Isolation of a New Flavonone Glycoside from Eria marginata

Experimental

General Experimental Proceduces. Optical rotations were determined on a Horiba SEAP-300 spectropolarimeter. UV data were obtained on a Shimadzu UV-2401PC spectrophotometer (Shimadzu, Kyoto, Japan). CD spectra were recorded on a Chirascan Circular Dichroism spectrometer (Applied Photophysics, Surrey, United Kingdom). IR was measured on a Perkin-Elmer 241 polarimeter. NMR spectra were recorded on a Bruker DRX-AV-500 spectrometer at 500 MHz for 1H and 125 MHz for 13C using standard pulse sequence programs. All chemical shifts were recorded with respect to TMS as an internal standard. MS was obtained on Finnigan MAT 95 instrument and VG Auto Spec-3000 spectrometer. Column chromatography was carried out on silica gel (200-300 mesh, Qingdao Haiyang Chemical Factory, Qingdao, China), MCI gel CHP20P (75-150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan), and Sephadex LH-20 (25-100 mm, Pharmacia Fine Chemical Co. Ltd., Uppsala, Sweden).TLC was performed on silica gel GF254 (Yantai Jiangyou Silica Gel Co. Ltd, Yantai, China). Semi-preparative HPLC was carried out using a system composed of a Waters 600 pump, with an Agilent 1100 detector and Sunfire C18 reversed phase column (10 × 150 mm, detected at UV of 254 nm). Solvents were of industrial purity and distilled prior to use.

Plant Material. The whole plants of E. marginata were collected from Pingbian County of Yunnan Province, China in December, 2008 and identified by Dr. Guangwan Hu, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China and the voucher specimen (No. 0812004) is deposited at the Herbarium of School of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming, China.

Extraction and Isolation. The air-dried powdered whole plants of E. marginata (0.2 Kg) were extracted with MeOH (2L × 4) at room temperature and the concentrated extract (31 g) was subjected to MCI gel chromatography and eluted with 80% aq. MeOH, 90% aq. MeOH, MeOH and acetone successively to provide 4 fractions, in order of elution. Frac-tion 1 (16.8 g) was isolated on silica gel chromatography eluting with gradient CHCl3:MeOH to afford 6 subfractions (1a-1f). Subfraction 1a (198 mg) was purified on Sephadex LH-20 eluting with 60% CHCl3 in MeOH to give 4 (20 mg) and 7 (25 mg). Subfraction 1b (541 mg) was separated on Sephadex LH-20 eluting with 60% CHCl3 in MeOH to produce 3 subfractions (1b1-1b3). Subfraction 1b1 (101 mg) was purified by reversed-phase HPLC with 57% aq. MeOH to yield 9 (6 mg) and 13 (3 mg). Subfraction 1b2 (57 mg) was isolated on Sephadex LH-20 (MeOH) to produce 5 (9 mg). Subfraction 1b3 (83 mg) was purified by reversedphase HPLC with 66% aq. MeOH to afford 6 (8 mg) and 8 (3 mg). Subfraction 1c (157 mg) was purified by Sephadex LH-20 (MeOH) to obtain 3 (3 mg). 1 (2.1 g) and 14 (200 mg) were obtained from 1d (4.3 g) and 1e (476 mg) respectively by repeated recrystallization. Fraction 2 (2.5 g) was subjected to silica gel chromatography eluting with CHCl3: acetone (30:1→0:1) to produce 2 subfractions (2a and 2b). Subfraction 2a (1.9 g) was purified by Sephadex LH-20 (60% CHCl3 in MeOH) to obtain 2 (1.3 g). Fraction 3 (3.5 g) was separated on silica gel chromatography eluting with gradient petroleum ether:acetone to afford 4 subfractions (3a-3d). Subfraction 3b (296 mg) was isolated on silica gel chromatography eluting with petroleum ether:acetone (5:1) to afford 10 (6 mg). Subfraction 3c (147 mg) was purified on Sephadex LH-20 (60% CHCl3 in MeOH) to give 11 (20 mg) and 12 (3 mg).

(2S)-Pinocembrin 7-O-[β-D-apiosyl(1→6)]-β-D-glucoside (1): A colorless amorphous powder, mp 112-114 ℃, −99.9 (c 0.0038, MeOH). ESI-MS m/z 573 (M+Na)+, HR-EI-MS m/z 550.1679 [M]+ (calcd for C26H30O13: 550.1686); UV λmax nm: 330 (sh) (3.31), 284 (4.16); IR (KBr): 3424, 1648, 1577, 1294, 1087, 1012; 1H and 13C NMR, see Table 1. CD (c 3.22 × 10−4, MeOH): [θ]330 +5483, [θ]284 −36347.