Pack c18 column

HPLC analyses of crude sample and HSCCC peak fractions were performed using a Waters Alliance 2695 liquid chromatography system, equipped with a reverse-phase YMC-Pack C₁₈ column (150 × 4.6 mm ID, 5 μm, YMC Co. Ltd., Tokyo, Japan) at 25 °C. The purity of baf A1 was determined by ¹H-NMR spectroscopy and an HPLC external standard method. HPLC-purified baf A1 was selected as external standard. For all HPLC analyses, the mobile phase was acetonitrile and water with a flow rate of 0.8 mL/min in isocratic elution mode (acetonitrile 80%, 25 min), and the eluent was evaluated by UV detection at 247 nm. A Waters Alliance 2695 liquid chromatography system equipped with a larger YMC-Pack C₁₈ column (250 × 10 mm ID, 5 μm, YMC Co. Ltd., Tokyo, Japan) was used for semi-preparative purification of baf A1 from the previous HSCCC enriched fractions. The flow rate was 2 mL/min, and acetonitrile and water (acetonitrile 80%, 30 min) was used as the mobile phase in isocratic elution mode. The wavelength of UV detection and the column temperature were identical to those of the analytical HPLC protocol. The compound identification of baf A1 in HSCCC peak fractions and semi-preparative HPLC fractions was conducted by ESI-MS, as well as ¹H-NMR and ¹³C-NMR analyses.

Optical rotations were obtained on a PerkinElmer 341 digital polarimeter. IR spectra were recorded on Shimadzu FTIR-8400S spectrometers. NMR spectra were obtained with a Bruker AV III 600 NMR spectrometer (chemical shift values are presented as δ values with TMS as the internal standard). HR-ESIMS spectra were performed on a LTQ-Obitrap XL spectrometer. Preparative HPLC was performed on a Lumtech K-1001 analytic LC equipped with two pumps of K-501, a UV detector of K-2600, and an YMC Pack C₁₈ column (250 × 10 mm, i.d., 5 μm, YMC Co. Ltd., Japan) eluted with CH₃OH-H₂O at a flow rate of 2 ml/min. C₁₈ reversed–phase silica gel (40–63 μm, Merk, Darmstadt, Germany), MCI gel (CHP 20P, 75–150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan), and silica gel (100–200 mesh, Qingdao Marine Chemical plant, Qingdao, the People’s Republic of China) were used for column chromatography. Pre-coated silica gel GF254 plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, the People’s Republic of China) were used for TLC. All solvents used were of analytical grade (Beijing Chemical Works).

Optical rotations were obtained on a PerkinElmer 341 digital polarimeter. IR spectra were recorded on Shimadzu FTIR-8400S spectrometers. NMR spectra were obtained with a Bruker AV III 600 NMR spectrometer (chemical shift values are presented as δ values with TMS as the internal standard). HR-ESIMS spectra were performed on an LTQ-Orbitrap XL spectrometer. Preparative HPLC was performed on a Lumtech K-1001 analytic LC equipped with two pumps of K-501, a UV detector of K-2600, and an YMC Pack C₁₈ column (250 mm × 10 mm, i.d., 5 μM, YMC Co. Ltd., Kyoto, Japan) eluted with CH₃OH-H₂O at a flow rate of 2 mL/min. C₁₈ reversed-phase silica gel (40~63 μM, Merk, Darmstadt, Germany), MCI gel (CHP 20P, 75~150 μM, Mitsubishi Chemical Corporation, Tokyo, Japan), and silica gel (100~200 mesh, Qingdao Marine Chemical plant, Qingdao, China) were used for column chromatography. Pre-coated silica gel GF254 plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, China) were used for TLC. All solvents used were of analytical grade (Beijing Chemical Works).