Two New Isoflavone Glycosides from the Extracts of the Fungus Monascus pilosus-Fermented Black Soybean

Experimental Section

General Procedures. Optical rotations were recorded on a Jasco P-200 digital polarimeter. UV and IR spectra were obtained using a Shimadzu UV-1650PC and a Jasco FT/IR- 4100 spectrometer, respectively. 1D and 2D NMR spectra were measured on a Bruker Ascend 700 MHz NMR spectrometer with tetramethylsilane as an internal standard, and chemical shifts expressed in terms of δ values. Electrospray ionization (ESI) mass spectra were obtained on a LTQ Orbitrap XL (Thermo Scientific) mass spectrometer. Preparative HPLC was performed using a Shimadzu system (LC-8A pump and SPD-20A UV/VIS detector) and a YMC-Pack ODS A column (250 × 20 mm i.d.), using a mixed solvent system of methanol-water at a flow rate of 8 mL/min. Medium pressure liquid chromatography (MPLC, Combi Flash RF, Teledyne ISCO) separations were performed using a RediSep Rf silica column (40 g) with a flow rate of 40 mL/min. Open column chromatography was performed using a silica gel (Kieselgel 60, 70-230 mesh, Merck) and Diaion HP-20 (Mitsubishi Kasei, Tokyo); and thin layer chromatography (TLC) was performed using a pre-coated silica gel 60 F254 (0.25 mm, Merck) and RP-18 F254S (0.25 mm, Merck Co., Darmstadt). All other chemicals and reagents were analytical grade.

Extraction and Isolation. The dried RMBS (10 Kg) were extracted with 80% EtOH (3 × 18 L) at room temperature. After filtration and evaporation of the solvent in vacuo, the EtOH extract was suspended in distilled water and then partitioned, in turn, with n-Hexane, CH2Cl2, EtOAc and n- BuOH. The EtOAc-soluble fraction (23 g), which showed the most potent activity, was subjected to further chromatographic separation. The EtOAc-soluble fraction was subjected to Diaion HP-20 column chromatography with the mixture of methanol/water (0%, 20%, 40%, 60%, 80%, 100% MeOH in water) to give six fractions (G44-56-31 ~ 36). G44-56-35 was subjected to MPLC on silica gel eluting with the mixture of CHCl3/MeOH (1:0 → 0:1, RediSep Rf silica column, 40 g, flow rate 40 mL/min) to give 11 subfractions (G44-92-1 ~ 11). Subfraction G44-92-7 was further purified by means of semi-preparative HPLC eluting with acetonitrile/water (30 to 50% acetonitrile) at a flow speed of 8 mL/min to yield compounds 1 (8 mg), 2 (12 mg), 3 (25 mg) and 4 (120 mg).

Acid Hydrolysis of Compound 2 and Determination of Stereochemistry of Ribofuranose. A solution of compound 2 (5 mg) in 9% aq. HCl (1 mL) was reacted for 2 h at 80 ℃. The reaction mixture was extracted with EtOAc repeatedly to remove the aglycone fraction, which was identical to genistein. The H2O layer was then concentrated to furnish the sugar residue (1.1 mg). The rotation recorded for the ribose isolated in this study was -25.3° (c 0.03, H2O), which closely matched that for the D-ribose (ref. -18.5°).

6''-O-Crotonylgenistin (1). White amorphous powder; -76.9° (c 0.1, MeOH); IR λmax 3516, 3455, 2918, 2853, 1709, 1656, 1511, 1442, 1370, 1239, 1182, 1073 cm-1; UV (MeOH) λmax nm (log ε): 260 (4.05), 329 (3.25); 1H (700 MHz) and 13C (175 MHz) NMR spectroscopic data, see Table 1; ESI-MS (negative mode) m/z 499 [M-H]-, 545 [M+COOH]-, 999 [2M-H]-, 1045 [2M+COOH]-; HRESIMS (negative mode) m/z 499.1233 (calcd for C25H23O11, 499.1235).

Genistein-7-O-α-D-ribofuranoside (2). White amorphous powder; +78.8° (c 0.2, MeOH); IR λmax 3366, 2931, 2353, 1651, 1612, 1445, 1368, 1244, 1039 cm-1; UV (MeOH) λmax nm (log ε): 260 (4.11), 331 (3.52); 1H (700 MHz, CD3OD) and 13C (175 MHz, CD3OD) NMR spectroscopic data, see Table 1; 1H NMR (700 MHz, DMSO-d6) and 13C (175 MHz, DMSO-d6) NMR spectroscopic data, see Supporting Information; ESI-MS (negative mode) m/z 269 [M–ribose]-, 401 [M-H]-, 447 [M+COOH]-; HRESIMS (negative mode) m/z 401.0868 (calcd for C20H17O9, 401.0867).