Evaluation of Phytochemical and Pharmacological Activities of a Hill

Tract Plant– Callicarpa arborea

Family: Verbenaceae

A project report submitted in partial fulfillment of the requirements for

the Degree of four years Bachelor of Pharmacy (B. Pharm)

B. Pharm. Project

Submitted by
Examination Roll No.: 161125
Examination Session: 2018-2019

Pharmacy Discipline
Life Science School

13th August, 2020 Khulna University

Khulna-9208

Bangladesh
 Dedicated To
My Beloved Parents
 Declaration

This is to certify that the project work entitled “Evaluation of Phytochemical and
Pharmacological Activities of a Hill Tract Plant- Callicarpa arborea” has been carried
out by As-Sazzad Mahmud in the Pharmacy Discipline, Khulna University, Khulna,
Bangladesh. The above project work or any part of this work has not been submitted in
anywhere for the award of any degree or diploma.

Signature of Candidate Signature of Supervisor

As-Sazzad Mahmud Professor Dr. Md. Saifuzzaman

i
 Approval

This is to certify that the thesis work submitted by As-Sazzad Mahmud entitled
“Evaluation of Phytochemical and Pharmacological Activities of a Hill Tract Plant–
Callicarpa arborea” has been approved by the Examination Committee for the partial
fulfillment of the requirements for the degree of Bachelor of Pharmacy (B. Pharm) in the
Pharmacy Discipline, Khulna University, Khulna, Bangladesh in August, 2020.

Board of Examiner (or Examination Committee)

1. ………………………………… Chairman of the Committee

Name :
Designation :
University :
2. ………………………………… Member

Name :
Designation :
University :
3. ………………………………… Member

Name :
Designation :
University :
4. ………………………………… Member

Name :
Designation :
University :

i
i
 Acknowledgement

All praises be to the Supreme of this universe, the Almighty God, Who enabled me to
undertake and complete this research work and finally write up the outcome project work
leading towards the fulfilment of the degree of Bachelor of Pharmacy.

Individual efforts alone can never contribute in totality to a successful completion of any
venture. I would be failing in my duty if I did not state my gratitude and appreciate to the
following individuals who have made valuable contribution toward the project work.

I wish to express my profound sense of gratitude to my most respectable teacher and

supervisor, Professor Dr. Md. Saifuzzaman, Pharmacy Discipline, Khulna University, for
his inspiration, constant guide and valuable suggestions made throughout the course of
study, without which this project work would not have taken its present shape. I also want
to express my profound sense of gratitude to my co-supervisor, Dr. Asish Kumar Das for
his cooperation.

I have the pleasure in expressing my thanks and regards to my respectable teacher

Professor Dr. Ashis Kumar Das, Head of Pharmacy Discipline, Khulna University, for his
continuous support and assistance during the course of this research work.

I have the pleasure in expressing my thanks and regards to all other honourable teachers
of Pharmacy Discipline, Khulna University, who allowed me to exercise thoughtful and
intelligent freedom to proceed with the research work.

My special thanks extend to Kasifa Afrin, Muslima khatun, Srabani Ghosh and all of my
classmates of Pharmacy Discipline, Khulna University for their friendly cooperation and
occasional help.

I would like to express my gratitude to my parents and my family members for their
admiration and encouragement.

I want to give my cordial thanks to Mr. Rabiul Islam, Assistant Registrar (Lab), Mr.
Ashiqul Islam, Lab Technician, and Mr. Nasir Hossain, Assistant lab technician,
Pharmacy Discipline, Khulna University.

I also extend my gratitude towards those who helped me in one way or other for
providing the help during the research work.

The Author August, 2020.

iii
 ABSTRACT

Callicarpa arborea (family – Verbenaceae), a hill tract plant has been traditionally used
to cure fever, influenza, boils, sores, etc. In order to ascertain the therapeutic uses of the
plant, leaves were subjected to explore for phytochemical nature and selected
pharmacological activities. In phytochemical analysis the ethanolic extract of the leaves
indicated the presence of reducing sugars, flavonoids, tannins, phenolics, glycosides,
gums, saponins, steroids, carbohydrate, acidic compounds and terpenoids. In the TLC
based qualitative antioxidant assay using DPPH, C. arborea extract showed the free
radical scavenging properties indicated by the presence of yellow spot on a purple
background on the TLC plates. In the quantitative study, DPPH free radical scavenging
assay showed SC50 value of ~34 µg/mL which is comparable to ascorbic acid (SC 50 ~14
µg/mL). Total phenolic, flavonoid and tannin contents were found to be almost 158 mg
GAE/g, 941 mg QE/g and 193 mg GAE/g of the dried leaf extract, respectively. Total
antioxidant capacity was also determined and found to be about 323 mg AAE/gm.
Moreover, the extract exhibited significant analgesic effect (44.12% and 58.82%
inhibition of writhing at the doses of 250 mg/kg and 500 mg/kg body weight,
respectively) in mice using acetic acid induced writhing method. For in vivo anti-
inflammatory activity, the extract showed a good level of decrease in formaldehyde
induced edema in mice using the same doses as above. The extract also showed moderate
level of decrease in yeast induced pyrexia while applying the same doses in mice. In
anti-hyperglycemic test, the plant extract was screened and compared with
Glibenclamide standard. The extract was found to reduce the blood glucose level to a
significant extent. Therefore, the present study revealed strong antioxidant, as well as
significant analgesic, anti-inflammatory, antipyretic and anti-hyperglycemic activities in
C. arborea leaves which support its folkloric uses.

iv
 CONTENTS Page No.

Declaration i

Approval ii

Acknowledgement iii

Abstract iv

Contents v

List of Tables xiii

List of Figures xv

List of Abbreviations xvii

Chapter One: Introduction 1-10

1.1 Overview 1

1.2 Medicinal plant 2

1.3 Traditional approach 3

1.4 Procedure of drug development from plant source 3

1.5 Rationale for medicinal plant research in Bangladesh 4

1.6 Rationale for Selecting hill tract plant 5

1.7 Plant Preview 5

1.8 literature review 7

1.9 Project Design 9

1.10 References 10

v
Chapter Two: Extraction 12-16

2.1 Introduction 12

2.2 Plant material 13

2.3 Materials & Methods 14

2.4 Extraction at a glance 15

2.5 Yield determination 15

2.6 Conclusion 15

2.7 References 16

Chapter Three: Phytochemical Tests 18-27

3.1 Introduction 18

3.2 Test material 18

3.3 Preparation of reagents 18

3.4 Methods of phytochemical tests 19

3.5 Observations 23

3.6 Result of chemical group tests 26

3.7 Conclusion 26

3.8 References 27

Chapter Four: Test for Antioxidant Activity 29-58

4.1 Introduction 29

4.2 Test for Qualitative Antioxidant Activity 30

4.2.1 Principle 30

4.2.2 Apparatus 30

vi
4.2.3 Reagents 31

4.2.4 Solvent system 31

4.2.5 Method 31

4.2.6 Observation 32

4.2.7 Result 34

4.3 Test for Quantitative Antioxidant Activity 36

4.3.1 DPPH Scavenging Assay 36

4.3.1.1 Principle 36

4.3.1.2 Apparatus 37

4.3.1.3 Reagents 37

4.3.1.4 Method 37

4.3.1.5 Data presentation 38

4.3.1.6 Result 40

4.3.2 Determination of Total Phenolic Content (TPC) 40

4.3.2.1 Principle 40

4.3.2.2 Apparatus 41

4.3.2.3 Reagents 41

4.3.2.4 Method 41

4.3.2.5 Data presentation 42

4.3.2.6 Determination of phenolic content 43

4.3.2.6 Result 44

4.3.3 Determination of Total Flavonoid Content (TFC) 44

vii
4.3.3.1 Principle 44

4.3.3.2 Apparatus 44

4.3.3.3 Reagents 45

4.3.3.4 Method 45

4.3.3.5 Data presentation 46

4.3.3.6 Determination of flavonoid content 47

4.3.3.6 Result 48

4.3.4 Determination of Total Tannin Content (TTC) 48

4.3.4.1 Principle 48

4.3.4.2 Apparatus 48

4.3.4.3 Reagents 49

4.3.4.4 Method 49

4.3.4.5 Data presentation 50

4.3.4.6 Determination of tannin content 51

4.3.4.6 Result 52

4.3.5 Determination of Total Antioxidant Content (TAC) 52

4.3.5.1 Principle 52

4.3.5.2 Apparatus 53

4.3.5.3 Reagents 53

4.3.5.4 Method 53

4.3.5.5 Data presentation 54

4.3.5.6 Determination of total antioxidant capacity 55

viii
4.3.5.6 Result 56

4.4 Discussion 56

4.5 Conclusion 57

4.6 Reference 57

Chapter Five: Evaluation of Analgesic Activity 60-67

5.1 Introduction 60

5.2 Methodology 60

5.2.1 Principle 60

5.2.2 Materials 61

5.2.3 Experimental animal 61

5.2.4 Study Design 61

5.2.5 Preparation of sample suspension 62

5.2.6 Working Procedure 63

5.3 Data presentation 63

5.4 Result & Discussion 66

5.5 Conclusion 67

5.6 References 67

Chapter Six: Evaluation of Anti-inflammatory Activity 69-78

6.1 Introduction 69

6.2 Methodology 69

6.2.1 Principle 70

6.2.2 Materials 70

ix
6.2.3 Experimental animal 70

6.2.4 Preparation of sample 70

6.2.5 Study Design 71

6.2.6 Working Procedure 72

6.3 Data presentation & Calculation 72

6.4 Result & Discussion 77

6.5 Conclusion 77

6.6 References 78

Chapter Seven: Evaluation of Anti-pyretic Activity 80-86

7.1 Introduction 80

7.2 Methodology 80

7.2.1 Principle 80

7.2.2 Apparatus and reagents 81

7.2.3 Experimental animal 81

7.2.4 Study Design 81

7.2.5 Preparation of sample suspension 82

7.2.6 Working Procedure 83

7.3 Data presentation 84

7.4 Result & Discussion 85

7.5 Conclusion 86

7.6 References 86

Chapter Eight: Evaluation of Anti-hyperglycemic Activity 88-94

x
8.1 Introduction 88

8.2 Methodology 88

8.2.1 Principle 88

8.2.2 Materials and reagents 89

8.2.3 Experimental animal 89

8.2.4 Study Design 89

8.2.5 Preparation of sample suspension 90

8.2.6 Working Procedure 91

8.3 Data presentation 92

8.4 Result & Discussion 93

8.5 Conclusion 93

8.6 References 94

Chapter Nine: Discussion 96-99

Chapter Ten: Conclusion 101

Chapter Eleven: Appendix 103

xi
 List of Tables
Table No. Title Page No.
3.1 Different chemical group tests for standard and C. arborea 24
extract

3.2 Result of chemical group tests 26

4.1 Solvent system used for TLC 31
4.2 DPPH Scavenging Assay of Ascorbic Acid (Standard) 38
4.3 DPPH Scavenging Assay of C. arborea Extract 39
4.4 UV Absorbance of gallic acid (standard) at 765 nm 42
4.5 UV absorbance of sample at 765 nm 43
4.6 Determination of phenolic content of C. arborea extract 44
4.7 UV Absorbance of Quercetin (standard) at 510 nm 46
4.8 UV absorbance of sample at 510 nm 47
4.9 Determination of flavonoid content of C. arborea extract 48
4.10 UV Absorbance of gallic acid (standard) at 725 nm 50
4.11 UV absorbance of sample at 725 nm 51
4.12 Determination of tannin content of C. arborea extract 52
4.13 UV Absorbance of ascorbic acid (standard) at 695 nm 54
4.14 UV absorbance of sample at 695nm 55
4.15 Determination of total antioxidant capacity of C. arborea extract 56
4.16 SC50 values of different assays and total content of secondary 57
metabolites (phenolic, flavonoid and tannin) of sample extract

Experimental profile to assess the effect of C. arborea extract on

5.1 acetic acid induced writhing of mice 62

5.2 Effects of C. arborea extract on acetic acid induced writhing of 64

mice

5.3 Statistical evaluation of effects of the extracts on acetic acid 65

induced writhing

xiii
6.1 Experimental profile to assess the effect of C. arborea extract 71

6.2 The measurement of paw thicknesses of mice for 10 days 73

6.2 Increase in paw thickness for the C. arborea extract on the 74
formaldehyde induced paw edema method in mice

6.3 Percent of inflammation and Percent inhibition of inflammation of 75

C. arborea extract at different days

7.1 Experimental profile to assess the effect of C. arborea extract on 82

pyrexia

7.2 Effects of C. arborea extract on pyrexia of mice 84

8.1 Experimental profiles to assess the anti-hyperglycemic activity 90
8.2 Oral Glucose Tolerance Test result of C. arborea extract 92

xiv
 LIST OF FIGURES

Figure No. Title Page No.

1.1 C. arborea whole plant 5

1.2 C. arborea leaves 5

1.3 C. arborea fruit 6

1.4 C. arborea flower 6
4.1 Observation of TLC plate under UV (shorter wave length) 33
4.2 Observation of TLC plate under UV (longer wave length) 33
4.3 Observation of TLC plate after spraying 0.02% DPPH 34
4.4 Observation of TLC plate after applying H2SO4 34
4.5 Percent (%) scavenged of DPPH vs. log concentration graph for standard 38
(ascorbic acid)

4.6 Percent (%) scavenged of DPPH vs. log concentration graph for C.
arborea extract 39

4.7 Comparison of Percent (%) scavenged of DPPH by ascorbic acid 40

(standard) and sample

4.8 Total phenolic content determination of C. arborea extract with the help
of gallic acid standard calibration curve 41

4.9 Total flavonoid content determination of C. arborea extract with the help 47
of Quercetin standard calibration curve

4.10 Total tannin content determination of C. arborea extract with the help of
gallic acid standard calibration curve 51

4.11 Total antioxidant capacity determination of C. arborea extract with the 55

help of ascorbic acid standard calibration curve

5.1 Mean Writhing vs. treatment of Diclofenac Na with C. arborea extract

on acetic acid induced writhing in mice 65

5.2 Percent (%) inhibition of writhing vs. treatment of Diclofenac Na and C. 66

arborea extract on acetic acid induced writhing in mice

Change in paw thickness (mm) in every hour for negative control,

6.1 positive control and C. arborea extract (500mg/kg and 250mg/kg) 76

xv
6.2 Percent (%) of Inflammation vs time of standard drug and C. arborea 76
extract on formaldehyde induced paw edema method in mice

6.3 Percent (%) inhibition of inflammation vs time of standard drug and C. 77

arborea extract on formaldehyde induced paw edema method in mice

7.1 Change of rectal temperature at different times (for different samples) 85

8.1 Blood glucose level verses time 93

xvi
 List of Abbreviations

Abbreviated forms Elaborations

Conc. Concentration
DPPH 2, 2-diphenyl-1-picryl hydrazyl
gm Gram
kg Kilogram
µg Microgram
min Minute
ml Milliliter
mm Millimeter
NSAID Non-steroidal Anti-inflammatory Drug
PGE Prostaglandin-E
SD Standard Deviation
SE Standard Error
SEM Standard Error Mean
UV Ultra-violet
wt. Weight
b.wt. body weight
No. Number
TLC Thin Layer Chromatography
Vs. Versus

xvii
CHAPTER ONE
INTRODUCTION
 Chapter 1: Introduction

CHAPTER ONE
Introduction
1.1 Overview
Phytochemicals (from the Greek word Phyto, meaning plant) are biologically active, naturally
occurring chemical compounds found in plants that provide health benefits for humans. They
protect plants from disease and damage and contribute to the plant’s color, aroma and flavor.
In general, the plant chemicals that protect plant cells from environmental hazards are called
as phytochemicals. Recently, it is clearly known that they have roles in the protection of
human health, when their dietary intake is significant.[1] Disease is as old as life itself, and
man has always been in search of agents to cure diseases. Plants and herbs have been in use
for eradication of diseases and human sufferings since antiquity. Man has been experimenting
with the plants. Discovery of medicines began through serendipity and man’s quest for drug
in nature. Since plants existed on earth from the very beginning of human evolution, the use
of plants in the daily lives and in alleviating human sufferings is as old as human civilization
on earth. In many countries traditional medicines or herbs are considered primary treatment
option.[2] The Indo-Aryans noted down the use of medicinal plants in Rig-Veda at around
4500-1600 BC.[3]
Bangladesh being a country of this Indian subcontinent also possess a great diversity in
plants. Around two thousand medicinal plants in this sub-continent and 449 medicinal plants
are enlisted in Bangladesh. [4]
Though the exact number of used plants is unknown there are some common medicinal plants
which are in use by kavirajes, traditional medicines for a long time. The existence of various
tribes like Chakma, Marma, Rakhain, Tripura, Garo, Khashia with cultural diversity are also
enriched the use of medicinal plants. This traditional attempt for treatment has survived the
enormous take-over of modern medicine systems because of the belief of people in mother-
nature. As a consequence, the knowledge behind the use of medicinal plants have passed down
from ancestors to predecessors. Some definite preparations and validation protocols for the use of
some medicinal plants have been determined. Still the rural, tribal and folklore society admire the
traditional use of plant extract or simple preparations like infusion, decoction, powder from plants
due to the great availability of the plants. One of the major uses of medicinal plants is in
infectious diseases to treat them or to reduce the symptoms. The most common use is still in
treating common cold, sinus infection and skin infections. Besides, with the advancement of
science people have come to know that the plants at our

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 Chapter 1: Introduction

reach contain magically bioactive components like alkaloids, glycosides, flavonoids, tannins,
terpenes, resins, gums, mucilage, antioxidants at small dose. Therapeutic uses of plants had in
effect stored at the very beginning of human life on earth when the primitive man, out of
necessity and by intuition, resorted to use plants to alleviate his sufferings from injuries and
diseases.
The medicinal plants have been used in traditional medicine for hundreds of years with
reputation as efficacious remedies although these may not have sufficient scientific data to
substantiate their efficacy. Of these, surprisingly large number are still of importance in
modern medicine.
Medicinal plants are rich sources of bioactive compounds and thus serve as important raw
material for drug production. It has now been established that the plants synthesize and
accumulate some secondary metabolites like alkaloids, glycosides, tannins, volatile oils etc
that may possess a great potential for biological activity and can be a curative agent in
therapeutic purposes.
Today, based on the historical information and traditional use, extensive work on finding the
actual constituents which is responsible for the medicinal properties of the plant is performed.
Isolation of different constituents from the different plants and the rationale of the use of this
plant in traditional medicine have been checked.

1.2 Medicinal plant

The plants that possess therapeutic properties or exert beneficial pharmacological effect on
the living body are generally known as “Medicinal Plant”. In another word, A medicinal
plant is any plant which contains substances that can be used for therapeutic purposes or
which is a precursor for synthesis of useful drugs. Medicinal plants may be defined as a
group of plants that possess some special properties or virtues that qualify them as article of
drugs and therapeutic agents and are used for medicinal purposes.

There are three ways in which plants have been found useful in medicine. [5]

➢ First, they may be used directly as teas or in other extracted forms for their
natural chemical constituents.
➢ Second, they may be used as agents in the synthesis of drugs.
➢ Finally, the organic molecules found in plants may be used as models for
synthetic drugs.

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 Chapter 1: Introduction

1.3 Traditional approach to obtain medicine from medicinal plants

A generalized approach to obtain medicine from medicinal plants is given below- [5]

Identification of plant parts

Collection of plants at suitable time and session

Drying of plants/plant parts in a suitable size

Grinding and sieving

Collection and storing of grinded materials in a cool and dry place

Extraction with suitable solvent

Fractionation of extracts

Phytochemical tests

Pharmacological tests

Identification of adverse or beneficial activity

Identification of the active principle

Determination of chemical structure Synthesis of active substance

1.4. Procedure of drug development from plant source

Modern drug development from plant source is carried out according to a systemic
investigation as described- [6]

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 Chapter 1: Introduction

o Selection and correct identification of the proper medicinal plant and its selected
part(s).
o Extraction with suitable solvent(s).
o Detection of biological activity of crude extract and establishment of a bioassay
system to permit the identification of the active fractions and rejection of the inactive
ones.
o Fractionation of crude extracts using the most appropriate chromatographic
procedures, biological evaluation of all fractions and separation of the active
fractions.
o Repeated fractionation of active fractions to isolate pure compound(s).
o Elucidation of chemical structure of pure compound(s) using spectroscopic methods.
o Evaluation of biological activity of pure compound(s).
o Investigation of other pharmacological properties of the active agent(s).
o Toxicological tests with pure compound(s).
o Production of drug in appropriate dosage forms.

1.5 Rationale for medicinal plant research in Bangladesh

More than 500 of such medicinal plants have so far been established as growing in
Bangladesh.[7] Some of these indigenous medicinal plants are extensively used in the
preparation of unani, ayurvedic and homeopathic medicines in Bangladesh. But the fact is
that almost all of them are lacking scientific evaluation of their therapeutic uses. So, standard
pharmacological and phytochemical methods should be applied in order to:

❖ Establish scientific evidence of their pharmacological use.

❖ Isolate the active constituent(s). This may offer a local natural source of a commonly used
drug or a novel therapeutic agent.

Bangladesh is a developing country of low economic growth. So scientific exploration and

standardization of potential crude drugs are needed to revolutionize our drug sector. Diverse
bioactive metabolites like steroids, terpenoids, flavonoids, alkaloids, glycosides, etc in plants
have formed the therapeutic basis of herbal medication. Thus, emphasis is given on the
biological screening of medicinal plants for further exploration of their active constituents.

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 Chapter 1: Introduction

1.6 Rationale for Selecting hill tract plant:

As the existence of various tribes like Chakma, Marma, Rakhain, Tripura, Garo, Khashia
with cultural diversity are enriched with the use of medicinal plants, it can be a great source
of research for the investigation of new drug. In hill tract, many more plants are yet to be
investigated and have no proper scientific data. That’s why my purpose is to introduce
unexplored hill plant with proper scientific data which will help to go for further research for
new medicine discovery.
As in Bangladesh, there are plenty of unexplored medicinal plants particularly in tropical
area, this can be the area of interest in discovering new remedies for diseases. Considering
this a hill plant named Callicarpa arborea is chosen for primary screening in term of
phytochemical and pharmacological investigation. Different parts like leaves, roots, stem
barks are used traditionally for different remedies. Scientific screening of this plant can go a
long way for the betterment of mankind.

1.7 Plant Preview (Callicarpa arborea)

1.7.1 Botanical features: Callicarpa arborea is commonly known as beauty berry. Other
common names may be included as Khoja, Bormala etc.[4] Generally, it is found in Dry
forests of Chittagong, Chittagong Hill Tracts, Cox's Bazar, Sylhet, Dhaka, North Bengal and
Mymensingh. [4]

1.7.2 Scientific classification (NCBI Taxonomy)

Kingdom: Plantae
Phylum: Tracheophyta
Class: Magnoliopsida
Figure 1.1: C. arborea whole plant.
Order: Lamiales
Family: Verbenaceae
Genus: Callicarpa
Species: Callicarpa arborea

Figure 1.2: C. arborea leaves.

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 Chapter 1: Introduction

1.7.3 Morphology
It is an evergreen tree that can grow up 8 to 20 meters tall. Branchlets, inflorescences, and
flower-stalks are densely velvet-hairy. Morphology of the specific parts are given below- [4]

Leaves: Leaves are elliptic, oblong-elliptic, or ovate, 13-37 x 7-13 cm, leathery, densely
yellow-brown velvety on the underside, dark green and shiny above, base wedge-shaped to
rounded, margin entire.

Flowers: Purple flowers are borne in cymes 6-11 cm across. Cymes are carried on 4-angled
stalks, longer than leaf-stalks. Sepal tube is cup-shaped, outside densely grey velvety.
Flowers are purple, about 3 mm. Stamens much longer than flower-tube. Ovary is densely
velvety.

Fruits: Fruit is purple-brown, about 2 mm in diameter, succulent, dark purple when ripe.

Flowering: May - July.

Figure 1.3: C. arborea fruit. Figure 1.4: C. arborea flower.

1.7.4 Chemical constituents

C. arborea has been reported to contain β-sitosterol, β-amyrin, lupeol, epilupeol, ursolic acid,
oleanolic acid, L(+)-α-amino-β-(p-methoxyphenyl) propionic acid, masnilic acid, betulinic
acid and baurerol. [8]

1.7.5 Medicinal uses

Bark is used to stimulate the digestion and also in the treatment of skin complaints. The juice
of the bark, combined with the bark juice of Cordia dichotoma and Psidium guajava is used
to treat indigestion. The powdered bark, combined with a pinch of tumeric (Curcuma longa)
is used to relieve fevers.[9]

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 Chapter 1: Introduction

A decoction of the leaves is used as a wash to treat cutaneous diseases. The root is chewed to
treat boils on the tongue. A decoction of the leaves is taken to relieve a stomachache. The
leaves are used as a poultice to heal sores. The plant is used to treat influenza.[10]

1.8 literature review

1.8.1 Wound healing activity of leaves:

Leaves of C. arborea Roxb was used for investigating wound healing property on rats. The
study was performed by excision, incision and dead space wound models by administering
-1 -1
the methanolic extracts at the dose of 300 mgkg day p.o and topically applying alcoholic
extracts (5% w/w) formulated as an ointment prepared by Indian Pharmacopoeia method.
Complete wound contraction was shown by the plant in the study period. In excision, incision
and dead space wound models, all the test drugs showed significant (P<0.0001) wound
healing activities compared to the control. Moreover, the ointment formulation of C. arborea
Roxb. had been observed to have equipotent wound healing activity as of the standard drug
Framycetin. Histopathological examination confirmed the mechanism of wound healing by
increased deposition of collagen, fibroblast on the granulation tissue and neovascularization.
[8]

1.8.2 Preliminary phytochemical and HPLC screening of triterpenoids fraction from

bark extract

Triterpenoids were found through Phytochemical screening test of C. arborea Roxb. bark
extract & The compounds have a semi-polar polarity direction. One dominant triterpenoid
compound was observed in the observation by HPLC. There may have a scope for the next
stage of structure elucidation determination through the use of preparative HPLC. [11]

1.8.3 Antidiabetic activity of hydro-alcoholic stem bark extract with antioxidant

potential in diabetic rats

Streptozotocin-induced diabetic rats were given dose 250 and 500 mg/kg body weight orally.
Results of antidiabetic activity study informed that hydro-alcoholic extract of C. arborea
stem bark possesses significant (p<0.05) hypoglycemic activity compared to normal control
group in experimental rats. Histological observations of treated pancreas and liver tissues
confirmed the antidiabetic efficacy of hydro-alcoholic extract. hydro-alcoholic extract also

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 Chapter 1: Introduction

revealed significant radical scavenging activity. So, finally it was said that hydro-alcoholic
extract of C. arborea stem bark may have possible role as herbal antioxidants in the treatment
of oxidative stress-induced diabetes mellitus for which phenolic and flavonoid contents were
responsible. [12]

1.8.4 Analgesics activity of stem bark extract

The bark of C. arborea contains terpenoids (bauerenol, betulinic acid etc.) and steroid (β-
sitosterol), and the analgesic activities showed by the plant extracts may be due to the
presence of these compounds as terpenoids and steroidal compounds have been reported to
possess good analgesic activity by inhibiting prostaglandin. In the acetic acid-induced
writhing inhibition assay, the ethanol extract and its chloroform and ethyl acetate soluble
fractions at a dose of 200 mg/kg body weight significantly inhibited writhing response with
42.7%, 70.3% and 32.8% of inhibition, respectively. [13]

1.8.5 Antioxidant activity of stem bark extract

The free radical scavenging activity of the plant extractives on the stable radical 1,1-
diphenyl-2 picrylhydrazyl (DPPH) was determined. In the antioxidant assay by DPPH,
significant free radical scavenging activity with SC50 values of 41.53, 14.59 and 71.2 μg/mL,
respectively was demonstrated using the ethanol extract and its pet ether and chloroform
soluble partitionates. [13]

1.8.6 Antimicrobial activity of stem bark extract

In the disc diffusion method, potential antimicrobial activity was revealed with zone of
inhibition ranging from 19-22 mm at a concentration of 400 μg/disc. On the other hand, the
pet ether soluble fraction induced significant clot lysis (30.43%). [13]

The methanol extracts of the stem bark of C. arborea was prepared to test for antibacterial
activity using disk diffusion method. Four Gram-negative bacteria such as Escherichia coli,
Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella typhimurium; and two
Gram-positive bacteria such as Micrococcus luteus and Bacillus subtilis were used. C.
arborea extract was effective against all the bacteria tested. These reveals that C. arborea is a
good source of antibacterial compound. [14]

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 Chapter 1: Introduction

1.8.7 Discussion:
After reviewing literatures, it is clear that extract of stem bark of C. arborea showed
antimicrobial, antioxidant, antidiabetic activities and compounds like triterpenoids are also
observed from that extract. On the other hand, we have only scientific data of wound healing
activity of leaves. So, the leaves of C. arborea may possess more opportunities to reveal such
activities with proper scientific data that are yet to be screened.

1.9 Project Design:

After reviewing literatures, it is seemed that leaves of C. arborea possess more scopes to be
screened for phytochemical and pharmacological evaluation. So, the aim of this project work
was to search bioactive metabolites and to evaluate the pharmacological activities of the
leaves of C. arborea.

For performing the investigation, the study was arranged in the below order:

Part A: Phytochemical tests

• Identification of different chemical groups present in the leaves of this plant
• Qualitative antioxidant assay.
• Quantitative antioxidant assay by free radical scavenging activity (DPPH)
• Determination of total phenolic content (TPC).
• Determination of total flavonoid content (TFC).
• Determination of total tannin content (TTC).
• Determination of total antioxidant capacity (TAC).

Part B: Pharmacological assessment

• Evaluation of anti-pyretic activity.
• Evaluation of anti-inflammatory activity
• Evaluation of analgesic activity.
• Evaluation of anti-hyperglycemic activity.

Page | 9
 Chapter 1: Introduction

1.10 References

1. Saxena, M., et al., Phytochemistry of medicinal plants. Journal of pharmacognosy and

phytochemistry, 2013. 1(6).
2. Ali, M., et al., Medicinal plants used in traditional system of medicine in Bangladesh.
1990, PHC Publication-2, VU University Press: Amsterdam. p. 55-64.
3. Tucakov, J.J.B.C., Healing with plants–phytotherapy. Beograd: Culture, 1971: p. 180-
90.
4. Ghani, A.J.D., Bangladesh, Medicinal Plants of Bangladesh, Asiatic Society of
Bangladesh. 2003: p. 500-504.
5. Rana, M.P., et al., The use of plants in indigenous health care practice of the hajong
tribe community in North Eastern Bangladesh. Journal of Forest Science, 2009. 25(1):
p. 25-33.
6. Lewis III, R.R.J.E.e., Ecological engineering for successful management and
restoration of mangrove forests. Ecological engineering, 2005. 24(4): p. 403-418.
7. Lithgow, D., G. de la Lanza, and R.J.E.E. Silva, Ecosystem-based management
strategies to improve aquaculture in developing countries: case study of Marismas
Nacionales. Ecological Engineering, 2019. 130: p. 296-305.
8. Kar, P.K., et al., Evaluatio of wou d-heali g activity of leaves of urtica parviflora roxb
ad Callicarpa arborea roxb i rats. Pharmacologyonline, 2009.
9. Manandhar, N.P., Plants and people of Nepal. 2002: Timber press.
10. Wiart, C., Medicinal plants of Asia and the Pacific. 2006: CRC Press.
11. Amin, M., et al., Preliminary phytochemical and HPLC screening of triterpenoids
fraction from bark extract of Callicarpa arborea Roxb. Journal of Chemical
Pharmaceutical Research, 2015. 7(10): p. 848-851.
12. Junejo, J.A., et al., Antidiabetic activity of hydro-alcoholic stem bark extract of
Callicarpa arborea Roxb. with antioxidant potential in diabetic rats. Biomedicine
pharmacotherapy 2017. 95: p. 84-94.
13. Shihan, M.H., et al., Pharmacological Evaluation of Stem Bark of Callicarpa arborea
Roxb. Dhaka University Journal of Pharmaceutical Sciences, 2015. 14(1): p. 111-116.
14. Roy, S., et al., Screening of Callicarpa arborea and Hemigraphis alternata for
antibacterial activity. Science Vision, 2020.

Page | 10
CHAPTER TWO
EXTRACTION
 Chapter 2: Extraction

CHAPTER TWO

Extraction

2.1 Introduction

Solid-liquid extraction is defined as an operation to separate elements contained in a solid

body by solubilization with a solvent and it may be followed by purification. A plant extract
must, by definition, be obtained from a solid-liquid extraction. Extraction is the crucial first
step in the analysis of medicinal plants, because it is necessary to extract the desired chemical
components from the plant materials for further separation and characterization.

2.1.1 Choice of procedure

Generally, two types of procedure are used for obtaining organic constituents-

1. Cold extraction and

2. Hot extraction

Cold extraction is preferred because of doing no harm to the active constituents available
within the plant material. On the other hand, hot extraction is generally avoided because of
the chance of thermal degradation of active constituents. [1]

2.1.2 Choice of solvent

The factors [2] affecting the choice of solvent are quantity of phytochemicals to be extracted,
rate of extraction, diversity of different compounds extracted, diversity of inhibitory
compounds extracted, ease of subsequent handling of the extracts, toxicity of the solvent in
the bioassay process, potential health hazard of the extractant. The choice of solvent is
influenced by what is intended with the extract. The choice will also depend on the targeted
compounds to be extracted. In traditional medicine practice, ethanol [2] is most widely used
extractant. The bioactive components of medicinal plants are usually unknown and the nature
of the extractant used affects the composition of the crude extract.

Alcohol used to prepare extract serves three [2] specific functions. First, alcohol is the only
edible solvent that will extract and preserve many of the naturally occurring herb constituents
that are poorly soluble in water, such as essential oils, resins, balsams and many alkaloids.
Second, alcohol is an excellent natural preservative, which maximizes the shelf life of the
extracts. Third, alcohol is a great carrying agent, which facilitates the absorption of the herb’s

Page | 12
 Chapter 2: Extraction

constituents into the bloodstream. The higher activity of the ethanol extracts as compared to
the aqueous extract can be attributed to the presence of higher amounts of polyphenols as
compared to aqueous extracts. It means that they are more efficient in cell walls and seeds
degradation which have non-polar character and cause polyphenols to be released from cells.
More useful explanation for the decrease in activity of aqueous extract can be ascribed to the
enzyme polyphenol oxidase, which degrade polyphenols in water extracts, whereas in
methanol and ethanol they are inactive. Moreover, water is a better medium for the
occurrence of the micro-organisms as compared to ethanol. The higher concentrations of
more bioactive flavonoid compounds were detected with ethanol 70% due to its higher
polarity than pure ethanol. Additionally, ethanol was found easier to penetrate the cellular
membrane to extract the intracellular ingredients from the plant material. Since nearly all of
the identified components from plants active against microorganisms are aromatic or
saturated organic compounds, they are most often obtained through initial ethanol or
methanol extraction. Methanol is more polar than ethanol but due to its cytotoxic nature, it is
unsuitable for extraction in certain kind of studies as it may lead to incorrect results. [2]

The solvent-to-sample ratio affects the quantity and quality of constituents obtained. In some
studies solvent to sample ratios of 10 mL solvent : 1 g to dry weight ratio has been used and
reported as ideal.[3]

2.2 Plant material

Fresh or dried plant material can be used as a source for secondary plant components.
However, most scientists working on the chemistry of secondary plant components have
tended to use dried plant material for several reasons. Differences in water content may affect
solubility of subsequent separation by liquid-liquid extraction and the secondary metabolic
plant components should be relatively stable, especially if it is to be used as an antimicrobial
agent. Furthermore, many plants are used in the dry form (or as an aqueous extract) by
traditional healers. Plants are usually air dried [4] to a constant weight but other researchers
o
dry the plants in the oven at about 40 C for 72h [5]. Also, plants will have different
constituents depending on the climatic conditions in which it is growing. The choice of plant
material used in the extract preparation is usually guided by the traditional use of the plant
and the ease of handling of the different plant parts like the leaves, stems etc [3].

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 Chapter 2: Extraction

2.3 Materials & Methods

The leaves of C. arborea were the raw material for the extraction process.

2.3.1 Plant Collection & Identification

For this present investigation the leaves of Callicarpa arborea was collected from Laltila,
Moulvibazar, Sylhet, Bangladesh on September, 2018 and was identified by the experts at
Bangladesh National Herbarium, Mirpur, Dhaka-1216, where a voucher specimen was
submitted (Accession Number: DACB-48442) for future reference.

2.3.2 Preparation of Crude Extract

2.3.2.1 Drying & Grinding

The collected plants were separated from undesirable materials or plants or plant parts. Those
were dried by shade drying to ensure the active constituents free from decomposition. Then
these were ground into a coarse powder with the help of a suitable grinder. The powder was
stored in an airtight container and kept in a cool, dark and dry place until analysis
commenced.

2.3.2.2 Cold extraction

C. arborea powder (350 gm) was taken in clean, flat-bottomed glass containers and soaked in
1300 ml ethanol. The containers with its contents was sealed and kept for a period of 15 days
accompanying occasional shaking and stirring.

2.3.2.3 Filtration of the solvent

The whole mixtures were then passed through a coarse filtration by a piece of clean cloth.
Then it was filtered through cotton & then filter paper.

2.3.2.4 Evaporation of the solvent

The filtrate obtained (ethanol extract) was evaporated through rotary evaporator. Then
concentrated extract was taken in beaker, the opening of beaker was wrapped by a sheet of
aluminum foil to which perforation was done for evaporation of ethanol and was kept in dry
and cool place for some days.

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 Chapter 2: Extraction

2.4 Extraction at a glance

Plant collection

Drying & Milling

Powder of the dried part

Macerated with ethanol for 15 days

Filtration by cotton plug

Filtration by whatman filter paper

Evaporation

Crude dried ethanol extract

2.5 Yield determination

42.22 gm crude extract was obtained from 350 gm of dried powder material.

Therefore, yield value (%) = 42.22/350×100

=12.06 %
~ 12%

2.6 Conclusion
A good amount of crude extract (42.22 gm) was obtained. As a standard precaution against
loss of material, concentrated extract was stored in the refrigerator for preceding
phytochemical and pharmacological activities.

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 Chapter 2: Extraction

2.7 References

1. Sasidharan, S., et al., Extraction, isolation and characterization of bioactive

compounds from plants’ extracts. African Journal of Traditional, Complementary
Alternative Medicines, 2011. 8(1).
2. Tiwari, P., et al., Phytochemical screening and extraction: a review. Internationale
pharmaceutica sciencia, 2011. 1(1): p. 98-106.
3. Ncube, N., A. Afolayan, and A. Okoh, Assessment techniques of antimicrobial
properties of natural compounds of plant origin: current methods and future trends.
African journal of biotechnology, 2008. 7(12).
4. Bariş, Ö., et al., Biological activities of the essential oil and methanol extract of
Achillea biebersteinii Afan.(Asteraceae). Turkish Journal of Biology, 2006. 30(2): p.
65-73.
5. Salie, F., P. Eagles, and H. Leng, Preliminary antimicrobial screening of four South
African Asteraceae species. Journal of ethnopharmacology, 1996. 52(1): p. 27-33.

Page | 16
 CHAPTER THREE
PHYTOCHEMICAL TESTS
 Chapter 3: Phytochemical Tests

CHAPTER THREE
Phytochemical Tests
3.1 Introduction
The plant kingdom represents an enormous reservoir of biologically active compounds with
various chemical structures and protective/disease preventive properties (phytochemicals).
These phytochemicals, often secondary metabolites present in smaller quantities in higher
plants, include the alkaloids, steroids, flavonoids, terpenoids, tannins, and many others.
Nearly 50% of drugs used in medicine are of plant origin[1], and only a small fraction of
plants with medicinal activity has been assayed. There is therefore much current research
devoted to the phytochemical investigation of higher plants which have ethnobotanical
information associated with them. The phytochemicals isolated are then screened for different
types of biological activity.

Callicarpa arborea is a tropical plant. These plants have many traditional usages. These
traditional usages may be due to some phytochemical compounds such as flavonoids,
glycosides, saponins, tannin, alkaloid etc. The crude extract was qualitatively tested for the
presence of chemical constituents using the significant reagents and chemicals.

3.2 Test Material

➢ C. arborea extract

3.3 Preparation of Reagents

Preparation of reagents was carried our using standard formulation as described by [2]

Mayer’s Reagent
To 1.36 gm mercuric iodide, 60 ml of water was mixed with a solution containing 5 gm of
potassium iodide in 20 ml of water.

Dragendroff’s Reagent
To 1.7 gm basic bismuth nitrate and 20 gm tartaric acid, 80 ml water was mixed. This
solution was mixed with a solution containing 16 gm potassium iodide and 40 ml water.

Page | 18
 Chapter 3: Phytochemical Tests

Fehling’s Solution A
To 34.64 gm copper sulphate, a mixture of 0.50 ml of sulfuric acid and sufficient water was
mixed to produce 500 ml.

Fehling’s Solution B
A mixture of 176 gm of sodium potassium tartarate and 77 gm of sodium hydroxide were
dissolved insufficient water to produce 500 ml. Equal volume of above solution were mixed
at the time of use.

Benedict’s reagent
A mixture of 1.73 gm cupric sulphate, 1.73 gm sodium citrate and 10 gm anhydrous sodium
carbonate were dissolved in water and the volume was made up to 100 ml with water.

Molish Reagent

To 2.5 gm of pure α-naphthol, 25 ml of ethanol was dissolved.

Wagner’s Reagent

A mixture of 0.4 g of iodine and 1.2 g of potassium iodide were dissolved in 20 ml of

distilled water.

Hager’s Reagent

To 0.5 g of picric acid, 50 ml of distilled water was dissolved.

3.4 Methods of Phytochemical Tests

Testing of different chemical groups present in extract represents the preliminary

phytochemical studies. In each test 5% (w/v) solution of extract in ethanol was taken unless
otherwise mentioned in individual test. The chemical group test, which are performed as
follows-

3.4.1 Tests for reducing sugar

Tests for reducing sugar such as Benedict’s & Fehling’s test were carried out as described by
[3]

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 Chapter 3: Phytochemical Tests

3.4.1.1 Benedict’s test

In a test tube, 0.5 ml of aqueous extract of the plant material was taken. 5 ml of Benedict’s
solution was added to the test tube, boiled for 5 minutes and allowed to cool spontaneously.
A red color precipitate of cuprous oxide indicates the presence of a reducing sugar.

3.4.1.2 Fehling’s test (standard test)

To 2 ml of an aqueous extract of the plant material, 1 ml of a mixture of equal volumes of

Fehling’s solutions A and B was added and boiled for few minutes. A red or brick red color
precipitate formation indicates the presence of a reducing sugar.

3.4.2 Tests for combined reducing sugar

An amount of 1 ml of aqueous extract of plant material was boiled with 2 ml of dilute

hydrochloric acid for 5 minutes, then cooled and neutralized with sodium hydroxide solution
and then Fehling’s test was performed as described above. A red or brick red color precipitate
formation indicates the presence of a combined reducing sugar.

3.4.3 Tests for phenolic compounds

Presence of phenolic compound was confirmed through Ferric Chloride & Lead Acetate Test
which is carried out as described by [4, 5]

3.4.3.1 Ferric Chloride Test

An aliquot of 2 ml aqueous solution of the extract was taken in a test tube. Then 1 ml of 5%
(w/v) aqueous Ferric chloride solution was added in the test tube. Dark green or bluish black
color precipitate indicates the presence of phenolic hydroxyl groups. Here aqueous solution
of gallic acid was used as standard.

3.4.3.2 Lead Acetate Test

An aliquot of 2 ml aqueous solution of the extract was taken in a test tube. Then 1 ml of 10%
(w/v) aqueous lead acetate solution was added in the test tube. White color precipitate
indicates the presence of phenolic compounds. Here aqueous solution of gallic acid was used
as standard.

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 Chapter 3: Phytochemical Tests

3.4.4 Tests for tannins

Presence of tannin was confirmed through Ferric Chloride & Potassium Dichromate Test
which is carried out as described by [4]

3.4.4.1. Ferric Chloride test

In a test tube, 5 ml solution of the extract was taken. Then 1 ml of 5% Ferric chloride solution
was added. Greenish black precipitate indicates the presence of tannins.

3.4.4.2. Potassium Dichromate Test

In a test tube, 5 ml solution of the extract was taken. Then 1 ml of 10% Ferric chloride
solution was added. Greenish black precipitate indicates the presence of tannins. The
formation of a yellow precipitate indicates the presence of tannins.

3.4.5 Test for flavonoids

Test for flavonoids was carried out as described by [2] [3]
1. An amount of 5 ml of dilute ammonia solution was added to a portion of the aqueous
filtrate of plant extract followed by addition of concentrated H 2S04. A yellow
coloration observed in each extract indicates the presence of flavonoids. The yellow
color disappears on standing.
2. An amount of 0.2 gm extract was dissolved in dilute sodium hydroxide and then
neutralized with dilute hydrochloric acid. Formation of yellow color and
disappearance of color indicate the presence of flavonoid.

3.4.6 Test for Saponins

Presence of saponins was tested as described by [5]
An amount of 1 ml solution of the extract was diluted with distilled water to 20 ml and
shaken in a graduated cylinder for 15 minutes. A layer of foam above the solution indicates
the presence of saponins.

3.4.7 Test for gums/ carbohydrates

Presence of gums/carbohydrates was tested as described by [5]

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 Chapter 3: Phytochemical Tests

An amount of 5 ml solution of the extract was taken and then Molish’s reagent and sulfuric
acid were added. Red violet ring produced at the junction of two liquids indicates the
presence of gums & bluish violet ring indicates the presence of carbohydrates.

3.4.8 Test for Steroids

Test for steroids was carried out through sulfuric acid as described by [5]

3.4.8.1 Sulfuric acid test

An amount of 1 ml solution of chloroform extract was taken and then added 1ml sulphuric
acid. A red color indicates the presence of steroid.

3.4.9 Test for alkaloids

’ ’
Presence of alkaloids was tested through Mayer s, Dragendroff s, Wagner Test, Hager Test as
described by [5]

’
3.4.9.1 Mayer s test
An amount of 2 ml solution of the extract and 5 ml of dilute hydrochloric acid (1%) were
’
taken in a test tube. Then 1 ml of Mayer s reagent was added. A white or creamy white color
precipitate indicates the presence of alkaloids.

’
3.4.9.2 Dragendroff s test

An amount of 2 ml solution of the extract and 5 ml of dilute hydrochloric acid (1%) were
’
taken in a test tube. Then 1 ml of Dragendroff s reagent was added. Orange brown precipitate
indicates as the presence of alkaloids.

3.4.9.3 Wagner Test

An amount of 2 ml of solution the extract was added into 0.2 ml of dilute hydrochloric acid.
Then 1 ml of Wagner’s reagent added into previous extract solution. Brown or reddish-brown
precipitate indicates the presence of alkaloid.

3.4.9.4 Hager Test

An amount of 2 ml of solution the extract was added into 0.2 ml of dilute hydrochloric acid.
Then 1 ml of Hager’s reagent added into it. Yellow color precipitate indicates the presence
of alkaloids.

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 Chapter 3: Phytochemical Tests

3.4.10 Test for Glycosides

Test for Glycosides was carried out as described by [3]
A small amount of an alcoholic extract was taken in 1 ml of water. A few drops of aqueous
NaOH were added. A yellow color indicates the presence of glycosides.

3.4.11 Tests for terpenoids

Test for terpenoids was carried out as described by [5]

3.4.11.1 Salkowski test

To 0.5 g of the extract, 2 mL of chloroform was added; Conc. H 2SO4 (3 mL) was carefully
added to form a layer. A reddish-brown coloration at the interface indicates the presence of
terpenoids.

3.4.12 Tests for proteins-xanthoprotein

Test for proteins-xanthoprotein was carried out as described by [5]

To 1 mL of extract, few drops of nitric acid were added by the sides of the test tube and
observed for formation of yellow color. This indicates the presence of xanthoprotein.

3.4.13 Tests for acidic compounds

Test for proteins-xanthoprotein was carried out as described by [6]

To the alcoholic extract, sodium bicarbonate solution was added and observed for the
production of effervescences. Production of effervescences indicates the presence of acidic
compound

3.5 Observations
The crude extract of C. arborea was subjected for chemical group tests and investigations of
different group tests compared with standards are given in table 3.1

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 Chapter 3: Phytochemical Tests

Table 3.1: Different chemical group tests for standard and C. arborea extract

Test For Reagent Observation Inference

Standard Extract
Fehling’s (Dextrose) Brick red Presence of
Brick red
Solution precipitate reducing sugar
Reducing precipitate
sugar
Benedict’s (Dextrose) Brick red Presence of
Brick red
reagent precipitate reducing sugar
precipitate

Combined Fehling’s (Dextrose) Brick red Presence of

Brick red combined
reducing sugar Solution precipitate
precipitate reducing sugar

Ferric chloride (Rose petal) Greenish black

Greenish black Presence of
solution precipitate
precipitate tannins
Tannins
Potassium (Rose petal) Yellow Presence of
dichromate Yellow precipitate precipitate tannins
(Quercetin) Appearance and
Concentrated Appearance and Presence of
disappearance of
H2SO4 disappearance of flavonoids
yellow color
Flavonoids yellow color

(Quercetin) Appearance and

Dilute HCl Appearance and Presence of
disappearance of
Dilute NaOH disappearance of flavonoids
yellow color
yellow color

Saponin Distilled water (Detergent) Layer of foam Presence of

Layer of foam saponin

Gums Molish’s (Liquid glue) Red-violet ring Presence of

reagent Red-violet ring gums

Steroids Sulphuric acid (Norgestrel) Red solution Presence of

Red solution steroids

Page | 24
 Chapter 3: Phytochemical Tests

’ (Nicotine) No white Absence

Mayer s test
. white precipitate precipitate of alkaloids
(Nicotine) No reddish-brown Absence
Wagner Reddish-brown
precipitate of alkaloids
Alkaloids precipitate

Dragendroff s
’ (Nicotine) No Orange brown Absence
Orange brown
test precipitate precipitate of alkaloids

Hagger (Nicotine) No yellow Absence

Yellow precipitate precipitate of alkaloids

Glycoside NaOH (Aloe vera) Yellow solution Presence of

Yellow solution glycoside

proteins- Nitric acid (Egg albumin) No yellow Absence of

xanthoprotein Yellow solution solution xanthoprotein

(Lemon) A reddish-brown Presence of

Terpenoids H2SO4 A reddish-brown
coloration at the
coloration at the terpenoids
interface
interface
Acidic Sodium Presence of
compounds bicarbonate (Salicylic acid) Effervescence acidic
solution Effervescence compounds

Tests for Molisch’s (Liquid glue) Bluish violet ring Presence of

Carbohydrates reagent Bluish violet ring Carbohydrates

(Gallic acid) Dark green or Presence of

Ferric chloride Dark green or phenolic
bluish black
Tests for test bluish black
precipitate compounds
Phenolic precipitate
Compounds Lead acetate (Gallic acid) Presence of
White precipitate phenolic
test White precipitate
compounds

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 Chapter 3: Phytochemical Tests

3.6 Result of chemical group tests

The experimental findings from the study C. arborea extract showed the presence or absence
of the following organic compounds as listed in table 3.2:

Table 3.2: Result of chemical group tests

Constituents Results for

C. arborea
Reducing sugar +
Combined reducing sugar +

Phenolic Compounds +

Tannins +

Flavonoids +

Saponin +
Gums +

Steroids +

Alkaloids _

Glycoside +

Xanthoproteins _
Terpenoids +
Acidic compounds +
Carbohydrates +
+ = Presence - =Absence

3.7 Conclusion

The phytochemical tests carried out on C. arborea extract revealed the presence of several
important constituents which might be responsible for its traditional medicinal uses.

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 Chapter 3: Phytochemical Tests

3.8 References
1. Harborne, J., Methods of extraction and isolation. Phytochemical methods, 1998. 3: p.
60-66.
2. Trease, G., W. Evans, and A.J.T.o.P.t.e.L.B.T. Pharmacognosy, physician’s guide to
herbal medicine. Textbook of Pharmacognosy. 13th ed. London: Bailiere Tindal,
1989: p. 176-180.
3. Ayoola, G., et al., Phytochemical screening and antioxidant activities of some
selected medicinal plants used for malaria therapy in Southwestern Nigeria. Tropical
Journal of Pharmaceutical Research, 2008. 7(3): p. 1019-1024.
4. Gowri, S.S. and K. Vasantha, Phytochemical screening and antibacterial activity of
Syzygium cumini (L.)(Myrtaceae) leaves extracts. Int J Pharm Tech Res, 2010. 2(2):
p. 1569-1573.
5. Sofowara, A., Medicinal plants and traditional plants in Africa. Chichester and New
York, 1982.
6. Amer, M.E., et al., Alkaloids and flavone acyl glycosides from Acanthus arboreus.
Journal of the Brazilian Chemical Society, 2004. 15(2): p. 262-266.

Page | 27
 CHAPTER FOUR
TEST FOR
ANTIOXIDANT ACTIVITY
 Chapter 4: Test for Antioxidant Activity

CHAPTER FOUR

Test for Antioxidant Activity

4.1 Introduction
Antioxidant is a molecule which neutralizes harmful compounds called free radicals that
damage living cells, spoil food, and degrade materials such as rubber, gasoline, and
lubricating oils. Antioxidants can take the form of enzymes in the body, vitamin supplements,
or industrial additives. They are routinely added to metals, oils, foodstuffs, and other
materials to prevent free radical damage. Free radicals are metastable chemical species
which, after being generated in vivo as byproducts of various biochemical reactions, tend to
rob electrons from the molecules in the immediate surroundings in order to replace their own
losses. These radicals may be regarded as molecular sharks, which if not scavenged
effectively on time, are capable of damaging crucial bio-molecules including those present in
cell membranes, mitochondria, DNA etc. and thus predisposing various pathophysiological
states. [1]

There is currently much interest in phytochemicals as bioactive components of food. The

roles of fruit, vegetables and red wine in disease prevention have been attributed, in part, to
the antioxidant properties of their constituent polyphenols (vitamins E and C, and the
carotenoids). Recent studies have shown that many dietary polyphenolic constituents derived
from plants are more effective antioxidants in vitro than vitamins E or C, and thus might
contribute significantly to the protective effects in vivo. [2] Phenolic compounds contribute to
the overall antioxidant activities of plants mainly due to their redox properties. Generally, the
mechanisms of phenolic compounds for antioxidant activity are neutralizing lipid free
radicals and preventing decomposition of hydroperoxides into free radicals.[3]

Phytochemical screening of C. arborea reveals the presence of polyphenolic compounds like

flavonoid, tannin etc. So, it may have some antioxidant properties based on the presence of
the different secondary metabolites. The aim of our study is to reveal the antioxidant
properties both qualitatively and quantitatively.

Page | 29
 Chapter 4: Test for Antioxidant Activity

4.2 Test for Qualitative Antioxidant Activity

Thin Layer Chromatography (TLC) is the medium in which qualitative antioxidant activity is
tested. It’s a chromatographic technique that is useful for separating organic compounds.
Because of the simplicity and rapidity of TLC, it is often used to monitor the progress of
organic reactions and to check the purity of products. TLC is a method of analysis in which
the stationary phase (a finely divided solid) is spread as a thin layer on a rigid supporting
plate and a liquid (mobile phase) is allowed to migrate across the surface of the plate. In this
process, the separation takes place on a planar surface and the mobile phase flow across the
plate by capillary action.[4]
The main purpose of this technique is to detect polar, non-polar and medium polar groups
present in the plant extract under UV light at short (254 nm) and long (360 nm) wavelength
and also to detect the presence of antioxidant through DPPH spraying. The presence of
H2SO4 reacting compounds was also detected.

4.2.1 Principle
Optimum diluted stock solutions are spotted on pre-coated silica gel TLC plates and the
plates are developed in solvent systems of different polarities (polar, medium polar and non-
polar) to resolve polar and non-polar components of the extract. The plates are dried at room
temperature and observed under UV light at short (254 nm) and long (360 nm) wavelength.
[4] The main purpose of this technique is to detect polar, non-polar and medium polar groups
present in the plant extract. After that, they are sprayed with 0.02% 2, 2-diphenyl-1-picryl
hydrazyl (DPPH) in ethanol. DPPH forms deep pink color when it is dissolved in ethanol.
When it is sprayed on the chromatogram of the extract, it forms pale yellow or yellow color
which indicates the presence of antioxidants. Bleaching of DPPH by the resolved bands is
observed for 10 minutes and the color changes (yellow on purple background) are noted. [4]

4.2.2 Apparatus
➢ TLC plates containing fluorescent material
➢ TLC tank
➢ Fine capillary tube
➢ Spirit lamp
➢ UV detector with short (254 nm) and long (360 nm) wavelength

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 Chapter 4: Test for Antioxidant Activity

➢ Spray gun
➢ Filter paper
➢ Forceps
4.2.3 Reagents
➢ DPPH (0.02 % w/v) solution in ethanol
➢ n-hexane
➢ Ethyl acetate
➢ Distilled water
➢ Methanol
➢ Chloroform
➢ Ascorbic acid

4.2.4 Solvent system

Solvent system used for TLC is given in table 4.1

Table 4.1: Solvent system used for TLC

Groups Solvent system Ratio
Non-polar n-hexane: Ethyl acetate 3:1
Medium polar CHCl3: CH3OH 5:1
Polar CHCl3: CH3OH: H2O 40:10:1

4.2.5 Method
The method was applied as described by [4]

4.2.5.1 TLC plate preparation

Commercially available TLC plate is cut into appropriate size of 10 cm long and 7 cm wide.

4.2.5.2 Sample application

➢ A fine capillary tube was used as spotter for sample application in the TLC plates.

➢ A very little amount of plant extract was taken in a small vial and diluted suitably
with ethanol.
➢ The sample was spotted in uniform size (about 0.3 cm) on TLC plates.
➢ The sample was applied several times in each spot to get better chromatogram.

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 Chapter 4: Test for Antioxidant Activity

➢ Each spot was dried before applying another volume of solution to the same spot.

➢ A little amount of ascorbic acid (as standard) was taken in a small vial and diluted
suitably with ethanol and was applied by spotter at the same way.

➢ Only sample was spotted on three TLC plates (one set) for the detection of UV
positive components and on another three plate’s (another set) ascorbic acid was also
spotted as standard for the detection of antioxidant positive component.

4.2.5.3 Development of chromatogram

➢ The chromatogram was developed by ascending technique
➢ Three solvent systems in the ratio mentioned before were kept in three jars.
➢ Plates were placed in each jar in such a manner that the sample spots were just above
the solvent surface.

➢ Filter papers were kept into each jar by wetting with respective solvent systems to
keep those jars saturated and the jars were closed tightly.
➢ The plates were then removed from the jars and dried with a current of air suitably.
4.2.5.4 UV positive components detection
After drying the plates, one set of plates were observed visually under UV light at longer
wavelength (360nm) and shorter wavelength (254nm) and various regions were marked by
pencil.

4.2.5.5 Antioxidant positive components detection

After developing the chromatogram, the other set of TLC plates were sprayed with 0.02%
DPPH solution of ethanol by a spray gun and observed. H 2SO4 solution was also sprayed on
another set of TLC plate for observation of antioxidant positivity.

4.2.6 Observation

The plates were viewed under UV detector both in short (254 nm) and long (360 nm)
wavelength. When the plates were viewed under UV detector a lot of colored and fluorescent
positive components were found in shorter and longer wavelengths respectively. After
applying DPPH on the TLC plate, yellow color on purple background was observed and
discoloration of developed site was noticed after the application of H2SO4 solution.

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 Chapter 4: Test for Antioxidant Activity

At shorter wave length:

Observation of TLC plate at shorter wave length(254nm) is given in figure 4.1

Ascorbic Extract Ascorbic Extract Ascorbic Extract

acid acid acid

[Non-polar] [Medium polar] [Polar]

Figure 4.1: Observation of TLC plate under UV (shorter wave length)

At longer wave length:

Observation of TLC plate at longer wave length(360nm) is given in figure 4.2
Ascorbic Extract Ascorbic
Ascorbic Extract Extract
acid acid acid

Non-Polar Medium Polar Polar

Figure 4.2: Observation of TLC plate under UV (longer wave length)

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 Chapter 4: Test for Antioxidant Activity

After DPPH application: Observation of TLC plate after DPPH application is given in
figure 4.3

Ascorbic Extract
Ascorbic Extract Ascorbic Extract
acid acid acid

Non-Polar Medium Polar Polar

Figure 4.3: Observation of TLC plate after spraying 0.02% DPPH

After H2SO4 application: Observation of TLC plate after H2SO4 application is given in
figure 4.4

Ascorbic Extract Ascorbic Extract Ascorbic Extract

acid acid acid

Non-Polar Medium Polar Polar

Figure 4.4: Observation of TLC plate after applying H2SO4

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 Chapter 4: Test for Antioxidant Activity

4.2.7 Result

In the TLC-based qualitative antioxidant assay using DPPH, C. arborea extract showed the
free radical scavenging properties indicated by the presence of yellow spot on a purple
background of the TLC plate. When the plates were viewed under UV detector a lot of
colored and fluorescent positive components were found in shorter and longer wavelengths
respectively. Discoloration of developed site was noticed after the application of H 2SO4
solution.

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 Chapter 4: Test for Antioxidant Activity

4.3 Test for Quantitative Antioxidant Activity

The presence of antioxidant leaded to the qualitative test for antioxidant activity. Various
antioxidant activity methods have been used to monitor and compare the antioxidant activity.
These analytical methods measure the radical-scavenging activity of antioxidants against free
radicals like the 2,2-diphenyl-1-picryl hydrazyl (DPPH) radical, the superoxide anion radical
(O2), the hydroxyl radical (OH¯), or the peroxyl radical. A rapid, simple and inexpensive
method to measure antioxidant capacity of plant extract involves the use of the free radical, 2,
2-diphenyl-1-picryl hydrazyl (DPPH). DPPH is widely used to test the ability of compounds
to act as free radical scavengers or hydrogen donors, and to evaluate antioxidant activity. It
has also been used to quantify antioxidants in complex biological systems. Antioxidant
activity has been expressed in various ways including the percentage of the reagent used, the
oxidation inhibition rate and so on. Here, five tests were done for the quantitative assays of
antioxidants of C. arborea extract. They are-
1. DPPH Scavenging Assay
2. Determination of Total Phenolic Content
3. Determination of Total Flavonoid Content
4. Determination of Total Tannin Content
5. Determination of Total Antioxidant Capacity

4.3.1 DPPH Scavenging Assay

4.3.1.1 Principle

The antioxidant potential of the ethanol extract was determined on the basis of their
scavenging activity of the stable 2, 2-diphenyl-1-picryl hydrazyl (DPPH) free radical. DPPH
is a stable free radical containing an odd electron in its structure and usually utilized for
detection of the radical scavenging activity in chemical analysis. 2 mL of each aliquot of the
different concentrations (2 – 512 μg/mL) of the extract was added to 6 mL of 0.004% w/v
methanolic solution of DPPH. Absorbance at 517 nm was determined after 30 min, and SC 50
(Scavenging conc. 50%) was determined. SC50 value denotes the concentration of sample
required to scavenge 50% of the DPPH free radicals. [5] [6]
The formula used for percent (%) scavenged ratio is-
Percent (%) scavenged = [(Blank absorbance - Sample absorbance) / Blank absorbance] X
100

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 Chapter 4: Test for Antioxidant Activity

4.3.1.2 Apparatus
➢ Test tubes
➢ Pipette

➢ Electronic balance

➢ Sonicator

➢ Volumetric flask
➢ Vortex mixer

➢ UV spectrophotometer
4.3.1.3 Reagents
➢ Methanol
➢ 0.004% DPPH in ethanol

➢ Ascorbic acid (Standard)

4.3.1.4 Method

i. At first, 21 test tubes were taken. They were washed properly and rinsed with ethanol.

ii. Among 21 test tubes, 9 were marked for the extract solution of different concentration
(2, 4, 8, 16, 32, 64, 128, 256 and 512 µg/mL) and 9 test tubes were taken to make
aliquots of 9 concentration (2, 4, 8, 16, 32, 64, 128, 256 and 512 µg/mL) for ascorbic
acid as standard and one for blank solution preparation.

iii. Plant extract and ascorbic acid were weighed and dissolved in methanol for the
preparation of stock solution.

iv. From the stock solution, various concentrations were prepared by dilution method for
the plant extract and standard.

v. DPPH was weighed and dissolved in methanol to make 0.004% (w/v) solution.

vi. 2 mL of each concentration for plant extract and standard was taken in a test tube and 6
mL of DPPH solution was added to it.

vii. Then test tubes were kept in dark place for 30 minutes at room temperature.

viii. DPPH solution was also added to the blank test tube at the same time where only
ethanol was taken as blank.

ix. After 30 minutes, absorbance of each test tube was measured by UV spectrophotometer
at 517 nm.

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 Chapter 4: Test for Antioxidant Activity

4.3.1.5 Data Presentation

Data obtained from DPPH Scavenging Assay of Ascorbic Acid (Standard) and Extract is
presented in table 4.2 & 4.3 respectively

Table 4.2 DPPH Scavenging Assay of Ascorbic Acid (Standard)

Concentration Log Absorbance Absorbance Average Percent SC50

(µg/mL) Conc. 1 2 Absorbance (%) of (µg/mL)
Scavenging

Blank - 0.889 0.880 0.8845 -

2 0.30 0.718 0.713 0.7155 19.11

4 0.60 0.648 0.645 0.6465 26.91
8 0.90 0.560 0.561 0.5605 36.63 14.26
16 1.20 0.403 0.404 0.4035 54.38
32 1.51 0.291 0.289 0.2900 67.21
64 1.81 0.175 0.179 0.1770 79.99

128 2.11 0.111 0.117 0.1140 87.11

256 2.41 0.058 0.057 0.0575 93.50
512 2.71 0.037 0.039 0.0380 95.70

y = 34.968x + 9.6365
% Scavenged Vs log concentration
110
100
90
80
% Scavenged

70
60
50
40
30
20
10
0
0 0.5 1 1.5 2 2.5 3
log concentration

Figure 4.5: Percent (%) scavenged of DPPH vs. log concentration graph for standard
(ascorbic acid)

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 Chapter 4: Test for Antioxidant Activity

Table 4.3: DPPH Scavenging Assay of C. arborea Extract

Concentration Log Absorbance Absorbance Average Percent SC50

(µg/mL) Conc. 1 2 Absorbanc (%) of (µg/mL)
e Scavengin
g
Blank - 0.889 0.880 0.8845 -

2 0.30 0.767 0.767 0.7670 13.28

4 0.60 0.735 0.733 0.7340 17.02

8 0.90 0.684 0.684 0.6840 22.67 34.04

16 1.20 0.613 0.611 0.6120 30.81

32 1.51 0.592 0.592 0.5920 33.07

64 1.81 0.477 0.477 0.4770 46.07

128 2.11 0.309 0.310 0.3095 65.00

256 2.41 0.259 0.259 0.2590 70.72

512 2.71 0.122 0.121 0.1215 86.26

% Scavenged Vs log concentration

100
80 y = 30.54x - 3.2129
% Scavenged

60

40

20

0 0.5 1 1.5 2 2.5 3

log concentration

Fig. 4.6: Percent (%) scavenged of DPPH vs. log concentration graph for C. arborea
extract

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 Chapter 4: Test for Antioxidant Activity

% Scavenged Vs log concentration

Standard C. arborea Extract
120

100 SC50 = 14(μg/ml)

% Scavenged

80
60
40 SC50 = 34(μg/ml)

20
0
0 0.5 1 1.5 2 2.5 3
log concentration

Fig. 4.7: Comparison of Percent (%) scavenged of DPPH by ascorbic acid (standard)
and sample.

4.3.1.6 Result
In the quantitative antioxidant assay, SC 50 value of C. arborea extract revealed free radical
scavenging activity in DPPH assay with approximate SC50 ~34 µg/ml while the SC50 of the
standard ascorbic acid was ~14 µg /ml.

4.3.2 Determination of Total Phenolic Content (TPC)

4.3.2.1 Principle
In the present study the total phenolic content (TPC) of C. arborea extract as determined by
using Folin-Ciocalteu (FC) reagent with analytical grade gallic acid as the standard. Standard
solution was prepared in different concentrations (0.02-0.15 mg/L) by using methanol as
diluting media and was put in separate test tubes. Plant sample was prepared as 1 mg/ml.
Then 5 ml FC reagent (10 times diluted with distilled water) was added in each 0.5 ml stock
solution. After 5 minutes; 10 ml 7% Na2CO3was added to the mixture and kept for 30
minutes at room temperature. Then absorbance was measured against blank at 765nm using
UV spectrophotometer. Total phenolic content of the extract was determined from the
standard curve and expressed as mg gallic acid equivalent (GAE)/g dried plant extract as
described by [7]

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 Chapter 4: Test for Antioxidant Activity

4.3.2.2 Apparatus
➢ Test tubes
➢ Beakers
➢ Pipette
➢ UV spectrophotometer
➢ Centrifuge apparatus
➢ Electronic balance
➢ Volumetric flask
4.3.2.3 Reagents
➢ Methanol
➢ Distilled water
➢ Folin-Ciocalteu (FC) reagent
➢ Gallic acid
➢ 7% Na2CO3

4.3.2.4 Method
The study was conducted using Folin-Ciocalteu (FC) reagent with analytical grade gallic acid
as the standard as described by [7]

4.3.2.4.1 Preparation of sample

0.005 g of plant extract was mixed with 5 ml of methanol.

4.3.2.4.2 Preparation of standard

0.01 gm gallic acid was dissolved in 10ml methanol. A standard solution 8 concentration of
the gallic acid was prepared by serial dilution method. These concentrations were 0.15, 0.1,
0.08, 0.06, 0.04, 0.02 mg/ml.
For 0.15 mg/ml: 0.75 ml from stock solution + 4.25 ml methanol
For 0.1 mg/ml: 0.5 ml from stock solution + 4.5 ml methanol
For 0.08 mg/ml: 0.4 ml from stock solution + 4.6 ml methanol
For 0.06 mg/ml: 0.3 ml from stock solution + 4.7 ml methanol
For 0.04 mg/ml: 0.2 ml from stock solution + 4.8 ml methanol
For 0.02 mg/ml: 0.1 ml from stock solution + 4.9 ml methanol

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 Chapter 4: Test for Antioxidant Activity

4.3.2.4.3 Dilution of Folin-Ciocalteu (FC) reagent

1 ml FC reagent was mixed with 9 ml distilled water to make 10 times dilution.

4.3.2.4.4 Working procedure

i. At first, 0.5 ml standard solution of each concentration (0.15, 0.1, 0.08, 0.06, 0.04,
0.02 mg/ml.) and 0.5 ml sample extract was taken separately into different test
tubes.
ii. Then 5 ml of dilute FC reagent (1/10) was added to every test tube.
iii. Then 4 ml solution of 7% Na2CO3 was added to it and vortex for 15 seconds.
iv. Then they were kept for 30 minutes at 40 temperatures.
v. After 30 minutes the UV absorbance was measured at 765 nm against blank for
each concentration, the absorbance was taken for two times and mean was used
for accuracy.
vi. Blank was prepared by following all the above steps except the addition of gallic
acid and sample.

4.3.2.5 Data presentation

Obtained data of UV Absorbance of gallic acid (standard) & sample at 765 nm are given in
table 4.4 and 4.5 respectively. Gallic acid standard calibration curve is given in figure 4.8

Table 4.4: UV Absorbance of gallic acid (standard) at 765 nm

Concentration Absorbance 1 Absorbance 2 Average
(mg/ml)

0.15 0.894 0.863 0.897

0.1 0.696 0.70 0.698

0.08 0.508 0.510 0.509

0.06 0.420 0.422 0.421

0.04 0.321 0.324 0.323

0.02 0.206 0.208 0.207

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 Chapter 4: Test for Antioxidant Activity

Gallic acid standard calibration curve

1 0.897

0.9
0.8 0.698
Absorbance at 765 nm

0.7 y = 5.42x + 0.1027

0.6 0.509 R² = 0.9875

0.5 0.421
0.4 0.323

0.3 0.207

0.2
0.1

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16

Concentration (mg/ml)

Figure 4.8: Total phenolic content determination of C. arborea extract with the help of
gallic acid standard calibration curve.

Table 4.5: UV absorbance of sample at 765 nm

Sample st nd Average
1 reading 2 reading
C. arborea 0.956 0.958 0.957
extract

4.3.2.6 Determination of phenolic content of in C. arborea extract

The following equation was obtained from a standard Gallic acid calibration
curve y = 5.42x – 0.1027;
Where y is the absorbance and x is the concentration of Gallic acid (mg/ml).
Based on the measured absorbance, Gallic Acid Equivalent (GAE) was read (mg/ml) from
the calibration line by using the following equation
Absorbance of sample − 0.1027
GAE =

5.42

Then total phenolic content (TPC) in plant ethanol extract in Gallic acid equivalents (GAE)
was calculated by using the following equation
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 Chapter 4: Test for Antioxidant Activity
TPC(mg GAE⁄g) = Sample concentration(g⁄ml)
GAE

Total phenolic content value is expressed in terms of mg of Gallic acid equivalent (GAE) per
gram of dry extract, which is a common reference compound. The determined phenolic
content of C. arborea extract is given in table 4.6

Table 4.6: Determination of phenolic content of C. arborea extract

Average absorbance GAE for dried Total phenolic content for dried
Sample at 765nm extract Extract (mg GAE/g)

C. arborea extract 0.957 0.1576 157.6

4.3.2.7 Result
Total phenolic content was ~158 mg GAE/g in C. arborea extract.

4.3.3 Determination of Total Flavonoid Content (TFC)

4.3.3.1 Principle
In the present study the total flavonoid content (TFC) of C. arborea extract as determined by
using Sodium nitrite, aluminum chloride, sodium hydroxide reagent with analytical grade
quercetin as the standard. Standard solution was prepared in different concentrations (0.25-
0.1 mg/L) by using methanol as diluting media and was put in separate test tubes. Plant
sample was prepared as 1 mg/ml. Then 4 ml of distilled water was added and 0.3 ml of 5%
w/v NaNO2 was added to every test tube and kept for 5 minutes. After that, aluminum
chloride, sodium hydroxide was added to every test tube and kept for 15 minutes. The
absorbance was measured against blank at 510nm using UV spectrophotometer. Total
flavonoid content of the extract was determined from the standard curve and expressed as mg
quercetin equivalent (QE)/g dried plant extract as described by [8]

4.3.3.2 Apparatus
➢ Test tubes
➢ Beakers
➢ Pipette

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 Chapter 4: Test for Antioxidant Activity

➢ UV spectrophotometer
➢ Electronic balance
➢ Volumetric flask
4.3.3.3 Reagents
➢ Methanol
➢ Distilled water
➢ 5% w/v Sodium nitrite (NaNO2)
➢ 10% w/v aluminum chloride (AlCl3)
➢ 1 M sodium hydroxide (NaOH)
➢ Quercetin

4.3.3.4 Method
The study was conducted using Sodium nitrite, aluminum chloride, sodium hydroxide reagent
with analytical grade quercetin as the standard as described by [8]

4.3.3.4.1 Preparation of sample

0.005 gm C. arborea extract was weighed and mixed with 5 ml methanol. It was sonicated
for 20 min.

4.3.3.4.2 Preparation of standard

0.01 gm Quercetin in methanol q.s. to 10 ml. Standard solution 5 concentrations of the

Quercetin was prepared by serial dilution method. These concentrations were 1, 0.75, 0.5,
0.25,0 mg/ml.

4.3.3.4.3 Preparation of 5% w/v NaNO2, 10% w/v AlCl3 &1 M NaOH

1.25 gm of NaNO2 was mixed with 25 ml of distilled water; 2.5 gm of AlCl 3 was mixed with
25 ml of distilled water and 1 gm NaOH mixed with 25 ml of distilled water to make the
following reagents at specified strength.

4.3.3.4.4 Working procedure

(i) At first, 1 ml standard solution of each concentration (1, 0.75, 0.5, 0.25,0 mg/ml.) and 1 ml
sample extract was taken separately into different test tubes.
(ii) Then 4 ml of distilled water was added and 0.3 ml of 5% w/v NaNO2 was added to every
test tube and kept for 5 minutes.

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 Chapter 4: Test for Antioxidant Activity

(iii) Then 0.3 ml of 10% w/v AlCl3 solution and 2 ml of 1 M NaOH was also added to every
test tube and added distilled water q.s. to10 ml of the solution.
(iv) Then they were kept for 15 minutes at room temperature.
(v) After 15 minutes the UV absorbance was measured at 510 nm against blank for each
concentration, the absorbance was taken for two times and mean was used for accuracy.
(vi) Blank was prepared by following all the above steps except the addition of Quercetin and
sample

4.3.3.5 Data presentation

Obtained data of UV Absorbance of quercetin (standard) & sample at 510 nm are given in
table 4.7 and 4.8 respectively. Quercetin standard calibration curve is given in figure 4.9

Table 4.7: UV Absorbance of Quercetin (standard) at 510 nm

Concentration Absorbance 1 Absorbance 2 Average
(mg/ml)

1.0 0.453 0.455 0.454

0.75 0.322 0.323 0.323

0.50 0.268 0.271 0.270

0.25 0.130 0.126 0.128

0 0.018 0.019 0.019

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 Chapter 4: Test for Antioxidant Activity

Quercetin standard calibration curve

0.5 0.454
0.45
0.4
0.35 0.323
Absorbance at 510 nm

0.3 0.27 y = 0.426x + 0.0258

0.25 R² = 0.9866

0.2
0.15 0.128

0.1

0.050.019
0
0 0.2 0.4 0.6 0.8 1 1.2
Concentration

Figure 4.9: Total flavonoid content determination of C. arborea extract with the help
of Quercetin standard calibration curve.

Table 4.8: UV absorbance of sample at 510 nm

Sample st nd Average
1 reading 2 reading
C. arborea 0.420 0.433 0.4265
extract

4.3.3.6 Determination of Total Flavonoid Content:

The following equation was obtained from a standard quercetin calibration curve-
2
y = 0.426x + 0.0258; R = 0.9866
Where, y is the absorbance of the sample at 510 nm, x is the concentration of Quercetin
2
(mg/ml) and R is the coefficient of determination.
Based on the measured absorbance, Quercetin Equivalent (QE) could be measured from the
calibration line by using the following equation-
Absorbance of sample − 0.0258
QE =

0.426

Then, total flavonoid content (TFC) in ethanolic plant extract in Quercetin Equivalent (QE)
was calculated by using the following equation-

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 Chapter 4: Test for Antioxidant Activity
TFC(mg QE⁄g) = Sample concentration(g⁄ml)
QE

Total flavonoid content value is expressed in terms of mg of Quercetin Equivalent (QE) per
gram of dry extract which is a common reference compound. The determined flavonoid
content of C. arborea extract is given in table 4.9

Table 4.9: Determination of flavonoid content of C. arborea extract

Average Quercetin equivalence Total flavonoid content

Sample absorbance at mg/ml for extract for extract (mg QE/g)
510nm

C. arborea extract 0.5265 0.9406 940.6

4.3.3.7 Result
Total flavonoid content was ~941 mg QE/g for the C. arborea extract.

4.3.4 Determination of Total Tannin Content (TTC)

4.3.4.1 Principle

In the present study the total flavonoid content (TFC) of C. arborea leaves extract is
determined by using Folin-Ciocalteu (FC) reagent with analytical grade gallic acid as the
standard. Standard solution was prepared in different concentrations by using methanol as
diluting media and was put in separate test tubes. Plant sample was prepared as 1mg/ml. Then
FC reagent (10 times diluted with distilled water) was added in each stock solution. After 5
minutes; Na2CO3 was added to the mixture and kept for 30 minutes at room temperature.
Then absorbance was measured against blank at 725nm using UV spectrophotometer. Total
tannin content of the extract was determined from the standard curve and expressed as mg
gallic acid equivalent (GAE)/ g dried plant extract as described by [9] [10]

4.3.4.2 Apparatus
➢ Test tube
➢ Beaker
➢ Pipette
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 Chapter 4: Test for Antioxidant Activity

➢ UV spectrophotometer
➢ Vortex machine
➢ Sonicator
4.3.4.3 Reagents
➢ Methanol
➢ Folin-Ciocalteu reagents
➢ Distilled water
➢ 35% Na2CO3
➢ Gallic acid (as positive control)

4.3.4.4 Method
The study was conducted using Folin-Ciocalteu (FC) reagent with analytical grade gallic
acid as the standard as described by [9] [10]

4.3.4.4.1 Preparation of sample

At first, 0.005 gm C. arborea extract was measured by electronic balance and mixed with 5 ml
of methanol.

4.3.4.4.2 Preparation of standard

0.01 gm gallic acid was dissolved in methanol to make final volume 10 ml, this is also called
stock solution. Standard solution of 5 concentrations of the gallic acid was prepared by serial
dilution method. These concentrations were 0.5, 0.4, 0.3, 0.2, 0.1 mg/ml. For 0.5 mg/ml: 2.5
ml from stock solution + 2.5 ml methanol
For 0.4 mg/ml: 2 ml from stock solution + 3 ml methanol
For 0.3 mg/ml: 1.5 ml from stock solution + 3.5 ml methanol
For 0.2 mg/ml: 1 ml from stock solution + 4 ml methanol
For 0.1 mg/ml: 0.5 ml from stock solution + 4.5 ml methanol

4.3.4.4.3 Dilution of Folin-Ciocalteu (FC) reagent

1 ml FC reagent was mixed with 9 ml distilled water to make 10 times dilute.

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 Chapter 4: Test for Antioxidant Activity

4.3.4.4.4 Working procedure

(i) At first, 0.1 ml standard solution of each concentration (0.5, 0.4, 0.3, 0.2, 0.1
mg/ml) and 0.1 ml extract sample was taken separately in different test tubes.
(ii) 7.5 ml of distilled water was added to each test tube.
(iii) Then 0.5 ml of FC reagent was added to the test tube.
(iv) 1ml of 35% Na2CO3 was added to the test tube and the solution was diluted q.s. to
10 ml with distilled water.
(v) Then all test tubes vortex for 15 second and kept at room temperature for 30
minutes.
(vi) Then absorbance of the solution was measured at 725 nm.
(vii) Blank was prepared by following all the above steps except the addition of gallic
acid and sample

4.3.4.5 Data presentation

Obtained data of UV Absorbance of gallic acid (standard) & sample at 725 nm are given in
table 4.10 and 4.11 respectively. Gallic acid standard calibration curve is given in figure 4.10

Table 4.10: UV Absorbance of gallic acid (standard) at 725 nm

Concentration (mg/ml) Absorbance 1 Absorbance 2 Average

0.5 0.382 0.383 0.381

0.4 0.317 0.318 0.318

0.3 0.207 0.207 0.207

0.2 0.131 0.132 0.132

0.1 0.059 0.058 0.059

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 Chapter 4: Test for Antioxidant Activity

Gallic acid standard calibration curve

0.45
0.4 0.381
0.35 0.318
0.3 y = 0.83x - 0.0296
Absorbance

0.25 0.207 R² = 0.9933

0.2
0.15 0.132
0.1 0.059
0.05
0
0 0.1 0.2 0.3 0.4 0.5 0.6
Concentration

Figure 4.10: Total tannin content determination of C. arborea extract with the help of
gallic acid standard calibration curve.

Table 4.11: UV absorbance of sample at 725 nm

Sample st nd Average
1 reading 2 reading

C. arborea extract 0.187 0.192 0.1895

4.3.4.6 Determination of tannin content of C. arborea extract

The following equation was obtained from a standard Gallic acid calibration curve
2
y = 0.83x -0.0296; R = 0.9933
Where, y is the absorbance and x is the concentration of gallic acid (mg/ml).

Based on the measured absorbance gallic acid Equivalent (GAE) was read (mg/ml) from the
calibration line by using the following equation-
GAE (mg/mL) =
Absorbance of sample − 0.0296
0.83

Then total tannin content (TTC) in methanolic plant extract in gallic acid equivalent (GAE)
was calculated by using the following equation-

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 Chapter 4: Test for Antioxidant Activity
TPC(mg GAE⁄g) = Sample concentration(g⁄ml)
GAE

Total tannin content value is expressed in terms of mg of gallic acid equivalent (GAE) per
gram of dry extract which is a common reference compound. The determined tannin content
of C. arborea extract is given in table 4.12

Table 4.12: Determination of tannin content of C. arborea extract

Average Total tannin content of

Sample Name absorbance at GAE of dried extract dried extract (mg
725nm GAE/g)

C. arborea
extract 0.1895 0.1927 192.7

4.3.4.7 Result
Total tannin content was ~193 mg GAE/g for the C. arborea extract.

4.3.5 Total Antioxidant Capacity (TAC)

4.3.5.1 Principle
In the present study the total antioxidant capacity (TAC) of C. arborea leaves extract is
determined by phosphomolybdate method [11] [12] using ascorbic acid as a standard.
Standard solution was prepared in different concentrations by using ethanol as diluting media
and was put in separate test tubes. Plant sample was prepared as 1mg/ml. A standard solution
of 5 concentrations of the gallic acid was prepared by serial dilution method. The fresh
working solution was prepared by mixing H 2SO4, Na3PO4 and Ammonium Molybdate in a
proportion of 4:2:4. The absorbance was measured against blank at 695nm using UV
spectrophotometer. Total antioxidant capacity of the extract was determined from the
standard curve. The phosphomolybdate method is quantitative, since the total antioxidant
capacity (TAC) is expressed as ascorbic acid equivalents.

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 Chapter 4: Test for Antioxidant Activity

4.3.5.2 Apparatus
➢ Test tubes
➢ Beakers
➢ UV spectrophotometer (single beam)
➢ Electronic balance
➢ Vortex machine
➢ Sonicator
➢ Measuring cylinder.
4.3.5.3 Reagents
➢ Ethanol
➢ H2SO4 (0.6 M)
➢ Ascorbic acid
➢ Na3PO4 (28 mM)
➢ Ammonium Molybdate (4 mM)

4.3.5.4 Method
The study was conducted using phosphomolybdate method as described by [11] [12]

4.3.5.4.1 Preparation of Sample

0.005 g of plant extract of C. arborea were mixed with 5 ml of methanol separately &
sonicated for 5 minutes.

4.3.5.4.2 Preparation of Standard

0.01 gm gallic acid was dissolved in 10 ml ethanol to prepare the stock solution. A standard
solution of 5 concentrations of the gallic acid was prepared by serial dilution method. These
concentrations were 0.5, 0.25, 0.125, 0.0625, 0.03125, 0 mg/ml. For 0.5 mg/ml: 2.5 ml from
stock solution + 2.5 ml ethanol
For 0.25 mg/ml: 1.25 ml from stock solution + 3.75 ml ethanol
For 0.125 mg/ml: 0.625 ml from stock solution + 4.375 ml ethanol
For 0.0625 mg/ml: 0.3125 ml from stock solution + 4.6875 ml ethanol
For 0.03125 mg/ml: 0.156 ml from stock solution + 4.84 ml ethanol

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 Chapter 4: Test for Antioxidant Activity

4.3.5.4.3 Preparation of Working Reagent

The fresh working solution was prepared by mixing H2SO4 (0.6 M), Na3PO4 (28 mM) and
Ammonium Molybdate (4 mM) in a proportion of 4:2:4.

4.3.5.4.4 Working Procedure

(i) 0.3 ml of extract and ascorbic acid used as standard and blank (ethanol) were combined
with 3 ml of reagent mixture separately and incubated at 95°C for 90 minutes.
(ii) After cooling to room temperature, the absorbance of each sample was measured at 695
nm against the blank.
(iii) All determinations were performed in duplicate.
(iv)The standard curve was prepared using 0; 0.5; 0.25; 0.125; 0.0625 and 0.03125 mg/ml
solutions of ascorbic acid in ethanol.

4.3.5.5 Data Presentation

Obtained data of UV Absorbance of ascorbic acid (standard) & sample at 695 nm are given
in table 4.13 and 4.14 respectively. Ascorbic acid standard calibration curve is given in figure
4.11

Table 4.13: UV Absorbance of ascorbic acid (standard) at 695 nm

Concentration (mg/ml) Absorbance 1 Absorbance 2 Average

0.5 0.911 0.913 0.912

0.25 0.761 0.763 0.761

0.125 0.511 0.519 0.515

0.0625 0.371 0.379 0.375

0.03125 0.188 0.192 0.190

0 0.001 0.002 0.001

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 Chapter 4: Test for Antioxidant Activity

Ascorbic acid standard calibration curve

1 0.912
0.9 0.761
0.8
0.7 y = 1.8309x + 0.0013
Absorbance

0.6 0.515
R² = 0.9964
0.5 0.375
0.4
0.3
0.19
0.2
0.10.001
0
0 0.1 0.2 0.3 0.4 0.5 0.6
Concentration

Figure 4.11: Total antioxidant capacity determination of C. arborea extract with the help of
ascorbic acid standard calibration curve.

Table 4.14: UV absorbance of sample at 695nm

Sample Absorbance 1 Absorbance 2 Avg. Absorbance

C. arborea 0.589 0.597 0.593

extract

4.3.5.6 Determination of Total Antioxidant Capacity of Plant Extracts

The following equation was obtained from a standard ascorbic acid calibration curve
y = 1.8309x + 0.0013; R² = 0.9964
Where y is the absorbance and x is the concentration of ascorbic acid (mg/ml).
Based on the measured absorbance, ascorbic acid equivalent (AAE) was read (mg/ml) from
the calibration line by using the following equation.
Absorbance of sample − 0.0013
AAE =

1.8309

Then total antioxidant capacity (TAC) in plant ethanol extract in ascorbic acid equivalents
(AAE) was calculated by using the following equation.
( ⁄ )=
( ⁄ )

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 Chapter 4: Test for Antioxidant Activity

Total antioxidant capacity is expressed in terms of mg of ascorbic acid equivalent

(AAE)/gram of dry extract, which is a common reference compound. The determined total
antioxidant capacity of C. arborea extract is given in table 4.15

Table 4.15: Determination of total antioxidant capacity of C. arborea extract

Sample Average absorbance AAE Total antioxidant capacity
at 695 nm (mg AAE/g)

C. arborea 0.593 0.3231 323.1

Extract

4.3.5.7 Result
The result of the present study showed that total antioxidant capacity of C. arborea extract
was found ~323mg AAE/gm of dry extract.

4.4 Discussion
In the qualitative antioxidant assay on TLC plate, any electron or hydrogen donating
molecule in mixture will react with and bleach DPPH. Thus, DPPH is reduced from purple to
a light-yellow component.[13] C.arborea extracts showed the free radical scavenging
properties indicated by the presence of light yellow spot on the TLC plate.
In DPPH radical scavenging activity test, purple colored DPPH has maximum absorption at
517 nm. Scavenging DPPH radical by antioxidants results decrease in absorption over time
which is proportional to the concentration of radicals being scavenged. In the DPPH radical
scavenging assay, C. arborea extract displayed DPPH radical scavenging activity with an
SC50 value ~ 34 µg/ml which showed a significant amount of antioxidant.
High content of phenols, flavonoids and tannins were also assayed in the current study. Here,
total phenolic content of C. arborea extract was ~ 157 mg GAE/g. However, total flavonoid
content was ~ 941 mg QE/g for the C. arborea plant extract. As far as total tannin content
was concerned, C. arborea dominated with a value of ~ 193 mg GAE/g.
In the determination of the total antioxidant capacity of the extract, quantitative
phosphomolybdate method was used where ascorbic acid was the standard and total
antioxidant capacity (TAC) was expressed as ascorbic acid equivalents. The study showed
that total antioxidant capacity of C. arborea was found ~ 323 mg AAE/gm of dry extract.

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 Chapter 4: Test for Antioxidant Activity

Table 4.16: SC50 values of different assays and total content of secondary metabolites
(phenolic, flavonoid and tannin) of sample extract.

Assay C. arborea extract

DPPH Radical Scavenging Activity (SC50 µg/ml) ~ 34
Total Phenolic Content (mg GAE/g) ~ 158
Total Flavonoid Content (mg QE/g) ~ 941
Total Tannin Content (mg GAE/g) ~ 193
Total Antioxidant Capacity (mg AAE/g) ~ 323

4.5 Conclusion

Based on obtained data of the present study it can be concluded that ethanolic extract C.
arborea contains large amounts of phenolic compounds, flavonoids and tannins, exhibits a
higher extent of antioxidant and free radical scavenging activities. These in vitro assays are
an indicator of these plant extracts as potential source of natural antioxidants, which might be
essential in the prevention of various oxidative stresses and related physiological disorders.

4.6 References

1. Diplock, A., et al., Functional food science and defence against reactive oxidative
species. British journal of nutrition, 1998. 80(S1): p. S77-S112.
2. Rice-Evans, C., N. Miller, and G.J.T.i.p.s. Paganga, Antioxidant properties of
phenolic compounds. Trends in plant science, 1997. 2(4): p. 152-159.
3. Javanmardi, J., et al., Antioxidant activity and total phenolic content of Iranian
Ocimum accessions. Food chemistry, 2003. 83(4): p. 547-550.
4. Sadhu, S.K., et al., Separation of Leucas aspera, a medicinal plant of Bangladesh,
guided by prostaglandin inhibitory and antioxidant activities. Chemical and
pharmaceutical bulletin, 2003. 51(5): p. 595-598.

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 Chapter 4: Test for Antioxidant Activity

5. Islam, M.N., et al., Total phenol, total flavonoid content and antioxidant potential of
methanol extract of boehmeria platyphylla d don leaves World J. Pharm, 2016. 5: p.
334-344.
6. Singh, S. and R.J.F.r.i. Singh, In vitro methods of assay of antioxidants: an overview.
Food reviews international, 2008. 24(4): p. 392-415.
7. Wolfe, K., et al., Antioxidant activity of apple peels. Journal of agricultural food
chemistry, 2003. 51(3): p. 609-614.
8. Miliauskas, G., P. Venskutonis, and T.J.F.c. Van Beek, Screening of radical
scavenging activity of some medicinal and aromatic plant extracts. Food chemistry,
2004. 85(2): p. 231-237.
9. Afroz, R., et al., Potential Antioxidant and Antibacterial Properties of a Popular
Jujube Fruit: A pple Kul (Z izyphus mauritiana). Journal of Food Biochemistry, 2014.
38(6): p. 592-601.
10. Killedar, S.G. and H.N.J.J.P.R. More, Estimation of tannins in different parts of
Memecylon umbellatum Burm. J Pharm, 2010. 3(3): p. 554-6.
11. Umamaheswari, M., T.J.A.J.o.T. Chatterjee, Complementary, and A. Medicines, In
vitro antioxidant activities of the fractions of Coccinia grandis L. leaf extract. African
Journal of Traditional, Complementary Alternative Medicines 2008. 5(1): p. 61-73.
12. Prieto, P., M. Pineda, and M.J.A.b. Aguilar, Spectrophotometric quantitation of
antioxidant capacity through the formation of a phosphomolybdenum complex:
specific application to the determination of vitamin E. Analytical biochemistry, 1999.
269(2): p. 337-341.
13. Masoko, P. and J.N. Eloff, Screening of twenty-four South African Combretum and
six Terminalia species (Combretaceae) for antioxidant activities. African Journal of
Traditional, Complementary and Alternative Medicines, 2007. 4(2): p. 231-239.

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 CHAPTER FIVE
TEST FOR
ANALGESIC ACTIVITY
 Chapter 5: Evaluation of Analgesic Activity

CHAPTER FIVE

Evaluation of Analgesic Activity

5.1 Introduction
Algesia or hyperesthesia is an unpleasant sensitivity to pain, evoked by external or internal
noxious stimulus. Excessive pain may be unbearable and causes other effects – sinking
sensation, apprehension, sweating, nausea, palpitation, rise or fall in BP. [1] In another word,
pain is an unpleasant sensory and emotional experience associated with actual or potential
tissue damage. Pain is an unpleasant sensation no doubt, but on the whole, it is usually
beneficial to man (or animal). It is mainly a protective mechanism for the body, occurs
whenever any tissues are being damaged, and it causes the individual to react to remove the
pain stimulus. Analgesic is an agent that reduces or eliminates pain by acting on the sensory
nervous system, either centrally or peripherally without significantly altering consciousness.

In modern world all tries to develop economically, less side effect containing but
therapeutically potent drugs. Our world is the rich source of many medicinal plants which are
the rich raw source of crude drugs. These crude drugs are the first materials for developing a
new potent drug. So, firstly it is needed to investigate the plant source which gives analgesic
activity by comparing with potent standard and then to determine and isolate those active
components.

A decoction of the leaves of C. arborea is taken to relieve a stomachache, traditionally.[2]

This leaded to the study of evaluating analgesics activity of this plant. The aim of the present
study was to evaluate analgesics activity of ethanolic extract of C. arborea leaves by acetic
acid induced writhing method.

5.2 Methodology of analgesic activity

Analgesic activity of C. arborea extract was tested using the model of acetic acid induced
writhing in mice as described by [3] [4] [5] [6]

5.2.1 Principle

The acetic acid induced writhing method is an analgesic behavioral observation assessment
method that demonstrates a noxious stimulation in mice. The test consists of injecting the

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 Chapter 5: Evaluation of Analgesic Activity

0.7% acetic acid solution intraperitoneally and then observing the animal for specific
contraction of body referred as ‘writhing’. A comparison of writhing is made between
positive control (Diclofenac Na), negative control and test sample given orally 30 minutes
prior to acetic acid injection. If the sample possesses analgesic activity, the animal that
received the sample will give lower number of writhing than the control, i.e. the sample
having analgesic activity will inhibit writhing.
.
5.2.2 Materials
➢ ➢
Hand gloves Balance
➢ ➢
1 ml syringe Feeding needle
➢ 3 ml syringe ➢
Beaker
➢ Acetic acid ➢
Distilled water
➢ Diclofenac Na

5.2.3 Experimental animal

Young Swiss-albino mice aged 4-5 weeks; average weight 16-22 gm were used for the
experiment. The animals were purchased from the animal house attached to the
pharmacology laboratory of Jahangirnagar University, Dhaka. They were kept in standard
environmental condition for one week in the animal house for adaptation after their purchase.
The animals were provided with standard laboratory food and tap water and maintained at
natural day night cycle.

5.2.4 Study design

Experimental animals were randomly selected and divided into four groups denoted as group-
I, group-II, group-III, group- IV consisting of 5 mice in each group. Each group received a
particular treatment i.e. negative control, positive control and the two doses of the extract.
Each mouse was weighed properly and the doses of the test samples and control substances
were adjusted accordingly. Dose was calculated on the basis of body weight of mice.
Experimental profile to assess the effect of C. arborea extract on acetic acid induced writhing
of mice is described in table 5.1

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 Chapter 5: Evaluation of Analgesic Activity

Table 5.1 Experimental profile to assess the effect of C. arborea extract on acetic acid
induced writhing of mice

Animal Dose Route

Group Treatment of administration

I 1% tween-80 10 mL/kg Oral

(Negative control) solution in water

II Diclofenac Na 25 mg/kg Oral

(Positive control)

III C. arborea extract 250 mg/kg Oral

(Test group-1)

IV C. arborea extract 500 mg/kg Oral

(Test group-2)

5.2.5 Preparation of sample suspension

5.2.5.1 Dose of extract: 500 mg/kg

To prepare suspension of the test samples at the dose of 500 mg/kg body weight, 250 mg of
extract was measured. The extract was triturated in unidirectional manner by the addition of
small amount of tween-80. After proper mixing of extract and tween-80, the distilled water
was slowly added. The final volume of the suspension was made 5 ml. To stabilize the
suspension, it was shaken well by vortex mixer. As the dose is 500 mg/kg, the mice of this
group were administered (0.01× body weight) ml solution.

5.2.5.2 Dose of extract: 250 mg/kg

To prepare suspension of the test samples at the doses of 250 mg/kg per body weight 250mg
of sample was measured and dissolved in 10 ml of distilled water. As the dose is 250 mg/kg,
the mice of this group were administered (0.01× body weight) ml solution.

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 Chapter 5: Evaluation of Analgesic Activity

5.2.5.3 Preparation of standard solution

Diclofenac Na injection (75 mg in 3 ml of a single ampoule) was used to prepare positive

control. From this ampoule 1 ml of Diclofenac Na was separated and then adjusted in 10 ml
distilled water to get the desired concentration of 2.5 mg/ml. As the dose of positive control is
25 mg/kg, the mice of positive control group were administered (0.01× body weight) ml
solution.

5.2.5.4 Preparation of 0.7% acetic acid

For preparation of 0.7% acetic acid solution, 0.35 ml glacial acetic acid was mixed with
49.65 ml of distilled water. Each mouse was injected (0.01× body weight) ml 0.7% acetic
acid solution.

5.2.5.5 Preparation of negative control solution

Few drops of tween-80 were mixed with 10 ml of distilled water which was used as negative
control. As the dose of negative control is 10 mg/kg, the mice of negative control group were
administered (0.01× body weight) ml solution.

5.2.6 Working Procedure

Test samples, positive and negative control solution were given orally by means of a feeding
needle. A thirty minutes interval was given to ensure proper absorption of the administered
substances. Then the writhing inducing chemical, acetic acid solution (0.7%) was
administered intraperitoneally to each of the animals of a group. After an interval of 5
minutes, which was given for absorption of acetic acid, number of squirms (writhing) was
counted for 15 minutes.

5.3 Data presentation

Each mouse of all groups was observed carefully to count the number of writhing that they
had made in 15 minutes. The animal does not always perform full writhing, because
sometimes the animals begin to produce writhing but they do not complete it. This
incomplete writhing was taken as half-writhing, so two half-writhing were taken as one full
writhing. The statistical significance threshold was set at 0.05. Effects & Statistical

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 Chapter 5: Evaluation of Analgesic Activity

evaluation of C. arborea extract on acetic acid induced writhing of mice are given in table
5.2 & 5.3 respectively.

Table 5.2: Effects of C. arborea extract on acetic acid induced writhing of mice
Administered Numbering Weight(gm) Dose Mean

dose to mice Writhing

of mice of mice (ml) Writhing
Group of mice

1 27 0.27 28

2 28 0.28 26

Negative control 3 29 0.29 30 27.2

4 27 0.27 30

5 27 0.27 22

1 23 0.23 9

Positive control 2 23 0.23 9

Diclofenac Na 3 22 0.22 10 9.2

(25 mg/kg)
4 26 0.26 11

5 22 0.22 7

1 33 0.33 18

C. arborea 2 30 0.30 10
extract
3 30 0.30 20 15.2
(250 mg/kg)
4 27 0.27 12

5 30 0.30 16

1 33 0.33 9

C. arborea 2 29 0.29 14
extract
3 33 0.33 12 11.2
(500 mg/kg)
4 29 0.29 10

5 28 0.28 11

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 Chapter 5: Evaluation of Analgesic Activity

Table 5.3: Statistical evaluation of effects of the extracts on acetic acid induced writhing

Animal group Mean of % Writhing % Inhibition of

Writhing ± SEM writhing
27.2 ± 1.50 100 -----
Negative control
Positive control
Diclofenac Na 9.2 ± 0.66 33.82 66.18
(25mg/kg) ***

C. arborea 15.2 ± 1.85 55.88 44.12

Extract (250 mg/kg) **
C. arborea 11.2 ± 0.86 41.18 58.82
Extract (500 mg/kg) ***

Significance: * P<0.05, ** p<0.01, ***p<0.001

Mean writhing Vs Treatment

35

30

25
Mean
Writh
ing 20
**
15
***
***
10

0
Negative Control Positive Control C.arborea 250mg/kg C.arborea 500mg/kg

Significance: * P<0.05, ** p<0.01, ***p<0.001

Figure 5.1: Mean Writhing vs. treatment of Diclofenac Na with C. arborea extract
on acetic acid induced writhing in mice.

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 Chapter 5: Evaluation of Analgesic Activity

% inhibition of writhing Vs Treatment

80

70 *** ***
60

50
% **
writhi
ng 40
inhibi
tion 30

20

10

Negative Control Positive Control C.arborea 250mg/kg C.arborea 500mg/kg

-10

Significance: * P<0.05, ** p<0.01, ***p<0.001

Figure 5.2: Percent (%) inhibition of writhing vs. treatment of Diclofenac Na and C.
arborea extract on acetic acid induced writhing in mice

5.4 Result & Discussion

The results of the test showed that C. arborea extract at dose of 250 mg/kg and 500 mg/kg
exhibited inhibition of writhing reflex by 44.12 % and 58.82 % respectively while the
standard drug Diclofenac Na inhibition was found to be 66.18 % at a dose of 25 mg/kg body
weight. The results of statistical analysis are as follow: Negative control Vs. Diclofenac
Sodium: Significant
Negative control Vs. C. arborea extract (250 mg/kg): Significant
Negative control Vs. C. arborea extract (500 mg/kg): Significant

So, it can be claimed that the analgesic activity of C. arborea extract was significant in
comparison with negative control animals when the statistical significance threshold was set
at 0.05. The extract, at the doses of 250 and 500 mg/kg body weight showed some level of
decrease in acetic acid induced writhing reflex of mice.

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 Chapter 5: Evaluation of Analgesic Activity

5.5 Conclusion
The result of acetic acid-induced abdominal constriction assay demonstrated a prominent
reduction in writhing reflux. The analgesic effect observed at 250 mg/kg & 500 mg/kg dose
was comparable with the NSAID standard drug diclofenac sodium.

These findings strongly recommend that extracts of C. arborea leaves have peripheral
analgesic activity and their mechanisms of action may be mediated through inhibition of local
peritoneal receptors via cyclooxygenase inhibition.

5.6 References

1. Tripathi, K., Essentials of medical pharmacology. 2013: JP Medical Ltd.

2. Wiart, C., Medicinal plants of the Asia-Pacific: drugs for the future? 2006:
World Scientific.
3. Hosen, S.Z., et al., VENT BY ACETIC ACID INDUCED WRITHING
METHOD. Bulletin of Pharmaceutical Research, 2011. 1(3): p. 63-7.
4. Ahmed, F., et al., Anti-inflammatory and antinociceptive activities of Lippia nodiflora
Linn. Die Pharmazie-An International Journal of Pharmaceutical Sciences, 2004.
59(4): p. 329-330.
5. Koster, R. Acetic acid for analgesic screening. in Fed proc. 1959.
6. Junior, O.D., et al., Investigation of the Anti-inflammatory and Analgesic Activities
of a Sample of Brazilian Propolis. Acta Farm. Bonaerense, 2004. 23(3): p. 285-291.

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 CHAPTER SIX
TEST FOR ANTI-
INFLAMMATORY ACTIVITY
 Chapter 6: Evaluation of Anti-inflammatory Activity

CHAPTER SIX
Evaluation of Anti-inflammatory Activity

6.1 Introduction
Inflammation is part of the complex biological response of body tissues to harmful stimuli,
such as pathogens, damaged cells, or irritants,[1] and is a protective response involving
molecular mediators. The function of inflammation is to eliminate the initial cause of cell
injury, clear out necrotic cells and tissues damaged from the original and the inflammatory
process, and initiate tissue repair. Depending upon the defense capacity of the host and
duration of response, it is classified as acute and chronic. Inflammation, pain and pyrexia
underlie several pathological conditions. Synthetic drugs, i.e. NSAIDs, opioids and
corticosteroids are clinically most important drugs used for the treatment of inflammatory
disorders, however their long-term use may induce toxic effects. Globally, efforts are
ongoing to introduce novel medicinal plants to develop effective, economic and innocuous
drugs.[1] Medicinal plants are believed to be an important source of useful compounds with
potential therapeutic effects. The research on plants with apparent folkloric use, as anti-
inflammatory agents, should therefore be regarded as a prolific and a rational research
strategy in the search for new anti-inflammatory drugs.

The development of novel compounds having anti-inflammatory activities with an improved

safety profile is still required. The synthetic ones are particularly expensive to develop as it
cost millions of dollars. This has prompted a new rush for herbal medicines as one of the best
potential sources of newer, safer and better drugs.

A decoction of the leaves of C. arborea is taken to relieve a stomachache and also used as a
poultice to heal sores, traditionally. [2] This has leaded to the study of evaluating anti-
inflammatory activity of this plant. The aim of the present study was to evaluate anti-
inflammatory activity of ethanolic extract of C. arborea leaves by formaldehyde induced paw
edema method.

6.2 Methodology of anti-inflammatory activity

Anti-inflammatory activity of C. arborea extracts were tested using the model of
formaldehyde induced paw edema method in mice as described by [3] [4] [5] [6]

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 Chapter 6: Evaluation of Anti-inflammatory Activity

6.2.1 Principle

Swiss albino mice were divided into four groups of five animals each. Animals were treated
orally with the extracts (250 and 500 mg/kg), indomethacin (10 mg/kg) and distilled water
(10 mL/kg). Formaldehyde is commonly used to activate the role of Phospholipase A 2 (PLA2)
in the pathophysiology of inflammation. Test samples, standard drug and control vehicle were
administered orally once a day for 10 days. After 1h on days 1 and 3 of treatment, these mice
were injected with 0.1ml of 2% formaldehyde into the foot pad of left hind paw [4]. Paw
edema was measured 1h before formaldehyde injection and at 4h after the injection on day 1
and every day at 1h after the treatment for 10 consecutive days.

6.2.2 Materials & Reagents

➢ Hand gloves ➢ Indomethacin
➢ 1 ml syringe ➢ Formaldehyde
➢ 3 ml syringe ➢ Distilled water
➢ Balance ➢ Tween-80
➢ Feeding needle ➢ Vortex mixer

6.2.3 Animals

Swiss albino mice of either sex weighing between 25-45 g were used. The animals were
obtained from the animal house attached to the pharmacology laboratory of Jahangirnagar
University, Dhaka. The mice were exposed to natural day and night cycles under ideal
ambient laboratory conditions. They were fed with mouse pellet feed and tap water.

6.2.4 Preparation of Sample

6.2.4.1 Dose of extract: 500 mg/kg

To prepare suspension of the test samples at the dose of 500 mg/kg body weight, 250 mg of
extract was measured. The extract was triturated in unidirectional manner by the addition of
small amount of tween-80. After proper mixing of extract and tween-80, the distilled water
was slowly added. The final volume of the suspension was made 5ml. To stabilize the
suspension, it was shaken well by vortex mixer. As the dose is 500 mg/kg, the mice of this
group were administered (0.01× body weight in gm) ml solution.

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 Chapter 6: Evaluation of Anti-inflammatory Activity

6.2.4.2 Dose of extract: 250 mg/kg

To prepare suspension of the test samples at the doses of 250 mg/kg per body weight, 250mg
of sample was measured and dissolved in 10 ml of distilled water. As the dose is 250 mg/kg,
the mice of this group were administered (0.01× body weight in gm) ml solution

6.2.4.3 Preparation of standard

For the preparation of standard indomethacin at the dose of 10mg/kg body weight, 10 mg
equivalent indomethacin was triturated with small amount of tween-80 and then adjusted
with distilled water to make final volume 10 ml. As the dose is 10 mg/kg, the mice of this
group were administered (0.01× body weight in gm) ml solution.

6.2.4.4 Preparation of negative control solution

Few drops of tween-80 were mixed with 10 ml of distilled water which was used as negative
control. As the dose of negative control is 10 mg/kg, the mice of negative control group were
administered (0.01× body weight in gm) ml solution.

6.2.4.5 Preparation of formaldehyde solution:

2 ml formaldehyde was dissolved in 98 ml distilled water to make a final volume of 100 ml.

6.2.5 Study Design

Experimental animals were randomly selected and divided into six groups denoted as group-
I, group-II, group-III, group- IV consisting of 5 mice in each group. Each group received a
particular treatment i.e. negative control, positive control and the two doses of the extract.
Each mouse was weighed properly and the doses of the test samples and control substances
were adjusted accordingly. Experimental profile to assess the effect of C. arborea extract in
Formaldehyde induced paw edema method is given in table 6.1

Table 6.1 Experimental profile to assess the effect of C. arborea extract

Animal Group Treatment Dose Route of

administration

I (Negative control) Distilled Water 10 ml/kg Oral

II (Positive control) Indomethacin 10 mg/kg Oral
III (Test group-1) C. arborea extract 250 mg/kg Oral
IV (Test group-2) C. arborea extract 500 mg/kg Oral
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 Chapter 6: Evaluation of Anti-inflammatory Activity

6.2.6 Procedure
➢ Tween-80 was used as a control drug. As the dose is 10 ml/kg, the mice of this group were administered (0.01× body weight in gm) ml solution once daily for 10
days.

➢ Test groups received extract at the doses of 250 mg/kg and 500 mg/kg body weight of each mouse. As the dose is 250 mg/kg
and 500 mg/kg, the mice of this group were administered (0.01× body weight in gm) ml solution once daily for 10 days.
➢ Indomethacin was used as a standard drug. As the dose is 10 mg/kg, the mice of this group were administered (0.01× body weight in gm) ml solution once daily
for 10 days.

➢ The paw volumes were measured before the first formaldehyde injection. After 1h of
administering each group of solution, 0.1ml of 2% formaldehyde was injected into the
st rd
left hind paw of each mouse on the 1 day and the 3 day of treatment. The thickness of
st
the injected paw was measured at 4h after the injection on day 1 day to generate
additional data on acute inflammation. Then the paw thickness was measured after 1h of
administration of each solution on each day of 10 consecutive days.

6.3 Data presentation and Calculation

The dose was calculated on the basis of body weight of mice. The measurement of paw
thicknesses of mice for 10 days, are tabulated in the table 6.2. The change in paw size was
calculated through the following formula and tabulated in the table 6.3. Percent of
inflammation and percent inhibition of inflammation are tabulated in the table 6.4

Change in paw thickness= (paw size after formaldehyde injection at pre-determined times -
paw size before formaldehyde injection).

Then percent inflammation and percent inhibition of inflammation was calculated through the
following formulas respectively. The data are shown in the table 6.2.4.

Percent inflammation of paw edema

= ℎ ×100 ℎ

Percent inhibition of inflammation= 100 - % inflammation

Page | 72
 Administered Sl. No. Body Required Paw thickness (mm)
dose to mice of wt. dose
Group mice (gm) (ml) Before Day Day Day Day Day Day Day Day Day Day
injecting 1 2 3 4 5 6 7 8 9 10

2 35 0.35 1.89 4.98 4.19 4.02 4.85 4.45 3.99 3.94 3.82 3.73 3.61
Negative 1 34 0.34 1.48 4.70 4.13 3.98 4.40 4.26 3.87 3.81 3.68 3.66 3.60
Control

2: T
3 35 0.35 2.09 4.35 3.86 3.62 4.71 4.11 3.69 3.60 3.51 3.48 3.39

e
h
4 35 0.35 1.85 3.29 3.19 2.95 3.58 3.23 2.49 2.47 2.40 2.26 2.23
4 33 0.33 1.65 4.52 3.89 3.69 4.63 4.01 3.63 3.55 3.55 3.49 3.42

pawofmeasurem
5 35 0.35 1.57 4.83 4.17 4.09 4.77 4.31 3.92 3.72 3.67 3.61 3.57
Positive 1 33 0.33 2.02 3.40 3.23 3.08 3.91 3.13 2.99 2.71 2.55 2.19 2.07
Control 2 37 0.37 2.08 3.53 3.27 2.67 3.74 2.96 2.7 2.69 2.43 2.36 2.29
(Indomethacin
10mg/kg) 3 35 0.35 1.78 3.26 2.90 2.85 3.38 3.11 2.64 2.63 2.51 2.49 2.35

thickne
5 36 0.36 1.68 3.30 3.13 2.69 3.72 3.69 2.81 2.64 2.34 2.11 2.05

sses

alu
Ev
on
ati
Extract of 1 35 0.35 1.87 4.92 3.64 3.46 4.60 3.61 3.50 3.45 3.45 3.43 3.40
C. arborea 2 37 0.37 1.98 4.38 4.14 3.98 4.73 3.78 3.70 3.66 3.59 3.57 3.29
(250mg/kg) 3 39 0.39 1.98 4.79 3.68 3.42 4.84 3.98 3.88 3.87 3.74 3.60 3.57

days10formic
4 40 0.40 2.30 4.95 4.40 4.31 4.79 4.04 3.90 3.89 3.68 3.61 3.48

inflammator
5 40 0.40 2.38 4.86 4.19 3.88 4.32 3.50 3.43 3.41 3.39 3.39 3.30

y-Antiof
Extract of 1 38 0.38 2.06 4.46 4.06 3.27 4.70 3.92 3.66 3.49 3.45 3.30 3.08

eof
C. arborea 2 35 0.35 1.84 4.72 3.97 3.78 4.80 3.92 3.57 3.48 3.38 3.29 3.02
(500mg/kg) 3 34 0.34 1.84 4.29 3.68 3.60 4.45 3.84 3.55 3.46 3.34 3.28 3.00
4 40 0.40 2.27 4.08 3.24 3.13 4.31 3.60 3.35 3.35 3.28 3.22 3.02
73|Page

5 32 0.32 1.73 4.19 3.89 3.68 3.80 3.73 3.70 3.25 3.20 3.14 2.89

vit
cti
A
y
 Chapter 6: Evaluation of Anti-inflammatory Activity

Table 6.3: Increase in paw thickness for the C. arborea extract on the formaldehyde
induced paw edema method in mice

Administered No. of Increased paw size/ Paw edema(mm)

dose to mice mice Day Day Day Day Day Day Day Day Day Day
Group 1 2 3 4 5 6 7 8 9 10

1 3.22 2.65 2.50 2.92 2.78 2.39 2.33 2.20 2.18 2.12
Negative 2 3.09 2.30 2.13 2.96 2.56 2.10 2.05 1.93 1.84 1.72
3 2.26 1.77 1.53 2.62 2.02 1.60 1.51 1.42 1.39 1.33
Control 4 2.87 2.24 2.04 2.98 2.36 1.98 1.90 1.90 1.84 1.77
5 3.26 2.60 2.52 3.20 2.74 2.35 2.15 2.10 2.04 2.00
Average 2.94 2.31 2.14 2.93 2.49 2.09 1.99 1.91 1.86 1.79
1 1.38 1.21 1.06 1.89 1.11 0.97 0.69 0.53 0.17 0.05
Positive 2 1.45 1.19 0.59 1.66 0.88 0.69 0.61 0.35 0.28 0.21
Control 3 1.48 1.12 1.07 1.60 1.33 1.86 0.85 0.73 0.71 0.57
(Indomethacin: 4 1.44 1.34 1.10 1.73 1.38 1.64 0.62 0.55 0.41 0.38
100mg/kg) 5 1.62 1.45 1.01 2.04 2.01 1.13 0.96 0.66 0.43 0.37
Average 1.48 1.26 0.97 1.78 1.34 0.86 0.75 0.56 0.40 0.32
1 3.05 1.77 1.59 2.73 1.74 1.63 1.58 1.58 1.56 1.53
Extract of 2 2.40 2.16 2.00 2.75 1.80 1.72 1.68 1.61 1.59 1.31
C. arborea 3 2.81 1.70 1.44 2.86 2.00 1.90 1.89 1.76 1.62 1.59
(250 mg/kg) 4 2.65 2.10 2.01 2.49 1.74 1.60 1.59 1.38 1.31 1.18
5 2.48 1.81 1.50 1.98 1.12 1.05 1.03 1.03 1.01 1.01
Average 2.68 1.91 1.71 2.56 1.68 1.58 1.55 1.47 1.42 1.32
1 2.40 2.0 1.21 2.64 0.86 1.60 1.43 1.39 1.24 1.02
Extract of 2 2.88 2.13 1.94 2.96 2.08 1.73 1.64 1.54 1.45 1.18
C. arborea 3 2.45 1.84 1.76 2.61 2 1.71 1.62 1.50 1.44 1.16
(500 mg/kg) 4 1.81 0.97 0.86 2.04 1.33 1.08 1.08 1.01 0.95 0.75
5 2.46 2.16 1.95 2.07 2.00 1.97 1.52 1.47 1.41 1.16
Average 2.40 1.82 1.54 2.46 1.65 1.61 1.46 1.38 1.29 1.05

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 Chapter 6: Evaluation of Anti-inflammatory Activity

Table 6.4: Percent of inflammation and Percent inhibition of inflammation of C. arborea

extract at different days
Treatment Day Day Day Day Day Day Day Day Day Day
Group 1 2 3 4 5 6 7 8 9 10

Avg. Change 2.94± 2.30± 2.14± 2.93± 2.49± 2.08± 1.98± 1.91± 1.85± 1.78±
Negative in Paw 0.18 0.157 0.181 0.093 0.139 0.143 0.138 0.134 0.134 0.136
Control thickness
(mm) ± SEM

% 100 100 100 100 100 100 100 100 100 100
inflammation

% inhibited ---- ---- ---- ---- ---- ---- ---- ---- ---- ----
inflammation

Positive Avg. Paw 1.48± 1.20± 0.97± 1.78± 1.34± 0.86± 0.75± 0.56± 0.40± 0.32±
control thickness in 0.039 0.058 0.095 0.080 0.189 0.216 0.065 0.145 0.091 0.088
Indometh mm ± SEM *** *** *** *** ** * *** *** *** ***
acin
% 50.34 54.54 45.32 60.75 53.82 41.14 37.67 29.31 21.51 17.87
(10 mg/kg) inflammation

% inhibited 49.66 45.46 54.68 39.25 46.18 58.86 62.33 70.69 78.49 82.13
inflammation

Avg. Paw 2.68± 1.91± 1.71± 2.56± 1.68± 1.58± 1.55± 1.47± 1.14± 1.32±
C. arborea thickness in 0.117 0.093 0.124 0.158 0.148 0.142 0.142 0.126 0.116 0.108
Extract mm ± SEM * * * * * *
(250
% 91.16 82.68 79.91 87.37 77.46 76.59 77.88 76.96 76.34 73.74
mg/kg) inflammation

% inhibited 8.84 17.32 20.09 12.63 22.54 24.41 22.12 23.04 23.66 26.25
inflammation

Avg. Paw 2.40± 1.82± 1.54± 2.46± 1.65± 1.61± 1.45± 1.38± 1.29± 1.05±
C. arborea thickness in 0.171 0.219 0.218 0.178 0.240 0.147 0.102 0.096 0.095 0.081
Extract mm ± SEM * * * * * ** **
(500
% 81.63 78.78 71.96 83.96 78.95 77.03 73.37 72.25 69.35 58.65
mg/kg) inflammation

% inhibited 18.37 21.21 28.04 16.04 21.05 22.96 26.63 27.75 30.65 41.34
inflammation

Significance: * P<0.05, ** p<0.01, ***p<0.001

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 Chapter 6: Evaluation of Anti-inflammatory Activity

Change in paw thickness (mm) in every hour vs treatment group

3.5
3

2.5
Paw thickness (mm)

2
1.5

0.5

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10
Negative Control Positive Control C. arborea( 250 mg/kg) C. arborea( 500 mg/kg)

Figure 6.1: Change in paw thickness (mm) in every hour for negative control,
positive control and C. arborea extract (500mg/kg and 250mg/kg)

% inflammation vs Time
120

100

%
inflam 80
matio
n
60

40

20

0
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10
Negative Control Positive Control C. arborea (250 mg/kg) C. arborea (500 mg/kg)

Figure 6.2: Percent (%) of inflammation vs time of standard drug and C. arborea
extract on formaldehyde induced paw edema method in mice

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 Chapter 6: Evaluation of Anti-inflammatory Activity

% Inhibition of inflammation vs time

90
% 80
Inhibi
tion
of 70
infla
mma
tion 60

50

40

30

20

10

0
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10
Negative Control Positive Control C. arborea (250 mg/kg) C. arborea (500mg/kg)

Figure 6.3: Percent (%) inhibition of inflammation vs time of standard

drug and C. arborea extract on formaldehyde induced paw edema
method in mice

6.4 Result & Discussion

Extract of C. arborea showed anti-inflammatory activity at the doses of 250 and 500mg/kg in
the observed 10days. Indomethacin (10mg/kg) was used as the standard drug. All of the
above values were compared with the negative control group. Based on the results, it can be
said that, dose of 500mg/kg showed more anti-inflammatory activity than dose of 250mg/kg.
Most of the values were statistically significant with comparing with the negative control
th
group. Significant values were observed from 5 day for the dose of 250 mg/kg body weight
th
while it was from 4 day for 500 mg/kg body weight.

6.5 Conclusion
So, it can be claimed that C. arborea extract showed fair decrease in formaldehyde induced
edema which indicates a significant level of anti-inflammatory activity of C. arborea extract.
Thus, it can be concluded that C. arborea possess some good level of anti-inflammatory
activity.

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 Chapter 6: Evaluation of Anti-inflammatory Activity

6.6 References
1. Ferrero‐Miliani, L., et al., Chronic inflammation: importance of NOD2 and NALP3 in
interleukin‐1β generation. Clinical Experimental Immunology 2007. 147(2): p. 227-
235.

2. Wiart, C., Medicinal plants of the Asia-Pacific: drugs for the future? 2006: World
Scientific.
3. Dharmasiri, M., et al., Anti-inflammatory and analgesic activities of mature fresh
leaves of Vitex negundo. Journal of ethnopharmacology, 2003. 87(2-3): p. 199-206.
4. Akindele, A. and O. Adeyemi, Antiinflammatory activity of the aqueous leaf extract
of Byrsocarpus coccineus. Fitoterapia, 2007. 78(1): p. 25-28.
5. Vasudevan, M., K.K. Gunnam, and M. Parle, Antinociceptive and anti-inflammatory
effects of Thespesia populnea bark extract. Journal of ethnopharmacology, 2007.
109(2): p. 264-270.
6. Singh, B., et al., Anti-inflammatory activity of Echinops echinatus. Journal of
ethnopharmacology, 1989. 25(2): p. 189-199.

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 CHAPTER seven
TEST FOR
ANTI-PYRETIC ACTIVITY
 Chapter 7: Evaluation of Anti-pyretic Activity

CHAPTER SEVEN
Evaluation of Antipyretic Activity

7.1 Introduction
Fever is a complex physiologic response triggered by infections or aseptic stimuli. Elevation
in body temperature occurs when the concentration of prostaglandin E 2 (PGE2) increases
within parts of the brain. Such an elevation contributes to a considerable alteration in the
firing rate of neurons that control the thermoregulation process in the hypothalamus. It is now
evident that most antipyretics exert their action by inhibiting the enzymatic activity of
cyclooxygenase and consequently reducing the levels of PGE2 within the hypothalamic
region. Recently, other mechanisms of action for anti-pyretic drugs have been presumed.
These mechanisms have shed the light on the ability of anti-pyretic agents to reduce pro-
inflammatory mediators, enhance anti-inflammatory signals at sites of injury or boost anti-
pyretic messages within the brain. [1] [2]
The development of novel compounds having anti-inflammatory activities with an improved
safety profile is still required. The synthetic ones are particularly expensive to develop as it
cost millions of dollars. This has prompted a new rush for herbal medicines as the only
potential source of newer, safer and better drugs.

The powdered plant part of C. arborea, combined with a pinch of tumeric (Curcuma longa)
is used to relieve fevers.[3] This leaded to the study of evaluating anti-pyretic activity of this
plant. The aim of the present study was to evaluate anti-pyretic activity of ethanolic extract of
C. arborea leaves by yeast-induced pyrexia method.

7.2. Methodology

Anti-pyretic activity of C. arborea extract was tested using the yeast-induced pyrexia in mice
as described by [4] [5] [6] [7]

7.2.1 Principle

Yeast induced pyrexia is called pathogenic fever and its etiology could be the production of
prostaglandins. The test consists of injecting the 15% yeast solution subcutaneously and then
after 24hr, the body temperature of animal is observed. A comparison of decreasing body
temperature is made between positive control (Paracetamol), negative control and test sample
given orally 1hr prior to measure body temperature. If the sample possesses antipyretic

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 Chapter 7: Evaluation of Anti-pyretic Activity

activity, the animal that received the sample will decrease body temperature than the control,
i.e. the sample having antipyretic activity will inhibit pyrexia. Paracetamol is used as the
reference standard drug.

7.2.2 Apparatus and reagents

➢ Hand gloves
➢ Electric balance
➢ Feeding needle
➢ Mortar & pestle
➢ Test tubes
➢ Distilled water
➢ Paracetamol
➢ Yeast

7.2.3 Experimental animal

Young Swiss-albino mice aged 7-8 weeks; average weight 25-40 gm were used for the
experiment. The animals were obtained from the animal house attached to the pharmacology
laboratory of Jahangirnagar University, Dhaka. They were kept in standard environmental
condition for one week in the animal house for adaptation after their purchase. The animals
were provided with standard laboratory food and tap water. All the experiments were
conducted on an isolated and noiseless condition.

7.2.4 Study design

Experimental animals were randomly selected and divided into four groups denoted as group-
I, group-II, group-III, group- IV consisting of 5 mice in each group. Each group received a
particular treatment i.e. negative control, positive control and the two doses of the extract.
Each mouse was weighed properly and the doses of the test samples and control substances
were adjusted accordingly. Experimental profile to assess the effect of C. arborea extract on
pyrexia is given in table 7.1

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 Chapter 7: Evaluation of Anti-pyretic Activity

Table 7.1 Experimental profile to assess the effect of C. arborea extract on pyrexia

Route of
Animal Group Treatment Dose administration

I (Negative control) Saline solution 10 mL/kg Oral

II (Positive control) Paracetamol 150 mg/kg Oral

III (Test group-1) C. arborea extract 250 mg/kg Oral
IV (Test group-2) C. arborea extract 500 mg/kg Oral

7.2.5 Preparation of sample suspension

7.2.5.1 Dose of extract: 500mg/kg

To prepare suspension of the test samples at the dose of 500 mg/kg body weight, 250 mg of
extract was measured. The extract was triturated in unidirectional manner by the distilled
water. The final volume of the suspension was made 5 ml. To stabilize the suspension, it was
shaken well by vortex mixer. As the dose is 500 mg/kg, the mice of this group were
administered (0.01× body weight) ml solution.

7.2.5.2 Dose of extract: 250 mg/kg

To prepare suspension of the test samples at the doses of 250 mg/kg per body weight 250 mg
of sample was measured and dissolved in 10 ml of distilled water. As the dose is 250 mg/kg,
the mice of this group were administered (0.01× body weight) ml solution.

7.2.5.3 Preparation of standard solution

For the preparation of standard paracetamol at the dose of 150 mg/kg body weight, 150 mg
equivalent paracetamol was triturated with small amount of distilled water and then adjusted
with distilled water to make final volume 10ml. As the dose of positive control is 150 mg/kg,
the mice of negative control group were administered (0.01× body weight) ml solution.

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 Chapter 7: Evaluation of Anti-pyretic Activity

7.2.5.4 Preparation of negative control solution

10 mg NaCl was mixed with 10 ml of distilled water which was used as negative control. As
the dose of negative control is 10mg/kg, the mice of negative control group were
administered (0.01× body weight) ml solution.

7.2.5.5 Working Procedure

The normal body temperature of each mouse was recorded using digital thermometer and
then pyrexia was induced in all mice by injecting 15% aqueous suspension of Brewer’s yeast
(10 ml/kg body weight) subcutaneously.
All groups were fasted overnight but allowed free accesses to drinking water and after 24h
rectal temperature of each mouse was recorded. The induction of pyrexia was confirmed by
rise in temperature more than 0.5°C, while animals showed rise in temperature less than
0.5°C were excluded from experiment.
Group I received saline (10 ml/kg) as a negative control, Group II received paracetamol (150
mg/kg) as a standard drug while the remaining groups III and IV received 250 and 500 mg/kg
extract orally. After drugs administration, rectal temperature was again recorded periodically
at 1, 2, 3 and 4 of drugs administration.

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 Chapter 7: Evaluation of Anti-pyretic Activity

7.3 Data presentation

Effects of C. arborea extract on pyrexia of mice is given in table 7.2

Table 7.2: Effects of C. arborea extract on pyrexia of mice

Administered No. of Weight 0
Body Temperature ( F)
dose to mice mice (gm) of
Group mice Normal After 1hr 2hr 3hr 4hr
24hr
1 24 98.9 101.1 100.0 101.2 101.3
100.2
2 23 98.5 101.3 101.2 101.1 101.2
100.2
Negative 3 23 98.3 99.9 100.0 100.0 99.9
100.0
Control 4 22 98.0 99.0 99.7 100.0 100.0
100.1
Saline solution 5 22 98.0 99.7 99.8 99.7 100.1
100.2
(10 mL/kg) Average ± SEM 100.1 ±
98.34 ± 100.2 ± 100.1 ± 100.4 ± 100.5 ± 0.040
0.169 0.436 0.271 0.311 0.308
1 22 98.2 101.5 99.6 99.2 98.9 98.3
Positive 2 22 98.2 101.8 98.0 98.0 98.3 98.1
control 3 21 97.9 100.0 98.4 98.2 98.1 98.3
Paracetamol 4 23 98.3 99.9 99.3 98.9 98.1 98.2
(150 mg/kg) 5 22 98.1 100.3 98.6 98.7 98.5 98.4
Average ± SEM 98.78 ± 98.60 ± 98.38 ± 98.26 ±
98.14 ± 100.7 ± 0.294 0.221 0.150 0.051
0.068 0.396 * ** *** ***
1 22 98.1 100.3 99.4 99.3 98.8 98.7
2 21 98.1 100.0 99.7 99.6 99.8 99.6
3 22 98.3 100.4 99.6 99.5 99.9 99.7
C. arborea 4 22 97.8 100.2 100.1 99.9 99.2 99.2
Extract (250 5 23 98.2 99.5 100.0 99.8 99.1 98.7
mg/kg) Average ± SEM 98.10 ± 100.1 ± 99.76 ± 99.62 ± 99.36 ± 99.18 ±
0.084 0.159 0.129 0.107 0.211 0.213
* * **
1 23 99.0 100.0 99.2 99.2 98.9 98.6
2 23 98.1 100.3 99.8 99.7 99.6 99.4
C. arborea 3 22 98.3 99.9 100.1 99.9 99.2 99.2
Extract (500 4 21 98.1 100.7 99.1 99.2 98.5 98.7
mg/kg) 5 22 98.5 100.1 99.3 99.1 99.3 98.5
Average ± SEM 98.40 ± 100.2 ± 99.50 ± 99.42 ± 99.10 ± 98.88 ±
0.167 0.141 0.192 0.159 0.187 0.177
* ** ***

Significance: * P<0.05, ** p<0.01, ***p<0.001

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 Chapter 7: Evaluation of Anti-pyretic Activity

Change of rectal temperature

at different times
101.5
101
100.5
100
Ractal temperature

*
99.5

*
*
*
*
*
*

99
98.5
98
97.5
97
96.5
96 Normal After 24 hr 1 hr 2 hr 3 hr 4 hr
Negative Control Positive Control C. arborea (250mg) C. arborea (500mg)

Significance: * P<0.05, ** p<0.01, ***p<0.001

Figure 7.1: Change of rectal temperature at different times (for different samples)

7.4 Result & Discussion

The above study was conducted to evaluate the anti-pyretic activity of the mice for a certain
period of time. The extract of C. arborea both 250 and 500 mg/kg showed antipyretic activity
by inhibiting the pyretic activity (reduction of rectal temperature of mice at the fixed time
duration). Paracetamol (150 mg/kg) was used as the standard drug. All of these values were
compared with the negative control group.
Subcutaneous injection of Brewer’s yeast induces pyrexia by increasing the synthesis of
prostaglandin. It is considered as a useful test for the screening of plants materials as well as
synthetic drugs for their antipyretic effect.[8] Yeast induced pyrexia is called pathogenic
fever and its etiology could be the production of prostaglandins. The inhibition of
prostaglandin synthesis could be the possible mechanism of antipyretic action as that of
paracetamol and the inhibition of prostaglandin can be achieved by blocking the cyclo-
oxygenase enzyme activity.[9] The oral administration of the extract significantly attenuated
rectal temperature of yeast induced febrile mice. Thus, it can be postulated that the extract of
leaves of C. arborea contained pharmacologically active principle(s) that interfere with the
release of prostaglandins.

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 Chapter 7: Evaluation of Anti-pyretic Activity

7.5 Conclusion
The result of yeast-induced pyrexia assay demonstrated a prominent reduction in pyretic
activity. The antipyretic effect observed at 250 mg/kg & 500 mg/kg dose was comparable
with the standard drug paracetamol. So, it can be claimed that the antipyretic activity of C.
arborea extract had significant antipyretic activity.

7.6 References
1. Simon, L.S., Role and regulation of cyclooxygenase-2 during inflammation. The
American journal of medicine, 1999. 106(5): p. 37S-42S.
2. Aronoff, D.M. and E.G. Neilson, Antipyretics: mechanisms of action and clinical use
in fever suppression. The American journal of medicine, 2001. 111(4): p. 304-315.
3. Manandhar, N.P., Plants and people of Nepal. 2002: Timber press.
4. Al-Ghamdi, M.J.J.o.e., The anti-inflammatory, analgesic and antipyretic activity of
Nigella sativa. Journal of ethnopharmacology, 2001. 76(1): p. 45-48.
5. Iwalewa, E., O. Iwalewa, and J.J.J.o.e. Adeboye, Analgesic, antipyretic, anti-
inflammatory effects of methanol, chloroform and ether extracts of Vernonia cinerea
less leaf. Journal of ethnopharmacology, 2003. 86(2-3): p. 229-234.
6. Vasundra, D., P.J.A.J.o.P. Divya, and c. research, Antipyretic activity of ethanol and
aqueous extract of root of Asparagus racemosus in yeast induced pyrexia. Asian
Journal of Pharmaceutical clinical research, 2013. 6(3): p. 190-193.
7. Muhammad, N., et al., Antipyretic, analgesic and anti-inflammatory activity of Viola
betonicifolia whole plant. BMC complementary alternative medicine, 2012. 12(1): p.
59.
8. Khan, I., et al., Anti-inflammatory activities of Sieboldogenin from Smilax china
Linn.: experimental and computational studies. Journal of ethnopharmacology, 2009.
121(1): p. 175-177.
9. Flower, R. and J. Vane, Inhibition of prostaglandin synthetase in brain explains the
anti-pyretic activity of paracetamol (4-acetamidophenol). Nature, 1972. 240(5381): p.
410-411.

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 CHAPTER eight
TEST FOR ANTI-
HYPERGLYCEMIC ACTIVITY
 Chapter 8: Evaluation of Anti-hyperglycemic Activity

CHAPTER EIGHT

Evaluation of Anti-hyperglycemic Activity

8.1 Introduction

Diabetes mellitus, is a chronic metabolic disorder characterized by a high blood glucose

concentration-hyperglycemia caused by insulin deficiency, often combined with insulin
resistance.[1] It has been reported that the chronic hyperglycemia is associated with
complications like renal failure, coronary artery disorder, neurological complications,
cerebra-vascular disease, blindness, limb amputation, long term dysfunctions and failure of
various organs and eventually premature death.[2]

Management of this disease may include lifestyle modifications, diet, exercise, and long–
term use of oral hypoglycemic agents or insulin therapy. Recently, phytomedicines gains
greater importance compared to synthetic antidiabetic drugs due to several adverse effects of
the synthetic drugs. It has been assumed that plant-based drugs are considered to be less toxic
and free from adverse effects in comparison to modern allopathic medicines.

Oral glucose tolerance test is a preliminary method to evaluate the antidiabetic activity of
plant extracts and other test samples. It is a medical test in which glucose is given and blood
samples are taken afterward to determine how it is quickly cleared from blood.

Antidiabetic activity of hydro-alcoholic stem bark extract was revealed with antioxidant
potential in diabetic rats. [3] This leaded to the study of evaluating analgesics activity of this
plant. The aim of the present study was to evaluate anti-hyperglycemic activity of ethanolic
extract of C. arborea leaves by oral glucose tolerance method.

8.2 Methodology
Anti-hyperglycemic activity of C. arborea extract was tested using the model of oral glucose
tolerance in mice as described by [4]

8.2.1 Principle
Oral glucose tolerance evaluation or oral glucose tolerance test (OGTT) is one of the
acceptable methods to evaluate the hypoglycemic activity of plant extracts and other test
samples. It is a medical test in which glucose is given and blood samples are taken afterward
to determine how it is quickly cleared from blood. Glibenclamide was used as standard drug.

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 Chapter 8: Evaluation of Anti-hyperglycemic Activity

Glucose tolerance test is a standard procedure that addresses how quickly exogenous glucose
can be cleared from blood. Specifically, uptake of glucose from the blood by cells is
regulated by insulin. Impairment of glucose tolerance (i.e., longer time to clear given amount
of glucose) indicates problems with maintenance of glucose homeostasis (insulin resistance,
carbohydrate metabolism, diabetes etc.).[4]

8.2.2 Materials & Reagent

➢ Hand gloves ➢ Forceps

➢ 1 ml syringe ➢ Needle
➢ 3 ml syringe ➢ Glucometer & strips
➢ Balance ➢ Powdered glucose
➢ Feeding needle ➢ Tween-80
➢ Beaker ➢ Glibenclamide
➢ Burner ➢ Distilled water

8.2.3 Experimental animal

For the present study, young Swiss-albino mice aged 4-5 weeks; an appropriate average
weight 20-40 gm was used. The mice were procured in Jahangirnagar University, Savar,
Dhaka-1342, Bangladesh. They were kept in standard environmental condition at an ambient
temperature. The animals were acclimatized to laboratory condition for one week prior to
experimentation.

8.2.4 Study Design

The experimental animals were tested in a fasting state (having no food or drink except water
for at least 10 hours but not greater than 16hours). The mice were randomly selected and
divided into four groups denoted as group-I, group-II, group-III and group-IV consisting of 5
mice in each group. Each group received a specific treatment i.e. Control, reference and the
test sample. Each mouse was weighed properly and the doses of the test sample, reference
and control substances were adjusted accordingly. The statistical significance threshold was
set at 0.05. Experimental profiles to assess the anti-hyperglycemic activity is given in table
8.1

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 Chapter 8: Evaluation of Anti-hyperglycemic Activity

Table 8.1: Experimental profiles to assess the anti-hyperglycemic activity

Animal Group Treatment Dose Route of administration

I 1% tween-80 10 ml/kg Oral

(Negative control) in water

II Glibenclamide 5 mg/kg Oral

(Positive control)

III 250 mg/kg Oral

(Test group-1) C. arborea extract

IV 500 mg/kg Oral

(Test group-2) C. arborea extract

8.2.5 Preparation of sample suspension

8.2.5.1 Preparation of test sample

To prepare the test sample at the doses of 500 mg/kg and 250 mg/kg bodyweight, 500 mg and
250mg of crude ethanol extract was measured. The extract was triturated in unidirectional
manner by the addition of 1% tween-80 as suspending agent. After proper mixing of extract
and tween-80, the distilled water was slowly added. The final volume was adjusted up to 10
ml. To stabilize the suspension, it was stirred well by vortex mixer.

8.2.5.2 Preparation of standard (reference) sample

For the preparation of standard Glibenclamide at the dose of 5 mg/kg body weight, 5 mg
equivalent Glibenclamide was measured and triturated with small amount of tween-80 and
then adjusted with distilled water to make final volume 10 ml.

8.2.5.3 Preparation of control

1 % tween-80 was mixed with 10 ml of distilled water which was used as control solution.

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 Chapter 8: Evaluation of Anti-hyperglycemic Activity

8.2.5.4 Preparation of glucose solution

For the preparation of glucose solution at the dose of 2 g/kg body weight, 2 gm glucose was
measured in the electric balance and dissolved in 10 ml of distilled water.

8.2.6 Working procedure

1. At first the mice were randomly selected and then weighed for control, reference and
test group.
2. After the selection and weighing the mice for control, standard and test groups the test
samples, standard and control solution, according to the respective dose, were given
orally by means of a feeding needle.
3. After 30 minutes later, to all groups (2gm/kg body weight) glucose solution was
administered orally with the help of a feeding needle.
4. Blood glucose levels were measured at 0 minutes, 30 minutes, 60 minutes and 90
minutes after glucose administration.
5. For determination of blood glucose level, blood sample was collected by cutting the tail
tips with a sharp blade. After collection of blood, the tail tips were exposed to povidone
iodine ointment to counteract the possibility of infection and inflammation. The blood
glucose level is determined by using the glucometer and compatible blood glucose
strips. The blood glucose level was measured in millimole per liter (mM/L).

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 Chapter 8: Evaluation of Anti-hyperglycemic Activity

8.3 Data Presentation

Oral Glucose Tolerance Test result of C. arborea extract is given in table 8.2

Table 8.2: Oral Glucose Tolerance Test result of C. arborea extract

Treatment Blood glucose level (m Mol / L)
(Per kg body weight)
At 0 min At 30 min At 60 min At 90 min
I 2.3 16.2 14.5 14.7

Negative Control 2.6 21.0 17.8 16.9

1% tween-80 in water 3.2 19.7 14.5 14.3
4.6 18.3 14.7 14.3
(10 ml/kg)
5.1 20.6 15.8 15.5

Average ± SEM 3.56±0.552 19.16±0.873 15.46±0.633 15.14±0.492

II 2.3 15.9 9.3 6.9

2.2 12.8 7.2 5.2

Positive control
3.5 13.5 8.3 6.0
Glibenclamide
4.7 14.3 10 6.4
(5 mg/kg)
3.9 15.8 8.5 5.9

3.32±0.478 14.46±0.615 8.66±0.474 6.08±0.282

Average ± SEM ** *** ***

2.9 18.6 11.2 10.3

III 2.4 20.2 12.8 12.3

3.9 18.0 12.5 10.6
C. arborea Extract (250
2.1 18.1 14.1 11.3
mg/kg)
3.2 17.6 13.3 10.9

2.90±0.315 18.50±0.454 12.78±0.479 11.08±0.347

Average ± SEM ** ***

2.3 14.7 14.1 8.9

IV 2.9 16.2 12.3 8.6

C. arborea Extract (500 2.8 17.4 11.6 7.2
mg/kg) 2.8 13.2 9.8 6.7
2.7 14.5 9.2 9.3

2.70±0.105 15.20±0.727 11.40±0.881 8.14±0.505

Average ± SEM ** ** ***

Significance: * P<0.05, ** p<0.01, ***p<0.001

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 Chapter 8: Evaluation of Anti-hyperglycemic Activity

Blood glucose level vs Time

25

20

**

*
*
Gluco
15
se

***
*
*
**
level
*
*
*

*
*
*
10

*
*
*
5

0
0 min 30 min 60 min 90 min
Time

Negative Control Positive Control C. arborea(250 mg) C. arborea(500mg)

Significance: * P<0.05, ** p<0.01, ***p<0.001

Fig. 8.1: Blood glucose level verses time

8.4 Result & Discussion

The results of the test showed that C. arborea extract at dose of 250 mg/kg and 500 mg/kg
exhibited significant clearance of glucose level from blood after oral ingestion of sample in
glucose loaded animal. Both doses of extract revealed comparable results against the standard
Glibenclamide drug. Significant activity was observed specially at 60 & 90 min in both
doses.

The oral glucose tolerance test does not distinguish between insulin resistance in peripheral
tissues and reduced capacity of the pancreas beta-cells to produce insulin. It only defines the
ability of the tested material about the clearance of glucose from blood.

8.5 Conclusion

The results of the present study indicated that C. arborea extract was found to reduce the
glucose level in glucose loaded animals. The active ingredients in the extract which reduce
the blood sugar was not known through the present study. Further investigation can be done
on knowing the active ingredients responsible for blood sugar reducing.

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 Chapter 8: Evaluation of Anti-hyperglycemic Activity

8.6 References

1. Taylor, C.R., et al., Improving diabetic retinopathy screening ratios using

telemedicine-based digital retinal imaging technology: the Vine Hill study. Diabetes
care, 2007. 30(3): p. 574-578.
2. Lyra, R., et al., Prevenção do diabetes mellitus tipo 2. Arquivos Brasileiros de
Endocrinologia & Metabologia, 2006. 50(2): p. 239-249.
3. Junejo, J.A., et al., Antidiabetic activity of hydro-alcoholic stem bark extract of
Callicarpa arborea Roxb. with antioxidant potential in diabetic rats. Biomedicine
pharmacotherapy 2017. 95: p. 84-94.
4. Joy, K. and R.J.J.o.E. Kuttan, Anti-diabetic activity of Picrorrhiza kurroa extract.
Journal of Ethnopharmacology, 1999. 67(2): p. 143-148.

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CHAPTER nine
DISCUSSION
 Chapter 9: Discussion

CHAPTER NINE
Discussion

The present work presented here was conducted to analyze some phytochemical and
pharmacological properties of leaves of C. arborea. Different parts of C. arborea are
traditionally used in the treatment of fevers, influenza, healing sores, boils on the tongue, skin
complaints, cutaneous diseases, stomachache, indigestions.[1] [2] There are some research article
on wound healing properties of leaves and anti-diabetic, anti-microbial, anti-oxidant, analgesics
properties of stem bark.[3] [4] [5] [6] So, present project work was designed to reveal medicinal
properties of leaves that were yet to be screened.

The leaves of C. arborea were collected from Laltila of Moulvibazar. During collection, any
type of adulteration was strictly prohibited. It was identified by experts at Bangladesh National
Herbarium, Mirpur, Dhaka, where a voucher specimen was submitted for future reference. The
collected sample was subjected to shade drying. After proper grinding and cold extraction with
ethanol, the yield was about 12% for C. arborea.

Phytochemical screening was conducted to reveal major functional groups in the mother
extract. There found no previous phytochemical screening data. The conducted phytochemical
screening revealed the presence of reducing sugar, alkaloids, flavonoids, tannins, phenolics,
glycosides, steroids terpenoids, carbohydrates, saponin and gums.

Qualitative antioxidant assay was performed by using thin layer chromatography (TLC)
technique in different solvent systems followed by DPPH spray. Upon DPPH spray, antioxidant
active compounds showed yellow spots. However, upon 10% H 2SO4 spray, H2SO4 reactive
compounds showed black spots.

Quantitative antioxidant study was carried out with DPPH scavenging assay, determination of total
phenolic, flavonoid, tannin & total antioxidant content. Previous study revealed a significant free
radical scavenging activity of stem bark with SC 50 values of 41.53, 14.59 and 71.2 μg/ml,
respectively which was demonstrated using the ethanol extract, it's pet ether and chloroform soluble
partitionates.[5] In the present study of DPPH scavenging assay, ethanolic extract of C.

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 Chapter 9: Discussion

arborea displayed a free radical scavenging activity with a value of SC 50 34.04 µg/m which is
comparable to that of ascorbic acid (SC 50 14.26 µg/ml), a well-known standard antioxidant.
However, total phenolic, flavonoid, tannin & total antioxidant content weren’t revealed yet. In the
present study, the amount of total phenolic content was determined with the Folin-Ciocalteu reagent
where gallic acid was used as standard and expressed as mg GAE/g of dried plant material. The
absorbance was taken at 765 nm and found a value of 158 mg GAE/g. In the determination of total
flavonoid content, aluminum chloride colorimetric method was applied where quercetin was used as
standard and expressed as mg QE/g of dried plant material. The absorbance was taken at 510 nm and
found a value of 941 mg QE/g. However, the amount of total tannin content was also determined
with the Folin-Ciocalteu reagent where gallic acid was used as standard and expressed as mg GAE/g
of dried plant material. The absorbance was taken at 725 nm and found a value of 193 mg GAE/g.
Finally, the amount of total anti-oxidant content was determined using phosphomolybdate method
where ascorbic acid was used as standard and expressed as mg AAE/g of dried plant material. The
absorbance was taken at 695 nm and found a value of 323 mg GAE/g.

Analgesic activity test of C. arborea leaves extract was performed using acetic acid induced
writhing method. The previous study performed by Shihan et al.[5] revealed that in the acetic acid-
induced writhing inhibition assay of stem bark of C. arborea, the ethanol extract and its chloroform
and ethyl acetate soluble fractions at a dose of 200 mg/kg body weight significantly inhibited
writhing response with 42.7%, 70.3% and 32.8% of inhibition, respectively which may be
responsible for terpenoids and steroidal compounds. At the present study, the results of the test
showed that ethanolic extract of C. arborea leaves at the dose of 250 mg/kg and 500 mg/kg body
weight exhibited inhibition of writhing reflex by 44.12 % and 58.82 %, respectively. However, the
standard drug Diclofenac Na was found to have an inhibition of 66.18 % at a dose of 25 mg/kg body
weight. So, comparable result at different doses with the previous study was noticed.

Anti-inflammatory activity test of C. arborea leaves extract was performed using formaldehyde
induced paw edema method. There was no exploration on anti-inflammatory activity of C. arborea
yet. However, in present study, ethanolic extract of C. arborea leaves showed anti-inflammatory
activity at the doses of 250 and 500mg/kg in the observed 10 days where indomethacin (10mg/kg)
was used as the standard drug. It was found that dose of 500mg/kg showed more anti-inflammatory

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 Chapter 9: Discussion

activity than dose of 250mg/kg & most of the values were significant while comparing with the
negative control group.

Anti-pyretic activity test of C. arborea leaves extract was performed using the yeast-induced
pyrexia method. Previously, anti-pyretic activity of C. arborea was unexplored. In present study,
ethanolic extract of C. arborea leaves showed anti-pyretic activity at the doses of 250 and
rd th
500mg/kg where paracetamol was used as the standard drug. During 3 and 4 hr. The extract
st
showed much activities while significant activity wasn’t observed in 1 two hours.

Anti-hyperglycemic activity of C. arborea leaves extract was tested using the model of oral
glucose tolerance. Previously, antidiabetic activity of hydro-alcoholic stem bark extract with
antioxidant potential in diabetic rats was revealed by Junejo, J.A., et al. [7] In that study,
streptozotocin-induced diabetic rats were given dose 250 and 500 mg/kg body weight orally and
found significant hypoglycemic activity compared to normal control group in experimental rats.
The result also revealed that it may have possible role as herbal antioxidants in the treatment of
oxidative stress-induced diabetes mellitus. However, the results of the present test showed that C.
arborea leaves extract at dose of 250 mg/kg and 500 mg/kg exhibited significant clearance of
glucose level from blood after oral administration of sample in glucose loaded animal. Both
doses of extract revealed comparable results against the standard Glibenclamide drug. Significant
activity was observed specially at 60 & 90 min in both doses.

Finally, we can say that the ethanolic extract of C. arborea leaves possess some phytochemical
& pharmacological activities.

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 Chapter 9: Discussion

Reference

1. Manandhar, N.P., Plants and people of Nepal. 2002: Timber press.

2. Wiart, C., Medicinal plants of the Asia-Pacific: drugs for the future? 2006: World
Scientific.
3. Kar, P.K., et al., Evaluatio of wou d-heali g activity of leaves of urtica parviflora roxb ad
Callicarpa arborea roxb i rats. Pharmacologyonline, 2009.
4. Junejo, J.A., et al., Antidiabetic activity of hydro-alcoholic stem bark extract of
Callicarpa arborea Roxb. with antioxidant potential in diabetic rats. Biomedicine
pharmacotherapy, 2017. 95: p. 84-94.
5. Shihan, M.H., et al., Pharmacological Evaluation of Stem Bark of Callicarpa arborea
Roxb. Dhaka University Journal of Pharmaceutical Sciences, 2015. 14(1): p. 111-116.
6. Roy, S., et al., Screening of Callicarpa arborea and Hemigraphis alternata for
antibacterial activity. Science Vision, 2020.
7. Junejo, J.A., et al., Antidiabetic activity of hydro-alcoholic stem bark extract of
Callicarpa arborea Roxb. with antioxidant potential in diabetic rats. Biomedicine
pharmacotherapy 2017. 95: p. 84-94.

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CHAPTER ten
CONCLUSION
 Chapter 10: Conclusion

CHAPTER TEN
Conclusion

In the present investigation, the ethanolic extract of C. arborea leaves has proven some
phytochemical & pharmacological activities.

Phytochemical analysis showed that leaves of C. arborea contained reducing sugar, alkaloids,
flavonoids, tannins, phenolics, glycosides, steroids, terpenoids, carbohydrates, saponin and
gums. It showed the presence of anti-oxidant compounds in TLC and the quantitative values of
anti-oxidant properties were evaluated by DPPH free radical scavenging assay, TPC, TFC, TTC
and TAC. Pharmacological assessments revealed that leaves of C. arborea possess good
analgesic, anti-inflammatory, anti-pyretic and anti-hyperglycemic activities.

The present study included several screenings and revealed that anti-oxidant, analgesic, anti-
inflammatory, anti-pyretic and anti-hyperglycemic activities are present in the leaves of the
plant. However, more pharmacological assessment involving isolation of bioactive compounds
and identification of their mechanism of actions are crucial for further study in this plant.

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CHAPTER eleven
APPENDIX
 Chapter 11: Appendix

CHAPTER ELEVEN
Appendix
Xn
1. Arithmetic Mean ( X ) =
Where, ΣX = Summation of Observed Value
n = No. of Observation

2. Standard Deviation (SD) = (X − X)2

n
Where, X = individual Value

X = Mean Value
n = No. of Observation

3. Standard Error (SE) = SD

(n −1)
Where, SD = Std. Deviation
n = No. of Observation
4. Student’s t-Test
t= M1−M2
(SE )2 + (SE )2
1 2

Where, M1 = Mean Value of Group- 1 (negative Control)

M2 = Mean Value of Group- 2 (Test)
SE1 = Std. Error of Group- 1
SE2 = Std. Error of Group- 2

5. Degree of Freedom = (n1- 1) + (n2-1)

Where, n1 = Number of observation’s of 1st sample n2
= Number of observation’s of 2nd sample
Or n*= (n-1), where, n=Number of mice in that group

6. % of Writhing = (Mean of Test / Mean of Control) Х 100

7. SE for % Writhing = (SE / Control Mean) X 100

8. % inhibition = {(Blank OD- Sample OD) / Blank OD} × 100

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