Contents of ·O2−, H2O2 and MDA were determined according to the methods described by Hu et al. [17 ] with slight modifications. The generation rate of ·O2− was determined using hydroxylamine method. Banana peel samples (0.50 ± 0.05 g) were ground with 3 mL of 50 mM Tris-HCl buffer (pH7.8) and the homogenate was centrifuged at 12,000 g at 4°C for 30 min. The reaction mixture (0.5 mL) contained 50 mM Tris-HCl buffer (pH7.5), 0.5 mM XTT [sodium, 3-1- (phenylamino-carbonyl)-3, 4-tetrazolium-bis(4-methoxy-6- nitro), and benzenesulfonic acid hydrate], and 50 μL of sample extracts. Corrections were made for the background absorbance in the presence of 50 U of superoxide dismutase (SOD). The rate of ·O2− production was expressed as μg·g−1 FW (fresh weight) · s−1.
For determination of H2O2, banana peels (0.50 ± 0.05 g) were ground and extracted in 3 mL cold acetone. The homogenate was centrifuged at 10,000 g at 4°C for 30 min and 0.5 mL of the supernatant fraction was mixed with 1.5 mL of CHCl3 and CCl4 (1:3, V/V) mixture, then 2.5 mL of distilled water was added and the mixture centrifuged at 10,000 g for 1 min and the aqueous phase collected for H2O2 determination. The reaction system included 0.5 mL aqueous phase, 0.5 mL of buffer (phosphate-buffered saline, 200 mM, pH7.8), and 20 μL (0.5 unit) of catalase as control or inactive catalase protein (catalase inactivated by heating in boiling water for 5 min). After incubation at 37°C for 10 min, 0.5 mL of 200 mM titanium 4-(2-pyridylazo) resorcinol (Ti-PAR) was added to the reaction mixture for further incubation at 45°C for another 20 min. Absorbance at 508 nm was measured and H2O2 content was indicated as μmol·g−1 FW (fresh weight).
For MDA analysis banana peel samples (0.50 ± 0.05 g) were ground in 3 mL of 0.1% trichloroacetic acid (TCA) and centrifuged at 10,000 g for 30 min, and 1.8 mL of the supernatant fraction was mixed with 1.8 mL of 20% TCA containing 0.5% thiobarbituric acid. The mixture was heated at 100°C for 30 min, cooled, and centrifuged at 15,000 g for 10 min. Absorbance was recorded at 532 nm and the value for nonspecific absorption at 600 nm was subtracted. An extinction coefficient of 155 mM−1·cm−1 was used to calculate MDA content and the content of MDA in banana peels was expressed as μmol·g−1 FW (fresh weight).
For determination of H2O2, banana peels (0.50 ± 0.05 g) were ground and extracted in 3 mL cold acetone. The homogenate was centrifuged at 10,000 g at 4°C for 30 min and 0.5 mL of the supernatant fraction was mixed with 1.5 mL of CHCl3 and CCl4 (1:3, V/V) mixture, then 2.5 mL of distilled water was added and the mixture centrifuged at 10,000 g for 1 min and the aqueous phase collected for H2O2 determination. The reaction system included 0.5 mL aqueous phase, 0.5 mL of buffer (phosphate-buffered saline, 200 mM, pH7.8), and 20 μL (0.5 unit) of catalase as control or inactive catalase protein (catalase inactivated by heating in boiling water for 5 min). After incubation at 37°C for 10 min, 0.5 mL of 200 mM titanium 4-(2-pyridylazo) resorcinol (Ti-PAR) was added to the reaction mixture for further incubation at 45°C for another 20 min. Absorbance at 508 nm was measured and H2O2 content was indicated as μmol·g−1 FW (fresh weight).
For MDA analysis banana peel samples (0.50 ± 0.05 g) were ground in 3 mL of 0.1% trichloroacetic acid (TCA) and centrifuged at 10,000 g for 30 min, and 1.8 mL of the supernatant fraction was mixed with 1.8 mL of 20% TCA containing 0.5% thiobarbituric acid. The mixture was heated at 100°C for 30 min, cooled, and centrifuged at 15,000 g for 10 min. Absorbance was recorded at 532 nm and the value for nonspecific absorption at 600 nm was subtracted. An extinction coefficient of 155 mM−1·cm−1 was used to calculate MDA content and the content of MDA in banana peels was expressed as μmol·g−1 FW (fresh weight).
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