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FACULTY of PHARMACEUTICAL SCIENCES Ameh Joseph

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FACULTY of PHARMACEUTICAL SCIENCES Ameh Joseph OKWUBIE, LAMBERT (PG / M.PHARM / 10/ 52771) EVALUATION OF OXIDATIVE STRESS INDICATORS ON FIVE MEDICINALPLANTS FROM CRUDE OIL POLLUTED ENVIRONMENT DEPARTMENT OF PHARMACOGNOSY AND ENVIRONMENTAL MEDICINE FACULTY OF PHARMACEUTICAL SCIENCES Digitally Signed by: Content manager’s Name DN : CN = Webmaster’s name Ameh Joseph Jnr O= University of Nigeria, Nsukka OU = Innovation Centre EVALUATION OF OXIDATIVE STRESS INDICATORS ON FIVE MEDICINALPLANTS FROM CRUDE OIL POLLUTED ENVIRONMENT BY OKWUBIE, LAMBERT (PG / M.PHARM / 10/ 52771) PROFESSOR S. I. INYA AGHA (SUPERVISOR) DEPARTMENT OF PHARMACOGNOSY AND ENVIRONMENTAL MEDICINE, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA. JANUARY 2014 i EVALUATION OF OXIDATIVE STRESS INDICATORS ON FIVE MEDICINAL PLANTS FROM CRUDE OIL POLLUTED ENVIRONMENT BY OKWUBIE, LAMBERT (PG / M.PHARM / 10/ 52771) BEING A PROJECT REPORT SUBMITTED TO THE DEPARTMENT OF PHARMACOGNOSY AND ENVIRONMENTAL MEDICINE, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MASTER OF PHARMACY DEGREE. PROFESSOR S. I. INYA AGHA (SUPERVISOR) DEPARTMENT OF PHARMACOGNOSY AND ENVIRONMENTAL MEDICINE, UNIVERSITY OF NIGERIA, NSUKKA. JANUARY 2014 ii CERTIFICATION Okwubie, Lambert, a postgraduate student in the department of pharmacognosy and environmental medicine; with Reg. No. PG/M.PHARM/10/52771 has satisfactorily completed the requirement for the course work and research for the Degree of Master of Pharmacy (M.Pharm) in Pharmacognosy and Environmental Medicines. 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 other University ___________________________ __________________________ Prof S.I. Inya Agha Dr. Mrs. U.E. Odoh Supervisor Head of Department ____________________________ ___________________________ Date Date iii DEDICATION This work is dedicated to GOD Almighty, my beloved wife, and sons. iv ACKNOWLEDGEMENT First and foremost, I thank the LORD GOD Almighty who gave me the ability to run this program. To HIM be all the glory in JESUS name Amen. I want to say a big thank you to my supervisor, Professor S.I. Inya Agha who gave her professional support and motherly advice, and Dr, Mrs. E.U Odoh for her wise contributions. I also appreciate the support of my wife and children, the management and staff of stratech laboratory, Enugu for their immense contribution to this work. To the following, Pharm. Elechi , Mrs D. Okoroafor of University of Port Harcourt , and Mr. and Mrs. Gilbert Nwaigwe, I say thanks to you all. v TABLE OF CONTENT Title page………………………………………………………………………………………..i Approval Page………………………………………………………………………………….ii Dedication………………………………………………………………………………………iii Acknowledgement……………………………………………………………………………..iv Table of contents………………………………………………………………………………..v List of tables……..……………………………………………………………………………..viii List of Figures……………………………………………………………………………….…..ix Abstract………………………………………………………………………………………..…x CHAPTER ONE 1 1.1 INTRODUCTION........................................................................................................1 1.2 Aim and Objective…………………………………………………………………………..4 1.3 Significance of the study…………………………………………………………………..4 CHAPTER TWO 5 2.1 literature review of crude oil pollution of environment in Nigeria……………………..5 2.2 What are medicinal plants?.................................................................................... 11 2.3 Literature review of medicinal plants use for this investigation………………………12 2.3 Effects of crude oil pollution on the environment………………………………………17 2.3 Crude oil spills; impacts on plants……………………………………………………….24 2.4 Oxidative stress; Phytopathology, Phytopathogenesis………………………………..26 vi 2.5 Environmental stress features…………………………………………………………...27 2.6 Biochemical feature in Oxidative stress formation…………………………………….28 2.4 The medicinal plants under study……………………………………………………….30 2.5 Antioxidant properties of the medicinal plants under study…………………………..48 CHAPTER THREE EXPERIMENTAL 49 3.1 materials and apparatus …………………………………………………………………49 3.2 Collection of test and control samples of medicinal plants……………………………49 3.3 Quantitative determination of ascorbic acid contents of the samples……………….50 3.4 Quantitative determination of the total phenolic compounds in the samples……….50 3.5 Quantitative determination of lipid peroxide content of the samples………………...51 3.6 Determination of the Ash values of the leaves of the samples……………………….51 3.7 Epidermal cells and stomata of the leaves of the samples…………………………...53 3.8 Phytochemical screening of the medicinal plants……………………………………..53 3.9 Statistical analysis…………………………………………………………………………56 CHAPTER FOUR: RESULTS 57 4.1 Quantitative determination of the ascorbic acid contents of the samples…….…….57 4.2 Quantitative determination of the Total phenolic compound of the samples……….58 4.3 Quantitative determination of lipid peroxide content of the samples………………...59 4.4 The Ash values of the leaves of the samples…………..………………………………60 4.5 Phytochemical screening of the medicinal plants……………..……………………….64 Vii 4.6 The epidermal examination………………………………………………………………65 CHAPTER FIVE: DISCUSSION AND CONCLUTION………………………………….70 REFERENCES…………………………………………………………………………………77 APPENDIX……………………………………………………………………………………..86 viii LIST OF TABLES 1: Result for the ascorbic acid contents of the samples.................................................57 2: Result for the total phenolic compounds in the samples...........................................58 3: Result for lipid peroxide content of the samples………………………………………59 4: Result for Total ash of the samples…………………………………………………… 60 5: Result for the water soluble ash…………………………………………………………61 6: Result for the acid insoluble ash………………………………………………………...62 7: Result for the sulphated ash……………………………………………………………..63 8: Result for Quantitative screening of the phytochemical contents of the medicinal plants………………………………………………………………………64 ix LIST OF FIGURES 1: A section of Imo River polluted by oil…………………………………………………….10 2: Matured Chromolaena odorata with flowers…………………………………………….30 3: A section of Psidium guajava (guava) tree………………………………………………36 4: Aspilia africana plant with flowers…………………………………………..…………….40 5: Picture of Manihot esculenta (cassava) plant……………………………………………43 6: Gongronema latifolium (Utazi) plant………………………………………………………46 7a: photomicrograph of upper epidermis of test sample of Aspilia africana .................. 65 7b: photomicrograph of upper epidermis of control sample of Aspilia africana..............65 8a: photomicrograph of lower Epidermis of test sample of Aspilia africana…………….65 8b: photomicrograph of lower epidermis of control sample of Aspilia africana…………65 9a: photomicrograph of upper epidermis of test sample of Chromolaena odorata……..66 9b: photomicrograph of upper epidermis of control sample of Chromolaena odorata…66 10: photomicrograph of lower epidermis of test sample of Chromolaena odorata……..66 11a photomicrograph of lower epidermis of test sample of Psidium guajava…………..67 11b photomicrograph of lower epidermis of control sample of Psidium guajava……….67 12a: Photomicrograph of upper epidermis of test sample of sample of Psidium guajava…………………………………………………………………………67 12b: Photomicrograph of upper upper epidermis of control Sample of Psidium guajava………………………………………………………………………….67 x 13a: Photomicrograph of test sample of Manihot esculenta lower epidermis…………..68 13b Photomicrograph of control sample of Manihot esculenta lower epidermis………..68 14a: Photomicrograph of test sample of Manihot esculenta upper epidermis…………..68 14b: Photomicrograph of control sample of Manihot esculenta upper epidermis………68 15a: Photomicrograph of test sample of Gongronema latifolium lower epidermis……..69 15b: Photomicrograph of control sample of Gongronema latifolium lower epidermis….69 16a: Photomicrograph of test sample of Gongronema latifolium upper epidermis……..69 16b: Photomicrograph of control sample of Gongronema latifolium upper epidermis…69 xi ABSTRACT In this work, evaluation of oxidative stress indicators on five medicinal plants collected from crude oil polluted environment was carried out. The medicinal plants evaluated are Psidium guajava, (L) (Myrtaceae ), Chromolaena odorata , (L) king and H.E Robins ( Asteraceae ), Aspilia africana , (Pers.) C.D.Adams, ( Asteraceae ), Manihot esculenta . Crantz (Euphorbiaceae ), and Gongronema latifolium Benth (Asclepiadaceae ) Oil pollution affect both the physiological, biochemical and physical states of the plants in their natural environments, invariably some of these changes may be due to oxidative stress on the plants brought about by the oil pollution of their natural environment The oxidative stresses in these five medicinal plants were evaluated using some oxidative stress indicators like ash values, effects on the epidermal cells and the stomata, the lipid peroxide, the total phenolic compounds contents and the ascorbic acid contents. Quantitative phytochemical screening was carried out, to examine the effect of the stress on the production of secondary metabolites by these medicinal plants. From the results it was observed that there were indication of oxidative stress with respect to the ascorbic acid contents and ash values, but with respect to the effect on the epidermal cells and stomata lipid peroxide and total phenolic compounds, some of the test samples indicate oxidative stress while others did not. On the overall Psidium guajava gave indication of oxidative stress
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