Inhibition of linoleic acid autoxidation.The antioxidant activity of WSE and purified fractions also was measured according to the method of Osawa and Namiki (31), with some modifications. After freeze-drying, 1.0 mg of each sample was suspended in 1.0 ml of 0.1 M phosphate buffer (pH 7.0) and added to 1 ml of linoleic acid (50 mM), which was previously dissolved in ethanol (99.5%). Incubation in a glass test tube, tightly sealed with a silicon rubber cap, was allowed at 60°C in the dark for 8 days. The degree of oxidation was determined by measuring the values of ferric thiocyanate according to the method described by Mitsuta et al. (30). One hundred microliters of the sample was mixed with 4.7 ml of 75% (vol/vol) ethanol, 0.1 ml of 30% (wt/vol) ammonium thiocyanate, and 0.1 ml of 0.02 M ferrous chloride, which had been dissolved in 1 M HCl. After 3 min, the degree of color development, which represents the oxidation of linoleic acid, was measured spectrophotometrically at 500 nm. BHT and α-tocopherol (1 mg/ml) also were assayed as antioxidant references. A negative control (without antioxidants) also was considered.