4.5.3. Free Radical Induced DNA Damage

The protective effect of acetone extract (AE) and compounds (**1**–**14**) on oxidative DNA damage was evaluated as per the previous method [40]. A total of 2 μL calf-thymus DNA mixed with 5 μL of 39 mM Tris buffer (pH 7.4) and 5 μL (10 μg) acetone extract and compounds (**1**–**14**) (10 μg of 2 mg/mL solution dissolved in DMSO) mixture was incubated at room temperature for 20 min. The reaction was initiated by adding 5 μL FeCl3 (500 μM) and 10 μL H2O2 (0.8 M) and incubated for 10 min at 37 ◦C. The reaction was stopped by adding 3 μL DNA loading dye. Finally, the mixture was subjected to 0.8% agarose gel electrophoresis in TAE (40 mM Tris, 20 mM acetic acid and 0.5 M EDTA) buffer (pH 7.2). A total of 3 μL of Ethidium bromide was added to agarose solution to stain DNA bands. The image was viewed under transilluminating UV light and photographed (Bio-Rad, ChemiDocTM XRS, Hercules, CA, USA with Image LabTM software (ver. 6.0.1, build34, standard edition, 2017). The band intensity of the DNA was measured by using ImageJ software (ver. 1.4.3.67, Broken Symmetry Software, Scottsdale, AZ, USA).
