Scopoletin

LX-2 cells were grown in Dulbecco’s modified Eagle’s medium (Invitrogen, Shanghai, China), with the addition of 10% fetal bovine serum (FSP500, ExCell bio, ShangHai), 100 μg/mL streptomycin, and 100 μg/mL penicillin in a humidified environment with 5% CO2 at 37 °C. scopoletin, caryophyllene oxide, and hydrocinnamic acid (≥ 99% purity by HPLC) were bought from TargetMol (USA) and dissolved in dimethyl sulfoxide (Sigma, USA). TGF-β1 was acquired from PeproTech, Inc. (Suzhou, Jiangsu, China).

The phytochemical components of Sinomenium acutum were identified as depicted above [16 (link),17 (link)] and compared with the reference retention time and mass spectrum. The latter included higenamine, gelsemine, magnoflorine, scopoletin, columbamine, jatrorrhizine, palmatine, syringin, and eleutheroside E, which were purchased from TargetMol (Wellesley Hills, MA, USA). UHPLC–MS/MS analysis was performed with a Dionex UltiMate 3000 system coupled with a Thermo Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The dried powder of Sinomenium acutum extract was added to 100% MeOH to attain a concentration of 20 mg/mL. Chromatographic separation was performed using an Acquity BEH C18 column (100 × 2.1 mm, 1.7 µm) with 0.1% formic acid in water and acetonitrile. The gradient eluent condition for UHPLC and the details for MS/MS were incorporated, as previously shown [38 (link)]. Data acquisition and processing were carried out withXcalibur and TraceFinder 5.1 software systems (Thermo Fisher Scientific).