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Analysis of Phytochemicals and Anti-Plasmodial ANALYSIS OF PHYTOCHEMICALS AND ANTI-PLASMODIAL ACTIVITIES OF EXTRACTS FROM HARRISONIA ABYSSINICA, LEUCAS CALOSTACHYS AND RUBIA CORDIFOLIA AGAINST PLASMODIUM FALCIPARUM MAGARA JEREMIAH (B.Ed. Sc.) I56/24461/2013 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF DEGREE OF MASTER OF SCIENCE (APPLIED PARASITOLOGY) IN THE SCHOOL OF PURE AND APPLIED SCIENCES OF KENYATTA UNIVERSITY DECEMBER, 2018 ii DECLARATION I, Magara Jeremiah, declare that this thesis is my original work and has not been presented in any other institution for a degree or any other award. Signature………………………………... Date………………………… Magara Jeremiah (B.Ed Sc.) Department of Zoological Sciences Supervisors’ Approval We confirm that the work reported in this thesis was carried out by the candidate under our supervision as the university supervisors. Signature………………………………. Date ………………………… Dr. Lucy Kamau Department of Zoological Sciences Kenyatta University Signature……………………………… Date………………………….. Dr. Hastings Ozwara Department of Tropical and Infectious Diseases Institute of Primate Research Signature……………………………….. Date…………………………. Dr. Grace Nyambati Department of Biomedical Sciences and Technology The Technical University of Kenya iii DEDICATION I dedicate this work to my wife Ann Monchari and sons Newton Orenge and Brighton Magara for their patience and inspiration. iv ACKNOWLEDGEMENTS This project was carried out at the Institute of Primate Research (IPR) under the supervision of Dr. Lucy Kamau, Dr. Grace Nyambati and Dr. Hastings Ozwara. I wish to thank them for the guidance at all stages of this work. My sincere appreciation to Dr. Grace Nyambati who did preliminary work on the three plants used in this study and Dr. Hastings Ozwara for allowing me to carry out the research in his laboratory at the IPR. I thank the entire technical staff in the Department of Tropical and Infectious Diseases (TID) at IPR for their commitment and technical assistance throughout the project. In particular I thank Ms Esther Kagasi, Fred Nyundo and Ruth Mumo. I am also grateful to Ms Agness Wangila Tsuma and the entire staff of Pharmacognosy Department, Kenyatta University for technical assistance during solvent extraction of the crude plant extracts. I thank my colleagues; Mr John Muchiri and Mr Irungu victor for tirelessly working with me for long hours. Their encouragement and criticism partly led to the success of this research. My sincere gratitude also goes to the entire Magara family: My late Dad for his contribution towards this study and my Mother, Brothers and sisters for their continuous support and prayers during this work. Lastly, I thank the Almighty God for granting me good health without which this work could not have been complete. May His Holy name be glorified forever! v TABLE OF CONTENTS DECLARATION ......................................................................................................... ii DEDICATION ............................................................................................................ iii ACKNOWLEDGEMENTS ....................................................................................... iv TABLE OF CONTENTS ............................................................................................ v LIST OF TABLES ....................................................................................................... x LIST OF FIGURES .................................................................................................... xi ACRONYMS AND ABBREVIATIONS ................................................................. xii ABSTRACT .............................................................................................................. xiv CHAPTER ONE: INTRODUCTION ........................................................................ 1 1.1 Background Information ............................................................................. 1 1.2 Problem Statement ....................................................................................... 2 1.3 Justification of the study .............................................................................. 2 1.4 Research Questions ..................................................................................... 3 1.5 Null Hypotheses .......................................................................................... 4 1.6 Objectives .................................................................................................... 4 1.6.1 General objective ......................................................................................... 4 1.6.2 Specific Objectives ...................................................................................... 4 1.7 Significance of the study ............................................................................. 5 CHAPTER TWO: LITERATURE REVIEW .......................................................... 6 2.1 Epidemiology of Malaria ............................................................................. 6 2.2 Economic cost of malaria ............................................................................ 8 2.3 General life cycle of Plasmodium species ................................................... 8 2.3.1 The vertebrate phase .................................................................................... 9 2.3.2 Invertebrate phase ...................................................................................... 10 2.4 The life cycle of P. falciparum .................................................................. 11 2.5 Control of malaria ...................................................................................... 13 2.5.1 Indoor residual spraying (IRS) .................................................................. 13 2.5.2 Use of long-lasting insecticide treated bed nets (LLIN) ........................... 14 2.5.3 Mosquito larval control ............................................................................. 14 2.5.4 Malaria chemotherapy ............................................................................... 15 vi 2.5.4.1 Aminoquinolines ....................................................................................... 15 2.5.4.1.1 Quinine ...................................................................................................... 15 2.5.4.1.2 Chloroquine ............................................................................................... 16 2.5.4.1.3 Amodiaquine ............................................................................................. 17 2.5.4.1.4 Primaquine ................................................................................................. 18 2.5.4.1.5 Mefloquine ................................................................................................ 19 2.5.4.1.6 Piperaquine ................................................................................................ 20 2.5.4.1.7 Lumefantrine ............................................................................................. 20 2.5.4.1.8 Halofantrine ............................................................................................... 21 2.5.4.2 Antifolate drugs ......................................................................................... 22 2.5.4.3 Artemisinins .............................................................................................. 22 2.5.4.4 Combined drug therapy ............................................................................. 23 2.5.5 Chemoprophylaxis ..................................................................................... 24 2.6 The practice of Herbal Medicine ............................................................... 24 2.7 Plants that have been studied for pharmacological activity ...................... 25 2.7.1 Ethnobotanical information on genus Harrisonia ..................................... 27 2.7.2 Ethnobotanical information on Leucas calostachys Oliv. ......................... 28 2.7.3 Rubia cordifolia L. .................................................................................... 29 CHAPTER THREE: MATERIALS AND METHODS ........................................ 30 3.1 Study site ................................................................................................... 30 3.2 Study design .............................................................................................. 30 3.3 Collection and preparation of plant materials ........................................... 30 3.4 Solvent extraction ...................................................................................... 31 3.4.1 Hexane extraction ...................................................................................... 31 3.4.2 Methanolic extraction ................................................................................ 31 3.4.3 Plant water extraction (Aqueous extract) .................................................. 32 3.5 The parasite ............................................................................................... 33 3.5.1 Preparation of human serum ...................................................................... 33 3.5.2 Processing of uninfected erythrocytes for culturing of the parasite .......... 34 3.5.3 Incomplete culture medium (ICM) ............................................................ 34 3.5.4 Complete culture medium (CCM) ............................................................. 35 3.5.5 Thawing of the malaria parasite and setting of the culture.
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