**3. Results**

#### *3.1. Free-Radical Scavenging Activity of the Powders*

In the present study, 17 powders produced by encapsulation of an OMWW polyphenolic extract by spray drying under different conditions in whey protein, maltodextrin, and gelatin were tested for their free-radical scavenging activity against DPPH• and ABTS•<sup>+</sup> radicals. All of them were able to scavenge DPPH• with IC50 values ranging from 295 ± 18 to 660 ± 73 μg/mL (Table 2). Moreover, all the extracts demonstrated a potent ABTS•<sup>+</sup> scavenging capacity with IC50 values ranging from 290 ± 9 to 710 ± 68 μg/mL (Table 2).


**Table 2.** Free-radical scavenging activity against DPPH• and ABTS•<sup>+</sup> radicals, protective activity against peroxyl radical (ROO)-induced DNA damage, and reducing power of the powders.

a Values are the mean ± SD of at least two separate triplicate experiments. b Values are the mean ± SD from three independent experiments. \* *p* < 0.05, indicates significant difference from the control values. c RACI: Relative antioxidant capacity index. ND: Non-detectable at the tested concentrations.

#### *3.2. Protection from ROO*•*-Induced Plasmid DNA Strand Cleavage*

All the powders protected from ROO-induced DNA damage with IC50 values ranging from 520 ± 41 to 2300 ± 227 μg/mL (Table 2).

#### *3.3. Reducing Capacity of the Extracts*

The estimation of the extracts' reducing capacity was based on the reducing power assay by determining the RP0.5AU values, which ranged from 434 ± 30 to 890 ± 18 μg/mL (Table 2). Samples 9, 12, 13, 15, 16, 17, 18, and 19 did not show absorbance of 0.5 at 700 nm at the tested concentrations (Table 2).

#### *3.4. Determination of Extracts' Non-Cytotoxic Concentrations in EA.hy926 Cells*

Based on the results from DPPH•, ABTS•+, ROO-induced DNA damage, and reducing power, the two most potent of the powders were selected in order to examine their antioxidants e ffects in endothelial EAhy.926 cells. Specifically, the selection was made according to the RACI value that each powder had in all the above assays (Table 2). Since the RACI values were based on the IC50 values, the lower the RACI value the higher the antioxidant capacity. Thus, the two most potent samples were 3 and 9. However, before examining the powders' antioxidant e ffects in EAhy.926 cells, their cytotoxicity was assessed using the XTT assay in order to select non-cytotoxic concentrations. The results showed that both powders had no significant cytotoxicity at concentrations up to 1600 μg/mL (Figure 1).

**Figure 1.** Cell viability following the treatment of EAhy.926 cells with samples 3 and 9. The results are presented as the means ± SEM of three independent experiments carried out in triplicate. \* *p* < 0.05 indicates significant di fference from the control value.

#### *3.5. E*ff*ects of Powders on GSH Levels in EA.hy926 Cells*

For assessing the e ffects of samples 3 and 9 on GSH levels in EA.hy926 cells, flow cytometry analysis was used. The results showed that treatment of EA.hy926 cells with sample 3 significantly increased GSH levels by 22%, 59%, and 82% at 400, 800, and 1600 μg/mL, respectively compared to control (Figure 2). When cells were treated with sample 9, GSH levels were significantly increased by 51% and 88% at concentrations of 800 and 1600 μg/mL, respectively, compared to the control (Figure 3).

**Figure 2.** The effects of sample 3 on glutathione (GSH) levels in EA.hy926 cells after treatment for 24 h, as assessed by flow cytometry. (**A**) The histograms of cell counts versus fluorescence of 10,000 cells after treatment with sample 3. (**B**) Bar charts demonstrate the GSH levels as % of control as estimated by the histograms in EA.hy926 cells after treatment. C1, C2, C3, C4: 200, 400, 800, and 1600 μg/mL, respectively. \* Statistically significant compared to the control cells. FL2: The detection of fluorescence using 488 and 580 nm as the excitation and emission wavelength, respectively.

**Figure 3.** The effects of sample 9 on GSH levels in EA.hy926 cells after treatment for 24 h, as assessed by flow cytometry. (**A**) The histograms of cell counts versus fluorescence of 10,000 cells after treatment with sample 9. (**B**) Bar charts demonstrate the GSH levels as % of control as estimated by the histograms in EA.hy926 cells after treatment. C1, C2, C3, C4: 200, 400, 800, and 1600 μg/mL, respectively. \* Statistically significant compared to the control cells. FL2: The detection of fluorescence using 488 and 580 nm as the excitation and emission wavelength, respectively.

#### *3.6. E*ff*ects of Powders on ROS Levels in EA.hy926 Cells*

Like GSH, the levels of ROS in EAhy.926 cells after treatment with samples 3 and 9 were evaluated by flow cytometry analysis. The results demonstrated that after treatment of cells with sample 3, ROS levels did not change at any concentration compared to control (Figure 4). Moreover, after cell treatment with sample 9, ROS were decreased by 25% at 1600 μg/mL, compared to control (Figure 5).

**Figure 4.** The effects of sample 3 on reactive oxygen species (ROS) levels in EA.hy926 cells after treatment for 24 h, as assessed by flow cytometry. (**A**) The histograms of cell counts versus fluorescence of 10,000 cells after treatment with sample 3. (**B**) Bar charts demonstrate the ROS levels as % of control as estimated by the histograms in EA.hy926 cells after treatment. C1, C2, C3, C4: 200, 400, 800, and 1600 μg/mL, respectively. \* Statistically significant compared to the control cells. FL2: The detection of fluorescence using 488 and 580 nm as the excitation and emission wavelength, respectively.

**Figure 5.** The effects of sample 9 on ROS levels in EA.hy926 cells after treatment for 24 h, as assessed by flow cytometry. (**A**) The histograms of cell counts versus fluorescence of 10,000 cells after treatment with sample 9. (**B**) Bar charts demonstrate the ROS levels as % of control as estimated by the histograms in EA.hy926 cells after treatment. C1, C2, C3, C4: 200, 400, 800, and 1600 μg/mL, respectively. \* Statistically significant compared to the control cells. FL2: The detection of fluorescence using 488 and 580 nm as the excitation and emission wavelength, respectively.
