4.4. Hemolytic Activity

To determine the toxicity of the peptides to normal mammalian cells, human erythrocytes were used to test the hemolytic activity of the peptides according to the method reported [53]. Blood was collected from the median cubital vein of healthy volunteers. The normal human erythrocytes were washed three times with Dulbecco’s phosphate-buffered saline (PBS; Biological Industries, Israel), with the resulting pellet was suspended in PBS and diluted to 5% (w/v). The sample peptide was diluted with physiological saline into various gradient concentrations, 200 μL of various gradient concentrations of samples and 200 μL diluted red blood cells were gentle mixed and incubated at 37 °C for 30 min. Parallel incubations of erythrocytes in the presence of normal saline or 1% Triton X-100 served as the negative and positive controls, respectively. After incubation, centrifugation was conducted at 2500 rpm/min for 5 min at room temperature. A total of 100 μL of the supernatant was taken to measure the absorbance at 540 nm (GeneQuant, Fairfield, CT, USA). The relative optical density was compared with the absorbance of 1% Triton X-100, which induce 100% hemolysis. The study was proved by the Research Ethics Committee of Kunming Institute of Botany, Chinese Academy of Sciences and the ethics approval number was Kib202103028. Informed consent was provided for blood donation.