2.10. β-Carotene/Linoleic Acid Method and Antioxidant Activities

Thymus antioxidant potency was measured using the β-carotene bleaching inhibition in the linoleic acid presence. β-carotene/linoleic acid emulsion was prepared by mixing 1 mg β- carotene, 25 μl of linoleic acid, and 200 mg Tween-20 in 1 ml of chloroform. The solvent was vacuum-evaporated using a rotary evaporator for 30 minutes at 40°C. After that, 100 ml of oxygenated water was composed with robust shaking, in order to form a stable emulsion. 350 μl of the essential oil dilutions in ethanol (ranging 0.01–1.28 mg/ml) was mixed with 2.5 ml of the β-carotene/linoleic emulsion gently, at the final stage. All tubes were placed in a shaker incubator (50 rpm) for 2 hours, in the light and at 50°C. The absorbance difference was recorded for 2 hours, using a spectrophotometer against a negative control (NC), which contains all the reagents except oils at 470 nm. The beta-carotene oxidation inhibition percentage (BIC %) was calculated as follows:

Appropriate routine reference substances amounts were also applied including vitamin C and butylated hydroxytoluene (0.5 to 128 μg/ml) for the antioxidant capacity evaluation. The relative antioxidative activities plot versus the oil concentrations was drawn, and BIC₅₀ was measured from the best fitted line equation in the scatter plot [16].