Hemolysis Test

In vitro hemolysis assays were conducted using red blood cells (RBCs). The hemolytic activity of samples was analyzed by quantification of the released hemoglobin by spectrophotometric measurement. Briefly, one New Zealand rabbit (male, 2.2 kg) was used in this study. We collected 18 mL blood by heart puncture, and placed it into a pre-prepared centrifuge tube, containing 2 mL 3.8% sodium citrate, to prepare fresh anticoagulated blood. Blood was slowly stirred using a glass rod to remove fibrin, then diluted in physiological saline (PS) at a 1:1 volume ratio, and centrifuged at 3000 rpm for 10 min. The supernatant was removed, the remaining sample diluted 1:10 in PS, and then purified and washed by centrifugation (3000 rpm, 10 min, 5 times) to collect the RBCs. Finally, precipitated RBCs were diluted with PS to prepare a 2% RBC suspension.²⁴

Before the experiment, fluid extracted from the precursor solution²⁵ was prepared by adding approximately 0.5 g of drug-free precursor solution in 5 mL PS, incubating at 37°C for 72 h, then filtering through a sterile filter (0.22 μm). To control for the effects of other components in the precursor solution, PYT, 64% ethanol, 32% ethanol, and 16% ethanol were treated in the same manner as described above. Groups used for the experimental design of the hemolysis experiment are shown in Table 1. Briefly, 1.5 mL of extracts were added to tubes labeled A–G, with 1.5 mL PS in test tube H, as a negative control, and 1.5 mL distilled water in test tube I, as the positive control group. Then, 2% RBCs suspension was added into each tube, the mixtures gently vortexed, and incubated for 4 h at 37°C. All samples were analyzed in triplicate.

Hemolysis Experiment Group Design

First, the presence or absence of hemolysis in each group of tubes was observed by visual inspection of RBC fragmentation and sedimentation. If RBCs sank, tubes were gently shaken to determine whether they could be resuspended and assess whether agglutination had occurred. Next, tubes were centrifuged at 1000 rpm for 10 min, 150 μL aliquots of supernatant form each sample transferred to a 96-well plate, and absorbance recorded using a microplate reader (Biotek, ELX800, USA). The maximum absorption wavelength (λ_max) was determined by UV/Vis spectroscopy as follows: supernatants from the positive control group were placed in quartz cuvettes with a 1 cm path in a total volume of 2.0 mL, then UV/Vis spectra (200–800 nm) were recorded to determine the λ_max value. To express the in vitro hemolytic properties of our samples, the percentage of hemolysis was calculated as follows:²⁶

Hemolysis (%) = (S_absorbance – H_absorbance)/(I_absorbance − H_absorbance) ×100

where S_absorbance, H_absorbance, and I_absorbance refer to the absorbance values of the sample, negative control group, and positive control group, which were the mean values of measured absorbance from test tubes A–G, H, and I, respectively.

The sediment collected in the tubes by centrifugation (as described above) was resuspended in PS, a drop placed on a glass coverslip, and another coverslip placed over it. The coverslips were then gently pulled apart horizontally and allowed to air dry. This technique provides uniform smear thickness and staining. Smears were then flooded with Wright’s stain for 2 min, followed by flooding with water for 4 min, drying, mounting on a glass slide, and examination by light microscopy.