2.11. β-Carotene-linoleic acid assay

We used β-carotene bleaching method (Mothana et al., 2012) for measuring the antioxidant activity of LSE. One milliliter of a 0.2 mg/mL β-carotene solution was mixed with 0.02 mL of linoleic acid and 0.2 mL of Tween-20 followed by removal of organic solvent (chloroform) by heating at 40 °C in a rotary evaporator. The residual mixture was diluted with 10 mL of distilled water and briefly mixed to form an emulsion. The blank was prepared without using β-carotene. The control contained 0.2 mL of 80% methanol instead of extract. An aliquot (5 mL) of the emulsion was mixed with 0.2 mL of sample at the concentration of 1.0 mg/mL. Rutin (1.0 mg/mL) was used as a positive control. The tubes were kept in a water bath at 40 °C for 2 h and the absorbance was recorded at 470 nm at 15 min intervals, using a UV–visible spectrophotometer (UV mini-1240, Shimadzu, Japan). The following equation was used to calculate the percent antioxidant activity:

% antioxidant activity = 1 − (Abs₀ − Abs_t)/(Abs°₀ − Abs°_t) × 100

where Abs₀ and Abs°₀ are absorbance values recorded at zero time for sample and control, respectively. Abs_t and Abs°_t are the absorbance readings for sample and control, respectively, measured at 120 min.