4.4. DNA Nicking Assay

The DNA nicking assay was performed using Fenton’s reagent [30]. The reaction mixture was prepared in triplicates by combining 5 µL Fenton’s reagent (41.5 mM pH 7.4 phosphate buffer, 0.2 mM FeSO₄, 980 mM H₂O₂), 0.5 µL pUC19 plasmid (1 µg/µL) and 4.5 µL of extract. Trolox (6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) and water were used as a positive and negative control, respectively. The concentrations of the extract added per reaction were 2.5, 5.0 and 10.0 µg/mL, and that of Trolox were 25, 50 and 100 µg/mL. The reaction mixtures were incubated at 37 °C for 30 min to allow the degradation of plasmid DNA by Fenton’s reagent. The above reaction mixtures were run at 1.5% agarose gel containing ETBR in TAE buffer (120 min at 50 V) to analyze the degree of DNA degradation. The gel image was visualized using a gel imaging system (ChemiDoc XRS+, Bio-Rad, CA, USA). The amount of nicked forms of pUC19 plasmid DNA was quantified using Image Lab 5.2.1 (Bio-Rad, CA, USA).