Nitric oxide (NO) scavenging assay

Nitric oxide (NO) scavenging activity was measured spectrophotometrically [16]. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generate nitric oxide, which interacts with oxygen to produce nitrite ions determined by the use of Griess reagents. A volume of 2 ml of 10 mM sodium nitroprusside and 0.5 ml of phosphate buffer saline (pH 7.4) were mixed with 0.5 ml of plant extract and ascorbic acid individually at various concentrations (500–3.9 µg/ml). The reaction mixture was incubated at 25 °C for 150 min. After 150 min, 0.5 ml of incubation solution was withdrawn and mixed with 1 ml of sulfanilic acid reagent (0.33 in 20% glacial acetic acid) and allowed to stand for 5 min at room temperature for completing diazotization. Then 1 ml of 0.1% w/v napthylethylenediamine dihydrochloride was added, mixed well and the mixture was incubated at room temperature for 30 min. The absorbance was taken at 540 nm. The amount of nitric oxide (NO∙) radical inhibited by the extract was calculated using the following equation:

where A_blank is the absorbance of the control reaction (containing all reagents except the test material).