2.8. Determination of superoxide radical scavenging activity
This activity was measured using NBT (nitro blue tetrazolium
reagent) method as described by Sabu and Ramadasan (2002).
The method is based on generation of superoxide radical (O2
−)
by auto oxidation of hydroxylamine hydrochloride in presence of
NBT, which gets reduced to nitrite. Nitrite in presence of EDTA
gives a color that was measured at 560 nm. Test solutions of extract
(20–200g/ml) were taken in a test tube. To this, reaction mixture
consisting of 1ml of (50mM) sodium carbonate, 0.4 ml of (24mM)
NBT and 0.2 ml of 0.1mM EDTA solutions were added to the test
tube and immediate reading was taken at 560 nm. 0.4 ml of (1mM)
of hydroxylamine hydrochloride was added to initiate the reaction
then reaction mixture was incubated at 25 ◦C for 15 min and
reduction of NBT was measured at 560 nm. Ascorbic acid was used
as the reference compound. Decreased absorbance of the reaction
mixture indicates increased superoxide anion scavenging activity.
All the extracts of S. rebuadiana were treated in the similar manner,
absorbance was recorded and the percentage of inhibition was
calculated according to the following equation:
%inhibition = A0 − At
A0
× 100.
where A0 was the absorbance of the control (blank, without extract)
and At was the absorbance in the presence of the samples of the
extract. All the tests were performed in triplicate and the graph
was plotted with the mean values.