Tramethylchroman- 2carboxylic acid) is used as a standard in comparison for the determination of the antioxidant activity of a compound. The results are also reported as the Trolox equivalent antioxidant capacity (TEAC), which is the molar concentration of the Trolox giving the same percentage decrease of absorbance of the ABTS as 1 mg/ml of the antioxidant testing extract, at a specific time point [21].2.5. DNA strand scission assaymutation is an ochre mutation, TAA, in the hisG gene which can be reverted by all six possible base-pair changes; both FCCP mechanism of action transitions and transversions. This mutation is also reverted by mutagens that cause oxidative damage [25].2.7. S9 preparationDNA damage and DNA protecting activity of extracts were detected on pBluescript KS DNA vector. Plasmid DNA was amplified and extracted from E.coli DH5a then oxidized with H2O2 +UV treatment in the presence or absence of the tested extracts and checked on 0.7 agarose in 1X TAE buffer (2 M Tris, 1M Sodium Acetate, 50 mM EDTA, pH = 8) according to the method described by Russo et al. [22] with some modifications. In brief, the experiments were performed in a volume of 9 l in an Eppendorf tube containing 2.34 g of plasmid DNA, H2O2 was added to a final concentration of 147 mM with and without 4 l of extracts at various concentrations. The reaction was initiated by UV irradiation and continued for 5 min on the surface of UV transilluminator (Bioblock Scientific, TF35 C, France) with intensity of 180W, at 254 nm under room temperature. After irradiation, the mixture was incubated at room temperature during 15 min. Finally, the reaction mixture along with gel loading PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28499442 dye was placed on 0.7 agarose gel for electrophoresis. Untreated pKS DNA was used as a control in each run of gel electrophoresis. Gel was stained with ethidium bromide and photographed with Bio-print (Vilbert lourmat, France).2.6. Bacterial strainsThe S9 microsome fraction is prepared from livers of rats treated with Aroclor 1254 [23]. The components of S9 mix were 8 mM MgCl2, 32.5 mM KCl, 5 mM G6P, 4 mM NADP, 0.1 M sodium phosphate buffer (pH = 7.4), and S9 fraction at a concentration of 0.68 mg/ml of mix. The S9 mix was prepared freshly for each assay.2.8. Salmonella-microsome assayOne hundred microliters of an overnight culture of bacteria (cultivated for 16h at 37 , approximate cell density (2-5) ?10 8 cells/ml) and 500 l of sodium phosphate buffer (0.2 M, pH 7.4 for assay without S9) or 500 l of S9 mix were added to 2 ml aliquots of top Agar (supplemented with 0.5 mM L-histidine and 0.5 mM D-biotine) containing different concentrations of each extract. The resulting complete mixture was poured on minimal agar plates prepared as described by Maron and Ames [23]. The plates were incubated at 37?C for 48 h and the revertant bacterial colonies of each plate were counted. Negative and positive control cultures gave numbers of revertants per plate that were within the normal limits found in the laboratory. An extract was considered mutagenic if the number of revertants per plate was at least doubled in S.typhimurium TA104 and TA 102 strains over the spontaneous revertant frequency [23,26]. Data were collected with a mean ?standard deviation of three plates (n = 3).2.9. Antimutagenicity testingSalmonella typhimurium strains TA102 and TA104 which are histidine-requiring mutants, were kindly provided by Pr.I. Felzen, (Universidade do Estado do Rio de Janeiro [UERJ], Rio de Janeiro, Brazil), and maintained as desc.
