Tyrosol

All standards of fatty acid methyl esters were purchased from Sigma (St. Louis, MO, USA). Methanol, n-hexane, petroleum ether, chloroform, potassium iodide, Folin–Ciocalteu reagent, sodium carbonate, starch, concentrated sulphuric, nitric and hydrochloric acid (37%) were purchased from Kemika (Zagreb, Croatia). Anhydrous sodium sulphate and 2,2-diphenyl-1-picryl-hydrazyl (DPPH) were purchased from Sigma (St. Louis, MO, USA).
Sodium and potassium hydroxides in the form of pellets, chloroform and analytical standards (dihydrocholesterol, campesterol, stigmasterol, β-sitosterol, eicosanol, docosanol, tricosanol, tetracosanol, pentacosanol, hexacosanol, heptacosanol, octacosanol, squalene and (±)-α-tocopherol) were purchased from Sigma (St. Louis, MO, USA). Ethanol and a silylating mixture according to Sweeley were purchased from Fluka (Buchs, Switzerland).
Hydroxytyrosol, tyrosol, oleuropein, lutein and apigenin were purchased from Extrasynthese (Genay, France). Caffeic acid and gallic acid were purchased from Sigma (St. Louis, MO, USA). Standard stock solutions of each compound were 1 mg/mL in Methanol.
All mineral standards were purchased from Merck (Darmstadt, Germany) and diluted to the appropriate obtained concentration.
All standards stored at +4 °C in amber vials and in a dark place until analysis.

For use as reagents, 6-Hydroxy-2, 5,7,8-tetramethylchromane-2-carboxylic acid (Trolox n 648471, Calbiochem San Diego, CA, USA,), 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS, A 1888, Sigma Aldrich, Taufkirchen, Germany)), 1,1-Diphenyl-2-picrylhydrazyl (DPPH, Sigma Aldrich), phosphate buffer solution (PBS), Ethanol (EtOH), Methanol (MtOH HPLC grade), Trifluoracetic acid, (TFA, Sigma Aldrich), nitric acid and acetic acid (HPLC gradient grade) were obtained from VWR Chemicals (Radnor, PA, USA). All other reagents were of analytical grade and the water was ultra-pure-grade. Phenolic standards: ferulic acid, tyrosol, isorhamnetin 3-O-rutinoside (narcissin) and kaempferol 3-O-glucoside were purchased from Extrasynthese (Genay, France). The in vitro digestion enzymes: α-amylase from Bacillus sp; pepsin, pancreatin, mucin, lipase from pigs; and bovine bile extract were obtained from Sigma-Aldrich (Milan, Italy).

Olea europaea L. wastes and by-product (free-oil olive pulp and leaves) were obtained from Leccino cultivar (Vinci, Florence, Italy). Leaves were collected in April 2019 during the pruning of olive trees; free-oil olive pulp in October 2019 after oil extraction. Poly (vinyl alcohol) (average MW 85–124 kg mol⁻¹, 99% hydrolyzed) was purchased from Sigma-Aldrich^® (Milan, Italy). Chemicals and HPLC grade solvents were purchased from Sigma-Aldrich (Milan, Italy). Tyrosol and oleuropein used as standards were supplied by Extrasynthèse (Genay, France). HydroxyTyrosol was synthesized according to an optimized and patented procedure [32 (link)].

All solvents for HPLC-DAD-MS analyses (HPLC grade), formic acid, and epigallocatechin gallate (analytical grade) were purchased from Sigma Aldrich Chemical Company Inc. (Milwaukee, WI, USA). Tyrosol, gallic acid, ellagic acid, catechin, and malvidin 3-O-glucoside were supplied by Extrasynthèse S.A. (Lyon, Nord-Genay, France). HydroxyTyrosol was synthetized in the laboratory [80 (link)]. HPLC-grade water was obtained via distillation and purification with a Labconco Water Pro PS polishing station (Labconco Corporation, Kansas City, MO, USA). Potassium sorbate, benzoic acid, potassium hydroxide, and hydrochloric acid 37% were purchased from Carlo Erba Reagents Srl (Cornaredo, Milan, Italy), Tween 20 from Biolife Italiana Srl (Milan, Italy), and Potato Dextrose Agar from Oxoid Ltd. (Basingstoke, Hampshire, UK). Sterile plasticware and cotton swabs were purchased from Unifo Srl (Zero Branco, Treviso, Italy).

Methanol (MeOH) and acetonitrile (AcN) were purchased from Merck (Darmstadt, Germany) and phosphoric acid from Sigma-Aldrich (St. Louis, MO, USA). Standards of apigenin, apigenin-7-O-glucoside, catechin, hydroxytyrosol, luteolin, luteolin-7-O-glucoside, oleuropein, rutin, tyrosol, and verbascoside were purchased from Extrasynthese (Genay, France). Deionized water was obtained by Siemens UltraClear (Siemens AG, München, Germany).
Multi-element standard solution from Perkin Elmer (NexION Setup Solution, Waltham, MA, USA) was used. Argon used to form plasma for the inductively coupled plasma mass spectrometer (ICP-MS) analysis was of purity 6.0 (Messer, Austria). Acetylene was supplied by Messer (Messer Croatia Plin d.o.o., Zaprešić, Croatia).
Deionized water was obtained by Siemens UltraClear (Siemens AG, München, Germany).

‘Hass’ avocado fruit and avocado oil (ETHNOS™) were purchased from a local market in Lleida, Spain. LMP was kindly donated by Herbstreith & Fox KG, Neuenbürg, Germany. The standards hydroxytyrosol, tyrosol, quercetin-3-O-glucoside, quercetin-3-O-rutinoside (rutin), kaempferol-3-O-glucoside, isorhamnetin, dimer B2 were purchased from Extrasynthese (Genay, France), whereas p-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid (protocatechuic acid), p-coumaric acid, caffeic acid, ferulic acid, chlorogenic acid (5-O-CQA), catechin, epicatechin were from Sigma-Aldrich (St. Louis, MO, USA). The digestive enzymes (pepsin and pancreatin from porcine pancreas) and bovine bile were purchased from Sigma-Aldrich, Inc. (St Louis, MO, USA). Ethanol and other solvents were purchased from Fischer Scientific (Leicestershire, UK) and Scharlau S.L. (Barcelona, Spain). The ultrapure water used to prepare all solutions of this study was obtained using a Milli-Q filtration system (18.2 mΩ, Merck Millipore, Madrid, Spain).

All-trans β-carotene (for biochemistry, purity 97%) was purchased from Merck (Darmstadt, Germany). Hydroxytyrosol, tyrosol, caffeic acid, luteolin-7-O-glucoside, p-coumaric acid, and oleuropein were obtained by Extrasynthese (Genay, France). Phosphoric acid and butylated hydroxytoluene (BHT) were from Sigma-Aldrich (Steinheim, Germany). Folin–Ciocalteu reagent and HCl as well as HPLC grade acetonitrile, acetone, n-hexane, ethyl acetate, and methanol were purchased from Chem-Lab NV (Zedelgem, Belgium). D-Glucose monohydrate, casein acid hydrolysate, yeast extract, L-asparagine, KH₂PO₄, MgSO₄·7H₂O, thiamine hydrochloride as well as emulsifiers Span 20 and Tween 80 were from Scharlau Chemie S. A. (Barcelona, Spain). D-Fructose was obtained by Panreac (Barcelona, Spain), while LCK 338 cuvette test was from Hach Lange (Düsseldorf, Germany). Potato dextrose agar (PDA) was supplied by Lab M Limited (Heywood, UK). All the other reagents and solvents of appropriate grade were purchased from various producers.