2.7. Lipid peroxidation inhibitory activity
The lipid peroxidation inhibitory activity of HPEL was determined
according to the method of Tsuda et al. (1994) with slight
modifications. Liposome sample (egg lecithin 6 mg/mL phosphate
buffer, pH 7.4) was sonicated using an Ultrasonicator (SB-5200DTD,
Xinzhi Biotech Co., Ningbo, China, 40 kHz) for 1 h. Then 0.1 mL of HPEL
samples was dissolved in 50% ethanol at different concentrations and
was added to 0.5 mL of the liposome mixture. The control was made
without the test samples. Lipid peroxidation was induced by adding
10μL of FeCl3(200 mM) and 10μLofL-ascorbic acid (200 mM). After
incubating for 1 h at 37 °C, the reaction was stopped by adding 2 mL of
0.25 N HCl containing 15% trichloroacetic acid (TCA) and 0.375% TBA
(Thiobarbituric acid). The reaction mixture was boiled for 15 min,
cooled and centrifuged, and the absorbance of the supernatant was
measured at 532 nm. The blank consisted of all the reagents but
without the lipid. The lipid peroxidation inhibitory activity was
calculated as inhibitory activity (%) =(1−absorbance of sample /
absorbance of control) × 100. The lipid peroxidation inhibitory activity
of BHT and CEL was also assayed for comparison. The EC50(50% of the
radicals scavenged by the test sample) values were also determined.
The lower the EC50value, the higher was the antioxidant activity.