Nitric oxide scavenging activity

Biologically, nitric oxide free radicals (NO.) are generated by the action of specific nitric oxide synthases that metabolize amino acids like arginine. In-vitro, sodium nitroprusside decomposes in aqueous solution at a physiological pH of 7.2 and releases NO free radicals that react with oxygen to produce stable nitrites and nitrates, which in turn, can be determined by Griess reagent⁴³. Here, 2 ml of 10 mM sodium nitroprusside dissolved in 0.5 ml phosphate buffer saline (pH 7.4) was mixed with 0.5 ml of various concentrations of Ag NPs (40, 80, 120, 160, and 200 μg/ml) suspended in 1:1 methanol and water. The mixture was then incubated at 25 ± 2 °C for 150 min and absorbance was measured at 546 nm for all the test samples that served as control. Then, 2 ml of the incubated solution was added to 2 ml of freshly prepared Griess reagent (equal quantities of 2% sulphanilamide in 1.47 M HCl and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in deionized water) and further incubated for 30 min. at room temperature. Absorbance at 546 nm was again measured after incubation with Griess reagent and NO inhibition was calculated as:

where A₀ is the absorbance before reaction and A_t is the absorbance after reaction with Griess reagent.

The results obtained were subjected to student’s ‘T’ test (to determine whether there is significant level of difference (‘p’ value) between the standard and the test samples) and regression analysis as well. From the regression equation, inhibitory concentrations were calculated.