Determination of SOD activity

SOD activity was measured using the nitro-blue tetrazolium (NBT) photoreduction method [14]. Assay of each sample includes a sample tube and two control tubes. The sample tube contained 10 µL of SOD enzyme extract homogenized in a substrate composed of 150 mL of 0.05 mol/L phosphate buffer solution, 20 µL of distilled water, 30 µL of 130 mmol/L Met solution, 30 mL of 750 µmol/L NBT solution, 30 mL of 100 µmol/L EDTA-Na₂ solution and 30 mL of 20 µmol/L solution, whereas the control tube contained the buffer without enzyme extract. One control tube was put in the darkness, while the other was placed under a 4000 lx fluorescent light for 20 min. The control tube in the dark is blank, control tube in the light is absorbance of illumination. The OD (optical density) value was determined at 560 nm using microplate reader (Synergy H1, Biotek, USA). One unit of SOD was defined as the amount of hemoglobin that inhibits the rate of NBT reduction by 50.0%. The SOD activity was calculated using the following formula:

where A_CK is absorbance of illumination for the control tube, A_E is absorbance of illumination for the sample tube, V is total sample volume (mL), V_t is sample volume for determination (mL) and W is sample fresh weight (g). The SOD value of each sample was measured in duplicate to ensure the accuracy of the assay. If the difference between two repeats was more than 10.0%, the experiments were carried out again.