In vitro Antioxidant Analysis and the DNA Damage Protective Activity of Leaf Extract of the Excoecaria agallocha Linn Mangrove Plant - Agricultural Chemistry - page 158

of the reaction mixture was removed and 0.5 ml of Griess reagent (1 % (w/v) sulfanilamide, 2
% (v/v) H 3 PO 4 and 0.1 % (w/v) naphthylethylene diamine hydrochloride were added. The
absorbance of the chromophore formed was measured at 546 nm.
4.4. Lipid peroxidation and thiobarbituric acid reaction
A modified TBARS assay [8] was used to measure the lipid peroxide formed using egg yolk
homogenate as lipid rich media [9]. Egg homogenate (0.5 ml of 10 %, v/v) and 0.1 ml of
extract were added to a test tube and made up to 1 ml with distilled water, 0.05 ml of FeSO 4
(0.07 M) was added to induce lipid peroxidation and the mixture was incubated for 30 min.
Then, 1.5 ml of 20 % acetic acid (pH 3.5) and 1.5 ml of 0.8 % (w/v) thiobarbituric acid in 1.1
% sodium dodecyl sulphate were added, the resulting mixture was vortexed and then
heated at 95 °C for 1 h. After cooling, 5.0 ml of butanol was added to each tube and
centrifuged at 3000 rpm for 10 min. The absorbance of the organic upper layer was
measured at 532 nm. Inhibition of lipid peroxidation percent by the extract was calculated as
100- [ (A 1 /A 2 ) x 100]; where A 1 is the absorbance value in the presence of extract and A 2 of the
fully oxidized control.
4.5. Determination of DPPH radical scavenging capacity
Quantitative estimation of the free-radical scavenging activity was measured by DPPH
assay [10]. The reaction mixture contained a different concentration (100-2000g/l) of test
extract and 2.9 ml of DPPH (60 M) in methanol. These reaction mixtures were taken in test
tubes and incubated at 37 o C for 30 min, the absorbance was measured at 517 nm. The
percentage of radical scavenging activity by the sample treatment was determined by
comparison with the methanol treated control group. BHT and ascorbic acid was used as a
positive control. The DPPH radical concentration was calculated using the following
equation: scavenging effect (%) = (DPPH ∙ ) T / (DPPH ∙ ) T=0 x 100, where (DPPH ∙ ) T is the
concentration of DPPH ∙ at 30 min time and (DPPH ∙ ) T=0 , the concentration at zero time (initial
concentration).
4.6. Total antioxidant activity
The assay is based on the reduction of Mo (VI) to Mo (V) by the extract and subsequent
formation of a green phosphate / Mo (V) complex at the acid pH [11]. The tubes containing
0.1 ml of the extract and the 1 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium
phosphate and 4 mM Ammonium molybedate) were incubated at 95 o C for 90 min. After the
mixture was cooled to room temperature, absorbances were taken at 695 nm against the
blank. The antioxidant capacity was expressed as AAE.
4.7. DNA nicking induced by hydroxyl radical
The DNA damage protective activity of E. agallocha L. extract was performed using super
coiled pCAMBIA1301 DNA. Plasmid DNA isolation was done using GenElute TM Plasmid