ANALGESIC, ANTI-INFLAMMATORY AND OTHER PHARMACOLOGICAL ACTIVITIES OF METHANOL LEAF EXTRACT OF OLAX SUBSCORPIOIDEA OLIV (OLACACEAE) IN LABORATORY ANIMALS


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TABLE OF CONTENTS

Title page
Abstract
Table of Contents
List of Abbreviations

CHAPTER ONE
1.0       INTRODUCTION
1.1       Statement of Research Problem
1.2       Justification of the Study
1.3       Aim and Objectives
1.3.1    Aim
1.3.2    Specific objectives
1.4       Research Hypothesis

CHAPTER TWO
2.0       LITERATURE REVIEW
2.1       Pain
2.1.1    Classification of pain
2.1.2    Pain pathways (mechanism)
2.1.3    Analgesics
2.1.4    Non-pharmacological management of pain
2.2       Inflammation
2.2.1    The inflammatory pathway
2.2.2    Anti-inflammatory drugs
2.3       Cytokines
2.3.1    Pathophysiological significance of cytokines
2.3.2    Cytokines, pain and inflammation
2.4       Traditional Medicine
2.4.1    Medicinal plants used for analgesic and anti-inflammatory purposes
2.4.2    Plants that have contributed to the development of modern analgesic and anti-inflammatory drugs
2.4.3    Olax subscorpioidea Oliv

CHAPTER THREE
3.0       MATERIALS AND METHODS
3.1       Animals
3.2       Materials and Equipment
3.3       Chemicals/Reagents
3.4       Collection and Identification of Plant Material
3.5       Extraction and Fractionation of Plant Material
3.6       Qualitative Phytochemical Analysis
3.6.1    Test for alkaloids
3.6.2    Test for flavonoids
3.6.3    Test for saponins
3.6.4    Test for cardiac glycosides
3.6.5    Test for tannins
3.6.6    Test for triterpenes
3.6.7    Test for steroids
3.6.8    Test for anthraquinones
3.6.9    Test for carbohydrates
3.7       Acute Toxicity Studies
3.8       Evaluation of Analgesic Activities in Mice
3.8.1   Acetic acid induced writhing test in mice
3.8.2    Formalin induced pain test in mice
3.8.3    Hot plate test in mice
3.9       Evaluation of Anti-inflammatory Activities
3.9.1    Acute anti-inflammatory study in rats
3.9.2    Sub-acute anti-inflammatory studies in rats
3.9.3    Investigating the involvement of inflammatory cytokines in the anti-inflammatory activities of Olax subscorpioidea
3.10     Investigation of Mechanisms of Analgesic Activities
3.11     Statistical Analysis

CHAPTER FOUR
4.0       RESULTS
4.1       Percentage Yield of the Leaf Extract and Fractions
4.2       Phytochemical Constituents
4.3       Median Lethal Dose
4.4       Analgesic Activities of Olax subscorpioidea
4.4.1    Effect of Olax subscorpioidea on acetic acid induced writhing in mice
4.4.2    Effect of Olax subscorpioidea on formalin induced pain test in mice
4.4.3    Effect of Olax subscorpioidea on hot plate test in mice
4.5       Anti-Inflammatory Activities of Olax subscorpioidea
4.5.1    Effect of Olax subscorpioidea on carrageenan-induced oedema in rats
4.5.2    Effect of residual aqueous and butanol fractions of Olax subscorpioidea on cotton-pellet induced granuloma formation in rats
4.5.3    Effect of residual aqueous and butanol fractions of Olax subscorpioidea onpro-inflammatory cytokines
4.5.4    Effect of residual aqueous and butanol fractions of Olax subscorpioidea onanti-inflammatory cytokines
4.6       Mechanisms of Analgesic Activities of Olax subscorpioidea

CHAPTER FIVE
5.0       DISCUSSION

CHAPTER SIX
6.0       SUMMARY, CONCLUSION AND RECOMMENDATIONS
6.1       Summary
6.2       Conclusion
6.3       Recommendations
REFERENCES
APPENDICES



ABSTRACT

Synthetic analgesic and anti-inflammatory drugs have major side effects such as constipation, nausea and vomiting, sedation and mental clouding, etc.,which have significantly limited their use.There is therefore, an intensification of search for newer analgesic and anti-inflammatory agents from the huge array of medicinal plant resources with better efficacy and fewer side effect profiles. Medicinal plants such as Olax subscorpioidea have been used traditionally for the management of pains, inflammatory diseases, yellow fever, cancer and rheumatism. The study aims at establishing the analgesic and anti-inflammatory potentials of methanol leaf extract of Olax subscorpioidea; and elucidating its possible mechanism of actions. The methanol extract and its fractions were subjected to phytochemical screening; oral and intraperitoneal median lethal dose (LD50) determination; evaluation of analgesic activities using acetic acid-induced writhing, formalin induced pain and hot plate tests in mice; and evaluation of anti-inflammatory activity using carrageenan-induced hind paw oedema model in rats. The doses (oral) used for these studies were 250, 500 and 1,000 mg/kg for the methanol extract, residual aqueous and butanol fractions; while doses of 150, 300 and 600 mg/kg were used for the hexane fraction. The residual aqueous and butanol fractions (1,000 mg/kg, orally) were subjected to sub-acute inflammation studies using cotton-pellet induced granuloma in rats; also the concentrations of inflammatory cytokines in the tissue exudates of rats following carrageenan induced paw oedema was investigated. The roles of opioidergic,

1, α2 and β)-adrenergic, serotonergic, ATP-sensitive potassium channels and nitric oxide-l-arginine pathways in the analgesic activities of the butanol fraction (1,000 mg/kg, oral) were further investigated. Results of the preliminary phytochemical screening of the methanol extract and the fractions indicated the presence of various phytochemicals such as carbohydrates, cardiac glycosides, tannins, flavonoids, alkaloids, saponins, steroid and triterpenes. The oral LD50 of the methanol extract, residual aqueous and butanol fractions was estimated to be greater than 5,000 mg/kg in both rats and mice; that of hexane fraction was estimated to be 2,200 and 3,800 mg/kg in mice and rats respectively. The intraperitoneal LD50 in mice was estimated to be 3,800 mg/kg for the methanol extract; 2,200 mg/kg for the residual aqueous fraction and 1,300 mg/kg for butanol and hexane fractions; it was estimated in rats to be 3,800 mg/kg for the methanol extract, residual aqueous and butanol fractions; and 2,200 mg/kg in the hexane fraction. The acetic acid induced writhes and the formalin induced pain licking effect were significantly (p<0.05, p<0.01 and p<0.001) reduced by the methanol extract and the fractions in a dose-dependent manner. The thermal pain latency was also significantly (p<0.05, p<0.01 and p<0.001) increased by the methanol extract and its fractions (except hexane fraction). The paw oedema was also significantly (p<0.05, p<0.01 and p<0.001) reduced by the methanol extract and the fractions across the time. The residual aqueous and butanol fractions (1,000 mg/kg) significantly (p<0.01 and p<0.001) reduced granuloma formation in the cotton pellet-induced granuloma studies in rats. The residual aqueous and butanol fractions significantly (p<0.05 and p<0.01) decreased the concentrations of vascular endothelial growth factor (VEGF); the butanol fraction significantly decreased ((p<0.05) concentrations of epidermal growth factor (EGF) and interleukin-1α (Il-1α). The residual aqueous and butanol fractions also significantly (p<0.05 and p<0.01) increased the concentration of Il-1β, IL-5 and interferon-γ (IFN-γ) while the residual aqueous fraction significantly(p<0.05) increased the concentration of IL-6 in the rats‘ paw tissue exudates. The pretreatment of mice with l-arginine and metergoline significantly (p<0.01 and p<0.05, respectively) decreased the analgesic effect of the butanol fraction; while pretreatment with naloxone, prazosin, yohimbine, propranolol and glibenclamide,each, had no significant effect on its analgesic activity. The results of the studies revealed that Olax subscorpioidea possesses marked analgesic and anti-inflammatory activities; the anti-inflammatory activity is mediated via the inhibition of pro-inflammatory cytokines such as IL-1α, IL-1β, VEGF and


EGF and/or via the stimulation of the synthesis of anti-inflammatory cytokines such as IL-5, IL-6 and IFN-γ. These results also suggest the possible involvement of serotonergic and nitric oxide pathways in the analgesic effect of Olax subscorpioidea.





CHAPTER ONE

1.0 INTRODUCTION

Pain is defined by the International Association for the Study of Pain (IASP) as an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage (Loeser and Treede, 2008). The British Pain Society (BPS) has defined pain as an emotion experienced in the brain; it is not like touch, taste, sight, smell or hearing. It can be perceived as a warning of potential damage, but can also be present when no actual harm is being done to the body (Moffat and Rae, 2010). It is the most common symptom of injuries and diseases (Haddad, 2007). It is a multidimensional sensory experience that is intrinsically unpleasant and associated with hurting and soreness. It may vary in intensity (mild, moderate or severe), quality (sharp, burning or dull), duration (transient, intermittent or persistent) and referral (superficial or deep, localized or diffuse). Although it is essentially a sensation, it has strong cognitive and emotional components; it is linked to or described in terms of suffering. It is also associated with avoidance motor reflexes and alterations in autonomic output. All of these traits are inextricably linked in the experience of pain (Woolf, 2004). Pain triggers various responses in the spinal cord and the brain, including reflexes, conscious perception, cognitive learning and memory processes, emotional reactions such as depression, and drug addiction (Gu et al., 2005).


Inflammation is considered as the primary physiologic defense mechanism that helps the body to protect itself against infection, burns, toxic chemicals, allergens or other noxious stimuli. An uncontrolled and persistent inflammation may act as an etiologic factor for......


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