2.6. Sheep Erythrocytes Hemolysis Assay

Sheep erythrocytes hemolysis assay was performed using the following protocol: sheep erythrocytes were washed three times with 5 volume of phosphate-buffered saline (PBS). Sheep erythrocytes at a final concentration of 5% were incubated with 4.4 mg/mL of Rigenase^® or ascorbic acid, used as reference compound [14], for 30 min at 37 °C. Then 200 mM AAPH was added to the mixtures and incubated at 37 °C; erythrocytes hemolysis was monitored every 20 min in the supernatants by spectrophotometric readings at A_{524 nm}. Hemolysis percentage was calculated by the formula: % hemolysis = [(A_sample/A_{positive control}) × 100]; whereas the positive control consisted of a mixture of erythrocyte suspension and water (to obtain 100% hemolysis) [14]. Reported results, expressed as mean ± SD, were the average of three independent experiments each made in triplicate. Statistical comparison between the reference compound and Rigenase^® samples was performed using the using the one-way Analysis of Variance (ANOVA). A p-value < 0.05 was considered statistically significant.