2.7.4. Inhibition of Lipid Peroxidation (TBARS Assay)

This assay was performed according to methods described by Damien Dorman et al. with slight modification [24]. Egg yolk rich in lipids was used as a source of an oxidable substrate. Briefly, 0.5 mL of 10% (w/v in 1.5% KCl) homogenate and 0.1 mL of sample solubilized in methanol were added to a test tube and made up to 1.0 mL with distilled water. An aliquot of 50 μL of FeSO₄ (0.07 M) was added to each sample to induce lipid peroxidation. To the mixture, 1.5 mL of 20% trichloroacetic acid (TCA) and 0.8% (w/v) thiobarbituric acid (TBA) in 1.1% (w/v) sodium dodecyl sulfate (SDS) stock reagent was added and then 1.5 mL of 20% acetic acid (pH adjusted to 3.5 with NaOH) was added. The resulting mixture was vortexed, followed by heating at 95°C for 60 min. After cooling, 5.0 mL of butanol was added to each tube and centrifuged at 3000 rpm for 10 min. The absorbance of the organic upper layer was measured at a wavelength of 532 nm. Butylated hydroxytoluene (BHT) and ascorbic acid were used as positive controls. The percentage inhibition of lipid peroxidation was calculated from the absorbance obtained using the following equation:

where A_c is the absorbance of blank and A_s is the absorbance of the sample mixture.

The IC₅₀ values (extract concentration needed to achieve 50% inhibition of lipid peroxidation) were obtained from a graph of % inhibition against concentration.