EVALUATION OF ANTIFUNGAL PROPERTIES OF EUPHORBIA PROSTRATA

EXTRACT AGAINST CANDIDA ALBICANS.

BY

AKOTH VIVIAN OCHIENG

P110/0406/2012

A RESEARCH PROJECT REPORT SUBMITTED TO THE DEPARTMENT OF

PHARMACY AND COMPLEMENTARY AND ALTERNATIVE MEDICINE IN
PARTIAL FULFILLMENT OF REQUIREMENTS FOR THE AWARD OF THE DEGREE
OF BACHELOR OF PHARMACY KENYATTA UNIVERSITY
DECLARATION
DEDICATION
ACKNOWLEDGEMENT
TABLE OF CONTENT
LIST OF TABLES
LIST OF FIGURES
LIST OF ABBREVIATIONS
 ABSTRACT

Medicinal plants have been used over centuries to improve human life primarily due to the
bioactive constituents such alkaloids, saponins, cardiac glycosides and tannins found in them.
These constituents have found use in treatment and lessening of symptoms since they are like
those in conventional drugs. Euphorbia prostata is wide that belongs to family, Euphorbiaceae,
is used widely in African traditional medicine for management of various diseases and
conditions. For instance, warmed leaves of the E. prostrata are crushed and squeezed onto the
body to treat fungal infection. The study was designed to investigate the antifungal properties of
of water and methanolic extracts of E. prostrata.

CHAPTER ONE: INTRODUCTION

1.0 Background information

The World health organization estimated about 80% of population living in Third world countries

exclusively uses traditional medicine to meet their primary health care needs. Since medicinal

plants are the backbone of traditional medicine, it can be deduced that over 3300 million of

people living in developing countries utilizes medicinal plants for lessening of symptoms and

treatment of various diseases and conditions. In Africa, the extensive use of medicinal plants is

highly linked to cultural beliefs and economic uses. Medicinal plants have been reported in

traditional medicine to be effective in treatment of animal and human mycoses and are currently

considered to be the valuable source of new antifungal drugs. Furthermore, methanol, ethanol

and water extracts of pomegranate peels were found to exhibit potent antifungal activity against

Candida alibicans . Medicinal plants have always been the best source to obtain conventional

drugs due their active biological constituents and are considered as an element in the complex

equation of healing. Even in allopathic medicine, 25% of the prescription consist of substances

derived from higher plants. Among the potential uses of medicinal plants are management of

infectious diseases caused by bacteria, fungi and virus, pain and inflammation. Several

medicinal plants have been investigated for their phytochemicals. On the other hand,

investigation on the chemical compounds present in medicinal plants responsible for specific
biological activity has become an interesting field of research. A few biological compounds

have been proved active even in controlled clinical evaluation.

According to Calderone & Fonzi (2001), Candida albicans also referred to as Monilia is

a fungus that often present on the skin and mucous membranes such as rectum, vagina and

mouth. When the fungus gets into the body, it can also travel through the bloodstream causing

infection of the throat, heart valves and intestines. The fungus can be present in the body or on

the skin without causing infection. However, Candida albicans become infectious agent when

there is change in body conditions that support it to grow out of control.

Most of the time, candida infections of the skin, mouth and vagina occur without

apparent reason but common cause of the infection is use of antibiotics which sometimes also

clear beneficial microorganism as well as harmful causing alteration of the body normal flora.

Changes in the body normal flora permit growth of candida out of control in their places

resulting in candidiasis or yeast infections. When C. albicans grows in the mouth it is referred to

as thrush, common in infants and yeast infection in the vagina. In addition, it causes candida

onychomycoses in the nails or paronychia when it grows next to the nail (Calderone & Fonzi,

2001).

Moreover, it can also infect the digestive drug and penis. Candida infection of the penis is

more common among uncircumcised men than circumcised men and often results from

unprotected sexual intercourse with an infected partner. In rare cases, when the body is

immunocompromised like in leukemia and HIV/AIDS, the fungus can enter the bloodstream and

causes infection of vital organs.

The symptoms associated with candida infections include creamy-white patches on the

tongue and on the lining of the throat or mouth. On the skin, it appears as diaper rash usually
reddish and sometimes scaly. On the other hand, vaginitis presents with white or yellowish

discharge, inflammation of vaginal walls and of vulva causing burning and itching sensation

while infection of the penis results in inflammation of glans of penis(balanitis). Infection of the

fingernails is characterized by reddish painful swelling around the nails. Pus may develop as the

disease progress (Calderone & Fonzi, 2001).

In cases of low immune response, where the microorganism can enter the bloodstream

and cause infection of the kidneys, heart valves, eyes, and lungs. The infection of the vital organs

manifest with fever and chills, anemia and shock although the symptoms vary with the organ

infected. For example, in kidney infection blood appears in the urine.

(Pappas et al, 2009), management of candida infection depends on the severity of the

infection. Mild infections can be treated with over the counter drugs or prescription medication.

These includes topical administration of antifungal such clotrimazole, miconazole, nystatin,

tioconazole or oral administration of fluconazole. For severe infections, intravenous medications

are given at the hospitals. Although antifungal agents usually clear up the trouble, most

infections recur due to resistance, thus the need to come up with newer and stable agents.

1.1 Plant morphology and distribution

Euphorbia prostrata is a weed belonging to the family, . E. prostrata is a native to West

Indies, but it has widely distributed throughout the tropics and subtropics. It occurs through

topical Africa and the Indian Oceans Islands. E. prostrata widely grows in gardens, disturbed

grounds roadsides (especially in sandy areas) and in cultivated lands from sea level to high

altitudes. The plant is a summer annual forming leafy stems up to 2 inches across. The plant has
simple leaves which are oppositely arranged. The edges of the leaf blade have teeth (Record,

2012).

The major constituents of Euphorbia prostrata include flavonoids, phenols, anthraquinone

glycosides, saponins, phlobotannins, tannins, polysaccharides and terpenoids. The plant contains

alkaloids in little proportions. A range of hydrolysable ellagitannins and flavonoids can be

isolated from the plants. Most of these bioactive compounds are found in the aerial parts (Saeed-

ul-Hassan, et.al, 2013).

1.2 Properties of Euphorbia prostrata

The latex of the plant possesses irritating and blistering properties to mucous membranes

and skin. Several hydrolysable ellagitannins can be isolated by using different fraction of extracts

from dried leaves. Among the hydrolysable ellagitannins are prostatins A, B, and C, euphorbins

G and H, tellimargradin I and II. Flavonoids can also be also isolated from the aerial parts of the

plant and include kaempferol, cosmosiin, rhamnetin-3-galactoside (Record, 2012).

Other compounds that can be obtain from the aerial parts are terpene alcohol beta-terpineol,

1,2,3-tri-O-galloyl-D-glucose and gallic acid. Myricyclic and tripterpenes (taraxerol, tirucallol)

are isolated from the roots. Both tannins and flavonoids have exhibited analgesic, antithrombic,

anti-inflammatory and hemostatic actions. Furthermore, flavonoids showed antiviral,

antioxidants and antiplatelet properties (Record, 2012).

1.3 Problem statement

 Fungal infections that are resistant to treatment with antifungal drugs are an emerging

public health problem, thus a major concern to prevent and control it. Antifungal resistance is a

primary concern for invasive infections with the fungus candida species. There is less public

know-how about the effects of antifungal resistance and its burden as compared to antibiotic-

resistant bacterial infections which are a widely-recognized public health threat. Since antifungal

resistance is relatively uncommon, the problem continues to evolve. This highlights the needs for

improve understanding of the reasons for their emergence and greater attention to methods that

can be adopted to prevent and control them. One of the methods that can be implemented is

development of newer drugs that are stable and safe against resistant- fungi species from

medicinal plants (Rodrigues, et. al, 2014).

Invasive infections that have developed resistance to first -line and second-line

antifungal medications namely fluconazole and echinocandins respectively, causing substantially

morbidity and mortality. Additionally, they are costly to manage and this highlights greater

concern to develop reliable and economical therapeutic options for the resistant fungi from

medicinal plant investigated and proved to have fungal inhibition potential. Some types of

candida species, especially Candida glabrata are resistance to fluconazole. Echinocandins

resistance, however, appears to be on the rise especially among candida species (C. glabrata).

The few remaining treatment options for candida infections that are both resistance to

fluconazole and echinocandins are expensive and can be toxic to patients thus, concern for

cheaper and safer antifungal medications (Rodrigues, et. al, 2014).

1.4 Objectives

1.4.1 Main objective

To investigate the antifungal activity of both methanolic and water extracts of Euphorbia

prostrata.

1.4.2 specific objectives

To compare the fungal growth inhibition potential of methanolic and water extract against

fluconazole as the standard

1.5 Research questions

This study is designed to answer the following questions.

1. Do the methanolic and water extract of Euphorbia prostrata derived from the whole plant

have antifungal activity.

2. Do both extracts have better antifungal efficacy than fluconazole
3. Which of the extracts have better efficacy than the other?

CHAPTER TWO: LITERATURE REVIEW

All parts of Euphorbia prostrata are widely used in various parts of the world for

treatment, control, prevention and improvement of symptoms, diseases and conditions. For

instance, in United States, the latex of the plant is applied to snakebites whereas in Mexico and

Venezuela it is used to treat tumors. in India, the extract of whole plant is used for management

of diabetes due to its hypoglycemic and anti-inflammatory properties. Furthermore, the plant has

find use in treatment of asthma and its infusion is taken as blood purifier (Record, 2012).

In Africa, the all part of the plant is used in traditional medicine for management of

various diseases and condition. In Burkina Faso, the leaves of E. prostrata are rubbed on wasp

and scorpion stings whereas in Benin, pounded leaves mixed with pounded shells are taken to

treat irregular menstruation. In addition, pounded leaves in water is taken to ease child birth. In

Togo, leaf decoction is drunk to treat threatened abortion while the small balls of ground plant
are inserted into the vagina to treat female infertility and dysmenorrhea. In DR Congo, the leaves

are warmed over fire, crushed and squeezed onto the body to cure insects stings and fungal

infections. Fungal infections were also cured by infusion or leaves decoction. The crushed whole

plant is taken together with bread to treat kidney stones (Record, 2012).

In Uganda, crushed leaves in water is used as mouthwash and gargle to cure oral thrush.

Moreover, pregnant women eat the boiled shoots mixed with sesame to reduce risk of

miscarriage. The plant juice induces labor during childbirth when taken orally. A bath infusion

the plant is recommended to treat insanity whereas in Angola, vapour bath of the whole plant

treats scabies while an infusion of the leaves or aerial parts is taken either alone or combined

with other plants to treat gonorrhea, stomach ache and dysentery in Indian Ocean Islands. In

Mauritius, the decoction of the whole plants is used as eye wash against conjunctivitis while the

plant latex is applied to warts and abscesses (Gaby, et. al, 2008).

In Kenya, the Maasai people chew the whole plant to treat gonorrhea. The plant is also

used in pain management in the previous study on analgesic properties of E. prostata, crude

extract of hexane and ethyl acetate proved to have significant analgesic properties (Biwott, et.al,

2015).

Different extracts of Euphorbia prostrata have showed antibacterial, antifungal as well as

inhibitory effects against HIV-1 Protease. On the previous studies to determine the antifungal

properties of E. prostrata, ethanol and water extract of the whole plant showed significant

activity against dermatophytes Trichophyton simii, Trichophyton targrophytes and Microsporium

gypseum in vitro and in vivo goats and rabbits. Both ethanol and water extract cured fungal

lesions in 3-4 weeks and were as effective as benzoic (Gaby, et. al, 2008). Dermatophytes are

fungi that require keratin for growth and causes superficial mycotic infections of the skin, nails,
beards, hair shafts and hair follicles. The pathogen causes acute or chronic disease with high

morbidity but not mortality. Dermatophytes have developed host specificity which ascribed to

difference in the composition of keratin. Based on their host specificity, dermatophytes are

classified into three ecological groups namely geophytes(soils), zoophiles(animals) and

anthropophytes(man) (Lakshmipathy, & Kannabiran, 2010).

Dermatophytes access entry into the host body via injured skin, burns or scars. Resting

hairs lack the essential nutrients required for growth of the organism hence hairs are not infected.

The pathogen invades the upper most non-living keratinized layer of the skin namely stratum

corneum and produces enzyme keratinase which induces inflammatory reactions at the site of

infection. Inflammation causes the organism to spread to other areas as it causes the pathogen to

move away from the site of infection and take residence at a new site. Dermatomycosis are

named as per the site of infection and commonly referred to as Tinea. Tinea capitis is the

infection of the scalp while Tinea corporis is an infection of the non-hairy, glabrous regions of

the body. Tinea pedis(foot), tinea ingium or onychomycosis(nails) and tinea barbae is infection

of beards (Lakshmipathy & Kannabiran, 2010).

The therapeutic regimen of antifungal infection is chosen based on site of infection,

etiological agent and penetrability of the drug. Duration of treatment mainly depend on the type

of infection and symptoms. However, 3-4 weeks of treatment is required for skin lesions whereas

feet inflammation requires 4-6 weeks of treatment. Terbinafine has showed fungicidal activity

against all genera of dermatophytes hence drug of choice for treatment of dermatophytoses.

Terbinafine and naftifin belong to allylamine groups of antifungal. These drugs act by blocking

the synthesis of ergo sterol, an essential component of fungal cell membrane through inhibition

of enzyme squalene epoxide. On the hand, Griseofulvin can be used to control the growth of
keratinized tissues. Griseofulvin act by interfering with polymerization of microtubules resulting

in abnormalities in cell division due to disruption of intracellular transport associated with

microtubules (Soares, et.al, 2013).

Currently, azole derivatives which consist of ketoconazole, clotrimazole, fluconazole

imidazole, itraconazole are commonly used to manage fungal infections due to their oral

bioavailability thus maintaining effective activity. Despite the advancement of science and

technology, the development of effective antifungal drugs is still lagging since the fungi are

eukaryotic and their mechanism is like that of human beings hence concern to develop antifungal

drugs from plants that only kill fungi without causing damage to human beings (Soares, et.al,

2013).

(Oliver- Bever, 1986), studies proved that ether extract of E. prostrata was found to have

powerful antifungal activity against Trichophyton mentagrophytes. Later, with the aim to

confirm antifungal activity, studies were repeated using petroleum ether and linseed oil extracts

of the plant against T. simii and M. gypseum. During studies to determine the biological

properties of Conyza and Euphorbia species, dichloromethane extract of euphorbia prostrata was

found to exhibit antifungal activities against Cladosporium cucumerinum, a fungus that attacks

ground parts of plants (Chaudhry, et. al, 2001).

CHAPTER THREE: METHODOLOGY

3.0 Introduction
 This study is designed to investigate the antifungal properties of methanol and water

extracts of Euphorbia prostrata against Candida albicans using agar diffusion method. Agar

diffusion method involves use of disks containing extract to be tested at known concentrations

into contact with an inoculated medium. Wells are punched in the medium to act as reservoirs of

plant solution to be examined at known concentrations and the standard. The diameter of

inhibition will be measured after the end of incubation period.

3.1 Collection of plants

The plant Euphorbia prostrata will be collected from Kenyatta University Medicinal

Garden and be authenticated by Mr. Karimi from Department of Botany, Kenyatta University.

The collected plants will then be sorted out, washed and air-dried at room temperature for two

weeks.

3.2 Materials and Instruments

Distilled water

Methanol

Fluconazole

Sabourauds dextrose agar

Candida albicans (test organism)

Petri dishes

Measuring cylinders

Conical flasks

Volumetric flasks, two 50mls and 500mls

Beakers

Cork borers

Micropipettes

Whatman filter papers

Oven

Rotary evaporator

Autoclave

Wire loop

Lamp

3.3 Extraction of plant materials

The well dried plants will be grounded and portions of resulting powder be soaked in

water and 40% absolute methanol respectively for three days with random shaking using rotary

machine. Methanol extract will be filtered using what man filter paper and then concentrated

with the help of rotary evaporator at 37 degrees Celsius to obtain crude extract. On the other

hand, water extract will be filter using a rotor and a Buchner funnel and then lyophilized to

obtain a crude powder.

3.4 preparation of medium

Sabourauds dextrose agar will be used for growth of organism, Candida albicans for the

preparation of inoculum. 6.5 grams of Sabourauds dextrose agar will be dissolved in 100ml of
distilled water and then heated to boiling. The medium solution will be autoclaved at 15psi for 15

minutes.

3.5 testing of antifungal activity

The prepared medium will be allowed to cool then candida albicans will be introduced

with the help of a wire loop and shook to attain a uniform growth. The seeded medium

containing candida albicans will be poured in petri plates (14cm) and then allowed to solidify.

Once the medium has solidified, three wells will be made using a cork borer of 8mm diameter.

Test solutions of 5mg/ml concentration will be poured in one hole while the standard solution of

fluconazole will be poured in another whole. One hole will be left blank. The samples will then

be incubated in an oven at 37 degrees Celsius for 48hours. The clear zone of inhibition will be

measured at the end of incubation period and potential fungal inhibition be compared.
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