A New Triterpene Saponin from the Tubers of Stachys sieboldii

Experimental Section

General Procedures. Optical rotations were measured on a Jasco P-1020 polarimeter in MeOH. IR spectra were recorded on a Bruker IFS-66/S FT-IR spectrometer. HR-FAB mass spectra were obtained on a JEOL JMS700 mass spectrometer. NMR spectra, including 1H-1H COSY, DEPT, HMQC, HMBC and NOESY experiments, were recorded on a Varian UNITY INOVA 700 NMR spectrometer operating at 700 MHz (1H) and 175 MHz (13C) with chemical shifts given in ppm (δ). Preparative HPLC was conducted using a Gilson 306 pump with Shodex refractive index detector and Econosil RP-C18 10 μm column (250 × 10 mm). Silica gel 60 (Merck, 70-230 mesh and 230-400 mesh) and RP-C18 silica gel (YMG GEL ODS-A, 12 nm, S-75 μm) were used for column chromatography. Spots were detected on TLC under UV light or by heating after spraying with 10% H2SO4 in C2H5OH (v/v). A Hewlett-Packard (HP) GC system 6890 Series equipped with a 5973 Mass Selective Detector (MSD) system. The system was controlled by the Enhanced Chem-Station Version B.01.00 program. The capillary column used for GC was an Agilent J&W HP-5MS UI (30.0 m × 0.25 mm i.d., 0.25 μm film thickness coated 5% diphenyl 95% dimethylpolysiloxane).

Plant Materials. The tubers of S. sieboldii were collected at Yecheon, Gyeongsangbuk-Do, Korea, in June 2012, and identified by one of the authors (K. R. Lee). A voucher specimen (SKKU-NPL 1211) was deposited in the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Korea.

Extraction and Isolation. The dried tubers of S. sieboldii (5 kg) were extracted with 80% MeOH three times at 60 °C. The resulting MeOH extract (1 kg) was suspended in distilled water (1.8 L) and partitioned with solvent to give n-hexane (7 g), CHCl3 (20 g), EtOAc (12 g), and n-BuOH (24 g) layers. The EtOAc-soluble layer (12 g) was separated on a RP-C18 silica gel column (230-400 mesh, 400 g), and eluted gradiently with MeOH-H2O (1:1, 1.5:1, 4:1, and 1:0) to afford eight fractions (fr. E1-E8) based on a TLC analysis. Fr. E5 (85 mg) was separated by Lobar-A column eluted with CHCl3-MeOH (20:1) and then purified by reversed-phase preparative HPLC with MeOH-H2O (12:7) at a flow rate of 2.0 mL/min (Econosil RP-C18 10 μm column; 250 × 10 mm; 10 μm particle size; Shodex refractive index detector) to obtain 1 (6 mg, tR = 16.0 min) and 3 (6 mg, tR = 11.5 min). The CHCl3-soluble layer (20 g) was chromatographed on a RP-C18 silica gel open column (230-400 mesh, 550 g) eluting with a gradient solvent system of MeOH-H2O (1:1 and 1:0), yielding nine subfractions (fr. C1-C9). Fr. C4 (8 g) was separated on a RP-C18 silica gel column (230-400 mesh, 350 g) with 80% MeOH and further separated by silica gel column using n-hexane-EtOAc (1:1) to give six subfractions (fr. C41-C46). Fr. C44 (17 mg) was purified by reversed-phase preparative HPLC with 65% CH3CN to yield 2 (5 mg, tR = 15.1 min) and 4 (3 mg, tR = 19.0 min). Fr. C46 (25 mg) was purified by reversed-phase preparative HPLC using 75% CH3CN to yield 5 (10 mg, tR = 13.6 min) and 6 (5 mg, tR = 16.6 min).

Sieboldii Saponin A (1): Colorless gum. +121.3 (MeOH); IR (KBr) νmax: 3421, 2936, 1739, 1368, 1216, 1055 cm−1; 1H (CD3OD, 700 MHz) and 13C-NMR (CD3OD, 175 MHz) see Table 1; HR-FABMS m/z 811.4480 [M + H]+ (calcd. for C42H67O15: 811.4480).

Acid Hydrolysis of 1: Compound 1 (1 mg) was treated with 1 N HCl (2 mL) at 80 °C for 1.5 h. After cooling, the hydrolysate was extracted with CHCl3 and the extract was evaporated in vacuo to yield 2 as a colorless gum. The sugar in water layer was identified as D-glucose by co-TLC (EtOAc-MeOH-H2O = 9:3:1, Rf value: 0.2) with D-glucose standard (Aldrich Co., U.S.A.).

2: +42.8 (MeOH); 1H-NMR (C5D5N, 700 MHz) δH 5.44 (1H, br t, J = 3.0 Hz, H-12), 5.00 (1H, br s, H-30a), 4.78 (1H, br s, H-30b), 4.27 (1H, m, H-2), 4.15 (1H, d, J = 2.4 Hz, H-3), 3.91(1H, d, J = 10.7 Hz, H-23a), 3.76 (1H, d, J = 10.7 Hz, H-23b), 2.73 (1H, d, J = 11.2 Hz, H-18), 1.09 (3H, s, H-27), 1.06 (3H, d, J = 6.4 Hz, H-29), 1.01 (3H, s, H-25), 0.98 (3H, s, H-26), 0.85 (3H, s, H-24); HR-FABMS m/z 487.3422 [M + H]+ (calcd. for C30H47O5: 487.3423).

Determination of the Sugar of 1. The sugar obtained from the hydrolysis of 1 was dissolved in anhydrous pyridine (0.1 mL) and L-cysteine methyl ester hydrochloride (2 mg) was added. The mixture was stirred at 60 °C for 1.5 h. After the reaction mixture was dried in vacuo, the residue was trimethylsilylated with 1-trimethylsilylimidazole (0.1 mL) for 2 h. The mixture was partitioned between n-hexane and H2O (0.3 mL each), and the organic layer (1 μL) was analyzed by GC-MS.13 The identification of D-glucose for 1 was detected by co-injection of the hydrolysate with standard silylated samples, giving single peaks at 16.429 min. Reten-tion time of authentic sample treated in the same way with 1-trimethylsilylimidazole in pyridine was 16.396 min.