Scholarly article on topic 'Complete 1H-NMR and 13C-NMR spectral analysis of the pairs of 20(S) and 20(R) ginsenosides'

Complete 1H-NMR and 13C-NMR spectral analysis of the pairs of 20(S) and 20(R) ginsenosides Academic research paper on "Chemical sciences"

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Journal of Ginseng Research
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{"less polar ginsenosides" / "nuclear magnetic resonance spectroscopy" / " Panax ginseng " / protopanaxadiol / protopanaxatriol}

Abstract of research paper on Chemical sciences, author of scientific article — Heejung Yang, Jeom Yong Kim, Sun Ok Kim, Young Hyo Yoo, Sang Hyun Sung

Abstract Background Ginsenosides, the major ingredients of Panax ginseng, have been studied for many decades in Asian countries as a result of their wide range of pharmacological properties. The less polar ginsenosides, with one or two sugar residues, are not present in nature and are produced during manufacturing processes by methods such as heating, steaming, acid hydrolysis, and enzyme reactions. 1H-NMR and 13C-NMR spectroscopic data for the identification of the less polar ginsenosides are often unavailable or incomplete. Methods We isolated 21 compounds, including 10 pairs of 20(S) and 20(R) less polar ginsenosides (1–20), and an oleanane-type triterpene (21) from a processed ginseng preparation and obtained complete 1H-NMR and 13C-NMR spectroscopic data for the following compounds, referred to as compounds 1–21 for rapid identification: 20(S)-ginsenosides Rh2 (1), 20(R)-Rh2 (2), 20(S)-Rg3 (3), 20(R)-Rg3 (4), 6′-O-acetyl-20(S)-Rh2 [20(S)-AcetylRh2] (5), 20(R)-AcetylRh2 (6), 25-hydroxy-20(S)-Rh2 (7), 25-hydroxy-20(S)-Rh2 (8), 20(S)-Rh1 (9), 20(R)-Rh1 (10), 20(S)-Rg2 (11), 20(R)-Rg2 (12), 25-hydroxy-20(S)-Rh1 (13), 25-hydroxy-20(R)-Rh1 (14), 20(S)-AcetylRg2 (15), 20(R)-AcetylRg2 (16), Rh4 (17), Rg5 (18), Rk1 (19), 25-hydroxy-Rh4 (20), and oleanolic acid 28-O-β-D-glucopyranoside (21).

Academic research paper on topic "Complete 1H-NMR and 13C-NMR spectral analysis of the pairs of 20(S) and 20(R) ginsenosides"

Accepted Manuscript

Complete H and C NMR spectral analysis of the pairs of 20(S) and 20(R)-ginsenosides

Heejung Yang , Jeom Yong Kim , Sun Ok Kim , Young Hyo Yoo , Sang Hyun Sung , Ph.D.

S1226-8453(14)00056-6 10.1016/j.jgr.2014.05.002

Reference: JGR 34

To appear in: Journal of Ginseng Research

Received Date: 8 October 2013 Revised Date: 11 March 2014 Accepted Date: 12 March 2014

Please cite this article as: Yang H, Kim JY, Kim SO, Yoo YH, Sung SH, Complete H and C NMR spectral analysis of the pairs of 20(S) and 20(R)-ginsenosides, Journal of Ginseng Research (2014), doi: 10.1016/j.jgr.2014.05.002.

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1 Complete 1H and 13C NMR spectral analysis of the pairs of 20(S) and 20(Ä)-ginsenosides

2 Running title: 1H and 13C NMR data of 21 ginsenosides

4 Heejung Yang1, Jeom Yong Kim2, Sun Ok Kim2, Young Hyo Yoo2, Sang Hyun Sung3'

5 1College of Pharmacy, Kangwon National University, Chuncheon 200-701, Korea

6 2Greencrosshs, Suntechcity, Sungnam 513-15, Korea

7 3College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University,

8 Seoul 151-742, Korea

9 10 11 12

20 *Correspondence

21 Professor, Ph.D., Sang Hyun Sung

22 College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University,

23 Seoul 151-742, Korea.

24 E-mail: shsung@snu.ac.kr

25 Phone: +82-2-880-7859

26 Fax: +82-2-877-7859

28 Ginsenosides, the major ingredients of Panax ginseng, have been under investigation for many

29 decades due to a wide range of pharmacological activities in Asian countries. The less polar

30 ginsenosides having one or two sugar residues are not present in nature and produced in the

31 manufacturing process including heating, steaming, acid hydrolysis and enzyme reactions. Up to now,

32 the :H and 13C NMR spectroscopic data for identification of the less polar ginsenosides are often not

33 available or scattered. We isolated 21 compounds including 10 pairs of 20(5) and 20(R) less polar

34 ginsenosides (1-20), and an oleanane-type triterpene (21) from the processed ginseng preparation and

35 tried to provide the complete 1H and 13C NMR spectroscopic data of the following compounds 1-21

36 for the rapid identification; 20(S)-ginsenosides Rh2 (1), 20(R)-Rh2 (2), 20(S)-Rg3 (3), 20(R)-Rg3 (4),

37 6'-0-acetyl-20(S)-Rh2 (20(S)-AcetylRh2) (5), 20(R)-AcetylRh2 (6), 25-hydroxy-20(S)-Rh2 (7), 2538 hydroxy-20(S)-Rh2 (8), 20(S)-Rh1 (9), 20(R)-Rh1 (10), 20(S)-Rg2 (11), 20(R)-Rg2 (12), 25-hydroxy-

39 20(S)-Rh1 (13), 25-hydroxy-20(R)-Rh1 (14), 20(S)-AcetylRg2 (15), 20(R)-AcetylRg2 (16), Rh4 (17),

40 Rg5 (18), Rk1 (19), 25-hydroxy-Rh4 (20), and oleanolic acid 28-O-^-D-glucopyranoside (21).

42 Keywords: Panax ginseng, NMR, protopanaxadiol, protopanaxatriol, less polar ginsenosides

Ginsenosides, major components in Panax ginseng Meyer, are mainly classified into two groups of the dammarane-type triterpenes, protopanaxadiol (PPD) and protopanaxatriol (PPT) [1]. The substitution of sugar chains at C-3 or C-20 in PPD, or at C-3, C-6 and C-20 in PPT give rise to a wide range of ginsenosides [2]. The PPD type representatively includes ginsenosides Rb1, Rb2, Rc and Rd, while the PPT type includes Re, Rf, Rg1 and Rg2, which have 3-5 sugar moieties, in harvested ginseng. In the process of steaming with heat and acidic solution or the microbial reaction, these polar ginsenosides are decreased and the less polar ones such as Rg2, Rg3, Rh1, and Rh2 are increased [35]. It was suggested that they could be generated by the elimination of sugar chains or dehydroxylation [6]. These reactions also can generate the irregular ^20(21) and ^20(22) ginsenosides such as Rg5, Rh3, Rh4, and Rk1 which are hardly found in nature [7]. Especially, 20(R)-ginsenosides including 20(R)-Rh2 and 20(R)-Rg3 are derived by the selective deglycosylation and dehydroxylation at C-20, followed by the biotransformation with the attack of hydroxyl group [8,9]. The acetylated ginsenosides are generated from the malonylated ginsenosides including malonyl (Mal)-Rb1, Rb2, Rc, Rd and Re by decarboxylation [10]. As the less polar ginsenosides can be easily absorbed into the blood vessel and act as the pharmacological agents to be potential drug candidates, the mass production or isolation of the less polar ones is the common interests in the field of ginseng industry [5].

Recent improvements in chromatographic technologies have led to analyze and isolate the stereoiosmers of minor ginesenosides in ginseng preparations [11]. Also, the structure-activity relationship between the diverse ginsenosides isolated by the improved techniques has been studied in cancer and non-cancer cells [12]. In the study, we isolated 21 minor ginsenosides from the processed ginseng preparation and unequivocally determined their structures by 1D and 2D NMR experiments, as well as comparison with the literature. We proposed that the NMR information of minor ginsenosides in the study will be useful for the study of the structure-activity relationship between some structural modifications such as number of sugar groups, sugar linkage at C-6, hydroxyl groups and stereoisomers of 20(5) and 20(R), as well as the identification of stereoisomers of ginsenosides.

MATERIAL AND METHODS General procedure

Column chromatography (CC) was carried out on Kiesgel 60 silica gel (40-60 /m, 230-400 mesh, Merck, USA), YMC-GEL ODS-A (5-150 /m, YMC) and Sephadex LH-20 (25-100 /M, Pharmacia, NJ, USA), and TLC was carried out on Kiesgel 60 F254 coated normal silica gel and RP-18 F254 coated reverse-phase silica gel. The !H and 13C NMR, !H-!H COSY, HSQC, and HMBC spectra were recorded on a Bruker AMX 500 or 600 spectrometer in pyridine-d5. Solvent signals were used as internal standards. The HPLC system consisted of a G-321 pump (Gilson Co. Ltd., USA), a G-151 UV detector (Gilson Co. Ltd., USA) and a YMC-Pack Pro C18 column (250 mm x 10 mm i.d.; 5 /m), and all chromatograms were monitored at 210 nm. HPLC grade solvents (Fisher Scientific, USA) were used in the MeOH-H2O or MeCN-H2O system. Ginseng Preparation

The processed ginseng preparation was gifted from Greencrosshs INC. (Sungnam, Korea). It was prepared with the patented technology and the previous reported study [13]. Briefly, the harvested ginseng was extracted with ethanol repeatedly followed by reacting with an enzyme containing ginsenoside-#-glucosidase. After performing the acid hydrolysis of the residue, the reactant was purified with HP-20 resin followed by washing out with distilled water and finally 95% ethanol. Isolation of ginsenosides from the processed ginseng preparation

Powders of the processed ginseng extract (90 g) were each subjected to normal silica column chromatography (CC) (20 x 5 cm) with a gradient elution of solvents (CHCl3:MeOH = 10:1, 7:1, 5:1, 3:1, 0:1, each 1 L) and it yielded 24 sub-fractions (GE1-24). 20(S/R)-AcetylRh2 (5, 6) (20 mg, Rt = 14.1 min) were obtained from GE-5 (2.8 g) sub-fraction by reversed phase (RP) silica gel CC (20 x 5 cm; MeOH:H2O = 9:1, 1 L), followed by preparative high performance liquid chromatography (HPLC) (MeOH:H2O = 65:35, 4 ml/min). Oleanolic acid 28-0-^-D-glucopyranose (21) (200 mg) was isolated by recrystallization (100% MeOH) from sub-fraction separated from GE-7 (6.5 g) sub-fraction by RP silica gel CC (10 x 3 cm; MeOH:H2O = 7:3, 2 L). Five sub-fractions (GE8-10 A-E) were obtained from GE8-10 (12.1 g) by RP silica gel CC (MeOH:H2O = 8.5:1.5, 4 L), and Rh4 (17) (5 mg, Rt = 19.1

min) was isolated from GE8-10 B and 20(S)-Rh2 (1) (300 mg, Rt = 5.7 min) and 20(R)-Rh2 (2) (210 mg, Rt = 6.1 min) were from GE8-10 C by preparative HPLC (MeCN:H2O = 55:45; 13 ml/min). The mixtures of 25-hydroxy-Rh4 (20) (35 mg, Rt = 11.1 min), 20S/R-Rh1 (9, 10) (90 mg, Rt = 13.2 min), 25-hydroxy-20(S)-Rh2 (7) (28 mg, Rt = 23.1 min) and 25-hydroxy-20(R)-Rh2 (8) (100 mg, Rt = 23.3 min) were prepared from GE12-14 (8.2 g) and they were isolated by RP silica gel CC (10 x 3 cm; MeOH:H2O = 7:3, 4 L), followed by preparative HPLC (MeCN:H2O = 50:50, 70:30; 13 ml/min). GE15-18 (10.1 g) was subjected to RP silica gel CC (MeOH^O = 6:4, 4 L) to give five sub-fractions (GE15-18 A-E). 20S-AcetylRg2 (15) (15 mg, Rt = 24.7 min) and 20R-AcetylRg2 (16) (8 mg, Rt = 25.1 min) were isolated from GE15-18 B, and Rk1 (19) (25 mg, Rt = 19.9 min) and Rg5 (18) (31 mg, Rt = 20.3 min) were from GE15-18 D by preparative HPLC (MeOH:H2O = 7:3, 10 ml/min), respectively. 20(S/R)-Rg2 (11, 12) (50 mg), 20(S)-Rg3 (3) (400 mg) and 20(R)-Rg3 (4) (400 mg) were obtained from GE19-20 (8.1 g) sub-fractions by RP silica gel CC (10 x 3 cm) with mixture of MeOH:H2O (3:1, 5 L). 20(S)-Rg2 (11) (10 mg, Rt = 13.1 min) and 20(R)-Rg2 (12) (15 mg, Rt = 13.4 min) were purified using preparative HPLC (MeCN:H2O = 35:65, 10 ml/min). GE21-22 (3.1 g) subfractions were further isolated to give the mixture of 25-hydroxy-20(S/R)-Rh1 (13, 14) (30 mg). RESULTS and DISCUSSION

The structures of compounds 1-21 were unequivocally determined by comparison of 1D and 2D NMR, and MS data with those in literature. They were 20(S)-ginsenosides Rh2 (1) [14], 20(R)-Rh2 (2) [15], 20(S)-Rg3 (3) [16], 20(R)-Rg3 (4) [16], 6'-0-acetyl-20(S)-Rh2 (20(S)-AcetylRh2) (5) [16], 20(R)-AcetylRh2 (6), 25-hydroxy-20(S)-Rh2 (7) [13], 25-hydroxy-20(R)-Rh2 (8) [13], 20(S)-Rh1 (9) [17], 20(R)-Rh1 (10) [17], 20(S)-Rg2 (11) [17], 20(R)-Rg2 (12) [18], 25-hydroxy-20(S)-Rh1 (13) [19], 25-hydroxy-20(R)-Rh1 (14) [19], 20(S)-AcetylRg2 (15) [20], 20(R)-AcetylRg2 (16) [20], Rk1 (17) [21], Rh4 (18) [17], 25-hydroxy-Rh4 (19) [18], Rg5 (20) [21], and oleanolic acid 28-O-^-D-glucopyranoside (21) in Fig. 1 [22]. Among them, compound 6 was not reported up to now. Compounds 5 and 6, and 13 and 14 were isolated as the mixture of the stereoisomers, and were not purified to each stereoisomer. Compounds 1-21 were categorized into their backbones (PPD-type; 1-6, 4 PPD derived-type; 7, 8, 18 and 19, PPT-type; 9-12, 15 and 16; PPD derived-type; 13, 14 and 17, and

an oleanane-type triterpene; 21) and :H and 13C NMR spectral data were listed in Table 1-6. The comprehensive :H and 13C NMR spectral data of compounds 1-21 will be worth for determining the structures of less polar ginsenosides. Some of their :H and 13C NMR spectroscopic data are often not available. As the others are scattered or old, it is hard to compare with the structures of the isolated compounds. In the study, the results were obviously assigned using 1D and 2D NMR spectroscopies methods and also confirmed by the comparison with those in the literature. Some signals, such as methyl groups of C-26 ~ C-30 and the saturated methylenes, which were not prepared in the literature were unambiguously determined with the aid of 2D NMR spectra including :H-H COSY, HSQC and HMBC spectra.

The 13C NMR spectral data in the study suggested the information for the structural elucidation of ginsenosides isomers. First, the chemical shifts of the characteristic peaks between 20(5) and 20(R)-ginsenosides provided the information for the identification of the stereoisomers. Especially, the changes of the chemical shifts between S- and R-forms at C-17, C-21 and C-22 in 13C NMR spectra were about AS (Ss - Sr) + 4.1 ± 0.1, + 4.3 ± 0.1 and - 7.4 ± 0.1 ppm, respectively (Table 2 and 4). Next, the presence of the signal (SC 88.8 ± 0.1 ppm) of the hydroxyl carbon at C-3, which was not overlapped with other hydroxyl groups in the backbone and the sugar moieties, easily indicated whether it was PPD- (1-8, 17 and 20) or PPT-type (9-16 and 18). Additionally, the signals of SC 170.6 ± 0.1 showed the existence of the acetyl groups (5, 6, 15 and 16) (Table 2 and 4). It was assumed that they were produced from malonyl moiety by decarboxylation during the manufacturing process and located at C-6 in the glucose group (5, 6, 15 and 16) [23]. Last, the chemical shifts of down-field signals indicated the types of the backbones. The values of a double bond at ^24(25) in 3, 12, 20-trihydroxydammar-24-ene and 3, 6, 12, 20-tetrahydroxydammar-24-ene (1-6, 9-12, 15 and 16) were Sc 126.1 ± 0.2 (C-24) and 130.1± 0.1 (C-25), respectively (Table 2 and 4). However, they were shifted into SC 124.2 ± 1.0 and 131.2 ± 0.0 due to the dehydration at ^20(21) (17) or ^20(22) (18 and 20) (Table 6). The differences between the chemical shifts of SC 155.5 and 108.1, and of SC 140.1 ± 0.1 and 123.4 ± 0.2 ppm exhibited the discrimination of 3, 12-dihydroxydammar-20, 24-diene (17) and 3, 12-dihydroxydammar-20(22), 24-diene (18 and 20). These results were in perfect agreement with the

literature [21,24,25]. Compound 21, an oleanane-type triterpene, might be produced by the selective

hydrolysis of sugar residues at C-3 in ginsenoside Ro [26] (Table 6).

ACKNOWLEDGEMENTS

This research was equally supported by Basic Science Research Program through the National

Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology

(2012R1A1A2039102) and the Global Leading Technology Program of the Office of Strategic R&D

Planning (OSP grant 10039320) funded by the Ministry of Knowledge Economy, Korea.

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Table 1. *H NMR spectroscopic data for compounds 1-8 in pyridine-^

No. 20(S)-Rh2a (1) 20(fl)-Rh2a (2) 20(S)-Rg3b (3) 20(fl)-Rg3b (4) 20(S/R)-AcetylRh2b, c (5 and 6) 25-hydroxy-20(S)-Rh2b (7) 25-hydroxy-20(fl)-Rh2b (8)

3S (J in Hz)

6a 6b 7a 7b 9

18 19 21 22a 22b 23a 23b 24 26

1.49 0.79 2.19

3.35 4.6, 0.72

1.47 1.21 1.40 1.58 1.11 3.89 2.01 2.01 1.49 1.88

1.39 2.35 0.77 0.94

1.40 2.01 1.68 2.57 2.29

5.29 1.63 1.60

1.30 0.97 0.94

1H, m) 1H, m) 1H, m)

1H, m)

1H, dd, J = 1.9)

1H, d, J = 2H, m)

m) m) m) m) m) m) m) m) m) m) m) m) s) s) s) m) m) m) m)

t-like)

s) s) s) s) s)

1.49 0.79 2.20

3.36 3.7, 0.73

1.49 1.23 1.42 1.58 1.13 3.91 2.00 2.11

1.51 1.91 1.35 2.38 0.80 1.00 1.38 1.70

2.52 2.45 5.30 1.68 1.63 1.30 0.98 0.98

1H, m) 1H, m) 1H, m)

1H, m)

1H, dd, J = 1.5)

1H, d, J = 2H, m)

1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 3H, s) 3H, s) 3H, s) 2H, m)

1H, m) 1H, m) 1H, t-like) 3H, s) 3H, s) 3H, s) 3H, s) 3H, s)

1.46 0.72 2.16

II.75 0.65 11.4

1.49 1.35 1.42 1.19

1.37 2.02 1.55 3.90 2.00

1.50 1.05 1.87

1.38 2.33 0.94 0.77 1.40 2.01 1.68 2.58

5.28 6.95)

1.60 1.60

1.27 1.08 0.92

1H, m) 1H, m) 1H, m)

1H, m)

1H, dd, J = 4.35) 1H, d, J =

1H, m)

1H, m)

1H, m)

1H, m)

1H, m)

1H, m)

1H, m)

1H, m)

1H, m)

1H, m)

1H, m)

1H, m)

1H, m)

1H, m)

3H, s)

3H, s)

3H, s)

1H, m)

1H, m)

1H, m)

1H, m) 1H, t, J =

3H, s) 3H, s) 3H, s) 3H, s) 3H, s)

0.72 2.17

II.75 0.66 11.5

1.41 1.21

1.37 2.00

1.55 3.91 1.97

1.56 1.04 1.93 1.35

2.38 0.99 0.80 1.37 1.71

2.52 2.47 5.30 1.68 1.64

1.27 1.09 0.96

1H, m) 1H, m) 1H, m)

1H, m)

1H, dd, J = 4.35) 1H, d, J =

1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 3H, s) 3H, s) 3H, s) 2H, m)

1H, m) 1H, m) 1H, t-like) 3H, s) 3H, s) 3H, s) 3H, s) 3H, s)

1.58 (1H, m) 0.88 (1H, m) 2.12 (1H, m) 1.78 (1H, m) 3.24 (1H, m) 0.71 (1H, m) 1.48 (2H, m)

1.49 (1H, m) 1.49 (1H, m)

0.75 (1H, m) 0.75 (1H, m)

2.20 (1H, m) 2.18 (1H, m)

1.38 (1H, m) 1.36 (1H, m)

3.36 1H, dd, J = 3.35 (1H, dd, J

4.4, 1 1.7) 4.4, 1 1.7)

0.73 (1H, m) 0.71 (1H, m)

1.58- .32 (2H, 1.52- .36 (2H,

m) m)

1.45 (1H, m) i y

1.20 (1H, m) i

1.41 (1H, m) i

1.52 (1H, m) i

1.02 (1H, m)

3.82 (1H, m)

1.94 (1H, m)

1.96 (1H, m)

1.42 (1H, m)

1.84 (1H, m)

1.44 (1H, m)

2.26 (1H, 2.30

m) (1H, m)

0.94 (3H, 0.91

s) (3H, s)

0.78 (3H, s)

1.32 (3H, s)

1.90 (1H, 1.62

m) (2H, m)

1.58 (1H,

2.46 (1H, 2.48

m) (1H, m)

2.16 (1H, 2.42

m) (1H, m)

5.24 (1H, 5.25

m) (1H, m)

1.62 (3H, s)

1.55 (3H, s)

1.20 (3H, s)

0.88 (3H, s)

0.96 (3H, s)

1.47 1.22 1.41

2.05 1.54 3.90

2.06 1.58 1.02 1.92 1.81 2.34 0.80 1.01 1.41 2.00 1.63 2.16 1.82 1.71

1.38 1.30 0.98 0.94

1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 3H, s) 3H, s) 3H, s) 1H, m) 1H, m) 1H, m) 1H, m) 2H, m) 3H, s) 3H, s) 3H, s) 3H, s) 3H, s)

1.48 1.22 1.41 2.03 1.52

3.90 2.00 1.57 1.02

1.91 1.80 2.40 0.81 1.00 1.38 1.71

2.10 1.98 1.71 1.40 1.40 1.30 0.98 0.94

1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 1H, m) 3H, s) 3H, s) 3H, s) 2H, m)

2H, m) 1H, m) 2H, m) 3H, s) 3H, s) 3H, s) 3H, s) 3H, s)

3-O-S-D-g

ucopyranosyl

4.92 7.8)

4.23 8.7)

1H, d, J =

1H, m) 1H, t, J =

4.92 7.60

4.02 4.22

1H, d, J =

1H, m) 1H, m)

4.90 7.55

4.23 4.21

1H, d, J =

1H, m) 1H, m)

4.91 7.6)

4.22 4.20

1H, d, J =

1H, m) 1H, m)

4.74 (1H, m) 3.92 (1H, m) 4.06 (1H, m)

4.93 7.8)

4.02 4.25

1H, d, J =

1H, m) 1H, m)

4.91 7.8)

4.22 8.8)

1H, d, J =

1H, m) 1H, t, J =

4' 4.18 (1H, t, J = 8.7) 4.18 (1H, m) 4.11 (1H, m) 4.13 (1H, m) 3.88 (1H, m) 4.20 (1H, m) 4.18 (1H, t, J = 8.8)

5 ' 3.90 (1H, m) 3.98 (1H, m) 3.88 (1H, m) 3.89 (1H, m) 3.87 (1H, m) 3.99 (1H, m) 3.98 (1H, m)

6'a 6% 4.56 (1H, d, J = 11.9) 3.67 (1H, dd, J = 5.5, 11.9) 4.57 (1H, d, J = 11.9) 4.37 (1H, dd, J = 5.5, 11.9) 4.53 (1H, m) 4.33 (1H, m) 4.54 (1H, m) 4.32 (1H, m) 4.79 (1H, m) 4.67 (1H, dd, J = 6.42, 11.88) 4.57 (1H, dd, J = 2.2,11.7) 4.37 (1H, dd, J = 5.4,11.7) 4.56 (1H, dd, J = 1.9,11.7) 4.37 (1H, dd, J = 5.4,11.7)

2 '-O-^-D-glucopyranosyl

1 5.35 (1H, d, J = 7.65) 5.36 (1H, d, J = 7.65)

2 4.10 (1H, m) 4.12 (1H, m)

3 4.29 (1H, m) 4.28 (1H, m)

4 4.32 (1H, m) 4.31 (1H, m)

5 3.91 (1H, m) 3.93 (1H, m)

6 a 4.46 (1H, m) 4.46 (1H, m)

6 b 4.45 (1H, m) 4.45 (1H, m)

COCH3 1.93 (3H, s)

232 1H NMR data were measured at a 600 or b 500 MHz, respectively. c 20(S/tf)-AcetylRh2; 6 '-O-acetyl-

233 20(S/R)-Rh2.

No. 20(S)-Rh2" (1) 20(R)-Rh2" (2) 20(S)-Rg3b (3) 20(R)-Rg3b (4) 20(S/R)-AcetylRh2b- c (5 and 6) 25-hydroxy-20(S)-Rh2b (7) 25-hydroxy-20(R)-Rh2b (8)

SC multiplicity

1 39.1 t 39.1 t 39.1 t 39.1 t 38.9 t 39.1 t 39.1 t

2 26.7 t 26.6 t 26.7 t 26.6 t 26.6 t 26.7 t 26.7 t

3 88.7 d 88.7 d 88.9 d 88.9 d 89.0 d 88.8 d 88.7 d

4 39.6 s 39.6 s 39.6 s 39.6 s 39.4 s 39.7 s 39.6 s

5 56.3 d 56.3 d 56.3 d 56.3 d 56.2 d 56.1 d 56.4 d 56.3 d

6 18.4 t 18.4 t 18.4 t 18.4 t 18.2 t 18.4 t 18.4 t

7 35.1 t 35.1 t 35.1 t 35.1 t 34.9 t 35.2 t 35.1 t

8 40.0 s 40.0 s 39.9 s 40.0 s 39.8 s 40.0 s 40.0 s

9 50.3 d 50.3 d 50.3 d 50.3 d 50.2 d 50.4 d 50.3 d

10 36.9 s 36.9 s 36.8 s 36.9 s 36.8 s 37.0 s 36.9 s

11 31.3 t 31.4 t 31.3 t 31.4 t 31.1 t 31.2 t 32.1 t 32.1 t

12 70.9 d 70.8 d 70.9 d 70.8 d 70.7 d 70.6 d 71.0 d 70.8 d

13 48.5 d 49.2 d 48.5 d 49.2 d 48.9 d 48.2 d 48.6 d 49.2 d

14 51.7 s 51.7 s 51.6 s 51.7 s 51.5 s 51.7 s 51.7 s

15 32.0 t 32.1 t 32.0 t 32.1 t 31.7 t 31.8 t 31.4 t 31.4 t

16 26.8 t 26.7 t 26.8 t 26.7 t 26.4 t 27.2 t 26.6 t

17 54.7 d 50.6 d 54.7 d 50.6 d 54.5 d 50.3 d 54.7 d 50.7 d

18 16.3 q 16.3 q 15.8 q 15.8 q 15.6 q 16.8 q 16.7 q

19 15.8 q 15.8 q 16.3 q 16.3 q 16.1 q 15.8 q 15.8 q

20 72.9 s 72.9 s 72.9 s 72.9 s 72.8 s 73.3 s 73.3 s

21 27.0 q 22.7 q 27.0 q 22.7 q 26.7 q 22.4 q 26.9 q 22.8 q

22 35.8 t 43.2 t 35.8 t 43.2 t 35.6 t 42.9 t 36.5 t 44.0 t

23 22.9 t 22.6 t 23.0 t 22.5 t 22.3 t 22.7 t 19.1 t 18.7 t

24 126.3 d 126 d 126.2 d 126.0 d 126.0 125.8 d d 45.7 t 45.5 t

25 130.7 s 130.7 s 130.7 s 130.7 s 130.5 s 69.6 s 69.7 s

26 25.8 q 25.8 q 25.7 q 25.8 q 25.7 q 25.6 q 30.2 q 30.1 q

27 17.6 q 17.7 q 17.0 q 17.2 q 17.1 q 16.8 q 29.9 q 29.9 q

28 28.1 q 28.1 q 28.1 q 28.1 q 27.9 q 28.1 q 28.1 q

29 16.7 q 16.7 q 16.5 q 16.5 q 16.5 q 16.4 q 16.3 q

30 17.0 q 17.3 q 17.6 q 17.6 q 17.5 q 17.0 q 17.3 q

3-O--ff-D-glucopyranosyl

1 106.9 d 106.9 d 105.0 d 105.1 d 106.6 d 106.9 d 106.9 d

2 75.7 d 75.7 d 83.4 d 83.4 d 74.5 d 75.8 d 75.7 d

3 78.7 d 78.7 d 77.9 d 77.9 d 78.1 d 78.7 d 78.7 d

4 71.8 d 71.8 d 71.6 d 71.6 d 71.3 d 71.9 d 71.8 d

5 78.3 d 78.3 d 78.2 d 78.2 d 75.1 d 78.3 d 78.3 d

6 63.0 t 63.0 t 62.8 t 62.8 t 64.4 t 63.1 t 63.0 t

2 '-O-^-D-glucopyranosyl

1 106.0 d 106.0 d

2 77.1 d 77.1 d

3 78.3 d 78.3 d

4 71.6 d 71.6 d

5 78.0 d 78.1 d

6 62.7 t 62.7 t

COCH3 170.5 s

COCH3 20.6 q

238 Multiplicity of 13C NMR data was determined by DEPT experiments. 13C NMR data were

239 measured at a 150 or b 125 MHz, respectively. c 20(S/R)-AcetylRh2; 6 '-0-acetyl-20(S/R)-Rh2.

No. 20(S)-Rh1" (9) 20(R)-Rh1a (10) 20(S)-Rg2 " (11) 20(R)-Rg2b (12) 25-hydroxy-20(S/R)-Rh1" (13 and 14) 20(S)-Acetyl-Rg2a c (15) 20(R)-Acetyl-Rg2a d (16)

Sh (j in Hz)

1a 1.66 (1H, m) 1.69 (1H, m) 1.61 (1H, m) 1.61 (1H, m) 1.67 (1H, m) 1.65 (1H, m) 1.64 (1H, m)

1b 1.02 (1H, m) 1.01 (1H, m) 0.92 (1H, m) 0.92 (1H, m) 1.02 (1H, m) 0.98 (1H, m) 0.97 (1H, m)

2a 1.89 (1H, m) 1.90 (1H, m) 1.83 (1H, m) 1.82 (1H, m) 1.89 (1H, m) 1.82 (1H, m) 1.82 (1H, m)

2b 1.80 (1H, m) 1.80 (1H, m) 1.76 (1H, m) 1.76 (1H, m) 1.82 (1H, m) 1.76 (1H, m) 1.76 (1H, m)

3 3.50 (1H, m) 3.50 (1H, br d) 3.43 (1H, m) 3.35 (1H, br s) 3.50 (1H, m) 3.46 (1H, m) 3.46 (1H, dd, J = 4.1, 11.2)

5 6 1.40 (1H, m) 4.40 (1H, td, J = 2.8, 10.5 ) 1.42 (1H, d, J = 10.5) 4.43 (1H, td, J = 2.9, 10.5 ) 1.37 (1H, m) 4.64 (1H, m) 1.39 (1H, m) 4.68 (1H, m) 1.42 (1H, m) 4.42 (1H, m) 1.37 (1H, m) 4.75 (1H, s) 1.39 (1H, m) 4.70 (1H, td, J = 3.3, 10.6)

7a 2.50 (1H, m) 2.51 (1H, m) 2.22 (1H, m) 2.23 (1H, m) 2.51 (1H, m) 2.14 (1H, m) 2.15 (1H, m)

7b 1.91 (1H, m) 1.93 (1H, m) 1.95 (1H, m) 1.96 (1H, m) 1.93 (1H, m) 1.97 (1H, m) 1.98 (1H, m)

9 1.53 (1H, m) 1.57 (1H, m) 1.51 (1H, m) 1.52 (1H, m) 1.58 (1H, m) 1.55 (1H, s) 1.56 (1H, s)

11a 2.11 (1H, m) 2.13 (1H, m) 2.04 (1H, m) 2.09 (1H, m) 2.13 (1H, m) 2.14 (1H, m) 2.15 (1H, m)

11b 1.56 (1H, m) 1.52 (1H, m) 1.51 (1H, m) 1.54 (1H, m) 1.56 (1H, m) 1.56 (1H, m) 1.57 (1H, m)

12 3.88 (1H, m) 3.91 (1H, m) 3.89 (1H, m) 3.90 (1H, m) 3.89 (1H, m) 3.93 (1H, m) 3.95 (1H, m)

13 2.01 (1H, m) 2.00 (1H, m) 1.97 (1H, m) 1.96 (1H, m) 2.02 (1H, m) 2.04 (1H, m) 2.01 (1H, m)

15a 1.59 (1H, m) 1.59 (1H, m) 1.51 (1H, m) 1.50 (1H, m) 1.63 (1H, m) 1.62 (1H, m) 1.62 (1H, m)

15b 1.07 (1H, m) 1.11 (1H, m) 0.83 (1H, m) 0.91 (1H, m) 1.10 (1H, m) 0.98 (1H, m) 1.02 (1H, m)

16a 1.76 (1H, m) 1.80 (1H, m) 1.73 (1H, m) 1.82 (1H, m) 1.32 (2H, m) 1.82 (1H, m) 1.88 (1H, m)

16b 1.30 (1H, m) 1.28 (1H, m) 1.28 (1H, m) 1.22 (1H, m) 1.38 (1H, m) 1.30 (1H, m)

17 2.26 (1H, m) 2.32 (1H, m) 2.25 (1H, m) 2.34 (1H, m) 2.28 (1H, m) 2.35 (1H, m) 2.31 (1H, m) 2.37 (1H, m)

18 1.16 (3H, s) 1.22 (3H, s) 1.18 (3H, s) 1.22 (3H, s) 1.03 (3H, s) 1.22 (3H, s) 1.25 (3H, s)

19 1.00 (3H, s) 1.04 (3H, s) 0.93 (3H, s) 0.96 (3H, s) 1.25 (3H, s) 0.99 (3H, s) 1.02 (3H, s)

21 1.37 (3H, s) 1.37 (3H, s) 1.38 (3H, s) 1.36 (3H, s) 1.38 (3H, s) 1.35 (3H, m) 1.35 (3H, s)

22a 2.01 (1H, m) 1.68 (2H, m) 1.98 (1H, m) 2.01 (1H, m) 2.00 (1H, m) 1.67 (2H, m) 2.04 (1H, m) 1.68 (2H, m)

22b 1.66 (1H, m) 1.62 (1H, m) 1.68 (1H, m) 1.63 (1H, m) 1.67 (1H, m)

23a 2.56 (1H, m) 2.48 (1H, m) 2.58 (1H, m) 2.57 (1H, m) 2.13 (1H, m) 2.02-1.99 (2H, m) 2.57 (1H, m) 2.49 (1H, m)

23b 2.25 (1H, m) 2.41 (1H, m) 2.23 (1H, m) 2.29 (1H, m) 1.86 (1H, m) 2.25 (1H, m) 2.41 (1H, m)

24 5.30 (1H, t-like) 5.28 (1H, t-like) 5.31 (1H, t-like) 5.29 (1H, t-like) 1.70 (2H, t-like) 5.29 (1H, t-like) 5.28 (1H, t-like)

26 1.63 (3H, s) 1.67 (3H, s) 1.63 (3H, s) 1.67 (3H, s) 1.38 (3H, s) 1.62 (3H, s) 1.68 (3H, m)

27 1.60 (3H, s) 1.61 (3H, s) 1.60 (3H, s) 1.62 (3H, s) 1.40 (3H, s) 1.59 (3H, s) 1.60 (3H, s)

28 2.05 (3H, s) 2.06 (3H, s) 2.06 (3H, s) 2.09 (3H, s) 2.05 (3H, s) 2.05 (3H, m) 2.03 (3H, m)

29 1.57 (3H, s) 1.59 (3H, s) 1.31 (3H, s) 1.34 (3H, s) 1.58 (3H, s) 1.29 (3H, s) 1.28 (3H, s)

30 0.79 (3H, s) 0.84 (3H, s) 0.91 (3H, s) 0.95 (3H, s) 0.82 (3H, s) 0.97 (3H, s) 1.00 (3H, s)

6-O--ff-D-glucopyranosyl

1 5.00 (1H, m) 5.03 (1H, m) 5.23 (1H, d, J = 6.9) 5.26 (1H, m) 5.02 (1H, m) 5.22 (1H, d, J=7.0) 5.22 (1H, d, J = 7.0)

2 4.08 (1H, m) 4.09 (1H, m) 4.32 (1H, m) 4.32 (1H, m) 4.07 (1H, m) 4.33 (1H, m) 4.32 (1H, m)

3 4.23 (1H, m) 4.25 (1H, m) 4.33 (1H, m) 4.36 (1H, m) 4.23 (1H, m) 4.29 (1H, m) 4.29 (1H, m)

4 4.19 (1H, m) 4.20 (1H, m) 4.19 (1H, m) 4.19 (1H, m) 4.07 (1H, m) 3.92 (1H, m) 3.94 (1H, m)

5 6 a 6 b 3.92 (1H, m) 4.51 (1H, m) 4.34 (1H, m) 3.95 (1H, m) 4.52 (1H, dd, J = 1.9, 11.4) 4.35 (1H, dd, J = 5.3, 11.4) 3.93 (1H, m) 4.49 (1H, m) 4.36 (1H, m) 3.95 (1H, m) 3.94 (1H, m) 4.50 (1H, m) 4.51 (1H, m) 4.37 (1H, m) 4.34 (1H, m) 4.01 (1H, t-like) 5.00 (1H, m) 4.61 (1H, m) 4.03 (1H, t-like, J = 8.2) 4.90 (1H, m) 4.63 (1H, m)

2 '-O-a-L-rhamnopyranosyl

1 2 3 6.47 (1H, br s) 4.75 (1H, m) 4.63 (1H, m) 6.47 (1H, s) 4.78 (1H, m) 4.66 (1H, m) 6.47 (1H, s) 4.68 (1H, dt, J = 3.2, 10.6) 4.64 (1H, m) 6.47 (1H, s) 4.75 (1H, m) 4.64 (1H, m)

4 4.30 (1H, m) 4.31 (1H, m) 4.34 (1H, m) 4.33 (1H, m)

5 6 COCH 4.92 (1H, m) 1.76 (3H, d, J = 6.2) 4.94 (1H, m) 1.78 (3H, br s) 4.98 (1H, m) 1.76 (3H, d, J=6.2) 2.04 (3H, s) 4.80 (1H, m) 1.77 (3H, d, J=6.1) 2.08 (3H, s)

242 1H NMR data were measured at a 500 or b 600, respectively. c 20(S)-AcetylRg2; 6 '-0-acetyl-20(S)-Rg2;

243 d 20(tf)-AcetylRg2; 6 '-0-acetyl-20(tf)-Rg2.

No. 20(S)-Rh1" (9) 20(fl)-Rh1" (10) 20(S)-Rg2 " (11) 20(fl)-Rg2b (12) 25-hydroxy-20(S/R)-Rh1" (13 and 14) 20(S)-Acetyl-Rg2a c (15) 20(fl)-Acetyl-Rg2a c (16)

dC multiplicity

1a 39.3 t 39.3 t 39.5 t 39.6 t 39.6 t 39.5 t 39.5 t

2 27.8 t 27.9 t 27.7 t 27.7 t 27.9 t 27.6 t 27.6 t

3 78.5 d 78.5 d 78.3 d 78.3 d 78.5 d 78.2 d 78.1 d

4 40.3 s 40.3 s 41.1 s 39.9 s 40.3 s 39.8 s 39.8 s

5 61.4 d 61.4 d 60.7 d 60.8 d 61.4 d 60.5 d 60.5 d

6 80.0 d 80.0 d 74.2 d 74.3 d 80.0 d 72.2 d 73.3 d

7 45.2 t 45.1 t 46.0 t 46.0 t 45.2 t 45.1 t 46.1 t 46.1 t

8 41.0 s 41.1 s 41.1 s 41.1 s 41.0 s 39.2 s 39.2 s

9 50.1 d 50.1 d 49.7 d 49.7 d 50.2 d 49.6 d 49.6 d

10 39.6 s 39.6 s 39.9 s 39.9 s 39.3 s 41.1 s 41.1 s

11 32.0 t 32.2 t 32.0 t 32.1 t 32.1 t 32.0 t 32.0 t

12 71.0 d 70.9 d 71.0 d 70.9 d 71.0 d 70.9 d 70.8 d

13 48.2 d 48.8 d 48.1 d 48.8 d 48.2 d 48.9 d 48.2 d 48.8 d

14 51.6 s 51.7 s 51.6 s 51.7 s 51.6 s 51.6 s 51.6 s

15 31.2 t 31.3 t 31.2 t 31.3 t 31.3 t 31.2 t 31.3 t

16 26.7 t 26.6 t 26.8 t 26.6 t 26.8 t 26.7 t 26.5 t

17 54.7 d 50.5 d 54.6 d 50.5 d 54.6 d 50.7 d 54.7 d 50.4 d

18 17.3 q 17.3 q 17.6 q 17.6 q 17.6 q 17.0 q 17.1 q

19 17.6 q 17.6 q 17.5 q 17.5 q 17.3 q 17.5 q 17.4 q

20 72.9 s 73.0 s 72.9 s 72.9 s 73.3 s 72.9 s 72.9 s

21 26.9 q 22.7 q 27.0 q 22.7 q 27.1 q 22.7 q 26.9 q 22.6 q

22 35.8 t 43.2 t 35.7 t 43.2 t 36.4 t 43.9 t 35.8 t 43.1 t

23 22.9 t 22.5 t 22.9 t 22.5 t 19.1 t 18.6 t 22.9 t 22.5 t

24 126.2 d 126.0 d 126.3 d 126.0 d 45.7 t 126.2 d 125.9 d

25 130.7 s 130.7 s 130.7 s 130.7 s 69.7 s 130.7 s 130.7 s

26 25.7 q 25.8 q 25.8 q 25.8 q 30.1 q 25.7 q 25.7 q

27 17.6 q 17.6 q 17.6 q 17.6 q 17.6 q 17.6 q 17.6 q

28 31.6 q 31.7 q 32.1 q 32.1 q 31.7 q 31.9 q 32.0 q

29 16.3 q 16.3 q 16.8 q 17.2 q 16.8 q 17.4 q 17.5 q

30 16.7 q 17.0 q 17.1 q 17.1 q 17.0 q 16.9 q 17.0 q

6-O-a-L-rhamnopyranosyl

1 106.0 d 106.0 d 101.9 d 101.9 d 105.9 d 101.2 d 101.2 d

2 75.4 d 75.4 d 79.4 d 79.4 d 75.4 d 78.2 d 78.2 d

3 79.6 d 79.6 d 78.5 d 78.5 d 79.6 d 79.0 d 79.0 d

4 71.8 d 71.8 d 72.4 d 72.4 d 71.8 d 72.3 d 72.3 d

5 78.1 d 78.1 d 78.3 d 78.3 d 78.1 d 75.3 d 75.3 d

6 a 63.0 t 63.0 t 63.0 t 63.1 t 64.8 t 64.8 t

2 '-O-a-L-rhamnopyranosyl

1' 101.7 d 101.7 d 102.0 d 102.0 d

2 '' 72.2 d 72.2 d 73.3 d 72.2 d

3 72.5 d 72.6 d 72.2 d 72.2 d

4 74.1 d 74.1 d 74.0 d 74.0 d

5 69.4 d 69.4 d 69.3 d 69.3 d

6 '' 18.7 q 18.7 q 18.6 q 18.6 q COCH3 170.7 s 170.7 s COCH3 20.8 q 20.8 q

247 Multiplicity of 13C NMR data was determined by DEPT experiments. 13C NMR data were

248 measured at a 125 or b 150 MHz, respectively. c 20(S)-AcetylRg2; 6 '-0-acetyl-20(S)-Rg2; d 20(R)-

249 AcetylRg2; 6 '-0-acetyl-20(R)-Rg2.

No. Rk1 (17) Rh4 (18) 25-hydroxy-Rh4 (19) Rg5 (20) oleanolic acid 28-O-P-D- glu (21)

SH (J in Hz)

1a 1.49 (1H, m) 1.67 (1H, m) 1.68 (1H, m) 1.47 (1H, m) 1.50 (1H, m)

1b 0.74 (1H, m) 1.01 (1H, m) 1.03 (1H, m) 0.75 (1H, m) 0.97 (1H,m)

2a 2.18 (1H, m) 1.85 (1H, m) 1.88 (1H, m) 2.18 (1H, m) 1.80 (2H, m)

2b 1.80 (1H, m) 1.80 (1H, m) 1.82 (1H, m) 1.78 (1H, m)

3 3.27 (1H, dd J = 4.3, 11.7) 3.49 (1H, dd, J = 4.7, 11.6) 3.50 (1H, dd, J = 11.6, 4.6) 3.27 (1H, dd, J = 4.3, 11.6) 3.42 (1H, dd, J = 5.2, 10.8)

5 0.67 (1H, d, J = 11.2) 1.40 (1H, m) 1.41 (1H, m) 0.67 (1H, d, J = 11.1) 0.83 (1H, m)

6a 1.47 (1H, m) 4.40 (1H, td, J = 3.2, 10.3) 4.41 (1H, td, J = 10.6, 2.8) 1.51 (1H, m) 1.51 (1H, m)

6b 1.36 (1H, m) 1.36 (1H, m) 1.34 (1H, m)

7a 1.47 (1H, m) 2.49 (1H, m) 2.51 (1H, dd, J = 12.7, 2.8) 1.43 (1H, m) 1.52 (1H, m)

7b 1.24 (1H, m) 1.92 (1H, m) 1.93 (1H, m) 1.21 (1H, m) 1.40 (1H, m)

9 2.80 (1H, m) 1.53 (1H, m) 1.55 (1H, m) 1.38 (1H, m) 1.64 (1H, m)

11a 1.91 (1H, m) 1.95 (1H, m) 1.56 (1H, m) 1.91 (1H, m) 2.08 (2H, m)

11b 1.40 (1H, m) 1.41 (1H, m) 1.46 (1H, m) 1.41 (1H, m) 5.44 (1H, m)

12 3.89 (1H, m) 3.88 (1H, m) 3.88 (1H, m) 3.90 (1H, m)

13 2.06 (1H, m) 2.71 (1H, m) 1.97 (1H, m) 2.77 (1H, m)

15a 1.45 (1H, m) 1.52 (1H, m) 1.71 (1H, m) 1.64 (1H, m) 2.35 (1H, m)

15b 1.06 (1H, m) 1.11 (1H, m) 1.18 (1H, m) 1.09 (1H, m) 1.16 (1H, m)

16a 2.06 (1H, m) 1.45 (2H, m) 1.46 (2H, m) 1.98 (1H, m) 2.36 (1H, m)

16b 1.57 (1H, m) 1.96 (1H, m) 2.72 (1H, m) 1.52 (1H, m) 1.92 (1H, m)

17 1.40 (1H, m) 1.20 (3H, s) 0.81 (3H, s) 1.98 (1H, m)

18 1.01 (3H, s) 1.01 (3H, s) 1.02 (3H, s) 1.01 (3H, s) 3.19 (1H, dd, J = 2.8, 10.8)

19a 0.80 (3H, s) 0.81 (3H, s) 1.74 (1H, m)

19b 1.27 (1H, m)

21a 5.14 (2H, s) 1.77 (3H, s) 1.79 (3H, s) 1.81 (3H, s) 1.33 (1H, m)

21b 1.05 (1H, m)

22a 2.48 (1H, m) 5.43 (1H, t, J = 7.0) 5.55 (1H, t, J = 6.7) 5.50 (1H, t, J = 6.6) 1.83 (1H, m)

22b 2.38 (1H, m) 1.74 (1H, m)

23 2.32 (1H, m) 2.72 (2H, m) 2.33 (2H, m) 2.77 (2H, m) 1.22 (3H, s)

24 5.28 (1H, m) 5.18 (1H, m) 1.71 (2H, m) 5.22 (1H, t, J = 7.2) 1.01 (3H, s)

25 0.87 (3H, s)

26 1.66 (3H, s) 1.59 (3H, s) 1.33 (3H, s) 1.62 (3H, s) 1.12 (3H, s)

27 1.59 (3H, s) 1.56 (3H, s) 1.33 (3H, s) 1.58 (3H, s) 1.21 (3H, s)

28 1.27 (3H, s) 2.02 (3H, s) 2.04 (3H, s) 1.28 (3H, s)

29 1.09 (3H, s) 1.55 (3H, s) 1.58 (3H, s) 1.10 (3H, s) 0.91 (3H, s)

30 0.95 (3H, s) 0.80 (3H, s) 1.22 (3H, s) 0.95 (3H, s) 0.89 (3H, s)

3-O--S-D-glucopyranosyl 6-O-S-D-glucopyranosyl 6-O--S-D-glucopyranosyl 6-O--S-D-glucopyranosyl 28-O-S-D-glucopyranosyl

1 4.89 (1H, m) 4.98 (1H, m) 5.01 (1H, d, J = 7.8) 4.91 (1H, d, J = 7.5) 6.31 (1H, d, J = 8.1)

2 4.20 (1H, m) 4.04 (1H, m) 4.06 (1H, m) 4.22 (1H, m) 4.18 (1H, m)

3 4.21 (1H, m) 4.20 (1H, m) 4.23 (1H, m) 4.23 (1H, m) 4.01 (1H, m)

4 4.11 (1H, m) 4.16 (1H, m) 4.19 (1H, m) 4.13 (1H, m) 4.33 (1H, m)

5 3.89 (1H, m) 3.91 (1H, m) 3.92 (1H, m) 3.90 (1H, m) 4.26 (1H, m)

6 a 6 b 4.53 (1H, m) 4.32 (1H, m) 4.48 (1H, dd, J = 2.6, 11.6) 4.32 (1H, dd, J = 5.4, 11.6) 4.51 (1H, dd, J = 11.5, 2.5) 4.33 (1H, dd, J = 11.5, 5.4) 4.55 (1H, dd, J = 2.0, 11.7) 4.32 (1H, m) 4.45 (1H, m) 4.37 (1H, m)

2'-O-y»-D-glucopyranosyl 2'-O-fi-D-glucopyranosyl

1 5.33 (1H, d, J = 7.6) 5.35 (1H, d, J = 7.6)

2 4.09 (1H, m) 4.12 (1H, m)

3 4.28 (1H, m) 4.32 (1H, m)

4 4.28 (1H, m) 4.30 (1H, m)

5 3.89 (1H, m) 3.90 (1H, m)

6 a 4.47 (1H, m) 4.46 (2H, m)

6 b 4.43 (1H, m)

252 1H NMR data were measured at 500 MHz, respectively.

No. Rk1 (17) Rh4 (18) 25-hydroxy-Rh4 (19) Rg5 (20) oleanolic acid 28-O-fi-D-glucopyranoside (21)

dC multiplicity

1 39.3 t 39.4 t 39.5 t 39.3 t 39.0 t

2 26.7 t 28.7 t 27.9 t 26.7 t 28.1 t

3 88.9 d 78.5 d 78.5 d 88.9 d 78.1 d

4 39.7 s 40.3 s 40.3 s 40.2 s 39.4 s

5 56.4 d 61.4 d 61.4 d 56.4 d 55.8 d

6 18.4 t 80.0 d 80.0 d 18.4 t 18.8 t

7 35.3 t 45.2 t 45.3 t 35.3 t 33.1 t

8 40.2 s 41.3 s 41.3 s 39.7 s 40.0 s

9 48.2 d 50.5 d 50.5 d 50.8 d 48.1 d

10 37.0 s 39.7 s 39.7 s 37.0 s 37.4 s

11 32.6 t 32.2 t 32.2 t 32.2 t 23.4 t

12 72.4 d 72.5 d 72.6 d 72.6 d 122.9 d

13 52.4 d 50.3 d 50.8 d 50.4 d 144.1 s

14 51.2 s 50.6 s 50.6 s 50.9 s 42.1 s

15 32.6 t 32.5 t 32.5 t 32.6 t 28.3 t

16 30.7 t 27.8 t 28.7 t 28.1 t 23.6 t

17 50.8 d 50.7 d 50.5 d 51.0 d 47.0 s

18 15.8 q 17.3 q 16.8 q 15.8 q 41.8 d

19 16.4 q 17.6 q 17.7 q 16.6 q 46.2 t

20 155.5 s 140.0 s 139.5 s 140.2 s 30.8 s

21 108.1 t 13.0 q 13.0 q 13.1 q 34.0 t

22 33.8 t 123.5 d 125.5 d 123.2 d 32.5 t

23 27.0 t 27.4 t 23.6 t 27.4 t 28.8 q

24 125.3 d 123.8 d 44.2 t 123.5 d 16.5 q

25 131.2 s 131.2 s 69.5 s 131.2 s 15.6 q

26 25.7 q 25.6 q 29.9 q 25.7 q 17.5 q

27 17.7 q 17.6 q 29.7 q 17.7 q 26.1 q

28 28.1 q 31.6 q 31.7 q 28.8 q 176.4 s

29 16.5 q 16.3 q 16.3 q 16.4 q 33.2 q

30 17.0 q 16.7 q 17.4 q 17.0 q 23.8 q

3-O-fi-D-glucopyranosyl 6-O-fi-D-glucopyranosyl 6-O-fi-D-glucopyranosyl 6-O-fi-D-glucopyranosyl 28-O-fi-D-glucopyranosyl

1 105.1 d 105.9 d 106.0 d 105.1 d 95.7 d

2 83.4 d 75.3 d 75.4 d 83.4 d 74.1 d

3 77.9 d 79.5 d 79.6 d 78.2 d 79.3 d

4 71.6 d 71.7 d 71.8 d 71.6 d 71.1 d

5 78.2 d 78.0 d 78.1 d 77.9 d 78.9 d

6 62.8 t 63.0 t 63.1 t 62.7 t 62.2 t

2 '-O-fi-D-glucopyranosyl 2 '-O-fi-D-glucopyranosyl

1 106.0 d 106.0 d

2 77.0 d 77.1 d

3 78.3 d 78.3 d

4" 71.6 d 71.7 d

5 78.0 d 78.1 d

6 62.7 t 62.8 t

256 Multiplicity of 13C NMR data was determined by DEPT experiments. C NMR data were measured at

257 125 MHz, respectively.

Name R1 R2 R3

20(S)-Rh2 (1) O-Glua H P-OH

20(R)-Rh2 (2) O-Glu H a-OH

20(S)-Rg3 (3) O-Glu-2 '-O-Glu H p -OH

20(R)-Rg3 (4) O-Glu-2 '-O-Glu H a-OH

20(S/R)-AcetylRh2 (5 and 6) O-AcetylGlub H a- and P-OH

20(S)-Rh1 (9) OH O-Glu *V-OH

20(R)-Rh1 (10) OH O-Glu a-OH

20(S)-Rg2 (11) OH O-Glu-2 -O-Rhac P-OH

20(R)-Rg2 (12) OH O-Glu-2 -O-Rha a-OH

20(S)-AcetylRg2 (15) OH O-AcetylGlu-2 -O-Rha P-OH

20(R)-AcetylRg2 (16) OH O-AcetylGlu-2 -O-Rha a-OH

CH rcH

Name R1 R2 R3

25-hydroxy-20(S)-Rh2 (7) O-Glc H a-OH

25-hydroxy-20(R)-Rh2 (8) O-Glc H a-OH

25-hydroxy-20(S/R)-Rh1 (13 and 14) OH O-Glc a- and P-OH

Rk1 (17)

Name Ri R2

Rh4 (18) OH O-Glu

Rg5 (20) O-Glu-2 '-O-Glu H

OH ^OH

hO '' OGlc 25-hydroxy-Rh4 (17)

278 Fig. 1. The structures of compounds 1-21 isolated from the processed ginseng extract.

oleanolic acid 28-0-#-D-glucopyranose (21)

aGlu: ^-D-glucose, bAcetylGlu: fS-D-6 '-O-acetyl-glucose, cRha: a-L-rhamnose