BIOCHEMICAL CHARACTERIZATION OF THE ANTIINFLAMMATORY CONSTITUENT AND ANTIOXIDANT ACTIVITY STUDIES ON STEM BARK EXTRACT OF CRATAEVA ADANSONII
In the last decade intensive research into the mechanisms of inflammation has implicated production of prostaglandins and reactive oxygen species as central to the progression of many inflammatory diseases. This study aimed at investigating/elucidating the biochemical mechanisms of antiinflammatory action and antioxidant activities of the bioactive phytoconstituents of stem bark extract of C. adansonii. The mineral contents, phytochemical analysis and oral acute toxicity (LD50) of the extract were determined. The extract was subjected to solvent-guided fractionation in a silica gel column successively eluted with n-hexane, ethylacetate, dichloromethane, and methanol (100%). The extract and fractions were subjected to biological activity guided studies for antiinflammatory activity using the egg-albumin induced rat hind paw-edema method as activity guide. The ethylacetate fraction was subjected to further separation in silica gel column eluted with graded mixtures of n-hexane and ethylacetate to obtain ethylacetate subfractions (EF1) and (EF2) a white crystalline solid (C.adansonii compound 1; CA1). The antiinflammatory activity of the isolate (CA1) was confirmed using the activity guide. The extract, bioactive fraction (EF) and isolated compound (CA1) were further subjected to antiinfammatory activity tests (formaldehyde-induced chronic inflammation test in rats, red blood cell membrane stabilization activity test and cyclooxygenase inhibition assays) and antioxidant tests (phosphomolybdate test for total antioxidant capacity, reducing power test, nitric oxide and hydrogen peroxide scavenging activity tests). The molecular structure of CA1 was established using nuclear magnetic resonance (NMR), infra-red, gas and mass spectrophotometers. The n-hexane, ethylacetate and methanol fractions tested positive to glycosides, terpenoids and flavones. Mrineral analysis of stem bark showed that it contained selenium, sodium, iron, zinc, calcium, potassium, manganese, and magnesium. Acute toxicity test on the extract gave an oral LD50 > 5 g/kg. Ethylacetate fraction caused the greatest inhibition of rat paw edema. Phytochemical tests on CA1 gave positive reaction to terpenoids. CA1 significantly (P < 0.05) suppressed acute edema. The ethylacetate fraction and CA1 significantly (P < 0.05) inhibited erythrocyte lysis. Analysis of the blood samples from formaldehyde-induced chronic inflammatory rats, indicated that the extract (200 and 500 mg/kg) caused non-significant (P > 0.05) difference in lymphocyte levels, total white blood cells, platelet count and % PCV compared with untreated control group, but exhibited significant decrease in % haemoglobin and % neutrophil levels. In relation to the baseline values, the extract exhibited significant (P < 0.05) increase in lymphocyte level, total white blood cells and platelet count, but significant decrease in % neutrophils level. The results on radical scavenging activities showed that ethylacetate, methanol and n-hexane fractions significantly (P < 0.05) exhibited reducing power. The total antioxidant capacity indicated that n-hexane and ethylactate fractions have significant (P <0.05) inhibition capacity. All the fractions exhibited significant (P < 0.05) percentage inhibition of in vitro hydrogen peroxide and nitric oxide scavenging activities when compared with vitamins E and C. Formalin-induced paw edema formations were significantly inhibited by the extract. The mean decrease in serum zinc was statistically significant in relation to control and the baseline values. Structural elucidation indicated that CA1 is lupeol with M.W. of 426.70 g, molecular formular of C30H50O and melting point of 215°C. Lupeol at the tested concentrations significantly (P < 0.05) and selectively inhibited cyclooxygenase-2 enzymes.
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