PHYTOCHEMICAL AND ANTIMALARIAL STUDIES OF THE LEAVES OF UVARIA CHAMAE P.BEAUV. (ANNONACEAE) BY HASSAN ALI BILA P13PHMC8010 DEPARTMENT OF PHARMACEUTICAL AND MEDICINAL CHEMISTRY AHMADU BELLO UNIVERSITY, ZARIA-NIGERIA April, 2016 i PHYTOCHEMICAL AND ANTIMALARIAL STUDIES OF THE LEAVES OF UVARIA CHAMAE P.BEAUV.(ANNONACEAE) BY HASSAN ALI BILA, BSc. Chemistry (ADSU) 2012 P13PHMC8010 A DISSERTATION SUMMITTED TO THE SCHOOL OF POST GRADUATE STUDIES AHMADU BELLO UNIVERSITY, ZARIA-NIGERIA IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF MASTER OF SCIENCE DEGREE IN PHARMACEUTICAL AND MEDICINAL CHEMISTRY DEPARTMENT OF PHARMACEUTICAL AND MEDICINAL CHEMISTRY AHMADU BELLO UNIVERSITY, ZARIA-NIGERIA ` ii DECLARATION I declare that the work in this dissertation entitled “PHYTOCHEMICAL AND ANTIMALARIAL STUDIES OF THE LEAVES OF UVARIA CHAMAE P.BEAUV. (ANNONACEAE)”has been carried out by me in the Department of Pharmaceutical and Medicinal Chemistry. The information derived from literature has been duly acknowledged in the text and a list of references provided. No part of this dissertation was previously presented for another degree or diploma at this or any other institution. Hassan Ali Bila ___________________________ _________________ ______________ Name of Student Signature Date iii CERTIFICATION This Dissertation “PHYTOCHEMICAL AND ANTIMALARIAL STUDIES OF THE LEAVES OF UVARIA CHAMAE P.BEAUV. (ANNONACEAE)” by Hassan Ali Bila meets the regulations government the award of the degree of Master of Science in Pharmaceutical and Medicinal Chemistry of the Ahmadu Bello University, Zaria and is approved for its contribution to knowledge and literary presentation. Prof. M. Ilyas ____________________________ _________________ ______________ Chairman, Supervisory Committee Signature Date Dr. A.M. Musa _____________________________ _________________ ______________ Member, Supervisory Committee Signature Date Dr. A.M. Musa _____________________________ _________________ ______________ Head of Department Signature Date Prof. K. Bala _____________________________ _________________ ______________ Dean School Of Postgraduate Studies Signature Date iv DEDICATION This research is dedicated to the Almighty God. For He has been the source of my strength. v ACKNOWLEDGEMENT First and foremost I am eternally grateful to Almighty God for making it possible for me to carry out this research work. He has been my anchor. My sincere gratitude goes to my supervisors, Prof. M. Ilyas and Dr. A.M. Musa for their assistance, guidance, instruction and advice throughout the course of this work. My profound gratitude also goes to my father for his love, care, support and prayers, May God reward him mightily. To my brothers and sisters, I know, I can’t thank you enough for your support but I know that you will not lack help and support when you need it. I wish to express my sincere gratitude to Dr. Y.M. Sani, MallamaSakynah and Dr. I.M.Maje for their continues encouragement and support during the course of this work, May God Almighty reward you abundantly. My profound gratitude also goes to Prof. Simon Gibbons, University of London for assisting with the NMR analysis and also Mallam Husseini, Department of Parasitology, Faculty of Veterinary Medicine Ahmadu Bello University, Zaria for assisting in the biological studies. If I continue to mention names I will not finish. I want to express my sincere gratitude to all the staff and students of the Department of Pharmaceutical and Medicinal Chemistry, Ahmadu Bello University, Zaria. I really enjoyed a good learning and working relationship with you all. God bless you. vi ABSTRACT Uvaria chamae P.Beauv. belong to the Annonaceae family of flowering plants. It is distributed in the savannah and secondary forest. The plant is used in ethnomedicine for the treatment of malaria, inflammation, gonorrhea, dysentery; pile and fever. The ethyl alcohol leaves extract of the plant was subjected to phytochemical as well as antimalarial studies. Phytochemical studies were carried out using techniques including preliminary phytochemical tests, thin layer chromatographic analysis (TLC), column chromatography and gel filtration. The antimalarial activity of the crude ethyl alcohol leaves extract was evaluated using two models, suppressive and curative tests. The result of the preliminary phytochemical screening revealed the presence of saponins, flavonoids, carbohydrates, tannins, alkaloids and terpenoids. A flavonoids was isolated from the extract. The structure of the compound isolated was elucidated using UV, IR, 1D NMR and ESI-MS. The compound was found to be an epicatechin (3, 3, 4, 5, 7- pentahydroxyflavan). The leaf extract caused no lethality in mice at oral LD50 value of greater than 5000 mg/kg body weight. This indicated that the extract is safe for oral use. In the suppressive test the extract exhibited good antimalarial property that was dose dependent. At doses of 25, 50 and 100 mg/kgbody weight the extract produced a significant (P< 0.05) chemosuppression of 48, 53.3 and 65% respectively. Chloroquine the positive control drug produced the highest parasite chemosuppression at 79%. In the curative test the extract at doses of 25, 50 and 100 mg/kg body weight produced significant (P< 0.05) chemosuppression at 68, 70 and 73% respectively. Chloroquine the positive control drug produced the highest parasite chemosuppression of 98%. The result of this studies has established the rationale for the use of this plant in ethnomedicine. The isolated compound might be responsible for the observed biological activity. vii TABLE OF CONTENTS Cover page- - - - - - - - - - - i Title Page - - - - - - - - - - ii Declaration - - - - - - - - - - iii Certification - - - - - - - - - - iv Dedication - - - - - - - -- - - v Acknowledgement - - - - - - - - - vi Abstract - - - - - - - - - - vii Table of Contents - - - - - - - - - viii List of Tables - - - - - - - - - - xiii List of Figure - - - - - - - -- - - xiv List of Plate - - - - - - - - - - xv List of Appendix - - - - - - - - - xvi List of Abbrebration - - - - - - - - - xvii CHAPTER ONE 1.0 Introduction - - - - - - - - - 1 1.1 Natural product- - - - - - - - - 1 1.2 Traditional medcine - - - - - - - - 2 1.3 Malaria - - - - - - - - - 3 1.3.1 Resistance to Malaria Chemotherapy- - - - - - 4 1.4 Medicinal plants use `for malaria treatment - - - - - 5 1.5 Sources of Drugs - - - - - - - - 8 1.5.1 Natural Source - - - - - - - - 9 1.5.2 Synthetic Drugs - - - - - - - - 9 1.5.3 Biosynthetic Sources - - - - - - - - 10 viii 1.6 Statement ofResearch Problem - - - - - - 10 1.7 Justification of the Study - - - - - - - 11 1.8 Aim of the Study - - - - - - - - 11 1.9 Specific Objectives - - - - - - - - 11 1.10 Hypothesis - - - - - - - - - 12 CHAPTER TWO 2.0 Literature Review - - - - - - - - 13 2.1 The Plants - - - - - - - - - 13 2.2 Habitat - - - - - - - - - 13 2.3 Taxonomy/ Nomenclature of the Plant - - - - - 15 2.3.1 Common and Local Names - - - - - - - 15 2.4 Botanical Description - - - - - - - - 15 2.5 EthnomedicinalUses - - - - - - - 16 2.6 Pharmacological Action of Plants from the Genus Uvaria - - - 16 2.7 Chemical Constituents- - - - - - - - 17 2.7.1 Flavonoids- - - - - - - - - - 19 2.7.1.1 Flavones- - - - - - - - - - 20 2.7.1.2 Flavonols- - - - - - - - - - 20 2.7.1.3 Flavanones- - - - - - - - - - 21 2.7.1.4 Flavanonols- - - - - - - - - - 22 2.7.1.5 Isoflavones- - - - - - - - - - 22 2.7.1.6 Neoflavonoids- - - - - - -` - - 23 2.7.1.7 Flavanols or flavan-3-ols or catechins- - - - - - 23 2.7.1.8 Anthocyanidins- - - - - - - - - 24 2.7.1.9 Chalcones- - - - - - - - - - 25 ix CHAPTER THREE 3.0 Materials and Methods - - - - - - - 26 3.1 Materials - - - - - - - - - 26 3.1.1 Solvents/Reagents and Chromatography Materials - - - - 26 3.1.2 Equipment - - - - - - - - - 26 3.1.3 Experimental Animals - - - - - - - 26 3.1.4 Malaria Parasites - - - - - - - - 27 3.2 Methods - - - - - - - - - 27 3.2.1 Collection and Identification of Plant Material - - - - 27 3.2.2 Extraction and Partitioning - - - - - - - 27 3.2.3 Preliminary phytochemical Screening - - - - - 28 3.2.3.1 Test for Anthraquinones - - - - - - - 28 3.2.3.2 Test for Alkaloids - - - - - - - - 28 3.2.3.1Test for Carbohydrates - - - - - - - 29 3.2.3.4 Test for Cardiac Glycosides - - - - - - - 29 3.2.3.5 Test for Saponins - - - - - - - - 30 3.2.3.4 Test for Flavonoids - - - - - -- - 30 3.2.3.7 Test for Tannins - - - - - - - - 31 3.2.3.8 Test for Steroids/Triterpenes- - - - - - - - 31 3.2.4 Chromatographic procedures - - - - - - - 32 3.2.4.1 Thin Layer Chromatographic Analysis. - - - - - 32 3.2.4.2 Column chromatography of ethyl acetate fraction - - - - 23 3.2.4.3 Gel Filtration Chromatography - - - - - - 33 3.2.4.4 Melting Point determination - - - - - - - 33 x 3.2.4.5 Test for catechins- - - - - - - - - 33 3.2.5 Pharmacological studies - - - - - - - 34 3.2.5.1 Acute toxicity studies - - - - - - - - 34 3.2.5.2 Antimalarial Studies - - - - - - - - 34 CHAPTER FOUR 4.0 Results - - - - - - - - - - 36 4.1 Extraction Yield - - -- - - - - - 36 4.2 Phytochemical constituent of the leave extract of Uvaria chamae-. - - 37 4.3 Thin Layer Chromatography- - - - - - - - 38 4.3.1 Thin-layer Chromatography of the crude extracts and partitioned fractions- 38 4.4 Column Chromatography of Ethyl acetate Fraction- - - - 40 4.5 Gel- Filtration of column fraction F4 - - - - - - 43 4.5.1TLC Profile of HB developed in different solvent system- - - 44 4.6 Solubility Profile of HB - - - - - - 44 4.7 Melting Point of HB - -- - - - - - - 44 4.8 Chemical Test on HB - - - - - - - - 45 4.9 UV Spectra