i

OMOBOYOWA, DAMILOLA ALEX

(PG/M.Sc/09/51443)

PG/M. Sc/09/51723

ACTIVITY OF CHLOROFORM-ETHANOL EXTRACTS OF CASHEW (Anacardium occidentale) KERNEL IN CASTOR OIL-INDUCED

DIARRHOEA IN RATS

BIOLOGICAL SCIENCES

A THESIS SUBMITTED TO THE DEPARTMENT OF BIOCHEMISTRY, FACULTY OF

BIOLOGICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA

Webmaster

Digitally Signed by Webmaster’s Name

DN : CN = Webmaster’s name O= University of Nigeria, Nsukka

OU = Innovation Centre

APRIL, 2011

ii

TITLE

ACTIVITY OF CHLOROFORM-ETHANOL EXTRACTS OF

CASHEW (Anacardium occidentale) KERNEL IN CASTOR

OIL-INDUCED DIARRHOEA IN RATS

A DISSERTATION SUBMITTED IN PARTIAL FULFILMENT

OF THE REQUIREMENTS FOR AWARD OF DEGREE OF

MASTER OF SCIENCE (M.Sc) IN PHARMACOLOGICAL

BIOCHEMISTRY, UNIVERSITY OF NIGERIA,

NSUKKA

BY

OMOBOYOWA, DAMILOLA ALEX

(PG/M.Sc/09/51443)

DEPARTMENT OF BIOCHEMISTRY

UNIVERSITY OF NIGERIA

NSUKKA

SUPERVISOR: PROF. O. F. C. NWODO

APRIL, 2011

iii

CERTIFICATION

OMOBOYOWA, Damilola Alex, a postgraduate student of the Department of

Biochemistry with the Reg. No PG/M.Sc/09/51443, has satisfactorily completed his

requirement for research work for the degree of Master of Science (M.Sc) in Pharmacological

Biochemistry. The work embodied in this project (dissertation) is original and has not been

submitted in part or full for any other diploma or degree of this or any other university.

PROF. O. F. C. NWODO PROF. L.U.S. EZEANYIKA (Supervisor) (Head of Department)

EXTERNAL EXAMINER

iv

DEDICATION

This work is dedicated to the Almighty God for his undeserved grace and mercy.

v

ACKNOWLEDGEMENT

To the glory of God, for a dream come through!

I wish to express my profound and immeasurable gratitude to my supervisor, Prof. O. F.

C. Nwodo for the painstaking supervision, encouragement, tolerance, patience and support

throughout the course of this work. I will not forget his fatherly consideration and utmost

interest of allowing me pass through the academic mentorship in order to model me. I am born

a lucky gem for having wonderful, caring, supportive and loving parents who had taken such

responsibility to sponsor my education from nursery to Master’s degree level. I appreciate the

love of God in my parents, Mr. and Mrs. J.O. Omoboyowa. May God give you long life to eat

the fruit of your labour.

This project would never have been accomplished without the encouragement and

contribution of many people; in such endless list are Dr. P. E. Joshua of the Department of

Biochemistry, University of Nigeria, Nsukka, Dr. V. Omoja, of the Department of

Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Nigeria, Nsukka,

Dr. C.E.C.C. Ejike of the Department of Biochemistry, College of Natural Sciences, Michael

Okpara Univeristy of Agriculture, Umudike and Pastor Ngozi Anyangbulam of the Department

of Science Education, University of Nigeria, Nsukka.

To the lecturers in my Department who gave me a chance and believed in my ability, I

say thank you, a million. Special appreciation goes to the Head of Department, Prof. L.U.S.

Ezeanyika for his words of encouragement. I am indebted to Prof. F.C. Chilaka, my

enzymology lecturer; Prof. P.N. Uzoegwu, my nucleic acid teacher; Prof. E.O. Alumanah, my

porphyrin teacher; Prof. I.N.E. Onwurah, my Industrial Biochemistry teacher, Prof. O. Obidoa,

my pharmacology teacher; Prof. O.U. Njoku, my cholesterol teacher; Dr. H.A. Onwubiko, Dr.

V.N. Ogugua, Dr. S.O.O. Eze, Mr. O.C. Enechi, Dr. B.C. Nwanguma, Dr. (Mrs.) C.A.

Anosike, Dr. P. E. Joshua, Mrs. U.O Njoku, Mr. V.E.O. Ozougwu.

As I began to think of all the people to whom I would like to express my

appreciation for their support, suggestions, and hard work in making this research possible, the

list continued to grow. My appreciation also goes to the family of Mr. and Mrs. J.I Adeyomoye,

my caring Uncle CH. Lebi Omoboyowa, my lovely sister Mrs. Fayobi Olaiya, Mrs. W.O.

Adeyomoye, the Proprietress, Glory of God Nur/Pry School, Ibadan and all the members of

Redeem Christian Church of God, Unity Parish, Nsukka for their love and support financially,

spiritually and morally. May God continue to bless you.

vi

To my younger ones Sharon, Gbemi, Joke, Tolu, Segun Funmilayo, Opeyemi, Funke,

Folakemi, may God preserve you for your own time.

It is a great priviledge to have loving friends. I appreciate the encouragement of my

friends: Bamidele Omolara, B.O. Ezema, and Mrs. Vivian Kelechi-Ebisike,

vii

ABSTRACT

The anti-diarrhoeal activity of ethanol-chloroform extracts of Anacardium occidentale kernel at

the doses of 21 and 84 mg/kg body weight were studied using rat models of diarrhoea,

enteropooling and gastro-intestinal motility induced castor oil. The results of the qualitative

phytochemical analysis showed that the ethanol-chloroform extracts (ethanol, chloroform and

middle layers) tested positive to flavonoids, alkaloids, saponins, reducing sugars, glycosides

and steroids, while chloroform layer and middle layer tested positive to fat and oil. Acute

toxicity and lethality studies on ethanol-chloroform extracts revealed an oral LD50 equal to or

more than 5000 mg/kg body weight in mice. The results showed that over a period of 5 hours

the extracts significantly (p<0.05) reduced the watery texture and number of faecal droppings

in treated groups compared with the untreated group. The extracts also significantly (p<0.05)

reduced the volume and weight of intestinal content compared to the control animals. On

gastro-intestinal motility, the extracts significantly (p<0.05) reduced the small intestinal transit

of charcoal meal in rats induced with castor oil. The extracts also significantly (p<0.05)

reduced the intestinal sodium and potassium ion (Na+ and K

+) concentrations compared with

animals in the untreated groups. These results showed that kernels of A. occidentale possess

anti-diarrhoeal properties through inhibition of hyper-secretion, enteropooling and gastro-

intestinal motility which can substantiate its use in the treatment of diarrhoea in traditional

medicine.

viii

TABLE OF CONTENTS

PAGE

Title Page … … … … … … … … … … i

Certification … … … … … … … … … … ii

Dedication … … … …. … … … … … … iii

Acknowledgement … … … … … … … … … iv

Abstract … … … … … … … … … vi

Table of Contents … … … … … … … … … vii

List of Tables … … … … … … … … … x

List of Figures … … … … … … … … … xi

List of Abbreviation … … … … … … … … … xii

CHAPTER ONE: INTRODUCTION

1.0 Preamble … … … … … … … … 1

1.1 Anacardium occidentale: Description of the plant … … … … 2

1.2 Taxicity of medicinal plants … … … … … … … 4

1.3 Toxicity of herbal remedies … … … … … … … 4

1.4 Taxonomy of cashew plant … … … … … … … 5

1.5 Traditional, medicinal and industrial uses of A. occidentale … … … 6

1.5.1 The leaf … … … … … … … … … 6

1.5.2 The cashew nut shell liquid … … … … … … … 6

1.5.3 The seed … … … … … … … … … 7

1.5.4 The bark … … … … … … … … … 7

1.5.5 The fruit … … … … … … … … … 7

1.6 Previous chemical and pharmacological investigations on A. occidental … 8

1.7 Diarrhoea … … … … … … … … … 10

1.7.1 Overview … … … … … … … … … 11

1.7.1.1 Symptoms of diarrhoea … … … … … … … 11

1.7.1.2 History of diarrhoea … … … … … … … 12

1.7.2 Physiology and pathophysiology of diarrhoea … … … … 13

1.7.3 Chronic diarrhoea … … … … … … … … 14

1.7.3.1 Inflammatory chronic diarrhoea … … … … … … 14

1.7.3.2 Osmotic chronic diarrhoea … … … … … … … 15

1.7.3.3 Secretory chronic diarrhoea … … … … … … … 16

1.7.3.4 Iatrogenic chronic diarrhoea … … … … … … … 16

1.7.3.5 Motility disorder causing chronic diarrhoea … … … … … 17

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1.7.4 Causes of diarrhoea … … … … … … … … 18

1.7.5 Diagnosis of diarrhoea … … … … … … … 21

1.7.5.1 Laboratory method … … … … … … … … 21

1.7.6 Management of diarrhoea … … … … … … … 23

1.7.7 Medication for chemo-therapy induced diarrhoea … … … … 25

1.7.7.1 Loperamide … … … … … … … … … 25

1.7.7.2 Atropine-diphenoxylate (Lomotil) … … … … … … 25

1.7.7.3 Octreotide … … … … … … … … … 25

1.7.7.4 Antibiotics … … … … … … … … … 25

1.8 Aim and Objectives of the research … … … … … … 26

CHAPTER TWO: MATERIALS AND METHODS

2.1 MATERIALS … … … … … … … … … 27

2.1.1 Plant materials … … … … … … … … … 27

2.1.2 Animals ... … … … … … … … … 27

2.1.3 Chemicals ... ... … … … … … … … 27

2.1.4 Equipment … … … … … … … … 27

2.2 METHODS … … … … … … … … … 28

2.2.1 Preparation of charcoal meal … … … … … … … 28

2.2.2 Preparation of 20% potassium hydroxide … … … … … 28

2.2.3 Extraction procedure … … … … … … … 28

2.2.4 Experimental analysis … … … … … … … 28

2.2.4.1 Phytochemical tests … … … … … … … 28

2.2.4.1.1 Test for carbohydrates … … … … … … … 29

2.2.4.1.2 Test for alkaloids … … … … … … … 29

2.2.4.1.3 Test for glycosides … … … … … … … 29

2.2.4.1.4 Test for saponins … … … … … … … 30

2.2.4.1.5 Test for tannins … … … … … … … 30

2.2.4.1.6 Test for flavonoids … … … … … … … 30

2.2.4.1.7 Test for steroids … … … … … … … 31

2.2.4.1.8 Test for reducing sugars … … … … … … … 31

2.2.4.1.9 Test for oils … … … … … … … 31

2.2.4.2 Acute toxicity and lethality (LD50) test … … … … … 32

2.2.4.3 Anti-diarrhoea studies (castor oil-induced diarrhoea test) … … … 32

x

2.2.4.4 Castor oil induced enteropooling test … … … … … 33

2.2.4.5 Castor oil-induced gastro-intestinal motility test … … … … 33

2.2.4.6 Preparation of samples for electrolyte test ... ... ... ... ... 34

2.2.4.7 Determination of sodium ion (Na+) concentration (Teco diagnostic kit) ... 35

2.2.4.8 Determination of potassium ion (K+) concentration (Teco diagnostic kit) ... 36

2.2.5 Statistical analysis ... … … … … … … … 36

CHAPTER THREE: RESULTS

3.1 Yield of the extract … … … … … … … … 37

3.2 Phytochemical composition of ethanol-chloroform extract … … … 37

3.3 Lethal dose (LD50) result … … … … … … … 39

3.4 Effect of extracts on wetness of the faeces … … … … … 39

3.5 Effect of extracts on frequency of defaecation … … … … 41

3.6 Effect of extracts on gastro-intestinal motility … … … … 43

3.7 Effect of extracts on gastro-intestinal enteropooling … … … … 45

3.8 Effect of extract on weight of the gastro-intestinal tract … … … 47

3.9 Effect of ethanol and chloroform layers of Anacardium occidentale

Kernel extract on intestinal sodium ion (Na+) concentration ... ... ... 49

3.10 Effect of ethanol and chloroform layers of Anacardium occidentale

Kernel extract on intestinal potassium ion (K+) concentration ... ... 51

CHAPTER FOUR: DISCUSSION

Discussion … … … … … … … … … 53

Suggestions for Further Studies … … … … … … … 55

References … … … … … … … … … 56

Appendices … … … … … … … … … 63

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LIST OF TABLES

Table 1: Qualitative phytochemical composition of extracts … … … 38

Table 2: Effect of extracts on wetness of faeces … … … … … 40

Table 3: Effect of extracts on frequency of defaecation … … … … 42

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LIST OF FIGURES

Fig. 1: Cashew tree … … … … … … … … … 3

Fig. 2: Three groups of antibiotic-associated diarrhoea detected by endoscopy … 20

Fig. 3: Bacterial culture of a diarrhoeal stool or rectal swap … … … … 22

Fig. 4: Effect of extracts on gastro-intestinal motility … … … … 44

Fig. 5: Effect of extracts on gastro-intestinal enteropooling … … … … 46

Fig. 6: Effect of Extracts on Weight of the Gastro-Intestine Tract … … ... 48

Fig. 7: Effect of extract on intestinal sodium ion concentration ... ... ... 50

Fig. 8: Effect of extract on intestinal potassium ion concentration ... ... ... 52

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LIST OF ABBREVIATIONS

AAD Antibiotic-associated diarrhoea

BDCP Bioresources development and conservation programme

B.W Body weight

CDD Control of diarrhoea diseases

CID Chemotherapy-induced diarrhoea

CNSL Cashew nut shell liquid

COX 1 Cyclooxygenase 1

COX 2 Cyclooxygenase 2

DNA Deoxyribonucleic acid

EIEC Enteroinvasive E. coli

ETEC Enterotoxigenic E. coli

GIT Gastro-intestinal tract

IBD Irritable bowel diseases

K+ Potassium ion

LD50 Lethal dose 50

LOX Lipoxygenase

Na+ Sodium ion

ORT Oral rehydration therapy

ORS Osmolarity oral rehydration salt

xiv

CHAPTER ONE

INTRODUCTION

Plants and their derivatives play key role in world health and have long been known to

possess biological activity. Thirty percent of all modern drugs are derived from plants (Riaz et

al., 2010). According to the World Health Organization, about 80% of the world’s population

relies essentially on plants for primary health care (Mckay and Blumberg, 2007).

In our traditional civilization, the healers used to occupy a special place within the

community; but with the advent of modern orthodox need, they are fast losing relevance.

Nigeria and indeed black Africa has been witnessing the gradual disappearance of traditional

healers, as well as a decline of their knowledge. By putting much effort into research on plants

in the sphere of ethno-medicine, it is possible to revive knowledge of traditional mode of

healing (Mustapha and Hafsat, 2007).

Diarrhoea is a major health problem especially in developing countries. In Africa,

diarrhoea remains a major contributor to the high rate of child mortality. In Nigeria, diarrhoeal

infection is probably the number one killer disease among children under 5 years (Magaji et al.,

2010). Each year, there are approximately 4 billon cases of diarrhoea world wide leading to 4

million deaths especially among children in this age group (Azubuike and Nkagineme, 2007).

Also, in this age group, 38% of all deaths are associated with diarrhoeal diseases (WHO, 1990)

with 7-12 month-old babies being the most susceptible (Audu et al., 2010). Diarrhoea is

defined by the World Health Organization as the presence of three or more loose or liquid

stools per day, or as the presence of more stools than is normal for that person. The incident of

diarrhoeal diseases still remains high despite the intervention of government agencies and

international organizations to halt the trend (Magaji et al., 2010). The use of herbal drugs in the

treatment of diarrhoea is a common practice in many African countries (Agunu et al., 2005).

Despite immense technological advancement in medicine, many people in developing countries

still rely on traditional healing practices and medicinal plants for their health care needs

(Ojewole, 2004). The World Health Organization (WHO) encourages studies onto traditional

medicinal prevention of diarrhoeal diseases (Alta and Mouneir, 2004). Therefore, there is

urgent need for intensification of research into medicinal plants claimed to be effective in the

management of diarrhoeal diseases (Mohammed et al., 2009).

The cashew (Anacardium occidentale) is a well known member of the Anacardiaceae

family and is commonly found in Northeast Brazil. The cashew nut has been commercially

exploited since colonization. Brazil, India and Mozambique are the leading cashew nut

producers in the world (Pimentel et al., 2009).

xv

Anacardium occidentale, an evergreen, multi-branched shrub of the Anacardiacea

family, commonly known as cashew, attains a height of about 12 meters and has a spread of 25

m. The kernels of the plant have been reported to have antidiarrhoeal effect (Orwa et al., 2009).

1.1 Anacardium occidentale: Description of the plant

The cashew (Anacardium occidentale) plant is a well known member of the

Anacardiaceae family and is commonly found in northeast Brazil (Paramashiva et al., 2001). A

medium-sized tree, much branched, grows to a height of 12 meters. When growing on lateritic

gravel or coastal sandy areas, it rarely exceeds 6m and develops a spreading habit and globose

shape with crown diameter to 12 cm. When growing on loamy soil, it reaches 15m and is much

branched, with smaller (94-96 m) crown diameter. The root system of a mature Anacardium

occidentale when grown from the seed consists of a very prominent tap root, a well developed

and extensive network of lateral and sinker roots. Its leaves are simple, alternate, glabrous,

round at ends, 10-18 × 8-15cm with short petiole (Orwa et al., 2009). When young, the leaves

are pale green or reddish and are dark green when mature. The inflorescence is a terminal

panicle-like cluster commonly bearing male and hermaphroditic flowers. The male flowers

usually bear one exerted stamen and nine small inserted ones. Anacardium occidentale

normally comes into flowering in 3-5 years. It has light grey back that is smooth when young

but develops small cracks with increased age, it has potential as a soil stabilizing plant because

it tolerates drought and is able to grow on relatively dry infertile soils and still provides some

financial returns (Orwa et al., 2009).

The thin-skinned edible cashew fruit has a light yellow spongy flesh, which is very

pleasantly acidic and slightly astringent when eaten raw and highly astringent when green. The

generic name was given by Lineaus and refers to the vaguely health shaped look of its false

fruit (Orwa et al., 2009). Cashew apple juice is a rich source of vitamin C (ascorbic acid) and

presents functional properties such as prevention of cancer and Helicobacter pilori – bacteria

that cause severe gastritis; besides, presenting antioxidant properties (Joelia et al., 2006). In the

main producing areas of East Africa and India, 95% or more of the apple crop is not eaten, as

the taste is not popular. However, in some parts of South America and West Africa, local

inhabitants regard the apple rather than the kernel as a delicacy (Orwa et al., 2009). The nut

which is the true fruit dries and does not split open. Inside the poisonous shell is a large curved

seed, nearly 2.5cm long which is the edible cashew kernel. As the nut matures, the stalk

(receptacle) at the base enlarges rapidly within a few days into the fleshy fruitlike structure,

broadest at the apex, popularly known as the fruit (Orwa et al., 2009).

xvi

In 1970s, Africa was the largest producer of cashew nuts accounting for 67.5% of world

production. This subsequently declined to 35.6% by 2000, with Nigeria, Tanzania and

Mozambique being largest producers. The production in Asia during the same periods

increased from 26.8% to 49.5% with the major producer being India, Indonesia and Vietnam.

Similarly, the production in South and Central America also rose from 4.5% in 1970 to 14.5%

in 2000 with Brazil and El Salvador being the leading producers (Hammed et al., 2008).

Fig. 1: Cashew tree

1.2 Toxicity of Medicinal Plants

Plants have long been recognized as a valuable source of medicines for treating a

variety of different diseases and complaints. Most, if not all civilizations, have used plants as

medicines. The use of plant natural therapeutics in Asia is wide spread, being used in the

treatment of numerous disorder including eczema, gastro-intestinal disorder, malaria and

respiratory disorder (Hoareau and DaSilva, 1999). Africa also has a long history of herbal

medication. For example, Phytolacca dodecandina is used as a molluscide in the control of

schistosomiasis (Reich et al., 1999). Vinblastine and vincristine derived from Catharanthus

xvii

roseus are currently used in the treatment of tumours (Sersa et al., 2001). Europe and America

also have a history of herbal medication. Studies demonstrate the myriad of medicinal plant

uses by indigenous North, Central America and South Americans (Roth et al., 2002).

Approximately 15000 medicinal plant species are currently in use in Europe (Hoareau and

DaSilva, 1999). The Eastern region of the Mediterranan has been distinguished throughout the

generations with a rich inventory of natural medicinal herbs (Saad et al., 2005).

Parallel with recent increase in the use of herbal medicine for the prevention and

treatment of various illnesses, there is increasing concern about the safety of medicinal plants.

There are general and herb-specific concerns regarding medicinal plants and their ability to

produce toxicity and adverse effects. Accidental herbal toxicity occurs not only as a result of a

lack of pharmaceutical quality control in harvesting and preparation, but also because herbal

remedies are believed to be harmless (Said et al., 2002).

1.3 Toxicity of Herbal Remedies

Most reports of toxic effects due to the use of herbal medicines and dietary supplements

are associated with hepatotoxicity although other toxic effects on the kidney, nervous system,

blood, cardiovascular and dermatological systems may result to mutagenicity and

carcinogenicity (Woolf, 2003). Hepatic impairment resulting from the use of conventional

drugs is widely acknowledged, but there is less awareness of the potential hepatotoxicity of

herbal preparations and other botanicals, many of which are believed to be harmless and are

commonly used for self-medication without supervision. Although regulation by the Food and

Drug Administration may be part of the solution, increasing public awareness and education

programmes for healthcare professionals about the potential dangers of herbal preparations will

need to be implemented (Chitturi and Farrell, 2000).

The reported toxicity of herbal formulations may be the result of several factors,

including the contamination with pesticides, microbes, heavy metals, toxins or adulteration

with orthodox drugs (Fugh-Berman, 2000). On the basis of various case reports, the liver injury

from herbal remedies has ranged from mild elevation of liver enzymes to liver failure (Wolf,

2003).

Veno-occlusive disease has been associated with pyrrolizidine alkaloids in Senecio

species, Heliotropium species and comfrey (Symphytum officinale). Chapparal (Larrea

divericata) leaf ingestion has led to the development of fulminant liver failure or cirrhosis.

xviii

Kava (Piper methysticum) has been associated with acute hepatitis. Many traditional Chinese

preparations have also been described to cause hepatotoxicity and liver failure (Woolf, 2003)

The effects of contaminations and dosage variations are higher in children than adults

(Woolf, 2003). The teratogenic effects of herbs are not known in many cases. It is possible that

herbal chemicals may be transported through the placenta to cause toxic effects on the foetus.

Roulet et al. (1988) reported the case of a newborn whose mother drank senecionine-containing

herbal tea daily for the duration of her pregnancy. The infant was born with hepatic veno-

occlusive disease and died. Senecionine is one of the pyrrolizidine alkaloids associated with

hepatic venous injury.

1.4 Taxonomy of the Cashew Plant

Kingdom: Plantae

Division: Magnoliophyta (Flowering plant)

Class: Magnoliospsida

Order: Sapindales

Family: Anacardiaceae

Genus: Anacardium

Species: occidentale

Botanical name: Anacadium occidentale

(Evans, 2001).

Local Names: Cashew has different names in different language groups. Habb al-biladhir

(Arabic); Hijuli, Hijlibadam (Bengali); Thiho Thayet si (Burmese); Yao kuo (Chinese); Kasjoe,

Mereke (Dutch); Cashew, Cashew nut (English); Kasoy,balogo, Bulabad (Filipino);

Anacardier, Acajou, Pommier cajou, Pomme d' cajou,anacadier, Noix d' cajou, Anacarde,

Pomme acajou (French); Acajubaum, Kaschubaum, cashwenuß, elefantenlaus (German); kaju

(Gujarati); bojan, kashu-mavu, kaju, hijuli (Hindu); jambu monyet, jambu mede (Indonesian);

acagia (Italian); kashu nattsu (Japanese); jambu mede, jambu monyet (Javanese); svaay chanti

(Khmer); gajus, jambu monyet (Malay); kasuowo, kasuwu (Mandinka); kaaju (Nepali); caju

(Portuguese); kajutaka (Sanskrit); cashú, merci, marañón, cacajuil, casho, cajuil, acaya

(Spanish); mkanju, mkorosho, mbibo (Swahili); mindiri (Tamil); yaruang, mamuang,

xix

himmaphan, mamuang letlor (Thai); cashew nut (Trade name); cây diêù, dào lôn hôt

(Vietnamese) (Orwa et al., 2009).

1.5 Traditional Medicinal and Industrial Uses of Anacadium occidentale

Cashew is a useful tree as different parts of it are used either individually or

collectively to treat several diseases. Fresh or hot water extract of different plant parts is used

orally as aphrodisiac (arouse sexual desire), anti-dysentric, antihaemorrhagic and externally as

anti-inflammatory (Evan, 2001).

1.5.1 The Leaf

Hot water leaf extracts are used orally for diabetes in Brazil and Thailand, diarrhoea in

Tanzania, and externally to wash ulcer in West Indies (Evan, 2001). Buds and young leaves are

used for skin diseases (Orwa et al., 2009).

1.5.2 The Cashew Nutshell Liquid (CNSL)

A by-product of processed cashew is mostly composed of anacardic acids (Alexander,

2008). These acids have been used effectively against tooth abscesses due to their lethality to

Gram-positive bacteria. They are also active against a wide range of other Gram-positive

bacteria. Cashew nut shell oil is anti-hypertensive and purgative; it is used for blood sugar

problems, kidney troubles, cholera, and cracks on soles of feet, hookworms, corns and warts

Evans, 2001). Old cashew liquor in small doses cures stomach-ache. The oil obtained from the

shell by maceration in spirit is applied to cure cracks on the sole of the feet common among the

villagers (Orwa et al., 2009). Anacardic acid is also used in the chemical industry for the

production of cardanol, which is used for resins, coatings, and frictional materials (Alexander,

2008).

1.5.3 Cashew Seed

The seeds of Anardium occidentale are consumed orally in Colombia as aphrodisiac

and to cure impotency, whereas in Cuba, seeds are first toasted and then their powder is mixed

with sugar to be consumed as an aphrodisiac. Hot water extract of the seed is used orally in

Peru as anti-dysentric, anti-haemorrhagic, purgative and respiratory stimulant and used

externally as anti-inflammatory. It is common in Peru to use the hot water extract of the seed to

xx

cure warts (Evan, 2001). The seeds are ground into powders and used as anti-venom for snake

bites (Orwa et al., 2009).

1.5.4 Bark

Bark is astringent, counter irritant, rubefacient, vesicant, and used for ulcer (Orwa et al.,

2009). It also yields a gum used in varnish. Hot water extract of the bark is used to treat

amenorrhoea in Haiti, to increase fertility in women in Ghana and help people manage diabetes

in Jamaica (Orwa et al., 2001). In other African countries; the water extract of the bark is taken

orally as anti-dysenteric, hypotensive and hypoglycemic (in Madagascar) (Orwa et al., 2009).

In Panama, the hot water extract is used externally as an anti-inflammatory agent. People

consume it orally to treat diarrhoea in Panama and Senegal (Evan, 2001).

1.5.5 Fruit

The fruit is reddish yellow and has a pleasant sub-acid stringent taste. The expressed

juice of the fruit makes a good wine, and if distilled, a spirit much better than rum. The fruit

itself is edible, and its juice has been found useful in uterine complaints and dropsy. It is a

powerful diuretic (Kubo, 2006).

Hot water extracts of both ripe and unripe fruits and of dried kernel are used to treat

several ailments in different countries including (Orwa et al., 2009). Indians use the hot water

extract of the dried kernel as an aphrodisiac while decoction of dried kernel is used for diabetes

mellitus in Europe. The unripe fruit is taken orally to treat haemorrhage and diarrhoea and the

juice of the unripe fruit is taken orally as a diuretic and anti-scorbutic in Guinea (Evan, 2001).

In Ghana, extracts of dried fruit prepared with hot water is used as a wash to treat yaws disease.

Exudates of the fresh pericarp are used externally as an emollient for chilblains and also as an

insecticide to prevent termite attack in Indian (Evan, 2001). The kernel is a demulcent, an

emollient and is used for diarrhoea (Orwa et al., 2009).

1.6 Previous Chemical and Pharmacological Investigation on Anacardium occidentale

Cashew (Anacardium occidentale) has high demand in the world market due to wide

range of applications (Kanna et al., 2009). It was probably introduced in Nigeria in the 16th

century, at the same time it was introduced in other African countries by the Portuguese

(Aikpokpodion et al., 2009). It is used as folk medicine in south Cameroon and other African

countries for the treatment of diabetes mellitus, diarrhoea and hypertension (Tedong et al.,

xxi

2006). The cashew tree (Anacardium occidentale) represents one of the cheapest sources of

non-isoprenoid phenolic lipids which have a variety of biological properties: they act as

molluscicides, insecticides, fungicides, have anti-termite properties, have medicinal application

and demonstrate antioxidant activity in vitro (De Lima et al., 2008). The medicinal properties

of phytochemicals present in cashew nut shell liquid have been reported to have cytotoxic

activity against tumuor cell lines, anti-diabetes, anti inflammatory and analgesic effects (Kanna

et al., 2009). Cashew gum is a complex polysaccharide which on hydrolysis, results in high

galactose content. The Brazilian cashew gum yields 70% galactose, 5% arabinose, 11%

glucose, 4% rhamnose, 1% mannose and 6% glucuronic acid (Mothe et al., 2008).

The commercial importance of cashew kernel is due to its richness in nutrients that

comprise of 47% fat, 21% protein and 22% carbohydrate, vitamin and all essential amino acids

especially thiamine (Kanna et al., 2009).

The efficacy of hexane fraction of Anacardium occidentale in reducing diabetes-

induced function and histological alterations in the kidney was studied by Tedong et al. (2006).

They aimed at identifying biochemical and renal histological abnormalities that occur with the

evolution of streptozotocin-induced diabetes in rats and to appreciate their possible reversal

after the establishment of good metabolic control with the hexane extract of Anacardium

occidentale.

The antifungal properties of Anacardium occidentale seed coats against five various

fungal isolates were studied and related to the phytochemicals of cashew nut shell liquid. The

study reported that all the three extracts possess antifungal activity and are effective against A.

niger and A. flavus. Curvalaria spp was resistant to them (extracts). The acetone extract of A.

occidentale was reported to consist of more phytochemicals than ethanol and ethyl acetate

extracts but the antifungal spectrum was comparatively less than that of ethanol extract (Kanna

et al., 2009).

The antioxidant activity of anacardic acid [6-pentadec (en)ylsalicylic acid] isolated

from the cashew nut and apple was reported by Kubo et al. (2005). The anacardic acid from

both nut and apple was found to possess preventive antioxidant activity while salicylic acid did

not show this activity. These anacardic acids prevent the generation of superoxide radicals by

inhibiting xanthine oxidase without radical scavenging activity (Kubo et al., 2005).

Olatunji et al. (2005) investigated the antidiabetic activity of cashew plant (Anacardium

occidentale) stem-bark methanol extract on fructose-fed (diabetes) and normal rats. They

reported that the administration of the extract significantly prevented changes in plasma

concentration of glucose, triacylglycerol, total cholesterol/HDL ratio, malondialdehyde, urea

xxii

and creatinine. This observation was due to or induced by enriched fructose diet. Treatment

with the extract did not have significant effect on plasma alkaline phosphatase activity. These

results show that chronic oral administration of methanol extract of Anarcardium occidentale

stem-bark at a dose of 220.0 mg/kg body weight may be a safe alternative antihyperglycaemic

agent that has beneficial effect on diabetes induced in rats, this is associated with a reduced

Lipid peroxidation (Olatunji et al., 2005).

The in vitro activity of the different fractions of the leaf extract of Anarcardium

occidentale on some bacterial isolates, namely; Escherichia coli, Straphylococcus aureus and

Pseudomonas aerugnosa was investigated by Mustapha and Hafsat (2007). The leaf extract of

A. occidentale (especially the chloroform fraction) has a good potential for the development of

antimicrobial drugs (Mustapha and Hafsat, 2007).

Melo-cavalcante et al. (2008) reported the antimutagenic activity of cashew apple fresh

juice and processed juice against methyl methane sulfonate, 4-nitroquinoline, N-oxide and

benzo(α)pyrene. Both fresh and processed cashew apple juice may be useful for protection

against direct and indirect mutagens (Melo-cavalcante et al., 2008). The antimutagenic activity

of these juices may have been mediated by some or all of the following; the generation of

scavenger reactive species in treatment with 4-nitroquinoline; the induction of adaptive systems

in treatment with methyl methane sulfonate; the enhancement of DNA repair in treatment with

4-nitroquinoline and benzo(α)pyrene; the inhibition of metabolic activation in treatment with

benzo(α)pyrene; and the interaction with mutagen or pro mutagen in treatment with methyl

methane sulfonate and benzo(α)pyrene. Therefore, both fresh and processed cashew apple juice

may not be merely a source of nutrients but also a complex mixture of chemical compounds

with beneficial properties for improving genomic stability (Melo-cavalcante et al., 2008). It has

been reported that cashew apple juices have antitumour, antimicrobial, urease inhibitory and

lipoxygenase activities (De-Brito et al., 2007), as evidenced by their ability to scavenge free

peroxyl radicals. The total radical-trapping antioxidant potential study showed lowered

oxidative damage-induced mutagenesis by co- and post - treatment with the juice (Melo-

cavalcante et al., 2003).

1.7 DIARRHOEA

Diarrhoea is an alteration in a normal bowel movement characterized by an increase in

the water content, volume or frequency of stools. A decrease in consistency (i.e. soft or liquid)

and an increase in frequency of bowel movements to ≥ 3 stools per day has often been used as a

definition for epidemiological investigations (Chitme et al., 2004). “Infectious diarrhoea” due

xxiii

to an infectious aetiology, is often accompanied by symptoms of nausea, vomiting or

abdominal cramps. “Acute diarrhoea” is an episode of diarrhoea of ≤ 14 days duration. Some

experts refer to diarrhoea that last for ≥30 days as “chronic” (Guerrant et al., 2001).

Acute diarrhoea is a common problem that usually lasts 1-2 days and goes away on its

own without special treatment. Prolonged diarrhoea persisting for more than 2 days may be a

sign of a more serious problem and poses the risk of dehydration. Chronic diarrhoea may be a

feature of a chronic disease. Diarrhoea can cause dehydration which means the body lacks

enough fluid to function properly. Dehydration is particularly dangerous in children and older

people and it must be treated promptly to avoid serious health problems (National Digestive

Diseases Information Clearinghouse, 2007).

Diarrhoea and the associated faecal urgency and inconsistence result from an imbalance

between the absorptive and secretory mechanisms in the intestinal tract accompanied by

hypermotility; this condition results in excess loss of fluid and electrolytes in faeces (Chitme et

al., 2004). It is an important health problem especially in developing countries where it is

hyper-endemic with parasitism (Ezenwali et al., 2010).

1.7.1 Overview

1.7.1.1 Symptoms of Diarrhoea

Diarrhoea is characterized by frequent loose stools. The consistency of the stool can be

anything from soft and pasty to completely watery. The colour can range from brown to clear.

Symptoms related to any diarrhoeal illness are often those associated with any injury to the

gastrointestinal tract such as fever, nausea, vomiting, and abdominal pain. All or none of these

may be present depending on the disease causing the diarrhoea. The number of bowel

movements can vary up to 20 or more per day. In some patients, blood or pus is present in the

stool (National Digestive Information, 2005).

Patients with diarrhoea present with various clinical features depending on the

underlying cause (Guerrant et al., 2001). Diarrhoea, due to small-intestinal disease, is typically

high in volume, watery and often associated with malabsorption and dehydration is frequent.

Diarrhoea due to colonic involvement is more often associated with frequent small-volume

stools, with the presence of blood and a sensation of urgency (National Digestive Information,

2005). Patients with acute infectious diarrhoea typically present with nausea, vomiting,

abdominal pain, fever, and frequent stools, which may be watery, malabsorptive, or bloody

depending on the specific pathogen (Lim and Wallace, 2004). In general, small-intestinal

pathogens are noninvasive, and ileocolonic pathogens are more likely to be invasive. Patients

xxiv

ingesting toxins or those with toxigenic infection typically have nausea and vomiting as

prominent symptoms along with watery diarrhoea but rarely have a high fever. Vomiting that

begins within several hours of ingesting a food should suggest food poisoning due to preformed

toxin. Parasites that do not invade the intestinal mucosa, such as Giardia lamblia and

Cryptosporidium, usually cause only mild abdominal discomfort (National Digestive

Information, 2005). Giardiasis may be associated with mild steatorrhoea, gaseousness, and

bloating. Dehydration can occur if diarrhoea is severe and oral intake is limited due to nausea

and vomiting, particularly in very young and elderly patients. It is manifested as increased

thirst, decreased urinary output with dark urine, inability to sweat and orthostatic changes

(Guerrant et al., 2001). In severe cases, it may lead to acute renal failure and mental status

changes like confusion and drowsiness (National Digestive Information, 2005).

1.7.1.2 History of Diarrhoea

Human society has suffered from infectious diarrhoea since pre-literate time, although

historians themselves generally did not devote much attention to it until fairly recently. This

may be because of the ubiquity of the sickness and death caused by diarrhoea, regardless of

culture and climate (Guerrant et al., 2001). It is only within the last century that inhabitants of

advanced societies could safely assume that life-threatening diarrhoea would spare their

households. History and current experience demonstrate irrefutably that relatively minor

disruptions in the normal functioning of society can lead to the swift return of epidemic

diarrhoea (Lim and Wallace, 2004).

Prehistoric man is thought to have suffered relatively from epidemic diarrhoea because

of the small absolute size of hunter-gather communities and low overall population density, it is

unlikely that epidemic diarrhoea could have circulated to any large extent (Porter, 1997).

Furthermore, the mobility necessitated by the hunter-gatherer existence ensured that water

supplies remained on the whole, unpolluted and food was not stockpiled to any significant

degree (Porter, 1997). Although, occasional parasitic illness (e.g. caused by Giadia and

Cryptosporidium) certainly did occur; in general, it seems likely that diarrhoea in the

Paleolithic period was a short-term nuisance rather than a real threat to community health (Lim

and Wallace, 2004).

Diarrhoea was documented from the beginning of civilization, but exact descriptions

are generally lacking. This is not surprising, because modern diagnostic technology did not

exist, and the techniques of careful history taking, physical examination and verbal description

of clinical illness took centuries to evolve (Porter, 1997).. Furthermore, many of the diseases

xxv

recorded in early history struck populations without pre-existing immunity, and could be

expected to demonstrate remarkably severe effects. Philosophically, the ancient world tended to

view illness as a deviation from some idealized state of health (in fact, the very word,

‘‘disease,’’ akin to ‘‘discomfort,’’ implies that illness is a variation from natural harmony and

serenity). The notion that a specific, identifiable cause might lead to a discrete set of symptoms

was not easily compatible with the ancient world’s philosophic understanding of the role of

humans in the universe. Diarrhoea, rather than being seen as a gastrointestinal disease, was

regarded as indicative of a deeper problem, such as disequilibrium of the humours. Likewise,

the body was seen as a mirror of the larger universe (Guerrant et al., 2001).

Intravenous rehydration for diarrhoea was reintroduced in the 1920s. Although

effective, it generally required hospitalization and was very expensive; it remained impractical

in impoverished areas, where diarrhoea was most prevalent. Recognizing this, many physicians

advocated oral rehydration solutions, but efforts at developing an effective formula were

hindered by poor understanding of the physiology of diarrhoea and the natural progression of

the disease, particularly with respect to the changes in intestinal epithelium. It was assumed, for

example, that fasting was a necessary component of treatment, because the gut required a rest

period before it was capable of absorbing nutrients (Lim and Wallace, 2004). Many infant

feeding solutions contained hypertonic salt or sugar, which exacerbated diarrhoea by causing

osmotic shifts into the intestinal lumen. The first studies on the electrolyte composition of

diarrhoea and efforts to treat diarrhoea based on specific electrolyte replacement were

performed by Darrow in the 1940s (Ruxin, 1999). He reported considerable success with an

oral solution of potassium, lactate, and glucose; however, he still viewed oral therapy as an

adjunct to be used after intravenous resuscitation. For two decades, interest in oral rehydration

therapy waxed and waned, because most practitioners and clinical researchers believed that the

advantages and efficacy of intravenous therapy were so manifest as to make further research

into oral therapy unnecessary or even unethical (Lim and Wallace, 2004).. Among the many

objections raised to oral therapy were the perceived inability of ignorant natives in developing

countries to measure fluid input and output; a failure to understand the physiology of diarrhoea

(particularly toxigenic diarrhoea); and concern occasioned by some spectacular failures in early

field trials. For example, one early attempt using hypertonic oral solutions in combination with

intravenous hydration resulted in 5 of 30 patients dying from congestive heart failure (Lim and

Wallace, 2004).

1.7.2 Physiology and Pathophysiology of Diarrhoea

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Intestinal Ion and Water Transport

Ions and water can move bi-directionally across the intestinal mucosa, i.e., from luminal

(mucosal) to blood (serosal) sides and vice versa. The difference between the 2 unidirectional

fluxes, or the “net” ion flux, determines the direction of net transport. The active transport of

ions (principally Na+, Cl

-, and HCO3

-) across the small intestinal epithelium provides the

electrical and chemical forces that drive the coupled absorption of nutrients as well as the net

absorption or secretion of water. A variety of absorptive and secretory agents, including local

and systemic hormones, neurotransmitters (Cooke, 2000), toxins released by enteric pathogens,

and other molecules that gain access into the intestinal lumen, stimulate ion and water

transport. Solutes are absorbed by active transport mediated by membrane-associated channels

and transporters. In addition, solutes can traverse the intestinal epithelium through 2 other

mechanisms, often collectively referred to as intestinal permeability. These are endocytotic

uptake from the lumen, followed by exocytotic delivery to the basolateral compartment and

intercellular transport through the tight junctions that separate enterocytes (the paracellular

pathway). Although the traditional view is that 80% of water transport uses the paracellular

pathway this transport is achieved by the creation of local osmotic gradients within the

paracellular channels. Hence, specific enterocyte membrane transporters (e.g. GLUT 1) that

induce solute transport ultimately drive absorption of water and ions. These transporters require

energy hence, the use of the term active transport. Net movement of ions and fluid across the

small intestinal epithelium in the basal state varies considerably along the length of the small

intestine (Camilliri, 2004).

1.7.3 Chronic Diarrhoea

The causes of chronic diarrhoea include inflammatory, osmotic, secretory, iatrogenic,

motility, and functional diseases. In general, no single cause of chronic diarrhoea is truly uni-

factorial from a perspective of pathophysiology. Thus, for example, prototype secretory

disorders such as cholera might be associated with secretion and altered motility. Secretion,

inflammation, and motility contribute to diarrhoea caused by Clostridium difficile toxin (Krose

et al., 1994).

1.7.3.1 Inflammatory Chronic Diarrhoea

Inflammatory diseases cause chronic diarrhoea with exudative, secretory, or

malabsorptive components. The pathogenesis of irritable bowel diseases (IBD) is increasingly

understood in terms of the molecular mechanisms that result in changes in both innate and

xxvii

acquired immunity (Krose et al., 1994). The response of the gut to bacteria and other antigens

in the lumen results from the interaction between the host, the genetic environment, dietary,

and bacterial antigens. Inflammatory causes of chronic diarrhoea might present with features

that suggest malabsorption or rectal bleeding. The nature of the malabsorption depends on the

regions affected (e.g. proximal vs. distal small bowel), and rectal bleeding is usually a

manifestation of colonic or rectal ulcerations (Cooke, 2000). The presence of an inflammatory

cause of chronic diarrhoea is indicated by the presence of mucus and blood in the stool, by

concomitant abdominal pain, or by symptoms referable to the skin, eyes and joints. Typically,

bowel imaging and tissue biopsies provide the diagnosis of the condition. Inflammatory chronic

diarrhoea is rapidly diagnosed when associated with rectal bleeding or malabsorption. In the

case of microscopic colitis, a therapeutic trial might be needed to determine whether it is the

cause of the chronic diarrhoea when no other cause is identified (Camilliri, 2004).

1.7.3.2 Osmotic chronic Diarrhoea

In osmotic diarrhoea, osmotically active substances draw fluids from the plasma into

the jejunum along the osmotic gradients through the highly permeable jejunal epithelium.

Examples of chronic diarrhoea due to an osmotic cause include malabsorption states such as

celiac disease, bacterial overgrowth, osmotic laxatives (including salts, polyethylene glycol,

and lactulose), and mal-digestion as they occur commonly in disaccharidase deficiency, and

pancreatic exocrine insufficiency (Cooke, 2000). Osmotic diarrhoea might result in

steatorrhoea and azotorrhoea (passage of fat and nitrogenous substances into the stool), but

typically they do not cause any rectal bleeding. With osmotic diarrhoea, the volume of stool is

reduced during fasting, but the osmotic gap of the stool is greater than 50 mOsm/kg (Camilliri,

2004). Measured osmolarity of the water from a fresh stool sample is a useful measurement to

identify such an osmotic gap. Normally, the stool water is electroneutral, that is, an equivalent

number of anions and cations are present. Thus, measurement of the sodium and potassium

concentrations multiplied by 2 should be almost equivalent to the osmolarity of plasma (280

mOsm/kg). When the measured osmolarity exceeds 2 times the sodium and potassium

concentration by more than 50 mOsm/kg, an osmotic factor is contributing to the diarrhoea.

When the stool water pH is below 5 and the osmotic gap is more than 50 mOsm/kg, the

osmotic factor is likely to be a disaccharide that has undergone bacterial fermentation in the

colon to produce acid residues that reduce stool water pH. Thus, a high osmotic gap with stool

water pH of less than 5 is indicative of disaccharidase deficiency (Camilliri, 2004).

xxviii

1.7.3.3 Secretory Chronic Diarrhoea

In secretory diarrhoea, there is secretion of iso-osmolar fluid into the intestine. In this

situation, other electrolyte abnormalities might coexist. Thus, hypokalemia and acidosis are

associated with Verner-Morrison syndrome. Examples of secretory diarrhoea include

congenital abnormalities such as congenital chloridorrhea, in which an abnormality in the

genetic control of chloride-bicarbonate exchange in the ileum results in the loss of chloride into

the stool. Another example is the loss of α2-adrenergic function in enterocytes of patients with

autonomic neuropathy caused by diabetes mellitus (Camiliri, 2004). Colonic secretion might

occur as a result of bile acid malabsorption or the effects of hormones secreted by

neuroendocrine tumuors including gastrin, serotonin, calcitonin, prostaglandins, and others.

The literature is replete with examples of exogenous infusions of these substances resulting in

intestinal secretion. More recently, the effects of the secretagogues have also been

demonstrated on explants of human intestinal epithelium (Hope et al., 2001). Secretory

diarrhoea might be associated with altered motor functions induced by the hormone or

transmitter produced by the tumuor, as in carcinoid diarrhoea (Camiliri, 2004).

1.7.3.4 Iatrogenic Causes of Chronic Diarrhoea

Chronic diarrhoea might follow abdominal surgery. After cholecystectomy, about 5%–

10% of patients develop diarrhoea; the mechanism is still not completely understood. Some of

these patients respond to sequestering bile acid with cholestyramine, but this is not a universal

response. Chronic diarrhoea might result from vagal injury and ileal resection (Hope et al.,

2001). In current practice, vagal injury results from fundoplication or gastric bypass

procedures. Testing for vagal malfunction includes the pulse rate and electrocardiographic R to

R interval response to deep breathing or measurement of pancreatic polypeptide in plasma in

response to modified sham feeding (Camiliri, 2004). The β-cells in the pancreas (source of

pancreatic polypeptide) are under control by vagal cholinergic neurons and sham feeding

stimulates the vagal nuclei in the brainstem. Thus, heart period response to deep breathing

should be greater than 10 per minute, although there are variations as a result of age and gender

(Hope et al., 2001). With abdominal vagal injury caused by surgery, the cardiac index of vagal

function is usually normal. Pancreatic polypeptide response to modified sham feeding should

increase by more than 25 pg/mL (or pg/L) if there is an intact vagal function. There are,

however, pitfalls in the interpretation of the pancreatic polypeptide response to sham feeding.

For example, there might be an inadequate sham feeding stimulus because of failure to follow

instructions or high baseline levels of pancreatic polypeptide as a result of uraemia. More

xxix

importantly, false negatives result from increased plasma levels of pancreatic polypeptide

induced by swallowed food, which activates the enteric phase of pancreatic polypeptide

secretion. Iatrogenic chronic diarrhoea might also follow ileal resection. The extent of resection

determines the mechanisms and manifestations (Camiliri, 2004).

1.7.3.5 Motility Disorders Causing Chronic Diarrhoea

Rapid transit delivers fluid secreted during digestion to the distal small bowel or colon;

this prevents reabsorption of normally secreted fluid in the small bowel, or it overwhelms the

reabsorptive capacity of the colon (Hope et al., 2001). On the other hand, reduced motility

leading to slow transit might result in bacterial overgrowth with bile acid deconjugation, poor

micelle formation, and steatorrhea. The clinical manifestations of chronic diarrhoea caused by

motility disorders include steatorrhea, usually up to 14 g per day (Camiliri, 2004).

Motility disorders causing chronic diarrhoea are usually not associated with any blood

loss per rectum. On the other hand, the clinical features indicate a neuropathic- or myopathic-

underlying disease. Systems review might identify symptoms caused by the classic triopathy of

diabetes mellitus (peripheral neuropathy, retinopathy, and nephropathy) or the peripheral

manifestations associated with collagenoses (e.g. affecting skin, eyes, mouth and joints).

Diabetes is associated with a number of diseases that cause diarrhoea and steatorrhoea. These

include exocrine pancreatic insufficiency, celiac sprue, small bowel bacterial overgrowth and,

rarely, bile acid mal-absorption (Camiliri, 2004). In patients with systemic sclerosis, chronic

diarrhoea might occur because of small bowel dilation, wide mouthed diverticulosis, or

bacterial overgrowth (Weston et al., 1998). Pneumatosis intestinalis occurs in a minority of

patients, but the diarrhoea might also be associated with incontinence, typically nocturnal

because of the weakness of the internal anal sphincter when the volitional external sphincter

cannot compensate during sleep. Bacterial overgrowth and diverticulosis require aspirate or

breath tests and small bowel follow-through with upright images to demonstrate air fluid levels

or a computed tomographic scan with careful evaluation for air fluid levels in the horizontal

position. Transit measurements are often undertaken in patients with suspected motility

disorders as a screening test for dumping syndrome (accelerated gastric emptying) and fast or

slow transit at all levels of the gut. Transit measurements evaluate the severity of the motor

abnormality (Weston et al., 1998).

However, it is important to realize that fast transit might be as a result of an underlying

disease, not only a primary neuromuscular disorder. For example, disease of the mucosa, such

as coeliac disease, might also result in accelerated transit (Camiliri, 2004).

xxx

1.7.4 Causes of Diarrhoea

Causes of diarrhoea include infective agents, certain medications, plant and animal

toxins, gastrointestinal tract (GIT) disorders and substances that increase GIT secretions. It can

also be caused by the ingestion of poorly absorbable materials or inflammatory and dysmotility

problems of the GIT (Otimenyin et al., 2008). The major causes of this illness include limited

access to or poor quality of water, poor food hygiene and sanitation among others. The bacteria

pathogens usually responsible include E. coli, Shigella, Samonella, Campylobacter, Yersinia

and Aeromonas (Nweze, 2009).

Acute diarrhoea is usually related to bacteria, viral or parasitic infections, chronic

diarrhoea are usually related to functional disorders such as irritable bowel syndrome or

inflammatory bowel disease. A few of the more common causes of diarrhoea include:

Bacterial Infection: Several types of bacteria consumed through contaminated food or

water can cause diarrhoea. Common culprits include Campylobacter, Salmonella,

Shigella and E. coli.

Viral Infections: Many viruses cause diarrhoea; They include Rotavirus, Norkalk virus,

Cytomegalovirus, Herpes simplex virus and viral hepatitis.

Food Intolerances: Some people are unable to digest food components such as artificial

sweeteners and lactose (sugar found in milk).

Parasites: Parasites can enter the body through food or water and settle in the digestive

system. Parasites that cause diarrhoea include Giardia lamblia, Entamoeba histolitica

and Cryptosporidium.

Intestinal Diseases: Inflammatory bowel disease, colitis, Crohns disease and coeliac

disease often lead to diarrhoea.

Reaction to Medicines: Antibiotics, blood pressure medications, cancer drugs and

antacids containing magnesium can all cause diarrhoea (National Digestive Diseases

Information Clearinghouse, 2007).

Antibiotic-associated diarrhoea (AAD) is defined as diarrhoea that occurs in association

with the administration of antibiotics (Bartlett, 2002). The direct toxic effects of antibiotics on

the intestine can alter digestive functions secondary to reduced concentrations of the normal

gut bacteria or cause pathogenic bacteria overgrowth, understanding the different mechanisms

that cause antibiotics-associated diarrhoea may help to prevent this condition, improve

medical care and reduce medical cost (Song et al., 2008). Clostridium difficile is widely

known to be responsible for approximately 10-20% of cases of antibiotic-associated diarrhoea

xxxi

and almost all cases of pseudomembranous colitis. However, Klebsiella oxytoca, enterotoxin

producing Clostridium perfringens, Staphylococcus aureus, Candida species, Salmonella

speices and Pseudomonas aeruginosa might also contribute to the development of antibiotic-

associated diarrhoea (Beaugerie and Petit, 2004).

Fig. 2: Three groups of antibiotic-associated diarrhoea detected by endoscopy. Normal (A),

non-specific colitis (B, C, D), and pseudomembranous colitis (E, F). (Song et al., 2008)

A B

C D

E F

xxxii

Some people develop diarrhoea after stomach surgery or removal of the gall bladder.

The reason may be due to a change in how quickly food moves through the digestive system

after stomach surgery or an increase in bile in the colon after gall bladder surgery. People who

visit foreign countries are at risk for traveller’s diarrhoea which is caused by eating food or

drinking water contaminated with bacteria, virus or parasite (Song et al., 2008).

1.7.5 Diagnosis of Diarrhoea

Bacterial and viral enteric infections are two of the major health problems in most

developing countries and a good diagnostic laboratory is important in the control of diarrhea

(Wilson, 2005). In connection with the programme launched by World Health Organization

(WHO) for the control of diarrhoea and epidemiological infection, information on the nature

and extent of the diarrhoea problem in a country is necessary which can only be realized with

the help of the laboratory. Without the laboratory, it is difficult to diagnose an unusual infection

(Santiago, 1983).

Diarrhoea is most common due to viral gastro-enteritis with rotavirus accounting for

40% of cases in children under five; in travellers however, bacterial infections predominate. It

can also be the part of the presentation of a number of medical conditions such as: Crohn’s

disease or mushroom poisoning (Wilson, 2005). In the treatment of cases, the laboratory is just

as important, because, while antimicrobials are not used routinely in diarrhoea, certain

diarrhoea such as Cholera, Shigellasis, Campylobacter diarrhoea, Amoeblasis and Giardiasis

require specific treatment especially if severe (Santiago, 1983).

1.7.5.1 Laboratory Methods

In diarrhoeal disease, the preferred specimen is the faeces. It should be properly

collected in the right container; a rectal catheter can be used. The stool should be freshly passed

and sent to the laboratory at once (Wilson, 2005). Although, the stool is preferable to the rectal

swab specimen, in practice, there are situations in which a rectal swab must be used such as

when it is desirable to collect the faeces immediately in the absence of a bowel movement,

when transport of the stool to the laboratory are for practical purpose, when too many stool

samples are to be collected at one time also, carefully collected rectal swab specimen may

xxxiii

sometimes be preferred for bacteria, which invade the mucosa of the lower intestine because

the swab sample is collected with a scrubbing motion of the intestinal mucosa (Santiago, 1983).

A sample transport medium in the glycerol saline mixture for V. cholera, one can use

alkaline peptone water for Salmonella and probably Shigella, selenith broth.

Fig. 3: Bacterial culturing in a diarrhoeal stool or rectal swab

These transport broth can at the same time promote growth of the specific pathogen. For

rotavirus examination, a small amount of stool or rectal swab is put into 1ml phosphate

buffered saline solution and frozen at -200C (Santiago, 1983).

Laboratory examination

Direct microscopic examination

Faecal leucocytes: Their presence especially, if many are seen, indicate an invasive

bacteria caused like Shigella, Yersinia, Campylobactera, Enteroinvasive E. coli (EIEC),

Salmonella, amoebic colitis causes idiopathic inflammatory bowel diseases or

pseudomembranous colitis. Absence of faecal leucocytes indicates non- invasive bacteria

caused, like cholera, enterotoxigenic E. coli (ETEC) or viral gastroenteritis. Giardiasis and

parasitic infection generally do not produce faecal leucocytes (Santiago, 1983). Other findings

xxxiv

revealed that fats and oils should point towards one of the diseases that cause chronic mal-

absorption as in chronic pancreatitis, sprue or other small bowel disease (Wilson, 2005).

Parasite and ova should be routinely looked for in gastro-entritis since they can also cause

diarrhoea. Electron microscopic examination could be used for Rotavirus and Norwalk virus

determination since both viruses are hard to culture. However, this examination is impractical

to use routinely because it is a very slow and expensive procedure. Any of the following test

methods can be used for the test: Sereny test, rabbit ileal loop assay, infant mouse assay, tissue

culture assay and ELISA test (Santiago, 1983).

The laboratory is very important in the diagnosis of diarrhoea disease, because, without

it many aetiologic causes of diarrhoea cannot be identified and the medical Practitioners might

have a hard time controlling the spread of diarrhoeal diseases (Santiago, 1983).

1.7.6 Management of Diarrhoea

Diarrhoea diseases are some of the major killers of children in the developing countries.

Each year, there are approximately 4 million cases of diarrhoea worldwide leading to 4 million

deaths especially among children aged 5 years or less (Azubuike and Nkagineme, 2007); in this

age group, 38% of all deaths are associated with diarrhoea diseases. In Southeast Asia and

Africa, diarrhoea is responsible for as much as 8.5 and 7.7% of all deaths respectively (WHO,

1990). Studies in Nigeria show among hospitalized children less than 5 years of age (Azubuike

and Nkagineme, 2007).

Early identification and appropriate management of cases by replacement of lost fluids

and electrolytes are crucial to reducing mortality and morbidity especially in under five

(Aguwa et al., 2010). Despite the fact that the control of diarrhoea disease (CDD) programme

of the World Health Organization (WHO) was launched in 1980 with the aim of promoting oral

rehydration therapy (ORT) and nutritional case management of diarrhoea disease, 3-4 million

children are still dying of diarrhoea every year (Sodemann et al., 1999). It has been argued that

the control of diarrhoea disease programme has emphasized the use and popularization of oral

rehydration salts rather than educating the public and health staff in the process of rehydration

and practice of rehydration (Sodemann et al., 1999).

The WHO meeting of experts concluded in 2001 that there are programmatic

advantages of using a single rehydrating solution globally for all cases of diarrhoea in all ages.

Evidence from large, well conducted and randomized controlled trials including those in India

showed that low osmolarity oral rehydration salt (ORS) with 75mEq/L of sodium and

xxxv

75mmol/L of glucose, osmolarity of 245osmol/L is effective in children with non-cholera and

in adults and children with cholera (Bhatnagar et al., 2010). A number of trials in India and

other low middle income countries have documented faster recovery and reduced severity from

zinc supplement during acute diarrhoea (Bhutta et al., 2000). Zinc deficiency is common in

children living in such settings due to low intake of animal foods, high dietary phytate content

and overall inadequate diets. This led to the WHO recommendation of supplemental zinc syrup

or tablets during acute diarrhoea (Bhatnagar et al., 2010). Therefore, adequate fluid and

electrolyte replacement and maintenance are keys to managing diarrhoea illness (Guerrant et

al., 2001).

Patients receiving chemotherapy or radiation are at high risk for developing diarrhoea,

which is estimated to be as high as 45% with chemotherapeutic drugs like irinotecan or 5-

fluorouracil (5FU). This side effect often leads to delay in treatment, dose reduction or

discontinuation of treatment. There is a small but significant mortality associated with

chemotherapy-induced diarrhoea (CID), especially when it occurs concomitantly with

mucositis and neutropenia (Wadler et al., 1998). Chemotherapy-induced diarrhoea (CID) can

be a serious complication and requires prompt assessment. The steps in this assessment are as

follows:

(1) Rule out other or concomitant causes of diarrhoea: - Other causes of diarrhoea must be

ruled out. These include medication (e.g. stool softeners, laxatives, antacids etc). Infection by

C. difficile or Candida species, partial bowel obstruction, mal-absorption, faecal impaction,

acute radiation reaction and surgery (short bowel syndrome). Diets high in fibre or lactose may

aggravate diarrhoea.

(2) Dietary modifications during diarrhoea: mild diarrhoea may be managed with diet to

decrease the frequency of stools. Patients should be advised to increase intake of clear fluids

(e.g. water, sport drinks, broth, gelatin, clear juices, decaffeinated tea and caffeine-free soft

drinks) (Walder et al., 1998).

1.7.7 Medications for Chemotherapy-Induced Diarrhoea

Patients who develop diarrhoea while on irinotecan chemotherapy should be treated

promptly with high dose of loperamide and dietary management alone is inadequate.

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1.7.7.1 Loperamide

Loperamide is indicated for diarrhoea that persists for more than 12-24 hours (Grade 1)

or for moderate diarrhoea (Grade 2). The standard dose of loperamide is 4mg followed by 2mg

every 4 hours or after each uniformed stool (maximal dose is 16mg/day). This dose may be

increased in patients with mild to moderate diarrhoea (Grade 1 or 2) that persists for more than

24 hours. Loperamide should be continued for 12 hours following resolution of the diarrhoea

and re-establishment of a normal diet. High dose of loperamide (4mg followed 2mg every 2

hours) is also recommended at the onset of any diarrhoea in patients receiving irinotecan

chemotherapy (Walder et al., 1998).

1.7.7.2 Atropine-diphenoxylate (Lomotil)

It may be added every 6-8 hours to loperamide therapy for grade 1 or 2 diarrhoea.

1.7.7.3 Octreotide

For grades 1 and 2 diarrhoea is lasting more than 24 hours despite high dose loperamide

± Atropine-diphenoxylate, octreotide 100-150mcg. Sc. TID may be considered. For grades 3

and 4 diarrhoea, octreotide is indicated, if there is no improvement in the diarrhoea after 24

hours, the dose of octreotide should be increased to 300-500mcg sc TID. The duration of

octreotide therapy should be individualized, but can be discontinued 24 hours after the end of

diarrhoea and re-establishment of normal diet.

1.7.7.4 Antibiotics

In the presence of concomitant neutropenia, (granulocyte < 1×106/L), antibiotics (e.g.

Ciprofloxacin 500mg BID) should be considered until resolution of diarrhoea and recovery of

granulocytes counts (Walder et al., 1998).

1.8 Aim and Objectives of the Study

The aim of this study was to investigate the anti-diarrhoeal activity of ethanol-

chloroform extract of cashew (A. occidentale) kernel in rats. The specific objectives

included:

xxxvii

to determine anti-diarrhoeal activity of the ethanol-chloroform extract of A.

occidentale in castor oil-induced diarrhoea rats.

to determine the effect of the ethanol-chloroform extract of A. occidentale on

enteropooling activity in castor oil-induced diarrhoea in rats.

to determine the effect of the ethanol-chloroform extract of A. occidentale on

gastro-intestinal motility of rats.

to determine the effect of ethanol-chloroform extract of A. Occidentale on the

concentrations of sodium and potassium ions in the intestinal solution of rats.

CHAPTER TWO

MATERIALS AND METHODS

2.1 MATERIALS

2.1.1 Plant Materials

xxxviii

Cashew (Anacardium occidentale) nuts used were collected from Obolloafor, Enugu

State, Nigeria. They were authenticated by Mr. A. Ozioko of the Bioresource Development and

Conservation Programme (BDCP) Research Centre in Nsukka.

2.1.2 Animals

Adult female Wistar albino rats of 10-16 weeks old with average weight of 160 ± 14g

were obtained from the Animal House of the Faculty of Pharmaceutical Sciences, University of

Nigeria, Nsukka. The animals were acclimatized for 10 days under standard environmental

conditions, with a 12 - hour light and dark cycle, maintained on a regular feed and water ad

libitum.

2.1.3 Chemicals

The chemicals used for this study were of analytical (reagent) grade and include the

following:

Chloroform and ethanol (were supplied by Sigma, England), tetraoxosulphate (vi) acid,

potassium hydroxide, sodium hydroxide, activated charcoal, gum acacia, lead acetate, castor

oil, tween 20, α-naphthol, iodine, potassium iodide, ferric chloride, ammonium and nitric acid.

2.1.4 Equipment

Weighing balance (Brand Scientific & Instrument Company); rotatory evaporator; Oven

(Gallenkamp, England); Water-bath and Chemical balance (Gallenkamp, England); Centrifuge

(Pic, England) and Spectrophotometer (Jenway 6405 uv/vis).

2.2 METHODS

2.2.1 Preparation of Charcoal Meal

A volume of 0.5ml of 10% charcoal (10g of charcoal dissolved in 100ml of distilled

water) was suspended in 10% gum acacia (10g of gum acacia dissolved in 100ml of distilled

water) to give charcoal meal.

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2.2.2 Preparation of 20% Potassium Hydroxide

To prepare 20% potassium hydroxide, 20g of potassium hydroxide pallet was added to

100ml of distilled water.

2.2.3 Extraction Procedure

The dried cashew kernels were isolated using a simple cutter knife, which was used to

split each nut open. A pointed knife employed to remove the kernel immediately from the shell

to minimize contamination with the cashew nut shell liquid (CNSL). The kernel was then

subjected to roasting at 80°C for one hour to remove the testa as described by Onilude et al.

(2010). The roasted kernel was ground into coarse form.

The pulverized kernel (1071g) was macerated in 3L mixture of chloroform and ethanol

(2:1) for 48 hours. The macerate was passed through Whatman No 4 filter paper. The filtrate

was shaken with 20% of distilled water to obtain three (3) layers.

The upper layer (ethanol layer) was drawn out; the middle layer was also separated

from the lower layer (chloroform layer). The three layers were concentrated with a rotary

evaporator and dried in a boiling water bath. The weight of the plant was taken after drying.

The extract yields were 5.7g (0.53%), 15.9g (1.48%) and 7.9g (0.74%) for upper, middle and

lower layers respectively.

2.2.4 EXPERIMENTAL ANALYSIS

2.2.4.1 Phytochemical Test

Basic qualitative phytochemical screening of the ethanol, chloroform and middle layers

of the water-treated extract of the kernel sample was carried out by testing for the presence or

absence of the following plant constituents: flavonoids, tannins, saponins, glycosides, fat and

oil, sterol, alkaloids, reducing sugar and carbohydrate. The phytochemical analysis of the

sample was carried out using procedures outlined by Harborne (1989) and Trease and Evans

(1989).

2.2.4.1.1 Test for Carbohydrate (Molish’s Test)

The extract (0.1g) was boiled with 2ml of distilled water and filtered. To the filtrate,

few drops of α-naphthol solution in ethanol (Molisch’s reagent) were added. Concentrated

sulphuric acid was then gently decanted down the side of the test tube to form a lower layer. A

purple interfacial ring indicated the presence of carbohydrate.

xl

2.2.4.1.2 Test for Alkaloids (General Tests)

The extract (0.5g) was boiled with 5ml of 2% hydrochloric acid on a water bath for 5

minutes, and then filtered. A one ml portion of the filtrate was treated with 2 drops of the

following reagents.

(i) Dragendroffs Reagent: (Bismuth potassium iodide solution). An orange to a red

precipitate indicated the presence of alkaloids.

(ii) Mayer’s Reagent (Potassium mercuric iodide solution): A creamy white precipitate

indicated the presence of alkaloids.

(iii) Wagner’s Reagent (Iodine in potassium iodide solution): A reddish brown precipitate

indicated the presence of alkaloids.

(iv) Picric acid (1%): A yellow precipitate indicated the presence of alkaloids.

2.2.4.1.3 Test for Glycosides (Fehling’s Test)

Dilute suphuric acid (5ml) was added to 0.1g of the extract in a test tube and boiled for

15 minutes in a water bath, then cooled and neutralized with 20% potassium hydroxide

solution. Ten milliliters (10ml) of a mixture of equal parts of Fehling’s solution A and B was

added and boiled for 5 minutes. A dense brick red precipitate indicated the presence of

glycoside.

2.2.4.1.4 Test for Saponins

Distilled water (20ml) was added to 0.25g of the extract and boiled in a water bath for 2

minutes. The mixture was filtrated while hot and allowed to cool. The filtrate was used for the

following tests.

(a) Frothing Test

The filtrate (5ml) was diluted with 15mls of distilled water and shaken vigorously. A

stable froth (foam) upon standing indicated the presence of saponins

(b) Emulsion Test

Two drops of olive oil were added to the froth solution and the contents shaken

vigorously. The formation of emulsion indicated the presence of saponins.

(c) Fehling’s Test

To 5ml of the filtrate was added 5ml of Fehling’s solution. Equal parts of A and B were

added and the mixture was heated on a water bath. Reddish precipitate which turned brick red

on further heating with sulphuric acid indicated the presence of saponins.

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2.2.4.1.5 Test for Tannins

One gramme (1g) of the extract was boiled with 20ml of water, filtered and used for the

following tests.

(a) Ferric Chloride Test

Few drops of ferric chloride were added to 3ml of the filtrate. A greenish black

precipitate indicated the presence of tannins.

2.2.4.1.6 Test for flavonoids

The extract (0.5g) was heated with 10ml of ethylacetate in boiling water bath for 3

minutes, the mixture was cooled, filtered and the filtrate was used for the following tests.

(a) Ammonium test

Four milliliter (4ml) of the filtrate was shaken with 1ml of dilute ammonia solution.

The layers were allowed to separate and the yellow colour in the ammoniacal layer indicated

the presence of flavonoids.

(b) 1% Aluminium Chloride Solution Test.

Another 4ml portion of the filtrate was shaken with 1ml of 1% aluminium chloride

solution. The layers were allowed to separate. A yellow colour in the aluminium chloride layer

indicated the presence of flavonoids.

2.2.4.1.7 Test for steroids

Ethanol (9 ml) was added to one gramme (1g) of the extract and refluxed for a few

minutes and filtered. The filtrate was concentrated down to 2.5 ml in a boiling water bath and

5ml of hot water was added to it and mixed thoroughly. The mixture was allowed to stand for 1

hour after which the waxy matter was filtered off. The filtrate was shaken with 2.5 ml of

chloroform in a separating funnel. The lower layer was drawn out. To 0.5 ml of the chloroform

extract in a test tube was carefully added one ml of concentrated sulphuric acid to form a lower

layer.

Another 0.5 ml of the chloroform extract was evaporated to dryness on a water bath and

heated with 3 ml of concentrated sulphuric acid for 10 minutes on a water bath. A reddish

brown interface indicated positive result for steroids

2.2.4.1.8 Test for Reducing Sugar

xlii

A volume of five milliliters (5ml) of a mixture of equal parts of Fehling’s solution A

and B was added to 5ml of the extract and heated in a water bath for 5 minutes. Brick red

precipitate showed the presence of reducing sugar.

2.2.4.1.9 Test for Oils

One hundred milligrammes (100mg) of the extract was pressed between filter paper and

the paper was observed. A control was prepared by placing two (2) drops of olive oil on filter

paper. Transparency of the filter paper indicated the presence of fats and oil.

2.2.4.2 Acute Toxicity and Lethality (LD50) Test

The acute toxicity and lethality of chloroform-ethanol extracts of the cashew kernel was

determined using the modified method of Lorke (1983). The test was divided into two stages.

In stage one, twenty-seven (27) randomly selected adult mice were divided into nine groups,

three per group (n=3) and received 10, 100 and 1000 mg/kg body weight of each of the ethanol,

middle layer and chloroform extracts respectively and the signs of toxicity and number of death

for a period of 24 hours were recorded. After 24-hour observation, the doses for the second

phase were determined based on the outcome of the first phase. Since there was zero death, a

fresh batch of animals was used following the same procedure in phase I but with higher dose

ranges of 1900, 2600 and 5000mg/kg body weight of the extract. The animals were also

observed for 24 hours for signs of toxicity and possible number of death. The LD50 was

calculated as the geometric mean of the high non-lethal dose and lowest lethal dose (Lorke,

1983).

2.2.4.3 Anti-Diarrhoeal Studies (Castor Oil-Induced Diarrhoeal Test)

The effect of chloroform-ethanol extracts on diarrhoea was evaluated in rats using the

method of Awouters et al. (1978), as modified by Nwodo and Alumanah (1991). Thirty-two

(32) Adult female albino Wistar rats, which had previously been feed on standard Pfizer diet

and allowed free access to water were used. Rats were fasted for 18 hours and divided into

eight groups with four rats per group (n=4). Group I (control) rats were administered with

Tween 20 (vehicle for dissolving the extract) using suitable stomach tube; Group II rats were

administered (oral) 2.5mg/kg body weight of lomotil (diphenoxylate and atropine sulphate);

which served as a standard drug; while Groups III, IV, V, VI, VII and VIII rats were orally

administered 21mg/kg and 84kg/mg body weight of extracts of Anacardium occidentale kernel.

xliii

After one-hour administration (oral) of the varying doses of the ethanol-chloroform extracts of

Anacardium occidentale kernel, the animals received 1ml of castor oil orally and each of the

animals was at this stage separated into their respective individual cages. The rats were

observed for consistency of faecal materials and the frequency of defaecation. Faeces were

collected on a white sheet of paper that was placed beneath individual cages. The number of

both wet and dry droppings was counted every 1 hour for a period of 5 hours and the white

paper was changed periodically for each evaluation. The level of inhibition (%) of wetness of

faeces and frequency of stooling caused by ethanol-chloroform extracts was calculated relative

to the control using the formula below:

Inhibition of defaecation (%) = %100

ControlofFaecesofNoMean

GroupTreatedofFaecesofNoMeanControlofFaecesofNoMean

2.2.4.4 Castor Oil-Induced Enteropooling Test

The effect of ethanol-chloroform extracts of Anacardium occidentale kernel was

evaluated using the castor oil–induced enteropooling method (Robert et al., 1976). Thirty-two

(32) rats were fasted for 18 hours with free access to water which were divided into eight (8)

groups with four rats per group (n = 4).

Group I rats were administered (oral) tween 20 (vehicle), which was used to dissolve

the extract; Group II rats were administered (oral) 0.29mg/kg body weight of hyoscine butyl

bromide as the standard drug; Groups III and IV were orally administered 21 mg/kg and 84

mg/kg b.w. of the ethanol layer of the extracts, group V and VI were administered 21 mg/kg

and 84 mg/kg b.w of the middle layer of the extracts (oral), while VII and VIII rats were

administered (oral) 21mg/kg and 84mg/kg body weight of the chloroform layer of the extracts

respectively by stomach intubation. One-hour post treatment, 1ml of castor oil was

administered to each rat by oral gavages. After 1-hour administration of castor oil, the rats were

sacrificed by cervical dislocation, the small intestine was located and tied at the pyloric

sphincter and caecal junction and cut out; the small intestine was weighed. The content of each

intestine was milked into a graduated test tube and the volume recorded. The empty small

intestine was reweighed and the difference in weight between the full and empty small intestine

was recorded as the weight of the small intestine content. The rate of reduction in the volume

and weight of intestinal content was calculated relative to the control.

2.2.4.5 Gastro-Intestinal Motility Test

xliv

The effect of ethanol-chloroform extracts of Anacardium occidentale kernel on gastro-

intestinal motility was evaluated using the method of Mascolo et al. (1994) with little

modification. Thirty-six adult female Wistar albino rats were fasted for 18 hours and separated

into nine groups of four rats per group (n=4). Each rat was administered 1ml of castor oil. After

a duration of 1 hour, rats in group I were administered (p.o.) 5mg/kg body weight of tween 20

the vehicle which is used in dissolving the extract, serving as the control; Group II rats were

administered (p.o.) 2.5mg/kg body weight of lomotil and group III rats were administered (p.o.)

0.29mg/kg body weight of hyocine which served as standard drug, Groups III and IV were

orally administered 21 mg/kg and 84 mg/kg b.w. of the ethanol layer of the extracts, groups V

and VI were administered 21 mg/kg and 84 mg/kg b.w of the middle layer of the extracts (p.o.),

while groups VII and VIII rats were administered (p.o.) 21mg/kg and 84mg/kg body weight of

the chloroform layer of the extracts respectively by stomach intubation. After one-hour

administration of the extract, animals were administered (p.o.) 0.2ml of charcoal meal (0.5ml

of 10% charcoal suspended in 10% gum acacia). After a duration of 1-hour, the animals were

sacrificed by overdose of anaesthesia and the small intestine was carefully separated from the

mesenterum in order to avoid being stretch. The length of the intestine from the pyloric

sphincter to the ileo-caecal junction (caecum) and the distance travelled by the charcoal meal

was measured for each animal. The gastro-intestinal transit was calculated as the percentage

distance travelled by the charcoal meal relative to the length of the intestine.

2.2.4.6 Preparation of Sample for Electrolyte Test

Female albino Wistar rats were fasted for 18 hours with water ad libitum and were

divided into six groups. Each rat was administered (p.o.) 1ml of castor oil. After 1 hour, rats in

group 1 were administered (p.o.) tween 20 (vehicle) for dissolving the extract; group II rats

were administered (p.o.) 2.5 mg/kg b.w of lomotil as a standard anti-diarrhoea drug; group III

and IV rats were administered 21 mg/kg and 84 mg/kg b.w of ethanol layer of the extract, while

group V and VI rats were administered 21 mg/kg and 84 mg/kg b.w of chloroform layer of the

extract. After 1 hour of the treatment, the rats were sacrificed by cervical dislocation, the small

intestines were located and tied at the pyrolic sphincter and ileo caecal junction and cut out, the

small intestines were weighed. The content of each intestine was milked out into a graduated

test tube and the volume was recorded. The effluents from the intestinal loops (serosal solution)

were centrifuged at 300G for 39 minutes. The supernatants were obtained and anlysed for Na+

and K+.

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2.2.4.7 Determination of Sodium Ion (Na+) Concentration (Teco Diagnostic Kit)

The method described by Maruna (1958) in which sodium is precipitated as the triple

salt, sodium magnesium uranyl acetate, with excess uranium then reacted with ferrocyanide,

producing a chromophore whose absorbance varies universally as the concentration of sodium

in the test sample was used.

Reagent composition

(1) filtrate reagent: Uranyl acetate 2.1 mM and magnesium acetate 20 mM in ethyl alcohol

(2) Acid reagent: a diluted acetic acid

(3) Sodium colour reagent: potassium ferrocyanide, non-reactive stabilizers.

(4) Sodium standard: sodium chloride: 150 mEq/L of sodium.

Procedure

(1) Test tubes were labeled: blank, standard, control and samples

(2) A volume of 1.0ml of filtrate reagent was pipette into all tubes

(3) A volume 50µl of sample was added to all tubes and distilled

(4) All the tubes were shaken vigorously and mixed continuously for 3 minutes.

(5) The tubes centrifuged at high speed (1500×g) for 10 minutes and the supernatant fluids

were tested.

(6) A volume of 1.0ml of acid reagent was pipette into the tubes.

(7) A volume of 50µl of supernatant was added to respective tubes and mixed.

(8) A volume of 50µl of colour reagent was added to all tubes and mixed

(9) The spectrophotometer was balanced with distilled water

(10)The absorbance of all the content of the tubes were read and recorded.

Calculation

ionConcentratdardSofAbsorbanceBlankofAbsorbance

SampleofAbsorbanceBlankofAbsorbance

tan

2.2.4.8 Determination of Potassium Ion (K+) Concentration (Teco Diagnostic Kit)

The amount of potassium ion was determined using sodium tetraphenylboron in a

specially prepared mixture to produce a colloidal suspension

xlvi

Reagent composition

(1) Potassium reagent: sodium tetraphenylboron 2.1 mM, preservative and thickening

agents

(2) Potassium standard: equivalent to 4mEq/L.

Procedure

(1) Test tubes were labeled: standard, control and samples

(2) A volume of 1.0ml of potassium reagent was added to all tubes

(3) A volume of 0.01ml of the samples were added to respective tubes

(4) After three (3) minutes the spectrophotometer was balanced with distilled water at

500nm. The absorbance of the content in each tube was read and recorded.

Calculation

dardSofionConcentratdardSofAbsorbance

UnknownofAbsorbancetan

tan

2.2.5 Statistical Analysis

The data obtained from the laboratory result of the tests were subjected to both one way

and two way analysis of variance (ANOVA). Significant differences were observed at p≤0.05.

The results were expressed as mean and standard error of mean (SEM). These analyses were

carried out using computer software known as Statistical Package for Social Sciences (SPSS),

Version 16.

CHAPTER THREE

RESULTS

3.1 Yield of the Ethanol-Chloroform Extracts of Anacardium occidentale Kernel

The three layers were concentrated in a rotatory evaporator and dried in a boiling water

bath. The extract yields: for ethanol layer was 5.7 g (0.53%), for the middle layer was 15.9 g

(1.48%) and for the chloroform layer was 7.9 g (0.74%).

xlvii

3.2 Phytochemical Composition of Ethanol, Chloroform and Middle Layers of the

Extract of A. occidentale Kernel

The qualitative phytochemical compositions (Table 1) showed relatively moderate

presence of bioactive compounds such as flavonoids and reducing sugar in the three layers. The

chloroform and middle layers showed relatively moderate presence of alkaloids, saponins, fat

and oil. Glycosides and steroids were relatively present in low concentration while ethanol

layer showed moderate presence of steroids only and low concentrations of glycosides,

saponins and alkaloids were apparently present. The bioactive compounds found to be

relatively absent in the extracts were tannins and carbohydrate as shown in Table 1.

Table 1: Qualitative phytochemical constituents of the ethanol-chloroform extract of A.

occidentale

Phytochemical

Constituents Ethanol layer Chloroform layer Middle layer

Flavonoids ++ ++ ++

Alkaloids + ++ ++

Carbohydrates ND ND ND

Saponins + ++ ++

Tannins ND ND ND

Fat and oils ND ++ ++

Reducing sugars ++ ++ ++

xlviii

Glycosides + + +

Sterols ++ + +

Key: ND= Not Detected, + = Low, ++ = Moderate

3.3 Lethal Dose (LD50) Result

In the investigation, there was no lethality or behavioural change in the three groups of

the mice that received 10, 100 and 1000 mg/kg body weight of the extracts at the end of first

experiment. Based on this result, further increased doses of 1900, 2600 and 5000 mg/kg body

weight of the extracts were administered. There was neither death nor behavioural change in

the animals that received 1900 and 2600 mg/kg body weight. Those that received 5000 mg/kg

body weight of the extract showed weakness but no death was observed within 24 hours of

administration. This result showed that the extracts were respectively safe at dose below

5000mg/kg body weight.

3.4 Effect of Ethanol, Chloroform and Middle Layers of A. occidentale Extract on

Wetness of Faeces

The extracts showed non-significant (p>0.05) effect on the wetness of the faeces after

one and two hours of administration of the extracts compared with the untreated group. Three

hours after the administration of the extracts, the chloroform layer (84 mg/kg b.w.), middle

layer (21 mg/kg b.w.) and middle layer (84 mg/kg b.w.) had significantly (p<0.05) reduced the

wetness of the faeces compared with the wetness of the faeces of control animals. Four- and

five-hour post-treatments of the animals with the chloroform and middle layers, the extract

significantly decreased (p<0.05) the wetness of the faeces compared with the wetness faeces of

the animals treated with the standard drug. The ethanol layer of the extract significantly

decreased (p<0.05) the wetness of the faeces compared with that of the faeces of the animal

administered Tween 20 only (Table 2).

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Table 2: Effect of ethanol, chloroform and middle layers of the extract on wetness of faeces

Treatment Dose 1hr 2hrs 3hrs 4hrs 5hrs Mean ± SD

(%)

Tween 20 5 ml/kg 0 0 4.5 2.5 5.3 2.46 ± 2.47

(NIL)

Lomotil 2.5 mg/kg 0 0 0 0 0 0

(100)

Ethanol layer 21 mg/kg 0 0 4.0 2.3 4.0 2.26 ± 2.21

(8.1)

Ethanol layer 84 mg/kg 0 0 4.2 2.5 4.6 2.06 ± 2.00

(16.3)

Chloroform layer 21 mg/kg 0 0 3.8 0.5 0 0.86 ± 1.66

(65)

Chloroform layer 84 mg/kg 0 0 0 0 0 0

(100)

Middle layer 21 mg/kg 0 0 0 0 0 0

(100)

Middle layer 84 mg/kg 0 0 0 0 0 0

(100)

Zero (0) means no wet faeces

Values in parenthesis = Percentage inhibition of defaecation

3.5 Effect of Ethanol, Chloroform and Middle Layers of the A. occidentale Extract on

Frequency of Defaecation

One- and two-hour pre-treatments of the rat with the extracts, as observed in Table 3,

showed that all the extracts exhibited neither significant decrease nor increase (p>0.05) in the

frequency of defaecation compared with the frequency of defaecation of animals treated with

l

the standard drug. Significant decrease (p<0.05) was observed in the frequency of defaecation

of the animals in all test groups after four- and five-hour treatment of the extracts with the

exception of chloroform (84 mg/kg b.w.) and middle (21 mg/kg b.w.) layers compared with

the frequency of defaecation of the animals in the control group (Table 3).

Table 3: Effect of ethanol, chloroform and middle layers of the A. occidentale extract on

frequency of defaecation

Treatment Dose 1Hr 2Hrs 3Hrs 4Hrs 5Hrs Mean±SD

(%)

Tween 20 5ml/kg 2 0.5 0.5 1 1 1.00± 0.61

(NIL)

Lomotil 2.5mg/kg 0 0.5 0 0 0 0.10 ± 0.22

(90)

Ethanol layer 21mg/kg 1 0.33 1 0 1.33 0.73 ± 0.55

(27)

Ethanol layer 84mg/kg 1.33 0.33 0.6 0 0 0.45 ± 0.55

(55)

Chloroform layer 21mg/kg 1 0 0 0 1.66 0.53 ± 0.76

(47)

Chloroform layer 84mg/kg 0 0 0.5 0.5 0 0.20 ± 0.27

(80)

li

Middle layer 21mg/kg 0.33 0 0 1.33 0 0.33 ± 0.58

(67)

Middle layer

84mg/kg 0.5 0 0 0.5 0.5 0.30 ± 0.27

(70)

Zero (0) means no defaecation

Values in parenthesis = Percentage inhibition of wetness of faeces

3.6 Effect of Ethanol, Chloroform and Middle Layers of A. occidentale Kernel Extract

on Gastro-Intestinal Motility

The charcoal meal test was used to determine the propulsive movement of the GIT. As

shown in Fig.4, the extracts administration to rats significantly (p<0.05) suppressed the

propulsive movement or transit of charcoal meal through their gastrointestinal tract compared

with the propulsive movement of the GIT of rats in the untreated group. The rats in lomotil

group showed significant (p<0.05) decrease of the intestinal motility compared with the rats in

hyocine group. The ethanol, middle and chloroform layers of the extract except ethanol layer at

21 and 84 mg/kg b.w. produced significant (p<0.05) decrease in the gastro-intestinal motility

compared with the gastro-intestinal motility of rats in hyocine group. Lomotil treatment

significantly (p<0.05) suppressed the gastro-intestinal transit of rats compared with the extract

(Fig. 4).

lii

97.75 96.5

79.75

87

95.25

90.38

102

82.5

98.25

77.1

46.82

54

61.88 61.25

55.7558.25

40.13

55.75

0

20

40

60

80

100

120

Tween

5ml/kg

Lomotil

2.4mg/kg

Hyocine

(0.29mg/kg

Ethanol

(21mg/kg)

Ethanol

(84mg/kg)

Chloroform

(21mg/kg

Chloroform

(284mg/kg

Middle

layer

(21mg/kg)

Middle

layer

(84mg/kg)

Groups

Dis

tan

ce (

m)

Total Length ofIntestine

Distance Travelled

Fig. 4: Effect of A. occidentale extracts on gastro-intestinal motility

3.7 Effect of Ethanol, Chloroform and Middle Layers of A. occidentale Kernel Extract

on Enteropooling of the Gastro-Intestine Tract

Fig. 5 shows significant (p<0.05) reduction in the volume of intestinal content using

two doses (21 and 84 mg/kg b.w.) of the different layers of the extract compared with the

volume of the intestinal content of the animals in the untreated group. The middle and

chloroform layers of the extract significantly (p<0.05) reduced the volume of the intestinal

content compared with the volume of the intestinal content of the animals administered the

ethanol layer of the extract. The middle layer of the extract at both doses and chloroform layer

of the extract at 21 mg/kg b.w. significantly (p<0.05) decreased the volume of the intestinal

content to the same extent as the standard drug (hyocine) while the chloroform layer (84 mg/kg

liii

b.w.) of the extract significantly (p<0.05) decreased the volume of intestinal content of the

animals compared with the volume of intestinal content of the animals administered hyocine.

There was non-significant difference (p>0.05) in the intestinal content of the animals

administered ethanol layer of the extract compared with the intestinal content of the animals

administered hyocine (Fig. 5).

2

1.33

1.53

1.43

1.31.27

1.331.3

0

0.5

1

1.5

2

2.5

Tween 5ml/kg Hyocine

(0.29mg/kg

Ethanol

(21mg/kg)

Ethanol

(84mg/kg)

Chloroform

(21mg/kg

Chloroform

(84)mg/kg

Middle layer

(21mg/kg)

Middle layer

(84mg/kg)

GROUPS

VO

LU

ME

OF

IN

TE

ST

INA

L C

ON

TE

NT

Tween 5ml/kg

Hyocine (0.29mg/kg

Ethanol (21mg/kg)

Ethanol (84mg/kg)

Chloroform (21mg/kg

Chloroform (84)mg/kg

Middle layer (21mg/kg)

Middle layer (84mg/kg)

Fig. 5: Effect of ethanol, chloroform and middle layers of A. occidentale kernel extract on

enteropooling of the gastro-Intestinal tract of rats.

liv

3.8 Effect of Ethanol, Chloroform and Middle Layers of A. occidentale Kernel Extract

on Weight of the Gastro-Intestinal Tract

Fig. 6 shows significant (p<0.05) reduction in the weight of the gastro-intestinal tract

using two doses (21 and 84 mg/kg b.w) of the different layers of the extract compared with the

weight of the gastro-intestinal tract of the animals in the untreated group except ethanol layer at

21 mg/kg b.w. The animals in the hyocine group showed significant (p<0.05) reduction in the

weight of the intestine compared with the animals in the untreated group. Hyocine treatment

significantly decreased the weight of the intestine of rats compared with the extract. The middle

layer of the extract significantly (p<0.05) reduced the intestinal weight of rats compared with

the ethanol and chloroform layers (Fig. 6).

2.8

2.37

2.93

2.672.73

2.8

2.47 2.47

0

0.5

1

1.5

2

2.5

3

3.5

Tween 5ml/kg Hyocine

(0.29mg/kg

Ethanol

(21mg/kg)

Ethanol

(84mg/kg)

Chloroform

(21mg/kg

Chloroform

(84)mg/kg

Middle layer

(21mg/kg)

Middle layer

(84mg/kg)

GROUPS

WE

IGH

T O

F T

HE

IN

TE

ST

INE

Tween 5ml/kg

Hyocine (0.29mg/kg

Ethanol (21mg/kg)

Ethanol (84mg/kg)

Chloroform (21mg/kg

Chloroform (84)mg/kg

Middle layer (21mg/kg)

Middle layer (84mg/kg)

lv

Fig. 6: Effect of ethanol, chloroform and middle layers of A. occidentale kernel extract on

weight of the gastro-intestinal tract of rats.

3.9 Effect of Ethanol and Chloroform Layers of Anacardium occidentale Kernel Extract

on Intestinal Sodium Ion (Na+) Concentration

Fig. 7 indicates that ethanol and chloroform extracts of A. occidentale at both doses (21

mg/kg and 84 mg/kg b.w) caused significant (p<0.05) reduction in rat intestinal sodium ion

concentration compared with the value obtained for the untreated group. Lomotil (the standard

anti-diarrhoea drug) significantly (p<0.05) reduced sodium ion concentration compared with

the value obtained for rats in the untreated group. The rats administered ethanol layer of the

extract at 84 mg/kg b.w showed similar trend of reduction in sodium ion concentration as the

rats administered the standard anti-diarhhoea drug (Fig. 7).

lvi

267.75 267.75

190.4 190.4

219.5

198.9

230.35

202

0

50

100

150

200

250

300

21 mg/kg 84 mg/kg

GROUPS

SODI

UM IO

N CO

NCEN

TRAT

ION

(mEq

/L)

Tween 20

Lomotil

Ethanol

Chloroform

Fig. 7: Effect of ethanol and chloroform layers of Anacardium occidentale kernel extract on

intestinal sodium ion (Na+) concentration

lvii

3.10 Effect of Ethanol and Chloroform Layers of Anacardium occidentale Kernel

Extract on Intestinal Potassium ion (K+) Concentration

The ethanol and chloroform extracts of Anacardium occidentale kernel extract at

varying doses (21 mg/kg and 84 mg/kg b.w) caused significant (p<0.05) reduction of potassium

ion concentration compared with the value obtained for rats inthe untreated group. The rats

administered the standard anti-diarrhoea drug (Lomotil) showed significant (p<0.05) decrease

in potassium ion concentration of intestinal fluid compared with the rats in the untreated group.

At 21 mg/kg b.w, the extracts were less effective than lomotil. The rats administered

chloroform layer (84 mg/kg b.w) of A. occidentale kernel extract showed similar reduction in

potassium ion concentration with the rats administered lomotil (Fig. 8).

lviii

13.5 13.5

6.77 6.77

10.72

10.32

9

7.77

0

2

4

6

8

10

12

14

16

18

20

21 mg/kg 84 mg/kg

GROUPS

PO

TA

SSIU

M I

ON

CO

NC

EN

TR

AT

ION

(m

Eq/

L)

Tween 20

Lomotil

Ethanol

Chloroform

Fig. 8: Effect of ethanol and chloroform layers of Anacardium occidentale kernel extract on

intestinal potassium ion (K+) concentration

lix

CHAPTER FOUR

DISCUSSION

Usually the chloroform-ethanol extract yields two layers after partitioning with water.

Provided the filtration is properly done. Surprisingly, in this study, partitioning of the

chloroform-ethanol extract yielded three layers consistently (n=3). Even in the presence of

potassium chloride (KCl) or sodium chloride (NaCl) there were still three layers. Generally,

either salt resolves the interface into either or both the upper and lower layer.

The qualitative phytochemical composition of the extracts revealed the presence of

flavonoids. Flavonoids are known to modify the production of cyclooxygenase 1 and 2 (COX-

1, COX-2) and lipo-oxygenase (LOX) (Moroney et al., 1998; Otimeyin et al., 2008). Though

several constituents are present in the extracts, it is possible that flavonoids, acting singly or in

combination with other constituents, produced the observed anti-diarrhoeal effect of A.

occodentale kernel.

Acute toxicity test on the extract in mice established a high LD50 value of less or equal

to 5000 mg/kg body weight which suggests that the kernels may be generally regarded as safe

with a remote risk of acute intoxication. The high degree of safety is also consistent with the

popular use of the kernel as food (Ezenwali et al., 2010).

In all animals, oral administration of castor oil induced/produced diarrhea. Evaluation

of the effect of ethanol-chloroform extract of kernel of cashew on diarrhoea experimentally

induced by castor oil in rats showed that it markedly or significantly (p<0.05) reduced the

frequency of defaecation, number of diarrhoea stools and wetness of the faecal droppings. This

result is consistent with the findings of Chitme et al. (2004) who observed that the castor oil-

induced diarrhoea model in rats allows the observation of measurable changes in the number of

stools and is a consequence of the action of ricinoleic acid librated from castor oil by lipase

enzymes (Chitme et al., 2004). The freed ricinoleic acid irritates the intestinal mucosa causing

inflammation and release of prostaglandins which stimulate gastro-intestinal secretion, motility,

epithelial permeability and edema of the intestinal mucosa (Zavala et al., 1998) thereby

preventing the reabsorption of sodium, chloride and water. Active intestinal secretion is driven

predominantly by net secretion of chloride or bicarbonate, inhibition of net sodium absorption

or increase in luminal osmotically active molecules (osmotic pressure) (Shah, 2004) which can

all give rise to diarrhoea.

lx

The methanol, chloroform and middle layers of the extract appeared to act on all parts

of the intestine; thus, the layers reduced the intestinal propulsive movement in the charcoal

meal treated model. Previous studies conducted by Levy (1982) and Vrushabendra et al. (2005)

showed that activated charcoal avidly absorbs drugs and chemicals on the surface of the

charcoal particles thereby preventing absorption (Levy, 1982; Vrushabendra et al., 2005). Pre-

treatment with the extracts suppressed the propulsive transit through the gastro-intestinal tract

which indicates that the kernel extracts reduced the frequency of stooling in diarrhoea. Delay in

gastric motility caused further re-absorption of water from faeces and additionally contribute to

reducing its watery texture. It might be that the layers of the extract inhibited gastro-intestinal

hypermotility in diarrhea induced by castor oil through anticholinegic effect. Anticholinegic

agents are known to inhibit gastro-intestinal (GI) hypermotility (Saralaya et al., 2010). The

above result is supported by the findings of Brown and Taylor (2005) who stated that castor

oil-induced gastro-intestinal hypermotility has been suggested to be indirectly mediated by the

cholinergic system since it is inhibited by atropine, a known anticholinegic agent (Brown and

Taylor, 2005). The result of this study showed that the layers of the extract reduced watery

texture of diarrhoeal stools as well as GI hypermotility; thus leading to the desired reduction of

stooling in diarrhoeal disease.

Studies on gastro-intestinal enteropooling showed that the layers of the extract

significantly (p<0.05) reduced both the weight and volume of the intestinal contents. These

effects, which are direct consequences of reduced water and electrolytes secretion into the

small intestine, might indicate that the extracts could enhance electrolyte and water absorption

from the intestinal lumen. According to Duggan et al. (2002), electrolyte absorption determines

the efficiency of nutrient absorption; this may be attributed to the enhanced electrolyte

absorption by the layers of the extract which might encourage the absorption of other intestinal

contents. If the volume of the intestinal content is the same with the weight of the intestine

then, water would have been the only transported material within the GIT. As the weight of the

intestine is greater than the volume of the intestinal content, other substance is secreted into the

lumen in addition.

The specific constituent responsible for the anti-diarrhoeal properties of cashew kernel

is yet to be identified. None of the several phytochemical constituents identified from the

extracts has been reported to possess anti-diarrhoeal properties. Although, the result of the

qualitative phytochemical analysis observed in this study showed moderate presence of such

bioactive compounds as flavonoids and reducing sugars in the three layers; alkaloids, saponins

and fats and oil in the chloroform and middle layers, the bioactive compound that was not

lxi

detected in the three layers of the extract was tannins. However, the findings from this study

are insufficient to directly ascribe the anti-diarrhoeal activity to any of the phytochemicals

present in the three layers of the extract. But flavonoids have been shown to possess anti-

diarrhoeal activity attributed to their ability to modify the production of COX-1, COX-2 and

LOX (Moroney et al., 1988); thereby, inhibiting intestinal motility.

Result from the study on electrolyte transport showed that the extract caused absorptive

efflux of K+ and Na

+ from the serosal solution to varying extents and antagonized the ion

transport alteration effects of castor oil on K+ and Na

+ fluxes. From these, one might deduce

that, the extract induced hyperpolarization by antagonizing the secretory effect of castor oil on

the flux of potassium ion. Net movement of K+ in jejunum and ileum occurs only down the

electrochemical gradient i.e. largely passive transport (Currie et al., 1992). Potassium diffuses

primarily through the lateral spaces and tight junction. It is apparent from the results that the

ability of the extract of cause hyperpolarization resulting from enhanced extrusion of Na+ and

K+ may contribute for its inhibition of castor oil-induced diarrhea. The extract may therefore

exert its anti-diarrhoeal effects by promoting water and electrolytes absorption.

In conclusion, the present study revealed some of the pharmacological basis for the

ethnomedical use of cashew (A. occidentale) kernel in the treatment of diarrhoea. Results of

this study showed that the constituents of cashew (A. occidentale) kernel possess anti-

diarrhoeal properties mediated through inhibition of castor oil-induced diarrhoea, hyper-

secretion, gastro-intestinal enteropooling and gastro-intestinal motility.

4.1 Suggestion for Further Studies

Further studies should be carried out to show the effect of the extract on the ion

movement, cyclooxygenase and bacteria growth and the parts of the gastro-intestinal tract the

extract act on are encouraged.

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