Hydroxyl
radical scavenging activity (HRSA) of the extractives was determined by the
method of Klein et al. [30]
with a slight modification. 0.5 ml of extractives/standard at different
concentration was taken in test tubes. 1 ml of Fe-EDTA solution (0.13% ferrous
ammonium sulphate and 0.26% EDTA), 0.5 ml of 0.018% EDTA solution, 1 ml of
0.85% DMSO solution and 0.5 ml of 22% ascorbic acid were added into the test
tubes. The test tubes were capped tightly and warm at 85°C for 15 minutes into
the water bath. After incubation, the test tubes were uncapped and 0.5 ml ice
cold TCA (17.5%) was added to each of test tubes immediately. 3 ml of nash
reagent (7.5 gm of ammonium acetate, 300 μl glacial acetic acid and 200 μl
acetyl acetone were mixed and made up to 100 ml) was added to all the tubes and
incubated at RT for 15 minutes. Absorbance was taken in UV-spectrophotometer at
412 nm wave length. Percentage hydroxyl radical scavenging (% HRSA) activity
was calculated using the following equation,