**4. Discussion**

The composition and the amounts of OMWW cause serious environmental problems in areas of olive oil production, especially when they are discharged without any previous treatment [15]. On the other hand, OMWWs contain high quantities of polyphenols with important bioactivities such as antioxidant property [1,11]. Thus, polyphenolic extracts from OMWW can be used as natural alternatives to commercial synthetic antioxidants with applications in the food industry and in the development of nutraceutical products [3,4,11,16].

However, when polyphenols are added in foods, there are problems regarding bad or bitter taste and discoloration. The encapsulation of polyphenolic extracts has been suggested as a method to overcome these problems as well as to improve polyphenols' stability, half-life, and bioavailability [10,17]. Thus, the aim of this study was to evaluate the effects on the redox status of endothelial cells of an OMWW extract encapsulated under different conditions and encapsulation carriers using spray drying. Apart from the encapsulation carrier, the spray drying conditions of the different samples differed in the inlet and outlet temperature, percentage of pump function, and use of Tween 80. Spray drying is a process widely used for encapsulation of oils and flavors [18]. Some of the advantages of this procedure are the generation of sample powders with good quality, low water activity, and easier handling and storage, while it also protects the active material against undesirable reactions [19]. The encapsulation carriers used were maltodextrin, maltodextrin/gelatin (5:1), whey protein, and whey protein/gelatin (5:1). Maltodextrin, a hydrolyzed starch, offers advantages for encapsulation such as relatively low cost, neutral aroma and taste, low viscosity at high solid concentrations, and protection against oxidation [20]. However, the most serious drawback of this material is its low emulsifying capacity; thus, it is desirable to use it in combination with other surface-active biopolymers such as gelatin [21]. Whey proteins, like other milk proteins, are hydrocolloids exhibiting good solubility and behavior and are used over the last years in food industry due to the increasingly need for natural products [22]. There have so far been only a few studies on the encapsulation of polyphenols from OMWW. For example, it has been reported that OMWW polyphenols encapsulated in maltodextrin and maltodextrin/acacia fiber (1:1) using spray drying exhibited antioxidant and antiglycative activities as well as inhibited Maillard reaction in milk [23–25]. Moreover, Caporaso et al. [22] have produced antioxidant powder by encapsulating OMWW polyphenols in whey protein/xanthan gum.

At first, in vitro antioxidant assays were applied in order to select the most potent samples that were used for the examination in endothelial cells. The findings showed that although all the 17 samples exhibited free-radical scavenging activity in DPPH• and ABTS•<sup>+</sup> assays, there was a grea<sup>t</sup> variation in their effect up to about two- and threefold, respectively. In ABTS•<sup>+</sup> assay, the samples encapsulated in whey protein were more potent at lower inlet/outlet temperature than at higher ones. However, inlet/outlet temperature did not seem to affect the potency of samples encapsulated in maltodextrin. Moreover, the scavenging activity against ABTS•<sup>+</sup> radical was not dependent on the encapsulation carrier. For example, in the ABTS•<sup>+</sup> assay, the first (No. 9) and third (No. 2) most potent samples were encapsulated in maltodextrin and whey protein, respectively. Finally, in the ABTS•<sup>+</sup> assay, the use of gelatin did not significantly affect the antioxidant potency of samples encapsulated either in whey protein or maltodextrin. Unlike the ABTS•<sup>+</sup> assay, in the DPPH• assay, inlet/outlet temperatures had no effect on the potency of samples encapsulated in whey protein. Moreover, like the ABTS•<sup>+</sup> assay, these temperatures did not seem to affect scavenging activity against DPPH• of samples encapsulated in maltodextrin. Furthermore, in the DPPH• assay, all the samples encapsulated in whey protein exhibited higher antioxidant activity than the samples encapsulated in maltodextrin (except No. 9). In the DPPH• assay, the addition of gelatin decreased antioxidant activity in samples encapsulated in whey protein, while it did not affect the activity of samples encapsulated in maltodextrin. Moreover, some samples (e.g., No. 9) exhibited high potency in both DPPH• and ABTS•<sup>+</sup> assay, while other samples (e.g., No. 5) had high potency in one assay and low in the other. The observed differences between DPPH• and ABTS•<sup>+</sup> may be explained by the different solvents used in these assays, that is, methanol and water, respectively. Thus, lipophilic compounds are more active in the DPPH• assay, while hydrophilic compounds are more active in the ABTS•<sup>+</sup> assay.

Apart from the free-radical scavenging activity, the samples' reducing capacity was assessed using the reducing power assay, since many antioxidants act as hydrogen donors. Again, the reducing capacity varied up to twofold between the samples exhibiting the higher and the lower activity. The type of encapsulation carrier did not affect reducing activity, since samples of both whey protein (e.g., No. 3) and maltodextrin (e.g., No. 9) demonstrated high potency. Moreover, reducing activity did not seem to depend on inlet/outlet temperatures in samples encapsulated either in whey protein or maltodextrin. It was also remarkable that the addition of gelatin in the encapsulation mixture decreased reducing activity in both whey protein and maltodextrin samples.

Furthermore, all the samples protected from ROS-induced DNA damage, but, like the other assays, the IC50 varied greatly up to about 4.4-fold. Samples encapsulated in both whey protein (e.g., No. 3) and maltodextrin (e.g., No. 9) exhibited high protection. The samples' protective activity was independent of inlet/outlet temperatures. As in the DPPH• assay, the use of gelatin decreased the potency of samples encapsulated in whey protein but not in maltodextrin. Previous studies have shown that OMWW extracts inhibited ROS-induced DNA damage using pure DNA or cells [26,27]. However, to the best of our knowledge, this is the first study reporting the protective e ffect of an encapsulated OMMW polyphenolic extract against DNA damage caused by free radicals.

Based on the RACI value that each sample had in all the above assays (i.e., DPPH•, ABTS•+, ROO-induced DNA damage, and reducing power), the two most potent samples were selected in order for their antioxidant activity to be examined in endothelial cells at non-cytotoxic concentrations. These two samples were No. 3 encapsulated in whey protein and No. 9 encapsulated in maltodextrin. For assessing the samples' antioxidant activity, GSH and ROS levels were evaluated by flow cytometry in endothelial cells. Cell treatment with both encapsulated samples showed significantly increased GSH levels, one of the most important antioxidant molecules [28]. The OMWW sample-induced increase in GSH may be due to the rescue of GSH from reaction with ROS by their direct scavenging, since OMWW encapsulated samples were shown to possess free-radical scavenging activity. Moreover, the observed increase in GSH may be attributed to increase in activity of enzymes involved in GSH synthesis and metabolism. For example, polyphenols found in olive oil have been reported to increase the expression and/or activity of glutathione peroxidase (GPx) and glutathione reductase (GR) [29]. The expression of such enzymes is mainly regulated by the transcription factor nuclear factor (erythroid-derived 2)-like2 (Nrf2) [30]. Hydroxytyrosol, one of the polyphenols identified in our OMWW extract used for the encapsulation [11], has been reported to activate Nrf2 in mouse heart [31]. Interestingly, we have also demonstrated that administration of feed containing polyphenolic extract from OMWW increased GSH in di fferent tissues including the heart of farm animals [3,4]. Moreover, ROS levels were decreased in endothelial cells after treatment with encapsulated OMWW samples. This decrease in ROS was in agreemen<sup>t</sup> with the OMWW sample-induced increase in antioxidant mechanisms such as GSH in the endothelial cells as well as with OMWW samples' free-radical scavenging activity. However, this ROS decrease was observed only in cells treated with sample No. 9 encapsulated in maltodextrin. The absence of ROS decrease in cells treated with whey protein sample No. 3 may be explained by the fact that samples' antioxidant e ffects were examined in naïve cells, that is, cells that were not treated with an oxidative agent. Thus, in such cells, the baseline ROS levels are low. Moreover, sample No. 9 exhibited higher potency in free-radical scavenging assays than sample No. 3. Overall, the above findings suggested the use of OMWW polyphenolic extract encapsulated either in maltodextrin or whey protein for the development of food supplements or biofunctional foods possessing antioxidant activity, especially protection from oxidative stress-induced pathologies associated with the cardiovascular system. Interestingly, OMWW polyphenolic extract has been reported to decrease cholesterol levels in rats [32].
