DETERMINATION OF THE ANTIMICROBIAL ACTIVITY OF LEAVES AND STEMS OF

GUARD PLANT (Lagenaria siceraria) AGAINST Escherichia coli, Staphylococcus aureus and

Candida albicans

NEREAH OMBWAYO MUSHIYI

A research project submitted to the Department of Medical Microbiology in the School of

Biomedical Sciences in partial fulfillment of the requirements for the award of the degree of
Bachelor of Science in Medical Microbiology of Jomo Kenyatta University of Agriculture and
Technology.

2020

i
 DECLARATION

I hereby declare that this project report is my original work and has not been presented for award
of degree at any other university.

Signature……………………………. Date…………………

NEREAH OMBWAYO MUSHIYI

HSB221-0357/2016

Declaration by the supervisor

This research project has been submitted for examination with my approval as a supervisor.

Dr. Ednah Song’oro, Department of Medical Microbiology, JKUAT

Signature…………………………. Date…………………..

ii
 DEDICATION

This research project is dedicated to my family for the moral support and encouragement.

iii
 ACKNOWLEGDEMENTS

First and foremost, I wish to acknowledge the guidance of the Almighty God for his protection,
and good health bestowed upon me throughout my work.
Secondly, I would like to thank my supervisor, Dr. Ednah Song’oro for her patience, advice and
dedication. I would also like to thank the staffs of JKUAT MMB laboratory for the most
productive correspondence I had during the research study, their input was invaluable.
All this would not have been possible without the generous support from my parents and my
fellow students and not forgetting MR. Njue and Lorna for providing the samples.
It is impossible to mention everyone I am immensely indebted to for their help in and outside of
the study. To those I didn’t mention, thank you and may God bless you.

iv
Contents
DECLARATION....................................................................................................................................ii
DEDICATION.......................................................................................................................................iii
ACKNOWLEGDEMENTS....................................................................................................................iv
LIST OF TABLES................................................................................................................................vii
LIST OF FIGURES..............................................................................................................................viii
LIST OF GRAPHS.................................................................................................................................ix
LIST OF ABBREVIATION....................................................................................................................x
ABSTRACT...........................................................................................................................................xi
CHAPTER ONE......................................................................................................................................1
INTRODUCTION...................................................................................................................................1
1.1 Background Information....................................................................................................................1
1.2 Statement of Problem........................................................................................................................2
1.3 Justification.......................................................................................................................................3
1.4 Research Questions............................................................................................................................3
1.5 Objectives..........................................................................................................................................3
1.5.1 General Objectives..........................................................................................................................3
1.5.1 Specific Objectives.........................................................................................................................3
CHAPTER TWO.....................................................................................................................................5
Literature Review....................................................................................................................................5
2.1 General Overview..............................................................................................................................5
2.2 Description of Lagenaria siceraria.....................................................................................................9
2.3 Taxonomy of Lagenaria siceraria....................................................................................................10
2.4 Geographical distribution................................................................................................................11
2.5 Economic Importance......................................................................................................................11
2.6 Test Organisms................................................................................................................................11
2.6.1 Staphylococcus aureus..................................................................................................................11
2.6.2 E. coli............................................................................................................................................12
2.6.3 Candida albicans...........................................................................................................................12
CHAPTER THREE...............................................................................................................................13
Materials and Methods..........................................................................................................................13

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3.1 Study site.........................................................................................................................................13
3.2 Study Design...................................................................................................................................13
3.3 Sampling criteria..............................................................................................................................13
3.4 Collection of samples......................................................................................................................13
3.5 Laboratory Procedures.....................................................................................................................13
3.6 Phytochemical Analysis...................................................................................................................14
3.6.1 Alkaloids......................................................................................................................................14
3.6.2 Flavonoids....................................................................................................................................14
3.6.3 Steroids.........................................................................................................................................14
3.6.4 Tannins.........................................................................................................................................14
3.7 Test Organisms................................................................................................................................15
3.7.1 Preparation of inoculum................................................................................................................15
3.7.2 Preparation of standard inoculum.................................................................................................15
3.8 Antimicrobial Susceptibility testing.................................................................................................15
3.9 Minimum inhibitory concentration..................................................................................................15
3.9 Data Processing and Presentation....................................................................................................16
CHAPTER FOUR.................................................................................................................................17
RESULTS..............................................................................................................................................17
4.1 Phytochemical test...........................................................................................................................17
4.1.1 Test for alkaloids..........................................................................................................................17
4.1.2 Test for flavonoids........................................................................................................................17
4.1.3 Test for steroids............................................................................................................................17
4.1.4 Test for tannins.............................................................................................................................17
4.2 Antimicrobial susceptibility testing.................................................................................................19
CHAPTER FIVE...................................................................................................................................27
DISCUSSION.......................................................................................................................................27
5.1 Phytochemical test.....................................................................................................................27
5.2 Antimicrobial susceptibility testing................................................................................................28
5.3 Minimum inhibitory concentration.................................................................................................29
CONCLUSION.....................................................................................................................................30
RECOMMENDATION.........................................................................................................................31
REFERENCE........................................................................................................................................32

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 LIST OF TABLES

Table 4.1: Table of phytochemical test results for stem ethanol extract……………………17

Table 4.2: Table of phytochemical test results for stem water extract…………………...…18

Table 4.3: Table of phytochemical test for leaf water extract………………………………18

Table 4.4: Table of phytochemical test for leaf ethanol extract……………………………..18

Table 4.5: Zone of inhibition of leaf ethanol extract…………….…………………..20

Table 4.6: Zone of inhibition of leaf water extract……………………………...……21

Table 4.7: Zone of inhibition of stem ethanol extract……………………………...…22

Table 4.8: Zone of inhibition of stem water extract………………………….………..23

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 LIST OF FIGURES

Figure 2.1 flower of Lagenaria siceraria……………………………………………….……..10

Figure 2.2 leaves and fruit of Lagenaria siceraria…………………………………………….10

Figure4.3: phytochemical test……………………………………………………………….....19

viii
 LIST OF GRAPHS

Graph 4.1 Graph of zones of inhibition of E.coli……………………………………………24

Graph 4.2 Graph of zones of inhibition of S.aureus………………………………………….25

Graph4.3: Graph of zones of inhibition of C.albicans…………………………………..……26

ix
 LIST OF ABBREVIATION

E.coli Escherichia coli

S. aureus Staphylococcus aureus

ANOVA Analysis of Variance

AMR Antimicrobial resistance

MHA Muller Hinton Agar

MMB Medical Microbiology

MRSA Multi-resistant Staphylococcus aureus

WHO World Health Organization

C.albicans Candida albicans

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 ABSTRACT

Medicinal plants play a critical role in the health of individuals. Many herbal remedies have been

employed in various medicinal systems for the treatment and management of different diseases.

The plant Lagenaria siceraria, a plant belonging to the family Cucurbitaceae, has been used in

the different systems of traditional medication for the treatment of diseases.

Lagenaria siceraria is an annual herbaceous climbing plant with a long history of traditional

medication used in many countries. Since ancient times, it has been known for its curative

properties, and has been utilized for treatment of various ailments including; jaundice, diabetes,

ulcers, piles, colitis, insanity, and hypertension and skin disease.

Its fruit pulp is used both as an emetic and purgative, and for its cooling, diuretic, antibilious, and

pectoral properties. Boiled in oil this pulp is used to treat rheumatism and insomnia. The flowers

are an antidote to poison. Leaf juice is used for baldness. A wide range of chemical compounds

including flavonoids have been isolated from the species.

The plant was sourced from kisii and the experiment was set up in the medical microbiology

laboratory. A phytochemical analysis procedure was conducted on the leaves and stems against

alkaloids, flavonoids, steroids and tannins. The experiment results were then presented in tables

accompanied with description.

A susceptibility test was conducted on the leaves and stems against E.coli, S.aureus, and

Candida albicans. Then minimum inhibitory concentration was conducted on leaves and stem

xi
extract. The experiment results will be presented in graphs and tables accompanied with

description.

xii
 Chapter OneHAPTER ONE

2.0 IntroductionNTRODUCTION

1.1 Background Information

With the rise of emerging and re-emerging diseases (Talaro et al, 2002) we need new drugs.

There is a growing need to explore the possibilities of various alternative plants and as well as

alternative system of medicine (Kannan et al, 2012).

Currently there is need for the use of plant and plant extracts and products for the development

of drugs. Importance of plant material in the treatment of diseases is due to the phytochemical

compounds that are present in the plant.

Phytochemical also known as secondary metabolites have been investigated as a source of

medical agents with minimal side effects and lower microorganism resistance. L. siceraria

commonly known as Bottle gourd is official in Ayurvedic Pharmacopoeia. It is one of the

excellent fruit for human being made and gifted by the nature having composition of all the

essential constituents that are required for normal and good human health [Badmanaban et al.,

2010].

It’s a vine grown for its fruit, which can be harvested young to be consumed as a vegetable.

When it’s fresh the fruit has a light green smooth skin. It belongs to kingdom Plantae, clade

Angiosperm, clade Eudicots, clade Rosids, order cucurbit ales, family cucurbitaceae, genus

Lagenaria, and species Lagenaria siceraria. Fruits can be huge and rounded, small and bottle

Shaped, or slim and can grow to be over a metre long. The bottle gourd may have been carried

from Africa to Asia, Europe and the America in the course of human migration, or by seed

1
floating across the ocean inside the gourd. It has been proven to have existed in the new world

prior to the arrival of Christopher Columbus.

1.2 Statement of Problem

MDaily many people die in Kenya due to infectious diseases. Microorganisms cause different

diseases and treatment of the diseases has become difficult due to the multidrug antibiotic

resistance of first line antibiotics (Shah et al 2010)

This creates the need to do extensive research on the potential plants that act as antimicrobial

agents

There is a growing need to explore the possibilities of various alternative plants and as well as

alternative system of medicine (Kannan et al, 2012). Plants are a good alternative as they are

easily available and have a great ability to solve the antimicrobial resistance to main stream

medication. Herbal medicine is easily accessible, cost effective and has fewer side effects as

compared to the synthetic or semi-synthetic medicines.

Candida albicans causes superficial infections involving the oral and vaginal mucosa. Candida

albicans causes deep tissues infections and disseminated blood stream infections. The azoles

have been used for the treatment of the bacterial with fluconazole being a significant anti-

bacterial medicine. However, research currently shows that there are strains of candida albicans

that are azole resistance ( maubon, garnaud, sanglard& cornet, 2014). Hence, need for alternative

medicine for the management of the microbe.

E.coli can cause various diseases, including pneumonia, urinary tract infection and diarrhoea.

2
S.aureus has recently acquired resistance to many drugs as it has a multi-resistance strain. The

MRSA is now a critical part in community and hospital acquired infection. The MRSA limits

treatment options with the modern medicine. Hence, MRSA is really difficult to manage which

has necessitated extensive research on the treatment options that are available hence need for

herbal medicine.

E.coli can cause various diseases, including pneumonia, urinary tract infection and diarrhoea.

1.3 Justification

Plants are a major source of alternative microbial agents e.g. the phytochemical compounds like
the tannin, flavonoids, and alkaloids.
Lagenaria siceraria have the phytochemical compounds, and are a good alternative as they are
easily available and have a great ability to solve the problem of antimicrobial resistance to main
stream medication.
Gouuard plant is readily available. Therefore, the success of this study will provide an alternative

source of drug for infections.

1.4 Research Questions

1. What are the phytochemicals present in gouourd plant.

2. Does the L.siceraria have antimicrobial activity against S. aureus, E.coli and Candida

albicans?

1.5 Objectives

1.5.1 General Objectives

1. To determine antimicrobial activity of Lagenaria siceraria against Staphylococcus

aureus, Escherichia coli, Candida albicans.

3
1.5.1 Specific Objectives

1 To determine the phytochemicals present in Lagenaria siceraria.

2 To determine antimicrobial activity of gourd plant against Staphylococcus aureus,
Escherichia coli and Candida albicans.

4
 CHAPTER Chapter TwoWO

2.0 Literature Review

2.1 General Overview

Natural products are a source of new chemical diversity and are the choice of today’s world.

Plants and its products are more reliable for its renewability and therefore, considered as catalyst

for human welfare. The therapeutic potential of plant products can be traced back over five

thousand years ago as there is evidence of its use in the treatment of diseases and for revitalizing

body systems in India, Egyptian, Chinese, Greek and roman civilizations (Mahesh and satish,

2008).

Initially, the ancient communities used to believe that plants possessed magical powers.

Therefore, medicinal herbs were an integral part of traditional medicine. The modern medicine

would be labeled as to be superior to the plant medicine. Recent studies have however shown

that herbal medicines have advantages of availability, cost, and reduced toxicity and reduced side

effect (parasuraman, thing, dhanaraj, 2014). Therefore there is advocacy in used of herbal

medicine. Besides, the general concern of resistance of the many available antibacterial drugs

has given herbal medicine chance for a comeback as alternative medicine (ventola, 2015).

Cucurbitaceae family is commonly known as the gourd, melon or pumpkin family. This
family is composed of 118 genera and 825 species, which are widely distributed in the warmer
regions of the world. The plants of Cucurbitaceae family provide the major contribution for
economically important domesticated species and are cultivated for medicinal and nutritional

5
value. Among all plants of the Cucurbitaceae family, Lagenaria species is the most popular.
The bottle gourd belongs to the genus Lagenaria that is derived from the word lagena,
meaning the bottle. In the older literature, it is often referred to as Lagenaria vulgaris
(common) or Lagenaria leucantha (white flowered gourd), but it is now generally agreed
that the correct name is Lagenaria siceraria (Mol.) Standl. It seems that bottle gourd was
originated from India because its wild races are still found in Dehradoon (high humid
area) and Malabar costal area. Old Indian script reveals its cultivation around 2000 B.C.
Archeological survey supports man s association with bottle gourd in Peru from 1100 to 13000
years B.C.

The bottle gourd is a favorite warm-season vegetable. Genus Lagenaria to which bottle gourd
belongs is characterized by following key features: The fruits are fleshy and multi seeded,
flowers are solitary and chalky white. Both the male and female flowers open at the same
time. Male flowers remain open only for a few hours, after which the petals get withered, thus
the flowers are short lived. Being a monoecious crop, bottle gourd is strictly cross pollinated.
Bees are the major pollinators.

Lagenaria siceraria, commonly known as Bottle gourd is one of the excellent fruits
gifted by the nature to human beings having composition of all the essential constituents
that are required for normal and good human health. Two varieties of this fruit viz., sweet
and bitter are available. Botanically both belong to the same genus. Nevertheless, the difficulty
in procuring and losing interest in cultivation of wild variety, the sweet and edible variety is now
being used in medicine as well.

Family: Cucurbitaceae.

English: bottle gourd

Punjab, Haryana, Delhi: lauki, dudhi or ghiya.

Kerala: Churakka.

Assam: atilao.

Andhra Pradesh: Sora kaaya.

6
Karnataka: Sorekayi.

Ayurvedic: Laghu, Ruska.

All parts of the plant including ripe fruits, leaves, stems and flowers are used for various ailments
(Porterfield GJ, 2005).

2.1.1 Chemical profile of Lagenaria siceraria

Analysis of edible portion of the fruit gave following values: moisture, 96.3; protein, 0.2; fat
(ether extract), 0.1; carbohydrates 2.9; mineral matter 0.5; calcium 0.02; and phosphorus <
0.01%. Other mineral elements reported to be present are: iron (0.7 mg/ 100g.), sodium (11.0
mg. /100g). Potassium (86.0 mg/100g.) And iodine (4.5 mcg/ kg.). Glucose and fructose
have been detected. The amino acid composition of the fruit is as follows: leucines
0.8; phenylalanine 0.9; valine 0.3; tyrosine 0.4; alanine 0.5; threonine 0.2; glutamic acid 0.3;
serine 0.6; aspartic acid 1.9; cystine 0.6; cysteine 0.3; arginine 0.4; and proline 0.3mg/g.
The fruit is a good source of B vitamins and a fair source of ascorbic acid. Bitter fruits yield
0.013% of solid foam containing cucurbitacins B, D, G and H, mainly cucurbitacin B; these
bitter principles are present in the fruit as aglycones. Leaves contain cucurbitacin B and roots,
cucurbitacins B, D and E 2 (Rahman AS, 2003).

Photochemical screening of the fruit revealed two steroids were isolated from the petroleum
ether fraction and they were identified as fucosterol and campesterol 18. Sugar and phenolic
content of the fresh product were assayed, providing a partial nutritional characterization of
this vegetable. Glucose and fructose (about 1:1 ratio) and traces of sucrose were found; in
addition, a small amount of unidentified mono- and di- caffeoylquinic acid derivatives was
detected.5 Flavonoid complexes occurring in the medicinal plants Lagenaria siceraria were
found to be flavone C- glycosides 20. Four new D:C-friedooleanane-type triterpenes
isolated, 3b -O-(E)-feruloyl- D:Cfriedooleana-7,9(11)-dien-29-ol, 3b -O-(E)- coumaroyl-
D:Cfriedooleana-7,9(11)-dien-29-ol, 3b-O- (E)-coumaroyl-D:Cfriedooleana-7,9(11)-dien-29-oic
acid, and methyl 2b ,3b - dihydroxy-D:C-friedoolean- 8-en-29-oate 21.7 A water-soluble
polysaccharide, isolated from fruiting bodies of Lagenaria siceraria, is composed of methyl-α-d-
galacturonate, 3-O-acetyl methyl-α-d-galacturonate, and β-d-galactose in a ratio of nearly

7
1:1:1. This polysaccharide showed cytotoxic activity in vitro against human breast
adenocarcinoma cell line (MCF-7) 22 (Chopra RN, Chopra IC, Verma BS, 2008).

Seed: Seeds are reported to contain saponin. Analysis of seed kernels (68% of seed wt.)
gives following values: moisture, 2.47; protein,30.72; oil,52.54; carbohydrates,8.3; fiber, 1.58;
ash ,4.43; CaO,0.11; and P2O3 , 2.46%. The oil obtained from seed kernals is clear and pale
yellow. Kernels from ripe seeds gave 45% of oil with the following characteristics: n40d ,
1.4711; sap.equiv., 301.6; iodine value ,126.5; free fatty acids,0.54%; and unsaponified
matter , 0.67% . The components of free fatty acids are: linoleic +.36% acids 64.0%; oleic,
18.2%; and saturated fatty acids, 17.8% 2. Seeds are reported to contain Lagenin16. (Rastogi
RP, Melhrotra BN, 2001).

Uses

2.1.2.1 Traditional Uses

The fruits, leaves, seeds, stems are used as medicine in the treatment of jaundice, diabetes,
ulcers, piles, colitis, insanity, hypertension, congestive cardiac failure, and skin diseases.

The fruit pulp is used as emetic, sedative, purgative, cooling, and diuretic. The flowers are an
antidote to poison. The stem bark and rind of the fruit are diuretic. Extracts of the plant have
shown antibiotic activity. Leaf juice is widely used for baldness (kirtikar KR, Basu BD, 2005)

2.1.2.23 Miscellaneous uses

Lagenaria siceraria is a common sight everywhere in the tribal dominated pockets of

Khammam district, where the ethnic groups mainly use the dry shells for carrying country

liquor (mahua drink, toddy), honey and water. Domestic utensils like bottles, bowls, milk pots,

spoons and containers of several types are made out of the dried shells. In some of the

pockets, it is being used for making stringed and wind musical instruments and pipes.

8
2.2 Description of Lagenaria siceraria

L. siceraria is a large pubescent, climbing or tailing herb with stout 5- angled stems and bifid

tendrils, found throughout India, either wild or cultivated.

Leaves are long, petiole, 3-5 lobed, 7-10×10-12 cm, hirsute.

Fruits are large, up to 1.8m long, bottle shaped with a hard shell-like epicarp when ripe.

Flowers are white, solitary, axillary unisexual. Male flowers possess botanical description of

calyx and campanulate, tube narrow, lobes 5, linear; petals 5, white; stamens 3, Female flowers

possess botanical description of calyx and corolla as in male flowers.

Ovaries are densely villous, style thick, stigmas 3, and bilobed 7.

Seeds are white, smooth, 1.6- 2.0 cm. long, horizontally compressed with marginal groove 2.

9
 Figure 2.1 flower of Lagenaria siceraria

Figure 2.2 leaves and fruit of Lagenaria siceraria

2.3 Taxonomy of Lagenaria siceraria

Lagenaria siceraria belong to the kingdom Plantae, subkingdom Tracheobionta, super

division Spermatophyta, division Magnoliophyta, class Magnoliopsida, subclass Dilleniidae,

order Violales, family Cucurbitaceae, genus Lagenaria ser., species Lagenaria siceraria.

10
2.4 Geographical distribution

Lagenaria siceraria is one of the earliest domesticated plants and has a pan tropical distribution.

It may have been spread by ocean currents to the shore of the new world or by human migration

in prehistoric times. It’s known to have been cultivated in An Africa, Asia and the new world in

pre-Columbian times.

It prefers well drained, moist, rich soil. It requires plenty of moisture in the growing season and a

warm, sunny position, sheltered from the wind.

2.5 Economic Importance

Immature fruit can be cooked and used as a vegetable.

The flowers are an antidote to poison.

It is also used as herbal medicine to treat diseases.

2.6 Test Organisms

2.6.1 Staphylococcus aureus

This is a normal commensal of the skin.

It is a member of Genus Staphylococcus. It comprises of gram positive cocci.

S. aureus is one of the most virulent causes of purulent infections. S. aureus grows best

aerobically even though they are facultative anaerobes (Yettou, Desrochers, & Champoux,

2006). The bacteria causes bacteremia, abscess and boils.

11
2.6.2 E. coli

E coli are a normal flora of the human gastro-intestinal tract. The bacterium belongs to the

Enterobacteriacea family. E coli are gram positive bacilli, a lactose fermenter and a major

cause of intestinal infections.

2.6.3 Candida albicans

Candida albicans is part of the natural microflora that is present in the gastrointestinal tract,

mouth and vagina. Normally it is harmless until there is an overgrowth. An overgrowth

causes infections at the sites where it is normally harmless.

12
 CHAPTER THREEChapter Three

3.0 Materials and Methods

3.1 Study site

The leaves and stems were obtained from kisii.

3.2 Study Design

A lab experimental study was carried out to investigate the antimicrobial activity of gourd plant

against Staphylococcus aureus, Escherichia coli and Candida albicans.

3.3 Sampling criteria

The leaves and stems were picked randomly from the farm.

3.4 Collection of samples

The leaves and stems were collected from the farm by hand picking then transported to MMB

laboratory.

3.5 Laboratory Procedures

3.5.1 Water extraction

Washed Tthe leaves and stems were washed with distilled water. Then they were cut them into

small pieces using a sterile scapel. Then crushed the leaves and stems were crushed separately

using a motor and pestle. TPlaced the crushed samples were placed into different beakers, and

added water and t. Then boiled.

13
The solution was aAllowed it to cool and then filtered and kept into plates and placed then in the

oven for 24hours. It was then rReconstituted and placed in the refrigerator for later use.

3.5.2 Ethanol extraction

Dried the leaves and stems. Crushed them and placed them in separate beakers. Then added

ethanol to each beaker and placed them in water bath for 48hours.

Filtered and kept into plates and placed them in the oven for 6hours. Reconstitute and placed in

the refrigerator for later use.

3.6 Phytochemical Analysis

The extracts of gourd plant were analyzed for the presence of the following phytochemicals.

3.6.1 Alkaloids

Each 2 ml of extract was put in test tubes and 2 drops of Meyer’s reagent and dragendorff

reagent was added on each test tube of extract. The formation of orange-red and buff-colored

precipitate indicated a would be positive results for alkaloids.

3.6.2 Flavonoids

Each 5ml dilute ammonia was added to 2ml of the extracts. After which H2SO4 was added to

each and shook. The layers were allowed to separate. A yellow coloration at the ammonia layer

indicateds the presence of flavonoid.

14
3.6.3 Steroids

Each 2ml of extract was put in a test tube.2ml of chloroform was added. After which 2ml

concentrated H2SO4 was added.

3.6.4 Tannins

Each 2ml of extract was put in a test tube, and was diluted with distilled water. After which, 2

drops of ferric chloride solution was added. A transient greenish to black color indicated the

presence of tannins.

3.7 Test Organisms

Pre-bacteria cultures were obtained from the MMB laboratory.

3.7.1 Preparation of inoculum

The plates were incubated aerobically for 24 hours at 370C for the bacterial isolates and for 72-
120 hours at room temperature for the fungal isolates. The discrete colonies that grew were sub
cultured on freshly prepared Nutrient Agar and Potato dextrose agar respectively to obtain pure
cultures.

3.7.2 Preparation of standard inoculum

Pure isolates of S. aureus, E coli and C albicans were diluted in sterile saline.

15
3.8 Antimicrobial Susceptibility testing

About 0.1 of suspensions of E.coli, C.albicans and S. aureus was inoculated on MHA and left to

diffuse for 5 minutes. The culture was done on thirteen plates, one with the leaves extracts, stem

extract, one for the control, from 100 upto105.

Two 6mm diffusion discs were placed in each plate for each microbe. The plates were incubated

at 37 0C degrees for 24 hours. The test was done three times.

The diameters of the zones of inhibition were measured and recorded.

3.9 Minimum inhibitory concentration

Broth dilution method was used for the minimum inhibitory concentration of Lagenaria

siceraria leaves and stem on E.coli and S.aureus. The extracts were diluted using distilled water

and 1ml was put in universal bottle and labeled 10-0. About .1ml was put in the second sterile

universal bottle and 1ml of distilled water added to it and labeled 10-1.I ml was drawn from the

second universal bottle and put in the third sterile universal bottle and labeled 10-2.Iml was drawn

from the third universal bottle and put in a fourth sterile universal bottle and labeled 10-3.1ml of

the solution was taken from the fourth bottle by a sterile syringe and put in a fifth sterile

universal bottle and labeled 10-4.1ml of the solution was drawn from the fifth bottle and put in

the sixth sterile universal bottle and 1ml of distilled water added to it and labeled 10-5.A bacteria

colony was picked using a sterile loop and placed in Mueller Hinton broth . 1ml of each solution

in the universal bottle was taken and added in the bottle containing the broths and incubated at

37 0c 24 hours and turbidity was checked.

16
3.9 Data Processing and Presentation

The data obtained was entered into Microsoft excel spreadsheets where it was subjected to

exploratory data analysis and then analyzed statistically using ANOVA. The results were

presented in table form and graph.

CHAPTER FOURChapter Four

4.0 RESULTSResults

4.1 Phytochemical test

4.1.1 Test for alkaloids

UObservation- upon adding 2 drops of Meyer’s reagent and dragendorff reagent to 2ml of the
extract, an orange-red precipitate and a buff-colored precipitate was observed

Inference- Alkaloids present

4.1.2 Test for flavonoids

UObservation-upon adding 5ml dilute ammonia, concentrated H2SO4 to 2ml extract, a yellow
precipitate was observed

17
Inference-Flavonoids present

4.1.3 Test for steroids

Observation- Uupon adding 2ml of chloroform, 2ml concentrated H2SO4 to 2ml of extract; a
greenish yellow precipitate was formed.

Inference- steroids present

4.1.4 Test for tannins

UObservation- upon adding 2 drops of ferric chloride solution to 2ml extract, a greenish
precipitate was formed.

Inference- tannins present.

Table 4.1: Table of phytochemical test results for stem ethanol extract

Phytochemical tested Inference

Steroids +
Flavonoids +
Tannins +
Alkaloids +

Table 4.2: Table of phytochemical test results for stem water extract

Phytochemical tested Inference

Steroids +
Flavonoids +
Tannins +
Alkaloids +

Table 4.3: Table of phytochemical test for leaf water extract

Phytochemical tested Inference

Steroids +
Flavonoids +
Tannins +
Alkaloids +

Table4. 4: Table of phytochemical test for leaf ethanol extract

Phytochemical tested Inference

18
Steroids +
Flavonoids +
Tannins +
Alkaloids +

Figure4.3: phytochemical test

4.2 Antimicrobial susceptibility testing

Serial 100 10-1 10-2 10-3 10-4 10-5

dilution

power

TEST 1

E.coli 9 8 8 7 7 7

S.aureus 12 10 9 8 7 7

C.albicans 13 11 10 7 7 6

19
TEST 2

E.coli 7 7 7 6 6 6

S.aureus 12 10 9 8 7 7

C.abicans 14 14 12 12 11 11

TEST 3

E,coli 10 9 9 8 7 7

S.aureus 12 11 11 9 9 9

C.albicans 14 14 12 12 11 11

Table 4.5: Table of zones of inhibition of ethanol extract of leaves

Serial 100 10-1 10-2 10-3 10-4 10-5

dilution

power

TEST 1

E.coli 10 9 9 8 7 7

S.aureus 9 8 8 7 6 6

C.albicans 12 12 11 6 6 6

20
TEST 2

E.coli 12 12 12 10 10 6

S.aureus 9 8 8 7 6 6

C.abicans 12 11 11 11 9 8

TEST 3

E,coli 12 11 10 7 7 7

S.aureus 12 10 9 7 7 6

C.albicans 12 12 11 11 9 8

Table 4.6: Table of zones of inhibition of water extract of leaves

Serial 100 10-1 10-2 10-3 10-4 10-5

dilution

power

TEST 1

E.coli 12 11 10 6 6 6

S.aureus 13 7 7 6 6 6

C.albicans 12 11 9 6 6 6

21
TEST 2

E.coli 14 14 13 7 7 7

S.aureus 13 7 7 6 6 6

C.abicans 12 10 10 7 7 6

TEST 3

E.coli 14 13 13 10 10 9

S.aureus 14 12 11 8 8 7

C.albicans 14 10 9 7 7 6

Table 4.7: Table of zones of inhibition of ethanol extract of stem

Serial 100 10-1 10-2 10-3 10-4 10-5

dilution

22
power

TEST 1

E.coli 13 12 11 6 6 6

S.aureus 7 6 6 6 6 6

C.albicans 6 6 6 6 6 6

TEST 2

E.coli 13 11 11 9 9 9

S.aureus 7 6 6 6 6 6

C.abicans 6 6 6 6 6 6

TEST 3

E,coli 12 10 7 6 6 7

S.aureus 11 10 6 6 6 6

C.albicans 8 8 8 8 8 8

Table 4.8: Table of zones of inhibition of water extract of stem

23
 Sum of df Mean F Sig.
Squares Square
Between 22.867 3 7.622 1.766 .231
Groups
Within 34.519 8 4.315
Groups
Total 57.386 11

Table 4.9: Table of ANOVA

There was no statistically significant difference in antimicrobial activity of Lagenaria siceraria

between the different plant extracts as determined by one-way ANOVA (F (3, 8) =1.766,

P<0.231)

*The mean difference is significant at the 0.05 level.

24
 14

12

10

8
STEM(ETHANOL)
STEM(WATER)
LEAVES(ETHANOL)
6
LEAVES(WATER)

0
10^0 10^1 10^2 10^3 10^4 10^5

Graph4.1: Graph of zones of inhibition of E.coli

25
 14

12

10

8
STEM(ETHANOL)
STEM (WATER)
LEAVES(ETHANOL)
6
LEAVES(WATER)

0
10^0 10^1 10^2 10^3 10^4 10^5

Graph4.2: Graph of zones of inhibition of S.aureus

26
 16

14

12

10
STEM(ETHANOL)
8 STEM(WATER)
LEAVES(ETHANOL)
LEAVES(WATER)
6

0
10˄0 10˄1 10˄2 10˄3 10˄4 10˄5

Graph4.3: Graph of zones of inhibition of C.albicans

27
 CHAPTER FIVEChapter Five

5.0 DISCUSSIONDiscussion

5.1 Phytochemical test

Lagenaria siceraria extracts were subjected to phytochemical screening. The results showed the

presence of steroids, alkaloids, tannins and flavonoids.

These results are similar to the study carried out by (Xie, Yang, Tang, Chen, & Ren, 2015). In

the study conducted by (J.O Ayorinde, B.S. Audu, T.O, Ogundeko &A.L.Ujah.) it showed the

absence of tannins and steroids.

The study found that Lagenaria siceraria extract containing tannins inhibit the growth of E.coli

and S.aureus. Tannins chelate iron thus making it unavailable for the microorganisms.

This result agrees with the study carried out by (Ivanov, 2014) that microorganisms need iron for

the normal functioning.

The study found that Lagenaria siceraria extract containing alkaloids interfere with protein

synthesis thus resulting to the death of gram positive and gram negative bacteria.

This result agrees with the study carried out by (chattopadhay et.al. 2001).

This study found that Lagenaria siceraria extracts containing flavonoids and steroids were

effective against S.aureus, E.coli, and Candida albicans.

28
Steroidal compounds are of importance and interest in pharmacy. They are related to sex

hormones and could, by serving as potent starting materials in the synthesis of sex hormones,

ensure such hormal balance. (2001).

This result agrees with related studies of antimicrobial activity which showed significant activity

against various strains of Staphylococcus aureus and Candida albicans by (Chattopadhay et al.,

2001).

5.2 Antimicrobial susceptibility testing

The extract was effective on the three test microorganisms; Ecoli, S.aureus and C.albicans. The

extract was effective upon dilution at 25mg/ml. Results demonstrated that more ethanol extract

had inhibitory effect against the test organisms than water extract.

In Candida albicans, leaf ethanol extract had more inhibitory effect compared to the other

extract.

In E.coli, stem ethanol extract had more inhibitory effect compared to the other extracts.

In S.aureus, leaf ethanol extract had more inhibitory effect compared to the other extracts.

Similar studies were conducted by (Y.P Nagaraja et.al.,2011) that shows in E.coli and S.aureus

leaf ethanol extract had more inhibitory effect compared to the other extract.

29
The Inhibition zones started to increase at 0.25mg/ml showing the maximum inhibition zone

after which the strength of the extract reduced thus reducing the inhibition zones.

The diluted extract was not as effective as the initial concentration of the extract.

This results agrees with related studies of antimicrobial activity that the mechanism of the extract

maybe due to pore formation on the cell wall’ leakage of the cytoplasmic constituents by

components such as alkaloids that were present in the extract.

There were more zones of inhibition in the ethanol extract compared to the water extract. This

result is similar to the study done by (Ghosh et, al., (2008).

There was no statistically significant difference in antimicrobial activity of Lagenaria siceraria

between the different plant extracts as determined by one-way ANOVA (F(3,8)=1.766,

P<0.231)*The mean difference is significant at the 0.05 level.

5.3 Minimum inhibitory concentration

MIC results were indicative of the extract’s activity against bacteria at low doses. MIC is the

lowest concentration of the extract that is able to inhibit growth of Ecoli, S.aureus and

C.albicans. The difference in MIC was recorded by checking turbidity of different broths against

a control.

In E.coli water extract it showed MIC at 2.5mg/ml and ethanol extract it showed MIC at

0.25g/ml.

30
In S.aureus water extract it showed MIC at 0.25g/ml and ethanol extract it showed MIC at

2.5g/ml.

CONCLUSION

The Lagenaria siceraria leaves possess the antioxidant substance which may be potential

responsible for the treatment of jaundice and other oxidative stress related diseases.

Of the 4 extract tested, leaves ethanol extract exhibited greatest antimicrobial activity. Stem

water extract had the least antimicrobial activity.

E.coli was the most susceptible organism tested, having been inhibited by 4 of the extract, while

Candida albicans was the most resistant organism, having been inhibited by 3 of the extracts.

The study concluded that tThere is presence of phytochemicals in the plant.

The fact that some of the organisms susceptible to the extract have shown multi-drug resistance

to conventional drugs, confirms the potential held by herbal medicines as the best alternative for

therapy of the emerging diseases.

31
RECOMMENDATION

There is need for conducting more studies to identify and characterize the medicinal principles in

Lagenaria siceraria, which may serve as novel compounds for development of new and more

effective antimicrobial drugs. This would prove very useful especially in this era when drug

resistance is a major issue.

Since this study involves the evaluation of specific part of the plant, it would be interesting to

investigate other parts of the plant to establish their activity as well.

It would also be valuable to perform synergistic studies to evaluate the performance of

Lagenaria siceraria when combined with conventional medicine.

There should be sensitization on the use of Lagenaria siceraria for their medicinal use as they

are highly nutritious.

32
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