*3.5. Antioxidant Assays*

The antioxidant ability of the extract was evaluated taking into account their capacity to protect the cells against a further exposure of H2O2 or their capacity to revert the damage induced by this substance after a previous exposure by measuring both ROS and GSH levels. No significant changes were recorded when cells were exposed to 0.3% of DMSO (data not shown).

The results showed that the extract was able to protect HepG2 cells against an induced oxidative stress at all concentrations studied, showing a marked decrease of ROS content at both treatment times (Figure 7A). Similarly, after the pre-treatment with H2O2 for 2 h, the extract presented a greater reversion role in a concentration and time-dependent manner. This reduced ROS content even lower than basal levels at the highest concentrations tested for 24 h and after 48 h of exposure of all studied concentrations (Figure 7B). By contrast, in both protection and reversion assays, GSH levels of the hepatic cells were not affected by the administration of 7.8 μg/mL during 24 h and 5.1 μg/mL for 48 h of the extract, while they experienced a significant increase when they were exposed to the highest concentration (Figure 7C,D). After the pre-treatment with 15.6 μg/mL and 10.3 μg/mL during 24 h and 48 h respectively and a later exposure of H2O2, the results showed higher GSH levels than basal content.

**Figure 7.** ROS (**A**) and GSH content (**C**) in HepG2 cells first pretreated with 0–15.59 μg/mL or with 0–10.28 μg/mL of the stilbene extract (45%) for 24 h or 48 h, respectively, and a later exposure to H2O2 100 μM for 2 h. ROS (**B**) and GSH (**D**) content in HepG2 cells exposed to H2O2 100 μM first and a later 24 h or 48 h-treatment with 0–15.59 μg/mL or 0–10.28 μg/mL, respectively. Cells exposed to H2O2 100 μM 2h were used as control. All values are expressed as mean ± SD. Differences were considered significant compared to the control group from *p* < 0.01 (\*\*) and *p* < 0.001 (\*\*\*).

The cell line Caco-2 presented lower protection and reversion capacity when compared to the effects observed in HepG2 cells. In the protection assay, the extract was able to significantly reduced ROS content with respect to the control group treated with H2O2 at all concentrations assayed after both pre-treatment times (Figure 8A). Similar results were obtained when Caco-2 cells were exposed to H2O2 prior the extract (Figure 8B). At the highest concentrations assayed ROS content was reduced down to basal levels. With respect to GSH levels, in both reversion and protection assays, GSH content were higher than basal levels after both times of treatment at the highest concentration assayed. The results showed a significant increase between control group with H2O2 treatment and those exposed to the highest concentration tested of the extract for 24 h and 48 h prior H2O2 (Figure 8C). Moreover, after the exposure to H2O2 for 2 h, only Caco-2 cells treated with 22.9 μg/mL during 24h or with 19.5 μg/mL for 48 h presented a significant increase of GSH levels (Figure 8D).

**Figure 8.** ROS (**A**) and GSH content (**C**) in Caco-2 cells first pretreated with 0–27.88 μg/mL or with 0–19.51 μg/mL of the extract for 24 h or 48 h, respectively, and a later exposure to H2O2 100 μM for 2 h. ROS (**B**) and GSH (**D**) content in HepG2 cells exposed to H2O2 100 μM first and a later 24 h or 48 h-treatment with 0–27.88 μg/mL or 0–19.51 μg/mL respectively. Cells exposed to H2O2 100 μM2h were used as control. All values are expressed as mean ± SD. Differences were considered significant compared to the control group from *p* < 0.01 (\*\*) and *p* < 0.001 (\*\*\*).
