Isolation of Ginsenoside Isomers from Processed Vietnamese Ginseng by Preparative HPLC

Abstract

Isolation of Ginsenoside Isomers from Processed Vietnamese Ginseng
by Preparative HPLC
Our recent studies of processed Vietnamese ginseng pointed out that heating processing changes its saponin composition by increasing the content of less polar ginsenosides. Most of those less polar ginsenosides exist as pairs of isomers and it is quite difficult to separate by normal column chromatography. The aim of this study is to isolate these stereoisomers using preparative-HPLC (prep-HPLC). An ethyl acetate extract of processed Vietnamese ginseng was used as the starting material to isolate ginsenoside isomers by recrystallization followed by column chromatography and pre-HPLC. Structures of isolated ginsenosides were identified by nuclear magnetic resonance (NMR) and mass chromatography (MS). Three pairs of ginsenoside isomers including ginsenosides-Rk3 and -Rh4, 20S-ginsenosides-Rh1 and 20R-Rh1, and 20R-ginsenosides-Rg3 and 20S-Rg3 were successfully isolated from prep-HPLC together with daucosterol, notoginsenoside-R2 (N-R2), and pseudoginsenoside-Rt­4 from processed Vietnamese ginseng. Among the isolated compounds, daucosterol, 20R-ginsenoside Rg3, 20S-ginsenoside-Rg3, and notoginsenoside-R2 were reported from Panax vietnamensis for the first time.
Keywords: Panax vietnamensis, Ginsenoside isomer, Processed Vietnamese ginseng, Preparative HPLC.

Full text

Le Thi Hong Van1, Nguyen Thi Minh Tam1, Bui Xuan Huong1,
Nguyen Ngoc Khoi1, Jeong Hill Park3, Nguyen Minh Duc1,2,*
1University of Medicine and Pharmacy, Ho Chi Minh City, Vietnam
2Ton Duc Thang University, Ho Chi Minh City, Vietnam
3College of Pharmacy, Seoul National University, Korea.
*Corresponding author: nguyenminhduc@tdt.edu.vn
(Received September, 7th, 2015)
Summary
Isolation of Ginsenoside Isomers from Processed Vietnamese Ginseng
by Preparative HPLC
Our recent studies of processed Vietnamese ginseng pointed out that heating processing changes its saponin
composition by increasing the content of less polar ginsenosides. Most of those less polar ginsenosides exist as
pairs of isomers and it is quite difficult to separate by normal column chromatography. The aim of this study is to
isolate these stereoisomers using preparative-HPLC (prep-HPLC). An ethyl acetate extract of processed
Vietnamese ginseng was used as the starting material to isolate ginsenoside isomers by recrystallization followed
by column chromatography and pre-HPLC. Structures of isolated ginsenosides were identified by nuclear
magnetic resonance (NMR) and mass chromatography (MS). Three pairs of ginsenoside isomers including
ginsenosides-Rk3 and -Rh4, 20S-ginsenosides-Rh1 and 20R-Rh1, and 20R-ginsenosides-Rg3 and 20S-Rg3 were
successfully isolated from prep-HPLC together with daucosterol, notoginsenoside-R2 (N-R2), and
pseudoginsenoside-Rt4from processed Vietnamese ginseng. Among the isolated compounds, daucosterol, 20R-
ginsenoside Rg3, 20S-ginsenoside-Rg3, and notoginsenoside-R2 were reported from Panax vietnamensis for the
first time.
Keywords: Panax vietnamensis, Ginsenoside isomer, Processed Vietnamese ginseng, Preparative HPLC.
1. Introduction
Ginseng saponins are the main components in Panax species, which were intensively studied. In our recent
study on processed Vietnamese ginseng (Panax vietnamensis. Ha et Grushv., VG), saponin constituents have
been changed remarkably during steaming process. Polar protopanaxadiol (PPD) and protopanaxatriol (PPT)
ginsenosides were decomposed to their less polar analogues rapidly [1]. It has been reported that these could be
come from the elimination of sugar chains or by dehydroxylation to generate the irregular D20(21) - or D20(22)-
ginsenosides as well as 20R-epimers [2]. However, it is painstaking to isolate ginsenoside isomers by using
normal chromatography methods. In our previous studies, prep-HPLC method was shown efficiency for isolation
of ginsenosides from processed VG. Therefore, in this study, one-step prep-HPLC was successfully applied to
isolate 3 pairs of ginsenoside isomers together with 2 other saponins.
2. Materials and methods
Materials
Processed VG (as described in our precious study [3]) which was steamed at 105oC for 8 hours and then dried at
about 50oC until constant weight.
Apparatus
TLC was performed with silica gel GF254 (40-63 µm, Merck) with CHCl3-MeOH-H2O (65:35:10; lower layer) as
solvent. Spots were detected by spraying with 10% H2SO4 in EtOH followed by heating. Column chromatography
(CC) was performed with silica gel (200-300 mesh) Lichroprep (Merck, Darmstadt, Germany).
Preparative HPLC (Prep-HPLC) was performed with LC-8A Shimadzu instrument using a Discovery HS C18
column (25 cm ´ 21.2 mm, 10 mm). Injection volume was 10 µL and the analysis was done at room temperature.
The absorbance was measured at the wavelength of 203 nm for the detection of saponins.
HPLC was performed on a Perkin Elmer series 200 HPLC (Perkin Elmer, Inc., Waltham, MA, USA) system
equipped with Alltech ELSD 2000 (Evaporative Light Scattering Detector; Alltech, Deerfield, IL, USA) and Sunfire
C18 column (250 mm × 4.6 mm. i.d., 5 μm) (Waters Corporation, Milford, MA, USA) to check the purity of isolated
compound. 1H and 13C-NMR spectra were recorded in pyridine-d5 with a Bruker AV-500 spectrometer. Q-TOF-
MS were recorded MicroTOF-QII LC/MS (Bruker Daltonics, Bremen, Germany) spectrometer.
Extraction and isolation
The ethyl acetate fraction (40 g) which was separated from previous study [4]was applied to silica gel CC with
CHCl3-MeOH (50:1; 20:1; 10:1; 8:1; 4:1; 3:1; 2:1: 1:1, v/v) as a mobile solvent system in increasing polarity to

afford ten fractions (1-10). Fraction 3 (3 g) was dissolved in mixture of MeOH and H2O to get precipitate. It was
then crystalized in MeOH-H2O to obtain 50 mg crystals and further purified by silica gelCC with CHCl3–MeOH–
H2O (85:15:0.1, v/v) to yield 25 mg daucosterol (1). Fraction 4 (12 g) was then subjected to silica gel CC and
eluted with EtOAc-MeOH (50:1; 40:1; 30:1; 25:1; 1:1, v/v) to yield 8 sub-fractions (4.1-4.8). Sub-fraction 4.3 (1.29
g) was further fractionated over silica gel CC with EtOAc-MeOH (9:1) to obtain sub-fraction contain a mixture of
ginsenosides-Rk3 and -Rh4 (183 mg). Ginsenoside-Rk3 (2) (36 mg) and ginsenoside-Rh4 (3) (18 mg) were
obtained by prep-HPLC (MeOH-H2O (60:40, v/v), 15 ml/min), respectively. Sub-fraction 4.5 (1.8 g) was further
subjected to silica gel CC with EtOAc–MeOH (25:1, v/v) to yield 8 sub-fractions (4.5.1 − 4.5.8). 20S-ginsenoside-
Rh1 (4) (132 mg) and 20R-ginsenoside-Rh1 (5) (290 mg) were obtained from sub-fraction 4.5.4 (625 mg) followed
by repeated prep-HPLC [MeOH-H2O (65:35, v/v), 15 ml/min]. Pseudo-ginsenoside-PRt4 (6) (400 mg) was
obtained by crystallization in EtOAc-MeOH from subfraction 4.5.7 (500 mg). 20R-ginsenoside-Rg3 (7) (130 mg)
was crystallized in MeOH from fraction 4.8 (1.71 g). The residue (1.54 g) was then separated by silica gel CC
with CHCl3– MeOH–H2O (85:15:0.1, v/v) to give 6 sub-fractions (4.8.1-4.8.6). 20S-ginsenoside-Rg3 (8) (30 mg)
was obtained from subfraction 4.8.4 by prep-HPLC [ACN-H2O (44:56, v/v), 15 ml/min]. Fraction 7 (1.76 g) was
then subjected to silica gel CC with EtOAc-MeOH (50:1; 30:1; 20:1; 10:1; 5:1; 1:1, v/v) to obtain seven sub-
fractions (7.1-7.7). Sub-fraction 7.4 (138 mg) was then purified by prep-HPLC [ACN-H2O (40:60, v/v), 10 mL/min]
to afford notoginsenoside-R2 (9) (18 mg) which was crystallized in MeOH-H2O.
3. Results and Discussion
3.1. Isolation of ginsenosides by prep-HPLC
The ethyl acetate extract (40 g) was chromatographed on silica gel column to obtain 10 fractions. Among these
fractions, fractions 4 and 7 were selected for further isolation of ginseng saponin by silica gelcolumn,
crystallization, and prep-HPLC to afford three pairs of isomers G-Rk3 (2, 36 mg), G-Rh4 (3, 18 mg); 20S-
ginsenoside-Rh1 (4, 132 mg) and 20R-ginsenoside-Rh1 (5, 290 mg); 20R-ginsenoside-Rg3 (7, 130 mg) and 20S-
ginsenoside-Rg3 (8, 30 mg) together with daucosterol (1, 25 mg), pseudoginsenoside-Rt4 (6, 400 mg), and
notoginsenoside-R2 (9, 18 mg). Prep-HPLC chromatograms of those ginsenosides are shown in Fig. 1
.

3.2.Structure and elucidation
Compound 1 was obtained as colorless crystal. The 1H NMR spectrum showed an olefinic proton signal at d 5.33
(d, J=18.0 Hz, H-6) and six methyl signals as follows 0.66 (3H, s, Me-18), 0.94 (3H, s, Me-19), 0.97 (3H, d, J=6.3
Hz, Me-21), 0.87 (3H, d, Me-26), 0.85 (3H, d, Me-27), and 0.87 (3H, m, Me-29). The doublet at d 5.06 (d, J=7.7
Hz) for anomeric proton signal. The 13C NMR spectrum showed the presence of 35 carbon atoms in the
structure. Among these, 6 carbon signals were glycosidic group corresponding to a hexose moiety, 29 carbon
signals for an aglycone moiety corresponding to sitosterol. These data and comparison with those of literature
[5]suggested that compound 1 was daucosterol.
The structures of compound 2-6 were determined as ginsenosides-Rk3 (2), ginsenoside-Rh4 (3), 20S-
ginsenoside-Rh1 (4), 20R-G-Rh1 (5), and pseudoginsenoside-Rt4 (6), respectively, on their basis of ESI-MS, 1H
and 13C NMR spectra data, and comparison with those reported in the literature [6,7].
Compound 7 was obtained as a white amorphous powder. It showed pseudo-ion peak [M+Na]+ at m/z807.4 in the
positive ESI-MS and [M-H]- at m/z 783.4 in the negative ESI-MS which were attributable to the molecular formula
C42H72O13. The 1H-NMR spectrum showed eight methyl signals at dH 0.76, 0.94, 0.97, 1.06, 1.23, 1.36, 1.62, 1.64
(each, 3H, s) and two anomeric proton signals due to two β-glucosidic linkages at δH 4.93(1H, d, J = 7.5 Hz, H-1'
of glucose at C-3 of aglycone) and at δH 5.36 (1H, d, J = 7.5 Hz, H-1″ of glucose at C-3 of aglycone).
Compound 8 was obtained as a white amorphous powder. It showed pseudo-ion peak [M+Na]+ at m/z807.4 in
the positive ESI-MS and [M-H]- at m/z 783.4 in the negative ESI-MS which were attributable to the molecular
formula C42H70O12. 1H-NMR spectrum showed eight methyl signals at dH 0.79, 0.94, 0.95, 1.09, 1.28, 1.41, 1.61,
1.63 (each, 3H, s) and two anomeric proton signals due to two β-glucosidic linkages at δH 4.91 (1H, d, J = 7.74
Hz, H-1' of glucose at C-3 of aglycone) and at δH 5.36 (1H, d, J = 7.8 Hz, H-1″ of glucose at C-3 of aglycone).
13C-NMR spectra of compound 7 and 8 are corresponded to that in the literature for the structure of ginsenoside
Rg3 [7]. Moreover, it was reported that the chemical shifts of neighbouring carbons to C-20 could be used to
clearly distinguish between 20R and 20S isomers [7]. The chemical shifts of the C-17, C-21, and C-22 signals in
the 13C-NMR spectrum of compound 7 were 50.6, 22.8, and 43.3; and compound8 were 54.8, 27.1, and 39.5,
respectively. 1H-NMR and 13C-NMR data of compound 7 and 8 were in accordance to those in the literature for
the structure of 20R- and 20S-ginsenoside- Rg3, respectively (Table 1). Therefore, the structure of
compound 7 was elucidated as 20R-ginsenoside-Rg3 and compound8 was elucidated as 20S-ginsenoside-Rg3.
Compound 9 was obtained as colorless needles. It showed [M+Na]+ peak at m/z 793 in the positive EI-MS
spectrum and [M-H]- peak at m/z 769 in the negative EI-MS spectrum which attributed to the molecular formula of
C41H70O13. 1H-NMR spectrum showed 8 methyl signals at dH 0.77, 0.93, 1.13, 1.31, 1.43, 1.60, 1.63, and 2.05
(each, 3H, s) and one olefinic proton signal at 5.31. Moreover, there were 2 anomeric proton signals due to two
β-glucosidic linkages at 4.91 (1H, d, J = 7.0 Hz) and at 5.75 (1H, d, J = 7.0 Hz). Two olefinic carbon signals at
δC 126.3 and 130.7 suggested one double bond in the structure of this compound. 1H-NMR and 13C-NMR of
compound 9 were in accordance for the structure of notoginsenoside-R2 (Table 1) [8]. Therefore,
compound 9 was identified as notoginsenoside-R2. Structures of isolated ginseng saponins are shown in Fig. 2.
Saponin
Backbone
R1
R2
Ginsenoside-Rk3 (2)
A-(d)
-H
-OGlc
Ginsenoside-Rh4 (3)
A-(c)
-H
-OGlc
20S-Ginsenoside-Rh1 (4)
A-(a)
-H
-OGlc
20R-Ginsenoside-Rh1 (5)
A-(b)
-H
-OGlc

24S-Pseudoginsenoside-Rt4 (6)
B
-Glc
-H
20S-Ginsenoside-Rg3 (7)
A-(a)
-Glc6-Glc
-H
20R-Ginsenoside-Rg3 (8)
A-(b)
-Glc6-Glc
-H
Notoginsenoside-R2 (9)
A-(a)
-H
-Glc-Xyl
Figure 2. Structures of saponins isolated from VG
Table 1. 1H-NMR and 13C-NMR spectroscopic data of the isolated ginsenosides (δ ppm, in pyridine-d5)
No. C
20R-G-Rg3 (7)
20S-G-Rg3 (8)
N-R2 (9)
δ 13C
δ 1H (500 MHz)
δ 13C
δ 1H (600 MHz)
δ 13C
δ 1H (500 MHz)
1
39.1
1.48; 0.73
39.1
1.47; 0.74
39.4
0.96; 1.60
2
26.6
2.17; 1.81
26.7
2.18; 1.80
26.8
1.30; 1.74
3
88.9
3.28 (1H, dd, J=4.1; 11.7 Hz)
88.9
3.28 (1H, dd, J=4.1; 11.7 Hz)
78.7
3.45 (1H, dd, J=5.0, 11.5 Hz)
4
39.7
39.7
40.1
5
56.4
0.67 (1H, d, J=11.5)
56.4
0.67 (1H, d, J=11.46)
61.3
1.35
6
18.5
1.51; 1.38
18.4
1.48
79.4
4.32
7
35.2
1.46; 1.23
35.2
1.44; 1.21
45.0
2.37; 1.91
8
40.0
40.0
41.1
9
50.4
1.38
50.4
1.38
50.1
1.50
10
36.9
36.9
39.6
11
31.4
1.65; 1.05
31.3
1.57; 1.02
32.1
1.48; 2.09
12
70.9
3.91
71.0
3.90
71.0
3.86
13
49.2
2.00
48.6
2.02
48.2
1.99
14
51.8
51.7
51.6
15
32.2
2.02; 1.55
32.1
2.02; 1.56
31.2
1.58; 1.10
16
26.7
1.91; 1.35
26.8
1.89; 1.41
27.7
1.78; 1.84
17
50.6
2.39
54.8
2.35
54.7
2.27
18
15.8
0.97(3H, s)
16.6
0.94 (3H, s)
17.3
1.13 (s)
19
16.4
0.81 (3H, s)
15.8
0.79 (3H, s)
17.6
0.93 (s)
20
73.0
72.9
73.0
21
22.8
1.38 (3H, s)
27.1
1.41 (3H, s)
26.9
1.37 (s)
22
43.3
1.71
35.9
2.02; 1.68
35.7
2.00; 1.64
23
22.6
2.53; 1.38
23.0
2.59; 2.28
22.9
2.57; 2.25
24
126.1
5.31 (1H, t, J=6.68)
126.7
5.30 (1H, t, J=7.11)
126.3
5.31 (t)
25
130.8
130.7
130.7
26
25.8
1.67 (3H, s)
25.ư8
1.63 (3H, s)
25.5
1.63 (s)
27
17.3
1.64 (3H, s)
17.7
1.61 (3H, s)
17.6
1.60 (s)
28
28.1
1.29 (3H, s)
28.1
1.28 (3H, s)
31.7
2.05 (s)
29
16.5
1.11 (3H, s)
16.3
1.09 (3H, s)
16.7
1.43 (s)
30
17.6
1.00 (3H, s)
17.0
0.95 (3H, s)
16.8
0.77 (s)
Glc-1’
105.1
4.93 (1H, d, J=7.5 Hz)
105.1
4.91 (1H, d, J=7.74 Hz)
103.5
4.91 (1H, d, J =7 Hz)
2
83.5
4.25
83.5
4.23
80.2
4.36
3
78.0
4.24
78.4
4.30
78.8
4.13
4
71.6
4.14
71.6
4.12
71.7
4.15
5
78.3
3.91
78.1
3.92
79.3
3.83
6
62.9
4.55; 4.33
62.9
4.53; 4.32
62.9
4.46 (dd, J = 2, 11.5 Hz); 4.28
Glc-1’’
106.1
5.36 (1H, d, J= 7.5 Hz)
106.1
5.36 (1H, d, J = 7.8 Hz)

2
77.2
4.12
77.2
4.11
3
78.3
4.29
78.2
4.30
4
71.7
4.31
71.7
4.32
5
78.1
3.93
78.0
3.92
6
62.7
4.48 (2H)
62.7
4.46; 4.32
Xyl-1
104.8
5.75 (1H, d, J = 7 Hz)
2
75.8
4.16
3
79.8
4.34
4
71.2
4.23
5
67.2
4.30; 3.63 (t)
Beside the conventional methods for isolation of natural compounds, prep-HPLC recently becomes the most
frequently used technique for isolation and purification of ginsenosides to obtain the highest purity, in particular
isomer ginsenosides [9]. Steaming process has been shown to change saponin constituents of Vietnamese
ginseng. The polar PPD (protopanaxadiol) and PPT (protopanaxatriol) ginsenosides such as ginsenosides-Rb1,-
Rd, and -Rg1 were converted to less polar ginsenosides such as G-Rk1, G-Rk3, G-Rh1, G-Rh4, G-Rg3, and G-
Rg5 during the heating process [2]. The formation of 20R-ginsenoside-Rg3 and its 20S isomer come from the C-
20 deglycosylation of polar PPD ginsenosides during heating process [2, 10]. 20R-ginsenoside-Rg3 is sparingly
soluble in methanol and easily soluble in CHCl3-MeOH-H2O in appropriate proportion while its isomer, 20S-G-
Rg3 is soluble in methanol, ethanol. This physicochemical property may be used in isolation and purification of
this 20R isomer. Prep-HPLC was shown to be an efficient method for the separation of four pairs of isomer from
processed VG including ginsenoside-Rk3and G-Rh4; 20S-G-Rh1 and 20R-G-Rh1; 20R-G-Rg3 and 20S-G-Rg3; and
G-Rk1 and G-Rg5 (reported in our previous study [4]).
According to the literature, notoginsenoside-R2, a PPT-type ginsenoside isolated from P. notoginseng, had not
been isolated from VG. Furthermore, it is not an artifact formed by steaming process [11]. Thus, it can be
concluded that notoginsenoside R2 was first isolated in VG.
4. Conclusion
Our study demonstrated that prep-HPLC is an efficient and simple method for the isolation and purification of not
only ginseng saponins but also ginsenoside isomers which are difficult to separate by normal chromatographic
methods. In the study, eight saponins and daucosterol were isolated and identified. Among them, 20R-
ginsenoside-Rg3 and 20S-ginsenosides-Rg3, daucosterol and notoginsenoside-R2 were first isolated from VG.
References
1. Le T. H. V., Lee S. Y., Lee G. J., Nguyen N. K., Park J. H., Nguyen M. D. (2015), Effects of steaming on
saponin compositions and antiproliferative activity of Vietnamese ginseng, Journal of Ginseng Research,
2015;39(3), 274-8. 2. Le T. H. V., Lee S. Y., Kim T. R., Kim J. Y., Kwon SW, Nguyen NK, et al. (2014), Processed
Vietnamese ginseng: Preliminary results in chemistry and biological activity. Journal of Ginseng Research,
38(2),154-9. 3. Le T. H. V., Nguyen N. K., Nguyen M. D. (2011), Isolation of ginsenoside-Rh1 in higher yield from
process Vietnamese ginseng, Journal of Medicinal Plants, 16(3), 187-93. 4. Le T. H. V., Nguyen T. D. N. N. K.,
Park J. H., Nguyen M. D. (2015), Ginsenoside-Rk1 and ginsenoside-Rg5 isolated from processed Vietnamese
ginseng, Journal of Medicinal Plants, 20(3), 149-55. 5. Sultana N., Afolayan A. J. (2007), A novel daucosterol
derivative and antibacterial activity of compounds from Arctotis arctotoides, Natural Product Research, 21(10),
889-96. 6. Nguyen M. D., Kasai R., Ohtani K., Ito A., Nguyen T. N., Yamasaki K., et al. (1993), Saponins from
Vietnamese ginseng, Panax vietnamensis Ha et Grushv. Collected in central Vietnam. I. Chemical and
Pharmaceutical Bulletin, 41(11), 2010-4. 7. Yang H., Kim J. Y., Kim S. O., Yoo Y. H., Sung S. H. (2014),
Complete 1H-NMR and 13C-NMR spectral analysis of the pairs of 20(S) and 20(R) ginsenosides, Journal of
Ginseng Research, 38(3):194-202. 8. Teng H. L., Chen J., Wang D., He Y., Yang C. (2002), Complete
assignment of 1H and 13C NMR data for nine protopanaxatriol glycosides, Magnetic Resonance in Chemistry, 40,
483-8. 9. Kim I. W., Sun W. S., Yun B. S., Kim N. R., Min D., Kim S. K. (2013), Characterizing a full spectrum of
physico-chemical properties of (20S)- and (20R)-ginsenoside Rg3 to be proposed as standard reference
materials, Journal of Ginseng Research, 37(1), 124-34. 10. Park I. H., Kwon S. W., Lee Y. J., Cho S. Y., Park M.
K., Park J. H. (2002), Cytotoxic dammarane glycosides from processed
ginseng, Chemical and Pharmaceutical Bulletin,50(4), 538-40. 11. Yang W. Z., Hu Y., Wu W. Y., Ye M., Guo D.
A. (2014), Saponins in the genus Panax L. (Araliaceae): a systematic review of their chemical
diversity, Phytochemistry, 106:7-24.