Folin cioc lteu s phenol reagent

OFE, botanical origin Olea europaea L. (trademark name: Oleaselect^®), was kindly provided by Indena s.a.s. (France). Oleaselect^® is a standardized extract obtained from the fresh pulp of a variety of selected Italian Olea europaea that are particularly rich in polyphenols.
For initial screening, five other olive extracts were examined. In particular, another olive fruit extract (Olive powder extract Opextan^®, Indena) and four OLE (i.e., Olea europaea leaves powder extract >6, >12, >20% oleuropein, purchased from Farmalabor, Italy, and >80% oleuropein, Sabinsa Europe GmbH, Langen, Germany) have been taken into account.
Standard hydroxytyrosol for HPLC analysis, gallic acid (GA), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), potassium persulfate, sodium carbonate, and Folin–Ciocâlteu’s phenol reagent were supplied by Sigma Aldrich (Milan, Italy). For the hydrogel and cream formulations, pharmaceutical-grade carboxymethyl cellulose sodium salt was supplied by Eigenmann e Vernally S.p.A. (Milan, Italy), whereas the other cosmetic ingredients reported in Section 3.3 were purchased from ZenStore (Salerno, Italy).

A reference standard of madecassic acid was obtained from J&K Scientific GmbH (Pforzheim, Germany), and methyl γ-linolenate was obtained from Sigma-Aldrich (Taufkirchen, Germany). Methanol, methylene chloride, and acetonitrile were obtained from Chemsolute (Th. Geyer GmbH & Co. KG, Renningen, Germany). Formic acid was obtained from Honeywell GmbH (Seelze, Germany). For GC analyses, the methylation reagent trimethylsulfonium hydroxide (TMSH) was from Fluka (Buchs, Switzerland). A fatty-acid methyl ester (FAME) reference mixture C16–C24 for the identification of fatty acids was purchased from Restek Corporation (Bellefonte, PA, USA), and tert-butyl methyl ether (TBME) was obtained from Merck (Darmstadt, Germany). Folin–Ciocâlteu’s phenol reagent, gallic acid monohydrate, and sodium carbonate were from Sigma-Aldrich (Taufkirchen, Germany).

Total phenolics were measured using a previously described method [72 (link)] with some modifications. For preparation of the extraction solution, 2 mL of EtOH was mixed with 1 mL of 0.1 M HCl (final pH was 4.0). The extraction solution (0.2 mL) was added to 50 mg of plant material and kept in the freezer for 48–72 h. After freezing, each tissue sample was homogenized, and then 0.3 mL EtOH was added. Samples were centrifuged at 10,000 rpm for 15 min. One milliliter of supernatant from each of the centrifuged samples was assayed for total phenolic content. Then, 0.25 mL of EtOH and 1.25 mL of H₂O were added to 0.25 mL of extract along with 0.125 mL of 1 N Folin–Ciocâlteu’s phenol reagent (Sigma Chemical Co., Burlington, Massachusetts, USA), vortexed, and allowed to incubate for 5 min. Next, 0.25 mL of 5% (w/v) sodium carbonate solution was added to each sample, vortexed, covered, and incubated in the dark for 1 h. The absorbance was read at 725 nm on a UV/Vis spectrophotometer (Shimadzu UV-1601PC spectrophotometer, Shimadzu Corp., Kyoto, Japan). A calibration curve was prepared with gallic acid (5–100 mg/mL), and the results were expressed as mg of gallic acid per g of FW.