Protective effect of Vitex nigundo on Freunds complete adjuvant induced arthritis by regulating antioxidant and inflammatory mediators

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The Vitex nigundo leaf powder was procured from local market and authenticated by Prof. A. B. Seerwani. Extract of V. nigundo was prepared by adding 1.0 g of powdered leaves to 25 ml ethanol: water (1:1) mixture. This mixture was left overnight at room temperature in dark and subsequently centrifuged for 30 min at 13000 xg. The supernatant was filtered and used as the test sample. 2.3. Total phenolic content The total phenol content (TPC) of the extract was estimated by the method of Singleton and Rossi (1965) using Folin-Ciocalteau reagent. This reagent oxidizes phenols forming molybdenum-tungsten blue. The total amount of phenolic compounds was calculated using propyl gallate (1 mg/ml) as standard. 2.4. Total triterpenoid content Total triterpenoid content (TTC) was estimated by the method of Chang and Lin (2011), using ursolic acid (1 mg/ml in methanol) as standard. 2.5. Total flavonoid content To determine the total flavonoid content (TFC), the aluminium chloride method was followed (Zhang et al., 2011). The method is based on the formation of flavonoid-aluminium complex, which shows absorption at 510 nm. Quercitrin hydrate (1 mg/ml in ethanol) was used as standard. 2.6. Free radical scavenging activity (FRSA) Free radicals scavenging activity was assessed by the method of Mellors and Tappel (1966). The method is based on the reduction of an ethanolic solution of 2, 2- diphenyl-1-p-picryl hydrazyl (DPPH) by hydrogen donating groups of antioxidant substance. BHT (0 -180 µg) was used as a positive control. Percentage of DPPH radical scavenging activity (%) was calculated using the following formula Radical scavenging activity (%) = (A0 – A1/ A0) × 100 where A0 and A1were the absorbance of control and test sample respec... ... middle of paper ... ...o, 2, 4 dinitrobenzene (CDNB) mediated by GST (Habig et al., 1974). 2.19. Determination of glutathione content Reduced GSH content was measured by the method of Beutler et al (1963) in tissue homogenate and whole blood. The method was based on the development of a stable yellow color with Ellman’s reagent due to the reduction of 5, 5,’ dithiobis 2- nitrobenzoic acid (DTNB) with GSH. 2.20. Protein carbonyl measurement Protein carbonyl content in liver and plasma was determined by the method of Levine et al., (1990). Carbonyl content was determined at 390 nm using molar absorption coefficient of 22000 M-1 cm-1. 2.21. Statistical analysis Microsoft Excel 2007 and the SPSS software package version 20.0 were used for statistical analysis. The mean values obtained for the different groups were compared by one-way ANOVA, followed by post hoc-LSD test.
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