ROS levels were assessed using 2′,7′-dichlorofluorescin diacetate (H2DCFDA). The extract was incubated with 5 μM H2DCFDA for 30 min at 37 ± 1 °C. ROS quantity was monitored by fluorescence (excitation wavelength of 350 nm and an emission wavelength of 600 nm).
The activity of the antioxidant enzymes catalase (CAT) (Sigma-Aldrich Co., St Louis, MO, USA), Superoxide dismutases (SOD) (19160, Sigma, St Louis, MO, USA), and glutathione S-transferases (GST) (CS0410, Sigma St Louis, MO, USA) were measured following the kit instructions. The level of ROS and the antioxidant activity enzyme was detected using a microplate reader (Bio-Rad Laboratories Inc., Hercules, CA, USA) [35 (link)]. For each sample, 3 replicates were performed.