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EVALUATION OF THE ANTINOCICEPTIVE AND ANXIOLYTIC-LIKE ACTIVITIES OF LEAF EXTRACT AND FRACTION OF Phyllanthus muellerianus KUNTZE BY UMEZINWA, OGOCHUKWU JENNIFER (PG/MSc/13/66461) DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA NSUKKA DECEMBER, 2015 EVALUATION OF THE ANTINOCICEPTIVE AND ANXIOLYTIC-LIKE ACTIVITIES OF LEAF EXTRACT AND FRACTION OF Phyllanthus muellerianus KUNTZE BY UMEZINWA, OGOCHUKWU JENNIFER (PG/MSc/13/66461) A PROJECT REPORT SUBMITTED TO THE DEPARTMENT OF PHARMACOLOGY & TOXICOLOGY, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA, IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF MASTER OF SCIENCE (M.Sc) DEGREE IN PHARMACOLOGY & TOXICOLOGY SUPERVISOR: PROF CHARLES O. OKOLI DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA NSUKKA DECEMBER, 2015 i CERTIFICATION This is to certify that Umezinwa, Ogochukwu Jennifer, a post graduate student in the department of Pharmacology and Toxicology with the Reg. No. PG/M.Sc./13/66461 has satisfactorily completed the requirements for the award of Masters of degree in Pharmacology and Toxicology. 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. Charles O. Okoli Dr. A. C. Ezike (Supervisor) (Head of Department) ii DEDICATION This work Is Dedicatedfirst and foremost to God Almighty, Secondly to my One in a Million parents, Pastor and Mrs Innocent Umezinwa Nwaizu. iii ACKNOWLEDGEMENT The success of any project work is attributed to divine providence financially and humanly. All thanks to God Almighty for His divine provisions, guidance and sustenance throughout the period of this study.Warmest appreciation is expressed to all who stood as props to make this study a comely success. Firstly, my sincere gratitude goes to my amiable, able, caring and very rare to find supervisor, Prof. Charles Okoli, who was always available to offer immediate response to any issue pertaining to the research work and always made me know that there was no turning back from this programme and I must finish what I had already started. He was a father to me throughout this programme and will always be, as he was not only an academic adviser but also a mentor. I am also grateful to the Head of Department, Dr.A. C. Ezike for the role she played as a teacher and an adviser, and also to all my lecturers especially Prof P. A. Akah who was a father, Dr. T. C. Okoye who was very supportive and treated me like a daughter, Dr. C. S. Nworu, who was a motivator and others that may not be mentioned here. My appreciation also goes to Dr. Michel Tchimene, the pharmacognosist that helped and guided me with my extraction and fractionation process and for his constant motivation. I won’t also forget Mr Ozioko and Mr. Ikechukwu A. Agbo who also played roles in directing me in some aspects of this work. I would also love to appreciate Henry Ugwu, Job Isah, Ukaegbu Chimere Young, Arome Solomon, Emmanuel Nnamonu, Ezechukwu Chiemeka, Lanre Durojaiye, Cosmas Samuel, Geraldine Anokwuru and Primrose Ochuba who assisted me in some ways during the period of this study.who were friends and also sisters. Also to my wonderful unforgettable iv roommates Nneoma G. Ogbusua and Amara P. Ndukwe-Ani and Mrs Amaka Evelyn, you were just awesome and I love you. And to the entire house of Graduate Students’ Fellowship, I thank God for making me part of you in this programme. Unfailingly, to my wonderful parents Pastor Innocent Nwaizu and Mrs. Favour Nwaizu for their unflinching spiritual, academic and financial support. For their incessant prayers and encouragement that had always kept me abreast with the challenges that accompanied the pursuit of this programme and the challenges ahead of life. I assure you that God will keep you to reap the fruit of your labour. Also to my siblings and other members of the family who upheld me in prayers and all forms of encouragement. This will not be complete if I fail to appreciate my special friends like Amara Chike, David Nwosu, Daniel Okike, Charles Esukpa, Pastor Aaron Nwabueze for their support and prayers. And finally, to the pastor and all members of the Watchman Catholic Charismatic Campus Fellowship, UNN, for their spiritual backup and prayers towards actualizing this goal. v ABSTRACT The anxiolytic and antinociceptive properties of the leaf extract and fraction of Phyllanthus muellerianus Kuntze (Phyllanthaceae) were investigated.The extract (MME; 500.18 g; 10%w/w), obtained by cold maceration in 1:1 mixture of methylene chloride: methanol for 48 h, was fractionationed in a silica gel column which yielded the ethyl acetate fraction (EF; 199.2 g; 39.83% w/w). The extract and fraction were subjected to phytochemical analysis. Acute toxicity of MME was determined in mice using the oral route. Anxiolytic-like activity of MME and EF was investigated in mice using behavioural paradigms of anxiety on the Elevated Plus Maze (EPM) and the Light and Dark box tests. Antinociceptive activity was evaluated using abdominal writhing induced by acetic acid in rats and paw-licking induced by formalin in mice. The results showed that MME and EF gave positive reactions for alkaloids, carbohydrates, flavonoids, glycosides, reducing sugar, saponins, steroids, tannins, terpenoids and resins. Acute toxicity test on MME established an oral LD50>5000 mg/kg in mice. MME and EF caused a significant (P<0.05) increase in the duration of stay, time spent on the open arm and central platform of the EPM, the number of entries into the open arm and rearing behaviour. They also caused a significant (P<0.05) decrease in the duration of stay and number of entries in the closed arm, grooming activity, number of head dips, stretch attend posture and number of urine streaks and fecal boli. In the Dark and Light box, MME and EF significantly (P<0.05) increased the time spent in the light areaand decreased the time spent in the dark area, They also significantly (P<0.05) increased the number of line crossings and transition between the light and dark areas, the rate of peeking out of the dark box, and number of entries into the light chamber. They also caused significant (P<0.05) reduction in the number of fecal boli produced in the light and dark box. MME and EFsignificantly (P<0.05) reduced the number of abdominal writhes induced by acetic acid and the duration of paw-licking in the two phases of formalin-induced paw-licking test. A comparison of the magnitude of activity in all the tests showed the order of potency EF>MME. vi TABLE OF CONTENTS Title page................................................................................................................................ i Certification........................................................................................................................... ii Dedication.............................................................................................................................. iii Acknowledgement................................................................................................................. iv Abstract.................................................................................................................................. vi Table of contents................................................................................................................... vii List of figures......................................................................................................................... xi List of tables........................................................................................................................... xiii List of appendices.................................................................................................................. xiv CHAPTER ONE: INTRODUCTION 1.1 The central nervous system………………………………...…………......... ...... 1 1.2 The concept of anxiety……………………...……………………………........... 1 1.2.1 Definition………...…………………………………………………………....... 1 1.2.2 Classification of anxiety disorders…...………………………………………..... 2 1.2.3 Etiology and diagnosis of anxiety disorders………………………………...... 9 1.2.4 Pathophysiology of anxiety disorders................................................................... 13 1.2.5 Mechanisms of anxiety………………………………………………………..... 14 1.2.6 Management of anxiety disorder……………………………………………...... 17 1.2.6.1 Pharmacotherapy of anxiety disorders………………………………………...... 18 1.2.6.2 Anti-anxiety drugs (Anxiolytics)……………………………………………...... 19 1.2.6.3 Mechanisms of action of anti-anxiety drugs (Anxiolytics)…………………...... 19 1.2.6.4 Psychotherapy for anxiety and panic disorders……………………………........ 25 1.2.7 Advances and novel therapeutic approach in the management and treatment of 26 anxiety disorders…………………………………………………………........... 1.3 The concept of pain…………………………………………………………....... 28 1.3.1 Definition……………………………………………………………………...... 28 1.3.2 Etiology and diagnosis of pain………………………………………………...... 29 vii 1.3.3 Pathophysiology of pain……………………………………………………....... 30 1.3.4 Pain transmission and modulation…………………………………………….... 31 1.3.5 Neurophysiology of pain……………………………………………………...... 32 1.3.6 Mechanisms of pain…………………………………………………………..... 34 1.3.7 Classification of pain………………………………………………………. ...... 35 1.3.8 Management of pain…………………………………………………………..... 38 1.3.8.1 Pharmacological management of pain………………………………………...... 38 1.3.8.2 Non-
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