ETHNOBOTANICAL STUDY AND ANTIMICROBIAL SCREENING OF

MEDICINAL PLANTS FOUND IN CHEREBES AND ENDO VILLAGE KEIYO

COUNTY

PRESENTED BY: KOSGEY JANET CHERUIYOT

SC/PGB/049/09

A RESEARCH PROPOSAL WRITTEN AND SUBMITTED TO THE SCHOOL

OF SCIENCE, DEPARTMENT OF BIOLOGICAL SCIENCE AS A PARTIAL

COMPLETION OF A DEGREE COURSE IN MASTER OF SCIENCE IN

MICROBIOLOGY

2011

1

DECLARATIONI, Kosgey Janet Cheruiyot, duly declare that this is my original work and has not been

presented in any other university for any other awards. Works from other sources have

been dully acknowledged.

STUDENT

NAME: KOSGEY JANET CHERUIYOT

SIGN:……………………………………,

DATE: 22nd NOV, 2010

UNIVERSIITY SUPERVISOR

NAME: Dr. NJENGA

HEAD DEPARTMENT OF BIOLOGICAL SCIENCE

SCHOOL OF SCIENCE, MOI UNIVERSITY

SIGN:………………………………

DATE: 22nd NOV, 2010

FIELD SUPERVISOR

NAME: Dr. MUTAI

CENTRE FOR TRADITIONAL MEDICINE,

KENYA MEDICAL RESEARCH INSTITUTE (KEMRI),

SIGN:…………………………

DATE: 22nd JUNE, 2011

TABLE OF CONTENTS

DECLARATION.................................................................................................................2

TABLE OF CONTENTS....................................................................................................3ABSTRACT........................................................................................................................4CHAPTER ONE..................................................................................................................5

1.0 INTRODUCTION.................................................................................................51.1 PROBLEM STATEMENT....................................................................................51.2 JUSTIFICATION..................................................................................................61.3 OBJECTIVES........................................................................................................71.4 HYPOTHESIS.......................................................................................................8

CHAPTER TWO.................................................................................................................92.0 History of Drug Development...............................................................................92.1 Medicinal Plants....................................................................................................92.2 Plants as an Alternative Source of Therapeutic Agents.......................................102.3 Plants and Plant Parts Used.................................................................................112.4 Documentation.....................................................................................................112.5 Plant Parts Used...................................................................................................122.6 Cultivation of Medicinal Plants...........................................................................122.7 Synergsm.............................................................................................................132.8 Activity of Some Medicinal Plant.......................................................................13

3.0 METHODOLOGY......................................................................................................143.1 Preliminary experimental design.........................................................................143.2 Collection of the plants from the field.................................................................143.3 Reagents...............................................................................................................143.4 Extraction of plant materials................................................................................153.5 Microbial Test Organisms...................................................................................153.6 Test strains...........................................................................................................153.7 Preparation of test organisms...............................................................................173.8 Preparation of McFarland Standard.....................................................................173.9 Antimicrobial Screening Test..............................................................................183.10 Determination of Minimum inhibitory concentration.......................................183.11 Cell toxicity.......................................................................................................193.14 Statistical data analysis......................................................................................203.15 Conservation......................................................................................................20EXPECTED OUTPUT..............................................................................................20

BUDGET...........................................................................................................................21WORKPLAN.....................................................................................................................24REFERENCES..................................................................................................................26

ABSTRACTTraditional herbal remedies are an important component in the provision of primary

health care. They serve as an alternative to conventional medicines which are normally

too expensive for most Kenyans, but the rate at which these herbal remedies are

disappearing from their natural habitat is alarming. There is little or no documentation on

the uses of these plants. Moreover, the indigenous knowledge on medicinal properties of

these plants is secretly guarded by the herbalists and is not available to most Kenyans.

The methods that were previously used to pass the information are presently not

applicable. The research seeks to validate the claims attributed to the medicinal plants

found in Keiyo County based on the indigenous knowledge and also to document the

medicinal plants found in the same area. The roots, stems and leaves of these plants will

be collected from Kerio valley Cherebes and Endo village in Keiyo County. The samples

will be extracted using water, ethyl acetate, hexane and methanol solvent. The crude

extract’s phytochemical composition will be determined. The extracts will be tested for

antimicrobial activity using Staphylococcus aureus, Klebsiella pneuformans, Escherichia

coli, Salmonella typhi, Cryptococcus neoformans, Candida albicans, Microsporum

gypsium and Trichopyton metangropyte. The minimum inhibition concentration of the

herbs will be determined by incubating microorganisms in different concentrations of the

extract, for 24 hours at 37oC then cultured in solid media for bacteria and yeast and at

27oC for 48 hours for moulds. The data obtained will be analysed using two way

ANOVA. Means will be separated using Tukey’s HSD test at 5 % level of significance.

The results will show the rich biodiversity we have and the need to conserve it.

CHAPTER ONE

1.0 INTRODUCTION

Traditional medicine has and still remains the main source for a large majority(80%) of

people in Ethiopia for treating health problems and medicinal consultancy including

consumption of the medicinal plants has a much lower cost than modern medicine

attention(Tildhun and Giday, 2007). Traditional medicine is used throughout the world as

it is dependent on locally available plants, which are easily accessible, and capitalizes on

traditional wisdom-repository of knowledge, simple to use and affordable. (Tesfaye and

Demissew, 2009). The traditional methods, especially the use of medicinal plants, still

play a vital role to cover the basic health needs in the developing countries too and more

over, the use of herbal remedies has increased in the developed countries in the last

decades. In this connection, plants continue to be a rich source of therapeutic agents. The

remarkable contribution of plants to the drug industry was possible because of the large

number of phytochemical and biological studies all over the world. The Indian

subcontinent is endowed with rich and diverse local health tradition, which is equally

matched with rich and diverse plant genetic source. A detailed investigation and

documentation of plants used in local health traditions and ethno-pharmacological

evaluation to verify their efficacy and safety can lead to the development of invaluable

herbal drugs or isolation of compounds of therapeutic value. (Kesaran et al, 2007)

1.1 PROBLEM STATEMENT

The documentation of medicinal plants in Kenya is very poor .A few of documentation

have been done on Plants from Central Kenya as Veterinary Medicine by Ngugi. Also

few plants have been studied from Kakamega rain forest; hence there is need to document

medicinal plants found in Keiyo County. Resistance to drugs by microorganisms has

increased. This resistance have been attributed to overdose and under dose of drugs due

to over counter prescription of drugs, ability of microorganisms to undergo genetic

variability (mutation), use of antibiotics in food preservation and general misuse of drugs

(Gislene, et a1 2000) hence there is need to come up with sensitive and effective drugs.

Keiyo County provides a myriad of the medicinal plants. However, there is need to carry

out proper identification of the medicinal plants, their antimicrobial activity and know

their phytochemical composition. This information will be necessary later for

conservation purposes and cheap drug manufacturing in Kenya.

1.2 JUSTIFICATION

Natural products have provided biologically active compounds for many years and many

of today’s medicines are either obtained directly from natural source or were developed

from a lead compound originally obtained from a natural source (Graham 2001).

In Kenya 75 plants species from 34 families are used to cure 59 ailments in traditional

medicine of central Kenya, 80% of South Africans use herbal remedies for their physical

and psychological health care at different stages of their life. Also in United State, 36 of

the 101 plants species implicated in drugs discovery are weedy species found mainly in

disturbed habitant (Lewis, 2009). There is good reason to be optimistic about the

potential future usefulness of plants based on natural products as a continued source of

potential lead compounds. Within many thousands of years of trial-and –error by

evolution on her side, Mother Nature is a vastly superior experimentalist to any mere

human organic chemist (Thomas, 2005). Many of this lead compounds are useful drugs

in themselves e.g. morphine and quinine, while other have been the basis for synthetic

drugs e.g. local anaesthetics developed from cocaine. Plants still remain a promising

source of new drugs and still continue to do so. Occasionally useful drugs which have

recently been isolated from plants include the anticancer agents, toxol, from the jaw tree

and the anti malarial agents artemisinin from Chinese plant (Graham, 2001.) In South

Africa different plant species are used to treat several diseases especially among the rural

population where western medicine is either not accessible or affordable. Today, about

200,000 traditional healers practice herbal medicine in South Africa and a high

percentage of the population use traditional medicine as their primary source of health

care (Lewis, 2009). Most biological active natural products are secondary metabolites

with quite complex structures. This has the advantage that they are extremely novel lead

compounds. In general natural products are particularly good at providing many new

chemical structure which no chemist would dream of synthesizing. For example, the anti-

malarial drug artemisinin is one such example, containing an extremely unstable-looking

trioxane, ring-one of the most unlikely structure to have appeared in recent years

(Grahams, 2001). Plant evolution has culminated in a wide variety of bio-molecules that

affect any animal that may choose to eat them; it is biologically advantageous for other

plants to produce noxious chemicals to decrease the likelihood of their being eaten.

Because of these diverse biological activities, any of these non-human biosynthetic

molecules could in principle, be a lead compound for human drugs discovery (Thomas,

2005). Considering the debt medicinal chemistry owes to natural world, it is soberly to

think that very few plants have been fully studied, the vast majority have not been studied

at all. The rainforests of the world are particularly rich in plant species which have still to

be discovered, let alone studied. Who knows how existing new lead compounds await

discovery for the fight against cancer, AIDs or other many of human afflictions? This is

why the destruction of rainforest and other ecosystems is so tragic. Once these

ecosystems are destroyed, unique plants species are destroyed with them. Medicine has

lost potentially useful plants for ever. For example, siphion a plant cultured near Cyrene

Is now extinct. It is almost certain that many more useful plants have become extinct

without medicine ever being aware of them (Graham, 2001)

1.3 Specific objectives

1 To identify and document traditional medicinal plants found Endo and Cherebes

villages in Keiyo County.

2 To determine the antimicrobial activity of these medicinal plants.

3 To determine the phytochemical constituents of medicinally active plants.

4 To determine toxicity of these medicinal plants

5 To provide a scientific rationale to validate the claimed therapeutic properties of

the medicinal plants.

1.4 HYPOTHESIS

NULL HYPOTHESIS

1. Medicinal plants from Keiyo county has no significant antifungal activity

and antibacterial activity

2. Medicinal plants from Keiyo county high toxicity level

3. Medicinal plants from Keiyo county has very high minimum inhibition

concentrations

4. Medicinal plants from Keiyo county has no active ingredient

CHAPTER TWO

2.0 History of Drug Development

In primitive times, surgical ‘therapies’ for epilepsy included ‘trephining’ holes through

the patients’ skull in order to release ‘evil humors and devil spirits’. The ancient also

employed ad hoc ‘medicinal’ therapies, ranging from rubbing the body of an epileptic

with genitals of a seal, to inducing episodes of sneezing at sunset. Also human blood was

widely regarded as curatives.

Charlatans would massage the heads of epileptic, thereby re-aligning the bone plates of

the skull, taking pressure off the brain and alleviating the curse of epilepsy.

By the Middle Ages, scientific foundations of epilepsy therapy were formed. They used

‘magical prescriptions’, which ranged from taking dogs bile to human urine. During

Renaissance magical treatment was subjected by medicinal profession in favor of

‘rational and scientific’ Gaelic therapies, which relied upon forced vomiting and bowel

purging with concomitant oral administration of peony extracts. During this time

epileptic were treated by castration, circumcision and clitoridectory since epilepsy was

perceived as secondary hyper-sexuality. This mode of treatment failed or killed

unfortunate patients. During late Renaissance inorganic salts were used as putative

therapies.

Antiepileptic copper salts were introduced in the first century with reported success; this

led to attempts of lead, bismuth, tin, silver, iron and mercury giving rise to

metallotherapy. This led to widespread failure due to lack of efficiency and excessive

toxicity. In mid 19th century quackery prospered as a treatment of epilepsy. Phlebotomy

was adopted and king CharlesII was among the unfortunate patient treated by this

method, he bled to death. (Nogrady and Weaver, 2005)

2.1 Medicinal Plants

The economic crisis, high cost of industrialized medicines, inefficient public access to

medicinal and pharmaceutical care, in addition to the side effects caused by synthetic

drugs are more of the factors contributing to the control role of medicinal plants in health

care. There is currently an increased in number of immune-compromised individuals due

to advances in medical technology and a pan-epidemic of HIV infections with the rise in

at-risk patients, the number of invasive fungal infections has dramatically increased in

both in developed and developing countries. (Susana et al, 2007).

Also many infectious microorganisms are resistant to synthetic drugs, hence an

alternative therapy is much needed (Swapna and Kannabiran, 2006). In the present

scenario, an emergence of multiple drug resistance in human pathogens and small

numbers of antimicrobials classes available stimulates research directed towards the

discovery of novel antimicrobial agents from other sources. (Susana et al, 2007)

Among the 887 medicinal plant species in Ethiopia, about 26 species are endemic and are

becoming increasingly rare and are at verge of extinction. (Tesfaye and Dimissew, 2009)

the solution is to practice cultivation and conservation of endangered species. Another

factor that contributes to the problem is the explosive growth in the use of plant-base

products in the last ten years. Hundreds of plant species are being converted into dozens

of different product types, for use across a wide variety of markets. These plants and

derived plant products are traded locally, regionally and internationally, as neuraticals,

dietary supplements, phytomedicines, homeopathic drugs, aromatherapy, oils, flavors,

fragrance and food additive. Because of the vast growing multi-million trade, there is

need to protect the medicinal plants by forming laws that governs harvesting of medicinal

plants.

2.2 Plants as an Alternative Source of Therapeutic Agents

Ethno-botanical studies are often significant in revealing locally important plant species

especially for the discovery of crude drugs. Documentation of traditional knowledge,

especially on the medicinal use of plants has provided many important drugs of modern

days (Tildhun and Giday, 2007). Over 50% of all modern clinical drugs are of natural

product origin and natural products play an important role in drug development programs

of pharmaceutical industry. In developing countries, especially in rural contexts people

usually turn into traditional healers when in diseased conditions and plants of ethno-

botanical origin are often presented for use. (Olatunde, 2003)

Also in developing countries, the world health organization (WHO) estimates that about

80% of the population relies on plant based preparations used in their traditional

medicine system and as basic needs for human primary health care. In recent years there

is need to study the plants having different values in their medicinal plants have been

evaluated for possible antimicrobial activity and potential cures from a variety of

ailments especially of microbial origin (Kesaran et al, 2007)

2.3 Plants and Plant Parts Used

Fifty thousand flowering plants are used for medicinal purposes. In Ethiopia about 800

species of plants are used in the traditional health care system to treat nearly 300 mental

and physical disorders (Tildhun and Mirutse, 2007). A total of 124 medicinal plants

which belong to 107, genera and 49 families of vascular plants were recorded in Kafa

Zone Ethiopia. These people used these plants to treat about 18 ailments of human and

domestic origin (Tesfaye and Sebsebe, 2009). a total of 71 plant species from 28 families,

mostly the Combretaceae (14%), Anacardiaceae (8%), Mimosaceae (8%), and Ebanaceae

(7%), were used to treat conditions such as herpes zoster, diarrhoea, coughing, malaria,

meningitis, and tuberculosis. (Chinsembu and Hedimbi, 2010)

2.4 Documentation

In Ethiopia, either the knowledge from herbalists is passed secretively from one

generation to the next generation by the word of mouth or their descendants inherit the

medico-spiritual manuscripts (Tildhun and Mirutse, 2007). Equally threatened is the

knowledge based on which the traditional system is based, as the ethno-botanical

information is not documented and remains in the memory of the elderly practitioners

(Tesfaye and Sebsebe, 2009). The purpose of documentation in ethno-botany is to try and

find out how people have traditionally used plants, for whatever purpose and how is still

doing so. Thus, ethno-botany tries to preserve valuable traditional knowledge for both

future generations and other communities (Tesfaye and Sebsebe, 2009).

2.5 Plant Parts Used

Herbs account the highest proportion of the type plants used as traditional medicine

followed by shrubs and trees (Tesfaye and Sebsebe, 2009). The people use various parts

of medicinal plants. Leaves contribute about 50%of parts used and followed by seeds

15% and roots 10%. There are instances where different parts of the same plant are being

used for different purposes (Tesfaye and Sebsebe, 2009). Contrary in southern Ethiopia

the most frequently utilizes plant parts were the underground part (root/rhizome/bulb)

42% (Tildhun and Mirutse, 2007). The most plant parts used were leaves (33%), bark

(32%), and roots (28%) while the least used plant parts were fruits/seeds (4%).

(Chinsembu and Hedimbi, 2010)

2.6 Cultivation of Medicinal Plants

The medicinal plants in southern Ethiopia are always cultivated on the upper slope f the

home garden behind the house. If the medicinal plants are grown in home garden quarters

with high soil nutrients, they grow faster, complete their life cycle within a relatively

shorter period and then die, a situation not appreciated by farmers. Instead the farmers

want the medicinal plants under stress conditions that subdue plant growth (Tesfaye and

Sebsebe, 2009).

Also in South Africa, plants traded and marketed for medicinal value are sourced from

crafting or cultivation or both. Artemisia a Chinese plant is cultivated worldwide for

antimalarial agent artemisinin. And also yew tree is cultivated for anticancer agent toxol

(Graham, 2001).

Aloe plant (the ‘wand of heaven’) is a cactus like-plant found in deserts of Africa,

Arizona and has long been used for its curative properties. Hence people have cultivated

it in home gardens (Nogrady and Weaver, 2005). Siphion is a plant cultivated near

Cyrene in North Africa and famed as contraceptive agent in ancient Greece and is now

almost an extinct (Graham, 2001).

2.7 Synergsm

There are cases where more than one plant is used to treat many ailments. Headache is

for example, treated with a combination of either six or nine or twelve medicinal plants.

There are also cases where a particular plant is used to treat many ailments. For instance,

both Clerodenrum myricoide (Hoist) RBr ex Vatke (Verbenaceae) and Croton

macrostachyus Del (Euphorblacetoae) are used to treat seven ailments (Tesfaye and

Sebsebe, 2009). Some plant products have a wide variety of different active principles

which act together to produce a beneficial effect (Nogrady and Weaver, 2005).

2.8 Activity of Some Medicinal Plant

Many plant products have been tested for their activity. Among them are Nigerian

medicinal plant Aspilia africana and Bryophylum pinnatum. They have alkaloids,

saponins, flavanoids, phenols and tannins. They also have ascorbic acid, riboflavin

thiamine and niacin. The minerals they have are Ca, P, K, Mg, Na, Fe and Zn. Unripe

pulp of Carica papaya have pronounced bacteriocidal activity against Stapylococcus

aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa and Shigella

flexneri(Oloyede, 2005). Oil derived from Dacrodes edulis (G. Don) has better activity

against bacterial species than yeast (Obame et al, 2008). Extracts from stem back, wood,

or whole root of Terminaria brownii inhibit standard strains of Staphylococcus aureus,

Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Salmonella typhi and

Bacillus anthracis and fungi Candida albicans and Cryptococcus pneuformans. Aqueous

extracts exhibit strong activity against both bacteria and fungi. Extracts of root and stem

back has relative mild cytotoxic activity against brine shrimp larvae with LC50 value of

114-36 ug/ml respectively. The stem wood extract has the highest toxicity against the

shrimp LD50 value 2.6ug/ml while a standard cydophosphamide anticancer drug has

16.3ug/ml. hence T. brownie has been successfully used against diarrhea and gonorrhea

(Mbwambo et al, 2007).

3.0 METHODOLOGY

3.1 Preliminary experimental design

Laboratory experimental design for bioassays;

Test groups will include selected organisms (ATCC strains and clinical isolates)

and crude extracts at different concentrations. This will be to determine whether

the extracts will be active either as antifungal or antibacterial agents.

Positive controls: This will consist of both antifungal and antibacterial drugs

(gentamicin and fluconazoles). This will be to determine whether the test

organisms used will be sensitive to common drugs or will be resistant.

Negative controls: This will consist of solvents and sterile distilled water, this will

be necessary to ensure that the solvents used for extraction and dissolution will

have no inhibitory actions on their own.

All experiments will be carried out in triplicates in order to minimize

experimental error.

3.2 Collection of the plants from the field

Kenyan Keiyo county medicinal plants will be collected. Selection of the plants will be

based on available ethnobotanical information from traditional health practitioners

consulted during the pilot study as well as available literature. The plants are used to treat

both skin condition as well as stomach problem. The herbalist advised on the samples to

be collected as the plant part being used for treatment. The plants parts collected will

include leaves, roots and stem bark. The study plant materials will be photographed and

collected for authentification at the East African Herbarium. The samples for extractions

will be collected in paper bags and taken to the Medical chemistry Laboratory, Centre for

Traditional Medicine and Drug Research for processing and extractions.

3.3 Reagents

Analytical grade organic solvents; n-hexane, dichloromethane, chloroforms, ethyl acetate,

methanol, acetone, and ammonia will be sourced from Kobian, Nairobi, Kenya.

Chloroform, methanol, vanillin, sulphuric acid and potassium hydroxide will also be

used.

3.4 Extraction of plant materials

Plants materials will be dried at room temperature, grounded into a fine powder using

laboratory grinding mill and stored in a cool and dry place. Total extraction of each plant

material will be prepared by mixing with solvent in the ratio of 1: 10 (plant

material/solvents). This procedure will be done successively using hexane,

dichloromethane and methanol where the plant materials will be soaked in 1000ml

solvent in conical flasks for 12 hours. The extracts will be filtered using Whatman No. 1

filter paper and solvents removed through evaporation under reduced pressure at 450C

using a rotary evaporator. The extracts will be kept in stoppered sample vials at 4 0 C until

they will be used (Ana et al., 2005; Chhabra et al., 1984; Mcloud et al., 1988). Total

water extract of each plant material will be done by soaking a weighed amount of the dry

powder (27-50g) in distilled water and shaking it for two hours with an electric shaker at

650C. The suspension will be filtered and the filtrate will be kept in a deep freezer before

it will be evaporated to dryness by freeze drying. The lyophilized dry powder will be

collected in stoppered sample vials, weighed and kept in a desiccator, to avoid absorption

of water, until they will be used.

3.5 Microbial Test Organisms

Selection of microbial test organisms will be based on the ethnobotanical information

collected on the target diseases. The biossay procedures will be conducted using methods

recommended by the National Committee for Clinical Laboratory Standards (NCCLS,

2003). The standard reference microbial organisms and clinical isolates will be obtained

from the Centre for Microbiology Research – KEMRI, all preserved under -800C.

3.6 Test strains

Test strains will be chosen in consideration based on the ethno botanical exploitation of

the plants. Standard organisms as well as clinical isolates that will be used in this study

include:-

Bacterial isolates

Gram positive

Staphylococcus aureus ATCC 25923

Methicillin resistant Staphylococcus aureus (MRSA) clinical isolate

Gram negative

Escherichia coli ATCC 25922

Pseudomonas aeruginosa ATCC 27853

Klebsiella pneumonia. (Clinical isolate)

Fungi isolates

Yeast

Candida albicans ATCC 90028

Candida parapsilosis ATCC 90029

Candida krusei ATCC 90026

Cryptococcus neoformans ATCC 32602

Dermatophytes (Clinical isolates)

Microsporum gypseum

Trychophyton mentagrophytes.

3.7 Preparation of test organisms

Stocked bacterial strains will be sub-cultured on Muller Hinton agar no. CM0337. (Oxoid

Ltd, Basingstoke, Hampshire, England). Incubation will be done at 240C for 12 – 18

hours to obtain freshly growing strains. Yeast and molds will be subcultured onto

Sabouraud Dextrose Agar no. CM 004 (Oxoid Ltd, Basingstoke, Hampshire, England).

Each media was prepared according to the manufacturer’s instructions. Yeasts will be

incubated for 24 hours while molds will be incubated for 72 hours at 300C to obtain

freshly growing culture (Rajakaruna et al., 2002).

3.8 Preparation of McFarland Standard

Exactly 0.5 McFarland equivalent turbidity standards will be prepared by adding 0.6ml of

1% barium chloride solution (BaCl2.2H2O) to 99.4ml of 1% sulphuric acid solution

(H2SO4) and mixed thoroughly. A small volume of the turbid solution will be transferred

to capped tube of the same type that will be used to prepare the test and control innocula.

It will be then stored in the dark at room temperature (25°C). Exactly 0.5 McFarland

gives an equivalent approximate density of bacteria 1x10-6 colony forming units

(CFU)/ml (Ana et al., 2005).

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3.9 Antimicrobial Screening Test

Five millimeters of sterile distilled water will be used to make suspension. From an

overnight growth of the test organism, 4-6 colonies will be emulsified and the suspension

was adjusted to match the 0.5 McFarland's standard. Respective plates were inoculated

using a sterile cotton wool swab. Antimicrobial susceptibility test was done using disk

diffusion methods. Briefly, 1mg of each extract was dissolved in 1ml of the appropriate

solvents and 10 µl of the mixture was impregnated onto 6mm sterile filter paper disk and

air dried. The disk was placed aseptically onto the inoculated plates. The bacterial and

yeast cultures was incubated at 370C for 24 and 48 hours respectively while mould

fungal cultures were incubated at 250C for 72 hours. After incubation, inhibition zone

diameter was measured in millimeters and recorded against the corresponding

concentrations as described by Elgayyar et al., (2000). Positive controls were set against

standard antibiotics and antifungal drugs while negative controls were set using disk

impregnated with extraction solvents.

3.10 Determination of Minimum inhibitory concentration

Broth micro dilution method will be used to determine minimum inhibitory concentration

for the active crude extracts against the test microorganisms. The method is

recommended by the National Committee for Clinical Laboratory Standards now Clinical

Laboratory Standard Institute (CLSI) (NCCLS, 2002). The tests will be performed in 96

well-micro-titer plates. Plants extracts dissolved in respective solvents will be transferred

into micro-titer plates to make serial dilutions ranging from101, 102, 103……..1010 . The

final volume in each well will be 100 μl. The wells will be inoculated with 5μl of

microbial suspension. The yeast and bacteria will be incubated at 370C for 24 hours while

molds will be incubated at 250C for 3-7 days in ambient air. The MIC will be recorded as

the lowest extract concentration demonstrating no visible growth as compared to the

control broth turbidity (Michael et al., 2003). Wells that will not be inoculated will be set

to act as control. All the experiments will be done in triplicates and average results will

be recorded.

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3.11 Cell toxicity

The cytotoxic concentration causing 50% cell lysis and death (CC50) will be determined

for the extracts by a method described by Kurokawa et al. (2001). The extracts of the

active plants will be tested for in vitro cytotoxicity, using human embryonic lung

fibroblast (HELF) Vero-199 cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) assay (Mossman, 1983). The HELF cells will be

cultured and maintained in minimum essential medium (MEM) supplemented with 10%

Featal bovin serum (FBS). The cells will be cultured at 37◦C in 5% CO2, harvested by

trypsinization, pooled in a 50ml vial. Approximately 100ul cell suspension (1×105

cells/ml) will be added to each well in a 96-well micro-titer plate. Each sample will be

replicated 3 times and the cells incubated at 37o in 5% CO2 for 24 hrs for attachment.

150ul of the highest concentration of each of the test samples (a serial dilution, prepared

in MEM) will be added into the same row and a serial dilution done. The experimental

plates will be incubated further at 37◦C for 48hrs. The cells in media without drugs will

be used as controls. After 48hrs of incubation, MTT (10µl) will be added into each well.

The cells will be incubated for 4 hrs or until a purple precipitate will be clearly visible

under a microscope. The medium together with MTT will be aspirated off from the wells

and dimethly sulfoxide (DMSO) (100µl) will be added and the plates shaken for 5 min.

The absorbance for each well will be measured at 562nm in a micro-titre plate reader

(Mossman, 1983) and percentage cell viability (CV) will be calculated via an excel

program with the formula;

%CV = Average abs of duplicate drug wells − Average abs of blank wells x 100%

Average abs of control wells

A dose–response curve will be plotted to enable the calculation of the concentrations that

kill 50% of the Vero-199 cells (IC50) (Kigondu, 2007).

19

3.14 Statistical data analysis

The results will be subjected to statistical analysis for qualification of variability.

Statistical packages for social scientist SPSS Version 12.0 will be utilized which enables

the analysis of variance by one way ANOVA to establish the significance variability

between and within groups (Plants, Solvents and organisms). Bioactivity will be used as

an independent variable to establish significance at 0.05 level of confidence. The

cytotoxic concentration causing 50% cell lysis and death (CC50) will be determined for

the extracts and analyzed accordingly. Three experiments will be performed for each of

the In-vivo study. In-vivo toxicity will be checked for significant differences in lethal

dose resulting to fifty percent deaths (LD50) values between the tests and the controls

groups. In vivo toxicity data will be analyzed by comparing the significance difference in

the LD50 of the treatment groups with respect to different plant extracts. The data will be

presented inform of tables, graphs and photographs.

3.15 Conservation

It will be done by establishing nurseries. The seeds or any other propagation part will be

collected during field work. These parts will be grown in native nurseries. The land will

be acquired by purchasing. When the project terminates it will be handed over to the local

authorities. The local authority will be expected to continuously manage the nurseries and

even establish native plants arboretum. Along with the written work it will be

accompanied by a document indicating all medicinal plants and their claimed therapeutic

action. The plants will be indicated if it’s endangered or not. A herbarium will be also

established as resource centre. During field work all plant parts will be collected, clearly

labeled, pressed and preserved in herbarium for future reference. The herbalists will be

trained on how to collect plants without destroying the entire plant, how to process and

package the products in a presentable manner. The herbalists will be highly encouraged

to form an association in which they will manage the herbarium and nurseries with the

local authorities.

20

EXPECTED OUTPUT

The herbalist who will be the source of the indigenous knowledge will be trained

on how to package the ground herbs and how to minimize spoilage but

maintaining its activity.

The herbalist will also benefit by being trained about the right dosage to be

administered to patients and the level of toxicity of the herb.

During collection of the herbal plants from the field their seeds will be collected

as well so that, they will be placed in the botanical garden and the seedling will be

provided to the residents to avoid extinction of the endangered herbal plants.

In-situ conservation site will be developed within the county to ensure that there

is continuous supply of medicinal plants to the herbalist to minimize the risk of

harvesting plants from the wild. And in turn this will also create employment.

Herbarium will be established as a reference laboratory for future research.

The herbalists association will be formed to manage the herbarium, medicinal

plants nursery and all areas related to medicinal plant activity.

The document containing these herbal plant species and their claimed therapeutic

action will be deposited in the county office to safeguard the herbal knowledge

from extinction.

Publications is expected at the end of this work.

BUDGET8.1 Equipment COST IN US$

Digital Camera(Sony) 225

Photographic microscope 962.5

Cool boxes@125 US$ 500

Vacuum pump 875

External hard disc 125

Sub-total 2687.5

21

8.2 Expendable supplies COST IN US$

Media @ 100 US$ 500

Solvents 2500

Glassware 1000

Rats food 325

Reagents 625

American Type Culture Collection(ATCC)@125 US$ 625

Sub-total 5575

8.3 Documentation COST IN US$

Books 187.5

Stationary 312.5

Printing, photocopy and internet 312.5

Communication (credit cards) 312.5

Sub-total 1125

8.4 Local travel COST IN US$

Fuel to the field 437.5

Accommodation and field allowances 375

Hiring of vehicles 625

Sub-total 1437.5

8.5 Extra personnel COST IN US$

herbalist 125

Forest tracker 125

Technician and animal attendant 12 months@87.5 US$ 1050

Sub-total 1300

22

8.6 Other costs COST IN US$

Meeting and discussions 375

Ideaconnection administration fee 1250

Credit card/paypal processing fees 375

Sub-total 375

TOTAL PROJECT BUDGET (US$):14,125

23

WORKPLANPHASE 1 PHASE 2

TASK Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4

J F M A M J J A S O N D J F M A M J J A S O N D

Sample

collection-the

plants will be

collected from

their natural

habitat

Documentation

Drying the

samples

Preliminary assay

for antimicrobial

assay

Toxicity tests

Minimum

Inhibition

Concentration

(MIC) tests

Phytochemical

assay

Establishing

24

herbarium

Analysis of data

Publications

25

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