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A New Triterpene Saponin from the Tubers of Stachys sieboldii
A New Triterpene Saponin from the Tubers of Stachys sieboldii
Bulletin of the Korean Chemical Society. 2014. May, 35(5): 1553-1555
Copyright © 2014, Korea Chemical Society
  • Received : December 10, 2013
  • Accepted : January 06, 2014
  • Published : May 20, 2014
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About the Authors
Hyeon Kyung Cho
Chung Sub Kim
Kyeong Wan Woo
Kang Ro Lee

Abstract
Keywords
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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 1 H- 1 H COSY, DEPT, HMQC, HMBC and NOESY experiments, were recorded on a Varian UNITY INOVA 700 NMR spectrometer operating at 700 MHz ( 1 H) and 175 MHz ( 13 C) with chemical shifts given in ppm (δ). Preparative HPLC was conducted using a Gilson 306 pump with Shodex refractive index detector and Econosil RP-C 18 10 μ m column (250 × 10 mm). Silica gel 60 (Merck, 70-230 mesh and 230-400 mesh) and RP-C 18 silica gel (YMG GEL ODS-A, 12 nm, S-75 μ m) were used for column chromatography. Spots were detected on TLC under UV light or by heating after spraying with 10% H 2 SO 4 in C 2 H 5 OH (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), CHCl 3 (20 g), EtOAc (12 g), and n -BuOH (24 g) layers. The EtOAc-soluble layer (12 g) was separated on a RP-C 18 silica gel column (230-400 mesh, 400 g), and eluted gradiently with MeOH-H 2 O (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 CHCl 3 -MeOH (20:1) and then purified by reversed-phase preparative HPLC with MeOH-H 2 O (12:7) at a flow rate of 2.0 mL/min (Econosil RP-C 18 10 μm column; 250 × 10 mm; 10 μm particle size; Shodex refractive index detector) to obtain 1 (6 mg, t R = 16.0 min) and 3 (6 mg, t R = 11.5 min). The CHCl 3 -soluble layer (20 g) was chromatographed on a RP-C 18 silica gel open column (230-400 mesh, 550 g) eluting with a gradient solvent system of MeOH-H 2 O (1:1 and 1:0), yielding nine subfractions (fr. C1-C9). Fr. C4 (8 g) was separated on a RP-C 18 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% CH 3 CN to yield 2 (5 mg, t R = 15.1 min) and 4 (3 mg, t R = 19.0 min). Fr. C46 (25 mg) was purified by reversed-phase preparative HPLC using 75% CH 3 CN to yield 5 (10 mg, t R = 13.6 min) and 6 (5 mg, t R = 16.6 min).
Sieboldii Saponin A (1): Colorless gum.
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+121.3 (MeOH); IR (KBr) ν max : 3421, 2936, 1739, 1368, 1216, 1055 cm −1 ; 1 H (CD 3 OD, 700 MHz) and 13 C-NMR (CD 3 OD, 175 MHz) see Table 1 ; HR-FABMS m / z 811.4480 [M + H] + (calcd. for C 42 H 67 O 15 : 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 CHCl 3 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-H 2 O = 9:3:1, Rf value: 0.2) with D-glucose standard (Aldrich Co., U.S.A.).
2:
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+42.8 (MeOH); 1 H-NMR (C 5 D 5 N, 700 MHz) δ H 5.44 (1H, br t, J = 3.0 Hz, H-12), 5.00 (1H, br s, H-30 a ), 4.78 (1H, br s, H-30 b ), 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-23 a ), 3.76 (1H, d, J = 10.7 Hz, H-23 b ), 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 C 30 H 47 O 5 : 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 H 2 O (0.3 mL each), and the organic layer (1 μ L) 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.
Acknowledgements
This research was supported by the Basic Science Research Program through the National Re-search Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A5A2A28671860). We thank the Korea Basic Science Institute (KBSI) for the MS spectral measurements.
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