Unity 400 spectrometer

Isolation procedures and purity of compounds were checked by thin layer chromatography (TLC, Merck Millipore, Billerica, MA, USA) visualized by UV light, and sprayed with Ce(SO₄)₂·2(NH₄)₂SO₄·2H₂O. All ¹H, ¹³C, and 2D-NMR experiments were recorded on acetone-d₆, on a Varian Unity 400 spectrometer (Varian, Palo Alto, CA, USA), at 400 MHz for ¹H-NMR, and at 100 MHz for ¹³C-NMR. EI-EM spectra were recorded on a JEOL JMX-AX 505 HA mass spectrometer (Jeol Ltd., Tokyo, Japan). All the reagents and solvents used were of analytical grade. Tannic acid (CAS: 1401-55-4) was purchased from Sigma Aldrich (St. Louis, MO, USA).

The supernatant extract was applied to a reverse phase silica gel Polygoprep 100–50 C18 VFC (vacuum flash chromatography) system, using stepwise gradient elution from H₂O to MeOH. The active fraction (2.72 g) was eluted with H₂O-MeOH 1:9 and was subjected to preparative reversed-phase HPLC equipped with a Symmetry C18 column (19 × 150 mm, 7 μ) and using a linear gradient of H₂O/CH₃CN from 5% to 100% of CH₃CN in 60 min at a flow rate of 15 mL/min. The active fractions were further purified by a semi-preparative HPLC on a Symmetry C18 column (7.8 × 150 mm, 7 μ) using isocratic elution with H₂O/CH₃CN 55:45 at a flow rate of 3 mL/min to yield 80 mg of the pure compound 1, 94 mg of compound 2 and 44 mg of compound 3. NMR spectra were recorded on a Varian “Unity 500” spectrometer at 500/125 MHz (¹H/¹³C) and on a Varian Unity 400 spectrometer at 400/100 MHz (¹H/¹³C). Chemical shifts were reported in ppm using residual CDCl₃ (δ 7.26 ppm for ¹H and 77.0 ppm for ¹³C) as an internal reference. Two-dimensional experiments COSY, TOCSY, HSQC, and HMBC were performed using standard pulse sequences. Data were processed using MestReNova 14.0.1 software. (+)-ESIMS spectra obtained on an Agilent 1100 Series LC/MSD spectrometer. High-Resolution Mass Spectroscopy (HRMS) was performed on an Agilent 6230 TOF LC/MS system using the ESIMS technique.