ORAC Assay for Essential Oils and Hydrosols

The assay was performed on a LS55 spectrofluorimeter (Perkin-Elmer, Leatherhead, UK) using 96-well white polystyrene microtiter plates (Porvair Sciences, Leatherhead, UK) according to a method described by Fredotovic et al. [73 (link)] and Nazlić et al. [74 (link)]. Each reaction contained 180 µL of fluorescein (1 µM), 70 µL 2,2′-Azobis(2-methyl-propionamidine) dihydrochloride (AAPH, Acros Organics, Fair Lawn, NJ, USA) (300 mM), and 30 µL of plant extract or reference standard Trolox (6.25–50 µM) (Sigma–Aldrich, St. Louis, MO, USA). All experimental solutions were prepared in a phosphate buffer (0.075 mM, pH 7.0). The extract of essential oil was prepared in acetone (20 mg/mL). This solution was further diluted 400× with the phosphate buffer for the experiments. For the hydrosol analyses we used total hydrosol diluted 10x. Measurements were performed in triplicate. ORAC values for essential oil were expressed as µmol of Trolox equivalents (TE) per g of essential oil and for hydrosol as µmol of Trolox equivalents (TE) per L of hydrosol.