IJMS 2021 vol.

8(1): 37-58
International Journal of Multidisciplinary Studies (IJMS)
Volume 8, Issue 1, 2021

Qualitative Phytochemical Screening, Anti-Bacterial Activity and

TLC Profiling of Different Parts of Three Medicinal Plants
S. Arasaretnam1*, H. R. P. Prasadini1 and U. Mathiventhan2

1
Department of Chemistry, Faculty of Science, Eastern University, Chenkalady, Sri Lanka
2
Department of Botany, Faculty of Science, Eastern University, Chenkalady, Sri Lanka

ABSTRACT

The aim of this study was to carry out phytochemical screening Anti-bacterial activity and TLC profiling
of sequentially extracted petroleum ether, dichloromethane, ethyl acetate and methanol extracts of leaf
and barks of the Nerium oleander, Leaf, bark and seeds of Cascabela thevetia and Cerbera odollum.
The phytochemical analysis of the extracts obtained from different solvents was carried and Ant-
bacterial activity was carried out for all the extractions at 25 mg/ml and 50 mg/ml test concentration
against E.coli and S.aureus bacteria by well diffusion method. All the tested extractions showed low
activity against both bacteria at 25mg/ml compared with 50mg/ml. Results were subjected to one way
(ANOVA) and followed by Tukey’s test (P < 0.05). All the extractions of all plant materials showed in
bioactivity against E.coli and S.aureus bacteria at 50 mg/ml among them. The statistical analysis
revealed that anti-bacterial activity of the ethyl acetate extracts of C.thevetia leaves and N.oleander
barks against E.coli bacteria and Methanol extract of Nerium oleander leaves and ethyl acetate extracts
of C.thevetia bark against S.aureus bacteria were comparable with the positive control Amoxicillin.
TLC profiling was carried out using a various solvent system of varying polarity for all sequential
extractions and obtained different retention factor (Rf) values of different phytochemicals.

KEYWORDS: Phytochemical analysis, Antibacterial activity, Thin-layer chromatographic

profiling, Sequential extractions, Cascabela thevetia and Cerbera odollum

Corresponding author: S. Arasaretnam, Email: s_arasaretnam@yahoo.co.uk

S. Arasaretnam et al.

1 INTRODUCTION treatment of Nervous system, Hemiplegia,

Epilepsy and skin diseases like Leprosy or
Plants have the capability to biosynthesize ringworm infection (Dey & Chaudhuri
a wide variety of chemicals, some of which 2014). Extract of the plant also possesses
play an important role in primary anti-bacterial and anti-fungal activities.
metabolic activities while others are part of The bark of the plant is used as intermittent
plant’s secondary metabolism. fever and cathartic, febrifuge and because
Phytochemicals are biosynthesized as of its toxic nature, the powerfully resolving
secondary metabolites, which are being roots are used in the form of plasters and is
synthesized in various parts of the plant usually applied externally and have been
such as a leaf, bark, roots, fruits and stem used against corns, warts, cancerous ulcer,
etc. (Pichersky & Gang 2000) These carcinoma, hard tumours. (Alobaidi 2014)
phytochemicals are the basic sources of
pharmaceutical industries and also used for The cardiac glycosides obtained from bark,
identifying the crude drugs and also kernels and flowers of Cascabela thevetia
phytochemical screening is important for are useful for heart diseases. The plant or
identification of therapeutically and its individual parts can be used for the
industrially important compounds such as treatment of various disorders in human
alkaloid, steroid, etc.(Akindele & beings such as diabetes, liver toxicity
Adeyemi 2007). fungal infection, microbial infection,
inflammation, pyrexia and relive pain
The phytochemical screening is mainly (Laphookhieo et al. 2004)
applied to the quality control of traditional
medicine. Thus, it is anticipated that Cerbera odollum is also known for various
phytochemicals with adequate anti- medicinal properties. The oil from the
bacterial efficacy will be used for the seeds is used as a cure for itching or
treatment of bacterial infections. applied to the hair as an insecticide.
(Renisheya et al. 2011). Even at present, (Kirtikar & Basu 1918) The bark and leaf
medicinal plants play a major role in the of the plant are traditionally used as emetic
primary health care of many developing and cathartic. Kernels are used as an
countries. (Vaghasiya et al. 2009) emetic. The fruit is used as a cure for
Therefore, in order to isolate and hydrophobia. (Rollet 1981) Its bark and
characterize novel antimicrobial fruits are purgative and used for the
compounds, scientists are interested in treatment of rheumatism. (Ahmed et al.
screening on medicinal plant extracts. 2006), (Guruswami et al. 1970) The
extraction methods of plant materials may
Nerium oleander, Cascabela thevetia, and influence the inhibitory potentiality when
Cerbera odollum belong to Family testing against microbes (Valgas &
Apocynaceae. All parts of these plants are Machado 2007). The sequential extraction
poisonous. Also, they play a vital role in technique has become a popular method
traditional medicine. The leaves of Nerium for the extraction of active components
oleander are most commonly used for the from natural sources as the sequential

38
 Qualitative Phytochemical Screening, Anti-Bacterial Activity and TLC Profiling of Different Parts of
Three Medicinal Plants

extraction procedure involves different from the shell and removed remnants outer
solvents of various polarities that can shell, twinges and pebbles. Then seeds
provide the optimum effect of extraction were opened manually to obtain kernels.
and better activity than direct solvent All the plant parts of plants were washed
extraction (Ken & Stevenson 2007) thoroughly with running tap water
(freshwater) to remove dust particles,
In the present study, it was attempted to contaminants, impurities and then all the
test the antibacterial activity of different samples were transported to the laboratory
solvent extractions of leaves, seeds and at the Department of Chemistry, Eastern
bark of three plants in family Apocynaceae University Sri Lanka, and then, all the
including Nerium oleander, Cascabela samples were finally cleaned with distilled
thevetia, and Cerbera odollum obtained by water. Collected plant materials were
sequential extraction methods tested authenticated using herbarium, at the
against gram positive and gram-negative Department of Botany, Eastern University
bacteria and also studied the Sri Lanka, and Voucher specimens were
phytochemical constituents present in the deposited in the herbarium of the
test extracts and TLC profile of each Department of the Botany, Eastern
extracts. The obtained results provide University, Sri Lanka. Then all the samples
support for the use of these plants in were air-dried for ten days until a constant
traditional medicine and further weight was achieved. Then the samples
investigation and isolation of were ground to a 0.5 mm particle size
Phytoconstituents. powder and weighed using electrical
balance and stored in airtight container.
2 MATERIALS AND METHODS

2.1 Samples collection

Three different plant species belonging to

family Apocyanacea were collected from
Puttalam district, Sri Lanka [Fig. 1]. The
aerial parts of the plant (Leaves, Barks) in
Nerium oleander plant and leaves, bark
and seeds of the plants in Cascabela
thevetia and Cerbera odollum plants were
collected from Puttalam district during the
month of November- December. The seeds
of Cascabela thevetia and Cerbera
odollum plants were carefully separated
Figure 1: Map of plant samples collection
locations (Bangadeniya, Mahawewa, and
Madurankuliya)

39
S. Arasaretnam et al.

2.2 Plant samples

Table 1: Abbreviation of Different part of different plant used for the study

Plant samples Abbreviations

Nerium oleander leaves NOL
Nerium oleander bark NOB
Cascabla thevetia leaves CTL
Cascabela thevetia bark CTB
Cascabela thevetia seeds CTS
Cerbera odollum leaves COL
Cerbera odollum bark COB
Cerbera odollum seeds COS

2.3 Preparation of plant extracts 2002). The yield percentage of each extract
was calculated using the results obtained.
Each powdered plant part of NOL, NOB,
CTL, CTB, CTS, COL, COB and COS 2.4 Phytochemical analysis
(Table 1) was successively extracted with
different organic solvents in increasing The phytochemical analysis involved
polarity order. 50 g of each powder was standardized chemical test in evaluating
macerated separately in 250 ml of light presence or absence of several active
petroleum ether (PE) with intermittent phyto constituents such as alkaloids,
shaking for two days. Then they were first flavonoids, Terpenoids, Steroids,
filtered through double-layer muslin cloth Saponins, Phenols, Tannins, Quinone,
Glycosides and Cardiac glycosides. All
and then using Whatman no 1 filter paper.
the test samples were subjected to analysis
The residue was further extracted three using modified and standard protocols.
times by using the same fresh solvent and (Maung Tin-Wa et al. 1960), (Sofowora
all the filtrates were pooled together. The 1993), (Evans 1996).
resulting residue was air-dried and further
extracted with Dichloromethane (DCM) 2.5 Bacterial culture
and followed by Ethyl acetate (EA) and
Methanol (MeOH) similar to the procedure Gram positive bacteria Staphylococcus
carried out for the PE extraction. Finally, aureus and Gram-negative bacteria,
from each 32 filtrates the solvent was Escherichia coli were kindly provided by
removed by using a rotary evaporator the Department of Microbiology, Faculty
under reduced pressure and low of Medicine, Eastern University, Sri
temperature. The yield of each crude Lanka. All the test bacteria were
extracts was weighed and stored at 4 °C maintained on nutrient agar slope at 4 ºC.
until used (Giridharan & Somasundaram Then, these were subcultured for 24 hours
before use.

40
 Qualitative Phytochemical Screening, Anti-Bacterial Activity and TLC Profiling of Different Parts of
Three Medicinal Plants
2.6 Preparation of bacteria cultures 2.8 Statistical analysis

Stock cultures were maintained at 4 °C on The diameter of zone of inhibition of

slopes of nutrient agar. Active cultures of replicates (four samples) was expressed as
the experiment were prepared by Mean ± standard deviation (SD). The data
transferring a loop full of cells from stock were subjected to One- way analysis of
cultures to nutrient agar medium plate for variance (ANOVA), followed by Tukey’s
preparation of subculture and those were test (P < 0.05) by using software Minitab
incubated for 24 hours at 37 °C. A 18 to 24 17 for windows version.
hours. Single colonies of sub cultured agar
plates were used to prepare bacterial 2.9 Thin layer chromatography analysis
suspension with turbidity of 0.5 for sequential plant extracts
McFarland (equal to
8
1.5× 10 colony forming units CFU/ml). For TLC analysis, plate with Silica gel 60
F254 TLC (Merck, Germany), 10×10 cm
2.7 Antibacterial screening
was cut with ordinary household scissors.
Plate markings were made with a soft
In vitro antibacterial activity of different pencil. Glass capillaries were used to spot
crude extracts obtained by sequential the sample for TLC applied sample volume
extraction was studied using agar well 1-μl of the sample by using capillary at a
diffusion method (Nair & Kalariya 2005). distance of 1 cm and developed in a TLC
Autoclaved nutrient agar was cooled down chamber using suitable mobile phase. The
to 40 °C, and around 15-20 ml of Nutrient developed TLC plates were air-dried and
agar was poured on glass Petri dishes and observed under ultraviolet light UV at both
allowed to solidify. Then 1 ml of bacterial 254 nm and 366 nm and exposed to freshly
suspension (1× 60 cells/ml) was poured prepared anisaldehyde spaying reagent and
into agar surface and spread by sterile glass dried for 1 minute for the development of
spreader. Wells were made on it using a colour in separate bands. The movement of
sterile cork borer of 5 mm diameter, and the analyte was expressed by its retention
each well was filled with 100 μl of each factor (Rf).
extract of test samples at 50 mg/ml test
concentration. Then, the plates were The different solvent systems used to
allowed to standby for 30 min and were observe the TLC profiles were chloroform,
incubated at 37 °C for 24 hours. DCM ethanol and 5 % methanol in
Amoxicillin was used as a reference. The acetonitrile, 2 % methanol in acetonitrile,
antibacterial activity was recorded by 1 % methanol in acetonitrile and 0.5 %
measuring the zone of inhibition in mm. methanol in acetonitrile.
around the well. Each experiment was
replicated twice and the mean value was
calculated.

41
S. Arasaretnam et al.

3 RESULTS & DISCUSSION

3.1 Weight of each plant material during ten days of air-dry.

Table 2: Weight of each plant material in each day during air dry.

Days Nerium oleander Cascabela thevetia Cerbera odollum

Leaves Bark Leaves Bark Seeds Leaves Bark Seed
Kernels Kernels
Day 198.50 142.60 309.88 531.58 180.29 317.05 566.93 185.21
01
Day 167.55 130.64 289.09 516.45 178.15 315.05 547.52 174.91
02
Day 159.54 118.29 250.48 475.54 176.02 295.65 511.25 172.55
03
Day 149.39 99.89 214.31 432.79 175.30 195.65 478.35 170.66
04
Day 126.54 95.76 150.65 398.98 174.09 180.70 254.45 164.73
05
Day 115.65 93.20 147.98 248.97 173.00 120.87 198.67 162.02
06
Day 90.91 91.97 142.80 201.56 171.95 110.54 187.50 159.43
07
Day 89.39 90.90 138.98 190.87 169.30 105.98 173.87 158.40
08
Day 84.70 89.56 130.56 184.78 169.00 104.80 171.29 157.35
09
Day 80.65 89.19 129.98 183.01 169.00 101.15 170.56 157.30
10

Drying and weighing were continued at the materials and reduce its water activity,
same time interval until the constant which inhibits microbial growth and
weight was obtained. Normally, the air minimize deteriorative biochemical
drying takes from 3-7 days to months reactions (Buchaillot et al. 2009).
depending on the types of samples dried. However, drying can have adverse effects
According to these results, weight loss of on phytochemical and nutritional
each plant material during the days 9 and components, especially heat-sensitive
10 days were negligible, and they were compounds (Hajimehdipoor et al. 2012).
taking almost 10 days to get constant There are different drying methods such as
value(Table 2). Drying is a process to freeze-drying, hot air drying, vacuum
remove moisture from fresh plant drying and microwave drying. These

42
 Qualitative Phytochemical Screening, Anti-Bacterial Activity and TLC Profiling of Different Parts of
Three Medicinal Plants
drying methods are linked with various for specific type of plant samples. The
energy consumption, and have a present study used the air-dried (Shade
significant effect on the phytochemicals of drying) method to dry the plant materials.
the samples (Nadi & Mey 2017).
Therefore, it is necessary to identify the
most suitable drying method and condition
Table 3: Yield percentage of crudes of sequentially extracted each plant extracts

Extract Percentage yield (%)

type NOL NOB CTL CTB CTS COL COB COS
PE 1.90 3.06 3.54 2.90 28.3 5.92 2.14 30.82
DCM 2.30 1.02 2.80 7.10 10.70 2.32 0.82 3.80
EA 4.20 1.08 3.02 1.28 2.78 1.50 2.44 3.66
MeOH 5.46 8.02 5.48 6.98 6.42 0.30 8.22 7.12

The objective of the sequential extraction flavonoids because these plant materials
process is to separate bioactive fractions contain high level of polar compounds that
according to the polarity of each are soluble in the solvent with high polarity
compound from different solvents which such as MeOH and EA. But in the case of
have different polarity. In the present CTS, COL and COS in PE extract showed
study, the extraction of each plant material higher yield percentage than other extracts
was done sequentially using solvents from solvents. This may be due to high level of
low polarity to high polarity in order of PE, non-polar compounds rather than polar
DCM, EA, and MeOH, which is high polar compounds. Thus, the plant materials
solvent. which were extracted from PE extract
According to the present study, result yield except CTS, COL and COS showed low
percentage (Table. 3) of each plant yield and low yield percentage. This could
material from sequential extractions be due to low level of non-polar
demonstrated that various solvents bring compounds that are soluble in non-polar
about different extraction yields. This is solvents such as PE. Yield percentage of
because differences in polarity of the different solvent extracts obtained from
extraction solvents could cause a wide sequential extraction clearly revealed that
variation in the level of bioactive fractions compounds dissolved in polar solvents are
in the extract. MeOH and EA extracts of quantitatively higher than compounds
NOL, NOB, CTL, CTB and COB showed dissolved in low polar solvents in most of
higher percentage yield. This could be due the plant materials. (Table 3)
to the presence of higher number of

43
 Qualitative Phytochemical Screening, Anti-Bacterial Activity and TLC Profiling of Different Parts of
Three Medicinal Plants

3.2 Phytochemical analysis tests

Table 4. Results of phytochemical analysis of Nerium oleander Plant

Phytochemicals Name of the test NOL NOB

MeOH

MeOH
DCM
DCM

EA
PE
EA
PE
Alkaloids Wagner’s reagent + + + + + + + +
Mayer’s reagent - - - - - - - -
Confirmatory test + + + + + + + +
Flavanoids Conc HCl - + + + - + + -
NaOH - + - - - + - +
Conc H2SO4 - - + + + - + -
Tri-terphenoids Liebermann- - + - - - - + -
Burchard test
Unsaturated Steroids Liebermann- + - + - - + - -
Burchard test
Salkowski test + + + + + + + +
Saponins Froth test - - - + - - - -
Phenols Lead acetate test - - - + - - - +
Ferric chloride test - + + + - - + +

Tannins Ferric chloride test - + - + - + + +

Glycoside Liebermann’s test - + + + - - - -

Cardiac Glycoside Killer-kilani test + + + + + + + +
Quinone Conc H2SO4 + + + + + + + +
Total number of phytochemicals 5 9 7 9 6 6 8 7
Note. PE-Petroleum Ether, DCM-Dichloromethane, EA-Ethyl Acetate, MeOH-Methanol

Table 5: Results of phytochemical analysis of Cascabela thevetia Plant

Name of the CTL CTB CTS

Alkal Phytoche

test
MeOH

MeOH

MeOH
oids micals

DCM

DCM

DCM
EA

EA

EA
PE

PE

PE

Mayer’s - - - - - - - - - - - -
reagent

45
S. Arasaretnam et al.

Wagner’s + + + + + + + + + + + +
reagent
Confirmato + + + + + + + + + + + +
ry test
Conc HCl - + + - - - + + - - + +
Flavan
oids

NaOH - + - - - + + + - + - -
Conc - - - - + - - - + - + -
H2SO4
Lieberman - - - - + + + - + + - -
Triterph
enoids

Burchard
test
Lieberman + + + - - - - + - - + +
Burchard
Sapo Unsaturated

test
steroids

Salkowski + - - - + + - - + + - -
test
Froth test - - - + - - - - - - - -
nin

Lead - + - + - - - + - - - +
acetate test
Glyc Tannin Phenols

Ferric - + + + - - - - - - - -
chloride test
Ferric - - - + - - - - - - - -
chloride test
Glycoside oside s

Liebermann - - + + - - - - - - + +
’s test
Killer- + + + + + + + - + + + +
kilani test
Quinones Cardiac

Conc - - - + + + - + + + + +
H2SO4

Total number of 3 5 7 8 6 6 4 5 6 6 6 7
phytochemicals

46
 Qualitative Phytochemical Screening, Anti-Bacterial Activity and TLC Profiling of Different Parts of
Three Medicinal Plants

Table 6. Results of phytochemical analysis of Cerbera odollum Plant

Name of the COL COB COS

Phytoch
emicals

test

MeOH

MeOH

MeOH
DCM

DCM

DCM
EA

EA
PE

PE

PE
EA
Mayer’s
- - - - - - - - - - - -
reagent
Wagner’s
+ + + + + + + + + + + +
Triterph Flavanoi Alkaloids

reagent
Confirmator
+ + + + + + + + + + + +
y test
Conc HCl - - - + - - - + - - - +
NaOH - + + - - + + - - + - -
Conc H2SO4 - - - - + - + - + - + -
enoids ds

Lieberman
Burchard - - - - + + + - + + + +
test
Lieberman
Burchard + + + - - - - + - - - +
Sapo Unsaturated

test
steroids

Salkowski
+ - - - + + + - + + - -
test
Froth test
- - - + - - - - - - - -
nin

Lead acetate
- + - + - + + + - - - +
test
Phenols

Ferric
- + + + - + - + - - - -
chloride test

Ferric
Glyc Tann

- - - + - - - - - - - -
chloride test
oside ins

Liebermann
- - - - - - - - - - + +
’s test

47
S. Arasaretnam et al.

Killer-

ones Glycoside
Quin Cardiac kilani test + + + + + + + + + + + +

Conc H2SO4
+ - + + - + + + + + + +

Total number of
4 5 6 7 5 7 7 6 6 6 6 7
phytochemicals
According to the results of present study, phytochemicals. But in the case of cardiac
alkaloids were present in all the sequential glycosides and Quinone, all the extractions
extractions of all tested plant materials. of most of the plant part showed positive
The results revealed that Alkaloids content results for both phytochemicals. (Table 4)
of these plant materials was high. DCM
extracts of all the tested plant materials According to the results of the study of
showed presence of flavonoids and also qualitative phytochemical analysis among
most of the plant materials in EA and three plant Leaf of N. oleander showed
MeOH showed positive results for more availability of phytochemicals in the
flavonoids when compared with PE tested samples. And also, some
extracts. It revealed that the presence of phytochemicals were available in low
flavonoids in DCM, EA and MeOH polarity solvent and some were available in
extractions may be due to the polarity of a polar solvent. The present study of the
flavonoids. qualitative phytochemical analysis
revealed that availability of
More terpenoids and steroids are existing phytochemicals depends on plant type, part
in the PE extracts when compared with of the plant, the solvent used, the polarity
other extracts. In most cases, steroids and of the solvent, plant extraction method, and
terpenoids are soluble in the non-polar also depends on the polarity of the
solvent than in the polar solvents. For the phytochemicals. (Tables 4-6)
Saponin test, only the MeOH extracts of
NOL, CTL and COL showed positive 3.3 Anti-bacterial screening
results. It revealed that the solubility of
saponins was more effective compared The present study was carried out to
with polar solvents and also only the leaves evaluate the in vitro anti-bacterial activity
of all the tested plant showed positive of different parts of three different plant
results for saponin which may be a high materials (Table 7) which were
content of saponins in leaves of each plant sequentially extracted with different
compared with other plant parts. Phenol, organic solvents of PE, DCM, EA and
tannin and glycoside showed more MeOH in increasing polarity order using
availability in the DCM, EA MeOH agar well diffusion method (Sukanya
extracts when compared with PE extracts. 2009) at 25mg/ml and 50mg/ml test
It may be due to the polarity of the solvent concentration against the human pathogen
as well as the polarity of both such as gram-negative E.coli and gram-

48
 Qualitative Phytochemical Screening, Anti-Bacterial Activity and TLC Profiling of Different Parts of
Three Medicinal Plants
positive S.aureus. (The 25 mg/ml test utilized in this study showed a varying
concentration was selected from the degree of antimicrobial activity against the
preliminary investigation). Amoxicillin gram-negative E.coli and gram-positive
was used as a standard positive control. S.aureus.
The study showed that all plant extract
Table 7: Mean diameters of inhibition of each solvent extract of each plant material against
E.coli bacteria at 50 mg/ml

Plant type/ The diameter of inhibition zone of inhibition(mm)

standard
SE PE SE DCM SE EA SE MeOH
NOL 19.750 ±1.708 18.000 ±0.816 26.500±0.577 25.500±0.577
NOB 14.750 ±0.957 12.500 ±0.577 30.500±1.291 21.000±0.00
CTL 12.750 ±0.957 13.500 ±0.577 32.000±0.00 20.250±0.957
CTB 17.750 ±0.957 17.750 ±0.957 20.000±0.816 20.000±0.816
CTS 13.750 ±0.957 14.500 ±0.577 13.250±0.957 20.000±0.816
COL 19.750 ±0.957 16.500 ±0.577 16.000±0.816 23.750±0.957

COB 14.500 ±1.291 18.500 ±0.577 12.500±0.577 19.000±0.816

COS 20.000 ±1.826 16.500 ±1.291 19.750±0.957 19.000±0.816
Amoxicillin 32.25± 0.957

Note: Values are means of 4 replicates ± Standard deviation, SE-Sequentially extracted

Table 8. Mean diameters of Zone of inhibition against S.aureus bacteria of each solvent
extract of each plant material at 50mg/ml

Plant type/ The diameter of inhibition (zone of inhibition(mm)

Standard
SE PE SE DCM SE EA SE MeOH
NOL 18.250±0.957 18.000±0.816 26.500±0.577 29.250±4.50
NOB 12.500±1.291 18.000±0.000 30.500±1.291 22.000±0.816
CTL 12.500±1.291 33.000±0.00 32.00±0.00 21.500±1.291
CTB 17.500±0.577 25.250±1.258 34.000±0.816 20.750±1.708
CTS 12.500±1.291 33.000±0.000 13.250±0.957 21.000±0.816
COL 17.000±0.816 15.500±0.577 16.000±0.816 29.500±1.291
COB 13.000±0.816 16.000±0.000 12.500±0.577 24.000±0.816
COS 18.000±0.816 12.500±1.291 19.750±0.957 20.500±1.291
Amoxicillin 34.50±0.577
Note: Values are means of 4 replicates ± Standard deviation

49
S. Arasaretnam et al.

According to the statistical analysis from other PE extracts of NOB, CTL, CTS and
one way ANOVA test for anti-bacterial COB.
activity of different extracts which were
sequentially extracted with above- Among the mean diameter of inhibition of
mentioned solvent types against gram- SE DCM extracts against E.coli bacteria,
negative bacteria of E.coli and gram- N. oleander showed high zone of
positive bacteria S.aureus at concentration inhibition, but when considered leaf and
of 50 mg/ml showed the p-value was lower bark, Leaf showed the highest zone of
than 0.05. So, there is a significance inhibition (18.000 ±0.816). Plant parts of
(p=0.00) at 95% confident level among the C. thevetia showed zone of inhibition but
diameter of inhibition zone of each test considered leaf, bark and seeds; bark
sample which was extracted from the showed the higher diameter of inhibition
respective solvent for both bacteria. (Table (17.750 ±0.957). From the leaf, bark and
8) seeds of C. odollum, bark showed higher
effect (18.500 ±0.577). From that,
According to the one-way ANOVA Tukey sequentially extracted DCM extracts of
pairwise comparison, sequentially NOL, CTB, COB shows higher effect to
extracted PE extracts of the mean diameter inhibit E.coli when compared with NOB,
of inhibition against E.coli bacteria, N. CTL, CTS, COL and COS. From that
oleander showed zone of inhibition, but revealed most of the active semi-polar
when considering leaves and bark, leaves phytochemicals extracted to DCM solvent
showed highest inhibition zone (19.750 in each NOL, CTB, COB and these
±1.708). Among plant parts of C. thevetia compounds effective to inhibit the growth
also showed higher zone of inhibition of E.coli bacteria when compared with
considering leaves, bark and seeds, bark other plant materials.
has shown highest diameter of (17.750
±0.957). From the leaves, bark and seeds Among the mean diameter of inhibition of
of the C. odollum, both leaves and seeds SE EA extracts against E.coli bacteria, leaf
show a higher effect (19.750 ±0.957, and bark of N. oleander plant, NOB
20.000 ±1.826). From that NOL, CTB, (30.500± 1.291) showed higher effect.
COL and COS extracted from PE solvent Among the CTL, CTB and CTS in C.
by sequential extraction process showed thevetia plant CTL (32.000±0.00) showed
higher effect to inhibit E.coli when a higher diameter of inhibition. From the
compared with other plant materials. From SE EA extracts of leaf, bark and seeds of
that revealed most of the non-polar C. odollum, COS (19.750± 0.957) showed
phytochemicals extracted to PE extracts of a higher effect when compared with the
NOL, CTB, COL and COS and these other two. From that NOB, CTL and COS
compounds might be more effective to extracted from EA solvent by sequential
inhibit the growth of E.coli bacteria when extraction process showed highly effective
compared with compounds extracted to to inhibit E.coli when compared with other
plant materials. From that revealed most of
the higher active polarity phytochemicals

50
 Qualitative Phytochemical Screening, Anti-Bacterial Activity and TLC Profiling of Different Parts of
Three Medicinal Plants
extracted to EA solvent in each NOB, CTL S.aureus when compared with other plant
and COS and these polar compounds might materials. From that revealed, most of the
be effective to inhibit the growth of E.coli non-polar phytochemicals extracted to PE
bacteria when compared with compounds extract in each NOL, CTB and COS and
those extracted EA extracts of NOL, CTB, these compounds might be more effective
CTS, COL and COB. to inhibit the growth of S.aureus bacteria
Among the mean diameter of inhibition of when compared with compounds those
SE MeOH extracts against E.coli bacteria, extracted to PE extracts of NOB, CTL,
leaf and bark of N. oleander, NOL showed CTS, COL and COS.
(25.500±0.577) higher effect to inhibit the
growth of E.coli bacteria when compared Among the mean diameter of inhibition of
with NOB. Among CTL, CTB and CTS, SE DCM extracts against S.aureus bacteria
from them, all the extracts showed a higher of NOL and NOB of N. oleander plant,
diameter of inhibition (20.250±0.957, both of NOL and NOB shows a higher
20.000±0.816, 20.000±0.816). From the effect to inhibit (18.00±0.82
COL, COB and COS, higher effect was and18.000±0.00). Among CTL, CTB and
showed in COL (23.750±0.957). From that CTS, from each extract, CTL and CTS
SE MeOH extracts of NOL, CTL, CTB, showed a higher diameter of inhibition
CTS and COL showed higher effect to (33.000±0.00, 33.000±0.00). From the
inhibit E.coli when compared with other COL, COB and COS, higher effect showed
plant materials. From that revealed most of in COB (16.000±0.000). From that NOL,
the highest active polarity phytochemicals NOB, CTL, CTS and COB extracted from
extracted to MeOH solvent in each NOL, DCM solvent by sequential extraction
CTL, CTB, CTS, COL and these polar process showed highly effective to inhibit
compounds might be effective to inhibit S.aureus when compared with other plant
the growth of E.coli bacteria when materials. From that revealed most of the
compared with compounds those extracted active semi-polar phytochemicals
to MeOH extracts of NOB, COB and COS. extracted to DCM solvent in each NOL,
NOB, CTL, CTS and COB and these
Among the mean diameter of inhibition of compounds might be effective to inhibit
SE PE extracts against S. aureus bacteria, the growth of S.aureus bacteria when
N. olender shows a high effect of compared with compounds extracted to
inhibition, when considering leaf and bark, DCM extracts of CTB, COL and COS.
leaf shows the highest inhibition
(18.25±0.96). Among C. thevetia plant, Among the mean diameter of inhibition of
when considering leaf, bark and seeds SE EA extracts against S.aureus bacteria
from each extract CTB showed a higher of NOL and NOB of N. oleander plant
diameter of inhibition (17.500±0.577). NOB shows higher inhibition zone against
From the COL, COB and COS, higher S.aureus bacteria (30.500±1.291). Among
effect showed in COS (18.000±0.816). the SE EA extracts of CTL, CTB and CTS,
From that SE PE extracts of NOL, CTB from each extract CTB shows a higher
and COS show a higher effect to inhibit diameter of inhibition against S.aureus

51
S. Arasaretnam et al.

bacteria (34.000±0.816). From the COL, According to the Tukey test, among all the
COB and COS higher effect shows in COS extracts compared with their mean
(19.750±0.957). From that NOB, CTB and diameter of inhibition against E. coli and S.
COS extracted from EA solvent by aureus with standard amoxicillin and
sequential extraction process showed finally revealed that EA extracts of NOB,
highly effective to inhibit S. aureus when CTL against E.coli and MeOH extract of
compared with other plant materials. From NOL and EA extracts of CTB for S.aureus
that revealed most of the highly active were comparable with standard
polarity phytochemicals extracted to EA Amoxicillin.
solvent in each NOB, CTB, COS and these
polar compounds are effective to inhibit In the present study, the plant materials
the growth of S.aureus bacteria when were sequentially extracted with different
compared with compounds those extracted organic solvents in increasing polarity
of EA extract of NOL, CTL, CTS, COL order. The sequential extraction method
and COB. ensures the extraction of active compounds
from plant material according to their
Among the mean diameter of inhibition SE polarity. Previous studies reported that
MeOH extracts of NOL and NOB of N. Cold extraction and well diffusion method
oleander plant, NOL shows a higher effect was better when compared with Soxhlet
to inhibit the growth of S.aureus bacteria extraction and disc diffusion method.
(29.250±4.50). Among the SE MeOH During the Soxhlet extraction, due to the
extracts of CTL, CTB and CTS, all the high-temperature treatment, some of the
extracts of CTL, CTB and CTS show a active biomolecules might escape from the
higher diameter of inhibition against extract. It has also been reported that the
S.aureus bacteria (21.500±1.291, agar well diffusion method is better than
20.750±1.708, 21.000±0.816). From the the disc diffusion method, because, the free
COL, COB and COS, higher effect was hydroxyl groups present in the disc may
shown in COL (29.500±1.291) From that prevent the diffusion of cationic polar
NOL, CTL, CTB, CTS and COL extracted molecules. The present study
from MeOH solvent by sequential demonstrated the inhibitory effect of cold
extraction process showed highly effective sequential extracts of present plant
to inhibit S.aureus when compared with materials against test bacteria of E.coli and
other plant materials. From that revealed S.aureus at two different test
most of the highest polarity concentrations of 25 mg/ml and 50mg/ml.
phytochemicals extracted to MeOH When compared with the activity of both
solvent in each NOL, CTL, CTB, CTS and test concentrations, 25 mg/ml showed very
COL and these polar compounds were poor activity against both bacteria when
effective to inhibit the growth of S. aureus compared with 50 mg/ml test
bacteria when compared with NOB, COB concentrations. Only the PE, EA and
and COS in MeOH extracts. MeOH extracts of NOL and NOB, PE
extracts of CTL showed somewhat higher

52
 Qualitative Phytochemical Screening, Anti-Bacterial Activity and TLC Profiling of Different Parts of
Three Medicinal Plants
inhibition zone at 25mg/ml against E.coli extraction revealed that in the most cases,
bacteria when compared with other compounds that dissolve in polar solvents
extractions. And also, the diameter zone of (Table 3) were quantitatively higher than
inhibition of all the solvent extracts of each compounds that dissolve in low polar
plant materials at 25 mg/ml against solvents except CTS, COS. The efficacy of
S.aureus showed very poor results an anti-bacterial effect of plant material
compared with E.coli. From those results, depends not only on the type of solvent,
further statistical analysis was carried out type of assay and the dose used but also on
only considering the anti-bacterial activity the method of extraction of plant materials.
at 50 mg/ml.
From the results of present study, all the
According to the results of the present examined plant parts of each three plants
study, SE EA of CTL and NOB showed a of family Apocyanacea such as N.
higher diameter of inhibition for E.coli oleander, C. thevetia and C.odollum
bacteria when compared with standard showed anti-bacterial activity against
Amoxicillin and also SE MeOH of NOL E.coli and S.aureus bacteria. Among them,
and SE EA of CTB showed a higher leaves and barks of N. oleander and Leaves
diameter of inhibition for S.aureus bacteria and barks of C. thevetia showed more
when compared with standard effects for each bacterium. From that, it
Amoxicillin. From this result, it revealed revealed that these extracts have a good
that most of the polar solvent extracts of potential for developing bio-inspired anti-
MeOH and EA showed better activity than bacterial drugs. There is a good scope to
low polar solvents such as PE and DCM. develop natural drugs to fight against
The higher inhibitory effect of polar bacterial pathogens. But care should be
solvent extracts is due to more solubility of taken as these plants have toxic properties
active components in polar solvents and also. Further investigation is
the polarity of solvents may play an recommended for isolation and
important role in the inhibitory effect of development of non-poisonous anti-
plant extract. Yield percentage of different bacterial pharmaceutical compounds from
solvent extracts obtained from sequential crude extracts from these plant extracts.

53
S. Arasaretnam et al.

3.4 Thin layer chromatography analysis

a b
Figure 2: TLC profile of each PE extracts of each plant material [a-
Elute: Dichloromethane, b- Elute: Chloroform]

a b
Figure 3: TLC profile of each a- DCM extract [Elute: Chloroform], b- EA extracts
[Elute: Chloroform]

54
 Qualitative Phytochemical Screening, Anti-Bacterial Activity and TLC Profiling of Different Parts of
Three Medicinal Plants

a b

c d
Figure 4: TLC profile of each MeOH extract [Elute: a- 0.5 % b- 1
% , c- 2 %, d- 5 % MeOH in Acetonitrile]

The sequential extraction method was NOB, COL and COB showed the same
carried out to confirm the nature of each compound with Rf value 0.686. NOL,
plant material by analyzing TLC NOB, CTB, CTS, COL and COB showed
chromatograms and compared with the same compound with Rf value 0.456.
bioactivity of each plant material in each The same compound with Rf value 0.424
plant extract. showed in NOB, CTB, and COL.
Compound with Rf value 0.784 showed
According to the TLC profile (Figs.2-4) of only in NOB. CTS and COS showed the
each PE extract of each plant material same compound with Rf value 0.320.
eluted by dichloromethane, all the plant According to the TLC profile (Figs. 2-4) of
materials except NOB showed same each plant extract of each plant material
compound with Rf value 0.928. And also, eluted by Chloroform; all the plant
NOB, CTL, CTB and COB showed same materials showed the same compound with
compound with Rf value 0.880 and NOB, Rf value 0.961. And also, NOB, CTB, COL
CTB, CTS, COL and COB showed the and COB showed the same compound with
same compound with Rf value 0.824. Also, Rf value 0.695. In the case of NOB, CTB,

55
S. Arasaretnam et al.

CTS, COL and COB and COS showed the present study, in each extraction, some
same compound with Rf value 0.495. CTS compounds might be the reason for the
and COS showed the same compounds increasing or decreasing of the bioactivity.
with the Rf values 0.291, 0.390, 0.495 and When compared with the TLC profile for
0.961, respectively. Only NOB showed each extract in respective solvents and
compound with Rf value 0.914. bioactivity for each extract, availability of
the compounds in each extract might be the
According to the TLC profile (Figs. 2-4) of reason to increase or decrease bio activity
each DCM extract of each plant material in each extract. For example, TLC profile
eluted by chloroform, all the plant of PE extracts of NOL and NOB eluted by
materials except CTB showed the same dichloromethane shows a higher number
compound with Rf value 0.981. And also, of compounds compared with NOB and
NOL, NOB, CTB, COB and COS showed NOL which shows higher bioactivity
the same compound with Rf value 0.973. compared with NOB against both bacteria.
And also, NOB, CTL CTB showed the Antagonist effect of these compounds
same compound with Rf value 0.928. might be the reason to decrease the
NOB, CTL, CTB, COL and COB showed bioactivity of PE extracts of NOB
the same compound with Rf value 0.562. compared with NOL. And also, some
NOB, CTL, CTB, CTS, COL and COS compounds in each extract might be the
showed the same compound with Rf value reason to increase the bioactivity
0.348. compared with other extracts. When
compared with TLC profile and bioactivity
According to the TLC profile (Figs. 2-4) for each solvent extract for respective plant
COL, COB and COS showed the same parts, some compounds showed in TLC
compounds with Rf values 0.404, 0.571 might increase bioactivity against E. coli
and 0.857. In the present study TLC and some compounds might decrease the
profiling of 04 sequentially extracted PE, bioactivity against S.aureus. Therefore,
DCM and EA and MeOH extracts give an these active extracts could be subjected to
impressive result directing towards the further isolation and purification of active
presence of a number of phytochemicals. compounds to discover novel lead anti-
Various phytochemicals give different Rf bacterial agents.
values in a different solvent system. This
variation in Rf values of the 4 CONCLUSIONS
phytochemicals provides a very important
clue in the understanding of their polarity Phytochemical analysis of different
and also helps in the selection of the solvent extracts of leaves and barks of N.
appropriate solvent system. This oleander and leaves, barks and seeds of
information will help selection of both C. thevetia and C.odollum of family
appropriate solvent system for further Apocynaceae revealed the presence of
separation of the pure compound from alkaloids, flavonoids, steroids, cardiac
these plant extracts by column glycosides, Quinone, phenol in all plant
chromatography. In the results of the materials and saponins only presence in the

56
 Qualitative Phytochemical Screening, Anti-Bacterial Activity and TLC Profiling of Different Parts of
Three Medicinal Plants
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