3.6. Inhibition of Lipid Oxidation in a Liposomal Model System

The antioxidant activity of gallic acid esters (galloyl phytol, galloyl phytanol, and galloyl eicosanol) on the lipid peroxidation was determined according to a previous study [23]. Liposomes were prepared using the thin-film hydration method. Lecithin (200 mg) was completely dissolved in chloroform (10 mL) and evaporated under vacuum at 25 °C. The lecithin film was preheated to 70 °C with a dropwise addition of 10 mL PBS (10 mM, pH 6.0) under magnetic agitation. Then, the mixture was stirred for 20 min to afford the fresh liposomes with a final lecithin concentration of 20 mg/mL.

To test the antioxidant activity, 0.5 mL sample at a fixed antioxidant concentration of 50 μM in a PBS buffer (0.01M, pH 6.0) was added to 1 mL liposome dispersion, followed by the addition of 0.5 mL AAPH (10 mM) solution. The mixture was incubated at 37 °C for 1 h. The PBS buffer was employed as blank. After the oxidation process, an aliquot (200 μL) of the mixture was withdrawn and reacted with 2-thiobarbituric acid (TBA) working liquid (300 μL) in the kit at 95 °C for 30 min. The reaction mixture was centrifuged at 13,000 rpm for 10 min after cooling down to an ambient temperature using ice. The absorbance values of supernatant (200 μL) at 532 and 600 nm were determined in a 96-well plate using a multimode microplate reader (Spark 10M, Tecan Group Ltd., Männedorf, Switzerland) and then the amount of thiobarbituric acid reactive substance was employed to estimate the antioxidant activity.