Metal chelating activity was determined in EDTA equivalence.2.6.6. Nitric Oxide Radical Scavenging Activity The procedure is based on the method, where sodium nitroprusside in aqueous solution at physiological pH spontaneously generates Vismodegib price nitric oxide, which interacts with the oxygen to produce nitrite ions that can be estimated using Griess reagent. Scavengers of nitric oxide compete with oxygen leading to reduced production of nitrite ions. The nitric oxide scavenging activity of different solvent extracts of C. dipsaceus on nitric oxide radical was measured according to the method of Sreejayan and Rao [28]. Sodium nitroprusside (10mM) in phosphate buffered saline, was mixed with different concentrations (50�C250��g) of C. dipsaceus extracts and incubated at room temperature for 150min.

Griess reagent (0.5mL) containing 1% sulphanilamide, 2% H3PO4, and 0.1% N-(1-naphthyl) ethylene diamine dihydrochloride was added to the mixture after incubation time. The absorbance of the chromophore formed was read at 546nm. BHT and rutin and the same mixture of the reaction without C. dipsaceus extracts were employed as positive and negative control. Radical scavenging activity was expressed as the inhibition percentage of free radical by the sample and was calculated using the following =(Control??OD?Sample??ODControl??OD)��100.(2)2.6.7.?formula:%radical??scavenging??activity Assay of Superoxide Radical (O2??) Scavenging Activity The ability to inhibit formazan formation by scavenging superoxide radicals by the extracts was studied by the method of Beauchamp and Fridovich [29].

Each 3mL reaction mixture (50mM sodium phosphate buffer (pH 7.6), 20��g riboflavin and 12mM EDTA, and 0.1mg NBT) with extracts in each test tubes was illuminated for 90s. Illuminated reaction mixture served as negative control, while the mixture without extract in dark was taken as blank. Immediately after illumination, the absorbance was measured at 590nm. The activity was compared to BHT and BHA. The percentage inhibition of superoxide anion generation was calculated using the following formula:%inhibition=(??Control??OD?Sample??ODControl??OD)��100.(3)2.6.8. Phosphomolybdenum Assay The antioxidant activity of samples was evaluated by the phosphomolybdenum method [30]. Sample solution was combined with 1mL of reagent solution (0.6M sulphuric acid, 28mM sodium phosphate, and 4mM ammonium molybdate).

The reaction mixture was incubated in a water bath at 95��C for 90min. After cooling to room temperature, the absorbance of the mixture was measured at 765nm against a blank. The results were reported in ascorbic acid equivalents per gram AV-951 extract (AEAC).2.7. Statistical AnalysisThe results were statistically analyzed and expressed as mean (n = 3)�� standard deviation. Values are analyzed by Duncan’s multiple test range (SPSS, ANNOVA statistical software, TULSA, USA).3. Result and Discussion3.1.