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EVALUATION of Uvaria Chamae AQUEOUS and ETHANOLIC EXTRACTS FOR EVALUATION OF Uvaria chamae AQUEOUS AND ETHANOLIC EXTRACTS FOR ACTIVITY AGAINST MULTIDRUG RESISTANT BACTERIA BY ONAKPA, IDACHABA E. PG/M.Sc/07/42926 DEPARTMENT OF MICROBIOLOGY UNIVERSITY OF NIGERIA, NSUKKA AUGUST, 2010 i TITLE PAGE EVALUATION OF Uvaria chamae AQUEOUS AND ETHANOLIC EXTRACTS FOR ACTIVITY AGAINST MULTIDRUG RESISTANT BACTERIA BY ONAKPA, IDACHABA EMMANUEL PG/M.Sc/07/42926 TO THE SCHOOL OF POST GRADUATE STUDIES, UNIVERSITY OF NIGERIA, NSUKKA IN PARTIAL FULFILMENT OF THE REQIREMENTS FOR THE AWARD OF MASTER’S DEGREE (M.Sc) IN MEDICAL MICROBIOLOGY SUPERVISOR: DR. (MRS) I.M. EZEONU AUGUST, 2010 ii CERTIFICATION Mr. Onakpa, Idachaba Emmanuel, a postgraduate student in the Department of Microbiology, majoring in Medical Microbiology, has satisfactorily completed the requirements for course work and research for the degree of Master in Science (M.Sc) in Microbiology. The work is embodied in his dissertation original and has not been submitted in part or full for either diploma or degree of this university or any other university. Dr. (Mrs.) I. M EZEONU Dr. (Mrs.) I.M. EZEONU Head, Supervisor, Department of Microbiology, Department of Microbiology University of Nigeria, University of Nigeria, Nsukka. Nsukka. iii DEDICATION This work is dedicated to my mother –Mrs. Habiba Onakpa –for being a constant source of quiet strength. iv ACKNOWLEDGEMENT My gratitude first goes to God Almighty for His gift of life and health. I wish also to acknowledge with humility the motherly support of my supervisor Dr. (Mrs.) I.M. Ezeonu without whom this work would have been impossible. Words fail me to express my gratitude for her patience, attention and motivation as she was always there to guide and direct me. I wish to thank Mrs. Nwuike of Medical Diagnostic Laboratory of the Microbiology Department, University of Nigeria, Nsukka and the staff of the Microbiology Section of the Enugu State University Teaching Hospital, Parklane Enugu for providing me with the clinical specimens used for the work. My thanks also go to Dr. E.A Eze, Mr. Ngene and Mr. Uchenna Nwodo for also furnishing me with the other strains of bacteria used. I must also not fail to thank Prof. C.U. Iroegbu for his constant and often critical observations. He gave me reasons to believe in myself, my work and its implications for the ageless annals of scientific collections and hence, the need to work diligently. I wish to also thank Prof. J.C. Ogbonna, Prof. (Mrs.) J.I Okafor and Dr. J.I Ihedioha for their cooperation, understanding and assistance throughout the duration of this research. Finally, I wish to thank my mother –Mrs. Habiba Onakpa, my father and siblings viz: Onakpa Aruwa, Asibi, Okutepa, Tijani, Shaibu, Idoko, Opaluwa, Uchogwu and Adi (egg) for their financial and moral support. v ABSTRACT The antimicrobial activities of ethanolic and aqueous extracts of Uvaria chamae root bark, stem bark and leaves were evaluated. The root bark extract of the plant is used in ethnomedicine as a prescription for piles, haematuria, treatment of fevers classed locally as „yellow- fever‟, and almost any indisposition accompanied by jaundice. Thirty eight bacterial strains resistant to a variety of routinely used antibiotics were used for the assays; four strains were from American Type Culture Collection (ATCC), one strain from Scottish Salmonella Reference Laboratory (SSRL), one strain from Northern Regional Research Laboratory (NRRL) and thirty two were clinical isolates. Agar well diffusion technique was used for the antibacterial assays. Generally, the extracts had antimicrobial activity against most of the test organisms. Inhibition was a direct function of concentration of extracts with 100% of organisms inhibited at 250mg/ml, albeit to different degrees. The alcoholic extracts were significantly (P<0.05) more active than the aqueous extracts, with the ethanolic stem bark extract having higher activity than the root bark and leaf extracts. The minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) varied with test isolates as well as with various extracts. For instance, the MIC of the ethanolic extracts of the stem bark varied from 7.81 mg/ml for Proteus mirabilis to 100 mg/ml for Klebsiella with their respective MBC‟s varying from 37.03 mg/ml to 200 mg/ml. The ethanolic extracts were successively partitioned using n-hexane(A), chloroform (B), ethyl acetate (C), acetone (D), and methanol (E). Five fractions were obtained for each of root bark, stem bark, and leaves and were designated Ar to Er, As to Es and AL to EL respectively. Fractions Ds, Es, and Cr had activity against 100, 90 and 73% of test strains respectively. The least active fractions were El (27%), Al (36%), Dl (45%), Ar (45%) and As (45%). Acute toxicity studies using LD50 assays showed the alcoholic stem bark extract to be virtually non-toxic ( LD50 vi >5000mg/kg body weight). Phytochemical analyses of crude extracts and fractions indicated the presence of flavonoids, saponins, tannins, alkaloids amongst others in relative proportion. vii TABLE OF CONTENTS Title Page… … … … … … … … … … i Certification… … … … …. … … … … ii Dedication… … … … … … … … … … iii Acknowledgement… … … … … … … … … iv Abstract.. … … … … … … … … … v Table of Contents.. … … … … … … … … vii List of Tables .. … … .. .. .. .. .. … x List of Figures … … .. … … …. … … … xi CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW… 1 1.1 Specific aims and objectives … … … … … .. 3 1.2 Literature review … … … … … … .. 3 1.2.1 Historical development of ethno medicine … …. … … 3 1.2.2 Antibiotic resistance.. … …. … … … … … 8 1.2.3 Natural products in drug discovery … … …. … …. 9 1.2.4 Plant and antibacterial production … … …. …. …. 11 1.2.5 Safety issues in herbal medicine: Implications for the health professions 14 1.2.6 Taxonomy and systematic of Uvaria.. … … … .. 17 1.2.7 The Plant- Uvaria chamae … .. … … … … 20 1.2.8 Uvaria chamae in alternative medicine.. … … … … 20 viii CHAPTER TWO: MATERIALS AND METHODS… … …. …. 24 2.1 Collection of plant materials. .. … … … … … 24 2.2 Extraction… … … … … … … .. … 26 2.3 Microorganisms… …. …. … .. … … … 26 2.4 Test for sensitivity or resistance to standard antibiotics… … … 27 2.5 Determination of antimicrobial activity of crude extracts.. … … 28 2.6 Determination of minimum inhibitory concentration… … … 29 2.7 Determination of minimum bactericidal concentration… …. … 29 2.8 Solvent-solvent fractionation… …. …. …. … … 29 2.9 Antibacterial assay of extract fractions… … … … … 32 2.10 Phytochemical evaluation of crude extracts and fractions.. … … 32 2.10.1 Test for saponins… … … … … … … … 32 2.10.2 Test for tannins… … … … … … …. … 33 2.10.3 Test for alkaloids.. .. … … … … … … 33 2.10.4 Test for flavonoids.. … … … … … .. … 34 2.10.5 Test for fats and oils… … … … … … … 34 2.10.6 Test for reducing sugar… … … … … … … 34 2.10.7 Test for glycosides … … … … … … … 35 2.11 Acute toxicity studies.. … … … … … .. 35 2.12 Statistical Analysis… … … … … … … … 36 ix CHAPTER THREE: RESULTS.. … … … … .. … 37 3.1 Antimicrobial susceptibility profile of test bacteria … .. … 37 3.2 Aqueous and ethanolic extractions.. … … .. … .. 40 3.3 Antimicrobial activities of crude extract… .. …. … … 42 3.4 Minimum inhibitory and minimum bactericidal concentrations of extracts 53 3.5 Yield from solvent-solvent fractionation… . … … … 56 3.6 Antimicrobial activities of extract fractions… … … … 58 3.7 Phytochemical composition of crude extract and fractions… …. 84 3.8 Acute toxicity profile.. … … … … … … 89 CHAPTER FOUR: DISCUSSION … … …. …. … .. 90 REFERENCES … … … … … .. .. .. 97 APPENDICES … … …. …. .. .. 102 x LIST OF TABLES Table 1: Plant derived widely employed in western medicine… … … … 7 Table 2: Resistance pattern of Gram negative organisms to conventional antibiotics.. 38 Table 3: Resistance pattern of Gram positive organisms to conventional antibiotics.. 39 Table 4: Minimum inhibitory comcentration… …. …. … … … 54 Table 5: Minimum bactericidal concentration… … …. … … … 55 Table 6: Preliminary phytochemical screening of crude extracts… … … … 85 Table 7: Phytochemical screening of root bark fractions … … … … … 86 Table 8: Phytochemical screening of stem bark fractions …. …. … … 87 Table 9: Phytochemical screening of leaf fractions … …. … … … 88 xi LIST OF FIGURES Figure 1: Extraction of crude plant parts …. …. …. .. 41 Figure 2: Activity of crude aqueous root bark extract … … .. 43 Figure 3: Activity of crude alcoholic root bark extract … .. .. 45 Figure 4: Activity of crude aqueous stem bark extract … .. .. 47 Figure 5: Activity of crude alcoholic stem bark extract … … ... 48 Figure 6: Activity of crude aqueous leaf extract … … .. .. 50 Figure 7: Activity of crude alcoholic leaf extract … … .. .. 52 Figure 8: The percentage recovery of crude extract fractions using solvent – solvent Fractionation … … .. … .. … … 57 Figure 9: Activity of root bark acetone fraction … … .. .. 59 Figure 10: Activity of stem bark acetone fraction … … .. .. 61 Figure 11: Activity of leaf acetone fraction … … .. .. 63 Figure 12: Activity of root bark ethyl acetate fraction … … .. .. 65 Figure 13: Activity of stem bark ethyl acetate fraction … … .. .. 67 Figure 14: Activity of leaf ethyl acetate fraction … … .. .. 69 Figure 15: Activity of root bark methanol fraction … … .. .. 71 Figure 16: Activity of stem bark methanol fraction … … .. .. 73 Figure 17: Activity of leaf methanol fraction … … .. .. 75 Figure 18: Activity of leaf chloroform fraction … … .. .. 77 Figure 19: Activity of root bark n – heaxane fraction … … .. .. 79 Figure 20:
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