C18 column

AFB1 and the subsequent degradation products were analyzed using HPLC (Waters Alliance 2695, Milford, MA, USA) equipped with a YMC C18 column (250 × 4.6 mm, 5 μm) and a UV detector set at 360 nm [62 (link)]. A volume of 10 μL of samples was injected and eluted with the mobile solvent consisting of water/methanol/acetonitrile (48:40:12, v/v/v) at a constant flow rate of 0.8 mL/min at 30 °C. The limit of detection (LOD) was 0.01 μg/mL AFB1 and the limit of quantitation was 0.05 μg/mL AFB1 under the experimental conditions used. The concentration of AFB1 was determined using external calibration curves of the standard AFB1 solution at different concentrations. The percentage of AFB1 degradation was calculated using the following formula: AFB1 degradation (%) = (1 − remaining AFB1 in sample/total AFB1 in control sample) × 100.

The HR-ESI-MS spectra were performed on Waters Xevo G2-XS QTOF mass spectrometer (Waters, Milford, MA, USA). NMR spectra were measured on a Bruker Avance-600 spectrometer (Bruker, Karlsruhe, Germany) in DMSO-D₆ operating at 600 MHz for ¹H NMR, HMBC and HMQC, 150 MHz for ¹³C NMR, respectively. Tetramethylsilane (TMS) was used as internal standard. Column chromatography was performed with silica gel (200–300 mesh) purchased from Qingdao Ocean Chemical Group Co. Ltd (Qingdao, China). Thin-layer chromatography was conducted on silica gel G plates (Qingdao Marine Chemical Inc.). Semi-preparative HPLC with 1525 binary pump combined Waters 2998 UV detector (Waters Co., Milford, MA, USA.) and YMC C₁₈ column (5 μm, 20 mm × 10 mm; YMC Co., Ltd., Japan) were also used to isolate the compounds. Methanol (MeOH) and acetonitrile (ACN) were HPLC grade (Fisher, USA). Purified water was purchased from YiBao Co, Ltd (Shenzhen, China). Other solvents used were analytical grade (Beijing Chemical Works, Beijing, China).

Optical rotations were measured on a Perkin-Elmer 341 automatic polarimeter (Perkin-Elmer). UV spectra were recorded in MeOH (1 mg/50 mL) on a UV 210A spectrophotometer (Shimadzu). NMR spectra were recorded on a Bruker DRX-500 and DRX-400 spectrometer (Bruker). The chemical shifts (δ) are reported in ppm using tetramethylsilane as an internal standard and the coupling constants (J) are given in Hertz (Hz). HRESI-MS spectra were recorded on Agilent G3250AA (Agilent) and AutoSpec Premier P776 spectrometers (Waters). Column chromatography (CC) was performed on self-packed open columns with silica gel from Qingdao Haiyang Chemical Co., Ltd (QHCC). Thin layer chromatography (TLC) analyses were conducted on glass sheets of silica gel GF₂₅₄ from QHCC and detected under a UV lamp at 254 or 365 nm and visualized by spraying 8 % phosphomolybdic acid-EtOH solution (w/v) or 5 % vanillin-H₂SO₄ (w/v) followed by heating, or visualized with iodine (I₂). Semi-preparative high performance liquid chromatography (HPLC) (Agilent 1200 Series) was performed on an YMC C₁₈ column (250 mm × 10 mm, 5 μm, YMC Karasuma-Gojo Bldg.). Fractions from all columns were collected by an auto-collecting apparatus and were combined according to TLC analyses. All other solvents and reagents were commercially purchased from Beijing Greenherbs Science and Technology Development Co., Ltd. and distilled prior to use.