β-Carotene bleaching assay

The antioxidant activity of the extracts was determined using the β-carotene/linoleic acid test (Dapkevicius et al. 1998). Approximately 10 mg of β-carotene (type I synthetic) were dissolved in 10 mL of chloroform. Then, 0.2 mL of this solution was added to a boiling flask containing 20 mg linoleic acid and 200 mg Tween 40. Chloroform was removed using a rotary evaporator at 40 °C for 10 min. Then, 50 mL of oxygenated distilled water was added slowly with vigorous stirring to form an emulsion. The emulsion (5 mL) was added to a tube containing 0.2 mL of extract solution prepared according to Choi et al. (2000). The absorbance was immediately measured at 470 nm against a blank consisting of an emulsion without β-carotene. The tubes were placed in a water bath at 50 °C and the oxidation of the emulsion was monitored spectrophotometrically by measuring absorbance at 470 nm over a 60 min period. Samples containing 0.2 mL of ethanol were also monitored and used as control. The same procedure was carried out for the stable antioxidant reference BHT (butylated hydroxytoluene) as positive control and blank. The antioxidant activities of the extracts were expressed as an inhibition percentage with reference to the control sample after 60 min of incubation, using the following formula (Duarte-Almeida et al. 2006):

where, AA = antioxidant activity; DR_C = degradation rate of control = [ln (a/b)/60]; DR_S = degradation rate of sample = [ln (a/b)/60]; a = absorbance at time 0; b = absorbance at 60 min.