**3. Results**

In this work, two extracts derived from OMW were assessed for their phenol contents and their potential biological activity on two human vascular cells. The two extracts were obtained employing a desolvented ethyl acetate extraction followed by water recovery.

Hydroxytyrosol and tyrosol were the principal quantified compounds in both OMW extracts, with significantly greater abundance in the S extract, mainly related to the first phenolic alcohol (4.55 mg g<sup>−</sup>1). The tyrosol concentration varied between 0.54 mg g−<sup>1</sup> in L extract and 0.58 mg g−<sup>1</sup> in L extract. The S extract also showed higher amounts of caffeic acid and oleuropein, respectively, of 0.36 and 0.15 mg g−1. Finally, among the identified flavonoids, apigenin-7-O-glucoside was more abundant in L extract with a concentration of 0.15 mg g−<sup>1</sup> (Figure 1).

**Figure 1. Chromatogram of phenolic compounds in S olive mill wastewater (OMW) extract**. (1) Hydroxytyrosol; (2) tyrosol; (3) vanillic acid; (4) caffeic acid; (5) ferulic acid; (7) apigenin-7-O-glucoside; (8) oleuropein; (9) luteolin-7-O-glucoside.

> Next, we proceeded with the extracts study by assessing their potential biological activities on two human vascular cells, HUVECs and HSMC, respectively, endothelial and vascular smooth muscle cells. Based on data from our previous studies [27–30], cell treatments were performed using the following extracts' concentrations: 10, 25, 50, and 100 μg/mL.

> Although widely accepted as a provider of health benefits, natural antioxidants, including phenolic compounds, based on both dosages and redox environment status, have been reported to act as prooxidants, thus increasing intracellular ROS levels and inducing cell death [23,25,26,31–34]. Moreover, extracts deriving from food processing waste have been reported to be unsafe for the environment [7]. For this reason, we first investigated the potential harmful effects of OMW extracts by assessing their ability to affect cell viability, metabolic activity, and intracellular ROS production, three aspects tightly interconnected in maintaining the physiological functions of vascular cells [35,36]. As reported in Figure 2, 24-h exposition of HUVECs (Figure 2A) and HSMCs (Figure 2B) to the indicated concentrations of OMW failed to induce cell death in either cellular model. To further investigate potential harmful effects and corroborate the absence of cell toxicity, we assessed the extract's effect on cellular metabolic activity. Figure 2C,D indicate that only the highest dose tested (100 μg/mL) was able to significantly affect the cell metabolic activity, while no effects were observed at the other tested concentrations.

> Since the two OMW extracts showed a remarkable quantity of antioxidant phenolic compounds, we sought to investigate their potential antioxidant activity by assessing the ability to counteract H2O2-elicited oxidative changes. First, we evaluated the ability of our ROS assay to reveal intracellular ROS level variations within a range of selected H2O2 concentrations. As reported in Figure 3A,B, the probe displayed both a significant dynamic range and linear response when challenged with increasing concentrations of H2O2, a

well-known cellular prooxidant in these vascular cells. [37,38]. Therefore, based on these results, 75 μM was the dosage of H2O2 employed for the experiments concerning potential extract antioxidant activity. In this experiment, cells were treated for 4 h with the four extract concentrations followed by a 2 h-treatment with 75 μM H2O2. Then, the levels of intracellular ROS were measured as indicated in the Section 2.

**Figure 2. Effect of OMW on HUVECs and HPASMCs viability and metabolic activity**. Cells were exposed for 24 h in the absence (CTRL) or presence to the indicated concentrations of OMW extracts. Cell viability (**A**,**B**) and metabolic activity (**C**,**D**) were assessed as reported in the materials and methods. HUVECs, human umbilical vein endothelial cell; HPASMCs, human pulmonary artery smooth muscle cells; L, liquid extract; S, solid extract; CTRL, untreated cells; \* significantly different from CTRL; Values are shown as mean ± SD and expressed as a percentage of the CTRL. (*n* = 4).

Exposure of the HUVECs to increasing concentrations of the two extracts significantly counteracted the H2O2-elicited increase of ROS in all the tested concentrations (Figure 3C). Similarly, compared to H2O2-treated cells, exposure of HSMCs to the two extracts induced a significant antioxidant effect in the whole concentration range (Figure 3D). Since oxidative stress is recognized to cause cell injury and even death, we next hypothesized that the observed extracts' antioxidant effects could provide cellular protection against cells damaged elicited by H2O. To this end, cells were pre-treated for 3 h with the indicated extract concentrations, and then 75 μM H2O2 was added for 24 h before cell viability determination. In agreement with the displayed antioxidant effect on H2O2-increased ROS production (Figure 3C,D), cell pre-treatments with increasing concentrations of the two OMW extracts significantly protected both cell lines from H2O2-elicited oxidative cell death.

**Figure 3.** (**A**,**B**) **Dose–response effect of H2O2 on intracellular ROS levels of HUVECs** (**A**) **and HPASMCs** (**B**)**.** Cells were exposed for 6 h to the indicated concentrations of H2O2. Intracellular ROS levels were assessed as reported in Materials and Methods. (**C**,**D**) OMW extracts counteract H2O2-induced ROS increase in both HUVECs (**C**) and HPASMCs (**D**). Cells were exposed for 3 h to the indicated concentrations of OMW extracts and then incubated for 6 h in the presence of 75 μM H2O2. Intracellular ROS levels were assessed, as reported in Materials and Methods. HUVECs, human umbilical vein endothelial cell; HPASMCs, human pulmonary artery smooth muscle cells; L, liquid extract; S, solid extract; CTRL, untreated cells; H2O2, hydrogen peroxide; \* significantly different from CTRL; § significantly different from each other; # significantly different from H2O2. Values are shown as mean ± SD and expressed as a percentage of the CTRL. (*n* = 5).

#### **4. Discussion**

World globalization requires sustainable growth. In this context, the processing of different types of waste, derived from food treatment or processing, to produce valueadded products is becoming a challenging reality that we must pursue. Waste processing or transformation may involve using different technologies, and the obtained products may or may not be safe for the environment and human health. However, since transformation may give rise to new, unknown, or unwanted compounds, it is useful to assess their safety, especially in terms of their impact on human health.

The olive (*Olea europaea L*.) is native to the Mediterranean area where it can be found in the wild form in the Middle East. *Olea europaea* L. is widely spread throughout the world, especially in the Mediterranean regions, where it accounts for nearly 96 percent of global olive production (FAOSTAT Food and Agriculture Data [20]). The production of olive oil forms a significant quantity of residue, such as aqueous waste called olive mill wastewater (OMW), and olive leaves. Other wastes are also produced during the filtration processes and during the storage time when the solid components migrate to the tank bottoms, generating sediments. These waste products are rich in bioactive compounds and can be

therefore considered valuable byproducts to be exploited for further uses [39,40]. Although OMWs are effluents capable of degrading soil and water quality, with serious negative effects on the environment [7], studies have revealed that they contain high concentrations of antioxidants, such as phenolic molecules [41]. However, since the quality of OMW compounds differs according to the olive variety, the technological process of olive oil production, storage time, and climatic conditions [40], it is imperative to evaluate the technological processes employed in OMW processing in order to obtain valuable bioactive compounds. In this regard, phenolic compounds derived from OMW have been reported to show interesting biological properties [10,39,40]. Nonetheless, yet a large amount of crusher residues remain without actual application, since only small quantities are used as natural fertilizers, biomass fuel, additives in animal feed, and activated carbon. However, these residues are a precious starting material to produce extracts of phenolic molecules that could be used in various industrial fields [7]. Different technologies are proposed rather than classical solid–liquid extraction to enhance phenolic compounds' extraction from different olive oil byproducts.

We used a liquid–liquid extraction procedure followed by acidic hydrolysis [18], which provided two extracts, L and S, harboring a remarkable amount of total phenol content. The quantitative analysis indicated the S extract had a higher phenol content compared to the L extract. Moreover, hydroxytyrosol and tyrosol had the primary phenols in both OMW extracts, with hydroxytyrosol significantly more abundant in the S extract than the L extract (Table 1).


**Table 1.** Main phenolic compounds in liquid (L) and freeze dried (S) OMW extracts (mg g<sup>−</sup>1).

Data are mean (*n* = 2) ± standard deviation. \*\* Significance for *p* < 0.01; \* significance for *p* < 0.05.

In the cellular experiments, we first evaluated potential extracts' harmful effects by treating endothelial and smooth muscle cells with different OMW dosages for 24 h. As reported in Figure 2A,B, the extracts were unable to induce cell death at all the concentrations tested. However, sometimes compound effects may affect cell metabolism without resulting in cell death. For this reason, we also assessed the extract effect on the two cells' metabolic activities. In this analysis, the extract showed no effect, except for the highest concentration, 100 μg/mL, where we found a slight but significant decrease in cell metabolic activity, an aspect that requires further investigations as it has been reported for other phenol compounds [23,25,26,31–34]. However, the finding that the concentration of 100 μg/mL affects only the cell metabolism without inducing cell death indicated that the extracts, even at high concentrations, appeared not to be toxic, being unable to induce cell death. Considering hydroxytyroso as the main compound present in the OMW, our findings align with In Vivo experiments reporting no toxic effects in rodents fed with HT concentrations as high as 2 g/kg b.wt. [42,43]. Similarly, no adverse clinical, biochemical, or gross necropsy effects were also reported in rats orally administered with HIDROXTM, a hydrolyzed aqueous olive pulp extract with a phenol composition closer to our OMW [44]. Moreover, In Vitro experiments, performed with different cell lines, also confirmed our findings reporting lack of cytotoxicity with HT concentrations as high as 500 μM [45–47].

Although chemical tests such as TBARS, ABTS, and DPPH are often used to determine compounds' antioxidant properties, a likely better method to investigate the antioxidant activity/effect of a molecule or extract would be In Vivo or in a cellular model. Indeed, a cellular model would provide the proper environment for studying potential interactions (e.g., compound(s)–cellular signal transduction pathway(s), compound(s)–cellular receptor(s)) that would be otherwise missed in a solely chemical setting. In this regard, the intracellular oxidation of H2DCF-DA is mainly caused by H2O2, an oxidizing molecule physiologically present in the cell and therefore functional to induce oxidative insults and study their effects on cell functions [48,49]. Thus we created H2O2-induced oxidative stress and investigated whether the extracts could counteract it by following the variation of intracellular levels of DFC, the oxidized form of H2DCF-DA, and assessing cell viability (Figures 3 and 4). In our experimental conditions, the different extracts' concentrations significantly inhibited the H2O2-induced increase of ROS in both HUVECs (Figure 3C) and in HAPVSCs (Figure 3D), bringing the oxidative stress values back to those of the controls. Consonant with observed extracts' antioxidant activity are the findings reported in Figure 4. The extracts were indeed capable of dose-dependently counteracting H2O2-induced cell death and restoring cell viability completely at the dosages of 50 and 100 μg/mL. Our current findings support and confirm previously performed experiments; indeed, similarly to EVOO-contained phenolic compounds [9,10,45,46,50], olive oil processing byproducts such as OMW are a source of valuable compounds harboring health benefit properties.

**Figure 4. OMW extracts protect HUVECs** (**A**) **and HPASMCs** (**B**) **from H2O2-induced cell death**. Cells were exposed for 3 h to the indicated concentrations of OMW extracts and then incubated in the absence (CTRL) or presence of 75 μM H2O2. Cell viability was assessed as reported in Materials and Methods. HUVECs, human umbilical vein endothelial cell; HPASMCs, human pulmonary artery smooth muscle cells; L, liquid extract; S, solid extract; CTRL, untreated cells; H2O2, hydrogen peroxide; \* significantly different from CTRL; # significantly different from H2O2. Values are shown as mean ± SD and expressed as a percentage of the CTRL. (*n* = 4).

#### **5. Conclusions**

The OMW extracts obtained by the production of olive oil showed interesting antioxidant activity on both the employed cell models. The tested extracts were capable of effectively protecting cells from oxidative stress-indued cell death, failing indeed to interfere with cell viability and even with the metabolism, except for the highest tested concentration. Our results indicates a different point of view concerning the food processing residues, which should not be considered waste but precious material containing molecules capable of modulating essential functions of cellular biological models, such as the vascular model. Further studies are required to establish these substances' fates in the organism and to understand their biological functions and fine molecular mechanisms.

**Author Contributions:** Conceptualization, A.M.P., A.P. (Antonio Piga) and G.P.; Formal analysis, A.P. (Amalia Piscopo) and G.P.; Funding acquisition, A.P. (Antonio Piga) and G.P.; Investigation, A.M.P., A.C. and R.G.; Methodology, A.M.P., A.C., R.G. and A.P. (Amalia Piscopo); Resources, A.P. (Antonio Piga) and G.P.; Validation, G.P.; Writing—original draft, A.M.P. and A.P. (Amalia Piscopo); Writing—review & editing, A.M.P., A.P. (Amalia Piscopo), W.M.A.-R., A.P. (Antonio Piga) and G.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the AGER 2 Project, grant no. 2016-0105, and by the Fondo UNISS di Ateneo per la Ricerca 2020 to G.P. and A.P.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All the data are reported in the article.

**Acknowledgments:** The authors thank the firm Olearia San Giorgio for supplying OMW.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

