Isolation and Characterization of a New Ginsenoside from the Fresh Root of Panax Ginseng

A new saponin, malonylginsenoside Ra3, was isolated from the fresh root of Panax ginseng, along with four known ginsenosides. The new compound was identified as (20S)-protopanaxadiol-3-O-(6-O-malonyl-β-D-glucopyranosyl(1→2)-β-D-glucopyranoside-20-O-β-D-xylopyranosyl(1→3)-β-D-glucopyranosyl(1→6)-β-D-glucopyranoside on the basis of extensive 1D and 2D NMR as well as HRESI-MS spectroscopic data analysis.


Introduction
Panax ginseng C.A. Meyer has been used in China for thousands of years as a traditional medicine and proved to exhibit wide pharmacological properties, such as anti-fatigue, anti-diabetes, as well as activity in the prevention of cancer and the ageing process [1][2][3][4]. The major components contributing to its pharmacology activities were considered to be the ginsenosides, a group of steroidal saponins. Around 40 ginsenosides have been isolated and characterized till now, including the recent identified ginsenosides Ki and Km [5]. Among these known compounds, malonylginsenosides are natural ginsenosides that exist in both fresh and air-dried ginseng roots and which contain malonyl residues attached to the glucose units of the corresponding neutral ginsenosides [6]. Kitagawa et al. and Yamaguchi et al. reported the presence of four acidic ginsenosides both in Asian and American ginseng [7,8]. Our previous pharmacology results showed that total malonyl-ginsenosides exhibit hypoglycemic effects on streptozotocin-induced diabetic mice [9]. During our continued studies on bioactive compounds from Panax ginseng [10][11][12], a novel ginsenoside, namely malonylginsenoside Ra 3 (compound 1), was isolated from a methanolic extraction of the fresh roots of Panax ginseng. This paper describes the isolation and structure determination of the new compound 1 (Figure 1).

Results and Discussion
A crude methanolic extract of the fresh roots of Panax ginseng was subjected to open column chromatography on silica gel and then purified by preparative HPLC, to yield five ginsenosides, one of which, namely malonylginsenoside Ra 3 (compound 1), was new. The other four saponins were identified as known malonylginsenoside-Rb 1 (compound 2), malonylginsenoside-Rb 2 (compound 3), malonylginsenoside-Rc (compound 4) and malonylginsenoside-Rd (compound 5) by comparison of NMR data with those in the literature [6] and by comparison with authentic sample by ESI-MS, optical rotation and TLC.  . IR (KBr) ν max /cm -1 : 3423 cm -1 (OH), 1732 cm -1 (C=O), 1608 (C=C) and 1386 cm -1 (-CH 3 ). Since compound 1 can't be dissolved in pyridine-d 5 , we added 0.1 mL of D 2 O in 0.5 mL of pyridine-d 5 as NMR solvent. Ginsenoside m-Rb 1 (compound 2) and the alkaline hydrolysis product of 1, ginsenoside Ra 3 (compound 1a) were also dissolved in the same mixture solvent for NMR measurement. Analysis of the 13 C-NMR spectrum (Table 1) and DEPT experiments, allowed the identification of eight methyl groups and six quaternary carbons. The 1 H-and 13 C-NMR spectroscopic data of compound 1 were similar to those of ginsenoside-Ra 3 [13], except the data attributed to a malonyl group (δ H 3.70, δ C 172.1, δ C 174.6). The malonyl group was assigned to C 3 -glc-C-6'' position by HMBC experiment (Figure 2), which the protons of C 3 -glc-H-6'' showed HMBC correlations with malonyl group (δ C 172.1). Malonyl group connection also caused a 2.7 ppm lower-field shift for C 3 -glc-C-6'' (δ C 66.1) than seen in ginsenoside-Ra 3 . Alkaline hydrolysis of compound 1 yield compound 1a, which showed the structure identical to ginsenoside Ra 3 by 1D NMR analysis ( Table 1). The absolute configurations of the sugar moieties were further determined to be β-D-glucose and β-D-xylose by chiral GC analysis. The 20 position was determined as S conformation due to its similar NMR data with the known compounds 1a and 2. All the data above led us to identified the structure of 1 as (20S)-protopanaxadiol

General
The 1 H-and 13 C-NMR spectra were measured on a Bruker Avance DRX 500 NMR spectrometer, using TMS as an internal standard. Chemical shifts (δ) are expressed in parts per million (ppm), with the coupling constants (J) reported in Hertz (Hz). The ESI-MS spectra were recorded on a triple quadrupole mass spectrometer Quattro (VG Biotech, Altrincham, England) and the HRESI-MS spectra on a Bruker FT-ICRMS spectrometer. Column chromatographies were carried out with silica gel 60 M (200-300 mesh), Lichrospher RP-18 (20 μm); TLC was performed with silica gel plates (Macherey-Nagel, SilG/UV 254 , 0.20 mm), with spots detected by UV 254 and H 2 SO 4 (10%). HPLC were carried out with a Agilent 1100 system.

Plant material
The fresh root of Panax ginseng was collected from Fu-Song, Jilin, China, in August 2003, and identified by one of the authors, Prof. Yi-Nan Zheng. A voucher specimen (ZYC-RS-03-08) has been deposited in College of Chinese Medicinal Material, Jilin Agricultural University.

Alkaline hydrolysis of compound 1
A solution of 1 (20 mg) in MeOH (3 mL) was treated with 5% KOH-MeOH (0.1 mL) and the whole mixture was stirred at room temperature (22 ºC) for 30 min [6]. The reaction mixture was neutralized with cation exchange resin (SP20ss, Resindion S.R.L., Rome, Italy) and filtered. Removal of the solvent from the filtrate under reduced pressure gave a product which was purified by column chromatography with reversed-phase silica gel (Zorbax SB-C 18 ) to furnish compound 1a, which was determined to be identical with an authentic sample [6] Table 1.

Acid hydrolysis of compound 1
To determine the stereochemistry of sugar moiety, compound 1 (2.0 mg) was refluxed with 6 N HCl (5 mL) at 100 ºC for 2 h [14,15]. The mixture was extracted with CHCl 3 to afford the aglycone, and the aqueous layer was neutralized with Na 2 CO 3 and filtered. The aqueous layer was dried under vacuum and the residue was re-dissolved in H 2 O for sugar analysis by TLC with n-BuOH-AcOH-H 2 O (4:1:2) as the solvent. The sample spots were detected by spraying aniline hydrogen phthalate reagent (100 mL n-BuOH saturated by H 2 O, 0.96 g aniline and 1.66 g phthalic acid) and heating at 120 ºC. D-Glucose and D-xylose were used as authentic standards. The absolute configuration of glucose was further determined by chiral GC analysis using a SatoChrom GC and a 0.25 mm × 25 m Hydrodexb-6-TBDM chiral capillary column (Macherey-Nagel, Germany). β-D-Glucose and β-D-xylose were used as an authentic GC standard. The aqueous layer residues mentioned above were re-suspended in dichloromethane (1 mL), and trifluoroacetic anhydride (50 µL) was added. The mixtures were allowed to react at room temperature overnight and dried under a stream of nitrogen at room temperature. The sugar derivatives were separated using the following temperature program: inlet temperature was set at 240 ºC, with hydrogen carrier gas and a 1/20 split, using nitrogen makeup gas. Column temperatures started at 120 ºC, ramped to 220 ºC at 50 ºC·min -1 and were maintained for 12 min.