2.8. Hemolytic assay

2 mL of human blood (Sigma Aldrich, St. Louis, MO, USA) were mixed with 48 mL of phosphate-buffered saline to achieve a final concentration of 4 % RBC, then centrifuged at 2000 rpm for 5 min. The procedure was repeated twice, each time aspirating the supernatant and replacing it with a new buffer. 2 mL of various peptide solution concentrations were then added to 2 mL of erythrocyte suspension and incubated at 37 °C for 1 h. Triton X-100 (Santa Cruz Biotechnology, Dallas, TX, USA) was used as a positive control. After incubation, tubes were gently vortexed and 1 mL of each sample was transferred, then centrifuged at 2000 rpm for five minutes, then 200 μL of each supernatant was transferred into a 96-well plate and their absorbance was measured at 450 nm. The following equation was used to calculate the percentage of hemolysis (Au - Evans, Au - Nelson et al. 2013):

Where: “A” is the absorbance value of the tested solutions, “A0″ is the absorbance value of the negative control, and ”AX“ is the absorbance value of the positive control.