14 Butylated hydroxy anisole (BHA) (Himedia, India) was used as standard. The extract in methanol was tested at 20–250 μg/ml. DPPH solution was used at 20 μmol/l. DPPH dilution with methanol without extract was control. Percentage of scavenging was calculated as follows, DPPHscavengingactivity(%)=[(Acontrol−Asample)/Acontrol]×100 The data was presented as mean of triplicate. The concentration required for 50% reduction of DPPH radical (IC50) was determined graphically. Lipophilic antioxidants in the extract was measured Selleckchem Gefitinib using β-carotene–linoleic acid system.15
The extract and quercetin in DMSO were tested at 100 μg/ml, 500 μg/ml and 1000 μg/ml. Total reaction volume was 3 ml. The absorbance was recorded at 470 nm at regular time intervals from 0 to1500 min. The control contained 0.2 ml DMSO without extract. The reagent without β-carotene was served as blank. The data is presented as mean of triplicate readings. The antioxidant activity (AA) was expressed as percentage inhibition and calculated using the following equation: AA(%)=[(Degradationrateofcontrol−degradationrateofsample)/Degradationrateofcontrol]×100where
degradation rate = ln (a/b) × 1/t, where ln = natural log, a = initial absorbance (470 nm), b = absorbance (470 nm) after time ‘t’ (in min). A modified thiobarbituric acid 17-AAG clinical trial reactive species (TBARS) assay was used.9 The extract and quercetin were tested at 60 μg/ml, 120 μg/ml, and 600 μg/ml in 250 μl aliquots. The absorbance was measured at 532 nm. The reaction without extract or quercetin served as the control. The test blank contained linoleic acid emulsion without peroxidation treatment. The assay was carried out as described previously with modifications.16 10 μl of extract or quercetin dilutions of 100 μg/ml, 200 μg/ml and 500 μg/ml concentrations incubated for 30 min with 5 μl of calf thymus of DNA (Genei, India. 1 mg/ml) treated with Fenton reagent. Then, the reaction was terminated by adding 30 μl loading buffer (2.5 μg/ml bromophenol blue, 60% sucrose in 1 ml TBE buffer 10 mmol/l and pH 8.0) and 15 μl of which was electrophoresed at 60 eV potential for 30 min in submerged 1% agarose gel. The intact bands without shearing in
the electrophoretogram indicates the DNA protection. HPLC was performed using analytical HPLC system (Agilent Technologies assembled 1100 and 1200 series) equipped with quaternary pump and UV–visible detector. Reversed phase chromatographic analysis was carried out in isocratic conditions using RP-C18 column (4.6 mm × 250 mm) packed with 5 μm diameter particles. The separation was carried out in water-acetonitrile-acetic acid (80:20:3, v/v/v) as mobile phase at flow rate of 0.8 ml/min. Quercetin, gallic acid, 4-hydroxy benzoic acid, vanillic acid, epicatechin, ferulic acid, p-coumaric acid, phloroglucinol and chlorogenic acid (Sigma Aldrich, Germany) were used as reference standards at 300 ppm in methanol. The injection volume was 10 μl. Detection was done at 280 nm and 320 nm.