EVALUATION OF ANTIOXIDANT ACTIVITIES OF SOME NIGERIAN MEDICINAL PLANTS BY THE USE OF 2, 2- DIPHENYL-1- PICRYLHYDRAZYL (DPPH) FREE RADICAL. BY UCHE, FIDELIA IJEOMA PG/M.PHARM/07/43544 DEPARTMENT OF PHARMACOGNOSY FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA, NSUKKA. FEBRUARY, 2010 1 Title page EVALUATION OF ANTIOXIDANT ACTIVITIES OF SOME NIGERIAN MEDICINAL PLANTS BY THE USE OF 2, 2- DIPHENYL-1- PICRYLHYDRAZYL (DPPH) FREE RADICAL. BY UCHE, FIDELIA IJEOMA PG/M.PHARM/07/43544 A THESIS SUBMITTED TO THE DEPARTMENT OF PHARMACOGNOSY, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA, IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF MASTER OF PHARMACEUTICAL SCIENCES (M. PHARM) DEGREE IN PHARMACOGNOSY. SUPERVISOR: DR. CHRIS O. EZUGWU DEPARTMENT OF PHARMACOGNOSY, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA. 2 FEBRUARY, 2010. Certification Uche, Fidelia Ijeoma is a postgraduate student in the Department of Pharmacognosy, with registration number PG/M. Pharm/ 07/43544. She has satisfactorily completed the requirement for research work for the of Master of Pharmaceutical sciences in Pharmacognosy. The work embodied in this project report is original and has not been submitted in part or full for any other diploma or degree of this or any other University. ---------------------------- ------------------------------ --- Supervisor Internal Examiner/ Head of Department. 3 DEDICATION This is dedicated to the sweet memory of my beloved parents, late Chief Felix N and Laura Okorom. 4 ACKNOWLEDGEMENT I am very grateful to almighty God for His endless mercy, grace, enduring love and divine assistance in this study. I am equally heavily indebted to my supervisor, Dr C.O. Ezugwu, who did not relent in instruction and direction on this study. My deep appreciation also goes to my beloved husband, Hon Uche Nwosu, whose encouragement and financial support led to the successful completion of this work. I appreciate so much the sub-grant given to me for this study by Mac Arthur Foundation through the University of Port Harcourt. 5 Moreover, the contributions of the entire staff and the Head, Department of Pharmacognosy, are highly appreciated. Finally, I very much appreciate the moral support given to me by distinguished lecturers such as: Professors O.K. Udeala, S.I. Ofoefule and F. C. Ohiri; Drs A.W. Obianime, E.C. Ibezim, O Okorie and C.E.C. Ugwoke. I express my sincere gratitude to them all. LIST OF TABLES Pages Table 1: Botanical names, Families and local/common names of the plants under study…………………………………………………….6 Table 2: Results of the Phytochemical screening of the 6 plants under study…………............................................................................62 Table 3: Results of the Phytochemical screening of the plants under study………………………………………………………………….63 Table 4: Antioxidant activities and reducing potentials of the plants under study……………………………………………….….64 LIST OF FIGURES 7 P a g e s Figure 1: The digital photograph of Kalanchoe pinnata in its natural habitat……………………………………………………….8 Figure 2: The digital photograph of Aspilia africana in its natural habitat……………………………………………………………11 Figure 3: The digital photograph of Mucuna pruriens flowers, fruits and leaves……………………………………………………………………14 Figure 4: The digital photograph of Mucuna pruriens in its 8 natural habitat…………...........................................................................15 Figure 5: The digital photograph of Emilia coccinea in its natural habitat…………………………………………………………….18 Figure 6a and b: The digital photograph of Laportea ovalifolia in its natural habitat……. ……………………………………………………….21 Figure 7: The digital photograph of Cucurbita moschata in its natural habitat…………………………………………………………….25 Figure 8: The seeds of Cucurbita moschata……………………………………….26 9 Figure 9: The digital photograph of Celosia trigyna in its natural habitat……35 Figure 10a: The digital photograph of Asystasia gangetica in its natural habitat…………….......................................................................40 Figure 10b: The diagram showing different stages of development of Asystasia gangetica……………………………………………………………41 Figure 10c: The digital photograph of Asystasia gangetica in its natural habitat………………………………………………………………..42 Figure 11: Antioxidant activities of Nigerian medicinal plants…………………….66 Figure 12: Antioxidant activities of Nigerian Vegetables……………………………67 10 Figure 13: Reducing potentials and antioxidant activities of the plants under study…………………………………………….…………………68 Figure 14: IC50 inhibitory concentration and antioxidant activity………………….69 Figure 15: Relationship between antioxidant activity and IC50……………………..70 Figures16a and 16b: Reducing potential and IC50…………………………………..71 Figures 17a and 17b: Relationship between reducing potential and Antioxidant activities of the plants…………………………………………………72 Figures 18a and b: Percentage antioxidant activities and IC50…………………….73 Figure 19: Effects of concentration of A.G, C.M and C.I on Antioxidant activities………………………………………………………….75 Figure 20: Effects of concentration on antioxidant activities 11 of L.O, E.C and M.P…………………………………………………………..76 Figure 21: Effects of concentration on antioxidant activities of A.A, K.P and ascorbic acid……………………………………………………77 ABSTRACT The methanol extracts of the leaves of Kolanchoe pinnata, Aspilia africana, Mucuna pruriens, Emilia coccinea, Laportea ovalifolia, Celosia trigyna, Cucurbita moschata and Asystasia gangetica were evaluated for antioxidant activities by the use of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical assay. The reducing potentials of these plants were also evaluated. The phytochemical screenings of the medicinal plants were equally carried out. The percentage antioxidant activity values for the plants are: 31.0 ± 1.80%, 58.4 ± 1.26%, 59.1 ± 1.60%, 60.0 ± 1.05%, 60.8 ± 1.28%, 62.4 ± 1.28%, 64.8 ± 2.10% and 75.7 ± 2.60% for A. gangetica, C. moschata, C. trigyna, L .ovalifolia, E. coccinea, M. pruriens, A. africana and K. pinnata respectively. These values were dose – dependent and statistically significant at P < 0.05 (ANOVA). The results indicated that K. pinnata has the highest antioxidant activity value 75.7 ± 2.60% while A. gangetica has the least value 31.0 ± 1.80%. The 12 percentage antioxidant activities of the plants were comparable to the standards used, the ascorbic acid and - tocopherol which were found to be 86.7 ± 1.08% and 97.2 ± 1.06% respectively. The reducing potentials of the plants were found to be proportionally correlated to the antioxidant activities of the plants. Phytochemical screenings revealed the presence of alkaloids, flavonoids, terpenoids, cardiac glycosides, steroids, saponins and tannins in the medicinal plants. TABLE OF CONTENTS Pages Title page…………………………………………………………………………..ii Certification……………………………………………………………………….i ii Dedication………………………………………………………………………....i v 13 Acknowledgement……………………………………………..………………..… v List of Tables……………………………………………………………..…….... vi List of Figures…………………………………………………………….……….vii Abstract..………………………………………………………………….…….…. x Table of contents………………………………………………………………….xii CHAPTER ONE: INTORDUCTION 1. The Significance of antioxidant…………………………………………….1 Types of antioxidants……………………………………………....………2 Activities of antioxidants………………………………………….……….4 Review of medicinal plants under study…………………...……….……...6 1.3.1. Pharmacognostic profile of the plants under study……..……..….……..9 1.4. Justification of the study…………………………………………………50 14 1.5. Objective of study……………………….……………………………….51 CHAPTER TWO: Materials and Methods. 2.1. Materials/Chemicals used……………………………………………..……….52 2.2. Collection and drying……………………………………………....………….52 2.3. Preparation of extracts…………………………………………......…………..53 2.4. Phytochemical screening………………………………………..……………..53 2.5. Antioxidant activity assay………………………………….…..………….......57 2.5.2 . Determination of reducing potentials……………………………………….58 2.6. Statistical analysis………………………………………….………………….59 15 CHAPTER THREE 3.1. Results ……………………………………………………………………..….60 3.2. Discussion/Conclusion………………………………………………….……..78 Reference………………………………………………………………….………. 81 Appendix………………………………………………………………………… …90 16 CHAPTER ONE INTRODUCTION 1. The Significance of Antioxidant Chemical compounds with unpaired radicals such as powerful oxidants and free radicals are capable, when present in the body, to damage lipids, proteins, and also DNA and consequently may bring about mutation (Ellinaim et al., 2003). Free radicals play a prominent role in human health. Free radical reactions have been implicated in the etiology and pathogenesis of chronic diseases that are life limiting such as cancer, hypertension, cardiac infarction, arteriosclerosis, diabetes etc. Antioxidants are compounds that inhibit or delay the oxidation of other molecules by inhibiting the initiation or propagation of oxidizing chain reactions (Velioglu, et al., 1998). Antioxidant activity is a very important pharmacological property. Many of the pharmacological functions such as antimutagenicity, anticarcinogenicity, anti-aging etc originate from this property. (Cook and Samman, 1996: Huang et al., 1992) The most important free radicals in the body are the reactive oxygen and nitrogen species, such as super oxide, hydroxyl and nitric oxide radicals. They are 17 generated in the body as a consequence of cellular and metabolic activities. They also arise from exogenous sources (exposure to ionizing radiations, injury, oxidative drugs, pollutants, etc). Excessive production and leakages from their site of generation