3 hydroxytyrosol

All reagents used were analytical-grade and HPLC-grade purity. Methanol, water, acetonitrile, and H₃PO₄ were obtained from Chem-Lab NV (Zeldegem, Belgium). Folin–Denis reagent, DPPH, and caffeic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Oleuropein (CAS: 32619-42-4) and 3-hydroxytyrosol (CAS: 10597-60-1) standards were obtained from Tokyo Chemical Industry Co. (TCI, Tokyo, Japan). Oleocanthal (CAS: 289030-99-5) and oleacein (CAS: 149183-75-5) standards were purchased from Carbosynth (Compton, UK) and PhytoLab (Vestenbergsgreuth, Germany), respectively.

To prepare the HIDROX solution, 1 g HIDROX^® 12% (Oliphenol LLC., CA, USA) powder was dissolved in 10 mL phosphate-buffer saline (PBS) and centrifuged at 850× g for 10 min. The aqueous layer was stored at −30 °C and used as the 100 mg/mL HIDROX stock. To prepare the HT solution, 1 g 3-hydroxytyrosol (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was dissolved in 10 mL PBS and the 100 mg/mL HT stock was stored at −30 °C. HIDROX and HT solutions were prepared by diluting the stock solutions with PBS before use. OLIVENOL^TM plus⁺ Healing Moisturizer (Oliphenol LLC.) containing 2%, 5%, and 10% HIDROX was prepared and used as HIDROX-containing cream. HIDROX-free (0%) ingredients of OLIVENOL^TM plus⁺ Healing Moisturizer were used as the base control.