Ferric chloride

Ethanol absolute was obtained from Carlo Erba (Milan, Italy). Acetic acid, acetonitrile, methanol, and water (high performance liquid chromatography—HPLC grade) were purchased from VWR (Milan, Italy).
α-pinene, β-pinene, linalool, β-myrcene, terpinolene, caryophyllene, humulene, and β-bisabolene, gallic acid, p-OH benzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, ferulic acid, and rosmarinic acid (from Sigma-Aldrich, Darmstadt, Germany) standards were employed. Working standard mixtures were prepared by appropriate dilution of the standards in methanol. All solutions were stored at −20 °C in the dark.
Folin–Ciocâlteu’s reagent, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2-diphenyl-1-picrylhydrazyl (DPPH˙), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS˙⁺) were purchased from Sigma-Aldrich (Darmstadt, Germany). Sodium carbonate, potassium persulfate, potassium hexacyanoferrate(III), trichloroAcetic acid, ferric chloride, and potassium phosphate monobasic were obtained from Carlo Erba (Milan, Italy).

The ferric reducing antioxidant power (FRAP) assay measures the antioxidant potential of compounds or of mixtures of compounds through the reduction of ferric iron (Fe³⁺) to ferrous iron (Fe²⁺) [114 (link),115 (link)]. This method was used to compare the antioxidant potential of α-tocopherol, γ-tocopherol, and OA as compared with Trolox used as the positive reference. Briefly, α-tocopherol, γ-tocopherol, OA and Trolox were prepared in a range of concentrations from 0 to 6 mg/mL in phosphate buffer (PB: 0.2 M, pH 6.6) and potassium ferrocyanide (PF: 1% w/v; Fluka) mixed in equal volumes (PB (200 μL) + PF (200 μL)). The mixture was incubated at 50 °C for 20 min. Then, 200 μL of trichloroacetic acid (TCA: 10% w/v) (Sigma) was added to the reaction mixture. After centrifugation (1000× g, 10 min), 500 μL deionized water and ferric chloride (0.5 mL; 0.1% w/v) (Carlo-Erba, Val-de-Reuil, France) were added. After 30 min, the absorbance was measured at 700 nm (Sunrise spectrophotometer, Tecan, Lyon, France). The antioxidant potential of α-tocopherol, γ-tocopherol, and OA evaluated with the FRAP method was estimated in Trolox equivalent (1 mole of α-tocopherol, γ-tocopherol, and OA is equivalent to X mole of Trolox; X was determined with the calibration curve obtained with Trolox).