2.2.7. Inhibition of linoleic acid oxidation

Linoleic acid oxidation was measured using a slight modification of the method described by Girgih et al. (2011). Samples at final assay concentrations of 0.25, 0.5, and 1.0 mg/ml were dissolved in 1.5 ml of 0.1 M sodium phosphate buffer, pH 7.0. Each mixture was added to 1 ml of 50 mM ethanolic linoleic acid and stored in a glass test tube kept at 60 °C in the dark for 7 days. On a daily basis, 100 μl of the sample mixture was removed and mixed with 4.7 ml of 75% aqueous ethanol, 0.1 ml of ammonium thiocyanate (30%, w/v) and 0.1 ml of 0.02 M acidified ferrous chloride (dissolved in 1 M HCl). An aliquot (200 μl) of the resulting solution was added and the degree of color development was measured using the spectrophotometer at 500 nm after 3 min incubation at room temperature. An increased absorbance implied an increase in the level of linoleic acid oxidation.