2.8. Nitric Oxide (NO) Scavenging Assay

The nitric oxide scavenging assay was carried out as previously described [28]. First, 2 mL of sodium nitroprusside was mixed with 0.5 mL of phosphate buffer pH 7.4 and 0.5 mL of different concentrations of extract (0.0031–1.0 mg/mL). The mixture was incubated at 25 °C for 150 min, and an initial absorbance (A0) was taken at 540 nm. Thereafter, 0.5 mL of the incubated mixture was mixed with 1 mL of a sulfanilic acid reagent and 1 mL of naphthylethylenediamine dichloride (0.1% w/v), and incubated at room temperature for 30 min, before another absorbance (A1) was taken at 540 nm. The same reaction mixture without the extract but with the equivalent amount of methanol served as the negative control. Ascorbic acid and DDM at various concentrations were used as the standard. All of the experiments were in triplicates. The percentage nitrite radical scavenging activity of the extracts and standard were calculated using the following formula:

where A0 is the absorbance before the reaction, and A1 is the absorbance after the reaction.