ANTITRYPANOSOMAL ACTIVITIES OF ACACIA NILOTICA AND KHAYA SENEGALENSIS AND THE HAEMATOLOGICAL PROFILE OF TRYPANOSOMA BRUCEI BRUCEI INFECTED WISTAR RATS

ABSTRACT

This study was aimed at investigating antitrypanosomal activities and haematological profile of crude extract and fractions of the stem bark of Acacia nilotica and Khaya senegalensis plants against Trypanosoma brucei brucei infected Wistar rats with a view to determining the phytochemical constituents and LD₅₀ of Acacia nilotica and Khaya senegalensis, antitrypanosomal activities of crude extract of Acacia nilotica and Khaya senegalensis against Trypanosoma brucei brucei infected Wistar rats, antitrypanosomal activities of the plant fractions administered to Trypanosoma brucei brucei infected Wistar rats and haematological profile of Trypanosoma brucei brucei infected Wistar rats, before and after administration of crude extract and fractions. The phytochemical constituents and toxicity of the stem bark of both plants were determined by the standard method and the LD₅₀respectively. The methanolic extracts and fractions of the plants was administered to the Wistar rats intraperitoneally daily and the parasitaemia count was determined using the rapid matching method. PCV, WBC and differential counts were determined before and after the administration to ascertain any significant differences. The phytochemical constituents of the stem barks of Acacia nilotica and Khaya senegalensis crude extracts and fractions revealed the following secondary metabolites; Alkaloids, tannins, glycosides, cardiac glycosides, saponins, triterpene, carbohydrates and flavonoids. The LD₅₀ for the crude extract of the stem bark of Acacia nilotica was 707.1mg/kg body weight while the LD₅₀ for the fractions (N-hexane, ethyl acetate and N-butane) was 547.7 mg/kg, 387.3 mg/kg and 707.1 mg/kg body weight respectively. The LD₅₀ for the crude extract of the stem bark of Khaya senegalensis was 547.7mg/kg body weight while the LD₅₀ for the fractions (N-hexane, ethyl acetate and N-butane) was 387.3 mg/kg, 547.7 mg/kg and 223.6 mg/kg body weight respectively. The stem barks of Acacia nilotica and Khaya senegalensis crude extracts (100, 200, 300 and 400mg/kg body weight) and fractions (50, 100, 150 and 200mg/kg body weight) had antitrypanosomal activity. Parasites were cleared from circulation within 12 days of treatment. Haematological indices of Acacia nilotica and Khaya senegalensis in Trypanosoma brucei brucei infected Wistar rats showed that there was no statistical significant change in the packed cell volume, white blood cells and differential counts before and after treatment with all doses of the crude extracts and fractions. The findings in this study provide very useful source for biopharmaceutical industries for the development of antitrypanosomal agents from the stem bark of Acacia nilotica and Khaya senegalensis for therapeutic intervention in the control of African trypanosomiasis. There is need for further extensive work on these plants using different Trypanosoma species in the management of African trypanosomiasis.

TABLE OF CONTENTS

Title Page………………………………………………………………………………………i

Declaration…………………………………………………………………………………….ii

Certification…………………………………………………………………………………..iii

Dedication…………………………………………………………………………………….iv

Acknowledgement……………………………………………………………………………..v

Abstract……………………………………………………………………………………….vi

Table of Contents……………………………………………………………………………viii

List of Figures………………………………………………………………………………..xii

List of Tables………………………………………………………………………………..xiii

List of Plates………………………………………………………………………………….xv

List of Appendices…………………………………………………………………………..xvi

List of Abbreviations……………………………………………………………………….xvii

CHAPTER ONE

1.0         INTRODUCTION……………………………………………………………………1

1.1         Statements of the Research Problem…………………..……………………………3

1.2         Justification…………………………………………………………………………...4

1.3         Aim…………………………………………………………………………………….5

1.4         Objectives……………………………………………………………………………..5

CHAPTER TWO

2.0         LITERATURE REVIEW……………………………………………………………6

2.1         The Parasite: Trypanosoma brucei…………………………………………………..6

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2.2         The vector: Glossina spp.……………………………………………………………9

2.3         Infection and Symptoms of Trypanosomiasis……………………………………..12

2.4         Disease Management………………………………………………………………..12

2.5         Treatment……………………………………………………………………………13

2.6         Epidemiology and Control………………………………………………………….16

2.7         Taxonomic Classification of Acacia nilotica……………………………………….16

2.7.1     Plant description……………………………………………………………………...18

2.7.2     Traditional claims…………………………………………………………………….20

2.7.3    Medicinal uses of different parts of Acacia nilotica…………………………………20

2.8         Taxonomic Classification of Khaya senegalensis………………………………….21

2.8.1     Plant description……………………………………………………………………...23

2.8.2     Medicinal uses………………………………………………………………………..25

CHAPTER THREE

3.0         MATERIALS AND METHODS…………………………………………………...27

3.1         Collection and Authentication of Plant Materials………………………………...27

3.2         Preliminary Processing and Preparation of Plant Materials…………………….27

3.3         Preparation of Crude Plants Extracts……………………………………………..27

3.4        Preparation of Plant Fractions using Partitioning Fractionation (Solvent-Solvent Extraction)………………………………………………………..27

3.5         Experimental Animals……………………………………………………………...28

3.6         Trypanosome Stock…………………………………………………………………28

3.7         Phytochemical Screening of Plant Extracts……………………………………….28

3.7.1.    Detection of alkaloids………………………………………………………………...28

3.7.2.    Detection of tannins………………………………………………………………….29

3.7.3.    Detection of glycosides………………………………………………………………29

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3.7.4.    Detection of cardiac glycosides………………………………………………………29

3.7.5.    Detection of saponins………………………………………………………………...30

3.7.6.    Detection of steroids………………………………………………………………….30

3.7.7.    Detection of triterpenes………………………………………………………………30

3.7.8.   Detection of carbohydrates…………………………………………………………...30

3.7.9.    Detection of flavonoids………………………………………………………………30

3.7.10. Detection of anthraquinones………………………………………………………….31

3.8.       Determination of LD₅₀………………………………………………………………31

3.9.       Determination of Antitrypanosomal Activities of the Crude Extract…………...32

3.10.    Determination of the Antitrypanosomal Activities of the Plant Fractions using Partitioning Fractionation (Solvent-Solvent extraction)…………………………33

3.11.    Determination of Haematological Profile………………………………………….33

3.11.1. Packed Cell Volume (PCV)………………………………………………………….33

3.11.2. White blood cell (WBC)……………………………………………………………..34

3.11.3. Differential count…………………………………………………………………….34

CHAPTER FOUR

4.0         RESULTS……………………………………………………………………………35

4.1         Phytochemical Constituents of Crude and Fractions of Acacia nilotica

(stem bark)and Khaya senegalensis (stem bark)………………………………...35

4.2         LD₅₀ Crude and Fraction Extracts Analysis of Acacia nilotica and

Khaya senegalensis…………………………………………………………………..39

4.3         Antitrypanosomal activities of crude extract and fractions of Acacia nilotica and Khaya senegalensis against Trypanosoma brucei brucei infected Wistar rats.......39

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CHAPTER FIVE

5.0         DISCUSSION……………………………………………………………………….66

5.1         Phytochemical Constituents of the Crude Extract and Fraction of the

Stem Bark of Acacia nilotica and Khaya senegalensis…………………………….66

5.2         The LD50 of the Crude Extract and Fraction of the Stem Bark of

Acacia nilotica and Khaya senegalensis against Adult Wistar rats………………67

5.3         The Antitrypanosomal activity of the Crude Extract and Fraction of the Stem Bark of Acacia nilotica and Khaya senegalensis against

Trypanosoma brucei brucei infected Wistar rats…………………………………..67

5.4         The Haematological profile for the Trypanosoma brucei brucei infected Wistar before and after crude extract and fraction of stem bark of

Acacia nilotica and Khaya senegalensis administration…………………………..69

CHAPTER SIX

6.0         CONCLUSION AND RECOMMENDATIONS………………………………….71

6.1         Conclusion…………………………………………………………………………...71

6.2         Recommendations…………………………………………………………………..72

6.3         Contribution to Knowledge………………………………………………………...72

REFERENCES……………………………………………………………………………...73

APPENDICES………………………………………………………………………………80

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4.19      Antitrypanosomal activity t of Khaya senegelensis (N-hexane fraction) on

Parasite Count in Wistar Rats infected with Trypanosoma brucei brucei……...……56

4.20      Antitrypanosomal activity of Khaya senegelensis (Ethylacetate fraction) on

Parasite Count in Wistar Rats infected with Trypanosoma brucei brucei…………...57

4.21    Antitrypanosomal activity of Khaya senegelensis (N-butane fraction) on

Parasite Count in Wistar Rats infected with Trypanosoma brucei brucei…………...59

4.22      Effect of Administration of Stem Bark Extract of Acacia nilotica N-hexane

fraction on Haematological Indices of Wistar Rats………………………………….60

4.23      Effect of Administration of Stem Bark Extract of Acacia nilotica Ethylacetate fraction on Haematological Indices of Wistar Rats……………………………….....61

4.24      Effect of Administration of Stem Bark Extract of Acacia nilotica N-butane

fraction on Haematological Indices of Wistar Rats………………………………….62

4.25      Effect of Administration of Stem Bark Extract of Khaya senegalensis

N-hexane fraction on Haematological Indices of Wistar Rats……………………….63

4.26      Effect of Administration of Stem Bark Extract of Khaya senegalensis

Ethylacetate fraction on Haematological Indices of Wistar Rats…………………….64

4.27      Effect of Administration of Stem Bark Extract of Khaya senegalensis N-butane fraction on Haematological Indices of Wistar Rats………………………………….65

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LIST OF PLATES

Plate                                                                                     Title                                                                                    Page

I                     Photogragh of Tsetsefly……………………………………………………...............11

II            The stem bark, root, seeds and leaves of Acacia nilotica…………………………….19

III          The stem bark, flower, fruit, leaves and seeds of Khaya senegalensis……................24

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LIST OF APPENDICES

Appendix                                                                          Title                                                                                    Page

I                     Photograph of Trypanosoma brucei brucei in blood film of Wistar Rats………….80

II            Baseline for Haematology and Chemistry values for Charles River Wistar Rats.....81

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LIST OF ABBREVIATIONS

AAT

WHO

HAT

VSG

NITR

PBS

PCV

rpm

WBC

bw

IUCN

TDR

OECD

-African Animal Trypanosomosis

-World Health Organization

-Human African Trypanosomiasis

-Variant Surface Glycoprotein

-Nigerian Institute for Trypanosomiasis Research

-Phosphate Buffered Solution

-Packed Cell Volume

-Revolution per minute

-White blood cell

-Body weight

-International Union for Conservation of Nature and Natural Resources

Tropical Disease Research

Organistion for Economic Cooperation and Development

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CHAPTER ONE

1.0                                                                         INTRODUCTION

African Trypanosomiasis (African sleeping sickness) is caused by trypanosomes which is found in Sub-Saharan Africa and is threatening more than sixty million lives on daily basis (Abdullahi and Emmanuel, 2012). Trypanosomes are protozoan parasites in the family Trypanosomatidae. Most trypanosomes are transmitted by the vector, tsetse flies (Glossinia spp) which are found only in Sub-Saharan Africa, between latitudes 14^o N and 20^o S (Bernard and Alain, 2012). The parasites include Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, (cause Human African Trypanosomiasis). Other trypanosomes primarily affect animals include Trypanosoma congolense, Trypanosoma vivax, Trypanosoma brucei brucei, Trypanosoma simiae and Trypanosoma godfreyi (Bernard and Alain, 2012).

Nigeria‟s natural habitation is made up of both savannah and tropical rainforest, which falls within the endemic area in Africa i.e. between latitude 15⁰Nand 29⁰ S. The diverse flora offers a wide spectrum of unique medicinal plants. There are varieties of studies of Nigerian plants used in the traditional management of trypanosomiasis, indicating significant anti-trypanosomal activity (in-vitro/in-vivo); some of which the metabolites responsible have been isolated and reported (Atawodi et al, 2003).

Trypanosoma brucei brucei are unicellular parasites transmitted by the tsetse fly. They are the causative agent of African animal trypanosomosis (AAT), also known as Nagana. Trypanosoma brucei brucei is the etiological agent for sleeping sickness which is one of the most serious protozoan diseases in Africa (Antia et al., 2009; Simarro et al., 2011).

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The disease results in acute, sub acute or chronic disease characterized by intermittent fever, anaemia, occassional diarrhoea, rapid loss of consciousness and often terminates in death (Olukunle et al., 2010). On the basis of mortality, Human African Trypanosomiasis is ranked ninth among 25 infectious diseases in Africa (Bernard and Alain, 2012).

The current chemotherapy of the human trypanosomiasis relies on six drugs namely; Suramin, Pentamidine, Melarsoprol, Eflorinithine, Arsobal and Mel B, five of which were developed over 30 years ago (Steverding and Tyler, 2005). All of the current therapies are unsatisfactory for various reasons, including unacceptable toxicity, poor efficacy, undesirable route of administration, drug resistance and high cost (Fairlamb, 2003). Natural products derived from plants offer novel possibilities to obtain new drugs that are active against trypanosomes (Hoet et al., 2004). The local use of natural plants as primary health remedies is due to their pharmacological properties. Many plant extracts owe their potency to the presence of metabolites. These metabolites are usually found in various parts of the plants like roots, leaves, shoots and barks. Many plants have therefore become sources of important drugs and as such the pharmaceutical industries have exploited medicinal plants as a source of bioactive agents that can be used in the preparation of synthetic medicine (Kinghorn, 1994).

Acacia nilotica belongs to the family Leguminosae. It is called „Bagaruwa‟ in Hausa, „Gaude‟ in Fulfulde and „Kangar‟ in Kanuri. It is commonly found in Guinea and Sudan savannah. Ethanol extract of the leaves have been reported to have anticancer and antioxidant activity (Kalaivani et al., 2010). The plant parts are commonly used, particularly in the Northern and South-Western Nigeria, as broad spectrum antimicrobials, especially in the treatment of dysentery. Khaya senegalensis is a tree in the Meliaceae family. It is widely distributed in the Sub-Saharan savannah. It is called “madachi” in Hausa, “dalechi” in Fulani, “ogonwo” in Yoruba and “ono” in Igbo.

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The bark is dark-grey to grey-brown. It is used for carpentry and construction. The bark is used for a variety of medical purposes such as malaria, stomach complaints and headaches. It is applied externally to skin rashes, wounds or any abnormality (Keay et al., 1989).

1.1.        Statement of the Research Problem

Trypanosomiasis, a disease of major importance in human and animals has continued to threaten human health and economical development (Kuzoe, 1993; WHO, 2014). The population at risk being about 69 million with one third of this number being at a „very high‟ to „moderate‟ risk and the remaining two thirds at a „low‟ to „very low‟ risk (WHO, 2014).

Trypanosomiasis is one of the most severe medical problems in Africa, infecting around 50,000 people every year (Ohaeri, 2010), and also affects 50-70 million animals (Ogbadoyi et al., 2007). Trypanosomiasis affects millions of people in Sub-Saharan Africa and is responsible for the death of about half a million patients per year (Barrett, 1999; WHO, 2014).

Anaemia is the most outstanding clinical and laboratory feature of African trypanosomiasis and also the primary cause of death (Bizimana et al., 2006).

Chemotherapy is the most widely used means of controlling the trypanosomiasis. The few registered trypanocides are often associated with severe side effects and required lengthly parenteral administration, lack efficacy and are unaffordable for most of the patients (Legros et al., 2002). Chemotherapy and chemoprophylaxis, which form the most important aspect of control and eradication of trypanosomiasis in African countries, are beset with problems. These include limited repertoire of compounds, resistance to drugs, drug toxicity and protracted treatment protocols (TDR, 1984). Poor clinical

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efficiency, drug resistance and toxicity are some of the limitations facing programmes targeted at controlling the disease (Onyeyili and Egwu, 1995; Geerts and Holmes, 1998; Legros et al., 2000; Kamuanga, 2003).

Previous studies have shown that the crude extracts of Acacia nilotica and Khaya senegalensis have antitrypanosomal effects (antitypanocidal or antitrypanostatic). The subject of this study is to show that fractions of these plant extracts could exhibit different and indeed more effective results.

1.2.        Justification

The high costs and toxicity of synthetic drugs have stimulated renewed interest in plant substitutes. With the emergence of drug resistant trypanosomes, cost-effective new drugs in the treatment of sleeping sickness are required.

Medicinal plants are less expensive and less hazardous to the health than their synthetic counterpart hence medicinal plants with anti-trypanosomal properties have paramount importance for the treatment of trypanosomiasis.

It has been known that plant products play an important role in certain disease control; for example Hoet et al., (2004), stated that natural products derived from plants offer novel possibilities to obtain new drugs that are active against trypanosomes.

Chemotherapy is the most widely used means of controlling the trypanosomiasis. The few registered trypanocides are often associated with severe side effects (Guttering, 1985) and require lengthy parenteral administration, lack efficacy and are unaffordable for most of the patients (Legros et al., 2002). Thus there is the urgent need to source for new, cheap effective non hazardous plants products that can help control trypanosomiasis.