Coumarin

The reagents used in cell culture were purchased from Gibco. An insulinoma cell line (INS-1) from rats was cultured in RPMI-1640 medium. A pancreatic beta cell line (MIN6) from mice was grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 1% penicillin–streptomycin, and 0.2% β-mercaptoethanol. Cells were maintained at 37°C in an atmosphere of 5% CO₂ in humidified air. To establish a model of oxidative stress in the β cell, INS-1 cells were cultured with alloxan (18 mM; Sigma, United States) or MIN6 cells were cultured with H₂O₂ (150 μM; Sigma) for 24h, which formed the oxidative stress (OS) model groups (Kimoto et al., 2003 (link); Li et al., 2020 (link)).
A stock solution of EA (40 mg/mL) was prepared. The main ingredients coumarin, cinnamyl alcohol, cinnamic acid, cinnamaldehyde (purity: HPLC ≥98%) were purchased from Shanghai Yuanye Bio-Technology (Shanghai, China) and prepared as stock solutions (50 mM) and diluted to a final concentration of 50 µM. The concentration required for experimentation was prepared with medium immediately before use.

The test R. solanacearum strain (Biovar3, Phylotype I) was obtained from Laboratory of Natural Product Pesticide of Southwest university, which was identified to be a highly pathogenic strain. The strain was cultured on NA agar medium (yeast extract 1.0 g, beef extract 3.0 g, peptone 5.0 g, glucose 10.0 g, agar 20.0 g, deionized water 1000 mL, pH 7.0) at 30 °C for 48 h. Single colonies were picked and transferred into NB liquid medium (NA medium without agar) for 12–16 h. The cells were harvested, by centrifugation, at an optical density at 600 nm (OD₆₀₀) of 1.0 and stored at −80 °C before use.
Resveratrol and coumarin used in this study were purchased from Shanghai Yuanye Bio-technology (Shanghai, China) and had ≥98% purity (by HPLC analysis). Stock solutions (20 mg/mL Resveratrol or coumarin) were prepared in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) and filter-sterilized through a bacterial filter before use.

Liquiritin, isoLiquiritin, cinnamic acid, glycyrrhizic acid, coumarin, cinnamic alcohol, cinnamaldehyde, and atractylenolide III were obtained from Shanghai Yuanye Bio-Technology Company (Shanghai, China). The purity of all standard compounds has met the analytical requirements (>98%). The four herbs of LGZGD were purchased from Anhui Puren Chinese Medicine Co., Ltd. (Bozhou China). Acetonitrile and methanol were of chromatographic grades (Merck Company, Germany). Rat IL-1β ELISA kit was purchased from Meimian (Jiangsu, China). Antibodies (NLRP3, GSDMD, GAPDH) were purchased from Abcam (Cambridge, MA, USA). Caspase-1 p20 was purchased from Affinity Biosciences (Cincinnati, OH, USA).