1 EVALUATION OF THE ANTIPLASMODIAL ACTIVITY OF METHANOL LEAF EXTRACT AND FRACTIONS OF LANDOLPHIA OWARIENSIS P. BEAUV (APOCYNACEAE) IN MICE BY OKONKWO, CHINELO HENRIETTA (PG/M.PHARM/10/52905) DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA, NSUKKA SEPTEMBER, 2015 2 EVALUATION OF THE ANTIPLASMODIAL ACTIVITY OF METHANOL LEAF EXTRACT AND FRACTIONS OF LANDOLPHIA OWARIENSIS P. BEAUV (APOCYNACEAE) IN MICE BY OKONKWO, CHINELO HENRIETTA (PG/M.PHARM/10/52905) A PROJECT REPORT PRESENTED TO THE DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA, NSUKKA IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF MASTER OF PHARMACY (M.PHARM) DEGREE DR ADAOBI C. EZIKE (SUPERVISOR) DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA, NSUKKA SEPTEMBER, 2015 3 TITLE PAGE EVALUATION OF THE ANTIPLASMODIAL ACTIVITY OF METHANOL LEAF EXTRACT AND FRACTIONS OF LANDOLPHIA OWARIENSIS P. BEAUV (APOCYNACEAE) IN MICE 4 CERTIFICATION OKONKWO, CHINELO HENRIETTA, a postgraduate student of the Department of Pharmacology and Toxicology with Registration Number PG/M.PHARM/10/52905, has satisfactorily completed the requirements for the award of the degree of Master of Pharmacy (M.Pharm) of the Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka. The work embodied in this project is original and has not been submitted in part or full for any other diploma or degree of this or any university. …………………………… …………………………… DR ADAOBI C. EZIKE DR ADAOBI C. EZIKE Supervisor Head of Department 5 DEDICATION This work is dedicated to God Almighty “for nothing is impossible with him” (Luke 1:37) and also to my husband and three children as well as my parents. 6 ACKNOWLEDGEMENTS I express my profound gratitude to God who out of His abundant love saw me throughout my education in this university and my research into this project “for in Him we live and move and have our being” (Acts 17: 28). I am sincerely grateful to my supervisor Dr Adaobi C. Ezike for her motherly love, untiring suggestions, constructive and objective criticisms which have led to the successful completion of this work. With heartfelt gratitude, I esteem highly my husband, Emmanuel and children Chisom, Chibuikem and Chidubem as well as my parents Mr & Mrs G.O. Ojielo, my brothers and sisters who are Okey, Maureen, Chioma, Chibueze, Chinedu, Nneka and Chinwe for their love, understanding, patience, encouragement, prayers, financial and moral support. My special thanks to Pharm Collins Onyeto and Prof. M. Emeje of NIPRD, for their prayers, encouragement and relentless effort in making this work a reality. I also appreciate Mr Austin Okorie for all his efforts and assistance in the laboratory, Mr Alfred Ozioko and Dr Michelle of InterCEDD, Rev Ezea of Pharmacognosy as well as Dr B. Adzu, Dr S. Ameh and Mrs Gloria of National Institute of Pharmaceutical Research and Development (NIPRD) for their various contributions to the successful completion of this work. I am particularly grateful to all my lecturers in the department especially Prof A Akah, my pharmacology father, Prof Charles Okoli, Dr C.S. Nworu, Dr T.C. Okoye, for the vast knowledge I acquired from them which has contributed towards the success of this work as well. I am also grateful to my friends Pharm (Mrs.) F Mbaoji, Pharm (Mrs) Ifeoma and the entire staff of the Department of Pharmacology and Toxicology especially Mr Bonny Ezeh, Mrs Ego and Mrs Ugwu for their assistance and encouragement. I am also thankful to my former Head of Department at National Hospital Abuja, Pharm Abdu Msheliza and his family and also Pharm C.A. Umezulike, Pharm (Mrs) A. 7 Ajemigbitse, Dr A. Akinmola, Dr Ogunfowokan, Dr Orilade, Allison all of National Hospital Abuja for their understanding, suggestions and encouragement in seeing to the successful completion of this work. I register my gratitude too to all my friends in the university as well as classmates in the Dept of Pharmacology and Toxicology among who are Ben, Dr Ugwu, Dr Chibueze, Ufere, Chinenye and all others not mentioned for their encouragement in their various capacities to my success. I appreciate Revds Sam Etim (MSP), Iffiok E Inyang (MSP), Festus Ejiofor (MSP), Srs Cecilia Azuh, Lucy and Evarista (MMM) for their encouragement, moral and spiritual support given to me marvelously in different ways. I equally appreciate all my friends namely: Lucy, Ben, Chinelo, Juliet Okonkwo, Ewi, Ohi, Immaculata, Dr Ogunleye and many others not mentioned here for their various contributions and support. Finally, I am sincerely grateful to all the staff of NIPRD, Idu, Abuja, for their various assistance and granting me access to their laboratory and also Dr Aina of National Institute of Medical Research (NIMR), Lagos for providing the parasite used for this work. 8 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 9 representing different components. The results of this study suggest that the leaf extract and fractions pose significant antiplasmodial activity. TABLE OF CONTENT Page Title page …...................................................................................................................... ii Certification ………………………………………………………………..................... iii Dedication ……………………………………………………………………………… iv Acknowledgement ………………………………………………………....................... v Abstract …………………………………………………………...……….………….. vii Table of content ............................................................................................................. viii List of tables ………………………………………………………………................... xii List of figures …………………………………………………………………………. xiii CHAPTER ONE: INTRODUCTION 1.0 Introduction ………………………………………………………………………... 1 1.1 Background ……………………………..……...…….…………………………….. 1 1.2 Definition of Malaria ……………………..………….…………………………….. 2 1.3 Epidemiology of Malaria …..….…………………………………………………… 2 1.4 Etiology of Malaria .............................................................................................…... 3 1.4.1 Vector ……………………………………………………………………………… 4 1.4.2 Biology of Malaria Infection (Life cycle of Malaria Parasite) .……………….…... 5 10 1.4.3 Pathogenesis of Malaria ...................................................................................…... 8 1.5 Clinical Manifestation of Malaria ...…………..………...………………………….. 9 1.6 Diagnosis of Malaria ………………..………………………….…………………. 10 1.7 Classification of antimalarials ………………………….......................................... 11 1.8 Chemotherapy of Malaria .................................................................................…... 13 1.8.1 Chloroquine …………………………………………………………………....... 14 1.8.2 Amodiaquine …………………...……………………………………………… 16 1.8.3 Quinine and quinidine ............................................................................…........... 16 1.8.4 Mefloquine .…………………………………………….……………………...... 17 1.8.5 Halofantrine .……………………………………………………………………. 17 1.8.6 Lumefantrine ..................................................................................................…... 18 1.8.7 Primaquine ……………………………………………..………………………. 19 1.8.8 Sulphadoxine - Pyrimethamine ......................................................................…... 19 1.8.9 Antibacterial agents ......................................................................................….... 20 1.8.10 Artemisinins …………………………………….……………………............... 20 1.8.10.1 Antimalarial Properties of Artemisinins .....…………………………………... 21 1.8.10.2 Artemisinin Based Combination Therapy (ACT) …………………………….. 22 11 1.9 Prevention and Control of Malaria ……………………………………………….. 24 1.10 Review of Plants with Antimalarial Activity …………………………………….. 26 1.11 Botanical Profile of Landolphia owariensis ……………………………………… 30 1.11.1 Plant Taxonomy …………………………………………………………………