3.5.2. TBARS Assay

The TBARS (thiobarbituric acid reactive substances) assay estimated the concentration of malondialdehyde, a product of lipid peroxidation, as a measure of the antioxidant capability of polyphenols in the seed coat extracts to protect the lipid bilayer against peroxidation in liposomes as a simulated model for a biological cell membrane. This assay was conducted according to the method proposed by Subramanian et al. [74]. Preparation of liposomes [75,76] is described in the supplementary materials (Procedure S1). To measure the antioxidant activity of pulse seed coat extracts, 200 µL of liposome solution (1 mg/mL) was added to 40 µL of diluted extracts (10% methanol) and the mixture was shaken for 15 min. The control solution was prepared by mixing 10% methanol only with the liposome solution. Lipid oxidation was initiated by adding 40 µL of each of the following solutions: 0.5 mM FeSO₄ solution, 5 mM ascorbic acid and water into the liposome-sample solution. The mixture was shaken and incubated in a 37 °C Thermomixer for 2 h. A volume of 40 µL of 3% sodium dodecyl sulfate and 400 µL of 0.375% TBA, 15% TCA, 0.25 M HCl (prepared by dissolving 0.375 g of thiobarbituric acid (TBA) and 15 g of trichloroacetic acid (TCA) in 100 mL of 0.25 M HCl solution) were added to the mixture, followed by vortexing and heating in a glycol bath at 95 °C for 30 min. After cooling, 800 µL of 1-butanol was added to each reaction mixture, vortexed for 20 s, and centrifuged at a speed of 16,200× g for 10 min. An aliquot of 200 µL of the organic (upper) layer was pipetted into a 96-well plate that was read using a microplate reader at 532 nm. Serial dilutions (2–52 µg/mL) of 1,1,3,3-tetramethoxypropane (TMP) in 10% methanol were used to construct a calibration curve for MDA [75]. An online IC₅₀ calculator tool was used to calculate the concentration of each extract (µg/mL) required to inhibit 50% of the MDA formation (lipid peroxidation product) in the reaction mixture (IC₅₀) [77]. Its reciprocal, the antiradical power (ARP, ARP = 1/IC₅₀) was then calculated to facilitate comparing the results of different assays.