DETERMINATION OF ANTIBACTERIAL ACTIVITY OF WHOLE PLANT EXTRACT

OF Phyllantltus amarus Schum and thonn (kabamba maliba) AGAINST Shigella

dysenteriae.

BY

NAME: MAGIMBI ARTHUR

REG.NO: BPH/0009/141/DU

A RESEARCH REPORT SUBMITED TO THE SCHOOL OF

PHARMACY OF KAMPALA INTRERNATIONAL UNIVERSITY

IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE A WARD OF BACHELOR

DEGREE OF PHARMACY.

SUPERVISOR: DR. ODDA JOHN

MAY2018

DECLARATION

I, MAGIMBI ARTHUR declare that this research is my original work and has not been

presented for the award of any degree elsewhere.

Signature ....... . .. ~ .... . .. . .... . ...... . Date .... . ..... ~"1/P.S/ ?:-PJ.{ ........ .

BPH/0009/141/DU

DEDICATION

I dedicate this to God , all my family members and dear friends for the spiritual, financial and

moral support they have given me through this period. I do this with out forgetting all lecturers

and finally my supervisor Dr John Odda for the effort they have put in to help me finish my

course.

ii

APPROVAL

I hi'> r~ ... carch prnposal is suhmilled to the school of pharmacy with the approvnl or the

... up~.:rv i:,or.

Sig11allll\' ... ~ .. • ............... .. Dale . . M.. 0.1 .... l.f.; ... ~. J. &' lk < >DDJ\ JOliN

1\.aulpala lnlcrnntiunal University western campus

TABLE OF CONTENTS

DECLARATION ............................................................................................................................ i

DEDICATION ............................................................................................................................... ii

APPROVAL ................................................................................................................................. iii

ACKNOWLEDGMENT ......................................... , ................................................................... iv

LIST OFT ABLES ...................................................................................................................... vii

ABBREVIATIONS .................................................................................................................... viii

CHAPTER ONE ........................................................................................................................... 1

1.0 INTRODUCTION ................................................................................................................. 1

1.1 BACKGROUND INFORMATION ....................................................................................... I

I .2 Statement of the pro b !em ...................................................................................................... 2

1.3 Justification for the study ...................................................................................................... 2

I .4 Research questions ............................................................................................................ 3

I .5 .2 Specific objectives .......................................................................................................... 3

CHAPTER TWO .......................................................................................................................... 4

2.0 LITERATURE REVIEW ...................................................................................................... 4

2.5 Methods for antibacterial susceptibility ................................................................................ 8

2.6 Minimum Inhibitory Concentration .................................................................................... l 0

2. 7 Minimum Bactericidal Concentration ................................................................................. I 0

CHAPTER THREE ...................................................................................................................... 11

3.0 MATERIALS AND METHODS ......................................................................................... 11

3 .I Study Design .. . . . .. .. . . .. .. .. . . . .. .. . . .. . .. . . .. .. . .. .. .. . .. .. .. .. .. .. . .. .. .. .. .. .. . . . .. .. .. .. .. .. .. .. . . .. .. .. . . .. .. .. .. . .. .. .. . .. .. ll

3.2 Study area ............................................................................................................................ ll

3.3 Plant collection and identification ....................................................................................... 11

3.4 Storage, drying and pulverization ....................................................................................... 11

3.5 Plant extraction .................................................................................................................... 12

3.6 Determination of extract yield(% yield) ............................................................................. 12

3.8 Determination of Minimum Inhibitory Concentration (MIC) using Broth dilution method

··················································································································································· 15 3.9 Determination of Minimum Bactericidal Concentration (MBC) ........................................ 16

3.10 Quality control ................................................................................................................... 17

v

3.14 Data analysis ..................................................................................................................... 18

3.15 Ethical considerations ....................................................................................................... 18

CHAPTER FOUR ....................................................................................................................... 19

4.0 RESULTS ............................................................................................................................ 19

4.1 The results for susceptibility test of Shigella dysenteriae to ethanol whole herb extract of Phyllanthus amarus ................................................ : .................................................................. 19

CHAPTER FIVE ........................................................................................................................ 23

5.0 DISCUSSION, CONCLUSION AND RECOMMENDATIONS ...................................... 23

5.1 DISCUSSION ..................................................................................................................... 23

5.2 CONCLUSION ................................................................................................................... 25

5.3 RECOMENDATIONS ........................................................................................................ 26

REFERENCES ............................................................................................................................ 27

APPENDICES ............................................................................................................................. 30

APPENDIX 1: TIME FRAMEWORK AND WORK PLAN ................................................. 30

APPENDIX 2 ............................................................................................................................... 31

Figure I: susceptablity testing agar well diffusion.( zones of inhibition) ................................. 3 I

vi

LIST OFT ABLES

Table I: Scientific classification of Shigella dysenteriae.

Table2: The results of susceptibility sample to Shigella dysenteriae on Phyllanthus amarus

extracts and controls.

Table3: The results showing minimum inhibitory concentration (MIC) of Shigella dysenteriea

against Shigella dysenteriae bacteria.

Table4: Minimum bactericidal concentration (MBC) of Phyllanthus amarus against Shigella

dysenteriae.

vii

MBC

MIC

NCCLS

!CLS

BSAC

K!U

ABBREVIATIONS

Minimum bactericidal concentration

Minimum inhibitory concentration

National Committee for Clinical Laboratory Science

Institute of Clinical Laboratory Standards

British Society for Antibacterial Susceptibility Testing

Kampala International University

viii

CHAPTER ONE

1.0 INTRODUCTION

l.lBACKGROUND INFORMATION

Medicinal plants over the century have been used as herbs for the treatment of several human

diseases and have been very important in the health care delivery at one stage or the other of

every nation (Oiuma et al., 2004). Research of recent has focused on natural plant products as

altematives to for existing drugs for disease therapy in developing countries (Aiyegoro et al.,

2007). Medicines derived from Plant have been part of traditional health care in most areas of

the world for centuries and there is increasing interest in them as sources of agents to fight

microbial diseases (Ajayi and Akintola, 2010). The emergence of multiple antibiotic resistant

organisms has constituted a global problem as far as treatment of some infectious diseases is

concerned.

The vehicles of transmission of this etiologic agent are mainly food and water. Several other

disease-causing organisms of medical importance have also developed resistance to these

conventional antibiotics. Infectious diseases still remain an impm1ant cause of morbidity and

mortality in man, especially in developing countries. In Africa today, many resort to the use of

locally made herbal medicines prepared as infusions in hot water, decoction in cold water,

concoction with food and as tinctures with alcohol as an alternative therapy for bacterial

infections (Oluduro and Omoboye, 2010). Plant parts such as the leaves, roots, bark, shoots, fruit

peels, immature and unripe fruits have been used in most herbal preparations. According to

George and Pamplona-Roger (1998) the therapeutic value of some common plants have been

used in the treatment of various ailments including enteric fever, dysentery, diarrhoea,

convulsions, common cold, malaria, jaundice, yellow fever, dental caries, intestinal parasites,

gastroenteritis, viraL bacterial and protozoan diseases. Antiseptic, diuretic, and anti­

inflammatory antibacterial properties have equally been reported (Alunas et al., 2005).

Medicines of herbal sources is readily available in our diverse vegetation, cheap and above all

carries the potential for introducing new templates into modern medicine (Akinyemiet et al.,

2005). Many countries in the world, including Ghana, practitioners of herbal medicine are still

consulted as a first choice in the treatment of ailments, due to the fact that traditional medicine

1

blends readily with the socio-cultural life of the people, and the truth that rigt medicine are more

expensive to procure and some right pharmaceutical preparations are many times faked (Abase et

a!., 20 II). There is a vast array of medicinal plants used singly or in combination with other

medicinal plants that confer synergistic effect in the treatments of various ailments.

In central uganda, Phyllanthus amarus is used for treatment of bacterial dysentery and this is

done by boiling fresh leaves of Phyllanthus amarus in water for about 30 minutes and when cool

the patient is given the resultant soup orally amounting to mug cup of about 500ml twice a day

for about a week.

Despite the wide herbal use of Phyllanthus amarus in the treatment of diarrheal diseases to

include dysentery there is a lack of scientific publication as to its efficacy against dysentery

caused by Shigella dysenteriae. The present work is an eff01i to determine the activity of leaf

extract of Phyllanthus amarus against Shigella dysenteriae.

1.2 Statement of the problem

It is estimated that 80% of people worldwide rely on herbal medicine for health care (iyenga .,

2012) and Phyllanthus amarus preparation is used to control several diseases derived for from

microbial infections which include bacterial dysentery.

The acceptance now of traditional medicine as an alternative form of health care and the

development of resistance to available antibiotics have led to widespread investigation into the

antibacterial activity of medicinal plants (Bisignano et a/.,!996). Despite the herbal use of

Phyl/anthus amarus in treatment of diarrheal diseases to include dysentery, there is lack of

scientific publication as to its efficacy hence there is need to study the antibacterial activity of

extract of Phyllanthus amarus used in central Uganda for traditional treatment of bacterial

dysentery.

1.3 Justification for the study

Phyl/anlhus amarus is a medicinal herb considered efficient for the cure of various ailments.

Despite its wide herbal use in the treatment of diarrheal diseases to include dysentery, there is

lack of scientific publication as to its efficacy against Shigella dysenteriae hence this study is

necessary to determine the activity of whole plant extract of Phyllanthus amarus against

Shigella dysenteriae

2

1.4 Research questions

What is the sensitivity of Shigella dysenteriae to various concentrations of crude extract of

Phyllanthus amarus?

What is the Minimum inhibitory concentration of whole plant extract of Phyllanthus amarus

against Shigella dysenteriae?

What is the Minimum bactericidal concentration of whole plant extract of Phyllanthus amarus

against Shigella dysenteriae?

1.50bjectives

1.5.1 Main objective

The main objective of this study is to investigate the antibacterial properties of whole plant

extract of Phyllanthus amarus on Shigella dysenteriae in vitro.

1.5.2 Specific objectives

•!• To determine the sensitivity of Shigella dysenteriae to various concentrations of crude

extract of Phyllanthus amarus.

•!• To determine Minimum inhibitor concentration (MIC)of whole plant extract of

Phyllanthus amarus against Shigella dysenteriae.

•!• To determine Minimum bactericidal concentration (MBC) of whole plant extract of

Phyllanthus amarus against Shigella dysenteriae.

3

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Phyllanthus amarus description

Phyllanthus amarus is a member of the Euphorlliaceae family (Spurge family), with over

6500species in 300 genera. Euphorbiaceae family consist of upright or prostrate herbs or shrubs,

often with milky acrid juice and is mainly a pan-tropical family with some species either more or

less temperate. The fruit is a 3-lobed capsule containing 6 seeds and extends from the cup with

a long stalk pendant about l-2mm. The leaves are simple, alternate or opposite and leathery, and

borne on petioles 0.3 to 0.5 mm long. The flowers are very small and diclinous, often in clusters

borne in greenish cup-shaped structures with glands. The plants are monoecious or monogamous.

It has smooth cylindrical stem thick and deciduous horizontal branchlets 4-12cm long and 0.5cm

thick, with 15 to 30 leaves.

2.1.1 Origin and distributon of Phyllanthus amarus

The plant is indigenous to tropical Americas, the Philippines and India (Chavellier, 2001). Plants

in the genus Phyllanthus are found around all tropical regions of the world; from Africa to Asia,

South America and the West Indies. The genus contains about 550-750 species in 10-11

subgenera (Unundar,l998). Some related species with medicinal importance include.

epiphyllanthus, Phyllanthus niruri Phyllanthus surinaria, Phyllanthus acuminatus and

l'hyllanthus emblica (Tirimana, 1987). The plant can be found along roads, valleys, on

riverbanks and near lakes. Phyllanthus amarus is sometimes mistaken and wrongly identified

with the closely related Phyl/anthus niruriL. In appearance, phytochemical structure and history

of use. Phyl/anthus niruri also reaches a height of 60 em with larger fruits and dark brown and

warty seeds than that of Phyllanthus amarus (Morton, 1981 ).

2.2 Traditional uses of Phy/lanthus amarus

Phyllanthus species are used ethnobotanicaly and in folk remedies in many countries.

Phyl/anthus amarus, Phyl/anthus nururi and Phyl/anthus urinaria have also been used in the

treatment of kidney related problems, gallstones, appendix inflammation and prostate problems

(Haydet, 1990). According to Foo and Wong (1992), the aerial patt of Phyllanthus amarus is

4

highly valued in traditional medicine for its healing properties. Fresh leaf juice of the plant can

be applied externally for the treatment of cuts and bruises. It is also good for treating Arthritis

and Asthma in patients (Adebisi, 1999).It has a long history of use in the treatment of liver,

kidney and bladder problems, diabetes and intestinal parasites (Foo, 1993). Phyllanthus amarus

has also shown to work as an antifungal, antibacterial and antiviral agent (Houghton et al.,

1996).Adeneye et al., (2006) reported that Phyllanthus amarus was used in traditional

medicine for its hepatoprotective, anti-diabetic, anti-hypettensive, analgesic, anti­

inflammatory and antimicrobial propetties. Joy and Katted (1994) also rep01ted the anti­

nociceptive, anti-lipidemic, anti-diabetic, anti-inflammatory, anti-lithic and anti-bacterial

properties of the plant. It is used traditionally in the treatment of liver ailments and kidney

stones. Whole plant is employed in some genitourinary infections. The young tender shoots are

used in chronic dysentery and the juice of the stem is mixed with oil in ophthalmology for eye

treatments.

Odugbemi (2008) and Chaudhury (2007) reported that the plant is effective for treating

gonorrhoea, genito-urinary diseases, asthma, diabetes, typhoid fever, jaundice, stomach-ache,

dysentery, ringworm, and hypertension. Kokarso (1996) confirmed the use of the plant in the

treatment of stomach disorders, skin diseases and cold. Nanden (1998) also repotted that the herb

is used to combat fever, flu and asthma in combination with other herbs. The plant when boiled

with the leaves is considered to be a diuretic and can be used in treating diabetes, dysentery,

hepatitis, menstrual disorders, and skin disorders. The herb can also be used for constipation. The

extracts from the roots can be used to treat jaundice (Heyde, 1990). In recent years, the plant has

been used successfully as a liver-protectant/detoxifier for conditions such as jaundice and

hepatitis B and can rapidly restore full functioning of a damaged liver. It has widely been

reported to offer good treatment for leprosy, hiccup, and peptic ulcer. It is anti-spasmodic, good

laxative, blood tonic, treatment of itch, flu, fever, dyspepsia, blennorrhagia, tenesmus,

gonorrhoea, malaria, uterus complaints, constipation, anorexia, carminative, tumour, colic; it

has HIV inhibitory activity, good anti - inflammation of appendix, bladder disorder (Obianime

and Uche, 2009). Meixa et al. (1995) confirm the anti-viral activity of Phyllanthus amarus

against hepatitis B virus. Phyllanthus amarus which is otherwise called Eyin-olobe in South­

Western Nigeria has healing effects on hypertensive patients. It was equally found efficacious for

5

treating malaria, diabetes, kidney stones and jaundice Phyllanthus amarus can be taken for

weight loose and help to increase male fertility.

2.3 Phytochemical properties of Phyllanthus amarus

Houghton et al. (1996) isolated securing type alkaloids by Column Chromatography (CC) and

preparative Thin Layer Chromatography (TLC). Hydro-alcoholic extract of Phyllanthus amarus

showed the presence of various phyla-constituent such as alkaloids, flavonoids, saponins and

tannins (Ambaba et al.,20 II). Experiments conducted into the phytochemicals present in

Phyllanthus amarus using UV, IR, Mass and NMR spectroscopy revealed that alkaloids,

flavanoids, hydrolysable tannins, major lignans and polyphenols were present in the plant Foo

and Wong(l992). Previous studies have reported some of these phyla-components to elicit a

wide range of biological activities, which include hypolipidemic, hypoglycemic, hypoazotemia

among others. Saponin is known to elicit serum cholesterol lowering activity by causing resin­

like action, thereby reducing the enterohepatic circulation of bile acids (Topping et al., 1980). In

the process, the conversion of cholesterol to bile acid is enhanced in the liver resulting in

concomitant hypocholesterolemia (Kritchevsky, 1977). The lignan constituents in the plant

consist of phyllanthin and hypophyllathin, amarinic acid, nynphyllin, phyllarurin and

ncolynan. Geranin a phytochemical in the plant has hypertensive effects and account for it's used

in hypertension conditions. This chemical can inhibit several neurotransmitter processes that

relay and receive pain signals in the brain. Geranin also has anti-ulcerous properties

(Chaudhury, 2007). Alkaloids and tannins contain in the plant contribute to the plants

effects as anti-malaria, anti-diarrhoea and analgesic agents.The major lignans, Phyllanthin and

Hypophyllanthin have been reported to exhibit anti-hepatotoxic activity.

2.4 SHIGELLOSIS

Shigellosis is a current health burden which is endemic and estimated to affect 80-165mllion

individuals annually. Ninety nine percent of infections caused by shigella occur in developing

countries, and the majority of cases and cases of deaths occur among children less than 5 years of

age ( kotloff et al., WHO. 2005). Shigella are gram negative intracellular bacterial pathogens that

inhibit the gastro intestinal tract of humans and are the causative agent of shigellosis.

6

2.4.1Shigella diseases and symptoms

Shigellosis is a disease affecting humans is usually acquired through contaminated food and

water sources (DuPont et al., 1989). The high incidence of shigella in developing countries is

considered to be attributed to the lack of clean water, poor sanitation, malnutrition and the cost of

antibiotic treatment (Jennison&verma, 2004). Shigella infect through the oral faecal route and

infection is transmissible with as few as 100 micro-organisms due to the bacterium's ability to

survive the acidity of stomach (Small et a/.,1994). Infection is known to produce a range of

symptoms and range from watery diarrhoea to severe dysentry. Severe dysentery is characterised

by fever, abdominal pain and acute permanent bloody and mucoid diarrhoea

(Phalipon&sansonetti, 2007). In the absence of effective treatment, patients with shigellosis can

develop secondary complicatons such as septicaemia and pneumonia

2.4.2Pathogenesis and Pathology of Shigella

The initial step in pathogenesis is clearing bacterial invasion or penetration of the colonic

mucosa, the resulting focus of Shigella infection is characterized by degeneration of the

epithelium and by an acute inflammatory elements, and dependent upon the ileocecal flow. As a

result, the patient will pass frequent, scanty, dysenteric stools (Hale, 1991 ).

The virulence factors include;

A. Lipopolysaccharide (LPS): LPS plays an important role in resistance to nonspecific

host defense encountered during tissue invasion. These genes help in invasion,

multiplication, and resistance to phagocytosis by tissue macrophages. LPS enhances the

cytotoxicity of Sh ET on human vascular endothelial cells.

B. Toxins Shigella dysenteriae produce an exotoxin, Shiga toxin. Also enterotoxins

designated shETJ and shET2 have been identified, and the genetic loci encoding these

toxins have been localized to the chromosome and plasmid respectively

C. Siderophores: Ability to secrete iron from chelating compounds, 'siderophores'

which chelate iron from intestinal fluids and then are taken up to release iron

inside the bacterium for its metabolic needs.

7

2.4.3 Shigella dysenteriae description

Shigella dysenteriae is a species of rod shaped bacterial of genus shigella( Ryan, et al., 2004).

Shigella species can cause shigellosis and Are gram negative, non spore forming, facultative

anaerobic non motile bacteria(Hale, et al., 1996).

Table I: Scientific classification of Shigella dysenteriae.

KINGDOM

Phylum

Class

Order

Family

Genus

Specie

BACTERIA

Proteobacteria:

Gammaproteobactera

Enter()ba'ctefiliies< ··· ··

Enterobacteriaceae

Shigella i {/. ;;t,;J,.~'?it¥i~@;,i:, Shige/ladysenteriae

Shigella dysenteriae is spread by contaminated water and food, causes the most severe dysentery

because of its most potent and deadly shiga toxin, but other species may also be dysentery agents

(Herold. If et al.,2004).

Most commonly observed signs associated with shigella dysente1y include colitis, malnutrition,

rectal prolapsed, tenesmus, reactive arthritis and central nervous system problems. Further,

S.dysenteriae is associated with the development of hemolytic uremic syndrome, which includes

anemia, thrombocytopenia and renal failure.

2.5 Methods for antibacterial susceptibility

Antibacterial susceptibility testing standard tests can be conveniently divided into diffusion and

agar dilution methods. Agar diffusion susceptibility testing is regarded as the golden standard for

all other susceptibility testing methods.

It is important to prepare agar plates in such a way that the antibacterial concentrations in the

plates are exactly or very close to the desired concentrations. When preparing the antibacterial

solutions and agar plates for agar dilution susceptibility testing, the following guidelines will be

8

recommended as given by the national committee for clinical laboratory science (NCCLS)

(Rhoad, 2004).

The dilution procedure is going to seems to be complicated, but this method will ensure that

there is minimal risk of making out of scale dilutions for the smallest concentrations. The ethno

pharmacology research the Antibacterial Susceptibility Testing (AST) (Tyler and Eric, 2014)will

be used to determine the efficacy of potential antibacterial from biological extracts against a

number of different bacterial species. In clinical research in vitro susceptibility tests is going to

be impmiant if an organism is suspected to belong to a species that has shown resistance to

frequently used antimicrobial agents.

They might also impotiant epidemiological study on susceptility and in comparison of new and

existing antimicrobial agents (EUCAST 2003). Successful discovery of novel natural

antimicrobials has necessitated the development of new bioassay techniques which are sensitive

enough to detect small amounts of biologically active chemicals (Lampinen, 2005).

Standardized in vitro tests it will be essential for screening plants extracts or compounds by

using MlC determination of natural products in order to get results that are comparable to those

of currently used antibiotics (Devienne and Raddii, 2002). When evaluating the performance of a

susceptibility tests it should include criteria like ease of use and the ability to yield the same

result when repeated testing is done, test sensitivity and specificity (Struelens, et a/1995).

Although current standard methods, approved by various bodies like the National Committee for

Clinical Laboratory Science(NCCLS) now known as Institute of Clinical Laboratory Standards

(ICLS), British Society for Antibacterial Chemotherapy (BSAC) (Atihur 1953) and the Europe

Committee for Antibacterial Susceptibility Testing (EUCAST), exist for guidelines of

antimicrobial susceptibility testing of conventional drugs, these might not be exactly applicable

to plant extracts and modification have to be made (Hammer et al, 1999).

Dilution methods includes agar dilution and broth micro/macro dilution. The broth and agar

based methods are the conventional reference methods for AST (Tenover eta!., 1995).

9

2.6 Minimum Inhibitory Concentration

Minimum inhibitory concentration (MIC) is defined as the lowest concentration antimicrobial

agent (mg/l) that will prevent the appearance of visible growth of microorganism within a period

time. Dilution method are used to determine the MIC of antibacterial susceptibility testing. The

MlC is used in resistance surveillance, the comparative testing of new agents to establish the

susceptibility of organisms that give equivocal results in discs test. Broth dilution is a technique

use for MlC in which containers holding identical volume of broth with antibacterial solution

(Mann and Markham, 1997).

2.7 Minimum Bactericidal Concentration

Minimum bacterial concentration (MBC) is method used to determine if the extract can kill or

inhibit the growth of bacteria pathogens. The minimal bactericidal concentrations is determined

after the MlCs results were obtained. This will be done by selecting the tube that shows no

growth during the M!Cs determination. A loop (sterile wire loop) from the tube containing the

media and the extract are inoculated into a sterile nutrient broth and Sabouraud's dextrose liquid

of freshly prepared media. These will be future inoculated for another 72hrs at 37°c for bacteria,

after which they will be examined for any bacterial growth. The lowest concentration at which

no growth is observed on the plate gives the MBC (Yamamoto and Loren, 2003).

10

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Study Design

The study to determine Phyllanthus amarus extract activity on Shigella dysenteriae was

experimental in the laboratory and it was conducted in JAN- APRIL 20 I 8 at KIU microbiology

laboratory.

3.2 Study area

This study was conducted at Kampala International University-Western campus at the hospital

microbiology and pharmacology laboratories because of availability of trained personnel and

equipment's that were needed and experiment was conducted for three (3) months. The

extraction was carried out in pharmacology laboratory while the susceptibility of Shigella

dysenteriae on Phyllanthus amarus extract was done in the hospital microbiology laboratory in

KIU teaching hospital, according to European Committee for Antibacterial Susceptibility Testing

(EUCAST) and European Society of Clinical Microbiology and Infectious Disease (ESCMID)

2003 procedure.

3.3 Plant collection and identification

The fresh whole plant of Phyllanthus amarus was collected in January 2018 where it grows

naturally in a banana garden in wakiso district central Uganda. The collected herb was deposited

at the department of botany of Mbarara and identified by a botanist and was given a voucher

number.

3.4 Storage, drying and pulverization

The collected herb sample was washed, chopped in small pieces and dried in a shade to avoid

direct sunshine that could degrade phytochemical due to ultra violet light because the active is

not known. The herbs were air dried at room temperature by displaying it on a dry cemented

table in pharmacology lab and it was turned daily to prevent fungal attack until complete dryness

that was confirmed by weighing every week for three weeks and the constant weight was

achieved then the plant was confirmed dry.

11

The dried sample was ground into powder by grinding it using a blender (Paola and collectors,

20 II). The reason for this is to increase the surface area contact between the plant particles and

the solvent during extraction, it is because the powder can easily be mixed with the solvent,

reduce particle size powder also facilitates crude extraction and more so also increase dissolution

rate.

3.5 Plant extraction

The plant extracts was prepared by using the method of (Alade&Irobi 1993). Extraction was

performed by macerating air-dried, powdered of Phyllanthus amarus in 70% ethanol.

Plant material was grinded and weighed, the initial weight of powder was 375g and it was

soaked in 3. 7 litres of 70% ethanol and put on a mechanical shaker for 72hours. After 72 hours

of agitation on mechanical shaker, the mixture was sieved using a clean sieve and after the

filtration was made using DRs Watts filtering paper of (0.5~tm) pore size in a funnel into a

measuring cylinder which was weighed mean while the residue which remained on the filter

paper was collected and air dried pending for extraction incase if the extract was not going to be

enough.

The filtrate was put in the drying oven at 50°c for concentration, after concentration the weight

of the concentrate (extract) was 17.6g then it was stored in the refrigerator at 4°C until the time it

was needed.

3.6 Determination of extract yield (% yield)

According to Paola et al (2011) the yield% w/w from all the dried extracts for example ethanol

extract was calculated as:

% yield =weight of extract X I 00% Percentage yield for ethanol=l7.6 x 100% = 4.7%

Weight of the powder 375

12

3.7 Determination of susceptibility activity of Phyllanthus amarus on Shigella dysenteriae

The isolation of the organism from KIU teaching hospital using stool from the patient

sample

Stool culture was collected and inoculated in Deoxycholate citrate agar (DCA) and salmonella

shigella agar (SSA) a selective media for the isolation of Shigella and Salmonella.

Cultural technique was done following aseptic precautions for avoidance of any contamination

fi·mn the surrounding environment. A sterile wire loop was used for inoculation of the sample on

a sterile dried culture media mentioned above. Following inoculation, culture plates were

incubated at 37°C for 24 hour.

Identification

A cultural characteristic of the isolates was observed for size, colour, texture, and appearances;

Shigella colonies appeared colourless on the media.

Morphological characteristics

Gram stain was done by making a smear of the colonies on clean dried glass slide, using a sterile

wire loop. The slide was allowed to air dried and the smear fixed over a Bunsen burner flame

three (3) times. The slide was taken to the staining rack and was stained with crystal violet for I

minute, treated with Jugal's iodine for 30 seconds, decolourize with 50% acetone alcohol under

slow running tap water until no more colour was observed flowing from the slide. The slide was

counterstained with I% Neutral red for 2 minutes; it was washed with water, drain dried and

examined with X I 00 oil immersion lense.

Gram negative rods bacteria were observed which the morphological characteristics of Shigella

species are.

13

Biochemical test

A biochemical test was done to further confirm the isolates by inoculating the colonies on triple

sugar iron (TSI) agar.

Result

Shigella species was observed by fermentation of the butt producing acid which changed the

colour of the media to yellow while the slant remained red showing non fermentation which

indicates alkalinity of the slant.

The preparation oftest extract stock solution

The stock solution of the plant extract was achieved by diluting 512mg of the extract in IOOOml

in order to achieved 512mg/L concentration of extract.

The preparation of Mueller Hinton agar

The broth was prepared by weighing 9.5g of the powdered media that was dissolved in 250mls

of distilled water and it was sterilized in the autoclave in the temperature of 121 'c at

I 00kpa(15psi) above atmospheric pressure for IS minutes.

The sterilized agar media was mixed in the heat preparation from the bursen burner and it was

dispensed in the sterilized culture plates where it was left to solidify in the covered plates. The

test microorganism was aseptically inoculated (approx. 1.0 x 106 colony forming units/ml) on

sterile Mueller !-linton agar by surface spreading to make uniform microbial inoculums. Using

sterile glass cork borers (6 mm in diameter), wells were carefully made on the agar plate without

distorting the media; to contain test extract.

A separate agar plate was used to test the control drugs; ciprofloxacin SJ.tg/ml against the bacteria

because it is first line for shigellosis as positive control and negative control contains solvent

used for dilution (distilled water)

Two hundred micro liters (200J.tl) of ethanol extracts in each well was dispensed into the well.

Controls were also dispensed and the plates were left on the bench for I 0 minutes in order for the

extracts not to pour away. The culture plates were then incubated at 37°C for 24 hours.

14

Using a metric ruler, the diameter of the zones of inhibition were taken in millimeters (mm) (the

diameter of the area of no growth of the microorganism around the disc) was measured for the

control in antibiotics and extract (Baris et al., 2006). The zone of inhibition was >6mm the

bacteria was said to be susceptible to the extracts and that's why the MIC was done. Since the

extract passed by satisfying the set criteria hence it was subjected to the MICas showed down

3.8 Determination of Minimum Inhibitory Concentration (MIC) using Broth dilution

method

Minimum inhibitory concentrations (MIC) of Phyllanthus amarus extract were performed on

organism Shigella dysenteriae using tube dilution method (Koneman et al., 1997). The bacterium

was inoculated in the serial dilution of eight tubes of Phyllanthus amarus extracts for ethanol

with 1 mg/ml of nutrient broth.

The preparation of MIC set np

It involved the use of I 0 test tubes in total for both ethanol extract that were dried in the hot air

drier and the test tubes were labeled from test tube I to I 0. In each test tube lml of the broth was

introduced.

Test tube I , I mls of the extract from stock of 512f,Lg/ml was introduced and it was mixed

thoroughly with I ml of the broth to give the concentration of 256 f.Lg/ml and I ml of the mixture

was transferred to test tube 2 and mixed to give the concentration of 128f,Lg/ml.

The 128f,Lg/ml of the solution was again picked and transferred test tube 3 to give the

concentration of 64f.Lg/ml. The 1 ml of 64f,Lg/ml was again picked and transferred to test tube 4 to

give the concentration of 32f.Lg/ml after thorough mixing.

The l ml of 32f.Lg/ml was again picked and transferred to test tube 5 and mixed thoroughly to give

the concentration of l6f.Lg/ml.

The I ml of l6f.Lg/ml was picked again and it was transferred to test tube 6 and it was mixed

properly to give the concentration of 8f.Lg/ml. then lml of Sf.Lg/ml was picked and transferred to

test tube 7 and it was mixed thoroughly to give the concentration of 4f,Lg/ml and finally test tube

8 I ml of 4f.Lg/ml was picked and transferred and mixed thoroughly to give the concentration of

2flg/ml.

15

Concentrations used from test tube J-8 (256flg/ml, 128J.lg/ml, 64J.lg!ml, 32J.lg/ml, l6J.lg/ml,

8J.lglml, 4J.lg/ml, 2J.tg/ml)

The bacteria were prepared as follows;

The test bacteria was made by getting the colonies from agar plate and dissolving in 2m! of

normal saline while comparing with McFarland standard giving the bacteria concentration of

l.Ox!06cfu/ml (cheesbrough et al2002). Then Jml of this bacterial solution was added to the first

test tube that contained I ml of broth which was containing 256J.lg/ml of the test extract. This

process resulted into the bacteria concentration in the first test tube (original bacterial

concentration in the broth) to become 5.0xl 05cfu/ml. This procedure of serial dilution was

repeated for all the test tubes giving the test extract concentration of 128J.lg/ml, 64;tg/ml,

32flg/ml, I6J.lg/ml, 8flg/ml, 4J.lg!ml ,2J.lg!ml and I J.lg/ml from test tube 1-8.

There were two controls i.e. Control I and control 2 which were also prepared as follows:

For control one (CJ) that had broth and bacteria and had no drug in order to find out whether the

media supported the growth of organism. On the other hand, control 2 (C2) had only broth and

no bacteria and no drug to determine whether the broth was contaminated by other organisms or

not.

Thereafter, the tubes were incubated at 37"C for 24 hours (Koneman et al., 1997). After

incubation, the tube with no turbidity next to the one showing turbidity (Microbial growth) was

considered as containing the MIC of the extract in question. The tubes that did not have growth

were showed by no turbidity in them.

All extracts that exhibited MIC of I OOJ.lg/ml and below were considered wotih further

investigation and Vice versa (Oiila., et al2001).

3.9 Determination of Minimum Bactericidal Concentration (MBC)

Following the MIC determination using the Broth dilution method, after the recommended

period of incubation, the Mueller Hinton agar was prepared and using a wire loop (O.Oiml)

samples were picked from the broth dilution that didn't have any growth after the incubation and

were spread on the agar plate that containing Mueller Hinton agar aseptically. This was followed

by incubation at a temperature of 37°C for 24 hours. The MBC was determined as the lowest

16

concentration of the extract that allowed less than 0.1% of the original inoculums of 5xl05cfu/ml

to grow. The agars that were showing no growth of the microorganism was considered as the

Minimum Bactericidal Concentration (MBC). The purpose of this test was to determine the

lowest concentration at which the ethanol extracts was able to kill Shigella dysenteriae (Baris et

al., 2006).

3.10 Quality control

Voucher specimen was prepared and deposited in the Herbarium of botany department for

correct botanical identification. Fresh plant materials was collected and dried under shade to

prevent loss of phytochemical due strong ultra violet and direct and can also cause evaporation

of volatile oil. The ethanolic extract was stored in sterile bottle to prevent contamination. The

bacteria specimen was carefully cultured and the media was prepared according to the

manufacturer's standards and it was tested to find whether it supports growth of bacteria and the

bacteria was isolated from patient specimen and it was tested for susceptibility to the standard

drug, the plates were carefully covered to avoid contamination from the environment organism

and the loop was carefully sterilized before the organism was picked.

3.11 Reliability of the study

After test microorganism was isolated, it was tested for effectiveness before the main research

was carried out in order to avoid the use of resistant type of the organism

The experiment had two controls that control l and control 2

3.12 limitation of the study

I didn't do phytochemical analysis due to time and financial constrains

I didn't do toxicity, safety and efficacy in lab animals and humans due to time period was shOJi.

[ also failed to determine the mechanism by which plant extract inhibit bacterial cells due time

limit and financial factors.

3.13 Time limit

The research study took 3months beginning from January of201 to April of2017.

17

3.14 Data analysis

The data collected was entered into Ms Excel and then analyzed in STATA vl4 using the

ANOVA test. A p value less than 0.05 was considered statistically significant. A post hoc

analysis (Bonferroni test) was carried out to identify the sources of differences. The results were

presented as Mean±SEM and as graphs .Based on microbiology guidelines if the mean diameter

of the zone of inhibition was >6mm then the plant extract was proceeded for the MIC.

ForMIC and MBC it was analyzed according to cutoffs.

3.15 Ethical considerations

The study was carried out after presenting research proposal and approved by research

committee of KIU-WC. All protocols in the different elaborations were observed and obeyed

such as handling of laboratory microorganisms was followed.

18

CHAPTER FOUR

4.0RESULTS The starting material was 375g of dried powered material from Phyllanthus amarus for the

extraction. The dried whole herb of Phyllanthus amarus was subjected to different 70% ethanol

solvent maceration. The percentage yield obtained was 4.7%.

4.1 The results for susceptibility test of Shigella dysenteriae to ethanol whole herb extract of Pltyllantltus amarus This was determined by agar diffusion method where the diameter zones of inhibition in mm

were measured after 24hours incubation at 37'c and it was done in triplicate (3times)

The zone of inhibition for ciprofloxacin was greater than that of all extracts since the

ciprofloxacin is the standard drug that is used as a first line in the treatment of bacterial

dysenteriae according to the (UCG 2016). The negative controls shows that solvent without

extract had no inhibitory effect on Shigella dysenteriae.

These results are summarised in the table I below.

TABLE 2 The results of susceptibility sample to Shigella dysenteriae on Phyllanthus

amarus extracts and controls

-----------Test drug Zone of Inhibition (mm) Activity Index P value

Mean±SEM

0.0000 ----~-- . --··-·-- ----- .

l28ftg/ml 11.5±0.5 0.47

256ftg/ml 12.5±0.5' 0.51

512ftg/ml 13.5±0.5' 0.55

Cipro (ftg/ml) 24.5±0.5' 1.00

Distilled water 0±0

'Indicates significant differences between the test drug and the negative control (distilled water)

19

l

0:9

0.8

&j 0.7

"' ..5 0.6

~0.5

~ OA <( 03

0.2

O.t.

0

Activity indices of the different test drugs

256 pg/m1 5:12 flalm Cipro

Test drug

MINIMUM INHIBITORY CONCENTRATION

Conventionally when test microbe is susceptible to the test extract, the MIC is usually

performed. In this study the MIC was performed according to the tube dilution method for both

ethanol and aqueous extract.

The concentration used for MIC determination was128flg/ml, 64flg/ml, 32flg/ml, l6flg/ml,

8f1g/ml, 4f1glml, 2flg/ml and lflg/ml.

The results in this study were considered only valid when there was growth in control one

(media supports growth) and no growth in control two (meaning no cross contamination of the

broth). The details of this MIC results are showed in the table 3

20

TABLE 3 The results showing minimum inhibitory concentration (MIC)of Shigella

dysenteriea against Shigella dysenteriae bacteria

Type

extract

Ethanol

Control!

Control2

128 64

+

Key; + means there was growth,

Means there was no growth.

+

Control A had broth and bacteria but without drug extract.

Control B only broth without bacteria or crude drug in it.

21

+ +

MINIMUM BACTERICIDAL CONCENTRATION

It is usually recommended after determining the MIC, the MBC is determined. In this case since

the extract exhibited MIC above the. cut off being below lOOflg/ml (Olila., et a! 200 I). MBC of

the plant extract was determined. The results of the MBC are presented in table four below.

Table 4: Minimum bactericidal concentration (MBC) of Pltyllantltus amarus against

Shigella dysenteriae.

Type of

extracts

Ethanol

Control A

Control B

tllbes. ()lg/ml)

128 64

+ + +

The original bacteria concentration was I xI 06cfu/ml as determined by comparison to Mcfarland

standard solution hence test tube I contained 5xl 05cfu/ml. since the MBC is the lowest

concentration of the extract that allowed less than 0.1% of the original inoculums of 5x105cfu/ml

to grow and colony forming unit per milliliter ( cfu/ml) on the plate at 32flg/ml is 600cfu/ml, this

makes 64fjg/ml to be the MBC since it showed no growth.

Key; + Means there was growth

-Means there was no growth

Control B the agar with no bacteria and no crude drug extract.

The results in table four, the MBC for ethanol extract were 64fjg/ml.

22

CHAPTER FIVE

5.0 DISCUSSION, CONCLUSION AND RECOMMENDATIONS

5.1 DISCUSSION This chapter presents discussion of findings of the research of antibacterial activity of

Phyllanthus amarus based on objectives of the study.

Bacterial susceptibility testing

The sensitivity tests of Phyllanthus amarus to Shigella dysenteriae showed that ethanolic

extract of the plant material showed antimicrobial activity on the test organism. The test

pathogen, Shigella dysenteriae was more susceptible to the ciprofloxacin as the positive control

with zone of inhibition at 24.5±0.5mm higher than the ethanol extract at all concentrations. The

highest susceptibility was recorded with the ethanolic extract at 5l2J.tg/ml with zone of

13.5±0.5mm followed by 256J.!g/ml at 12.5±0.5mm and finally 128J.tg/ml with zone at

1 1.5±0.5mm

According to the results above, it was realized that the sensitivity increased with increasing

concentration which was in agreement with what (Oiuduro and Omoboye, 201 0). According to

Oluduro and Omoboye (20 1 0) the antibacterial activities of most plant extracts are concentration

dependent as zone of growth inhibition increased with increasing concentration of the extracts.

Ekwenye and Elegalam (2005) and Azu and Onyeagha (2007) also reported that the efficacy of

most plant extracts is concentration dependent.

Umbare et al. (2009) assessing the quality of Phyllanthus amarus leaves extract for its

hypolipidemic activity found the presence of four phyto-constituent namely alkaloids,

flavonoids, saponins and tannins in the plant sample. Flavonoids, tannins, alkaloids, steroids,

terpenoids, saponins and glycosides were also obtained by Obianime and Uche (2009) in

their comparative study of the methanol extract ofP.amarus leaves.

Oluduro and Omoboye (2010) indicated that the presence of phytochemicals in plant

extracts is a function of their antimicrobial activities against the test pathogen as they play

important roles in bioactivity of medicinal plants. They further explained that phytochemicals

Z3

exeti antimicrobial activity through different mechanisms. Chonoko and Rufai (2011) also

indicated that there was a link between the antibactei·ial activity exhibited by the plant extracts to

the presence of steroids flavonoids, tannins, alkaloids and saponins. Tannins, for example, act

by iron deprivation, hydrogen binding or specific interactions with vital proteins such as

enzymes in microbial cells (Scalbert, 1991; Akinpelu et al., 2008).

Basing on the discussion above, the difference between the activity of the extract and the

standard antimicrobial drug may be due to the mixtures of bioactive compounds which probably

have antagonistic effects against the major bioactive(s) present in the extracts compared to the

pure compound contained in the standard antibiotic ciprofloxacin. The standard drug which has

the highest zone of inhibition while distilled water had no zone of inhibition it is because water

has no active ingredients.

The policy implication of these findings is that more research should be carried out probably

using bioassay guided fraction to determine the lead compound(s) in the extract.

Minimum Inhibitory Concentration of Plzyllantlzus amarus

Following the sensitivity analysis by a standard microbiology, the MIC is usually performed if

the test organism is sensitive to the test extract /drug. In this case since Shigella dysenteriae

bacteria were active against herb extract, the MIC was carried out according to the Mann and

Markham method (Mann and Markham, 1997).

The quality control, some measures were put in place for this results to be valued included two

controls i.e. control 1 and control 2 which were also prepared as follows:

For control one that had broth and bacteria and had no drug in order to find out whether the

media supported the growth of organism. On the other hand, control 2 had only broth and no

bacteria and no drug to determine whether the broth was contaminated by other organisms or not.

Since the controls were working then the results were considered valid.

The analysis of the results showed that the MIC of ethanolic extract was 64J.lg/ml. For test

extract whose MIC is <1 OOJ.!glml is considered as good candidates to be developed into new

drugs (Olila., et al 2001). Since our MIC value was below 100J.!glml, this herb is a good

candidate for drug development.

24

The policy implication of these findings is that more research should be carried out probably

using bioassay guided fraction for lead compound in the extract.

Minimum bactericidal Concentration (MBC) of Phyllanthus amarus

During drug development for a test material as an antibacterial agent against a given bacteria if

the MIC is <I OOJlg/ml then the next step it is to do the MBC.MBC is the lowest concentration of

the extract that allowed less than 0.1% of the original inoculum of 5xl 05cfu/ml.

The analysis of the results showed that the MBC for ethanol was 64jlg/ml. This was because

original bacteria concentration was lxl06cfu/ml as determined by comparison to Mcfarland

standard solution hence test tube I contained 5xl 05cfu/ml. Since the MBC is the lowest

concentration of the extract that allowed less than 0.1% of the original inoculum of 5xl 05cfu/ml

to grow which was at 500cfu/ml (being the cut off value below which MBC is considered and

vice versa).The colony forming unit per milliliter ( cfu/ml) on the plate at 32J1glml is 600cfu/ml,

hence this makes 64Jlg/ml to be the MBC since it showed no growth.

The policy implication of these findings is that more research should be carried out probably

using bioassay guided fraction for lead compound(s) in the extract.

5.2 CONCLUSION This study found that the whole plant extract ofphyllanthus amarus for 70% ethanolic extract

was active against Shigella dysenterie at 512ftg/ml with zone of 13.5±0.5mm followed by

256Jlglm! at 12.5±0.5mm and finally l28j1g/ml with zone at 11.5±0.5mm.

Furthermore the MIC of the extract was 32Jlg/ml and the MBC for was 64Jlg/ml which indicates

that Phyllanthus amarus whole plant extract has antibacterial activity against Shigella

dysenteriae.

Based on these results, this study has scientifically validated the ethnobotanical use of

Phyllanthus amarus to treat bacterial dysentery in central Uganda.

25

5.3 RECOMENDATIONS I recommended that;

I. The more research should be carried out to determine lead compound in the Phyllanthus

amarus extract responsible for the anti-microbial activity against Shigella dysenteriae.

2. The mechanism of action by which a plant extract inhibits bacterial cells should be

studied.

3. Plant extract should be tested on different microorganisms that cause dysentery to find

out its activity against them and spectrum.

26

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Oluma, H.O, Umoh, E.U, Onekutu, A. and Okolo, J. (2004). Antibacterial potentials of eight

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29

APPENDICES

APPENDIX 1: TIME FRAMEWORK AND WORK PLAN

Concept presentation

Proposal writing

Proposal presentation

Plant collection/extraction

Antibacterial evaluation

Data analysis

Report writing

Report submission

/IIIII/

II/III! 111!!111

1111/llf

30

11/11/ll

ll!fl/1

/!IIIII/

!!11/!l/ /!/1!11

I filii/

APPENDIX2

Figure I: susceptibility testing agar well diffusion.( zones of inhibition)

Figure 2: Minimum inhibitory concentration (MIC) Set up.

31