EVALUATION OF THE ANTIPLASMODIAL ACTIVITY OF METHANOL LEAF EXTRACT AND FRACTIONS OF LANDOLPHIA OWARIENSIS P. BEAUV (APOCYNACEAE) IN MICE

TABLE OF CONTENT

Page Title page

Certification

Dedication

Acknowledgement

Abstract

Table of content

List of tables

List of figures

CHAPTER ONE: INTRODUCTION

1.0       Introduction

1.1       Background

1.2       Definition of Malaria

1.3       Epidemiology of Malaria

1.4       Etiology of Malaria

1.4.1    Vector

1.4.2    Biology of Malaria Infection (Life cycle of Malaria Parasite)

1.4.3    Pathogenesis of Malaria

1.5       Clinical Manifestation of Malaria

1.6       Diagnosis of Malaria

1.7       Classification of antimalarials

1.8       Chemotherapy of Malaria

1.8.1    Chloroquine

1.8.2    Amodiaquine

1.8.3    Quinine and quinidine

1.8.4    Mefloquine

1.8.5    Halofantrine

1.8.6    Lumefantrine

1.8.7    Primaquine

1.8.8    Sulphadoxine - Pyrimethamine

1.8.9    Antibacterial agents

1.8.10  Artemisinins

1.8.10.1 Antimalarial Properties of Artemisinins

1.8.10.2 Artemisinin Based Combination Therapy (ACT)

1.9       Prevention and Control of Malaria

1.10     Review of Plants with Antimalarial Activity

1.11     Botanical Profile of Landolphia owariensis

1.11.1  Plant Taxonomy

1.11.2  Description of Plant

1.11.3  Geographical Distribution of Plant

1.11.4  Ecology

1.11.5  Ethnomedicinal Uses

1.12     Literature Review

1.13     Aim

CHAPTER TWO: MATERIALS AND METHODS

2.1       Materials

2.1.1    Animals

2.1.2    Drugs

2.1.3    Chemicals and Reagents

2.1.4    Equipment

2.2       Methods

2.2.1    Collection, Authentication and Preparation of Plant Materials

2.2.2    Preparation of Crude Extract

2.2.3    Solvent-guided Fractionation of Crude Extract

2.2.4    Determination of Yield (%)

2.2.5    Phytochemical Analysis of Extract and Fractions

2.2.5.1 Test for Saponins

2.2.5.2 Test for Tannins

2.2.5.3 Test for Flavonoids

2.2.5.4 Test for Resins

2.2.5.5 Test for Steroids and Terpenoids

2.2.5.6 Test for Alkaloids

2.2.5.7 Test for Glycosides

2.2.5.8 Test for Fats and Oils

2.2.5.9 Test for Carbohydrates

2.2.5.10 Test for Reducing Sugar

2.2.6 High Performance Liquid Chromatography (HPLC) Analysis

2.3 Pharmacological Studies

2.3.1    Determination of Acute Toxicity (LD50) of ME

2.3.2    Rodent Parasite (Plasmodium berghei berghei NK65)

2.3.3    Parasite Innoculaton

2.3.4    Evaluation of Activity on Early Malarial Infection (4-Day Suppressive Test)

2.3.5    Evaluation of Activity on Established Infection (Curative or Rane Test)

2.3.6    Evaluation of Prophylactic Activity (Repository Test)

2.4 Statistical Analysis

CHAPTER THREE: RESULTS

3.1       Extraction and Fractionation

3.2       Phytochemical Analysis of extract and fractions

3.3       High Performance Liquid Chromatography (HPLC) Analysis

3.4       Acute Toxicity (LD50) of ME

3.5       Pharmacological Studies

3.5.1 Effect of ME and Fractions on Early Malaria Infection (4-Day Suppressive Test) 55 3.5.2 Effect of ME and Fractions on Established Infection (Rane Test)

3.5.3 Prophylactic Effects of ME and Fractions against P. berghei berghei infected mice

CHAPTER FOUR: DISCUSSION AND CONCLUSION

4.1       Discussion

4.2       Conclusion

References

ABSTRACT 

Landolphia owariensis P. Beauv (Apocynaceae) is a woody liane commonly used in Africa for the treatment of gonorrhea, worm infestation and malaria. The methanol leaf extract (ME) of L. owariensis was obtained by cold maceration and then fractionated into nhexane (nHF), ethylacetate (EF) and methanol (MF) fractions. The methanol extract and fractions were tested against chloroquine-sensitive Plasmodium berghei berghei in early, established and repository models of infection using Peter’s 4-day suppressive model, Rane’s curative model and Peters prophylactic model respectively. The antiplasmodial activity was evaluated by determining the parasitemia, body weight and survival time of each of the eighty-four mice comprising six mice per group. Groups 1-12 were given graded doses of 200, 400 and 800 mg/kg body weight of extract or fractions respectively while group 13 and 14 received 5 mg/kg/day of chloroquine and 3% Tween 80 respectively. All administration was orally.

Acute toxicity was studied using modified Lorke’s method. Phytochemistry of extract and fractions as well as HPLC fingerprinting of ME, EF and MF were also carried out. The methanol extract and all the fractions exhibited significant (P<0.05) but varying levels of antiplasmodial activity comparable to the group treated with chloroquine. MF elicited the highest chemosuppression of 96.04% at 800 mg/kg with the prophylactic model while nHF elicited the least activity with chemosuppression of 29.38-58.75% at 200 -800 mg/kg respectively. The phytochemical screening of the extract and fractions revealed the presence of secondary metabolites. The LD50 was estimated to be greater than 5000 mg/kg p.o in mice. HPLC analysis of ME, EF and MF showed different peaks representing different components. The results of this study suggest that the leaf extract and fractions pose significant antiplasmodial activity.

CHAPTER ONE

1.0  INTRODUCTION

1.1   Background

Malaria presents a global devastating burden. The latest estimates released December 2013 indicate that about 207 million cases of malaria were reported in 2012 with an uncertainty range of 135 million to 287 million and an estimated 627 000 deaths with an uncertainty range of 473 000 to 789 000 (WHO, 2014). A child dies every minute from malaria in Africa. Malaria though a complex disease is preventable and curable (WHO, 2008a; WHO, 2014). It is a disease caused by infection with single-celled protozoan parasites of the genus Plasmodium.

There has been a dramatic increase in both international and domestic funding for malaria control since the last decade as well as an increasing national political commitment to controlling malaria and intensified efforts in endemic countries (WHO, 2008b; Pigott et al.,

2012). Malaria control has also featured high on the world’s health and development agenda since the launch of Roll Back Malaria Initiative by World Health Organization (WHO) in 1998. The inspiration behind this initiative was to alleviate poverty and strengthen health systems in malaria endemic countries in such a way that the enormous public health problem caused by malaria will be fundamentally addressed (WHO, 2008b). Despite the intensified efforts and initiative, eradication of malaria still poses a global challenge and threat. Malaria occurs in 109 countries of the world in which Nigeria is one of the 5 main contributors of 50% of global death and 47% of malaria cases. Global strategy consists of three components that will ensure reduction in mortality and morbidity of malaria. This includes control with....

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