Hydroxytyrosol

All the chemicals used in this study were of analytical grade. Water was daily deionized by using a Milli-Q system from Millipore (Bedford, MA, USA). Ethanol was supplied by J.T. Baker (Deventer, The Netherlands). Methanol and acetonitrile, both of LC-MS grade, were purchased from Prolabo (Paris, France). Acetic acid and pure standards of apigenin, apigenin-7-glucoside, hydroxytyrosol, luteolin, luteolin-7-glucoside, pinoresinol, rutin, tyrosol and vanillic acid were acquired from Sigma-Aldrich (St. Louis, MO, USA); whereas oleuropein was purchased from Extrasynthese (Lyon, France).
A stock standard solution was prepared by dissolving the appropriate amount of each compound in methanol. Then, diluted working solutions were obtained at nine different concentrations (0.5 mg/L; 1 mg/L; 2.5 mg/L; 5 mg/L; 12.5 mg/L; 25 mg/L; 50 mg/L; 100 mg/L and 200 mg/L) and were stored at −20 °C. If any other concentration level was required for a particular sample or to establish the analytical parameters of the method, it was logically prepared.

For in vitro analyses, we used the MCF-7 cell line, representing the molecular subtype of human breast cancer Luminal A, and MDA-MB-231 cells, representing the triple-negative subtype. Cell lines were obtained from the American Type Culture Collection (ATCC; LGC Standards, Middlesex, UK). MCF-7 cells were grown in EMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and supplemented with 10% fetal bovine serum (FBS; Gibco) and 0.01 mg/mL of insulin. MDA-MB-231 cells were grown in DMEM (Gibco) and supplemented with 10% FBS. Cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. The cell cultures were subjected to Mycoplasma screen tests periodically.
For fatty acid treatments, oleic acid- or linoleic acid-albumin from bovine serum (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) were used at 0 µM (control), 1 µM, 10 µM, 100 µM, or 1 mM concentration. All the solutions contained 0.1 mg/mL of BSA (Sigma-Aldrich).
For treatments with EVOO minor compounds, we used hydroxytyrosol (PHL80152, Sigma Aldrich) at 0 µM (control), 100 µM, 250 µM, and 400 µM; oleuropein (12,247, Sigma Aldrich) at 0 µM (control), 10 µM, 30 µM, and 50 µM; and lutein (L9283, Sigma-Aldrich) at 0 µM (control), 5 µM, 10 µM, and 30 µM [25 (link),26 (link),27 (link)]. All the solutions contained 0.1% of DMSO.

Glucose (≥99.5%), lactic acid (90%), formic acid, sulfuric acid (95.0–98.0%), methanol and acetonitrile of HPLC grade, methanol and ethanol of analytical grade, hydroxytyrosol, and tyrosol were purchased from Sigma-Aldrich (Sigma-Aldrich Co. LLC, St. Louis, MO, USA). Ultrapure water was obtained from an Elga Purelab Option R system (Veolia Environnement S.A., Paris, France). Extra virgin olive oil (EVOO) samples (n = 26) were obtained from producers and research laboratories. They differed by geographical origin (different Italian regions), cultivar, olive maturity, and extraction technology.