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

Oxidative stress is involved in several acute and chronic pathological processes due to its ability in activating inflammatory pathways [37,38]. The antioxidant activity of plum polyphenolic compounds has been previously investigated in different system models, such as ABTS, DPPH, FRAP, and ORAC assays [5,16]. However, due to the absence of information on the scavenger activity of ROS in biological models, we investigated the ability of PEP in reducing intracellular oxidative stress. The experiment was carried out using similar concentrations of PEP to those used in the antibacterial activity assay (1 mg/mL). This concentration did not significantly affect cell viability at 24 h after treatment (data not shown). As shown in Figure 2, HT-29 cells, exposed to TBHP, increased the ROS level (oxidative control). Pre-treatment of intestinal cells with PEP showed a significant (*p* < 0.05) reduction in cellular ROS generation, stimulated by TBHP, compared with the oxidative control, except when the cells were pre-treated with VD 40 ◦C sample. VD 80 ◦C and SPD caused the highest protection against oxidative damage in stressed cells, inducing an inhibition percentage of ROS production close to 37% respect to the oxidative control cells. Although, FD and VD 60 ◦C had a significant antioxidant activity with respect to the experimental control, this was lower than the samples obtained with treatments carried out at higher temperatures. Previously, others have shown that drying processes can influence the antioxidant capacity of the dried products, observing an increase in the antioxidant capacity of plum extracts dried by microwave vacuum at high temperatures [3]. Apparently, the temperature applied during plum juice drying is related to the increase in the content of polyphenolic compounds able to scavenge ROS, as the highest values of those compounds were noted after drying at 80 ◦C. In this regard, the drying temperature of the PEP and the subsequent polyphenolic composition affected the antioxidant capacity of the obtained powders, therefore, the extracts exposed to higher temperatures during the drying process showed a higher content in the methyl 3-caffeoylquinate (Table 2) and significantly improved antioxidant activities. It is well known that phenolic acids can prevent oxidative damage because of their ability to scavenge ROS [39]. Within the phenolic acids, chlorogenic acid and their derivatives, such as methyl 3-caffeoylquinate acid, act as potent ROS scavengers by donating hydrogen atoms to reactive molecules, transforming them to less active radicals, and maintaining an optimal cellular oxidative balance [40–42].

**Figure 2.** Protective effect of PEP (1 mg/mL) on intracellular ROS production. HT-29 cells were incubated with the powders for 24 h and then treated with 2.5 mM TBHP for 3 h, and ROS production was determined. Values are expressed as a percentage relative to the control conditions and are represented by mean ±SD (*n* = 3). Bars with different letters indicate significant differences on ROS production by ANOVA post hoc LSD Tukey test (*p* ≤ 0.05). Freeze drying (FD); Vacuum drying 40 ◦C (VD 40 ◦C), 60 ◦C (VD 60 ◦C), 80 ◦C (VD 80 ◦C); Spray drying (SPD).

Our findings show that PEP could induced cellular protection against TBHP-induced oxidative stress by preventing intracellular ROS accumulation. However, the level of antioxidant capacity of the PEP may be determined by the conditions applied during the drying process.
