Lipid Peroxidation Inhibition Assay

The lipid peroxidation inhibition assay was conducted following the method described by Haenen and Bast [21 (link)]. Thiobarbituric acid (TBA)-reactive species were responsible for the lipid peroxidation (LPO) activity. To induce LPO, 0.005 mL of FeSO₄ (0.07 M) and approximately 1 mL of distilled water were added to the mixture, which was then incubated for 30 min. Afterward, the mixture was heated to 95 °C for 1 h. Subsequently, 1.5 mL of 0.8% (w/v) TBA, 1.5 mL of 20% acetic acid, 0.5 mL of 20% trichloroacetic acid (TCA), and 1.1% SDS were added. Another set of samples was treated similarly but without TBA. After cooling, each tube was filled with 5.0 mL of butanol and centrifuged for 10 min at 5000 rpm. The percentage of lipid peroxidation inhibition in the samples was calculated using the following formula: