ANTIMICROBIAL EFFECTS OF LEAF AND ROOT EXCRACTS OF Crotalaria brevidens ON Candida

albicans, Staphylococcus aureus AND Escherichia coli

RESEARCH PROPOSAL IN PARTIAL FULLFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF BACHELOR
OF SCIENCE, WITH INFORMATION TECHNOLOGY OF MASENO UNIVERSITY

BY,

MUTEMBEI JOEL KABERIA

REG. NO. SC/00517/011

COURSE: SBT 413A- RESEARCH PROJECT 1

PROPOSED SUPERVISORS: 1.0 DR. D. M MUSYIMI OF DEPARTMENT OF RESEARCH

2.0 DR. G. OPANDE OF THE DEPARTMENT OF RESEARCH

CONTACT ADDRESS: MASENO UNIVERSITY

DEPARTMENT OF RESEARCH

PRIVATE BAG 40105,

MASENO.

2015

1
MASENO UNIVERSITY

SCHOOL OF BIOLOGICAL AND PHYSICAL SCIENCES

DEPARTMENT OF RESEARCH

ANTIMICROBIAL EFFECTS OF LEAF AND ROOT EXCRACTS OF Crotalaria brevidens ON Candida albicans,
Staphylococcus aureus AND Escherichia coli

ABSTRACT

Crotalaria brevidens(slenderleaf) is one of the African leafy vegetables that has been grown and
consumed in Kenya for a long time, its young leaves and shoots are used as cooked vegetable. It also
acts as an agent to promote suicidal germination of striga, a parasitic plant that is a major problem
weed for maize and millet growers. slenderleaf young leaves and shoots constitute to 100% of the daily
dietary requirements for vitamin A, vitamin C, iron, calcium and 40% of proteins when 100g of the fresh
weight are consumed. There has been increased resistance of pathogens to drugs and high cost of the
available drugs hence advocating this study. The vegetable has medicinal value where it has been used
in treating stomach related ailments and malaria thus can be a potential candidate for this study. The
objective of this study will be to investigate the antimicrobial effects of slenderleaf on candida albicans,
staphylococcus aureus and E.coli, and to determine presence of
phenols,steroids,glycosides,saponins,quinones,tannins,terpenoids and flavonoids. The study seeks to
address slenderleaf as an alternative remedy to the resistance of the available drugs and their high cost.
To achieve all this; the plant roots and leaves will be collected, shade dried and crushed to obtain fine
powder by extraction with petroleum ether, chloroform, acetone and methanol as the solvents using
soxhlet apparatus . Different concentrations of the plant extracts will be prepared by diluting the stock
extracts to petroleum ether, chloroform, acetone and methanol in the following concentrations :25%),
(50%), 75%and 100%.This will be replicated four times in a complete random design for accuracy and
precision. A control will be done with water as the plant extract. The screening of antimicrobial activity
of crude extracts will be carried out by measuring the zone of inhibition using agar diffusion method.
Data obtained will be subjected to analysis of variance (ANOVA) and means separated and compared
using least significance difference (LSD) at (p<0.05). The results obtained will be used to recommend for
extraction of active components of slenderleaf to manufacture natural drugs.
BY,
MUTEMBEI JOEL KABERIA REG NO. SC/00517/011 SIGNATURE____________
DATE_________________
SUPERVISORS,
DR. DAVID MUSYIMI SIGNATURE_______________
DATE____________________

DR. GEORGE OPANDE SIGNATURE_______________

DATE____________________

2
TABLE OF CONTENTS PAGE

Title------------------------------------------------------------------------------------------------------------------------------- 1

Abstract-------------------------------------------------------------------------------------------------------------------------- 2

Table of contents-------------------------------------------------------------------------------------------------------------- 3

Chapter one-------------------------------------------------------------------------------------------------------------------4

1.0 Introduction------------------------------------------------------------------------------------------------------------------- 4

1.1 Problem statement---------------------------------------------------------------------------------------------------------- 5

1.2 Justification------------------------------------------------------------------------------------------------------------------- 5

1.3 Objectives--------------------------------------------------------------------------------------------------------------------- 6

1.4 Hypothesis--------------------------------------------------------------------------------------------------------------------- 6

Chapter two-------------------------------------------------------------------------------------------------------------------7

2.0 Literature Review----------------------------------------------------------------------------------------------------------- 7

Chapter three----------------------------------------------------------------------------------------------------------------12

3.0 Materials and methods-------------------------------------------------------------------------------------------------- -12

Chapter four------------------------------------------------------------------------------------------------------------------14

4.0 Data analysis---------------------------------------------------------------------------------------------------------------- 14

4.1 Expected outcome--------------------------------------------------------------------------------------------------------- 14

4.2 References------------------------------------------------------------------------------------------------------------------- 15

4.3 Appendix 1------------------------------------------------------------------------------------------------------------------- 18

4.4 Appendix 2------------------------------------------------------------------------------------------------------------------- 19

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 CHAPTER ONE

1.0 INTRODUCTION

Slenderleaf is a plant in the family Leguminosae/Fabaceae that is consumed in Africa

(Schippers,2002;maundu et al.,1999;Abukutsa-onyango,2004). The plant grows in grassland and
bushland, often on termite moulds at roadsides, in cultivated lands, disturbed forests and near
seasonally flooded areas. It grows from 500m-2700m above sea level and is propagated by seeds. In
Kenya its leaves are widely used as a vegetable in western Kenya(Luo, Luhya, west Pokot) and this
species is bitter(Maundu et al., 1999). Slenderleaf has bluish-green leaves and grows to a height of
210cm, has bright yellow flowers and the seed color normally contain anthocyanins(Abukutsa-
onyango,2004). It occurs in the wild from northern Nigeria eastwards to Ethiopia and south to southern
Tanzania. Slenderleaf is reported as cultivated and eaten as a vegetable in Kenya, Sudan, Uganda and
Tanzania(Chweya and Ezyguire;1999,Chweya, 1997).Presently about 4 billion people (approximately 60
- 80 %) of the world population and 90 % in Africa use herbal medicine for their primary health care
(Ramzi et al., 2008, Musyimi et al., 2008, Parekh et al., 2006, Hamann, 1991) with slenderleaf falling in
this category.

Medicinal plants have long been the subject of human curiosity and need. Plant derived products are
present in 14 of 15 therapeutic categories of pharmaceutical preparations that are currently
recommended by medical practitioners and they form an important part of the health-care
system(Caceres et al,1993). A number of medicinal applications have been reported on slenderleaf. In
Kenya the leaves are used to cure stomach-aches, swellings and malaria(Olembo et al.,1995; Abukutsa-
Onyango;2004,2007). Slenderleaf has been reported to have several agronomic advantages that include:
ability to produce seeds under tropical conditions, performs well in nitrogen stressed soils due to their
ability to fix nitrogen in the atmosphere, drought tolerance and intercropping suitability and as a fodder
crop and as green manure (Chweya,1997; Abukutsa-Onyango;2004,2007).

Slenderleaf is rich in vitamins, minerals, trace elements, dietary fibre and proteins(Humphrey et
al.,1993; Mathenge,1997;Maundu,1997; Nordeide,1997). The composition of this vegetable per 100g
edible portion is: water 74.5g, protein 8.8g, calcium 222mg, iron 0.8mg(Leung et al.,1996). Effectively
the vegetable is important in food security, during times of drought or poor harvest and are also vital for
income generation. Withstanding their value as food, the vegetables also serve as source of medicines
hence important in their ecological, agronomical and cultural values(Geissler et al., 2002;
Abbiw,1997;Okafor,1997).

Slenderleaf has a bitter taste which is attributed to presence of alkaloids and phenolic
compounds(Schippers,2002;Abukutsa-onyango,2007) hence the need to carry out phytochemical
screening to investigate the phytochemicals present. The nutritional and antioxidant potential of
Slenderleaf is of health or nutraceutical significance and thus should help encourage its
consumption(Mibei,2011; Manzoor and Maksuda, 2000).

4
The development of drug resistance in human pathogen against commonly used antibiotics has
necessitated the need for finding potential new compounds with therapeutic uses (Dahale et al,2010).
There has been increased resistant strains of clinically important pathogens which have led to the
emergence of new bacterial strains that are multi-resistant (Aibinu et al.,2003). The non-availability and
high cost of new generation antibiotics with limited effective span have resulted in morbidity and
mortality (William,2000). Economic circumstances and widespread belief in the effectiveness of many
traditional therapies may have also contributed to the high dependence on herbal medicine. On
average, herbalists charge between Ksh. 20 and Ksh. 150 for cases, and often the patients pay in kind.
Lambert et al., (2005) reported that in Sub-Saharan Africa, 480 million people (60%) do not have access
to modern health care and pharmaceuticals. Khalil (2007) indicated that people s economic status,
population growth and prices of pharmaceutical drugs are factors driving communities into using herbal
medicines. Hence a need to look into effective antimicrobial agents among materials of plant origin
arose such as slenderleaf with an aim of discovering potential active ingredients that can be used as
alternative sources of new antimicrobial drugs(Pretorius,2003; Moreillion et al.,2005).

Slenderleaf causes suicidal germination of striga weed (Striga hermonthica) which has been reported
worldwide as an obnoxious cereal weed and thus has the capability to reduce the striga weed
population in the soil (Abukutsa-Onyango,2007).

Studies of antimicrobial effects of other species within the genus like C. burhia and the closely related C.
ochroleuca have been done but hardly on slenderleaf. However, studies on traditional medicines,
indigenous leafy vegetables have shown its therapeutic effects hence advocating for this research.
Studies have shown that countries that retain indigenous vegetable diet and had high consumption of
these vegetables are much less likely to be affected by cardiovascular diseases, diabetes and other
adverse consequences of the nutrition in transition(Johns and Sthapit,2004). Findings by johns musingizi
et al.,(2000) found that there was a potential relationship of indigenous vegetables and ability to treat
diabetes, gastrointestinal tract infections and protozoan parasites in Kenya and Tanzania(Musingizi et
al.,2000). Slenderleaf thus being one of them has inhibitory potential and has indicated by (Kimiywe
etal.,2007), there is need to do further investigation to ascertain the perceptions. This has now created a
need to investigate inhibitory effects of slenderleaf on selected microbes.

1.1 PROBLEM STATEMENT

In this 21st century, there has been hiking of prices of medicines used to diagnose bacterial and fungal
infections. This has been cumbersome for the local people to access medicines with regard to their
economic status which calls for a quick intervention. Furthermore, in the last few years the number of
immunocompromised and immunosuppressed patients who frequently develop opportunistic, systemic
and superficial mycoses(Caceres et al.,1993) such as candidiasis, dermato-mycosis,fungal infections have

5
increased dramatically(Ribbon,1988;Diamond,1991). This mainly is due to non-availability of effective
antifungal drugs for systemic infections and toxicity of available drugs like Amphotericin-B(Sarah,1991).
On the other hand there has been increased resistant strains of clinically important pathogens which
have led to the emergence of new bacterial strains that are multi-resistant. Thus there is an increased
need for development of alternative antipathogenic substances. One possible approach is to screen
local medicinal plants in search of suitable antipathogenic ,antibacterial and antifungal substances.
Slenderleaf has not been exploited as antimicrobial agent and thus could be the possible remedy for this
since its attributed to contain active antimicrobial ingredients hence medicines can be developed from
it.

1.2 JUSTIFICATION

Medicinal plants have long been the subject of human curiosity and need. Plant derived products are
present in 14 of 15 therapeutic categories of pharmaceutical preparations that are currently
recommended by medical practitioners and they form an important part of the health-care
system(Caceres et a,1993). Previous studies have been done on indigenous medicinal leafy
vegetables(Kimiywe et al.,2007),herbal remedies(Timothy et al.,1990), cultural and nutrition significance
on slenderleaf but not on its antimicrobial effects. Previous studies on closely related species C.
ochlereuca has proved to have inhibitory effects on various microbes. Personal communication with
some of the people residing in western, Kenya, has revealed slenderleaf as potential vegetable that
heals stomach ailments and malaria. Olembo et al.,(1995) stated that traditional leafy vegetables have
medicinal properties for treatment of HIV/AIDS,stomach-related ailments and other diseases. The
potential source of antimicrobial agents developing from plants species is an alternative strategy for the
production of safe and standardization of a phytomedicines against harmful microbes. The plant based
antimicrobial agents have enormous therapeutic potential while they dont have any major side effects
to the human beings (Leung et al.,1996). This study aims at investigating antimicrobial activity of
slenderleaf on Escherichia coli, Candida albicans , and Staphylococcus aureus. This would help solve the
problem of acquiring synthetic antibiotics and thus diversify its use globally.

.3 1OBJECTIVES

1.3.1 General objectives

To determine the antimicrobial effects of root and leaf extracts of Crotalaria brevidens on candida
albicans, staphylococcus aureus and Escherichia coli and its phytochemical compounds.

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 1.3.2 Specific objectives

(i) To determine the inhibitory effects of roots and leaf extracts of C. brevidens on
Staphylococcus aureus
(ii) To determine the inhibitory effects of roots and leaf extracts of C. brevidens on
E. coli
(iii) To determine the inhibitory effects of roots and leaf extracts of C. brevidens on
Candida albicans
(iv) To determine the phytochemical compounds of C. brevidens

1.4 HYPOTHESES

Crotalaria brevidens roots and leaf extracts have inhibitory effects on Staphylococcus aureus, Candida
albicans and Escherichia coli.

Crotalaria brevidens possesses many phytochemical compounds in its roots and leaves.

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 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Growth habit, taxonomy and classification of slenderleaf

Slenderleaf is a plant in the Fabaceae family that is eaten in Africa. It is an erect herb with many
branches usually 0.5m to 1.2m high. The leaves are devided into three narrow leaflets typically10cm
long by 2cm wide. Flowers are yellow with very conspicuous reddish purple veins. The fruits are long
narrow pods as large as 5cm long by 0.7cm wide which are black when dry. The seeds are yellowish
orange to dark red(Maundu et al.,1999). The herbal plant and a vegetable is referred to as Rattle pod,
Rattlebox(Ethiopia), Sunhemp or slenderleaf. In Luyha it is called Miroo while in Luo its named as Mitoo.
Other names includes: Crotalaire, Sonnette(French) and Marejea in Swahili.

The scientific classification is as shown below:

Kingdom : plantae

Subkingdom:Tracheobionta (vascular plants)

Superdivision:Spermatophyta (seed plants)

Division:Magnoliophyta (flowering plants)

Class:Magnoliopsida (dicotyledons)

Subclass:Rosidae

Order:Fabales

Family:Fabaceae/Leguminosae

Genus:Crotalaria L

Species:Crotalaria brevidens Benth

Synonym : Crotalaria intermedia.

2.2 Ecological distribution of slenderleaf

8
The centre of diversity of the species is believed to be of Africa. The species is commonly cultivated and
consumed throughout East Africa such as Ethiopia, Uganda, and Kenya but less in West Africa like
Nigeria. In fact its much abundant in western Kenya where it is eaten as a staple vegetable. It also sold
in markets in major towns of Western Kenya.

2.3 Medicinal value of slenderleaf and phytochemical compounds of slenderleaf

2.3.0 Medicinal value of slenderleaf

slenderleaf is one of the most important African indigenous vegetable whose young leaves and shoots
are consumed and contributes 100% of the daily dietary requirement for vitamin A, vitamin C, iron,
calcium and 40% proteins when 100g of fresh weight are consumed (Maundu et al.,1990; Abukutsa-
Onyango,2003). It has medicinal Implications where it treats malaria and stomach related ailments
(Olembo et al.,1995; Abukutsa-Onyango, 2004). In Luo land it is also used to treat boils, swellings and
improves appetite(Personal communication ,Aloo Omondi).

Slenderleaf is consumed boiled or fried in oil and is also used as a potherb in stews or soups. Its often
boiled with Jutes mallow (Corchorus autorius L.) to reduce the bitter taste, tomatoes, onions, oil and/or
milk are added. In Kenya leaves are used to treat sore-throat and mouth thrush (Chweya,1999;
Abukutsa-Onyango,2003). Henceforth, inhibitory effects on Candida albicans, Escherichia coli and
staphylococcus aureus is necessary.

2.3.1 Phytochemical compounds of slenderleaf

Schippers,(2002) associated the bitterness of slenderleaf to possession of alkaloids and phenols.

Moreover, Abukutsa, (2007) in her study of response of slenderleaf to nitrogen application also
indicated that slenderleaf has a nasty taste when consumed and attributed it to presence of
phytochemicals. However, they did not investigate or carry out the phytochemical composition to
ascertain the allegations.Preliminary phytochemical screening of closely related species Crotalaria
ochreleuca showed presence of alkaloids, saponins, cardenolides, flavonoids and polyphenols(Orech et
al,2005).

The medicinal values of plants and their component phytochemicals such as alkaloids, tannins,
flavonoids and other compounds have been found to produce a definite physiological action on human
body (Taiye and Iyamah,2014). A systematic search for useful bioactivities from medicinal plants is now
considered to be a rational approach in nutraceutical and drug research. The presence of active
phytochemicals in some herbs used in curing many diseases affecting people in African communities has
been documented(Odetola and Bassir,1986). More particularly, the phytochemical properties of some
plants used in forklore medicine in Nigeria has been investigated(W.H.O,1978). Therefore, there is

9
increased need to do phytochemical screening on slenderleaf to justify the medical value implicated on
it.

2.4 Nutritional value of slenderleaf

The composition of leaves of slenderleaf and the closely related C. ochroleuca per 100g of fresh edible
portion is water 74.5g, proteins 8.8g, calcium 222mg, iron 0.8mg (Leung et al.,1996; Maundu et al.,
1999, Abukutsa-Onyango, 2003). The composition is comparable to other dark green vegetables, except
that the dry matter content is higher than the average. The leaves are bitter due to the presence
chemicals such as pyrrolizidine, alkaloids, ditarpenes and phenolic compounds. This has necessitated the
need to carry out phytochemical screening at various plant extracts concentrations

2.5 Review of plant pathogens under study

They are pathogenic fungi, a Gram-positive bacteria and a Gram-negative bacteria. Gram-positive
bacteria are those bacteria that give a positive result in the gram-stain test. Gram-positive bacteria take
up the crystal violet (Dark blue dye) after an alcohol wash and appears to be purple-colored when seen
through a microscope. This is due to the thick peptidoglycans layer in the bacterial cell wall. On the
hand, Gram-negative cannot retain the violet stain after the decolorisation step with alcohol since it
degrades the outer membrane of Gram-negative cells making the cell more porous and incapable of
retaining the crystal violet stain. Due to their thinner peptidoglycan and being sandwiched between
inner cell membrane and the bacterial outer membrane, it then takes the counter stain (safranin) and
appears red or pink. Since Gram-positive are more receptive to antibiotics than Gram-negative is our
subject of study and this is attributed to its cell wall. This is how inflammation cycle starts in the body of
an organism. The organisms for study are pathogenic fungi (Candida albicans), Gram-positive
(Staphylococcus aureus) and Gram-negative (E.coli).

2.5.1 Biology of Candida albicans

Candida albicans is a diploid fungus that grows both as yeast and filamentous cells and a causal agent of
opportunistic oral and genital infections in humans (Ryan,2004., Hube,2007). Although often referred as
dimorphic, C. albicans is in fact polyphenic. When cultured in standard yeast laboratory medium, it
grows as ovoid yeast cells. However mild environmental changesin temperature and ph can result in
morphological shift to pseudo-hyphal growth (Sudbury, 2011). Pseudo-hyphae share many similarities
with yeast cells, but their role during candidiasis remains unknown. When Candida albicans are grown in
a medium that mimics the physiological environment of a human host, they grow as true hyphae. Its
ability to form hyphae has been proposed as virulence factors as these structures are often observed
invading tissue and stains that are unable to form hyphae are defective causing infection. Candida
albicans can also form chlamydospores, the function of which remains unknown (Berman,2002;
Staib,2007). The fungi is normally present on the skin and the mucous membranes such as the vagina,
mouth or rectum. The fungus can also travel through the blood stream and affect the throat, intestines,
and heart valves. Candida albicans becomes an infection agent when there is alteration of the normal

10
flora of the body allowing its growth. When growth persists it weakens the intestinal walls, penetrating
through into the blood stream and releasing its toxic byproducts throughout the body as they spread;
these toxins cause damage to body tissues and organs making immune susceptible (Lederber, 2004).

2.5.2 Pathology of Candida albicans

C. albicans is a commensal and a constituent of the normal gut flora comprising microorganisms that live
in the human mouth and gastrointestinal tract. The fungus lives in 80% of the human population without
causing harmful effects, although its overgrowth causes candidiasis which is commonly observed in
immune-compromised individuals such HIV infected patients. A common form of candidiasis restricted
to the mucosal membrane in mouth or vagina is thrush which can be easily cured to patients who have
their immune not compromised . To infect host tissue, the usual unicellular yeast-like form of candida
albicans react with environmental cues (or different compounds such as N-acetylglucosamine or proline)
and switches into an invasive, multicellular, filamentous form, a phenomenon called dimorphism (Ryan
and Ray,2004).

There is evidence that prolonged treatment with broad-spectrum antibiotics such as chloramphenicol
and the tetracyclines may predispose to the development of Candidiasis perhaps by killing off normal
microbial antagonists to the fungus. Candidiasis of the mouth is called thrush which is common in bottle
fed babies and in persons with terminal illness. Candidiasis of the mucous membrane of the vagina is
called vaginitis, and is most common in diabetic or pregnant women. Candidiasis of the skin is most
likely to occur in the folds, especially in moist areas such as the armpits, between the buttocks , and in
the navel. The folds between the fingers and around the nails are often affected in persons whose hands
are frequently wet. Occassionally the fungus invades the respiratory system, producing a
bronchopulmonary infection that stimulates miliary tuberculosis. The infection rarely becomes
systematic but when it does so it may damage the lining of the heart or the meninges(Scheinfield and
Lambiase , 2010).

2.5.3 Biology of Staphylococcus aureus

Staphylococcus aureus is a gram positive bacteria that is a member of firmcutes, and is frequently found
in the human respiratory tract and on the skin. It is positive for catalase and nitrate reduction.it is
estimated that 20% of the human population are long term carriers of S.aureus which can be found as
part of the normal skin flora and in anterior naves of nasal passages (kluytmans et al.,1997; cole et
al.,2001). This bacteria appears as grape like clusters when viewed through a microscope and has large,
round, golden yellow, colonies often with hemolysis when grown on blood agar plates(Ryan and
Ray,2004).

2.5.4 Pathology of staphylococcus aureus

S. aureus causes staph infections and is a successful pathogen due to a combination of nasal carriage
and bacterial immune-evasive strategies. S.aureus can cause a range of illnesses ranging from minor skin

11
infections such aspimples, imperigo, boils(furuncles), cellutis, folliculitis, carbuncles, scalded skin
syndrome and abscesses, to a life threatening diseases such as pneumonia, meningitis, osteomyelitis,
endocarditis, toxic shock syndrome(TSS), bactereremia and sepsis. Its incidence ranges from skin, soft
tissue, respiratory, bone,joint, endovascular to wound infections. It is still one of the five most common
causes of nosocomial infections and is often the cause of postsurgical wound infections. Each year, some
500,000 patients in united states hospitals contract a staphylococcal infection(Bowersox and
John,1999).

S . aureus can infect tissues when the skin or mucosal barriers have been breached. S. aureus can
spread through contact with pus from an infected wound , skin to skin contact with an infected
person by producing hyaluronidase that destroys tissues and contact with objects such as towels,
sheets, clothing or athletic equipment used by an infected person. S .aureus is one of the causative
agent of mastitis in dairy cows. Its large polysaccharide capsule protects the organism from recognition
by the cows immune system defences (Jarraud,2001).

2.5.5 Biology of Escherichia coli

E .coli is a gram negative , facultatively anaerobic, rod- shaped bacteria of the genus Eschereria that is
commonly found in the lower intestines of warm blooded organisms(endoderms). The bacteria is one of
the most diverse bacterial species with several pathogenic strains with different symptom and with only
20%of the genome, common to all strains (Lukjancenko, 2010).

E .coli and related bacteria constitute about 0.1%of gut flora and faecal. Oral transmission is the major
route through which pathogenic strains of the bacterium cause disease ( Lan and Reeves, 2002). Virulent
strains of E. coli can cause gastro enteritis, UTI and neonatal meningitis, in rare case, virulent strains are
also responsible for haemolytio-uremic syndrome, peritonitis, mastitis, septicemia and gram negative
pneumonia(Vogt and Dippold, 2005).

2.5.6 Pathology of E.coli

Most of E. coli strains are harmless, but some serotypes are pathogenic and can cause serious food
poisoning in humans, and are occasionally responsible for product recalls (Vogt and Dippold, 2005). The
harmless strains are part of the normal flora of the gut and can benefit their host by producing vitamin
k2(Bentley et al; 1982) and by preventing the establishment of pathogenic bacteria within the intestine
(Hydault et al.,2001; Reid et al.,2001).The pathogenesis of E. coli can well be explained as;

EHEC(Enter hemorrhagic) which is a syndrome represented by a single strain(serotype 0157:H7) and

which causes diarrhea distinct from some others(including shigela)in that there is copious bloody
discharge and no fever. The life threatening situation is its toxic effects on the kidneys(hemolytic
uremia). The E. coli does not invade the intestinal mucosa as readily as shigella but does not produce
two toxins that are virtually identical to shiga toxin. The toxins play a role in the intense inflammatory
response and are enhanced by iron deficiency (Parry, 2007).

12
Another aspect is the enteropathogenic whereby it induces a watery diarrhea. The organisms to adhere
to the intestinal mucosa are moderatively invasive which causes an inflammatory response. The
diarrhea and other symptoms are probably caused by invasion of the host cells and interference with
cellular process rather than by production of toxins.

Enterotoxigenic which are important in causing diarrhea in infants and travelers especially in regions of
poor sanitation. The disease may vary from minor discomfort to severe cholera like disease. Disease
requires colonization and excretion of one or more toxins. The toxins lead to secretion fluid and
resulting diarrhea(Reed,2005).

Entero-invasive is also an aspect which is a type which closely resembles shigellosis in its mechanism.
These organisms penetrate the cell wall of the colon causing cell destruction and extreme diarrhea.
They do not produce shiga toxin. To sum up is the nature of being enteroaggregative which has its
distinguishing feature as the ability of the organism to attack to tissue culture cells in aggregative
manner. These strains are associated with persistent diarrhea in young children. They adhere to the
intestinal mucosa and cause non-bloody diarrhea and inflammations. This may be related to toxin
production(Parry,2007).

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 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Collection of plant materials

Plant material of slenderleaf will be obtained from Maseno university amphitheatre between
April and May 2015. The material will be will be cleared off adhered soil particles in the field by
shaking and placed inside polythene paper bags where it will be carried along to the university
Botany laboratory and then will be washed by running water to get rid of the adhering soils and
other external microorganisms. The plant roots and leaves will be allowed to dry under shade
for about 10 days(Musyimi et al.,2008), and then roots and leaves will be powdered using an
electric blender and stored in a sterile bottle containing 25ml of sterile distilled water at room
temperature for 6 hours(Bonjar,2004). Both aqueous,petroleum ether,chloroform,acetone and
methanol extraction will be carried out by the help of soxhlet extractor(quick fit U.K). 50g of
powdered leaves will be extracted with 250ml of 40% solvent(Akinyemi et al;2005). Extracts of
roots and leaves will be separated. In organic extraction, Soxhlet apparatus will be employed in
obtaining extracts from powdered plant materials by polar methanol ,chloroform, ether and
acetone as solvents.
3.2 Sources and maintenance of organisms
A fungal yeast Candida albicans , gram negative bacteria(Escherichia coli) and gram positive
bacteria Staphylococcus aureus will be obtained from Maseno university and will be maintained
on Mueller Hinton Agar(Oxoid, UK) medium.

3.3 Culture media preparation

Mueller Hinton Agar will be prepared according to manufacturers instructions, autoclaved and
dispensed at about 25ml per plate in 12X12cm petri dishes. These set plates will have a
thorough incubation overnight to ensure they are sterile before use.
3.4 Antimicrobial Bioassays
Plant extracts will be diluted in distilled water and methanol respectively to make stock solution.
Different concentrations of the plant extracts will be prepared by diluting the stock extracts to
water,petroleum ether, methanol, chloroform and acetone in the concentrations (25%), (50%),
(75%) and 100%. Each labeled medium plate will be uniformly inoculated with Candida albicans,
Escherichia coli and Staphylococcus aureus by using a sterile cotton swab rolled in the
suspension to streak the plate surface in a form that lawn growth can be observed.
Susceptibility testing will be carried out by measuring the inhibitory zone diameters on the
Mueller Hinton (Oxoid, UK) using convectional paper by disc method. Inhibitory zone distances
will be measured and rounded off to the nearest whole numbers (mm) for analysis. The

14
 measurements will be measured by rulers. The treatments will be replicated four times for
accuracy and precision.
To measure the MIC values, various concentrations of the stock , 10, 20, 30mg/ml will be
assayed against the test bacteria. The minimum inhibitory concentration will be defined as the
lowest concentration able to inhibit any visible bacterial growth (Prescott et al.,1999; Bonjar,
2004).

3.5 Disc method

Circular discs, 8mm diameter each will be cut from Whatman No.1 filter paper using a paper
punch and each dipped in a known concentration of the extracts for about 2 minutes, then will
be gently transferred to the centre of the inoculated agar media. Petri dishes inoculated with
bacteria and fungi will be kept for incubation for 24hrsat 370c and 250c respectively. The
diameter of inhibition zones will be measured with a ruler and compared with the n-hexane
control disc(Musyimi et al., 2008)
3.6 Phytochemical extraction method
Extract preparation: the dried leaves powder (25gm) will be extracted in soxhlet apparatus by
using 25ml of different solvents having polarities of ethanol, methanol, ethyl acetate, acetone
and n- hexane for 48hrs and then concentrated by evaporation. These prepared extracts will be
used for phytochemicals analysis. All chemicals used in the study were analytical grade.
Phytochemical screening will be done according toMusyimi et al.,(2008), Harborne, (1998),Mibei
et al.,2012) and Akinyemi et al.,(2005) as explained below :-
3.6.1 Determination of alkaloids: Two grams of the extract will be extracted by warming it for 2
minutes with 20ml of 1% H2SO4 acid in a 50ml conical flask on a water bath, with intermittent
shaking. It will then be centrifuged and the supernatant will be pipetted off into a small conical
flask. One drop of Meyers reagent will be added to 0.1ml supernatant in a semi-micro tube. A
cream precipitate will indicate the presence of alkaloids.
3.6.2 Determination of flavonoids: Five milliliters of dilute ammonia solution will be added to a
portion of the aqueous filtrate of the extract followed by addition of concentrated H2SO4. A
yellow coloration will indicate the presence of flavonoids.
3.6.3 Determination of tannin: Tannin will be determined by the Folin-Denis colorimetric
method described by Harborne, (1998). About 0.5 g of the dried powdered samples will be
boiled in 20ml of water in a test tube and then filtered through Whatman No. 42 filter paper. A
few drops of 0.1% ferric chloride will be added. A brownish green or a blue-black coloration will
indicate the presence of tannins(Akinyemi et al.,2005).
3.6.4 Determination of phenols: Ferric chloride test will be carried out where the extract will be
diluted to 5ml with distilled water. To this, a few drops of neutral 5% Ferric chloride solution will
be added. A dark green or a blue-black color will indicate the presence of phenolic
compounds(Mibei et al.,2012).
3.6.5 Determination of steroids: Two ml of acetic anhydride will be added to 0.5 g ethanolic
extract of each sample with 2ml H2S04. The color change from violet to blue or green in some
samples will indicate the presence of steroids (Harborne, 1998).

15
3.6.6 Determination of saponin: About 2 g of the powdered sample will be boiled in 20ml of
distilled water in a water bath and filtered. Ten milliliters of the filtrate will be mixed with 5ml of
distilled water and shaken vigorously to form a stable persistent froth. The froth will be mixed
with 3 drops of olive oil and shaken vigorously, then will be observed for the formation of
emulsion(Musyimi et al.,2008).
3.6.7 Test for terpenoids : Five milliliters of each extract will be mixed with 2ml of chloroform,
and concentrated sulphuric acid will be carefully added to form a layer. A reddish brown
coloration that will form at the interface will indicate presence of terpenoids (Harborne, 1998).
3.6.8 Test for anthraquinones :Powdered plant material will be boiled with 10% HCl for a few
minutes, then filtered and allowed to cool. This will then partitioned against equal volume of
chloroform. Formation of rose-pink color upon addition of 10% aqueous ammonium solution
will indicate the presence of anthraquinones (Harborne, 1998).
The presence of various phytochemicals in prepared extracts will be reported after the
experiments for data analysis.
3.6.9 Cardiac glycosides : Five ml of extract will be treated with 2ml of glacial acetic acid
containing a drop of FeCl3 solution. This will then underplayed with 1ml conc. H2SO4. A brown
ring of the interface will indicates a deoxy sugar characteristics of cardenolides.

16
 CHAPTER FOUR

4.0 DATA ANALYSIS

4.1 Experimental data analysis
Data from the study obtained for the relationships between the plant part extracts,
concentrations and microorganisms will be subjected to analysis of variance (ANOVA) using SPSS
statistical package. Means will be separated and compared at (p<0.05) using the less significant
difference.

4.2 EXPECTED OUTCOME

a. There will be no growth of E.coli ,S taphylococcus aureus, and Candida albicans on the
region concentrated with slenderleaf extract
b. There will be documentation of specific phytochemicals present in the extracts of
slenderleaf

17
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APPENDIX 1:BUDGET

ITEM QUANTITY PRICE

petri dishes 38 380
plant material 5kg 650
Filter paper 4pkts 200
Distilled water 5 liters 1000
Methanol 3 liters 2500
Acetic 1 liter 1000
Sulphuric acid 2 liters 2000

Chloroform 1 liter 1000

Ethanol 1 liter 1000

Ethyl acetate 1 liter 1000

Sodium hydroxide 2 liters 2000
Iron chloride 1 liter 1000
Test tubes 25 4000
Mueller Hinton agar 4000
Mark pens 3 pieces 180
Labels 350
Photocopying, printing and 2150
binding
Assistant researcher 1200
Transport 60
TOTAL 22470

23
APPENDIX 2: WORK PLAN
ACTIVITY APRIL 2015 MAY 2015 JUNE 2015 JULY 2015
Collection of
materials
Laying of
experiment
Observation of
inhibitory activity
Reviewing on the
experimental
progress
Data analysis
Report writing and
literature review
Binding and
submission

DOING SMALL THINGS IN A GREAT WAY- ANY DREAM IS VALID JUST DONT ABORT IT.

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