β-Carotene-Linoleic Acid Assay

β-Carotene bleaching assay was done according Wettasinghe and Shahidi (1999). One milliliter of β-carotene solution (0.2 mg/mL in chloroform) was pipetted into a round bottom flask containing 0.02 mL of linoleic acid and 0.2 mL of 100% Tween-20. The mixture was evaporated in a rotary vacuum evaporator for 10 min to remove chloroform. The mixture was immediately diluted with 100 mL of distilled water with vigorous shaking to form an emulsion. Varying concentrations of extract and standard (100–500 μg/mL) was added to 5 mL of the emulsion in different test-tubes and the mixture was kept at 37°C for 1 h. Absorbance of sample and control was measured at time t = 0 and t = 60 min. Total antioxidant activity was calculated based on the following equation: AA = [1 - (A₀ -A_t) × (A₀⁰ -A_t⁰)^-1] × 100, where AA is antioxidant activity, A₀ and A₀⁰ are the absorbance values measured at the initial incubation time for samples and control, respectively while A_t and A_t⁰ are the absorbance values measured in the samples or standards and control at t = 60 min. Mean of three determinations was recorded.