Standard calibration mix

All substrates were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Samples were routinely analyzed on an Agilent 1260 Infinity II HPLC instrument (Santa Clara, CA, USA). The HPLC was equipped with an Agilent Eclipse Plus C₁₈ RRHD column (1.8 µm, 2.1 mm × 50 mm) and eluted with a linear gradient of 10–95% of acetonitrile-water for 20 min, 95% acetonitrile-water for 10 min and drop down to 10% in 5 min, then 10% acetonitrile-water for 5 min at a flow rate of 0.5 mL/min. ¹H-, ¹³C-, HSQC- and HMBC-NMR spectra were recorded on an Agilent 600 DD2 spectrometer. Chemical shift values (δ) are given in parts per million (ppm) and the coupling constants (J values) are in Hz. Chemical shifts were referenced to the residual solvent peaks of DMSO-d₆. HPLC-HRESIMS spectra were acquired on an Agilent 1290 Infinity II HPLC coupled with an Agilent QTOF 6530 instrument operated in negative ion mode using capillary and cone voltages of 3.6 kV and 40–150 V, respectively. For accurate mass measurements the instrument was calibrated each time using a standard calibration mix (Agilent) in the range of m/z 150–1900.

The purified compounds were individually subjected to spectrum analysis for structure identification. The high-resolution electrospray ionization mass spectrometry (HRESIMS) spectrum of the compound was recorded with an Agilent QTOF 6545 instrument operated in a positive-ion mode at capillary and cone voltages of 3.6 kV and 40 to 150 V, respectively. The collision energy was optimized from 15 to 50 V. For accurate measurement of metabolite mass, the instrument was calibrated each time using a standard calibration mix (Agilent) in the range of m/z 150 to 1,900. The 1D and 2D NMR (nuclear magnetic resonance) spectrum data for each compound (including ¹H, ¹³C, heteronuclear single quantum coherence [HSQC], correlation spectroscopy [COSY], nuclear Overhauser effect spectroscopy distortionless enhancement by polarization transfer [NOESY DEPT], and/or heteronuclear multiple-bond correlation [HMBC]) were recorded at 25°C using a Bruker Avance III-500 spectrometer equipped with a 5-mm Pabbo BB-¹H/D probe (60 (link)). The chemical shift values (δ) are given in parts per million, and the coupling constants (J values) are shown in hertz. Chemical shifts were referenced to the residual solvent peaks of CD₃OD-d₄. All spectra were processed with the MestRe Nova program (version 9.0.1; Metrelab Research, Santiago de Compostela, Spain).