3.7.2. DCFH-DA Assay

In order to evaluate a potential antioxidant effect of the HI extract, three concentrations (25, 50, 75 μg/mL) that did not affect the cellular viability were selected and further tested on the two cancerous cell lines and on the normal HGF cells (Figure 6). The treatment with the HI extract alone modified the oxidative status of all three cell lines, with a statistical decrease of ROS being observed at the highest tested doses. Similar to the HI extract, NAC treatment alone led to a decrease in the basal oxidative status compared to the negative control. Exposure to H2O2 alone resulted in an approximately threefold increase of the signal when compared to the negative control, while pre-exposure to the antioxidant NAC decreased the ROS generation by approximately 50%. Pre-incubation with HI extract decreased the ROS in a dose-dependent manner, reaching statistical significance at the two highest doses of 50 and 75 μg/mL. In the case of T47D-KBluc cells, at the highest dose, the reduction of oxidative stress was almost equal to the one exerted by NAC.

**Figure 6.** Antioxidant effect of the HI extract evaluated using DCFH-DA assay on T47D-KBluc, A549, and HGF. The cellular model was pre-exposed to the extract (25, 50, and 75 μg/mL) or NAC (20 mM) for 24 h, and further incubated with 50 μM DCFH-DA. The antioxidant effect of the HI extract was assessed after 2 h from oxidative stress induced by exposure to 250 μM H2O2. The results are expressed as relative means ± standard deviations (six technical replicates for each of the three biological replicates) where the negative control (DMSO 0.2%) is 100%. (\*) indicates significant differences compared to H2O2 exposure alone; (#) indicates significant differences compared to negative control (ANOVA + Dunnett's; *p* < 0.05).

The obtained results are in agreement with the previously detailed antioxidant activity observed in the non-cellular assays, but due to insufficient data present in literature, no direct comparison was possible.
