*2.5. Antioxidant Activity of the Ethanolic Extract*

The nitric oxide (NO) scavenging activity of the extract was measured according to the method described by Mayur et al. [16]. In the wells of a 96-well microtiter plate, the ethanolic extract of *S. perfoliata* was allowed to react with an equal volume of sodium nitroprusside (10 mM) for 90 min at room temperature. To the wells of the test sample, 100 μL of Griess reagent was added and to the blank color control wells, dH2O was added. The final concentration of the extract ranged from 15.6 to 2000 μg/mL and. L-ascorbic acid was used as the positive control. The nitrite content was measured after 5 min at 546 nm and the percentage scavenging activity was determined.

To determine the DPPH scavenging activity 20 μL of extract, L-ascorbic acid (positive control) and 100% ethanol (negative control) were allowed to react with 90 μL of 90 μM DPPH (2,2-Diphenyl-1-picrylhydrazyl) methanolic solution in a 96-well microtiter plate. To the blank extract color control wells, DPPH was substituted with dH2O. The final concentrations of the sample and L-ascorbic acid ranged from 7.8 to 500 μg/mL and 1.6 to 100 μg/mL respectively. The plate was incubated for 30 min in the dark and the absorbance measured at 515 nm using a BIO-TEK Power Wave Multiwell plate reader (Analytical and Diagnostic Products, Weltevreden Park, South Africa).

The total antioxidant capacity was assayed by the phosphomolybdenum (PM) method [17].The extract and gallic acid were prepared to stock solutions of 5 mg/mL. In test tubes, solutions of 0.3 mL of the samples were added to 1 mL of 0.6 M sulphuric acid (H2SO4), 1 mL of 4 mM ammonium molybdate and 1 mL of 28 mM sodium phosphate (total of 3 mL reagent). The blank was prepared by adding 0.3 mL of methanol instead of the extract. The test tubes were incubated at 95 ◦C for 90 min. Absorbance was measured at 695 nm. The fifty percent inhibitory concentration (IC50) concentration was calculated for each of the antioxidant assays.
