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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
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DEDICATION
To all those who it has pleased Almighty God to make my teachers
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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.
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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-3α- 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.
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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 ……………………………………………13 1.5.2 Pathophysiology of inflammation…………………………….…….13 1.5.3 Inflammatory exudates ……………………………………………..14 1.5.4 Mediators of inflammation ………………………………………...15 1.5.5 Cells involved in inflammation …………………………….………17 1.5.6 Molecular Mechanisms of inflammatory response ………………...18 1.5.6.1 Adhesion molecules ………………………………………………..19 1.5.6.2 Leukocyte mobility and chemotaxis ……………………………….20 1.5.7 Disorders associated with pain and inflammation …………………20 1.5.8 Pain, inflammation and infection …………………………………..21 1.6.0 Management of pain and inflammatory disorders …………………23 1.7.0 Quest for natural products …………………………………….……29 1.7.1 Management of pain and inflammation using natural products ……………………………………………….…....30 1.7.2 Plants with promising analgesic and anti-inflammatory activities ……………………………………………………………30 1.7.3 Plant secondary metabolites with antinociceptive and anti-inflammatory effects………………………………………30 1.7.3.1 Flavonoids with antinociceptive and anti-inflammatory activities …………………………………………..………………..33 1.7.3.2 Classification and nomenclature of flavonoids …………………….36 1.7.3.3 Pharmacological activities of flavonoids …………………………..36 1.7.3.4 Mechanism of biological activities of flavonoids…………………..41 1.7.4 Terpenoids and steroids with antinociceptive and anti-inflammatory effects………………………………….………..41 1.7.4.1 Classification and nomenclature of steroids of plant origin …….…45 1.7.4.2 Review methods of isolation and purification of steroids …..……. 45 1.8 Review of botanical profile of Lupinus arboreus……………..………48 1.8.1 Taxonomy of Lupinus arboreus ……………………………………48 1.8.2 Description and distinctive features of the plant.……………….…..50 1.8.3 Geographical spread……………………………………………...…50 1.8.4 Values of Lupine……………………………………………………51 ix
1.9 Aim and scope of the work……………………………………..……..……53
CHAPTER TWO: MATERIALS AND METHODS 2.1 Plant materials …………………………………………………...…………54 2.2 Solvents and Reagents………………………………………………...……54 2.3 Equipment …………………………..……………………………………...54 2.4 Animals………… ………………………………..………………….…..…55 2.5 Methods ………………………………………………..……….…………..55 2.5.1 Extraction and concentration of plant materials ……………..……..……...55 2.5.2 Determination of extractive yield ………………………...……………..…55 2.6 Phytochemical analysis ……………………………...………..……………55 2.6.1 Test for carbohydrate ………………………………………………..……..55 2.6.2 Test for alkaloids……………………………………………………..……..56 2.6.3 Test for reducing sugar………………………………………….………….56 2.6.4 Test for glycosides………………………………………………….…...….56 2.6.5 Test for saponins……………………………………………………..…..…56 2.6.6 Test for tannins …………………………………………………..……..….57 2.6.7 Test for flavonoids ………………………………………………...….…....57 2.6.8 Test for resins ……………………………………………………….……...58 2.6.9 Test for proteins………………………………………………….…………58 2.6.10 Test for fats and oil…………………………………………………..……..59 2.6.11 Test for steroids and terpenoids………………………………….…………59 2.6.12 Test for acidic compounds…………………………………….………...….59 2.7 Bioassay-guided isolation of the active constituents of L. arboreus.……………………………………………...………..59 2.7.1 Column chromatographic separation of the methanol extract .…….…...... 59 2.7.2 Isolation and purification of the active constituents of hexane fraction (HEF)………………………………………….…..……..60 2.7.3 Isolation and purification of the active constituents of ethylacetate fraction (EAF) ………………………….………….…...…..60 2.8 Pharmacological test …………………………………….………….………61
2.8.1 Acute toxicity and lethality test (LD50)and preliminary screening ….……..61 2.8.2 Determination of antinociceptive activities ……………………………...... 61 2.8.2.1 Thermally-induced pain (Hot plate test) in mice……………………...….....61 x
2.8.2.2 Acetic acid-induced pain (writhing reflex test) in mice……………….……61 2.8.2.3 Pressure-induced pain (tail immersion test) in rats………………………....62 2.8.3 Inflammatory test………………………………………………………...…62 2.8.3.1 Acute inflammation test (egg albumin–induced inflammation)…………....62 2.8.3.2 Chronic inflammation test (formaldehyde-induced inflammation)…..…….63 2.8.4 The isolated active constituents ……………....……………………………65 2.8.4.1 Phytochemical analysis of fractions, AHF1, AHF2, AEF1 and AEF2….…65 2.8.4.2 Determination of melting point …………………………………………….65 2.8.4.3 IR spectral analysis ……………………………………………………...…65 2.8.4.4 UV spectral analysis…………………………………………………….….67 2.8.4.5 GC-MS analysis of AHF1, AHF2 and AEF1………………………..……..67 2.8.4.6 H-NMR (ID and 2D cosy) and C13-NMR analyses ………………….……67 2.9 Statistical Analysis ………..…………….………………….……..…….…..67
CHAPTER THREE: RESULTS 3.1 Extraction yield………………………………………………………….....68 3.2 Phytochemical analysis ……………………………………….….….…..…68 3.3 Acute toxicity and lethality ……………………………………...………...68 3.4 Antinociceptive and anti-inflammatory effects………….………...……….68 3.4.1 Antinociceptive effect…………………………………….…..……….…...68 3.4.2 Effect of the extract and fractions on egg albumin- induced (acute) oedema in rats ……………………….……………..…...………..…75 3.4.3 Effect of extracts and fractions on formaldehyde -induced (chronic) oedema in rats…….. ……………………………...……...……....75 3.5.1 Effect of AHF1, AHF2 and AEF1 on egg albumin-induced (acute) oedema in rats………………………………………..…………...... 75 3.5.2 Effect of AHF1, AHF2 and AEF1 on formaldehyde-induced (chronic) oedema in rats………………………………………...….…….....76 3.6 Isolation and solvent fractionation ……………………………..………...... 76 3.6.1 Percentage yield of fractions and their phytochemical constituents ……………………………………………...... 76 3.7 Isolation and characterization of the bioactive constituents ……………………………….……………….……..76
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3.8 Elucidation of the structures of the isolated active constituents…………………………………...………..…...84 3.8.1 AHF1 ………………………………………………………….…...….....84 3.8.2 AHF2 ……………………………………………………….……..…...... 85
3.8.3 AEF1 ……………………………………………………….……..…...... 86 CHAPTER FOUR: DISCUSSION AND CONCLUSION 4.1 Discussion …………………………………………….…………...... …87 4.2 Summary and conclusion ………………...…………………….………90 References …………………..………………………………….…...…..92 Appendices ……………………………………………….……....……116
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LIST OF FIGURES Figure 1: Basic structures of some flavonoids…………..……………....….…..44 Figure 2: Basic structures of some steroids ………………..………….....…..…47 Figure 3: The photograph of Lupinus arboreus leaves…………………...….…49 Figure 4: Chemical structure of AHF1 (stigmastene 3, 6-dione)…….…….…...84 Figure 5: Chemical structure of AHF2 (ursolic acid)………..…………..……...85 Figure 6: Chemical structure of AEF1 (tetrahydroxyflavone-3- α-rhamnoside) ...…………………..………86 Figure 7: Effect of crude methanol extract on acetic acid-induced pain………116 Figure 8: Effect of fractions and active constituents on Acetic acid-induced writhing ……………………………………….117 Figure 9: Effect of fractions on egg albumin-induced (acute) paw oedema…………………………………………………118 Figure 10: Effect of fractions on formaldehyde-induced (chronic) paw oedema……………………………………....………119 Figure 11: UV spectral scan for AHF………………………………….....….…120 Figure 12: UV Spectral scan for AHF2…………………………………………121 Figure 13: IR spectral scan for AEF1……………………………………….…..122 Figure 14: IR spectral scan for AEF2…………………………….……………..123
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LIST OF TABLES Table 1: Therapeutic uses and adverse effects of opioid analgesic………….…...... 25 Table 2: Therapeutic uses and adverse effects of NSAIDs …………………………………………….....28 Table 3: Plant with reported analgesic/anti inflammatory activities ……………………………………………………...31 Table 4: Classification of naturally occurring flavonoids based on their skeleton .………………..…………………...... 37 Table 5: Classification of naturally occurring steroids based on their structures…………………..………………..…...46 Table 6: Solvent systems employed in the column chromatographic separation of HEF …...……...………...64 Table 7: Solvent systems employed in the column Chromatographic separation of EAF………………...…...66 Table 8: Phytochemical constituents of methanol leaf extract of L. arboreus………………………………………………….70 Table 9: Phytochemical constituents of fractions and active Substances of L.arboreus…………...……………………71 Table 10: Effect of extract of L. arboreus on Hot plate-induced pain in mice……………………..……………………….72 Table 11: Effect of extract of L. arboreus on acetic acide-induced writhing……………………..…………….73 Table 12: Effect of extract of L. arboreus on pressure-induced (tail immersion) pain in rats………..…………………….74 Table 13: Effect of methanol extract of L. arboreus on egg albumin-induced (acute) inflammation in rats...... 77
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Table 14: Effect of methanol leaf extract of L. arboreus on formaldehyde-induced (chronic) inflammation in rats …………….……………..78 Table 15: Effect of solvent fraction AHF1 and AEF1 and ACF2 in mouse writhing model…………..………….…..79 Table 16: Effect of solvent fraction on egg albumin-induced (acute) oedema in rats…………………………..…….…..80 Table 17: Effect of solvent fraction on formaldehyde-induced (chronic) oedema in rats…………………..…………...... 81 Table 18: Effect of AHF1, AHF2 and AEF1 on egg albumin-induced (acute) paw oedema in rats……..……...82 Table 19: Effect of AHF1, AHF2 and AEF1 on formaldehyde-induced (chronic) oedema in rats…...….....83
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LIST OF ABBREVIATIONS AHF1: An active analgesic and anti-inflammatory Steroid isolated from L. arboreus leaves. AHF2: An active analgesic and anti-inflammatory terpene isolated from L. arboreus leaves. AEF1: An active analgesic and anti-inflammatory flavonol glycoside isolated from L. arboreus leaves AEF2: Tetrahydroxy aromatic compound isolated from L. arboreus HEF: Hexane fraction EAF: Ethyl acetate fraction MAF: Methyl alcohol (methanol) fraction APP: Acute phase protein APR: Acute phase reactant APT: Attached proton test ARDs: Acute respiratory distress syndrome CAM: Cell adhesion molecules COX-1: Cyclooxygenase 1 COX-2: Cyclooxygenase 2 DMARDs: Disease-modifying anti-rheumatic drugs IFN: Interferon Ig: Immunoglobulin IL: Interleukin LPS: Lipopolysaccharide LT: Leukotriene LX: Lipoxin NSAIDs: Non steroidal anti-inflammatory drugs PG: Prostaglandin PMN: Polymorphonuclear leukocytes RA: Rheumatoid arthritis ROI: Reactive oxygen intermediates TNF: Tumor necrosis factor TX: Thromboxane
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LIST OF RELEVANT PUBLICATIONS
1. Ohadoma, S.C; Nnatuanya, I.; Amazu, L.U; Okolo, C.E. (2014): Antimicrobial activity of the extract and fractions of Lupinus arboreus. J. Med. Plants Res. 8(8): 386-91.
2. Ohadoma, S. C., Akah, P. A (2014). Pharmacological assessment of the antinociceptive and anti-inflammatory effects of the extract and fractions of Lupinus arboreus. Asia Pacific Journal of Tropical Medicine (in press).
CHAPTER ONE xvii
INTRODUCTION 1.1 Scientific background
There exists tremendous need for scientists to explore the therapeutic values of medicinal plants (WHO, 1986). Apparently, this is in recognition that more than 80% of the world’s population uses or has at various time resorted to herbal remedy for treatment of health disorders (WHO, 1983). This is because the plant kingdom holds many species which contain substances of high medicinal value. In the African continent alone, over 5,000 species of plants are known to occur in the forest region and most of them have been used for several centuries in traditional medicine for prevention and treatment of disease (Iwu, 1993).
The history of healing arts in Africa can be traced back to 3200 BC during the reign of Menes, the first Pharaoh of Egypt who with his son was credited with many scientific preparations. The honour of the first African physician in a scientific sense actually belongs to the great Imhotep, who lived about 2980 BC during the reign of Pharaoh Zosar of the third dynasty. He was a scribe, a high priest and renowned healer who by 525 BC had become identified as the god of medicine (Ghalioungui, 1973). All these ancient African healers had elaborate mateFrial medica which consisted mainly of mixtures of herbal preparations from African medicinal plants. It is unfortunate that few African medicinal plants are recognized in modern pharmacopeias even when there are numerous African varieties of such ‘official’ drugs that are of higher medicinal value. A good example is the African Rauwolfia vomitoria which has a higher content of the antihypertensive alkaloid reserpine and the antiarrythmic drug ajmaline. (Iwu, 1993). Despite a large number of research publications on the constituents and biological activity of medicinal plants from Africa, the development of therapeutic agents from African medicinal plants has remained a neglected area.
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The general knowledge of African medicinal plants is very limited and their documentation is becoming increasingly urgent because of their rapid loss due to westernization, deforestation and anthropogenic activities. Documentations have proved that Africa has the highest rate of deforestation in the world, Cote d’ lvore and Nigeria having 6.5 and 5.0% respectively per year compared with the global rate 0.6% (McNeely,1990). It is necessary to note that many species of plants of potent medicinal value in Africa are yet to be discovered and a large number of them are being screened for their possible pharmacological activities. As earlier stated, over 5,000 plants are known to be used for medicinal purposes in Africa but only a few have been described or studied. This is one of the reasons for this current investigation on the antinociceptive and anti inflammatory activities of L. arboreus.
1.2.1 Definition of pain The International Association for the Study of Pain’s widely used definition states: “Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or desicribed in terms of such damage” (IASP, 2008). Perception provides information on the pain location, intensity as well as the nature of the pain (Lasch, 2002). The concept of pain in its widest sense encompasses both sensation and perception, leading to various conscious and unconscious responses including the emotional response. Pain may arise from an illness. It may accompany a psychological condition, such as depression, or may even result in the absence of a recognizable trigger. Of all the numerous etiology of pain, injury is a major cause (Beers and Berkow, 2002).
1.2.2 Types of pain Pain can broadly be classified into two namely: acute pain and chronic pain.
1.2.2.1 Acute pain This is the type of pain that usually disappears as the cause or stimulus is removed. Acute pain often manifests from tissue damage, such as broken xix bone or a skin burn. It can also be associated with muscle cramps or headaches (Mark and Robert, 2002). Nerve endings, or receptors are at the front end of pain sensation. Nerve cells (neurons) perform general purpose function of providing an interface between the brain and body. This general purpose remains constant but the capabilities vary widely implying that certain types of neurons are capable of transmitting pain signal to the brain. It is therefore, necessary to appreciate the neurons that support it in order to properly understand acute pain. Virtually every surface or organ of the body is connected or wired with nociceptors, having the central portion of these cells located in the spine sending thread like projections to every part of the body (Martin, 2003). Classification of nociceptors is according to the stimulus associated with transmission of a pain signal. Thermoreceptive nociceptors are stimulated by temperatures which are potential cause of tissue damage (Martin, 2003). Polymodal nociceptors can respond to both temperature and pressure and are the most sensitive. Polymodal nociceptors also respond to chemicals released by the cells in the area from which the pain emanates. It is the mechanoreceptive nociceptors that respond to pressure stimuli that may be cause by injury (Martin, 2003). Stimulation of nerve endings of the nociceptor releases a cascade of neurotransmitters in the spine. Substance P for example, is prominent in relaying pain message to neurons leading to the spinal cord and brain. Each neurotransmitter has a purpose. Neurotransmitters may also stimulate neurons leading back to the site of the injury a response that prompts cells in the injured area to release chemicals that in addition to triggering an immune response influence the intensity and duration of the pain.
1.2.2.2 Chronic pain This depicts pain that persists after an injury heals, and long-term pain from an unidentifiable cause. In the event of continued stimulation of nociceptors, changes occur within the nervous system. At the molecular level, changes are dramatic and may include alterations in genetic transcription of xx neurotransmitters and receptors (AAN, 2008). Changes may also happen in the absence of an identifiable cause which is one of the frustrating aspects of chronic pain – the stimulus may be unknown. As an example, the stimulus cannot be identified in as many as 85% of individuals suffering lower back pain (Wheeler et al., 2004). Chronic pain may be caused by the body’s response to acute pain (Wheeler et al., 2004). One in three people in the United States is estimated to experience chronic pain at some point in their lives with approximately 50 million of them either partially or completely disabled (ACPA, 2008). According to IASP (2008), allodynia simply refers to a feeling of pain in response to a normally harmless stimulus. Some patients who have suffered nerve damage as a result of viral infection experience unbearable pain from just the light weight of their clothing. Related to allodynia is hyperalgesia in the sense that the response to a painful stimulus is extreme. A mild pain stimulus, in this case, such as a pin prick, causes a maximum pain response. It is after a limb is amputated that phantom limb pain occurs. (IASP, 2008)
1.2.3 Location and severity of pain The intensity and quality of pain varies. There exists mild, moderate, or severe pain. With regard to quality, pain may vary from a dull ache to sharp piercing, burning, pulsating, tingling, or throbbing sensations. For instance, the pain from hitting a finger on a needle feels different from the pain of touching a hot iron, despite the fact that both injuries involve the same part of the body (IASP, 2008). The nerve cells in the dorsal horn transmit the pain message rapidly if the pain is severe. If the pain is relatively mild, the pain signals along a different set of nerve fibres, are transmitted at a slower rate. The location or site of the pain is very important in affecting its severity on a person’s emotional and cognitive response. This can be buttressed by the fact that pain related to the head or other vital organs is usually more disturbing than pain of equal intensity in a toe or finger (IASP, 2008). xxi
Another factor that affects pain perception is gender. Sex hormones in mammals have been shown in recent research to affect the level of tolerance to pain (AAPM, 2008). The pain threshold in experimental animals is elevated by the male sex hormone, testosterone, while estrogen, appears to elevate the animal perception of pain (AAPM, 2008). However, humans are influenced by their personal antecedence of history and cultures and by body chemistry (AAPM, 2008). In similar studies (AAPM, 2008) of adult volunteers, it was found that women tend to recover from pain more quickly than men, cope more effectively with it, and are less likely to allow pain to control their lives. One explanation of this difference came from research with a group of analgesics known as Kappa-opioids, which work better in women than in men (Altman, 1998). It is the thinking among some researchers that female sex hormones may increase the effectiveness of some analgesic medications, while male sex hormones may make them less effective (Altman, 1998). Adding to this, women appear to be less sensitive to pain when their estrogen and progesterone levels are high, as happens during pregnancy and certain phases of the menstrual cycle (Altman, 1998). As an exception, women with irritable bowel syndrome (IBS) often experience greater pain from the disorder during their menstrual periods (Yates, 2004).
Apart from gender, another factor that influences pain perception in humans is family upbringing (Harstall and Maria, 2003). While some parents comfort children who are hurting, others even punish or ignore them for crying or expressing pain. In some families, only female members are allowed to express pain but expect males to “keep a stiff upper lip”. People who suffer from chronic pain as adults may be helped by recalling their family’s spoken and unspoken messages about pain, and working to consciously change those messages (Harstall and Maria, 2003).
As an extension of nuclear family consideration, a person’s cultural and ethnic background can shape his perception of pain. Lasch (2002) in a periodical titled “culture and pain” indicated that people who have been xxii exposed to western explanation and treatment for pain may seek mainstream medical treatment more readily than otherwise. In another periodical, “Atypical facial pain”, Halsey, (2004) showed there are also differences among various ethnic groups within Western societies regarding ways of coping with pain. According to the report, one study of African American, Irish, Italian, Jewish, and Puerto Rican patients being treated for chronic facial pain found differences among the groups in the intensity of emotional reactions to the pain and the extent to which the pain was allowed to interfere with daily functioning. The report however, suggested that much more work on larger patient samples was needed to understand the many ways in which culture and society affect people’s perception of and response to pain.
1.2.4 Demography of pain Particularly in its milder cases, acute pain is a common experience in the general population. At least one occasion in a day, there is the tendency for one to remember when one felt a little muscle soreness, a brief tension headache, or had minor injury such as cuts when shaving (ACPA, 2008). Conversely, chronic pain is more widespread than is generally thought. It is estimated that 86 million people in the United States of America suffer from and are partially rendered disabled by chronic pain (ACPA, 2008). Specific disorders are implicated in the demographics of chronic pain. Singh et al., (2004) revealed that chronic pelvic pain (CPP) is more common in women than in men. Worldwide, it is said to affect about 14% of adult women. Chronic pelvic pain (CPP) is most common among women of productive age, particularly, those between 26 and 30 years, in the United States of America (ACPA, 2008). Among African Americans, CPP appears to be more common than among Asian Americans or Caucasians (ACPA, 2008). Lower back pain (LBP) is among the specific disorder upon which the demographics of chronic pain depend. LBP is perhaps, the most common chronic disability in persons younger than 45 years. It is estimated that 80% of people in the United States of America experience an episode of LBP at xxiii some point in life (Wheeler et al., 2004). This same report has indicated that about 3-4% of adults are disabled temporarily each year by LBP, with another 1% of the working age population disabled completely and permanently. LBP becomes a chronic syndrome in 5% of the population while 95% recover within 6 to 12 weeks. Halsey (2004) showed that atypical facial pain is a less-common chronic pain syndrome. It affects about two persons per 100,000 populations each year. Almost entirely, atypical facial pain is a disorder of adults. It is believed to affect both men and women equally, and to occur with equal frequency in ethnic groups and all races. This is another disorder upon which the demographics of chronic pain depend. Another specific disorder is headaches known to be very common in the adult population in North America; about 95% of women and 90% of men in the USA and Canada have had at least one headache in the past one year; mostly tension headaches. Migraine headaches are less common than tension headaches affecting about 11% and 15% of the population in the USA and Canada respectively (NINDS, 2004). According to this report, migraines occur most frequently in adults between 25 and 55 years old; the gender ratio is about 3 (female): 1 (male). Cluster headaches are the least common type of chronic headaches. It affects about 0.4% of adult males and 0.08% adult females in USA, with the gender ratio 7.5-5 (male): 1 (female).
1.2.5 Physiological pain Physiological pain exists in several forms, depending on the pain source and nociceptors or pain detecting neurons. The experience of physiological pain can be grouped thus (NINDS, 2004):
1.2.5.1 Cutaneous pain Refers to pain occasioned by injury to the skin or superficial tissues. The cutaneous tissue nociceptors terminate just below the skin because of high concentration of nerve endings causing a well-defined, localized and short xxiv term pain. Instances of injuries that produce cutaneous pain include first- degree (Minor) burn, paper cuts, lacerations and exterior wound (NINDS, 2004):
1.2.5.2 Somatic pain This originates from ligaments, bones, tendons, blood vessels, blood vessels, and even nerves themselves. Somatic pain receptors in these areas are few, so the pain is a dull and of longer duration than cutaneous pain. Sprains and broken bones are examples of somatic pain (NINDS, 2004).
1.2.5.3 Visceral pain This pain emanates from the body’s viscera, or organs. Visceral pain is extremely difficult to localize, and is often called “referred pain”, meaning that the sensation is unrelated to the injury site (NINDS, 2004). Perhaps, the best known example of referred pain is myocardial ischaemia, which is the loss of blood flow to a part of the heart muscle tissue (NINDS, 2004). This sensation can occur in the upper chest as a restricted feeling or as an ache in the left shoulder, arm or even hand; referred pain can be elucidated via the findings that pain receptors in the viscera as well excite spinal cord neurons that are excited by cutaneous tissue. Recognizing that the brain normally associates firing of these spinal cord neurons with stimulation of somatic tissues in skin or muscle, pain response emanating from the viscera are interpreted by the brain as originating from the skin. Ruch’s Hypothesis is coined for the theory that visceral and somatic pain receptors converge and form synapses on the same spinal cord pain-transmitting neurons (NINDS, 2004).
1.2.5.4 Phantom limb pain This refers to the sensation of pain from a limb that has been lost may be by amputation or in quadriplegia, where a person no longer feels physical pain signals (NINDS, 2004).
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1.2.5.5 Neuropathic pain This is also referred to as “neuralgia”. It can occur as a result of injury or disease to the nerve tissue. This can disrupt the ability of the sensory nerves to transmit correct information to the thalamus, making the brain to interpret painful stimuli even when there is no obvious or physiological cause for pain (NINDS, 2004).
1.3 Common causes of pain Common causes of pain include: trauma, temporal arteritis, infection, irritation, headache, cancer, cerebral aneurysm, sinusitis, meningitis, heart attack, pancreatitis, hernia, ulcer, hepatitis, appendicitis, pelvic inflammatory disease (PID) in women, kidney stone, muscle strain, rheumatoid arthritis (BRMC, 2008).
1.4 Pain receptors and their stimulation
It should be noted that the brain itself has no pain receptors hence not sensitive to pain. However, a headache comes from the membrane surrounding the brain and spinal cord known as the dura mater. Headache is thought to be produced by stimulation of these dural nociceptors. The physiology of pain shows that the body’s pain receptor can respond to mechanical, such as stretch gone too far, thermal (extreme heat or cold) or chemical stimuli. The chemical can be externally ingested, or may be body products released during trauma or inflammation. For instance, lactic acid causes muscle pain after heavy exercise (BRMC, 2008). The pain receptors in the skin and other tissues are all free nerve endings (NWE, 2009). They are widespread in the superficial layers of the skin as well as certain internal tissues such as the peritoneum arterial walls and joint surfaces. Pain is not simple, it can be elicited by multiple types of stimuli, and can be quite complex to treat. Pain receptors exist throughout the body. When the body feels pain, whether from touching a hot stove or low back pain, the pain signals nociceptor. In less than a second, the spinal cord and brain begin responding. The brain processes the pain stimulus, and sends an impulse xxvi down the spinal cord to the neurons in pain. In general, fast pain is elicited by the mechanical and thermal types of stimuli whereas the slow pain can be elicited by all three types (NWE, 2009). In pathological conditions, tissue injury is the immediate cause of pain and this results in the release of a varity of chemical agents which are assumed to act on the nerve terminals, either activating them directly or enhancing their sensitivity to other forms of stimulation. In most cases, stimulation of pain endings in the periphery is chemical in origin (NWE, 2009). Excessive mechanical or thermal stimuli can obviously cause acute pain, but the persistence of such pain after the stimuli have been removed and the pain resulting from inflammatory or ischaemic changes in the tissues generally reflects a chemical stimulation of the pain afferents (Rang et al., 2003). Pain receptors do not adapt to stimulus. In some conditions, excitation of pain fibres becomes greater as the pain stimulus continues, leading to a condition called hyperalgesia. Two main types of nociceptors, Aδ and C fibres, mediate fast and slow pain respectively (NWE, 2009). Opioids on the hand function by stimulating mu, delta and kappa receptors. Other receptors have been postulated but probably do not exist such as sigma and epsilon. The status of Mµ 1, Mµ 2, delta 2 and so on that constitute opioid receptor subtypes is still controversial because a look at the genes for these receptors does not appear to be the presence of subtypes. This does not however, mean that post – translational modification may not have created distinct subclasses of, say, the Mu receptor (BRMC, 2008). The identification of a so called “Orphan” receptor called “Opiate- like receptor 1 “(ORL-1) is of interest. It has about 60% sequence homology with the other opiate receptors. An endogenous ligand that acts on ORL-1 has recently been identified and called nociceptin, otherwise known as orphanin F2 (BRMC, 2008).
1.4.1. Neurotransmitters xxvii
Some of the main groups of chemicals that excite the chemical type of pain will be highlighted though there exist a plethora of neurotransmitters that mediates transmission of the sensations of pain in both brain and spinal cord.
1.4.2 Excitatory neurotransmitters In this group glutamate and the tachykinins, (substance P, neurokinin A and neurokinin B) and other agents that act at the various neurokinin receptors; vasoactive intestinal polypeptide, somatostatin, bombesin and calcitonin gene-related peptide are among substances that transmit pain impulses from incoming nerves in the dorsal horn (Rang et al., 2003). Neurotransmitters involved in “descending pain regulation” have norepinephrine with alpha-2 stimulatory effects, and the effects of serotonin are very prominent. These chemical substances are especially important in stimulating the slow suffering type of pain that occurs after tissue injury (Rang et al., 2003)
1.4.3 Inhibitory neurotransmitters There exist several inhibitory neurotransmitters, but in the CNS, gamma amino butyric acid (GABA) is known to dominate. It is reputed as being responsible for over 40% of inhibition that takes place in CNS (Dworkin et al., 2003). Glycine is another example.
1.4.4 Specific neurotransmitters (a) Histamine, acetylcholine and serotonin (5-HT) are among the members of chemicals that excite the chemical type of pain. (Dworkin et al., 2003) (b) Prostaglandins and substance P enhance the sensitivity of pain nerve endings but do not directly excite them (Dworkin et al., 2003). (c) Substances such as lactic acid, adenosine triphosphate and potassium are potent mediators of inflammation (Dworkin et al., 2003). (d) Capsaicin- This is a substance in chillipepper that gives them their pungent effect. It is selectively known to excite nociceptor nerve terminals xxviii leading to intense pain if injected into the skin or applied to selective structure such as the cornea of the eye (BRMC, 2008). (e) Tachykinins- Neurokinin receptors do mediate pain in the spinal cord. Substance P binds to the NK-1 receptor while neurokinins A and B bind respectively to the Nk-2 and Nk-3 receptors (Dworkin et al., 2003). These receptors are G-protein coupled, and raise intracellular calcium levels, thereby triggering gene transcription (Dworkin et al., 2003). (f) GABA- Is widespread in the spinals cord and brain, together with glycine. Interneurones in Laminae 1, 11, and 111 are rich in GABA, and mediate gate control in the dorsal horn by synapsing on neurons that contain substance P (Rang et al., 2003). (g) Glutamate- The predominant of the glutamate receptors is NMDA amongst at least two others- the AMPA receptor and metabotropic receptor. The NMDA receptor mediates a host of spinal responses to serve painful stimulation (Dworkin et al., 2003). (h) Gene c-fos- This is the most recent and perhaps the most significant discovery ever in the field of pain. Seemingly crucial to the profound central nervous system changes that occur when an animal or man feels pain is the cellular analogue of a viral oncogene, a rather special gene and its cellular product, the protein called Fos (Dworkin et al., 2003). CNS c-fos expression correlates extremely well with painful stimulation. C-fos is a proto-oncogene meaning that it can promote vast intracellular changes including cellular marker for pain. At cellular level, fos generically, is one of the inducible transcription factors (ITFs) that controls mammalian gene expression (Dworkin et al., 2003).
1.4.5 Transmission of pain signals in the central nervous system
The perception of pain occurs when nociceptors in the skin, muscles, or internal organs detects pressure, inflammation, a toxic substance or another harmful agent upon stimulation and transmit to spinal cord. The pain message is filtered by specialized nerve cells that act as gate keepers (Dworkin et al., xxix
2003). Based on the cause and severity of pain, the nerve cells in the spinal cord may either activate motor nerves, which govern the ability to move away from the pain source or release chemicals that raise or reduce the strength of the original pain message on its way to the brain. The dorsal horn is the part of the spinal cord that receives and processes the pain message reaching the brain. It is relayed to an egg- shaped central structure called the thalamus, the part of the brain in which pain perception occurs. From the thalamus, the information is transmitted to three specialized area within the brain; - the somatosensory cortex, which interprets physical sensations; the limbic system, which forms a border around the brain stem and governs emotional responses to physical stimuli and the frontal cortex which handles thinking (Meier, 2003). The somatosensory cortex in the cerebrum is exactly the point where an individual becomes fully aware of the pain. But the activation of these three regions explains why human perception of pain is a complex combination of sensation, emotional arousal and conscious thought (Meier, 2003). There are two pathways for the transmission of pain in the central nervous system. These are the paleospinothalamic tract for slow pain, and fast pain which travels via Aδ fibres to terminate on Lamina 1 of the dorsal horn follows neospinothalamic tract (NWE, 2009). It then means that the two pathways correspond to the two types of pain:- a fast sharp pathway and a slow – chronic pain pathway. The fast sharp pain signals are elicited by either mechanical or thermal pain stimuli, they are transmitted in the peripheral nerve to the spinal cord by the small type Aδ–fibres at velocities between 6 and 30 m/second. Conversely, the slow chronic type of pain mostly elicited by chemical stimuli is transmitted by type C fibres at velocities between 0.5 and 2 m/second (Greenspan, 1997). A sudden painful stimulus often gives a “double” pain sensation due to this double system of pain innervations. The neospinothalamic tract transmits pain from the fast type Aδ–fibres with glutamate as the neurotransmitter secreted in the spinal cord at the Aδ-fibre nerve endings. The paleospinothalamic pathway transmit pain mainly from xxx the peripheral slow chronic type C pain fibres although it does transmit some signals from type Aδ – fibres as well as with substance p and other related peptides being the transmitter substances concerned with slow chronic pain (Dickson et al., 1997).
1.5 Inflammation principles Inflammation could be defined as the complex biological response of vascular tissue to harmful stimuli such as pathogens, damaged cells or irritants (Mitchell and Cotran, 2004). It is a protective attempt by the organism to remove injurious stimuli as well as initiate the healing process for the tissue. This is achieved by diluting, destroying or otherwise neutralizing harmful agents (e.g. microbes or toxins) with the subsequent activation of events that lead to eventual healing and reconstitution at site of injury (Mitchell and Cotran, 2004). In the absence of inflammation, wounds and infections would never heal and progressive destruction of tissue would compromise the survival of the organism. Inflammation may become aberrant and harmful like other vital processes (Ringler, 1997). The cardinal signs of inflammation are rubor et tumour cum calore et dolore which when translated means redness and swelling with heat and pain. Function laesa (loss of function) is the next cardinal sign of inflammation (Virchow, 1985). Redness arises due to great increased blood flow to the inflamed part while the swelling is as a result of increased flow of blood as well as additional presence of substances that have exuded from the blood vessel into the surrounding tissues (exudates). The heat arises from the increased flow of blood carrying warmth to the periphery from higher interior temperature of the body. The pain results from increased pressure upon nerve endings and irritating effects of the toxic products of injurious agent, loss of function is due to the pain initiated reflex inhibition of muscle movement, mechanical swelling and tissue destruction (Ihedioha, 2003).
1.5.1 Causes of inflammation xxxi
The main causes of inflammation include pathogenic organisms, chemical poisons, mechanical and thermal injuries and immune reactions (Ringler, 1997).
1.5.2 Pathophysiology of inflammation Inflammation occurs in three phases viz: acute, immune response and chronic inflammation. Acute inflammation arises within seconds of the tissue injury and last for some minute or hours. It is characterized by local vasodilatation and increased capillary permeability due to alterations in the vascular endothelium which leads to increased blood flow (hyperemia) that causes redness (erythema) and the entry of the into the tissue (edema) (Roberts and Morrow, 2001). As the inflammatory process continues, macrophages predominate, actively removing damaged cells or tissue. If the cause of injury is eliminated, acute inflammation may be followed by a period of tissue repair. Blood clots are removed by fibrinolysis and damaged tissue are regenerated or replaced with fibroblast, collagen or endothelial cells. However, inflammation may become chronic leading to further tissue destruction and fibrosis (Roberts and Morrow, 2001). The immune response is initiated when immunologically competent cells are activated in response to foreign organism or antigenic substances liberated during the acute or chronic inflammatory response (Furst and Munster, 2001)
Chronic inflammation occurs after some days and is characterized by the appearance of mononuclear cell infiltrate composed by macrophages, plasma cell and lymphocytes. In this phase, the host’s response to inciting stimulus usually productive or proliferative, is activated. Cells at the site proliferate and produce matrix substances that add structural (collagen) and nutritional support (new blood vessels to the lesion). Chronic inflammatory reactions are often observed in auto immune diseases many of which are induced by the host’s sensitivity to the inciting agent. In most cases, the cells in chronic xxxii inflammatory reactions are different from those in acute inflammatory reactions (Ringler, 1997). In addition to macrophages and lymphocytes and plasma cells, resident parenchyma cells proliferate: fibroblasts become more numerous and produce more collagen, the single largest component of connective tissue: endothelial tissue divides and make more blood vessels (Ringler, 1997).
1.5.3 Inflammatory exudates Inflammatory exudates produced during inflammatory reactions are commonly classified into the fluid and cellular exudates. a. Fluid exudates: The pressure in post capillary venules may overcome the osmotic pressure of plasma proteins in acute inflammation. Therefore fluid and low molecular substances have the tendency to penetrate into the surrounding area. The increased capillary permeability for plasma proteins is the key factor for the production of inflammatory exudate. In the interstitial area, high- molecular proteins may be split into smaller fragments that participate in the raising of osmotic pressure of interstitial fluid (Stvrtinova et al., 1995) b. Cellular exduates: These are formed during the second and the third phases of inflammation -acute and chronic cellular responses. During the acute response, neutrophils are prevalent, whereas mononuclear cells (macrophages and lymphocytes) overcome chronic response. Cell composition of exudates differs not only depending on the phase of inflammation but also on the type of inflamed tissue and factors triggering inflammatory process (Stvrtinova et al., 1995).
1.5.4 Mediators of inflammation Inflammatory mediators are soluble and diffusible molecules that act locally at the site of tissue damage and infection, and at distant sites. The xxxiii mediators of inflammation include histamine, bradykinin, complement components (cytokine-3-alpha-c3a.cytokine-3-beta-c36 and cytokine-5alpha- c5a). prostanoids (prostaglandin E2- PGE2 – prostaglandin D2PGD2 and prostanoids I2 PGl2).
Leukotriene (LTB4 LTC4 LTF4) platelet activating factor, neuropeptide (e.g. substance P. calcitonin gene- related peptide) nitric oxide, cytokines (e.g. interleukins, tumor necrosis factor B- chemokines) neutral protease hydro peroxide, hydroxyl; and superoxide (Lee, 1998). Specially, acute inflammation is mediated by histamine, serotonin, bradykinin, prostaglandins, leukotrienes while interleukins 1-3 and 3 granulocytes macrophage colony stimulating factor, tumour necrosis factor alpha platelet derived growth factor mediate chronic inflammation. (Furst and Munster, 2001). Histamine in man, and 5-HT in rodents are the most important autacoids that are stored in mast cell and basophil granules (Stvrtinova et al., 1995).
Different cells of the body have receptors for histamine which can be classified into three types namely: H1, H2, and H3 (Garrison, 1990). The H1 receptors mediate inflammation, acute vascular effects together with smooth muscle constriction in the bronchi and the stimulation of eosinophil chemotaxis. The H2 receptors on the other hand, mediate secretions; inhibition of eosinophil chemotaxis but causes the vasodilation. Finally, the H3 receptors are mainly involved in the control of histamine release by different producing cells (Magno et al., 1961; Stvrtinova et al., 1995). 5-HT does the function of increasing vascular permeability, dilating capillaries and producing contraction of nonvascular smooth muscle. 5-HT is mostly stored in the gastrointestinal tract and central nervous system (Stvrtinova et al., 1995). The COX pathway produces thromboxanes, prostacycline and prostaglandins. The lipoxygenase pathway produces in one branch leukotrienes and in the second branch lipoxins (Stvrtinova et al., 1995). Prostaglandins unlike histamine do not exist free in tissues. They are synthesized and released in response to an appropriate stimulus (Miller, 2006). PGE2 is known to increase sensitivity to pain; enhances vascular permeability, is pyrogenic, and stimulates xxxiv leukocyte cAMP with recognizable suppressive effect on the release of mediators by mast cells, phagocytes and lymphocytes (Miller, 2006).
Monocytes, macrophages, and platelets are known to produce thromboxane A2 (TXA2). It causes platelets aggregation and constricts blood vessels and respiratory airway. A potent vasodilator prostacyclin (PGI2) antagonizes these effects of TXA2 (Stvrtinova et al., 1995). The production of
Leukotriene LTB4, can be inhibited by colchicine- a potent anti-inflammatory agent employed for the treatment of gout (Stvrtinova et al., 1995). The slow reacting substances of anaphylaxis (SRS-A) which is a combination of LTC4, LTD4 and LTE4, are produced by a wide variety of cells, such as monocytes and macrophages. They are known to exert spasmogenic and smooth muscle contraction, mainly in the bronchus. They also mediate mucus secretion (Ogawa and Calhoun, 2006).
Lipoxins LXA4, induces rapid dilation of arteriole and can also oppose LTD4- induced vasoconstriction. LXA4 and LXB4 stimulate microcirculation changes. Hence, LXA4 may regulate the action of vasoconstrictor leukotrienes. It can block neutrophil chemotaxis induced by both LTB4 and N-formyl-oligopetides. Cytotoxicity of natural killer cells can be inhibited by
LXA4 and LXB4 (Serhan, 1989; Aliberti et al., 2002).
Other autacoids that play a critical and broad role in human health and diseases, especially those related to inflammation and resolution include resolvins and protectins (Serhan, 2002). These mediators were first identified in resolving inflammatory exudates and in tissues with abundant DHA. As novel mediators, they were generated from eicosapentanoic acid (EPA) and dicosahexanoic acid (DHA) that exert protent bioactions (Serhan et al., 2000). Bioactive members from DHA with conjugated triene structures which are immunoregulatory (Serhan et al., 2002) and neuroprotective (Marcheselli et al., 2003) are called protectins/neuroprotectins. Platelet-activating factors (PAFs) refer to a group of acetyl-alkyglycerol ether analogs of phosphatidylcholine produced by platelets. PAFs potentiate the stickiness of xxxv endothelial cells for leukocytes. PAFs cause platelet aggregation and are potent phagocyte chemoattractants and stimuli of lysosomal enzyme release and reactive oxygen product formation by eosinophils, neutrophils, and macrophages (Stvrtinova et al., 1995)
1.5.5 Cells involved in inflammation There exists numerous cells that are involved in inflammation and inflammatory responses in the body. Playing a central role in inflammation and immediate allergic reactions are mast cells and basophils. From them are potent inflammatory mediators released. Degranulation known as the extracellular release of the mediators can be induced by: (i) chemical substances, such as toxins, venoms, proteases (ii) Physical factors, such as high temperature, ionizing radiation, mechanical trauma (iii) immune mechanisms which may be IgE-dependent or IgE-independent, (iv) endogenous mediators, including cationic proteins derived from eosinophils and neutrophils (Abraham and Malaviya, 1997; Galli et al., 1999) The mean generation time for eosinophils in the bone marrow is approximately 2 to 6 days (Stvrtinova et al., 1995). Another name for which neutrophils are known is polymorphonuclear leukocytes (PMN). They account for 50 to 60 per cent of the total circulating leukocytes and constitute the “first line of defence” against infectious agents or “nonself” substances that penetrate the body’s physical barriers (Stvrtinova et al., 1995). It is worthy to note that the first cells to be recruited to sites of injury or infection once an inflammatory response is initiated are the neutrophils. Their targets include bacteria, viruses, fungi, protozoa, virally infected cells and tumour cells. Despite neutrophils being essential to host defence, they have been implicated in the pathology of many chronic inflammatory conditions as well as ischemia-reperfussion injury (Wagner and Roth, 2002). Also, neutrophils are involved in the production of free radicals in the tissue (Weissmann et al., 1980; Perez and Weissmann, 1981).
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The original classification of macrophages and menocytes were as cells of the reticulo-endothelial system (RES) (Aschoff, 1924). Later, Van Furth et al., (1972) proposed the emononuclear phagocyte system (MPS), placing monocytes and macrophages as the basic cell types of this system. The development of the cells takes place in the bone marrow.
1.5.6 Molecular mechanisms of inflammatory response Within the microcirculation, the accumulation of leukocytes in inflamed tissue results from adhesive interactions between leukocytes and endothelial cells. Postcapillary venule is one region of the microvasculature where these adhesive interactions and the excessive filtration of fluid and protein that accompanies an inflammatory response are largely confined. It is not a simple process but quite complicated and broadly involve four distinct components namely; Circulation adhesion, diapedesis, and migration. Initially, leukocytes must surmount haemodynamic forces in order to adhere to the endothelial cell surface lining the typical vessel wall (Stvrtinova et al., 1995). Thereafter, there is the necessity of crawling their way along the endothelial cell surface, migrate through junctions between endothelial cells, and penetrate the basement membrane before gaining entry into, and migrating through the tissue spaces. A variety of factors such as expression of adhesion molecules on leukocytes and/or endothelial cells, signaling by cytokines and chemotactic factors, products of leukocyte (Superoxide) and endothelial cell (nitric oxide) activation, and the physical forces generated by the movement of blood along the vessel wall, determine the nature and magnitude of the leukocyte- endothelial cell adhesive interactions that take place within postcapillary venules (Stvrtinova et al., 1995).
1.5.6.1 Adhesion molecules There are less receptors and their ligands during an inflammatory response hence adhesion molecules serve to enhance pairing betweent them, xxxvii and of course, transmit signals that direct specific effector functions. Cell adhesion molecules (CAMs) have at least four super families that participate in these events of the selectins, the intergrins, certain members of the immunoglobulin (Ig) super family and cadherins (Stvrtinova et al., 1995).
The three family members of the selectin- Endothelial (E)- selectin, leukocyte (L)- selectin, and platelet (P)- selectin are named according to the cells with which they were originally discovered. The selectins are a family of divalent cation- dependent glycoproteins. They are carbohydrate-binding proteins, binding fucosylated carbohydrates, especially, sialytated Lewisx, and mucins (R α D systems, 2008).
According to the particular β submit they possess, which is shared by all members of the group, the integrins represent a large family of heterodimeric glycoprotein which can be subdivided. They are expressed mainly by leukocytes, giving rise to their alternative name- the leukocyte intergrins. In general the others are more widely distributed (Rojas and Ahmed, 1999). Cell adhesion molecules with calcium- independent transmembrane glycoproteins are the Ig superfamily. These include intercellular adhesion molecules (ICAMs), Vascular cell adhesion molecule (VCAM-1), platelet endothelial- cell adhesion molecule (PECAM-1) as well as Neural-cell adhesion molecule (NCAM). Notable is that each Ig superfamily CAM has an extracellular cysteine residue, a transmembrane domain, and an intracellular domain known to interact with the cytoskeleton (Rojas and Ahmed, 1999). They typically, bind integrins or other superfamily CAMs. Endothelial CAMs play an important role in immune response and inflammation (R & D Systems, 2008). The three most common cadherins-calcium-dependent adhesion molecules are neural (N) – cadherin, placental (P) cadherin, and epithelial (E) – cadherin (Gumbiner, 2000). The adhesive tendencies of the cadherins are dependent upon the ability of the intracellular domains to interact with xxxviii cytoplasmic proteins such as the catenins cadherins are intimately involved in embryonic development and tissue organization (R & D system, 2008)
1.5.6.2 Leukocyte mobility and chemotaxis The influx of circulating leukocytes from the blood into inflamed tissues involve the following processes:
i. rolling of leukocytes along the vasculature, mediated via transient interactions between selectin proteins and their carbohydrate ligands. ii. activation of both neutrophils and endothelial cells and a high affinity interaction between integrins and glycoproteins of immunoglobulin superfamily. iii. extravasation (crawling along the endothelium, diapedesis, and migration into tissue) in response to a chemoattractant gradient (Stvrtinova et al., 1995).
1.5.7 Disorders associated with pain and inflammation Strictly speaking, pain itself is not a disease condition but a symptom or sign or manifestation of a disorder or disease condition. For instance, in hot weather an individual without fluid replenishment may become dehydrated which may cause painful muscular cramp referred to as stroker’s cramp. As a remedy, replenishing the lost fluid and administering sodium chloride can eliminate the pain (Cassel, 1998). There are therefore, a variety of disease conditions associated with pain and inflammation. These ranges from disorders caused by infections like tetanus (severe muscular spasm normally occurs), pneumonia, chronic lung disease in premature infants, chronic asthma to autoimmune disorders such as systemic lupus erythematosus, lupoid hepatitis, haemolytic anaemia, thrombocytopenic purpura, agranulocytosis, myasthemia gravis, insulin-dependant diabetes mellitus, good pasture syndrome, bullux penghigoid disease, poly arthritis Nodosa diseases, agranulocytosis, acute lymphoid leucomia, chronic lymphocytic xxxix thyroiditis, and rheumatoid arthritis (Cassel, 1998) which is perhaps, the most popular and common, hence will be discussed in this thesis.
Rheumatoid arthritis (RA) is an autoimmune disease with recurrent systemic and chronic inflammation primarily involving the joints. This is due to inability or failure to distinguish “self” tissue and foreign “non-self antigen cells”. The pathological consequences of this reactivity constitute what is called autoimmune disease (The Arthritis foundation, 2008). Being a systemic disease, rheumatoid arthritis can affect organs and other body portions other than the joints. For instance, Sjogren’s syndrome is inflammation of the glands of the eyes and mouth leading to dryness of these areas. (The Arthritis foundation, 2008). Pleuritis which is reheumatoid inflammation of the tissue surrounding the heart can cause a chest pain with deep breathing and coughing. Pericarditis which is inflammation of the tissue surrounding the heart can cause a chest pain that typically changes in intensity when lying down or leaning forward. The lung tissue itself can also become inflamed, and sometimes nodules of inflammation referred to as rheumatoid nodules, develop within the lungs (The Arthritis foundation, 2008). The rheumatoid disease is implicated in anemia since it can reduce the number of red blood cells and white blood cells (The Arthritis foundation, 2008). As a consequence, decreased white blood cells can be associated with an enlarged spleen. This is called felty’s syndrome. Rheumatoid nodules- firm lumps under the skin, can occur around the elbows and fingers where there is frequent pressure (The Arthritis foundation, 2008). Vasculitis is a rare but serious complication, usually with long standing rheumatoid disease. It is a blood vessel inflammation. It can interrupt blood supply to tissues and lead to death (William, 2003). Symptom of vasculitis is most often initially visible as tiny black areas around the nail beds or as leg ulcers. Rheumatoid arthritis rarely, can even affect the joint that is responsible for the tightening of our vocal cords to alter the tone of the voice; when this xl cricoarytenoid joint is infamed, hoarseness of voice can occur (Ruddy et al. 2000; William, 2003). 1.5.8 Pain, inflammation and infection Infections are often involved in cell damage. In pain and chronic inflammatory diseases of unknown etiology, the belief that infectious agents are a cause is universal. Virulence of microorganisms and the induction of inflammation depend on their ability to replicate in human or animal and to devastate cellular structures. Mycoplasmas were proposed as far back as the 1930s, as a cause of rheumatoid arthritis in animals (Cassell and Cole, 1981). Endotoxin otherwise referred to as lipolysaccharide (LPS) is responsible for many pathophysiological symptoms observed during Gram-negative bacterial infections (Cassell and Cole, 1981). Microorganisms can produce and release different exotoxins, during growth and multiplication, which are potent injurious agents. Other microorganisms, after lysis or destruction, release from phospholipids and lipopolysaccharide envelops the toxins called endotoxins (Cassel and Cole, 1981).
Pathophysiological symptoms associated with endotoxin include pyrogenicity which is the ability to cause an increase in body temperature. Leukocytopenia/Leukocytosis (changes in the number of circulating leukocytes; complement activation, activation of macrophages, aggregation of platelets, increase of capillary permeability, and so on. Endotoxin shock is produced upon release of a higher dose of endotoxin. Endotoxin induces an immune response and all these biological events are mediated via the endogenous mediator known as tumour necrosis factor -α (TNF - α) (Vousden and Farrel, 1994).
There is an exception in this case, viruses are typical intracellular parasites and use cells for their own replication hence do not produce exotoxins or endotoxins (Vousden and Farrel, 1994). During this replication, damage of cell structure leading to the cell death is observed. Also, viruses may be xli responsible for the timorous transformation of cells (Vousden and Farrel, 1994; Morris et al., 1995).
Three different types of pathogen have theoretically been documented for pain and chronic inflammatory diseases of unknown origin, namely:
a. Those pathogens that are fastidious and previously recognized but because of their fastidiousness or lack of appreciation of their disease- producing potential are not included in the differential diagnosis. Mycoplasma is an example. Mycoplasmas may cause chronic lung disease in newborns and chronic asthma in adults (Lindsey et al., 1971; Allegra et al., 1994).
b. Those pathogens previously not recognized that therefore go undetected. Chlamydia pneumoniae is an example. It is the common cause of acute respiratory infection, and has been associated with atherosclerosis (Kuo et al., 1993; 1995). Infection from either group can result in misdiagnosis and lack of treatment. Depending on the biology of the organism and intrinsic and extrinsic factors of the host the organism can persist, resulting in chronic inflammation.
c. Those pathogens that elicit an autoimmune response resulting in persistent inflammation without the persistence of the inciting agent (Kuo et al., 1998).
1.6 Management of pain and inflammatory disorders Analgesics are agents that relieve pain. Through their actions on the nervous system, analgesics reduce or abolish sufferings from pain without producing unconsciousness or sleep (Cassel, 1998). Agents employed in the Management of pain and inflammatory disorders are classified broadly into:
(a) Narcotics or opioid analgesics xlii
Opioid analgesics act in the central nervous system and some cause drowsiness. Multiple areas of brain have been shown to have opiate receptors (Jensen, 1997). Receptor-binding studies and subsequent cloning confirmed the existence of three main receptor types mu, delta, kappa and their subtypes. A fourth member of the opioid peptid receptor family, nociceptin/orphanin
FQ (N/QFQ) receptors was cloned in 1994 (Jensen, 1997). Analgesic effects of opioids arise from the ability to inhibit directly the ascending transmission of nociceptive information from the spinal cord dorsal horn to activate pain control circuits that descend from the mid brain through the rostral ventromedial medulla, to the spinal cord dorsal horn. Opioid peptides and their receptors are found through these descending pain control circuits (Jensen, 1997). The opioid analgesics are classified into three categories 1. Pure agonists: morphine, levorphanol, pethidine, fentanyl, meperidine, methadone. 2. Partial agonists: pentazocine, butorphanol, nalbuphine, buprenorphine, cyclazocine. 3. Antagonists: naloxone, naltrexone, nalorphine, levallorphan. Pure agonists are known to combine with the receptors and provoke a response. Usually, they are represented thus: affinity + intrinsic activity = 1. Antangonists are represented as affinity + intrinsic activity = 0, meaning that they combine with the receptors but do not provoke a response. Agent acting as a weak agonist on its own and as an antagonist in the presence of another agonist acting on the same receptor is called partial agonist or dualist.
xliii
Table 1: Therapeutic uses and adverse effects of opioid analgesic:
Drugs Uses Adverse effects Pure agonist
Codeine Antitussive Behavioral restlessness,
Fentanyl Anaesthetic adjuvant Respiratory depression, Management of Chronic Nausea, vomiting, Malignant pain Constipation, postural Meperidone Analgesic Hypotension and urinary Retention. Methadone Relief of chronic pain
Morphine Treatment of opioid abstinence
syndrome. Relief of acute pulmonary oedema, analgesic. Partial agonist Pentazocine Analgesic Sedation, sweating, Buprenorphine Maintenance drug for Nausea, weakness, Opioid dependent subject Dizziness, anxiety, Hallucinations, xliv
Antagonists Naloxone Treatment of opioid Naltrexone Induced toxicity, Especially respiratory Depression, diagnosis of Physical dependence on Opioid, therapeutic Agents in treatment of Compulsive users of Opioids.
(b) Steroidal anti-inflammatory agents The steroidal anti-inflammatory drugs are the glucoccorticoids. Prednisolone, prednisone, dexamethasone, triananolone, betamethasone and beclomethasone are common examples of corticosteroids used clinically for their anti- inflammatory action (Bennett and Brown, 2003).
The mechanisms by which corticosteroids induce anti-inflammatory effects are multiple and not completely understood (Cronstein and Weissmann, 1995). These include stabilization of lysosomal and other cellular membranes (Weissmann and Thomas, 1963); allosteric effects in proteins, redirection of lymphocyte traphic (Samuels and Tomkins, 1970; Parillo and Fauci 1979); direct inhibition of various phospholipases, the induction proteins as lipocortin (Blackwell et al., 1979) inhibition of transcription of various cytokins by endotherlial cells (IL-1, IL-3 and TNFa) and inflammatory cells (Bochner et al., 1987, Dinarello and Mier, 1987) and metalloprotease and inflammatory enzymes (inducible nitric oxide synthetase) (Shapiro et al., 1991).
The anti-inflammatory steroids inhibit phospholipase A2 indirectly by inducing the synthesis of an inhibitory protein lipocortin (Flower, 1988). The xlv inhibition of this enzyme responsible for the release of arachidonic acid is the major mechanism of anti-inflammatory activity of corticoteroids ((Haynes, 1990; Vane and Botting, 1992). Inhibition of this enzymes result in inhibition of synthesis of prostaglandins, thromboxanes and leucotrienes and this may explain why steroids are more potent anti-inflammatory agents than NSAIDs (Vane and Botting, 1992). Corticosteroid medications can be given orally by inhalation or injected directly into tissues and joints. They are more potent than NSAIDs in reducing inflammation and in restoring joint mobility and function. Corticosteroids are useful for short periods during severe flares of disease activity or when the disease is not responding to NSAIDs. However, corticosteroids can have serious side effects, especially when given in high doses for long periods of time. These side effects include weight gain, facial puffiness, thinning of the skin and bone, easy bruising, cataracts, risk of infection, muscle wasting, and destruction of large joints, such as the hips (Haynes, 1990). Corticosteroids also carry some increased risk of contracting infections (Haynes, 1990). Abruptly discontinuing corticosteroids can lead to flare of the disease or other symptoms of corticosteroid withdrawal and is discouraged. Thinning of the bones due to osteoporosis may be prevented by calcium and vitamin D supplements (The Arthritis foundation, 2008).
(c) Non steroidal anti-inflammatory drugs (NSAIDs) The non steroidal anti inflammatory drugs are heterogenous group of compounds, often chemically unrelated which share the same therapeutic actions and side effects. They have anti-inflammatory, analgesic and anti- pyretic activities. The prototype drug is aspirin. They are also called aspirin- like drugs. The NSAIDs act by inhibition of the enzyme prostaglandin endoperoxide synthase or fatty acid cyclooxygenase which converts arachidonic acid into prostaglandins an important mediator of pain and inflammation (Vane, 1971). There are two forms of cyclooxygenase termed cyclcooxygenase-1(Cox-1) and cyclooxygenase-2 (Cox-2). Cox-1 is a constitutive isoform found in most xlvi normal cells and tissues, while Cox-2 is induced in setting of inflammation by cytokines and inflammatory mediators. Cox-1 is constitutively expressed in the stomach. This accounts for the markedly reduced occurrence of gastric toxicity with the use of selective inhibitors of Cox-2. (Vane, 1971).
All NSAIDs, including selective Cox-2 inhibitors are antipyretic, analgesic and inflammatory (Vane, 1971). One important exception is acetaminophen, which is an antipyretic and analgesic but is largely devoid of anti inflammatory activity. This can be explained by the fact that acetaminophen effectively inhibits cycloxgenase in the brain but not at sites on inflammation in peripheral tissues (Vane, 1971).
Table 2: Therapeutic Uses And Adverse Effects of NSAIDS Drugs Uses Side effects
Aspirin Antipyretic, analgesic, Gastric ulceration ,
Rheumatoid arthritis, Gastrointestinal bleeding, Coronary artery disease, Allergic reactions, Deep vein an thrombosis Hepatitis.
Sulfasalazine Rheumatoid arthritis, Anorexia, abdominal Acute gout, rheumatoid Pain, ulceration, hepatitis,
Indomethacin Arthritis, osteoarthritis Dizziness, tinnitus, rashes and Gl upset. Diclofenac Rheumatoid arthritis, G l bleeding, and xlvii
Osteoarthritis, post Ulceration, allergic Operative Reactions, fluid retention Pain, dymenorrhea And oedema.
1.7 Quest for natural products The use of medicinal plants in Nigeria and other countries of black Africa dates back to many centuries ago. Information in the form of folklore and practices showed that many plants and plant materials were used by the aborigines for curative purposes, long time before the conquest by the Europeans. The extracts and infusion from Cymbopogan citrates (Gramineae), Azadirachta indica (Meliaceae), Carica papaya (Caricaceae) and Magnifera indica (Anacardiaceae) were used by rural inhabitants in Nigeria as remedy against malaria attack (Ojinnaka, 1998). Most of these plants are still used for the same purpose. Medicinal plants were used by people of ancient cultures, without knowledge of their active ingredients. No reason could be offered why some plants were used to cure more than one xlviii ailment, or why two similar plants had different effects. The curative action of the medicinal plants became known by the introduction of European scientific methods. As such, many of the reported medicinal plants were investigated, leading to extraction and characterization of their active ingredients. Plants are found to be sources of many chemical compounds, most of which account for their various uses by man. The most important of these compounds are steroids, flavonoids, alkaloids, terpenoids, tannins, phenols and glycosides.
These compounds are products of secondary metabolism of plants and they are called natural products (Harborne, 1998; Finar, 1980). The recent upsurge of interest in alternative medicine in the United States is one reflection of dissatisfaction with a one-dimensional “Scientific” approach to pain (Lasch, 2002). More than 80% of the world’s population use or has at various times resorted to herbal remedy for treatment of health disorders (WHO, 1983). The need for scientists to explore the therapeutic values of medicinal plants becomes necessary (WHO, 1986). The quest for naturally occurring compounds of herbal or plant origin that could be of benefit as analgesic, stimulated our interest in Lupinus arboreus leaf.
1.7.1 Management of pain and inflammation using natural products There exist numerous scientific evidences buttressing successful management of pain and inflammation using natural products. In almost each family in the plant kingdom, there are representative analgesic and anti- inflammatory herbs (Okoli et al., 2003).
1.7.2 Plants with promising analgesic and anti-inflammatory activities. Lupinus arboreus is of great medicinal value with proven analgesic and anti-inflammatory activities (Ohadoma et al., 2010). Some other xlix medicinal plants with analgesic or/and anti-inflammatory activities are presented in Table 3. For some of these plants, there are proven and documented evidences for their use in the treatment of pain and inflammatory disorders in traditional medicine (Okoli et al., 2003). For some other plants, antinociceptive and antinflammatory activities are inferred from other identified pain and inflammatory activities related to the modulation of complex pain and inflammatory response (Okoli et al., 2003).
1.7.3 Plant secondary metabolites with antinociceptive and anti- inflammatory effects Alkaloids, terpenoids, tannins, phenols, steroids, glycosides and flavonoids are the products of secondary metabolism by plants and they are called natural products (Finar, 1980; Harborne, 1994). Extraction of the metabolites is either by maceration or by refluxing the plants material in an organic solvent, to obtain a solution known as extract. Characterization of extracts of medicinal plants is encouraged due to its numerous benefits to science and society. The information obtained from the process makes detailed pharmacological studies possible.
Table 3: Plant with reported analgesic/anti inflammatory activities. Plant Family Used/studied References part Agerantum conyzoides Asteraceae Leaf Abena et al., (1996) Sambucus ebulus Caprifoliaceae Rhizome Ahmadiani et al., (1998) Caralluna tuberculata Asclepiadacea Flower, leaf Ahmed, (1993) Diodia scandens Rubiaceae Aerial part Akah et al., (1993) l
Ficus platyphlla Moraceae Bark Amos et al., (2002) Turner ulmifolia Turnderaceae Leaf Antonio et al., (1998) Bryophyllum pinnatum Crassulaceae Leaf Arrigoni-martelli (1977) Icacina trichantha Icacinaceae Tuber Asuzu et al., (1999) Siderites spp Lamiaceae Flowers Bally, (1937) Euphorbia royleana Euphorbiaceae Latex Bani et al., (2000) Curcuma longa Zingiberaceae Rhizome Chandra and Gupta, (1972) Pothomorphe peltata Piperaceae Leaf Desmarchelier et al., (2000) Mitracarpus scaber Rubiaceae Leaf Ekpendu et al., (1994) Taxodium distichum Taxodiaceae Fruit El Tantawy et al., (1999) Moringa oleifera Moringaceae Stem Ezeamuzie et al., (1993) Anthurium cerrocampuse Araceae Stem Gado and Gigler, (1991) Centaurea cyamus Asteraceae Flowers Garbacki et al., (1999) Heterotheca inuloides Asteraceae Flowers Gene et al., (1998) Dalbergia sissoo Fabaceae Leaf Hajare, (2001) Terminalia ivorensis Compositae Leaf Iwu, (1993) Scutelleriae biocalensis Scutellaceae Leaf Kubo, (1984) Calotropis spp Asclepiadacea Flowers Kumar and Basin, (1994) Dicliptera chinensis Acanthaceae Aerial part Lin et al., (1993) Tithonia diversifolia Compositae Aerial part Lin et al., (1993) Calligonum comosum Polygonaceae Aerial part Liu et al., (2001) Agerantum conyzoides Asteraceae Leaf Magalhae et a.l, (1997) Agerantum conyzoides Asteraceae Leaf Marques et al., (1988) Alchornea cordifolia Euphorbiacea Leaf Marva- manga et a.l, (2008) Butea frondosa Papilionaceae Leaf Mengi and Despande (1999) Chasmanthera dependens Menispermaceae Leaf Morebise et al., (2001) Emilia sonchifolia Asteraceae Leaf Muko and Ohiri, (2000) Tanacetum parthenium Asteraceae Leaf Murphy et al., (1988) Syzygium cumini Myrtaceae Bark Muruganadan et al., (2001) Premna herbacea Verbanaceae Root Narayanan et al., (2000) Anthurium Araceae Leaf Nishida and Tomezawa, cerrocampanese (1980) Culcasia scandens Araceae Leaf Okoli and Akah, (2004) li
Aspilia africana Compositae Leaf Okoli et al., (2007) Bryophyllum pinnatum Crassulaceae Leaf Olajide et al., (1998) Entada abyssinica Mimosaceae Leaf Olajide and Olada, (2001) Aspilia africana Compositae Leaf Oyedapo et al., (1997) Holmskiodia sanguinea Verbanaceae Leaf Pal et al., (1996) Opuntia fiscusindica Cactaceae Stem Park et al., (2001) Croton lecheri Rubiaceae Leaf Perdue et al.,, (1979) Orbignya phalerata Aracaceae Fruit Pereira da silva and Parente, (2001) Rheo spathaceae Commelinacea Leaf Perez, (1996) Ambrosia artemisiaefolia Compositae Leaf Perez, (1996) Cissus trifoliate Vitaceae Root Perez, et al., (1993) Teucrium buxifolium Lamiaceae Arial part Puntero, (1997) Ficus elastica Moraceae Root Sackeyfio and Lugeleke, (1988) Psidium guianense Myrtaceae Leaf Santos et al., (1997) Anthurium Araceae Leaf Segura et al., (1998) cerrocampanese Cedrus deodora Pinaceae Wood Shinde et al., (1999) Anthurium Araceae Leaf Tarayre et al, (1989) cerrocampanese Aegle marmelos Rutaceae Root, bark Udupa et al., (1994) Moringa oleifera Liliaceae Fresh juice, gel Udupa et al., (1994)
1.7.3.1 Flavonoids with antinociceptive and anti-inflammatory activities Flavonoids are one of the largest classes of naturally-occurring polyphenolic compounds (Geissman and Crout, 1969). This group of plant pigments is largely responsible for the colors of many fruits and flowers, and over 4,000 flavonoid compounds have been characterized and classified according to chemical structure (Murray, 1996). The word “flavonoid” comes lii from the Latin flavus which means yellow; however some flavonoids are red, blue, purple or white (Mills and Bone, 2000). Chemically they are C6-C3-C6 compounds in which the two C6 groups are substituted benzene rings, and the C3 is an aliphatic chain which contains a pyran ring (Robbinson, 1991). Flavonoids occur as O- or C-glycone (Mills and Bone, 2000). Numerous medicinal plants contain therapeutic amounts of flavonoids, which are used to treat disorders of the peripheral circulation (Mills and Bone, 2000), to lower blood pressure (Blumenthal, 2003), to improve aquaresis (Robbers and Tyler, 2000), as anti-inflammatory (Mills and Bone, 2000), antispasmodic (Robbers and Tyler, 2000) and anti-allergic agents (Mills and Bone, 2000). The many pharmacological effects of flavonoids are linked to their ability to act as strong antioxidants and free radical scavengers, to chelate metals, and to interact with enzymes, adenosine receptors, and biomembrane (Mills and Bone, 2000). Some flavonoids also possess antimicrobial activity (Harborne and Williams, 2000).
The flavonoid glycoside, chrysoeriol 7-0-β-D glucopyranosyl (2 1) –D-apiofuranoside isolated from Dalbergia volubilis exhibited anti- inflammatory activity (Hye and Gafur 1975). A flavonoid from Hedychium spicalum showed a significant activity with less ulcerogenic index than phenylbutazone (Srimal et al., 1984). Two new flavanone glycosides, diinsininol and diinsinin from rhizomes of Sacropthyte piriei
(Balanophoraceae), showed IC50 values of prostaglandin synthesis inhibition 9-20 µm and 13-14 µm respectively and in the inhibition of platelet-activating-factor-induced exocytosis, IC50 values of 49 and 39 µm, respectively (Ogundaini et al., 1996).
Three flavonoids identified as (i) 4’ hydroxy3’, 5’ liprenylisoflav anone (ii) 4’ hydroxy 6, 3’, 5’ triprenylisoflavano-ne (iii) 3, 9-dihydroxy- 2, 0 diprenylprterocarpene (erycrystagallin) (Hedge, 1997) were isolated from a methanol extract of bark of the Samoan medicinal plant Erythrina liii variegate (Leguminosae). The compounds were shown to possess phospholipase A2 (PLA2) inhibitory activity. The compounds-Dicadalenol, Caryolane-1, 9 β-diol and quercetin isolated from aerial parts were identified as the active principle of Boswellia resin, inhibiting the key enzyme in leukotrine biosynthesis, 5-lipooxygenase (5-LOX). Of the boswellic acids characterized, 3-0-acetyl-11-keto-β- boswellic acid (AKBA) proved to be the most potent inhibitor of 5-LOX (Stephan, 2000). A new diterpenoid, tolypodial has been established from the terrestrial Cyanobacteria tolypothrix nodosa (HT-58-2) (Jiang, 2000). Tolypodiol and its monoacetate derivative show potent anti-inflammatory activity in mouse ear oedema assay (Jiang, 2000). The antipyretic and anti- inflammatory activity of a new sesquiterpene, spartidienedione isolated from Psila spartioides (Asteraceae) were evaluated in rabbits and guinea pigs (Delporte, 1996). At a dose of 25 mg/kg, this substance showed anti- inflammatory activity and antipyretic activity (Delporte, 1996). Two new sesquiterpene cyclopentenones, dysidenones A, B and a new sesquiterpene aminoquinone, dysidine, all containing the same rearranged drimane skeletone have been isolated along with bolinaquinone from Sponge dysidea species (Clelia 2001). Bolinaquinone, dysidine and a 1:1 mixture of dysidenone A and B significantly inhibited human synovial phospholiphase A2 (PLA2) at µm concentration (Clelia 2001).
The oleoresin fraction of Commiphora mukul exhibited significant anti- arthritic and anti-inflammatory activities (Satyavati et al., 1969; Shukla, 1986). A steroidal compound isolated from C. mukul displayed a significant activity which is dose dependent and more potent than the resin fraction present in C. mukul (Shukla, 1986). β- sitosterol isolated from Cyperus rotendus possessed potent anti-inflammatory activity against carrageenan and cotton pellet-induced oedema in rats and was comparable to hydrocortisone liv and oxyphenbutazone (Singh 1970). The compound also possesses significant membrane Heterotheca inuloide (Asteraceae) (Guillerno, 2001) were shown to display dose dependent anti-inflammatory activities. Quercetin, quercetin 3-0-rhamnoside (quercitrin) and quercetin 3-0-rutinoside (rutin) from 80% MeOH extract of leaves of Morinda morindoides (Rubiaceae) also showed similar inhibition of classical pathway complement system (Climanaga, 1995).
The dichloromethane extract of the aerial parts of Tanacetum microphyllum (Compositae) yielded two anti-inflammatory flavanoids: 5, 7, 3’ trihydroxy-3, 6, 4’- trimethoxy flavone (Centaureidin) and 5, 3’-dihydroxy-4’-methoxy-7- carbomethoxyflavonl (Abad et al., 1993). Three flavonoids, 7-0 methylaromadendrin, rhamnocitrin, and 3-0 acetylpadmatin, isolated from Inula viscose (Asteraceae) dichloromethane extract were shown to have 12-0 tetradecanoylphorbol-13-acetate induced oedema inhibitory activity in mice (Selvador, 1999). Some other flavonoid subtypes, eg. Coumarins and xanthones, have been shown to inhibit the COX-1/COX-2 catalyzed prostaglandin (PG) biosynthesis in vitro (Moroney, 1988; Kavimani et al., 2000; Jachak, 2001). The coumarin Calophylolide from the nuts of Calophyllum inophyllum (Clusiaceae) effectively reduced the increased permeability induced by the chemical mediators involved in inflammation, like histamine, serotonin and bradykinin. The ED50 was found to be 144.1, 250 and 135.5 mg/kg p.o., respectively against these mediators (Bhalla, 1980; Gopalkrishnan, 1980). Four coumarins, 7-methoxycoumarin (herniarin), 6, 7-dihydroxycoumarin (aescuetin), 6-methoxy-7- glucosidyl coumarin (Scopolin), and 6-hydroxy-7- methoxycoumarin (Scopoletin), were isolated from the ethanol extract of the flower tops of Santolina oblongifolia Boiss. (Compositae) (Silvan, 1996). The isolated compounds showed marked activity as inhibitors of eicosanoid-release from ionophore stimulated mouse peritoneal lv macrophages (Silvan, 1996). Magniferin, a xanthon C-glucoside from Canscora decussatta, mangostin and related compounds from Cerinia mangostana (Shankarnarayan et al., 1979) and xanthones from Calophyllum scohyllum and Mesua ferrea are shown to have anti- inflammatory activity (Gopalkrishnan, 1980).
1.7.3.2 Classification and nomenclature of flavonoids Flavonoids have been classified into the several skeletal types. Some of the known skeletal types are given (Table 5) below, while the structures of representative groups are given in Figure 1
Flavonoids have long been recognized to possess antiallergic, anti- inflammatory, activiral antiproliferative and carcinogenic activities as well as effect on some aspect of mammalian metabolism (Cody et al., 1988). Flavonoids have been shown to affect a large scavenging activity, to chelate certain metal ions, to have antioxidant properties, to affect cellular membranes and more importantly affect cellular protein phosphorilation. Some or all of these properties may account for the multiple activities of these natural products Middleton and Kandaswami, (1994).
1.7.3.3 Pharmacological activities of flavonoids Antiatherosclerotic effects: Because of their antioxidative properties, flavonoids are likely have a major influence on the vascular system. Oxygen radicals can oxidize LDL, which injures the endo the wall and thereby promotes atherosclerotic changes. A few clinical studies have pointed out that flavonoid intakes protest against coronary hearty disease (Hertog et al., 1993; 1995).
Analgesic and anti- inflammatory effects: Cyclooxygenase and lipoxygenase play an important role for inflammatory mediators. They are involved in the lvi release by arachidonic acid, which is a starting point of a general inflammatory response. Neutrophils containing lipoxygenase create chemotactic compounds from arachidonic acid, they also provoke the release of cytokines. Selected phenolic compounds were shown to inhibit both the cyclooxygenase and 5- lipoxygenase pathways (Ferrandiz et al., 1990; laughton et al., 1991). This inhibition reduces the release of arachidonic
Table 4: Some naturally occurring flavonoids based on their skeleton Skeletal type Examples
Flavones and their glycosides Luteolin, Apigenin
Flavonols, dihydroflavonols and their glycosides Quercetin, Kaempferol
Flavanones and their glycosides Narigin
Isoflavones and isoflavanones Diadzein
Biflavonoids and triflavonoids Kavaflavone
Chalcones and dihydrochalcone Buetin
Xanthones Maniferin
Aurone Aureusidin
Anthocyanins Cyanidine-3-glucoside
Flavans and proanthocyandins Kazinol H
Neoflavonoids Nivegen
lvii
acid (Yoshimoto et al., 1983). The exact mechanism by which flavonoids inhibit these enzymes is not clear. Quercetin, in particular, inhibits both cyclooxygenase and lipoxygenase, thus diminishing the formation of these inflammatory metabolites (Kim et al., 1998). Another anti-inflammatory feature is the ability of flavonoid to inhibit eicosanoid biosynthesis (Damas et al., 1985). Eicosanoids such as prostaglandins, are involved in various immunologic responses (moroney et al., 1988) and are the end products of cyclooxygenase and lipoxygenase pathways. Flavonoids also inhibit both cytosolic membranal tyrosine kinase (Fornica and Regelson 1995).
Antitumour effects: The antitumour activity of flavonoids is still a point of discussion. Antioxidant systems are frequently inadequate, and damage from reactive oxygen is proposed to be involved in carcinogenesis (Loft and Poulsen 1996). Reactive oxygen species can damage DNA, and division of cells with unrepaired or misrepaired damage leads to mutations. If these changes appear in critical genes such as oncogenes or tumor, suppressor gene initiation or progression may result (Robert et al., 2001). Reactive oxygens species interfere directly with cell signaling and growth. The cellular damage caused by reacting oxygen can induce mitosis, increasing the risk that damage DNA will lead to mutations, and can increase the exposure of DNA to mutagens. It has been stated, flavonoids are antioxidants, can inhibit carcinogenesis (Stefani et al., 1999). Some flavonoids- such as fisetin, apeginin luteolin are stated to be potent inhibitors of cell proliferation lviii
(Fotsis et al., 1997). Quercetin and apigenin inhibited melanoma growth and influenced the invasive and metastatic potential in mice (Caltagirone et al., 2000). This finding may offer new insights about possible therapies for metastic disease.
Antithrombogenic effects: Platelet aggregation contributes to both the development of atherosclerosis and acute platelet thrombus formation, followed by embolization of stenosed marteries (Robert et al., 2001). Activated platelets adhering to vascular endothelium generate lipid peroxides and oxygen free radicals, which inhibit the endothelial formation of prostacyclin and nitrous oxide. It was shown in the 1960s that tea pigment can reduce blood coagulability, increase fibrinolysis, and prevent platelet adhesion and aggregation (Lou et al., 1989). Selected flavonoids, such as quercetin, kaempferol, and myricetin were shown to be effective inhibitors of platelet aggregation in dogs and monkeys (Osman et al., 1998). Flavonols are particularly antithrombotic because they directly scavenge free radicals, thereby maintaining proper concentrations of endothelial prostacyclin and nitric oxide (Gryglewki et al., 1987). One study showed that flavonoids are powerful antithromobotic agents in vitro and in vivo because of their inhibition of the activity of cyclooxygenase and lipoxygenase pathways (Alcaraz and Ferrandiz, 1987). It is well known that arachidonic acid, which is released in inflammatory conditions, is metabolized by platelets to form prostaglandin, endoperoxides, and thromboxane A2, leading to platelet activation and aggregation (Tzeng et al., 1991). The main antiaggregatory effect of flavonoids is due to by inhibition of thromboxane A2 formation. Flavonoids affect arachidonic acid metabolism in different ways. Some flavonoids specifically block cyclooxygenase or lipoxygenase, whereas others block both enzymes (Landolfi et al., 1984). Antibacterial activity of flavonoids: A number of flavones, flavonols, flavanones, and isoflavones, as well as some of their methoxy, isoprenyl, and acylated derivatives, show antibacterial activity (Harborne and Willaims, lix
2000). The flavone chrysin (5,7-dihydroxyflavone), in the amount of 5 mcg was reported to inhibited the growth of the Gram-negative bacilli Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) at a rate comparable to that of streptomycin (Ali et al., 1998). Wage and Hedin (1985) reported that among the quercetin mono-and diglycosides tested, quercetin-3-O-rhamnoside exhibited the strongest activity against Pseudomonas maltophilia (P. maltophilia) and E cloacae, while the other quercetin-O-glycosides (3-O-glucoside, 3-O- galactoside, 3’-O-galactoside, 3-O-rutinoside, 3-O-galactoglucoside, and 7-O- glucoside) exhibited weak activity or no activity against these bacteria. Malterund et al., (1985) tested six flavonones for antibacterial activity (which included naringenin, taxifolin, and dihydrokaempferide); only naringenin showed activity against E. coli, S. aureus, and E. faecalis. In another study, Bojase et al., (2002) showed that the isoflavonones containing phenyl groups had the highest activity against Gram-positive bacteria such as S. aureus and B. subtilis. This activity was greatest when the phenyl groups were located at position C-6 or C-8 in ring A and C-3’ or C-5’ in ring B. 27.
Antiviral effects: The antiviral activity of flavonoids was shown in a study by Wang et al (1998). Some of the viruses reported to be affected by flavonoids are herpes simplex virus, respiratory syncytial virus, parainfluenza virus, and adenovirus. Quercetin was reported to exhibit both antiinfective and antireplicative abilities.
Xanthine oxidase: The xanthine oxidase pathway has been implicated as an important route in the oxidative injury to tissues, especially after ischemia-reperfusion (Sanhueza 1992). Both xanthine dehydrogenase and xanthine oxidase are involved in the metabolism of xanthine to uric acid. Xathine oxidase reacts with molecular oxygen, thereby releasing superoxide free radicals. Some flavonoids, e.g quercetina nd silibin, inhibit xathine oxidase activity, thereby resulting in decreased oxidative injury (Shoskes, 1998; Chang et al., 1993). lx
Leukocyte immobilization: The immobilization and firm adhesion of leukocytes to the endothelial wall is another major mechanism responsible for the formation of oxygen-derived free radical, but also for the release of cytotoxic oxidants and inflammatory mediators and further activation of the complement system. Oral administration of a purified micronized flavonoid fraction was reported to decrease the number of immobilized leukocytes during reperfusion (Friesenecker et al., 1994). The decrease in the number of immobilized leukocytes by flavonoids may be related to the decrease in total serum complement and is a protective mechanism against inflammation-like conditions associated with, for example, reperfusion injury (Friesenecker et al., 1994; 1995). Some flavonoids can inhibit degranulation of neutrophils without affecting superoxide production (Ferrandiz, 1996). The inhibitory effect of some flavonoids on mast cell degranulation was shown to be due to modulation of the receptor-directed Ca2+ channels in the plasma membrane (Bennett et al., 1981).
Figure 1 Basic structure of some flavonoids
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1.7.3.4 Mechanism of biological activities of flavonoids Antioxidative effects and direct radical scavenging: The best-described property of almost every group of flavonoids is their capacity to act as antioxidants. The flavones seem to be the most powerful flavonoids for protecting the body against reactive oxygen species (Robert et al., 2001). Flavonoids can prevent injury caused by free radicals in various ways. One way is the direct scavenging of free radicals. Flavoniods are oxidized by radicals resulting in a more stable, less reactive radicals. In other words flavonoids stabilize reactive oxygen species by reaching with the reactive compound of the radical.
Nitric oxide: Several flavonoids including quercetin cause a reduction in the ischemia reperfusion injury by interfering with inducible nitric-oxide synthase activity (Shoskes, 1998). When flavonoids are used as lxii antioxidants, free radicals are scavenged and therefore can no longer react with nitric oxide, resulting in less damage (Shutenko et al., 1999).
1.7.4 Terpenoids and steroids with antinociceptive and anti- inflammatory effects Several triterpenoids and steroids have been isolated from extracts of plants with anti-inflammatory effect. In most instances, the anti- inflammatory activity of such extracts has been attributed to the isolated compounds.
The isolation of seven novel naturally occurring triterpene alchohols from non saponifiable lipids of the seeds of Camellia japonica and Camellia sasanqua has been reported (Akihisa et al., 1998). The anti- inflammatory activity of these compounds, tirucall-5, 7, 24-triene-3β-ol, lemmaphylla-7, 21-diene-3β-ol, isoeuphol, isotirucallol, (24R)-24, 25- epoxybutrospermol and its 24S-epimer, and isoaglaoil, was studied in mouse ear edema model (Akihisa et al., 1998). Lupeol was isolated from the hexane stem bark extract of Crataeva religiosa Forst. (Caparidaceae) as the anti-inflammatory component. In an extensive anti-inflammatory studies, lupeol exhibited anti inflammatory effect in a variety of acute and chronic anti inflammatory test models in rat and mice (Singh and Pandey, 1997). Lupeol ester, lupeol, lineolate, obtained by esterification of lupeol with linleoyl chloride was shown to exhibit antiarthritic activity greater than that of lupeol.
The triterpenoids of the oleanene and ursine series were found to be active against carrageenan induced oedema, formaldehyde-induced arthritis in rats. It has been suggested that the anti-inflammatory activity of the triterpenoids of the oedema series vary with the polarity of compounds which is enhanced by the number of hydroxyl groups in the molecule (Bhargava, 1970). Oleanolic acid 3-β-glucoside isolated from the seeds of Randia dumertorum lxiii
(25-500mg/kg, p.o) showed a significant anti-inflammatory activity in the exudative and proliferative phases of inflammation in rats (Ghosh, 1983). Salai guggal, the oleogum of Boswellia serrata, has been shown to possess anti-inflammatory and anti-arthritic activities. It was shown to be effective in controlled clinical trials in arthritic patients. Its activity may be due to the boswellic acids present in the olegum (Atal 1980; Singh et al., 1984). Two new triterpene saponins having phospholipase inhibitory activity were isolated from methanol extract of the leaves of Myrsine australis (Hedge, 1997). These are 3-0{-β-D-xylopyranosy-(1→2)-0-β-D-glucopyranosyl- (1→4) {0-β-D-glucopyrnosyl- (1→2) {-a-L-arabinosyl} 16 a-hydroxy-13β, 28-epoxyoleanane and 3 β-0-{-β-D-rhamnopyranosyl}-16-a-hydroxy-13,28- epoxyoleanane. Both compounds showed IC50 values of 3 and 3um, respectively, versus phorbol 42-myristate 13-acetate stimulated phospholipase in human promylocytic lukemic (HI-60) cells (Hedge 1997). Two oleane type triterpene saponin, zanhasaponins A and B and the cyclitol pinitol isolated from the methanol extract of root bark of Zanha africana (Sapindaceae) were active as inhibitors of phospholipase A2 (Selvador, 1999). Pentacyclic triterpenes from the 11-keto-boswellic acid series has stabilizing effect, inhibition of leucocytes migration and antipyretic activity (Okoli and Akah, 2004; Gupta 1971). The steroid, α- spinasterol, obtained from the stem-bark of Symplocos spicata showed a significant activity against acute inflammation induced by arrageenan in rats and was more potent than phenylbutazone but less potent than betamethasone (Froton 1983). Six steroidal saponins were isolated from Anemarrhena asphodeloides Bunge (Liliaceae), a traditional Chinese medicine, and named anemarrhenasaponin I (An-I), anemarrhenasaponin Ia (An-Ia), timosaponin B-I (TB-I), timosaponin B-II (TB-II), timosaponin B-III (TB-III), and timosaponin A-III (TA-III). All these compounds provoked remarkable inhibiting effect on platelet aggregation (Zhang et al., 1999).
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Steroids consist of an essentially lipophilic (or hydrophobic, non-polar) cyclopentanoperhydrophenanthrene (Figure 3) nucleus modified on the periphery of the nucleus or on the side chain by the addition of hydrophilic (or lipophobic, polar) groups.
For lead optimization through derivatization/semisynthesis, the challenge of any drug discovery effert is to identify and develop compounds with properties that are predictive of good efficacy and safety in humans. In this regard, organic synthesis plays a pivotal role. Once lead services with some desirable profiles are identified, the compounds can progress to lead optimization, entailing structural modifications with the goal of achieving optimal efficacy and pharmacokinetic/pharmacodynamic properties.
The frequent occurance of natural products as complexes of structurally related analogues be exploited by the natural-product investigator as a guide for intial SAR (Structure-activity Relationship) experiments. In this regard, even simple synthetic modifications, as those obtained through “shotgun” transformations (key functional groups required for biological activity can be identified by allowing the parent compound to react with conspecific derivatizing reagents, such as alkyl halides, anhydrides, acyl halides) can be instrumental in leading to an optimized semi-synthetic analogue. The knowledge gained through understanding the natural SARand the shotgun approach can provide an early foundation on which an overall synthetic strategy could be developed.
1.7.4.1 Classification and nomenclature of steroids of plant origin Structurally, steroids are classified on the number of carbon present in the molecules. The major classes of steroids are given in Table 4.
1.7.4.2 Reviewed methods of isolation and purification of steroids The ideal method to utilize for the extraction of lipid from a tissue should be one which will remove all the required lipophylic compounds efficiently lxv without losses or artifact formation due to hydrolysis, autoxidation or other degradation. Chloroform-methanol mixture has been widely applied to obtain lipids from animals and have also been adopted for the extraction of lipids from microorganisms and plants (Goad and Akihisa, 1997). The advantage of using chloroform-methanol system is based on the ability of the mixture to form a monophasic system with the water in the tissue which will then disrupt the membrane structures and remove the lipid from the tissues. Plant materials are often subjected to extraction without any preparation other than coarse chopping after drying and powdering. Oven-or air-drying tissue before extraction may cause changes in the composition and amount of material to be extracted. These could result from oxidation or enzyme catalytic reaction which could occur during the dry process.
Table 5: Some occurring steroids and their structures Number of carbons Class Example 17 Gonane 18 Estranges Estradiol 19 Androstanes Testosterone lxvi
21 Pregnanes Progesterone 24 Cholanes Cholic acid 27 Cholestanes Cholesterol 28 Ergostane Ergosterol 29 Stigmastanes Stemasterol 30 Tetracyclic Triterpenoids Lanosterol
Figure 2: Basic structures of some steroids
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1.8 Review of botanical profile of Lupinus arboreus 1.8.1 Taxonomy of Lupinus arboreus (Kinnane, 2009) Kingdom: Plantae lxviii
Division: magnotiophyta Class: Magnoloipsida Order: Rosales Family: Leguminosae – or Fabaceae Genus: Lupinus Species: arboreus Common names: yellow bush lupine, coastal bush lupine
The genus Lupinus is made up of about 300 species of annuals, perennials, and evergreen sub-shrubs or shrubs (Kinnane, 2009). These plants are found mostly in the Mediterranean region, North Africa, and North, Central and South America where they are grown on dry hilly grassland or coastal sands or cliffs or along the banks of streams and rivers. Lupinus should be grown in a sunny site where the soil is slightly acidic with a pH in the range of 4.5-7.5. (Kinnane, 2009)
Figure 3: The Photograph of Lupinus arboreus leaves lxix
The soil should be light, moderately fertile and well draining. Lupinus can tolerate some alkaline soils up to pH 8.0 provided the soils are free draining lxx and not subjected to prolonged waterlogging. Waterlogging should be avoided because plants are very prone to root-rot. If planted in heavier soil plants will grow and flower but tend to die out after a few years. Even in their ideal soil these plants tend to be somewhat short-lived. Plants can easily be raised from seed and will flower the year after planting. Lupinus are susceptible to heliothis damage. Other insect pests include Lucerne seed-web moth, aphids, thrips and blue oat mites. They are also prone to the following diseases- cucumber mosaic virus, brown leaf spot, phomopsis stem blight and gray leaf spot (Kinnane, 2009).
1.8.2.1 Description and distinctive features of the plants Lupinus arboreus is a bushy shrub up to six feet tall, usually with bright yellow (sometimes blue) sweet-smelling flowers and green, sparsely pubescent (appearing glabrous), palmately compound leaves. It occurs as an invasive species in northern California coastal dunes. It appears to be native to California from Sonoma country south. Where plants often have a hairy upper leaf surface. Yellow bush Lupinus is predominantly a dune species, but it can be found along roadsides and in disturbed areas. Yellow bush Lupinus hybridizes with the native Lupinus littoralis, which is smaller (less than two feet) and more prostrate-decumbent, has purple and white flowers, and always has a hairy upper leaf surface. Intermediate Lupines usually are smaller and more prostrate than Lupinus arboreus with blended yellow, purple, and white flowers (Wear, 1998). Yellow bush lupine can be easily distinguished from blue bush lupine (Lupinus chamissonis), which can achieve the same height but has silver, densely hairy leaves that appear gray-blue and light violet to blue flowers (Wear, 1998).
1.8.3 Geographical spread Native to southern and central California, yellow bush lupines was introduced to many dune systems as a sand stabilizer during the early to mid- lxxi
1900s. Pickart and Miller (1998) traced the introduction of yellow bush lupine to the Humboldt Bay dune system. According to the account, in 1908, the operator of a fog signal station on the north spit of Humboldt Bay gathered seeds of yellow bush lupine from the presidio where it had previously been introduced and planted them around the station. In 1917 seeds from the new signal station population were collected and scattered beside railroad tracks along the spit. From these and subsequent plantings, the extent of yellow bush lupine has risen from 244 acres (97 ha) in 1939 to over 1,000 acres (400 ha) (Pickart and Miller 1998). Yellow bush lupine now dominates 28 percent of total vegetation cover on Humboldt Bay dunes (Pickart and Miller 1998). In Nigeria, the spread of yellow bush lupine is attributed to the quest for aesthetic and environmental beauty where it serves as ornamental flowers.
1.8.4: Values of Lupine Multifarious uses to which the lupine might be put include:
i. Aesthetic value This is perhaps, the greatest use of lupine especially L. arboreus in Africa, and Nigeria in particular. It adorns streets, institutions, and residential buildings as ornamental plant.
ii. Nutritional values The major physiological and biochemical feature of lupine is the capability to synthesize plenty of protein. (Kurlovich, et al., 2000). Leguminous crops, including lupine, are an important source of protein and other nutritious substances. There is classical relationship between legumes and nitrogen-fixing bacteria. As legumes such as the lupine sprout, they send out roots that encounter nitrogen-fixing bacteria in the soil. The bacteria infect the roots and stimulate them to form nodules. Nodule cells then engulf the bacteria and provide oxygen and energy from photosynthesis in return for nitrogen needed to make protein (Simms, et al., 2005). Study showed that the different strains of the bacteria Bradyrhizobia that interact with roots of the lxxii six species of lupine have been identified (Simms, et al., 2005). It was confirmed that the larger the root nodule, the more nitrogen –fixing bacteria it contained.
The lupine is grown for fodder, and it, is found to be highly nutritive and wholesome. In fact, the lupine is one of the few legumes that come close to soybean in protein content (Rachel, 2006). Among the rich special diversity of lupine, there are species, varieties and forms, which accumulate large amounts of protein, oil and other useful substances. As it is characteristic of all fodder crops, the nutritive value of protein in lupine is reduced because of an unbalanced amino acid composition. There is a deficiency in methionine- an essential sulfur- containing amino acid (Kurlovich et al., 2000). In the words of Pliny (2009) “No kind of fodder is more wholesome and light of digestion than white lupine. If taken commonly at meals, it will contribute a fresh colour and cheerful countenance”. iii. Medicinal value The ancients employed the lupine medicinally (Pliny 2009). Women use lupines mingled with lemons to make soft ointment. Topically, lupines are used against deformities of the skin, scabies, ulcers, scald heads, and other cutaneous distempers (Pliny, 2009).
Though Pliny (2009) reported that the ancients employed lupine medicinally, there exist very few scientific investigations to back up the claims. Among the few scientific findings is the isolation of L-asparaginase from developing seeds of Lupinus arboreus (Lough, 1992). Asparaginase, purified from these developing seeds was resolved into three isoforms, designated asparaginases A,B and C. (Lough, 1992). Polyclonal antibodies raised against asparaginase A and B precipitated asparaginases activity from a partially purified Lupinus arboerus seed extract. L. asparaginases are known to catalyse the formation of the neuroactive amino acid L. aspartate by deamination of asparagines lxxiii
(Lough, 1992). The major pathophysiological significance of L- asparaginase activity is in its chemical use for the treatment of acute lymphatic leukaemia and neoplasias that require asparagines (Lough, 1992).
Another value of lupine is on leukocyte immobilization. The immobilization and firm adhesion of leukocytes to the endothelial wall is another major mechanism responsible for the formation of oxygen-derived free radical (Lough, 1992), but also for the release of cytotoxic oxidants and inflammatory mediators and further activation of the complement system. Oral administration of a purified micronized flavonoid fraction was reported to decrease the number of immobilized leukocytes during reperfusion (Friesenecker et al., 1994). The decrease in the number of immobilized leukocytes by flavonoids may be related to the decrease in total serum complement and is a protective mechanism against inflammation-like conditions associated with, for example, reperfusion injury (Friesenecker et al., 1994; 1995).
1.9 Aim and scope of the work The aims of this study are: i. To investigate the antinociceptive and anti-inflammatory activities of Lupinus arboreus leaf using standard scientific methods ii. To isolate the active constituents of Lupinus arboreus leaf responsible for the antinociceptive and anti-inflammatory effect via bioactivity- guided fractionation iii. To identify and characterize the isolated active constituents by a combination of phytochemical analysis, melting point (m.p) determination, ultraviolet (UV), Infrared (IR), nuclear magnetic resonance (NMR), and gas chromatography/mass spectral (GC/MS) analyses.
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CHAPTER TWO MATERIALS AND METHODS 2.1. Plant material Leaves of Lupinus arboreus were collected from Owerri, Imo State, Nigeria in January 2009. Official identification was done by Pharm F. N. Osuala, of Pharmacognosy Department, Madonna University where a voucher specimen has been deposited in the Herbarium, specimen No. MHE 093. The leaves were air-dried at room temperature for 28 days and ground to fine powder.
2.2. Solvents and reagents Hexane, ethyl acetate, methanol, silica gel (60-230 mesh), precoated silica gel G254 (Merck). Napthol solution in ethanol (Molisch reagent), concentrated sulphuric acid, 3% sulphuric acid in 50% ethanol, potassium mercuric iodide solution (Mayer’s reagent), bismuth potassium iodide solution (Dragendroff’s reagent), iodine in potassium iodide solution (Wagner’s reagent), picric acid solution (1%), dilute ammonium solution, solution of crystalline CuSO4 in sulphuric acid (Fehling’s solution I), solution of Rochelle salt and potassium hydroxide (Fehling’s solution II), 20% potassium hydroxide solution, olive oil, ferric chloride solution, lead acetate solution, ethylacetate, 1% aluminum chloride solution, acetone, concentrated hydrochloric acid, mercuric nitrate in nitric acid containing a trace of nitrous acid (Millon’s reagent), concentrated nitric acid, dilute sodium hydroxide, crystal of copper sulphate. Egg albumin, lxxv pentazocine, diclofenac, normal saline, acetic acid, piroxicam. All laboratory reagents were freshly prepared and freshly distilled water was used when needed.
2.3.0 Equipment Water bath, separating funnel, test tubes, beakers, filter papers, funnels, litmus papers spatula, measuring cylinders, stop watch, bijou bottles, cornical flasks, retort stands, measuring tape, miller (grinder), hot-air oven (Gallenkamp, U.K) rotary evaporating dish, hand gloves, electronic weighing balance, glass columns for column chromatography, 20 x 20 cm glass chromatoplates and chromatographic tanks. Digital plethysmometer (LE 7 150) at Faculty of Pharmacy, Madonna University., FT-IR spectral were recored on a FT-IR spectrometer (SHIMADZ) NARICT Zaria, Nigeria. UV spectra were obtained in a UV 2102 PC spectrophotometer (UNICO®, USA) at Department of Industrial Chemistry, University of Nigeria, Nsukka. FT-IR and UV spectra repeated at Usmanu Dan Fodiyo University, Sokoto, Nigeria. GC/MS analyses were carried out using Agilent 5973N mass selective detector coupled to Agilent 6890N gas chromatograph at Usmanu Dan Fodiyo University, Sokoto.
2.4 Animals: Albino mice of both sexes weighing 20-32 g and Wistar rats of both sexes weighing 200-320 g were used. The animals were maintained at the Animal House, Department of Pharmacology and Toxicology, Madonna University, Elele Campus, Rivers State, Nigeria.
2.5 Methods 2.5.1 Extraction and concentration of plant materials: The leaves ground to powder (4 kg) were extracted by cold maceration using absolute methanol for 48 h. After filtration, the resulting solution was concentrated using rotary evaporator, and further oven- dried at a temperature of 50 oC. The dried concentrate was stored in a refrigerator for future use.
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2.5.2 Determination of extractive yield The oven-dried residue was allowed to cool and the extractive yield calculated using the formula: Yield % = (weight (g) extract/weight (g) of plant material) x 100.
2.6 Phytochemical analysis Phytochemical analysis of the methanol extract (ME) and fractions were carried out using the procedure outlined by Trease and Evans (2002).
2.6.1 Test for Carbohydrate Molisch Test The extract (0.1 g) was boiled with 2 ml of distilled water and filtered. To the filtrate, few drops of naphthol solution in ethanol (Molisch’s reagent) were added. Concentrated sulphuric acid was then gently poured down the side of the test tube to form a lower layer. A purple interfacial ring indicates the presence of carbohydrate.
2.6.2 Test for Alkaloids Sulphuric acid 3% (20 ml) in 50% ethanol was added to 2 g of the extract and heated on a boiling water bath for 10 minutes. Cooled and filtered. 2 ml of the filtrate was tested with a few drops of Mayer’s reagent (potassium mercuric iodide solution). Dragendorff’s reagent (bismuth potassium iodide solution), Wagner’s reagent (iodine in potassium iodide solution), and picric acid solution (1%). The remaining filtrate was placed in 100 ml separatory funnel and made alkaline with dilute ammonia solution. The aqueous alkaline solution was separated and extracted with two 5 ml portions of dilute sulphurie acid. The extract was tested with a few drops of Mayer’s, Wagner’s Dragendorff’s reagents and pieric acid solution. Alkaloids give milky precipitate with few drops of Mayer’s reagent; reddish brown precipitate with few drops of Wagner’s reagent; yellowish precipitate with few drops of pieric acid and brick red precipitate with few drops of Dragendorff’s reagent.
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2.6.3 Test for Reducing sugar A mixture (5 ml) of equal parts of Fehling’s solution I and II were added to 5 ml of aqueous extract and then heated on a water bath for 5 minutes. A brick red precipitate shows the presence of reducing sugar.
2.6.4 Test for Glycosides Dilute sulphuric acid (5 ml) was added to 0.1 g of the extract in a test tube and boiled for 15 minutes on a water bath, then cooled and neutralized with 20 % potassium hydroxide solution. 10 ml of a mixture of equal parts of Fehling’s solution I and II was added and boiled for 5 minutes. A more dense brick red precipitate indicates the presence of glycoside.
2.6.5 Test for Saponins Distilled water (20 ml) was added to 0.25 g of the extract and boiled on a hot water bath for 2 minutes. The mixture was filtered while hot and allowed to cool and filtrate was used for the following tests. a Frothing Test The filtrate (5 ml) was diluted with 15 ml of distilled water and shaken vigorously. A stable froth (Foam) upon standing indicates the presence of saponins. b. Emulsion Test To the frothing solution was added 2 drops of olive oil and the contents shaken vigorously. The formation of emulsion indicates the presence of saponins. c. Fehling’s Test The filtrate (5 ml) was added 5 ml of Fehling’s solution (equal parts of Fehling’s solution I and II) and the contents were heated on a water bath. Reddish precipitate which turns brick red on further heating with sulphuric acid indicates the presence of saponins.
2.6.6 Test for Tannins lxxviii
The powered material (1 g) was boiled with 20 ml of water, filtered and used for the following test. a. Ferric chloride Test To the filtrate (3 ml), few drops of ferric chloride were added. A greenish black precipitate indicates the presence of tannins. b. Lead Acetate Test To a little of the filtrate was added lead acetate solution. A reddish colour indicates the presence of tannins.
2.6.7 Test for Flavonoids Ethyl acetate (10 ml) was added to 0.2 g of the extract and heated on a water bath for 3 minutes. The mixture was cooled, filtered and the filtrate was used for the following tests a. Ammonium Test The filtrate (4 ml) was shaken with 1 ml of dilute ammonia solution. The layers were allowed to separate and the yellow colour in the ammoniacal layer indicates the presence of flavonoids.
b. Aluminium Chloride solution (1%) Test Another 4 ml portion of the filtrate was shaken with 1 ml of 1% Aluminium chloride solution. The layers were allowed to separate. A yellow colour in the Aluminium chloride layer indicates the presence of flavonoids.
2.6.8 Test for Resins a. Precipitation Test The extract (0.2 g) was extracted with 15 ml of 96% ethanol. The alcoholic extract was then poured into 20 ml of distilled water in a beaker. A precipitate occurring indicates the presence of resins.
b. Colour Test lxxix
The extract (0.2 g) was extracted with chloroform and the extract was concentrated to dryness. The residue was redissolved in 3 ml of acetone and another 3 ml concentrated hydrochloric acid was added. This mixture was heated in a water bath for 30 minutes. A pink colour which changes to magenta red indicates the presence of resins.
2.6.9 Test for Proteins The extract (0.5 g) was extracted with 20 ml of distilled water and the filtrate was used for the following tests. a. Million’s Test To a little portion of the filtrate in a test tube, two drops of Million’s reagent were added. A white precipitate indicates the presence of proteins.
b. Xanthoproteic Reaction Test The filtrate (5 ml) was heated with few drops of concentrated nitric acid. A yellow colour which changes to orange on addition of an alkali (dilute Sodium hydroxide) indicates the presence of protein.
c. Pieric Acid Test To a little portion of the filtrate was added a few drop of pieric acid. A yellow precipitate indicates the presence of proteins. d. Biuret Test A crystal of copper sulphate was added to 2 ml of the filtrate, and then 2 drops of potassium hydroxide solution was added. A purple or pink colour shows the presence of proteins.
2.6.10 Test for Fats and Oil The extract (0.1 g) was pressed between filter paper and the paper was observed. A control was also prepared by placing 2 drops of olive oil on filter paper. Translucency of the filter paper indicates the presence of fats and oil.
2.6.11 Test for Steroids and Terpenoids lxxx
Ethanol (9 ml) was added to 1 g of the extract and refluxed for a few minutes and filtered. The filtrate was concentrated to 2.5 ml on a boiling water bath. 5 ml of hot distilled water was added to the concentrated solution, the mixture was allowed to stand for 1 hour and the waxy matter was filtered off. The filtrate was extracted with 2.5 ml of chloroform using separating funnel. To 0.5 ml of the chloroform extract in a test tube was carefully added 1 ml of concentrated sulphuric acid to form a lower layer. A reddish brown interface shows the presence of sterioids. Another 0.5 ml of the chloroform extract was evaporated to dryness on a water bath and heated with 3 ml of concentrated sulphuric acid for 10 minutes on a water bath. A grey colour indicates the presence of terpenoids.
2.6.12 Test for Acidic Compounds The extract (0.1 g) was placed in a clear dry test tube and sufficient water added. This was warned in a hot water bath and then cooled. A piece of water-wetted litmus paper was dipped into the filtrate and the colour change on the litmus paper was observed. Acidic compounds turn blue litmus paper red.
2.7 Bioassay-Guided Isolation of the active constituents of L. aboreus leaf.
2.7.1 Column chromatographic separation of the methanol extract:
The dried methanol extract was separated using column chromatography. Two hundred grammes of activated silica gel (70-230 mesh) was packed to two-third the length of a glass column (150 x 1.5cm,). Ten grammes of the dry methanol extract was dissolved in methanol-water mixture (1:2) v/v and introduced into the column. The column was eluted with 1.5 L Hexane, 1.2 L of ethylacetate and 1.0 L methanol in succession, to yield, n- Hexane (HEF), ethyl acetate (EAF) and methanol (MEF) fractions. The fractions HEF, EAF and MEF were screened for analgesic and anti-inflammatory activities.
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2.7.2 Isolation and purification of the active constituents of hexane fraction (HEF). A portion of the hexane fraction (4.0 g) was seperated using silica gel column chromatography. The column was eluted with gradient mixtures of hexane: ethyl acetate; and hexane: ethylacetate: methanol; (Table 6). Aliquots of 20 ml were collected and monitored with phytochemical reactions, TLC and UV spectral analysis. Similar fractions were combined. The fractions were screened for antinociceptive and anti-inflammatory activities.
Fractions 1-74 and 75-86 eluted with hexane: ethylacetate (90:10 v/v) yielded
AHF1 (65 mg) and AHF2 (60 mg) respectively. The compounds were recrystallized in acetone and the purity examined by TLC precoated with 0.25 mm silica gel 60 GF 254 from Merck and eluted with hexane: ethyl acetate 90:10 v/v.
2.7.3 Isolation and purification of the active constituents of ethylacetate fraction (EAF) A portion of the ethylacetate fraction (4.0 g) was separated using silica gel column chromatography. The elution was carried out with gradient mixtures of hexane: ethylacetate; ethylacetate: methanol and hexane: ethylacetate: methanol. (Table 7). Aliquots of 25 ml were collected and monitored with TLC, phytochemical reactions and UV spectal analysis. Major fractions AEF1 (50-69) and AEF2 (70-79) were screened for antinociceptive and anti- inflammatory activities.
Fractions 50-69 (AEF1) eluted with ethylacetate: methanol 4:1 was purified by PTLC on silica gel plate (0.5 mm) developed with ethylacetate as mobile phase to isolate 60 mg of AEF1 2.8. Pharmacological Test
2.8.1 Acute toxicity and lethality test (LD50) and Preliminary Screening The extract was subjected to acute toxicity testing in mice using the method described by Lorke (1983). And, the crude methanol extract of L. lxxxii arboreus (CME) was subjected to preliminary antinociceptive and anti- inflammatory screening.
Animals were divided into three groups of three animals each. Groups A, B, and C received a single i.p dose of 10 mg/kg, 100 mg/kg and 1000 mg/kg of the extract respectively. After a period of 24 hours, the number of death in each group was recorded. From the result of the first stage, the second stage was carried out. In this stage, three groups of one animal in each group were involved. Groups A, B and C received a single i.p dose of 30 mg/kg, 60 mg/kg and 120 mg/kg of the extract respectively. The animals were monitored for a period of 24 hours and the number of death in each group recorded.
2.8.2 Determination of antinociceptive activities 2.8.2.1 Thermally-induced pain (Hot plate test) in mice Using the method described by Janssen and Jageneau (1957), hot plate test was performed on 4 groups of 5 mice each. Mice were treated intraperitoneally with 2 different doses (30, 60 mg/kg) of test extract. The animals were placed on a hot plate maintained at 55±0.5 oC,. Licking paws or jumping which indicate latency or discomfort reaction, was measured for each mouse just prior to extract administration, and later at 30, 60 and 90 minutes after administration. A control group received diclofenac sodium (50 mg/kg).
2.8.2.2 Acetic acid-induced pain (writhing reflex test) in mice Mice of either sex were randomly divided into 4 groups of six each. Distilled water was given to one group serving as the negative control. The other two groups received two doses of L.aboreus extract (30 mg/kg and 60 mg/kg) i.p. Thirty minutes later, 0.01% of acetic acid was injected i.p. A positive control group received diclofenac (50 mg/kg). The writhing movement (abdominal contraction) was observed and the number recorded for 15 minutes, starting from 5 minutes after injection of acetic acid. The percentage inhibition of writhing movement relative to the control animals was then calculated using the method of Oriowo (1982) and Otimenyin (2004).
lxxxiii
2.8.2.3 Pressure- induced pain (tail immersion test) in rats
Using the method described by Sanchez-Blazques and Garzon (1989) the animals prior to the antinociceptive experiment, were screened for sensitivity test by immersing the tip of their tails into hot water maintained at 55oC. The rats that lifted the tail within 5 seconds were selected for this study on 4 groups of 6 rats each. The rats were then treated thus:
Group 1 - Pentazocine (0.5 mg/kg i.p) Group 2 - Extract (30 mg/kg i.p) Group 3 - Extract (60 mg/kg ip) Group 4 - Normal saline
The reaction time which is the time taken to lift tail was measured at 15, 30, 45, 60, 75 and 90 minutes.
2.8.3 Inflammatory test 2.8.3.1 Acute Inflammation test (egg albumin induced- inflammation) The rat pedal oedema method (Akah and Njike, 1990; Hess and Milong, 1972; Perez, 1996) was employed in studying the anti-inflammatory effect of the extract; solvent fractions, column fractions and isolated compounds on acute inflammatory model. Adult albino Wistar rats weighing 200-320 g of both sexes were grouped with five animals in each group. They had free access to food and water. The phlogistic agent (egg albumin 0.0l ml) was injected into the subplantar surface of the hind paw 30 minutes after treatment was administered.
Groups 1 and 2 received 30 and 60 mg/kg respectively of crude methanol extract (CME); HEF; EAF; and MEF; AHF1; AHF2, AEF1 and AEF2; intraperitoneally. Groups 3 and 4 received piroxicam (0.5 mg/kg) or aspirin (100 mg/kg) solubilized with 10 % Tween 80, and negative control i.p, respectively. Using water displacement method, anti-inflammatory effect was evaluated by the effect of different treatments on egg albumin-induced lxxxiv inflammation by measuring changes in volume of water displaced by the inflamed hind paw over time. From the values obtained from measuring changes in volume of water displaced, percentage inhibition of inflammation were calculated as follows:
% inhibition = (Vo - Vt ) x 100 Vo
Where Vt = volume displaced at a time point
Vo = volume displaced of vehicle (control) treated rats at the same time.
2.8.3.2 Chronic-inflammation Test (formaldehyde-induced inflammtion) Adult Wistar rats (n=5, per group) of both sexes, weighing 200-320 g were used in studying the anti-inflammatory effect of extract, solvent fractions, column fractions and isolated compound (Seyle, 1949) receiving 30 or 60 mg/kg i.p.
Day 1, after 1 hour of administration, inflammation was induced by subplanta injection of 0.1 ml of 2.5% formaldehyde solution and repeated on day 3. Inflammation was assessed by measuring the rats paw volume by water displacement method before the induction of inflammation and once daily for 10 days, starting from day 1, after induction of inflammation. Drug administration was continued once daily for the first 5 days and once every other day for the next 5 days. Control animals received either i.p administration of piroxicam (0.5 mg/kg) or indomethacin (5 mg/kg) solublished with 10 % Tween 80 or equivalent volume of vehicle (10% Tween 80).
The percentage inhibition was calculated thus: % inhibition = (Vo – Vt) x100 Vo Where Vt = volume displaced at a time point,
and Vo = volume displaced of vehicle/control treated rats at the same time.
lxxxv
Table 6: Solvent systems employed in the column chromatographic separation of HEF
System Ratio of components Volume of system used (ml) Hexane Ethyl acetate Methanol 1 18 2 0 2000 2 8 2 0 750 3 7 3 0 750 4 3 1 0 500 5 2 1 0 500 6 1 2 1 500
lxxxvi
2.8.4 The isolated active constituents 2.8.4.1 Phytochemical analysis of fractions, AHF1, AHF2,AEF1 and AEF2 The phytochemical tests on the extracts, fractions and isolated compounds were carried out using standard procedures (Trease and Evans, 1989; Harborne, 1998). Small quantities of the extract, fractions or isolated compounds were dissolved in a suitable solvent in a test tube and a given quantity of the reagent added. The mixtures were shaken and the presence or absence of alkaloids, flavonoids, steroids, terpenoids, saponins, tannins, carbohydrates, reducing sugar, proteins fats and oils etc observed. The following reagents were used Dragendorf’s (alkaloids). Aqeuous ammonia, ferric chloride (flavonoids), Libermann-Buchard reagent (sterols and triterpenoids), ferric chloride, lead subacetate (tannins), Moliseh’s test (carbohydrate). Fehling’s test (reducing sugars). Frothing and emulsion tests were used to detect the present of saponnins.
2.8.4.2 Determination of melting point The isolated compounds were filled in narrow capillary tubes sealed at one end and paced into the melting point apparatus (Electrothermal ®, England). The capillary was observed through the magnifying glass and the temperature at which the material started flowing was taken as the melting.
2.8.4.3: IR spectral analysis lxxxvii
The IR spectra of AHF1 and AHF2, AEF1 and AEF2 were determined. The compounds were prepared in KBr disc and the spectra recorded in FTIR, Shimadu.
Table 7: Solvent systems employed in the column chromatographic separation EAF.
System Ratio of components Volume of system used (ml) Hexane Ethyl acetate Methanol 1 4 1 0 300 2 3 1 0 250 3 2 1 0 250 4 1 2 1 150 5 1 2 1 150 6 0 1 3 125
lxxxviii
2.8.4.4 UV spectral analysis UV Spectra of AHF1 and AHF2, AEF1 and AEF2 (in chloroform) were recorded with UV 2102 PC spectrophotometer (UNICO) by using 1 cm quartz cells.
2.8.4.5 GC-MS Analysis of AHF1, AHF2 and AEF1 Gas Chromatography/Mass spectral (GC/MS) analysis of AHF1, AHF2, AEF1 and AEF2 were carried out using Agilent 5973N mass selective detector coupled to Agilent 6890N gas chromatograph, equipped with a cross-link 5% PH-ME siloxane HP5-MS capillary column (30m x 0.25 mm, film thickness of 0.25µm). Operating conditions follow thus: carrier gas, helium with a flow rate of 2ml/min; column temperatures, 60-275oC at 4oC/min; injector temperatures, 280oC; injected volume 2 µl; split ratio, 1:50. The MS operating parameters were as follows: ionization potential, 70 eV; ionization current, IA; ion source temperature, 200oC and resolution of 1000. Identification of components in AHF1, AFH2, AEF1 and AEF2 were based on comparison of the retention times and computer matching of MS fragments with the NISTOL 2.L library.
2.8.4.6 H-NMR (1D and 2D cosy) and C13-NMR analyses lxxxix
HNMR (200MHz) and 13CNMR (50MHz) spectra were measured in
CDCl3 at ambient temperature. Chemical shifts were recorded in ppm relative to TMs as internal standard. Homounclear (proton-proton) correlation spectroscopy (1H 1H-COSY) experiment was also carried out on the NMR of AHF1, AHF2, AEF1 and AEF2.
2.9: Statistical Analysis Results obtained were expressed as mean± S.E.M, analysed using one way analysis of variance (ANOVA) by SPSS version II. Difference between means were considered significant at p<0.05 and post – hoc tests were then performed using the Dunnet test.
CHAPTER THREE RESULTS
3.1 Extraction yield Extraction of L. arboreus ground leaves (4 kg) with methanol gave 500 g of dry methanol extract (ME) representing 12.5% w/w.
3.2 Phytochemicals analysis
Phytochemical studies showed that crude methanol extract had the abundance of saponins, glycosides, steroids, terpenes and flavonoids. Resins, protein and reducing sugar occurred in moderate amounts, while alkaloids appeared but in trace amount (Table 8). Hexane fraction (HEF) contained steroids and terpenes; Ethylacetate fraction (EAF) contained flavonoids and glycosides while methanol fraction (MEF) contained tannins, saponins and glycosides. Bioactive fraction AHF1 contained steroids; AHF2 contained steroids and terpenes; AEF1 had flavonoids and glycosides and AEF2 contained flavonoids (Table 9).
3.3 Acute toxicity test:
xc
The i.p toxicity test (LD50) of methanol leaf extract of Lupinus arboreus was calculated to be 84.95 mg/kg .
Stage Dose Number of death I 10 mg/kg 0/3 100 mg/kg 1/3 1000 mg/kg 3/3
II 30 mg/kg 0/1 60 mg/kg 0/1 120 mg/kg 1/1
LD50 = a x b Where a = The highest dose that does not produce death b = The lowest dose that does produce death
Thus: LD50 = 60 x 120 = 84.85
LD50 = 84.85 mg/kg
3.4 Antinociceptive and anti inflammatory effects. 3.4.1 Antinociceptive effect
The crude methanol extract (CME) (30 and 60 mg/kg, i.p) exhibited dose- dependent inhibition of thermally induced pain and this was significant (p<0.01) compared to diclofenac 50 mg/kg at the 90th mins (Table 10). On acetic acid-induced writhing test CME possessed antinociceptive activity with 71.13 percentage inhibition at 60 mg/kg; 47.80 percentage inhibition at 30 mg/kg while positive control, diclofenac (50 mg/kg) exhibited 47.21 percentage inhibition (Table 11). Tail immersion method exhibited significant (p<0.05) antinociceptive effect. This effect was time-dependent but not dose- xci dependent. The effect was observed to be more potent at 45 to 60 minutes (Table 12). The extract at 30 and 60 mg/kg produced activity comparable to pentazocine. The fractions HEF, EAF, and MEF on acetic acid-induced writhing test exhibited significant (p<0.05) inhibition of 73, 64 and 24 percent respectively, compared with the positive control diclofenac, 63 percent. HEF and EAF were therefore, the most active fractions and were selected for further studies.
AHF1 (30 mg/kg) and AHF2 (30 mg/kg) fractionated from HEF significantly (p<0.05) inhibited pain by 75 and 71 % respectively. AEF1 (30 mg/kg) fractionated from EAF also significantly (p<0.05) inhibited pain by 71 % (Fig. 8).
Table 8: Phytochemical constituents of methanol leaf extract of Lupinus arboreus.
Phytochemical constituents Relative abundance Saponins +++ Glycosides +++ Flavonoids +++ Steroids +++ Terpenes +++ Tannins ++ xcii
Resins ++ Protein ++ Reducing sugar ++ Alkaloids +
+++ = Abundantly Present. ++ = Moderately Present. + = Present in trace amount.
Table 9: Phytochemical constituents of fractions and active substances of L. arboreus:
Fractions Phytochemical Constituents
HEF Steroids***, Terpenes, * ** EAF Flavonoids** *, Glycoside *** MEF Tannins*, Saponins*, Glycosides * xciii
AHF1 Sterioids *** AHF2 Steroids***, Terpenes *** AEF1 Flavonoids***, Glycosides*** AEF 2 Flavonoids**
*** - Abundant ** - Moderate * - traces
Table 10: Effect of extract of L. arboreus on Hot plate - induced pain in mice
Treatment Reaction time at minutes after treatment (mg/kg, i.p) xciv
0 min 30min 60min 90min Distilled water 0.3 ml 5.60±0.35 4.01±0.31 3.70±0.31 2.07±0.31 Extract 30 5.66±0.41 8.54±0.09* 8.86±0.89* 8.11±0.14* Extract 60 5.95±0.28 9.03±1.09* 9.31±0.41* 9.64±0.25** Diclofenac 50 5.56±0.61 6.58±0.89 6.12±0.67 7.12±0.62
Key:*=p<0.05; **= p<0.01; Values are mean± SEM, n=5; zero minute= reaction time prior/before treatment, values significantly higher than the negative control and diclofenac.
Table 11: Effect of extract of L. arboreus on acetic acid-induced writhing
Treatment (mg/kg, i.p) N No of writhes Inhibition %
Control (water) 8 49.23±5.1 - Extract (30) 6 25.70±3.2* 48±3.2 xcv
Extract(60) 6 14.12±3.7* 71.13±3.7 Diclofenac (50) 6 18.12+3.7* 63±3.7
No. of writhes are mean ± S.E.M.. * p<0.05, N= 6-8 per group. Inhibition values significantly higher than negative control and diclofenac.
Table 12: Effect of extract of L. arboreus on pressure – induced (tail immersion) pain in rats
Latency time (minutes) xcvi
Treatment (mg/kg, ip)
15 30 45 60 75 90 Pentazocine (0.5) 3.00± 0.58 7.33± 0.66 6.67± 0.33* 6.33± 0.66* 4.00± 0.58 2.67± 0.33 Extract (30) 3.67± 0.67 2.33± 0.33 5.33± 0.67* 5.33± 0.88* 4.33± 0.33 2.32± 0.33 Extract (60) 2.33± 0.33 4.67± 0.23 5.67± 1.20* 5.0± 1.00* 3.33± 0.33 2.66± 0.33 Normal saline 2.33± 0.33 2.67± 0.30 2.33± 0.30 2.33± 0.30 2.33± 0.30 2.33± 0.33 *P<0.05, n= 6 per group. values significantly higher than negative control and comparable to pentazocine
3.4.2 Effect of extracts and fractions on egg albumen-induced (acute) oedema in rats
Table 13 shows that the crude methanol extract (30 and 60 mg/kg) produced a dose-dependent inhibition of egg albumin-induced oedema over a period of 4 hours. At 30 and 60 mg/kg, i.p, however, it produced a significant (p<0.05) anti-inflammatory effect with inhibition of oedema 81.10 and 91.50 % respectively at 4 hours, comparable to piroxicam the standard control.
In a similar way the hexane fraction (HEF) (60 mg/kg) produced a significant (p<0.05) anti-inflammatory effect with oedema inhibition of 79 % at 4 hours. The ethylacetate fraction (EAF) (60 mg/kg) produced a significant (p<0.05) xcvii anti-inflammatory effect with oedema inhibition of 40% at 4 hours. The methanol fraction (MEF) (60 mg/kg) did not significantly inhibt oedema formation (14% inhibition) (Table 16). The inhibitory effect of HEF and EFF are higher than and equal respectively, to the inhibition caused by standard anti-inflammatory drug, aspirin (100 mg/kg) (Table 16). HEF and EFF are therefore, selected for further studies.
3.4.3 Effect of extracts and fractions on formaldehyde-induced oedema in rats
The crude methanol extract (CME) (30 and 60 mg/kg) inhibited the oedematous response to formaldyde-induced arthritis. The inhibition was dose-dependent, over a period of 4 hours; showing significant (p<0.05) 68 and 69 % inhibition respectively for CME (30 and 60 mg/kg). The values were however, not higher than the inhibition caused by piroxicam (0.5 mg/kg) which recorded 73 % inhibition (Table 14). The result of the effect of HEF and EAF is shown in Table 17. Both of them at 60 mg/kg i.p, inhibited the oedematous response to formaldehyde-induced arthritis. The inhibition induced by HEF (85.7 %) was higher than that caused by the standard anti- inflammatory drug, piroxicam (0.5 mg/kg, i.p) 76.1 %. And EAF caused less inhibition (64.2 %) than piroxicam. 3.5.1 Effects of AHF1, AHF2 and AEF1 on egg albumen-induced (acute) oedema in rats The results of effect of AHF1, AHF2 and AEF1 on egg albumen- induced oedema in rats are shown in Table 18. At 30 mg/kg (i.p), AHF1, AHF2 and AEF1 produced very high significant inhibition of oedema, 78%, 72% and 66% respectively, than aspirin (100 mg/kg) 46% inhibition.
3.5.2 Effect of AHF1, AHF2 and AEF1 on formaldehyde-induced (chronic) oedema in rats. At 30 mg/kg, i.p, AHF1, AHF2 and AEF1 showed high significant inhibition of oedema: 79%, 72% and 65% respectively. The values indicated xcviii higher anti-inflammatory effect than indomethacin which has 48% inhibition (Table 19).
3.6 Isolation and solvent fractionation: Of the isolated fractions, hexane fraction (HEF), Ethyl acetate fraction (EAF), and methanol fraction (MAF), significant antinociceptive and anti- inflammatory effects were exhibited by HEF and EAF. This is shown in Table 15, Table 16 and Table 17. Therefore, HEF and EAF were selected for further studies.
3.6.1 Percentage yield of fractions and their phytochemical constituents
Fractions (g) yield (% w/w) phytochemical constituents HEF 4.10 x 100 = 20.5 steroids, terpenes 20 AEF 2.30 x 100 = 11.5 Glycosides, flavonoids, 20 MEF 2.20 x 100 = 11.0 Tannins, alkaloid, saponins, 20 glycosides
3.7.0 Isolation and characterization of the bioactive constituents: Bioactivity guided separation of HEF and EAF led to the isolation of AHF1(65 mg) AHF2, (60 mg) and AEF1 (60 mg) respectively as the active antinociceptive and anti-inflammatory constituents. AEF2 (45 mg) was also isolated from EAF.
Table 13: Effect of the methanol extract of L. arboreus on egg albumin-induced acute inflammation in rats.
xcix
Treatment (mg/kg) i.p Mean displaced volume (ml)
0 h 0.5 h 1 h 2 h 3 h 4 h Extract (30) 0.82±0.02 1.30±0.01 1.13±0.02 1.05±0.09 0.95± 0.10* 0.91± 0.07* (25.40) (50.80) (63.50) (72.40) (81.10) Extract (60) 0.79±0.06 1.15±0.05 1.05±0.05 0.98±0.03 0.88± 0.03 0.83± 0.05 (42.90) (58.80) (69.90) (80.90) (91.50) Piroxicam (0.5) 0.91±0.03 1.50±0.04 1.35±0.07 1.08±0.05 1.08± 0.05 0.98± 0.05 (06.40) (30.20) (73.10) (73.80) (85.10) Normal saline 0.70±0.03 1.33±0.02 1.33±0.02 1.33±0.02 1.17± 0.03 1.17± 0.03 (0.5 ml) h = time in hours * P<0.05 n = 5 per group, values significantly lower than the negative control. Values in parenthesis represent percentage inhibition of oedema
Table 14: Effect of the methanol extract of L. arboreus on formaldehyde- induced (chronic) inflammation/arthritis in rats. c
Treatment (mg/kg) i.p Displaced volume (ml)
0 h 0.5 h 1 h 2 h 3 h 4 h Extract (30) 0.82±0.04 0.47±0.05 0.34±0.03* 0.36±0.05* 0.32±0.80** 0.31± 0.03*. (40) (62) (61) (65) (68) Extract (60) 0.78±0.05 0.45±0.04 0.32±0.06* 0.33±0.06* 0.31± 0.16* 0.30± 0.03* (43) (64) (65) (67) (69) Piroxicam 0.89±0.06 0.48±0.05 0.30±0.03* 0.28±0.05 0.29± 0.40 0.26± 0.05* (0.5) (39) (67) (69) (69) (73) Normal 0.71±0.02 0.79±0.01 0.91±0.02 0.93±0.02 0.93± 0.09 0.96± 0.08 saline (0.5 ml)
h = time in hours n = 5 per group, * P<0.05, values significantly lower than the negative control. Values in parenthesis represent percentage inhibition of odema
Table 15: Effect of solvent fractions, AHF1 and AHF2 and AEF1 in mouse writhing model of pain ci
Treatment (mg/kg, i.p) No of writhes Inhibition %
Control (water) 49.23±5.0 - Diclofenac (50) 18.12+3.7* 63 HEF (60) 13.12±3.2** 73 EAF (60) 17.70±3.5* 64 MEF (60) 37.65±3.1 24 AHF1 (30) 12.30±3.2** 75 AHF2 (30) 14.24±3.1** 71 AEF1 (30) 14.16+3.2** 71
n = 6 – 8 per group * p<0.05, **p<0.01, n/grp= number per group (given) values significantly different from the negative control.
Table 16: Effect of solvent fraction on egg albumin induced (acute) oedema in rats
cii
Treatment (mg/kg) i.p Mean volume displaced
1 h 2 h 3 h 4 h HEF (60) 0.24+0.07** 0.19+0.06** 0.16+0.06** 0.16+0.08** (72) (78) (78) (79) EAF(60) 0.64±0.09 0.52±0.07* 0.44±0.09* 0.45±0.07* (27) (42) (40) (40) MEF (60) 0.78±0.08 0.74±0.09 0.65±0.08 0.64±0.07 (11) (17) (12) (14) Aspirin (100) 0.78±0.10 0.55±0.12 0.40±0.12 0.45±0.13* (11) (38) (45) (40) Normal Saline 0.88±0.04 0.90±0.06 0.74±0.09 0.75±0.08 (0.4 ml)
*p<0.05, **p<0.01, n=5 per group, values significantly different from the negative control. Values in parenthesis represent percentage inhibition of oedema
Table 17: Effect of solvent fraction on formaldehyde- induced (chronic) oedema in rats. ciii
Treatment (mg/kg) i.p Mean volume displaced(ml)
1 h 2 h 3 h 4 h
HEF (60) 0.14±0.06** 0.13±0.05** 0.13±0.05** 0.12±0.04** (83.7) (85.3) (84.5) (85.7) EAF (60) 0.78±0.10 0.64±0.11 0.62±0.11 0.30±0.03* (09.3) (28.0) (26.1) (64.2) MEF (60) 0.79±0.03 0.77±0.04 0.77±0.06 0.76±0.05 (10.4) (8.9) (3.5) (4.7) Piroxicam (0.5) 0.77±0.02 0.6±0.06 0.51±0.02* 0.20±0.02** (10.3) (31.9) (39.2) (76.1) Normal Saline (0.4 ml) 0.86±0.04 0.89±0.06 0.84±0.09 0.84±0.07
*p<0.05, **p<0.01, n=5 per group, values significantly different from the negative control. Values in parenthesis represent percentage inhibition of oedema
Table 18: Effect of AHF1, AHF2 and AEF1 on egg albumin - induced (acute) paw oedema in rats
civ
Treatment (mg/kg) mean volume displaced (ml)
1 h 2 h 3 h AHF1 (30) 0.24±0.06**(73) 0.19±0.07**(78) 0. 16±0.07*** (78) AHF2 (30) 0.23+0.11**(73) 0.22+0.04 (75) 0.20+0.06*** (72) AEF1 (30) 0.44±0.09* (50) 0. 32±0.07*(64) 0.24±0.05** (66) ASA (100) 0.78±0.11 (11) 0.55±0.12*(38) 0.40±0.13* (46) Normal Saline 0.88±0.03 0.90±0.05 0.74±0.07 (0.4 ml)
* P<0.05, ** P<0.01, *** P<0.001, n= 5 ASA = Acetyl salicylic acid, values significantly different from the negative control; Values in parenthesis represent percentage inhibition of oedema.
Table 19: Effect of AHF1, AHF2 and AEF1 on formaldehyde- induced (chronic) oedema in rats cv
Treatment (mg/kg) i.p Means oedema (ml)
1 h 2 h 3 h AHF1 (30) 0.18±0.01**(74) 0.17±0.03**(76) 0. 15±0.02*** (79) AHF2 (30) 0.23±0.03**(67) 0. 22±0.04**(69) 0.20±0.03*** (72) AEF1 (30) 0.29±0.03**(58) 0. 27±0.04**(61) 0.25±0.04** (65) Indomethacin (5) 0.38±0.05*(45) 0.38±0.02*(46) 0.37±0.05* (48) Normal Saline 0.70±0.05 0.71±0.07 0.71±0.08 (0.5 ml)
* p<0.05, ** p<0.01, *** p<0.001, n= 5, values significantly different. Values in parenthesis represent percentage inhibition of oedema.
3.8 Elucidation of the structures of the isolated active constituents 3.8.1 AHF1: White amorphous solid: m.p:138 cvi
CHCl UV λ 3 max nm(ε):242 (31.8), 272 (30.6) kBr -1 IR V max Cm : 2950-2849, 1700, 1429, 1369, 1150, 815, 820 1 H-NMR(CDCl3, 200 MHz):δ4.05(1H t, J=5.69 Hz, H- 23) δ 0.72 (3H-s, Me-18), δ0.87(3H t, Me-29), δ0.95(3H d, Me- 27), δ1.00(3H d, Me-26), δ1.18 (3H d, Me-21), δ2.10(1H, s,H- 5)
13 C-NMR (CDCl3, 50MHz): δ99.4(C-23 and C-24) δ206.8(C-3 and C-6). AHF1 was elucidated as stigmastene 3, 6-dione.
0 0
Figure 4: Chemical structure of AHF1 (stigmastene 3, 6-dione).
cvii
3.8.2 AHF2: White crystalline solid: m.p:202 CHCL UV λ 3 max nm(ε):227.2 (1.321), 362.6 (0.07), 493.4(0.034), 3267(OH), kBr -1 IR υ max cm : 3300-2600 (broad, OH), 1678 (C=O), 1287, 1266, 1248, 1231, 1179, 1024 and 922, 1 H-NMR(CDCl3, 200 MHz):δ5.15(1H, bs),
4.49(1H,t, J=7.4(HZ), 2.01 (3Hs-OCOCH3), 1.60(3H s),
0.96(6H s), 0.83 (9H,s) and 0.76 (3H, s).
AHF2 was elucidated as ursolic acid. COOH
H0
Figure 5: Chemical structure of AHF2 (ursolic acid)
cviii
3.8.3: AEF1:Yellow semisolid EtoH UV λ max nm:257, 302sh, 352 kBr -1 IR V max cm : 3500- 3200(OH), 2900, 2800, 1650(c=O) 1600(aromatic €=c),1280, 1150, 750,730 (aromatic)
NMR(CD3OD, 200 MHz):δ6.20(1H d, J=2.2Hz, H-6) δ6.37(1H d, J=2.2Hz, H-8) δ7.34(1Hd, J=2.2Hz, H-21),
1 δ7.31(1Hdd, Jab=8Hz, Jac=2.2Hz, H-4 ), δ6.93(1H dd, J=2.2H, J=8.8Hz, H-5”), δ7.76(1H, d, J=8.8Hz, H-61), δ5.34(1Hd, J=1.4, H-111), δ8.5(1H dd, H-2”), δ4.2(1H dd, H-3”), δ3.7(1H dd, H- 4”), δ3.30(1H m, H-5”), δ0.93(1H d, J= 5.8Hz, Me-6”) AEF1 was elucidated as Tetrahydroxyflavone-3α-rhamnoside
0H
H0 0 0H
0H 0 0 0 0H 0H CH 3 Figure 6: Chemical structure of AEF1 (Tetrahydroxyflavone -3-α- rhamnoside)
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CHAPTER FOUR DISCUSSION AND CONCLUSION 4.1 Discussion There has been a growing interest in the treatment of pain and inflammation by traditional medicine practitioners using Lupinus arboreus in liquid form which hitherto has enjoyed recognition as an ornamental plant for environmental beautification (Ohadoma, et al., 2010). Although this species has a wide usage in ethnomedical management of pain and inflammatory disorders, there is however, no scientific report validating these activities of Lupinus arboreus.
Acute toxicity study on the methanol extract in mice has established an intraperitioneal LD50 of 84. 85 mg/kg. The phytochemical constitutents of the methanol extract (CME) and fractions HEF and AEF were identified as proteins, carbohydrates, tannins, resins, alkaloids, saponins, glycosides, flavonoids, steroids and terpenes. The composition of fractions HEF and AEF are less complex being made up of only steroids, terpenes; flavonoids and glycosides. The active constituents AHF1, AHF2 and AEF1 contained steroids, terpenes, flavonoids and glycosides. Some flavonoids however, are potent prostaglandins inhibitors as well as inhibitors of phosphodiesterases (Manthey et al., 2001). Generally, these typical plant constitutents are known to be biologically active, provoking a vaerity of pharmacological actions such as antinociceptive and anti inflammatory effects (Collier et al., 1968; Bentley et al., 1983; Mansour and Watson, 1993; Jensen, 1997; Ahmadiani et al., 1998, 2000; Miller, 2006; Galli et al., 1999; Turner, 1965).
From this study, the leaf extract exhibited antinociceptive effect in hot plate, writhing and tail immersion tests, which is indicative that both spinal and supraspinal mechanisms may be involved. Thermal pain is mediated via the cx supraspinal level (Campos et al., 2002); the activation of the µ-opioid receptors mediates antinociceptive effects (Shah et al., 1994); the spinal and supraspinal mechanism of pain are mediated by mµ and sigma-δ receptors. The characterization of AHF1, AHF2 and AEF1, had revealed that the isolated active constituents are stigmastene 3, 6- dione (a stigmast steroid), ursolic acid (a triterpene steroid) and tetrahydroxyflavone 3-α-rhamnoside (a flavonol glycoside) respectively. Stigmastene 3, 6 – dione was crystallized as creamy white amorphous solid, melting point 138 0C and tested positive for steroid (Harborne, 1998). IR spectrum (1369 and 1150) indicating isopropyl moiety and NMR spectra (3 H, d) indicating ethyl group strongly suggests the presence of stigmast nucleus (Gaspar and Das Neves, 1993; Miles et al., 1994). The ketone nature of the compound was evident from the strong IR 1 absorption at υmax 1700; there was no H-NMR evidence for aldehyde and no IR band for hydroxyl group. The physical data of active constituent agree well with that reported for the 3, 6- dione (Gaspar and Das Neves, 1993; Miles, 1994).
Ursolic acid was crystallized as white crystalline solid, melting point 202 OC and tested positive for steroid and terpenes (Harborne, 1998). IR spectrum (3300-2600) indicating broad and hydroxyl group NMR spectra 3H,s strongly suggest a triterpenoid nucleus. The physical data of this active constituent agree well with that of a triterpene hydroxyl acid reported for ursolic acid (Morah, 1985).
Tetrahydroxy flavones 3 - α- rhamnoside was purified from AEF and tested positive for flavonoid (Harborne, 1998). IR spectrum showed broad peak at 3500- 3200 cm-1 (OH group), 1650 (C=0), 1600 (C = C, aromatic). The presence of a methyl signal at δ 0.93, which coupled with H-5” indicated rhamnose thus, with physical data confirmed tetrahydroxy flavone 3 -α- rhamnoside (Markham and Geiger, 1994).
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The results obtained in this study showed that the methanol leaf extract of Lupinus arboreus, HEF, AEF, AHF1, AHF2 and AEF1 possessed remarkable antinociceptive and anti-inflammatory effects. Positive antinociceptive effect is considered when the animals fail to respond to painful stimulus for a period corresponding to the pretreatment reaction time (PTRT) plus 4 seconds (Woolfe and Macdonald, 1964). The antinociceptive and anti-inflammatory activities were observed in both acetic acid-induced writhing, tail immersion, hot plate; formaldehyde, and albumin-induced oedema tests in animals. Hot plate test is a specific central antinociceptive test (Parkhouse and Plewry, 1979; Ramezani et al., 2001). Since pain and inflammation are characterized by the release of mediators like histamine, 5-HT and prostanoids (Damas et al., 1990; White 1999), the compounds may have inhibited the action of these mediators. In addition to the inhibition of arachidonic acid metabolism, these compounds have been reported to inhibit increase in vascular permeability (Pereira da Silva and Parente, 2001) as well as exhibit anticomplementary activity (Srivastava and Kulshreshta 1989). The antinociceptive and anti- inflammatory activities of stigmastene 3, 6 – dione is in harmony with steroids isolated from plants as shown in several studies to exhibit significant analgesic and anti-inflammatory activities (Shukla et al., 1986; Gupta et al., 1971; Froton et al., 1983; Zhang et al., 1999; Marva- Manger et al., 2008). Some stigmast steroids isolated from Vernonia colorata leaves, for example, have also been shown to exhibit anti- inflammatory activities (Cioffi et al., 2004).
Ursolic acid, a triterpene hydroxyl acid is biogenetically related to α- amyrin. The occurrence of ursolic acid as the major secondary metabolite of many plants such as Strychnos spinosa and Stachytarpheta indica is of interest (Morah, 1985). Ursolic acid is known and documented to have anti- inflammatory properties as well as a chain of other physiological activities (Morah, 1985).
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Tetrahydroxy flavones 3 -α-rhamnoside is a flavonol glycoside. It is well documented for its antinociceptive and anti-inflammatory activities (Oteiza et al., 2005). Various mechanisms have been adduced to the anti-inflammatory properties of flavonoids. These mechanisms derive from the ability of flavonoids to interact with cellular membranes in specific ways (Oteiza et al., 2005). Two possible revelant interactions are: (a) the partitioning of the polyphenol in the non-polar core of the membrane, associated with the hydrophobic nature of the flavonoid; and (b) the interaction of the hydrophilic flavonoids and oligomers with the polar headgroups of lipids at the lipid- water interface, mainly associated with the formation of hydrogen bond. These interactions significantly contribute to the antioxidant and membrane stabilizing properties of flavonoids (Oteiza et al., 2005). Antioxidant/free radical scavenging activity may in effect be another important mechanism by which AEF1 elicits its antinociceptive and anti-inflammatory activities. It is known that flavonoids can display antioxidant activity in numerous biological systems (Lotito and Fraga, 1998; Rice-Evans, 2001). This antioxidant activity has been attributed mainly to their capacity to scavange oxygen and reactive nitrogen species (Bors et al., 1990) and to chelate redox- active metals (Van Acker et al., 1998).
In the developing world, the search for alternative means of managing inflammatory disorders in general and rheumatoid arthritis in particular has led to extensive investigations into the world’s rich vegetation for newer and better anti-inflammatory agents.
From this study as well, the leaf extract of Lupinus arboreus showed antinociceptive and anti-inflammatory properties. This finding may not be unrelated to the presence of flavonoids (Hotellier et al., 1979), and steroids responsible for the antinociceptive and anti-inflammatory properties of many medicinal plants.
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4.2 Summary and conclusion There is a great deal of interest in the discovery of novel drugs in the management of pain and inflammatory disorders since these are among the most common and perhaps, difficult disease conditions to treat. Many promising drug candidates for pain and inflammatory disorders continue to be investigated by scientists all over the world. In addition to toxicity associated with most of the drugs that are of synthetic origin, drugs which are affordable to the generality of the people in developing countries remain elusive. This fact necessitates the need of exploiting alternative sources for lead analgesic and anti-inflammatory compounds, recognizing that natural products have been in use by man for centuries.
This present study therefore, validates the antinociceptive and anti- inflammatory properties of Lupinus arboreus leaves in animal models of pain and inflammation. The extract and fractions were screened for antinociceptive properties using different models of pain; and anti-inflammatory activity using both acute and chronic models of inflammation. Bioactivity- guided studies on the active fraction HEF and EAF, led to the isolation of antinociceptive and anti-inflammatory compounds AHF1-a stigmast steroid (stigmastene 3, 6-dione), AHF2- a triterpenoid (ursolic acid) from HEF. And AEF1- a flavonol glycoside (tetrahydroxyflavone-3α- rhamnoside) from EAF.
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