Lichroprep rp c18

The dried seeds of PC were purchased from the Kyungdong Oriental Drug Market in 2013 (Seoul, Korea). A voucher specimen (No. SPH 13003) was deposited in the herbarium of Sookmyung Women’s University (Seoul, Korea). The air-dried plant materials (8.8 kg) were extracted with n-hexane (1.1 kg), and the remaining material was extracted with methanol. After the extracted solution was filtered and evaporated in vacuo, the residue (1.6 kg) was suspended in water and successively partitioned with hexane and ethyl acetate. The ethyl acetate fraction (153 g) was subjected to silica and RP-C18 (LiChroprep RP-C18, 40–63 µm, Merck, Darmastadt, Germany) column chromatography and six compounds were isolated: bavachin (1) [32 (link)], bavachinin (2) [32 (link)], 7,8-dihydro-8-(4-hydrxyophenyl)-2,2-dimethyl-2H,6H-benzo-[1,2-b:5,4-b′]dipyran-6-one (3) [32 (link)], corylin (4) [33 (link)], kanzonol B (5) [34 (link)], and bakuchiol (6) [33 (link)]. Purity of all compounds was confirmed by reverse-phase high performance liquid chromatography analysis and the nuclear magnetic resonance (NMR) spectrum. Structures were elucidated by IR, mass, and NMR spectroscopic data analysis.

The phytoestrogen standards used in the analytical method were purchased from ExtraSynthese (Lyon, France), except for puerarin, equol and calycosin, which were obtained from TCI (Tokyo Chemical Industry; Tokyo, Japan), and calycosin-7-O-β-D-glucoside, lariciresinol, matairesinol and secoisolariciresinol, which were obtained from Biosynth Carbosynth (Compton, UK). The molecule of 2-(4-chlorophenyl) malonaldehyde, used as the internal standard (I.S.), was purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile (ACN) and water (LC-MS grade) were purchased from J.T. Baker (Phillipsburg, NJ, USA), while the formic acid (LC-MS grade), dimethyl sulfoxide (DMSO) and analytical-grade solvents used for the sample extractions (acetonitrile, methanol and acetic acid) were obtained from Fischer Chemicals (Hampton, NH, USA). Magnesium sulphate anhydrous (MgSO₄) and sodium chloride (analytical grade) were obtained from Chem-Lab (Zedelgem, Belgium), while LiChroprep RP-C18 (40–63 µm) was provided by Merck (Rahway, NJ, USA).

NMR spectra were recorded in CD₃OD on a Bruker Avance III 500 spectrometer (Bruker, Karlsruhe, Germany). HR-ESI-MS analysis was conducted using a Micromass Q-TOF micro instrument (Micromass, Manchester, UK). Vacuum Liquid Chromatography (VLC) was carried out on Lichroprep RP-C18 (40–63 µm) Merck using a sintered glass No. 4 and vaccum for the elution. Flash chromatography was carried out on a Grace Reveleris system equipped with dual UV and ELSD detection using Grace^® cartridges (Silica gel or RP-C₁₈) (Grace, Epernon, France). HPLC separations were performed on a Dionex apparatus equipped with an ASI-100 autosampler, an Ultimate 3000 pump, a STH 585 column oven, a diode array detector UVD 340S, and a Chromeleon software. A prepacked RP-C₁₈ column (Phenomenex 250 × 10 mm, Luna 5 µ) was used for semi-preparative HPLC. The eluting mobile phase consisted of H₂O with TFA (0.0025%) and CH₃CN with a flow rate of 5 mL/min and the chromatogram was monitored at 205 and 210 nm. Thin-layer chromatography (TLC) was carried out using silica gel 60 F₂₅₄ pre-coated aluminum plates (0.2 mm, Merck, Darmstadt, Germany). After developing with solvent systems, spots were visualized by spraying with 50% H₂SO₄ followed by heating.