3.6.3. Superoxide Radical (SO) Scavenging Activity Assay

The SO assay evaluated the ability of the tincture to inhibit the formation of the formazan by the reduction of the nitro blue tetrazolium (NBT) radical, by scavenging the superoxide radicals generated in the riboflavin-light-NBT system. The percentage of SO radical scavenging activity was calculated using the following formula: % Superoxide radical scavenging activity = (A₀ − A₁/A₀) × 100, where A₀ = absorbance of control (blank) and A₁ = absorbance of tincture. The superoxide anion radicals (SO) were generated in a mixture of 2.0 mL of Tris-HCl buffer (16 mm, pH 8.0) with 2.0 mL of nitroblue tetrazolium (NBT, 0.3 mm) and 2.0 mL nicotinamide adenine dinucleotide solution (NADH, 0.936 mm). Then, 0.1 mL tincture was diluted to 100.0 mL with water and 1 mL of this solution was added to this mixture. Next, 2.0 mL phenazine methosulfate solution (PMS, 0.12 mm) were then added to all this to initiate the reaction and the mixture was incubated at 250 °C for 5 min. Absorbance was measured at 560 nm using a blank prepared from 2.0 mL Tris-HCl buffer, mixed with 2.0 mL NBT and 2.0 mL NADH solution, 4.0 mL water and 2.0 mL PMS solution. As a reference, 4.0 mL of 1.152 mg/mL Trolox solution was used and the results were expressed as µm Trolox equivalents/g. This solution was added to 2.0 mL Tris-HCl buffer, mixed with 2.0 mL NBT solution, 2.0 mL NADH solution and 2 mL PMS solution (0.12 mm). All tests were performed in triplicate [53].