A

MAJOR PROJECT REPORT

ON
“EVALUATION OF PHYTOCHEMICALS AND ANTIMICROBIAL
ACTIVITY OF ETHANOLIC EXTRACTS OF LEAVES, FRUIT AND
SEEDS OF Momordica charantia”

Submitted for the partial fulfilment of the requirement

for the award of the degree of
Bachelor of Technology
In
Biotechnology
Of
Dr. A.P.J. Abdul Kalam Technical University
Formerly Uttar Pradesh Technical University

Submitted By: Supervised By:

Madhavi Singh (1706854025) Dr. Avinash Singh
Associate Professor
Department of Biotechnology
MIET, Meerut

Department of Biotechnology
Meerut Institute of Engineering and Technology, Meerut
2021

i
 CERTIFICATE

Date……………

This is to certify that Ms. Madhavi Singh, roll no. 1706854025 of B. Tech. (Biotechnology)
VIII semester has carried out a project work on Evaluation of phytochemicals and
antimicrobial activity of ethanolic extracts of leaves, fruit and seeds of Momordica
charantia under my supervision for the partial fulfillment of the award of the degree of
Bachelor of Technology in Biotechnology at Department of Biotechnology, Meerut
Institute of Engineering and Technology (MIET), Meerut (Affiliated to Dr. A.P.J. Abdul
Kalam Technical University, Lucknow). The above said project is a bonafide record of
work done by them during the session 2020-2021.

Supervisor:

Dr. Avinash Singh

Associate professor

Department of Biotechnology

ii
 DECLARATION

I hereby declare that this submission is my own work and that, to the best of my knowledge
and belief, it contains no material which to a substantial extent has been accepted for the
award of any other degree or diploma of the university or other institute of higher learning.

Signature:

Name: Madhavi Singh

Roll No.: 1706854025

iii
 ACKNOWLEDGEMENT

I would like to express my sincere gratitude to my supervisor Dr. AVINASH SINGH for
providing their invaluable guidance, comments and suggestions throughout the course of
the project. I would specially thank Dr. NITIN SHARMA for constantly motivating me to
work harder.

Submitted By:

Madhavi Singh (1706854025)

iv
 TABLE OF CONTENTS

Title Page No.

LIST OF FIGURES vii
LIST OF TABLES viii
ABBREVIATION x
ABSTRACT xi

CHAPTER 1: INTRODUCTION 1-2

1.1: SCIENTIFIC IDENTIFICATION 2

1.2: CHEMICAL STRUCTURE 2

1.3: PHYTOCHEMICAL CONSTITUENT 3

1.4: MORPHOLOGICAL CHARACTERISTICS 4-6

1.5: PHARMACOLOGICAL ACTIVITY 7-9

1.6: OBJECTIVE 9

CHAPTER 2: REVIEW OF LITERATURE 10-14

CHAPTER 3: MATERIAL AND METHODS

3.1: COLLECTION OF PLANT MATERIAL 15-16

3.2: PREPARATION OF EXTRACT 16-17

3.3: EXTRACTION METHOD 18

3.4: PHYTOCHEMICAL SCREENING 18-26

3.5: ANTIMICROBIAL ACTIVITY 27

CHAPTER 4: RESULT AND DISCUSSION

4.1: PHYTOCHEMICAL SCREENING 28-29

4.2: DETERMINATION OF ANTIMICROBIAL ACTIVITY 30-31

v
4.3: CONCLUSION AND FUTURE SCOPE 32

REFERENCES 33-34

vi
 LIST OF FIGURES

FIGURE NO. NAME PAGE NO.

1.1 Momordica charantia fruit 1
1.2 Chemical structure of 2
momordin

1.3 Chemical structure of 2

charantin

1.4 M. charantia stem 4

1.5 M. charantia root 4
1.6 M. charantia leaves 5
1.7 M. charantia flowers 5
1.8 M. charantia fruit 6
1.9 M. charantia seed 6
3.1 L.S. of Momordica 15
charantia

3.2 Momordica charantia 15

leaves

3.3 Pestle mortar 15

3.4 Momordica charantia 16
seeds and fruit

3.5 Parts of momordica 16

charantia

3.6 Filtration of extracts 17

vii
 LIST OF TABLES:

TABLE NO. NAME PAGE No.

1 Plant parts and their phytochemical 3

Constituents

2 Morphological Characteristics 4-6

3 Alkaloids Test;(Mayer’s, Wagner’s, 18-19

Dragendroff’s, Hager’s Test)

4 Carbohydrate Test;(Molisch’s, 20-21

Benedict’s, Fehling’s Test)

5 Saponins Test;(Froth, Foam Test) 22

6 Tannins Test;(Gelatin Test) 22

7 Flavonoid Test;(Alkaline reagent, Lead 23

acetate Test)

8 Amino acid Test;(Ninhydrin Test) 23-24

9 Phenol Test;(Ferric chloride Test) 24

10 Steroid’s test;(Acetic anhydride Test) 24

viii
11 Terpenes Test;(Copper acetate Test) 25

12 Glycosides Test;(Legal’s Test) 25-26

13 Phytosterols Test;(Salkowski’s Test) 26

14 Phytochemicals Screening of Seeds, 29

Leaves and Fruit of Momordica
Charantia

15 Diameter (mm) of 70 of inhibition 30

ix
ABBREVIATIONS

1. MC - Momordica charantia
2. DMSO - Dimethyl sulfoxide
3. E. coli - Escherichia coli
4. S. aureus - Staphylococcus aureus
5. DW - Distilled water
6. Ppt – Precipitate

x
 ABSTRACT
This research work is based upon extraction and estimation of phytochemical content of
Momordica charantia and evaluation of corresponding antimicrobial activity due to their
presence in leaves, fruit and seeds of the plant. It has been used as a food and natural
medicine for treatment of diabetes, cancer, wound inflammation, fever and many infectious
diseases. This project work mainly focused upon 2 objectives- firstly, to obtain the plant
extract samples of various plant parts of Momordica charantia (leaves, fruit and seeds
separately) followed by their phytochemical screening and secondly, to separately check
the antimicrobial activity of each plant extract obtained from leaves, fruit and seeds of MC
plant. The results showed the presence of phytochemicals like alkaloids, carbohydrates,
saponins, tannins, flavonoids, amino acid, phenols, glycosides, steroids, fatty acid and
phytosterol in ethanolic extracts of plant samples. It is also revealed that MC acts as a
source of carbohydrates, amino acids and fatty acids, which are basic nutrients to maintain
general health of the body. In the present study, antimicrobial activity was determined for
Staphylococcus aureus and Escherichia coli by using stokes disc diffusion method and
analysing the resultant zone of inhibition. On a comparative basis, it is also observed that
since leaves and fruits contain a higher variety of phytochemicals, they exhibit much
effective antimicrobial activity than seeds. Moreover, every plant extract sample showed
higher efficacy on S. aureus, which is a Gram-positive bacterium, than E. coli, a Gram-
negative bacterium.

Keywords: Momordica charantia, phytochemicals, antimicrobial activity, ethanolic

extract.

xi
 CHAPTER 1

INTRODUCTION
Momordica charantia, a member of Cucurbitaceae family, commonly known as Bitter
melon, bitter gourd or karela has long been used as a food and natural medicine.
Momordica charantia contains different biologically active phytochemicals, which
includes proteins, triterpenes, saponins, flavonoids, steroids, alkaloids, and fatty acids. This
plant has long been used for the treatment of microbial infections, wound inflammation,
diabetes mellitus, abdominal pain, tumour proliferation, cancer, fever and hypertension and
its fruit has been used as a vegetable for thousands of years. There are several classes of
secondary metabolites, such as tannins, flavonoids, and alkaloids in the fresh and dried
leaves extracts of Bitter melon with antimicrobial activity. The fruits and leaves of
Momordica species are rich in phytochemicals. This phytochemical composition of both
the leaves reported antimicrobial activity against different bacteria, like Salmonella, E. coli,
Bacillus and Streptococcus. In traditional medication, fruits and leaves are used to cure
several diseases like: gout, rheumatism, colic, worms, illness of liver and spleen.[1]

Fig.1.1 - Momordica charantia

Besides, both in vitro and in vivo studies have demonstrated that bitter melon may also
elicit toxic or adverse effects under different conditions. Its pharmacological properties are

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 attributed to each part of the plant like seeds, roots, leaves and particularly the unripe fruits.
Momordica charantia is used as a blood purifier due to its bitter tonic properties.

1.1 SCIENTIFIC IDENTIFICATION:

SCIENTIFIC NAME – Momordica charantia
KINGDOM – Plantae
FAMILY – Cucurbitaceae
GENUS -Momordica
SPECIES – charantia

1.2 CHEMICAL STRUCTURE:

Fig.1.2 Chemical structure

of momordin

Fig. 1.3 Chemical structure of charantin

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1.3 PHYTOCHEMICAL CONSTITUENTS:

The fruits of bitter melon are utilized as vegetables whereas the whole plant parts like:
fruits, leaves, roots and seeds used as medicine.

Table 1- Plant parts and their phytochemical constituents

SOURCE PHYTOCHEMICALS REFERENCES

Momorcharins, momordin, charantin,

Plant Body cucurbitins, eleostearic acids. [2]

Glucosides, saponins, alkaloids,

proteins, triterpenes and steroids.

Momordicine, charantin, polypeptide-

p insulin

Fruit Amino acids- aspartic acid, serine, [3]

glutamic acid, threonine.

Fatty acids- palmitic, stearic, oleic,

linoleic, linolenic acid.

Enzyme- Urease
Seeds [4]

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 Amino acids- valine, threonine,
methionine, isoleucine, glutamic acid,
phenylalanine.

1.4 MORPHOLOGICAL CHARACTERISTICS:

Table 2- PLANT PARTS AND THEIR DESCRIPTION

PART DESCRIPTION IMAGE

Stem It is a creeping prostrate herb, sometimes

climbing. It grows up to 5 m high. The
stem is slender, slightly pubescent, grooved
and light green and it branches at the base

Fig.1.4 - M.
charantia stem

Root It has a primary root that extends to the

vertex where the stem is born

Fig.1.5- M.
charantia root

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Leaves Leaves are round and dark green in colour,
with a diameter range varying from 1 to 3.5
inches. They are simple type and with
alternate phyllotaxy. They are palmate and
deeply lobed, hence having 3-7 palmate
Fig.1.6 - M.
lobed with sharply toothed teeth and are
charantia leaves
borne on long channelled petiole, which
has a simple tendril at its base.

Flowers are solitary, axillary at the end of a

Flowers peduncle, 5 to 15 cm long. It is a
monoecious species i.e., male flowers and
Fig.1.7 - M.
female flowers separately. Female flowers
charantia flower
are smaller than male flowers

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 Fruit is fleshy, broadly ovoid oblong to
Fruit fusiform, 4 to 20 cm long and 2.5 to 4 cm
wide, dehiscent at the top by 3 valves. It is
covered with irregular tubercles. It is
yellow-orange to scarlet. It contains many
seeds.

Fig. 1.8 - M.
charantia fruit

Seeds are oval elliptic, almost toothed at

Seed the top, 10 to 16 mm long and 7-9 mm
wide and 2-3 mm thick. Seeds are covered
by red mucilage.
Fig.1.9 - M.
charantia seed

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1.5 PHARMACOLOGICAL ACTIVITY:

Various preparations of M. charantia extracts from fruit juice to dried fruit bits have been
employed traditionally worldwide, particularly for blood-sugar lowering effects
(Welihinda et al., 1986). Contemporary scientific research has proven that bitter melon
auspiciously possesses different pharmacological activities including antidiabetic,
anticancer, antimicrobial, antihepatotoxic, antioxidant, antiviral, antiulcerogenic, and
larvicidal activities (Anjum et al., 2012). Several bioactive compounds like tannins,
alkaloids, terpenoids, steroids, carbohydrates, flavonoids, etc. naturally exist in medicinal
plants and they are responsible to produce definite pharmacological actions in the human
body (Prakash et al., 2015). Different nutrient and non-nutrient constituents of the plant
materials possess anticancer properties that were established in various in vitro and in vivo
models and by utilizing these constituents and cancer prevention strategies had emphasized
(Bulbul et al., 2018).

Following pharmacological activities are supposed to be shown by the extracts of various

plant parts of M. charantia [5].

● Antimicrobial activity – Antimicrobial activity can be defined as a collective term

for all active principles (agents) that inhibit the growth of bacteria, prevent the
formation of microbial colonies, and may destroy microorganisms. Crude extracts
(from which natural compounds were isolated) were tested for possible
antimicrobial activity by using the disc diffusion technique (Chowdhury et al.
2015).

● Antioxidant activity – Antioxidant activity can be defined as a limitation or

inhibition of nutrient oxidation (especially lipids and proteins) by restraining
oxidative chain reactions. The antioxidant efficacy of methanol extract of seeds of
MC was evaluated by DPPH-free radical scavenging activity by means of the
approach of Brand-Williams et al. (1995), where ascorbic acid was employed as
standard.

● Cytotoxicity activity – Cytotoxicity is an in vitro test to determine whether the

given chemical substance will cause any cell death due to leaching of toxic
substances or from direct contact. This experiment was conducted on brine shrimp
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 nauplii. Four distinct test solutions were used to evaluate the cytotoxicity of the
extract, where vincristine sulphate was used as a positive control (McLaughlin et
al., 1998).

● Anthelmintic activity – The property of a chemical substance to kill or drive out

parasitic worms from the intestines is defined as its anthelmintic activity. The
anthelmintic assays were executed as per the approach of Ghosh et al. with minor
modifications (Ghosh et al. 2005).

● Antidiarrheal activity – Having the property of opposing or correcting diarrhoea

is referred to as anti-diarrheal activity. Castor oil-induced diarrhoea was induced
according to the method of Uddin et al., 2005.

● Thrombolytic activity – A drug or chemical substance that causes a blood clot to

break up is called thrombolytic agent. Thrombolytic activity was conducted on the
basis of a method described by Furie and Furie, 2008.

Antimicrobial activity and agents:

Antimicrobial activity refers to the process of killing or inhibiting the growth of a variety
of microorganisms and it may be anti-bacterial, antifungal or antiviral activity. Various
antimicrobial agents are used for this purpose and they all have different modes of action
by which they act to suppress the infection.

Mechanism of action of phytochemicals:

● Inhibit microorganisms
● Interfere with metabolic processes
● Modulate gene expression
● Modulate signal transduction pathways

It may also include chemotherapeutic with chemo-prevention referring to the use of agents
to inhibit, reverse or retard tumorigenesis.

Factors affecting the antimicrobial activity:

● Concentration of the agent

● Temperature

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 ● Contact time
● pH
● Nature of microorganism
● Growth phase of microorganisms.
● Presence of special structure (spore, capsule)
● Number of microorganisms
● Presence of extraneous materials (blood,pus,etc)

Commonly used terms are:

1. BACTERICIDAL: It is defined as a chemical agent capable of killing bacteria,

but not necessarily bacterial spores.
2. BACTERIOSTATIC: It is defined as the chemical agent capable of preventing
the growth of bacteria but not of killing them. Here reproduction and replication is
prevented.
3. MINIMUM INHIBITORY CONCENTRATION (MIC): It is defined as the
lowest concentration of the drug or antibiotic that inhibits the growth of the test
organism.
4. MINIMUM BACTERICIDAL CONCENTRATION (MBC): It is defined as the
minimum concentration of the drug or antibiotic that kills the given test organism.

1.6 OBJECTIVE:

The current project work is done with the following objectives:

● Obtaining the plant extract samples of various plant parts of Momordica charantia
(leaves, fruit and seeds separately) followed by their phytochemical screening.
● To separately check the antimicrobial activity of each plant extract obtained from
leaves, fruit and seeds of MC plant.

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 CHAPTER 2

LITERATURE REVIEW
Various plants around the world are used for different medicinal purposes. Due to the
presence of a versatile range of phytochemical constituents present in them, they act as a
source for numerous drugs which are used in the treatment of several diseases. Some may
be used in health supplements as well, to deliver the nutrients that may not be consumed in
sufficient quantities along with the food. MC is regarded as one of the most potent and
powerful plants used to cure a wide range of ailments and health problems, according to
some traditional medicine systems that exist worldwide, including Ayurveda.

Different parts of this plant are consumed because they act as a storehouse of a variety of
minerals, vitamins, amino acids, fatty acids and a wide range of phytochemicals that
includes alkaloids, tannins, saponins, flavonoids, phenols, phytosterols, etc. As a result of
the presence of these phytochemical constituents, the different plant parts of MC have
shown to exhibit miscellaneous pharmacological properties. The study and analysis of
following research articles, their results and observations act as an evidence to the veracity
of the facts and statements mentioned here. Thorough study of these undermentioned
research works gave us a proper perspective on how to proceed and operate on our project
work.

Zahan S. et al. [5] carried out the research work with the purpose to investigate the in vivo
(analgesic, antidiarrheal, neurological, and cytotoxic) and in vitro (antioxidant,
antimicrobial, thrombolytic and anthelmintic) activity of different fractions of methanolic
extract of Momordica charantia. In preliminary phytochemical screening, crude
methanolic extract of MC showed positive (+) response for the presence of alkaloids,
carbohydrates, glycosides, saponins, phytosterols, phenols, tannins, flavonoids, proteins
and amino acids. The overall results of the study tend to suggest that the methanolic extract
and its fractions have promising pharmacological activities. The study was entirely focused
on the seed value. It is thought worthy to select this extract for further studies.

Mada S. B. et al. [6] performed the study with the objective to investigate the antimicrobial
activity and to carry out phytochemical screening of aqueous and ethanol extracts of the

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leaves of Momordica charantia L. The result of the phytochemical screening revealed the
presence of alkaloids, tannins, saponins, flavonoids and cardiac glycosides. However, the
antimicrobial activity was investigated by agar-well diffusion methods against
Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa.
The result showed that at a concentration of 100 mg/ml, both aqueous and ethanol extracts
inhibited the growth of all the tested pathogenic bacteria, though with varying degrees of
susceptibility of the bacterium. The diameter of zones of inhibition obtained ranged from
17 to 14 and 15 to 11 mm for ethanol and aqueous extracts, respectively. The results
obtained in this study support the use of M. charantia in herbal medicine and it could be
used as a source of broad spectrum oral antimicrobial agent for the treatment of diseases
associated with these pathogenic bacteria investigated.

Hlaing S. et al. [7] stoked upon 5 different species of genus Momordica. They carried out
extraction and isolation of the active Momordica glucoside charantin compound on the
fruit of M.charantia L. The compound charantin was confirmed by thin layer
chromatography, melting point, and U.V, IR spectroscopic methods. The test indicated that
all the fruits showed the presence of alkaloid, glycosides, reducing sugar, saponin
glycoside, steroids, phenolic compound, a-amino acid, carbohydrates, organic base or
neutral and tannin. The preliminary phytochemical examination was carried out on the
fruits of five species. The tests showed that the cyanogenic glycoside was absent from all
fruits. The active compound charantin was extracted from the fruits of M.charantia L. The
compound charantin showed activity on diabetes.

Khan N.H. et al. [8] executed the research work with the objective to perform preliminary
and extended investigation on Momordica charantia (bitter gourd). Two extraction
methods were carried out on the fruit of MC, which are Soxhlet method and maceration
method by using absolute ethanol as solvent. The Soxhlet ethanolic extract showed the
presence of alkaloids, triterpenoids and carbohydrates. Macerated ethanolic extract was
divided into 2 portions, filtrate and residue. The filtrate showed the same active compounds
with Soxhlet ethanolic extract whereas the residue showed presence of alkaloids, saponins,
tri-terpenoids and phenol. Antimicrobial activity was carried out against different bacteria
which include Bacillus subtilis and Staphylococcus pyogenes as Gram-positive bacteria
and Pseudomonas aeruginosa and Escherichia coli as Gram-negative bacteria. A diffusion

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method had been used to screen for antimicrobial activity. All the extracts showed no zone
of inhibition on Mueller-Hinton agar against the tested bacteria and hence this study
reported that Momordica charantia fruit did not show antimicrobial activity.

Sood A. et al. [9], in their study, dealt with the antimicrobial activity and phytochemical
screening of seeds extract of five plants of Cucurbitaceae family- Momordica charantia
(Karella), Cucumis sativus (Cucumber), Praecitrullus fistulosus (Tinda), Cucurbita pepo
(Kaddu), Lagenaria siceraria (loki) that are commonly available and readily consumed in
India. The study reveals that these plants under study can be used for the treatment of
cancer, congestive heart failure, lowering of cholesterol levels in blood, healing of wounds,
endotoxemia etc. since they contain various phytochemicals that are known to treat above
mentioned diseases. The demonstration of broad spectrum of antibacterial activity by
Momordica charantia (Karela), Cucumis sativus (Cucumber), and Praecitrullus fistulosus
(Tinda) may help to discover new chemical classes of antibiotic substances that could serve
as selective agents for infectious disease, chemotherapy and control. With the evidence of
antibacterial and antifungal activities of the extracts of preparations under study, it can be
rationally suggested that further work needs to be done to identify the chemical natures of
the active principles as well as their modes of actions on bacterial cells and their roles in
curing diseases.

Leelaprakash G. et al. [10] performed the research work with the aim to determine the in
vitro antimicrobial and antioxidant activity of aqueous and methanol extracts of
Momordica charantia leaves. In preliminary phytochemical analysis we observed
glycosides, alkaloids, phytosterols, saponins, phenolic compounds, proteins, fats and fixed
oils and flavonoids, and thin layer chromatography was also performed. Antimicrobial
activity was evaluated for Pseudomonas aeruginosa, Escherichia coli, Klebsiella
pneumoniae and Bacillus subtilis by using stokes disc diffusion and well diffusion
methods. Aqueous extract of leaves showed milder antimicrobial activity compared to
methanolic extract, which certainly indicates that methanolic extract contain higher
concentration of active antimicrobial agents such as alkaloids, glycosides, volatile oils,
which are all found in more abundant in MC. Preliminary results of the activity of
antimicrobial agents such as plant active components, antibiotics are usually expressed in
vitro as zones of inhibition around the chemical.

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Mushtaq W et al. [11]. presented their study with the aim to explore antidiabetic potential
of wild fruit of Momordica charantia L. (Family: Cucurbitaceae) from local germplasm of
District Bhimber Communities, Kashmir, India. The purpose was to evaluate the herbal
recipe of food folklore of the remote rural area, where the majority population relies on
herbal therapeutics. Ethnomedicinal knowledge was collected through Rapid Appraisal
Approach (RAA) along with structured and semistructured interviews with local people
and herbalists. Pharmacological analysis was conducted in the laboratory using Rabbits as
model organisms, and diabetics were induced by use of alloxan. The study demonstrated
that ethanolic extract of Momordica charantia has potential antidiabetic properties in
Type2 diabetes mellitus, thus justifying the traditional usage of the plant as food medicine.
Its phytochemical analysis ethanolic extract demonstrated the presence of various
compounds like alkaloids, saponins, sterols, steroid, terpenoids, flavonoids, tannins,
phlobatannins and cardiac glycosides. There is hitherto space to evaluate the fruit using
more solvents and also investigate other cultivars/populations from different villages of the
area and country to broaden the horizon of ethnopharmacological research on this plant.

Roopashree TS et al. [12] conducted their research work as we know in view of increasing
resistance to existing antimicrobial agents, herbal drugs are being looked at as a very
important source for discovery of new agents for treating various ailments related to
bacterial infections. Cassia tora, Calendula officinalis and Momordica charantia are well
known plants in Asia including India which possess a wide range of pharmacological
activities. These drugs have been used in India as folk remedy in the form of decoctions
and infusions to treat bacterial infections and also claimed to be effective against a variety
of skin conditions like psoriasis, acne, wounds etc. The present investigation was carried
out to study the unexplored area of these drugs towards their antibacterial activity with
respect to their traditional use as antipsoriatic agents. The herbs were subjected to
successive extraction using different solvents and the extracts were subjected to
antibacterial evaluation against both gram positive and gram-negative organisms by cup
plate technique. Among the various extracts, aqueous extracts were found to be more
effective against all the bacteria. Staphylococcus aureus was more susceptible to the
aqueous extracts among the tested organisms.

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Zaidan et al. [13] carried out the study with the knowledge of the fact that medicinal plants
have many traditional claims including the treatment of ailments of infectious origin. In
the evaluation of traditional claims, scientific research is important. The objective of the
study was to determine the presence of antibacterial activity in the crude extracts of some
of the commonly used medicinal plants in Malaysia, Andrographis paniculata, Vitex
negundo, Morinda citrifolia, Piper sarmentosum, and Centella asiatica. In this preliminary
investigation, the leaves were used and the crude extracts were subjected to screening
against five strains of bacteria species, Methicillin Resistant Staphylococcus aureus
(MRSA), Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and
Escherichia coli, using standard protocol of Disc Diffusion Method (DDM). The
antibacterial activities were assessed by the presence or absence of inhibition zones and
MIC values. M. citrifolia, P. sarmentosum and C. asiatica methanol extract and A.
paniculata (water extract) have potential antibacterial activities to both gram positive S.
aureus and Methicillin Resistant S. aureus (MRSA). None of the five plant extracts tested
showed antibacterial activities to gram negative E. coli and K. pneumoniae, except for A.
paniculata and P. sarmentosum which showed activity towards P. aeruginosa. A.
paniculata being the most potent at MIC of 2 µg/disc. This finding forms a basis for further
studies on screening of local medicinal plant extracts for antibacteria properties.

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 CHAPTER 3

MATERIALS AND METHODS

3.1 COLLECTION OF PLANT MATERIAL:

Plant material – Leaves, Seeds and fruit of MC plant.

Equipment & Instrument Used – Conical flask, aluminium foil, measuring cylinder,
spatula, pestle mortar, distilled water, oven, refrigerator, autoclave, Whatman no.1 filter
paper, shaker.

Plant samples were collected from the village of Pawati, Meerut, U.P., India. Fruits and
leaves of MC plants were separated. After separation, all the different plant parts were
washed with DW. The fruits were then cut longitudinally into small pieces. Then peel,
seeds, flesh were separated from this and along with the leaves, they were dried in shade.
After drying, each of the dried samples was crushed by using pestle mortar (Yesilada et al.,
1999). To get fine powder, the crushed sample was sieved using a sieve (Mesh size=100).

Fig.3.1- Inner part of Momordica charantia

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Fig 3.2 Momordica charantia leaves Fig 3.3 Pestle mortar

Fig 3.4 Momordica charantia seeds and fruit

3.2 PREPARATION OF EXTRACT:

1 litre of 50% ethanol solution was prepared by mixing equal amounts of pure ethanol and
water. 50 grams of fine dried powder of each constituent (seed, fruit and leaves of plant)
was taken and each of them was mixed with 250ml of prepared solvent (50% ethanol)
contained in a separate flask. The flasks were then covered with aluminium foil and kept
in a shaker for 48 hrs at 120 rpm.

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After 48 hrs, the flasks were taken out and then the extract was filtered by using Whatman
No.1 filter paper. After collection of each of the filtrates, the solvent was evaporated at
37℃. As the extract became dried, it was stored so that it could be used for further
phytochemicals screening. [17]

Fig. 3.5 – Parts of momordica charantia

Fig. 3.6 – Filtration of extracts

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3.3 EXTRACTION METHOD:

❖ MACERATION –

Maceration is one of the extraction methods that soak raw materials in the extracting
solvent. In this, powdered extract is kept with the solvent, and is allowed to stand at room
temperature with continuous agitation. After some time, the active constituents will be
extracted out from the extract and the final product is obtained [11].

3.4 PHYTOCHEMICAL SCREENING:

Detection of Alkaloids- Extracts (2ml) were dissolved individually in 1% dilute

hydrochloric acid and filtered. The filtrates were used to test for the presence of
alkaloids.

● MAYER’S TEST: Filtrates were treated with few drops of Mayer’s reagent
(potassium mercuric iodide). Formation of a yellow colour precipitate indicated the
presence of Alkaloid.
● WAGNER’S TEST: Filtrates were treated with Wagner’s reagent (Iodine in
Potassium Iodide). Formation of brown/reddish precipitate indicates the presence of
alkaloids.
● DRAGENDROFF’S TEST: Filtrates were treated with Dragendroff’s reagent
(solution of Potassium Bismuth Iodide). Formation of red precipitate indicates the
presence of alkaloids.
● HAGER’S TEST: Filtrates were treated with Hager’s reagent (saturated picric acid
solution). Presence of alkaloids confirmed by the formation of yellow coloured
precipitate.

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Table 3- Alkaloids Test; (Mayer’s, Wagner’s, Dragendroff’s, Hager’s Test)

TEST COMPOSITION COLOUR

Mayer’s Test Mayer’s reagent is freshly prepared by Appearance of

dissolving a mixture of mercuric yellow colour
chloride (1.36 g) and of potassium ppt.
iodide (5.00 g) in water (100.0 ml).

Wagner’s Test 2.5 gm iodine is dissolved in 12.5 gm of Appearance of

potassium iodide and add 250 ml of brown/reddish
water to produce solution. ppt.

Dragendroff’s Test Dragendroff's reagent is prepared by Appearance of

mixing a concentrated solution red ppt.
of potassium iodide with a solution
of bismuth subnitrate in a
diluted acid (acetic acid or tartaric
acid, hydrochloric acid or sulfuric
acid is rarely being used) as a low pH is
mandatory for this reagent.

Hager’s Test Take 1ml of extract and add 3ml of Appearance of

saturated aqueous solution of picric acid. yellow colour
ppt

Detection of Carbohydrates- Extracts were dissolved individually in 5 ml distilled

water and filtered. The filtrates were used to test for the presence of carbohydrates.

Page | 19
● MOLISCH’S TEST: Filtrates were treated with 2 drops of alcoholic α-naphthol
solution in a test tube. Formation of the violet ring indicates the presence of
Carbohydrates.

● BENEDICT’S TEST: Filtrates were treated with Benedict’s reagent and heated
gently. Formation of orange red precipitate indicates the presence.

● FEHLING’S TEST: Filtrates were hydrolysed with dil. HCl, neutralized with alkali
and heated with Fehling’s A & B solutions. Formation of red precipitate indicates
the presence of reducing sugars.

Table 4- Carbohydrate Test;(Molisch’s, Benedict’s, Fehling’s Test)

TEST COMPOSITION COLOUR

Molisch’s Test Take 2ml distilled water and add Formation of violet
Molisch reagent; dissolve 3.75 g of rings.
α-naphthol in 25 ml of Ethanol
99%. This should be prepared
fresh. Then add conc. sulphuric
acid in it.

Benedict’s Test One litre of Benedict’s solution can Formation of orange

be prepared from 100 g of red ppt.
anhydrous sodium carbonate, 173 g
of sodium citrate and 17.3 g of
copper (II) sulphate pentahydrate.

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 Fehling’s Test 2 different solutions: Fehling's A Formation of red ppt.
and Fehling's B.

Fehling's A is a blue copper (II)

sulphate containing solution.

Fehling's B is a clear liquid

consisting of potassium sodium
tartrate (Rochelle salt) and a
powerful alkali, normally sodium
hydroxide. Solutions A and B are
separately prepared and stored
during the evaluation.

In order to get the final Fehling

solution that is deep blue, the two
solutions are later combined in
equal amounts. Cu2 complex is the
deep blue ingredient. The tartrate
tetra-anions in the solution act as a
chelating agent.

Detection of Saponins- These are glucosides with foaming characteristics.

● FROTH TEST: Extracts were diluted with distilled water and were shaken in a
graduated cylinder for 5-15 minutes. Formation of 1 cm layer of foam indicates the
presence.

● FOAM TEST: Extract was shaken with 20 ml of water. If foam produced persists
for ten minutes it indicates the presence of saponins.

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 Table 5- Saponins Test;(Froth, Foam Test)

TEST COMPOSITION COLOUR

Froth Test 3 mL of the aqueous solution of the Formation of foam

extract were mixed with 10 mL of which last for more
distilled water in a test-tube. The test- than 10 minutes.
tube was shaken vigorously for about 5
min, it was allowed to stand for 30 min
and observed for honeycomb froth.

Foam Test 1ml solution of extract was diluted with Formation of foam
distilled water to 20 ml and shaken in a which last for more
graduated cylinder for 15 minutes than 10 minutes.

Detection of Tannins-

● GELATIN TEST: To the extract, 1% gelatin solution containing sodium chloride

was added. Formation of white precipitate indicates the presence of tannins.

Table 6- Tannins Test;(Gelatin Test)

TEST COMPOSITION COLOUR

Gelatin Test Aqueous solution of gelatin Formation of white ppt.

and sodium chloride are
added.

Detection of Flavonoid-

● ALKALINE REAGENT TEST: Extracts were treated with few drops of sodium
hydroxide solution. Formation of intense yellow colour, which becomes colourless
in addition to dilute acid, indicates the presence of flavonoids.
Page | 22
● LEAD ACETATE TEST: Extracts were treated with few drops of lead acetate
solution. Formation of yellow colour precipitate indicates the presence of flavonoids.

Table 7- Flavonoid Test;(Alkaline reagent, Lead acetate Test)

TEST COMPOSITION COLOUR

Alkaline Reagent 2 ml of 2.0% NaOH mixture was Formation of

Test mixed with aqueous plant crude intense yellow
extract, concentrated yellow colour colour
was produced, which became
colourless when we added 2 drops of
diluted acid to mixture.

Lead Acetate Test Extract treated with lead acetate Formation of

solution. yellow colour

Detection of Amino acid-

● NINHYDRIN TEST: The extract was heated with 2% ninhydrin reagent in a boiling
water bath for 10 min. Formation of blue colour indicates the presence of amino acid.

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Table 8- Amino acid Test; (Ninhydrin Test)

TEST COMPOSITION COLOUR

Ninhydrin Test A 2% solution of ninhydrin must be Formation of

prepared by dissolving 0.2 grams of blue colour.
ninhydrin in 10ml of either ethanol
or acetone. Now a 1% solution of the
amino acid (analyte) in distilled
water must be prepared. A few drops
of the 2% ninhydrin solution must be
added to this solution.

The test tube must be kept in a warm

water bath for approximately 10
minutes.

Detection of Phenols-

● FERRIC CHLORIDE TEST: Extracts were treated with 3-4 drops of ferric
chloride solution. Formation of bluish black colour indicates the presence of phenols.

Table 9- Phenol Test; (Ferric chloride Test)

TEST COMPOSITION COLOUR

Ferric Chloride Test Extract treated with Appearance of bluish

aqueous ferric chloride. black ppt.

Page | 24
Detection of Steroids-
● ACETIC ANHYDRIDE TEST- 1ml of the extract is treated with concentrated
sulphuric acid in acetic anhydride. This formation of blue-green colour indicated the
presence of steroids.

Table 10- Steroid’s test; (Acetic anhydride Test)

TEST COMPOSITION COLOUR

Acetic Anhydride Test Add several drops of acetic Formation of blue-green

anhydride and then 2 drops colour ppt.
of concentrated H2SO4 and
mix carefully.

Detection Of Fatty acids-

● It was done by filter paper press test in which extracts were pressed in filter
paper and results were observed.

Detection Of Terpenes-

● COPPER ACETATE TEST: Extracts were dissolved in water and treated with 3-
4 drops of copper acetate solution. Formation of green colour indicates the presence
of terpenes.

Table 11- Terpenes Test; (Copper acetate Test)

TEST COMPOSITION COLOUR

Copper Acetate Test The extract is dissolved in Appearance of green colour

water and treated with 3–4 ppt.
drops of copper
acetate solution.

Page | 25
 Detection Of Glycosides- Each of the extracts were hydrolysed with dil. HCl and then
subjected to a test for glycosides by treating the extracts with Legal’s Reagent.

● LEGAL’S TEST: Extracts were treated with sodium nitroprusside in pyridine and
sodium hydroxide. Formation of pink to blood red colour indicates the presence of
cardiac glycosides.

Table 12- Glycosides Test; (Legal’s Test)

TEST COMPOSITION COLOUR

Legal’s Test Dry 5 ml of the above Formation of pink to blood

extract on a water bath, and red colour ppt.
dissolve the residue in the
mixture of 1 ml of water, a
few drops of 10% sodium
hydroxide and 1 ml of 0.3%
nitroprusside sodium
reagent.

Detection of Phytosterols:

● SALKOWSKI’S TEST: The extracts were treated with chloroform and filtered.
The filtrates were then treated with few drops of concentrated sulfuric acid and
allowed to stand and golden yellow colour shows the presence. [12]

Page | 26
Table 13- Phytosterols Test;(Salkowski’s Test)

TEST COMPOSITION COLOUR

Salkowski’s Test Extract was taken in 2 ml of Appearance of golden

chloroform and in it 2 ml of yellow colour
concentrated sulphuric acid
was added from the side of
the test tube. The test tube
was shaken for a few
minutes.

Page | 27
3.5 ANTIMICROBIAL ACTIVITY:

For determination of antimicrobial activity of various parts of MC plants, the disc diffusion
method was employed in accordance with the standard method by Bauer et al. (1966) to
assess the presence of antibacterial activities of the plant extracts. A bacteria culture (which
has been adjusted to 0.5 McFarland standard), was used to inoculate Muller Hinton agar
plates evenly using a sterile swab. The plates were dried for 15 minutes and then used for
the sensitivity test. The discs which had been impregnated with a series of plant extracts
were placed on the Mueller Hinton agar surface. Each test plate comprises six discs. One
positive control, which is a standard commercial antibiotic disc, one negative control, and
four treated discs. The standard antibiotic disc used was chloramphenicol 20 µg for two
bacterial strains, on which the antimicrobial activity was tested - S. aureus and E. coli. S.
aureus is a type of Gram-positive bacteria and E. coli is a Gram-negative type. They acted
as the positive control whereas negative control was DMSO (100%). Besides the controls,
each plate had four treated discs placed about equidistant to each other. The plate was then
incubated at 37°C for 18 to 24 hours depending on the species of bacteria used in the test.
After the incubation, the plates were examined for the inhibition zone. The inhibition zone
were then measured using callipers or threads. [13]

Page | 28
 CHAPTER 4

RESULT AND DISCUSSION

4.1 Phytochemical screening:

The results of the phytochemical analysis are represented in table 3. It is quite evident from
the figures obtained that the fruit of the MC plant contains a maximum number of different
types of phytochemicals, followed by leaves and seeds. The ethanolic extract of fruit
produced a positive response for all the phytochemicals tested for, that include alkaloids,
carbohydrates, tannins, flavonoids, glycosides, saponins, phytosterols, phenols, terpenes,
fatty acids, sterols and amino acids. These results which were observed in our study are
almost similar to research findings of the work of Mada et. al. (2013) [6].

The phytochemical analysis of ethanolic extract of seeds reveals that it exhibits positive
response for the presence of alkaloids, carbohydrates, tannins, flavonoids, glycosides,
saponins, phytosterols, phenols and amino acids. Result of phytochemical screening also
shows that the ethanolic seed extract produced a negative response for terpenes (in copper
acetate test), fatty acids and sterols. Similar results were obtained in the study conducted
by Zahan et. al. (2019) [5].

For ethanolic extract of leaves of MC plant, it is observed that it shows positive response
for alkaloids, carbohydrates, tannins, flavonoids, glycosides, saponins, phytosterols,
phenols, terpenes and amino acids. Although it also showed a negative response for fatty
acids and sterols. The observations recorded in the present study were comparative to the
research work of Hlaing et. al. (2005) [7].

Page | 29
Table 14-

Phytochemicals Screening of Seeds, Leaves and Fruits of Momordica Charantia

PHYTOCHEMICALS LEAVES SEEDS FRUITS

Alkaloids + + +

Carbohydrates + + +

Saponins + + +

Tannins + + +

Flavonoids + + +

Amino acids + + +

Phenols + + +

Steroids - - +

Fatty acid - - +

Terpenes + - +

Glycosides + + +

Phytosterol + + +

(+) = presence of phytochemicals

(-) = absence of phytochemicals

Page | 30
4.2 Determination of Antimicrobial activity:
In this study, Staphylococcus aureus was taken as Gram positive (+) while Escherichia coli
was taken as Gram negative (-) bacterial strains and Ampicillin was taken as standard
antibiotic for positive control and DMSO as negative control.

Ethanolic extracts of leaves, fruit and seeds of MC plant were investigated separately for
checking the antimicrobial activity, by determination of zone of inhibition in Disc diffusion
method. The results for the same are shown in table 4.

The observations indicated varying zones of growth inhibition. Ampicillin and various
extracts showed greater effect on Staphylococcus aureus as compared to Escherichia coli,
whereas no zone of inhibition was observed by DMSO as negative control. Almost similar
results were also obtained in the research work done by Ahmed et. al. (2019) [14] and
Ozusaglam et. al. (2013) [15].

Table 15 - Diameter (mm) of Zone of inhibition

Test Leaf Extract Fruit Extract Seed Extract Ampicillin DMSO

Bacteria μg/ml μg/ml μg/ml (+ve) (-ve)

10 20 10 20 10 20 10𝜇𝑔 5%

S. aureus 11 13 9 12 8 10 18 0

E. coli 7 11 8 11 7 9 15 0

Page | 31
 Chart for S. aureus
20
18
16
Zone of inhibition

14
12
(mm)

(mm)

10
8
6
4
2
0
Leaf extract Seed extract Fruit extract Ampicillin 10μg

10μg/mL 20μg/mL

Chart for E.coli

16
14
Zone of inhibition

12
10
(mm)

8
6
4
2
0
Leaf extract Seed extract Fruit extract Ampicillin(10μg)

10μg/mL 20μg/mL

Page | 32
 CONCLUSION AND FUTURE SCOPE

The presence of antimicrobial activity led us to the conclusion that the plant extract
contains bioactive compounds. Qualitative tests revealed the fact that methanolic extract
of seed of MC contains major phytochemicals viz. carbohydrate, glycosides, phenolics,
flavonoids, phytosterols, tannins, proteins and amino acids, saponins, terpenoids and
alkaloids. Therefore, plants could be used as a potential source for the development of an
effective antimicrobial agent. Moreover, it may also be used as a potential source of a
natural preservative in the pharmaceutical and food/feed industry.

It is also observed that ethanolic extracts of all sample plant parts showed better
antimicrobial activity on S. aureus, which is Gram positive bacteria, as compared to E. coli,
which is Gram negative bacteria.

This finding also forms the basis for further studies to prepare an optimized preparation of
the extracts of various plant parts of MC to further evaluate them against a wider range of
bacteria strains.

Further studies are recommended that will involve various parts of the plant from distinct
areas, select different fractions of crude extracts and purify the most active antimicrobial
components. Toxicity studies should also be done to determine their safety.

Page | 33
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