Anti-inflammatory activity in vitro

For the extraction of total extracts, 10 g of dried medicinal powder from each of the four species was added to 150 mL of 70% ethanol solution. The solutions were extracted three times using heating and reflux (2 h per extraction). The extracts were then combined and concentrated under reduced pressure The total extracts of DY, YM, SZ, and ES were obtained as 1.7723, 1.7963, 1.4352, and 2.7994 g, and the extraction rates were 17.723, 17.963, 14.352, and 27.994%, respectively.

For cell culture and passaging, frozen RAW 264.7 cells were removed from liquid nitrogen and quickly thawed in a 37°C water bath. The supernatant was then removed by centrifugation, 1 mL of RPMI 1,640 was added to the complete medium containing 10% fetal bovine serum to resuspend the cells, after which the cell suspension was transferred to a Petri dish to be cultured in a incubator at 37°C, 5% CO₂. The cell status was observed the next day. When the cells grew to 80–90% of the culture dish, they could be passaged. From the beginning of recovery, it is recorded as the first generation. Generally, cells from three to eight generations are selected for experiments. When the cell density was moderate, they were removed from the medium and fresh medium was added after centrifugation to make a cell suspension. The cells were then transferred to a Petri dish in a certain proportion to continue culturing in the incubator at 37°C, 5% CO₂ for later use.

For MTT assays, cells in the logarithmic growth phase were selected, and the cell concentration was adjusted to 1 × 10⁴ cells/mL, then inoculated into a 96-well plate, 100 μL/well, and cultured in a 37°C, 5% CO₂ incubator for 24 h. Different concentrations of the total extracts in the medium were added to cells in the experimental group, while equal volumes of the medium were added to cells in the control group. The cells were incubated with the extracts for 24 h. About 10 μL of MTT solution at 5 mg/mL was added per well and incubated in the dark for 4 h. The supernatant was then removed and 100 μL/well dimethyl sulfoxide (DMSO) was added and mixed evenly. After the purple crystal (formazan) was completely dissolved, the optical density (OD) of each well was measured using a microplate reader at the wavelength of 570 nm. The experiment was repeated three times to calculate the cell survival rate of each group.

For the establishment of the inflammatory cell model, RAW 264.7 cells in the logarithmic growth phase were selected, and the cell concentration was adjusted to 5 × 10⁵ cells/mL, and then inoculated in a 24-well plate, 500 μL/well, and cultured in a 37°C, 5% CO₂ incubator for 24 h. The experimental group was incubated with different concentrations of lipopolysaccharide (LPS) (0.01, 0.1, 1, and 10 μg/mL), while the control group received only a culture medium. The NO concentrations were measured at 12, 24, and 48 h using a kit, according to the instructions. The experiment was repeated three times.

For the effect of LPS-induced RAW 264.7 cells on NO production and inflammatory factors, RAW 264.7 cells in the logarithmic growth phase were selected, and the cell concentration was adjusted to 5 × 10⁵ cells/mL, and then inoculated in a 24-well plate, 500 μL/well, and cultured in a 37°C, 5% CO₂ incubator for 24 h. The cells were pretreated with different concentrations of total extracts for 1 h and then LPS was added for a total of 24 h. The positive drug group was added with LPS and indomethacin (INM, 100 μM). The model control group received only LPS, the blank control group received only the medium, and the NO concentrations were determined according to the instructions of the NO, TNF-α, and IL-6 kit. The experiment was repeated three times.