32.1)
SPSS Statistics Version 23. Frequencies and percentages
Indian 4 (19.1) 90.4 (15.4)
were used for categorical variables, and descriptive
Education Level, n (%)
statistics, including mean and standard deviation (SD),
No formal education 1 (2.1) 50.0 (<0.01)c
were used for continuous variables describing the study
Primary School 11 (23.4) 39.6 (33.3)
population. The MMSE cut-off score for cognitive
Secondary School 21 (44.7) 80.4 (19.2)
impairment is 23 (31). The game cut-off score for
cognitive impairment was determined by ROC curve, and Diploma 13(27.7) 96.6 (11.4) (<0.01)d

sensitivity and specificity tests. The trade-off value from Post-graduation 1 (2.1) 98.0

the ROC curve was used to categorise the game values Marital Status, n (%)
into cognitive impairment, and normal cognition. The root Single 8 (17) 79.3 (23.9) (0.276)c
mean-square error (RMSE) and normalised RMSE Married 38 (80.9) 75.4 (30.9)
(NRMSE) were used to evaluate the estimation Divorced 1 (2.1) 28
performance. High R2, eg R2 > 0.6 and RMSE less than Co-morbidities, (0.345)a
10% ensures the model fits the data well (32). Mean of n comorbidities 1.1 (1.1)
(SD)
3. Results and Discussion
a=Pearson's correlation, b =Independent t-test, c = One-way
In total, 25 females and 22 males between the ages of
18 and 78 years were included in the study. The MMSE ANOVA, d= Spearman's correlation

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DOI : 10.5530/ctbp.2020.4s.15

There was a significant negative correlation between optimization of the Holey Moley game in predicting
age and game score (r = -0.43a, p<0.01) indicating that cognitive impairment is summarized
with increasing age, game scores decreased. Additionally,
there were no significant differences in game score
between sexes. The majority of participants were Malay
(n = 19), and Chinese (n = 19), and the remaining
participants were Indian (n = 9). However, no significant
differences in mean game score were found among races.
Participants represented a variety of education levels (No
formal education = 1, Primary School = 11, Secondary
School = 21, Diploma = 13, Post-graduation = 1). There
was a significant correlation between game score and
level of education (r= 0.674, p<0.01). Number of
comorbidities varied among participants (range 0-4), and
there was a negative correlation between comorbidity and
game score (r = -0.141, p =0.345).

Predicting cognitive status using mobile game score

The Holey Moley game was then validated against
the MMSE, which is a gold standard for cognitive
assessment. The game cut-off score for cognitive
impairment was 63, which was determined by ROC curve
(figure 1). A sensitivity and specificity test was performed Fig. 1 : Area under the ROC curve for the validation of
to ensure reliability as shown in table 4. Validation and game scores

Table 4 : Mobile game and MMSE sensitivity and specificity cross tabulation
Mobile Game and MMSE Cross tabulation
MMSE Total
Cognitive Normal
impairment cognition
Count 11 1 12
Mobile Cognitive % within MMSE 100% 2.8% 25.5%
Game impairment
Count 0 35 35
Normal % within MMSE 0% 97.2% 74.5%
cognition

Count 11 36 47
Total % within MMSE 100% 100% 100%

Table 5 : Validation and optimization of the Holey Moley game in predicting cognitive impairment

Model n Trade-off Sensitivity Specificity AUC 95% CI P-value

value (%) (%) ROC

Validation 47 63.5 100 97.2% 0.953 0.894 1.0 <0.01

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DOI : 10.5530/ctbp.2020.4s.15

The RMSE between the actual MMSE scores In this study, participants came from different
administered by physicians and the estimated scores based educational backgrounds, and game score correlated
on mobile game was 1.8. The normalised RMSE was significantly with level of education. Participants with
computed by dividing the RMSE by the value range of low levels of education performed poorly on the game
actual MMSE score (i.e., 30-9+1 = 22), was 8.1 %, despite having good cognitive status as assessed by
showing that the proposed system could yield an accurate MMSE. These findings demonstrate that patient
evaluation of MMSE (33). There was a significant characteristics, such as younger age and good educational
positive correlation between MMSE and game scores background, are able to be tested using mobile devices.
(Pearson's correlation r= 0.92, P <0.01) (Figure 2). This Number of comorbidities varied among patients (range
indicates that our game-specific variables can capture the 0-4), and there was a significant negative correlation
varying degrees of cognitive functions measured by the between comorbidities and game score. It is already
MMSE. Based on MMSE scores, there were 12 proven that physiological properties of comorbidities can
participants with impaired cognitive function. MMSE reduce one's cognitive function (35). This reflects that
scores ranged from 9 to 30. patient's comorbidities can impair cognitive function.
The correlation of the Holey Moley game score with
existing methods of clinical cognitive assessment (i.e.
MMSE) is strong and may be useful in the detection of
cognitive impairment. High R2, eg R2 > 0.6 and RMSE
less than 10% ensures the model fits the data well. Thus,
game-based assessment is a promising instrument for
cognitive screening in clinical settings after proper
validation. Our findings demonstrate that games can
potentially revolutionise cognitive assessment in clinical
settings, allowing for more frequent, affordable, and
enjoyable assessments. Ideally, a suitably modified
Fig. 2 : Correlation between MMSE scores and game mobile game would be able to detect risk of cognitive
scores impairment, and disease-related deterioration. Since the
game-based assessment can be delivered independently,
patients may be able to self-monitor. The game
The goal of this study was to demonstrate the
performance provided to healthcare providers may lead
feasibility of a game-based cognitive assessment
to appropriate interventions and/or investigations to
delivered on tablet technology that can be self-
ensure optimal treatment care.
administered without the supervision of trained staff
against standard mental status assessment tools. The Limitations
Holey Moley game was selected based on its quick and Further research is needed to generalise these results
user-friendly interface, time efficiency (60 seconds), and to different clinical conditions and settings. The design
cost effectiveness. It was then validated against the of this study was cross-sectional, each participant was
MMSE. only studied during their clinic visit, and played the game
Our findings showed that there was a significant only once. Future research may assess the reliability of
negative correlation between age and game score, the game when played repeatedly by the same patient in
indicating that with increasing age, game score decreased. clinic during follow-up to investigate the effects of prior
These findings are similar to a study which reported that, exposure.
in general, older adults were less likely to use technology
4. Conclusion
than younger adults. The relationship between age and
adoption of technology was mediated by cognitive The freely available Holey Moley game is a promising
abilities, computer self-efficacy, and computer anxiety instrument for cognitive screening in clinical settings.
(34). This could be due to physiological decline in This work demonstrates the validity of games for
cognitive function, or lack of interest in mobile games cognitive screening that can be self-administered with
among elderly participants. minimal supervision from trained staff.

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A validation study using gamification technique

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DOI : 10.5530/ctbp.2020.4s.16

Risk Assessment of Sleep Apnoea and Quality Of Sleep Among

General Public in Klang Valley
Muhammad Qamar1, Leong Mun Yee2, Muhammad Ahsan Iftikhar Baig2,
Muhammad Haseeb Tariq3, Muhammad Junaid Farrukh2 *
1Department of Clinical Pharmacy, MAHSA University, 42610 Jenjarom, Selangor, Malaysia.
2Department of Clinical Pharmacy, UCSI University, 56000 Cheras, Wilayah Persekutuan Kuala Lumpur, Malaysia.
3Department of Clinical Pharmacy, School of Pharmaceutical Sciences, UniversitiSains Malaysia, Penang, Malaysia

*Corresponding autor : junaid@ucsiuniversity.edu.my

Abstract sleepers over a period of time. Study findings will help

Untreated Obstructive Sleep Apnoea (OSA) OSA can healthcare providers and policymakers to educate and
lead to various health complications and increase the risk spread awareness about OSA among the public. This will
of an automotive accident. It is believed that there is a be beneficial in the early diagnosis and treatment of OSA
significant correlation between OSA and quality of sleep. before it complicates to other co-morbidities.
individuals with OSA should be diagnosed and treated as Key words : Obstructive sleep apnoea (OSA); Quality
early as possible to improve their quality of life of sleep; Berlin questionnaire; PSQI Questionnaire;
Malaysia.
Thus, this study aimed to identify the level of risk of OSA
and quality of sleep among the general public in Klang 1. Introduction
Valley, Malaysia and to find out the association, difference
Obstructive Sleep Apnoea (OSA) is a common sleep-
and correlation between them. A cross-sectional study was
related breathing disorder, characterizedby a repetitive
carried out among 420 respondents who were recruited
episode of partial or complete upper airway obstruction
through convenience sampling from shopping malls in
during sleep, despite the persistent effort to breath (1,2).
Klang Valley, Malaysia.Participants aged 18 years and
World Health Organization (WHO) declared that OSA
above who agreed to participate in the survey were
has influenced more than 100 million people worldwide
included in the study. Self-administered established
and is reported to be highly prevalent (3). The prevalence
questionnaires were used to assessOSA and Sleep quality.
of OSA is reported from as low as 15% in Singapore to
Data were analyzed using descriptive and inferential as much as 78% in Brazil (3,4). The prevalence of OSAin
analysis.Mean (SD) age of the respondents was 38.52 community-dwelling adults aged between 30 - 70 years
(14.19) and 40% of them aged between 30 and 49. The in Malaysia is determined to be 8.8% and 5.1% in males
majority of the study population were at low risk of having and females respectively. These results obtained in
OSA (81.7%) and were poor sleepers (65.5%). It was Malaysia are almost twice as compared to those reported
found that gender, age categories, BMI categories, by Young et al.(5).
education level, employment status, co-morbidities and
OSA is typically asymptomatic in its initial
smoking were significantly associated with OSA (p<0.05)
stages;therefore, the patients remain unaware unless
and there was a significant difference in the mean global
noticed by the family members (6). The actual signs and
PSQI score (p<0.05) between the races, age categories,
symptoms appear in the later course of the disease when
and religions. The risk of OSA was significantly
abnormal breathing pattern is observed by the family
associated with quality of sleep (p=0.011, contingency
members and suspected by the physician on routine check-
coefficient=0.123) where poor sleepers (21.8%) were at
up (7). The substantial signs and symptoms of OSA
higher risk of OSA. OSA was significantly correlated with
include heavy snoring, frequent nocturnal awakenings
the quality of sleep (r=0.124, p=0.011).The majority of
leading to fragmented sleep, impaired quality of sleep
the study population was at a low risk of OSA. However,
triggering excessive daytime sleepiness (2,8). Prolonged
individuals at high-risk OSA were found to have poor
untreated OSA is associated with significant morbidity
sleep quality. Therefore, OSA may develop in poor
comprising cardiovascular, metabolic, and neurocognitive

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complications (2,8) and may result in impaired working severity of daytime sleepiness and history of high blood
performance and a higher risk of road accidents (9). pressure and obesity Respondents are considered having
An individual's Quality of Life (QOL) is highly a high risk of OSA if they scored positive for two or more
dependent on the quality and duration of sleep, therefore, categories and low risk if they scored positive for one or
changes in quality of sleep may affect an individual's QOL none of the categories among the three and Section C
(10). OSA is one of the risk factors that may contribute consisted of Pittsburgh Sleep Quality Index (PSQI)
to poor quality of sleep. Individuals with OSA should, questionnaire to assess the quality of sleep of the
therefore, be diagnosed and treated as early as possible respondents(16).It consists of nineteen self-rated
to improve their quality of life and prevent the occurrence questions used for PSQI scoring. These nineteen self-
of various complications and risks for a road traffic rated questions are combined to yield seven component
accident. scores;each of them ranges between zero to three
points.All the seven component scores are then added up
Few studies have been carried out in Malaysia,
to obtain the global PSQI score, which ranges between 0
reporting the prevalence of OSA among community-
to 21 points. For the results, the global PSQI score that is
dwelling adults and bus drivers (11,12) and quality of
lower than five indicates that the respondent is a good
sleep among medical students (13). None of the
sleeper while greater than five indicates that the
studiesdetermined the association of OSA and quality of
respondent is a poor sleeper. Higher points indicate the
sleep in Malaysia. Therefore, this study was aimed to
worse quality of sleep experienced by the respondent.
determine the risk of sleep apnea, quality of sleep and
Permission to use Berlin and PSQI questionnaires were
evaluate the association between them.
taken from both the authors.
2. Materials and Methods
Data collection and ethics
Study design and setting Respondents were explained about the purpose of this
A cross-sectional survey was done on 420 participants. study and written informed consent was obtained before
They were recruited conveniently from different shopping the survey begins. Ethical approval was taken from the
malls in KlangValley, Malaysia. Those participants who Research Management Centre (RMC), MAHSA
were Malaysian, 18 years or older and agreed to University. Patients who refused to participate in this
participate in the survey were included. study were excluded.
Sample size Data analysis
According to the Department of Statistics Malaysia, Statistical analyses were performed using the IBM
the population size of Klang Valley, Malaysia is SPSS Statistics Version 23. The data collected were
approximately 7.9 million. The total sample size for this expressed as descriptive statistics such as frequencies,
study was 385, and it was calculated using Sample Size percentages, mean as well as inferential statistics.
Calculator by Raosoft®, Inc. with a margin of error of Categorical data were expressed as proportions, n (%)
5%, 95% confidence interval and response distribution whereas continuous data were expressed as means ±
of 50%(14).A total of 420 respondents were chosen, standard deviation (SD). The normality of the data was
approached individually and enrolled in this study. checked and determined by using the normality test. Since
Survey items the data obtained was normally distributed, parametric
A pre-validated, self-administered questionnaire was tests were used for data analysis. The tests employed were
adapted from previous studies and translated into independent sample t-test, one-way ANOVA and
BahasaMelayu and Chinese by a certified translating Pearson's correlation.
agency, MSB Venture (SA0352850-U). The The association between the risk of having OSA and
questionnaires comprised of three main sections. Section categorical data was evaluated using the chi-square test
A captured the basic socio-demographic data of the and differences with continuous variables were estimated
respondents. Section B consisted of the Berlin using independent t-test. Besides, the differences between
Questionnaire to assess the risk of having OSA(15). It global PSQI score (continuous variable) and socio-
consists of ten closed-ended questions, and they are demographic data wereassessed using independent t-test
categorizedinto three categories according to the factors and one-way ANOVA, whereas the correlation between
evaluated, such as snoring frequency and severity, the them wasexamined using Pearson's correlation.

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3. Results and Discussion

Pilot study
To check the clarity and appropriateness of the
translated questionnaire, the Malay version of the
Pittsburgh Sleep Quality Index (PSQI-M) and Berlin
Questionnaire (BQ-M) was pre-tested in a pilot study of
30 individuals. The internal consistency of the translated
PSQI-M in the pilot study, measured by Cronbach's alpha,
was 0.68 while Cronbach's alpha value for the Category
1 and 2 of BQ-M was 0.76 and 0.86 respectively.
Socio-demographic data of the respondents Figure 2: Results of Pittsburgh Quality of Sleep (PSQI)
Out of 450, 420 participants gave their consent and
voluntarily enrolled in this survey and return the of having OSA (Table 1). Significant differences were
questionnaire to the principalinvestigator;therefore, the reported for mean age (p <0.001) and mean BMI (p
response rate of this study was 93% (420/450) that <0.001) between low and high-risk OSA groups. It was
surpassed the good index of response rate. It can be reported that individuals with greater mean age
observed that most of the respondents who enrolled in (44.31±15.05 years) and greater mean BMI (29.44±5.31
this study were within the mean age 38.5 (+14.1)and the kg/m2) were at a higher risk of having OSA.
majority of them were female (59%) and Malay (43.1%). Score of Pittsburgh Sleep Quality Index (PSQI)
Most of the respondents were non-smoker (91%), non-
alcoholic (81.7%) with no co-morbidities (78.3%). The PSQI score ranged from 0 (minimum) to 15
(maximum) with a mean score of 5.56 ± 2.80. Out of
Risk of obstructive sleep apnoea (OSA) and Quality 420, 65.5% (n=275) of the respondents achieved the score
of sleep 5 and more than 5, indicates poor quality of sleep while
The result of berlin questionnaireshows that 18.3% 34.5% (n=145) attained the score less than 5 which
(n=77) of the general public was at high-risk for OSA suggestive of good quality of sleep (Table 2).
(Figure 1). While results from PSQI, 65% (n=275) was Table 2 : Scores of Pittsburgh Sleep Quality Index
identified as poor sleepers (Figure 2).
Pittsburgh Sleep Quality Index Score Frequency (%)
Total PSQI score
Minimum 0
Maximum 18
Mean (SD) 5.86 ± 2.80
Good <5 145 (34.5)
Poor 275 (65.5)

Differences between the socio-demographic factors and

PSQI score
There were significant differences in the mean PSQI
score between age categories (p=0.038), race (p=<0.001),
Figure 1 : Prevalence of risk of OSA
and religion (p=0.017) as shown in Table 3.
Association and differences of risk of obstructive sleep
apnoea (OSA) across the various socio-demographic Correlation between sleep quality with age and BMI
characteristics of the respondents Regarding the correlation between the quality of sleep
It has been observed that there were statically with age and BMI, there was a significant weak negative
significant association between gender (p=0.001), age correlation between the age and mean global PSQI score
categories(p=0.001), BMI categories (p<0.001), (r= -0.113, p=0.016) (Table 4).
education levels (p=0.015), employment status (p=0.013), OSA risk and quality of sleep
co-morbidities (p <0.001), smoking (p=0.042)with risk
On univariable analysis to determine the association

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DOI : 10.5530/ctbp.2020.4s.16

Table 1: Association and difference between risk of OSA across the various socio-demographic characteristics of the
respondents (n=420)
Variables Risk of having OSA p-value
Low risk High risk
Gender n (%) 0.001a
Male 127 (73.8) 45 (26.2)
Female 216 (87.1) 32 (12.9)

Age Mean (+SD) 37.22 (+13.68) 44.31 (+15.05) <0.001b

Age categories (years) n (%) 0.001a
18-29 126 (87.5) 18 (12.5)
30-49 141 (83.9) 27 (16.1)
50-64 64 (73.6) 23 (26.4)
>65 9 (42.9)
12 (57.1)
BMI (kg/m2) Mean (+SD) 24.11 (+4.13) 29.44 (+5.31) <0.001b
BMI categories n (%) <0.001a
21 (100.0) 0 (0.0)
Underweight 185 (92.0) 16 (8.0)
Normal 103 (82.4) 22 (17.6)
Overweight 37 (58.7)
Obese 26 (41.3)
Race n (%) 0.261a
153 (84.5) 28 (15.5)
Malay 145 (81.5) 33 (18.5)
Chinese 44 (73.3) 16 (26.7)
Indian 0 (0.0)
Kadazan 1 (100.0)
Religion n (%) 0.318a
154 (85.1) 27 (14.9)
Muslim 35 (72.9) 13 (27.1)
Hindu 28 (73.7) 10 (26.3)
Christian 122 (82.4) 26 (17.6)
Buddhist 1 (100.0) 0 (0.0)
Sikh 1 (25.0)
Freethinker 3 (75.0)
Marital status n (%) 0.053a
151 (86.3) 24 (13.7)
Single 187 (77.9) 53 (22.1)
Married 5 (100.0) 0 (0.0)
Divorced
Education n (%) 0.015a
No formal education 1 (100.0) 0 (0.0)
Primary school 8 (61.5) 5 (38.5)
Secondary school 82 (75.2) 27 (24.8)
Diploma 81 (77.1) 24 (22.9)
Bachelors 142 (88.2) 19 (11.8)
Masters 26 (92.9) 2 (7.1)
PhD 0 (0.0)
3 (100.0)

Employment status n (%) 0.013a

226 (81.0) 53 (19.0)
Employed 16 (88.9) 2 (11.1)
Unemployed 18 (62.1) 11 (37.9)
Retired 24 (80.0) 6 (20.0)
Housewife 52 (94.5) 3 (5.5)
Student 2 (22.2)
Own business 7 (77.8)

a
= Chi-square (p-value <0.05 = significant association (p-value <0.05 = significant difference) OSA: Obstructive Sleep Apnoea

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Table 3 : Differences between the socio-demographic characteristics and PSQI score
Variables PSQI Score
Mean (+SD) 95% CI p-value
Gender 0.449a
5.78 (2.89)
Male -0.68 – 0.40
Female 5.92 (2.75)
Age category (years) 0.038c
6.41 (2.76) 5.96 – 6.86
18 – 29 5.63 (3.04) 5.16 – 6.09
30 – 49 5.48 (2.31) 4.99 – 5.98
50 – 64 5.67 (2.59)
> 65 4.49 – 6.85
BMI category 0.881c
6.00 (2.28) 4.96 – 7.04
Underweight 5.87 (2.99) 5.45 – 6.28
Normal 5.73 (2.74) 5.24 – 6.21
Overweight
Obese 6.06 (2.42) 5.46 – 6.67
Race <0.001c
6.30 (2.91) 5.88 – 6.73
Malay 5.20 (2.51) 4.83 – 5.57
Chinese 6.55 (2.93) 5.79 – 7.31
Indian
Kadazan 4.00 (-) -
Religion 0.017c
6.28 (2.92) 5.85 – 6.71
Muslim 6.23 (3.05) 5.34 – 7.12
Hindu 6.05 (2.25) 5.31 – 6.79
Christian 5.22 (2.64) 4.79 – 5.64
Buddhist 7.00 (-)
Sikh 2.03 – 7.47
Freethinker

Marital status 0.060c

6.23 (2.89) 5.80 – 6.66
Single 5.59 (2.70) 5.25 – 5.93
Married 1.90 – 11.30
Divorced 6.60 (3.78)
Employment status 0.133c
5.70 (2.86) 5.36 – 6.04
Employed 6.28 (1.93) 5.32 – 7.24
Unemployed 5.21 (2.21) 4.37 – 6.05
Retired 6.20 (2.46) 5.28 – 7.12
Housewife 6.71 (2.98) 5.90 – 7.51
Student
Own business 6.11 (3.48) 3.44 – 8.79

Gross monthly income 0.191c

6.32 (2.76) 5.85 – 6.79
< RM 1000 5.81 (3.02) 5.17 – 6.46
RM 1000 – 2999 5.60 (2.91) 5.04 – 6.16
RM 3000 – 4999 5.89 (2.68) 5.15 – 6.63
RM 5000 – 6999 5.06 (2.21) 3.96 – 6.15
RM 7000 – 8999 4.25 (1.98) 2.59 – 5.91
RM 9000 – 10,000
> RM 10,000 5.79 (2.15) 4.54 – 7.03

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Co-morbidities 0.384a
6.08 (2.87) -0.36 – 0.93
Yes 5.79 (2.75)
No
Smoking 0.976c
5.91 (2.79) 5.63 – 6.19
Non-smoker 5.07 (2.55) 4.08 – 6.06
Light smoker 5.78 (3.23) 3.29 – 8.26
Moderate smoker 5.00 (-)
Heavy smoker -
Alcohol intake 0.063c
6.03 (2.85) 5.73 – 6.33
Non-alcoholics 5.29 (2.23) 4.76 – 5.83
Social drinker 3.13 (1.96) 1.49 – 4.76
Moderate drinker
Heavy drinker 4.00 (-) -

a = Independent t-test (p-value <0.05 = significant difference); b = Pearson's Correlation (p-value <0.05 = significant difference);
c = One-way ANOVA (p-value <0.05 = significant difference); CI= Confidence Interval

Table 4 : Correlation between sleep quality with age and Our study revealed a significant association
BMI betweenthe risk of having OSA among males (26.2%) as
Age = -0.113 0.021b compared to females (12.9%). This finding is consistent
with several studies supporting that the male gender is
significantly associated with developing the risk of
BMI = -0.018 0.710b OSA(1,2,17).A study from Iran reported that ;male
respondents had a higher risk of OSA (51.4%) as
compared to females (26.5%)(18). Similarly, males
between quality of sleep with risk of OSA. Good sleepers showed higher prevalence of OSA (12.6%, n = 95) as
(88.3%) had a low risk of OSA as compared to poor compared to females (3.3%, n = 27) in a recent North
sleepers (78.2%)(Table 5). Similarly, poor sleepers were West Adelaide Health Study (NWAHS), 2018 (19).
at higher risk of having OSA (21.8%)(p=0.011). This
Age has a linear relationship with OSA risk (20-22);
indicated that there was a significant positive correlation elderly tends to have a higher risk of OSA because of
between the variables (r=0.124, p=0.011). The correlation
reduced respiratory efficiencydue to the aging
between OSA risk and quality of sleep was expressed
processwhich increases the prevalence of OSA in the
using Phi and Cramer's V value. Both the values were older population(23).Jordan et al. proposed that the upper
found to be the same, which was 0.124.
airway dilator muscles in older individuals might not work
Table 5 : Association between OSA risk with quality of as efficiently as in the younger (2). The aged population
sleep might experience airway collapse easily due to loss of
Quality of sleep Good sleepers (<5) Poor sleepers p-value collagen or might arouse easily due to ,more mediocre
n (%) (>5)
quality of sleep (2).
Several studies have reported that obesity is one of
OSA risk n (%)
the main risk factors that contributes in the development
Low risk n (%) 128 (88.3) 215 (78.2) 0.011a of OSA (1,2,17,18). Obese participants (58.7%) were at
High risk n (%) 17 (11.7) 60 (21.8)
higher risk of having OSA as compared to non-obese in
a the present study. In a Wisconsin cohort study with a 4-
= Chi-square (p-value <0.05 = significant association)
year follow-up, the risk of developing OSA among those
who do not have OSA at the beginning was six-fold higher
The current study aimed to identify the risk of OSA
as the weight increases by 10%(23). Excessive fats
and quality of sleep through validated research tools
deposition at the neck region could increase the likelihood
among the general public. Our result showed that 18.3%
of airway obstruction, especially during supine sleep
and 65.5% of enrolled respondents had a high risk of
position with the pull of gravity(2).
OSA and poor sleep quality, respectively.
Muhammad et al
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The level of education is also significantly associated games, which may lead to irregular sleep timings.
with developing the risk of OSA. Foroughi et al. reported According to the statistics in Malaysia, 28.0% of the
that respondents with a low educational background were gamers were maleaged between 21 - 35(34).Peracchia et
at higher risk of OSA (18). Likewise,Sunwoo et al. also al. concluded that exposure to video games for a long
recorded high OSA risk among the respondents with a period, especially in the evening, can significantly cause
middle or low level of education (OR=1.60; 95% CI, 1.24 sleep problems to arise and subsequently leads to poor
- 2.07), (24). Individuals with a higher level of education quality of sleep(35).Correlation analysis showed a weak
are more concerned about their health as compared to negative association of age with mean global PSQI score
those with a lower level of education (25). (r= -0.113, p=0.021).Therefore, the younger the age of
Individuals with self-reported co-morbidities (42.9%) an individual, the higher the mean global PSQI score
were significantly at higher risk of having OSA as would be, and thus more inferior their quality of sleep.
compared to those without co-morbidities (11.6%) Moreover, young adults aged 18 - 29 were mostly
(p=<0.001). Mild (56.2%), moderate (67.6%) and severe students in tertiary educational institutes or fresh
OSA (70.0%) OSA was observed in people with co- graduates,who are at the beginning of their careers and
morbid conditions by Pinto et al in 2016.Tveit et al. also professional life. Poor sleep quality among such a group
reported that the prevalence of some cardio-metabolic of people might be due to work stress or vice versa, as
diseases (e.g. hypertension, diabetes mellitus, obesity) indicated by Valerio et al. (p=<0.001)(36).
were higher with greater severity of OSA(27). Higher There was a significant difference in the mean PSQI
mortality risk was identified among OSA patients with score between different religions. Muslims had a higher
comorbidities as compared to those without co-morbid mean PSQI score (6.28±2.92) as compared to the non-
condition in Taiwan by Chiang et al(HR: 11.01, 95% CI Muslims. Prayer time was one of the factors affecting
4.00-30.33, p = <0.01) (28). Evidence suggests that co- the Muslim's sleep schedule(37). According to the
morbidities might cause changes in the physiological religious practice of Muslims, praying five times a day,
functions of the body systems and subsequently leads to starting with the first prayer (Fajr) during the dawn, which
OSA. is roughly estimated to be one or one and half hours before
Smoking had also shown as one of the risk factors of sunrise(38). Hence, Muslims might be experiencing a
developing the OSA. Smoking caused sleep disturbance, shorter duration of sleep as compared to individuals of
nicotine-related relaxation of the upper airway, and other religions due to the early rise for prayers,
inflammation in the upper airway because of inhalation specifically those who went to bed late.
of smoke (2,31) Our results depicted risk of OSA was Univariate analysis showed that respondents with
significantly associated with smoking. Active smokers good quality of sleep had a low risk of OSA and vice
had a high OSA risk than non-active smokers in Nigeria versa, which is in line with Sokwalla et al.study results
(OR=28.67, 95% CI, 1.43 - 576.31, p=0.028) (29). that concluded a significant association between the risk
According to the Wisconsin cohort study, there wasthree of OSA and poor quality of sleep among individuals from
times higher risk of developing OSA in current smokers Kenya. Good sleepers (70.2%) had lower risk of OSA as
as compared to the former or never smokers (30). compared to the poor sleepers (43.9%)(39) and OSA
Majority of our study participants had a poor quality affects the quality of sleep negatively(40).
of sleep which is consistent with the previous literature
Limitations
(32). Quality sleep is essentialfor all age groups,
regardless of ethnicity. Poor quality sleep was Few limitations should be considered when
significantly prevalent among Indians in the present study. interpreting the results from this study. Given that our
According to NHMS 2014, Indians were highly prevalent study is a cross-sectional study design, so the results can't
(28.1%) to be obese, which is also one of the risk factors be generalized and represent all state of Malaysia since
of OSA as discussed above, followed by Malays (22.0%) the study was conducted in 12 selected shopping malls
(33). in different areas of KlangValley. Other limitations
included the time and budget constraints. Besides, the
Younger adults aged 18 - 29 were having the highest
findings of this study may be biased in terms of memory
mean PSQI score as compared to the other age categories.
and information because the data obtained such as the
The younger generation nowadays is addicted to video
subjective sleep quality and the major variables were self-

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Current Trends in Biotechnology and Pharmacy 154
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reported. Thus, it might differ from the actual situation. 5. Young T, Peppard PE and Gottlieb DJ. (2002).
Other than that, the willingness of the respondents to Epidemiology of Obstructive Sleep Apnea. Am J
provide information in some short open-ended questions Respir Crit Care Med, 165(9):1217-39.
and the accuracy of the data should be considered because 6. Sánchez-de-la-Torre M, Campos-Rodriguez F and
they might potentially affect the study results. Last but Barbé F. (2013). Obstructive sleep apnoea and
not least, sleep measurement was based on subjective cardiovascular disease. Lancet Respir Med,1(1):61-
descriptions rather than objective assessment. There 72.
might be a memory gap between certain variables such
7. Punjabi NM. (2008). The Epidemiology of Adult
as total sleep time and sleep onset, subsequently
Obstructive Sleep Apnea. Proc Am Thorac Soc,
influencing the classification of people with good or poor
5(2):136-43.
sleep quality based on the total score of PSQI and the
results of this study. 8. Dewan NA, Nieto FJ and Somers VK. (2015).
Intermittent hypoxemia and OSA: Implications for
4. Conclusion
co-morbidities. Chest, 147(1):266-74.
The majority of the study population was at low risk
of OSA, even though most of them were poor sleepers. 9. Spicuzza L, Caruso D and Di Maria G. (2015).
However, high-risk OSA individuals were found to have Obstructive sleep apnoea syndrome and its
poor sleep quality. Therefore, OSA may develop in poor management. Ther Adv Chronic Dis, 6(5):273-85.
sleepers over some time. Study findings will help 10. Medic G, Wille M and Hemels MEH. (2017). Short-
healthcare providers, community pharmacists and and long-term health consequences of sleep
policymakers to educate and spread awareness about OSA disruption. Nature and Science of Sleep, 9, 151.
and quality of sleep among the general public. Early 11. KAMIL MA, TENG CL and HASSAN SA. (2007).
diagnosis and treatment of OSA,and managing the quality Snoring and breathing pauses during sleep in the
of sleep before it complicates to other co-morbid Malaysian population. Respirology, 12(3):375-80.
conditions is deemed necessary.
12. Mohd Yusoff MF, Baki MM and Mohamed Net al.
Acknowledgment (2010). Obstructive sleep apnea among express bus
All the authors would like to thankthe general public drivers in Malaysia: Important indicators for
for participating in this study. screening. Traffic Inj Prev, 11(6), 594-599
Funding 13. Zailinawati AH, Teng CL, Chung YC, Teow TL, Lee
The study received funding from UCSI. PN and Jagmohni KS. (2009). Daytime sleepiness
Conflict of Interest and sleep quality among Malaysian medical students.
Med J Malaysia, 64(2):108-10.
The authors declared no conflict ofinterest.
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Current Trends in Biotechnology and Pharmacy 156
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DOI : 10.5530/ctbp.2020.4s.17

Anti-Angiogenic Effect of Ethanolic Extract and its Phenolic Rich Fraction of

Acacia auriculiformis Bark in the Chick Embryo Chorioallantoic
Membrane Model
Chong Wei Chean1, Sasikala Chinnappan1*, Mogana R.1, Ashok Kumar B1
1Faculty of Pharmaceutical science, UCSI University, Kuala Lumpur, Malaysia 56000

*Email : sasikala@ucsiuniversity.edu.my

Abstract observed the limited development of tumour with no blood

Acacia auriculiformisplant is widely used in traditional vessels. The maximum diameter of avascular tumour are
medicines for treatment of various diseases. The main less than 1mm(1). Cancer cells are unable to get into the
objective of this study is to evaluate the anti-angiogenic circulation before the development of blood vessel on
effect of ethanolic extract and its phenolic rich fraction the tumour. Vascularized tumor has a higher probability
of A. auriculiformis bark in the chick embryo of metastasizing than prevascular tumor(2).Angiogenic
chorioallantoic membrane model. Dried powdered bark activators promote angiogenesis and angiogenic inhibitors
of A. auriculiformis was extracted with 70% ethanol and inhibit angiogenesis. Examples of angiogenic activators
the resultant was partitioned with hexane, ethyl acetate are vascular endothelial growth factor A (VEGFA),
and aqueous. Folin-Ciocalteu assay was used to quantify fibroblast growth factors (FGFs), platelet-derived growth
the phenolic content in fractions of A. auriculiformis bark. factor (PDGF) and epidermal growth factor (EGF).
The anti-angiogenic effect of ethanolic extract and Examples of angiogenic inhibitors are thrombospondin
phenolic rich fraction were evaluated by using in-ovo 1, angiostatin, endostatin and tumstatin. Development of
chorioallantoic membrane (CAM) model. The reduction new vessels are controlled by a balanced mixture of
in total blood vessels number in the CAM model was angiogenic activators and angiogenic inhibitors(1).
considered as positive indicator of anti-angiogenic effect. Acacia auriculiformis belongs to the family
Ethyl acetate fraction showed the highest phenolic content Fabaceae.A. auriculiformis is a multipurpose leguminous
which was 621±16.20 mg of gallic acid equivalent per tree that is important in medicinal and forestry fields.
gram of fraction. CAM treated with ethanolic extract (250 Many therapeutically active constituents are derived from
g, 500 g), ethyl acetate fraction (10 g & 50 g) and this plant(3).A. auriculiformis is used traditionally as
prednisone (250 g) showed significant reduction antimalarial medication and to treat eyes conditions &
(p<0.05) in total blood vessel (TBV) 46.4±0.89, skin diseases(4).A. auriculiformis contains carbohydrates,
36.4±2.30, 47.6±3.05, 37.6±1.82 & 37.0±2.00 compared tannins, anthocyanidins, flavonoids and saponins(3).A.
with negative control group (61.7±2.52). The anti- auriculiformis has a unique saponin due to presence of
angiogenic effect shown by the ethanolic extract and ethyl tridesmoside saponins. The saponins are Proacaciaside-
acetate fraction of A. auriculiformis might be due to the I, Proacaciaside-II, acaciamine, Acaciaside A &
presence of phenolic compound. A. auriculiformis can Acaciaside B. Acaciaside A &Acaciaside B are proved to
be a new source of anti-angiogenic agent in anticancer be responsible for the antifilarial, antimicrobial,
therapy. spermicidal activities(3).Acacia auriculiformis is chosen
Key words : Anti-angiogenesis, A. auriculiformis, as a potential candidate to provide phytoconstituents with
ethanolic extract, phenolic rich fraction, CAM assay antioxidant activity. From the current studies, the bark of
A. auriculiformis is known to have the antioxidant activity
1. Introduction due to itsphenolic content(5). The extract from the bark
Angiogenesis is the development of new blood vessel has showed quenching capacity on DPPH, ABTS, OH-,
from pre-existing capillaries.Angiogenesis is involved in O2- and NO(3).A. auriculiformis is a potential source of
tissue repair and granulation tissue formation. In healthy antioxidants which could be useful as pharmaceutical
adults, angiogenesis takes place during tissue repair and products(6).
the female reproductive cycle(1). Angiogenesis is The main objective of this study is to evaluate the anti-
essential for tumour growth and metastasis. Folkman(1) angiogenic effect of ethanolic extract and phenolic rich

Anti-angiogenic effect of Acacia auriculiformis

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fraction of A. auriculiformis bark in the chick embryo The total phenolic content of fraction was expressed
chorioallantoic membrane model. in gallic acid equivalents (GAE) using the formula(10):
2. Materials and Methods (C)( V )
A
Collection and authentication of plant materials m
The barks of Acacia auriculiformis were collected A = total phenolic content of fraction, mg/g plant extract
inCheras, Malaysia. Authentication of plant (UPM/IBS/ in GAE
UB/H23/19)was obtained from Dr. Mohd Hafizi Adzmi C =concentration of gallic acid established from the
Hanafi from the Biodiversity unit of University Putra calibration curve (mg/ml)
Malaysia (UPM). V = volume of extract in millilitre
Preparation of crude extract m = weight of dry plant extract in gram
Fresh bark was collected and cleaned with water, dried Preparation of test samples
in dark and dust-free environment. The dried bark was Negative control : 10 L of phosphate buffer solution
then powdered using a blender and 70% ethanolic extract (PBS) /pellet
was prepared by maceration method(7). About 100g of Positive control : 250 g prednisone/pellet
powered dry bark was soaked with 2 liter of 70% ethanol
Extract : 250 g/pellet and 500 g/pellet
and the mixture was stirred twice daily for 4 days. The
Phenolic rich fraction : 10 g/pellet and 50 g/pellet
extract was filtered through the filter paper and the excess
solvent was removed by using rotary evaporator. Finally, Circular pellet were cut from a piece of filter paper
supernatant solution was lyophilized and dried crude with a paper puncher(11).Circular discs were sterilized
extract was kept in airtight container(7). by exposing them under UV light for at least 5 minutes.
Preparation of phenolic rich fraction (PRF) The pellet was infused with different solution and used
5g dried ethanolic extract was dissolved in 100ml of as an implant. Prednisone, ethanolic extract & phenolic
water and sequentially fractionated in a separatory funnel rich fraction were dissolved in PBS in different
with 100 ml hexane and 100 ml ethyl acetate. The solvent concentration. Then the solution was added drop wise
in these fractions was evaporated by the rotavapor to on the pellet using the micropipette. The pellet infused
prepare the dried fractions. Extract and fractions were with solution was lightly placed on the Chorioallantoic
dissolved with 70% ethanol todetermine the phenolic membrane (CAM) using sterile forceps.
content(7). Chorioallantoic membrane (CAM) assay
Folin-Ciocalteu assay 30 fertilized fresh eggs (within 7 days postlaying) were
Folin-Ciocalteu method was used to determine the obtained from Lay Hong Sdn Bhd, Klang, Malaysia.
total phenolic content (TPC) of the above fractions. The These eggs were assigned into 6 treatment groups (n=5)
concentration of phenolic content in each fractionwas for each treatment group. Eggshell surface was cleaned
derived from a gallic acid calibration curve. To prepare a with moist tissue paper to remove any dirt. Then, eggshell
calibration curve, 20, 40, 60, 80 and 100 micrograms of surface was cleaned with 70% ethanol. Eggs were placed
gallic acid were dissolved in 1ml of 70% ethanol and horizontally on suitable egg tray. No rotation of the eggs
mixed with 5 mL of Folin-Ciocalteu reagent (10%v/v) was required throughout the experiment(11). Eggs were
respectively. These mixtures were incubated in the dark incubated at 37-degreeCelsius and 60% humidity for 2
at room temperature for 3 minutes. Then, 3.0 g of days.
anhydrous Na2CO3 in the form of 4?mL of Na2CO3
On incubation day 3, 2-3ml of albumin was removed
(7.5%w/v) was added to each mixture. These mixtures
from each egg though the acute pole of the egg using
were further incubated in the dark for 90 minutes. Then,
25G hypodermic needle and 3ml syringe. Removal of
the absorbance was read at 765 nm using double beam
albumin dissociated the CAM from the eggshell
UV spectrophotometer(8). The calibration curve was the
membrane by creating a false air sac directly over the
graph of absorbance value against the respective
CAM.On incubation day 4, a square window
concentration of gallic acid. The absorbance of the
(approximately 10 x 10 mm) was opened on the shell. A
fractions was read using a similar procedure as descried
rotating carborundum disc was used to cut the shell using
for the gallic acid. All determinations were performed in
without damage the underlying eggshell membrane.
triplicate(9).

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DOI : 10.5530/ctbp.2020.4s.17

Several drops of phosphate buffer solution were CAM assay

applied to moisten the eggshell membrane and wash away The total number of blood vesselsand percentage
the shell dust. Forceps were used to remove whole piece
inhibition are used as the main criteria for analysis of
of shell(12).The window was sealed with paper clinical
anti-angiogenic effect. Reduction of blood vessel in test
tape and eggs were returned the into the incubator(13).On
groups are significantly (P< 0.05) different compared to
day 10 of incubation, implant was placed onto the
negative control group. The 500 µg of ethanolic extract
CAM.CAMs were photographed with a camera after 24
hours. Total number of blood vessel was counted in an showed most least number of TBV (36.4±2.30) compared
area of 3x3cm on the CAM membrane. with 250 µg of ethanolic extract (46.4±0.89) and 10 µg
All the equipment, instrument and working area were & 50 µg of ethyl acetate fraction (47.6±3.05, 37.6±1.82)
cleaned with 70% ethanol before any procedure. Eggs (Graph 1 and 2). The reduction of blood vessels on the
were cleaned before incubation to remove egg outer CAM confirmed the anti-angiogenic potential of the
surface debris(13). The making of square window on the extract and fractions. Percent vascularity inhibition (PVI)
eggshell and the placing of the implantation were was calculated to determine the degree of inhibition of
performed in sterile condition (within the laminar flow blood vessel formation exerted by the A. auriculiformis
hood in the clean room). All these precautions were to extract and fraction.500µg of ethanolic extract treatment
reduce the risk of contamination. group showed highest PVI (41.0±3.73%) compared with
Percentage inhibition calculation 250 µg of ethanolic extract PVI (24.8±1.47) and 10 µg,
The total blood vessel (TBV) number was the sum of 50 µg of ethyl acetate fraction PVI (22.8±4.94, 39.1±2.94)
number of primary, secondary and tertiary blood (Fig 1).
vessels(14). The percentage inhibition was derived from
total blood vessel number.
Calculation of percentage inhibition using the
following formula (14) :
Percentage inhibition (%) = [(TBV of CAM treated by
phosphate buffer- TBV of CAM treated by extracts/
fraction)/(TBV of CAM treated by phosphate buffer)] ×
100%
Statistical analysis
All the values was recorded as mean ± standard error
of mean (S.E.M.) the data was analyzed using one-way
ANOVA with the differences of the means considered Graph 1 : Quantitation of blood vessels in treatment groups
significant at p<0.05. * Significantly different at P<0.05 by Tukey's HSD test
compared with negative control group.
3. Results and Discussions
Folin-Ciocalteu assay
Total phenolic content in different fraction of A.
auriculiformis was expressed as milligrams of gallic acid
equivalents (GAE). Ethyl acetate fraction was the highest
(621±16.20) followed by hexane fraction (398±2.89) and
aqueous fraction (267±5.69) mg of gallic acid per gram
of aqueous fraction as shown in Table 1.
Table 1: Total phenol content in mg of gallic acid
equivalent per gram of each fraction
Fractions Total phenol (mg gallic acid/g)
Aqueous 267±5.69
Graph 2 : Percentage inhibition in treatment groups
Hexane 398±2.89
* Significantly different at P<0.05 by Tukey's HSD test
Ethyl acetate 621±16.20 compared with negative control group.

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the highest phenolic content which was equivalent to

621±16.20mg of gallic acid per gram of dried fraction
powder. The ethyl acetate as the phenolic rich fraction
was having the highest potential in showing the highest
significant anti-angiogenesis activity.
The reduction of the number of blood vessels was
considered as positive indication of anti-angiogenic
potential(14). Quantitative analysis of the present study
revealed that all embryos treated with the different
concentrations of ethanolic extract and ethyl acetate
fraction of A. auriculiformis significantly inhibited the
outgrowth of new blood vessels in the chorioallantoic
membrane. The anti-angiogenic property shown might
be due to high phenol content present in extract and
fraction. Several polyphenols have reported for their
antiangiogenic effects(20).
Many studies suggest that angiogenesis is vital for
the development of solid tumors. Angiogenesis is also
involved in metastasis by allowing cancer cells to spread
from primary tumor site. The development of tumor and
Fig 1: Representative image of CAM from each treatment metastasis requires angiogenesis because the blood
groups. vessels formed provide nutrient and oxygen for the
CAM treated with negative control (A), 250µg of prednisone tumor(21). To conclude, inhibition of angiogenesis can
as positive control (B), 250µg of ethanolic extract (C), 500µg control tumor growth and prevent metastasis(14).
of ethanolic extract (D), 10µg of ethyl acetate fraction (E) &
Antiangiogenesis therapy is a promising approach to the
50µg of ethyl acetate fraction (F).
treatment for cancer(21).
Cancer causes a huge number of morbidity and Reactive oxygen species (ROS) causes many human
mortality worldwide and is predicted to be responsible diseases including cancer. ROS induces oxidative stress
for 9.6 million deaths in 2018(15). Chemotherapy is the which is involved in the cancer progression. ROS involves
mainstream choice in management of a range of cancers. in VEGF signaling pathway. VEGF is a very potent
Chemotherapy also affects all rapidly dividing normal angiogenic growth factor that functions in both in both
tissues(16). Chemotherapy can cause many side effect the physiological and pathological conditions. VEGF
like bone marrow toxicity, neurotoxicity and signaling is essential in normal vascular development and
cardiotoxicity(17, 18). Moreover, the cancer cells develop homeostasis. VEGF signaling also promotes the growth
resistance to cytotoxic drugs by mutation during cytotoxic of tumor by promoting development of tumor vasculature.
treatment. This mutation creates tumour cells that are less Exogenous ROS induces expression of VEGF in many
susceptible to the drugs(16).Epidemiological studies cell types such as endothelial cells, smooth muscle cells,
suggest diet with high number of antioxidants can reduce and macrophages. VEGF can raise the level of
the risk of cancers significantly. The intake of dietary intracellular ROS. Then, the intracellular ROS induces
antioxidants has now received further attention because endothelial cell migration and proliferation. The close
of universal acceptability based on its safety and less side relationship between ROS and angiogenesis is proven
effects potential(19).Various extracts of this plant have by extensive studies(22).
shown antioxidant benefit(6).
The main role of antioxidants is to neutralize free
Folin-Ciocalteu assay was performed to quantify the radical to stop the angiogenic effect of ROS(23).Phenolic
phenolic content of the fractions of A. auriculiformis(8). compounds can neutralize free radicals to reduce the
Folin assay reveled that phenolic compound was present pathological angiogenesis process. Several studies have
in different concentration in each fraction. The ethyl proven that organic extracts from plant with high content
acetate fraction as the phenolic rich fraction was having of phenolic compounds are having potent antioxidant

Chong et al
Current Trends in Biotechnology and Pharmacy 160
Vol. 14 (5) 156-161, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.17

activities(24).Phenolic compounds can reduce the 6. Singh, R., Singh, S., Kumar, S. and Arora, S. (2007).
generation of free radicals by reducing oxidative Evaluation of antioxidant potential of ethyl acetate
processes such as lipid peroxidation(21). The capability extract/fractions of Acacia auriculiformis A. Cunn.
of inhibiting angiogenesis shown by this extract could Food and chemical toxicology, 45(7):1216-1223.
be attributed to phenolic compound which have potent 7. Kumar, M.Y., Tirpude, R., Maheshwari, D., Bansal,
antioxidant capacity(21). A. and Misra, K. (2013). Antioxidant and
Present study proved that the ethanolic extract and antimicrobial properties of phenolic rich fraction of
ethyl acetate fraction of A. auriculiformis bark have anti- Seabuckthorn (Hippophaerhamnoides L.) leaves in
angiogenic effect on CAM model which could be due to vitro. Food chemistry, 141(4):3443-3450.
its high phenolic content.This observed vascular 8. Singleton, V.L. and Rossi, J.A. (1965). Colorimetry
inhibitory effect of A. auriculiformis suggests its anti- of total phenolics with phosphomolybdic-
cancer property(14). phosphotungstic acid reagents. American journal of
4. Conclusion Enology and Viticulture, 16(3):144-158.

The ethyl acetate fraction as the phenolic rich fraction 9. Kabir, H., Shah, M., Hossain, M.M., Kabir, M.,
was having the highest phenolic content which was Rahman, M. and Hasanat, A. (2016). Phytochemical
equivalent to 621±16.20mg of gallic acid per gram of screening, Antioxidant, Thrombolytic, ?-amylase
dried fraction powder. Ethanolic extract of A. inhibition and cytotoxic activities of ethanol extract
auriculiformis and its ethyl acetate fraction showed of Steudneracolocasiifolia K. Koch leaves. Journal
inhibition in the blood vessel formation in CAM. From of Young Pharmacists, 8(4):391-397.
the study we concluded that A. auriculiformis plant is a 10. Kabir, M.S.H., Hossain, M.M., Kabir, M.I., Ahmad,
potential source for anti-angiogenic compound. Future S., Chakrabarty, N. and Rahman, M.A. (2016).
studies are planned in the bioassay guided isolation, Antioxidant, antidiarrheal, hypoglycemic and
purification and identification of the anti-angiogenic thrombolytic activities of organic and aqueous
constituent in the ethyl acetate fraction & ethanolic extract extracts of Hopeaodorata leaves and in silico PASS
of A. auriculiformis(25). The identification of the active prediction of its isolated compounds. BMC
compounds is essential to discover the underlying complementary and alternative medicine, 16(1):474-
mechanism of anti-angiogenic activity of A.auriculiformis 483.
(24). 11. Naik, M., Brahma, P. and Dixit, M. (2018). A Cost-
5. References Effective and Efficient Chick Ex-Ovo CAM Assay
Protocol to Assess Angiogenesis. Methods and
1. Geiger, T.R. and Peeper, D.S. (2009). Metastasis
protocols, 1(2):19-27.
mechanisms. Biochimica et Biophysica Acta (BBA)-
Reviews on Cancer, 1796(2):293-308. 12. UMass Amherst Libraries. Experiments on the Chick
Embryo: Tools and Techniques: UMass Amherst
2. Klein, G.J. and Weinhouse, S. (1985). Advances in
Libraries; [updated 2019 May 9; cited 2019 Sept 1].
cancer research. Academic Press, 43:33-45.
Available from: https://www.youtube.com/user/
3. Sharma, N., Singh, S. and Singh, S.K. (2016). UMassAmherstLibrary/about.
Review on Phytopharmacological Properties of
13. Ribatti, D., Nico, B., Vacca, A. and Presta, M. (2006).
Acacia auriculiformis A. Cunn. ex. Benth. Planta
The gelatin sponge-chorioallantoic membrane assay.
Activa, 1:1-6.
Nature protocols, 1(1):85-92.
4. Girijashankar, V. (2011). Micropropagation of
14. Mamutuk, R.L. and Usman, C.M. (2017) ANTI-
multipurpose medicinal tree Acacia auriculiformis.
ANGIOGENICITY AND TERATOGENICITY OF
Journal of Medicinal Plants Research, 5(3):462-466.
HYPTIS SUAVEOLENS LEAF ETHANOLIC
5. Sathya, A. and Siddhuraju, P. (2012). Role of EXTRACT IN MALLARD DUCK (ANAS
phenolics as antioxidants, biomolecule protectors PLATYRHYNCHOS) EMBRYOS. Science
and as anti-diabetic factors-Evaluation on bark and International, 29(4):817-822.
empty pods of Acacia auriculiformis. Asian Pacific
15. Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R.L.,
journal of tropical medicine, 5(10):757-765.
Torre, L.A. and Jemal, A. (2018) Global cancer

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statistics 2018: GLOBOCAN estimates of incidence 21. Hulikere, M.M., Joshi, C.G., Ananda, D., Poyya, J.
and mortality worldwide for 36 cancers in 185 and Nivya, T. (2016). Antiangiogenic, wound healing
countries. CA: a cancer journal for clinicians, 68(6): and antioxidant activity of Cladosporium
394-424. cladosporioides (Endophytic Fungus) isolated from
seaweed (Sargassum wightii). Mycology, 7(4):203-
16. Rang, H.P., Dale, M.M., Ritter, J.M. and Flower, R.J.
211.
(2007). Cancer chemotherapy. Rang & Dale's
Pharmacology(6th edition), Churchill Livingstone., 22. Kim, Y.W. and Byzova, T.V. (2014). Oxidative stress
London, pp.718-723. in angiogenesis and vascular disease. Blood,
123(5):625-631.
17. Wonders, K.Y. and Reigle, B.S. (2009). Trastuzumab
and doxorubicin-related cardiotoxicity and the 23. Morry, J., Ngamcherdtrakul, W. and Yantasee, W.
cardioprotective role of exercise. Integrative cancer (2017). Oxidative stress in cancer and fibrosis:
therapies, 8(1):17-21. Opportunity for therapeutic intervention with
antioxidant compounds, enzymes, and nanoparticles.
18. Gaurav, K., Goel, R., Shukla, M. and Pandey, M. Redox biology, 11:240-253.
(2012). Glutamine: A novel approach to
chemotherapy-induced toxicity. Indian journal of 24. Lee, J.S.,Shukla, S., Kim, J.A. and Kim, M. (2015).
medical and paediatric oncology: official journal of Anti-angiogenic effect of Nelumbo nucifera leaf
Indian Society of Medical &Paediatric Oncology, extracts in human umbilical vein endothelial cells
33(1):13-20. with antioxidant potential. PLoS One, 10(2):1-17.
25. Habib-Martin, Z.A., Hammad, H.M., Afifi, F.U.,
19. Dai, J. and Mumper, R.J. (2010). Plant phenolics:
Zihlif, M., Al-Ameer, H.J., Saleh, M.M., Abaza, I.F.
extraction, analysis and their antioxidant and
and Nassar, Z.D. (2017). In vitro and in vivo
anticancer properties. Molecules, 15(10):7313-7352.
evaluation of the antiangiogenic activities of
20. Kamble, S.S. and Gacche, R.N. (2019). Evaluation Trigonella foenum-graecum extracts. Asian Pacific
of anti-breast cancer, anti-angiogenic and antioxidant Journal of Tropical Biomedicine, 7(8):732-739.
properties of selected medicinal plants. European
Journal of Integrative Medicine, 25:13-19.

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Current Trends in Biotechnology and Pharmacy 162
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DOI : 10.5530/ctbp.2020.4s.18

Anti-Angiogenic Effect of Ethanolic Extract and its Phenolic Rich Fraction of

Filicium decipiens in the Chick Embryo Chorioallantoic Membrane Model
Looi Kah Xin, Sasikala Chinnappan*, Mogana R, Ashok Kumar B
Faculty of Pharmaceutical Sciences, UCSI University, Kuala Lumpur, Malaysia

*Corresponding Author: sasikala@ucsiuniversity.edu.my

Abstract were observed in ethanolic extract (250µg and 500µg)

Cancer has been reported to be the 4th most common and ethyl acetate fraction (50µg and 100µg).
causes of mortality in Malaysia, in year 2018. Although Ethanolic extract and ethyl acetate fraction of FD bark
there are various cancer treatments available, side effects showed anti-angiogenic activity that may have
are always be the limitation of these treatments. Various chemotherapeutic potentials.
medicinal plants have been studied extensively for their Key words : Anti-angiogenic; Filicium decipiens; chick
anti-angiogenic activity. Moreover, natural sources are embryo chorioallantoic membrane
safer and produce lesser side effects. This study aimed to
search for alternative cancer treatment from medicinal 1. Introduction
plant, by examining anti-angiogenic activity of ethanolic Cancer has been turned up to be the 4th most common
extract and its phenolic rich fraction of Filicium decipiens causes of mortality in Malaysia in year 2018. There was
(FD)in the chick embryo chorioallantoic membrane 12.6% of cancer death reported in government hospital,
(CAM) assay. and this rate was even higher in private hospitals, which
The plant extract was prepared by maceration in 70% was evidenced by a contribution of 26.7% to the death.
ethanol andits fractions (hexane, ethyl acetate and Overall, cancer had caused 39.3% of death in Malaysia,
aqueous) were prepared from dry ethanolic extract. Total which is almost half of the death causes. To add on to the
phenolic content (TPC) of the fraction was assayed by previous point, approximately of 37,000 cancer cases are
using Folin-Ciocalteu method. CAM in-ovo method was newly diagnosed every year, and it amount will be more
used to evaluate the anti-angiogenic activity of ethanolic than 55,000, by year 2030(1).
extract of FD bark (250µg, 500µg) and its phenolic rich Angiogenesis is defined as the generation of new blood
fraction (50µg, 100µg). Prednisone (250µg) was used as vessels from its pre-existing blood capillaries via
positive control. Qualitative observation of reduction in "sprouting" of endothelial cells which expands the
the thickness of blood vessels and quantitative analysis vascular tree. It isregulated and coordinated by
in the reduction of the number of total blood vessels and variousendogenous systemic or local chemical signals,
percentage of blood vessels inhibition were measured to that are vital in regulating smooth muscle cells as well as
determine the anti-angiogenic activity of the extract and endothelial cells function for repairing damaged blood
fraction. vessels. It is a normal, yet crucial process needed by
Ethyl acetate fraction contained the highest total human body as it helps in human development,
phenolic content (349.59mg ± 0.29) than aqueous reproduction, and wound repair(2).However, angiogenesis
(123.17mg± 0.25), hexane (175.31mg ± 0.18) fractions. is also one of the contributing factors to the development
Ethanolic extract (250µg, 500µg) and ethyl acetate of cancer.
fraction (50µg, 100µg) showed significant reduction Nowadays, interest has been developed to study and
(P<0.05) in the total number of blood vessels (43, 14, 46 investigate the mode of action of various phytochemicals
and 33) compared with negative control (62). Ethanolic from different medicinal plants. Several studies have been
extract (250µg and 500µg) and ethyl acetate fraction conducted to reveal their antioxidant activity. Plants such
(50µg and 100µg) showed percentage of blood vessels as Tragopogon porrifolius, Lasiosiphonerio-
inhibition of 30.6%, 76.4%, 25.8% and 46.5% cephalusdecne, Leea indica and other medicinal plants
respectively. Reduction in the thickness of blood vessels have been studied extensively for their antioxidant

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Current Trends in Biotechnology and Pharmacy 163
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DOI : 10.5530/ctbp.2020.4s.18

activity(3,4,5). Natural products or secondary metabolites completely dried. Maceration technique was employed
such as lignans, terpenoids, coumarins, tannins, phenolic by immersing 180g of dried FD bark powder in 70%
acids, quinones, alkaloids, and flavonoids discovered ethanol, at a ratio of 1:10 for 4 days (12). After 4 days,
from medicinal plants are showing significant antioxidant the ethanolic extract was filtered through a Whatman filter
effect, which is playing very vital role to the treatment of paper, and the filtrate was collected and evaporated by
cancer (6). Phytochemicals such as polyphenolic acids, using rotary evaporator (Buchi, R-200 Switzerland).
phenolic diterpenes, tannins, and flavonoids with versatile Finally, the resulting concentrated ethanolic extract was
biological activities are the potential source of natural lyophilised using freeze dryer, and the dried ethanolic
antioxidants. Plant polyphenols are being recognized as extract was stored in airtight container for further use.
potential choice of therapeutic agents in targeting
Preparation of phenolic rich fractions
cardiovascular disease, pathological angiogenesis, and
cancer, in the next decade (7). There is strong evidence 5 g of dried ethanolic extract was weighed and
and recommendations from associated meta-analyses as dissolved in 100 ml of water, and then fractionated
well as the support from epidemiological studies that sequentially using a separatory funnel with 100 ml of
people are being protected from diseases such as diabetes, hexane and 100 ml ethyl acetate respectively. The
neurodegeneration, osteoporosis, cardiovascular diseases, resultant fractions were evaporated and concentrated. The
and cancer development if they consume plant different concentration of ethanolic extract and its
polyphenols-rich diet in a long term basis (8, 9). phenolic rich fractions were used for anti-angiogenic
study (13).
Filicium decipiens (FD) originates from the family of
Sapindaceae, and its common name in English is known Total phenolic content (TPC) assay
as fern-leaf tree, while in Malaysia, it is known by its Folin-Ciocalteu method was employed to determine
local name, Payung. FD grows in tropic zones such as and analyse total phenolic content (TPC) of the fractions
Asia and Africa, whereit can be found in areas with a obtained. 1 ml of sample solution was mixed with 5 mL
height up to 1000 meters. It is a 25-meter tall treewith of Folin-Ciocalteu reagent (diluted tenfold) and incubated
grey brown stem (10). FD had been studied for its in the dark at room temperature for 3?min. Then, 4 mL
antidiabetic, hypolipidemic, anti-inflammatory and of saturated 7.5% Na2CO3 was added to the mixture and
antioxidant activity. Its phytochemistry found such as the final volume was 10mL. The mixture was then further
sitosterol, kaempferol and quercetin raised the interest incubated for 90 minutes in the dark and its absorbance
for this study (11). Hence, this study aims to evaluate the value was taken at a wavelength of 765?nm using double
anti-angiogenic effect of ethanolic extract and its phenolic beam UV spectrophotometer. A standard curve was
rich fraction of Filicium decipiens, which may be a obtained by mixing 1 mL aliquot of 10 µg/mL, 20 µg/
beneficial finding for cancer treatment in the future. mL, 30 µg/mL, 40 µg/mL and 50 µg/mL gallic acid
solution with 5 mL of FolinCiocalteu reagent and 4 mL
2. Materials and Methods
of NaCO3 solution. The results were expressed as gallic
Chemicals and reagents acid equivalents (GAE), which is the amount of gallic
70% Ethanol, ethyl acetate, n-hexane, Folin-Ciocalteu acid (mg) per gram of extract. The total phenolic contents
reagent, phosphate buffer saline, sodium carbonate, of the fractions were calculated by using the formula,(14)
prednisone, and gallic acid were purchased from C = C1 × V/m
Medigene Sdn. Bhd. (Selangor) where C = total phenolic content in GAE (mg/g)
C1 = concentration of gallic acid obtained from the
Collection and authentication of plant materials calibration curve in (mg/ml)
The barks of FD were collected in Cheras, Malaysia V = volume of extract in (ml)
and authentication of the plant was obtained from Mohd m = weight of the plant extract in (g).
Hafizi Adzmi Hanafi, Biodiversity Unit of University The TPC determination of each fraction was
Putra Malaysia (UPM). (UPM/IBS/UB/H24/19) performed in triplicate, to get their average result, and
the data were reported as mean ± SD.
Preparation of ethanolic extract
Fresh barks of FD were cleaned thoroughly by using Preparation of test samples
distilled water, then dried under shade in a dust-free, clean The stock solution of standard prednisone (10µmg/
environment and grinded into fine powder after they were mL), ethanolic extract (4 mg/0.2 mL), and ethyl acetate

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Current Trends in Biotechnology and Pharmacy 164
Vol. 14 (5) 162-167, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.18

fraction (1 mg/0.2 mL) were prepared by dissolving in using rotary cutter to expose the blood vessels on the
1% w/v phosphate buffer solution. Filter papers of 5mm CAMs. Parafilm tape was used to seal the window created
diameter were punched out and sterilized properly. and the eggs were continued to be incubated until day 10
Different volume of solutions were applied drop wise on of the experiment. All procedures were done under
the sterilized filter papers according to the required dose laminar air flow hood.
to be tested(15). The concentration of prednisone, At day 10 of incubation, different concentration of
ethanolic extract and ethyl acetate fraction used as follow: ethanolic extracts (250 µg/pellet and 500 µg/pellet), its
Prednisone : 250 µg/pellet (25 µL) ethyl acetate fractions (50 µg/pellet and 100 µg/pellet)
Ethanolic extract : 250 µg/pellet (12.5 µL) and and prednisone (250 µg/pellet) were loaded on sterile
500 µg/pellet (25 µL) filter paper with a diameter of 5mm and placed on the
Ethyl acetate fraction : 50 µg/pellet (10 µL) and CAM. An egg without applying any test sample was set
100 µg/pellet (20 µL). as negative control of this study.
All procedures were performed under laminar air flow
In Vivo chick embryo chorioallantoic membrane (CAM)
hood to ensure surrounding environment is sterile, to
assay
minimize the contamination. CAMs were observed 24
30 fertilized chicken eggs were purchased from local hours later and photographed with a digital camera for
hatchery, Hing Hong Sdn. Bhd. detailed images. The thickness of the blood vessels was
Inclusion criteria observed, and the primary, secondary and tertiary blood
vessels number were counted and recorded. The result
Three days old fertilized fresh eggs.
of experiment groups was compared with negative control
Eggs without any sign of cracking. group. To ensure consistency, the largest blood vessel
Exclusion criteria from the heart was designated as primary blood vessel
(PBV), blood vessels that branch out from the primary
More than three days old fertilized chicken
blood vessel were designated as secondary blood vessels
embryos.
(SBV), and for blood vessels that branch out from the
Cracked eggs. secondary blood vessels are counted as tertiary blood
Double yolk embryos. vessels (TBV)(17). The total blood vessels (TTV) were
calculated by adding PBV, SBV and TBV together. The
Pharmacological test
following formula was employed to calculate the
All eggs were cleaned by using 70% ethanol to remove percentage of inhibition(18).
dust and impurities. Then, the eggs were incubated in a
Percentage inhibition= [(N of CAM treated in negative
horizontal position under a constant humidity around 60%
control group - N of CAM treated by extracts/ fraction) /
at 37 °C(16). The eggs were divided into the following
(N of CAM treated in negative control group)] × 100%
groups, and each group consists of 5 eggs:
Whereby N= Total blood vessels (TTV)
Group 1: Negative control (without drug)
Group 2: Prednisone (250 µg/pellet) Statistical analysis

Group 3: Ethanolic extract of FD (250 µg/pellet) The data was analysed using one-way ANOVA
followed by Tukey's post hoc test, and all results were
Group 4: Ethanolic extract of FD (500 µg/pellet) recorded as mean ± standard deviation. The results were
Group 5: Ethyl acetate fraction of ethanolic extract of considered significant when P value is <0.05.
FD (50 µg/pellet)
3. Results and Discussions
Group 6: Ethyl acetate fraction of ethanolic extract of
FD (100 µg/pellet) Total phenolic content
On incubation day 3, a small hole was created by using TPC of each fraction was calculated by using the
a sterile pin at one end of the eggs to remove about 2 to standard curve with the equation of y = 0.0140x + 0.0459,
3ml of albumin by using sterile syringe with needle to where R2 = 0.9993 (Graph 1), and the values were
detach the developing CAM(16). On incubation of day expressed as mean ± standard deviation as shown in Table
4, a square window of about 2cm x 2cm was created by 1.

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Current Trends in Biotechnology and Pharmacy 165
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DOI : 10.5530/ctbp.2020.4s.18

It was shown that ethyl acetate fraction (0.1 mg/mL) (Figure 1 A-F) There was significant reduction (P<0.05)
contains the highest phenolic content compared to water in the thickness of the blood vessels in prednisone
(0.1 mg/mL) and hexane fractions (0.1 mg/mL). (Table (250µg), ethanolic extract (250 µg, 500 µg) and ethyl
1) Ethyl acetate fraction contains about twice the amount acetate fraction (50 µg, 100 µg) treated group, when
of phenolic contents (349.59 ± 0.29mg) than hexane compared to the negative control group. Different
fraction (175.31 ± 0.18mg), and almost thrice the amount concentrations of extracts and fractions showed different
of phenolic content in water fraction 123.17 ± 0.25mg). extent of reduction in the thickness of blood vessels.
Water fraction contains the lowest amount of phenolic (Figure 1 A-F) The number of primary, secondary, tertiary
content among the three fractions(19). (Table 1) blood vessels and percentage of inhibition were
Table 1. TPC of water, hexane and ethyl acetate fractions calculated.
Fractions TPC (GAE, mg/g)
Water 123.17± 0.25
Hexane 175.31 ± 0.18
Ethyl acetate 349.59 ± 0.29

Chick Chorioallantoic Membrane Assay

Qualification and Quantification of Blood Vessels
The condition and thickness of blood vessels in CAM
treated by different groups were observed and shown.
Graph 1. Standard curve of gallic acid
The presence of bioactive compounds, which is known
as secondary metabolites is always associated with
medicinal and pharmacological actions of medicinal
plants. Secondary metabolites like alkaloids, waxes,
terpenoids, fatty acids, phenolics (simple phenolics and
flavonoids), glycosides and their derivatives are reported
to have medicinal properties, such as inhibition of the
process of angiogenesis(20). Angiogenesis contributes to
the growth and metastasis of tumor as it supplies essential
nutrients oxygen needed by the growing tumors.
As supporting evidence to the potential anti-
angiogenic activity of FD, other studies have

CAM treated with negative control (A), 250µg of prednisone Graph 2. Number of blood vessels among different
as positive control (B), 250µg of ethanolic extract (C), 500µg
experiment groups
of ethanolic extract (D), 50µg of ethyl acetate fraction (E)
&100µg of ethyl acetate fraction (F). * means significant results with p<0.05compared with
Fig 1: Image of CAM from each treatment groups. negative control group.

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Current Trends in Biotechnology and Pharmacy 166
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DOI : 10.5530/ctbp.2020.4s.18

Based on the present study, by using chick embryo

CAM model, the new pharmacological effect of FD has
been confirmed, proven by the inhibition of angiogenesis
in term of total blood vessels number, as well as thickness
of the blood vessels. FD bark ethanolic extract and its
ethyl acetate fraction showed significant antiangiogenic
activity at all doses of treatment studied, where both
ethanolic extract and ethyl acetate fraction reduced the
thickness and number of total blood vessels in CAM. To
date, there have been extensive studies on natural product
compounds as well as extracts that showed potent anti-
angiogenic activity, in conjunction to having good
Graph 3. Percentage of blood vessels inhibition among antioxidant activities(17). We can therefore conclude that
different experiment groups the anti-angiogenic activity of ethyl acetate fraction from
* means significant results with p<0.05compared with FD bark in this study may arise from its anti-oxidative
negative control group. capacity, primarily due to the presence of phenolic
compounds(17, 23), whereby antiangiogenic effect of
demonstrated that selected phytochemical constituents
ethanolic extract from FD may due to the presence of
(phenolics) extracted from this plant showed anti-oxidant
other phytoconstituents in addition to phenolic
activity (21). Furthermore, a correlation between
compounds(24).
antioxidant and anti-angiogenesis was observed, and it
is reported that angiogenesis in vitro is stimulated by vital 4. Conclusion
endogenous reactive oxygen species (ROS), for example, The significant reduction in the thickness and total
hydrogen peroxide. Too much ROS will give rise to number of blood vessels revealed the anti-angiogenic
oxidative stress, and hence promoting various human activity of ethanolic extract and its ethyl acetate fraction
ailments such as inflammation, hypertension, of FD. The anti-angiogenic activity of ethanolic extract
atherosclerosis, as well as cancer(17). Antioxidants can and its ethyl acetate fraction are due to the presence of
scavenge reactive oxygen species which damage lipids, phenolic compounds. Hence, it can be concluded that FD
proteins and DNA. could provide a new source of chemical agents for anti-
In CAM, chicken allantoic fluid secretion provides angiogenic cancer therapy and warrants further studies.
natural endogenous growth factors to it. These proteins
5. References
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ethanolic extract (250 µg, 500 µg) and its ethyl acetate Malaysia, Health Ministry survey shows. Malaymail.
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of total blood vessels and percentage inhibition in the cancer-fourth-biggest-killer-in-malaysia-health-
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Current Trends in Biotechnology and Pharmacy 168
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DOI : 10.5530/ctbp.2020.4s.19

Knowledge and Awareness About Blood Pressure, Stroke and Prevalence of

Hypertension : A Cross-Sectional Study in a Private University, Kuala Lumpur
Chuan Sheng Yap1*, Zi Xuan Khor1, Peng Nam Yeoh1, R. Mogana1, Yook Chin Chia2
1Faculty of Pharmaceutical Sciences, UCSI University, Malaysia.
2Department of Medical Sciences, School of Healthcare and Medical Sciences, Sunway University, Malaysia.

*Corresponding author : YapChuanSheng@outlook.com

Abstract premature death if not detected and treated early.(1)

Prevalence of hypertension is rising in Malaysia. This Lewington S et al. reported that an increase in 20mmHg
study aimed to assess the prevalence of hypertension systolic blood pressure and an increase in 10mmHg of
among students and staff of a private university in diastolic blood pressure may double the risk of mortality
Malaysia. As most of the risk factors of hypertension is caused by stroke and ischemic illnesses.(2) As a
modifiable in nature, this study also aimed to determine developing country, Malaysia's population is still trying
the knowledge of university students and staff on to adapt the unprecedented economic growth and
hypertension, stroke, and linkage between these two modernization in which both directly and indirectly affect
diseases. Convenient sampling used to conduct a survey the health-related behaviour of the population.(3)
during a blood pressure screening campaign. A total of The prevalence of hypertension in Malaysia has been
803 responds were collected, and the prevalence of increasing for the previous years.(3) A Malaysian study
hypertension was found to be 5.5% (45 out of 803 in 2016 reported that up to 32.7% of Malaysian adults
respondents). Hypertension was more prevalent in male have hypertension.(4) Due to the significant health care
respondents (9.1%) than female respondents (1.8%) (P < burden of hypertension, its increasing prevalence indicates
0.05). Other factors associated to hypertension include that more attention in Malaysian healthcare community
smoking habits and high body mass index (BMI). Weak must be allocated to this non-communicable disease.
positive correlations were observed between the BMI
Various factors, such as high salt intake, excessive
category and blood pressure (r=.396 for systolic blood
adrenergic tone, sedentary lifestyle, genetic influences,
pressure, r=.317 for diastolic blood pressure). 17.3% of
poor sleep habits, smoking, alcoholism, and others have
male respondents were found to be active smokers, while
been identified to be associated with essential
34.9% of total respondents were either overweight or
hypertension. (5-7) Furthermore, other demographic
obese. The overall knowledge of students and staff on
differences such as educational level, gender, and body
hypertension and stroke was good, however, certain
mass index (BMI) are also associated with increased blood
knowledge insufficiencies were identified. Factors
pressure. (8,9) As most of the risk factors are modifiable
affecting knowledge includes the educational field of
in nature, it is vital for researchers to assay the population's
students and the academic qualifications of respondents.
awareness and knowledge in identifying new strategies
As a conclusion, our study found knowledge gap and
to reduce prevalence of hypertension.
potential interventions which university and healthcare
team may target to reduce the prevalence of high blood Gooding HC and colleagues reported that most of the
pressure in university settings. young adults had poor awareness of hypertension, and
this puts them at increased risk of this disease.(10) Another
Keywords : hypertension knowledge, stroke knowledge,
investigation by Zhang YY and Moran AE found that
hypertension prevalence, university
adults aged 18 to 39 had significantly lower awareness
1. Introduction of hypertension when compared to elder adults.(11) They
Hypertension is one of the most common medical also documented that male participants had generally
conditions seen in primary healthcare settings. It may lead lower hypertension awareness than female participants.
to myocardial infarction, stroke, renal failure, and (11) From our literature reviews, there are insufficient

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Current Trends in Biotechnology and Pharmacy 169
Vol. 14 (5) 168-175, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.19

studies or reports on the prevalence of hypertension in in four sections. Section one comprised of 11 questions
adults from a university setting in Malaysia. on participants' demographic data, including field of
Hence, this study aimed to investigate the prevalence study, gender, smoking status, and others. Section two
of hypertension among university students and staff. The assessed participants' most recent blood pressure
study also assessed the current level of knowledge and measurement and current health status. Section three had
awareness about hypertension and stroke among the study 16 questions evaluating participants' knowledge of
population, as well as the relationship of hypertension to hypertension, while section four evaluated participants'
stroke. knowledge of stroke. For the last two sections, one mark
was given for each correct answer, and no mark was given
2. Materials and Methods for incorrect answers.
Study design The questionnaire's face and content validity were
This was adescriptive cross-sectional study conducted assessed by expert reviewers, and a pilot test was
at UCSI University, Malaysia. In this study, a blood conducted on 50 samples to determine its reliability. The
pressure screening campaign was conducted from June final iteration of questionnaire was disseminated to the
to July 2018 to measure the blood pressure of university eligible respondents who gave their informed consent for
students and staff. During the campaign, all participants this study.
aged 18 years old and above were presented with Respondents who replied more than 75% correct
Respondent Information Sheet and invited to fill up the answers for the knowledge questions were deemed to
questionnaire. Participants who refused to provide have good knowledge in that section. Respondents who
informed consent were excluded from the study. scored 50% to 74% correct answers were categorized as
The screening processes were carried out by having moderate knowledge, while those who scored less
undergraduate pharmacy students under the supervision than 50% were categorized as having poor knowledge.
of pharmacists. The students were trained to follow the Data analysis
blood pressure measurement protocol specified by the
Data was entered in Microsoft Excel 2016 and
Malaysia Society of Hypertension (MSH) before the
analysed using Statistical Package for the Social Science
commencement of screening campaign. The blood
(SPSS) software version 23. Descriptive data such as
pressure was measured from participants' non-dominant
demographic characteristics were described as
arm twice at 1 minute apart, and the mean values were
percentages. The sample population's average blood
taken as participants' blood pressure. If the difference
pressure and knowledge scores were described as mean
between the first and second readings are 20 mmHg or
with 95% confidence interval (CI).
greater, then a third reading was taken. To ensure
consistency of the measurements, 'Rossmax X5 Digital Mann-Whitney U Test, Kruskal Wallis Test, and Chi-
Blood Pressure Monitor' was used throughout the Square Test were used to compare between groups, and
research. Spearman's correlation was used to measure association
between factors. A P-value of P > 0.05 is considered to
Sample size and sampling technique
be statistically significant.
During the sampling period, there were around 2500
active science stream and 3000 art stream students in 3. Results and Discussion
UCSI University, with 362 academic staff and 326 non- Demographic data, blood pressure, and BMI of
academic staff. By using a margin error of 0.05 at 95% respondents
confidence interval, the computed sample size for science
A total of 677 students and 126 staff consented to the
and art stream students were 334 and 341 subjects
study. 51.8% of respondents were male (n = 416).
respectively. The sample size required for academic and
Majority of respondents are non-smoker (88.6%), while
non-academic staff were 187 and 177 subjects
59.2% did not drink any alcoholic beverage.
respectively. Convenient sampling was used to attain the
participants. Blood pressure profile of respondents were classified
according to the Malaysia Clinical Practice Guideline:
Research instrument Management of Hypertension 5th edition.(12) From Table
The questionnaire adopted from MSH is available in 1, 45 out of 803 respondents (5.5%) had systolic blood
English, Malay, and Chinese. It consisted of 53 questions pressure exceeding 140mmHg and/or diastolic pressure

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Current Trends in Biotechnology and Pharmacy 170
Vol. 14 (5) 168-175, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.19

exceeding 90mmHg. The average systolic blood pressure students' 116.61/73.18 mmHg [95% CI: Systolic
of the study population was 117.24mmHg [95% CI: (115.55,117.67), Diastolic (72.51,73.86)] (P < 0.05). A
(116.24,118.23)] and the average diastolic blood pressure possible reason for such observation could be due to the
was 74.12mmHg [95% CI: (73.47,74.78)]. overwhelming workload and emotional stress of
Table 1: Demographic data, blood pressure, and BMI of university staff. A study by Adedoyin RA et al. observed
respondents that university staff were often occupied by lecture classes
with tight schedule, meetings and other duties, leading
Variable to psychological stresses which contribute to an increase
University student (n, %) 677 (84.3) in blood pressure.(13)
334 (41.6)
Science stream 343 (42.7) Table 2 : Blood pressure of respondents based on gender
Art stream and occupation.
University staff (n, %) 126 (15.7) Variable Gender Population
67 (8.3)
Academic staff Blood Pressure Male Female Students Staff
59 (7.3) Classification
Non-academic staff (n, %) (n = 416) (n = 387) (n = 677) (n = 126)
Mean age in years
21.8
Students 36.6 Optimal 174 (41.8) 296 (76.5) 407 (60.1) 63 (50.0)
Staff Normal 130 (31.3) 60 (15.5) 167 (24.7) 23 (18.3)
Gender (n, %) At risk
Stage 1 74 (17.8) 24 (6.2) 69 (10.2) 29 (23.0)
416 (51.8)
Male Stage 2 31 (7.5) 7 (1.8) 28 (4.1) 10 (7.9)
387 (48.2)
Stage 3
Female 6 (1.4) 0 (0) 5 (0.7) 1 (0.8)
Alcoholic beverage drinker (n, %)
1 (0.2) 0 (0) 1 (0.1) 0 (0)
328 (59.2)
Non-drinker 289 (36.0)
Social drinker 34 (4.2)
For university students, the prevalence of hypertension
Moderate drinker 5 (0.6) was 4.9% (n = 34). This finding was lower than a previous
Heavy drinker study from Shah Alam, Malaysia, where 10% of the
Smoking status (n, %) students were found to be hypertensive.(14) Nevertheless,
712 (88.6) it is alarming to see that 1 in 20 young adults in this
Non-smoker 64 (8.0) university were already having high blood pressure.
Social smoker 17 (2.1)
Moderate smoker 10 (1.2) Our data also found that high blood pressure was more
Heavy smoker prevalent in male (9.1%) than female respondents (1.8%)
Blood pressure classification (n, %) (P < 0.05). The mean systolic and diastolic blood pressure
470 (58.5) of male respondents was 122.68mmHg [95% CI:
Optimal 190 (23.7)
Normal (121.35,124.02)] and 75.44mmHg [95% CI:
98 (12.2)
At risk 38 (4.7) (74.51,76.37)] respectively, whereas systolic and diastolic
Stage 1 6 (0.7) blood pressure for female was 111.39mmHg [95% CI:
Stage 2 1 (0.1) (110.14,112.63)] and 72.71mmHg [95% Cl:
Stage 3 (71.81,73.60)] (P < 0.05).This finding is consistent with
Body mass index (n, %) other prevalence studies in Malaysia which reported
128 (15.9) higher hypertension prevalence among men than
Underweight 392 (48.8)
Normal women.(14-16) A previous study reported that differences
200 (24.9)
Overweight 78 (9.7) in blood pressure levels can be evident among males and
Obese females even in their twenties.(17) Possible factors
include the better BMI profile of female, and the lesser
Table 2 shows respondents' blood pressure category likelihood of female becoming a smoker.(17) Indeed, in
based on their gender and occupation. The average our study there were more smokers among male
systolic/diastolic blood pressure of university staff was respondents than female (17.3% of male vs 4.9% of
120.61/79.19 mmHg [95% CI: Systolic (117.86,123.36), female respondents) (P < 0.05). The difference in body
Diastolic (77.39,80.99)], which is higher than that of composition and insulin resistance has also been

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Current Trends in Biotechnology and Pharmacy 171
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suggested as contributing factors for lower blood pressure Table 3 : Average systolic and diastolic blood pressure
among female adolescents.(8) Furthermore, estrogen may in each BMI category.
act as a protective factor against hypertension.(18) Variable Average systolic BP Average diastolic BP
Smokers were found to have higher systolic blood (mm Hg) (mm Hg)

pressure than non-smoker (P < 0.05). Possible reasons Body mass index
include the enhanced atherogenesis in large capacitance
vessel among smokers, leading to increased systolic blood Underweight 110.12 (108.04,112.20)* 70.92 (69.52,72.32)*
pressure.(19) Nicotine may also increase peripheral Normal
115.06 (113.80,116.33)* 72.48 (71.68,73.29)*
vasoconstriction via sympathetic pathway, and increase Overweight
Obese 121.22 (119.28,123.15)* 76.41 (75.04,77.77)*
serum lipid level which gives rise to atherosclerosis.(20)
The percentage of smokers in our study population 129.46 (125.79,133.14)* 82.08 (79.58,84.58)*

(17.3% of male respondents) suggested that smoking

*Values presented as mean with 95% confidence interval.
cessation campaign can be beneficial in university
settings. The negative consequences of smoking had been Variable Average systolic BP (mm Hg) Av e r a g e
well-documented in most of the practice guidelines diastolic BP (mm Hg)
around the world, but smokers who volunteered for Spearman's correlation was carried out to determine
smoking cessation remained low in Malaysia.(21) the relationship between average blood pressure level
University can play a role by establishing policies or with BMI. Result revealed statistically significant positive
enforcing rules that include punitive measures on students
correlation between average systolic and diastolic blood
who were found to be smoking in the campus. Education
pressure with body mass index. (P < 0.05) Weak positive
and reminders should be given to students on frequent
correlation was found between BMI and average systolic
basis. Furthermore, abolishing smoking area in university
blood pressure (r=.396), and between BMI and average
for students to smoke can be effective as well.
diastolic blood pressure (r=.317).
Our study did not find any significant difference in
This is similar to the findings from REDISCOVER
blood pressure across different categories of alcohol
consumption. This finding is dissimilar with other studies Study of hypertension, where obese respondents were
which reported dose-related relationship between alcohol reported with higher prevalence of high blood pressure.(4)
consumption and blood pressure.(22) It is possible among BMI has been reported to be a good predictor of blood
the drinkers in our study population, majority (88%) of pressure, where an increment of 1 unit BMI can contribute
them were light drinker, possibly due to financial to an average increase of 2.0mmHg in systolic blood
restriction of university students in obtaining costly pressure and 1.3mmHg in diastolic blood pressure.(24)
alcoholic drinks. High BMI is one of the common modifiable risk
BMI profile of respondents were classified according factors for many disease, and appropriate prevention can
to Malaysia Clinical Practice Guidelines: Obesity.(23) help to reduce healthcare burden to the society. Besides,
The mean BMI of university students was 21.70kg/m2 physical inactivity, poor dietary habit may also contribute
[95% CI: (21.40,21.99)] and the mean BMI of university to the high BMI levels.
staff was 24.88kg/m2 [95% CI: (24.10,25.65)] (P < 0.05). National Health and Morbidity Survey 2015 reported
Male respondents have significantly higher BMI value that Malaysians had poor daily intake of vegetables and
than female respondents (P<0.05). The average BMI for
fruits.(21) Students' hectic and heavy workload may be
male and female was 22.85kg/m2 [95% CI: (22.45,23.27)]
associated with unhealthy eating practice too. Deliens T.
and 21.49kg/m2 [95% CI: (21.11,21.87)] respectively.
and colleagues documented that university students' busy
This could be one of the contributing factors for higher
involvement in faculty events and academic activities may
blood pressure level among male respondents.
have driven students to neglect healthy diet, and seek for
Correlation between blood pressure and BMI fast and convenient food.(25) Although Malaysian
Table 3 shows that as BMI class increased, the average Dietary Guideline is available for the public, many
systolic and diastolic BP increased. The blood pressure Malaysian were not aware of it.(26) Healthcare
level was significantly different in each class of body professionals and lecturers should take a more active role
mass index (P < 0.05). in promoting healthy diet to students and public.

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Current Trends in Biotechnology and Pharmacy 172
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Knowledge about hypertension students. Mean knowledge score for art stream and
The knowledge about hypertension was also compared science stream students was 12.25 [95% CI:
(11.94,12.56)] and 13.29 [95% CI: (13.03,13.55)]
between science stream and art stream students. From
respectively (P < 0.05). This may be due to the exposure
Table 4, 81.1% of science stream students have good
of science stream students to biology subjects and human
knowledge, compared to only 72.3% of art stream
physiologies in the academic curriculum.

Table 4 : Respondents' knowledge on hypertension

Variable Student Staff
Knowledge on Art Stream Science stream Academic Non-academic
hypertension (n, %)
(n = 343) (n = 334) (n = 67) (n = 59)

Poor
25 (7.2) 12 (3.6) 2 (3.0) 0 (0)
Moderate
Good 70 (20.4) 51 (15.3) 4 (6.0) 17 (28.8)
248 (72.3) 271 (81.1) 61 (91.0) 42 (71.2)

Additionally, there are more academic staff (91.0%) the right answer (P < 0.05). There was also significant
who had good knowledge when compared to non- difference between university staff and students (P <
academic staff (71.2%) (P < 0.05).This may be explained 0.05), where 15.6% of university students and 7.1% of
by the differences in education level between academic university staff considered stressful lifestyle to not affect
and non-academic staff. 73.2% of the academic staff have blood pressure level. Good stress management practice
post-graduate qualifications, while 71.2% of non- should be instilled in all university students and staff.
academic staff only attained academic qualifications of University students are susceptible to high level of stress
bachelor's degrees or below. due to multiple examinations, overwhelming homework
Smokers and alcoholic drinkers were not found to have and other assignments.(27) Nichter M. discovered that
significant differences in knowledge when compared to students has a tendency to take cigarette smoking in an
their respective counterparts. attempt to handle stress.(28) Such unhealthy practices
should be stopped and prevented at the university level
The study also investigates respondents' responses to through proper education and awareness.
individual questions. For the question relating high salt
intake to risk of hypertension, the majority of the study Majority of the respondents (55.2%) were not aware
population (n = 637, 79.3%) answered correctly. that a sedentary lifestyle can increase the risk of
However, among university staff, it was discovered that hypertension.Only 39.4% of student respondents
30.5% of the non-academic staff were not aware that high answered this question correctly, compared to 65.1% of
salt intake is one of the risk factors of hypertension. staff (P < 0.05). This shows a poor awareness of the study
Furthermore, it was found that among 166 respondents population on the linkage between sedentary lifestyle and
who were not aware that high salt intake can increase the high blood pressure.
risk of hypertension, 42.2% of them were either 771 respondents (96%) were aware that the public
overweight or obese. This suggested poor dietary should measure their blood pressure at least once per year.
awareness. However, out of these 771 respondents, only 54.6% of
86.7% of study population were aware that frequent them did measure their blood pressure within past one
stressful lifestyle can increase the risk of hypertension. year. Besides, a small percentage of them (9.9%) never
There was significant difference between science stream had their blood pressure measured before, even though
and art stream students (P<0.05) on the choice of answer they believed that it is important to measure blood
for this question, where 88% of science stream answered pressure on yearly basis. Many teenagers and young
correctly but only 80.8% of the art stream students chose adults are prone to have poor attitudes and practices for

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Current Trends in Biotechnology and Pharmacy 173
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Table 5 : Respondents' answer on selected hypertension knowledge questions

Question Science Student Art Students Academic Staff Non-academic Staff
(n = 334) (n = 343)
(n, %) Correct Incorrect Correct Incorrect Correct Incorrect Correct Incorrect

Risk factors of
hypertension
High salt intake 272 (81.4) 62 (18.6) 254 (74.1) 89 (25.9) 60 (89.6) 7 (10.4) 41 (69.5) 18 (30.5)
Stressful lifestyle 294 (88.0) 40 (12.0) 277 (80.8) 66 (19.2) 62 (92,5) 5 (7.5) 55 (93.2) 4 (6.8)
Sedentary lifestyle 152 (45.5) 182 (54.5) 115 (33.5) 222 (66.4) 45 (67.2) 22 (32.8) 37 (62.7) 22 (37.3)

BP Screening
Is it important to 317 (94.9) 17 (5.1) 330 (96.3) 13 (3.7) 67 (100) 0 (0) 57 (96.6) 2 (3.4)
measure BP at least
once a year?

health screenings, they believe that most of the non- Our study found that 3.4% of student respondents (n
communicable diseases are age-related rather than = 23) thought stroke affects kidney, lung, or liver.19.9%
lifestyle-related. Evans N. and colleagues revealed that of the students and 15.9% of staff thought that stroke
the health risks possessed by adolescence these days were primarily affects the heart rather than the brain. This was
unmatched with their health priorities.(29) In another consistent with studies conducted in India and Nepal
study conducted by Cao QQ. and colleagues, they whereby the knowledge on the stroke-affected organ was
reported that many patients only found out that they had poor.(31,32) The observation in our study may be due to
hypertension when stroke occurred.(30) a lack of educational campaigns on stroke awareness in
Most of the respondents (87.2%) were able to identify Malaysia.
stroke as a potential complication of hypertension. There Table 6 : Respondents' knowledge on stroke
was no statistically significant difference in blood Variable Student Staff
pressure of responders who were aware of such linkage, Knowledge on Art Science Academic Non-academic
and those who were not aware of it. However, we did stroke (n, %) Stream stream
(n = 67) (n = 59)
observe a trend where respondents who were aware of (n = 343) (n = 334)
such complications are more likely to have optimal blood 22 (6.4) 13 (3.9)
Poor
2 (3.0) 1 (1.7)
pressure (59.4%) compared to respondents who disagreed Moderate 63 (18.4) 55 (16.5)
Good 8 (11.9) 14 (23.7)
(53.2%). Understanding the complication of chronic 258 (75.2) 266 (79.6)
57 (85.1) 44 (74.6)
diseases is an important factor in active prevention of
the diseases.
Majority of the respondents (87.7%) agreed that
Knowledge about stroke lowering blood pressure can reduce the risk of stroke.
77.4% of university students had good knowledge This showed good awareness on the link of high blood
about stroke (77.4%) with a mean knowledge score of pressure to stroke. According to RE-LY trial, every 10
14.24 [95% CI: (13.99,14.50)] out of a total of 19 mmHg increase in mean BP will lead to 6 to 7% increased
questions. Similarly, university staff were able to answer risk of stroke.(33) It was hoped that as the public aware
knowledge-based questions about stroke as well. Majority on the linkage between blood pressure and stroke, they
of the university staff (80.2%) have good knowledge can provide better self-discipline in management of
about stroke. In comparison of stroke knowledge score, hypertension. In fact, among all common risk factors of
university staff {14.89 [95% CI: (14.37,15.40)]} has stroke, hypertension can be considered as the easiest risk
greater knowledge score than university students (P < factors to be managed.(34)
0.05).There was no significant difference between 87.9% of respondents also understood that stroke
knowledge score of academic and non-academic staff. patients should be sent to hospital immediately, ideally
Science stream students (mean knowledge score 14.63 within the first 4 and a half hours after stroke occurred.
[95% CI: (14.30,14.96)]) had significantly greater However, for respondents who were not aware that brain
knowledge than art stream students (mean knowledge is the primary organ affected by stroke, half of them
score 13.86 [95% CI: (13.47,14.25)]) (P < 0.05).This is (49.7%) did not feel that it is necessary for stroke patients
similar for the knowledge of hypertension in current to be admitted immediately. One of the first line
study.
Yap et al
Current Trends in Biotechnology and Pharmacy 174
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DOI : 10.5530/ctbp.2020.4s.19
management of stroke, alteplase, provides better outcome 2. Lewington S, Clarke R, Qizilbash N, Peto R, Collins
and health benefits if patients were admitted within the R. (2002). Age-specific relevance of usual blood
first 4 and a half hours.(35) It is important to increase pressure to vascular mortality: A meta-analysis of
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This study found that the knowledge about prospective studies. Lancet.
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were good and there was no difference in the blood K. (2016). Hypertension in Malaysia: An analysis
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level was not associated with raised blood pressure among 4. Abdul-Razak S, Daher AM, Ramli AS, Ariffin F,
university staff and students, therefore future study should Mazapuspavina MY, Ambigga KS, et al. (2016).
also assess the attitude and practice of study population Prevalence, awareness, treatment, control and socio
to investigate other reasons of the poor health practices. demographic determinants of hypertension in
4. Conclusion Malaysian adults. BMC Public Health.16(1):1-10.
Overall, 5.5% of university staff and students had 5. Seyed Mehrdad Hamrahian M. (2017).
systolic blood pressure exceeding 140mmHg and/or Pathophysiology of Hypertension: Pathogenesis of
diastolic blood pressure exceeding 90mmHg. Male Essential Hypertension, Factors Influencing BP
respondents have significantly higher average blood Regulation, Etiology of Essential Hypertension.
pressure than female respondents. Smoking habit and Medscape.
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The prevalence of overweight and obese in university treatment. Journal of the American Society of
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University students and staff were found to have Richer L, Veillette S, et al. (2009). Sex differences
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However, knowledge insufficiency was found in certain composition and metabolism in adolescence. Arch
areas, such as the link between sedentary lifestyle and Pediatr Adolesc Med.
hypertension. Science stream students have significantly 9. Gyamfi D, Obirikorang C, Acheampong E, Danquah
higher knowledge than art stream students on these KO, Asamoah EA, Liman FZ, et al. (2018).
healthcare-related topics. As for university staff, the Prevalence of pre-hypertension and hypertension and
knowledge about hypertension and stroke was found to its related risk factors among undergraduate students
be significantly higher in academic staff compared to non- in a Tertiary institution, Ghana. Alexandria J Med.
academic staff. This may be due to the higher level of 10. Gooding HC, McGinty S, Richmond TK, Gillman
education and knowledge sharing culture in academic MW, Field AE. (2014). Hypertension awareness and
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interventions which university and healthcare team may Intern Med.
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Comparison of Blood Pressure Patterns of Teaching 25. Deliens T, Clarys P, De Bourdeaudhuij I, Deforche
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Investigation on Antibacterial Activity of Pyrogallol in Methicillin

Resistant Staphylococcus aureus
Yik-Ling Chew1*, Joo-Kheng Goh2*, Chairunnisa Arasi2
1Faculty of Pharmaceutical Sciences, UCSI University, No. 1 Jalan Menara Gading, UCSI Heights, 56000 Kuala Lumpur, Malaysia
2School of Science, Monash University Malaysia, Jalan Lagoon Selatan, 47500, Bandar Sunway, Selangor Darul Ehsan, Malaysia
*Corresponding author : ence: chewyl@ucsiuniversity.edu.my, goh.joo.kheng@monash.edu

Abstract on the appropriate usage of antibiotics. Ongoing

The aims of this study areto study the antibacterial development of new antibiotics, active surveillance efforts
activity of pyrogallol towards MRSA strains and evaluate and advances in infection prevention are urgently required
the effects of various concentrations of pyrogallol over as MRSA remains a prominent pathogen with persistently
time in relation to the stages of the growth of the bacteria. high mortality (2). Massive research efforts on the
Antibiotic susceptibility of pyrogallol was assessed using discovery on effective antibiotics against MRSA is needed
disc diffusion and microbroth dilution method. It was to combat successive waves of resistant pathogens and to
found that the minimum inhibitory concentration (MIC) meet the challenges of resistance development.
and minimum bactericidal concentration (MBC) of Antibiotics from natural products are reported to have
pyrogallol were 15.6 µg/mL. Time-kill kinetic assay complex architectural scaffolds and active functional
performed showed that lower concentration of pyrogallol groups which could interact with biological targets. It is
could exhibit some extent of bacteriostatic effect towards believed that natural product-derived antibiotics are
the bacteria, whereas higher concentrations were lethal synthesized as secondary metabolites due to survival
from lag phase onwards. Pyrogallol exhibited strong advantages to the organisms. These metabolites are
antibacterial activity against MRSA. It exhibited MIC and synthesized as organism defense mechanism against pest
MBC at 15.6 µg/mL. Time-kill kinetic assay showed and pathogens. Natural derived antibiotics could inhibit
pyrogallol could slightly inhibit the exponential growth microoganisms by four classical targets, namely bacteria
of MRSA, and bacteria was lethal at higher cell-wall biosynthesis, protein biosynthesis, DNA
concentrations. Possible bactericidal mechanism of replication and folate coenzyme biosynthesis (3-5).
pyrogallol was thoroughly discussed in this study. More Examples of natural products derived antibiotics are
detailed studies on the mechanism of action is in progress. polymyxin B, valinomycin, daphtomycin, novobiocin,
Key words : Pyrogallol; Methicillin resistant erythromycin, etc.
Staphylococcus aureus; antibacterial; polyphenols; Pyrogallol (IUPAC name 3,4,5-trihydroxybenzoic
mechanism acid) is present naturally in numerous plants (Fig 1). It is

1. Introduction
Antibiotic resistance bacteria infections have become
an alarming concern and widely spread around the world.
Bacteria developed resistance towards antibiotics. This
causes massive increment mortality caused by infectious
diseases. Methicillin-resistance Staphylococcus aureus
(MRSA) has higher mortality rate than human
immunodeficiency virus (HIC) or acquired immune
deficiency syndrome (AIDS) (1). There were more than
80000 severe infections reported in the USA in 2011. Also
more than one-half of hospital-related S. aureus infections
reported in most Asian countries were found to be related
to MRSA infection (2). The prevalence of MRSA infection
remains high in Asia countries due to the poor awareness Figure 1 Chemical structure of pyrogallol

Antibacterial activity of pyrogallol

Current Trends in Biotechnology and Pharmacy 177
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also an important functional group in many polyphenol by Kubo et al.(11) and Kang et al (12) with slight
compounds. Literatures have reported that polyphenols modifications. Pyrogallol was diluted with sterile distilled
which carry pyrogallol group have stronger bioactivities, water to achieve 4 different concentrations: ¼ MIC, ½
compare to those which consist of similar structures, i.e. MIC, MIC and 2 MIC. Pyrogallol at these four
catechol and resorcinol rings (6-8). It has been reported concentrations and control group were added into an
that pyrogallol moieties in polyphenols is responsible to initial inoculum of 1 × 108cfu/mL. All samples were
exhibit broad spectrum antibacterial activity. VN Lima incubated at 370C. Samples were withdrawn at selected
et al (9) recently reported that pyrogallol could time points (0, 4, 6, 8, 10, 12 and 24 hours), serially diluted
synergistically exhibit stronger antimicrobial activity with using sterile saline before samples were plated onto
antibiotics against S. aureus and Candida spp. Although MHA. The plates were then incubated at 370C for 20
literatures have reported that pyrogallol could exhibit hours, and colony forming units were estimated.
antimicrobial activity towards various bacteria, studies Triplicates were performed for each sample.
on the antimicrobial action and killing pattern of
Statistical analysis
pyrogallol on MRSA are still not well defined.
The experimental results for disc diffusion were
The main objectives of this study are to study the
expressed as mean ± standard deviation. The data were
antibacterial activity of pyrogallol towards MRSA strains
analysed using one-way analysis of variance (ANOVA)
and evaluate the effects of various concentrations of
using SPSS version 20.
pyrogallol over time in relation to the stages of the growth
of the bacteria. 3. Results and Discussion

2. Materials and Methods Disc diffusion assay demonstrated that both strains
of MRSA were susceptible to pyrogallol at 0.1 mg, and
Bacteria culture the MICs determined were 15.6 µg/mL. MBC for MRSA
Pyrogallol (Sigma Aldrich) was tested on two strains ATCC 33591 was 15.6 µg/mL. MRSA hospital strain was
of methicillin resistance Staphylococcus aureus (MRSA): slightly more resistant to pyrogallol, MBC was 31.3 µg/
ATCC 33591 and a hospital strain, gifted by Assoc. Prof. mL.
Dr. Vasantha Kumari Neela from Universiti Putra
Time kill studies was performed on MRSA ATCC
Malaysia. Both bacteria strains were cultivated onto
33591 at four increasing concentrations of pyrogallol to
nutrient agar (Oxoid) at 37 °C for 16- 20 hours.
determine the killing pattern and time required. Time kill
Antibacterial susceptibility assays assays could evaluate the activity of pyrogallol against
Antibacterial susceptibility of pyrogallol was tested the MRSA and determine the bactericidal or bacteriostatic
with agar diffusion assay (10) and microbroth dilution activity of an agent over time in relation to the stages of
method (1). In disc diffusion assay, 0.1 mg of pyrogallol, the growth of the bacteria (at lag, exponential, stationary
dissolved in 100 µL methanol was loaded onto sterile phase).
blank disc (6 mm diameter; Oxoid) and the disc was It was found that pyrogallol slowed down the growth
impregnated onto Mueller Hinton agar (MHA; Oxoid), at lower concentrations (¼ MIC and ½ MIC) but could
pre-inoculated with bacteria 1 × 108 coliform units (cfu)/ not completely inhibit and kill the microorganism. Longer
mL, standardised using Miles and Misra technique (1). lag phase was noticed and the number of cells increased
The plates were then incubated for 16 - 20 hours at 37°C after 6 hours of treatment (Fig 2). Although stationary
and the diameter of the inhibition zones was measured. phase was noticed in growth curve for ¼ MIC and ½
Minimum inhibitory concentration (MIC) and minimum MIC, the total number of cells was significantly lower
bactericidal concentration (MBC) of pyrogallol was than the control. This showed that lower concentration
assessed using microbroth dilution method. MIC was of pyrogallol could exhibit some extent of bacteriostatic
recorded as the lowest concentration of pyrogallol which effect towards the bacteria.
completely inhibit bacteria growth. MBC was determined Reduction in cells number was observed after
when no visible growth seen on the first streak on MHA treatment at MIC and higher concentration. The pattern
of the clear wells. The test was repeated three times. of antibacterial activity of pyrogallol was bactericidal.
Time-kill kinetics assay Cell number remained constant for treatment at MIC in
Time-kill kinetic assay was performed as described the first 6 hours, followed by reduction in number. Higher

Yik-Ling et al
Current Trends in Biotechnology and Pharmacy 178
Vol. 14 (5) 176-180, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.20

showed greater antibacterial activity. On the other hand,

extracts which consisted of phytochemicals (procyanidins
or flavonoids) bearing the catechol and resorcinol groups
as major constituents would exhibit weaker activity.
These findings showed the importance of pyrogallol
group in exhibiting stronger bioactivity than other similar
hydroxyphenol groups.
There were studies which proposed the two possible
antibacterial mechanisms: (1) oxidative stress, and (2)
disruption on membrane fluidity. Pyrogallol has been
reported to exhibit dual antioxidant/prooxidant properties
(14, 15). The dual antioxidant/prooxidant property enable
the compound to either scavenge or produce radicals
Figure 2 Time kill curve of various concentrations of depending on the environment (16). In this study, it is
pyrogallol against MRSA ATCC 33591 likely that the compound had generated reactive oxygen
species, such as hydrogen peroxides (H2O2) and
dosage of pyrogallol (2 MIC) would result in significant superoxide (15, 17) to inhibit the bacterial growth.
decrease in viability of MRSA and onset of detectable Increase in H2O2 concentration was noticed in pyrogallol
killing in shorter period of time. Reduction in cell number containing media (15). Lim et al(14) reported that
at higher dosage was noticed from t = 0 hour onwards pyrogallol inhibited the growth of Vibrio vulnifus, where
(Fig 2). the oxidative stress-related protein in bacteria was
Taguri et al.(6) reported that pyrogallol exhibited the upregulated with the presence of pyrogallol. However,
strongest antibacterial activity among the selected presence of antioxidants could reduce the inhibitory effect
polyphenols studied, namely epicatechin, catechol, by pro-oxidants generated by pyrogallol.
epigallocatechin, caffeic acid, epigallocatechin-3-O- Numerous studies have reported that polyphenol
gallate etc. In addition, pyrogallol is active towards most compounds targeted on bacteria cell membrane (18-21).
of the Gram-positive and Gram-negative bacteria selected. Polyphenols was reported to mediate the antibacterial
This showed that it is a broad-spectrum antibacterial activity by adsorbing on to the surface of the bacterial
agent. Pyrogallol has also been reported could exhibit cell wall before exhibiting the antibacterial activity (6).
antibacterial activity against Pseudomonas putida, Literatures had reported that polyphenols could inhibit
Pseudomonas pyocyanea, Corynebacterium xerosis (13). bacteria in three stages: (1) cell membrane attachment;
Interestingly, pyrogallol has also been reported could (2) cell membrane fluidity modification; and (3) cell
exert synergistic effect with Norfloxacin and Gentamicin membrane structure disruption (1, 22-24). The binding
in inhibiting S. aureus. The antimicrobial activity of these of polyphenols to bacteria cell membrane could disrupt
antibiotics together with pyrogallol against S aureus was the membrane architecture. For instance, epicatechin
enhanced, where the MIC was reduced up to 20-fold (9). gallate inhibits MRSA by insertion into the bacteria
The number of pyrogallol rings is also well correlated cytoplasmic membrane and disruption of penicillin-
to the antibacterial activity. Authors reported that binding protein 2a-mediated ?-lactam resistance (22);
compounds with pyrogallol group attached were likely berberine and piperine could interfere the microbial
to exhibit stronger antibacterial activity than those with growth by intercalating the cell wall and DNA (23, 24);
catechol groups. For instance, prodelphinidins which and tannins damaging the bacterial cell membranes of
carries a pyrogallol groups exhibited stronger Listeria monocytogenes (25). Smith et al(26) reported
antibacterial activity than procyanidins (6), although both that polyphenols could reduce the membrane integrity,
compounds are structurally similar. Similar findings were inhibit oxidative phosphorylation and the cell transport
also noticed in other polyphenol compounds: between processes. It is believed that pyrogallol could exhibit the
gallic acid and protecatechuic acid, between myricitrin lipid peroxidation towards MRSA cell membrane, where
and rutin, and between pyrogallol and catechol. In the free radicals extracted electrons from the bacterial
addition, authors also reported that plant extracts which cell membranes (27), modified the cell membrane fluidity
consisted of major compounds with pyrogallol groups and resulted in cell membrane disruption. Chedea et al(27)

Antibacterial activity of pyrogallol

Current Trends in Biotechnology and Pharmacy 179
Vol. 14 (5) 176-180, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.20

reported that bactericidal of polyphenols which exhibited 4. Yoneyama, H. and Katsumata, R. (2006). Antibiotic
pro-oxidation effect were likely to exhibit bactericidal resistance in bacteria and its future for novel
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phosphatidylcholine and phosphatidylethanolamine and Biochemistry, 70(5):1060-1075.
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partition coefficients immersed in the phospholipid Mechanisms of Antibiotics and Antibiotic
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Our study demonstrated that pyrogallol exhibited
strong antibacterial activity against MRSA. It exhibited 6. Taguri, T., Tanaka, T. and Kouno, I. (2006).
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pyrogallol could slightly inhibit the exponential growth Biological and Pharmaceutical Bulletin,
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was thoroughly discussed in this study. However, it is and Ubukata, M. (2008). Pyrogallol structure in
necessary to conduct more detailed studies on the polyphenols is involved in apoptosis-induction on
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Acknowledgement
8. Monobe, M., Ema, K., Tokuda, Y. and Maeda-
The authors are thankful to Monash University
Yamamoto, M. (2010). Enhancement of phagocytic
Malaysia and UCSI University Kuala Lumpur for
activity of macrophage-like cells by pyrogallol-type
financial and facilities support, and Assoc. Prof. Dr.
green tea polyphenols through caspase signaling
Vasantha Kumari Neela from Universiti Putra Malaysia
pathways. Cytotechnology, 62(3):201-203.
for the gift of hospital isolate.
9. Lima, V.N., Oliveira-Tintino, C.D., Santos, E.S.,
Conflict of Interest
Morais, L.P., Tintino, S.R., Freitas, T.S., Geraldo,
All authors of this study declare that there are no Y.S., Pereira, R.L., Cruz, R.P. and Menezes, I.R.
conflicts of interest. (2016). Antimicrobial and enhancement of the
antibiotic activity by phenolic compounds: Gallic
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Analysis of the Effectiveness of Drug Awareness Campaigns

Using Google Trends
Deng Ruolan1, Muhammad Shahzad Aslam2*
1Department of Journalism, Xiamen University Malaysia, Sepang, 43900, Malaysia.
2School of Traditional Chinese Medicine, Xiamen University Malaysia, Sepang, 43900,

Corresponding author : aslam.shahzad@xmu.edu.my

Abstract 1. Introduction
Globally, drug-related problems have attracted much Globally, the drug has attracted much attention from
attention from the public because of the negative health the public because of the health problems it may cause.
effects and the huge social burden. Therefore, Substance abuse not only contributes to death but also
Policymakers, healthcare institutions, and the people are relates to short-term or long-term health effects(Johnston,
concentrating on improving drug awareness to eradicate O'malley, Miech, Bachman, & Schulenberg, 2016). To
the abuse of illicit or prescription drugs for the destiny of treat dependence on the drug could cause a huge press to
a healthier community. They spent a lot to designate drug the whole society as well. However, the cruel situation is
prevention campaigns as well as programs. However, the that the percentage of illegal drug users and drug abusers
previous study has not measured the effectiveness of drug is shockingly high and the number is continuing to
awareness campaigns comprehensively and accurately.
increase year by year based on the annual reports from
The public was also understudied previously where the
the global drug-focused institution (UNODC, 2010)Drug
public learning preference and knowledge loophole of
deal even becomes a powerful source to stimulate world
the drug are unclear yet. The foremost objective of this
economic advancement.It also finds that most of these
article is to figure out the effectiveness of drug awareness
drug abusers did not get medical or mental treatment at
campaigns using Google Trends. It also aims at revealing
all.
audiences' preference of search method when they
searching for the related information. This articleuses the Therefore, Policymakers, healthcare institutions, and
qualitative method to explore the effectiveness of drug the people worldwide are concentrating on how to
awareness campaign and the preferred search methods eradicate the abuse of illicit or prescription drugs for the
of the public to gain information about drugs by analyzing destiny of a healthier community(Fonseca et al.,
the data on Google Trends which tracks the public interest 2017)They designate drug prevention campaigns as well
of "drugs" over time worldwide. The result found that as programsto, improve the awareness of the public
the effect of the global drug awareness campaigns in 2018 towards the detrimental consequences of substance use.
is moderate and ephemeral and public prefers using the Drug awareness wasan essential concept in these
web search to collect information they want about "drugs". campaigns which is characterized as the understanding
Globally, "pharmaceutical drugs"is the hottest topic and knowledge of nature, mechanism, signs,
related to drugs during the last year. This article finds a consequences, prevention methods etc. of substance use
generally moderate influence of drug awareness (Schmitt et al., 2011). It is a key element to prevent
campaigns in 2018. The public prefers to use Web Search substance abuse because people who are more aware of
to find information about drugs. Moreover, the top 5 drug use are less likely to misuse drug(Jordan & Andersen,
countries where the "drugs" gains the highest attention 2017).
from the public is different when the search method is
different. To improve drug awareness worldwide, the United
Nations office sets June 26 as the World Drug Day when
Key words : Drug awareness, Drug prevention
various campaigns and activities will be held to celebrate
campaigns, Effectiveness, Drug-related problems, Search
this date. They also decided an Action Week from June
preference, Google Trends.

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24 to June 30 worldwide. On World Drug Day 2018, Day trend can be used to test the awareness of audiences
of Action campaign was put into effect globally. And each towards the drug. Google Trendsis a useful instrument
country also made specific campaigns nationally. West, which can track the search frequency of certain topics
central and South Africa carried out "Listen First" around the world through different search methods,
campaign to raise drug awareness; India implemented a including web search, image search, news search, google
shopping, and YouTube search. Choi and Varian (2012)
"Deep Dive" campaign to promote understanding and
also claimed that Google Trends is useful to help predict
communication(UNODC, 2018); the United States
trends(CHOI & VARIAN, 2012). Thus, drug awareness
executed the activity with the theme of publication;
can be measured globally by the public's interest in
QuitStigmaNow was launched by Canada("World Drug "drugs" on the search engine.
Day 2018 - Vienna NGO Committee on Narcotic Drugs,"
This article would come up with the following questions:
2018).
1. Is there a difference in public drug awareness
The previous research has systematically explored the
before and after the drug awareness
possible factors which may cause individuals to use the
campaignsglobally in 2018?
substance. O'Hara, Armelie, and Tennen (2015) found
the role of people's intention and social circle in this 2. What are the preferred search methods for gaining
process(O'Hara, Armeli, & Tennen, 2015). Yang and Xia information about drug issues during the last year
(2019) found that the lack of knowledge towards the globally?
possible harmful impact and the lack of punishment 3. What are the top countries where drug awareness
towards drug abuse are the two main reasons for drug is relatively higher during the last year?
misuse(Yang & Xia, 2019). It was argued that drug use
4. What related topics and queries appear the most
is initially a kind of social activity but ends with social
frequently worldwide during the last year?
isolation (Tam, Kwok, Lo, Lam, & Lee, 2018). Therefore,
based on these studies, drug prevention programs are The foremost objective of this article is to figure out
carried out to improve public awareness. the effectiveness of drug awareness campaigns using
Google Trends. It also aims at revealing audiences'
However, drug awareness campaigns are not easy to
preference of search method when they searching for the
implement because they consume a large amount of
related information. In addition, the article plans to find
money and human resources to set up the whole
out the countries with relatively higher drug awareness
plan(Substance Abuse and Mental Health Services
and the top hot topics and queries related to this topic
Administration, 2014). Several crucial features of
worldwide.
effective programs are identified, including highlighting
the harmful effect, offering information on how to resist The article tries to investigate some practical
temptation, and targeting audiences sharply (Botvin & implications for drug awareness campaigners to analyze
Griffin, 2007). the quality of the campaigns and to draw more effective
plans. Although the drug misuse issue is intricate and
While the prior study has only analyzed the
prominent, the campaigners and researchers are still
effectiveness of these campaigns by systematic reviews
dedicating to find the most viable way to improve public
(Das, Salam, Arshad, Finkelstein, & Bhutta, 2016). It has
drug awareness. Knowing the effectiveness of drug
not measured the effectiveness of more comprehensively
awareness campaigns can also assist drug campaigners
and accurately.The public was also understudied
to learn fromprevious experiences for the purpose of the
previously where the public learning preference and
advancement of the next program. Reflecting the past
knowledge loophole of the drug are unclear yet. As drug
campaigns whether they are successful or not can reduce
misuse is a global issue, it has not been studied from a
or eliminate the risk of failure considering the difficulty
global level but only was targeted from the specific
of setting up a campaign. The preferred search method
country.
of the public can suggest the correct media platforms and
Currently, with the advancement of communication content forms for the content producers to target on, which
technology, audiences tend to prefer search information can help increase the circulation and traffic of the content.
on the internet through websites because of its simple Therefore, it can reach a wider public to exert the best
operation and abundant resources (Chie et al., 2015). This power. The hottest related topics and queries can indicate

Deng and Muhammad

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DOI : 10.5530/ctbp.2020.4s.21

the weakness of the drug awareness campaign and Trends during the last year, including the date of World
education programs as well as point out the future Drug Day - June 26, 2018, and the Action Week (June 24
direction for campaigners to produce personalized to June 30). The location will be chosen as worldwide.
content. Knowing the audiences better can help optimize All categories will be selected. Each set of results from
the content. The comparison between countries globally web search, image search, news search, google shopping,
could indicate the strength of some countries so that the and YouTube search will be selected as the sample to
rest can learn from the success or evade from the failure detect whether there is any difference in the public interest
of these top countries. It would help solve drug issues of "drugs" before and after the drug awareness campaigns
from a global level and as a team. in 2018.
As the second objective of this research is to find out
2. Materials and Methods
the preferred search methods during last year globally,
This article will use the qualitative method to explore the level of public interest on each search methods will
the effectiveness of drug awareness campaign and the be compared. The term is still "drugs". The location is
preferred search methods of the public to gain information worldwide. The time period is between the past 12
about drugs by analyzing the data on Google Trends months. And all categories will be contained.
which tracks the public interest of "drugs" over time In order to understand the topic more
worldwide. Because the first purpose of this article is to comprehensively, the data concerning the interest by
analyze the effectiveness of the drug awareness campaign region of "drugs", related topics and queries will be
in 2018, the sample data will be drawn from Google collected under all these five search platforms. The
Table 1: Search Strategy
Search term Drugs
Location Worldwide
Time frame Past 12 months (March 19, 2018 – March 19, 2019)
Categories All categories
Search methods Web search, image search, news search, google shopping, YouTube search
Interest by region Include low search volume regions (region areas)
Related topics and Top
queries

keyword, location, time frame, and categories will still

be the same.

3. Results and Discussion

The Effectiveness of Drug Awareness Campaign
Worldwide
Figure 1 observes a stable and high-interest level of
"drugs" after June 26, 2018. During the whole year, the
Fig 2 : The Interest Trend of "Drugs" Worldwide Using
Image Search
interest level falls between 75 and 100. And it reports no
difference before and after the drug awareness campaign
in 2018 globally.
From Figure 2, a slight rise in the interest level of
"drugs" is indicated during the period of the global drug
awareness campaigns. However, the impact of the
campaigns is not striking. According to Figure 3, public
Fig 1 : The Interest Trend of "Drugs" Worldwide Using records a short period of moderately growing focus on
Web Search "drugs" from June 24 to June 30. Based on Figure 4, a
Drug awareness campaigns using google trends
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Fig 6 : Top 5 Regions Using Web Search

Fig 3 : The Interest Trend of "Drugs" Worldwide Using

News Search

Fig7 : Top 5 Regions Using Image Search

Fig 4 : The Interest Trend of "Drugs" Worldwide Using

Google Shopping

Fig 8 : Top 5 Regions Using News Search

Fig 5 : The Interest Trend of "Drugs" Worldwide Using

YouTube Search

sharp increase is witnessed from June 24 to June 30. Fig 9 : Top 5 Regions Using Google Shopping
While Figure 5 shows a continuously and slightly
increasing public interest level of "drugs".
Preferred search methods
By comparing the above five figures, it can be
concluded that web search is the most often used method
to find information about "drugs". The interest level of
"drugs" using web search is nearly 100 over the whole
year. While the interest levels using other search methods
Fig 10 : Top 5 Regions Using YouTube Search
are comparatively lower.
Top drug awareness countries
From the following figures, it is clear that the top 5 Grenada, Jersey, and Bermuda for News Search, Canada,
countries interested in "drugs" topic are Nigeria, Liberia, Philippines, Ireland, Singapore, and India for Google
Zambia, Ghana, and Tonga for Web Search, Tonga, Shopping, Guernsey, Anguilla, Isle of Man, Jersey, and
Dominica, American Samoa, Fiji, and St. Kitts & Nevis Netherlands for YouTube Search.
for Image Search, Antigua & Barbuda, Cayman Islands,

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DOI : 10.5530/ctbp.2020.4s.21

Top related topics & related queries

The following five figures illustrated the top related
topics and queries by using five search methods. Figure
11 shows that by Web Search, the top related topics are
"Drug - Topic" and "Pharmaceutical drug - Topic"; the
top related queries are "drug", "London drugs", and "what
is a drug". It can be seen from Figure 12 that "Drug -
Fig11 : Top Related Topics and Related Queries Using Topic" and "Pharmaceutical drug - Topic" are the top
Web Search related topics; "drug" and "no drugs" are the top related
queries by using Image Search. Based on Figure 13, the
top related topics by using News Search are still the same
from using Web Search and Image Search. The top related
queries are "war on drugs", "London drugs", "avicii
drugs", and "demi lovato drugs". Based on Figure 14,
the top related topics are the same as the above results.
While the top related queries are different. "London
drugs" is the only top query using Google Shopping.
Fig 12 : Top Related Topics and Related Queries Using According to Figure 15, the top three related topics are
Image Search "Drug - Topic", "Sex and drugs - Topic", "Pharmaceutical
drug - Topic" using YouTube Search. And the top related
queries are "on drugs" and "love drugs".
Generally, the effect of globaldrug awareness
campaigns in 2018 is moderate and ephemeral. After the
campaign, the interest in "drugs" only improved a little.
This little impact fades away and the interest level drops
down immediately after the Action Week. Weiss and
Tschirhart (1994) also draw a similar conclusion that drug
Fig 13 : Top Related Topics and Related Queries Using campaigns produce a "boomerang" influence on public
News Search awareness where the effect lasts shortly(Weiss &
Tschirhart, 1994). They found that the impact only existed
during the campaign while the situation came back to
the original point after the campaign. A survey conducted
in French received a similar result that generally, the
campaign increased recipients' drug awareness partially
and inconsistently (Cuchet-Chosseler, Bocoum, Camara,
Abad, & Yamani, 2011). The campaign is mildly effective
and lasts for a short period.
Fig 14 : Top Related Topics and Related Queries Using The result also finds that the public prefers using the
Google Shopping web search to collect information they want about
"drugs". The hotness of "drugs" was highest on the web
search among these five search tools. This is consistent
with the previous research where the web search gains
more and more attention from the public (Chuklin,
Markov, & Rijke, 2015). They also indicate that people
around the world have a tendency to find information by
using web search. One advantage of web search is that
all the relevant contents are available on this instrument
Fig 15 : Top Related Topics and Related Queries Using including images, videos, news, and links to shopping
YouTube Search products (Xie et al., 2017). That means all the information

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from the rest of the search methods can be found on web 2. Chie, Q. T., Tam, C. L., Bonn, G., Wong, C. P., Dang,
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Nielsen, 2018), pharmaceutical drugs non-medical use
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Abad, B., & Yamani, E. (2011). Results of a survey
is much more prevalent than illicit drugs globally(Hulme,
to evaluate the efficacy of a regional awareness
Bright, & Nielsen, 2018). Pharmaceutical drugs have
campaign on counterfeit street medicines in Bamako,
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Mali and Nouakchott, Mauritania. Medecine
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4. Conclusion 7. Fonseca, F., Torrens, M., Farré, M., McBride, K. E.,

Guareschi, M., Touzeau, D., Dart, R. C. (2017).
Generally, the top related topics are "Drug - Topic"
Patterns of prescription drug use and misuse in Spain:
and "Pharmaceutical drugs - Topic" among these five
The European Opioid Treatment Patient Survey.
different search methods. The top related queries are
Heroin Addiction and Related Clinical Problems.
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drug awareness campaigns. It can give practical solutions source and diversion of pharmaceutical drugs for
for content producers and health professionals to target non-medical use: A systematic review and meta-
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Mahavadi, S., Rao, R.S.S.K. and Murthy, K.S. (2007).
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Sun Protection Effect of 2-Hydroxy-4-(Octyloxy) Benzophenone

in Sunscreen Creams Formulations by a Combination of
Inorganic UV Filters
Asmiyenti Djaliasrin Djalil1*, Anisa Tri Susanti1, Bella Apriani1, Muhammad Arba2, Ika Yuni Astuti1
1Faculty of Pharmacy, Universitas Muhammadiyah Purwokerto, Indonesia
2Faculty of Pharmacy, Universitas Halu Oleo, Kendari, Indonesia

Corresponding author : asmiyentidjaliasrindjalil@ump.ac.id

Abstract 1. Introduction
Overexposure of ultraviolet (UV) radiation, especially Exposure of solar ultraviolet (UV) radiation in
UVB (280-320 nm) and UVC (200-280 nm) have a excessive conditions can cause skin problems including
harmful effect on the skin. Sunscreen such as derivatives sunburn, edema, erythema, immune suppression,
of benzophenone can protect the skin from these wrinkles, dermatitis, urticarial, aging, hypopigmentation,
detrimental effects. In this research, we evaluated the hyperpigmentation, and skin cancer [1-2]. The ozone layer
potency of 2-hydroxy-4-(octyloxy) benzophenone as a can absorb 100% UVC (200-290 nm), 90% UVB (290-
sunscreen and improved the ability by combining it with 320 nm), and a little amount of UVA (320-400 nm).
the physical blocker TiO2 or ZnO in the form of a cream However, depletion of the ozone layer causes increased
formulation. We use a D-optimal mixture design to obtain UV transmission to the earth's surface. Sunscreen is used
the cream formulation with high Sun Protection Factors to reduce skin damage from UV radiation. Sunscreen is
(SPF) and acceptable characteristics. Several cream offered in many formulas like gel, lotion, stick, cream,
formulations containing 2-hydroxy-4-(octyloxy) lip balm, and spray. Gel preparations are commonly used
benzophenone and TiO 2 or 2-hydroxy-4-(octyloxy) for sunscreens because the gel provides some advantages
benzophenone and ZnO were prepared with including a cooling effect on the skin, elastic, and
concentrations of 5-10%. The creams were tested for the transparent appearance.
physicochemical parameters such as pH, color, odor, Based on the basis of its mechanism of action, there
homogeneity, viscosity, and stability. The SPF was are 2 types of sunscreens including chemical absorbers
observed by spectrophotometry and the value was (organic) which absorb the UV radiation and physical
calculated using the Mansur equation. SPF value of 2- blockers (inorganic) which reflect and scatter UV
hydroxy-4-(octyloxy)benzophenone, TiO2, and ZnO were radiation [3-4]. Previously, sunscreen only focuses to
25.21±0.47; 24.74±0.35; 3.20±0.05, respectively. SPF cover UVB radiation. Currently, the FDA recommends
value of creams combining 2-hydroxy-4-(octyloxy) the use of broad-spectrum sunscreens which cover not
benzophenone and TiO2 were in the range of 4.140-6.326. only the entire spectrum of UVB but also the UVA. Zinc
Furthermore, the SPF value of creams combining 2- oxide, a physical blocker sunscreen, was able to protect
hydroxy-4-(octyloxy) benzophenone and ZnO were in the skin from UVA radiation [5]. However, physical blocker
range of 3.609-8.052. The creams meet the requirement is less proficient against UVB protection [6]. A
of physicochemical properties with acceptable combination of sunscreen that can protect the skin not
characteristics. They were stable when the creams kept only in the UVB region but also in the UVA regions, will
at room temperature for one month. In this current study, provide added value.
the formulation of sunscreen creams with high SPF and
The benzophenones derivative, 2-hydroxy-4-
acceptable characteristics obtained by a combination of
(octyloxy) benzophenone (HOB) is commercially
10% 2-hydroxy-4- (octyloxy) benzophenone and 5%
available. The compound has been used extensively in
titanium dioxide or ZnO.
organic synthesis. Based on the structure, we considered
Key words : 2-Hydroxy-4-(octyloxy) benzophenone . the molecule to be applicable as a UV filter. Furthermore,
Sunscreencream.Sun protection factor. TiO2.ZnO the compound was prepared to develop sunscreen cream

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formulations with satisfactory characteristics. The purchased from Cognis. Olive oil and ascorbic acid were
purpose of this study was to evaluate in-vitro sunscreen from Prima Chemical.
activity of a cream formulation containing HOB and Optimization of cream formulation : The experimental
improved the ability by combining it with the physical design was a two factor two-level general factorial
blocker TiO2 or ZnO. (Design Expert 7.0.0) and seven formulations were
prepared (Table 1). The amount of HOB (X1) and TiO2
2. Materials and Methods
or ZnO (X2) were selected as independent variables. The
Materials : Zinc oxide was from KOBO. 2-hydroxy-4- number of independent variables was optimized for
(octyloxy)benzophenone was obtained from Sigma- dependent variables: viscosity and SPF. The low (5%),
Aldrich. Methylparaben, propylparaben, mineral oil, and high (10%) are the values of HOB, TiO2 or ZnO,
propylene glycol, triethanolamine, glycerin, ZnO, and Mathematical equations were generated for each
aquadest were obtained from Brataco Chemica parameter. The mathematical models were studied for
(Indonesia). Cetyl alcohol, glyceryl monostearate were significance.

Table 1 : Composition of cream formulations with different amount of 2-hydroxy-4-(octyloxy) benzophenone, TiO2,
and ZnO
Ingredients Run1 Run2 Run3 Run4 Run5 Run6 Run7 Negative Control
Ingredients A
Cetyl alcohol (g) 1 1 1 1 1 1 1 1
Mineral oil (g) 10 10 10 10 10 10 10 10
Olive oil (g) 10 10 10 10 10 10 10 10
Propylparaben (g) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
Glyceryl monostearate(g) 16 16 16 16 16 16 16 16
Ingredients B 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
Methylparaben (g) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
Glycerin(g) 7 7 7 7 7 7 7 7
Ascorbic acid 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
HOB 5 10 10 6.25 8.75 7.5 5 0
TiO2/ZnO 10 5 5 8.75 6.25 7.5 10 0

Aquadest ad (ml) qs ad to 100 100 100 100 100 100 100 100

Cream formulation : The basic sunscreen formulation Physicochemical evaluation : Physicochemical

was prepared according to the formula recommended by evaluation of cream was tested for homogeneity, pH,
EIRI with some modifications [7]. The Ingredients B viscosity, and spreadable. Homogeneity was observed by
(methylparaben, glycerin) were first heated (70ºC) as applying the cream on a glass object. The cream must
well as ingredients A (glyceryl monostearate, cetyl give indicate of a non-grained appearance and no visible
alcohol, mineral oil, and propylparaben). The ingredients spot. The pH was measured using a pH meter. Viscosity
A were poured into a warm porcelain mortar and a was measured in Brookfield viscometer (DV-1 Prime)
required quantity of olive oil was added to the mixture. using a LV-4 spindles and a rotation rate of 60 RPM.
The water phase (B) was added slowly to the oil phase Finally, the spreadable test of the cream was measured
(A). The ascorbic acid was added and grinded well. After by applying the cream (500 mg) between two pieces of
that, ZnO or TiO2 was dissolved in hot distilled water circle glass plates (diameter 15 cm) for one-minute
and added to the mixture. Finally, HOB was added to compression. After that, the standard weight (50 g) was
form a homogeneous cream. applied to the upper plate. After one minute's
Organoleptic evaluation : All creams were observed for compression, the spreading diameter of the cream was
physical form including texture, color, and odor. These measured. All measurements were made in triplicate.
physical characteristics were observed by visual tested. Determination of SPF value of HOB, TiO2, ZnO, and
cream formulation : Samples were prepared according

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to the method recommended by Dutra [8]. One gram of HOB, ZnO, and TiO2 at UV region (200-400 nm) were
sample was weighed and diluted to 100 ml with ethanol. recorded and shown in Figure 1. It presented that the
The solution was ten milliliters. Afterwards 5.0 ml of absorbance of HOB in the UVB region was higher than
filtered solution was transferred to a 50 mL volumetric that of UVA and part of UVC. The absorbance of TiO2 in
flask, adjusted with ethanol. The absorbance of the final the UVA and UVC region were higher than that of HOB.
solution was measured by spectrophotometry in the range On the other hand, ZnO has relatively similar absorbance
of 290 to 320 nm for every 5 nm wavelength interval. throughout the UV region, and also ZnO has a greater
Finally, the SPF value was calculated using the Mansur absorbance in the UVA region than that of HOB. The
equation [9]. SPF value of HOB, TiO2, and ZnO were 25.2±0.5;
24.7±0.3; and 3.2±0.05 which is considered ultra, ultra,
2. Results and Discussion
and extra protection, respectively, in terms of sunscreen
Today, many strategies are proposed to obtain protection.
sunscreens with high SPF values and can cover a broad
spectrum of UV radiation. One strategy is to combine
UVA and UVB filters as well as chemical and physical
sunscreens. This sunscreen combination method tends
to be preferred because it only requires a preparation
process that is identical to the conventional one, and only
the required formulation ingredients are adjusted. In this
study, we used ZnO or TiO2 as a physical sunscreen and
HOB as a chemical sunscreen.
The previous study in our research group show that
the HOB has a higher SPF value of 25.2±0.5 compared
with benzophenone-3,3',4,4'-tetracarboxylate dianhydride Fig. 1. Absorption curve of the solution of 2-hydroxy-4-
(6.4±0.2) or 2-benzoylbenzoic acid (2.7±0.4) [10]. The (octyloxy)benzophenone, ZnO, and TiO2 (right). The
SPH value was also higher than that of SPF of natural concentration of 1% in ethanol.
sunscreen corn cob (4.95±0.86) [11]. The absorbance of

Table 2 : Physicochemical parameter evaluation of HOB-TiO2 creams

Formulation Color Odor pH Homogeneity Viscosity (cps) Spreadability

(cm)
Negative White Characteristic of 4.01±0.01 Homogenous 9136.66±106.92 4.66±0.07
control olive oil
Run1 White Characteristic of 5.95±0.02 Homogenous 9546.66±94.51 4.65±0.10
olive oil
Run2 White Characteristic of 5.90±0.02 Homogenous 7853.33±96.09 4.21±0.11
olive oil
Run3 White Characteristic of 6.56±0.02 Homogenous 8236.66±105.98 4.65±0.06
olive oil
Run4 White Characteristic of 5.58±0.03 Homogenous 8956.66±80.82 4.24±0.10
olive oil
Run5 White Characteristic of 5.09±0.03 Homogenous 8426.66±96.09 4.91±0.06
olive oil
Run6 White Characteristic of 5.86±0.03 Homogenous 8466.66±96.09 4.91±0.05
olive oil
Run7 White Characteristic of 6.19±0.02 Homogenous 9756.66±105.98 4.30±0.01
olive oil

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Table 3 : Physicochemical parameter evaluation of HOB-ZnO creams

Formulation Color Odor pH Homogeneity Viscosity (cps) Spreadability
(cm)
Negative White Characteristic 4.01±0.01 Homogenous 9136.66±106.92 4.66±0.07
control of olive oil
Run1 White Characteristic 7.04±0.020 Homogenous 8823.33±76.37 4.24±0.07
of olive oil
Run2 White Characteristic 7.02±0.015 Homogenous 8783.33±65.06 4.22±0.09
of olive oil
Run3 White Characteristic 7.00±0.015 Homogenous 8636.66±55.07 4.25±0.04
of olive oil
Run4 White Characteristic 6.92±0.030 Homogenous 8323.33±100.16 4.19±0.05
of olive oil
Run5 White Characteristic 7.10±0.023 Homogenous 7870±62.44 4.10±0.07
of olive oil
Run6 White Characteristic 7.08±0.025 Homogenous 8143.33±25.16 4.13±0.08
of olive oil
Run7 White Characteristic 7.05±0.011 Homogenous 8696.66±115.90 4.20±0.07
of olive oil

Table 4 : SPF of HOB-TiO2 and HOB-ZnO cream

The formulated sunscreen creams were evaluated for formulations
several physicochemical tests and the results were
displayed in Table 2-3. The formulate creams showed
SPF
good acceptable odor, characteristic odor of olive oil, and
Combination Combination
Formulation
of HOB and of HOB and
were white color. The viscosity of formulated sunscreen
TiO2 ZnO
creams ranged from 7853 to 9546 cps. The concentration
Negative
of TiO2 has a positive effect on the viscosity. It can be 0.580±0.005 0.498±0.014
control
concluded that the creams have good viscosity (2.000- Run1 4.872±0.018 4.888±0.067
50.000 cps, SNI). Viscosity is an important parameter Run2 6.326±0.013 8.052±0.022
for evaluating cream preparations and the characteristics Run3 6.164±0.017 7.606±0.018
are related to the spreadable of the creams. Run4 4.140±0.011 3.610±0.009
The pH value of formulated sunscreen creams Run5 6.389±0.018 5.694±0.037
combination of HOB and TiO2 ranged from 5.09 to 6.56. Run6 5.063±0.011 6.419±0.022
Whereas the pH value of formulated sunscreen creams Run7 4.489±0.018 4.531±0.034
combination of HOB and ZnO ranged from 6,92 to 7.08. To optimize the creams for viscosity and SPF value, a
Cream combination of HOB and TiO2 has a lower pH general factorial method is applied in this study. The
value than ZnO. All of formulated sunscreen creams meet amount of HOB and TiO 2 /ZnO was chosen as
the requirements for sunscreen preparation ranging from independent variables. A statistical model was used to
4.5-8.0 [12]. The creams would not irritate if applied to observe the responses.
the skin.
The ANOVA (analysis of variance) of HOB-TiO2
The SPF values of the sunscreen creams containing creams displayed that the viscosity was appropriate to
both HOB and TiO2/ZnO in various concentrations were explain the main effect model (P-value 0.0024) as well
displayed in Table 4. The results show that the SPF values as SPF (P-value 0.0408). By developing a normal
in the UVB region were directly dependent on HOB or probability plot of internally studentized residuals, a
TiO2/ZnO concentrations. The creams have a low SPF patterned was made for the normality statement (Fig. 2).
values compare with the active compounds, this is due to Furthermore, the ANOVA of HOB-ZnO creams
their low amount of active compounds. showed that the viscosity was appropriate to explain the
main effect model (P-value 0.0294) as well as SPF (P-

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DOI : 10.5530/ctbp.2020.4s.22

Y = 6,41A + 4,52B - 1,34AB (2)

Y = SPF
A = Concentration of HOB
B = Concentration of TiO2
The same way was used to observe the responses of
Fig. 2. Plot of internally studentized residuals vs. predicted viscosity and SPF to the transformed factors of HOB-
response of viscosity (left) and SPF (right) of HOB-TiO 2 ZnO creams. The results were shown in equation 3-4,
creams. and Fig 5.

value 0.0368). A normal probability plot of internally

studentized residuals was displayed in Fig 3. The
statement of normality is fulfilled when the residual plot
is approached along a straight line. The plot is acceptable,
so we conclude that the resulting general factorial
equation can be used in predicting SPF or viscosity of
HOB-TiO2 creams or HOB-ZnO creams. Fig. 5. The graph relates to the response of viscosity (left) and
SPF (right) to the transformed factors of HOB-ZnOcreams.

Y = 1,24A + 0,59B - 0,15AB (3)

Y = viscosity
A = Concentration of HOB
B = Concentration of ZnO
Fig. 3. Plot of internally studentized residuals vs. predicted
response of viscosity (left) and SPF (right) of HOB-ZnO creams. Y = 1,24A + 0,59B - 0,15AB (4)
Y = SPF
The fitted equation relating the responses viscosity to A = Concentration of HOB
the transformed factors of HOB-TiO2 creams was shown B = Concentration of ZnO
in equation 1 and Fig 4. The equation shows a positive
interaction with HOB of 8082.88 and TiO2 of 9628.80. It The desirability value was used to find out the best
means that HOB and TiO2 will increase viscosity, and viscosity and SPF of 7 runs. The optimized of HOB-TiO2
TiO2 has the greatest effect on increasing viscosity cream formulation obtained by using design experts was
compare with HOB. In addition, there is a negative a combination of 10% of HOB and 5% of TiO2, with
interaction with the combination of HOB and TiO2 with viscosity of 8083 cps, SPF 6.4, and desirability of 1.
an interaction coefficient of -1196.14. Furthermore, the Furthermore, the optimized of HOB-ZnO cream
fitted equation relating the responses SPF to the formulation was a combination of 10% of HOB and 5%
transformed factors is shown in equation 2 and Fig 4. of ZnO, with viscosity and SPF of 8651 cps and 7.8,
respectively. The desirability obtained by the software
was 0.940.

4. Conclusion
The 2-hydroxy-4-(octyloxy) benzophenone, TiO2, and
ZnO were exhibited sun protection activity. The
benzophenone combined with TiO 2 or ZnO can be
Fig. 4. The graph relates to the response of viscosity (left) and formulated as a cream with satisfying physical
SPF (right) to the transformed factors of HOB-TiO2 creams. characteristics. In this current study, the formulation of
Y = 8082,88A + 9628,80B - 1196,14AB (1) sunscreen creams with high SPF and acceptable
Y = viscosity characteristics obtained by a combination of 10% 2-
A = Concentration of HOB hydroxy-4-(octyloxy)benzophenone and 5% TiO 2 or
B = Concentration of TiO2 ZnO.

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Acknowledgement 7. EIRI Board of Consultants and Engineers. (2007).

The authors are very grateful to LPPM UMP for Cosmetics Processes and Formulations Hand Book
financial support through the Applied Research Grant with Herbal Cosmetics Technology and Formulae,
2019. Engineers India Research Institute, India.
Conflict of Interest 8. Dutra, E.A., da Costa e Oliveira, D.A.G. Kedor-
The Authors declare that they have no conflicts of Hackman, E.R.M., Santoro, M.I.R.M. (2004).
interest. Determination of Sun Protection Factor (SPF) of
Sunscreens By Ultraviolet Spectrophotometry. The
5. References Brazilian Journal of Pharmaceutical Sciences,
1. Polefka, T.G., Meyer, T.A., Agin, P.P., Bianchini, R.J. 40(3):381-385.
(2012). Effects of Solar Radiation on the Skin. 9. Mansur, J.S., Breder, M.N.R., Mansur, M.C.A.,
Journal of Cosmetic Dermatology, 11:134-143. Azulay, R.D. (1986). Determinação do Fator de
2. Donglikar, M.M. and Deore, S.L. (2016). Proteção Solar por Espectrofotometria'. Anais
Sunscreens: a Review. Pharmacognosy Journal, Brasileiros de Dermatologia. 61:121-124.
8(3):171-179.
10. Djalil, A.D., Ambarwati, T., Genatrika, E. (2018).
3. Gabros, S., Zito, P.M. (2019). Sunscreens and Characterization of Sunscreen Cream Containing
Photoprotection, StatPearls Publishing LLC, Benz op heno ne- 3 , 3 ' , 4 , 4 ' - t et r a ca r b ox yl a t e
Maryland. dianhydride. IOP Conference Series: Materials
4. Rai, R., Shanmuga, S.C., and Srinivas, C.R. (2012). Science and Engineering, 434: 012090.
Update on Photoprotection. Indian Journal of 11. Djalil, A.D., Chandra, T.B. (2018). Formulation and
Dermatology, 57(5):335-342. Characterization of Sunscreen Lotion From Corn
5. Kullavanijaya, P., Lim, H.W. (2005) Photoprotection. Cob (Zea mays L.) extract. in Resources
Journal of the American Academy of Dermatology, Development Toward Civil Society Based on Local
52(6):937-958. Wisdom, Headway Global Research Consultancy
PTE LTD, Singapore.
6. Latha, M.S., Martis, J., Shobha, V., Sham Shinde,
R., Bangera, S., Krishnankutty, B., Bellary, S., 12. [SNI] Standar Nasional Indonesia. (1996).
Varughese, S., Rao, P., Naveen Kumar, B.R. (2013). SediaanTabir Surya. Dewan Standarisasi Nasional,
Sunscreening Agents: a Review. The Journal of SNI 16-4399-1996.
Clinical and Aesthetic Dermatology, 6(1):16-26.

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Current Trends in Biotechnology and Pharmacy 194
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DOI : 10.5530/ctbp.2020.4s.23

In Silico Studies of Green Tea Catechins Against HER-2

Receptor in Breast Cancer
Fitriyani1 2*, Taufik M. Fakih3, Daryono H. Tjahjono2
1Faculty of Pharmacy, Universitas Muhammadiyah Purwokerto, Banyumas 53182, Indonesia
2School of Pharmacy, Bandung Institute of Technology, Bandung 40132, Indonesia
3Faculty of Mathematics and Natural Sciences, Universitas Islam Bandung, Bandung 40116, Indonesia
Corresponding author : fy.fitriyani19@gmail.com

Abstract type of cancer in Indonesia. In the development of breast

Green tea catechins have been widely studied and cancer, there are some protein roles. One of the proteins
known to have anticancer activity, including breast cancer. is HER-2. HER-2 is a transmembrane receptor tyrosine
Breast cancer is cancer with the highest prevalence in kinase that activates multiple proliferative signaling,
Indonesia, after cervical cancer. HER-2 (Human including PI3K/ Akt and Ras/ MAPK(1). In normal cells,
Epidermal Growth Factor Receptor-2) has a crucial role HER-2 has a role in cells' growth and proliferation. In the
in the development of breast cancer. Thus, this protein is case of HER-2 positive breast cancer, the excessive HER-
widely used as a therapeutic target. In this study, catechin 2 expression causes the increase of cancer cell activity,
activities on HER-2 Receptor Tyrosine Kinase (RTK) and the tumor which grows faster, is more aggressive, is
domain of breast cancer are investigated through in silico less sensitive to hormone therapy, and chemotherapy (2).
study. Four catechin compounds, namely EGCG, EGC, HER-2 has been widely studied as a therapeutic target
ECG, EC, and native ligand, were given docking and for HER-2 positive breast cancer. In HER-2 positive breast
molecular dynamic simulations. Molecular docking is cancer, the amount of HER-2 overexpresses around 20-
used to study the interaction of protein-ligand using 30%. Recently, most patients with advanced HER-2
AutodockTools. The stability of amino acid residue positive breast cancer do not recover from their illness.
interaction with catechin was identified through molecular They, on the contrary, getresistance to therapeutic agents
dynamics using GROMACS and the binding free energy that target HER-2 (3).
was calculated using MM-PBSA. Among the four There are many in vitro and in vivo researches showing
catechin compounds, EGCG has the best RMSD value. the correlation between consuming green tea and a
This indicates that EGCG has the best structural stability. reduced risk of breast cancer (4)(5). The ability of green
The value of binding free energy ( G) of catechin tea to protect against breast cancer seems to be mediated
compounds is greater than the native ligand, showing that by catechins, which are polyphenol (6)(7). The greatest
the compounds have a lower affinity for HER-2. The result compounds of catechin is epigallocatechin-3-gallate
reveals that catechin compounds have lower activity than (EGCG), epigallocatechin (EGC), epicatechin-3-
the native ligand. However, catechin compounds have the gallate(ECG), and epicatechin (EC) (8).
same active site, and three catechin compounds can also
Catechins have been known to have many therapeutic
interact with ASP863 which is an important residue in
activities, including anticancer activities(9). Green tea
HER-2. Therefore, catechin compounds, especially
catechins have been proven to inhibit breast cancer cell
EGCG, EGC, and ECG are potential to be developed into
proliferation and block carcinogenesis (10). In this
HR-2 inhibitors through structural modification.
research, the activity of catechins against HER-2 Tyrosine
Key words : Catechin. Molecular docking. Molecular Kinase domain of breast cancer is investigated through
dynamics. HER-2. Breast cancer. in silico study.
1. Introduction 2. Materials and Methods
Breast cancer is cancer commonly suffered by women. Materials: The crystal structure kinase domain of HER-
Based on IARC (International Agency for Research on 2(human epidermal growth factor receptor 2) receptor
Cancer) data in 2018, breast cancer is the most common were obtained from protein data bank (PDB code: 3PP0)

In silico studies of green tea catechins

Current Trends in Biotechnology and Pharmacy 195
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(11). Catechin structures (EGCG, EGC, ECG, EC) were chlorine and sodium atoms were added to neutralize the
made using ChemDraw Ultra 8.0, and Chem3D Ultra system. The next step is equilibration to make the whole
8.0software. system at constant temperature and pressure. All MD
simulations were conducted for 10 ns. The trajectories
The used software was AutoDock 4.2.6.and
were analyzed and visualized using Discovery studio.
AutoDockTools 1.5.6 (The Scripps Study Institute,
downloaded in http://www.autodock.scripps.edu/), 3. Results and Discussion
BIOVIA Discovery Studio 2017 (http://
The Human Epidermal Growth Factor Receptor type
www.accelrys.com/u), Gromacs v.5.1.1
2 (HER-2) is a member of the oncogenic proteins family,
(www.gromacs.org), ChemDraw Ultra 8.0 (https://
which is the main target of cancer therapy including breast
chemistry.com.pk/software/free-download-chemdraw-
cancer (16). The structure of tyrosine kinase of HER-2
ultra-8/), Gaussian 09 (https://gaussian.com).
can be seen in Fig. 1. In this research, the four catechin
Protein and ligand preparation : The crystal structures compounds of Camellia sinensis were docked into binding
of HER-2 kinase domain obtained from protein data bank pocket of HER-2 as their native ligands.
(12)(3PP0) were in the complex form and native ligand.
The protein was separated from the ligand, was removed
all its water molecules and was added with hydrogen
atom. In this research, catechins (EGCG, EGC, ECG, EC)
were used as the ligand. The ligand structures (2D and
3D) were made using ChemDraw Ultra 8.0, then were
determined their physicochemical parameters. The 3D
ligand structure was optimized through Gaussian using
DFT/ B3LYP (13) with a STO-3G basis set to reach its
stationary points.
Molecular docking : Molecular docking was conducted
through AutoDockTools v.4.2.3. Software. The validation
of molecular docking was conducted by re-docking the
native ligand (14). Crystal structures of HER-2 kinase
domain have 2-{2-[4-({5-chloro-6-[3- (trifluoromethyl)
phenoxy] pyridin-3-yl} amino) -5H-pyrrolo[3,2-
d]pyrimidin-5-yl] ethoxy} ethanol as the native ligand
(11) and the surrounding residues, which are defined as Fig. 1. The crystal structure kinase domain of HER-2
the active site with geometric position (X = 16.38, Y = Molecular docking
17.39, Z = 26.21 Å). The pose with the lowest binding Validation of the docking process was conducted
energy, obtained from the docking result, was then chosen through re-docking method using AutoDock. The
for MD simulation. validation was conducted in the active site of the native
Molecular dynamics simulation : All MD simulations ligand on crystallographic results. The re-docking result
were carried out using GROMACS v.2016.3 package with indicates RMSD value of 1.19 Å, meaning that the atom
AMBER 99SB force field (15). The partial charges and position inside the ligand of the re-docking result is not
topology files of ligands were produced by ACPYPE, a much different from the position of crystallographic
tool based on ANTECHAMBER, with the intermolecular ligand (14). This result indicates that this method can be
potential represented as a sum of Lennard-Jones (LJ) used for docking process.
force and pairwise Coulomb interaction, electrostatic The optimized catechin compounds were docked to
force was determined by the particle mesh Ewald (PME) HER-2 protein through Autodock Tools using the same
method. Initial atomic velocities were created on the basis procedures and coordinates as when being validated.
of Maxwellian distribution at the absolute temperature Catechin compound docking on Her-2 protein obtained
of 310 oK. Numerical integrations were calculated by ten poses. Then, the lowest binding energy value, showing
the velocity Verlet algorithm. The system was solvated the most stable binding, among the poses was selected.
in a cubic box with TIP3P water model, and then sufficient The docking result reveals that the binding energy

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between catechins and HER-2 protein is negative. The important residues in the native ligand, SER728,
However, the catechins had higher binding energy than MET801, THR862 and ASP863, can be seen in Table 2.
the native ligand. This indicates that the potential of Based on the docking result, catechin compounds can
catechins in binding the active sites of HER-2 is weaker interact with HER-2 protein through hydrogen bond in
than the native ligand, but catechins still have the ability SER728, MET801, THR862 and ASP863 amino acids
to bind HER-2 receptors. (shown by the green circle in Fig. 2). This indicates that
Table 1: Docking results between Catechins with HER- the active sites between the native ligand and the catechin
2 receptor in HER-2 protein are the same so that they will produce
the same activities as the native ligand in inhibiting HER-
2 protein. Visualization of the docking result and the
Ligand G (kcal/mol)
interaction between catechin with HER-2 protein are
Epigallocatechin-3-gallate (EGCG) -6.94 shown in Fig. 2 and Table 2.
From Ashtekar's research, it was found that ASP863
Epicatechin-3-gallate (ECG) -7.07
is a very important amino acid residue in HER-2. Of the
Epigallocatechin (EGC) -7.02 several HER-2 inhibitors, only Lapatinib and Neratinib
interact with ASP863 residue, which is why both drugs
Epicatechin (EC) -7.63
specifically target HER-2(17). In this study, information
Native ligand -10.48 was obtained that three catechin compounds namely
EGCG, EGC, and ECG can interact with ASP863 residue
The value of binding free energy is shown in Table 1.
Table 2 : Atomic Interaction Between Ligands with The Kinase domain of HER-2 receptor

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DOI : 10.5530/ctbp.2020.4s.23

ns) for the simulation is shown in Fig. 3. Based on the

Fig. 3, it can be seen that the native ligand complex has
lower RMSD value than the catechin-receptor complex.
This indicates that the structure of the native ligand
complex is more stable.

Fig. 2. Catechin interactions with the kinase domain of

HER-2
thus three compounds are potential hits to develop as
HER-2 inhibitors.
Fig. 4. RMSF plot of each amino acid residue during 10
Molecular dynamics
ns simulation. EC (black), ECG (red), EGC (green),
Molecular dynamic simulation is crucial for EGCG (blue), and native ligand (yellow).
understanding changes of protein conformation over time
(18). Molecular dynamics was conducted to evaluate the RMSF is calculated for every amino acid residue of
dynamic behaviour of catechins in the complex with protein, which is to see the extent of fluctuations in the
kinase domain of HER-2 (PDB ID: 3PP0) during 10 ns. movement of each amino acid residue during the
This study analyses some parameters such as RMSD simulation. The plot for ligand RMSF (nm) versus ligand
protein-ligand, RMSF and the formed protein-ligand atom index is shown in Fig. 4. The low flexibility of amino
interaction. acid residues shows the stable interaction in the active
RMSD analysis is used to obtain insight of the site bond to the test compound. This is because the atoms
structural conformation happened to protein during forming the amino acid residues tend not to change much
simulation. The plot for HER-2 protein versus time (10 from their position during molecular dynamic simulation.
From Fig. 3, it can be seen that native ligand complex
(native ligand-HER-2) has the lowest value compared to
catechin complex (Catechin-HER-2). This indicates that
native ligand?HER-2 complex has a better stability than
catechins complex.
To assess the affinity of each catechin compound
against HER-2, the calculation of binding free energy
was conducted using MM-PBSA method (19). Binding
free energy is the total result of several components,
namely electrostatic, van der Waals, and non-polar
desolvation energy, in the interaction of receptor-ligand
which can be calculated directly using some
conformations of the simulation result trajectory. Table
3 shows that bond free energy of receptor HER-2- native
ligand complex is lower than HER-2catechin complex.
Fig. 3. RMSD plot of each ligand-HER2 complex during This indicates that catechin compounds have a bad
10 ns simulations. EC (black), ECG (red), EGC (green), affinity against HER-2 receptor.
EGCG (blue), and native ligand (yellow).

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DOI : 10.5530/ctbp.2020.4s.23
Table 3 : Binding Free Energy
G) (kJ/mol)
Ligand
Molecular Docking Molecular Dynamic
Epigallocatechin-3-gallate
-29.03696 -64.709 +/- 11.880
(EGCG)
Epicatechin-3-gallate (ECG) -29.58088 -87.123 +/- 16.580
Epigallocatechin (EGC) -29.37168 -50.341 +/- 11.212
Epicatechin (EC) -31.92392 -58.757 +/- 14.551
Native ligand -43.84832 -153.205 +/- 12.199

There are differences in the value of binding free Acknowledgements

energy obtained from the docking and molecular dynamic The authors thank to Bandung Institute of Technology
results, see Table 3. Based on the energy value, for providing facilities to execute this work.
epicatechin-3-gallate (ECG) has the best binding energy
Conflict of Interest
among the other catechin compounds. This result is in
line with the number of the interaction between ECG and The author declaire that there is no conflict of interest.
HER-2 receptor. Even though the catechin compounds
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Bacus, S. (2007). HER2 therapy. Small molecule
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can interact with the HER-2 receptor, but the binding C., Lu, J-L., et al. (2016). Suppressive Effects of
free energy of the catechin-HER-2 receptor complex is Tea Catechins on Breast Cancer. Nutrients, 8(458):
higher than the native ligand-HER-2 receptor complexs. 1-15.
This indicates that the catechin compounds has a poor
5. Seely, D., Mills, E.J., Wu, P., Verma, S., Guyatt, G.H.
affinity for HER-2 receptors.The Catechin compounds
(2005). The effects of green tea consumption on
binding several amino acid residues that are the same as
incidence of breast cancer and recurrence of breast
in native ligands, this indicates that they are binding to
cancer: A systematic review and meta-analysis.
the same active site. In addition, three catechin
Integrative Cancer Therapy, 4(2):144-155.
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important residue in HER-2 (17). Therefore, catechin 6. Rafieian-Kopaei, M.and Movahedi, M. (2017).
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DOI : 10.5530/ctbp.2020.4s.23

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DOI : 10.5530/ctbp.2020.4s.24

Revisiting the Intractable Barriers Affecting Medication Adherence

Among Outpatients with Schizophrenia
Julaeha Julaeha1 2*, Umi Athiyah2*, Verra Yuliana3, J.P Ayuningtyas3, Andi Hermansyah2
1Faculty of Pharmacy,17 Augustus 1945 Jakarta University, Jakarta, Indonesia
2Department of Pharmacy Practice, Airlangga University, Surabaya, Indonesia
3Menur National Mental Hospital, Surabaya, Indonesia
*Corresponding Author : julaeha-2016@ff.unair.ac.id, umi-a@ff.unair.ac.id

Abstract be optimal to prevent patient from relapse and

Medication adherence is one of the foremost problems hospitalization (1). Low adherence has been evident in
affecting antipsychotic efficacy in schizophrenia patients. many patients with schizophrenia contributing to a
Medication nonadherence among schizophrenia patients number of severities including higher risk of suicide and
has been often estimated > 50%, leading to higher rates financial burden which affects not only patients but also
of relapse and hospitalization as well as to decreasing their families and care givers (2). Several publications
cognitive and functional prognosis. The purpose of the have reported significant portion of non-adherence in the
study is to identify the strategy for improving medication case of schizophrenia ranging from 40% to 70% (3). In
adherence in schizophrenia and evaluate adherence using fact, 75% of patient with schizophrenia stop taking their
Medication Adherence Rating Scale (MARS) and medication within 18 months (4).
determinant factors affecting adherence. Prospective Poor adherence in schizophrenia can be associated
study with cross sectional design was conducted from with a number of factors such as social isolation,
October to December 2019. Especially data from stigmatization and comorbidities substance misuse of
schizophrenia outpatients in one of national mental psychotropic medication (5). As adherence is a complex
hospital in Indonesia. Schizophrenia outpatients were phenomenon, these factors may be exacerbated by a wide
majority male (60%), the age range from 31-49 years were variety of other causes such as lack of illness awareness,
70%, most of patients are single (63,33%), 70% have the adverse effect of the medication, the long-term
secondary education, 70% of them are from Surabaya treatment and the fragmented health care services for
area, and half of them their duration of the disease from 1 patient with mental health issues. Such condition may be
to 5 years. This study showed that the pattern of
increased yet undetected in the outpatient setting as patient
prescription of antipsychotics are risperidone and
will need to undertake and be responsible for the
clozapine were the most antipsychotics prescribed. 40%
medication at their own risk (5).
of patients have good adherence, 40% of patients have
partial adherence, and only 20% of patients' poor The risk of non-adherence in the outpatient setting
adherence. Most of schizophrenia outpatients have cannot be neglected. A systematic review of longitudinal
experience in forget to take his/her medicine and careless studies reported that there were 27% of individuals with
at times about taking his/her medicine and less knowledge schizophrenia who had poor outcome after the first
about schizophrenia. In other hand, 100% patients have episode of psychosis (6). Another study indicated that
agreed by staying on medication, it can prevent getting 82% of patients would likely to suffer first relapse and
sick. The mental hospital should utilize educational 78% would continue to suffer the second relapse after
program to improve patient's awareness about their the first episode of psychosis (7).
disorder and their medications to improve their adherence. The causes of non-adherence include the patients
Key words : Schizophrenia,Antipsychotics, SGAs, factors as fear of adverse effects, physical and psychiatric
Medication Adherence, MARS, Mental Health conditions, forgetfulness, external distractions,
misunderstanding instructions, lack of insight and lack
1. Introduction of information about disorders. Treatment factors as
It is not easy to maintain medication adherence on numerous medications, enduring symptoms, partial or no
patient with schizophrenia. Although the advances in efficacy. Social economic factors as lack of income,
psychopharmacology have greatly improved the range of transportation, living alone, and stigma of mental illness
options for treating schizophrenia, the outcome may not (8).

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DOI : 10.5530/ctbp.2020.4s.24

Schizophrenia is one of nine chronic disease covered Instrument : The Medication Adherence Rating Scale
by national health covered in Indonesia (9). In Indonesia, (MARS) tool was used for measuring level of adherence.
the number of relapse on schizophrenia patients was It was formerly evolved and validated by Thompson et
reported. The number of relapsed had significant al., to evaluate treatment compliance specifically in
correlation with medication non-adherence. The common people under antipsychotic treatment. It was designed to
problems of medication non-adherence among assess both the patients attitude towards medication and
schizophrenia patients in Indonesia were social economic, also actual medication taking behavior. the reliability
attitudes to medication, knowledge, and family support analysis of the MARS using cronbach's alpha was 0.75
(10). (14). The validity and reliability of MARS with large
sample (N=319) by Fond et al., a coefficient were close
Medication non-adherence will escalate the risk of
to 0.6 (15). It was translate into Indonesia and validated
recurrences, hospital admission rate and medication
by Yuliana et al., with reliability result 1.107 (16).
expense (11). The cost of re-hospitalizations and non
adherence per year were 100 billion USD and 290 USD MARS consists of three parts questions/statements;
(12). In Indonesia, cost of illness schizophrenia was question 1-4 represent treatment adherence behavior,
estimated 32 million IDR/year/patient (13). The objective question 5-8 represent attitude toward taking medicines
of this study is to identify the strategy for improving and question 9-10 represent adverse effects and attitudes
medication adherence in schizophrenia and evaluate to antipsychotic treatment. Every question or statement
adherence using Medication Adherence Rating Scale should be answered with a 'YES' or 'NO' answer. A
negative response indicate with non-compliance is code
(MARS) and determinant factors affecting adherence.
as zero. Whereas a positive response indicate with
2. Materials and Methods compliance is coded as one.
Design : Prospective study with cross sectional design For questions 1-6 and 9-10 an 'disagree' answer is
was conducted from October to December 2019. This indicate of positive response and hence should be coded
study has been approved by the Ethic Committee of the as one. In opposed for questions 7-8 a 'agree' answer
Menur Mental Hospital with number of ethical approval pointing to positive response and hence should be coded
070/7556/305/2019. Especially data from schizophrenia as one. The whole of adherence scoring range between
outpatients in one of national mental hospital in Indonesia. nil (non-compliance) to ten (compliance), with a greater
Non probability sampling (purposive sampling) all score pointing good attitudes and behavior towards
schizophrenia patients who registered as an outpatient positive compliance. Patient with total score < 5 (non
national mental hospital in the chosen sitting and fulfill adherence), 5-7 (partial adherence), and ? 8 (good
the inclusion criteria was selected. adherence).

Subjects : The inclusion criteria are patient with Processing and analyzing data : Statistical Package for
schizophrenia, being adult aged 18 or older, who agree social Science (SPSS) version 24 was used. The following
to participate in the study, and patient who have insight. statistical measured were used as descriptive measures
The exclusion criteria are patient who have other mental as numbers, percentage, mean and standard deviation.
disorder and patients diagnosed with brain dysfunction Analytical statistics as T-test independent sample and
or cognitive impairment. The minimum sample size for Analysis of Variance one away.
descriptive quantitative research not less than 30. The 3. Results and Discussion
participants were 30 patients. Informed consent was
Characteristic of schizophrenia outpatients and
obtained from all participants after explaining the study
medication
and its objectives. Participants were included only after
they signed the informed consent. All researchers ensured Table 1 reveals that male were majority (60%), the
participant data confidentiality and compliance with the age range from 31-49 years were 70%, most of patients
Declaration of Helsinki. This study was conducted in one are single (63,33%), 70% have secondary education, 70%
of national mental hospital in Indonesia. Participants were of them are from Surabaya area, and half of schizophrenia
interviewed regarding their history of mental illness, patients have mental disorder with a range duration of 1
sociodemographic characteristic, and pharmacological to 5 years. Table 2 shows that the pattern of prescription
of antipsychotics are risperidone and clozapine were the
treatment.
most antipsychotics prescribed for schizophrenia

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Table 1. Characteristic of schizophrenia outpatients extrapyramidal syndrome and increased quality of life
Characteristics N % (17).
Gender Level of adherence and attitude towards medication
Male 18 60 The effectiveness of the medication is impacted and
Female 12 40 the chance of recurrence will be elevated when people
Age (year) with schizophrenia discontinue taking medication. The
18-30 4 13.3 results of this study, only six patients (20%) have poor
31-49 21 70 adherence (see figure 1). This results was in the line with
50-65 4 13.33 Kamali et al., who reported that schizophrenic subjects
>65 1 3.33 had poor adherence lower than schizophrenic subjects
Marital status had good adherence (18). Contrasting to other studies,
Single 19 63.33 who informed that lack of compliance was found in about
Married 10 33.33 half of people suffering from schizophrenia (19,20,21).
Divorced 1 3.33
Educational level
Elementary school 4 13.33
Junior high school 9 30
Senior high school 12 40
College or higher 5 16.67
Occupation
Full time 11 36.66
Part time 5 16.67
Not worker 14 46.67
Duration of treatment (year)
1-5 15 50
6-10 10 33.33 Figure 1. Number of level of patients adherence
11-15 5 16.67
Number of antipsychotic In other hand, table 3 indicated that most of patients
Monotherapy 4 13.33 with schizophrenia undergo in forget to take his/her
2 antipsychotics 23 76.67 medicine and careless at times about taking his/her
3 10 medicine, adverse effects and deficiency of insight and
MARS total score shortage of information about their disorder. From the
Minimum total score 4 sighting, this is might because the healthcare team did
Maximum total score 10 not provide the client and caregiver the comprehensive
Mean total score 7.20 information according to their treatment and illness.
Including intervention, dosage regiment, therapeutic
Table 2. Regimen of oral antipsychotics effect, and adverse effect. The results of current study
Regimen therapy N % indicated that half participants with schizophrenia
Risperidone 3 10 disorder did not clearly understand their illness and
Risperidone + Clozapine 12 40 medication.
Risperidone + Clozapine + 3 10 Many studies has reported that medication adherence
Trifluoperazine is related to knowledge and experience of, and insight
Clozapine + Trifluoperazine 8 26.67
into the illness, in addition to patient's attitudes toward
Haloperidol 1 3.33
the use of medication for the treatment of psychiatric
Haloperidol + Clozapine 2 6.67
disorders (22,23,24,25). One of study reported that when
Aripiprazole + Clozapine 1 3.33
patients were not fully informed about their illness and
outpatients. Currently, atypical antipsychotics became treatments, there were likely to discontinue medication
drug of choice in schizophrenia treatment considering therapy of their own volition without discussing the matter
more effective in relapse prevention, reduced risks of with healthcare professional (23).

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Table 3. Frequency of Attitude towards medications Table 5. Analysis of Variance One Away
Medication adherence YES NO
questions/statement N % N % Adherence
1. Do you ever forget to take 15 50 15 50 Studied N Adherenc SD Sig
your medication? variable e score
2. Are you careless at times 13 43.3 17 56.7 (mean)
about taking your medicine? Education lavel
3. When you feel better, do 6 20 24 80
Elementary 4 7.50 2.380 .942
you sometimes stop taking
your medicine? school
4. Sometimes, if you feel 4 13.3 26 86.7 Junior high 9 7.00 1.732
worse when you take the school
medicine, do you stop taking Senior high 12 7.33 1.303
it? school
5. I take my medicine only 2 6.7 28 93.3
when I am sick
College or 5 7.00 2.000
6. It is unnatural for my 3 10 27 90 higher
mind and body to be Total 30 7.20 1.627
controlled by medication Treatment duration
7. My thoughts are clearer 16 53.3 14 46.7 1-5 year 13 6.92 1.847 .307
on medication
6-10 year 12 7.75 1.357
8. By staying on medication, 30 100 0 0
I can prevent getting sick 11-15 year 5 6.60 1.517
9. I feel weird, like a 9 30 21 70 Total 30 7.20 1.627
‘zombie’, on medication Number of antipsychotic
10. Medication makes me 19 63.3 11 36.7 1 4 7.75 1.500 .307
feel tired and sluggish antipsychot
ic
Factor affecting medication non-adherence 2 23 6.96 1.692
The triggers of non adherence include the personal antipsychot
ics
factors, medication factors, and socio-economic
3 8.33 .577
environment factors. The results of this study there is no antipsychot
significant different adherence score between gender ics
group and educational level group (table 4). This finding Total 30 7.20 1.627
inline with Naafi et al., who reported there is no
meaningful difference between patients characteristic and during treatment has negative impact on their treatment
the patients medication adherence level (26). Conforming compliance. One of study reported that pharmacist
current study pointed out that there is no meaningful counseling there was meaningful difference adherence
different between treatment factors such as duration of level between pre and post pharmacist counseling
treatment and number of antipsychotics with treatment intervention (27). Therefore, the health care team should
compliance score (table 5). give the patient and/or the caregiver psycho-educational
program for compliance of treatment improvement
Table 4. Independent sample T-test gender different
(28,29,30).
Gender N adherence SD Sig.(2 There are several limitations in this study. Due to
score tailed)
limited sample size and lack of clinical data as adherence
(mean)
Male 18 7.33 1.847 .564 parameter. Prospectively study with various number
Female 12 7.00 1.279 sample size and objective parameter of adherence might
Total 30 7.20 1.627 be considered. Despite the several limitations, our study
provides preliminary finding to explore barriers affecting
This study contrasting to Dibonaventura et al., Dassa medication adherence in mental health disorder treatment.
et al., and Yang et al., who reported that the quantity of 4. Conclusion
medications may affects patient's toward compliance Adherence to medication is a critical issue for patients
(16,17,18). The results of this current study shows more with mental disorder. It cannot be overemphasized that
than 50% of participants has experiences with patients should have insights into their own mental
antipsychotic side effects. Lack information or education disorder and realize the necessity of taking medications
about heir medication and side effect might be occur to improve their chances of a successful recovery. The

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healthcare provider should empower counseling program of first-episode psychosis. Psychological Medicine.
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Acknowledgement with severe mental illness. Pharmacy Today. 2013
The authors thank the Indonesia Endowment Fund for June;19:69-80.
Education for funding support this study through 9. Social Insurance Administration Organization.
Beasiswa Unggulan Dosen Indonesia scheme. Beside Practical Guidelines of Referral Program for
that, the author thank the all participants and all staffs National Health Coverage Participant. 2014.
the national mental hospital for providing supports and Available from: https://bpjs-kesehatan.go.id/bpjs/
facilitating data collections. dmdocuments/4238e7d5f66ccef 4ccd89883c46
fcebc.pdf.
Conflict of interest
10. Sari SP, Suttharangsee W, Chanchong W. The effect
The authors declare no financial or commercial
of self-management with family participation on
conflict of interest. medication adherence among patients with
schizophrenia in Indonesia: A pilot study.
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A Production and Activity Test of Anti-bacterial Compounds

of Endophytic Fungi BR-S1 (a) Isolate Extract in
Different General Growth Media
Kurniawan1* and Mustiah Yulistiani2
1Department of Medical Laboratory Technology (DIV) Universitas Muhammadiyah Purwokerto, Indonesia
2Nursing Department (Undergraduate), Universitas Muhammadiyah Purwokerto, Indonesia
Corresponding author : kurniawan@ump.ac.id

Abstract
BR-S1 (A) isolate is an endophytic fungi isolated from Key words : Anti-bacterial compounds · endophytic fungi
the medicinal plant of tea parasite (Scurrulaoortiana) · and general growth medium
which is estimated to contain anti-bacterial compounds.
1. Introduction
The research questions are as follow; can an anti-bacterial
compound be produced in general growth medium, and Indonesia is one of the countries with the greatest
is it effective in inhibiting or killing MRSA bacteria biodiversity in the world which owns various types of
pathogen.The aim of this research is to find out the types plants, animals, fungi, and bacteria with uncover its
of general growth media that can be used to produce anti- potential. One real step to do is through the exploration
bacterial compounds and to determine the effectiveness and management of various types of plants as a source of
of these compounds in inhibiting or killing pathogenic medicine or medicinal raw materials.
MRSA bacteria.This research was conducted using Tea plant parasite is one of the plants being explored
laboratory experimental methods with the main variables and collected recently as a source of medicine or medicinal
in forms of three different types of general growth media, raw materials. Based on several results of previous studies,
namely the Potato Dextrose Broth (PDB) medium, the it is believed that the ability of the parasite plant in treating
CzapekDox Liquid Medium (CDLM), and the Malt various diseases is related to the contained active
Extract Broth (MEB) medium. The results show that the compounds.
three types of general growth media were not able to Although the potential of the tea plant parasite is
stimulate the production of anti-bacterial compounds covered, direct utilization of this plant as a source of
which were characterized by the absence of discoloration medicine or medicinal raw materials is apparently not
and medium turbidity and the absence of thick mycelium easy and is constrained by several factors such as the its
growth. The results of anti-bacterial activity tests on limited number, requires large biomass, and the nature or
pathogenic MRSA bacteria show no inhibition zone structure of the active compound which is very
formed around the disc paper added with endophytic fungi sophisticated. To overcome these obstacles, endophytic
extracts, whereas positive controls formed inhibitory/clear fungi which lives within these plants is utilized as a source
zones.The production process of BR-S1 (A) isolate anti- of medicine or medicinal raw materials.
bacterial compound was influenced by three factors, Endophytic fungi or molds are molds which have been
namely the composition and chemical properties of the whole or part of their life cycle by colonizing healthy
medium, age and number (concentration) of cells, and tissue from host plants both intercellularly and
environmental conditions (temperature and aeration) of intracellularly without causing disease with obvious
the production site. It can be concluded that the three types symptoms (2). As the most dominant group, endophytic
of general growth media of PDB, CDLM, and MEB fungi have great potential to be developed related to its
cannot be used as a medium for the production of anti- ability of several types of molds which are able to produce
bacterial compounds. The antibacterial compounds several active compounds such as antibiotics,
produced by BR-S1 (A) isolates are not effective in antiimmunosuppressive, antidiabetic, anticancer,
inhibiting pathogenic MRSA bacteria. antiinsecticidal, and antiviral in a wide range.

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The results of (5) discovered that in the tea plant 3. To present the effectiveness of antibacterial
parasite, 17 endophytic mold were successfully isolated. compounds produced by endophytic fungi isolates
The results of testing crude extracts from 17 isolates BR-S1 (A) in inhibiting the growth of MRSA
showed that there were only 5 isolates which presented bacteria
positive results in inhibiting bacterial growth. The ability This research is expected to provide information about
of endophytic molds to produce secondary metabolites the types of medium that can be used and the most optimal
(antibacterial compounds) on a laboratory scale (in vitro) in producing antibacterial compounds and to illustrate
is affected by two main factors; physical factors such as how much effectiveness in inhibiting the growth of
pH, temperature and incubation time and chemical factors MRSA bacteria.
such as nutrient content available in growth media such
as sources of N, C, amino acids, and other additional 2. Materials and Methods
nutrients. Material and research objectives : The toolsutilized in
Study on the correlation or effect between types of this study are markers with 0.5 cm and 1.8 cm diameter
fermentation media with the ability to produce holes, petri dishes, incubators (Memmert INB 400),
antibacterial compounds from endophytic fungi isolates Laminar Air Flow (LAF), test tubes, Erlenmeyer flask,
needs to be done. It is essential because this study will rotary shakers (Kottermann 4010), thermometer,
assist to find the most appropriate or optimal medium separating funnel, vacuum pump, tapered erlenmeyer,
for the production of antibacterial compounds. Later, the measuring cup, stirrer hotplate (Barnstead SPI 31320-
results of this production can be tested onMRSA bacteria, 33), oven (Memmert UNB 400), analytical balance (AND
a special strain of the bacterium Staphylococcus aureus GR-200), micropipettes (Boeco), refrigerator (LG
which has resistance to beta-lactam antibiotic group expresscool), water bath, calipers, autoclaves (All
which includes methicillin, oxacillin, penicillin, America 25X) and centrifuges.
amoxicillin, cephalosporin, and carbapenem. The materials used in this study included samples of
Based on the description above, a number of problems the tea plant parasite, tap water, 75% ethanol, 5.25%
can be formulated as follows : sodium hypochlorite (bayclin), sterile distilled water,
1. What types of fermentation medium can be used filter paper, cotton, 1% streptomycin antibiotics, Potato
by endophytic fungi isolates BR-S1 (A) in Dextrose Agar (PDA) medium (Oxoid), Manitol Salt
producing antibacterial compounds? Agar (MSA) medium (Oxoid), Plate Count Agar (PCA)
medium (Oxoid), Potato Dextrose Broth (PDB) medium
2. Which type of fermentation medium is the most
(Oxoid), Tryptic Soya Agar (TSA) medium (Merck),
optimal in producing antibacterial compounds
Tryptic Soya Broth (TSB) medium (Merck), CzapekDox
from endophytic fungi isolates BR-S1 (A)?
Agar (CDA) medium (Oxoid), Malt Extract Agar (MEA)
3. How effective are antibacterial compounds medium (Oxoid), CzapekDox Broth (CDB) medium
produced by endophytic fungi isolates BR-S1 (A) (Oxoid), Malt Extract Broth (MEB) medium (Oxoid),
in inhibiting the growth of MRSA bacteria? Mueller Hinton Agar (MHA) medium (Merck), paper disk
This study is expected to provide information about blank (Oxoid), ethanol 96%, antibiotic vancomycin, fungi
the types of medium which can be used and the most isolates endophytic BR-S1 (A) and MRSA bacteria.
optimal in producing antibacterial compounds and Research design and data analysis : This research was
discover its effectiveness in inhibiting the growth of conducted by laboratory experimental methods with
MRSA bacteria. independent variables in the form of different carbon
The objectives are as follow: sources (C) contained in growth media such as dextrose
1. To discover the types of fermentation medium (PDA/PDB), sucrose (CDA/CDB) and Malt extract
which can be used by endophytic fungi isolates (MEA/MEB). The dependent variable in this study was
BR-S1 (A) in the production of antibacterial the ability of BR-S1 (A) endophytic fungi isolates to
compounds produce antibacterial compounds.
2. To determine the most optimal type of Endophytic fungi cultivation in tea plant parasite (8)
fermentation medium in producing antibacterial with modification : Isolate endophytic fungi BR-S1 (A)
compounds from endophytic fungi isolates BR- were grown on PDA medium and incubated for 7-14 days
S1 (A) at room temperature assuming on the 14th day the entire

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surface of the medium was overgrown and covered by to obtain natant and supernatant. Supernatant was
mycelium. Then, the PDA medium was sampled using a collected and put into microtube for centrifugation at a
hole diameter of 0.5 cm to be inoculated on the PDA, rate of 13,000 rpm for 15 minutes. The obtained
MEA, and CDA medium with each medium planted with supernatant was then used to test antibacterial activity.
1 sample right in the middle of the petri dish and with the As a negative control, a sterile liquid medium was utilized
reverse position of the sample so that the mycelium by extracting the same method as above.
directly touched the surface of the medium. All
Antibacterial activity test of crude extract of
subsequent mediums were incubated at room temperature
endophytic fungi isolate BR-S1 (A) was carried out
with the petri dish turned upside down for 7-14 days and
through agar diffusion method. It was prepared a cup of
each medium was repeated 3 times.
MHA medium which had previously been inoculated with
Characterization and measurement of growth of tea 1 ml of overnight MRSA bacteria and allowed to solidify.
parasite molds (3) : The morphological character of BR-
On top of the medium, it was then placed 7 pieces of 6
S1 (A) endophytic fungi isolate in all types of cup medium
mm in diameter sterile disk paper, each dropped with 20
was observed both macroscopically and microscopically
µl filtrate/extract of endophytic mycelium fungi (6 disc
and their growth rate was measured every day.
paper) and vancomycin antibiotics (1 disc paper) as
Macroscopic observations were the color and texture of
positive control. The medium MHA cup was then
the surface of the colony, the color behind the colony,
incubated at 37°C for 1-2 x 24 hours and observed
the edge of the colony, the shape of the colony, the
whether there were any inhibitory zones (clear zones)
presence or absence of zoning, the growing area, radial
lines, exudate drops and its color, and the organs formed formed around the disk paper. Inhibition zones formed
(fruiting body, schlerotia, synnema, sporodochia, stroma, were measured by a calipers diameter and so did the
and setae). Microscopic observations were the presence diameter of the paper discs used. Based on the diameter
sectional hyphae, hyphae pigmentation, presence or data obtained, the areas of inhibition zone were calculated
absence of clam connections, forms of hyphae by the following formula :
modification (spiral, nodular organ, pectinate body, antler 2
hypha, racquet hypha), the presence of rhizoid , asexual
Lzhaw a Lzhak:Lzhaw- Lkc
spores (simple shape, special shape, size, arrangement),
2
and the presence of sexual spores like ascospores, Lkc b
basidiospores, andzigospores).
MRSA test bacteria reaction (6) with modification : Information :
Reaction of the bacterial inoculum test was carried out ra : Radius of inhibition zone (mm)
by the Standard Plate Count (SPC) method. 1 ml stock of rb : Radius of paper disk (mm)
MRSA bacteria was taken to be added to 25 ml of a sterile Lzhaw : Area of initial inhibition zone (mm2)
TSB medium and shaker at 100 rpm for 1x24 hours at 37 Lkc : Area of disc paper (mm2)
oC. From the TSB medium, the dilution was carried out Lzhak : Area of the final inhibitory zone (mm2)
up to 10-7 dilution level with the last two dilutions being : 3,14
duplicated plating by Pour Plate method on the MHA
medium. Then, the MHA medium was incubated for 1x24 3. Results and Discussion
hours at 37 oC and counted the number of colonies
Cultivation of endophytic fungi in tea plant parasite:
growing on each petri dish.
In this study, three types of fermentation media were PDA
Extraction and antibacterial activity test of tea plant
or PDB medium, MEA or MEB medium, and CDA or
parasite (8) with modification : All liquid media were
CDB medium. From the three types of mediums, it could
removed from the rotary shaker and the results of
be seen that the endophytic fungi of the tea plant parasite
cultivation were filtered by filter paper (Whatman No.
isolate BR-S1 (A) had the fastest growth in the PDA or
1) so that mycelium and filtrate would be obtained later.
PDB medium, followed by the CDA or CDB medium
The pH of the filtrate was measured by pH meter and
and finally the MEA or MEB medium (Figure 1)
then centrifuged at a speed of 3,000 rpm for 20 minutes

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Extraction and antibacterial activity test for endophytic Information table 1:

fungi of tea plant parasite Figure 1 Figure 2 1. Negative control of PDB extract
2. Negative control of CDB extract
3. Negative control of MEA extract
4. MEB Extract
5. CDB Extract
6. GDP Extract
7. Positive vancomycin control

Figure 1. Growth rate of endophytic fungi isolates BR-S1

(A) in several types of fermentation medium. A). PDA
medium; B). CDA medium; C) MEA medium; D). PDB
medium; E). CDB medium; F). MEB medium.

Table 1. Inhibition zones calculation of antibacterial

activity test for endophytic fungi BR-S1 (A) extract
against MRSA bacteria from three different types of liquid Figure 2. Results of endophytic fungi fermentation of BR-
fermentation medium. S1 (A) isolates in three different types of fermentation media.
Dis Repetitions (mm2) A). PDB medium; B). CDB medium; and C). MEB medium.
k avera
U1 U2 U3 The fermentation process of endophytic fungi isolate
No ge
1 0.00 0.00 0.00 0.00 0.00 BR-S1 (A) was carried out on PDB, CDB, and MEB
2 0.00 0.00 0.00 0.00 0.00 mediums with an incubation time of 7 days at 30 oC in a
3 0.00 0.00 0.00 0.00 0.00 100 rpm incubator shaker. This research selected liquid
4 0.00 0.00 0.00 0.00 0.00 fermentation medium considering the easy regulated and
5 0.00 0.00 0.00 0.00 0.00 determined liquid medium, its homogenecity and can
6 0.00 0.00 0.00 0.00 0.00 provide optimal conditions for cell growth of its
190. 205.3 252.1 648.1 216.0 composition and concentration, and the use of the medium
7
67 3 5 5 5 becomes more efficient.

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From figure 1 we know that these three types of than 4 weeks) and hadcome the phase of death (decline).
medium were the same common medium in fact and used In this phase, metabolic activity had decreased so that
for the cultivation of various types of mold or fungi. the nutrient content contained in the medium could not
However, its energy sources (carbon) had a different be converted and utilized by endophytic molds to
composition. The PDA or PDB medium was composed stimulate cell growth.
from potato extract and dextrose (glucose), the CDA or Another factor which also affected the fermentation
CDB medium was composed from sucrose as the only process was bad condition of the fermentation
source of carbon and the MEA or MEB medium was environment. During the fermentation process, the
composed from malt extract containing polysaccharides. temperature inside the incubator shaker had been set at
PDA or PDB medium was the medium which obtained room temperature (28oC), but in reality the temperature
the simplest carbon source, namely the monosaccharide inside the incubator shaker reached hotter (higher) than
group in the form of dextrose (glucose) which could be room temperature (28oC). Previous research suggested
directly absorbed and utilized by endophytic fungi to that temperature was one of the environmental factors
stimulate cell growth. It was different from CDA or CDB which could affect the life and growth of mold cells (11).
and MEA or MEB medium which collected carbon Fungi growth was affected by several factors and one of
sources in the form of disaccharides (sucrose) and them was environmental temperature. Normally, mold
polysaccharides (maltose) which could be utilized by could grow optimally in the temperature range of 25oC -
endophytic fungi to be broken down first into simpler 35oC. A higher temperature than the optimal temperature
monosaccharides. Previous research suggested that mold could cause mold cells to damage and die (1).
species could utilize some of the carbon sources contained The extraction process of fermented endophytic molds
in the medium for vegetative cell growth and the of BR-S1 (A) isolates was carried out through filtration
availability of simple carbon sources which would be of vacuum pump and filter paper. In this process, it was
relatively easily digested and could increase their obtained in the form of supernatant fluid without the
metabolic processes (11). filtrate of endophytic fungi mycelium. The results of this
The fermentation results of endophytic fungiisolates extraction process were further processed to test the
BR-S1 (A) could be seen in figures 2 above. From the antibacterial activity against MRSA bacteria.
picture, it could be understood that the fermentation Figure 3 and table 1 above described the extract of
process did not occur optimally, this could be seen from endophytic fungi isolate BR-S1 (A) from fermentation
the color of the fermentation medium which did not turn on three different types of liquid fermentation media had
out to be thicker or darker compared to the sterile no ability to inhibit/kill MRSA bacteria characterized by
fermentation medium. In addition, samples of endophytic the absence of inhibitory zones formed around disc paper.
fungi mycelium planted in the fermentation medium did Inhibition zones were only formed on paper disk No. 7
not grow and multiply. administered with vancomycin antibiotics as a positive
control.
The fermentation process of endophytic fungi was
affected by several factors such as the composition and The inability of endophytic fungiextract of BR-S1 (A)
chemical properties of the medium, age and number isolates to inhibit/kill MRSA bacteria might be affected
(concentration) of cells added to the fermentation by several factors. These factors were the type of
fermentation medium, the incubation period, the produced
medium, and the environmental conditions in which
specificity of secondary metabolite compounds and the
fermentation occurred. From these factors, age and
level of resistance of the test bacteria.
number (concentration) of cells and environmental
conditions which contributed the fermentation process The utilization of three different types of liquid
in this study did not optimally occur. fermentation medium in this study was initially intended
to determine the most optimum medium in producing
Considering from the number (concentration) of cells
secondary metabolite compounds from BR-S1 (A) isolate
used, this study was more than enough because every 1
endophytic molds. However, the test results indicated no
bottle of 150 ml volume fermentation medium had been
secondary metabolite compounds produced from the three
filled with 3 samples of endophytic fungi mycelium, each
types of liquid fermentation medium. Initial allegations
1 cm in diameter. On the contrary, considering from the
to the absence of secondary metabolite compounds
age of the cells, endophytic molds utilized were old (more produced were the physical condition of the environment
Production and activity test of anti-bacterial compounds
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vegetative cells, mostly mold or fungi but these were

unable to support the production of secondary
metabolites. The composition of the fermentation medium
greatly affected endophytic fungi in producing secondary
metabolites (12). This was supported by the statement of
(11) which stated that fermentation process by
commercially available liquid medium often resulted on
the yield of secondary metabolites which were limited.
This was also supported by the findings of (7) study which
revealed that the PDB medium was less able to stimulate
the production of antibiotics which were able to inhibit
both prokaryotic microbes of gram positive and
eukaryotic.
Another factor which affected the absence of
antibacterial activity ability from the extract of endophytic
fungi isolate BR-S1 (A) was the incubation period
selected to produce secondary metabolites. Each type of
mold had a life cycle which varied in duration from the
lag phase to the death phase. In this study, the incubation
period used was 7 days and it was assumed that the 7-
day growth of endophytic mold isolates of BR-S1 (A)
had not reached to the stationary phase so that the
production of secondary metabolite compounds was
unavailable. This was in line with the statement of (10)
which stated that the stationary phase was a phase in
which the number of living and dead cells were relatively
equal, the amount of nutrients begins to decrease so that
Figure 3. Antibacterial activity test results of BR-S1 (A) it stimulated endophytic molds to produce secondary
endophytic fungi isolate fungi against MRSA bacteria from metabolites as a survival mechanism. Secondary
three different types of liquid fermentation medium. A). 1st metabolites were usually produced only in the stationary
repetition; B). 2nd repetition, and C). 3rd repetition phase of growth.
Information figure 3:
1. Negative control of PDB extract Endophytic fungi isolate BR-S1 (A) was not able to
2. Negative control of CDB extract inhibit/kill MRSA bacteria suspected because secondary
3. Negative control of MEA extract metabolite compounds produced by these endophytic
4. MEB Extract fungi obtained narrow specificity for certain types of
5. CDB Extract bacteria and beyond MRSA bacteria type. In addition, it
6. GDP Extract
was also possible that MRSA bacteria had high resistance
7. Positive vancomycin control
to secondary metabolites produced by endophytic mold
isolates BR-S1 (A). This was in line with the statement
(temperature) of incubation, too old endophytic mold by (9) which stated that each endophytic fungi was able
isolates, and the composition of the fermentation medium. to produce secondary metabolites which varied with
Previous research stated that one of the important factors different specificity and effectiveness. MRSA bacteria
influencing mold growth is the conditions of growth, if were Gram positive bacteria with a cell wall structure
molds are in suitable conditions, the enzymatic expression composed of layers of peptidoglycan, polysaccharides,
to produce secondary metabolites is also maximum (4). and theatricic acid. These three cell wall components were
PDB, CDB, and MEB medium were synthetic/ covalently bonded to produce large and sophisticated cell
semisynthetic medium selected for isolation, cultivation, walls which affected the antibacterial compounds tested
and enumeration of molds or fungi in general. These three could not penetrate the cell wall and did not enter the
types of medium were able to support the growth of cell.

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4. Conclusion 4. Khairiah, N. danNintasari, R. (2017). Isolasid

Based on the results of the research and discussion above, anujiaktivitasantimikrobakapangendofitdarikayuulin
several conclusions can be drawn as follows: (EusideroxylonzwageriTeijsm&Binn.). Jurnal Riset
IndustriHasilHutan, 9(2): 65-74
1. Three types of commercial medium,PDB, CDB and
5. Kurniawan,Ratnaningtyas, N.I.danIrianto, A.(2014).
MEB medium, cannot be utilized as a fermentation
Efektivitasekstr akkapangendofittanamanbe
medium for endophytic fungi isolates BR-S1 (A) in
naluteh(Scurrulaoortiana) d'alammenekanpertum
the production of bacterial compounds
buhanbakterimethicillin resistant Staphylococcus
2. Three types of PDB, CDB and MEBcommercial aureus (MRSA) secarain vitro.Tesis. Post Graduate
mediumare not optimum as a fermentation medium P r og r a m, Un i v er s i t a s J en d er a lS o ed i r ma n ,
for endophytic fungi isolates BR-S1 (A) in the Purwokerto. (Not published). pp 50-60
production of antibacterial compounds 6. Kurniawan dan Ratnaningtyas, N.I. (2018).
3. Antibacterial compound extract of endophytic fungi Efektivitasekstrakkapangendofitisolat BR-S1 (A)
isolate BR-S1 (A) results in the three types terhadapbakteriMethicillin-Resistant Staphy
fermentation process of PDB, CDB, and lococcus aureus (MRSA). Meditory: The Journal of
MEBcommercial media are ineffective in inhibiting Medical Laboratory, 6(2): 99-107
or killing MRSA bacteria 7. Margino, S. (2008). Produksimetabolitsekunder
Acknowledgement (antibiotik) olehisolatjamurendofit Indonesia.
MajalahFarmasi Indonesia, 19(2): 86-94
We as researchers would like to express our gratitude
to the Universitas Muhammadiyah Purwokerto for 8. Mu'azzam, K.A.A.R., Taufiq, M.M.J., Azlina, N.I.,
funding this research and also giving permission to use Noorhazira, S. and Darah, I. (2015). Screening of
an integrated laboratory from the beginning to the end of antibacterial activity of endophytic fungi isolated
this research. from different leaf ages ofCurcuma mangausing
different growth media. International Journal of
Conflict of Interest Research in Medical and Health Sciences, 5(02): 1-
The authors have no conflicts of interest to declare. 10
All co-authors have seen and agree with the contents of 9. Nurhidayah, Hasanah, U., danIdramsa. (2014).
the manuscript and there is no financial interest to report. Pengaruhekstrak metabolitsekunderjamurendofit
We certify that the submission is original work and is not tumbuhan Cotylelobiummelanoxylondal
under review at any other publication. ammenghambat pertumbuhanmikrobapatogen.
Prosiding Seminar Nasional Biologidan Pembela
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jarannya, JurusanBiologi FMIPA Universitas Negeri
1. Babay, L.(2013). Pengaruhsuhudan lama Medan, Medan, Indonesia. pp 308-317.
penyimpananter hadapjumlahkapangpada roti tawar
10. Prahesti, A., Pujiyanti, S. danRukmi, M.G.I.(2018).
(suatupenelitian di industrirumahtanggapangan Kota
Isolasi, ujiaktivitasdanoptimasi inhibitor ?-amilase
Gorontalo). Skripsi. Program StudiKesehatan
isolate kapangendofittanamanbinahong (Anredera
Masyarakat, FakultasIlmu-IlmuKesehatandan
cordifolia) (Ten.) SteenisDiani. JurnalBiologi7
Keolahragaan, UniversitasNegeriGorontalo,
(1):43-51
Gorontalo (Not published): 1-9
11. Septiana, E. danSimanjuntak, P.(2017). Pengaruh
2. Elfita, Muharni, Munawar and Aryani, S. 2012.
kondisikultur yang berbedaterhadapaktivitasantioksi
Secondary metabolite from endophytic fungi danmetabolitsekunderkapangendofitasalakarkunyit.
Aspergillusniger of the stem bark of kandisgajah Traditional Medicine Journal. 22(1): 31-36
(Garciniagriffithii). Indonesia Journal of Chemistry,
12. Suciatmih. 2010. Pengaruhkon sentrasiantimi
12(2): 195-200.
kroorganisme, media fermentasi, danwaktuinku
3. Ilyas, M. (2006). Isolasidanidentifikasikapang basiterhad appertumbuhanAbsidiacorymbifera
padarelungrizosfirtanaman di kawasancagaralam (Cohn) Sacc. & Trotter darijamurendofit
GunungMutis, Nusa Tenggara Timur. Biodiversitas: Fusariumnivale (Fr.) Ces. Media Penelitian dan
Journal of Biological Diversity, 7(3): 216-220. Pengembangan Kesehatan, XX(1): 17-25

Production and activity test of anti-bacterial compounds

Current Trends in Biotechnology and Pharmacy 213
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DOI : 10.5530/ctbp.2020.4s.26

Phytochemical Investigation, Cytotoxicity and Anti-Diabetic Activity of

Whole Fresh and Dry Ethanolic Extracts of Sudanese Portulaca quadrifida
Layla Fathi Yassin1*, Ayat Ahmed Alrasheid2, Khalid Abdallah Enan3,
Ali Abdalla Adam1, MazinYousif Babiker4
1Department of Pharmaceutical chemistry, Faculty of Pharmacy, University of Medical Sciences and Technology. Khartoum, Sudan.
2Department of Pharmacognosy, Faculty of Pharmacy, University of Medical Sciences and Technology, Khartoum, Sudan.
3Department of Virology, Central laboratory, Ministry of Higher Education and Scientific Research, Khartoum, Sudan.
4Department of Pharmacology and Texocology, Faculty of Pharmacy, International University of Africa, Khartoum, Sudan.

*Corresponding Author : laylaelbadry@hotmail.com

Abstract standing people's interest in medicinal plants has brought

Historically, natural products have been used since about today's modern fashion of medicinal plant
ancient times and in folk medicine for the treatment of processing and usage 4.Many people especially in
many diseases and illnesses. Portulacaquadrifida developing countries resort to using medicinal plants and
popularly called Chicken-weed belongto family herbs instead of medicines due to the worse economic
Portulacaceae. In Sudanese folk medicine, this plant has conditions and high prices of medicines. One of these
been used to treat diabetes, and as a poultice to treat medicinal herbs is Portulacaquadrifida popularly called
neuralgia in herpes zoster. Pharmacological activities of Chickenweed,it is an annual much branched mat-forming,
P. quadrifida include anti-microbial, anti hyperglycemic, succulent species in the family Portulacaceae5.P.
antioxidant,anti-inflammatory, anti- nociceptive, anti quadrifida stem is succulent, diffuse, and purple in color
convulsion properties, anti epileptic and anti cancer at maturity, less than a millimeter in diameter and up to
activity.In this study the phytochemical, Cytotoxicity and 50 cm; rooting at the nodes6. The leaves are edible,
anti-diabetic activity of whole fresh and dry ethanolic frequently used in salads.Different parts are also used for
extracts of Sudanese P. quadrifida L were investigated. various curative purposes. P. quadrifida have similar
The results showedhigh content of tannin 28.07 - 13.68 medicinal uses, but less widely used as P. oleracea7.P.
ppm for fresh and dry samples respectively. The P. quadrifidadistributed in Africa and tropical Asia;
quadrifidaextracts were found to be rich source of vitamin introduced into the warmer areas of the Americas, not
C (1.16 - 1.76 ppm) for fresh and dry respectively. In the existed in Australia5. The chemical constituents of P.
cytotoxicity test using Microculture tetrazolium (MTT) quadrifida containing alkaloids, flavonoids, triterpenoids,
assay, the IC50 values were 858 and 155.3092 ppm for glycosides, carbohydrates, tannins, amino acids and
dry and fresh samples extracts respectively. The Alpha saponins8.P.quadrifidawas used traditionally to
amylase inhibition of both extracts showed high activity treaturinary discharges, inflammations, asthma, cough,
with inhibition percentage 95 and 91 % for dry and fresh and ulcers; a poultice of the plant is applied in abdominal
extracts respectively at concentration 1000 ppm compared complaints and hemorrhoids9. In Sudanese folk medicine,
to the positive control (Acarbose). P.quadrifidais used to treat diabetes, and as poultice to
treat neuralgia in herpes zoster. Thepharmacological
Key words : Portulacaquadrifida, Tannin,Cytotoxicity,
activities of P.quadrifidaincludeantimicrobial, anti
Anti-diabetic activity, Sudan.
hyperglycemic, antioxidant10,anti-inflammatory, anti
1. Introduction nociceptive activity11,anti convulsion;anti epileptic12and
anti-cancer activities13 have been revealed in several
Historically, natural products have been used since
studies.
ancient times and in folk for the treatment of many
diseases and illnesses1.The first records that Plants were 2. Materials and Methods
the basis of sophisticated traditional medicine system
Samples preparation and extraction procedure
written on clay tablets in cuneiform, are from
Mesopotamia and date from about 2600 B.C?2. During P. quadrifida plant was collected directly from
the 17th and 18th centuries recognition of plant use thefieldin Khartoum, Sudan. The plant was identified and
derived medicines grew rapidly3.The continuous and authenticated by the herbarium unit of the Medicinal and

Phytochemical investigation of Sudanese Portulaca quadrifida

Current Trends in Biotechnology and Pharmacy 214
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DOI : 10.5530/ctbp.2020.4s.26

Aromatic plants Institute Research Center. After washed using micro-plate readers. The optical density was
by running tap water,samples were divided into two measured at 540 nm and the percentage of viable cells
groups, Whole plant fresh and whole plant dry (air dried was calculated as relative ratio of optical densities.
at room temperature). Both samples were extracted with
In vitro anti-diabetic activity
absolute ethanol at room temperature for 72 hours. After
filtration by Whatman filter paper Number 4,all samples Serial concentrations of test samples (100-1000?g/ml)
were allowed to dryness. and standard drug (Acarbose) were prepared.About 500
?l of each prepared solutions were transferred into another
Total Tannin Content (TTC) tests tube containing ? -amylase (0.5mg/ml) in 500 ?l of
The total tannin content of the extracts was determined 0.20 mM phosphate buffer (pH 6.9) solution and
as described byShanmukhaet al.14. About 1ml of Fecl3 incubated at room temperature for 10 min. After these,
(1%)and 1ml of K3Fe(CN)6(1%) were added to 1 ml of 500 ?l of 1% starch solution in 0.02 M sodium phosphate
each extract (1mg/ml) and completed the volume to 10 buffer (pH 6.9) were added to each tube and incubated at
ml with distilled water. Mixtures were shaken and left to room temperature for 10 min. The reaction was stopped
stand at room temperature for 15 minutes and the UV- with 1.0 ml of 3,5 dinitrosalicylic acid (DNS)colour
VIS absorbance was measured at 510 nm. Standard curve reagent. The test tubes were incubated in a boiling water
of tannic acid was used to quantify theP. quadrifida TTC. bath at 100 0C for 5 min, cooled to room temperature.
The standard curve was obtained by preparing set of 5 The reaction mixture was then diluted after adding 10 ml
dilutions 100, 200, 300, 400, and 500 ppm tannic acid distilled water and absorbance was measured at 540 nm
solutions from 1000 ppm stock solution. Absorbance was by Shimadzu Spectrophotometer UV double beam.17
measured at 510 nm using Shimadzu spectrophotometer.
Calculation of Inhibitory Concentration (IC50)
Quantitative determination of Ascorbic Acid (Vitamin
The concentration of the plant extracts required to
C) using HPLC
inhibit 50% of the ?-amylase enzyme (IC50) were
Ascorbic acid content in the P. quadrifida extracts was Calculated by using of Microsoft office Excel 2007.
determined by HPLC chromatographic method15 Inhibitionpercentage (%) was calculated by: % = (Ac-
performed on a HPLCShimadzu, ODS-3 column (6 mm As)/Ac X 100
x 150) reversed phase matrix (5 ?m) and elution was
Where : Ac; is the absorbance of the control and AS;is
carried out in a gradient system with0.1% (v/v) acetic
the absorbance of the sample.
acid :(95:5%)methanolwith 1.0 ml/min flow rate under
ambient temperature. The volume of injection was 20 3. Results and Discussion
?land the UV detector was set at 254 nm.Ascorbic acid
Total Tannin Content (TTC)
standard solutions was prepared in concentration of 1,
10, 100 ppm and the standard curve was obtained. Total tannin content was expressed as milligram of
tannic acid equivalent per gram. From the tannic acid
Cytotoxicityusing MTT assay standard curve(Figure 1) the TTC content was calculated.
The experiment was performed according to method The total tannin content of whole fresh and whole
described by Berridge et al16. Vero cells were cultured dry samples ethanolic extracts ofP. quadrifidawas
in a 96 -well plate for overnight CO2 environment at evaluated. The highest content was found in fresh sample
37°C. Supernatant was removed, and 20 µl of serially
diluted extracts (range from 0.01 to 100 µg/ml) and 80
µl complete medium DMEM supplemented with 5% (v/
v) fetal bovine serum, penicillin (100 units/ml), and
streptomycin (100 µl/ml) were added to each well. After
incubation, the culture medium was aspirated carefully
and 50 µl of 3-(4, 5-dimethylthiazol) -2, 5diphenyle-
tatrazolium bromide (MTT) solution (2 mg/ml PBS) was
added to each well and further incubated for 4 hours.
MTT solution was aspirated. The plate was agitated at
room temperature for 15 min then read at 540 nm by
Figure 1. Tannic acid standard curve

Layla et al
Current Trends in Biotechnology and Pharmacy 215
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DOI : 10.5530/ctbp.2020.4s.26

extract (28.07 ppm) followed by dry sample extract (13.68 quadrifidaextract was determined using HPLC from the
ppm). The tannin compounds are widely distributed in Ascorbic acid standard calibration curveand results are
many species of plants. Determination of the preliminary presented in Table 1. The fresh and dried whole plant
phytochemicals of P. quadrifida in several solvents extracts were found to be rich source of vitamin C. Both
demonstrates the presence of tannin in petroleum ether, extracts showed comparable results of vitamin C
chloroform and ethanol extract18.Severalstudies determination (Figure 2,3,4).
confirmed that the tannins exhibit antioxidant, Table 1. Ascorbic acid Content in whole fresh and dry
antimicrobial, anti-inflammatory, antidiarrheal, and for extracts of P. quadrifida
heal burns and treat other diseases.19
Extract Ascorbic acid Content (ppm)
Quantitative determination of ascorbic acid (vitamin
C) Fresh P. quadrifida 1.160
Ascorbic acid content in whole fresh and dryP. Dry P. quadrifida 1.760

Figure 2. Spectrumof standard ascorbic acid

Figure 3. Spectrum of ascorbic acid in freshP. quadrifida extract

Figure 4. Spectrum of ascorbic acid in dry P. quadrifida extract

Phytochemical investigation of Sudanese Portulaca quadrifida

Current Trends in Biotechnology and Pharmacy 216
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DOI : 10.5530/ctbp.2020.4s.26

MTT assay In vitro antidiabetic activity

Cytotoxicityof ethanolic extract of whole dry and fresh Medicinal plants play an important role in the
P. quadrifida at different concentrations (500, 125 and management of diabetes mellitus especially in developing
62.5 ppm) was evaluated against normal Vero cell using countries. Moreover, during the past few years some of
MTT assay, results are presented in Table 2. The toxicity
the new bioactive drugs isolated from plants showed anti-
of both extracts was concentration dependent. The extract
diabetic activity used in clinical therapy.21 In anti-
of dry P. quadrifidadisplayed low toxicity effect against
diabetic activity evaluation by alpha amylase inhibition,
Vero cells, while the fresh P. quadrifida extract showed
high toxicity with highest inhibitory percentage (92.09 both extracts showed highest activity with inhibition
and 73.35 %) at concentration 500 and 125 ppm percentage 95 and 91 % for dry and fresh extracts
respectively.The IC50 value was calculated and the results respectively. Results are presented in Table 3. The anti-
were 858 and 155.3092 ppm for dry and fresh P. diabetic drugs from plants in current clinical use are
quadrifida sample extracts respectively. Extracts which preferred mainly due to lesser side effects and low cost.
revealed IC50<90 ppm were considered toxic20. Thus, Results indicated that both fresh and dried P.quadrifida
it was clear both extracts of P. quadrifida were safe.
possessed anti-diabetic activity,besides stronger activity
Table 2. Cytotoxicity of P. quadrifidaextracts was observed in the fresh herb. These findings provided
Sample Concentration Inhibition % IC50(ppm) evidence for the application and development of fresh
extract in the treatment of diabetes mellitus18. Comparing
Whole Dry 62.5 -22.4476 858.7197 the results from the two extracts, the whole dry and the
125 -18.8811 whole fresh ofP.quadrifida shows comparable values of
500 33.84615 vitamin C content and anti- diabetic activity. The whole
Whole Fresh 62.5 -12.8671 155.3092 dry extract showed lesser toxicity than whole fresh extract
125 73.35664 while the fresh whole extract showed measurable higher
500 92.0979 tannin content compared to the dry whole extract.

Table 3. -amylase inhibition percentage of P.quadrifidaextracts

Sample Inhibition % IC50 (ppm)

250 ppm 500 ppm 1000 ppm

Dry P.quadrifida 58.28274 87.33738 95.4033 179.5797

Fresh P.quadrifida 58.54293 85.16912 91.4137 173.0966

Control +ve (Acarbose) 90.80659 %

4. Conclusion as a source of medicinal agent. Further investigations are

Generally, natural sources are rich in chemical important for the identification of active principles to
constituent that observe beneficial medical uses. development new drugs against various diseases.
P.quadrifida is one of these plants that show medicinal Conflict of interests
uses but less widely investigated. The high Tannin,
Authors declareno conflict of interests.
vitamin C levels,less toxicity, and anti-diabetic activity
for both whole fresh and the whole dry extracts of 5. References
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A continuing source of novel drug leads. Pure and 13. Mulla, S. K andSwamy, P. A. (2012). Anticancer
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8. Das, M and Kumar, A. (2013). Phyto-
pharmacological review of Portulacaquadrifida Linn. 18. GeethaPriya, G. (2014). Evaluation of
Journal of Applied Pharmaceutical Research, 1(1), InvitroAndInvivo Anti Diabetic Activity of Ethanolic
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9. Kirtikar, K. R andBasu, B. D. (1987). Indian
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Phytochemical investigation of Sudanese Portulaca quadrifida

Current Trends in Biotechnology and Pharmacy 218
Vol. 14 (5) 218-221, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.27

Analysis of Prednisone in Indonesian Uric Acid Herbs Using

High Performance Liquid Chromatography
Pri Iswati Utami1*, Elza Sundhani2, Deka Maulyani1
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Muhammadiyah Purwokerto,
Purwokerto, Central Java, Indonesia.
2Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Universitas Muhammadiyah
Purwokerto, Purwokerto, Central Java, Indonesia.

*Corresponding Author : priiswatiutami@ump.ac.id.

Abstract manufacturer has to be given label JAMU and a unique

The problem of adulteration of herbal medicines logo of jamu in the package. Based on Indonesia's
product with active pharmaceutical ingredients (API) has regulations, traditional medicines are prohibited from
existed for years in Indonesia. According to the containing isolated or synthetic medicinal chemicals.
government's rules, it is not allowed to add API to However, the existence of active pharmaceutical in herbal
traditional herbal medicine. Uric acid herbs are one of products is still found (1).
the most popular herbal medicine products. Prednisone Steroid, including prednisone, was one of the most
is one of the corticosteroids that has been reported to be frequent adulterants. An examination of distributed jamu
detected in herbal products. The purpose of this study is thus becomes an important issue to prevent harmful side
to identify prednisone in uric acid herbs using High effects due to adulterated herbal medicine.
Performance Liquid Chromatography (HPLC). The Some methods reported have been published to
stationary phase used in this study was C18 analyze prednisone in medicinal herbal medicine products
Puroshper®STAR RP-18e LiChroCART® column (250 such as Thin Layer Chromatography (2); Solid Phase
- 4.6 mm; 5 µm i.d.), while the mobile phase was methanol Extraction-High Performance Liquid Chromatography/
: water (60 : 40 v/v). The mobile phase flow rate was set HPLC (3);Ultra-Performance Liquid Chromatography-
at 1 ml/min. Prednisone in the sample was detected at Mass Spectrometry (4), and Gas Chromatography-Mass
wavelength 243 nm using a UV detector. Validation Spectrometry (5). The purpose of this study is to identify
methods in this study consisted of precision, linearity, prednisone in uric acid herbs using simple High
the limit of detection (LOD), the limit of quantitation Performance Liquid Chromatography (HPLC) method
(LOQ), and accuracy. The precision is indicated by the with an ultraviolet detector.
relative standard deviation (RSD) value of 0.33% (<2%).
The correlation coefficient value (r) of 0.9955 obtained 2. Materials and Methods
from the prednisone calibration curve shows the method's The Separation was carried out on a set of HPLC
linearity. The recovery values of 100.11 ± 0.82 % indicates instruments (Shimadzu Prominence-i LC-2030C)
the accuracy of the method that meets the requirements. equipped with Puroshper®STAR C18 columns (250 mm
The LOD and LOQ values were 2.96 and 9.85 µg/ml, x 4.6 mm i.d. 5µm). The absorption spectrum was made
respectively. Method validation parameters have been using a Shimadzu 1800 UV-VIS Spectrophotometer. The
proven that meet the requirements. The HPLC method different brands of uric acid herbs were taken from several
can be used to analyze prednisone in uric acid herb shops in Purwokerto, Central Java, Indonesia. Sample
samples. The application of the method for analysis of from eight different companies were coded as A, B, C, D,
eight herbal products taken from the market shows that E, F, G, and H.
prednisone was detected in two products. Preparation of the mobile phase : The mobile phase was
Key words : prednisone · uric acid· herbs · HPLC made from a mixture of methanol and water at a ratio of
60:40 v/v. The mixture is then filtered and sonicated for
1. Introduction 20 minutes.
Jamu / Herbs is a herbal preparation, an Indonesian Preparation of standard solution : The prednisone
traditional medicine. Herbs that produced by the reference standard was carefully weighed as much as 10
Analysis of prednisone using HPLC
Current Trends in Biotechnology and Pharmacy 219
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DOI : 10.5530/ctbp.2020.4s.27

mg and put in a 10 ml volumetric flask, then dissolved of 243 nm was used for the detection of the drug in this
with a mobile phase quantitatively to obtain a 1000 g/ HPLC method.
ml solution. From there, the solution was pipetted 1.0 ml The optimum composition of the mobile phase : In this
and put into a 10 ml volumetric flask, and then diluted study, prednisone analysis in uric acid herbs was carried
quantitatively with a mobile phase so that a solution of out by HPLC with a stationary phase of RP-18. Based on
100 g / ml is obtained. the results of the optimization of the mobile phase, the
Determination of the maximum wavelength of most optimal separation is produced by a mixture of the
prednisone :The 10 g/ml prednisone reference solution mobile phase of methanol : water (60:40 v/v).
was scanned at a wavelength of 200 - 400 nm using a System suitability : System suitability is an integral part
UV-Vis spectrophotometer. The wavelength with of the analysis procedure. The test is based on the concept
maximum absorption was determined from the spectrum that equipment, electronics, analysis procedure, and the
obtained. samples to be analyzed are the whole system so that they
Optimization of the composition of the mobile phase: can be evaluated (6). Table 1 shows the results of the
Some mobile phase compositions used are: buffer pH 4 system suitability test results. From repeated injection of
prednisone solution in HPLC, the RSD values for the
and methanol (80:20 v/v); buffer pH 4 and acetonitrile
parameters of retention time, peak area, and tailing factor
(80:20 v/v); methanol and water (50:50 v/v); and
are <1.0%, respectively. The tailing factor of 1.639 shows
methanol and water (60:40 v/v).
the shape of the peak that meets the criteria of asymmetric
System suitability test : A prednisone standard solution aspects because its value is less than 2.0 (7). The system
of 25 g/ml was prepared 6 times and then injected into suitability test results show that the conditions used to
HPLC with a volume of 20 l. The mobile phase flow determine prednisone levels have a good system
rate was set at 1.0 ml/min. Retention time, peak area, suitability based on the RSD value <2% (7).
and tailing factor were recorded. Then the average, SD,
Table 1 : System Suitability
and RSD were calculated.
Retention Tailing
Preparation of prednisone calibration curve : Injection No. Area
time (min) factor
Prednisone solution in the mobile phase with a 1 6.567 822,764 1.615
concentration of 10; 15; 20; 25; 30 and 35 g/ ml were 2 6.517 822,175 1.643
prepared. Each solution was filtered and sonicated for 3 6.615 827,412 1.626
10 minutes. Then each of them was injected into the
4 6.610 827,821 1.645
HPLC. The peak area shown on the chromatogram was
5 6.609 823,670 1.651
recorded. The plot between prednisone concentration and
6 6.611 828,167 1.655
peak area was made.
Average 6.588 825,334.83 1.639
Validation of analytical methods :The validation
SD 0.039 2,752.53 0.015
parameters of the tested analytical methods include
RSD (%) 0.594 0.33 0.944
selectivity, linearity, the limit of detection and limit of
quantitation, precision, and accuracy. Validation was done Prednisone calibration curve : Figure 1 shows a
according to ICH guidelines (6). prednisone calibration curve made in the range of 10 -
35 µg/mL. The calibration curve obtained is used to
Determination of prednisone in the sample : Uric acid calculate prednisone levels in the sample.
herbs samples of brands A, B, C, D, E, F, G, and H were
carefully weighed 150; 4500; 75; 40; 560; 187.5; 750;
and 2500 mg, respectively. Then, put in a 10 mL
volumetric flask, dissolved with the mobile phase, then
filtered and sonicated for 20 minutes. The solution was
injected into the HPLC. The determination was carried
out in triplicate.
3. Results and Discussion
The maximum wavelength of prednisone : Based on
the UV absorption spectrum of prednisone, a wavelenght Fig. 1. Prednisone calibration curve

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DOI : 10.5530/ctbp.2020.4s.27

Table 2 : Method validation result Around the prednisone peak, which is retention time
around 6, 5 minutes, there is no potential for interference
Parameter Result from other components of the mobile phase or sample
matrix.Table 2 shows the results of the analysis method
Retention time 6.588 ± 0.039 minute validation. The prednisone retention time was 6.588 ±
Linearity r = 0.9955 0.039 minutes. Linearity is indicated by the correlation
coefficient close to 1.0 (r=0.9955). The linear relationship
Range 10-35 µg/mL between concentration and peak area was proven in the
range of 10-35 µg/mL.The detection limit and the
LOD 2.95 µg/mL quantitation limit were determined by mathematical
LOQ 9.85 µg/mL calculation of the calibration curve. The detection limit
and the limit of quantitation are 2.95 µg/mL and 9.85 µg/
Precision RSD = 0.33 %. mL, respectively. Precision is indicated by the RSD value
of less than 2.0 %. Table 3 shows the accuracy of the
Table 3: Accuracy of Prednisone Quantitative Analysis HPLC method for Prednisone analysis in uric acid herbs.
Method in Herbs The recovery value is from 92.26 to 103.05% (average
recovery 100.11 ± 0.82 %). The accuracy criterion for
Standard Standard the analysis method is that the average recovery value is
Recovery RSD
Sample added found SD
(%) (%) 100 ± 2% (7).
(µg/mL) (µg/mL)
A 35 32.41 92.26 2.24 2.42
Table 4 : The results of the analysis of prednisone in uric
acid herb products
B 35 36.06 100.94 1.70 1.65
Sample Prednisone content (µg/mg)
C 35 35.01 100.04 0.07 0.07
A n.d.
D 35 33.59 95.96 0.95 0.99 B n.d.
E 35 35.78 102.24 0.18 0.18 C 21.76 ± 0.44
D 50.07 ± 0.30
F 35 35.82 102.34 0.40 0.39
E n.d.
G 35 35.80 102.27 0.36 0.35 F n.d.
H 35 36.07 103.05 0.66 0.65 G n.d.
H n.d
Validation of analytical methods : Figure 2 shows the
selectivity of the HPLC method that meets the criteria. n.d. = not detected
The results of the analysis of prednisone in uric acid
herb products : The results of the analysis of uric acid
herbs samples shown in Table 4 shows that the
adulteration of active pharmaceutical ingredients,
especially prednisone, is still found in two products taken
from the market in Indonesia. The presence of
adulteration in this study is an addition to the previous
findings. In previous studies it has been reported that in
antidiabetic jamu still found the active pharmaceutical
ingredients of glibenclamide (8). In "kuat lelaki" jamu
found sildenafil as an adulterant (9), whereas in "pegal
linu" jamu there was no paracetamol detected (10).
Another study also reported that sibutramine as adulterant
was detected in herbal slimming products collected from
the market in Depok City, West Java, Indonesia (11).
The active pharmaceutical ingredients are also found
Fig. 2. Chromatogram of (a) Blank; (b). Prednisone
in various herbal supplement products in several countries
reference; and (c) Uric acid herbs product.

Analysis of prednisone using HPLC

Current Trends in Biotechnology and Pharmacy 221
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DOI : 10.5530/ctbp.2020.4s.27

such as the presence of cyproheptadine and intraditional Chinese medicine and dietary
dexamethasone in weight gain product in Iran (12); supplements using hydrogen as a carrier gas. PLoS
sildenafil, tadalafil, and vardenafil hydrochloride in ONE, 13(10): e0205371. https://doi.org/10.1371/
herbal medicine and food samples collected in Sultanate journal.pone.0205371.
of Oman (13).
6. International Conference on Harmonization (2005)
4. Conclusion Q2(R1): Validation of Analytical Procedures: Text
and Methodology.
The HPLC method was successful in clearly
identifying and quantifying prednisone present in uric 7. Shabir, G. A. (2003). Validation of high-performance
acid herbs. From eight herbs, showed that two samples liquid chromatography methods for pharmaceutical
(25%) confirmed the presence of prednisone as an analysis Understanding the differences and
adulterant. This also calls for a thorough focus on making similarities between validation requirements of the
the regulation systems for this jamu stricter. The US Food and Drug Administration, the US
regulations related to licensing and labeling of jamu Pharmacopeia and the International Conference on
should be as strong as to ensure 100 % product integrity. Harmonization. J. Chromatogr. A, 987:57-66.
Acknowledgements 8. Utami, P.I. Firman, D.and Djalil, A.D. (2019).
The technical assistance by technical staff, Identification of glibenclamide in antidiabetic jamu
Department of Analytical Chemistry, Department of by high performace liquid chromatography method:
Biological Chemistry, Faculty of Pharmacy, Universitas Study in Purwokerto, Indonesia. J. Phys.: Conf. Ser.,
Muhammadiyah Purwokerto, Indonesia. 1402 055065.

Conflict of Interest 9. Sarigih, A.T.W. Kusuma, A.M.and Utami, P.I. (2010).

Analisis Sildenafil Sitrat Pada Jamu Tradisional Kuat
The authors declare no conflict of interest.
Lelaki Merk A dan B dengan Metode Kromatografi
5. References Cair Kinerja Tinggi. Pharmacy Pharmaceut J
1. www.pom.go.id. (2020). Public Warning Badan Indones., 7(2):24-34. 10.30595/pji.v7i1.554.
Pengawas Obatdan Makanan Republik Indonesia 10. Firdaus, M.I.and Utami, P.I. (2009). Analisis
tentang Obat Tradisional mengandung Bahan Kimia Kualitatif Parasetamol Pada Sediaan Jamu Serbuk
Obat, No: B.HM.01.01.1.44.11.18.5411, accessed on Pegal Libu yang Beredar di Purwokerto. Pharmacy
May 20. Pharmaceut J Indones., 6(2):1-5. 10.30595/
2. Hon, K-L.E. Lee, V.W.Y. Leung, T-F. Lee, K.K.C. pji.v6i2.408.
Chan, A.K.W. Fok, T-F.and Leung, P-C. (2006). 11. Hayun, H. Maggadani, B. P. and Amalina, N. (2016).
Corticosteroids are not Present in a Traditional Determination of Sibutramine Adulterated in Herbal
Chinese Medicine Formulation for Atopic Dermatitis Slimming Products Using TLC Densitometric
in Children. Ann Acad Med Singapore, 35:759-63 Method. Indonesian Journal of Pharmacy, 21(1):15-
3. Ku, Y-R. Liu, Y-C.and Lin, J.H. (2001). Solid-phase 21.
Extraction and High-performance Liquid 12. Saberi, N. Akhgari, M. Bahmanabadi, L. Bazmi, E.
Chromatographic Analysis of Prednisone and Mousavi, Z. (2018). Determination of synthetic
Adulterated in a Foreign Herbal Medicine. J Food pharmaceutical adulterants in herbal weight gain
and Drug Analys, 9(3):150-152. supplements sold in herb shops, Tehran, Iran. DARU
4. Yu, K. Powell, M. Maziarz, M. and Patel, Journal of Pharmaceutical Sciences, 26:117-127.
D.M.(2016). Analysis of an Adulterated Herbal 13. Al Lawati, H. A. J. Al Busaidi, I. Kadavilpparampu,
Medicinal Product Using Ultra-Performance Liquid A. M. and Suliman, F. O. (2017). Determination of
Chromatography Coupled with QTOF Mass
Common Adulterants in Herbal Medicine and Food
Spectrometry. World J Tradit Chin Med, 2(3):1-9. Samples using Core-shell Column Coupled to
5. Lin, Y-P. Lee, Y-L. Hung, C-Y. Chang, C-F. and Chen, Tandem Mass Spectrometry. Journal of
Y. (2018). Detection of adulterated drugs Chromatographic Science, 55(3):232-242.

Pri et al
Current Trends in Biotechnology and Pharmacy 222
Vol. 14 (5) 222-225, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.28

An Analysis of Rat Meat with FTIR and GC / MS

for Halal Authentication
Wiranti Sri Rahayu1*, Pri Iswati Utami1, Irfan Nugraha1, Rati Janah1
1Faculty of Pharmacy, Universitas Muhammadiyah Purwokerto, Indonesia

*Corresponding author : wirantisrirahayu@ump.ac.id or wirantisrirahayu@gmail.com

Abstract Non halal component in food products can be analyzed

Fourier Transform Infra Red (FTIR) and Gas with several methods, such as Gas Chromatography-Mass
Chromatography Mass Spectrophotometry (GCMS) Spectrometry/GCMS (3,4), Fourier Transform Infra Red/
method were developed to determine rat fat which is FTIR (5,6,8), Liquid Chromatography/HPLC (9) and
extracted from rat meat. Fat was extracted with methanol Polymerase Chain Reaction/PCR (7). FTIR method is fast
and chloroform as a solvent. Derivatization process was and consistent method, even in low analyte concentration
conducted to convert fat into a methyl ester form using (10), non-destructive, sensitive, and does not require
sodium methoxide and boron trifluoride. GCMS analysis complicated sample preparation (11).
was used to identify the fatty acid composition from rat However, the FTIR method has limitations, that is it
fat. The FTIR spectral bands correlated with bovine, pork, cannot certainly identify the content type of the sample's
chicken and rat fat were scanned, interpreted, and each fatty acid component (8). Fatty acid analysis can be
identified. Qualitative differences between FTIR spectra done by GCMS method. Fatty acid composition of meat
were proposed as a basis tools for differentiating between can be used for distinguishing rat meat from other meat.
rat fat and other fat. Principal Component Analysis (PCA) The fatty acid composition is determined as methyl ester.
at combining wavenumber regions of 1250-1100 cm-1 The purpose of this research is to identify rat meat in
and 3010-2850 cm-1 was capable of distinguishing rat food products by using FTIR and GCMS methods.
meat from other meat. GCMS chromatogram showed the
2. Materials and Methods
fatty acid composition in rat meat which five major
compounds were hexadecenoic acid, 9,12- Lipid Extraction :
octadecadienoic acid, 9-hexedecenoic acid, tetradecanoic Black rats were obtained from local farm in Banyumas
acid and 7-hexadecanoic acid. PCA based on fatty acid regency Indonesia. Bovine, pork and chicken meat was
composition can distinguish rat meat from other meat. obtained from local market in Banyumas Indonesia. Fat
Key words : Rat meat · FTIR · GCMS · was extracted using chloroform: methanol by Bligh &
Dyer methods.
1. Introduction
GC-MS Analysis :
A muslim is majority population in Indonesia, whose
must consume halal food products. Thus, it is important Fat was hydrolyzed with alkaline to produce fatty acid,
to ensure the halals of food products. Adulteration non and followed with methyl esterification.
halal meat in food product become increase and some Transesterification performed by the BF3-MeOH method
food products are found to have been adulterated with to form fatty acid methyl ester. Approximately 50 µL oil
non halal ingredient, such as rat meat in meatball (1,2). samples were added with 1.0 mL n-hexane and 200 µL
0.2 N NaOCH3 solutions and heated at 60°C for 10 min.
Meatballs are a popular food in Indonesia where the
Then, the mixture was added to 1.5 mL BF3-methanol
main component is meat. The meat can be from beef,
reagent, and heated at 60°C for 10 min. After it was cool,
chicken, or fish. Some food manufacturers replace halal
1 mL of saturated NaCl solution was added, and shake.
meat with non halal meat such as rat, because it is easier
The resulting hexane layer was used as a sample solution
to get and cheaper. The aim to reduce the cost. However,
for GC-MS. Subsequently, 1 µL of the clear supernatant
it is unfavorable for consumers and harmful to health
was taken and injected into a GC-MS (Shimadzu QP2010,
because rat can cause some diseases like Salmonellosis,
Shimadzu Corp., Tokyo, Japan). The column used is a
Leptospirosis and Plague (1).
Analysis of rat meat using FTIR and GC / MS
Current Trends in Biotechnology and Pharmacy 223
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DOI : 10.5530/ctbp.2020.4s.28

SH-Rxi-5Sil MS (5% diphenyl/95% dimethyl MS) which present in the lipid extracted from rats and
polysiloxane) capillary column (30 m x 0.25 mm ID, 0.25 other meat. Saponification with alkaline and followed
m film thickness). Helium was used as the carrier gas by BF3-catalyzed methylation were used to form fatty
at flow rates of 1.0 mL/min. The injector temperature acid methyl ester. Peak identification of fatty acids methyl
was 280°C. The oven temperature was set at 100°C for 5 ester in the analyzed samples was conducted by
min, increased to 240°C at a rate of 4°C/min and held at comparing the retention time and molecular mass of mass
the final temperature for 30 min. The GC-MS operation spectra of standard mass spectra, which were obtained
was controlled by Lab Solution software. MS spectra from library (Wiley9.lib) of the GCMS instrument and
were obtained in wide range of m/z 10- 500. FAME peaks also confirmed by comparing the mass spectrometric
were identified by comparing their retention time with fragmentation pattern with the standard.
the FAME standard and similarity index (SI more than Composition of fatty acid can be used to differentiate
90%). rat fat from other species. GCMS chromatogram at figure
Fat analysis using FTIR : 1 revealed that hexadecanoic acid has the highest level
fatty acid from rat fat. The other major constituents are;
Fat from each meat was dropped on the ATR crystal,
9,12-octadecadienoic acid(tr 34.424min); 9-hexadecenoic
which was placed in a controlled temperature (20°C) as
much as 1 drop. Then, the fat was scanned for 32 times
at the wave number of 4000-650 cm-1 with a resolution
of 4 cm-1 and was recorded in the form of absorbance.
FTIR spectra were analyzed using chemometrics in the
form of PCA using Horizon MB software.

3. Results and Discussion

Fatty acid composition was identified and measured
with gas chromatography with mass spectrometry (GC Figure 1. Rat's fat GC chromatogram

Table 1: Fatty Acid Compositions of Lipid Extracted from Bovine, Rat, Chicken and Pork Obtained By GC-MS
Method
Fatty Acid Fatty Acid Percentage (%)
Rat Bovine Chicken Pork
Dodecanoic Acid 0 0 0.07 0.2
pentadecanoic acid 0.2 0.61 0.06 0.09
tetradecanoic acid 1.79 3.18 0.56 1.82
9-octadecenoic acid) 0 24.52 46.57 37.62
cis-9-tetradecenoic acid 0.05 1.32 0 0
7-hexadecanoic acid 1.31 0.1 0.28 0.42
hexadecanoic acid 25.09 23.75 24.62 24.19
9-hexedecenoic acid 1.79 4.64 3.7 2.27
octadecanoic acid 0.32 12.75 7.74 13.15
heptadecanoic acid 0.29 1.17 0.1 0.44
cis-10-heptadecenoic acid 0.1 1.02 0.04 0.25
9,12-octadecadienoic 16.11 1.57 15.59 17.36
5,8,11,14-eicostate 0 0 0 0.22
6,9,12-octadecadienoic 0 0 0 0.13
eicosenoic acid 0.15 0.07 0.06 0.18
methyl eicosanoic acid 0.56 0 0 0
10-nonadecenoic acid 0 0.24 0 0
11,13-eicosadienoic 0.06 0 0 0.45
11-octadecenoic acid 0 47.43 0 0

Wiranti et al
Current Trends in Biotechnology and Pharmacy 224
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DOI : 10.5530/ctbp.2020.4s.28

acid (tr 29.594 min); tetradecanoic acid (tr 24.64 min);

and 7-hexadecanoic acid (tr 29.140 min). From Table 1
it can be seen that rat has a larger percentage of saturated
fatty acids than beef, chicken and pork. The differences
in the degree of unsaturated fatty acid in animal fats could
be due to the individual fatty acid distribution pattern
(12). The presence of methyleicosanoic acid is found in
lipid extracted from rats, but it is not found in bovine. In
contrast, 11- octadecenoic acid (47.30%) exists only in
lipid extracted from bovine. Hexadecanoic acid is found
to be approximately equal in all analyzed lipid types. The Figure 4. PCA Score Plot From Rat, Chicken, Pork And
quantity of 9.12-Octadecadienoic acid in bovine (1.57%) Bovine Fat at wavenumber 1250-1100 cm-1 and 3010-
is much lower than in rats (16.11 %). The fatty acid 2850 cm-1
composition of lipid extracted from rats were unique
compared to bovine, chicken and pork. From the GC- peak at wave number 3010-2950 cm-1. The absorption
MS analysis, it is found that the major constituents of pattern at wave number 3008.95 cm-1 (peak a) for rat fat
lipid extracted form rats were fatty acids with chain shows relatively higher peaks compared to bovine fat.
lengths of 15 to 21 carbon atoms (mainly C17 and C19). The high peak of rat fat absorbance in the area shows the
PCA score plot in Figure 2 showed that fatty acid presence of unsaturated fatty acid content which
composition can distinguish rat fat from other fat. Rat contributes to the high absorbance value, namely the C-
fat is located at different quadran from other fat. H stretching vibration area of the cis double bond.
Fingerprint area (1500-700 cm -1 ) that is at
wavenumber of 1118.71 cm-1 (i) shows the typical
spectrum of rat fat that is the C-O stretching vibration
area. The third point of difference is located in
wavenumber 1026.13 cm-1 (k) and 972.12 cm-1 (l) in
which this area does not show any absorption in the rat
fat spectrum.
Figure 4 showed that PCA can be accomplished to
classify between rat, pork, chicken and bovine fat. Rat
fat stand far from others fat, chicken and pork stand at
same quadrant which it showed they have similarity.

Figure 2. PCA Score Plot from rat, chicken, pork and bovine 4. Conclusion
fat based on fatty acid composition This study investigated application of GC-MS and
FTIR to identify rat meat based on fat and fatty acid
profile. The high constituents of lipid extracted from rats
are 9-Octadecenoic acid; hexadecanoic acid; 9,12-
octadecadienoic acid; octadecanoic acid; 9-hexadecenoic
acid; tetradecanoic acid; and 7 hexadecanoic acid. The
major constituents are fatty acids with chain lengths of
15 to 21 carbon atoms (mainly C17 and C19) and
unsaturated fatty acid higher than saturated fatty acid.
Figure 3. FTIR spectrum rat and bovin fat
GCMS method can be used to authenticate rat meat with
FTIR analysis was conducted based on the differences fatty acids content.
between the functional groups of fat from rat, chicken, Application of multivariate statistical analysis such
pork and bovine meat, which were measured at wave as PCA would be required to determine source of the
number 4000-650 cm-1. Figure 3 shows that the difference origin. Hence, this study showed that fatty acid data
of FTIR spectrum between rat and bovine fat is a typical allowed separation rat fat from other animal fats.

Analysis of rat meat using FTIR and GC / MS

Current Trends in Biotechnology and Pharmacy 225
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DOI : 10.5530/ctbp.2020.4s.28

The difference of rat and beef fat is located in Meatball Formulation, Asian Journal of
wavenumber 1026.13 cm-1 (k) and 972.12 cm-1 (l) in Biochemistry, 10(4): 165-172.
which this area does not show any absorption in the rat 6. Rahayu W.S., Rohman A., Sudjadi, Martono S.,
fat spectrum. FTIR spectroscopy at wavenumber region (2018), The potential use of infrared spectroscopy
1250-1100 cm-1 and 3010-2850 cm-1 combined with and multivariate analysis for differentiation of beef
chemometrics techniques can be used to determine rat meatball from dog meat for Halal authentication
meat. analysis, Journal of Advanced Veterinary and Animal
Acknowledgements Research, 5 (3): 307-314.
The technical assistance by technical staff, 7. Fibriana F, Widianti T., Retnoningsih A. and Susanti
Department of Analytical Chemistry, Faculty of (2012). Deteksi Daging Babi Pada Produk Bakso di
Pharmacy, Universitas Muhammadiyah Purwokerto, Pusat Kota Salatiga Menggunakan Teknik
Indonesia. Polymerase Chain Reaction, Biosaintifika, 4(2): 106-
112.
Conflict of Interest
8. Che Man Y B, Rohman A and Mansor T. S. T.(2011).
The authors declare no conflict of interest.
Differentiation of lard from edible oils by means of
5. References Fourier transform infrared spectroscopy and
chemometrics, Journal of the American Oil Chemists'
1. Widyasari I.Y., Sudjadi and Rohman A.(2015).
Society, 74:187-192.
Detection of Rat Meat Adulteration in Meat Ball
Formulations Employing Real Time PCR.Asian 9. Ahda, M. (2016). Application of HPLC (High
Journal of Animal Sciences,9(6): 460-465. Pressure Liquid Chromatography) for Analysis of
Lard in the Meatball Product Combined with PCA
2. Rahmania H.(2014).Analisis Daging Tikus dalam
(Principal Component Analysis). Asian J. Pharm.
Bakso Sapi Menggunakan Metode Spektroskopi
Clin. Res.9(6):120-123.
Inframerah yang Dikombinasikan dengan
Kemometrika, Skripsi,Universitas Gadjah Mada, 10. Hermanto, S., Muawanah, A., Harahap, R. (2008).
Yogyakarta, pp. 1-5. Profil dan Karakteristik Lemak Hewani (Ayam, Sapi
dan Babi) Hasil Analisa FTIR dan GCMS (Profile
3. Nurjuliana M., Che Man Y. B., Mat Hashim and
and Characteristics of Animal Fat (Chicken, Cow
Mohamed A. K. S.(2011). Rapid identification of
and Pork) FTIR and GCMS Analysis Results).
pork for halal authentication using the electronic nose
Valensi, 1, (3): 102-109.
and gas chromatography mass spectrometer with
headspace analyzer, Meat Sci,. 88:638-644. 11. Rohman, A., Che Man, Y. B. (2011). The Use of
Fourier Transform Mid Infrared (FT-MIR)
4. Rahayu W.S., Rohman A., Sudjadi, Martono S.
Spectroscopy For Detection and Quantification of
(2018).Identification of Dog for Halal
Adulteration in Virgin Coconut Oil. Food Chemistry,
Authentification with Gas Chromatography Mass
129 (2): 583-588.
Spectroscopy (GCMS) and Chemometrics,
Advanced Science Letter, 24 (1): 138-141. 12. Nizar N.N.A., Marikkar J.N.M., Hashim D.M.,
(2013), Differentiation of Lard, Chicken Fat, Beef
5. Guntarti A, Martono S, Yuswanto A. and Rohman
Fat and Mutton Fat by GCMS and EA IRMS
A.(2015). FTIR Spectroscopy in Combination with
Techniques, J.Oleo.Sci., 62 (7): 459-464.
Chemometrics for Analysis of Wild Boar Meat in

Wiranti et al
Current Trends in Biotechnology and Pharmacy 226
Vol. 14 (5) 226-232, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.29

Epidemiological Studies of Schistomiasis in Bauchi Central

Senatorial Zone, Nigeria
Usman, A.M
Biological Science Department, Nigerian Army University Biu, Borno State, Nigeria
Corresponding Author : usman.alhajimohammed55@gmail.com

Abstract Key words : Epidemiology, Schistosomiasis,

A twelve months Epidemiological Studies was Bulinusglobosus, Cercariae, Bauchi
conducted in Bauchi Central Senatorial Zone in 2016 to 1. Introduction
determine the prevalence, water contact activities, water
Schistosomiasis is a complex water-borne disease
quality and vector aspect of schistosomiasis in the study
caused by blood-dwelling trematode worms of the genus
area. Six hundred 600 samples of each urines and stools
Schistosoma and five species parasitizing humans include
were collected and examined microscopically for
Schistosoma haematobium, Schistosoma mansoni,
schistosomes eggs. The urine samples were examined
Schistosoma japonicum, Schistosoma intercalatum and
using sedimentation method while the stool samples were
Schistosoma mekongi. After malaria and intestinal
examined using formol-ether concentration technique.
helminthiasis, schistosomiasis is the third most
Twelve 12(2%) out of the entire urine samples examined
devastating tropical disease in the world, being a major
had eggs of Schistosoma haematobium and none of the
source of morbidity and mortality for developing countries
stool samples were positive with the egg of any intestinal
in Africa, South America, the Caribbean, the Middle East,
shistosomes. Two water bodies were randomly selected
and Asia (28). It is estimated that 200 million people are
from each selected local governments for surveyed of the
infected, of which 120 million are symptomatic and 20
intermediate hosts (snails) of the parasite. The
million have severe disease. In 74 countries, 600 million
intermediate host were collected and examined for
persons are at risk of infection (9,15) and the disease is
cercariae by exposing them to sunshine for 30 minutes in
principally for tropical and sub-tropical regions which
a beaker containing water and water samples were also
found in South and Central America, Africa, Asia and
collected for water quality studies such as Ph, temperature
South - East Asia. It is estimates suggest that 85% of all
and dissolved oxygen. Four hundred and twenty two (422)
schistosomiasis cases are now in Subsaharan Africa (9)
snails were collected and examined. Out of it, only
and centers for disease control and prevention in (2013)
21(4.9%) Bulinusglobosusshed cercariae and also the only
classified the disease as tropical neglected disease. The
vector of the parasite found in the area. Six hundred (600)
disease occurs in all the 36 state of Nigeria including the
questionnaires were distributed in order to determine the
Federal Capital Territory (12). Nigeria is an endemic area
participants' knowledge and perception about the parasite,
with an estimated 11 million people infected (18). (90)
sex, age, water source, toilet facilities and their
estimated that 101.28 million people are at risk of
occupations. The infection rates by the parasite in different
infection in Nigeria while 25.83 million people are
sexes is not statistically significant (p>0.05) while in
actually infected.
different age groups, individuals using different water
source, individuals using different types of toilet facilities Snails are the intermediate host of the parasite and
and individual with different occupational groups were snail ecologists have tried to correlate snail distribution
all statistically significant (P<0.05). The water quality with physico chemical factors and to discover the ranges
seemed to have an effect on the infectivity of the snail of these factors within which the snails thrive (33). Under
vectors as out of the 422 snails collected and examined natural conditions, snail are exposed to a range of varying
only 21(4.9%) snails were infected in the water with low and often interacting environmental factors which produce
pH value and high dissolved oxygen. From the results collective effect on them and it is usually difficult to
obtained, schistosomiasis is not endemic in the study area. separate the effect of any factor from others (4). It is well
Health education is recommended to maintain the non- documented that intermediate host snail inhabit a wide
endemic nature of the parasite in the study area. range of natural habitats (7, 25, 26, 29, 31). Man made

Epidemiological studies of schistomiasis

Current Trends in Biotechnology and Pharmacy 227
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habitats such as irrigation canals, pools behind dams, was adopted (16) to collect the snail and bottle for water
ponds along roads and ditches (temporal habitats) may samples.
become rapidly inhabited by these intermediate host
Identification and examination of snails
snails, thus contributing to disease transmission (2, 17,
24, 27). Therefore, understanding the ecology of the snail The snails collected were separated and identified on
intermediate host, geographical distribution of the snail the arrival to laboratory according to standard key
vectors borne disease and also their transmission process described by (8). Each species were placed in a separate
is very important in tackling the disease. glass beaker bearing labels showing the location of
collection; reference number and date of collection. 10
This study is aim at determining the prevalence of the
snails were placed in each beaker 500ml capacity. 100ml
parasite and other factors that influences the transmission
of water was added before exposing them to sun- light
of the parasite such as snail populations (intermediate
for 30 minutes to facilitate the shedding of cercariae by
host) and physio-chemical qualities of the water bodies,
the snails. Then, the water in the snail containers were
thereby providing adequate information that can be
examined for cercariaeunder a dissecting microscope.
utilized in designing a suitable programmed for effective
Each snail in the containers that is positive with cercariae
control of schistosomiasis in study area.
was separated and examined further, by placing each of
2. Materials and Methods them in a separate beaker. 10mls of water was added and
exposed to sunlight for another 30 minutes. The water
Study area
examined again for emergence.
The study was conducted in Bauchi Central Senatorial
Water sample analysis
Zone, Nigeria. Six local governments make up the zone
out of the twenty local government of the state which Water samples were collected from the selected sites
includes Ningi, Warji, Ganjuwa, Misau, Dambam and and taken to the laboratory and analyzed for temperature,
Darazo. The zone occupies a total land of 15627km Ph and dissolved oxygen. Temperature was determined
representing about 32.4% of the state's total land area. using portable Hanna instruments Dist 5 EC/TDS/
According to the 2006 census, the zone has a total Temperature Tester HI98311.o c, pH was determined
population of 1448386. The zone has two distinctive using Hanna instrument pHep(R) pH Tester - HI98107
vegetation namely Sudan and Sahel savannah. During while dissolved oxygen was determined by Wicklers
the dry season, pools of the varying sizes (lakes, ponds, method.
dams, ditches) are found in various parts of the zone Ethical clearance
which serve as a source of water for domestic,
Permission and consent was sought from the Bauchi
recreational, occupational and socio-cultural purposes
state government (MOH/GEN/S/1409/1) before
(35).
proceeding with the research after which the District
Sampling techniques heads of the selected local governments were approached
A random sampling technique was employed to select for same purpose as well as the consent of the individual
three (3) local governments out of the six (6) local participants.
governments in the zone and two water sites in each of Data analysis
the selected local governments for snail collections. The The data collected were subjected to Chi-square test
sample size of 600 was determined using the formula as the relationships between two variables were compared
describe by (19, 3) and the sample size of each local and simple percentage. p<0.05 were use to determined
government was also determined by (21). the level of significance.
Sample collection 3. Results and Discussion
Each selected individual was given two containers for A total of 422 snails were collected from January to
collection of stool and urine and a questionnaire. Before December, 2016 for the purpose of this study. During the
giving the containers, they were instructed on when and study only Bulinusglobosus species were found in the
how to collect the samples. Snail and water samples were area that can transmit the parasite and 21 (4.9%) out of
collected from the selected water bodies using long Bulinusglobosus were infected with the infective stage
handed dip net to scoop at each site for 10 minutes which of the parasite or shed cercariae. Table 1 showed the

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Table 1 : Prevalence of snails collected according to month during the study

Months No of snail collected No Infected (SNAIL) = Observed

(O) infectivity rate
(%)

January 42 2 4.8
February 39 0 0
March 12 0 0
April 18 0 0
May 7 0 0
June 0 0 0
July 0 0 0
August 0 0 0
September 37 2 5.4
October 78 7 8.9
November 103 6 5.8
December 86 4 4.7

Total 422 21 4.9

X2 calculated = 20.57; X2 tabulated = 19.68, df = 11, P>0.05

number of snails collected and examined in each month Table 3 shows the infection rate according to group
during the study. The month of October had the highest age of the participants, out of all the age group examined
infected snails with 8.9%, followed by November, 11-20 year age group recorded the highest prevalence
September, January, and December with 5.8%, 5.4%, rate with 9(5.2%) followed by 21-30, 31-40 with 2 (1.8%),
4.8%, and 4.7% respectively while in February, March, 1 (0.9%) respectively and there was no infection in age
April, May, June, July and August no was infected or group 4-10 and 41 - above. The infections rate among
snail shed cercariae. The infections rate among the snails different age group was statistically significant (P<0.05).
collected during the study in relation to month were no Also the infection rate according to gender, shows out of
significantly different at p>0.05. Also the prevalence rates the 600 samples examined (420 male and 180 female).
of the parasite in different locations (local government). Female had the highest infection rate when compare with
Was not significant at p<0.05. A total of 600 urine and male counterpart with 4(2.2%) and 8(1.9%). The infection
stool samples each were collected in the three selected rate between the sexes was significantly different
local government in zone. The samples collected in each (P>0.05) as shown in table 4.
of the local government are 225, 136 and 239 in Misau, Table 3 : Prevalence of Schistosomiasis in the Different
Dambam and Ganjuwa respectively. Ganjuwa local Age Groups of Inhabitants in Bauchi central senatorial
government had the highest prevalence rate with 8(3.3%) zone.
followed by Misau and Dambam had the lowest with
3(1.3%) and 1(0.7%) respectively as show in table 2. Age No No Prevalence
Examined Infected (%)
Table 2 : Prevalence of The Parasite in Three selected
local government in the Zone. 4-10 130 0 0
Local No No Prevalence 11-20 172 9 5.2
Govt. Examined Infected (%) 21-30 113 2 1.8
Misau 225 3 1.3 31-40 103 1 0.9
Dambam 136 1 0.7 41- above 82 0 0
Ganjuwa 239 8 3.3 Total 600 12 2
Total 600 12 2 X2 calculated = 27.97; X2 tabulated = 9.488, df= 4,
X2 Calculated = 3.83; X2 Tabulated = 5.991, Df= 2, P>0.05 P>0.05

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Table 4 : Prevalence of S.haematobium in Relation to Table 6 : prevalence of the parasite among inhabitant
Sex in the study population with different source of water.
Sex No No Prevalence Source of No No Prevalence
Examined Infected (%) Water Examined Infected (%)
Male 420 8 1.9 Pipe borne 130 1 0.8
Female 180 4 2.2 Pool/pond 40 2 5
Total 600 12 2 Borehole 250 0 0
X2 calculated = 0.06; X2 tabulated = 3.841, df= 1, P>0.05 River/stream 36 9 25

Table 5 : Prevalence of the parasite in relation to different Drawn well 144 0 0

occupational groups in the senatorial zone. Total 600 12 2
Occupation No No Prevalence X2 calculated = 163.07; X2 tabulated = 7.815, df= 4,
Examined Infected (%) P>0.05

Students 268 8 3.0 1(0.6%) respectively. During the study no individual

found using buckets as toilet facility. The prevalence rate
Civil servants 130 0 0
among the study population using different type of toilet
Farmers 124 3 2.4 facilities is significantly different (p<0.05).
Other 78 1 1.3
Table 7 : Prevalence of the parasites among different
Total 600 12 2 users of toilet facilities during the study.
X2 calculated = 13.38; X2 tabulated = 7.815, df= 3,
Toilet No No Prevalence
P>0.05
facility Examined Infected (%)
Table 5 show the infection rate of the parasite
W.C Toilet 160 1 0.6
according to different occupational group. The
Bucket 0 0 0
occupational groups are namely; students, farmers, civil
servants and others. Students had the highest prevalence Pit latrine 300 2 0.7
rate of 8(3.0%) followed by farmers 3(2.4%), others Bush 100 8 8
1(1.3%) and no civil servant were infected with parasite Anywhere 40 1 2.5
during the study. The prevalence rate of the parasite in Total 600 12 2
the different occupational groups was statistically
X2 calculated = 11.03; X2 tabulated = 7.815, df= 4,
significant (P<0.05). However, prevalence rate among
P>0.05
the available water sources in the area are pipe-borne
water, Borehole, pool/pond, River/Stream and drawn Table 8 shows relationship between numbers of snails
well. Individuals whose sources of water supply for that shed cercariae and the average water quality values.
domestic use were streams recorded highest prevalence The average pH, temperature and dissolved oxygen of
25(9%) followed by pool/pond 5(5%) and pipe bond the water samples collected were measure using
water had the least 1(0.8%) while no infection in borehole appropriate meters and found to range from 7.0 to 8.5,
and drawn well. The difference in the infection rate in 22.2 to 31.90C and 7.0 to 8.3mg/l respectively. Only one
different occupational group in the study area was type of intermediate host of the parasite (snails) were
statistically significant (P<0.05) as show in table 6. encountered during the study in this zone
(Bulinusglobosus). 422Bulinusglobosus was collected
Table 7 shows the Infection rates in an individual
and examined and only 21(4.9%) were found infected or
who's used different types of toilet facilities. The available
shed cercariae during the study. The month that
toilet facilities are namely water closet toilet, bucket, pit
cercarieawere shed had the lowest pH and temperature
latrine, bush and those who use anywhere to defecate.
values but highest dissolved oxygen. Table 10 shows
Individuals who use bush to defecate recorded highest
relationship between numbers of snails that shed cercariae
infection rate of 8 (8%) followed anywhere, pit latrine
and the average water quality values.
and water closet system with 1(2.5%), 2(0.7%) and
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Table 8 : Relationship between Snails Shedding Cercariae and average water quality values

Month No of snails No of snails Average Water Quality Values

collected Shed
cercariae
o
pH Temp C Dissolve oxygen
mg/l

January 42 2
7.0 22.2 8.3
February 39 0 7.0 24.7 7.9
March 12 0 7.2 27.8 7.5
April 18 0 7.8 31.9 7.0
May 7 1 8.1 29.0 7.4
June 0 0 8.2 28.6 7.4
July 0 0 8.0 25.1 7.8
August 0 0 8.5 24.3 8.0
September 37 2 7.7 25.0 7.9
October 78 6 7.4 26.0 7.7
November 103 6 7.2 24.1 8.0
December 86 4 7.1 22.3 8.3

Total 422 21

The entire Bauchi State experience two climatic indicates low endemicity of the parasite in the study area.
seasons, the dry season (October to April) and the wet These observations is in agreement with the earlier reports
season (May to September). This study includes both the by (6,10)in Bauchi and Yobe states respectively which
data in two seasons. The snail vectors species all are neighboring state to the study area. The low
encountered during the study showed a great seasonal prevalence of the disease, recorded in this study area may
variation in all selected site with the peak at the beginning probably be due to the fact that some of the localities are
of dry season. This observation agrees with the earlier urban settlements with improved water supply and toilet
reports (23, 31). Out of 422 Bulinusglobosus snails facilities. However the snail vector are not harbouring
examined only 21(4.9%) shed infective stage, (cercariae) the infective stage of the parasite as only 21(4.9%) of the
of the parasite. This means the snail vectors were not snail vectors shed cercariae in their streams. The infection
harbouring the infective stage of the parasite in this zone. rate is higher in male than female counterpart, infection
However, the infection in different location of the study rate in individual with different occupational group with
area showed that Ganjuwa had the highest prevalence students and farmers had the highest prevalence rate than
rate followed by Misau and Dambam local government other occupational groups and also is higher in different
had the least with 3.3%, 1.3% and 0.7% respectively. age group with age 11-20 had the highest prevalence.
This pattern of infection in different locations within the The different between infection rate was not significant
same study area was similar to the report (11, 30). The but individual with different occupational group and
major factors that might be responsible for these patterns different age a group were all statistically significant
are low literacy level, poor sanitation due to lack of basic (p>0.05). (34) report said the main groups at risk are
amenities such as water, inadequate and indiscriminate school age children, specific occupational group
disposal of human wastes, migration of infected (fishermen, irrigation workers, farmers) , woman and
individuals from endemic areas and high water contact other groups using infected water for domestic purposes.
activities such as irrigation activities, recreation and other (20) reported that infection in pre- school and school
related activities. children was primarily due to exposure occasioned by
The overall prevalence of 12(2%) of the parasite washing, bathing, dry season farming, and fishing
(schistosomiasis) was observed and only Schistosoma activities. Also the main reason of different between male
haematobium egg were found in the study area. This and female would be due to the greater water contact

Epidemiological studies of schistomiasis

Current Trends in Biotechnology and Pharmacy 231
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activities by male compared to their female counterpart. D. L. (1991). A Simplified General Method for
Female mature early when compare to the male therefore, Cluster-sample Surveys of Health in Developing
they restricted socially to water contact activities. These Countries. World Health Statistics Quarterly 44: 98-
agree with (1, 5, 14, 32) in Niger, Kundiga,Danjarima 106.
and Bauchi respectively. 4. Berrie, A.D. (1970). Snail problems in African
However, three physico-chemical parameters were Schistosomiasis. Advances in Parasitology
measured for the purpose of this study i.e pH, temperature , Dawes, B.E.B; Academic Press, New York 8.43
and dissolved oxygen. The average of these physico- 5. Biu, A.A., Kolo, H.B. and Agbadu E.T. (2009).
chemical parameter values of the selected water bodies Prevalence of Schistosoma haematobium
were taken and found were to be within the range that Infection in School Age Children of Konduga
can support snail breeding. These values are in LocalGovernment Area, Northeastern Nigeria. Int
consonance with those recorded by other researchers such journal of biomedical and Health Sciences.5:181-
as (22, 23) in Imo and Bauchi states respectively. Low 184.
populations of snails were found in water bodies with 6. Bolonwu, R. (2007). Prevalence and intensity of
low dissolved oxygen or even absent in some cases. This Schistosoma heamatobium Infection among Primary
study has corroborate that dissolved oxygen in water School Children Katagum Local Government,
bodies plays an important role in snail breeding, even if Bauchi State, Nigeria. Sahel Medical Journal Vol.10
all other parameters are within the normal range as no 1 pp11-12.
observed by (22). Only one species of snail were found
7. Brown, D.S (1994).Freshwater snails of Africa and
and collected during the study 422 Bulinusglobosus and
their Medical Importance (2ndedn). Taylor and
out of this only 21 (4.9%) where infected or shed
Francis Ltd: London.609p.
cercariae.
8. Brown, D.S. and Christensen, N. O. (1993). A field
In conclusion, as only 12(2%) out of 1200 samples
Guide to African Water Snails II (West African
(urine and stools 600 each) examined were positive with
Species) Danish Bilharziasis Laboratory Manual.
Schistosoma haematobium eggs and out of 422 snail
Chalottenlund, Denmark, 54pp.
vectors examined only 21(4.9%) were harbouring the
9. Chitsul, L., Engel, D., Monstresor, A and Aavioli, L.
infective stage of the parasite (cercariae). From the result
of this study, it is concluded that the disease had low (2000). The global status of Schistosomiasis and its
endemicity in the study area and is showing decline Control. Acta Tropical. 77(1): 41-49
pattern when compare with previous studies. Therefore, 10. Dawet, A., Benjamin, C.B. and Yakubu, D.P (2012).
proper health education to continue discourage people Prevalence and intensity of Schistosoma
from urinating and defecating in or near open water as haematobium among resident of Gwong and Kabong
well as periodic survey of the water bodies in the area in Jos north Local Government area, Plateau State,
for snail intermediate hosts control is recommended for Nigeria. International Journal of Tropical Medicine.
eradication of the disease and avoidance of further 7(2)69-73pp.
separation of the parasite in the study area. 11. Dunah, C. S. and Bristone, B. (2000). The Prevalence
of Schistosomahematobium Among Primary School
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24. Oladejo, S.O. and Ofoezie, I.E (2006). Unabated helminthiasis. World Health Organ Tech Rep:Ser,
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Current Trends in Biotechnology and Pharmacy 233
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DOI : 10.5530/ctbp.2020.4s.30

Anti-Osteoporotic Effects of Alendronate and Sitagliptin in Streptozotocin

Induced Type 2 Diabetes Mellitus in Ovariectomized Rats
Vadivelan Ramachandran1*, Gautam Adhikari1, Manogaran Elumalai2
1Department of Pharmacology, JSS College of Pharmacy, Ootacamund, The Nilgiris, Tamil Nadu-643001, India.
2Department of Pharmaceutical Biology, Faculty of Pharmaceutical Sciences, UCSI University (South Wing),
Cheras, Kuala Lumpur - 56000, Malaysia.

*Corresponding Author : vadivelanr@jssuni.edu.in

Abstract Key words : Ovariectomy, Diabetes,Alendronate,

Diabetes mellitus is a metabolic disorder identifies as Sitagliptin, Serum Calcium.
hyperglycaemia and osteoporosis is a bone disorder within 1. Introduction
which quality of bone and bone mineral density decline.
Diabetes in osteoporotic patients is remarkably increased Osteoporosis is a progressive chronic disease
risk of bone fracture. Alendronate, a bisphosphonate first characterized by decreased bone mass, bone quality,
line therapy for osteoporosis treatment which prevents resulting in bone fragility related to an enhanced risk
bone fracture by inhibiting osteoclast. Sitagliptin, an oral fracture(1). According to World Health Organization
antidiabetic agent used for the treatment of type II diabetes osteoporosis as a decrease in bone mass (50%) and bony
mellitus by inhibiting Dipeptidyl peptidase-4 activity. quality (50%). Bone loss happens once the cellular events
Sitagliptin may regulate bone homeostasis by inhibiting of bone formation are quantitatively larger [2]. A highest
osteoclast & supressing osteoclast differentiation. The bone mass at skeletal maturity is taken into account to be
present study was to investigate the anti-osteoporotic the most effective protection against age related bone loss
effect of sitagliptin and alendronate on bone mechanical and consequent fracture risk. Calcium is in every of the
properties in streptozotocin (STZ)induced diabetes in foremost vital determinants and nutrient factors to
overiectamized rats (OVX). 30 female Wistar rats determine the peak bone mass in young adults. There
weighing from 180-250 g were divided into five groups usually aren't any symptoms within initial stages of bone
each of 6 rats. Osteoporosis was induced by bilateral loss. However once bones are weakened by osteoporosis,
ovariectomy.After seven days of surgery the type 2 signs and symptoms that include back pain caused by a
diabetes mellitus was induced by single intraperitoneal fractured or collapsed vertebra, loss of height over time,
injection of streptozotocin (50 mg/kg) and nicotinamide bone fracture that occurs much more easily than expected
(110 mg/kg). Groups III, IV and V were treated with (3).
alendronate (3 mg/kg), sitagliptin (30 mg/kg) and
Diabetes mellitus has direct and indirect deleterious
combination respectively for 42 days. The body weight
effects on osteoblast function and bone formation. Clinical
of sitagliptin (30 mg/kg) and concurrent administration
evidences showed that bone mineral density (BMD) was
of alendronate (3 mg/kg) and sitagliptin showed
below normal range in patients with type 1 diabetes
significant increased compared to OVX-STZ groups.
mellitus, a subtype of diabetes caused by inability of
There is improvement in serum calcium and ALP in
pancreatic beta cells to secrete insulin (4). Diabetes
sitagliptin, alendronate and combination groups compared
associated decrease in BMD and weakened bone structure,
to OVX-STZ groups. Alendronate and combination
of difficult pathophysiology, is taken in to account to be
groups showed significant increase in bone weight
one of the major factors that cause bone fragility and
compared to STZ-OVX group. There is no significant
changed in bone length and diameter. The bone mineral elevated incidence of fractures in diabetic patients.
mass and three point bending test significantly increased Hyperglycemia can affect the bone density through
in alendronate and combination groups compared to different mechanisms.Toxic effects caused by high levels
OVX-STZ groups. The concurrent administration of of glucose could directly decrease the osteoblast function
alendronate and sitagliptin showed beneficial effects in and number.High levels of glucose might independently
STZ induced type 2 diabetes mellitus in OVX rats. alter the amount of osteoblast gene expression through

Effects of alendronate and sitagliptin in type 2 diabetes mellitus

Current Trends in Biotechnology and Pharmacy 234
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DOI : 10.5530/ctbp.2020.4s.30

the osmotic and non-osmotic pathways. These changes of glycemic management (9). Hence from the above
lead to inhibition of bone forming cell maturation and background, the present study was to investigate the anti-
bone mineralization.Impairment of osteoblast maturation, osteoporotic effects of alendronate and sitagliptin in STZ
caused by high glucose levels, lead to an impaired inducedtype 2diabetesin OVX rats.
response to 1, 25 hydroxy vitamin D3. This indirectly
2. Materials and Methods
causes the down regulation of vitamin D receptors
(5).High glucose levels, through non-enzymatic Animals
pathways, might induce glycationof numerous proteins 30 female Wistar rats weighing from 180-250 g were
and manufacture the products called advanced housed in plastic cages at maximum of 3 per cage. The
glycosylation end-products (AGEs). These products are animal was obtained from Central Animal House, JSS
seen in numerous tissues of diabetic subjects and are College of Pharmacy, Ootyand were maintained under
presupposed to be concerned in pathogenesis of diabetes. controlled environmental conditionson alternate 12 hours
It appears hyperglycemia and AGEs have a serious role dark/light, temperature 21 ± 2°C. commercial pelleted
in fragility of bones in both type of diabetes. In cortical feed and water ad libitumwas provided to animals. All
bone, accumulation of AGEs causes a rise in production the experiments were performed after obtaining prior
of cross-links between collagens. Though this method approval fromCPCSEA and IAEC (Approval No.: JSSCP/
can enhance the rigidity and hardness of collagen, it OT/MPharm/10/2018-2019. The rats were acclimatizedto
doesn't have an effect on the bone mineralization. Indeed the experiment conditions for 5 days before initiating the
there's a negative relation between AGEs and size and experimental procedure.
fragility of the human trabecular bone that might justify
Chemicals and reagents
the raised bone fragility and fracture in diabetic subjects.
Moreover, apart from the direct effects of high glucose, Streptozotocin (STZ) was purchased from HI-Media,
accumulation of AGEs encompasses a direct inhibitory India. Alendronate and sitagliptin were purchased from
effect on the proliferation and differentiation of bone cells Cipla and Sun Pharma respectively. Glucometer (Accu-
(6). Production and accumulation of AGEs will induce Chek) was purchased from (Roche Diabetes Care,
the cellular cell death through production of reactive India),ALP and calcium kits were purchased from Q-Line
oxygen species (ROS) and oxidative stress(7). Diagnostic Systems and Ensure Biotec. Pvt. Limited,
India. All other chemicals and reagents were analytical
Alendronate is a nitrogen containing bisphosphonates grade.
that's used for the treatment of some type of osteoporosis.
It's taken orally and is usually recommended along with Induction of osteoporosis
vitamin D, calcium supplementation, and lifestyle Osteoporosis induction in animal was by bilateral
changes. The mechanism of nitrogen-containing ovariectomy which involved the removal ofboth the
bisphosphonates (eg,pamidronate, alendronate, ovaries and type 2 diabetes mellitus was induced by STZ
risedronate, ibandronate, and zoledronate) inhibit a key and nicotinamideadministration.
enzyme, farnesyl pyrophosphate synthase, within the Surgery procedure
mevalonate pathway, thereby preventing the synthesis of
isoprenoid compounds that are important for the The animal was anaesthetized by 50 mg /kg ketamine
posttranslational modification of little guanosine and 40 mg/kg xylazine injectedthrough intra-peritoneal
triphosphate (GTP)-binding proteins (which are GTPases) route. The anesthetized animal was shaved on the ventral
like Rab, Rho, and Rac. The inhibition of protein region below the ribs.The position of the ovaries was
prenylation and also the disruption of the function of those located by feeling the fat pads surrounding it with
key regulatory proteins describe the loss of osteoclast thethumb.Incision was made in the middle of the rat using
activity(8). No. 23 scalpel blade.A small incision was made in the
muscle layer using a scalpel blade, fine scissors wasused
Sitagliptin is an oral anti-diabetic agent is often used to make incision.The ovary surrounded by a variable
as mono-therapy or in combined therapy with different amount of fat was pulled out of the incision.A ligature
oral antidiabetic drugs within the treatment of T2DM by was placed in the fallopian tube on both sides to control
inhibiting Dipeptidyl peptidase-4 activity. Sitagliptin bleeding. The ovarieswere removed above the ligature at
would decrease bone loss and increase bone strength in the junction of the oviduct.The abdomen was properly
diabetic rats by decreasing bone resorption independent closed by proper ligation and suturing(10).

Vadivelan et al
Current Trends in Biotechnology and Pharmacy 235
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Induction of type 2 diabetes mellitus digital vernier caliper. Right tibia and femur bones was
The rats were induced diabetic after two weeks of cleaned from the surrounding tissues and stored in 4%
ovariectomy.Single dose of STZ 50 mg/kg was injected formaldehyde. Bone mass was measured using digital
intraperitoneally after 15 min followed by balance.
intraperitonealadministration of nicotinamide (110 mg/ Bone mineral mass (15)
kg).The serum glucose level was measured after 72 h
Initial right femur bone weight was taken using
and serum glucose level more than 250 mg/dl were
balance. Bone was placed in crucible dish and ashed at
considered as diabetic rats (11).
the temperature of 600° C for 48hours in the incinerator.
Experimental design Ash was collected and weighed. The ratio of the mass of
After successful induction of osteoporosis along with bone to the bone mass was determined.
type 2 diabetes mellitus rats were divided into 5 groups Three point bone bending test
of 6 rats per group.
Bone was placed in the two triangle which serves as
Group I : Sham base 2 point and another point is present perpendicular
Group II : OVX-STZ to it. Constant force was applied to bone by rotating the
Group III : OVX-STZ+Alendronate (A) (3mg/kg p.o) screw present at top. Bone breaking time was noted for
Group IV : OVX-STZ+Sitagliptin(S)(30 mg/kg p.o) each group.
Group V : OVX-STZ+Alendronate(3mg/kg p.o)+ Statistical analysis
Sitagliptin (30 mg/kg p.o) The data are represented as mean ± SEM. Body weight
Group I served as sham and group II served as an and serum glucose of animals were analysed by two-way
OVX-STZ. Group III, IV and V were treated with ANOVA and biochemical except serum glucose,
Alendronate (3 mg/kg), Sitagliptin (30 mg/kg) and mechanical property wasanalysed by one-way ANOVA
Alendronate (3mg/kg p.o) + Sitagliptin (30 mg/kg followed by Bonferroni multiple post hoc test p values
p.o)respectively for 42 days. The body weights and serum (p<0.05) was considered notably. The analysis was carried
glucose level were measure at every 14 days. The blood using GraphPad prism 6 software.
was collected at the end of treatment for serum alkaline
3. Results and Discussion
phosphatase (ALP) and calcium estimation. At the end
of the treatment animals were sacrificed and bones (tibia Effects of alendronate and sitagliptin on body weight :
& femur) were collected and stored in 10% formalin. The results of body weight in OVX-STZ rats are given
Biochemical parameters in (Figure 1). The result showed that body weight
Rats were monitored for every 14 days regarding body
weight and serum glucose. On day 42, the rats were fasted
for 12 hours and anaesthetized using (50 mg/kg ketamine
plus 5 mg/kg diazepam, intraperitoneally), approximately
3 mL of blood was collected by cardiac puncture,
centrifuged at 4000 rpm for 10 min for separation of
serum. Each serum sample was stored in a clean sterile
micro centrifuge tubes at -800 C until analysis.
Analytical measurements (12, 13)
The serum levels of glucose was measured using Accu-
chek (Roche Diabetes Care, India), by tail vein puncture, Figure 1 : Effects of alendronate and sitagliptin on body
calcium and alkaline phosphatase (ALP) were assayed weight
The results were expressed as mean ± SEM.; n=6,
for each rat with specific enzyme kits, [Ensure Biotech a significantly different vs Sham (p<0.05),
Pvt limited and Q Line Diagnostic systems, India] that b significantly different vs OVX-STZ (p<0.05), csignificantly
were used according to the manufacturer's instructions different vs alendronate (p<0.05),
ns non-significant compared to OVX-STZ group
Bone length, diameter and mass (14)
Two-way ANOVA followed by Bonferroni multiple comparison
Right femur length and diameter was calculated using post-test

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significantly (p < 0.05) decrease in OVX-STZ group

when compared to Sham group. On 42 days chronic
treatment with sitagliptin and concurrent administration
of alendronate and sitagliptin showed significant increase
(p < 0.05) in body weight compared to OVX-STZ group
however alendronate showed no significant change in
body weight compared to OVX-STZ rats. Concurrent
administration of alendronate and sitagliptin showed no
significant changed in body weight when compared to
sitagliptin treated groups.
Effects of alendronate and sitagliptinon serum
glucose :
The results of serum glucose in OVX-STZ rats are Figure 3 : Effects of alendronate and sitagliptin on serum
given in (Figure 2). The result showed that serum glucose calcium
significantly (p< 0.05) increase in OVX-STZ group when The results were expressed as mean ± SEM.; n=6,
a significantly different vs sham (p<0.05),
compared to sham group. On 42 days chronic treatment b significantly different vs OVX-STZ (p<0.05),
with sitagliptin and concurrent administration of d significantly different vssitagliptin (p<0.05)
alendronate and sitagliptin, showed significant (p<0.05) One-way ANOVA followed by Bonferroni multiple comparison
decrease in serum glucose level when compared to OVX- post-test
STZ group however alendronate did not show any change
with alendronate, sitagliptin and concurrent
in serum glucose as compared to OVX-STZ group.
administration of alendronate and sitagliptin groups
Concurrent administration of alendronate and sitagliptin
showed significant (p<0.05) decrease in serum calcium
showed significant (p<0.05) decrease in serum glucose
when compared to OVX-STZ group. Concurrent
level compared to alendronate group.
administration of both drugs showed significant (p<0.05)
decrease in serum calcium compared to alendronate and
sitagliptin individual treated groups.

Figure 2 : Effects of alendronate and sitagliptin on serum

glucose
The results were expressed as mean ± SEM.; n=6,
a significantly different vs Sham (p<0.05),
b significantly different vs OVX-STZ (p<0.05),
c significantly different vs alendronate (p<0.05),
ns non-significant compared to OVX-STZ group

Two-way ANOVA followed by Bonferroni multiple comparison

post-test Figure 4 : Effects of alendronate and sitagliptin on serum
alkaline phosphatase
Effects of alendronate and sitagliptin on serum The results were expressed as mean ± SEM.; n=6,
calcium : a significantly different vs sham (p<0.05),
b significantly different vs OVX-STZ (p<0.05),
The results of serum calcium in OVX-STZ rats are c significantly different vs alendronate (p<0.05),
given in (Figure 3). The result showed that serum calcium d significantly different vssitagliptin (p<0.05)
significantly (p<0.05) increase in OVX-STZ group when One-way ANOVA followed by Bonferroni multiple comparison
compared to Sham group. On 42 days chronic treatment post-test

Vadivelan et al
Current Trends in Biotechnology and Pharmacy 237
Vol. 14 (5) 233-244, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.30

Effects of alendronate and sitagliptin on serum alkaline

phosphatase :
The results of serum alkaline phosphatase in OVX-
STZ rats are given in (Figure 4). The result showed that
serum ALP has been significant (p<0.05) increase in
OVX-STZ group when compared to Sham group. On 42
days chronic treatment with alendronate, sitagliptin and
combination of both drugs showed significant (p<0.05)
decrease when compared to OVX-STZ group. Concurrent
administration of alendronate and sitagliptin showed
significant (p<0.05) decrease in serum ALP compared to
alendronate and sitagliptin individual treated groups
however alendronate group show significant decrease in
Figure 6: Effects of alendronate and sitagliptin on tibia
serum ALP compared to sitagliptin group.
weight
Effects of alendronate and sitagliptin on bone weight : The results were expressed as mean ± SEM.; n=6,
a significantly different vs sham (p<0.05),
The results of bone weight in OVX-STZ rats are given b significantly different vs OVX-STZ (p<0.05),

in (Figure 5 and 6). The result showed that bone d significantly different vssitagliptin (p<0.05)
ns non-significant compared to OVX-STZ
weightsignificantly (p<0.05) decrease in OVX-STZ
group when compared to sham group. Alendronate and One-way ANOVA followed by Bonferroni multiple comparison
post-test
combination of both drugs showed significant (p<0.05)
increase in bone weight when compared to OVX-STZ Effects of alendronate and sitagliptinon bone length,
group however. Combination of both drugs showed diameter and mass :
significant (p<0.05) increase in bone weight compared
The results of bone length and diameter in OVX-STZ
to sitagliptin however no significantdifference when
rats are given in (Table 1). The result showed that there
compared to alendronate.
is no significant difference in bone length and diameter

Figure 5 : Effects of alendronate and sitagliptin on femur Figure 7 : Effect of alendronate and sitagliptinon bone
weight mineral mass
The results were expressed as mean ± SEM.; n=6, The results were expressed as mean ± SEM.; n=6,
a significantly different vs sham (p<0.05), a significantly different vs sham (p<0.05),
b significantly different vs OVX-STZ (p<0.05), b significantly different vs OVX-STZ (p<0.05),
d significantly different vssitagliptin(p<0.05) c significantly different vs alendronate (p<0.05),
ns non-significant compared to OVX-STZ d significantly different vssitagliptin (p<0.05)

One way ANOVA followed by Bonferroni multiple comparison One-way ANOVA followed by Bonferroni multiple comparison
post-test post-test

Effects of alendronate and sitagliptin in type 2 diabetes mellitus

Current Trends in Biotechnology and Pharmacy 238
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DOI : 10.5530/ctbp.2020.4s.30

Table 1: Effects of alendronate and sitagliptin on bone length and diameter

Groups Length Diameter
Femur length (mm) Tibia length (mm) Femur (mm)
SHAM 32.90 ± 0.485 35.32 ± 1.034 2.44 ± 0.092
ns ns
OVX – STZ 32.31 ± 0.849 34.53 ± 1.149 2.34 ± 0.078 ns
OVX-STZ + A 33.24 ± 0.577 ns 35.16 ± 0.926 ns 2.34 ± 0.094 ns
OVX-STZ + S 32.98 ± 0.519 ns 34.20 ± 1.119 ns 2.36 ± 0.078 ns
OVX-STZ + A + S 32.55 ± 0.526 ns 34.72 ± 0.992 ns 2.32 ± 0.103 ns
Data expressed as mean ± SEM.; n=6.
ns - non significant compared to all groups

One-way ANOVA followed by Bonferroni multiple comparison post-test

in OVX-STZ group when compared to sham group and Effects of alendronate and sitagliptin on three point
treatment groups. bending test :
The results of bone mineral mass in OVX-STZ rats The results of three-point bending test in OVX-STZ
are given in (Figure 7). The result showed that there is are given in (Figure 8).The result showed that there is
significant (p<0.05) decrease in bone mineral mass in significant (p<0.05) decrease in time in OVX-STZ group
OVX-STZ group when compared to sham group. when compared to sham group. Alendronate and
Alendronate and combination of both drugs showed concurrent administration of both drugs showed
significant (p<0.05) increase in bone mineral mass significant (p<0.05) increase time compared to OVX-STZ
compared to OVX-STZ group however sitagliptin did group however sitagliptin showed no significant
not showed significant difference compared to OVX-STZ difference in time compared to OVX-STZ group.
group. Combination group showed significant (p<0.05) Concurrent administration of both drugs showed
increase in bone mineral when compared to alendronate significant (p<0.05) increase in time compared to
and sitagliptin individual treated groups. alendronate and sitagliptin individual treated groups.

3. Results and Discussion

Effects of insulin insufficiency on the bone could also
cause bone and mineral deformity in diabetic patient.
Each type 1 and type 2 diabetes mellitus have been related
to bone diseases such as osteoporosis. Osteoporosis, a
metabolic bone disorder, is becoming more and more
prevailing in older population. Osteoporosis occurs due
to an inequality between osteoblast and osteoclast
function and the diabetes induced osteoporosis is unclear
(16, 17).The present study was performed to evaluate the
anti-osteoporotic effect of alendronate and sitagliptin in
STZ induced type 2 diabetes mellitus in ovariectomized
rats.
Figure 8 : Effect of alendronate and sitagliptin on three
point bending test Chan et al.,(18) reported that in diabetic osteoporosis
The results were expressed as mean ± SEM.; n=6, condition, there is decrease in body weight in OVX-STZ
a significantly different vs sham (p<0.05),
group. This is due to insufficient insulin which stops the
b significantly different vs OVX-STZ (p<0.05),
body to receiving glucose from the blood. When this
c significantly different vs alendronate (p<0.05),
d significantly different vssitagliptin (p<0.05),
occurs, the body starts utilizing fat and muscle for energy
ns-non significant compared to OVX-STZ and reduced in body weight. The similar result were found
One-way ANOVA followed by Bonferroni multiple comparison in the present study where body weight of all OVX-STZ
post-test rats were decreased initially but after 42 days of treatment,

Vadivelan et al
Current Trends in Biotechnology and Pharmacy 239
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DOI : 10.5530/ctbp.2020.4s.30

there was gradual increase in body weight in sitagliptin Concurrent administration of alendronate and sitagliptin
and combination treated groups. Whereas, alendronate showed increased in activity when compared to OVX-
treated group decreased the body weight even after the STZ, alendronate and sitagliptin treated rats.
treatment for 42 days. This indicates alendronate doesn't Diabetes in ovariectomized decreases the bone
have any effect on diabetes in ovariectomized rats. minerals which was detected in this study. The OVX-
Results of the present study showed significant STZ group showed a decrease in the ash values (23).
increase in blood glucose in OVX-STZ rats compared Alendronate and sitagliptin treatment increased ash
sham group. Farid et al. (19) were reported similar results. values compared to OVX-STZ rats. It suggests that
After 42 days of treatment, there was gradual decrease in alendronate and sitagliptin have bone protective action.
blood glucose in sitagliptin and combination treated Concurrent administration of alendronate and sitagliptin
groups. Whereas, Alendronate treated group was not showed increased in ash value when compared to OVX-
showing any change in blood glucose level as compared STZ and alendronate treated rats.
to OVX-STZ rats after the treatment for 42 days.
4. Conclusion
Calcium is one of the most vital minerals present in
The present study demonstrates a bone preserving
bone. It helps maintain bone strength, build and maintain
effect of concurrent administration of alendronate and
bones. It was suggested that calcium absorption plays a
significant role in bone turnover, and its deficiency results sitagliptin in OVX STZ induced type 2diabetes mellitus
and this effect may be due to suppression of
in a reduced bone mineralization (20). The present study
osteoclastogenesis. Our data gives apotential effect of
results showed significant increase in serum calcium in
combination of both drugs alendronate and sitagliptin by
OVX-STZ as compared to sham group and Hassan et al.
preventing osteoporosis of postmenopausal women with
(21)also reported similar results. The increase in serum
diabetes and further information need to be assessed in
calcium indicated that there is more bone resorption of
future preclinical and clinical studies.
in OVX-STZ. After 42 days treatment with alendronate,
sitagliptin and combination of both drugs showed Acknowledgements
significant decreases in serum calcium compared to We acknowledge the generous research infrastructure
OVX-STZ rats. and supports from Department of Pharmacology. (DST-
ALP is an enzyme that is mainly found in liver and FIST, Sponsored) from JSS College of Pharmacy, JSS
bone. Levels of this enzyme increases when bone cells Academy of Higher Education & Research, Rocklands,
are active. ALP helps in bone mineralization. Abnormal Ooty, The Nilgiris, Tamilnadu, India.
level of ALP in blood is due to bone disorder (22).
Conflict of Interest
Biochemical results revealed that serum ALP
concentrations were increased in OVX-STZ group as The authors have no conflict of interest
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DOI : 10.5530/ctbp.2020.4s.31

Perception and Satisfaction Among Single and Dual Users Malaysian Vapers
Towards Electronic Cigarettes. A One Year Observational Study
Aziz-ur-Rahman1*, Mohamad Haniki Nik Mohamed2, Syed Mahmood3, Ashok Kumar Balaraman4
1Department of Clinical Pharmacy, Faculty of pharmaceutical sciences, UCSI University, Kuala Lumpur, Malaysia
2Department of Pharmacy Practice, Kulliyyah of pharmacy, International Islamic University of Malaysia (IIUM),
Kuantan Campus, 25200, Pahang, Malaysia
3Department of Pharmaceutical Engineering, Faculty of engineering technology University Malaysia Pahang, Gambang, 26300
4 Department of Pharmaceutical Biology, Faculty of pharmaceutical sciences, UCSI University Kuala Lumpur, Malaysia
Corresponding author email: aziz@ucsiuniversity.edu.my

Abstract spreading worldwide. EC is a battery-operated device

In Malaysia and elsewhere in the world Electronic available in various sizes and shapes, such as conventional
cigarette (EC) consumers, use the product either alone cigarettes (CCs), pens and boxes. ECs do not release
(single users, SUs) or in combination with conventional smoke; instead, they vaporises e-liquids (e-juice) that
cigarettes (CCs) (dual users, DUs). However, the existing vapers inhale. Nicotine, propylene glycol (PG), glycerine
studies have limited to explore the long-term perceptions and a couple of other ingredients mostly tobacco or fruit
and satisfaction levels among SUs and DUs.The present flavours are the chief constituents of e-liquids. The aim
study aimed to evaluate the perceptions and satisfaction of developing ECs is to simply simulate the act of smoking
levels among SUs and DUs after one year of EC use. A via the nicotine contents of e-liquids while avoiding the
total of 218 SU and DUs participants were enrolled in noxious effects of tobacco smoke. Furthermore, ECs
the current study from the districts of Pekan and Kautan, produce vapours that resemble smoke and are associated
state Pahang, Malaysia. The 70 were SUs, as verified by with most of the social and behavioural features of
the carbon monoxide (CO) levels of <8 ppm, and the smoking like the hand to mouth action (1-3).
remaining were DUs, as verified by the CO levels of ?8 According to the Malaysian National Health and
ppm. The participants were interviewed after 1 year of Morbidity Survey (NHMS, 2015), smokeless tobacco
EC use regarding their perceptions and satisfaction levels consumption, including EC use, in Malaysia increased
to EC which were scored on a scale of 0-4, not sure to from 0.7% to 10.9% during 2011-2015. Furthermore, the
very sure. Overall, 33.3% of the participants were very report speculated that the sudden increase in the
much sure that ECs are an effective smoking cessation consumption of smokeless tobacco products among
aid. However, 43.8 % of SU participants were very much Malaysian adults (?15 years of age) was due to the
sure that ECs are an effective smoking cessation aid then increased consumption of ECs (National Health and
30.2% of DUs. While perceptions of SUs regarding the Morbidity Survey, 2015) (4). However, the 2016
safety of ECs was 78.9% as compared to 86.6% of DUs. Malaysian National E-cigarette Survey reported that the
After one year of EC use more SUs perceived positively prevalence of EC users among Malaysian adults ?18 years
satisfaction than DUs. However, there was no perception of age was 3.2% (3.3% in urban and 2.9% rural areas),
variance in both groups' users related to the safety of EC. while the prevalence of combined EC use with tobacco
Nevertheless, the EC understanding and its attainment cigarettes was 2.3% (5).
are recommended to be confirmed further in diverse Furthermore, in a cross-sectional provincial survey
populations. among 429 Malaysian EC users, 85% of the participants
Key words : Electronic cigarette, conventional cigarette, believed that ECs are not as bothersome as CCs and can
perception, satisfaction, carbon monoxide be used in public places (6). A survey by Factasia.org
reported that more than three-quarters of the 400
1. Introduction
participating Malaysian adult smokers believed that ECs
Electronic cigarette (EC) is a relatively novel product, are a promising substitute for CCs and were willing to
and its use as an electronic nicotine delivery system is use the product if it is legal, risk-free and as accessible as
Perception and satisfaction of electronic cigarette users
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DOI : 10.5530/ctbp.2020.4s.31

CCs (7). In Malaysia and elsewhere in the world, EC smoking prevalence of 22.8% (4). The above study sites
smokers use the product either alone (single users, SUs) were selected due to time and capital constraints.
or in combination with CCs (dual users, DUs). However, Furthermore, these sites were more convenient in terms
there are limited published data regarding the perceptions of accessibility to EC users.
and satisfaction levels among Malaysian SUs and DUs Participants were stratified into two groups based on
after long-term EC use. Both SUs and DUs represent their smoking status: SUs [use of EC alone and measured
significant populations of the existing vaper community, carbon monoxide (CO) levels of <8 ppm] and DUs (use
and their opinions after longterm EC use may reveal of both ECs and CCs and CO levels of >8 ppm) (Figure
perceived benefits and contrary effects related to EC 1).
usage. Therefore, the present study aimed to assess the
perceptions and satisfaction levels among SUs and DUs Data collection
after a year of EC use. Study data were collected between March 2015 and
June 2016. Initially, each study participant was questioned
2. Materials and Methods
independently for 25-30 minutes to collect
Study design sociodemographic details, smoking history (pack-years)
The prospective study was designed to assess the and EC use history. More than 90% of the study
satisfaction and perceptions levels among current SUs participants understood and spoke English. For
and DUs in a natural setting over 1 year period. participants who did not know English, a translator
(English to Bahasa Malay) was engaged. The same
Sample size
translator assisted in all interviews to avoid errors due to
A total of 218 participants who consented to follow prejudice and inconsistencies.
the study procedure were enrolled through convenience
At the final interview, each participant's perceptions
sampling. The participants were enrolled at a ratio of 2:1
and satisfaction levels towards ECs were reported on a
(148 DUs and 70 SUs) as the majority of the EC users
scale of 0-4 (0 = not sure, 1 = less sure, 2 = moderately
are DUs (4-5). Finally, data were collected from 82%
sure, 3 = very sure and 4 = very much sure). Participants'
(176) of the enrolled participants. Convenience sampling
responses to queries related to whether ECs help in
was implemented because random samples of SUs and
quitting smoking, reducing CC consumption, preventing
DUs from the main study population were difficult to
relapse to smoking, controlling cravings for smoking and
obtain. Thus, the above enrolment technique was the most
managing smoking-related withdrawal symptoms was
feasible in terms of time and funding constraints.
assessed. Participants were assessed for satisfaction levels
Inclusion and exclusion criteria with the use of ECs as a smoking cessation aid. They
The inclusion criteria were as follows: current single were also asked whether they would recommend the use
and dual-use of EC products since a minimum of 1 month, of ECs to their friends and kin as a smoking cessation
age of 18-65 years and good self-reported health. aid. Besides, we documented any case of EC dependence
Exclusion criteria were as follows: use of any smoking and physical harms associated with EC use.
cessation medicines, such as nicotine replacement therapy Ethical approval and consent to participate
or varenicline, currently or within the previous year and
The study questionnaire, protocols, consent forms,
dependency on any illegal drugs.
participant information sheet and study-related flyer to
Study questionnaire recruit the study subjects were approved by the Research
A pre-validated, English questionnaire was Ethics Committee (IREC) of Kulliyyah of Medicine,
administered via interviews to collect data. The International Islamic University Malaysia (IIUM),
questionnaire was developed and pilot-tested among Kuantan on 9th October 2014, IREC no. 302 and by the
Malaysian SUs and DUs of ECs (8). National Medical Research Registration (NMRR.NO:15-
180-24,825). Written consent was obtained from all the
Settings and stratification participants before their enrolment in the study
Study participants were selected from the semi-urban
Data analysis
districts of Kuantan and Pekan in the state of Pahang,
Malaysia. Smoking prevalence in the state of Pahang was Categorical variables were summarised as frequencies
25.5% in the year 2015, which is more than the national and percentages, and continuous variables were

Aziz et al
Current Trends in Biotechnology and Pharmacy 243
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Figure 1 : Number of particpants at baseline and at week 52 among the single and dual users vapers

calculated as medians. Chi-squared test was applied for 3. Results and Discussion
categorical variables, and independent t-tests were applied
Baseline characteristics of the participants
to compare mean differences between the groups. Mann-
Demographic characteristics of both groups did not
Whitney U test was used to compare nonparametric data
vary. In both, the groups, the median age was 23 years
between the groups. Statistical methods were two-tailed,
and nearly 98% of the participants were males. More DUs
and a p-value of <0.05 was considered significant.
than SUs were unmarried at enrolment. Both the groups
Intention to treat analysis was applied for study outcomes.
showed the same race distributions (p = 0.632). Majority
Data of all participants who were interviewed at least
of the study participants were Malays (80%), followed
once during the follow-up visits were included in the final
by Chinese (11.9%) and Indians (1.8%). Approximately
analysis. Those who missed more than two interviews
73% of the study participants were either studying or held
were excluded from the final analysis. Participants who
a diploma or degree. The two groups did not differ in
were lost to follow-up were excluded (n = 3). Analyses
terms of profession and income. At the initial visit,
were performed using the Statistical Package for Social
physical and behavioural dependences on ECs were the
Sciences (IBM®, SPSS® Inc., Chicago, IL) for Windows
same in both the groups (p = 0.668).
version 21.

Perception and satisfaction of electronic cigarette users

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DOI : 10.5530/ctbp.2020.4s.31

Table 1 Perception and satisfaction levels among SUs and DUs after one year period of EC use
Not Very Very much
Less sure, Moderately
Perception and Satisfaction Groups sure, sure, sure, Median P (2T)
n (%) sure, n (%)
n (%) n (%) n (%)
1. Did e-cigarette help in DUAL USERS (n = 119) 46 (38.7) 27 (22.7) 11 (9.2) 30 (25.2) 5 (4.2) 1.00
quitting smoking? SINGLE USERS (n = 57) 8 (14) 9 (15.8) 5 (8.8) 26 (45.6) 9 (15.8) 3.00 <0.001
TOTAL USERS (n = 176) 54 (30.7) 36 (20.5) 16 (9.1) 56 (31.8) 14 (8)
2. Did e-cigarette help in DUAL USERS (n = 119) 16 (13.4) 19 (16) 30 (25.2) 54 (45.4) 0 (0) 2.00
reducing tobacco smoking? SINGLE USERS (n = 57) 1 (1.8) 2 (3.5) 14 (24.6) 35 (61.4) 5 (8.8) 3.00 <0.001
TOTAL USERS (n = 176) 17 (9.7) 21 (11.9) 44 (25) 89 (50.6) 5 (2.8)
3. Did e-cig help in controlling DUAL USERS (n = 119) 28 (23.5) 24 (20.2) 30 (25.2) 36 (30.3) 1 (.8) 2.00
relapse from tobacco smoking? SINGLE USERS (n = 57) 0 (0) 12 (21.1) 10 (17.5) 35 (61.4) 0 (0) 3.00 <0.001
TOTAL USERS (n = 176) 28 (15.9) 26 (20.5) 40 (22.7) 71 (40.3) 1 (0.6)
4. Did e-cigarette help in DUAL USERS (n = 119) 9 (7.6) 26 (21.8) 47 (39.5) 36 (30.3) 1 (0.8) 2.00
controlling craving of smoking? SINGLE USERS (n = 57) 1 (1.8) 9 (15.8) 12 (21.1) 35 (61.4) 0 (0) 3.00 0.001
TOTAL USERS (n = 176) 10 (5.7) 35 (19.9) 59 (33.5) 71 (40.3) 1 (0.6)
5. Did e-cigarette help to DUAL USERS (n = 119) 9 (7.6) 24 (20.2) 48 (40.3) 38 (31.9) 00 2.00
managed withdrawal symptoms SINGLE USERS (n = 57) 0 (0) 18 (14) 12 (21.1) 37 (64.9) 00 3.00 <0.001
of smoking? TOTAL USERS (n = 176) 9 (5.1) 32(18.2) 60 (34.1) 75 (42.6) 00
6. Will you suggest e-cigarette DUAL USERS (n = 119) 8 (6.7) 39 (32.8) 35 (29.4) 37 (31.1) 0 (0) 2.00
to your loved ones who want to SINGLE USERS (n = 57) 1 (1.8) 8 (14) 15 (26.3) 30 (52.6) 3 (5.3) 3.00
<0.001
quit smoking with the help of e- TOTAL USERS (n = 176) 9 (5.1) 47 (26.7) 50 (28.4) 67 (38.1) 3 (1.7)
cig?
7. Your satisfaction towards e- DUAL USERS (n = 119) 10 (8.4) 47 (39.5) 26 (21.8) 30 (25.2) 6 (5) 2.00
cigarette as an effective quit SINGLE USERS (n = 57) 1 (1.8) 19 (33.3) 12 (21.1) 19 (33.3) 6 (10.5) 3.00 <0.001
smoking aid. TOTAL USERS (n = 176) 11 (6.3) 66 (37.5) 38 (21.6) 49 (27.8) 12 (6.8)
8. Have you scared about the DUAL USERS (n = 119) 2 (1.7) 98 (82.4) 17 (14.3) 2 (1.7) 00 1.00
side effects of e-cigarette? SINGLE USERS (n = 57) 0 (0) 45 (78.9) 11 (19.3) 1 (1.8) 00 1.00 0.316
TOTAL USERS (n = 176) 2 (1.1) 143 (81.3) 28 (15.9) 3 (1.7) 00
DUAL USERS (n = 119)
1 (0.8) 103 (86.6) 14 (11.8) 1 (0.8) 00 1.00
9. Have you scared about the SINGLE USERS (n = 57)
0 (0) 45 (78.9) 12 (21.1) 0 (0) 00 1.00 0.131
contents of e-cigarette liquids? TOTAL USERS (n =
1 (0.6) 148 (84.1) 26 (14.8) 1 (0.6) 00
176)
DUAL USERS (n = 119) 8 (6.7) 65 (54.6) 41 (34.5) 5 (4.2) 00 1.00
10. Did you scare of becoming
SINGLE USERS (n = 57) 7 (12.3) 22 (38.6) 20 (35.1) 8 (14) 00 1.00 0.255
addicted to the e-cigarette?
TOTAL Users (n = 176) 15 (8.5) 87 (49.4) 61 (34.7) 13 (7.4) 00
DUAL USERS (n = 119)
-- 3 (2.7) 69 (58) 46 (38.7) 1 (0.8)
11. Did you like the taste of e- SINGLE USERS (n = 57) 2.00
-- 0 (0) 38 (66.7) 18 (31.6) 1 (1.8) 0.609
cigarette? TOTAL USERS (n = 2.00
-- 3 (1.7) 107 (60.8) 64 (36.4) 2 (1.1)
176)
DUAL USERS (n = 119)
12. Did you like the smell of e- -- 3 (2.7) 71 (59.7) 44 (37) 1 (0.8)
SINGLE USERS (n = 57) 2.00
cigarette? -- 0 (0) 34 (59.4) 22 (38.6) 1 (1.8) 0.545
TOTAL USERS (n = 2.00
-- 3 (1.7) 107 (59.7) 66 (37.5) 2 (1.1)
176)
DUAL USERS (n = 119)
6 (5) 24 (20.2) 34 (28.6) 55 (46.2) 00
13. Did you like to have more SINGLE USERS (n = 57) 2.00
7 (12.3) 18 (31.6) 18 (31.6) 14 (24.6) 00 0.002
nicotine in your e- cigarette? TOTAL USERS (n = 2.00
13 (7.4) 42 (23.9) 52 (29.5) 69 (39.2) 00
176)
DUAL USERS (n = 119)
14. Did you like to have more -- 2 (1.7) 79 (66.4) 38 (31.9) 0 (0)
SINGLE USERS (n = 57) 2.00
concentrated vapour in your e- -- 1 (1.8) 32 (56.1) 23 (40.4) 1 (1.8) 0.162
TOTAL USERS (n = 2.00
cig? -- 3 (1.7) 111 (63.1) 61 (34.7) 1 (0.6)
176)
DUAL USERS (n = 119)
15. Is it easier to draw puff on e- -- 17 (14.3) 81 (68.1) 21 (17.6) 00
SINGLE USERS (n = 57) 2.00 0.323
cigarette compared to tobacco -- 3 (5.3) 44 (77.2) 10 (17.5) 00
TOTAL USERS (n = 2.00
cigarette? -- 20 (11.4) 125 (71) 31 (17.6) 00
176)
DUAL USERS (n = 119)
16. Did you scare that, if you 5 (4.2) 36 (30.3) 67 (56.3) 11 (9.2) 00
SINGLE USERS (n = 57) 2.00
stop e-cigarette use, you will 7 (12.3) 27 (47.4) 17 (29.8) 6 (10.5) 00 0.006
TOTAL USERS (n = 1.00
start smoking again? 12 (6.8) 63 (35.8) 84 (47.7) 17 (9.7) 00
176)

Perception and satisfaction of the 176 final visit participants were measured on a scale of 0-4 scales (0 = not sure; 1 = less sure;
2 = moderately sure; 3 = very sure; 4= very much sure). The data are nonparametric and expressed as median two-tailed p-value
are calculated by the Mann-Whitney U test.

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Perception and satisfaction of EC use effects associated with EC and e-liquids. Currently,
The measured perceptions and satisfaction levels although the safety of ECs remains controversial, the
among both the groups are summarised in Table 1. All majority of the study participants strongly expressed less
study participants were asked whether ECs helped in apprehension regarding the harms of ECs. This perception
efforts to quit smoking. Overall, 31.89% of the of the safety of ECs is consistent with reports of previous
participants were very sure that ECs helped them quit studies in which participants believed ECs to be less
smoking. However, more SUs (median = 3) than DUs harmful and less destructive than CCs (9-13). In terms of
(median = 1) answered positively to this query (p < 0.05). perceptions regarding addiction to ECs, nearly two-thirds
Similarly, all participants were asked whether ECs helped of the participants were moderately afraid of becoming
reduce CC consumption. More than 50.6% of the EC dependent. This result is also comparable with the
participants were sure that ECs helped them reduce CC results of previous studies in which vapers felt less
consumption. Again, more SUs (median = 3) than DUs dependent on ECs than on CCs (14). Experts on tobacco
(median = 2) answered positively (p < 0.05). use believe that dependency on CC among smokers is
due to not only nicotine but also some other chemicals in
Furthermore, the participants were asked whether ECs
tobacco smoke, which reinforce the nicotine effects and
contributed to preventing relapse to smoking. Nearly
lead to severe addiction to CCs (1,3).
40.3% of the participants were very sure that ECs helped
to prevent relapse to smoking. In addition, all participants Additionally, a severe addiction to CCs is due to the
were asked about their level of satisfaction towards ECs fast delivery of nicotine to the brain via pulmonary
as an effective smoking cessation aid. A total of 43.8 % absorption, providing immediate satisfaction to smokers.
of SU participants were very much sure that ECs are an During vaping, the nicotine supplied by ECs is poorly
effective smoking cessation aid then 30.2% of DUs. While absorbed in the lungs of vapers. Consequently, less
perceptions of SUs regarding the safety of ECs was 78.9% nicotine reaches the brain, leading to less satisfaction
as compared to 86.6% of DUs respectively. The views of among users. However, the technology of ECs is
both group users' related to other queries are reported in advancing each day, and novel EC models like JUUL®
table 1. deliver higher doses of nicotine than CCs, thus offering
more satisfaction to vapers (15-16).
The present study is the first of its kind in Malaysia.
This study revealed the perceptions and satisfaction levels More than three-quarters of the participants preferred
among SUs and DUs after 1 year of EC use. More than the taste and smell of ECs over those of CCs. However,
one-third of the participants believed that ECs helped three-quarters of the participants also expressed that ECs
them quit smoking, reduce CC consumption and prevent should produce thick vapours. Moreover, these
relapse to smoking. Furthermore, nearly the same participants stated that it was easier to draw puffs from
proportions of SUs and DUs expressed that ECs helped ECs than from CCs. Similarly, previous studies have
them cope with the urge to smoke and related withdrawal confirmed that majority of the users preferred the smell
symptoms. Besides, nearly one-third of the study and taste of ECs but desired higher vapour production to
participants were satisfied with EC use and rated it as an make vaping as enjoyable as smoking (10-11). However,
effective smoking cessation aid. However, to the queries previously published studies have expressed concerns
regarding the effectiveness of ECs in helping to quit regarding these fascinating characteristics of EC because
smoking and reduce CC consumption, more SUs than they may appeal to adolescents and non-smokers and
DUs responded positively. encourage them to start vaping, leading to the
development of nicotine addiction (17-19).
The findings of the present study are consistent with
those of previous studies reporting that vapers were More than two-thirds of the participants felt that ECs
satisfied with EC use and rated it as an effective smoking should supply more nicotine to gain higher smoking
cessation aid (9-11). However, we also found that the EC satisfaction. The insufficient delivery of nicotine may be
users differed in their perceptions of the effectiveness of the reason most users craved for CCs. Generally, the
ECs as a smoking cessation aid. The results indicated nicotine supplied by ECs depends on nicotine
that DUs were less satisfied with the effectiveness of ECs concentration and propylene glycol proportion in e-
even after 1 year of EC use, which warrants further liquids, puffing technique and device. Although the
research. More than three-quarters of the study majority of the participants used third-generation EC
participants reported that they were less afraid of the side devices, they used very low nicotine concentration (6 mg/

Perception and satisfaction of electronic cigarette users

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ml) (20) which may be one of the reasons for participants' of the present study may not be generalisable to the other
desire for a higher concentration of nicotine in ECs. populations with low educational qualifications and poor
In addition, low nicotine supply from ECs may be health status. Furthermore, the study participants
due to the improper selection of nicotine base in e-liquids. comprised those who were already striving to quit
The major ingredients of e-liquids include propylene smoking, which further limits any generalisation of the
glycol (PG) and vegetable glycerin (VG). E-liquids that results. Of note, the present study revealed the perceptions
have a 50 PG :50 VG formulation ratio deliver more of two types of EC users, particularly in terms of benefits
nicotine than those that contain a 25 PG:75 VG and undesirable effects of ECs, which have hardly ever
formulation ratio. High supply of nicotine from e-liquids been reported before in Malaysia. Finally, the results of
with 50 PG: 50 VG ratio is thought to be due to low the present study indicate that SUs had a more positive
boiling point of PG (187.6°C). Thus, e-liquids with a opinion of EC than DUs after 1 year of EC use.
higher proportion of PG evaporate faster than those with 4. Conclusion
a higher proportion of 25 PG:75 VG ratio where the
Both SUs and DUs generally perceived ECs well and
boiling point of VG is 290°C. Therefore, e-liquids with
were satisfied after 1 year of EC use. However, SUs were
high PG content deliver immediate nicotine supply and
more optimistic and more satisfied with EC than DUs.
increase smoking satisfaction in users than base e-liquids
Nevertheless, these study results warrant further
with high VG content (21).
validation in different populations.
Regarding technical problems and physical injuries
Acknowledgements : We sincerely thank our participants
associated with EC use, less than one-tenth of the users
for joining this study and providing information about
reported technical problems with EC use. These problems
electronic cigarettes.
included e-liquid leakage, device heating and rapid battery
discharge. These results are consistent with those reported Funding : This was a self-financed study, and all authors
by Etter & Bullen (2011) and Farsalinos et al. (2014). did not receive any funding from any sponsor or
However, technological problems can be rectified by organisation.
elevating product standards and by developing new, Availability of Data and Materials : The datasets
innovative EC models. generated and analysed during the current study are
No physical harm associated with EC use was reported available from the corresponding author on reasonable
by any participant. However, one case of e-liquid spilling request.
with no injury or side effects was reported. Previous Competing interests : The authors declare that they have
studies have reported accidental and intentional nicotine no competing interests.
poisoning cases among children and adults (22-23). For
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Investigation of Process Variables in the Development of

Nateglinide Nanocrystals
Ng Chia Huey1, Ashok Kumar Janakiraman1*,
Shiek Abdul Kadhar Mohamed Ebrahim Habibur Rahman1
1Faculty of Pharmaceutical Sciences, UCSI University, Cheras, 56000, Malaysia
Corresponding Author : ashok@ucsiuniversity.edu.my

Abstract insulinotropic agent, which acts to stimulate insulin

Nateglinide (NTG) classified under the secretion when needed( ).Nanocrystal formulation is one
biopharmaceutical classification system (BCS) class II, of the best among all strategies to improve the solubility
an oral antidiabetic drug. Solubility is one of the main of BCS class II drugs. The advantage of this formulation
factors to improve its bioavailability. The present research approach is that nanocrystals can help to increase the
developed Nateglinide nanocrystals to overcome its low dissolution velocity and the saturation solubility, which
solubility and high permeability by ultrasonic probe will subsequently increase the bioavailability of
method and characterized by coulter counter analysis, zeta Nateglinide( - ).For drugs such as Nateglinide, where the
sizer, zeta potential, Differential Scanning Calorimetry oral bioavailability is essential to reduce the glycemic
(DSC), Fourier Transformed Infrared Spectroscopy (FT- level, it is crucial to obtain a rapid and complete
IR), and Field Emission Scanning Electron Micrograph dissolution of the drug.
(FESEM). Coulter counter analysis serves as a method Drug nanocrystals are the particles consisting only of
to choose the best formulation based on the process pure drug with the size range from 200 to 800 nm. The
variables includes different surfactants and its advantage of drug nanocrystals is that it has a high drug
concentration and time of sonication. The formulation loading capacity. Practically the nanocrystals are
A4, B3, C4, D4, and E2 were chosen out of 25 nanocarriers with 100% drug loading capacity. The drug
formulations for Malvern zeta sizer based on coulter nanocrystals do not have any matrix material, but they
counter analysis data. Particle size results showed the can be stabilized by the addition of the surfactant layer or
formulations fall into the category of nanoscales were stabilizing polymer layer. Nanocrystals can be a dispersion
A4, C4, and E2, while C4 and E2 have excellent stability in either water or non-aqueous dispersion media, and these
due to its particle velocity. Based on the particle size and dispersions are known as nanosuspension( - ).The
zeta potential resultsE2 has been determined as the objective of this study was to formulate the Nateglinide
optimized formulation. NTG nanocrystals under the nanocrystals and optimize the process parameters using
optimized conditions gave rise to the mean diameter of the ultrasonication method as well as to improve the
181 nm, zeta potential value of 54 mV and polydispersity saturation solubility of Nateglinide adopting the concept
index 0.23. DSC results suggest the decreased crystallinity of nanonization.
of nanocrystals and its irregular shape of nanoparticles
2. Materials and Methods
confirmed by the FESEM image. To conclude,
nanocrystals are a promising method to improve the Materials : Nateglinide was purchased from Wuhan Vanz
solubility of BCS Class II drugs, further in vivo studies Pharm Inc, China. Poloxamer 188 solutions, Poloxamer
are required to claim the therapeutic potentials of 407 and Polyethyleneimine, was obtained from Sigma-
Nateglinide nanocrystals. Aldrich, Tween 80 was obtained from Chempur, Malaysia
and Sodium dodecyl sulphate were obtained from Chemiz,
Key words : Nateglinide;Nanocrystals; Surfactant;
Malaysia.
Ultrasonication
Selection of surfactant system : Atypical strategy includes
1. Introduction the addition of hydrophilic polymers and/or surfactants
Nateglinide is an oral antidiabetic drug that is under to the nanosuspensions to stabilize nanocrystal
the BCS class II which is having low solubility and high formulations. According to previous studies of
permeability. Nateglinide is a non-sulphonyl urea nanosuspension, hydrophilic non-ionic surfactants such

Development of nateglinide nanocrystals

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as Poloxamer 407 (P407) and Poloxamer 188 (P188), as distilled water, distilled water with 1% SDS, phosphate
Tween 80 (Tw80), cationic surfactants such as buffer saline, phosphate buffer saline with 1% SDS. The
Polyethyleneimine (PEI), and anionic surfactants such drug content was measured by using UV
as Sodium dodecyl sulfate (SDS) used to prepare spectrophotometer (PerkinElmer Lambda 25) (7).
nanoparticles and they successfully been incorporated
Characterization of Nateglinide nanocrystals
into nanocrystals production to improve the dissolution
of those drugs( - ). Coulter counter analysis : The particle size of
Nateglinide nanocrystals is measured using Coulter
Drug-excipient compatibility : To determine the drug
counter analysis (Beckman Coulter Z1 Particle Counter,
and excipient compatibility, Nateglinide is mixed
Fullerton, CA) of all prepared formulations. Best
physically with surfactants, and this physical mixture is
formulation from each surfactant was chosen based on
then analyzed by using FT-IR to determine if there is any
the particle size obtained through the coulter counter
intermolecular interaction between the drug and the
analysis result.
surfactants. Fourier transform infraredspectroscopy
(ThermoScientific iD5 ATR, Waltham, MA, USA) was Zeta sizer and zeta potential : Formulations A4, B3, C4,
used to analyze compatibility between the pure D4, and E2has been chosen form coulter counter result
Nateglinide and surfactants( ). A small calibration for the zeta sizer and zeta potential analysis by Malvern
standard powder was spread on the attenuated total zetasizer (Software v 7.03, AA Almelo, Netherlands) at
reflection (ATR) crystal and scanned. Between each a fixed temperature 25°C.The samples diluted with
measurement, the ATR crystal was carefully cleaned with distilled water before the analysis(8-9). Zeta sizer and
methanol and then air-dried. FT-IR spectra for zeta potential are measured to find out the average particle
Nateglinide, surfactants alone and physical mixtures size and charge of the Nateglinide nanocrystals.
between Nateglinide and the surfactants recorded in the Physically stable Nateglinide nanocrystals should have
range of 4000-400 cm-1 averaging 20 scans at a resolution Zeta potential of 30 mV as a minimum value. Zeta
of 4 cm-1 and air background using OMNIC software. potential of 30 mV is for stabilized nanosuspension in
electrostatic form whereas 20 mV is for combined
Solubility studies : The solubility study of the Nateglinide
electrostatic and steric stabilization( ).
is first determined by adding pure Nateglinide powder in
distilled water, methanol, water with 1% SDS. The Fourier transformed infrared (FT-IR) spectroscopy :
solution placed in an orbital shaker for 24 hrs. The FT-IR spectroscopy used to investigate the potential
resulting solution is then subjected to quantification by intermolecular interaction between Nateglinide and the
UV spectrophotometer at 211 nm. The same steps freeze-dried nanocrystals. The Nateglinide powder and
followed for the solubility of Nateglinide in different pH, freeze-dried nanocrystals (A4, B3, C4, D4 and E2) were
which is pH 1.2, 6.8, and 7.4( ). The Nateglinide solubility spread on the ATR crystal and scanned. Between each
of pH 1.2 assessed in HCl while pH 6.8 and pH 7.4 using measurement, the ATR crystal was carefully cleaned with
phosphate buffer saline (PBS) alone as well as PBS in methanol and then air-dried( ).
pH 7.4 with 1% SDS. Differential scanning calorimetry (DSC) : Thermal
Preparation of nateglinide nanocrystals : The analysis was obtained using a differential scanning
nanocrystals formulation prepared by adding 100mg of calorimeter (Perkin Elmer, USA) for Nateglinide and the
Nateglinide powder dissolved in 5 mL of methanol. The optimized freeze-dried nanocrystals E2 formulation.
solution then mixed with 0.5%, 1.0% and 5.0% of Equivalent to 20mg of sample was crimped in a standard
different surfactants concentration. This mixture placed aluminium pan. The thermograms were obtained at a
in the ultrasonicator (Q500 sonicator) for 5 and 15 min scanning rate of 10°C/min over a temperature range of
at an output power of 500 W ( ). The type of surfactants 30 to 300°C under a constant purging of nitrogen at 40
used, the concentration of surfactants and sonication time mL/min (7).
was the process variables in this study. The Field emission scanning electron micrograph
nanosuspension was then lyophilized to get solid (FESEM): Scanning electron micrograph of Nateglinide
nanocrystals for the FT-IR, DSC and FESEM analysis( ). and freeze-dried nanocrystals formulation (E2) was
Saturation solubility : The saturation solubility of obtained on titanium-coated samples with a Jeol JSM-
Nateglinide nanocrystal formulation was determined by 7600f Schottky Field Emission Scanning Electron
adding the nanocrystals into the different solution such Microscope,Tokyo, Japan.

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Statistical analysis:The experimental results are 3248.42 cm-1,Pani N et al.,reported that the (-N-H
expressed as mean ± SD. Statistical evaluation of the data stretching) is seen at wavenumber 3296 cm-1 according
was done using ANOVA. Statistically, significant to the characteristic bands of Nateglinide(13- ).Other
differences will be considered at a level of p < 0.005. relevant functional groups that were present include (-
CH2-cycloalkane) at wavenumber 2925.06 cm-1, the
3. Results and Discussion
carboxylic acid (-COOH) at wavenumber 1711.85 cm-1
Drug-excipient compatibility : The FT-IR of the physical
and carbonyl -(C=O) at 1645.33 cm-1 whereas the
mixtures of Nateglinide and all the surfactants are chosen
retaining functional group in polyethyleneimine were the
shows that there is no significant interaction between the
presence of aliphatic methyl (-CH3) at wavenumber
drug and the surfactants. Therefore, all the surfactant
2858.31 cm-1, aliphatic secondary amine (-NH) at
which has been chosen is suitable to be used in this
wavenumber 1601.67 cm-1 and primary amine carbon
study.FT-IR graph of Nateglinide, PEI and its physical
(C-N) at wavenumber 1031.45 cm-1which is similar to
mixture (Fig. 1) shows that the secondary amine
the study conducted by Hong H et al.,( ).
functional group of Nateglinide is seen at wavenumber

Fig. 1. FT-IR of Nateglinide, PEI and physical mixture of Nateglinide with PEI

Development of nateglinide nanocrystals

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Table 1 : Solubility of Nateglinide in different solve the highest solubility in methanol (95.99 ± 0.7476 mg/
mL),whereas water with the addition of 1% SDS (2.205
Solvent Concentration (mg/mL)
± 0.107 mg/mL) is the second followed by water (0.9492
Methanol 95.99 0.7476 ± 0.130 mg/mL), PBS with 1% SDS (0.9868 ± 0.003
Water 0.9492 0.130 mg/mL) and phosphate buffer saline (PBS) (0.8269 ±
0.004 mg/mL). Nateglinide has the highest solubility in
Water + 1% SDS 2.205 0.107
pH 7.4 that can be seen that (0.09982± 0.001mg/mL)
PBS 0.8269 0.004 among the different pH ranges.
PBS + 1% SDS 0.9868 0.003 Preparation of Nateglinide nanocrystals : Nateglinide
nanocrystals formulated using various surfactantswith
pH 1.2 0.09159 0.004
different concentrations at different sonication timesin
pH 6.8 0.09603 0.001 Table2. The alphabet of each formulation shows the
pH 7.4 0.09982 0.001
different surfactants, andformulation code A stands for
Poloxamer 188; B for Tween 80; C for Sodium Dodecyl
Sulphate; D for Poloxamer 407 and E for Polyethyl
Solubility study : Nateglinide is freely soluble in methanol eneimine. The numbering stands for surfactant
while it is practically insoluble in water( ). FromTable 1, concentration and sonication time. The number 1 stands
theNateglinide solubility value in water was 0.9492 for 0.5% surfactant at 5 min sonication time, number 2
0.130 mg/mL, which is insoluble. Maggi Let al., revealed stands for 0.5% surfactant at 15 min sonication time,
this is due to the lipophilic nature of Nateglinide as well number 3 stands for 1.0% surfactant at 5 min sonication
as its poor wettability. When Nateglinide powder placed time, number 4 stands for 1.0% surfactant at 15 min
in water, it tends to aggregate, and thereforedifficult to sonication time, and number 5 stands for 5.0% surfactant
measure its solubility in water( ). Nateglinide has had at 15 min sonication time.

Fig. 2. Saturation solubility of Nateglinide formulation in PBS + 1% SDS

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Table 2 : Formulation code for Nateglinide nanocr Table 3 : Coulter counter analysisdata
Concentration Sonication Surface mean Specific surface
Formulation Sample
Surfactant of surfactant Time diameter ( m) area (mm)
Code
(%) (min) 1 3.661 6.047 x 105
A1 Poloxamer 188 0.5 5 2 4.236 5.226 x 105
A2 Poloxamer 188 0.5 15
A 3 7.422 2.983 x 105
4 2.716 8.092 x 105
A3 Poloxamer 188 1 5 5 2.735 8.150 x 105
A4 Poloxamer 188 1 15 1 1.421 1.558 x 106
2 0.751 2.948 x 106
A5 Poloxamer 188 5 15
B 3 0.306 7.233 x 105
B1 Tween 80 0.5 5 4 1.116 1.984 x 106
5 3.024 7.319 x 105
B2 Tween 80 0.5 15
1 3.465 6.389 x 105
B3 Tween 80 1 5 2 4.632 4.779 x 105
B4 Tween 80 1 15 C 3 5.236 4.228 x 105
4 2.430 9.109 x 105
B5 Tween 80 5 15 5 4.223 5.241 x 105
C1
Sodium Dodecyl
0.5 5 1 4.312 5.314 x 105
Sulphate
2 3.283 6.743 x 105
Sodium Dodecyl
C2
Sulphate
0.5 15 D 3 3.319 2.220 x 108
C3
Sodium Dodecyl
1 5
4 2.651 8.350 x 105
Sulphate 5 3.401 6.510 x 105
Sodium Dodecyl
C4
Sulphate
1 15 1 1.939 1.141 x 106
Sodium Dodecyl 2 0.316 6.999 x 106
C5 5 15
Sulphate E 3 5.118 4.236 x 105
D1 Poloxamer 407 0.5 5 4 5.739 3.857 x 105
5 4.381 5.053 x 105
D2 Poloxamer 407 0.5 15

D3 Poloxamer 407 1 5 Table 4 : Results of particle size, zeta potential and

D4 Poloxamer 407 1 15 polydispersity index
Formulation Particle size Zeta Polydispersity
D5 Poloxamer 407 5 15
code (nm) potential index
E1 Polyethyleneimine 0.5 5
A4 602.56 -9.34 N/A
E2 Polyethyleneimine 0.5 15
B3 10.94 -5.5 0.185
E3 Polyethyleneimine 1 5

E4 Polyethyleneimine 1 15 C4 272.87 -60.5 0.431

E5 Polyethyleneimine 5 15 D4 835.31 -4.33 N/A

Saturation solubility of Nateglinide nanocrystals : E2 181.21 55.7 0.230

Nateglinide nanoformulation solubility was done in four
different dissolution media such as water, water with 1% media PBS pH 7.4 + 1% SDS( ). However, it can be seen
SDS, PBS and PBS with 1% SDS.Nateglinide has the that the solubility of formulation E (E1, E3, E4 and E5)has
highest solubility in buffer saline (PBS) with 1% SDS decreased in day 2, except E2. This phenomenon may
and pH7.4among different pH ranges. Therefore, PBS happen due to the antagonism of surfactants since the
pH 7.4 + 1% SDS is chosen as a medium for the surfactant used in formulation E is polyethyl
dissolution of Nateglinide nanocrystals.Based on Fig. 2, eneimine,whereas the surfactant used in the dissolution
formulation E has the highest solubility overall in PBS is sodium dodecyl sulphate. Antagonism of surfactant
pH 7.4 + 1% SDS dissolution media. Remko M,found will increase surface tension as well as critical micellar
that if adding 1% concentration of sodium lauryl sulphate concentration leading to a reduction in solubility( ). PEI
of surfactant into water will improve the solubility of is a surfactant with a positive charge,whereas SDS is a
Nateglinide. Therefore, it can be seen that formulation E surfactant with a negative charge.The statistical analysis
has the highest solubility in day one with dissolution of one-way ANOVA between 25 formulations showed a

Development of nateglinide nanocrystals

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Fig. 3. Particle size, zeta potential and polydispersity index for formulation E2

Fig. 4. FT-IR of freeze-dried formulation E2

p-value of <0.005, which indicates that there is a shown in Table 4. Formulation C4 and E2 formulation
significant difference between the formulations. were fit into the criteria based on their Zeta potential of -
Coulter counter analysis : The results obtained from 60.5 mV and +55.7 mV respectively. The differences
coulter counter analysis, formulation A4, B3, C4, D4 and between the positive and negative charge may be due to
E2 are selected for further analysis. These formulations the nature of the surfactant. The results suggest that the
are chosen based on the smallest particle size among all addition of the surfactant, which may mask the available
the formulation from each surfactant concentration. charge on the surface of Nateglinide ( ).Formulation C4
Generally, those formulations are selected as they have consists of sodium dodecyl sulphate, which is an anionic
the smallest particle size in their surfactant group. From surfactant whereas formulation E2 consists of
Table3, the selected formulations were highlighted, in polyethyleneimine, which is a cationic surfactant.
whichformulation A4, B3, C4, D4 and E2 had the size of The last parameter for the selection of the optimized
2.716 m, 0.306 m, 2.430 m, 2.651 m 0.316 m formulation is the polydispersity index (PDI), that would
respectively. be 0.2 and below required for the formulation of
Zeta sizer, zeta potential and polydispersity index nanocrystals withnarrow size distribution. Low PDI
(PDI) : Zeta sizer analysis of the five formulationsare indicates that the nanoparticles exhibita uniform

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distribution of particle size of the nanoparticles and the H polymorph is around the range of 130 to 140°C.
dispersion homogeneity of the nanoparticles ( Bruni Get al., discovered that the Nateglinide polymorph
).Formulation E2 has a size of 181.21 nm with a zeta H is having a melting point of 137.9 ± 0.5°C ( , ).The
potential of +55.7 mV and PDI of 0.230 in Fig. 3, which thermal curve of lyophilized Nateglinide nanocrystal
was prepared polyethyleneimine (PEI) at 0.5% showed a broad endothermic effect with 135.52°C and
concentration and 15 min sonication time. It has particle an associated fusion enthalpy of -178.80 Jg-1, indicative
size <200 nm with excellent stability to fulfil nanocrystals of its crystalline state. Endotherm of lyophilized
standards. Nateglinidenanocrystal revealed that it had shifted
backwards about 5°C with a significant reduction in peak
FT-IR of freeze-dried formulation : FT-IR graph of
intensity and it may be due to experimental conditions
freeze-dried formulation of Nateglinide with
such as ultrasonication speed, moisture content, sample
Polyethyleneimine by ultrasonication process does not
weight and concentration of PEI. It could alternatively
affect the essential functional groups of Nateglinide and
indicate that the shift in the melting point shows that there
surfactantdepicted inFig. 4. It is evident, with the retention
is a physicochemical change to Nateglinide in the Nano-
of the functional group at certain characteristic bands
level[18]. A further comparison of X-Ray Diffraction
ofNateglinide and Polyethyleneimine. The results suggest
(XRD) of pure drug and nanocrystal formulation of
that the surfactant does not show any significant
Nateglinide study will be needed to indicate if there are
interaction with Nateglinide even after the ultrasonication
any polymorphic changes in the pure drug after the
as well as the freeze-drying process ( ).
ultrasonication process( ).
Differential scanning calorimetry (DSC) : DSC
Field emission scanning electron micrograph : The
thermographs of the pure drug is seen in the upper graph
scanning electron microscope of pure Nateglinide shows
in Fig. 5 which exhibited sharp endotherm with a melting
the morphology of Nateglinide as cylindrical sticks in
point at 140.15°C an associated enthalpy of -94.34 Jg-1.
Fig. 6whereas the FESEM of formulation E2 in the
This melting point shows that pure Nateglinide has the
lyophilized form showed smaller rod shape that looks
polymorph H as the melting point of pure Nateglinide in

Fig. 5. DSC graph of (a) Nateglinide and (b) Formulation E2

Development of nateglinide nanocrystals

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Fig. 6. SEM image (a) Nateglinide and (b) Formulation E2

like aggregate. Kim Het al., reported that the Further, the in vivo studies is required to claim the
ultrasonication process might cause the nanocrystals to enhancement in the bioavailability of Nateglinide
undergo polymorphic changes in their structure[ nanocrystals with optimized process variables.
].Therefore, the morphology changes of Nateglinide may
Acknowledgement
indicate that there are polymorphic changes in Nateglinide
The authors gratefully acknowledge funding by the
after the ultrasonication process and lyophilization
Faculty of Pharmaceutical Science, UCSI University,
process, but further X-Ray diffraction will be needed to
Malaysia (PP 491 Project).
confirm the polymorphism of Nateglinide. XRD can be
used to determine if the structure has changed from its The authors have declared no conflict of interest.
crystalline to amorphous form (19). 5. References
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Current Trends in Biotechnology and Pharmacy 258
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DOI : 10.5530/ctbp.2020.4s.33

Evaluation of Antibacterial Activity Against Multidrug-Resistance (MDR)

Bacteria by the Fractions of Artabotrys suaveolens (Blume)
Jian-You C1*, R. Mogana1, Chandramathi SR2, Ashok Kumar B1, Sasikala C1 and Geethanjali K2
1Faculty of Pharmaceutical Sciences, UCSI University, Jalan Puncak Menara Gading, 56000, Kuala Lumpur, Malaysia
2Department of Microbiology, Faculty of Medicine, University of Malaya, Jalan Universiti, 50603, Kuala Lumpur, Malaysia.

Corresponding author : mogana@ucsiuniversity.edu.my

Abstract vancomycin (inhibition zones=10.67±0.58 mm;

Rising of antibiotic resistance is threatening the global MIC=0.78 mg/mL; MBC=0.78 mg/mL). It also inhibited
health care system and increasing the worldwide death MRSA (inhibition zones=11.33±0.58 mm; MIC=0.25 mg/
rate. Therefore, research and discovery of alternative mL; MBC>1 mg/mL) compared to vancomycin
antimicrobial agents from plant sources are encouraged. (inhibition zones= 11.00±0 mm; MIC=0.78 mg/mL;
The Artabotrys suaveolens (Blume) belongs to the MBC=0.78 mg/mL), followed by chloroform and water
Annonaceae family and mainly distributed in tropical and fraction. All three fractions were bacteriostatic against
subtropical regions of the world. It was indigenously used MSSA ATCC 29213 and MRSA based on the results.
to treat postnatal weakness and cholera infection. The finding in this study has confirmed Artabotrys
The study aims to provide evidence of the plant as an suaveolens (Blume) stem could be an alternative source
alternative source of antibacterial agent based for its of antibacterial agent and has provided evidence to the
folkloric use to treat infection. This study was undertaken traditional use of Artabotrys suaveolens (Blume) in
to fractionate the chloroform extract of the stem of infection. Future studies on the isolation and
Artabotrys suaveolens (Blume) and to investigate the in characterization of bioactive compounds from the
vitro antimicrobial activities of different solvent fractions fractions are required to confirm their activity.
against three ATCC and MDR bacteria. Key words : Antibiotic resistance, Artabotrys suaveolens,
bacteriostatic, Staphylococcus aureus
Liquid-liquid fractionation was performed resulting
with petroleum ether, chloroform and water fraction of 1. Introduction
the stem of Artabotrys suaveolens (Blume). Qualitative Rising of antimicrobial resistance is threatening the
phytochemical analysis was conducted for alkaloid, global health care system and increase the worldwide
cardiac glycoside, flavonoid, saponin, sterol and tannin. mortality rate, and it is originated from overuse and society
Antibacterial activity was ascertained by disc diffusion abuse of antibiotics (1,2). Anti-microbial resistance would
assay, minimal inhibitory concentration (MIC) and be a global issue in future, that could wipe out 10 million
minimum bactericidal concentration (MBC) against three of population by 2050 per year, much more than 8.2
ATCC strains (MSSA ATCC 29213, K. pneumoniae million death from cancer (3). Meanwhile, human has
ATCC 13883 and E. coli ATCC 35218) and three clinical been using the plants for their medicinal purpose for over
isolated strains (MRSA, K. pneumoniae, A. baumannii). past decades, due to the precious secondary metabolites
GraphPad Prism 8 was used for data analyses. Differences which probably possesses many pharmacological effects,
are statistically significant when p<0.05. inspiring an important element for future drug research
Qualitative phytochemical analysis revealed that both and development (4,5). Therefore, research and discovery
petroleum ether and chloroform fraction contained of alternative and affordable antimicrobial agents from
alkaloid, sterol and tannin, while water fraction contained plant sources is encouraged.
cardiac glycoside, saponin, and tannin. Petroleum ether The Artabotrys genus plants belong to the Annonaceae
fraction showed notable antibacterial activity against family, they comprised of more than 100 species
MSSA ATCC 29213 (inhibition zones=10.00±1 mm; worldwide, they are wood-climbing shrubs which
MIC=0.5 mg/mL; MBC>2 mg/mL) compared to distributed mainly in tropical and subtropical regions of

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the world (6), also in tropical region of Africa and Eastern ether was added into the funnel and shaken vigorously
Asia (7). The leaves of Artabotrys spp usually arranged with the cap closed to allow partitioning of extract
as simple, opposite and alternate; textured as coriaceous between water and petroleum ether. Two layers of
(leather like), glabrous (smooth surface) and glabrescent fractions were formed after shaking, petroleum ether
(hairless); appeared as glossy (shiny) and the leaves are fraction layer would be at top and water fraction layer at
usually attached to the petiole (stalk). Buds formation at bottom. Water fraction is collected and transferred into
the axils of leaves on the orthotropic branches can grow another separatory funnel, while petroleum ether fraction
out vegetative plagiotropic branches, develop into thorns was collected into a round bottom flask (Favorit). To the
commonly in shady condition or formed sympodial water fraction, 300ml of chloroform (Merck) was added
branches with hooks and flowers (8).The flowers are and shaken vigorously to allow partitioning of extract
generally white or greenish-yellow colour when ripe, between water and chloroform. Two layers were formed
fragrant odour, axillary, solitary, or in clusters of two or after shaking, water fraction layer would be at top and
three, the peduncles (supporting stalk that bearing chloroform fraction layer at bottom. Chloroform fraction
flowers) are sharply-hooked shape (9). The 3 sepals is collected into a round bottom flask (Favorit), followed
(green) and 6 petals (yellow) are usually nearly equal in by water fraction.
sizes, free and valvate in aestivation, united and concave The collected petroleum ether and chloroform
at the base. Carpel and stamens are countless and closely fractions were concentrated with a rotary evaporator
arranged, oblong and cuneate (wedge) in shapes. The (Buchi, R-200 Switzerland) to remove their respective
carpel contains 2 ovules in the ovary. The fruits are solvents. Water fraction was freeze dried (Alpha 1-4 LD
cylindrical or ellipsoid and the seeds is oblong shape(8). plus, CHRIST, Germany) to remove water. The
The decoction of the roots and barks of Artabotrys concentrated fractions without solvent were carefully
suaveolens was traditionally used for emmenagogue, and recovered and stored in suitable containers, then
postnatal weakness in Philippines by orally. In Indonesia proceeded to qualitative phytochemical analysis (10,11).
and India, the infusion and decoction of the leaves of
Phytochemical analysis
Artabotrys suaveolens were orally used to treat cholera
Qualitative phytochemical analysis of the fractions
(8).
for alkaloids, cardiac glycosides, flavonoids, saponins,
However, the in vitro antibacterial properties of sterols and tannins will be determined as follows: (12-
Artabotrys suaveolens have not been studied. Therefore, 17).
this study is intended to investigate the phytochemical
Dragendorff's test for alkaloid : 1.7g of Bismuth sub-
compounds and the antibacterial activity of the water
nitrate, 20ml of Glacial Acetic Acid (GAA), and 5ml of
(H2O), petroleum ether (PE) and chloroform (CHCl3)
fractions of the chloroform extracts from the stem of the Potassium iodide (KI) solution (50%w/v) are dissolved
in water, then make up the volume into 100ml as stock
Artabotrys suaveolens with the aim of establishing an
solution. From the stock solution, 10ml is mixed into 20
alternative source of antibacterial and the basis for its
folkloric use to treat infection (8). ml of GAA and make up to 100ml with distilled water as
working solution. 2 ml of solution of each fraction was
2. Materials and Methods filtered, then the filtrate was mixed with 0.2 ml of GAA
Plant samples collection in a test tube, followed by 1ml of working solution.
The barks of Artabotrys suaveolens were collected Orange-brown precipitate indicates positive of alkaloid.
from a forest in Perak, Malaysia (4°46'N, 100°56'E). The Keller-Killiani test for cardiac glycosides : A mixture of
plant was identified by the FRIM (Forest Research 4.0 ml of GAA and one drop of 2.0% Ferric Chloride
Institute Malaysia). A herbarium sample (PID-251215- (FeCl3) solution were added into 10 ml of plant extract,
10) has been deposited in the FRIM. followed by 1ml of concentrated Sulphuric acid (H2SO4).
Fractionation of plant samples Reddish brown ring appears between the layers
confirming the presence of cardiac glycosides.
The chloroform extraction of plant was previously
done. 50gm of chloroform extract of Artabotrys Shinoda test for flavonoids : 4mg of each fraction was
suaveolens was suspended in 300ml of purified water to dissolved in 2ml of absolute ethanol and filtered. Then
make a viscous suspension. Then the suspension was the filtrate was treated with 0.5 g of Magnesium turnings
transferred into a separating funnel. 300 ml of petroleum followed by a few drops concentrated hydrochloric acid

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(38% HCl). The presence of flavonoids is indicative if inoculating loop to MHB liquid medium and this liquid
pink or magenta-red colour developed after few minutes. culture was incubated at 37°C for 24 hrs. The growth of
Froth test for saponins : Each fraction was shaken turbidity of liquid culture was then adjusted equivalent
vigorously to froth and was then allowed to stand for 15- to McFarland 0.5 turbidity standard. The turbidity of the
20 min and classified for saponin content as follows: (no actively growing broth culture was adjusted with sterile
froth = negative; froth less than 1cm = weakly positive; 0.9% (w/v) saline solution to obtain turbidity optically
froth 1.2cm high = positive; and froth greater than 2cm comparable to McFarland 0.5 turbidity standard, that
high = strongly positive). indicated the broth culture containing approximately
1×108 CFU/ml for each tested strain(18).
Salkowski test for sterols : 40mg of fraction will be
dissolved in 2ml of chloroform and filtered. The filtrate Disc-diffusion test
is then added to 1mL of concentrated H 2SO 4. The The tests were performed using Mueller Hilton agar
presence of sterols is indicated by the 2 phases formation for bacterial strains using disc diffusion method following
with a red colour in the chloroform phase. the Clinical and Laboratory Standards Institute (CLSI)
Ferric chloride test for tannins : About 1mg of fractions guidelines(19).
were dissolved in 6ml of hot distilled water and filtered. 100mg/ml of each fraction sample solution were
The solution is divided in two test tubes. To the first test prepared by dissolving 100mg into 1ml of 99.9% DMSO.
tube, 1ml of 0.9% sodium chloride solution was added. Then 6mm sterile paper disc was impregnated with 10µL
Second test tube was added with 1ml of 0.9% sodium of each sample solution to yield 1mg/disc using
chloride solution and 1ml of 1% (w/v) gelatine solution, micropipette. Noted that all the impregnated paper discs
and to the third test tube few drops of 2% FeCl3 was should be completely dried before applying to the agar
added. Formation of a precipitate in the second treatment surface. Each bacterial culture was thoroughly streaked
suggests the presence of tannins, and a positive response onto the surface of Muller-Hinton agar Petri plates using
after addition of FeCl3 to the third portion which will a sterile swab to ensure complete coverage of the plates
result in a characteristic blue, blue-black, green, or blue- and a lawn of growth with uniform thickness. The agar
green colour supports this inference. plate was divided into four quarters: three fractions(1mg/
Antibacterial susceptibility testing (AST) disc) and one positive control(1µg/disc). The impregnated
The antibacterial activity of plant fraction was 6mm paper disc (1mg/disc) were applied to their
evaluated by testing against the clinical isolated bacteria: respective quarters at approximately equal distance to
Methicillin Resistant Staphylococcus aureus (MRSA), each other, negative control was placed at the centre of
Klebsiella pneumoniae (K. pneumoniae) and the agar plate. Within 15 min after discs were applied,
Acinetobacter baumannii (A. baumannii) and the the agar plates were inverted and incubated with ambient
American Type Culture Collection strains: Methicillin air at 37°C for 24hrs. The antibacterial activity was
Sensitive Staphylococcus aureus ATCC 29213 (MSSA determined after incubation, by measuring the diameter
ATCC 29213), Klebsiella pneumoniae ATCC 13883 (K. of zone of inhibition (in mm) include the 6mm disc size.
pneumoniae ATCC 13883) and Escherichia coli ATCC This experiment was repeated as triplicates, and the mean
35218 (E. coli ATCC 35218), with comparison to positive ± standard deviation (SD) of three replicates were
control: vancomycin for Gram-positive bacteria; presented (18).
gentamicin for Gram-negative bacteria, DMSO was used Determination of Minimum Inhibitory Concentration
as negative control. All tested bacteria were obtained from (MIC) and Minimum Bactericidal Concentration (MBC)
local public hospital: University of Malaya Medical The minimum inhibitory concentration (MIC) of the
Centre (UMMC). against ATCC reference strains and isolated clinical
Preparation of bacterial inoculum strains were determined by microdilution dilution, as
The stock culture of each of the six bacterial strains described by Eloff (20) and using the Clinical and
were sub-cultured on Mueller-Hinton agar (MHA) Petri Laboratory Standards Institute (CLSI) as guideline(19).
plates at 37°C for 24 hrs prior to inoculation into the Stock solution (64mg/ml) of respective plant fraction
Mueller-Hinton broth (MHB) solution. were prepared by dissolving 64mg into 1ml of 99.9%
Few colonies (2 to 3) of similar morphology of the DMSO. The advantages of using 99.9% DMSO as solvent
respective bacteria were transferred with a sterile are elimination of microbial contamination of the plant

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fraction and good solubility during the serial dilution for fractionation of chloroform extracts of the Artabotrys
procedures(21). MIC test of the plant fraction against six suaveolens stem, chloroform provided the highest yield
bacterial strains was aseptically done in 96-well plate as with fractionation yields of 61.92 %. Contrary, petroleum
duplicate by two-fold serial dilution method, 100µL of ether provided lowest yield with fractionation yield of
sterile water was prefilled into all the wells of 96-well 6.73 %. This suggest that the phytochemical components
microtiter plate, followed by 100µL of stock solution in the stem of Artabotrys suaveolens is more favourable
(64mg/ml) to yield 50% of the stock solution (32mg/ml) to non-polar chloroform (25). Results are showed in
in 200µL after adequate mixing. 100µL from the Table1.
50%(32mg/ml) plant fraction solution was transferred to Table 1 : Percentage yield of fractionation of the
the wells in next row, followed by mixing to yield 25% chloroform extract of stem of Artabotrys suaveolens by
(16mg/ml) of plant fraction solution. This procedure was various solvents.
repeated for the subsequent 6 rows, resulting in eight
Solvents Yield (%)
concentration ranging from 32mg/ml to 0.25mg/ml in 100
µL solution. Bacterial broth culture with turbidity of Petroleum ether 6.73
1×108 CFU/ml was adjusted to 100µL of 1×106 CFU/ Chloroform 61.92
ml turbidity before adding into wells, finally yielded
Water 31.35
concentration ranging from 16µg/ml to 0.125mg/ml. The
final concentration of positive control is ranging from Qualitative analysis of the P.C. of Artabotrys
25µg/ml to 0.2µg/ml while negative control is ranging suaveolens
from 25% to 0.2%. The microtiter plate was incubated
with ambient air at 37°C for 24 hrs. 40 mL of 0.4 mg/ml The results of phytochemical analysis were tabulated
in Table 2, water fraction contained cardiac glycoside,
INT (p-iodonitrotetrazolium, Sigma) INT dye was used
to detect bacteria cell viability, pink-purple colour saponin, and tannin, while petroleum ether and
indicates microbial growth. The MIC was visually chloroform fraction contained alkaloid, sterol and tannin.
determined, the lowest concentration that displaying no Table 2 : The results of the phytochemical analysis of
visible growth (no visible pink-purple colour) was the fractionation of Artabotrys suaveolens.
recorded as the MIC (22). Phytochemical Artabotrys suaveolens fractions
Only fraction that have MIC values of lower or Petroleum Ether Chloroform Water
constituents
equivalent to 0.5 mg/mL (strong inhibitors) were tested Alkaloid ++ ++ –
for the MBC values, the content in MIC value and two Cardiac Glycoside – – ++
wells above the MIC value were cultured on MHA. then Flavonoid + + –
incubated at 37°C with ambient air for 24 hrs. The MBC Saponin – – +
was considered as the lowest concentration that produced Sterol ++ + –
<10 bacteria colonies after incubation. The plant fraction Tannin – – ++
was considered bactericidal if the ratio MBC/MIC < 4,
The samples were observed for the colours or
while considered bacteriostatic if the ratio MBC/MIC >
precipitation produced by the reagent, and the scores
4(23).
would be given accordingly: (-) score: negative, (+) score:
Statistical analysis weakly positive, (++) score: positive.
The experimental results were expressed as mean ±
standard deviation (SD) of three replicates. One-way Antibacterial Susceptibility Testing (AST)
analysis of variance (ANOVA) and Tukey's testwere Disc-diffusion test
employed for the data analysis. P values less than 0.05
First of all, results revealed that all three fractions
were considered statistically significant.
were showing antibacterial activity against the gram-
3. Results and Discussion positive bacteria in this study (MSSA ATCC 29213 and
Fractionation of plant samples MRSA). Worth to be mentioned that petroleum ether
Extraction yield is important to measure the efficiency fraction demonstrated best activity against MSSA ATCC
of solvent to extract desired substances from the raw 29213 and MRSA. However, no activities were showed
material (24). Among the three different solvents used against the gram-negative bacteria in this study (K.

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Table 3 : Antibacterial activity of the fractions of chloroform extract of Artabotrys suaveolens (1 mg/disc) and
positive control (1 µg/disc) tested by disc diffusion assay against three ATCC bacteria and MDR bacteria.
Microorganisms Zone of Inhibition (mm)

AS Fractions (1 mg/disc) Control antibiotics (1 µg/disc)

PE CHCl3 H2 O Vancomycin Gentamicin

ATCC strains

MSSAATCC 29213 10.00±1 a 9.67±0.58 a 7.83±0.29 10.67±0.58 a

NA

K. pneumoniae ATCC
— — — NA 15.00±0
13883

E. coli ATCC 35218 — — — NA 15.00±1

Clinical isolated strains

b
MRSA 11.33±0.58 10.00±0 8.00±0 11.00±0 b NA

K. pneumoniae — — — NA 16.33±0.58

A. baumannii — — — NA —

AS: Artabotrys suaveolens, PE: Petroleum ether fraction, CHCl3: Chloroform fraction, H2O: Water fraction, NA: Not Applicable,
and -: no zone of inhibition (0 mm)

Table 4 : Minimum Bactericidal Concentration(MBC), Minimum Inhibitory Concentration(MIC) and MBC/MIC

ratio of the fractions of chloroform extract of Artabotrys suaveolens(mg/mL) and positive control antibiotics(µg/mL).
Microorganism AS Fractions Control antibiotics

MBC/MIC values (mg/mL) MBC/MIC values (µg/mL)

MBC/MIC ratio, (+) bactericidal, ( –) MBC/MIC ratio, (+) bactericidal, (–)

bacteriostatic bacteriostatic

PE CHCl3 H 2O Vancomycin Gentamicin

ATCC strains

>2/0.50±00 >2/0.50±00 >2/0.50±00 0.78/0.78±00

MSSA ATCC 29213 NA
(–) (–) (–) 1(+)

K.pneumoniae 0.2/0.2
NA NA NA NA
ATCC 13883 1(+)

6.25/1.56
E. coli ATCC 35218 NA NA NA NA
4(+)

Clinical isolated strains

>1/0.25±00 >2/0.50±00 >2/0.50±00 0.78/0.78±00

MRSA NA
(–) (–) (–) 1(+)

0.39/0.20
K. pneumoniae NA NA NA NA
2(+)

A. baumannii NA NA NA NA NA

AS: Artabotrys suaveolens, PE: Petroleum ether fraction, CHCl3: Chloroform fraction, H2O: Water fraction, NA: Not Applicable.

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pneumoniae ATCC 13883, E. coli ATCC 35218, K. Since chloroform and water fractions were inhibiting the
pneumoniae and A. baumannii). As expected, no zone of growth of MRSA and MSSA ATCC 29213 at 0.5 mg/mL
inhibition was produced by the plant fractions against (MIC), but the MBC was probably above 2 mg/mL, thus
the gram-negative bacteria in this study, because gram- indicating that MBC/MIC ratio was apparently >4. Worth
negative bacteria were usually more resistant to plant- to be mentioned that petroleum ether fraction alone has
derived antibacterial compounds than gram positive inhibited MRSA growth at 0.25 mg/mL (MIC), but the
bacteria (26, 27). The susceptibility distinction between MBC was probably above 1 mg/mL, suggested the MBC/
gram positive and negative bacteria could be explained MIC ratio was >4 also.
by their different cell wall structures and composition The zone of inhibition was reported as mean ± SD of
(28-31). three experiments including 6 mm disc (n=3). The values
Minimum Inhibitory Concentration (MIC), Minimum with the same alphabet character indicate there were no
Bactericidal Concentration (MIC) and the MBC/MIC significant differences in one-way ANOVA and Tukey
ratio multiple comparison test (p>0.05)
Since disc diffusion assay has showed only the gram- The relationship between phytochemicals from
positive bacteria (MRSA and MSSA ATCC 29213) were Artabotrys suaveolens (Blume) and their antibacterial
susceptible to the plant fraction, and gram-negative activity
bacteria are resistant. Therefore, MIC and MBC test were
Petroleum ether fraction of Artabotrys suaveolens was
proceeded for S. aureus only. MIC and MBC test were
showing most potent antibacterial activity against MRSA
conducted to determine the bacteriostatic or bactericidal
and MSSA ATCC 29213, antibacterial activity was
activities of plant fractions using 8 mg/mL as the endpoint
probably due to the presence of alkaloid (34), flavonoids
for MIC, whereby the plant fractions with MIC value at
(35) and sterols (36) found in the preliminary
most of 8 mg/mL was showing some significant inhibitory
phytochemical analysis.
action to the bacteria, any concentrations higher than 8
mg/mL were considered as insignificant or ineffective 4. Conclusion
(32).
Evaluation of the antibacterial activity from the
Chloroform and water fractions from the chloroform fractions of the chloroform extract of Artabotrys
extract of Artabotrys suaveolens were showing activity suaveolens (Blume) stem suggested it could be an
against MRSA and MSSA ATCC 29213 with MIC value important bacteriostatic source of alternative antibacterial
of 0.5 mg/mL, except petroleum ether fraction was agent in respect of its selective activity against S. aureus.
showing MIC value of 0.25 mg/mL against MRSA. The This validates the traditional use of Artabotrys suaveolens
antibacterial activity of plant fractions was further (Blume) in infection.
classified based on the obtained MIC values. MIC values
Hence, further studies on the isolation and
= 0.5 mg/mL and any lower concentrations indicate the
characterization of bioactive compounds from the
plant fractions are strong inhibitors(33). Based on the
fractions that responsible for the selective activity against
classification proposed above, the MIC values ranging
S. aureus are required. Further antibacterial studies on
from 0.25 mg/mL to 0.5 mg/mL by all fractions
other gram-positive bacteria, as well as time-kill assay
(petroleum ether, chloroform and water) indicate they
and synergistic assay are proposed for future studies.
were strong inhibitors against MRSA and MSSA ATCC
29213, especially the MIC value of 0.25 mg/mL obtained Acknowledgments
from petroleum ether fraction against MRSA, suggested The authors would like to thank the PhD candidates
its antibacterial activity was double than chloroform and of Department of Microbiology, Faculty of Medicine,
water fractions against MRSA. University of Malaya : Ms Geetha and Ms Amni for their
Antimicrobial substances were considered assistantce in microbiology works.
bacteriostatic when the MBC/MIC ratio >4, and
Conflict of Interest
bactericidal when the MBC/MIC ratio < 4(23). Based on
the MBC/MIC ratio, it suggested that all fractions were The authors declare that they have no conflict of
bacteriostatic against MRSA and MSSA ATCC 29213. interests.

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DOI : 10.5530/ctbp.2020.4s.34

In-Vitro Cytotoxic Activities of Brassica oleracea Var capitata

by Using Brine Shrimp Lethality Assay
Ashok Kumar Balaraman1*, Sasikala Chinnappan1, R. Mogana1,
Aziz Ur Rahman, and Tan Zhe Way2
1Faculty of Pharmaceutical Sciences, UCSI University, Kuala Lumpur, Malaysia.
2Faculty of Pharmacy, Asian Metropolitan University, Selangor, Malaysia.
Corresponding author : drashokbalaraman@gmail.com

Abstract cancer remain a high priority for the scientific community

The present study was aimed for phytochemical across the world.
screening and evaluates cytotoxic activity of ethanol Brassica oleracea (B. oleracea) belonging to the
extract of both leaves and seeds of Brassica oleracea var member of Brassicaceae family. There are various
capitata. Both leaves and seed were blended to get the varieties under Brassica oleracea (B. oleracea) which are
coarse powder and it was subject for extraction by B. oleracea var. acephala (kale), var. alboglabra(chinese
maceration technique. 95% ethanol was used as a solvent kale), var. botrytis (cauliflower), var.capitata (cabbage),
for extraction. The cytotoxic activity was determined in var. gemmifera (Brussels sprouts), var. gongylodes
terms of its ability to kill the brine shrimps. Both extracts (kohlrabi), var. italic (broccoli) and var. sabellica (Curly
were tested at different concentrations ranging from 10- and Portuguese kale). Cabbage is native to Coastal
1000 µg/ml with potassium dichromate served as Southern and Western Europe (2). It is the common
standard. Cytotoxic assay showing that leaves extract vegetable we consumed everyday which are inexpensive
having higher percentage of mortality than seeds extract. and rich in nutrients.
Both extracts having moderate cytotoxic activities with Cabbage is use as medicine since ancient time. In folk
LC50 valueof 301.67 µg/ml and 350.76 µg/ml for leaves medicine cabbage was used to cure hangover, sore throat,
and seeds, respectively. However, the mortality of brine abscess, and dysentery. Cabbages also used to cure simple
shrimps caused by both extracts was lesser as compared and complicated injuries, rheumatic pains, facial
with standard of potassium dichromate with LC50 value neuralgia, headaches, leg ulcer, anthrax, and many others.
35.67 µg/ml. The phytochemical screening of both During World War I, they were used to treat trench foot
extracts revealed the presence of alkaloids, sterols, of soldiers. Moreover, the water in which cabbage leaves
terpenoid, carbohydrates, flavonoids, glycosides and have been cooked has been used to treat rheumatism (3).
saponins. These phytochemicals play a prominent role in
During recent decades, there has been an increasing
cytotoxic activity. Since the plant was showing cytotoxic
demand for finding newer and safer chemotherapeutic
activity, it can be further subjected for isolation of the
agents. Besides, the current diseases are increasingly
therapeutically active compounds with cytotoxic activity
become resistant to the drugs. Numerous studies have
and for further pharmacological evaluations.
shown thatvarious natural products can interfere with
Key words : Brassica oleracea, phytochemical,cytotoxic. different stages of cancer which are cancer induction,
1. Introduction growth and progression therefore may effectively block
malignancy (4). Thus, it is important to discover new
Cancer is an abnormal growth of cells which can
drugs to combat with drug resistant and adverse side
invade and spread to other part of the body. There are effects ofsynthetic drugs.
more than 100 types of cancer. Cancer is important leading
causes of morbidity and mortality worldwide and poses The present study was undertaken to investigate
both economic and psychological challenges. According cytotoxic activity of leaves and seeds extract of this plant.
to World Health Organization (WHO), there are 14 million 2. Materials and Methods
new cases and 8.2 million of people died each year due
to cancer. Cancer contributed to about 13% of all people Materials
death worldwide (1). Therefore, cure and prevention of 95% ethanol, sodium chloride, sodium bicarbonate,

In-vitro aytotoxic activities of Brassica oleracea var capitata

Current Trends in Biotechnology and Pharmacy 267
Vol. 14 (5) 270-275, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.34

potassium dichromate, Fehling A reagent, Fehling B treated with few drops of diluted sodium hydroxide
reagent, chloroform, concentrated sulphuric acid, solution. Formation of intense yellow colour, which
concentrated hydrochloric acid, iron (III) chloride, becomes colourless on addition of dilute acid, indicates
Wagner's reagent, and sodium hydroxide. the presence of flavonoids.
Preparation of plant materials Test for phenols :
Fresh cabbage and dried seeds were purchased from Ferric chloride test : A Small quantity of extract was
local market in Cheras. After through washing, the leaves dissolved in 5ml of water and filtered/ Extracts were
were cut into small pieces and dried completely under treated with 3-4 drops of 10% ferric chloride solution.
shade at room temperature. The dried leaves were ground Formation of bluish black colour indicates the presence
to coarse powder by using blender. of phenols.
Extraction of plant material Test for saponin :
The extracts were obtained by using cold maceration Foam test : A Small quantity of extract was dissolved in
method. The powder was soaked in 95% ethanol. 10ml of distilled water. The solution was shaken
Periodical stirring of the mixture was performed for the vigorously and observed for a stable persistent froth for
next 48 hours. The extracts were filtered, and the 20 min.
extraction was repeated twice (5). Then, the filtrate
Test for tannins :
obtained was evaporated at 60°C by using water bath.The
extract was then dried and stored in desiccators. A semi- A Small quantity of extract was boiled in 10 ml of
solid extract was formed.The resultant yield of extract water in a test tube and then filtered. A few drops of 0.1%
obtained was 14.78% and 10.67% of dry weight for leaves ferric chloride was added and observed for brownish
and seeds, respectively. green or a blue-black colouration.

Preliminary phytochemical screening In-vitro cytotoxic activity

Phytochemical screening were performed using Brine shrimp lethality bioassay was carried out to
standard procedures (6, 7, 8). investigate the cytotoxicity of extracts of B. oleracea var
capitata. Artificial sea water was prepared by dissolving
Test for alkaloids
38gm of NaCl in 1 liter of distilled water for hatching
Wagner's test : small quantity of extract was dissolved the shrimp eggs. Small quantity of sodium bicarbonate
in 8ml of 1% HCl and warm for two minutes. It was was added to obtain the pH 8.4 as sea water. The seawater
filtered and few drops of Wagner's reagent was added. was put in a small plastic container (hatching chamber)
Formation of reddish-brown precipitate indicates the with a partition for dark (covered) and light areas. Shrimp
presence of alkaloids. eggs were added into the dark side of the chamber while
Test for steroids, terpenoids and cardiac glycosides: the lamp above the other side (light) was used to attract
Salkowski test : Small quantity of extract was dissolved the hatched shrimps. Oxygen was supplied through an
in 5ml of chloroform. It was filtered and 2 ml of air pump. Two days were allowed for the shrimp to hatch
concentrated H2SO4 was added to form a layer. The and mature as nauplii (larva). The crude extract was
formation of brown ring indicates presence of sterols. dissolved in artificial sea water to obtain 10mg/ml as stock
solutions. Then, a series of solution of varying
Test for carbohydrates concentrations (10µg/ml, 50µg/ml, 100µg/ml, 500µg/ml
Fehling's test : Small quantity of extract was dissolved and 1000 µg/ml) were prepared from the stock solution
in 5ml of water and filtered. Equal volume of Fehling A by serial dilution method. Ten nauplii were drawn through
and Fehling B reagents were mixed. The aqueous extract a capillary tube and place in each well containing 4.5ml
was added to boiling Fehling's solution in a test tube. A artificial sea water and added various concentrations of
brick red coloured precipitate of cuprous oxide forms crude extract. The final volume was made up to 5 ml
indicates the presence of carbohydrates. using artificial sea water. A parallel series of tests with
Test for flavonoids : the standard potassium dichromate solution (10-100 µg/
ml) were tested and the blank control was always
Alkaline reagent test : A Small quantity of extract was included. After 24 hours, surviving shrimps in each vial
dissolved in 5ml of water and filtered. Extracts were were viewed with a magnifying glass, counted and the

Ashok et al
Current Trends in Biotechnology and Pharmacy 268
Vol. 14 (5) 270-275, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.34

survival data recorded (9). Six replicates were used for Table 1 : Qualitative phytochemical analysis of ethanol
each treatment and control. The percentage of mortality extracts from leaves and seeds of Brassica oleracea var
(% M) is calculated as (10). capitata.
%M=number of dead nauplii/total number of nauplii×100
Phytochemical Ethanol Extract
In case where negative control deaths occur, the data
were corrected using Abbott's formula (11): components Leaves Seeds
% M= [(test - control)/control] x 100 Alkaloids + +
3. Results and Discussion Sterols + +
Preliminary phytochemical screening Terpenoid + +
Table 1 showed the result of phytochemical screening
Carbohydrates + +
of leaves and seeds extracts of Brassica oleracea var
capitata. Both extracts indicated the presence of the Flavanoids + +
following secondary metabolites; alkaloids, sterols,
Phenols - -
terpenoid, carbohydrates, flavonoids, glycosides and
saponins. The phenols and tannins were found to be Glycosides + +
absent in both extracts. According to previous research,
Saponins + +
the principle active compounds detected here which
include alkaloids, sterols, terpenoid, flavonoids and Tannins - -
saponins are known to poses cytotoxic activity of the brine
shrimps (12,13).
(+) Present, (-) Absent

Tab 2 : In-vitro cytotoxic activity of potassium dichromate and Brassica Oleracea var capitata extracts.

% of mortality
Serial Concentration
Groups Mean ± S.D. LC50 (µg/ml)
No. (µg/ml)
(n=6)
10 21.67±7.53
20 61.67±4.08
1. Potassium dichromate 40 71.67±7.53 35.67
80 80.00±8.94
100 93.33±5.16
10 25.00±5.48
50 33.33±8.17
2. Leaves extract 100 58.33±7.53 301.67
500 75.00±8.37
1000 96.67±5.16
10 20.00±8.94
50 40.00±6.32
3. Seeds extract 100 51.67±7.53 350.76
500 65.00±8.37
1000 91.67±7.53

In-vitro aytotoxic activities of Brassica oleracea var capitata

Current Trends in Biotechnology and Pharmacy 269
Vol. 14 (5) 270-275, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.34

In-vitro cytotoxic activity

In the present study, BSLA was used to evaluate
cytotoxic activities of B. oleracea leaves and seeds extract.
BSLA is an efficient, rapid and inexpensive assay for
testing biochemical activity of plant extract (14). It was
useful for preliminary screening the plant extract's
toxicity (15). LC50 value lower than 1000 µg/ml is
considered bioactive in toxicity evaluation of plant
extracts by BSLA11. Values of LC50 between 500 µg/
ml to 1000 µg/ml are categorized as weakly cytotoxic,
those between 100 and 500 µg/ml as moderately
Fig 2b : The toxicity effects of the Brassica Oleracea
cytotoxic, and those less than 100 µg/ml was considered var. capitata (seeds) using brine shrimp lethality assay
to have strong cytotoxic activity (16). after 24 hours.
The result of cytotoxic activity of ethanol extract of
concentration of the extract. Increasing in concentration
B. Oleracea and positive control potassium dichromate
of test samples increased mortality gradually. The
in terms of mortality of brine shrimps were presented in
maximum mortalities took place at the highest
Table 2 and graphically present in figure 1, 2a and
concentration of 1000µg/ml whereas the least mortality
2b.There are varying degrees of lethality observed with
took place at 10µg/ml in the evaluation for cytotoxicity
exposure to different concentrations of the test samples
using BSLA. In this investigation, both leaves extract
in brine shrimp lethality assay (BSLA). The degree of
and seeds extract exhibited cytotoxic activities with the
lethality was found to be directly proportional to the
LC50 values 301.67µg/ml and 350.76 µg/ml respectively.
These showed that both seed and leaves extract having
moderate cytotoxic activities. Besides, the leaves extract
having the highest percentage of mortality when
compared with seeds extract. However, the mortality of
brine shrimps caused by both extracts was lesser as
compared with potassium dichromate which serves as a
standard. When compared LC50 of leaves and seeds
extracts, leaves extract is more potent than seeds extract.
The cytotoxicity of the extract is contributed by
presence of alkaloids, flavonoids and terpenoids(12).
Moreover, the presence of saponins in extract also
Fig 1 : The toxicity effects of the potassium dichromate justified the potency as antitumor and anticancer
using brine shrimp lethality assay after 24 hours. agent(13).
Alkaloid has been shown to exhibit antiproliferation
and antimetastasis activities on various types of cancers
both in vitro and in vivo (17) . Flavanoid has been shown
to exhibit antimutagenic and antimalignant effects (18).
This was associated with flavonoid can interfere with
the initiation, development, and progression of cancer
by the modulation of cellular proliferation, differentiation,
apoptosis, angiogenesis and metastasis (19). Terpenoids
have been shown to suppress the growth of a variety of
cancer cells without exerting any toxicity in normal cells.
Terpenoids act at various stages of tumor development,
Fig 2a : The toxicity effects of the Brassica Oleracea inhibit initiation and promotion of carcinogenesis, induce
var. capitata (leaves) using brine shrimp lethality assay tumor cell differentiation and apoptosis, and suppress
after 24 hours. tumor angiogenesis, invasion, and metastasis through

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Current Trends in Biotechnology and Pharmacy 270
Vol. 14 (5) 270-275, ISSN No. 0973-8916 (Print), 2230-7303 (Online)
DOI : 10.5530/ctbp.2020.4s.34

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 Current Trends in Biotechnology and Pharmacy
ISSN 0973-8916 (Print), 2230-7303 (Online)

Editors
Prof.K.R.S. Sambasiva Rao, India Prof. Karnam S. Murthy, USA
krssrao@abap.co.in skarnam@vcu.edu

Editorial Board
Prof. Anil Kumar, India Dr.P. Ananda Kumar, India
Prof. P.Appa Rao, India Prof. Aswani Kumar, India
Prof. Bhaskara R.Jasti, USA Prof. Carola Severi, Italy
Prof. Chellu S. Chetty, USA Prof. Ursula Kües, Germany
Dr. S.J.S. Flora, India Dr. Govinder S. Flora, USA
Prof. H.M. Heise, Germany Prof. Huangxian Ju, China
Prof. Jian-Jiang Zhong, China Dr. K.S.Jagannatha Rao, Panama
Prof. Kanyaratt Supaibulwatana, Thailand Prof.Juergen Backhaus, Germany
Prof. Jamila K. Adam, South Africa Prof. P.B.Kavi Kishor, India
Prof. P.Kondaiah, India Prof. M.Krishnan, India
Prof. Madhavan P.N. Nair, USA Prof. M.Lakshmi Narasu, India
Prof. Mohammed Alzoghaibi, Saudi Arabia Prof.Mahendra Rai, India
Prof. Milan Franek, Czech Republic Prof.T.V.Narayana, India
Prof. Nelson Duran, Brazil Dr. Prasada Rao S.Kodavanti, USA
Prof. Mulchand S. Patel, USA Dr. C.N.Ramchand, India
Dr. R.K. Patel, India Prof. P.Reddanna, India
Prof. G.Raja Rami Reddy, India Dr. Samuel J.K. Abraham, Japan
Dr. Ramanjulu Sunkar, USA Dr. Shaji T. George, USA
Prof. B.J. Rao, India Prof. Sehamuddin Galadari, UAE
Prof. Roman R. Ganta, USA Prof. B.Srinivasulu, India
Prof. Sham S. Kakar, USA Prof. B. Suresh, India
Dr. N.Sreenivasulu, Germany Prof. Swami Mruthinti, USA
Prof.Sung Soo Kim, Korea Prof. Urmila Kodavanti, USA
Prof. N. Udupa, India

Assistant Editors
Dr.Giridhar Mudduluru, Germany Dr. Sridhar Kilaru, UK
Prof. Mohamed Ahmed El-Nabarawi, Egypt Prof. Chitta Suresh Kumar, India
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