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Anti-Inflammatory and Analgesic Activities of Ethanol Extract of Stem-Bark of Hymenodictyon Pachyantha (Udelose) in Experimental Animals

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Anti-Inflammatory and Analgesic Activities of Ethanol Extract of Stem-Bark of Hymenodictyon Pachyantha (Udelose) in Experimental Animals ANTI-INFLAMMATORY AND ANALGESIC ACTIVITIES OF ETHANOL EXTRACT OF STEM-BARK OF HYMENODICTYON PACHYANTHA (UDELOSE) IN EXPERIMENTAL ANIMALS BY AHAM, EMMANUEL CHIGOZIE (PG/M.Sc/16/81353) DEPARTMENT OF BIOCHEMISTRY UNIVERSITY OF NIGERIA NSUKKA NOVEMBER, 2018 i TITLE ANTI-INFLAMMATORY AND ANALGESIC ACTIVITIES OF ETHANOL EXTRACT OF STEM-BARK OF HYMENODICTYON PACHYANTHA (UDELOSE) IN EXPERIMENTAL ANIMALS A RESEARCH PROJECT REPORT SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF MASTER OF SCIENCE (M.Sc) DEGREE IN PHARMACOLOGICAL BIOCHEMISTRY, UNIVERSITY OF NIGERIA, NSUKKA BY AHAM, EMMANUEL CHIGOZIE (PG/M.Sc/16/81353) DEPARTMENT OF BIOCHEMISTRY UNIVERSITY OF NIGERIA NSUKKA SUPERVISOR: PROF. O. F. C. NWODO NOVEMBER, 2018 i CERTIFICATION Aham, Emmanuel Chigozie, a postgraduate student of the Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, with Registration Number; PG/M.Sc/16/81353, has satisfactorily completed the requirements for the award of degree of Master of Science (M.Sc) in Biochemistry (Pharmacological). The work embodied in this dissertation is original and has not been submitted in part or full for any other diploma or degree of this or any other university. _________________ _________________ PROF. O. F. C. NWODO PROF. F. C. CHILAKA (Supervisor) (Head of Department) ___________________ External Examiner ii DEDICATION This research work is dedicated to Late Mr. Aham Joe, whose tenets I aim at upholding. May perpetual light continue to shine on you as you continue to rest in the bossom of the Almighty. iii ACKNOWLEDGEMENTS My foremost gratitude goes to the Almighty God for sustaining me throughout this stage and making it possible for this research work to be successfully completed. I would also like to express my heartfelt gratitude to my supervisor, Prof. O. F. C. Nwodo who despite his tight schedule supervised this work. It was a privilege having him as a supervisor. He went beyond being a supervisor, but also played the role of a father, counselor and advisor. His scholarly expertise and constructive criticism was useful in achieving this great feat. I am really grateful sir. A sincere appreciation to the present administration of the Department of Biochemistry, University of Nigeria, Nsukka under the headship of Prof. F. C. Chilaka, whose words of encouragement and moral support kept me on the task that was set before me. Indeed, your administrative competence and skills have constituted a great blessing to my academic plight. With a deep sense of appreciation, I hold in esteem all my lecturers and staff particularly Prof. L. U. S. Ezeanyika, Prof. O. U. Njoku, Prof. I. N. E. Onwurah, Prof. H. A. Onwubiko, Prof. B. C. Nwanguma, Prof. S. O. O. Eze, Dr. P. E. Joshua, Dr. C. S. Ubani, Dr. O. C. Enechi, Dr. V. E. O. Ozougwu, Dr. A. L. Ezugwu, Dr. (Mrs.) C. A. Anosike, Dr. (Mrs.) O.U. Njoku, Dr. (Mrs.) F. N. Nworah, and Dr. (Mrs.) C. Nkwocha; not forgetting the assistance from Mr. I. U. Okagu, and Dr. E. Anaduaka including the entire staff of the Department of Biochemistry, University of Nigeria, Nsukka, for the knowledge they have bestowed in me; thereby making me a well ground Biochemist. I wish to express my heartfelt gratitude to my dear mother, Mrs. Aham Assumpta. She played the motherly role in encouraging me. In a special way I appreciate the family of Prof. C.A. Igwe, Mr. Uchenna A. Ibekwe and Mr. Patrick Onyi’s family, for the financial and moral supports they accorded me; may God reward you bountifully. I would not also forget the likes of Nath Francis, Akubunwa Emmanuel, Egwim Frank, Anyanwu Justus and to my numerous friends for their tireless efforts in seeing to the completion of this work. To all my classmates, Mr. Osuagwu Evans and those who donated their cherished blood to be used in the course of this work. I am grateful. May God reward you all abundantly. iv ABSTRACT This study was aimed at investigating the anti-inflammatory and analgesic activities of ethanol extract of Hymenodictyon pachyantha stem-bark as well as the possible mechanisms of anti- inflammatory action of the plant extract. The plant material was extracted using 3.5 litres of absolute ethanol. The anti-inflammatory activity of the ethanol extract was evaluated by determining its effect on egg albumin-induced rat paw oedema, phospholipase A2 activity, calcium chloride-induced platelet aggregatory response and membrane stabilization activity. The effect of the extract on acetic acid-induced writhing responses was also investigated. The percentage yield of the ethanol extract was 3.32%. Phytochemical analyses of the extract revealed the presence of flavonoids (1359.268 ± 0.02 mg/100g), terpenoids (2154.695 ± 0.01 mg/100g), steroids (3.782 ± 0.05 mg/100g), saponins (0.405 ± 0.03 mg/100g), alkaloids (268.856 ± 0.12 mg/100g), tannins (1375.930 ± 0.08 mg/100g) and phenols (2900.169 ± 0.15 mg/100g). The acute toxicity test of the extract showed no toxicity up to 5000 mg/kg body weight. The stem-bark extract at 100, 200 and 400 mg/kg body weight significantly (p < 0.05) and dose- dependently inhibited egg albumin-induced rat paw oedema in both early and late stages of inflammation when compared with the untreated control, sustained over a period of 0.5 to 5 hrs. The standard anti-inflammatory drug (indomethacin, 10 mg/kg b. w.) followed a similar trend. The extract also significantly (p < 0.05) inhibited phospholipase A2 activity in a dose-related manner when compared to the control, with a range of 0.1 to 0.5 ml; inhibiting the enzyme activity by 78.92 to 95.59%. The extract also significantly (p < 0.05) and concentration- dependently inhibited platelet aggregatory response when compared to the control. The extract significantly (p < 0.05) inhibited hypotonicity-induced red blood cell membrane lysis in a concentration- dependent manner, similar to the standard drug indomethacin. Ethanol extract of Hymenodictyon pachyantha stem-bark (100, 200 and 400 mg/kg b. w.) significantly (p < 0.05) reduced the number of writhings induced by 0.6% acetic acid solution in a dose dependent manner counted over a period of 20 mins. The results, therefore suggest that the mechanisms of the anti-inflammatory effect may be due to the stabilization of lysosomal membrane, by inhibiting phospholipase A2 and aggregation of platelets. Findings of this investigation provide empirical evidence for the use of Hymenodictyon pachyantha stem-bark extract in folkloric treatment of inflammatory disorders. v TABLE OF CONTENTS Title Page i Certification ii Dedication iii Acknowledgements iv Abstract v Table of Contents vi List of Figures xii List of Tables xiii List of Abbreviations xiv CHAPTER ONE: INTRODUCTION 1.0 Introduction 1 1.1 Description and uses of Hymenodictyon pachyantha 2 1.1.1 Taxonomic classification of Hymenodictyon pachyantha 3 1.1.2 Common names of Hymenodictyon pachyantha 3 1.1.3 Geographical distribution 3 1.2 Previous studies on Hymenodictyon pachyantha 3 1.3 Overview of inflammation 4 1.3.1 Inflammatory mediators 6 1.3.1.1 Cell derived mediators 6 1.3.1.1.1Vasoactive amines 6 1.3.1.1.2 Prostaglandins 7 1.3.1.1.3 Thromboxane A2 and prostacyclin 7 1.3.1.1.4 Cytokines 8 vi 1.3.1.1.5 Leukotrienes 8 1.3.1.1.6 Chemokines 9 1.3.1.1.7 Nitric Oxide 9 1.3.1.1.8 Platelet-activating factor 10 1.3.1.2 Plasma-derived mediators 10 1.3.1.2.1 Complement system 11 1.3.1.2.2 Clotting or coagulation system 11 1.3.1.2.3 Fibrinolytic system 11 1.3.1.2.4 Kinin system 12 1.3.2 Cells of inflammation 13 1.3.2.1 Agranulocytes 13 1.3.2.1.1 Macrophages 13 1.3.2.1.2 Monocytes 14 1.3.2.1.3 Lymphocytes 14 1.3.2.1.3.1 T-cells and B-cells 15 1.3.2.1.3.2 Natural killer cells 15 1.3.2.2 Granulocytes 16 1.3.2.2.1 Mast cells 16 1.3.2.2.2 Neutrophils 17 1.3.2.2.3 Basophils 17 1.3.2.2.4 Eosinophils 17 1.3.2.3. Platelets 18 1.3.3 Classification of inflammation 18 1.3.3.1 Acute inflammation 18 1.3.3.1.1 Mechanism of acute inflammation 19 vii 1.3.3.1.2 Responses in acute inflammation 20 1.3.3.1.3 Vasodilatation 21 1.3.3.1.2.2 Increased vascular permeability 20 1.3.3.1.2.2 Neutrophil extravasation 22 1.3.4 Resolution of the acute inflammatory response 24 1.4 Chronic inflammation 26 1.4.1 Types of chronic inflammation 27 1.4.1.1 Non-specific proliferative 27 1.4.1.2 Granulomatous inflammation 27 1.4.2 Inflammatory response in chronic inflammation 28 1.5 Inflammatory disorders 28 1.6 Anti-Inflammatory agents 28 1.6.1 Corticosteroids 29 1.6.2 Non-steroidal anti-inflammatory drugs (NSAIDs) 30 1.7 Aim and objectives of the study 32 1.7.1 Aim of the study 32 1.7.2 Specific objectives of the study 32 CHAPTER TWO: MATERIALS AND METHODS 2.1 Materials 33 2.1.1 Plant materials 33 2.1.2 Animals 33 2.1.3 Bacterial organism 33 2.1.4 Blood samples 33 2.1.5 Drugs 33 2.1.6 Chemicals and reagents 34 viii 2.1.7 Equipment and instruments 34 2.2 Methods 34 2.2.1 Preparation of plant extract 34 2.2.2 Qualitative phytochemical analysis 35 2.2.2.1 Test for alkaloids 35 2.2.2.2 Test for flavonoids 35 2.2.2.3 Test for saponins 35 2.2.2.4 Test for tannins 35 2.2.2.5 Test for terpenoids and steroids 36 2.2.2.6 Test for phenols 36 2.2.3 Quantitative phytochemical analysis 36 2.2.3.1 Estimation of the concentration of flavonoids 37 2.2.3.2 Estimation of the concentration of terpenoids 37 2.2.3.3 Estimation of the concentration of steroids 37 2.2.3.4 Estimation of the concentration of saponins 37 2.2.3.5 Estimation of the concentration of alkaloids 37 2.2.3.6 Estimation of the concentration of tannins 38 2.2.3.7 Estimation of the concentration of phenols 38 2.2.4 Acute
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