**3. Results**

### *3.1. Characterization of Olive By-Products and Waste*

The valorization of waste and by-products derived from the olive oil industry represents an attractive and sustainable challenge aiming to reduce the environmental impact and the disposal costs. Extracts derived from by-products can contain several compounds displaying bioactive activity. The phenolic compounds recovery from waste and byproducts is usually carried out using organic solvent extraction. The choice of the solvent to use is made according to the purpose of extraction, the polarity of the interesting components, overall cost, safety, etc. For this reason, in this study, the recovery of phenolic compounds was realized with hydro-alcoholic solutions for OP and OLs and ethyl acetate for OWW. The latter solvent showing a high extraction efficiency for phenolic extraction from OWW, and at the end of the extraction procedure is eliminated (through concentration), and the phenolic compounds were recovered with water.

Table 1 reports the results of total phenol content, antioxidant activity, and the chemical characterization of the main phenolics in the considered olive waste and by-products. Extracts obtained from OWW showed higher total phenolic content (about 9 mg·mL−1) compared to the pomace extract (4.5 mg·mL<sup>−</sup>1).

Among these, the main ones were: hydroxytyrosol, tyrosol, and oleuropein in agreement with our previous work [21]. Particularly, the OWW was rich in hydroxytyrosol (1.4 mg·mL−1), also if this content may vary according to several factors as oil extraction technique, OWW characteristics, cultivar, extraction method of phenolic content. Furthermore, for OP extract, the main phenolic compound was hydroxytyrosol, but in a lower amount (0.2 mg·mL<sup>−</sup>1). OWW extract showed a notably higher value of antioxidant activity compared to OP extract (3579 and 451 μmol TE mL<sup>−</sup>1, respectively), denoting a strong antioxidant activity against ABTS radical.

The OLE showed high total phenol content and antioxidant activity according to Ghasemi et al. [22]. Among phenols, oleuropein, verbascoside, apigenin-7-O-glucoside, luteolin-7-O-glucoside, tyrosol, and rutin were identified.

The most represented compounds were oleuropein with a concentration of 137 mg g−<sup>1</sup> in line with the results of other authors [22,23], in fact, generally, the amount of this secoiridoid is in the range of 25–30% of the total polyphenols in OLE [24].


**Table 1.** Total phenol content (TPC, mg GAE mL−<sup>1</sup> for OWW and OP; mg GAE g−<sup>1</sup> for freeze-dried OLE), antioxidant activity (μmol TE mL−<sup>1</sup> for OWW and OP; μmol TE g−<sup>1</sup> for OLE) and phenolic profile (mg·mL−<sup>1</sup> for OWW and OP; mg·g−<sup>1</sup> for OLE) of phenolic extracts.

GAE: gallic acid equivalents; OWW: olive mill wastewater; OP: olive pomace; TE: Trolox equivalents; OLE: olive leaves extract.

#### *3.2. Biological Characterization of Olive By-Products*

An increase in systemic oxidative stress related to abnormal production of ROS is often associated with aging and several diseases including hypertension, obesity, and cancer. A growing number of bioactive phytocompounds, known for their beneficial and antioxidant actions on human health, are more frequently recommended as therapeutic adjuvants in several diseases [25]. Here, we evaluated the effects of extracts obtained from olive by-products and wastes on cell viability in human colorectal carcinoma HCT8 cells. Furthermore, the potential antioxidant efficacy of olive by-products and wastes was assayed as well. Cells were incubated overnight with extracts derived from OWW at increasing concentrations (0.03 mg·L<sup>−</sup>1, 0.06 mg·L<sup>−</sup>1, and 0.12 mg·L−<sup>1</sup> of phenols). Compared to cells left under control conditions, treatment with OWW extracts does not alter cell viability at the concentration used (Figure 1a). Under similar conditions, ROS content was measured in HCT8 cells. Compared to untreated cells (CTR), ROS production increased in cells incubated with OWW extracts at 0.12 mg·L−<sup>1</sup> (Figure 1b). Indeed, compared to the positive control (tBHP), cells co-incubated with the oxidant tBHP, and OWW extracts showed a significant decrease in ROS generation induced by tBHP (Figure 1b).

Next, HCT8 cells were treated with extracts obtained from OP at increasing concentrations (0.03 mg·L−1, 0.06 mg·L−<sup>1</sup> and 0.12 mg·L−<sup>1</sup> of phenols). Compared to CTR cells, treatment with OP extracts at 0.12 mg·L−<sup>1</sup> of phenols displayed a cytotoxic effect (Figure 2a). Fluorimetric measurements revealed that cells co-incubated with the oxidant tBHP and OP extracts at 0.12 mg·L−<sup>1</sup> of phenols showed a significant decrease in ROS generation induced by tBHP. However, a relevant but not significant reduction in ROS content was measured in cells co-treated with tBHP and OP extracts at 0.03 mg·L−<sup>1</sup> and 0.06 mg·L−<sup>1</sup> of phenols. Incubation with the OP extracts alone, at increasing concentrations (0.03 mg·L<sup>−</sup>1, 0.06 mg·L<sup>−</sup>1, and 0.12 mg·L−<sup>1</sup> of phenols) does not alter ROS production.

**Figure 1.** Cell viability (**a**) and ROS content (**b**) in HCT8 cells exposed to olive wastewater (OWW). (**a**) Cells were left under basal condition or exposed to increasing concentrations (0.03 mg·L−1, 0.06 mg·L<sup>−</sup>1, and 0.12 mg·L−<sup>1</sup> of phenols) of OWW and were stained with crystal violet solution. Data are shown as means ± Standard Error Mean (S.E.M.) of 3 independent experiments and analyzed by one-way ANOVA followed by Dunnett's multiple comparisons test. (**b**) ROS content was measured using dihydrorhodamine-123 fluorescence in cells treated as previously described. As a positive control, cells were treated with tert-butyl hydroperoxide (tBHP). Data are shown as means ± S.E.M. of 3 independent experiments and analyzed by one-way ANOVA followed by Dunnett's multiple comparisons test. (\* *p*< 0.05 vs. CTR; ## *p* < 0.01 and # *p* < 0.05 vs. tBHP).

**Figure 2.** Cell viability (**a**) and ROS content (**b**) in HCT8 cells exposed to olive pomace (OP). (**a**) Cells were left under basal condition or exposed to increasing concentrations (0.03 mg·L<sup>−</sup>1, 0.06 mg·L<sup>−</sup>1, and 0.12 mg·L−<sup>1</sup> of phenols) of OP and were stained with crystal violet solution. Data are shown as means ± S.E.M. of 3 independent experiments and analyzed by one-way ANOVA followed by Dunnett's multiple comparisons test (\* *p* < 0.05 vs. CTR). (**b**) ROS content was measured using dihydrorhodamine-123 fluorescence in cells treated as previously described. As a positive control, cells were treated with tert-butyl hydroperoxide (tBHP). Data are shown as means ± S.E.M. of 3 independent experiments and analyzed by one-way ANOVA followed by Dunnett's multiple comparisons test. (# *p* < 0.05 vs. tBHP).

Similar evaluations were performed in cells treated with OLE. Specifically, compared to CTR, treatment with OLE does not alter cell viability at the used concentrations (Figure 3a). Moreover, compared to the positive control (tBHP), HCT8 cells coincubated with tBHP and OLE at 0.12 mg·L−<sup>1</sup> of phenols displayed a significant reduction in ROS content. Incubation with the OLE alone, at increasing concentrations (0.03 mg·L<sup>−</sup>1, 0.06 mg·L<sup>−</sup>1, and 0.12 mg·L−<sup>1</sup> of phenols) does not alter ROS content (Figure 3b).

**Figure 3.** Cell viability (**a**) and ROS content (**b**) in HCT8 cells exposed to olive leaves extract (OLE). (**a**) Cells were left under basal condition or exposed to increasing concentrations (0.03 mg·L<sup>−</sup>1, 0.06 mg·L<sup>−</sup>1, and 0.12 mg·L−<sup>1</sup> of phenols) of OLE and were stained with crystal violet solution. Data are shown as means ± S.E.M. of 3 independent experiments and analyzed by one-way ANOVA followed by Dunnett's multiple comparisons test. (**b**) ROS content was measured using dihydrorhodamine-123 fluorescence in cells treated as previously described. As a positive control, cells were treated with tert-butyl hydroperoxide (tBHP). Data are shown as means ± S.E.M. of 3 independent experiments and analyzed by one-way ANOVA followed by Dunnett's multiple comparisons test. (# *p* < 0.05 vs. tBHP).

#### **4. Discussion**

Extra virgin olive oil (EVOO) consumption displays protective effects on human health [26,27]. Acute intake of EVOO significantly ameliorates insulin sensitivity and glycemia in patients with metabolic syndrome by modulating the expression of genes involved in inflammation, metabolism, and cancer [28]. Furthermore, in high-fat-diet fed rats, administration of polyphenol-enriched olive oil improved insulin sensitivity, lipid profile, and interfered with inflammatory pathways [29]. Several compounds of EVOO, indeed, can modulate the NF-kB signal transduction pathway and decrease oxidative stress [30]. Importantly, the volume of wastes derived from the olive oil industry represents a worrying issue. While olive pomace can be treated to produce olive pomace oil or biomasses for fuel generation, olive mill wastewater represents a serious environmental matter and the disposal costs are very high. Notably, it has been reported that olive mill by-products and wastes can still contain numerous bioactive and valuable compounds useful in the pharmaceutical, food, and cosmetic industries. It is well established that the phenolic composition within the oil and its derivative by-products is complex and it is related to olive variety, the maturation stage, and seasons [31,32].

Oleuropein, which is found in olives, olive leaves, and oil can be degraded during olive oil production. Hydroxytyrosol and tyrosol are the main degradation products of oleuropein displaying several biological activities. Among the considered by-products, OLs are the main source of oleuropein, as reported in Table 1, since this waste is not subjected to the processes involved in oleuropein degradation during the olive oil production [33,34]. In pancreatic cancer cells, dose-dependently treatment with oleuropein or hydroxytyrosol significantly reduced cell viability and proliferation [32]. Moreover, oleuropein administration promotes apoptosis by upregulating the p53 signal pathway in breast and colon cancer cells [34,35]. Verbascoside is one of the most abundant phenolic compounds in the OLE; this compound displays beneficial effects for its antioxidant and anti-inflammatory and antitumor actions beyond the neuroprotective properties [35]. In the present report, incubation with an increasing concentration of extracts obtained from olive by-products and wastes does not alter cell viability which might be due to the low concentrations that were used. However, only the treatment with the extract obtained from the olive pomace, used at the highest concentration, caused a significant reduction in cell viability as assessed by the crystal violet assay. By comparing the extract composition of the OP with the ones of the other extracts, we found that OP extract contains higher amounts of vanillic acid and luteolin-7-glucoside. In colon HCT116 cells, exposure to vanillic acid induced G1 phase arrest and inhibited cell proliferation, possibly by inhibiting the expression of HIF-1, mTOR, and Raf/MEK/ERK signal transduction pathways [31]. Furthermore, luteolin-7-glucoside reduced keratinocyte proliferation by inhibiting IL22/STAT3 signaling [33]. Therefore, exposure to high levels of vanillic acid and luteolin-7-O-glucoside may explain the observed reduction of HCT8 cell survival exposed to the highest concentration of the OP extract.

Olive oil and olive by-products are thus a natural source of antioxidant compounds and their beneficial effects have been tested in several in vitro and in vivo experimentations [36–38]. Interestingly, it has been proposed that the concentration of polyphenols in the intestinal tract is higher than elsewhere because phenols can reach the colon directly or through the bile. However, relevant variability within volunteers has been also documented, which might be due to the difference in the expression level of metabolizing enzymes and to a different microbiota profile [39]. Recent reports revealed that selective phenols can interfere with oxidative and inflammatory pathways. Specifically, in a mouse model of acute colitis, administration of oleuropein-loaded lipid nanocarriers significantly reduced TNF-α release and intracellular ROS [40] strongly suggesting that phenols in the extracts may be considered valuable compounds for pharmaceutical applications. Here, with the same phenolic concentration, the extract derived from the olive wastewater displayed a higher antioxidant activity compared with the extracts derived from olive pomace and leaves. These responses might be due to the high concentration level of hydroxytyrosol and tyrosol detected in the olive mill wastewater [41]. Among phenolic compounds isolated from the olive tree, hydroxytyrosol is the most powerful antioxidant followed by oleuropein, caffeic acid and, tyrosol [42]. Conversely, at a high concentration level, antioxidants can react with molecular oxygen thereby acting as pro-oxidants [43]. Impairing antioxidant cellular defense and inducing the generation of reactive species may be a useful strategy to remove, and kill cancer cells. In human colon cancer DLD1 cells, hydroxytyrosol induced cell apoptosis, and the activation of PIPK3/AKT pathway through ROS production [44]. Accordingly, incubation with the only extract obtained from OWW, at the highest concentration, significantly increased the generation of ROS, which might be useful to activate specific intracellular signals leading to cell death as already shown by others [44,45]. Furthermore, we found that at the highest concentration level, incubation with the extract obtained from OP reduced cell viability. By contrast, treatment with phenols at a lower concentration does not exert a cytotoxic effect but it can reduce the ROS production induced by the synthetic oxidant compounds tBHP. Together, these findings suggest that phenols in the olive by-products can tightly modulate the intracellular ROS content that can act as signaling molecules cross-linking several intracellular pathways modulating cell viability.

#### **5. Conclusions**

This study underscores the crucial importance of the chemical and functional characterization of olive by-products. Using the ileo-carcinoma cell line HCT8 cells we found differential effects of the extracts on ROS generation and cell viability even though all extracts contain an enriched fraction of polyphenols. Therefore, further investigations would be needed to describe and identify the potential role and the possible applications and use of the bioactive compounds in the olive wastes in the pharmacological and food industry.

To conclude, according to the guiding principles of the circular economy, this study highlights and underlines the possibility of valorizing olive by-products and wastewater as they contain numerous bioactive compounds that individually or synergistically can bring beneficial effects to human health.

**Author Contributions:** Conceptualization, M.C., C.M., G.T. and M.R.; methodology, M.C., M.D., G.D., A.D.B. and A.D.M.; data curation, G.T., G.V. and F.C.; writing—original draft preparation, M.C. and G.T.; writing—review and editing, G.T., F.C. and M.P.; funding acquisition, F.C. 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.

**Acknowledgments:** The financial support of Marianna Ranieri is supported by "Intervento cofinanziato dal Fondo di Sviluppo e Coesione 2007–2013–APQ Ricerca Regione Puglia, Programma Regionale a Sostegno della Specializzazione Intelligente e della Sostenibilità Sociale ed Ambientale– FutureInResearch" (code CHVNKZ4). Annarita Di Mise is supported by "Attrazione e Mobilità dei Ricercatori, PON "R&I" 2014–2020, Azione I.2" (code AIM1893457).

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

### **References**

