Title page
Table of content
List of figures
List of tables
List of abbreviations
List of relevant publications from the thesis

1.1       Scientific background
1.2.1    Definition of pain
1.2.2    Type of pain Acute pain Chronic pain
1.2.3    Location and severity of pain
1.2.4    Demography of pain
1.2.5    Physiological pain Cutaneous pain Somatic pain Visceral pain Phantom limb pain Neuropathic pain
1.3.      Common causes of pain
1.4.0    Pain receptors and their stimulation
1.4.1    Neurotransmitters
1.4.2    Excitatory neurotransmitters
1.4.3    Inhibitory neurotransmitters
1.4.4    Specific neurotransmitters
1.4.5    Transmission of pain signals in the central Nervous system
1.5.0    Inflammation principles
1.5.1    Causes of inflammation
1.5.2    Pathophysiology of inflammation
1.5.3    Inflammatory exudates
1.5.4    Mediators of inflammation
1.5.5    Cells involved in inflammation
1.5.6    Molecular Mechanisms of inflammatory response Adhesion molecules Leukocyte mobility and chemotaxis
1.5.7    Disorders associated with pain and inflammation
1.5.8    Pain, inflammation and infection
1.6.0    Management of pain and inflammatory disorders
1.7.0    Quest for natural products
1.7.1    Management of pain and inflammation using natural products
1.7.2    Plants with promising analgesic and anti-inflammatory activities
1.7.3    Plant secondary metabolites with antinociceptive and anti-inflammatory effects Flavonoids with antinociceptive and anti-inflammatory activities Classification and nomenclature of flavonoids Pharmacological activities of flavonoids Mechanism of biological activities of flavonoids
1.7.4    Terpenoids and steroids with antinociceptive and anti-inflammatory effects Classification and nomenclature of steroids of plant origin Review methods of isolation and purification of steroids
1.8       Review of botanical profile of Lupinus arboreus
1.8.1    Taxonomy of Lupinus arboreus
1.8.2    Description and distinctive features of the plant
1.8.3    Geographical spread
1.8.4    Values of Lupine
1.9       Aim and scope of the work

2.1       Plant materials
2.2       Solvents and Reagents
2.3       Equipment
2.4       Animals
2.5       Methods
2.5.1    Extraction and concentration of plant materials
2.5.2    Determination of extractive yield
2.6       Phytochemical analysis
2.6.1    Test for carbohydrate
2.6.2    Test for alkaloids
2.6.3    Test for reducing sugar
2.6.4    Test for glycosides
2.6.5    Test for saponins
2.6.6    Test for tannins
2.6.7    Test for flavonoids
2.6.8    Test for resins
2.6.9    Test for proteins
2.6.10  Test for fats and oil
2.6.11  Test for steroids and terpenoids
2.6.12  Test for acidic compounds
2.7       Bioassay-guided isolation of the active constituents of L. arboreus
2.7.1    Column chromatographic separation of the methanol extract
2.7.2    Isolation and purification of the active constituents of hexane fraction (HEF)
2.7.3    Isolation and purification of the active constituents of ethylacetate fraction (EAF)
2.8       Pharmacological test
2.8.1    Acute toxicity and lethality test (LD50)and preliminary screening
2.8.2    Determination  of antinociceptive activities Thermally-induced pain (Hot plate test) in mice Acetic acid-induced pain (writhing reflex test) in mice Pressure-induced pain (tail immersion test) in rats
2.8.3    Inflammatory test Acute inflammation test (egg albumin–induced inflam mation) Chronic inflammation test (formaldehyde-induced inflammation)
2.8.4    The isolated active constituents Phytochemical analysis of fractions, AHF1, AHF2, AEF1   and AEF2 Determination of melting point IR spectral analysis UV spectral analysis GC-MS analysis of AHF1, AHF2 and AEF1 H-NMR (ID and 2D cosy) and C13-NMR analyses
2.9       Statistical Analysis

3.1       Extraction yield
3.2       Phytochemical analysis
3.3       Acute toxicity and lethality
3.4       Antinociceptive and anti-inflammatory effects
3.4.1    Antinociceptive           effect
3.4.2    Effect of the extract and fractions on egg albumin- induced (acute) oedema in rats
3.4.3    Effect of extracts and fractions on formaldehyde -induced (chronic) oedema in rats
3.5.1    Effect of AHF1, AHF2 and AEF1 on egg albumin-induced (acute) oedema in rats
3.5.2    Effect of AHF1, AHF2 and AEF1 on formaldehyde-induced (chronic) oedema in rats
3.6       Isolation and solvent fractionation
3.6.1    Percentage yield of fractions and their phytochemical constituents
3.7       Isolation and characterization of the bioactive constituents
3.8       Elucidation of the structures of the isolated active constituents
3.8.1    AHF1
3.8.2    AHF2
3.8.3    AEF1

4.1       Discussion
4.2       Summary and conclusion

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 not significant (p> 0.05). The oedema inhibition recorded by HEF and EAF were higher than the inhibition by aspirin (100 mg/kg). The CME (30 and 60 mg/kg) significantly inhibited formaldehyde- induced arthritis, in a dose-related, manner over a period of 4 hours (p< 0.05) (68 and 69 % inhibition respectively). Both HEF and EAF at 60 mg/kg i.p, significantly (p< 0.05) inhibited the oedematous response to formaldehyde-induced arthritis, causing 85.7 and 64.2 % inhibition respectively. The inhibitory effects of the isolates AHF1, AHF2 and AEF1 on egg albumin-induced (acute) oedema in rats (78; 72, and 66 % respectively) were significant and better than that of aspirin (100 mg/kg) (46 %). The effect of AHF1, AHF2 and AEF1, (30 mg/kg i.p) on formaldehyde-induced (chronic) oedema in rats were 79 %, 72 % and 65 % respectively. The isolated active compounds were identified as stigmastene 3, 6-dione (AHF1), ursolic acid (AHF 2), tetrahydroxyflavone-3a- rhamnoside (AEF1), and ellagic acid (AEF 2). In this study, the extract and fractions of L. arboreus leaves exhibited antinociceptive effect in different models of pain; and anti-inflammatory effects against both acute and chronic models of inflammation. The isolated compounds AHF1, AHF2 and AEF 1 appear to be responsible for the antinociceptive and anti-inflammatory effects. The compound AEF2 identified as ellagic acid, known for its antimicrobial activity, was concomitantly isolated. These compounds were isolated and characterized for the first time from L. arboreus.

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.

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....

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