EFFICACY OF SOME MEDICINAL PLANTS USED IN VARIOUS PARTS OF
KENYA IN TREATING SELECTED BACTERIAL AND FUNGAL PATHOGENS
INDIA JACQUELINE (B. ED. (Sc.)
REG. NO. 15611562112005
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE
(MICROBIOLOGy) IN THE SCHOOL OF PURE AND APPLIED SCIENCES OF
KENY ATTA UNIVERSITY
JANUARY, 2015
ii
DECLARATION
This thesis is my original work and has not been presented for an award of a degree in
any other University or any other award.
India Jacqueline
Reg. No. 156/15621/2005
Signed----~--~-52--------------
Declaration by Supervisors
This thesis has been submitted for examination with our approval as University
supervisors.
Prof. Paul Okemo
Department of Microbiology,
Kenyatta University
\~.Signature . J~ ~ !)...o (~~Date .
Dr. John Maingi
Department of Microbiology,
Kenyatta University
Signatur~
Dr. Christine Bii
Centre for Microbiology Research,
Kenya Medical Research Institute
S· Cc ~Ignature............. . . Date .. ~.~J~le?:-.~!-$. .
iii
DEDICATION
To my late father, Noah Juma, my mother Phelesia Juma, my husband John Okanda and
our children Blessing and Kinda for you I will undertake all that is desirable within my
reach.
iv
ACKNOWLEDGEMENTS
Special thanks go to the Almighty God for giving me knowledge, patience and strength to
accomplish this work. I would like to express my earnest appreciation to my supervisors,
Prof. Paul Okemo, Dr. John Maingi and Dr. Christine Bii for dedicating their time to
supervise this research work and for their constructive comments and guidance during the
entire time of the study. They all have been a tower of strength to my academic
achievement.
I am grateful to Prof. Jacob Midiwo of Chemistry Department, University of Nairobi,
Kenya; without whom I could not have attained efficient skills and good results on
extraction and phytochemical screening. My sincere gratitude goes to Mr. Richard Korir
of the Centre for Microbiology Department, KEMRI, Nairobi, for his time and expertise
throughout the research duration.
Very special thanks go to my husband, mother and the entire family for their tremendous
support throughout my studies. Special thanks to Eunice Jurna, Joshua Owiti, Mike
Musuya, Winnie Kore, Martha Miyandazi, and Josephine Dulo for their prayers and
inspiration.
vTABLE OF CONTENTS
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENTS iv
TABLE OF CONTENTS v
LIST OF TABLES x
LIST OF FIGURES xi
LIST OF PLATES xii
ACRONYMS AND ABBREVIA TIONS xiii
ABSTRACT xiv
CHAPTER ONE 1
INTRODUCTION 1
1.1 Background infonnation 1
1.2 Problem statement and justification 6
1.3 Research questions 8
1.4 Hypotheses 9
1.5 Objectives 9
1.5.1 General objective 9
1.5.2 Specific objectives 9
CHAPTER TWO 11
LITERATURE REVIEW 11
2.1 Antimicrobial agents 11
2.2 Antibacterial drugs 12
2.2.1 Penicillins 12
2.2.2 Aminoglycosides 12
2.2.3 Tetracyclines 13
vi
2.2.4 Quinolones 14
2.2.5 Macrolides 14
2.2.6 Sulphonamides 15
2.3 Antifungal agents 15
2.4 Medicinal plants 17
2.4.1 Medicinal plants in the world 19
2.4.2 Medicinal plants in Africa 21
2.4.3 Medicinal plants in Kenya 23
2. 4. 4 Plants under investigation 25
2.4.4.1 Ficus sycomorus Linn. (Moraceae) 25
2.4.4.2 Balanites aegyptica L. Drel (Balantiaceae) 25
2.4.4.3 Sesbania sesban Linn. (Papilionaceae) 26
2.4.4.4 Tamarindus indica L. (Fabaceae) 26
2.4.4.5 Albizia coriaria Welw. ex Oliv (Fabaceae) 26
2.4.4.6 Ormocarpum trichocarpum Taub. Engl. (Fabaceae) 26
2.4.4.7 Senna didymobotrya Fresen. (Caesalpinaceae) 27
2.4.4.8 Commiphora africana (A. Rich) Eng.Syn (Burseraceae) 27
2.4.4.9 Psiadia punctualata (DC) Vatke (Compositeae ) 27
2.4.4.10 Rhus natalensis Krauss (Anacardiaceae) 28
2.4.4.11 Ricinus communis Linn (Euphorbiceae) 28
2.4.4.12 Zanthoxylum chalybeum Engl. (Rutaceae) 28
2.4.4.13 Bosia angustifolia A. Rich (Capparaceae) 28
2.4.4.14 Melia volkensii Gurke (Meliaceae) 29
2.4.4.15 Zanthoxylum gilletii De Wild (Rutaceae) 29
2.4.4.16 Fuerstia africana T.C.E. Fries (Lamiaceae) 29
2.4.4.17 Urtica dioica Linn (Urticaceae) 30
vii
2.4.4.18 Vernonia amygdalina Del. (Asteraceae) 30
2.5 Bacterial infections 30
2.5.1 Escherichia coli 30
2.5.2 Salmonella typhi 31
2.5.3 Staphylococcus aureus 32
2.5.4 Methicillin resistant Staphylococcus aureus 33
2.5.5 Bacillus subtilis 34
2.6 Fungal infections 34
2.6.1 Dennatophytes 35
2.6.2 Candida albicans 36
2.6.3 Cryptococcus neoformans 37
2.6.4 Aspergillus niger 38
2.7 Phytochemicals in medicinal plants 38
2.7.1 Tannins 39
2.7.2 Saponins 40
2.7.3 Flavonoids 40
2.7.4 Alkaloids 40
2.7.5 Terpenoids 41
CHAPTER THREE 42
MATERIALS AND METHODS 42
3.1 Ethnobotanical survey 42
3.2 Collection and authentication of plant materiaL : 43
3.3 Preparation of plant materials 45
3.4 Extraction 45
3.5 Concentration into solid sample 45
3.6 Microbial test organisms ~ 46
viii
3.6.1 Bacterial isolates 46
3.6.2 Fungal isolates 46
3.7 Maintenance of microbial stock cultures 47
3.8 Antibacterial assays 47
3.9 Antifungal assays 48
3.10 Determination of minimum inhibitory concentration 49
3.11 Determination of Minimum Bactericidal/Fungicidal Concentrations (MBCs/ MFCs) ... 50
3.12 Phytochemical screening 51
3.12.1 Test for alkaloids 51
3.12.2 Cardiac glycosides 51
3.12.3 Test for saponins 52
3.12.4 Test for tannins 52
3.12.5 Flavonoids 52
3.12.6 Test for terpenoids 53
3.13 Statistical analysis 53
CHAPTER FOUR 54
RESULTS 54
4.1 Ethnobotanical survey 54
4.2 Antibacterial activity of the plant crude extracts against standard strains of bacteria 60~
4.2.1: Zone of inhibition plates 63~
4.3 Antifungal activity of the plant crude extracts against standard strains offungus 65~
4.3.1 Zone of inhibition plate against fungal strains 68~
4.4 The Minimum Inhibitory Concentrations (MICs) and the Minimum Bactericidal
Concentration (MBCS) 69~
4.5 Phytochem ical screening 73~
4.5.1 Tannins 73~
ix
4.5.2 Alkaloids 7~
4.5.3 Saponins 7~
4.5.4 Cardiac glycosides 75~
4.5.5 Terpenoids 75~
4.5.6 Flavonoids 76§6
CHAPTER FIVE 79~
DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS 79~
5.1 DISCUSSION 79~
5.2 CONCLUSIONS 100~
5.3 RECOMMENDATIONS 102~
REFERENCES 104~
APPENDICES 123~
xLIST OF TABLES
Table 1: Selected medicinal plants used by the communities from Mwingi North, Kisii
South and Rarieda Counties in treating various bacterial and fungal ailments----------- 55
Table 2: Zones of inhibition produced by the plant extracts against the selected bacterial
strains----------------------------------------------------------------------------------------------- 62
Table 3: Zones of inhibition produced by plants extracts against fungal strains--------- 67
Table 4: The minimum inhibitory concentration and the minimum bactericidal
concentration for bacterial test cultures-------------------------------------------------------- 71
Table 5: The minimum inhibitory concentration and the minimum fungicidal
concentration for fungal test cultures----------------------------------------------------------- 72
Table 6: Phytochemical screening ofthe plants extracts------------------------------------- 77
xi
LIST OF FIGURES
Figure 1: Map of Kenya showing Kisii, Bondo and Mwingi counties -------------------- 44
Figure 2: Percentage frequency of diseases treated using herbal drugs in Mwingi North,
Kisii South and Rarieda Counties --------------------------------------------------- 57
xii
LIST OF PLATES
Plate 1: Ricinus communis --------------------------------------------------------------------- 58
Plate 2: Ses bania ses ban ----------------------------------------------------------------------- 58
Plate 3: Fuerstia africana------------------------------------------------------------------------ 58
Plate 4: Urtica dioica ---------------------------------------------------------------------------- 58
Plate 5: Senna didymobotrya-------------------------------------------------------------------- 59
Plate 6: Zones of inhibition of Fuerstia africana, Senna didymobotrya extracts and that
of the positive and negative control against Bacillus subtilis --------------------- 60
Plate 7: Zones of inhibition of Fuerstia africana, Ficus sycomorus extracts and that of
the positive and negative control against Methicillin Resistant Staphylococcus
aureus ------------------------------------------------------------------------------------ 61
Plate 8: Zones of inhibition of Tamarindus indica extracts and that of the positive and
negative control against Staphylococcus aureus --------------------------------- 61
Plate 9: Zones of inhibition of Cammiphora africana extracts against Microsporum
gypseum----------------------------------------------------------------------------------- 65
Plate 10: Zones of inhibition of Rhus natalensii extracts and that of the positive control
against Microsporum gypseum ------------------------------------------------------- 65
Plate 11: Microtitre plates showing Minimum inhibition concentration. ----------------- 68
Plate 12: Reddish brown colouration of the interface indicated the presence ofterpenoid
in Fuerstia africana ------------------------------------------------------------------ 77
AIDS
ANOVA
ATCC
CI
CLSI
DCM
DMSO
GOI
IRN
KEMRI
MBC
MFC
MIC
NCAPD
NCCLS
PKF
UNICEF
UNIDO
xiii
ACRONYMS AND ABBREVIATIONS
Acquired Immune Deficiency Syndrome
Analysis ofVarience
American Type Culture Collection
Confidence Interval
Clinical Laboratory Standard Institute
Dichloromethane
Dimethyl Sulfoxide
Government of India
International Research Network
Kenya Medical Research Institute
Minimum Bactericidal Concentration
Minimum Fungicidal Concentration
Minimum Inhibitory Concentration
National Coordinating Agency for Population and Development
National Committee on Clinical Laboratory Standards
Pannell Kerr Forster
United Nations International Children's Emergency Fund
United Nations Industrial Development Organization
xiv
ABSTRACT
Medicinal plants have been used since time immemorial to treat and prevent human
ailments. WHO indicates that up to 80% of the world's population uses traditional
medicine. Infections caused by bacteria and fungi have become a major health problem
globally accounting for over 50,000 deaths every day. It is estimated that more than 70%
of the pathogenic bacteria are resistant to at least one of the antibiotics commonly used to
treat them. Conventional drugs are expensive and have side effects. Many plants have
been used by various communities in Kenya in the treatment of bacterial and fungal
infections but they have not been validated. The main aim of this study was to determine
the efficacy of some medicinal plants used by various communities in Kenya that treat
the selected bacterial and the selected fungal diseases in man. An ethnobotanical survey
was used to select and collect plants from Mwingi North, Kisii South and Rarieda
Districts based on their use to treat infectious diseases such as skin infection, diarrhea and
many others. Crude extracts from Zanthoxylum chalybeum, Boscia angustifolia, Melia
volkensii, Zanthoxylum gilletii, Fuerstia africana, Urtica dioica, Vernonia amygdalina,
Ricinus communis, Commiphora africana, Psidia puntulata, Senna didymobotrya,
Ormocarpum trichocarpum, Sesbania sesban, Balanites aegyptiaca, Albizia coriaria,
Ficus sycomorus, Rhus natalensis and Tamarindus indica believed to contain secondary
metabolites were screened against ten microorganisms, including the bacteria:-
Salmonella typhi ATCC 19430, Escherichia coli ATCC 25922, Bacillus subtilis,
Staphylococcus aureus ATCC 25923 and Methicillin Resistant Staphylococcus aureus.
The fungal strains that were used are; Aspergillus niger, Candida albicans ATCC 90028,
Microsporum gypseum, Crytococcus neoformans ATCC 18310 and Trichophyton
mentagrophyte. The plants were screened using Kirby Bauer disc diffusion method.
Phytochemical screening was carried out to identify the presence or absence of classes of
bioactive compounds. Data was analyzed using one way ANOV A, significant means
were separated using Tukey's test. Generally, Fuerstia africana, Zanthoxylum
chalybeum, Balanites aegyptiaca, Ormocarpum trichocarpum, Senna didymobotrya and
Tamarindus indica gave strong antibacterial results of between 14.5 mm and 20 mm as
Albizia coriaria, Ficus sycomorus, Commiphora africana Rhus, natalensis Senna
didymobotrya, Psidia puntulata, and Tamarindus indica produced strong antifungal
results of between 15.5 mm and 20.5 mm. The results ofMICs and the MBCslMFCs of
the extracts of Albizia coriaria, Ficus sycomorus, Senna didymobotrya, Psidia puntulata,
Fuerstia africana, Balanites aegyptiaca and Tamarindus indica showed a good activity
of 0.9375 mg/ml in some test cultures. Salmonella typhi ATCC 19430 and Escherichia
coli ATCC 25922 were the least sensitive bacteria while Candida albicans ATCC 90028
was the least sensitive fungus. The present study indicates that the majority of the plants
tested are an important source of antibacterial agents especially on Gram positive bacteria
(Staphylococcus aureus, Bacillus subtilis and Methicillin Resistant Staphylococcus
aureus) and antifungal agents against the dermatophytes especially Microsporum
gypseum. This study recommends that the plant extracts with good antimicrobial activity
be subjected to both pharmacological and toxicological studies.
1CHAPTER ONE
INTRODUCTION
1.1 Background information
Infectious diseases are the leading cause of death worldwide (Bandow et al., 2003;
Parekh and Chanda 2007). The major infectious diseases cause over 11 million deaths
annually (WHO, 2005). Half of all deaths in the tropical countries result from infectious
diseases (Okigbo and Mmeka, 2008; Assob et al., 2011). Infections caused by pathogenic
bacteria and fungi remain an important public health concern particularly in developing
countries because of factors such as: emergence of bacterial and fungal strains that are
resistant to most useful antibiotics (Abad et al., 2007; WHO, 2007) HIV/AIDS pandemic
(Wagate et al., 2008) and unavailability of vaccine (Assob et al., 2011). Conventional
drugs are expensive and the western health facilities are also inaccessible to rural people
(Matu and Staden, 2003; Wagete et a/., 2008).
The problem of multiple drug resistance has come about due to the haphazard use of the
antimicrobials and re-emergence of diseases caused by genetically versatile microbes
(Olila et al., 2001; Islam et al., 2006; Wagete et al., 2010). For instance, in many
countries antimicrobials are bought directly from chemists with no prescriptions or
advice from a health professional (WHO, 2005). About a third of the 150 million
prescriptions for antibiotics each year in human medicine, are not needed according to the
US centre for Disease Control and Prevention (Rangasamy et a/., 2007). Drug resistance
made it necessary for researchers to search for alternative antimicrobial agents from other-
sources like plants (Pirbalouti et al., 2010). In addition to that, the human body is much
2better suited to treatment with herbal remedies than chemical compounds since humans
have evolved with plants and our digestive system and physiology are geared to digesting
and utilizing plant based foods with medicinal value (Farooq, 2005). Considering all
these factors, the African Union in 2001, declared the years 2001 - 2010 as the decade for
African traditional medicine thereby acknowledging therapeutic potential and
endorsement, use and development of medicinal plants in developing countries (NCAPD,
2005).
Medicinal plants have been used since time immemorial to treat and prevent human
ailments because they have components of therapeutic value (Hassan et al., 2006;
Gulluce et al., 2006; Parekh and Chanda, 2007). Domesticated and non-domesticated
animals in ordinary settings unconsciously treat themselves when sick by eating various
parts of medicinal plants such as leaves, stems, barks and roots (Sindiga et aI., 1995).
They may also treat their skin conditions by briskly rubbing themselves against suitable
plants with curative properties (Sindiga et aI., 1995). Diets high in plant -based foods and
beverages are associated with a lower risk of chronic diseases (Njoroge et al., 2012).
WHO estimates that up to 80% of the world's population relies on plants for their
primary health care needs (Doughari, 2006; Turker and Usta, 2008; Verma et aI., 2011).
Such a large population depends on traditional medicine due to factors such as : Increase
in resistance to the commonly used antibiotics, high cost and inaccessibility to antibiotics
especially in ruralareas. It is however noted that medicinal plants are readily available,
they have little side effects and there is extensive local knowledge on herbal medicine
3amongst the communities (Rojas et al., 2006; Doughari et al., 2008). There are about
20,000 plant species used for medicinal purposes (Gulluce et al., 2006). From which at
least 121 chemical substances are extracted (Olila et al., 2007). Some of the known good
sources of pharmacologically active compounds are natural products from fungi and
higher plants (Olila et al., 2001). Many of the effective drugs such as anti-malarial, anti-
cancer, anti-diabetic and antibiotics such as atropine and ergometrine compounds have
been purified from medicinal plants (Olila et al., 2001; Samie et al., 2005). Medicinal
plants are also a source of many active ingredients in the pharmaceutical industries
(Maundu and Tengnas, 2005). Besides that, when the deadly disease, AIDS came up, the
Ministry of Health called on the herbalists to work together with them in search of a
treatment for the disease (Sindiga et al., 1995). Approximately two thirds of HIV /AIDS
patients in many developing countries seek symptomatic relief and manage opportunistic
infections through the use of traditional medicine (WHO, 2011). Therefore, it is sensible
to study plants from the local flora which are used for treatment of such infections
(Atindehou et al., 2002).
The use of medicinal plants for treatment is the most preferred form of traditional
medicine and is highly lucrative in the international market (WHO, 2008). The annual
sales from herbal medicine and other plant products in Western Europe totalled to US
dollar 5 billion in 2003-2004, US dollar 14 billion in China in 2005 and US dollar 160
million in Brazil in 2007 (WHO, 2008). There is also a large demand for crudely
prepared medicinal plant parts mainly in Europe and America which usually are
distributed as over the counter medications (Matu and Staden, 2003). In Africa parts of
4medicinal plants are sold at every market in urban and peri-urban centres (Rukangira,
2001). For instance in East Africa, the dried leaves of Securidaca longepedunculata is
used for treatment of wounds and sores, coughs, venereal diseases and snake bites. In
Malawi, the same plant is used for wounds, coughs, bilharzias, venereal diseases, snake
bites and headaches. While in Nigeria it is used for treatment of skin diseases (Rukangira,
2001).
The medicinal value of plants used for traditional medicine lies in the chemical
substances they contain to treat chronic and common bacterial infections (Duraipandiyan
et aI., 2006; Kareru et aI., 2008). These compounds represent secondary metabolites that
serve as defense agents against invading micro-organisms and predators (Ghdeib and
Shtayeh 1999; Nwodo et aI., 2010). They also help in regulation of growth in plants
(Nwodo et aI., 2010). The secondary metabolites include the alkaloids, steroids, tannins,
phenol compounds, flavonoids and fatty acids (Ashokkumar et aI., 2010). The numbers
of flowering plants occurring on earth are conservatively estimated at 250,000. About 6%
of these have been screened for biological activity and 15% are reported to have been
evaluated phytochemically. These figures depict the need for a study to uncover a
probable abundance of medicinal extracts in these plants (Turker and Usta, 2008).
The rate at which the incidence of new and re-emerging infectious diseases is increasing
is alarming. The development of resistance to the antibiotics that are in current clinical
use is also a problem (Parekh and Chanda, 2008). It is estimated that more than 70% of
the pathogenic bacteria are resistant to at least one of the antibiotics commonly used to
5treat them (Okemo et al., 2011). This calls for urgent and continued need to find new
antimicrobial compounds with varied chemical structures and new mechanisms of action
(Parekh and Chanda, 2007) and has necessitated the need to search for new antimicrobial
substances from other sources including plants (Doughari, 2006; Duraipandiyan et al.,
2006).
Many studies concerning the use of medicinal plants in Kenya have been carried out
targeting different communities (Maundu and Tengnas, 2005; Kokwaro, 2009) other
studies include: medicinal plants from Bomet district (Cheruiyot, 2009), ethnobotany of
the Samburu and Turkana (Bussman, 2006; Omwenga et a/., 2009) and traditional
medicine among the Embu and Mbeere people (Kareru et al., 20 07). However, the
ethnobotany in Kisii, Rarieda and Mwingi Districts is scarcely known. Many medicinal
plants are used by the herbalists from Kisii, Rarieda and Mwingi Districts but their
efficacy has not been established.
The ethnobotanical studies that are reported in Eastern province have focused on Embu,
Mbeere, Makueni and Machakos districts, leaving out Mwingi district which is interior
and likely to have more intact traditional knowledge (Njoroge et al., 2010). Therefore,
more studies should be done on plants from Mwingi district. The biodiversity of the
plants found in Kisii district offer great possibilities in the search for natural products
with antimicrobial compounds. The people from Rarieda district also have a strong
cultural belief in traditional medicine (Osewe, 2011).
61.2 Problem statement and justification
Infectious diseases have become a major health problem globally, accounting for over
50,000 deaths everyday (Doughari and Manzara, 2008). This has been brought about by
the increasing prevalence of multi-drug resistant bacterial and fungal strains to the
available conventional medicine (Parekh and Chanda, 2007; Doughari and Manzara,
2008). It is estimated that more than 70% of the pathogenic bacteria are resistant to at
least one of the antibiotics commonly used to treat them (Okemo et al., 2011). The
problem is major and unless planned action is taken in the whole world, we have the risk
of getting back to pre- antibiotic period where many children died of infectious diseases
and major surgery was not possible because of risk of infection (WHO, 2005).
Traditional medicine has fewer side effects, better patient tolerance, acceptance due to
long history of use, renewable in nature, the most affordable and available means of
medical treatment to the rural people (WHO, 2002; Joshi et al., 2011; Shagal et al.,
2013). Conventional medicine are expensive and have side effects (Okemo et aI., 2011)
such as toxicity, allergic reactions and disruption of normal bacterial flora which could be
life threatening (Saify et aI., 2000; Uzoigwe and Agwa, 2011). It was estimated that 2.22
million hospitalized patients had serious Adverse Drug Reactions (ADR) and 106,000
died in a single year in the U.S.A (Joshi et al., 2011; Issazadeh et al., 2012).
In recognition of the role of traditional medicine in primary health care, the Kenya
government, in the 1979-1983 development plan, called for research to evaluate the
functions and determine the extent of usefulness of traditional medicine (NCAPD, 2005).
A key goal of Kenyans vision 2030 is to supply adequate health care for all. This can be
7achieved faster by main streaming traditional health care practices into the national health
care systems (Kaingu et al., 2011). According to WHO (2008), there is lack of scientific
evidence to evaluate the safety and efficacy of traditional medicine. Therefore, there is
need to screen medicinal plants for better understanding of their properties, safety and
efficacy (Doughari et al., 2008) and also to validate their traditional uses and identify the
active compounds (Demet et al., 2008).
Many plants have been used by various communities in Kenya in the treatment of
bacterial and fungal infections but they have not been validated (Korir et al., 2012a).
Attempts have been made to gather information on traditional medicine and the people
who use them (Maundu and Tengnas, 2005; Kokwaro, 2009). However, only a small
number of plants have been identified and the efforts have not covered all the
communities in Kenya. Few studies have been carried out on the medicinal plants from
Kisii and Mwingi Districts by Mariita et al. (201Oa and 2011). However no research has
been done on MRSA, B. subtilis, C. neoformans, filamentous fungi and dermatophytes.
In studies done by Mariita et al., (2010a and 2011) stem barks were used but in this
research, leaves were used. In addition to that the solvent used for extraction was
different. Mariita et al. (2010a and 2011) used methanol for extraction. However in the
current study, the leaves were extracted using a mixture of dichloromethane and
methanol in the ratio of 1:1 according to (Midiwo, 2010).
According to Kokwaro, (2009) these plants have been documented to be used by different
communities without testing their efficacy. The efficacy of medicinal plants used by
8herbalists from Rarieda District had not been established. Such indigenous knowledge
also needs to be documented before it is lost with the passing on of the older generation
(Kareru et aI., 2007; Vaghasiya et aI., 2011). Traditional healers by their nature do not
keep records and most of the knowledge they have is passed on verbally from generation
to generation (Kaingu et aI., 2011). There is an increased loss of therapeutic plants as a
result of anthropogenic activities (Rukangira et aI., 2001).
Claims by the traditional healers about the efficacy of herbal preparations have little
validation and documentation yet they treat many people. It is therefore imperative that
chemical and bioassay analysis is carried out to find out the efficacy of the medicinal
plants. We are likely to lose most of the information because of inadequate
documentation and communication of the local health traditions through oral narratives.
It is vital therefore that the efficacy is validated and documented. This supports the main
objective of this study to investigate crude extracts of the selected plants using in vitro
models of relevance to validate and document scientific antifungal and antibacterial
activities of these plants.
1.3 Research questions
1. Do the communities from Mwingi North, Kisii South and Rarieda Districts have
information on the medicinal plants used in the treatment of selected bacterial and
fungal diseases?
11. Are the candidate plants active against the selected bacterial pathogens?
111. Are the candidate plants active against the selected fungal pathogens?
9IV. Do the plant extracts have phytochemical constituents that are responsible for
antimicrobial activity?
1.4 Hypotheses
1. The communities from Mwingi North, Kisii South and Rarieda Districts do not
have information on the medicinal plants used III the treatment of common
bacterial and fungal diseases.
11. The selected medicinal plants do not have activity against the selected bacterial
pathogens.
lll. The selected medicinal plants do not have activity against the selected fungal
pathogens.
IV. The phytochemical constituents present in the medicinal plants are not known.
1.5 Objectives
1.5.1 General objective
To determine the efficacy of some medicinal plants used by various communities in
Kenya that treats the selected bacterial and fungal pathogen.
1.5.2 Specific objectives
1. To investigate the ethnobotany of some medicinal plants used by the communities
from Mwingi North, Kisii South and Rarieda Districts.
11. To determine the antibacterial activity of the plant crude extracts against standard
strains S. typhi, E. coli, MRSA, S. aureus and B. subtilis.
10
111. To determine the antifungal activity of the plant crude extracts against standard
strains of C. albicans, A. niger, T mentagrophyte, C. neoformans and M gypseum.
IV. To determine the MICs, MBCs and MFCs of the crude extracts showing
antimicrobial activity against the selected bacterial and fungal pathogens.
v. To determine the phytochemicals present in the crude extracts.
11
CHAPTER TWO
LITERATURE REVIEW
2.1 Antimicrobial agents
An antimicrobial agent is a secondary metabolite produced by bacteria that has
inhibitory properties against microorganisms which includes antibiotics and synthetic
compounds but with minimal effects on mammalian cells (Elliott et al., 2007). The drugs
used in the treatment of infectious diseases are divided into two groups; chemical
antimicrobials which are synthetic chemical compounds and antibiotics which are
produced by bacteria and fungi (Elliott et al., 2007). The main mechanisms involved in
the action of antimicrobial drugs against microorganisms include; interference with cell
wall synthesis, disruption of cell membrane, inhibition of nucleic acid synthesis,
inhibition of protein synthesis, inhibition of enzymatic activity (Bhatia, 2008), inhibition
of metabolic pathways (Tenover, 2006) and inhibition of folate fatty acid (Hancock,
2007).
Currently, multiple drug resistance has been developed due to increased misuse of
antibiotics in the treatment of bacterial infections (Agyare et al., 2006; Khan et al., 2011).
Antimicrobial resistance is one of the world's most serious public health problem (WHO,
2010). With the increase in resistant bacterial and fungal strains, there is a corresponding
rise in demand for natural antimicrobial treatments (Assob et al., 2011) and herbal
medicine could be a reservoir of new antimicrobial substances waiting to be discovered
(Assob et al., 2011).
12
2.2 Antibacterial drugs
Antibiotics are organic substances produced by microorganisms and are capable of
inhibiting the growth of another microorganism at low concentration (Chinedu, 2005).
The overuse of antibiotics in the treatment of bacterial infections has led to increased
incidences of resistant pathogens to the available antibiotics (Agyare et al., 2006).
Bacterial resistance can only be combated by the discovery of new drugs and herbal
medicines are potential candidates for antimicrobial alternatives.
2.2.1 Penicillins
These antibiotics are based on a structure called the beta-lactam ring which prevents cell
wall synthesis by binding to an enzyme that makes peptidoglycan (Bhatia, 2008).
Benzylpenicillin (penicillin G) exhibits activity against staphylococci, streptococci,
neisseriae, spirochaetes and other organisms (Greenwood et al., 2002). Resistance to
penicillin was developed due to a beta - lactamase enzyme (penicillinase) which is able
to break the beta - lactam ring and destroy the antibiotics (Bhatia, 2008) or changes in
penicillin-binding protein as occurs in MRSA (Cheesbrough, 2000). Resistance to
penicillin is widespread in staphylococci and increasingly in others such as Streptococcus
pneumoniae and Neisseria gonorrhea (Bhatia, 2008).
2.2.2 Aminoglycosides
These are a group of antibiotics consisting of amino sugars and aminocyclitol ring held
together by glycoside bonds. Common examples of aminoglycosides are streptomycin,
neomycin, apramycin, kanamycin and gentamicin (Trene ry, 2011). Gentamicin is an
13
antibiotic widely used for the treatment of bacterial infections (Poormoosavi et al., 2010).
They inhibit formation of the ribosomal initiation complex and also cause misreading of
messenger RNA (Greenwood et al., 2002). Aminoglycosides are very poorly absorbed
when taken orally and for the treatment of systemic infections, they must be given
parenterally (Turnidge, 2003). Aminoglycosides are obligate nephrotoxins and renal
impairment is eventually detected in all patients if treated for long (Trenery, 2011). They
are mainly reserved for the treatment of severe sepsis caused by coliforms and other
Gram negative aerobic bacilli (Cheesbrough, 2000; Elliot et al., 2007). They have
antimicrobial activity on S. aureus, Streptococci and many Gram-negative species such
as neisseriae, Haemophilus and coliforms (Elliot et al., 2007).
2.2.3 Tetracyclines
Tetracycline is a major member of a group of antibiotics with a broad spectrum of
activity which is widely used in medicine and veterinary science to treat bacteria
infections (Ali et al., 2010). They have a broad spectrum of activity against many Gram-
negative and Gram positive bacteria, chlamydiae, mycoplasmae and rickettsiae (Elliott et
al., 2007). The tetracycline antibiotics inhibit binding of tRNA to ribosomes (Ali et al.,
2010). Resistance to tetracycline is common in Heamophilus influenzae, Streptococcus
pneumoniae and Streptococcus pyogenes (Cheesbrough, 2000). The most commonly used
tetracyclines are doxycycline and minocycline (Greenwood et al., 2002). The extensive
use of tetracycline in the therapy of human and animal infections is due to their
antimicrobial properties and lack of major adverse side effects (Chopra and Roberts,
2001).
14
2.2.4 Quinolones
These drugs are synthesized from nalidixic acid and inhibit the action of DNA gyrase, the
enzyme responsible for super coiling of DNA (Bhatia, 2008). Examples are: Nalidixic
acid, which has antimicrobial activity on enterobacteriaceae but with no activity on
Gram-positive organisms. Fluoroquinolones such as ciprofloxacin, ofloxacin and
norfloxacin with activity against coliforms and other Gram-negative bacilli (Elliott et al.,
2007). Ciprofloxacin is commonly used to treat urinary tract infections but it has been
associated with tendonitis and has the tendency of altering the normal flora in the colon
and also promotes the growth of bacteria, responsible for the development of
inflammation of the colon (Uzoigwe and Agwa, 2011).
2.2.5 Macrolides
Macrolides cover a family of different antibiotics produced by fungi of the genus
Streptomyces and some bacteria such as Arthrobacter spp. (Kwiatkowska and Maslinska,
2012). Its mode of action is to inhibit bacterial protein synthesis at ribosomes by binding
to 50s ribosomal subunit (Kwiatkowska and Maslinska, 2012). They are mainly used for
the treatment of staphylococcal infections, respiratory infections and urethritis
(Cheesbrough 2000). They are used as alternatives to people with penicillin
hypersensitivity (Bhatia, 2008; Kwiatkowska and Maslinska, 2012). Resistance may
occur with S. aureus, S. pyogenes and S. pneumoniae (Cheesbrough, 2000). Examples of
macrolides are erythromycin, clarithromycin and azithromycin (Elliott et al., 2007).
15
2.2.6 Sulphonamides
Sulphonamides are a group of drugs containing sulfur dioxide and nitrogen directly
linked to a benzene ring as a basic structure (Chantachaeng et al., 2011). They are
divided into two main groups according to chemical structure and therapeutic actions;
sulfonamide antibiotics such as sulfamethoxazole, sulfamethoxypyridazine and
sulfonamide non antibiotics such as furosemide, hydrochlorothiazide and glipizide
(Chantachaeng et al., 2011). They are broad-spectrum antibacterial agents which exhibit
antimicrobial activity on Streptococci, Neisseriae and H inj/uenzae (Elliott et al., 2007)
but resistance is common and the group is also toxic (Greenwood et al., 2002).
Sulphonamides were introduced in 1935 and about 10 years later, 20 % of clinical
isolates of Neisseria gonorrhea had become resistant to them (Chinedu, 2005). Due to the
emergence of resistance to fluoroquinolones in 2007 (CDC, 2011) CDC now
recommends dual therapy for gonorrhea with cephalosporin, ceftriaxone 250mg plus
either azithromycin or doxycycline (CDC, 2011).
2.3 Antifungal agents
Treatment with antifungal agents depend on site, level of infection, species involved, the
efficacy, safety profile and kinetics of the available drugs (Galgoczy et al., 2008). It is
unfortunate that at the moment, only a few classes of antifungals are available and they
lack potency or are toxic to human hosts (Lin, 2009; Sai et al., 2012). Modem antifungal
agents have become expensive; have side effects and fungal strains have developed
resistance to the already available fungal drugs (Koroishi et al., 2008; Zuzarte et al.,
2011). This has led to the reduction of the number of available and effective antifungal
16
agents thus necessitating the development of new antifungal drugs (Hamza et al., 2006).
These have inspired an extended search for new classes of antifungals and compounds
that inhibit resistant mechanisms because of the spread of multidrug resistant strains of
fungi and the toxicity during prolonged treatment with several antifungal drugs (Garcia et
al., 2003; Yigit et al., 2008). The above mentioned reasons have made it important to
search for newer drugs from plants for the treatment of opportunistic fungal infections.
One approach can be testing of plants which are used customarily for their antifungal
actions as possible sources of drug development (Abad et al., 2007).
Antifungals can be grouped into three classes based on their site of action: first, azole
which prevents the synthesis of ergosterol, for example fluconazoles which are active
against Candida species. Intraconazole is however active against Aspergillus species and
dermatophytes (Elliot et al., 2007; Ghannoum et al., 1999). The second are polyenes
which interact with fungal membrane and interfere with its sterol content for example,
nystatin which is used for the treatment of local infections of the vagina and skin and
amphotericin B which is used to treat histoplasmosis and blastomycosis. Lastly,
flucytosine (5-fluorocytosine) which has antimicrobial activity against most yeasts such
as Candida species (not C. krusei) and C. neoformans (Elliott et al., 2007).
Most of the clinically used antifungals have various disadvantages in terms of toxicity,
efficacy and cost. Their frequent use has led to the emergence of resistant strains (Abad et
al., 2007). Selective action against fungi without toxic side effects is not easy to get and
17
useful antifungal agents are few (Elliott et al., 2007). The currently available polyenes are
amphotericin B and nystatin, although differing safety profiles have reduced nystatin to
topical use (Thompson et al., 2009). In addition to that, there is limited scientific
evidence on efficacy of medicinal plants in treatment of fungal infections (Harnza et al.,
2006). Due to these short comings, the efficacy of eighteen medicinal plants against five
strains of fungi was determined.
2.4 Medicinal plants
Herbal medicines are defined as any preparations containing one or more active herbal
substances or herbal extractives (Agyare et al., 2006). The medicinal plants are the plants
whose parts (leaves, seeds, stems, roots, fruits and leaves) extract infusions, decoctions
and powders are used in the treatment of different diseases of humans, plants and animals
(Jamil et al., 2007). About 20,000 plant species used for medicinal purposes are reported
by WHO (Gullece et al., 2006; Khan et al., 2011). Plants have been used since antiquity
as a good source of anti-infective agents and during recent years, the search for plants
with antimicrobial activities has continued to gain importance (Assob et al., 2011).
Antimicrobial agents can easily be obtained from medicinal plants that act as their source
(Kubmarawa et al., 2007; Rangasamy et al., 2007). Therefore, there is need to screen
plants for their antimicrobial properties.
Currently about 30 % of the modem pharmacological drugs are gotten directly or
indirectly from plants (Doughari and Manzara, 2008). Plants have provided a rich source
of new drugs and cure for diseases (Adesina, 2005). Many such medicines including
18
strychnine, aspnn, vincristine and taxol which are made from plants (Agyare et al.,
2006). The antimicrobial drugs such as quinine and artemisine are either made from
plants or had plant derived chemical structures used as templates (Muregi et al., 2003).
Purgative drugs are usually a mixture of anthraquinone derivatives from cassava or
related species. Other plant derived drugs include anti-ulcers drugs, carbenoxole from
Glycyrrhiza glabra roots and the antimalarial drug quinine from Cinchona bark, just to
mention but a few (Adesina, 2005).
According to WHO, traditional medicine is one of the ways to achieve total health care
coverage of the world's population (Rukangira, 2001). Medicinal plants are used for
primary healthcare by about 70 %- 90 % of populations in developing countries (WHO,
2011). For instance, Nauclea latifolia smith (Rubiaceae) is used to manage venereal
diseases and constipation in Sierra Leone (Agyare et al., 2006). A root infusion of N
latifolia is used for the treatment of gonorrhea in Sudan. The roots are used as chewing
sticks for toothache and dental caries (Agyare et al., 2006). In Ghana, the roots and
leaves are used for stomach complaints and for treating sores while in Nigeria, the fruits
are sometimes used for the treatment of piles and dysentery (Agyare et al., 2006).
Medicinal herbs can even cure deadly diseases such as cancer and AIDS that have
resisted conventional drugs (Jamil et al., 2007).
Skin diseases, diarrhoea, malaria, respiratory infection, bacterial and fungal infections are
the most common health problem in rural areas (Pirzada et al., 2010). In resource limited
countries, a lot of medicinal plants are used traditionally for treatment against these
19
diseases (Pirzada et a/., 2010). The use of herbal and other alternative therapies are
attractive in many developing countries because of poor health care management systems
and the high cost of developing a new pharmaceutical drug that is estimated averagely
over US $ 800 million (Muhammad and Awaisu, 2008).
For many generations, naturally occumng substances and locally made herbs for
medication of wounds, including circumcision wounds and chronic skin ulcers have been
known (Mboto et a/., 2009). Drug inventions from ethnopharmacology and natural
products are still an important expectation in uplifting the deprived livelihoods of rural
communities (Nanyingi et a/., 2008). The antimicrobial activity exhibited by the extracts
of medicinal plants has been supported by several researchers (Rangasamy et a/., 2007;
Doughari et a/., 2008).
2.4.1 Medicinal plants in the world
In Africa, Asia and other developing worlds, medicinal plants provide a rich source of
raw materials for traditional medicine, particularly in the villages (Tsegaye, 2006). In as
much as conventional medicine is easily accessible, many Asian countries; China, India,
Japan and Korea still rely on traditional medicine (WHO 2002; Ramamorthy, 2010) ..
During the period of 2000, legislation on traditional medicines was developed by Canada,
Nigeria, Australia, Madagascar and United States of America (WHO, 2002). This was
formed since over 50 % of the population in the poorest parts of Africa and Asia do not
have access to essential drugs (Muhammad and Awaisu, 2008) therefore making
20
medicinal plants the only other alternative. Most of the western medicinal preparations
have one or more ingredients of plant origin (Nahak and Sahu, 2010).
About 25,000 effective plant base formulations are used in modem medicine and known
to rural communities all over India and about 10,000 designed formulations are available
in the local medicinal texts (GOI, 2000). The WHO estimates that not less than 25 % of
all contemporary medicines are obtained from medicinal plants either directly or
indirectly using modem technology (WHO, 2011). In India each plant is of use for man
and animal (Nahak and Sahu, 2010) therefore, medicinal plants constitute the principal
health care resources and making the consumption of modem medicine the lowest in the
world (GOI, 2000).
There is tremendous increase in demand, production and sale of traditional medicinal
products in the last decade due to expanded global market (WHO, 2011). There is a large
demand of crudely prepared medicinal plants mainly in Europe and America which
usually distributes them as over-the-counter medications (Matu and Staden, 2003).
Medicinal plants make a valuable source of foreign exchange for a lot of developing
nations and the universal market for herbs and medicinal plants run into several billion
dollars per year. Countries such as Bulgaria, Germany, Poland and others in Africa and
Asia are renowned as main exporters of plant based medicinal products and raw materials
for overseas medicinal plant industries (Muhammad and Awaisu, 2008).
21
2.4.2 Medicinal plants in Africa
Herbal medicine has been used in Africa for hundreds of years even before colonization.
Currently it is still being used and about 80 % of the population relies on it for basic
health care services (Okigbo and Mmeka, 2008). Even though colonialists discouraged
the use of herbal medicine in Africa, it is still used (Orwa et al., 2008). Localized
treatment for different diseases have been used since time immemorial in rural Africa and
herbal medicines possessing antimicrobial activity have significant advantages in the
societies that greatly used them (Parker et aI., 2007).
The proportions of traditional healers to conventional doctors in relation to the population
of the whole of Africa are a testimony. A good example is Ghana where for every
traditional healer there are 224 people as compared to one conventional doctor for about
21,000 people. This is also manifested in Swaziland where the ratio is 110 people per
traditional healer and 10,000 people per conventional doctor (Rukangira, 2001). This is
partially attributed to the fact that some conventional medicines are not effective or
unable to treat some new diseases such as HIV /AIDS or have side effects (Kareru et al.,
2007). In addition, plants form an integral part of life in many indigenous African
communities as readily and cheaply available alternative to allopathic medicine (Wagete
et aI., 2010).
A variety of naturally occurnng products in Africa are exported to pharmaceutical
companies in the West yearly in exchange for quick cash and partly due to lack of
machinery to develop them locally (Adesina, 2005). Most of the countries in Africa rely
22
on medicinal plants. Malaysia is cited as among the countries with commendable work in
natural products use that should be copied by other countries (Muhammad and Awaisu,
2008). They have shown increased numbers of good quality herbal products in their
market as a result of collaborative work with the government, drug industries, and other
stakeholders that laid a strong base for the development of this sector (Muhammad and
Awaisu, 2008).
In Ethiopia, medicinal plants have been used to treat different diseases by the local
people since antiquity (Yinerger and Yewhalaw, 2007). Herbal medicine in South Africa
is highly esteemed in value and over 70 % of the populace heavily relies on traditional
health practitioners in matters of health care (Samie et al., 2005).
Over 400 plant species are approximated to be applied to the management of regular
diseases in East Africa (Mariita et al., 2010a). In Uganda, the ratio of traditional medical
practitioners is between 1:200 and 1:400 in comparison to the allopathic practitioners,
whose ratio is 1:20,000 or less. The distribution of personnel is however not directly
proportional, with a greater percentage in urban setups rendering them inaccessible to the
rural populace (WHO, 2002). Treatment of measles has been achieved by using
medicinal plants (Olila et al., 2001).
Over 60 % of the rural population in Tanzania depends on traditional medicine in solving
their health care problems (Moshi et al., 2009). Those residing in urban setups where
many modem health care facilities are available still go to traditional healers since the use
23
of herbal medicine still has a strong cultural influence (Moshi et aI., 2006). It is
approximated that there is between 30,000 - 40,000 traditional practitioners compared to
about 600 medical doctors in Tanzania. In Malawi the situation is worse with about
17,000 traditional practitioners to only 35 medical doctors (Rukangira, 2001).
2.4.3 Medicinal plants in Kenya
There are over 10,000 species of indigenous flora in Kenya, of which about 1,200 are of
medicinal value (ONIDO, 2004). Studies and further proofs have demonstrated that there
is overwhelming application of herbal medicine in Kenya since the conventional drugs
are expensive (Orwa et al., 2008; Kitonde et al., 2013). This has been documented by
ethnobotanical surveys (Kareru et al., 2008; Nanyingi et al., 2008, Njoroge et al., 2010;
Mariita et al., 2010b, Okemo et al., 2011). In Kenya, traditional medicine is used in the
treatment of diseases caused by bacteria and fungal pathogens (Kokwaro, 2009). Not only
is the usage extended to rural areas but also in urban regions as demonstrated by
increased activities of traditional practitioners all over the country (Orwa et al., 2008).
The demand for herbal products in Nairobi has seen shelves of large supermarkets, drugs
stores and health shops packed with herbal teas made from leaves of Artemisia, Ajuga
and Prunus among others (Dharani and Yenesew, 2010). There are a lot of inequalities in
health care service provision and those who live in urban areas enjoy easy access to
health facilities. About 70 % of urban population has health facilities within 4 km, unlike
their rural counterparts at 30 % of population (PKF and IRN, 2005).
24
Although the Government of Kenya has expanded conventional health care in the last 10
years, it is still not readily accessible and many regions are still totally underserved. This
has made most communities to continue using traditional medicine which is affordable
and available (Bussman, 2006). In rural Kenya, traditional healers are preferred over
conventional trained doctors. Studies have shown that for one conventional doctor there
are about 8,500 people while for a traditional herbalist, there are less than 1,000 people to
each herbalist (Dharani and Yenesew, 2010). Data from the Ministry of Gender, Sports,
Culture and Social Services reveals that the number of traditional medical practitioners
registering their commercial enterprises/herbal clinics (usually in the urban areas) is on
the increase. Further, the number of patients treated in these herbal clinics is also on the
increase, sometimes reaching well over 500 patients per month (Njoroge, 2012). Kenya
has a research programme in herbal medicine; The Traditional medicine and Drug
Research Centre of Kenya Medical Research Institute, Nairobi (UNIDO, 2004). In
addition to that, the department of Chemistry of the University of Nairobi isolates
compounds from herbal medicine and supplies them to the pharmaceutical companies
such as Merck (USA) and Jansen (Belgium) to test them for biological activity and
process drugs from them.
In Kenya, many communities rely on traditional medicine, for instance in Eastern
Province, Embu and Mbeere districts are equipped with a diversity of native herbal
medicine. The plants are used in treating several diseases by the local herbalists (Kareru
et al., 2008). The Kenyan pastoralists of Samburu origin have also long kept their
knowledge on the use of local plants for a diversity of purposes (Nanyingi et al., 2008).
25
The Samburu still depend on a diet of milk, blood from animals and soups made from
natural herbs, berries and other wild fruits. This culture has made herbal knowledge to
remain widespread in the community (Bussmann, 2006). The Nandi community also
relies on medicinal plants to treat various human and livestock ailments (Jeruto et al.,
2008). Further, the Kambas are well known as one of the tribes in Kenya that have
greatly preserved their knowledge of the use of local plants for medicinal purposes
(Njoroge et al., 2010). Among the Luos, medicinal plants are effective in managing
certain cultural problems which may not respond to conventional practices like Chiira- a
condition manifesting as a curse due to broken taboos among the Luo community
(Personal communication Osewe, 2011). The Kipsigis community in Kenya uses many
plants preparations in managing various ailments (Korir et al., 2012a).
2. 4. 4 Plants under investigation
2.4.4.1 Ficus sycomorus Linn. (Moraceae)
It is traditionally used for treating stomach pains (Samie et al., 2010). It is reported to
have activity against S. aureus, S. typhi and MRSA (Olusesan et al., 2010). It possesses
anti-diarheal activity (Ahmadu et al., 2007).
2.4.4.2 Balanites aegyptica L. Drel (Balantiaceae)
This plant has a long history of traditional uses for many diseases (Chothani and
Vaghasiya, 2011). It is one of the most common plants which are neglected (Chothani
and Vaghasiya, 2011). The boiled roots are used to treat malaria and stomach pains
(Yitebitu, 2004). Its stem bark has a high antifungal activity against C. albicans
(Maregesi et al., 2008).
26
2.4.4.3 Sesbania sesban Linn. (Papilionaceae)
The fresh roots and leaves are used to treat scorpion stings, boils and abscesses (Yitebitu,
2004). The leaves of this plant have been studied and moderately inhibited the growth of
E. coli and A. niger (Hossain et al., 2007).
2.4.4.4 Tamarindus indica L. (Fabaceae)
It is used in many cultures for a wide range of applications (Caluwe et al., 2010). Its
fruits were found to be active against A. niger and C. albicans (Caluwe et al., 2010).
While stem bark demonstrated activity on E. coli, S. typhi, S. aureus and B. subtilis
(Doughari, 2006). Tamarindus indica L. from Rarieda District has not been tested against
E. coli, S. typhi, S. aureus, B. subtilis, A. niger and C. albicans.
2.4.4.5Albizia coriaria Welw. ex Oliv (Fabaceae)
This plant is highly regarded in the Luo community (Maundu and Tengnas, 2005). The
leaves and stem bark are used for the treatment of cough (Olila et al., 2007; Namukube et
al., 2011). From previous studies, it was found to be active against B. subtilis and E. coli
but had no activity on S. aureus (Olila et al., 2007).
2.4.4.6 Ormocarpum trichocarpum Taub. Engl. (Fabaceae)
The roots are used to treat stroke paralysis, bilharzias and for bone setting (Moshi et al.,
2006). This plant has been studied on Giardia lamblia (Johns et al., 1995). However, the
antimicrobial properties of this plant on the tested microorganism have not been done.
27
2.4.4.7 Senna didymobotrya Fresen. (Caesalpinaceae)
The leaves of the plant are used in the management of STDs and back pain in Central
Kenya (Njoroge and Bussman, 2009) and as a remedy for ringworm infection (Tsegaye,
2006). The plant extracts are used in the management of hypertension, microbial
infections, hemorrhoids, sickle cell aneamia, inflammation of the fallopian tubes and
fibroids (Geisler et al., 2002; Kamatenesi, 2004). This plant has been tested against
Plasmodium falciparum (Ramalhete et al., 2008) and Entamoeba histolytica (Raini,
2011). Despite well spelt ethnomedical application, very little bioassay guided studies
have been conducted on the extractives from this plant.
2.4.4.8 Commiphora africana (A. Rich) Eng.Syn (Burseraceae)
The stems are used as toothbrushes by Rendile and Kamba communities (Maundu and
Tengnas, 2005). From previous studies, the roots showed activities against S. aureus, E.
coli and C. albicans (Akor and Anjorin, 2009). It exhibited activity as an antihelminthic
(Gbolade, 2008).
2.4.4.9 Psiadia punctualata (DC) Vatke (Compositeae)
Is used for relief of colds and abdominal pains (Kokwaro, 2009). It showed a significant
anti-leishmanial activity (Githinji et al., 2009). It mildly inhibited the coffee berry disease
fungus though it did not show any mosquito larvicidal activity (Midiwo et al., 2002).
Antimicrobial properties on the tested microorganism in this study have not been
determined.
28
2.4.4.10 Rhus natalensis Krauss (Anacardiaceae)
Traditionally, it is used in the treatment of coughs, colds, headache and back pain
(Gathirwa et al., 2011). The root extract is employed in the treatment of diarrhea by the
Luo community (Personal communication, Osewe, 2011). It has been found to possess
antiviral activity (Parker et al., 2007).
2.4.4.11 Ricinus communis Linn (Euphorbiceae)
It is traditionally used against headache, rheumatism, colic and acute diarrhea (Kota and
Manthiri, 2011). The leaf extracts are active against B. subtilis, E. coli and S. aureus
(Kota and Manthiri, 2011). The seed extract showed moderate activity on S. aureus and
E. coli. (Jumbo and Enenebeako, 2007).
2.4.4.12 Zanthoxylum chalyheum Engl. (Rutaceae)
It is used in the treatment of malaria and other infectious diseases (Olila et al., 2001). The
stem barks have shown moderate antibacterial activity while the leaf extracts showed no
antibacterial activity (Matu and Staden, 2003). Though this plant has been used as human
and veterinary medicine, there still remains a lot that is unknown and therefore more
research should be carried out (Olila et al., 2001).
2.4.4.13 Bosia angustifolia A. Rich (Capparaceae)
It is used in the treatment of bacterial diseases (Hassan et al., 2006). Its roots have been
found to be active against S. aureus, E. coli but inactive against S. typhi (Hassan et al.,
2006). It has shown a strong antibacterial activity against S. aureus (Mariita et al., 2011).
29
According to Omwenga et al. (2009) this plant showed weak activity against S. aureus
and B. subtilis.
2.4.4.14 Melia volkensii Gurke (Meliaceae)
Its leaf extracts have shown weak activity against Vibrio cholerae but were active against
E. coli (Akanga, 2008). Ethanolic extracts of its fruit have been found to be lethal against
Southern green stink bug (Mitchell et al., 2004).
2.4.4.15 Zanthoxylum gilletii De Wild (Rutaceae)
It has been found to be inactive against S. typhi, S. aureus, E. coli and C. albicans.
(Mariita et al., 201Oa). This plant is used in the treatment of genitourinary and rheumatic
troubles, severe coughs, colds, healing of the womb in women after giving birth, chest
pains, mouth ulcers and sore throats (Kokwaro, 2009; Tarns et al., 2006).
2.4.4.16 Fuerstia africana T.C.E. Fries (Lamiaceae)
Traditionally, the species has been used as a galactagogue, female fertility agent and a
treatment for tapeworms and urinary problems (Koch et al., 2006). It has been found to
be inactive against S. typhi, E. coli and C. albicans but showed moderate activity against
S. aureus (Mariita et al., 2010a). However, not much has been done on the antimicrobial
properties of this plant.
30
2.4.4.17 Urtica dioica Linn (Urticaceae)
It has a long history of use as medicine (Uzun et al., 2004). It is used for treating
anaemia, enlarged prostate and childlessness. Its leaves have been found to be very active
against S. aureus but it showed moderate inhibitory activity against E. coli and was
inactive against C. albicans (Demet et al., 2008).
2.4.4.18 Vernonia amygdalina Del. (Asteraceae)
It is used in the treatment of nausea, diabetes, loss of apetite, dysentery and other
gastrointestinal tract problems (Nwanjo, 2005). It has been found to be active against S.
aureus, E. coli and C. albicans (Okigbo and Mmeke, 2008). This is contrary to Mariita et
al. (2010a) and Cheruiyot et al. (2009) who reported that this plant did not have the
ability to inhibit E. coli and C. albicans.
2.5 Bacterial infections
Bacterial infection is on the increase all over the world (Oyewole et al., 2010) and
HIV/AIDS pandemic, poor hygiene, overcrowding and resistance to conventional
antimicrobials among other factors have led to increased prevalence of bacterial
infections (Wag ate et al., 2008). Infectious diseases mostly diarrhoea threaten lives of
millions of people around the world (Samie et al., 2005).
2.5.1 Escherichia coli
Escherichia coli belong to the large group of Gram negative bacteria referred to as
enterobacteria. It causes urinary tract infections, infections of wounds, sepsis, meningitis
31
and diarhoeal diseases (Cheesbrough, 2000). Although E. coli is a normal flora in the gut
and is naturally harmless, it may cause gastro-intestinal diseases ranging from mild, self-
limiting diarrhea to haemorrhagic colitis (Greenwood et al., 2002). Serotype 0157:H7 is
enterotoxigenic and is the most important pathogen that causes diarrhea in infants,
children and adults (Chen et al., 2009). It is estimated that 1.5 million children below 5
years die from diarrhoea each year. In addition to that more than 80% of child deaths in
Africa and Asia is because of diarrhea (UNICEF/WHO, 2009). In Kenya 27,400 children
die from diarrhea annually (UNICEF/WHO, 2009; Ndongwe, 2011). A study conducted
on surveillance of antimicrobial resistance at a tertiary hospital in Tanzania revealed that
E. coli had a high resistance to several antimicrobial agents such as ampicillin,
trimethoprim-sulfamethoxazole, ceftazidine, tetracycline and sulfonamide (Blomberg et
al., 2004).
2.5.2 Salmonella typhi
Salmonella typhi is a Gram negative bacteria that causes enteric fever (typhoid and
paratyphoid) and bacteraemia (Cheesbrough, 2000). The genus Salmonella has three
species; Salmonella typhi and Salmonella paratyphi which cause enteric fever and
Salmonella enteritidis which cause enteritis (Elliot et al., 2007). Conventional
antisalmonella drugs are becoming more and more unavailable to the common man in
Africa due to increased cost (Gatsing and Adoga, 2007). Moreover, high morbidity and
mortality caused by typhoid fever is increasing with the emergence and worldwide spread
of S. typhi resistance to the most commonly used antibiotics (WHO, 2007). Typhoid fever
is a public health problem in developing countries (Gatsing and Adoga, 2007). In 2000, it
32
is estimated that over 2.16 million episodes of typhoid occurred worldwide, resulting in
216,000 deaths (Bhuta et aI., 1999; Ochiai et aI., 2008). Typhoid is a common and
serious disease amongst children and adults in Kenya (Mweu and English, 2008).
Antibiotic resistant strains of S. typhi are increasing rapidly, including multi drug resistant
strains and those less sensitive to ciprofloxacin (Roeck, 2007) and are also resistant to
chloramphenicol and cotrimoxazole, the antibiotics commonly prescribed for the
treatment of typhoid fever (Oluyege et aI., 2009). The emergence and spread of drug
resistance have challenged treatment options for typhoid in many countries (Chalya et al.,
2012).
2.5.3 Staphylococcus aureus
They are Gram positive bacteria which form clusters when grown on solid media. The
most important is S. aureus which causes superficial infections and systemic infections
such as endocarditis inflammation of bone or bone marrow (Parry et aI., 2004;
Deurenberg and Stobberingh, 2008; Haque et al., 2011). S. aureus is an opportunistic
pathogen and a leading cause of hospital acquired infections (Klein, 2007; Haque et al.,
2011) and may also cause more serious infections such as ulcers, burns, pneumonia,
osteomyelitis, septicaemia, mastitis, meningitis, food poisoning and toxic skin exfoliation
(Cheesbrough, 2000). S. aureus is carried in the nose of 40% or more of healthy people,
in feaces in about 20% of the people and skin carriage in 5-10% of the population
(Cheesbrough, 2000; Elliot et aI., 2007).
33
Staphylococcus aureus has been successful in developing resistance to anti-microbial
agents. Beta-lactamase producing strains of S. aureus emerged immediately after
penicillin was introduced and now almost all isolates of S. aureus in hospitals are
resistant to penicillin (Klein, 2007; Bhatia, 2008). A study done by Daini and Akano
(2009) found out that 62% of S. aureus isolates had multiple resistance to the commonly
used antimicrobial agents. It has a remarkable genetic diversity and can acquire new
exogenous genes, which allow it to adapt to various changing environmental conditions
to modulate its pathogenicity (Moellering, 2010). Infections caused by S. aureus are
increasingly becoming difficult to control because the organism has acquired new
antimicrobial resistance determinants (Jumbo and Enenebeaku, 2007; Oyetunji et al.,
2012). This necessitates a continuous search for alternative medicine, preferably from
plants since plants are less toxic, readily available and cheap.
2.5.4 Methicillin resistant Staphylococcus aureus
These are strains of S. aureus which are resistant to the antibiotic methicillin. Many
strains of MRSA are now resistant to multiple antibiotics (Elliot et al., 2007; Oyetunji et
al., 2012) such as ~-lactam agents, aminoglycosides and fluoroquinolones (Greenwood et
al., 2002). Infections caused by MRSA have become a major problem worldwide (Cuny
et al., 2010) and the risk of transmission and diseases caused by MRSA are greatest
amongst hospital patients ( Bhatia, 2008). MRSA have emerged as the predominant cause
of infections in debilitated patients. For instance those in intensive care units, where the
combinations of many antibiotics and the use of invasive devices contribute greatly to the
risk of acquisition (Greenwood et al., 2002). MRSA can cause sepsis ranging from world
34
infections to urinary tract infections and septicaemia (Elliot et al., 2007). The measures
taken to control MRSA in hospitals may be different depending on its prevalence and the
particular clinical areas affected (Greenwood et al., 2002). Vancomycin is however used
for the management of MRSA infections (Cheesbrough, 2000).
2.5.5 Bacillus subtilis
The genus Bacillus are Gram positive, rod-shaped bacteria that form spores (Hong et al.,
2009) Bacillus subtilis and Bacillus pumilis have been implicated in causing food
poisoning similar to that due to Bacillus cereus (Greenwood, 2007). They do not form
toxins but some strains produce antibacterial peptides such as the antibiotic bacitracin,
which could cause growth in the internal tract (Greenwood, 2007). Increase in out breaks
of food-borne diseases has brought up concerns over pathogenic and spoilage micro-
organisms in foods (Rahman and Kang, 2009). B. subtilis sometimes causes disease in
immunocompromised humans such as meningitis, endocarditis, endophthalmitis,
conjunctivitis or acute gastroenteritis (Bonjar and Farrokhi, 2004). B. cereus and B.
subtilis may be found in wounds and tissues of immunocompromised or burned patients
(Greenwood, 2007).
2.6 Fungal infections
Over the past few decades, fungal infections have increased rapidly (Yigit et al., 2008)
and this has been brought about by increased prevalence of immunocompromised
populations such as organ transplant recipients, cancer and HIV/AIDS patients (Garcia et
al., 2003; Abad et al., 2007). Fungal infection is one of the greatest killers of the
35
irnmunocompromised populations (Moyes and Naglik, 2011). Available literature
indicates that up to 90% of all HIV patients suffer from fungal infections at some point
during the course of the disease, of which 10-20% dies as a result of fungal infections
(Runyoro et aI., 2006; Samie et aI., 2010).
Many plants have been used by herbalists and elders in the treatment of several fungal
infections (Hamza et al., 2010). The plants which are traditionally used should be tested
for their fungal activities and drug developed from them since the fungal strains have
developed resistance to antifungal drugs which are also expensive (Abad et al., 2007;
Koroishi et aI., 2008).
2.6.1 Dermatophytes
These are a group of related filamentous fungi, also referred to as ringworm fungi, which
cause infection of the skin, hair and nails. They cause athlete's foot (Tinea pedis),
ringworm (Tinea capitis) and jock itch (Tinea cruris) (Greenwood et aI., 2002; Koroishi
et aI., 2008). They are mainly caused by three genera of fungi namely Trichophyton,
Microsporum and Epidermotophyton (Greenwood et al., 2002; Elliott et al., 2007).
The dermatophytes infect the hair, skin and nails due to their ability to obtain nutrients
from keratin for growth. Imrnunocompromised patients can be infected with more severe
and extensive lesions (Galgoczy et al., 2008). Dermatophytes cause a high morbidity in
patients undergoing organ transplants or suffering from AIDS (Brandao et al., 2010).
Dermatophytosis is a trivial disease but has a lot of psychological effect and a costly
36
disease in terms of treatment (Sai et al., 2012). Topical treatment with c1otrimazole and
terbinafine are commonly used for localized lesions. In widespread infections of Tinea
unguium and scalp ringworm, complete antifungal treatment with oral drugs such as
itraconazole, terbinafine and griseofulvin are necessary for systemic therapy (Galcoczy
et al., 2008, Koroishi et al., 2008).
2.6.2 Candida albicans
It occurs in the mouths of up to 80% of healthy individuals and is mostly associated with
normal oral flora (Williams et al., 2011). Candida are the most common fungal micro-
organism of humans and the causal agents of oral, gastrointestinal and vaginal
candidiasis, causing severe morbidity in millions of persons worldwide (Lazzell et al.,
2009; Moyes and Naglik, 2011). Clinically significant risk factors for individuals to
acquire Candida infections include: compromised immunity, over use of antibiotics, use
of central venous catheters, prolonged hospital stay and tumors (Fleming et al., 2011).
For instance oral candidiasis mostly occurs after over using a large variety of antibiotics
which facilitates later growth of Candida through lessening competitive pressure by
lowering the oral bacterial population (Williams et al., 2011).
Candida are the third leading cause of central venous catheters related infections with the
second highest colonization to infection rate and the overall highest crude mortality
(Lazzell et al., 2009). Candida vaginitis is a common infection during pregnancy
(Cheesbrough, 2000). Vaginal candidiasis alone affects about 75% of women at least
once during their reproductive age which is about 30 million infection episodes annually
(Moyes and Naglik, 2011). Candida infection are also the most common oral symptoms
37
of HIV infection, with half of HIV positive patients and 90% AIDS patients suffering
from oral candidiasis (Moyes and Naglik, 2011). Oral candidiasis is the first and most
frequent fungal infection in HIV patients (Hamza et al., 2006; Runyoro et aI., 2006).
Candida albicans is already resistant to the limited toxic and expensive anticandida drugs
available in the market (Runyoro et aI., 2006). Topical antifungal agents such as nystatin,
miconazole, fluconazole, intraconazole and amphotericin B, are commonly used in
treating oral candidiasis. However, a number of challenges such as a limited number of
effective antifungal agents affect the management of Candida infections (Runyoro et aI.,
2006).
2.6.3 Cryptococcus neoformans
Cryptococcus neoformans is responsible for about 1 million infections annually and over
600,000 deaths worldwide (Mansour et aI., 2011). The infection is transmitted by
inhalation of the fungus from soil which is contaminated with bird droppings (Elliott et
aI., 2007). It usually affects the lungs, brain and meninges and occasionally other parts of
the body (Cheesbrough, 2000). Those at highest risk of developing cryptococcal
infections are. individuals with AIDS or lymphoma or recipients of chronic
immunosuppressive medication (Barchiesi et aI., 2000; Mansour et al., 2011).
Candida albicans and, C. neoformans are the most common opportunistic infections in
HIV/AIDS patients (Samie et aI., 2010). It causes meningoencephalitis mainly in
immunocompromised host which is the most distressing symptom of cryptococcal
38
disease and is fatal unless treated (Florio et al., 2011). In resource limited settings,
cryptococcal meningitis can cause up to 30% mortality in AIDS patients (Lin, 2009). A
report by CDC found cryptococcal meningitis to have killed about 624,000 people
annually (Lin, 2009). Amphotericin B and triazoles are consecutively used to treat
cryptococcal infections (Barchiesi et a/., 2000).
2.6.4 Aspergillus niger
Aspergillus spp. have been found to be the cause of food borne diseases or food spoilage
(Pirbalouti et a/., 2010). One of the most important problems facing the food industries is
food spoilage, infact developing and developed counties also find food-borne illnesses as
a global problem (Rahman and Kang, 2009). Food -borne micro-organisms like A. niger
exist freely in nature causing a lot of deaths and sickness in the population and can also
be very dangerous agent to man as it can cause opportunistic infections (Sumathy et al.,
2010). For example in HIV-infected individuals, aspergillosis is caused by A. jlavus, A.
niger and A. fumigates (Runyoro et al., 2006).
2.7 Phytochemicals in medicinal plants
Phytoconstituents are the natural bioactive compounds found in plants. They work with
nutrients and fibres to form an integrated part of defense system against various diseases
and stress conditions (Koche et al., 2010). Phytochemicals are majorly divided into
primary and secondary groups according to their activity in plants metabolism. Primary
groups contain common sugar, amino acids, proteins and chlorophyll. While secondary
groups are made of alkaloids, flavonoids, saponins, tannins, phenolic compounds and
39
many others (Khan et a/., 2011). Secondary metabolites of plants act as defense
mechanisms against infestation by many microorganisms, insects and herbivores (Koche
et al., 2010). The medicinal value of plants. depend on the bioactive phytochemical
constituents which are found in parts of the plants. They give a definite physiological
action on human body (Chletri et al., 2008; Koche et al., 2010; Sule et al., 2011).
The most important of these bioactive compounds of plants are alkaloids, tannins and
phenolic compounds (Chletri et al., 2008). Phytochemical screening of plants has shown
the presence of various chemicals such as alkaloids, tannins, flavonoids, steroids,
glycosides and saponins (Koche et al., 2010) which are well known for their laxative and
pharmacological effects on humans and animals (Sule et al., 2011).
2.7.1 Tannins
Tannins are heterogeneous groups of high molecular weight phenolic compounds with
the capacity to form reversible and irreversible complexes with protein, polysaccharides
(cellulose, hemicelluloses, pectin) alkaloids and nucleic acids (Frutos et al., 2004).
Tannins are generally more common in parts of the plant that are considered precious to
it, for example new leaves and flowers which have high chances of being eaten by
herbivores (Frutos et al., 2004). Tannin content of plants is increased by high
temperature, poor soil quality, water stress and extreme light intesities (Frutos et al.,
2004). Tannin is known for many physiological activities such as stimulation of
phagocytic cells, host mediated tumor activity and various anti-infective actions (Cowan,
1999).
40
2.7.2 Saponins
Saponins are glycosides that are found commonly in plants. They are in large quantities
in many foods eaten by animals and man (Soetan et al., 2006). Saponin serve as lytic
agent because it has detergent properties and are well known to show anti-inflammatory
properties, while alkaloids and glycosides act as defense mechanism of the plant (Shital
et al., 2010). Saponins have been used commercially for various functions such as
medicine, detergents, adjuvants and cosmetics (Eskander et al., 2006). A lot of
pharmacological activities such as antibiotic, antifungal, antiviral, hepatoprotective, anti-
inflammatory and antiulcer have been reported on saponins (Soetan et al., 2006).
2.7.3 Flavonoids
Flavonoids are a large family of polyphenolic components produced by plants. Studies
have shown that flavonoids help reduce blood lipid and glucose and enhance human
immunity (Ghasernzadeh et ai., 2011). They also have various biological activities such
as antibacterial, antidiabetic, antiallergic, antiviral, antinflammatory, antimutagenic,
anticarcinogenic and are insecticidal (Orhan et al., 2010). Plants synthesise flavonoids
as they respond to microbial infection (Cowan, 1999; Bii et al., 2010).
2.7.4 Alkaloids
Alkaloid is derived from the Arabic word al-qali, which means the plant from which soda
was first obtained (Kutchan, 1995). Alkaloids are one of the largest groups of
phytochemicals in plants and are known to be toxic against cells of foreign organisms
(Issazadeh et al., 2012). Majority of natural products are physiologically active in
mammals. Among the monoterpenoid indole alkaloid pharmaceuticals that are still
41
commercially isolated from plant material are the antimalarial drug qumme, the
antineoplastic drug camptothecin, the rat poison and homeopathic drug strychnine and the
antineoplastic chemotherapeutic agents, vincristine and vinblastine (Kutchan, 1995).
Alkaloids have shown antimicrobial activity against Giardia and entomoeba species.
They also possess antidiarrheal effect (Cowan, 1999).
2.7.5 Terpenoids
Terpenoids are synthesized from acetate units and they share their origin with fatty acids.
Examples of common terpenoids are methanol and camphor (monoterpenes), famesol
and artemisin (sesquiterpenoids). They are known to be active against fungi, bacteria,
protozoa and viruses (Cowan, 1999).
42
CHAPTER THREE
MATERIALS AND METHODS
3.1 Ethnobotanical survey
A survey was carried out in Katse ( Mwingi North) Iyabe and Kerina (Kisii South),
Lweya and Ragengni Divisions in Rarieda Districts, Kenya (Figure 1) on the major
medicinal plants used by communities inthe treatment of bacterial and fungal diseases.
Selection of the plants was based on available ethnobotanical information from the
herbalists consulted as well as available literature. In order for the survey to obtain
indigenous knowledge on plants used, interviews were conducted using a semi-structured
questionnaire (Appendix 2). Informants who are knowledgeable practitioners (herbalists)
were identified with the help of local people and the local administration and selected as
respondents to the survey (Yinerger and Yewhalaw, 2007).
Before carrying out the interview however, a written consent was sought from every
informant (Kasolo et al., 2010) appendix 1. Oral interviews were held in their homes. In
the survey, the respondents were asked for the local names of the plants they used as
medicine, parts used, target diseases, mode of preparation, administration of the resulting
preparations and the plants availability.
Species choice value model was preferred because it is a percentage of all the informants
who cite a number of species for a particular category that is used in identifying the most
popularly used medicinal plant species (Yinerger and Yewhalaw, 2007). Therefore; each
prescription was considered authentic only after confirmation from three informants.
43
3.2 Collection and authentication of plant material
In this study, eighteen different medicinal plants which include; Zanthoxylum chalybeum
Engl, Boscia angustifolia A. Rich, Melia volkensii Gurke, Zanthoxylum gilletii De Wild,
Fuerstia africana T. C. E Fries (Figure 5), Urtica dioica Linn (Figure 6)" Vernonia
amygdalina Del, Ricinus communis Linn (Figure 3), Commiphora africana (A. Rich)
Eng. Syn, Psidia puntulata (DC) Vatke, Senna didymobotrya Fresen (Figure 7),
Ormocarpum trichocarpum Taub. Engl, Sesbania sesban Linn (Figure 4), Balanites
aegyptiaca L. Drel, Albizia coriaria Welw. ex Oliv, Ficus sycomorus Linn, Rhus
natalensis Krauss and Tamarindus indica L. were collected from Kisii, Rarieda and
Mwingi Counties. The plants collected were those for treatment of gastroenteritis,
respiratory system illnesses as well as skin conditions. Fresh parts of the plant like the
roots, leaves and barks were collected depending on the most commonly used by the
traditional healers. Photographs of the selected plant species were taken. Plant
identification was carried out by a plant taxonomist from Botany Department, Chiromo,
University of Nairobi, Kenya. Voucher specimens collected were prepared and deposited
at the University of Nairobi herbarium.
44
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Figure 1: Map of Kenya showing Kisii, Banda and Mwingi Districts.
45
3.3 Preparation of plant materials
The freshly collected parts of the plant were washed thoroughly with running tap water,
chopped into smaller pieces and then dried under shade at room temperature for a period
of two weeks until they were completely dry (Matu et al., 2012). The small pieces were
ground into powder using Wiley mill, (model no. 2, USA) at Botany Department,
Chiromo University of Nairobi.
3.4 Extraction
The extraction was carried out at the Chemistry Department Chiromo, University of
Nairobi. One hundred and fifty grams of each dry powder was mixed in a conical flask
with 1000 ml of methanol and dichlomethane (1: 1) mixture and left over night (Kitonde
et al., 2013; Midiwo, 2010). The extracts were then filtered through a whatmann filter
paper No.1.
3.5 Concentration into solid sample
Dry organic crude extracts were obtained after evaporating dichloromethane and
methanol using a rotary evaporator (Buchii B-205 Switzerland) in a water bath set at a
temperature of 40°C (Omwenga et al., 2009). The paste was collected into small vials
which were then placed in an evaquated decicator filled with anhydrous copper sulphate
to obtain dry powdered samples. All samples were stored in the refrigerator at a
temperature of 4°C if not bio-assayed immediately.
46
3.6 Microbial test organisms
A selection of microbial test organisms was based on the ethnobotanical information
collected on the target disease, their significance as opportunistic pathogens and their
resistance to conventional drugs. The chosen microorganisms also cause infectious
diseases which are common to man and are easily transmitted. The standard reference
microbial organisms and clinical isolates were obtained from the centre for Microbiology
Research - KEMRI, Nairobi, Kenya. Standard reference microbial organisms,
environmental microorganisms and clinical isolates used in this study were:
3.6.1 Bacterial isolates
Gram positive
• Staphylococcus aureus - ATCC 25923
• Methiciline resistant Staphylococcus aureus (MRSA) - Clinical isolates
• Bacillus subtilis - Environmental organism
Gram negative
Escherichia coli - ATCC 25922
Salmonella typhi -ATCC 19430
3.6.2 Fungal isolates
Yeast
Candida albicans - ATCC 90028
Cryptococcus neoformans - ATCC 18310
47
Dermatophytes
Microsporum gypseum - Clinical isolates
Trychophyton mentagrophytes - Clinical isolates
Filamentous fungi
Aspergillus niger - Environmental organism
3.7 Maintenance of microbial stock cultures
Stocked bacterial strains were subcultured on Muller Hinton agar no. CM0337. (Oxoid
Ltd, Basingstoke, Hampshire, England). Incubation was carried out at a temperature of37
°C for 24 h to obtain freshly growing strains (Omwenga et al., 2009). Yeasts and molds
were subculcultured onto Sabaraud Dextrose Agar No. CM 004 (Oxoid ltd Basingstoke,
Hampshire, England). The Yeasts were incubated at a temperature of 37°C for 24 h
(Cruz et al., 2007), filamentous fungus at 28°C for 48 h in humid chamber (Costa et al.,
2010) and the dermatophytes at a temperature of 25°C for 72 h (Korir et al., 2012b).
Both the bacterial and fungal strains were maintained at a temperature of 4°C.
3.8 Antibacterial assays
The antibacterial bioassay was performed by Kirby Bauer disk diffusion method (Omori
et al., 2012). Muller Hinton agar no. CM0337 (Oxoid Ltd, Basingstoke, Hampshire,
England) was prepared according to the manufacturers instructions for the purposes of
culturing bacteria. Normal saline solution was used to dilute fresh 24 h culture of
bacterial type cultures or clinical isolates to attain a 0.5 McFarland Standard which gives
an equivalent approximate density of 1x 108 of bacteria (Kitonde et al., 2013). Spread
48
plate method was used to culture 100 III of the bacterial suspension that was introduced
into the' petri dishes. Eighteen dry sterile discs (6 mm diameter) were soaked in 100 III
plant extract (made by dissolving 300 mg of each extract in 1000 III (lml) of DMSO).
The discs were air dried and placed aseptically onto the inoculated plates at a distance of
3mm apart. Discs .impregnated with DMSO and then air dried were used as negative
controls while commercially available discs of chloramphenical were used as positive
control for bacteria. Incubation was carried out at a temperature of 37°C for 24 h. All
tests were performed in triplicate. After incubation, bacterial growth inhibition was
determined by measuring the diameter zones in millimeters using a transparent ruler and
recorded against the corresponding plant extracts (Omwenga et al., 2009; Mariita et al.,
20 lOa).
3.9 Antifungal assays
To determine the antifungal activities of the plant extracts against fungal strains,
Sabourand Dextrose Agar no. CM 004 (Oxoid Ltd, Basingstoke, Hampshire, England)
was prepared using manufacturer's instructions for the purpose of culturing fungi.
Normal saline solution was used to dilute fresh 24 h culture of fungal type cultures or
clinical isolates to attain a 0.5 McFarland Standard. Spread plate method was used to
culture 100 III of the fungal suspension that was introduced into the petri dishes.
Eighteen dry sterile discs (6 mm diameter) were soaked in 100 III plant extract (made by
dissolving 300 mg of each extract in 1000 III (1 ml) of DMSO). The discs were air dried
and placed aseptically into the inoculated plates at a distance of 3 mm apart. Discs
impregnated with DMSO and then air dried were used as negative controls while
49
commercially available discs of miconazole were used as positive control (Bii et al.,
2010). The inocula were incubated at respective conditions where the yeast cultures were
incubated at a temperature of 37°C for 24 h (Cruz et al., 2007) filamentous fungi at a
temperature of 28°C for 48 h in humid chambers (Costa et al., 20 I0) and dermatophytes
at a temperature of25 "C for 72 h in humid chambers (Korir et al., 2012b). All tests were
performed in triplicate. After incubation, fungal growth inhibition was determined by
measuring the diameter zones in millimeters using a transparent ruler and recorded
against the corresponding plant extracts (Matu et al ., 2012).
3.10Determination of minimum inhibitory concentration
Plant extracts were made by dissolving 300 mg of each crude extract in 1000 III (Iml) of
DMSO. Broth micro dilution method was used to determine minimum inhibitory
concentration for the active crude extracts against the test microorganisms according to
the National Committee for Clinical Laboratory Standards (NCCLS) now Clinical
Standard Institute (eLSI) (Korir et al., 2012b).
The tests were performed in 96 well micro-titer plates. Serial doubling dilutions were
carried out so that the concentration in the next well was half of the concentration in the
previous well. The MIC was determined only where the plant extract showed strong
antimicrobial activity (~ 9 mm) by the disc diffusion method (Mariita et al., 20l0a). The
wells were filled with 50 III of the Muller Hinton broth for bacterial strains and
Sabourand Dextrose broth for fungi. Then, 50 III of the plant extract (made by dissolving
300 mg of each extract in 1000 III (lml) of DMSO for complete dissolution) were
50
dispensed into the first well before serial dilutions. The serial dilutions were achieved by
transferring 50 III of Muller Hinton broth or Sabourand Dextrose broth containing the
extract from the first well through the second, third and fourth wells. Then 50 III of the
test isolates were dispensed into each well. One row of wells was used as negative control
of the growth of the microorganisms in the medium, whereas 50 III of the antibiotic
(Chloramphenicallmiconazole) were used as a positive control. Micro titre plates were
covered. Bacteria and yeasts were incubated at 37°C for 24 h (Kitonde et al., 2013)
filamentous fungi at 28°C for 48 h in humid chambers (Costa et al., 2010) and
derrnatophytes at 25°C for 72 h in humid chambers (Korir et al., 2012b). Minimum
inhibitory Concentrations (MIC) were determined by recording the lowest concentration
of the active extracts that inhibited growth of micro-organisms as compared to the control
broth turbidity (Wagete et a!., 2010; Kitonde et al., 2013).
3.11 Determination of Minimum Bactericidal/Fungicidal Concentrations (MBCs/
MFCs)
The wells where there were no growth (not turbid) from MIC results, the bacteria were
subcultured on Mueller Hinton Agar and fungi on Sabourand Dextrose Agar. The
bacterial and yeast cultures were incubated at 37°C for 24 h and the dermatophytes at a
temperature of 25°C for 72 h (Korir et al., 20 12b). The lowest concentration of the plant
extracts that did not yield any colony on the solid medium after sub culturing and
incubating for 24 h for bacteria was taken as the MBC and 72 h was taken as the MFC
for derrnatophytes and 24 h for yeasts. All tests were performed in triplicate (Samie et a!.,
2010; Omori et al., 2012).
51
3.12 Phytochemical screening
3.12.1 Test for alkaloids
Two hundred milligrams of plant extract were dissolved in 10 ml methanol and filtered, 1
ml of the filtrate was mixed with 6 drops of Wagner's reagent (made by mixing 1.27 g
iodine and 2 g potassium iodide in 100 ml of water). A creamishl brownish-red/orange
precipitate indicated the presence of alkaloids. A low (+) concentration was recorded if
the addition of the reagent produced a faint turbidity, a moderate (++) concentration was
recorded if a light opalescence precipitate was observed and a high (+++) concentration
was recorded if a strong yellowish-white precipitate was observed (Omwenga et al.,
2009; Mariita et aI., 2011).
3.12.2 Cardiac glycosides
Five milligrams of the filtrate was treated with 2 ml of glacial acetic acid containing two
drops of ferric chloride solution. This was underlayed with 1 ml of concentrated sulphuric
acid. Green-blue colour indicated the presence of cardiac glycosides. A (+) reaction was
recorded when a faint green-blue colour was observed (indicating low concentrations of
detectable cardiac glycosides); A (++) reaction was recorded when a medium green-blue
colour was observed (indicating moderate concentrations of detectable cardiac
glycosides) and A (+++) reaction was recorded when a deep green-blue colour was
observed indicating high concentrations of detectable cardiac glycosides (Edeoga et al.,
2005; Omwenga et aI., 2009; Mariita et al., 2011).
52
3.12.3 Test for saponins
Zero point five milligrams of extract was added to a 5 ml of distilled water in a test tube.
The solution was shaken vigorously and observed for a stable persistent froth. Low (+)
saponins concentration was recorded when the froth reached a height of 50 mm, medium
(++) for the froth between 60-100 mm while high (+++) was represented by more than
IOOmm.
3.12.4 Test for tannins
Zero point five milligrams of the extract was boiled in 10 ml of water in a test tube and
then filtered. A few drops of 0.1% ferric chloride were added and observed for brownish
green or a blue black colouration. Heavy precipitate indicated a high concentration of
tannins (+++) , medium precipitate (++) for medium concentration while a slight
precipitate indicated a low concentration (+) (Mariita et al., 2011).
3.12.5 Flavonoids
Five milliliters of dilute ammonia solution were added to a portion of the aqueous filtrate
of each plant extract followed by addition of concentrated sulphuric acid. The yellow
colouration which disappeared steadily indicated the presence of flavanoids. By
observing a strong yellow colouration a high (+++) concentration was recorded, moderate
yellow indicated moderate (++) concentration while a pale yellow colour indicated a low
(+) concentration of flavanoids (Edeoga et al., 2005; Omwenga et al., 2009; Mariita et
al., 2011).
53
3.12.6 Test for terpenoids
Zero point five milligrams each of the extract was added to 2 ml of chloroform and 3 ml
of concentrated sulphuric acid was then added carefully to form a layer. The presence of
terpenoids was indicated by the formation of a reddish brown colouration of the interface.
3.13 Statistical analysis
Minitab Statistical Software 13.20, 2000 was used in analyzing the data. The data
analyzed were the average zones of inhibition values for each test cultures obtained from
the antibacterial and antifungal assays expressed as mean standard deviation means. One
way ANOV A at 95% CI was used in testing the significance among the groups. A
probability value of :s 0.05 was considered significant as Tukey's test was used in
analyzing significant differences among group means (Mariita et aI., 201Oa).
54
CHAPTER FOUR
RESULTS
4.1 Ethnobotanical survey
The results of ethnobotanical survey are presented in Table 1, in which the plants are
arranged in alphabetic synopsis according to families. The local names, parts used, drug
preparation methods, diseases treated and the areas where the plants were collected from
are also presented in table 1.
In this study a total of 18 species distributed in 15 families were identified. The family
reported with the highest number of medicinal plant species was Fabaceae (3 species),
this was followed by Rutaceae (2 species). Capparidaceae, Meliaceae, Lamiaceae,
Urticaceae, Asteracea, Moraceae, Balantiaceae, Papilionaceae, Caesalpinaceae,
Burseraceae, Compositae, Anacardiaceae and Euphobiaceae had one species each.
Eleven plants were collected from Lweya and Ragengni (Rarieda), 4 plants from Kerina
and Iyabe (Kisii South) and 3 plants from Mwingi North. Various parts were harvested
depending on the parts the communities prefer to use in the treatment of various ailments.
The most frequently used preparations for administration were concoctions and
decoctions. Only 2 plants were preferred by infusion.
55
The leaves (9 plants) were the most frequently used parts of the plant, followed by the
bark (5 plants) then roots (4 plants). The diseases that were most frequently treated using
herbal medicines are gastrointestinal (37%), respiratory (31%), other ailment (18%), skin
infection (6%) genital urinary tract (4%) and wounds (4%) (Figure 2).
Table 1: Selected medicinal plants used by the communities from Mwingi North, Kisii
South and Rarieda Counties in treating various bacterial and fungal ailments
Botanical Family name Local name Parts Methods Diseases Area collected
and Voucher treated
Name number used from
Rhus Anacardiaceae Sangla Roots decoction Coughs, colds, Lweya
lI11talensis 2011111018 (Luo) headache, (Rarieda)
Krauss
diarrhea
Vernonia Asteracea Omosabakwa leaves concoction Loss of appetite Kerina
amygdalina
2011111015 (Kisii) Gastro intestinal (Kisii South)Del.
Problems,
diarrhea
Balanites Balantiaceae Othoo bark decoction Stomach pains, Ragengni
aegyptiaca . L.
Drel. 2011IIJ05 (Luo) Dysentery (Rarieda)
Commiphora Burseraceae Arupiny bark decoction Diarrhea Lweya
africana (A. 2011IIJ02 (Luo) Fever (Rarieda)Rich) Engl.
Syn
Senna Caesalpinaceae Owinu(Luo) roots decoction Diarrhea Ragengni
didymobotrya 201111104 Ringworm (Rarieda)
Fresen
56
Boscia Capparidaceae Mwenzenze leaves concoction Chest pains Mwingi
angustt ifo li a
2011IIJ017 (Kamba) Stomachache North
A.Rich
Psidia Compositae Atilili roots decoction Stomachache Lweya
puntulata
2011IIJ03 (Luo) Colds, diarrhea . (Rarieda)(DC)Vatke
Ricinus Euphorbiaceae Odagwa leaves concoction Stomachache, Lweya
communis 201llIJOl (Luo) diarrhea, pains (Rarieda)
Linn.
Tamarindus Fabaceae Chwa Bark decoction Diarrhea,cough Ragengni
indica L.
20lllIJ010 (Luo) Fever, tonsils (Rarieda)
Albizia Fabaceae Ober Bark decoction Cough, diarrhea Lweya
coriaria 201llIJ08 (Luo) (Rarieda)
Welw.ex
Oliv.
Ormocarpum Fabac.eae Det leaves concoction Diarrhea, Lweya
trichocarpum
201111105 (Luo)
typhoid
(Rarieda)raub.Engl.
Fuerstia Lamiaceae Ekebunga leaves concoction Urinary Kerina
africana
2011111011 Baiseke
problems,
(Kisii South)T.C.EFries
(Kisii)
Tongue
infections,
diarrhea,
Skin infections
Melia Meliaceae Mukau Leaves concoction Pains In the Mwingi
volkensii 2011111016 (Kamba)
body
North
Gurke
Ficus Moraceae Ngowo(Luo) Bark decoction Stomach pains, Lweya
sycomorus
57
Linn. 201111109 Coughs, (Rarieda)
wounds
Sesbania PapiI ionaceae Oyieko(Luo) Roots decoction Diarrheal Ragengni
sesbanLinn. 201111106 Diseases, boils (Rarieda)
Zanthoxylum Rutaceae Mukenea Leaves concoction Diarrhea, Sore Mwingi
chalybeum
2011111013 (Kamba)
throat, coughs, NorthEngl. Chest pain
Zanthoxylum Rutaceae Egekoma Leaves Infusion Genitourinary, Kerina
coughs, mouthgillettii De 2011111014 (Kisii) ulcers, throat (Kisii South)
Wild
Urtica Urticaceae Rise (Kisii) Leaves concoction Blood Iyabe
dioica Linn. 2011111012 purification, ( Kisii South)
Anaemia, boils
58
Key
• Respiratory
• Gastrointestinal
• Genital urinal tract
• Skin
.WOlUlds
• Other ailments
Figure 2: Percentage frequency of diseases treated using herbal drugs in Mwingi North,
Kisii South and Rarieda Districts.
Plate 1. Ricinus communis Plate 2. Sesbania sesban
Plate 3. Fuerstia africana
Plate 5. Senna didymobotrya
59
Plate 4. Urtica dioica
60
4.2 Antibacterial activity of the plant crude extracts against standard strains of
bacteria
The results indicated that all the plant extracts tested had bacterial inhibitory effects.
However, the inhibition of bacteria by the plant extracts varied with different plants. The
zones of inhibition between 7 and 10 mm were reffered to as low/weak activity, between
11and 14 moderate activity and between 15 and 21 high! strong activity.
The plant extracts significantly inhibited the growth of B. subtilis and S. aureus ATCC
25922 compared to other strains of bacteria (Table 2). A total of five plant extracts had
high activity against B. subtilis. These include; Fuerstia africana T. C. E Fries (17.0
mm), Senna didymobotrya Fresen (16.0 mm) (Plate 6), Ormocarpum trichocarpum Taub.
Engl (15.5 mm), Zanthoxylum chalybeum Engl (15.0 mm) and Tamarindus indica L.
(15.0 mm), while nine plant extracts had a moderate activity of between 11 and 13
against B. subtilis, Ricinus communis Linn (13.0 mm) (Plate 1), Boscia angustifolia A.
Rich (13.0 mm), Albizia coriaria Welw. ex Oliv (12.0 mm), Balanites aegyptiaca L. Drel
(12.00 mm), Commiphora africana (A. Rich) Engl. Syn (12.0 mm), Rhus natalensis
Krauss (11.5 mm), Sesbania sesban Linn (11.00 mm), Psidia puntulata (DC) Vatke (11)
and Zanthoxylum chalybeum Engl (11.0 mm). The remaining four plant extracts showed
weak activity against B. subtilis. Melia volkensii Gurke, Ficus sycomorus Linn, Urtica
dioica Linn and Vernonia amygdalina Del produced a weak antibacterial activity of 10.0
mm, 9.5 mm, 8.0 mm and 7.0 mm respectively. The zones of inhibition were significantly
different (P < 0.05) (Table 2 and Apendix 7).
61
For the case of the S. aureus ATCC 25923, Fuerstia africana T. C. E Fries, Senna
didymobotrya Fresen, Tamarindus indica L. (Plate 8), Balanites aegyptiaca L. Drel,
Psidia puntulata (DC) Vatke, Rhus natalensis Krauss and Ficus sycomorus Linn gave
antibacterial activity of 19.0 mm, 16.0 mm, 14.5 mm, 14.0 mm and 11.5 mm respectively
(Table 2). On the other hand, six plants produced low activity against S. aureus ATCC
25923 with a zone of inhibition ranging betw een 7.5 -10mm. For example, Albizia
coriaria Welw. ex Oliv (10.0 mm), Commiphora africana (A. Rich) Engl. Syn (9.5 mm),
Ormocarpum trichocarpum Taub. Engl. (8.5 mm), Sesbania sesban Linn (8.0mm),
Zanthoxylum gilletii De Wild (8.0 mm) and Urtica dioica Linn (7.5 mm). All the
remaining plant extracts were completely inactive (6.0 mm). The zones of inhibition were
significantly different (P < 0.05) (Table 2 and Appendix 4).
Fuerstia africana T. C. E Fries is the only plant that showed strong activity with a zone
of inhibition of20.0 mm against Methicillin resistant Staphylococcus aureus (Table 2 and
Plate 7). Albizia coriaria Welw. ex Oliv, Commiphora africana (A. Rich) Engl. Syn and
Senna didymobotrya Fresen produced moderate activity of 13.0 mm, 11.5 mm and 11.0
mrn respectively while Vernonia amygdalina Del and Zanthoxylum gilletii De Wild gave
a low antibacterial activity of 9.00 mm. All the remaining 11 plant extracts were
completely inactive (6.0 mm) against this test organism. The zones of inhibition were
significantly different (P < 0.05) (Table 2 and Appendix 3). In reference to Table 2, all of
the plant extracts did not show any activity against E. coli ATCC 25922 and S. typhi
ATCC 19430. (Appendix 5 and 6).
62
Table 2: Zones of inhibition produced by the plant extracts against the selected bacterial
strains in mm
Medicinal plants MRSA S. aureus Salmonella E. coli B. subtilis
Boscia angustifolia 6.0a 6.0a 6.0a 6.0a 13.0b
Fuerstia africana 20.0d 19.0c 6.0a 6.0a 17.0c
Melia volkensii 6.0a 6.0a 6.0a 6.0a 10.0b
Urtica dioica 6.0a 7.5a 6.0a 6.0a 8.0a
Vernonia amygdalina 9.0b 6.0a 6.0a 6.0a 7.0a
Zanthoxylum chalybeum 6.0a 6.0a 6.0a 6.0a 15.0c
Zanthoxylum gilletti 9.0b 8.0a 6.0a 6.0a 11.0b
Albizia coriaria 13.0c 10.0b 6.0a 6.0a 12.0b
Balanites aegyptiaca 6.0a 14.5c 6.0a 6.0a 12.0b
Commiphora africana 11.5b 9.5a 6.0a 6.0a 12.0b
Ficus sycomorus 8.5b 11.5c 6.0a 6.0a 9.5a
Ormocarpum trichocarpum 6.0a 8.5a 6.0a 6.0a 15.5c
Psidia puntulata 6.0a 14.0c 6.0a 6.0a 11.0b
Rhus natalensis 6.0a 14.0c 6.0a 6.0a 11.5b
Ricinus communis 6.0a 12.0b 6.0a 6.0a 13.0b
Senna didymobofrya 1l.0b 16.0c 6.0a 6.0a 16.0c
Sesbania sesban 6.0a 8.0a 6.0a 6.0a 11.0b
Tamarindus indica 6.0a 16.0c 6.0a 6.0a 15.0c
+ve control 26.0d 24.0d 23.0b 25.5b 26.0d
-ve control 6.0a 6.0a 6.0a 6.0a 6.0a
Zones of inhibition in same column indicated by different letters are significantly
different
63
4.2.1: Zone of inhibition plates
Plates 6 to 8 show zones of inhibition produced by plant extracts against bacterial strains.
Senna didymobotrya
Chloramphenical
DMSO
Fuerstia africana
Plate 6: Zones of inhibition of Fuerstia africana, Senna didymobotrya and that of
chloramphenical and DMSO control against Bacillus subtilis
64
DMSO
Ficus
sycomorus
Chloramphenical
Fuerstia
africana
Plate 7: Zones of inhibition of Fuerstia africana, Ficus sycomorus and that of
chloramphenical and DMSO control against Methicillin Resistant Staphylococcus aureus
Tamarindus indica
Chloramphenical
DMSO
Plate 8: Zones of inhibition of Tamarindus indica and that of chloramphenical and
DMSO control against Staphylococcus aureus
65
4.3Antifungal activity of the plant crude extracts against standard strains of fungus
The antifungal activities of 18 plant species were assayed in vitro by Kirby Bauer disk
diffusion method against 5 fungal species. From the findings a strong antifungal activity
ranging between 9 -20.5 mm was recorded.
Table 3 summarizes the average microbial growth inhibition of the (DCM and methanol)
extracts of the screened plant species against five strains of fungi. It was observed that 7
plants had great activity against M gypseum with some producing very large mean zones
of inhibition like Psidia puntulata (DC) Vatke (20.5 mm), Commiphora africana (A.
Rich) Engl. Syn (17.5 mm) (Plate 9), Senna didymobotrya Fresen (17.0 mm),
Tamarindus indica L. (16.0mm), Albizia coriaria Welw. ex Oliv (16.0 mm), Ficus
sycomorus Linn (15.5 mm) and Rhus natalensis Krauss (15.5 mm) (Plate 10). On the
hand, Fuerstia africana T. C. E Fries gave a moderate activity of 13.00mm while Boscia
angustifocia A. Rich, Ricinus communis Linn and, Ormocarpum trichocarpum Taub.
Engl gave low average zones of inhibitions of 10.00 mm, 10.0 mm and 8.5 mm against
the strain respectively. The remaining plant extracts did not show any visible activity (6.0
mm) against M gypseum. The zones of inhibition were significantly different (P < 0.05)
(Table 3 and Appendix 12).
Ricinus communis Linn produced moderate activity of 11.5 mm against A. niger (Table
3) while Fuerstia africana T.C.E Fries, Sesbania sesban Linn and Tamarindus indica L.
gave low activity of 8.0 mm, 8.0 mm and 7.5 mm respectively. The remaining 14 plant
extract; Zanthoxylum chalybeum Engl., Melia volkensii Gurke, Zanthoxylum gilletii De
66
Wild, Fuerstia africana T.C.E Fries, Urtica dioica Linn, Vernonia amygdalina Del.,
Commiphora africana (A. Rich) Eng!. Syn, Psidia puntulata (DC) Vatke, Senna
didymobotrya Fresen, Sesbania sesban Linn, Balanites aegyptiaca L. Drel., Albizia
coriaria Welw. ex Oliv, Ficus sycomorus Linn., Rhus natalensis Krauss and Tamarindus
indica L. were inactive against A. niger (6.0 mm). The zones of inhibition were
significantly different (P < 0.05) (Table 3 and Appendix 10).
Only Fuerstia africana T. C. E Fries showed weak activity of 9.0 mm against C.
neoformans ATCC 18310 and moderate activity of 12.0 mm against T mentagrophyte
(Table 3, Appendix 9 and 10). All the remaining seventeen plant extracts were
completely inactive producing a mean zone of 6.0 mm (Table 3). For the case of C.
albicans ATCC 90028, all the eighteen plant extracts were inactive against this test
organism. The zones of inhibition were not significantly different between the plant
extracts but were significantly different between the plant extracts and the positive
control (P < 0.05) (Table 3 and Appendix 8).
67
Table 3: Zones of inhibition produced by plants extracts against fungal strains in mm
Medicinal plants C.albicans C. A. T. M.
neoformans niger mentagrophyte gypseum
Boscia angustifolia 6.0a 6.0a 6.0a 6.0a 10.Ob
Fuerstia Africana 6.0a 9.0b 8.0a 12.0b 13.0b
Melia volkensii 6.0a 6.0a 6.0a 6.0a 6.0a
Urtica dioica 6.0a 6.0a 6.0a 6.0a 6.0a
Vernonia amygdalina 6.0a 6.0a 6.0a 6.0a 6.0a
Zanthoxylum chalybeum 6.0a 6.0a 6.0a 6.0a 6.0a
Zanthoxylum gilletti 6.0a 6.0a 6.0a 6.0a 6.0a
Albizia coriaria 6.0a 6.0a 6.0a 6.0a 16c
Balanites aegyptiaca 6.0a 6.0a 6.0a 6.0a 6.00a
Commiphora africana 6.0a 6.0a 6.0a 6.0a 17.5c
Ficus sycomorus 6.0a 6.0a 6.0a 6.0a 15.5c
Ormocarpum 6.0a 6.0a 6.0a 6.0a 8.5b
trichocarpum
Psidia puntulata 6.0a 6.0a 6.0a 6.0a 20.5d
Rhus natalensis 6.0a 6.0a 6.0a 6.0a 15.5c
Ricinus communis 6.0a 6.0a 11.5b 6.0a 10.Ob
Senna didymobofrya 6.0a 6.0a 6.0a 6.0a 17.0c
Sesbania sesban 6.0a 6.0a 8.0a 6.0a 6.00a
Tamarindus indica 6.0a 6.0a 7.5a 6.0a l6.0c
+ve control 12.5b l3.0c 18,Oc 21c 22d
-ve control 6.0a 6.0a 6.0a 6.0a 6.0a
Zones of inhibition in same column indicated by different letters are significantly
different
68
4.3.1 Zone of inhibition plate against fungal strains
Plates 9 to 10 show zones of inhibition produced by plant extracts against fungal strains.
Commiphora
africana
Plate 9: zones of inhibition of Cammiphora africana against Microsporum gypseum
Rhus natalensis
Miconazole
Plate 10: Zones of inhibition of Rhus natalensis and that of miconazole control against
Microsporum gypseum
69
4.4 The Minimum Inhibitory Concentrations (MICs) and the Minimum Bactericidal
Concentration (MBCS)
The minimum inhibitory concentration of the plant extracts which had inhibition
diameters of 9 mm and above was determined by the broth micro dilution method in 96
well microtitre plates (plate 11) and the results are presented in table 4 and 5
Six plants had MIC and MBC against MRSA. Albizia coriaria Welw. ex Oliv and
Commiphora africana (A. Rich) Eng. Syn gave MIC that were equal to MBC of 1.875
mg/ml. Fuerstia africana T. C. E Fries gave MIC and MBC of 0.9375 mg/ml and 1.875
mg/ml respectively while Vernonia amygdalin a Del and Zanthoxylum gilleti De Wild
gave MIC and MBC of 3.75 mg/ml and 7.5 mg/ml respectively while Senna
didymobotrya Fresen gave MIC and MBC of 1.875 mg/ml and 3.75 mg/ml respectively.
All the tested plants were screened for MIC and MBC against B. subtilis except Urtica
dioica Linn and Vernonia amygdalina Del., Fuerstia africana T. C. E Fries and Senna
didymobotrya Fresen gave a low MIC and MBC against B. subtilis of 0.9375 mg/ml
which is very close to that of the positive control value 0.4688 mg/ml. Ficus sycomorus
Linn and Melia volkensii Gurke had a weak MIC and MBC of 3.75mg/ml and 7.5 mg/ml
respectively. Zanthoxylum gilleti De Wild Albizia coriaria Welw. ex Oliv, Balanites
aegyptiaca L. Drel, Psidia punctulata (DC) Vatke, Rhus natalensis Krauss, Ricinus
communis Linn, Senna didymobotrya Fresen, Fuerstia africana T. C. E Fries had MICs
that were equal to MBC.
Balanites aegyptiaca L, Psidia punctulata (DC) Vatke, Ricinus communis Linn, Senna
didymobotrya, Tamarindus indica L. and Fuerstia africana T. C. E Fries showed a low
MIC and MBC of 0.9375 mg/ml against S. aureus ATCC 25923. Although the MIC and
70
MBC for positive control was lower, these are promising plant extracts given that they
are crude extract compared to pure compounds of the positive control. Zanthoxylum
gilleti De Wild showed a high MIC and MBC of 3.75 mg/ml and 7.5 mg/ml respectively
and therefore exhibits the lowest activity.
Fungal strain especially M gypseum gave the best MIC and MBC results (Table 5).
These results were ranging between 0.9375 mg/ml to 3.75 mg/ml with 7 plants giving
MIC of 0.9375 mg/ml. M gypseum was therefore the most susceptible microbe amongst
the tested strains. Psidia punctulata (DC) Vatke, Albizia coriaria Welw. ex Oliv,
Commiphora africana (A. Rich) Engl. Syn, Senna didymobotrya Fresen and Tamarindus
indica L. produced similar activity in MIC and MFC of 0.9375 mg/ml.
Ficus sycomorus Linn.and Rhus natalensis Krauss gave different values for MIC and
MFC against M gypseum. The MIC was 0.9375 mg/ml and MFC 1.875 mg/ml for both
plants. Boscia angustifolia A. Rich and Ricinus communis Linn gave a low activity of
3.75 mg/ml for MIC and MFC againstM gypseum.
Among all the plant extracts tested against C. neoformans and T mentagrophyte only
Fuerstia africana T. C. E Fries was found to be active. Fuerstia africana T. C. E Fries
produced a similar MIC and MFC of 3.75 mg/ml against. T mentagrophytes and a weak
MIC and MFC of3.75 mg/ml and 7.5 mg/ml respectively against C. neoformans. Ricinus
communis Linn was the only plant screened against A. niger. It gave a similar MIC and
MFC of 3.75 mg/ml. No plant extracts were screened against C. albicans ATCC 90028
for MIC and MFC because all the plants were negative on it.
71
Table 4: The minimum inhibitory concentration and the minimum bactericidal
concentration for bacterial test cultures in mg/ml
MRSA S. aureus
Plant MIC MBC MIC MBC
B. angustifolia ND ND ND
F africana 0.9375 1.875 0.9375
M volkensii ND ND ND
U dioica ND ND ND
Vamygdalina 3.75 7.5 ND
Z chalybeum ND ND ND
Z gilletti 3.75 7.5 ND
A. coriaria 1.875 1.875 1.875
B. aegyptiaca ND ND 0.9375
C. africana 1.875 1.875 3.75
F sycomorus ND ND 1.875
0. trichocarpum ND ND ND
P.puntulata ND ND 0.9375
R. natalensis ND ND 1.875
R. communis ND ND 1.875
S. didymobotrya 1.875 3.75 0.9375
S. sesban ND ND ND
T indica ND ND 0.9375
Positivecontrol 0.4688 0.4688 0.4688
Negative control Growth observed in all the tubes
MBC
ND
0.9375
ND
ND
ND
ND
ND
3.75
0.9375
7.5
3.75
ND
0.9375
1.875
3.75
0.9375
ND
0.9375
0.4688
B. subtilis
MIC
1.875
0.9375
3.75
ND
ND
1.875
3.75
1.875
1.875
1.875
3.75
0.9375
3.75
3.75
1.875
0.9375
3.75
0.9375
0.4688
1.875
0.9375
7.5
ND
ND
3.75
3.75
1.875
1.875
3.75
75.0
1.875
3.75
3.75
1.875
0.9375
3.75
1.875
0.4688
ND- Not done, MRSA- Methicillin Resistant Staphylococcus aureus, B. subtilis- Bacillus
subtilis, MIC- minimum inhibitory concentration, MBC- minimum bactericidal
concentration.
72
Table 5: The minimum inhibitory concentration and the minimum fungicidal
concentration for fungal test cultures in mg/ml
Test Culture
Medicinal plants
C. neoformans
MIC MFC
A. niger
MIC MFC
B. angustifolia
F. africana
M volkensii
U dioica
V amygdalina
Z. chalybeum
Z. gilletti
A. coriaria
B. aegyptiaca
C. africana
F. sycomorus
0. trichocarpum
P. puntulata
R. natalensis
R. communis
S. didymobotrya
S. sesban
T indica
positive
control
Negative
control
ND ND
3.75 7.5
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
0.4688 0.4688
T. mentagrophyte
MIC MFC
ND ND
3.75 3.75
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
0.4688 0.4688
Growth was observed in all the tubes
M. gypseum
MIC MFC
3.75 3.75
1.875 3.75
ND ND
ND ND
ND ND
ND ND
ND ND
0.9375 0.9375
ND ND
0.9375 0.9375
0.9375 1.875
ND ND
0.9375 0.9375
0.9375 1.875
3.75 3.75
0.9375 0.9375
ND ND
0.9375 0.9375
0.4688 0.4688
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
3.75 3.75
ND ND
ND ND
ND ND
0.4688 0.4688
MIC- Minimum Inhibitory concentration, MFC- Minimum fungicidal concentration, ND-
Not done
73
Plate 11: Microtitre plates showing Minimum inhibition concentration. The plate shows
four extracts at different concentration beginning from 10-1 to 10-9 dilution factors
4.5 Phytochemical screening
All the eighteen medicinal plants extracts were screened for phytochemicals. Alkaloids,
tannins, terpenoids, saponins, cardiac glycosides and flavonoids were the phytochemicals
that had their presence determined and the results presented in table 6
4.5.1 Tannins
Tannins were found to be present in all plant extracts except in Balanites aegyptiaca L.
Drel (Table 6). Vernonia amygdalina Del, Zanthoxylum gilletti De Wild, Albizia coriaria
Welw.ex Oliv, Commiphora africana (A. Rich) Engl. Syn, Ficus sycomorus Linn, Rhus
natalensis Krauss, Ricinus communis Linn, Senna didymobotrya Fresen and Sesbania
sesban Linn were found to have high concentrations. Boscia angustifolia A. Rich,
74
Fuerstia africana T. C. E Fries, Zanthoxylum chalybeum Engl, Ormocarpum
trichocarpum Taub. Engl, Psidia puntulata (DC) Vatke and Tamarindus indica L. had
moderate amounts while low concentrations were noted in the extracts of Urtica dioica
Linn and Melia volkensii Gurke.
4.5.2 Alkaloids
The alkaloids were mostly found in moderate concentrations in most extracts. For
example they were found in Boscia angustifolia A. Rich, Fuerstia africana T.C.E Fries,
Vernonia amygdalina Del., Senna didymobotrya Fresen, Sesbania sesban Linn, Ficus
sycomorus Linn, Albizia coriaria Welw.ex Oliv and Tamarindus indica L. (Table 6)
They were found in low concentrations in Urtica dioica Linn, Zanthoxylum chalybeum
Engl., Zanthoxylum gilletii De Wild, Balanites aegyptiaca L. Drel., Psidia punctulata
(DC) Vatke but were absent in Ormocarpum trichocarpum Taub. Engl, Melia volkensii
Gurke, Commiphora africana (A. Rich) Engl. Syn, Ricinus communis Linn and Rhus
natalensis Krauss.
4.5.3 Saponins
The saponins were completely absent in Boscia angustifolia, Vernonia amygdalina Del,
Urtica dioica Linn, Zanthoxylum chalybeum Engl., Zanthoxylum gilletii De Wild, Psidia
punctulata (DC) Vatke, Senna didymobotrya Fresen, Ormocarpum trichocarpum Taub.
Engl and Ficus sycomorus Linn plant extracts (Table 6). In Sesbania sesban Linn,
Balanites aegyptiaca L. Drel., Albizia coriaria Welw.ex Oliv, Commiphora africana (A.
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Rich) Engl. Syn, Rhus natalensis Krauss and Tamarindus indica L. the saponins were
moderate. Fuerstia africana T. C. E Fries, Melia volkensii Gurke and Ricinus communis
Linn, had low concentrations of saponins in the plant extracts.
4.5.4 Cardiac glycosides
Most abundant cardiac glycosides were found to be present in Albizia coriaria Welw.ex
Oliv, Commiphora africana (A. Rich) Engl. Syn , Rhus natalensis Krauss, Fuerstia
africana T. C. E Fries, Tamarindus indica L. and Ficus sycomorus Linn plant extracts
(Table). Cardiac glycosides were found in moderate concentration in Melia volkensii
Gurke, Balanites aegyptiaca L. Drel, Psidia punctulata (DC) Vatke and Ricinus
communis Linn while in low concentrations in Vernonia amygdalina Del., Zanthoxylum
chalybeum Engl., Ormocarpum trichocarpum Taub. Engl and Sesbania sesban Linn.
They were absent in plant extracts of Boscia angustifolia A. Rich, Urtica dioica Linn,
Zanthoxylum gilletii De Wild and Senna didymobotrya Fresen.
4.5.5 Terpenoids
High concentrations of terpenoid were found in Albizia coriaria Welw.ex Oliv,
Commiphora africana (A. Rich) Engl. Syn, Rhus natalensis Krauss, Fuerstia africana T.
C. E Fries, Ormocarpum trichocarpum Taub. Engl, Balanites aegyptiaca L. Drel, Senna
didymobotrya Fresen and Ficus sycomorus Linn plant extracts (Table 6). This was
indicated by the formation of a reddish brown colouration of the interface (Plate 12).
Extracts of Melia volkensii Gurke and Psidia punctulata (DC) Vatke and Ricinus
communis Linn had low concentration. Terpenoids were found to be absent in Boscia
76
angustifolia, Urtica dioica Linn, Vernonia amygdalina Del., Tamarindus indica L. and
Sesbania sesban Linn.
4.5.6 Flavonoids
Flavonoids were found in all other plant extracts except in Melia volkensii Gurke (Table
6). Flavonoids concentrations were high in the extracts of Ormocarpum trichocarpum
Taub. Engl, Balanites aegyptiaca L. Drel, Zanthoxylum gilletii De Wild, Psidia
punctulata (DC) Vatke and Ricinus communis Linn. In Rhus natalensis Krauss, Fuerstia
africana T. C. E Fries, Tamarindus indica L., Ficus sycomorus Linn and Zanthoxylum
chalybeum Engl the co ncentration of flavonoids in the plant extracts was moderate.
Boscia angustifolia A. Rich, Vernonia amygdalina Del, Urtica dioica Linn, Senna
didymobotrya Fresen, Albizia coriaria Welw.ex Oliv, Commiphora africana (A. Rich)
Engl. Syn and Sesbania sesban Linn had low concentration of flavonoids.
77
Table 6: Phytochemical screening of the plants extracts
Medicinal plants Tannins Alkaloids Saponin Cardiac Terpenoids Flavanoids
glycosides
Boscia ++ ++ +
angustifolia
Fuerstia ++ ++ + +++ +++ ++
africana
Melia + + ++ ++
;,olkensii
Urtica dioica + + +
Vernonia +++ ++ + +
amygdalina
Zanthoxylum ++ + + + ++
chalybeum
Zanthoxylum +++ + + +++
gilletti
Albizia +++ ++ ++ +++ +++ +
coriaria
Balanites + ++ ++ +++ +++
aegyptiaca
Commiphora +++ ++ +++ +++ +
africana
Ficus +++ ++ +++ +++ ++
sycomorus
Ormocarpum ++ + +++ +++
trichocarpum
Psidia ++ + ++ ++ +++
puntulata
Rhus +++ ++ +++ +++ ++
natalensis
Ricinus +++ + ++ ++ +++
communis
Senna +++ ++ +++ +
didymobofrya
Sesbania +++ ++ ++ + +
sesban
Tamarindus ++ ++ ++ +++ ++
indica
+++ (High), ++ (Moderate), + (Low) and - (Not present).
78
Terpenoid
Plate 12: Reddish brown colouration ofthe interface indicated the presence ofterpenoid
in Fuerstia africana
79
CHAPTER FIVE
DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS
5.1 DISCUSSION
The present research provides information on 18 medicinal plants used in the treatment of
diarrheal diseases, respiratory system illnesses, skin infection, fever, wounds among
others. From this study, the most cited health problem was diarrhea (Figure 2). This may
have been contributed by consumption of contaminated water (Jeruto et al., 2008). The
ethnobotanical survey results (Table 1) it indicated that traditional medicine still plays a
significant role in meeting the basic healthcare needs of people from Mwingi North, Kisii
South and Rarieda Districts in treating various diseases.
The 18 medicinal plants studied are distributed in 15 families. The most frequently used
preparation for drug methods were concoctions and decoctions. It was observed that
some of the plants were prepared by mixing more than one plant to make a concoction.
This is supported by Nanyingi et al. (2008). The use of concoctions suggests that the
compounds could only be active in combination, due to synergistic effects of several
compounds (Omori et al., 2012). On the plant parts used, it was noted that the leaves (9
plants) were the most frequently used parts of the plant followed by the bark (5 plants)
then roots (4 plants) and this is in agreement with Nanyingi et al. (2008) findings. The
leaves may be preferred because they are the main photosynthetic organs in plants and all
the phytochemical compounds are made in them then later translocated to other parts like
barks and roots (Jeruto et al., 2008). The use of roots and barks is dangerous to the
80
existence of the harvested plant, since it contributes to the loss of plants obtained by such
destructive harvesting methods (Jeruto et al., 2011).
The results obtained in this study indicate a considerable difference in antibacterial
activity of different extracts from selected plant species. Medicinal plants used by
herbalists to treat non-fungal infections were also active against some strains of fungi
tested. (Tables 2 and 3). This indicated that these extracts could be used for both bacterial
and fungal infections. For example; Tamarindus indica L., Senna didymobotrya Fresen,
Rhus natalensis Krauss, Psidia puntulata (DC) Vatke, Ficus sycomorus Linn, Albizia
coriaria Welw. ex Oliv and Fuerstia africana T. C. E Fries exhibited strong activity
against both bacterial and fungal strains. In general among the tested microbial strains,
bacteria were found to be more sensitive to many of the tested agents compared to the
fungi (Table 2 and Table 3). This is similar to Korir et al. (2012b) findings, who reported
that the bacterial strains were more sensitive to most of the crude extracts than the fungal
strains.
The antibacterial activity was more pronounced on the Gram positive bacteria than on
the Gram negative bacteria, for example; Tamarindus indica L., Senna didymobotrya
Fresen, Rhus natalensis Krauss, Psidia puntulata (DC) Vatke, Albizia coriaria Welw.ex
Oliv, Balanites aegyptiaca L. Drel and Fuerstia africana T. C. E Fries produced high
antibacterial activity against S. aureus ATCC 25923 and B. subtilis but no plant extract
was active on E. coli ATCC 25922 and S. typhi ATCC 19430 (Table 2). This is in
agreement with previous reports by some researchers (Duraipandiyan et al., 2006;
81
Pirbalouti et al., 2010). The probable reason for the variation in the activities between
Gram negative and Gram positive bacteria could be due to their morphological difference
(Maregesi et al., 2008; Pirbalouti et al., 2010). The Gram negative bacteria have an extra
outer membrane in their cell wall which is richer in lipopolysaccharides and acts as a
barrier to foreign substances including antibiotic molecules (Maregesi et al., 2008;
Pirbalouti et al., 2010). It is also associated with the enzyme found in periplasmic space
which can break down the molecules introduced from outside (Pirbalouti et al., 2010).
B. subtilis and S. aureus ATCC 25923 were generally more sensitive to the plants
extracts (Plates 6 and 8). This may be explained by the cell wall composition of the Gram
positive bacteria (S. aureus and B. subtilis) which have a relatively thick layer of
peptidoglycan sheets of interconnected glycan chains made up of polymer which is fully
permeable to many substances and thus sensitive to most plant extracts (Kitonde et al.,
2013). This is supported by Samie et al. (2005) who reported that Bacillus spp and S.
aureus were most sensitive whereas E. coli and Salmonella anatum were more resistant
to crude extracts of plants.
Fuerstia africana T. C. E Fries was the most active plant extract. It is widely used among
the Kisii for a wide range of conditions including: diarrhea, mouth infections, urinary
tract problems and skin infections hence this practice is supported by the broad spectrum
of activity displayed by the plant extract (Table 1). It gave zones of inhibition of 20.00
mm, 19.00 mm, 17.00 mm, 9.00 mm, 12.00 mm and 13.00 mm against MRSA, S. aureus
ATCC 25923, B. subtilis, C. neoformans ATCC 18310, T. mentagrophyte and M
82
gypseum respectively (Table 2 and 3). These results suggest that, the extract of Fuerstia
africana T. C. E Fries could be used for management of bacterial diseases caused by S.
aureus such as boils, sores, wounds and diarrhea. This result is in agreement with another
study that found Fuerstia africana T. C. E Fries to be active against S. aureus and MRSA
(Nge'ny et al., 2011) but partly agrees with the findings of Mariita et al., (2010a) who
found that the plant showed a moderate activity on S. aureus but was inactive against E.
coli, S. typhi and C. albicans. The difference may have been as a result of harvesting
periods and solvents used in extraction of the plant (Samie et al., 2005; Samie et al.,
2010; Asob et al., 2011). Mariita et at. (2010a) used methanol as solvent for extraction
while in this study DCM and methanol in the ratio of 1:1 was used.
The phytochemical analysis indicated that all the tested compounds were present hence
its broad spectrum activity is attributed to the phytoconstituents it possesses (Doughari
and Manzara, 2008). The tannins and alkaloids Fuerstia africana T. C. E Fries possessed
are known to be cytotoxic to bacterial cells and could explain the broad spectrum activity
(Omwenga et al., 2009).
Amongst all the 18 plants tested, it's only Fuerstia africana T. C. E Fries that showed a
low activity (9.00 mm) against C. neoformans ATCC 18310 (Table 3 and Appendix 9).
This is supported by Korir et al. (2012a) who found that C. neoformans was resistant to
all plant extracts. This may be because of the presence of polysaccharride capsule which
is made of galactoxylomanna and glucuronoxylomannan (Susane et al., 2009; Teresa and
Alspaugh, 2012). The polysaccharide capsular material in C. neoformans is responsible
83
for virulence and antimicrobial resistance (Korir et aI., 2012a). For instance, the capsule
enlargement has been associated with protection of the host fungus against host defense
mechanism such as phagocytosis and oxidative burst (Susane et al., 2009). In addition,
capsular material also acts directly against the host. In macrophages, C. neoformans
releases polysaccharide from its capsule into vesicles around the phagome and
accumulation of these vesicles in the cytoplasm of the cell results in macrophages
dysfunction and lysis (Hansang and Robin, 2009).
Senna didymobotrya Fresen showed activity of 16.00 mm against S. aureus ATCC 25923
and B. subtilis and 11.00 mm against MRSA. It was also very active against M gypseum
(17.00 mm) but it was completely inactive against all the other tested fungal and bacterial
strains. This is contradictory to the previous findings in which Senna didymobotrya
Fresen was active against E. coli and C. albicans (Korir et aI., 2012b). This may be
attributed to the differences in locality of the plant species and time of collection (Matu
and Staden, 2003). Plants collected during the rainy season will not have the same
phytochemical concentrations as the ones collected during the dry season (Matu and
Staden, 2003). The phytochemical screening indicated that tannins and terpenoids were
present in high concentrations but the flavonoids were in low concentration. Tannins
obtained from the bark of the stem of Senna didymobotrya Fresen are known to have
antimicrobial activity (Chothani and Vaghasiya, 2011). Tannins act by reducing secretion
and making the intestinal mucus resistant through the formation of protein tannate
(Balogun et al., 2011).
84
Albizia coriaria Welw. ex Oliv produced antibacterial activity with a zone of inhibition
of 13.00 mm against MRSA, 12 mm against B. subtilis and 10.00 mm against S aureus
ATCC 25923 but was inactive against S typhi and E.coli (Table 2). Albizia coriaria
Welw. ex Oliv also exhibited a high antifungal activity with a zone of inhibition of
16.00mm against M gypseum but was inactive against the remaining tested fungal
strains. This is partly in agreement with the report by Olila et al. (2007) who found it to
be active against B. subtilis and E. coli but had no activity against Saureus. C. africana is
also one of the most active plants that showed antimicrobial activity against four micro-
organisms with a zone of inhibition of 11.50 mm, 9.5 mm, 12.00 mm and 17.5 mm
against MRSA, S aureus ATCC 25923, B. subtilis and M gypseum respectively. This is
partly in agreement with Akor and Anjorin (2009) who used the root extracts and found
that it exhibited activity against S aureus, E .coli and C. albicans. The difference in the
results might be attributed to the parts of the plants used, physical factors (temperature,
light and water) contamination by field microbes, substitution of plants and the locality
(Okigbo and Mmeke, 2008).
The phytochemical analysis of Albizia coriaria Welw. ex Oliv and Commiphora
africana (A. Rich) Engl. Syn indicated that tannins, terpenoids, saponin, cardiac
glycosides, and flavonoids were present in various concentrations except alkaloid that
were not present in Commiphora africana (A. Rich) Engl. Syn. Tannins possess
antibacterial properties because of their ability to react with proteins to form stable water
soluble compounds that kill bacteria by disrupting their cell membranes (Mariita et aI.,
2011).
85
Ficus sycomorus Linn has been reported to posses anti-diarrheal activity (Ahmadu et al.,
2007). In this study, the bark extract was found to be active against S. aureus ATCC
25923 and B. subtilis, though it also exhibited a mild activity on MRSA with zones of
inhibition of 11.50 mm, 9.5 mm and 8.5 mm (Table 2). It also exhibited a strong activity
of 15.5 mm against M gypseum. In previous research, crude ethanol extract of Ficus
sycomorus Linn showed activity against S. aureus, MRSA and S. typhii (Kubmarawa et
al., 2007). This is partially in agreement with the present study. However in the current
study, it was not active against S. typhi ATCC 19430. Ficus sycomorus Linn did not show
any activity in the yeast species but was very active against M gypseum and this is in
agreement with Samie et al. (2010) who did not find any antifungal activity on the yeast.
The difference might be attributed to differences in geographical location, type of
solvents used in extraction, the type of tested microorganisms, parts used, storage
conditions and methods of analysis (Wagete et al., 2008; Olusesan et al., 2010; Obeidat
et al., 2012).
The phytochemical analysis of Ficus sycomorus Linn revealed the presence of secondary
metabolites such as: tannin, cardiac gl ycosides, terpenoids and flavonoids. The high
concentration of terpenoids might have contributed to the antibacterial activity. Since this
is supported by Chiruvella et al. (2007) who isolated terpenoids from Soymida febrifuga
and reported that the antibacterial activity of terpenoids was due to membrane disruption
by the terpenes. The presence of some of these compounds has been demonstrated in
previous studies by other researchers. For example the presence of terpenes and tannins
in the leaves has been demonstrated (Ahmadu et al., 2007). The presence of glycosides,
86
tannins and flavonoids in the stem bark extract has been revealed (Kubmarawa et al.,
2007).
In this research, Zanthoxylum giletii De Wild was found active against B. subtilis, MRSA
and showed a mild activity on S. aureus ATCC 25923 but was inactive against E. coli
25922 and S. typhi ATCC 19430. It also had no antifungal activity on the tested fungi.
This is partially in agreement with Mariita et al. (2010a) who reported that the plant was
inactive against E. coli, S. typhi, S. aureus and C. albicans but contradicts the findings of
Agyare et al. (2006) who documented that the leaf extract of Zanthoxylum gilletii De
Wild, was active against E. coli, S. aureus, B. subtilis and C. albicans but inactive against
A. niger. The slight difference may be due to the solvent used for extraction and dosage
(Okigbo and Mmke, 2008) climatic and environmental factors (Kubmarawa et al., 2007;
Okigbo and Mmke, 2008). Plants collected at different times and from different regions
might have different activities against micro-organisms (Samie et al., 2005). On testing
for phytochemicals, it was found out that the plant possessed a high concentration of
tannins and flavonoids, moderate concentration of terpenoids and alkaloids though it
lacked saponins and cardiac glycosides. This finding partly agrees with the fmding of
Agyare et al., (2006) who found out that the plant possessed alkaloids, tannins and
saponms. The high concentration of flavonoids might have been responsible for the
observed antimicrobial activity since they have been reported to exhibit strong
antimicrobial activity (Olusesan et al., 2010).
87
In the present study, Balanites aegyptiaca L. Drel was found to be active against S.
aureus ATCC 25923 and B. subtilis but was inactive against MRSA, S. typhi ATCC
19430, E. coli ATCC 25922 and all the tested fungal strains. This is partially in
agreement with a research carried out by Bidawat et al. (2011) who reported that the
flavonoids fractions were active against E. coli, B. subtilis and S. aureus. On the contrary,
Balanites aegyptiaca L. Drel stem bark exhibited a high antifungal activity against C.
a/bicans (Maragesi et al., 2008) and the leaves demonstrated a high antityphoid activity
(Doughari et al,. 2007). Ethanol extracts of leaves were found to be more effective
against E. coli, B. subtilis, S. aureus and C. albicans compared with the extract of stem
bark (Gour and Kant, 2012). The difference could be as a result of extraction methods,
geographical areas where the plants were collected, the part of plant material used and
time of collection of the plant materials which in turn affected the amount of constituents
of the plants (Ibrahim et al., 2009; Okemo et al., 2011) besides, the extraction procedures
could also alter the phytochemical composition in plants (Korir et a/., 2012a).
The phytochemical analysis of Balanites aegyptiaca L. Drel revealed that the plant
contained flavonoids, terpenoids, glycosides, saponins and traces of alkaloids but did not
posses tannins. The antibacterial activity might have been due to the presence of
flavonoids and terpenoids (Banson and Adeyemo, 2007). The high concentration of
flavonoids could have contributed to the antimicrobial effect since flavonoids have been
found to kill enzymes involved in cell wall biosynthesis in pathogens (Negi et al., 2009).
Balanites aegyptiaca L. Drel has a long history of traditional uses for a wide range of
88
diseases (Chothani and Vaghasiya, 2011). This could have been contributed by the
presence of the secondary metabolites in various concentrations (Nwodo et al., 2010).
Tamarindus indica L. is a multipurpose plant whose most parts find at least some use
(Caluwe et al., 2010). In the current research, the stem bark extract of Tamarindus indica
L. demonstrated activity against S. aureus ATCC 25923, B. subtilis and M gypseum but
was inactive against MRSA, E. coli ATCC 25923, S. typhi ATCC 19430 and the other
tested fungal strains. This is partially in agreement with Doughari (2006) whose report
showed that Tamarindus indica L. showed activity against E. coli, S. typhi, B. subtilis and
S. aureus. According to Daniyan and Muhammed (2008) fruit extract of Tamarindus
indica L. was inactive against S. typhi but active against E. coli and S. aureus. This is
supported by Nwodo et al. (2010) whose findings revealed that the plant was inactive
against S. typhi but exhibited activity against E. coli, B. subtilis and S. aureus. The
differences might have been caused by the solvents used for extraction, plant collection
periods, age of the plant used, freshness of plant materials, adulteration and geographical
difference (Okigbo and Mmeka, 2008). It has been documented that different solvents
have diverse solubility capacities for different secondary metabolites (Doughari and
Manzara, 2008). The phytochemical constituents such as: tannins, alkaloids, saponins,
cardiac glycosides and flavonoids were present in the current study. The antimicrobial
activity is therefore explained by the presence of the phytochemical constituents (Nwodo
et al., 2010). Flavonoids have been reported to possess antimicrobial activity (Olusesan,
2010) and are known to prevent gastric ulcers due to the astringent and antimicrobial
89
properties which appear responsible for gastric - protective activity (Njoroge et al.,
2012).
In this study, Rhus natalensis Krauss and Psidia punctulata (DC) Vatke demonstrated
strong activity against B. subtilis, S. aureus ATCC 25923 and M gypseum. Rhus
natalensis Krauss gave zones of inhibition of 11.5 mm, 14.00 mm, and 15.5 mm
respectively (Table 2). While Psidia punctulata (DC) Vatke gave 14.00 mm, 11.00 mm
and 20.5 mm respectively. This signifies that, the extracts of could be used to produce
antifungal and antibacterial compounds for the treatment of diseases caused by M
gypseum, B. subtilis and S. aureus. Rhus natalensis Krauss has been reported to exhibit
antiplasmodial activity, (Gathirwa et al., 2011). It has also been found to give protection
frOII1 periodontopathic bacteria by preventing bacterial enzyme function (Parker et al.,
2007). Psidia punctulata (DC) Vatke has been reported to possess antileishmanial
properties (Githinji et al., 2009). It also demonstrated a mild activity against coffee berry
disease fungus (Midiwo et al., 2002).
The Phytochemical screenmg indicated that tannins, saponms, cardiac glycosides,
terpenoids and flavonoids were present in Rhus natalensis Krauss, but alkaloids were
absent. Psidia punctulata (DC) Vatke possessed tannins, alkaloids, cardiac glycosides,
terpenoid and flavonoids but lacked saponins. The known active phytochemical
constituents in Psidia punctulata (DC) Vatke are flavones and phenylpropenoids (Githinji
et al., 2010). The antibacterial activity of flavonoids is probably due to their ability to
complex with extracellular and soluble proteins and to complex with bacterial cell walls
90
(Banson and Mann, 2008). This could be attributed to the antimicrobial properties of the
plant.
Sesbania sesban Linn and Ormocarpum trichocarpum Taub. Engl. showed moderate
activity against B. subtilis by producing zones of inhibiting of 11.00 mm and 15.5 mm
respectively. They also exhibited a mild activity against S. aureus ATCC 25923 of 8.00
mm and 8.50 mm respectively. They did not demonstrate any activity against MRSA, E.
coli ATCC 25922, S. typhi ATCC 19430, the yeast and dematophytes though the leaf
extracts of Ormocarpum trichocarpum Taub. Engl., were slightly active against M
gypseum (8.5 mm) and the bark extract of Sesbania sesban Linn also showed mild
activity on A. niger of 8.00 mm. This result is partly in agreement with previous studies
which revealed that the leaves of Sesbania sesban Linn showed activity against B. cereus,
E. coli and A. niger (Hossain et al., 2007). Sesbania sesban Linn did not show any
activity on the yeast fungi but showed mild activity against A. niger (filamentous fungi)
this may be due to differences in the structure of the cell wall of the organisms (Paiva et
al., 2010) the protein on C. albicans, function as selective transport system used to
remove wastes and compounds that are dangerous to the cell (Nester et al., 2004). This is
of medical importance since it allows micro-organisms to oust antimicrobial medications
that are used to destroy them and therefore make them resistant. This explanation could
be one of the reasons why C. albicans ATCC 90028 was resistant to all crude extracts.
The phytochemical screening of Ormocarpum trichocarpum Taub. Engl indicated that
terpenoid and flavonoids were present in high concentrations, while alkaloids and
91
saponins were absent. Sesbania sesban Linn had a high concentration of tannin, but
alkaloids and saponins were moderate, cardiac glycosides and flavonoids were low and
terpenoids were absent. Tannins, cardiac glycosides, flavonoids and saponins have been
linked with antimicrobial activity (Nwodo et al., 2010). For instance saponins act by
breaking the cell membrane of bacteria (Omwenga et al., 2009).
Ricinus communis Linn exhibited activity against S. aureus ATCC 25923, B. subtilis, M
gypseum and A. niger. The inhibition zones were 12.00 mm, 13.00 mm, 11.50 mm and
10.00 mm respectively. But the plant did not show any activity against the tested Gram
negative bacteria and yeast. The activity of Ricinus communis Linn is in agreement with
the findings of Verma et al. (2011) where the root extract was found to have activity
against S. aureus, B. subtilis, and A. niger but contrasts with their findings that it was
active against E. coli. From a study carried out by Jumbo and Enenebeako (2007) the
fermented seed extract of Ricinus communis Linn was found to be active against S.
aureus and E. coli. This also partly agrees with the findings of the current research. The
difference could be attributed to the part of the plant used and geographical regions (Arya
et al., 2010).
The Phytochemical screemng of Ricinus communis Linn indicates that tannins and
flavonoids were present in high concentrations, cardiac glycosides and terpenoids in
moderate concentration, saponins in low concentration but alkaloids were absent. The
presence of tannins and flavonoids in high concentrations could be responsible for the
antimicrobial activities. Tannins have been reported to have antimicrobial properties
92
since they can react with proteins forming stable water soluble compounds thus killing
the bacteria by damaging the cell membrane directly (Okemo et al., 2011). Flavonoids
can also interact with mitochondria and interfere with pathways of intermediary
metabolism (Williams et al., 2004). Flavonoids and flavonoid - derived plant natural
products have for a long time been known to function as antimicrobial defense
compounds (Korir et al., 2012b) The presence of tannins could be responsible for the
antimicrobial activities because they have antifungal, anti-inflammatory and healing
properties (Moyo et al., 2010). This could explain the inhibition of A. niger.
In the present study, Vernonia amygdalina Del showed activity against MRSA but was
slightly active against B. subtilis. It was inactive against S. typhi ATCC 19430, S. aureus
ATCC 25923, E. coli ATCC 25923 and the tested dermatophytes, yeast and A. niger.
These findings are in agreement to those of Mariita et al. (2010) and Cheruiyot et al.
(2009) who reported that this plant did not have any ability to inhibit E. coli, S. typhi and
C. albicans. This is also supported by Uzoigwe and Agwa (2011) who found that E. coli
and S. aureus did not show susceptibility to both the leaf and stem extract of Vernonia
amygdalina Del. This is contrary to reports by Okigbo and Mmeka (2008) and Ogundare
(2011) in which Vernonia amygdalina Del was active against E. coli, S. aureus and C.
albicans. The phytochemical analysis revealed that the plant had a high concentration of
tannins, moderate concentration of alkaloids and traces of glycosides and flavonoids,
though it did not have saponins and terpenoids. It is possible that the antibacterial
activities of this plant may be due to the presence of bioactive components as noted by
Ogundare (2011).
93
Findings from the current study shows that the leaf extracts of Boscia angustifolia A.
Rich demonstrated activity against B. subtilis (13.00 mm) and M gypseum (10.00 mm)
only but was inactive against the rest of the microorganisms tested. This is partially in
agreement with the findings by Omwenga et al. (2009) which revealed that the plant
showed low activity against S. aureus and B. subtilis but was inactive against S. typhi and
E. coli. This finding is contradicted by Hassan et al. (2006) who found that the root
extracts were active against E. coli. The difference might have been attributed to different
parts used since the concentration of the active phytochemicals can vary significantly
between different parts of a given plant (Gathirwa et al., 2011). The phytochemical
screening revealed that the plant possesses tannins, alkaloids and flavonoids in various
concentrations but lacked saponins, cardiac glycosides and terpenoids. Tannins have been
found to form irreversible complexes with rich protein resulting in the inhibition of cell
protein synthesis (Issazadeh et al., 2012). Alkaloids are known to be toxic against cells of
foreign organism (Issazadeh et al., 2012).
The leaves extract of Melia volkensii Gurke and Zanthoxylum chalybeum Engl only
inhibited B. subtilis with zone of inhibition of 10.00 mm and 15.00 mm respectively but
they did not show any activity against the fungal strains (Tables 2 and 3). This is not in
agreement with Akanga (2008) whose report on Melia volkensii Gurke showed activity
against E. coli. The findings on Zanthoxylum chalybeum Engl is partially in accordance
with Matu and Staden (2003) whose observations revealed that the stem bark had
moderate antibacterial activity against S. aureus. Zanthoxylum chalybeum Engl has been
94
reported to have no antibacterial activity against E. coli and S. aureus. It also showed no
antifungal activity against C. albicans (Olila et al., 2001). The antidiarhoea activity could
be as a result of their synergy with components from other plants and some metabolites
(Doughari, 2006).
The preliminary phytochemical analysis of Melia volkensii Gurke revealed the presence
of cardiac glycosides and terpenoids in moderate concentration and traces of saponin,
while alkaloids and flavonoids were missing. This partially contradicts the phytochemical
analysis results of Akanga (2008) which indicated that Melia volkensii Gurke possesses
tannins, terpenoids, flavonoids, steroids, alkaloids, cardiac glycosides and saponin in
varying amounts. The phytochemical test on Zanthoxylum chalybeum Engl indicated that
the plant had tannin and flavonoids in moderate amounts, alkaloids, cardiac glycosides
and terpenoids in low amounts but saponins were lacking. Melia volkensii Gurke and
Zanthoxylum chalybeum Engl only inhibited the growth of B. subtilis and this might have
been brought about by the fact that the plants may have contained antibacterial
constituents. Besides this, the drying process may have caused conformational changes to
occur in some of the chemical constituents found in Melia volkensis Gurke and
Zanthoxylum chalybeum Engl (Parekh and Chanda, 2007).
Findings from the current study showed that Urtica dioica Linn exhibited mild activity
on S. aureus ATCC 25923 with a zone of inhibition of (7.5mm) and B. subtilis (8.00 mm)
but was inactive on all the tested fungal species. This is partially in agreement with Uzun
et at. (2004) whose findings revealed that the plant was active against S. aureus,
95
moderately inhibited E. coli but was inactive against C. albicans. Urtica dioica Linn may
not have any effect on the fungal strains tested but may participate in facilitating
diffusion of active compounds into the blood, stabilizing the body temperature and
reducing toxicity. This explains why herbal medicines are given in cocktail by herbalists
(Adesina, 2005; Samie et al., 2010). The low activity by Urtica dioica Linn against B.
subtilis (8.00 mm) and S. aureus (7.5 mm) could suggest presence of resistance of these
micro-organisms. The activity may be improved by increasing the concentration of the
crude extract. This is supported by Kitonde et al. (2013) who reported that if drugs show
less activity against the test organisms then that indicates the development of resistance
by the micro-organisms. The antimicrobial activity would be increased by increasing the
concentration of the extract (Kitonde et al., 2013), since diluting crude extracts normally
weaken their antimicrobial activity (Ramamoorthy et al., 2010). The phytochemical
screening of this plant revealed that the plant had traces of tannins, alkaloids and
flavonoids but lacked saponins, cardiac glycosides and terpenoids.
The low concentration of the bioactive compounds in this plant might have been
responsible for the poor antimicrobial activity (Nwodo et al., 2010). Besides the absence
of active ingredients, other reason that could explain the low antimicrobial activity
include: the parts of the plant used, solvent used for extraction, the location where the
plants were collected and the time when the plants were collected (Hamza et al., 2006).
The low activity by Urtica dioica Linn extract could also be due to low concentration of
diffusible compounds (Ibrahim et al., 2009). It is also possible that the drying process
96
could have caused disintergration reactions that lead to production of other non active
chemicals (Omwenga et al., 2009).
The absence of antimicrobial activity does not disapprove the ethnobotanical uses of the
plants since the herbalists combine several plants. The herbalists use more than one plant
to make a concoction for the management of a given disease because of the presence of
different phytochemicals in different plants (Omwenga et al., 2009). Synergy is a
common concept in herbal medicine, suggesting that plant containing compounds
potentiate each other's action (Koroishi et al., 2008) while in this study, the bioassay test
was done per plant. In addition to that the presence of some impurities has reduced the
efficacy of the crude extracts (Ogundare, 2011).
Minimum inhibitory concentration is a quantitative assay and provides more information
on the potency of the compounds present in the extracts (Korir et al., 2012b). It was
observed that there was no plant extract whose activity was equal to or more than that of
the positive control while growth was observed in all concentrations in the tubes in the
case of the negative control. The high MIC of crude extracts which was between 0.9375
mg/ml and 3.75 mg/ml compared to the standard drugs which was 0.4688 mg/ml is a
clear indication that the active element in the crude extract was in low concentration
which required the use of large amounts of crude extracts to increase the desired
therapeutic effects (Korir et al., 2012a). The lower the MIC the better the plant extract
against the micro-organism tested (Korir et al., 2012 b). Albizia coriaria Welw. ex Oliv
and Commiphora africana (A. Rich) Eng. Syn gave MICs that were equal to MBCs of
97
1.875 mg/ml against MRSA. This clearly indicates that the concentration that inhibits the
growth of MRSA is the same concentration of Albizia coriaria Welw. ex Oliv that kills
the test organism. Fuerstia africana T. C. E Fries gave an MIC and MBC of 0.9375
mg/ml and 1.875 mg/ml respectively against MRSA. while Vernonia amygdalina Del,
and Zanthoxylum gilletii De wild gave an MIC and MBC of 3.75 mg/ml and 7.5 mg/ml
respectively while Senna didymobotrya Fresen gave an MIC and MBC of 1.875 mg/ml
and 3.75 mg/ml respectively (Table 4). The crude extracts of medicinal plants at low
concentrations shorten the length of treatment, reduce over dose and toxicity or side
effects (Kitonde et aI., 2013).
All the tested plants were screened for MIC and MBC against B. subtilis except Urtica
dioica Linn and Vernonia amygdalina Del., Fuerstia africana T. C. E Fries and Senna
didymobotrya Fresen gave a strong MIC and MBC against B. subtilis of 0.9375 mg/ml
very close to that of the positive control value 0.4688 mg/ml. Ficus sycomorus Linn and
Melia volkensis Gurke had a weak MIC and MBC of 3.75 mg/ml and 7.5 mg/ml
respectively. Zanthoxylum gilletii De Wild Albizia coriaria Welw.ex Oliv, Balanites
aegyptiaca L. Drel, Psidia punctulata (DC) Vatke, Rhus natalensis Krauss" Ricinus
communis Linn, Senna didymobotrya Fresen and Fuerstia africana T. C. E Fries had
MICs that were equal to MBC. These antimicrobial effects may be attributed, possibly in
combination, to various phytochemicals detected during the extracts chemical screening
and which are known to cause damage to cell membranes, causing leakage of cellular
materials and ultimately the microorganism death (Marzouk et aI., 2010).
98
Balanites aegyptiaca L. Drel., Psidia punctulata (DC) Vatke, Ricinus communis Linn,
Senna didymobotrya Fresen, Tamarindus indica L. and Fuerstia africana T. C. E Fries
showed a strong MIC and MBC of 0.9375 mg/ml against S. aureus ATCC 25923. Low
MIC and MBC values are an indication of high efficacy against the organism in question
(Doughari et aI., 2008). This study therefore provides scientific evidence of their efficacy
against diarrhea caused by S. aureus. Zanthoxylum gilletii De Wild showed a high MIC
and MBC of 3.75 mg/ml and 7.5 mg/ml therefore exhibits the lowest activity.
Antimicrobial agents with low activity against a microbe have a high MIC while a very
active antimicrobial agent has a low MIC (Banson and Adeyemo, 2007).
No plant extracts were screened against E. coli 25922 and S. typhi ATCC 19430 for MIC
and MBC because all the plants were inactive on them. Lack of activity against E. coli
could be attributed to the fact that E. coli are extended spectrum ~-lactamase producers
(ESBLS) and ~-lactamase is one of the major causes of drug resistance (Mariita et al.,
2010a). Escherichia coli is known to quickly acquire drug resistance (Chandarana et al.,
2005). This could be the reason why all the plant extracts showed no efficacy against it.
Fungal strain especially M gypseum gave the best MIC and MFC results. Ranging
between 0.9375 mg/ml to 3.75 mg/ml with 7 plants giving MIC of 0.9375 mg/ml. M
gypseum was therefore the most susceptible microbe amongst the tested strains. Psidia
punctulata (DC) Vatke, Albiziacoriara Welw.ex Oliv, Commiphora africana (A. Rich)
Engl. Syn Senna didymobotrya Fresen and Tamarindus indica L. produced similar
99
activity on MIC and MFC ofO.9375mg/ml against M gypseum. These are very promising
plants against M gypseum, therefore supports the use of these plants for skin conditions.
Ficus sycomorus Linn and Rhus natalensis Krauss gave different values for MIC and
MFC. The MIC was 0.9375 mg/ml and MFC 1.875 mg/ml for both plants against M
gypseum. Boscia angustifolia A. Rich and Ricinus communis Linn gave a low activity of
3.75 mg/ml for MIC and MFC for both plants. Among all the plant extracts tested against
C. neoformans and T mentagrophytes only Fuerstia africana T. C. E Fries was found to
be active. It produced a similar MIC and MFC of 3.75 mg/ml against. T mentagrophytes
and a weak MIC and MFC of 3.75 mg/ml and 7.5 mg/ml respectively against C.
neoformans 18310 (Table 5).This may be due to the high concentration of terpenoids
which has cytotoxic activity against bacteria and fungi (Mamta and Jyoti, 2012). Ricinus
communis Linn was the only plant screened against A. niger which had a similar MIC and
MFC of 3.75 mg/ml. High values indicated that the micro-organism are less sensitive to
the plant extracts (Obeidat et al., 2012). No plant extracts were screened against C.
albicans ATCC 90028 for MIC and MFC because all the plants were negative on it. This
might have been brought about by the biofilms formed by C. albicans ATCC 90028
when single cells attach to a surface and grow into microcolonies which unite and form a
complex 3 -D structure that is held together by hyphae and an exopolymer matrix
(Lafleur, 2011).
The highest MIC and MBCIMFC values of the test microbes is an indication that either
the plant extracts have low activity on the tested bacterial and fungal strains or that the
100
microorganisms have the potential of developing antibiotic resistance, while the low MIC
and MBCIMFC values for the tested microbe is an indication that the plant extracts has
the potential to treat any diseases associated with the pathogenic microbes effectively
(Doughari and Manzara, 2008; Doughari, 2006). In this research all the active plant
extracts were bacteriostatic/fungistatic since antibacterial substances are considered as
bactericidal agents when the ratio MBCIMIC :s 4 and bacteriostatic agents when the ratio
ofMBCIMIC is ~4 (Gatsing and Adoga, 2007).
5.2 CONCLUSIONS
The result of the study revealed that knowledge about the diseases treated by various
plants is still maintained among the people of Kisii South, Rarieda and Mwingi North
Districts. The survey shows that the community from Kisii South, Rarieda and Mwingi
North Districts use medicinal plants to treat different ailments like gastrointestinal,
respiratory problems, skin infections and other ailments. The leaves were the most
frequently used parts of the plant and the most frequently used preparations for
administration were concoctions and decoctions.
The present study indicates that the majority of the plants tested are important source of
antibacterial agents especially on Gram positive bacteria and antifungal agents against the
dermatophytes especially M gypseum. Therefore supporting their uses by herbalists and
locals for treatment of infectious diseases and giving scientific proof of their claimed
efficacy against diarrhea
101
The plant extracts showed activity against MRSA, S. aureus and B. subtilis. Senna
didymobotrya, Commiphora africana (A. Rich) Engl. Syn, Albizia coriaria Welw. ex
Oliv and Fuerstia africana T. C. E Fries exhi bited activity against the above three
mentioned Gram positive bacteria. While Ficus sycomorus Linn., Rhus natalensis Krauss,
Psidia puntulata (DC) Vatke, Ricinus communis Linn and Tamarindus indica L. also
showed activity against S. aureus ATCC 25923 and B. subtilis. Zanthoxylum chalybeum
Engl., Melia volkensii Gurke, Sesbania sesban Linn and Ormocarpum trichocarpum
Taub. Engl. only inhibited S. aureus ATCC 25923. Therefore the plants can be used in
treating diseases caused by MRSA, S. aureus ATCC 25923 and B. subtilis but not E. coli
ATCC 25922 and S. typhi ATCC 19430.
The plant extracts were also active against M gypseum, T mentagrophyte, C. neoformans
ATCC 18310 and A. niger. Fuerstia africana T. C. E Fries was the most effective in
combating the pathogenic micro-organisms studied since it displayed antimicrobial
activity against almost all the bacterial and fungal test cultures. Fuerstia africana T. C. E
Fries showed a broad spectrum activity by inhibiting M gypseum, T mentagrophyte, C.
neoformans ATCC 18310. The extracts of Commiphora africana (A. Rich) Engl. Syn,
Psidia puntulata (DC) Vatke, Senna didymobotrya Fresen, Albizia coriaria Welw.ex
Oliv, Ficus sycomorus Linn., Rhus natalensis Krauss, Tamarindus indica L. and Ricinus
communis Linn showed activity against M gypseum. No plant extract was found to be
active against C. albicans ATCC 90028. Generally more extracts were found to be more
active against bacteria than fungi.
102
The extracts of Senna didymobotrya Fresen and Fuerstia africana T. C. E Fries gave the
strongest MIC and MBC of 0.9375 mg/ml against B. subtitis while Psidia puntulata (DC)
Vatke, Fuerstia africana T. C. E Fries, Senna didymobotrya Fresen, Tarnarindus indica
L. and Ricinus communis Linn also gave the strongest MIC and MBC against S. aureus
ATCC 25923. The plant extracts of Commiphora africana (A. Rich) Engl. Syn, Psidia
puntulata (DC) Vatke, Senna didymobotrya, Tamarindus indica L. and Albizia coriaria
Welw. ex Oliv produced similar activity on MIC and MBC of 0.9375 mg/ml against M
gypseum. Therefore the plants can be used for the treatment of diseases caused by M
gypseum.
The screening revealed the presence of various phytochemical compounds for example
alkaloids, tannins, cardiac glycosides, terpenoids, saponins and flavonoids. All medicinal
plants investigated were effective against B. subtilis. Fuerstia africana T. C. E Fries is
widely used among the Kisii for a wide range of conditions. Hence this was supported by
the broad spectrum of activity displayed by its crude extract making it the best amongst
the crude extract used. Melia volkensis Gurke and Urtica dioica Linn were the least
active plants.
5.3 RECOMMENDATIONS
1. Since this study has proved that the extracts of Fuerstia africana T. C. E Fries,
Albizia coriaria Welw. ex Oliv, Senna didymobotrya Fresen, Psidia puntulata
(DC) Vatke, Tamarindus indica L. and Rhus natalensis Krauss are readily
103
available sources of antifungal and antibacterial principles, their extracts should
be subjected to both pharmacological and toxicological studies.
11. The active principles responsible for antimicrobial activity in this study should be
extracted and identified then screened against the selected bacterial and fungal
pathogens used in their research.
111. Bioassays of combinations of plant extracts that exhibited moderate and low
activity for example Boscia angustifolia A. Rich, Zanthoxylum gilletti De Wild,
Ficus sycomorus Linn, Ormocarpum trichocarpum Taub. Engl, Rhus natalensis
Krauss, Balanites aegyptiaca L. Drel and Ricinus communis Linn should be
carried out to establish any synergism between them.
104
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123
APPENDICES
Appendix 1: Consent form
Introduction
Hallo, I am India Jacqueline a student at Kenyatta University School of pure and applied
sciences department of Microbiology. This information form seeks informed consent for
your participation in a study that seeks to determine the efficacy of some medicinal plants
used against infectious diseases caused by some selected bacteria and fungi.
Project title: Ef ficacy of some medicinal plants used in various parts of Kenya in
treating selected bacterial and fungal pathogens.
Purpose: This is a study to determine the efficacy of some medicinal plants used in
various parts of Kenya. The plants are used among the traditional communities in Kenya
as herbal concoction but their efficacy has not been established. The results will provide
scientific proof for their claimed medicinal use.
Procedure
If you consent to be in this study, I will ask you to answer survey questions in a private
place at or near your home. The survey will take approximately 40 minutes. I will ask
questions about medicinal plants and the diseases they treat. I will also ask how you
acquire the knowledge on traditional medicinal. Example questions are: "which plants do
you use routinely?", "How do you prepare these herbal drugs?", and "what diseases do
they treat or prevent?". You do not have to answer any questions you do not want to
answer.
Benefits of participation
You may not benefit directly from being in this study. However, the findings will provide
useful information on the efficacy of the medicinal plants against the diseases caused by
the selected bacterial and fungal pathogens.
Risks or discomforts
The risks of being in this study are low. Some of the questions are sensitive and you may
feel uncomfortable giving all the information. You may also be inconvenienced because
of the time involved.
124
Confidentiality
All the information in this study will be kept confidential. In order to protect your
privacy, all hard copy data will be stored in designated lockers and access will be limited
to the principle investigator. Using passwords only known to the principle will safeguard
electronic data. Only people working directly on the study will be able to look at these
records.
Voluntary participation
Being in this study is voluntary. You do not have to answer any question that you do not
want to. You are free to stop taking part in this study at any time.
Who to contact
If you have questions about this research or feel you have been harmed by the research,
please contact; India Jacqueline through 0722284276, the principle investigator of the
study or Kenyatta University board of postgraduate studies.
Statement of consent
I have read and / or had this form explained to me. I understand the reason for the study.
My questions have been answered. I agree to take part by my own choice.
Participant's name participant's signature/
Thumb print
date
Witness's name Witness's signature date
(Applies for illiterate participants only)
I have explained purpose of this study to the participant, including the purpose,
procedures, risks and benefits of the study. I have answered any questions she / he had.
Investigator's name Investigator's signature Date
125
Appendix 2: Questionnaire
1. NAME AGE (30-40) (40-50) (50-60) (70-ABOVE)
COUNTy .
LOCATION .
2. Which plants do you use routinely?
3. Name plants in your local language that you consider most medicinal.
4. How do you prepare these herbal drugs? Choose where applicable for each of the
drugs you have mentioned. (Boiling, Soaking, burning, pounding, roasting).
5. What mode of drug application do you use? Chose where applicable for each of the
plants you have chosen. (Inhaling, bathing, gurgling, swallowing, chewing, and
other).
6. Do you get them locally or from elsewhere?
7. Where did you get these plants in the past and at present?
8. What diseases do they treat or prevent?
9. Pease list the signs by which you recognize the diseases?
10. What part is used?
11. Do you mix with other plants?
12. Where do you get your clients?
13. How many patients do you treat per day?
14. What is the availability ofthese plants?
15. Where did you get these plants in the past and at present?
16. How do you preserve these plants for future use?
17. How did you acquire the knowledge on traditional medicine?
18. Do you have any plans to pass this knowledge to your children and/or grandchildren
etc?
19. How often do your customers come back after initial treatment?
126
Appendix 3 :One-way ANOVA, MRSA versus Plants
Analysis of Variance for MRSA
Source DF SS MS F P
Plants 19 1633.500 85.974
Error 40 0.000 0.000
Total 59 1633.500
* *
Individual 95% CIs For Mean
Based on Pooled StDev
Level N Mean StDev ----------+---------+---------+------
Albizia 3 13.0000 0.0000 *
Balanite 3 6.0000 0.0000 *
Boscia a 3 6.0000 0.0000 *
Commipho 3 11.5000 0.0000 *
Ficus sy 3 8.5000 0.0000 *
Fuerstia 3 20.0000 0.0000 *
Melia vo 3 6.0000 0.0000 *
Neg. con 3 6.0000 0.0000 *
Ormocarp 3 6.0000 0.0000 *
post. co 3 26.0000 0.0000 *
Psidia p 3 6.0000 0.0000 *
Rhus nat 3 6.0000 0.0000 *
Ricinus 3 6.0000 0.0000 *
Senna di 3 11.0000 0.0000 *
Sesbania 3 6.0000 0.0000 *
127
Tamarind 3 6.0000 0.0000 *
Urtica d 3 6.0000 0.0000 *
Vernonia 3 9.0000 0.0000 *
Zanthoxy 3 6.0000 0.0000 *
Zanthoxy 3 9.0000 0.0000 *
----------~---------~---------~------
Pooled StDev = 0.0000 12.0 18.0 24.0
Appendix 4: One-way ANOVA, S. aureus versus Plants
Analysis of Variance for S. aureus
Source DF SS MS F P
Plants 19 1436.813 75.622 * *
Error 40 0.000 0.000
Total 59 1436.813
Individual 95% CIs For Mean
Based on Pooled StDev
Level N Mean StDev --------~---------~---------~--------
Albizia 3 10.0000 0.0000 *
Balanite 3 14.5000 0.0000 *
Boscia a 3 6.0000 0.0000 *
Commipho 3 9.5000 0.0000 *
Ficus sy 3 11.5000 0.0000 *
Fuerstia 3 19.0000 0.0000 *
Melia vo 3 6.0000 0.0000 *
128
Neg. con 3 6.0000 0.0000 *
Ormocarp 3 8.5000 0.0000 *
post. co 3 24.0000 0.0000
Psidia p 3 14.0000 0.0000 *
Rhus nat 3 14.0000 0.0000 *
Ricinus 3 12.0000 0.0000 *
Senna di 3 16.0000 0.0000 *
Sesbania 3 8.0000 0.0000 *
Tamarind 3 16.0000 0.0000 *
Urtica d 3 7.5000 0.0000 *
Vernonia 3 6.0000 0.0000 *
Zanthoxy 3 6.0000 0.0000 *
Zanthoxy 3 8.0000 0.0000 *
--------~---------~---------~--------
Pooled StDev = 0.0000 10.0 15.0 20.0
*
Appendix 5: One-way ANOV A, Salmonella versus Plants
Analysis of Variance for Salmonella
Source DF SS MS F P
Plants 19 823.6500 43.3500 * *
Error 40 0.0000 0.0000
Total 59 823.6500
129
Individual 95% CIs For Mean
Based on Pooled StDev
Level N Mean StDev --------+------ + + _
Albizia 3 6.0000 0.0000 *
Balanite 3 6.0000 0.0000 *
Boscia a 3 6.0000 0.0000 *
Commipho 3 6.0000 0.0000 *
Ficus sy 3 6.0000 0.0000 *
Fuerstia 3 6.0000 0.0000 *
Melia vo 3 6.0000 0.0000 *
Neg. con 3 6.0000 0.0000 *
Ormocarp 3 6.0000 0.0000 *
post. co 3 23.0000 0.0000 *
Psidia p 3 6.0000 0.0000 *
Rhus nat 3 6.0000 0.0000 *
Ricinus 3 6.0000 0.0000 *
Senna di 3 6.0000 0.0000 *
Sesbania 3 6.0000 0.0000 *
Tamarind 3 6.0000 0.0000 *
Urtica d 3 6.0000 0.0000 *
Vernonia 3 6.0000 0.0000 *
Zanthoxy 3 6.0000 0.0000 *
Zanthoxy 3 6.0000 0.0000 *
--------+---------+---------+--------
Pooled StDev = 0.0000 10.0 15.0 20.0
130
Appendix 6: One-way ANOV A, E. coli versus Plants
Analysis of Variance for E. coli
Source DF SS MS F P
Plants 19 1083.713 57.038 1.0E+16 0.000
Error 40 0.000 0.000
Total 59 1083.713
Individual 95% CIs For Mean
Based on Pooled StDev
Level N Mean StDev -+---------+---------+---------+-----
Albizia 3 6.0000 0.0000 *
Balanite 3 6.0000 0.0000 *
Boscia a 3 6.0000 0.0000 *
Commipho 3 6.0000 0.0000 *
Ficus sy 3 6.0000 0.0000 *
Fuerstia 3 6.0000 0.0000 *
Melia vo 3 6.0000 0.0000 *
Neg. con 3 6.0000 0.0000 *
Ormocarp 3 6.0000 0.0000 *
post. co 3 25.5000 0.0000 (*
Psidia p 3 6.0000 0.0000 *
Rhus nat 3 6.0000 0.0000 *
Ricinus 3 6.0000 0.0000 *
Senna di 3 6.0000 0.0000 *
Sesbania 3 6.0000 0.0000 *
131
Tamarind 3 6.0000 0.0000 *
Urtica d 3 6.0000 0.0000 *
Vernonia 3 6.0000 0.0000 *
Zanthoxy 3 6.0000 0.0000 *
Zanthoxy 3 6.0000 0.0000 *
-~---------~---------~---------~-----
Pooled StDev = 0.0000 6.0 12.0 18.0 24.0
Appendix 7: One-way ANOVA, B. subtilis versus Plants
Analysis of Variance for B. subtilis
Source DF
Plants 19
Error 40
Total 59
SS MS F P
1059.413 55.759 9.8E~ 15 0.000
0.000 0.000
1059.413
Individual 95% CIs For Mean
Based on Pooled StDev
Level N Mean StDev -~---------~---------~---------~-----
Albizia 3 12.0000 0.0000 *
Balanite 3 12.0000 0.0000 *
Boscia a 3 13.0000 0.0000 *
Commipho 3 12.0000 0.0000 *
Ficus sy 3 9.5000 0.0000 *
Fuerstia 3 17.0000 0.0000 *
132
Melia vo 3 10.0000 0.0000 *
Neg. con 3 6.0000 0.0000 *
Ormocarp 3 15.5000 0.0000 *
post. co 3 26.0000 0.0000 *
Psidia p 3 11.0000 0.0000 *
Rhus nat 3 11.5000 0.0000 *
Ricinus 3 13.0000 0.0000 *
Senna di 3 16.0000 0.0000 *
Sesbania 3 11.0000 0.0000 *
Tamarind 3 15.0000 0.0000 *
Urtica d 3 8.0000 0.0000 *
Vernonia 3 7.0000 0.0000 *
Zanthoxy 3 15.0000 0.0000 *
Zanthoxy 3 11.0000 0.0000 *
-~---------~---------~---------~-----
Pooled StDev = 0.0000 6.0 12.0 18.0 24.0
Appendix 8: One-way ANOV A, Candida versus Plants
Analysis of Variance for Candida
Source DF SS MS F P
Plants 19 228.1500 12.0079 8.4E~ 15 0.000
Error 40 0.0000 0.0000
Total 59 228.1500
133
Individual 95% CIs For Mean
Based on Pooled StDev
Level N Mean StDev -+---------+---------+---------+-----
Albizia 3 6.0000 0.0000 *
Balanite 3 6.0000 0.0000 *
Boscia a 3 6.0000 0.0000 *
Commipho 3 6.0000 0.0000 *
Ficus sy 3 6.0000 0.0000 *
Fuerstia 3 6.0000 0.0000 *
Melia vo 3 6.0000 0.0000 *
Neg. con 3 6.0000 0.0000 *
Ormocarp 3 6.0000 0.0000 *
post. co 3 12.5000 0.0000
Psidia p 3 6.0000 0.0000 *
Rhus nat 3 6.0000 0.0000 *
Ricinus 3 6.0000 0.0000 *
Senna di 3 6.0000 0.0000 *
Sesbania 3 12.5000 0.0000
Tamarind 3 6.0000 0.0000 *
Urtica d 3 6.0000 0.0000 *
Vernonia 3 6.0000 0.0000 *
(*
(*
Zanthoxy 3 6.0000 0.0000 *
Zanthoxy 3 6.0000 0.0000 *
-+---------+---------+---------+-----
Pooled StDev = 0.0000 6.0 8.0 10.0 12.0
134
Appendix 9: One-way ANOV A, C. neoformans versus Plants
Analysis of Variance for C. neoformans
Source DF SS MS F P
Plants 19 159.0000 8.3684 * *
Error 40 0.0000 0.0000
Total 59 159.0000
Individual 95% CIs For Mean
Based on Pooled StDev
Level N Mean StDev ----------+---------+---------+------
Albizia 3 6.0000 0.0000 *
Balanite 3 6.0000 0.0000 *
Boscia a 3 6.0000 0.0000 *
Commipho 3 6.0000 0.0000 *
Ficus sy 3 6.0000 0.0000 *
Fuerstia J 9.0000 0.0000 *
Melia vo 3 6.0000 0.0000 *
Neg. con 3 6.0000 0.0000 *
Ormocarp 3 6.0000 0.0000 *
post. co 3 13.0000 0.0000 *
Psidia p 3 6.0000 0.0000 *
Rhus nat 3 6.0000 0.0000 *
Ricinus 3 6.0000 0.0000 *
Senna di 3 6.0000 0.0000 *
Sesbania 3 6.0000 0.0000 *
135
Tamarind 3 6.0000 0.0000 *
Urtica d 3 6.0000 0.0000 *
Vernonia 3 6.0000 0.0000 *
Zanthoxy 3 6.0000 0.0000 *
Zanthoxy 3 6.0000 0.0000 *
-----------r----------r----------r------
Pooled StDev = 0.0000 8.0 10.0 12.0
Appendix 10: One-way ANOV A, A. niger versus Plants
Analysis of Variance for A. niger
Source DF SS MS F P
Plants 19 474.1500 24.9553 1.8E-r16 0.000
Error 40 0.0000 0.0000
Total 59 474.1500
Individual 95% CIs For Mean
Based on Pooled StDev
Level N Mean StDev ----r----------r----------r----------r---
Albizia 3 6.0000 0.0000 *
Balanite 3 6.0000 0.0000 *
Boscia a 3 6.0000 0.0000 *
Commipho 3 6.0000 0.0000 *
Ficus sy 3 6.0000 0.0000 *
Fuerstia 3 8.0000 0.0000 *
136
Melia vo 3 6.0000 0.0000 *
Neg. con 3 6.0000 0.0000 *
Onnocarp 3 6.0000 0.0000 *
post. co 3 18.0000 0.0000 *
Psidia p 3 6.0000 0.0000 *
Rhus nat 3 6.0000 0.0000 *
Ricinus 3 11.5000 0.0000 *
Senna di 3 6.0000 0.0000 *
Sesbania 3 8.0000 0.0000 *
Tamarind 3 7.5000 0.0000 *
Urtica d 3 6.0000 0.0000 *
Vernonia 3 6.0000 0.0000 *
Zanthoxy 3 6.0000 0.0000 *
Zanthoxy 3 6.0000 0.0000 *
----r----------r----------r----------r---
Pooled StDev = 0.0000 7.0 10.5 14.0 17.5
Appendix 11: One-way ANOVA, T. mentagrophyte versus Plants
Analysis of Variance for T. mentagrophyte
Source DF
Plants 19
Error 40
SS MS F P
716.8500 37.7289 6.6E-r 15 0.000
0.0000 0.0000
Total 59· 716.8500
137
Individual 95% CIs For Mean
Based on Pooled StDev
Level N Mean StDev ---------+---------+---------+-------
Albizia 3 6.0000 0.0000 *
Balanite 3 6.0000 0.0000 *
Boscia a 3 6.0000 0.0000 *
Commipho 3 6.0000 0.0000 *
Ficus sy 3 6.0000 0.0000 *
Fuerstia 3 12.0000 0.0000 *
Melia vo 3 6.0000 0.0000 *
Neg. con 3 6.0000 0.0000 *
Ormocarp 3 6.0000 0.0000 *
post. co 3 21.0000 0.0000 *
Psidia p 3 6.0000 0.0000 *
Rhus nat 3 6.0000 0.0000 *
Ricinus 3 6.0000 0.0000 *
Senna di 3 6.0000 0.0000 *
Sesbania 3 6.0000 0.0000 *
Tamarind 3 6.0000 0.0000 *
Urtica d 3 6.0000 0.0000 *
Vernonia 3 6.0000 0.0000 *
Zanthoxy 3 6.0000 0.0000 *
Zanthoxy 3 6.0000 0.0000 *
---------+---------+---------+-------
Pooled StDev = 0.0000 10.0 15.0 20.0
138
Appendix 12: One-way ANOV A, M. gypseum versus Plants
Analysis of Variance for M. gypseum
Source DF SS MS F P
Plants 19 1763.212 92.801 4.1E+15 0.000
Error 40 0.000 0.000
Total 59 1763.213
Individual 95% CIs For Mean
Based on Pooled StDev
Level N Mean StDev ---------+---------+---------+-------
Albizia 3 16.0000 0.0000 *
Balanite 3 6.0000 0.0000 *
Boscia a 3 10.0000 0.0000 *
Commipho 3 17.5000 0.0000 *
Ficus sy 3 15.5000 0.0000 *
Fuerstia 3 13.0000 0.0000 *
Melia vo 3 6.0000 0.0000 *
Neg. con 3 6.0000 0.0000 *
Ormocarp 3 8.5000 0.0000 *
post. co 3 22.0000 0.0000 *
Psidia p 3 20.5000 0.0000 *
Rhus nat 3 15.5000 0.0000 *
Ricinus 3 10.0000 0.0000 *
Senna di 3 17.0000 0.0000 *
Sesbania 3 6.0000 0.0000 *
139
Tamarind 3 16.0000 0.0000
Urtica d 3 6.0000 0.0000 *
Vernonia 3 6.0000 O.QOOO*
Zanthoxy 3 6.0000 0.0000 *
Zanthoxy 3 6.0000 0.0000 *
---------1----------1----------1--------
*
Pooled StDev = 0.0000 10.0 15.0 20.0