*2.10. Antioxidant Activity Against Intracellular Reactive Oxygen Species (ROS) Production*

Human intestinal epithelial cell line HT-29 was used for the evaluation of oxidative stress. Intracellular ROS were measured by the DCFH-DA assay as previously was reported by Martín et al. [24]. Cells were seeded (5 <sup>×</sup> <sup>10</sup><sup>4</sup> cells per well) in 24-well plates and grown until they reached 70% of confluence. Cells were pre-treated with plum powders (1 mg/mL) dissolved in serum-free medium for 24 h. After that, the cells were washed with PBS and incubated with 20 μM DCFH-DA for 30 min at 37 ◦C. Then, cells were washed twice with PBS to remove the unabsorbed probe and treated with serum-free medium, containing 2.5 mM tert-butyl hydroperoxide (TBHP). ROS production was immediately monitored for 180 min in a fluorescent microplate reader Synergy HT using a λex 485 nm and λem 530 nm. After being oxidized by intracellular oxidants, DCFH-DA changes to dichlorofluorescein (DCF) and emits fluorescence. The cells treated with TBHP was used as oxidative control (100% of intracellular ROS production). All samples were analyzed in triplicate (*n* = 3). All experiments were carried out between passage 10 to passage 30 to ensure cell uniformity and reproducibility. Data were expressed as percentage of fluorescence generation relative to negative control cells.
