2.3.2. Olive Leaves (OLs)

Total phenol content, antioxidant activity, and single phenolic compounds identification were performed according to Difonzo et al. [10] with some modifications. For the total phenol content determination, 20 μL of the extract was added to 980 μL of ddH2O and 100 μL of FolinCiocalteu reagent. After 3 min, 5% Na2CO3 solution was added, following incubation at room temperature for 60 min. The absorbance was read at 750 nm using a Cary 60 spectrophotometer (Agilent, Milan, Italy). The TPC was expressed as gallic acid equivalents (GAE) in mg·L−<sup>1</sup> juice.

For the antioxidant activity assay ABTS, the radical was generated by a chemical reaction with potassium persulfate (K2S2O8). For this purpose, 25 mL of ABTS (7 mM in H2O) was spiked with 440 μL of K2S2O8 (140 mM) and allowed to stand in darkness at room temperature for 12–16 h (the time required for the formation of the radical). The working solution was prepared by taking a volume of the previous solution and diluting it in ethanol until its absorbance at *λ* = 734 nm was 0.70 ± 0.02. A Cary 60 spectrophotometer Agilent (Milan, Italy) was used. The reaction took place directly in the measuring cuvette: 50 μL of each sample was added to 950 μL of the final ABTS*·*<sup>+</sup> solution. After 8 min the decrease of absorbance was measured at 734 nm. The results are expressed in μmol Troloxequivalents (TE) g−<sup>1</sup> dry weight. Each sample was analyzed in triplicate.

The main identified phenolic compounds were quantified by means of HPLC-DAD (Dionex Ultimate 3000 RSLC, Waltham, MA, USA). Dionex Acclaim 120 C18 analytical column (150 × 3 mm i.d.) with a particle size of 3 μm (Thermo Scientific, Waltham, MA, USA) was used. The mobile phases were (A) water/acetic acid (98:2, *v*/*v*) and (B) acetonitrile at a constant flow rate of 1 mL·min−1. The column temperature was set at 35 ◦C. The gradient program was as follows: 5 min, 95% A; 10 min, 80% A; 15 min, 75% A; 20 min, 65% A; 25 min, 0% A; 35 min, 95% A. The identification of phenolic compounds was based on a comparison of retention times obtained by pure standard (Sigma-Aldrich Co. LLC, St. Louis, MO, USA) and literature data [10]. The quantification was carried out by means of external calibration curves with the relative standard for the selected compounds.

#### *2.4. Cell Culture and Treatment*

The human colon cancer cells HCT-8 were cultured as previously described [20]. Briefly, cells were grown in Advanced RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 100 i.u. mL−<sup>1</sup> penicillin, 100 <sup>μ</sup>g·mL−<sup>1</sup> streptomycin at 37 ◦C in 5% CO2. Cells were left under basal condition or treated overnight with the same phenolic concentration (0.03 mg·L<sup>−</sup>1, 0.06 mg·L<sup>−</sup>1, and 0.12 mg·L<sup>−</sup>1) obtained from extracts of olive leaves or olive pomace or olive mill wastewater.
