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
DECLARATION
I, 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 .................................................................................................... 3
ABSTRACT ........................................................................................................................ 4
CHAPTER ONE ................................................................................................................. 5
1.0 INTRODUCTION ................................................................................................ 5
1.1 PROBLEM STATEMENT ................................................................................... 5
1.2 JUSTIFICATION ................................................................................................. 6
1.3 OBJECTIVES ....................................................................................................... 7
1.4 HYPOTHESIS ...................................................................................................... 8
CHAPTER TWO ................................................................................................................ 9
2.0 History of Drug Development .............................................................................. 9
2.1 Medicinal Plants.................................................................................................... 9
2.2 Plants as an Alternative Source of Therapeutic Agents ...................................... 10
2.3 Plants and Plant Parts Used ................................................................................ 11
2.4 Documentation .................................................................................................... 11
2.5 Plant Parts Used .................................................................................................. 12
2.6 Cultivation of Medicinal Plants .......................................................................... 12
2.7 Synergsm............................................................................................................. 13
2.8 Activity of Some Medicinal Plant....................................................................... 13
3.0 METHODOLOGY ..................................................................................................... 14
3.1 Preliminary experimental design ........................................................................ 14
3.2 Collection of the plants from the field ................................................................ 14
3.3 Reagents .............................................................................................................. 14
3.4 Extraction of plant materials ............................................................................... 15
3.5 Microbial Test Organisms................................................................................... 15
3.6 Test strains .......................................................................................................... 15
3.7 Preparation of test organisms .............................................................................. 17
3.8 Preparation of McFarland Standard .................................................................... 17
3.9 Antimicrobial Screening Test ............................................................................. 18
3.10 Determination of Minimum inhibitory concentration....................................... 18
3.11 Cell toxicity....................................................................................................... 19
3.14 Statistical data analysis ..................................................................................... 20
3.15 Conservation ..................................................................................................... 20
EXPECTED OUTPUT ............................................................................................. 20
BUDGET .......................................................................................................................... 21
WORKPLAN .................................................................................................................... 24
REFERENCES ................................................................................................................. 26
ABSTRACT
Traditional 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 antimalarial 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 ethnobotanical 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 40 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).
17
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.
18
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.
EXPECTED OUTPUT
20

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.
BUDGET
8.1
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
21
2687.5
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
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
8.4
8.6
1125
Local travel
COST IN US$
Fuel to the field
437.5
Accommodation and field allowances
375
Hiring of vehicles
625
Sub-total
8.5
5575
1437.5
Extra personnel
COST IN US$
herbalist
125
Forest tracker
125
Technician and animal attendant 12 months@87.5 US$
1050
Sub-total
1300
COST IN US$
Other costs
22
Meeting and discussions
375
Ideaconnection administration fee
1250
Credit card/paypal processing fees
375
Sub-total
TOTAL PROJECT BUDGET (US$):14,125
23
375
WORKPLAN
PHASE 1
TASK
Q1
Q2
PHASE 2
Q3
Q4
Q1
Q2
Q3
J F MA M J J A S O N D J F MA M J J
Sample
collection-the
plants
will
collected
their
be
from
natural
habitat
Documentation
Drying
the
samples
Preliminary assay
for antimicrobial
assay
Toxicity tests
Minimum
Inhibition
Concentration
(MIC) tests
Phytochemical
assay
24
Q4
A S O
N D
Establishing
herbarium
Analysis of data
Publications
25
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