*2.6. Oxidative Stress and Antioxidant Ability Assays*

The oxidative stress endpoints measured, ROS content and GSH levels, were carried out following the methods described in Gutiérrez-Praena et al. (2012) [23]. The results of both assays were expressed as relative light units (RLU).

For the estimation of the protection and reversion abilities of extract, H2O2 100 μM was administered to induced changes in the cell membranes and antioxidant system in HepG2 and Caco-2 cells [31].

For the protection assay, after discarding the previous medium, exposure solutions (EC50/<sup>2</sup> and EC50/4) of the extract were first added to the cells and incubated at 37 ◦C for 24 h or 48 h. After the treatment time, the medium was discarded and then exposed to 100 μM H2O2 for 2 h. Similarly, Caco-2 and HepG2 were pre-treated with H2O2 for 2 h for the reversion assay and a later exposure of the extract for 24 or 48 h. Unexposed cells were included in the figures in order to compare the results with basal levels of ROS and GSH. A control of DMSO was also incorporated in all plates.

Then, both abilities were evaluated by measuring the ROS levels and GSH content as previously described.
