i PHARMACOLOGICAL ASSESSMENT OF Lupinus arboreus Sims (FABACEAE) METHANOL EXTRACT AND THREE ACTIVE CONSTITUENTS FOR ANTINOCICEPTIVE AND ANTI-INFLAMMATORY EFFECTS BY OHADOMA, SYLVESTER CHIKA (PG/Ph.D/07/42486) A THESIS SUBMITTED TO THE DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA NSUKKA IN FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF DOCTOR OF PHILOSOPHY (Ph.D) DEGREE IN PHARMACOLOGY PROF. P. A. AKAH (Ph.D) (SUPERVISOR) DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA NSUKKA JUNE 2014 ii CERTIFICATION This is to certify that Ohadoma, Sylvester Chika, a postgraduate student in the Department of Pharmacology and Toxicology, University of Nigeria, Nsukka, with registration number PG/Ph.D/07/42486 has satisfactorily completed the requirements for the award of the Degree of Doctor of Philosophy (Ph.D) in Pharmacology. The findings embodied in this Thesis “Pharmacological assessment of Lupinus arboreus Sims (Fabaceae) Methanol extract and three active constituents for antinociceptive and anti-inflammatory effects” are original and have not been submitted in part or full for the award of any other diploma or degree of this or any other university. _______________ Supervisor _____________________ Head of Department ___________________ External Examiner iii DEDICATION To all those who it has pleased Almighty God to make my teachers iv ACKNOWLEDGEMENT There are so many people to thank, so many friends to acknowledge, so many colleagues to appreciate and major mentors to extol. As I begin to put on paper the feelings I have towards them I sincerely lack words to convey my actual heartfelt happiness towards my mentor and supervisor, Professor P. A. Akah, for his caring and excellent supervision throughout the course of this thesis, and also for meticulously reading the whole of the manuscript. Happily, I am extremely grateful and very proud to associate with him as a lovable source of inspiration. My thanks also go to Professor P. C. Unekwe of the Department of Pharmacology and Therapeutics, Nnamdi Azikiwe University, Nnewi campus, who groomed and supervised me up to master degree level. I remain grateful to him. Worthy of mention are Dr. T. C. Okoye, Head, Department of Pharmacology and Toxiciology, University of Nigeria, Nsukka, Dr. C. S. Nworu and Professor C. O. Okoli for their guidance throughout the progress of this work. Professor Ajalli of blessed memory, deserves my acknowledgment. It is painful that late Prof. Ajalli of Pharmaceutical Chemistry Department, University of Nigeria, Nsukka who provided me with valuable materials did not live to see this day. I am indebted to Dr. Mathias Agbo and Mr. Mbaorji, all of Industrial Chemistry Department, UNN, for their commitment towards this work. Likewise, Dr. Mitchel and his team at the International Centre for Ethnomedicine and Drug Development (InterCEDD), Nsukka. All staff in the Department of Chemistry, Usmanu Dan Fodiyo University deserves my THANKS for their role towards the completion of this work. To a whole host of behind-the-scene supporters I say thank you. They include Dr. L. U. Amazu, Chris Okolo, Pharm F. N. Osuala, Dr. Isaac Nnatuanya, Mr. J. C. Enye, and Prof. P. J. C. Nwosu; not forgetting Madonna University and all staff of Pharmacology Department including those in charge of animal house for the opportunity of using their facilities and expertise. Finally, I remain grateful to God Almighty, the maker of all things there is, for His guidance, direction, and protection as well as making me a member of Ohadoma’s family. For typing out the manuscript correctly, I am grateful to Chinemenma Goodness of Jopec Computers. v ABSTRACT The methanol extract and chemical constituents of Lupinus arboreus leaf were investigated for antinociceptive and anti-inflammatory activities. The study was by experimental design. the extract was partitioned to yield hexane, ethylacetate, and methanol fractions. Phytochemical tests were done on the extract and fractions. Acute toxicity test (LD50) was carried out on crude methanol leaf extract (CME). Extract hexane fraction (HEF), ethylacetate fraction (EAF) and methanol fraction (MEF) were subjected to bioactivity guided fractionation using mice tail immersion, hot plate, acetic acid- induced tests and formaldehyde- and, egg albumin-induced rat paw oedema, as activity guide for antinociceptive and anti-inflammatory studies respectively. The active constituents were isolated by bioactivity-guided silica gel column chromatography eluted with gradient mixtures. The isolated active compounds were characterized using a combination of phytochemical analysis, m.p. determination, UV, IR, NMR and GC/MS spectral analyses. The intraperitoneal (i.p) LD50 of the crude methanol extract was 84.85 mg/kg. Phytochemical analysis of the methanol extract indicated the presence of steroids, flavonoids, glycosides, terpenes and saponins. Tannin, resin, reducing sugar and protein were moderately present. The hexane fraction contained steroids and terpenes while ethylacetate fraction contained flavonoids and glycosides. Two active compounds AHF1 and AHF2 were obtained from the hexane fraction while AEF1 was obtained from the ethyl acetate fraction. The AHF1 contained steroids. while AHF2 contained terpenes; AEF1 contained flavonoids. The crude methanol extract (CME) (30 and 60 mg/kg,) i.p produced dose-related resistance against thermal pain and significant (p< 0.01) inhibition of pain. On acetic-induced writhing test CME exhibited a dose- related antinociceptive activity with 71.13 and 47.80 % at 60 and 30 mg/kg respectively. Fractions HEF, and EAF exhibited significant (p < 0.05) pain inhibition of 73 and 64 % respectively while MEF produced 24 percent pain inhibition. AHF1 and AHF2 fractionated from HEF significantly (p< 0.05) exhibited pain inhibition of 75 and 71 % respectively at 30 mg/kg. AEF1 (30 mg/kg) also significantly (p< 0.05) inhibited pain reflex by 71 %. In egg albumin-induced (acute) oedema in rats, CME (30 and 60 mg/kg) produced a dose-related oedema inhibition of 81.10 and 91.50 % respectively at the 4th hour. Similarly, the hexane fraction (HEF) and ethylacetate (EAF) at 60 mg/kg produced a significant (p< 0.05) oedema inhibition of 79 and 40 % respectively at 4th hour. The effect of methanol fraction (MEF) (60 mg/kg) was vi not significant (p> 0.05). The oedema inhibition recorded by HEF and EAF were higher than the inhibition by aspirin (100 mg/kg). The CME (30 and 60 mg/kg) significantly inhibited formaldehyde- induced arthritis, in a dose-related, manner over a period of 4 hours (p< 0.05) (68 and 69 % inhibition respectively). Both HEF and EAF at 60 mg/kg i.p, significantly (p< 0.05) inhibited the oedematous response to formaldehyde-induced arthritis, causing 85.7 and 64.2 % inhibition respectively. The inhibitory effects of the isolates AHF1, AHF2 and AEF1 on egg albumin- induced (acute) oedema in rats (78; 72, and 66 % respectively) were significant and better than that of aspirin (100 mg/kg) (46 %). The effect of AHF1, AHF2 and AEF1, (30 mg/kg i.p) on formaldehyde-induced (chronic) oedema in rats were 79 %, 72 % and 65 % respectively. The isolated active compounds were identified as stigmastene 3, 6-dione (AHF1), ursolic acid (AHF 2), tetrahydroxyflavone-3a- rhamnoside (AEF1), and ellagic acid (AEF 2). In this study, the extract and fractions of L. arboreus leaves exhibited antinociceptive effect in different models of pain; and anti-inflammatory effects against both acute and chronic models of inflammation. The isolated compounds AHF1, AHF2 and AEF 1 appear to be responsible for the antinociceptive and anti-inflammatory effects. The compound AEF2 identified as ellagic acid, known for its antimicrobial activity, was concomitantly isolated. These compounds were isolated and characterized for the first time from L. arboreus. vii TABLE OF CONTENT Title page …………………………………………………………………..……..i Certification ………………………………………………………………..…….ii Dedication …………………………………………………………………..…..iii Acknowledgement …………………………………………………………..…..iv Abstract……………………………………………………………………...……v Table of content…………………………………………………...…………….vii List of figures……………………………………………………………...……xii List of tables ……………………………………………………………...……xiii List of abbreviations …………………………………………………...…....….xv List of relevant publications from the thesis …………………………...………xvi CHAPTER ONE: INTRODUCTION ……………………………………………………………...1 1.1 Scientific background………………………..………………………1 1.2.1 Definition of pain ……………………………………………………2 1.2.2 Type of pain …………………………………………………………2 1.2.2.1 Acute pain …………………………………………………………...2 1.2.2.2 Chronic pain …………………………………………………………3 1.2.3 Location and severity of pain …………………………….………4 1.2.4 Demography of pain ……………………...………...……….……5 1.2.5 Physiological pain ……………………………………………..…7 1.2.5.1 Cutaneous pain ………………………………………………...…7 1.2.5.2 Somatic pain …………………………………………………...…7 1.2.5.3 Visceral pain ……………………………………………...………7 1.2.5.4 Phantom limb pain ……………………………………..…………8 1.2.5.5 Neuropathic pain …………………………………...…………….8 1.3. Common causes of pain ….. ……………………………….…..8 1.4.0 Pain receptors and their stimulation ……………………...…..…..8 1.4.1 Neurotransmitters …………………………………………….10 1.4.2 Excitatory neurotransmitters ……………………………………10 1.4.3 Inhibitory neurotransmitters …………………………….………10 1.4.4 Specific neurotransmitters ………………………………………10 viii 1.4.5 Transmission of pain signals in the central Nervous system. ...……11 1.5.0 Inflammation principles ……………………………………………12 1.5.1 Causes of inflammation