4.7.3. β-Carotene/Linoleic Acid Bleaching Assay

The β-carotene/linoleic acid bleaching assay was performed following the method described by Miraliakbari and Shahidi, with slight modification [44]. A mixture of β-carotene and linoleic acid was prepared by dissolving 0.5 mg β-carotene in 1 mL chloroform and 25 µL linoleic acid in 200 mg Tween 20. The chloroform was then completely evaporated under vacuum, and 100 mL of distilled water was subsequently added to the residue, and then the mixture was shaken vigorously to form an emulsion. From this emulsion, 2.5 mL was transferred into different test tubes containing 350 µL of ALE at different concentrations. All samples were vortexed for 1 min and placed at 50 °C in a water bath for 2 h together with a negative control (blank), which contained the same volume of ethanol instead of the samples. The absorbance of samples was measured at 470 nm using a spectrophotometer at initial time (t = 0) against a blank (emulsion without β-carotene). A standard BHT was used as a positive control. Antioxidant activities (inhibitions percentage, I%) of the samples were calculated using the following Equation (2):

where A _{sample t=0} and A _{sample t=2h} are the absorbance values for the test sample at the beginning of the experiments and after 2 h assay, respectively. A _{control t=0} and A _{control t=2h} are the absorbance values for the control at the beginning of the experiments and after 2 h assay, respectively.