Antiplasmodial Activities and in Vivo Safety of Extracts and Compounds of Seven Indigenous Kenyan Medicinal Plants Used Traditionally to Treat Malaria
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i ANTIPLASMODIAL ACTIVITIES AND IN VIVO SAFETY OF EXTRACTS AND COMPOUNDS OF SEVEN INDIGENOUS KENYAN MEDICINAL PLANTS USED TRADITIONALLY TO TREAT MALARIA ONYOYO SAMWEL GUYA I56/8873/99 A RESEARCH THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE IN BIOCHEMISTRY, KENYATTA UNIVERSITY OCTOBER, 2020 ii DECLARATION AND DEDICATION I declare that this thesis is my original work and it has not been presented for a degree in any other University or for any other award. I dedicate this work to my late father mzee Festus Onyoyo Ouya who was the source of my aspiration as he had a special aspiration towards acquiring knowledge. Onyoyo Samwel Guya Reg/No: 156/8873/99 Signature______________________ Date_________________ We confirm that the work reported in this thesis was carried out by the candidate under our supervision Supervisors Prof Eliud NM Njagi Department of Biochemistry, Microbiology and Biotechnology Kenyatta University P.O Box 43844, Nairobi Signature______________________ Date_________________ Prof Anastasia N Guantai Department of Pharmacology and Pharmacognosy University of Nairobi P.O Box 19676, Nairobi Signature______________________ Date__________________ Prof Caroline C Langat-Thoruwa Department of Chemistry Kenyatta University P.O Box 43844, Nairobi Signature_____________________ Date___________________ iii TABLE OF CONTENTS DECLARATION AND DEDICATION .................................................................. ii TABLE OF CONTENTS ......................................................................................... iii LIST OF TABLES .................................................................................................... vi LIST OF FIGURES ................................................................................................ viii ACKNOWLEDGMENTS ........................................................................................ ix ABBREVIATIONS AND ACRONYMS ................................................................. x ABSTRACT ............................................................................................................ xvi CHAPTER ONE ........................................................................................................ 1 INTRODUCTION ..................................................................................................... 1 1.0 Background information ........................................................................................ 1 1.1 Malaria the disease ................................................................................................ 1 1.2 Control and treatment of malaria ........................................................................... 2 1.3 Drug discovery for malaria .................................................................................... 3 1.4 Phytotherapeutics .................................................................................................. 4 1.5 In vivo lethality test (Brine Shrimp Bioassay) ....................................................... 4 1.6 Statement of the problem ....................................................................................... 5 1.7 Justification of the study ........................................................................................ 7 1.8 Null hypotheses ..................................................................................................... 8 1.9 Study objectives ..................................................................................................... 9 1.9.1 General objective ................................................................................................ 9 1.9.2 Specific objectives .............................................................................................. 9 CHAPTER TWO ..................................................................................................... 10 LITERATURE REVIEW ....................................................................................... 10 2.1 Control and treatment of malaria ......................................................................... 10 2.1.1 Vector control and malaria prophylaxis ........................................................... 10 2.1.2 Antimalarial chemotherapy using conventional drugs ..................................... 11 2.2 Plants as sources of antimalarial agents .............................................................. 16 2.2.1 Extraction and purification of compounds ....................................................... 21 2.2.2 Liquid-solid (adsorption) chromatography ....................................................... 21 2.2.3 Gelfiltration (exclusion) chromatography ........................................................ 24 2.2.4 Preparative thin layer chromatography (PTLC) ............................................... 24 2.2.5 Structural elucidation of isolated compounds .................................................. 25 iv 2.3. Ethnomedicine survey ........................................................................................ 26 2.3.Reasons for conducting ethnomedicine survey in Kilifi and Homa Bay ............ 27 2.3.1 Kilifi County ..................................................................................................... 27 2.3.2 Homa-Bay County ............................................................................................ 28 2.3.3 Geographical distributiom and phytochemistry of selected medicinal plants .. 30 2.4. Evaluation of safety of crude extracts ................................................................ 38 2.4.1 Overview .......................................................................................................... 38 2.4.2 Safety screening methods ................................................................................. 39 CHAPTER THREE ............................................................................................... 43 MATERIALS AND METHODS ............................................................................ 43 3.1. Materials ............................................................................................................. 43 3.1.1 Test organisms .................................................................................................. 43 3.1.2 Medicinal plants (Test articles) ........................................................................ 43 3.1.3 Reagents ............................................................................................................ 43 3.1.4 Equipments ....................................................................................................... 43 3.2.0 Methods ............................................................................................................ 44 3.2.1 Preparation of in vitro Plamodium test ............................................................. 44 3.2.2 Preparation of the plant extracts ....................................................................... 46 3.2.3 Preparation of parasite culture system .............................................................. 47 3.2.4 Preparation of the working drug solutions for the tests .................................... 49 3.2.5 Harvesting the malaria parasites ....................................................................... 49 3.2.6 Calculation of percentage parasite growth inhibition ....................................... 50 3.2.7. Safety screening ............................................................................................... 51 3.3.0 Isolation and characterization of active chemicals/compounds from plants .... 58 3.3.1 Isolation of compounds .................................................................................... 58 3.3.2. Characterization of isolated compounds ......................................................... 65 CHAPTER FOUR ................................................................................................... 67 RESULTS ................................................................................................................. 67 4.0 Pant extracts ......................................................................................................... 67 4.1 Yield of plant extracts ......................................................................................... 67 4.3 Results of the Brine shrimp lethality test ............................................................ 68 4.3 In vitro antiplasmodial activity of the plant extracts ........................................... 69 4.4 In vivo subacute toxicity test of DCM extract of C limon root in rabbits ........... 70 v 4.3 Results on Histology ............................................................................................ 73 4.6 Results for isolation of compounds ..................................................................... 74 4.6.1 Compounds isolated from the DCM root of C. limon (CLR 2)........................ 74 4.6.1.1 Isolation of compounds from the DCM root of C. limon (CLR 2) ................ 74 4.6.2 Compounds isolated from Bridelia cathartica methanolic leaf extract (BCL-3)75 4.6.3 Compounds isolated from M. pyrifolia hexane leaf extract: Adsorption chromatography ......................................................................................................... 76 4.6.4 Structures of isolated pure compounds ............................................................. 78 4.6.5 NMR, IR and