Acquity beh c18 1.7 μm

Metabolites were separated in a UHPLC system (Dionex Ultimate 3000) equipped with a Waters Column (Acquity BEH C18 1.7 μm, 2.1 × 50 mm) at 40°C. The mobile phase consisted of water containing 0.1% formic acid and 2 mM ammonium formate (solvent a, v/v), and acetonitrile containing 0.1% formic acid and 2 mM ammonium formate (solvent b, v/v), with a flow rate of 250 μL/min, and the following gradient elution program: 0–1.0 min, 5% b; 1.0–5.0 min, 5% to 60% b; 5.0–8.0 min, 60% to 100% b; 8.0–11.0 min, 100% b; 11.0–14.0 min, 100% to 60% b; 14.0–15.0 min, 60% to 5% b; 15.0–18.0 min, 5% b. The Q-Exactive instrument (Thermo) equipped with electrospray ionization in positive and negative switching modes was utilized to detect the above samples, and the system was calibrated and controlled by Xcalibur 3.1 and Q-Exactive Tune software. The UHPLC Q-Orbitrap analysis can produce large amounts of raw data using TraceFinder software. The data was exported into Excel spreadsheets by Simca-P for PCA (principle components analysis), PLS-DA (partial least squares discriminant analysis), t-test, volcano plot, and VIP (variable importance in projection) plot analysis [26 (link)].

High-speed counter-current chromatography (HSCCC) was carried out with a TBE-1000A high-speed countercurrent chromatography system (Shanghai Tauto Biotech Co. Ltd., Shanghai, China) with three serially connected multilayer coil separation columns (tubing = 1.6 mm; total volume = 1000 ml) and an 80 ml sample loop. The system was equipped with an LPLC pump (TBP5002, Shanghai Tauto Biotech Co. Ltd., Shanghai, China), a Thermo Finnigan SSI 500 UV detector (Thermo Electron Co., San Jose, CA), and Autochro-WIN software (version 3.0, Younglin-Tech, Seoul, Korea). ¹H and ¹³C NMR spectra were recorded on a Bruker AM500 instrument (Billerica, Massachusetts, USA). Profiling and tentative identification of the compounds were achieved with ultrahigh-performance liquid chromatography coupled with electrospray ionisation and quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS; ACQUITY UPLC™ system coupled to a Micro mass QT Premier™, Waters, Milford, MA, USA) with a reversed-phase column (ACQUITY BEH C18 1.7 μm, 2.1 × 100 mm, Waters). Purified water was obtained from a Milli-Q Academic system (Merck Millipore, Burlington, MA, USA). The reagents required for UPLC-ESI-QTOF-MS analysis were acetonitrile (ACN) (Merck Millipore), formic acid, and leucine enkephalin (Sigma–Aldrich, St. Louis, MO, USA). Solvents for NMR were purchased from Cambridge Isotope Lab Inc. (Andover, MA, USA).