2202 uv vis spectrophotometer

FRAP assay was carried out by the method of Benzie and Strain [57 (link)]. FRAP reagent was prepared by mixing acetate buffer, TPTZ solution and FeCl₃.6H₂O solution in the ratio of 10:1:1. About 3 ml of FRAP reagent was dispensed into the test tubes followed by addition of 300 μl of test sample. Test tubes were shaken to mix the content well and incubated at 37 °C for 10 min. Absorbance of the reaction mixture was taken at 593 nm (Systronics 2202 UV–Vis Spectrophotometer, India). Trolox was used as standard antioxidant. Increase in the absorbance of the reaction mixture as compared to control is considered as increase in the reducing potential of test sample.

A modified thiobarbituric acid reactive species (TBARS) assay [55 (link)] was performed to determine the lipid peroxides produced using egg yolk homogenate as lipid rich media [56 (link)]. Various concentrations of test sample were added to the test tubes containing egg homogenate (0.5 ml of 10% v/v). About 50 μl of FeSO₄ solution was added to the test tubes to induce lipid peroxidation and incubated test tubes at 37 °C for 30 min. After ½ hour, 20% acetic acid (pH 3.5), 0.8% of TBA in 1.1% SDS and TCA (20%) were added. All the contents of the tubes were mixed properly and heated at 95 °C for 1 h. After heating, test tubes were cooled, followed by addition of 5 ml of butanol and centrifuged at 1036 × g for 10 min. The absorbance was taken at 532 nm (Systronics 2202 UV–Vis Spectrophotometer). Trolox was used as antioxidant standard.
Inhibition of lipid peroxidation (%) was calculated using the formula as given below: $\mathrm{Radical}\mathrm{scavenging}\mathrm{activity}\left(\%\right)=\left(1‐\mathrm{E}/\mathrm{C}\right)\times 100$ where,
C is the absorbance of fully oxidized control,
E is the absorbance in the presence of test sample.

The assay is based on the reduction of the green ABTS•⁺ to colorless ABTS [14 (link)]. The ABTS stock solution (7 mM) was added to 2.45 mM potassium persulfate to generate ABTS cation by allowing the mixture to be oxidized for 15–17 h at room temperature in the dark before use. The oxidized ABTS cation solution was diluted with ethanol to an absorbance of 0.70 (± 0.02) at 734 nm. After this, 300 μL of the fraction was added to 1.0 mL of the ABTS cation solution. The decrease in the absorbance was read after 5 min using a spectrophotometer (Systronics 2202 UV–Vis spectrophotometer, Gujarat, India). The ABTS cation solution was taken as a blank. The standard antioxidant used was L-ascorbic acid. The antiradical activity of fractions of C. fistula was expressed as percentage inhibition of ABTS•⁺, which was calculated according to the formula mentioned below.

where A₀ is the absorbance of ABTS•⁺ solution + vehicle solvent (control); A₁ is the absorbance of the reaction mixture (containing the different concentrations of a fraction and ABTS•⁺ solution).