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Inflammation (Coined from a Latin Word Inflammeve, to Set on Fire) Is Part of the Complex Biological Response of Vascular Tissue

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Inflammation (Coined from a Latin Word Inflammeve, to Set on Fire) Is Part of the Complex Biological Response of Vascular Tissue i OKONKWO A.C. (PG/M.PHARM/08/50349) AN EVALUATION OF THE ANTI-INFLAMMATORY PROPERTIES OF EXTRACTS AND FRACTIONS OF AERIAL PARTS OF Phyllanthus niruri L. A THESIS SUBMITTED TO THE DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY, FACULTY OF PHARCEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA PHARMACOLOGY AND TOXICOLOGY JUNE, 2011 Digitally Signed by Webmaster’s Name Webmaster DN : CN = Webmaster’s name O= University of Nigeria, Nsukka OU = Innovation Centre ii AN EVALUATION OF THE ANTI-INFLAMMATORY PROPERTIES OF EXTRACTS AND FRACTIONS OF AERIAL PARTS OF Phyllanthus niruri L. BY OKONKWO A.C. (PG/M.PHARM/08/50349) A PROJECT REPORT SUBMITTED TO THE DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY, FACULTY OF PHARCEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA, IN PATRTIAL FULFILMENT OF THE REQUIRMENT FOR THE AWARD OF MASTER OF PHARMACY (M.PHARM) DEGREE PROF. C. O. OKOLI (SUPERVISIOR) DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA JUNE, 2011 iii CERTIFICATION I, OKONKWO ANGELA CHIEMENAM, a postgraduate student in the Department of Pharmacology and Toxicology and with registration number PG/M.PHARM/08/50349 have satisfactorily completed the requirements for course and research work for the degree of Master of Pharmacy (M. Pharm.) in Pharmacology and Toxicology. The work embodied in this dissertation report is original and has not been submitted in part or in full for any other diploma or degree of this or any other university. ________________________ __________________________ Prof. Charles O. Okoli Prof. Charles O. Okoli (Supervisor) (Head of Department) iv DEDICATION This work is dedicated to God, for His abundant graces, and to my family. v ACKNOWLEDGEMENT I am grateful to God Almighty whose unfailing love and faithfulness saw me through to the completion of this work. My profound gratitude goes to my supervisor, Prof. Charles O. Okoli who patiently and tirelessly supervised this work. Thank you very much for your encouragement and unflinching support. Your sacrifices are immeasurable. I also wish to express my love and regards to my husband, Engr. Nwezza for his support and encouragement during the course of my work. You are simply wonderful. And I remain grateful to my parents, Mr. and Mrs. J. Okonkwo for their unalloyed support and contributions to the success of this work. I also wish to send warm regards to my siblings. The assistance and warm company I enjoyed from my lecturers are worthy of note. I wish to thank Prof Akah, Pharm Ndu, Mrs. Mbaoji and all the staff of the Department of Pharmacology and Toxicology for being part of this work. I also wish to thank specially Dr. (Mrs) A.C. Ezike who helped me with some of the materials for the work. My thanks go to Dr. S. Udegbunam of the Department of Veterinary Medicine for his professional assistance. I also cherish the friendship I enjoyed from my fellow postgraduates students and friends. And finally, I am greatly indebted to the various scholars and publishers whose works were consulted in the course of this study. vi TABLE OF CONTENTS Title page - - - - - - - - - - i Certification - - - - - - - - - - ii Dedication - - - - - - - - - - iii Acknowledgement - - - - - - - - - - iv Table of Contents - - - - - - - - - v List of Tables - - - - - - - - - ix List of Figures - - - - - - - - - x Abstract - - - - - - - - - - xi CHAPTER ONE: INTRODUCTION - - - - - - - 1 1.1 Inflammation - - - - - - - - 1 1.1.1 Acute Inflammation - - - - - - - 2 1.1.2 Chronic inflammation - - - - - - - 3 1.2 The inflammatory response - - - - - - 4 1.2.1 Macrophage and Neutrophil Responses during inflammation - 5 1.2.2 Resolution of Inflammation - - - - - - 7 1.3 Mediators of inflammation - - - - - - 8 1.3.1 Cell Derived Mediators - - - - - - 9 1.3.1.1 Histamine - - - - - - - - 9 1.3.1.2 Serotonin (5 -Hydroxytryptamine) - - - - - 10 1.3.1.3 Endotoxin - - - - - - - - 10 1.3.1.4 Nitric Oxide - - - - - - - - 10 1.3.1.5 Eicosanoids - - - - - - - - 11 1.3.1.6 Platelet Activating Factor (PAF) - - - - - - 12 1.3.1.7 Cytokines - - - - - - - - 12 1.3.1.7.1 Cytokines involved in acute inflammation - - - - 12 1.3.1.7.1.1 Interleukin-1 - - - - - - - - 12 vii 1.3.1.7.1.2 Tumor necrosis factor - - - - - - - 13 1.3.1.7.1.3 Interleukin-6 - - - - - - - - 13 1.3.1.7.1.4 Interleukin-11 - - - - - - - - 14 1.3.1.7.1.5 Interleukin-8/ chemokines - - - - - - 14 1.3.1.7.1.6 Colony Stimulating factor - - - - - - 14 1.3.1.7.2 Cytokines involved in chronic inflammation - - - - 15 1.3.1.7.2.1 Interleukin-2 - - - - - - - - 15 1.3.1.7.2.2 Interleukin-3 - - - - - - - - 15 1.3.1.7.2.3 Interleukin-4 - - - - - - - - 15 1.3.1.7.2.4 Interleukin-5 - - - - - - - - 16 1.3.1.7.2.5 Transforming growth factor-B - - - - - 16 1.3.1.7.2.6 Interferons - - - - - - - - 16 1.3.2 Plasma Derived Mediators - - - - - - 17 1.3.2.1 The Complement system - - - - - - 17 1.3.2.2 The kinin system - - - - - - - 18 1.3.2.3 The coagulation system - - - - - - 18 1.3.2.4 The fibrinolysis system - - - - - - 19 1.4 Disorders of inflammation - - - - - - 19 1.4.1 Rheumatoid arthritis - - - - - - - 19 1.4.2 Systemic Lupus Erythematosus - - - - - 20 1.4.3 Crohn’s disease (CD) - - - - - - - 21 1.4.4 Asthma - - - - - - - - 21 1.5 Anti-inflammatory agents - - - - - - 22 1.5.1 Non-steroidal anti-inflammatory drugs - - - - 22 1.5.2 Disease modifying anti-rheumatic drugs (DMARDs) - - - 24 1.5.3 Glucocorticoids drugs - - - - - - - 24 1.6 Anti-inflammatory medicinal plants - - - - - 25 viii 1.6.1 Botanical profile of Phyllanthus niruri - - - - 32 1.6.1.1 Taxonomy of plant - - - - - - - 32 1.6.1.2 Plant description - - - - - - - 33 1.6.1.3 Geographical distribution of plant - - - - - 33 1.6.1.4 Ethnomedicinal uses - - - - - - - 33 1.6.1.5 Literature Review of Phyllanthus niruri L. - - - - 34 1.6.1.6 Aim and scope of study - - - - - - 36 CHAPTER TWO: MATERIALS AND METHODS - - - - 37 2.1 Materials - - - - - - - - 37 2.1.1 Chemicals, solvents and reagents - - - - - 37 2.1.2 Animals - - - - - - - - 37 2.1.3 Equipment - - - - - - - - 37 2.2 Methods - - - - - - - - 38 2.2.1 Collection and preparation of plant materials - - - - 38 2.2.2 Extraction of plant material - - - - - - 38 2.2.3 Column chromatographic separation of the methanol extracts - 38 2.2.4 Phytochemical analysis of extracts and fractions - - - 39 2.2.5 Pharmacological tests - - - - - - - 43 2.2.5.1 Acute toxicity test - - - - - - - 43 2.2.5.2 Anti-inflammatory activity tests - - - - - 43 2.2.5.2.1 Studies on acute inflammation - - - - - 43 2.2.5.2.1.1 Topical edema induced by xylene in the mouse ear - - - 43 2.2.5.2.1.2 Carrageenaan induced pedal edema in rat - - - - 44 2.2.5.2.2 Studies on chronic inflammation - - - - - 45 2.2.5.2.2.1 Formaldehyde-induced arthritis in rats - - - - 45 2.2.5.2.2.2 Cotton-pellet granuloma test - - - - - - 46 2.3 Statistical analysis - - - - - - - 46 ix CHAPTER THREE: RESULTS - - - - - - - 47 3.1 Extraction and Fractionation - - - - - - 47 3.2 Phytochemical constituents of extracts and fractions - - - 47 3.3 Acute toxicity studies - - - - - - - 47 3.4 Effect of extract and fractions on topical (acute) inflammation - 47 3.5 Effect of extract and fractions on systemic (acute) inflammation - 51 3.6 Effect of extract and fractions on formaldehyde induced arthritis - 51 3.7 Effect of extract and fractions on cotton pellet granuloma - - 51 CHAPTER FOUR: DISCUSSION AND CONCLUSION - - - 58 References - - - - - - - - - - 61 x LIST OF TABLES Tables Page Some Medicinal Plants with Anti-inflammatory Ethnomedicinal uses 26 Phytochemical constituents of Extract and Fractions 48 Acute Toxicity (LD50) of Extract 49 Effect of Extract and Fractions on Acute Topical Edema of the Mouse Ear 50 Effect of Extract and Fractions on Acute Edema of the Rat Paw 52 Percent Inhibition of Edema caused by Carrageenan in Rats 53 Effect of Extract and Fractions on Formaldehyde Induced Arthritis in Rats 54 Percent Inhibition of Arthritis Induced by Formaldehyde in Rats 55 Global effect of Extract and Fractions on Formaldehyde Arthritis in Rats 56 Effect of Extract and Fractions on Cotton Pellet Granuloma 57 xi LIST OF FIGURES Figures Pages Figure 1 Extraction and Fractionation Scheme 39 xii ABSTRACT The effects of extract and fractions of aerial parts of Phyllanthus niruri Linn (Euphorbiacaea) on acute and chronic inflammation were studied. The methanol extract (210 g; 10.5 % w/w) obtained by cold maceration for 48 h was fractionated in a silica gel column successively eluted with dichloromethane and methanol (100%) to yield the dichloromethane (31 g; 25.8 % w/w) and methanol ( 31.5 g; % w/w) fractions. The effect of the extract and fractions on acute inflammation was studied using topical edema induced by xylene in the mouse ear and carrageenan-induced pedal edema of the rat paw. Effect on chronic inflammation was evaluated using cotton pellet-induced granuloma test and formaldehyde induced arthritis in rats. The acute toxicity of the methanol extract was further studied in mice using the oral route. The results showed that the extract and fractions significantly (P<0.05) inhibited the development of topical edema in the mouse ear and systemic edema of the rat paw. They also inhibited granuloma tissue growth and the global edematous response to formaldehyde. The dichloromethane fraction showed the greatest inhibitory effect in all the models. Acute toxicity test on the extract established an oral LD50 >5000 mg/kg in mice. Phytochemical analysis revealed the presence of alkaloids, carbohydrates, resins, saponins, tannins and terpenoids in the extract and fractions. The
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