Acylated protopanaxadiol-type ginsenosides from the root of Panax ginseng

Article abstract of DOI:10.1002/cbdv.201000196

Six new protopanaxadiol-type ginsenosides, named ginsenosides Ra ₄-Ra₉ (1-6, resp.), along with 14 known dammarane-type triterpene saponins, were isolated from the root of Panax ginseng, one of the most important Chinese medicinal herbs. The structures of the new compounds were determined by spectroscopic methods, including 1D- and 2D-NMR, HR-MS, and chemical transformation as (20S)- 3-O-{β-D-6-O-[(E)-but-2-enoyl] glucopyranosyl-(1→2)-β-D-glucopyranosyl}-20-O-[β-D-xylopyranosyl- (1→4)-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranosyl] protopanaxadiol (1), (20S)-3-O-[β-D-6-O-acetylglucopyranosyl-(1→2)- β-D-glucopyranosyl]-20-O-[β-D-xylopyranosyl-(1→4) -α-L-arabinopyranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (2), (20S)-3-O-{β-D-6-O-[(E)-but-2-enoyl]glucopyranosyl-(1→2)-β- D-glucopyranosyl}-20-O-[β-D-glucopyranosyl-(1→6)-β-D- glucopyranosyl]protopanaxadiol (3), (20S)-3-O-{β-D-6-O-[(E)-but-2-enoyl] glucopyranosyl-(1→2)-β-D-glucopyranosyl}-20-O-[α-L- arabinopyranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (4), (20S)-3-O-{β-D-4-O-[(E)-but-2-enoyl]glucopyranosyl-(1→2) -β-D-glucopyranosyl}-20-O-[α-L-arabinofuranosyl-(1→6) -β-D-glucopyranosyl]protopanaxadiol (5), (20S)-3-O-{β-D-6-O-[(E)-but- 2-enoyl]glucopyranosyl-(1→2)-β-D-glucopyranosyl}-20-O-[α-L- arabinofuranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (6). The sugar moiety at C(3) of the aglycone of each new ginsenoside is butenoylated or acetylated. Copyright

Full text of DOI:10.1002/cbdv.201000196

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
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Acylated Protopanaxadiol-Type Ginsenosides from the Root of Panax
ginseng
by Guo-Yuan Zhu, Ying-Wei Li, Desmond Kwok-Po Hau, Zhi-Hong Jiang, Zhi-Ling Yu, and
Wang-Fun Fong*
Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist
University, Kowloon, Hong Kong SAR, P. R. China
(phone: þ852-34112928; fax: þ852-34112902; e-mail: wffong@hkbu.edu.hk)
Six new protopanaxadiol-type ginsenosides, named ginsenosides Ra4 –Ra9 (1–6, resp.), along with
14 known dammarane-type triterpene saponins, were isolated from the root of Panax ginseng, one of the
most important Chinese medicinal herbs. The structures of the new compounds were determined by
spectroscopic methods, including 1D- and 2D-NMR, HR-MS, and chemical transformation as (20S)-
3-O-{b-d-6-O-[(E)-but-2-enoyl]glucopyranosyl-(1!2)-b-d-glucopyranosyl}-20-O-[b-d-xylopyrano-
syl-(1!4)-a-l-arabinopyranosyl-(1!6)-b-d-glucopyranosyl]protopanaxadiol (1), (20S)-3-O-[b-d-6-O-
acetylglucopyranosyl-(1!2)-b-d-glucopyranosyl]-20-O-[b-d-xylopyranosyl-(1!4)-a-l-arabinopyrano-
syl-(1!6)-b-d-glucopyranosyl]protopanaxadiol (2), (20S)-3-O-{b-d-6-O-[(E)-but-2-enoyl]glucopyra-
nosyl-(1!2)-b-d-glucopyranosyl}-20-O-[b-d-glucopyranosyl-(1!6)-b-d-glucopyranosyl]protopanaxa-
diol (3), (20S)-3-O-{b-d-6-O-[(E)-but-2-enoyl]glucopyranosyl-(1!2)-b-d-glucopyranosyl}-20-O-[a-l-
arabinopyranosyl-(1!6)-b-d-glucopyranosyl]protopanaxadiol (4), (20S)-3-O-{b-d-4-O-[(E)-but-2-
enoyl]glucopyranosyl-(1!2)-b-d-glucopyranosyl}-20-O-[a-l-arabinofuranosyl-(1!6)-b-d-glucopyrano-
syl]protopanaxadiol (5), (20S)-3-O-{b-d-6-O-[(E)-but-2-enoyl]glucopyranosyl-(1!2)-b-d-glucopyra-
nosyl}-20-O-[a-l-arabinofuranosyl-(1!6)-b-d-glucopyranosyl]protopanaxadiol (6). The sugar moiety
at C(3) of the aglycone of each new ginsenoside is butenoylated or acetylated.
Introduction. – Panax ginseng C. A. Meyer (Araliaceae) is a well-known oriental
medicinal plant that has been used as a general tonic for thousands of years. Previous
phytochemical studies on P. ginseng led to the isolation of ginsenosides (triterpenoid
saponin glycosides) [1–5], polyacetylenes [6][7], sesquiterpenoids [8], flavonoids [9],
and polysaccharides [10]. Protopanaxadiol- and protopanaxatriol-type ginsenosides
are the major biologically active components of P. ginseng that contribute to its diverse
pharmacological effects on the central nervous system, cardiovascular system,
endocrine secretion, immune-modulation, metabolism, stress, aging, and cancer [11–
13]. Due to the continued interests in the chemical diversity and structureꢀbioactivity
relationship of ginsenosides [14][15], we reinvestigated the root of P. ginseng and
obtained six new acylated protopanaxadiol-type ginsenosides, 1–6, and 14 known
protopanaxadiol-type ginsenosides. Here, we report the isolation and structure
elucidation of these new acylated protopanaxadiol-type ginsenosides.
Results and Discussion. – The concentrated 70% EtOH extract of P. ginseng root
was sequentially partitioned with petroleum ether/H2O, AcOEt/H2O, and BuOH/H2O.
The BuOH-soluble fraction was subjected to a series of column chromatography on
ꢀ 2011 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
silica gel, macroporous resin, and reversed-phase RP18. After purification by high-
performance liquid chromatography (HPLC), six new acylated protopanaxadiol-type
ginsenosides, 1–6, along with 14 known ginsenosides, i.e., ginsenosides Ra1 [1], Ra2 [1],
Rb1 [3], Rb2 [4], Rb3 [16], Rc [16], Rd [17], Rs1 [5], Rs2 [5], malonylginsenoside Rb1
[4], gypenoside XVII [17], pseudoginsenoside RC1 [18], quinquenoside R1 [19],
vinaginsenoside R16 [20], were identified. Known compounds were identified by
comparing their MS and NMR-spectroscopic properties with published data.
Compound 1 was isolated as a white amorphous powder with a molecular formula
C62H102O27 determined by HR-ESI-MS spectrum (m/z 1277.6519 ([MꢀH]ꢀ ). The IR
spectrum of 1 showed strong absorption bands at 3392 and 1078 cmꢀ1, suggesting an
oligoglycosidic structure, together with absorption bands at 1716 and 1654 cmꢀ1 due to
an a,b-unsaturated ester C¼O and olefin group. The UV spectrum showed significant
absorptions at 210 and 253 nm, which also indicated the presence of a,b-unsaturated
1
ester. The H-NMR spectrum of 1 (Table 1) displays signals due to eight tertiary Me
groups at d(H) 0.82–1.65, an olefin at d(H) 5.29, and five anomeric H-atoms at d(H)
4.89 (d, J¼7.2, HꢀC(1’)), 5.32 (d, J¼7.6, HꢀC(1’’)), 5.10 (d, J¼8.0, HꢀC(1’’’’)), 4.92 (d,
J¼6.8, HꢀC(1’’’’’)), and 5.09 (d, J¼8.0, HꢀC(1’’’’’’)). In the 13C-NMR spectrum of 1, 30
C-atom signals assigned to the aglycone and five sets of signals due to three b-
glucopyranosyl, one a-arabinopyranosyl, and one b-xylopyranosyl groups were observed
(Table 2). Comparison of the 13C-NMR chemical shifts of the aglycone moiety in 1 with
those of (20S)-protopanaxadiol and its glycosides [1][3][21] suggests that 1 is a (20S)-

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
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protopanaxadiol-type ginsenoside, which was confirmed by HMBC and NOESY
analysis (Figs. 1 and 2). On comparison of the 1H- and 13C-NMR signals of 1 with those
of ginsenoside Ra1 (7) [1], an additional set of signals due to a butenoyl unit at d(C)
166.7, 123.3, 144.8, and 17.8, and d(H) 5.96 (dd, J¼15.6, 1.6, HꢀC(2’’’)), 7.03 (dq, J¼
15.6, 6.8, HꢀC(3’’’)), and 1.64 (d, J¼6.8, Me(4’’’)) were observed. Based on the typical
AB-system coupling constant (J¼15.6) of HꢀC(2’’’) and HꢀC(3’’’), the configuration
of the butenoyl unit was determined as (E). HMBC Correlation between HꢀC(6’’)
(d(H) 5.00 and 4.88) and C(1’’’) (d(C) 166.7) as well as a downfield shift for C(6’’) (d(C)
64.4) of the glucosyl unit indicates that the butenoyl group is attached to C(6’’) of
terminal glucopyranosyl of disaccharide moiety linked at C(3) of the aglycone. The
linkage sites and sequences of the sugars and of the aglycone were determined by
HMBC long-range correlations HꢀC(1’)/C(3), HꢀC(1’’)/C(2’), HꢀC(1’’’’)/C(20),
HꢀC(1’’’’’)/C(6’’’’), and HꢀC(1’’’’’’)/C(4’’’’’). Furthermore, the alkaline hydrolysis of 1
with MeONa liberated ginsenoside Ra1 (7) which confirmed that 1 is an acylated
Fig. 1. Selected 1H,1H-COSY (—) and HMBC (H!C) correlations of compound 1
Fig. 2. Selected NOESY correlations of compound 1

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Table 2. 13C-NMR (100 MHz, C5D5N) Chemical Shift Assignments for Compounds 1–6
1
2
3
4
5
6
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
C(7)
C(8)
39.3
26.7
89.3
39.8
56.5
18.6
35.2
40.1
50.3
37.0
30.9
70.2
49.6
51.4
30.8
26.9
51.5
16.3
16.1
83.5
22.3
36.2
23.2
39.2
26.7
89.3
39.8
56.5
18.5
35.2
40.1
50.3
37.0
30.9
70.2
49.6
51.4
30.8
26.9
51.5
16.3
16.1
83.5
22.3
36.2
23.2
39.3
26.7
89.3
39.8
56.5
18.6
35.3
40.1
50.3
37.0
30.8
70.3
49.6
51.5
30.8
26.9
51.7
16.4
16.1
83.6
22.5
36.3
23.3
39.3
26.7
89.2
39.8
56.5
18.6
35.2
40.1
50.3
37.0
30.8
70.2
49.5
51.5
30.8
26.9
51.7
16.3
16.1
83.6
22.4
36.2
23.3
39.2
26.7
89.1
39.7
56.4
18.5
35.2
40.0
50.2
36.9
30.8
70.3
49.5
51.5
30.7
26.7
51.7
16.3
16.1
83.4
22.4
36.2
23.2
39.2
26.7
89.2
39.8
56.5
18.6
35.2
40.1
50.2
37.0
30.8
70.3
49.5
51.5
30.7
26.9
51.7
16.3
16.0
83.4
22.4
36.2
23.2
C(9)
C(10)
C(11)
C(12)
C(13)
C(14)
C(15)
C(16)
C(17)
Me(18)
Me(19)
Me(20)
Me(21)
C(22)
C(23)
C(24)
C(25)
Me(26)
Me(27)
Me(28)
Me(29)
Me(30)
126.0
131.2
25.9
17.9
28.0
16.6
17.5
126.0
131.2
25.9
17.9
28.0
16.5
17.5
126.0
131.1
25.9
18.0
28.1
16.6
17.5
126.0
131.2
25.9
17.9
28.0
16.6
17.4
126.1
131.0
25.8
17.9
28.1
16.7
17.4
126.1
131.1
25.8
17.9
28.0
16.6
17.4
3-Glc
105.0
84.4
78.5
71.5
3-Glc
105.0
84.4
78.5
71.5
3-Glc
105.0
84.5
78.6
71.5
3-Glc
105.0
84.4
78.6
71.5
3-Glc
105.0
83.2
78.4
71.7
3-Glc
105.0
84.4
78.6
71.5
C(1’)
C(2’)
C(3’)
C(4’)
C(5’)
C(6’)
78.2
62.9
78.2
62.9
78.0
62.9
78.0
62.9
78.2
62.9
78.0
62.9
2’-Glc
106.3
76.9
78.0
71.0
2’-Glc
106.3
76.8
78.0
71.1
2’-Glc
106.3
76.9
78.2
71.0
2’-Glc
106.3
76.8
78.2
71.0
2’-Glc
105.7
77.1
75.4
72.4
2’-Glc
106.3
76.9
78.2
71.0
C(1’’)
C(2’’)
C(3’’)
C(4’’)
C(5’’)
C(6’’)
75.6
64.4
75.5
64.8
75.6
64.5
75.6
64.4
76.3
62.2
75.6
64.4
6’’-Bu
166.7
123.3
144.8
17.8
6’’-Ac
171.1
21.0
6’’-Bu
166.7
123.3
144.8
17.9
6’’-Bu
166.7
123.3
144.8
17.8
4’’-Bu
166.1
123.2
145.2
17.8
6’’-Bu
166.7
123.3
144.7
17.8
C(1’’’)
C(2’’’)
C(3’’’)
Me(4’’’)

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Table 2 (cont.)
1
2
3
4
5
6
20-Glc
20-Glc
98.1
74.9
79.4
71.9
20-Glc
98.2
74.9
79.3
71.6
20-Glc
98.2
75.0
79.3
71.8
20-Glc
98.1
75.1
79.3
72.2
76.6
68.5
20-Glc
98.2
75.1
79.3
72.2
76.6
68.6
C(1’’’’)
C(2’’’’)
C(3’’’’)
C(4’’’’)
C(5’’’’)
C(6’’’’)
98.1
74.9
79.4
71.9
76.9
69.9
76.9
69.8
77.1
70.3
76.9
69.3
6’’’’-Ara(p)
105.2
73.0
74.0
78.8
6’’’’-Ara(p)
105.2
73.0
74.0
78.8
6’’’’-Glc
105.4
75.3
78.5
71.8
6’’’’-Ara(p)
104.7
72.2
74.2
68.7
6’’’’-Ara(f)
110.2
83.4
78.9
86.0
6’’’’-Ara(f)
110.2
83.4
78.9
86.0
C(1’’’’’)
C(2’’’’’)
C(3’’’’’)
C(4’’’’’)
C(5’’’’’)
C(6’’’’’)
65.8
65.8
78.4
62.9
65.7
62.7
62.7
4’’’’’-Xyl
107.0
75.5
78.6
71.1
4’’’’’-Xyl
107.1
75.4
78.6
71.1
C(1’’’’’’)
C(2’’’’’’)
C(3’’’’’’)
C(4’’’’’’)
C(5’’’’’’)
67.4
67.4
ginsenoside Ra1. All signals of the 1H- and 13C-NMR were assigned based on 2D-NMR
data including COSY, HSQC, HMBC, and NOESY. On the basis of the above results,
the structure of 1 was determined as (20S)-3-O-{b-d-6-O-[(E)-but-2-enoyl]glucopyr-
anosyl-(1!2)-b-d-glucopyranosyl}-20-O-[b-d-xylopyranosyl-(1!4)-a-l-arabino-
pyranosyl-(1!6)-b-d-glucopyranosyl]protopanaxadiol, and named ginsenoside Ra4 .
Compound 2 was obtained as a white amorphous powder and showed absorption
bands at 3392, 1739, 1636, and 1079 cmꢀ1 due to OH, ester, and olefin groups in its IR
spectrum. The molecular formula C60H99O27 was verified from its negative-ion-mode
HR-ESI-MS (m/z 1251.6356 ([MꢀH]ꢀ )). The alkaline hydrolysis of 2 with MeONa
also yielded ginsenoside Ra1 suggesting that 2 is also an acylated ginsenoside Ra1. The
1H- and 13C-NMR spectra (Tables 1 and 2) of 2 are very similar to those of 1 and
ginsenoside Ra1 [1], except for the signals due to an Ac group at d(C) 171.1 and 21.0,
and d(H) 2.03 (s, 3 H), which suggest that 2 is an acetyl derivative of ginsenoside Ra1.
The location of Ac group was assigned to C(6’’) of terminal glucopyranosyl moiety
linked at C(3) of the aglycone on the basis of an acetylation shift effect on C-atom
signals around C(6’’) (d(C) 64.8) and HMBC correlation between HꢀC(6’’) (d(H) 4.93
and 4.78) and C(1’’’) (d(C) 171.1). Thus, the structure of 2 was determined as (20S)-3-
O-[b-d-6-O-acetylglucopyranosyl-(1!2)-b-d-glucopyranosyl]-20-O-[b-d-xylopyrano-
syl-(1!4)-a-l-arabinopyranosyl-(1!6)-b-d-glucopyranosyl]protopanaxadiol,
and
named ginsenoside Ra5.
Compound 3 was isolated as a white amorphous powder. Its molecular formula was
established as C58H96O24 on the basis of its HR-ESI-MS (m/z 1175.6223 ([MꢀH]ꢀ ),
1221.6277 ([MþHCOO]ꢀ )). The alkaline hydrolysis of 3 yielded ginsenoside Rb1

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
suggesting that 3 is an acylated ginsenoside Rb1. The 1H- and 13C-NMR data (Tables 1
and 2) of 3 were found to be identical to those of ginsenoside Rb1 [3], except for the
signals ascribed to a (E)-butenoyl group at d(C) 166.7, 123.3, 144.8, and 17.9, and d(H)
5.96 (br. d, J¼15.6, HꢀC(2’’’)), 7.03 (dq, J¼15.6, 6.8, HꢀC(3’’’)), 1.65 (Me(4’’’)). The
acyl group was determined to be at C(6’’) on the basis of three-bond HMBC correlation
between HꢀC(6’’) (d(H) 4.87 and 4.99) and C(1’’’) (d(C) 166.7), as well as the acylation
1
shift of the C(6’’) signal. All signals of the H- and 13C-NMR were assigned based on
2D-NMR techniques; thus 3 was determined as (20S)-3-O-{b-d-6-O-[(E)-but-2-
enoyl]glucopyranosyl-(1!2)-b-d-glucopyranosyl}-20-O-[b-d-glucopyranosyl-(1!6)-
b-d-glucopyranosyl]protopanaxadiol and was named ginsenoside Ra6.
The molecular formula of compound 4 was determined as C57H93O23 from the
quasimolecular-ion peaks at m/z 1145.6084 ([MꢀH]ꢀ ) and 1191.6142 ([MþHCOO]ꢀ )
in its negative-ion-mode HR-ESI-MS. The IR spectrum of 4 showed a typical
absorption band at 1716 cmꢀ1 due to an a,b-unsaturated ester group which was
confirmed by UV absorptions at 210 and 253 nm. The alkaline hydrolysis of 4 with
MeONa liberated ginsenoside Rb2, suggesting that 4 is an ester of ginsenoside Rb2. The
1H- and 13C-NMR spectra of 4 (Tables 1 and 2) showed signals assignable to a
ginsenoside Rb2 moiety [4] and a (E)-butenoyl part. This was fully supported by 2D-
NMR data including COSY, HSQC, HMBC, and NOESY. In HMBC spectrum of 4, a
long-range correlation was observed between HꢀC(6’’) (d(H) 4.99 and 4.87) and C(1’’’)
(d(C) 166.7), which suggests that the butenoyl group is at C(6’’) of terminal
glucopyranosyl in C(3)-linked disaccharide moiety. Accordingly, the structure of 4
was determined as (20S)-3-O-{b-d-6-O-[(E)-but-2-enoyl]glucopyranosyl-(1!2)-b-d-
glucopyranosyl]-20-O-[a-l-arabinopyranosyl-(1!6)-b-d-glucopyranosyl]protopanax-
adiol, and was named ginsenoside Ra7.
Compound 5 and 6 have the same molecular formula, C57H94O23, as determined
from their HR-ESI-MS data. The alkaline hydrolysis of 5 and 6 yielded the same deacyl
product ginsenoside Rc, suggesting that 5 and 6 are the ester derivatives of ginsenoside
Rc. Detailed analysis of 1H- and 13C-NMR spectra (Tables 1 and 2) of 5 and 6 revealed
that both compounds possess a ginsenoside Rc part and a (E)-butenoyl moiety, but
there is a difference between the butenoyl moiety linkage position in 5 and 6.
Comparison of the 13C-NMR data (Table 2) of 5 with those of ginsenoside Rc [16]
revealed an esterification downfield shift at C(4’’) (d(C) 72.4) in 5, which suggests that
the butenoyl group is at C(4’’) of the terminal glucopyranosyl of disaccharide moiety
linked at C(3) of the aglycone. This was confirmed by the three-bond correlation
between HꢀC(4’’) (d(H) 5.79 (t, J¼9.6)) and C(1’’’) (d(C) 166.1) in HMBC spectrum of
5. Hence, compound 5 was characterized as (20S)-3-O-{b-d-4-O-[(E)-but-2-enoyl]glu-
copyranosyl-(1!2)-b-d-glucopyranosyl}-20-O-[a-l-arabinofuranosyl-(1!6)-b-d-glu-
copyranosyl]protopanaxadiol, named ginsenoside Ra8. On the other hand, the typical
acylation downfield shift of C(6’’) signal at d(C) 64.4 in13C-NMR spectrum of 6 and
HMBC correlation between HꢀC(6’’) (d(H) 4.99 and 4.86) and C(1’’’) (d 166.7)
suggests that the butenoyl moiety in 6 is at C(6’’) of terminal glucopyranosyl of C(3)-
attached disaccharide. Consequently, the structure of 6 was determined as (20S)-3-O-
{b-d-6-O-[(E)-but-2-enoyl]glucopyranosyl-(1!2)-b-d-glucopyranosyl}-20-O-[a-l-ara-
binofuranosyl-(1!6)-b-d-glucopyranosyl]protopanaxadiol, which is named ginseno-
side Ra9.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
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Compared to malonyl ginsenosides from P. ginseng [22], ginsenosides Ra4 –Ra9 (1–
6, resp.) reported in this article are very minor acylated ginsenosides. Occurrence of
these minor components illustrates the chemical diversity and complexity of saponins
in P. ginseng.
This project was supported by a Faculty Research Grant of Hong Kong Baptist University. We are
grateful to the State Key Laboratory of Chinese Medicine and Molecular Pharmacology (Shenzhen,
China), Hong Kong Polytechnic University, for the use of their NMR facilities.
Experimental Part
General. Column chromatography (CC): silica gel (SiO2, 200–300 mesh, Qingdao Haiyang Chemical
Group Co., Qingdao, P. R. China), D101 macroporous resin (Tianjin Pesticide Co., Tianjin, P. R. China),
and RP18 (20–45 mm, Merck). TLC: Precoated SiO2 60 F254 (Merck) and RP18 F254 (Merck) reversed-
phase (RP) plates. Semiprep. HPLC: PerkinꢀElmer Series 200 separation system equipped with 253C
diode array detector and YMC-Pack ODS-A semiprep. column (10 mm, 250ꢁ10 mm) with a flow rate of
5.0 ml/min. Optical rotations: Jasco P-1010 polarimeter (Na 589 nm). UV Spectra: Jasco V-530
spectrophotometer; lmax (log e) in nm. IR Spectra (KBr): PerkinꢀElmer Spectrum One FT-IR
spectrometer; ˜n in cmꢀ1. NMR: Bruker DMX-400 NMR spectrometer; d in ppm using the lowest field
signals of (D5)pyridine (1H, d(H) 8.71; 13C, d(C) 149.9) as an internal ref., J in Hz. 2D-NMR (COSY,
HSQC, HMBC, and NOESY): standard Bruker potocols. ESI-MS: Thermo Finnigan LCQ Advantage
mass spectrometer; in m/z. UPLC/HR-ESI-MS: Acquity Ultra Performance LC System (Waters)-Bruker
micrOTOF mass spectrometer.
Plant Material. The root of Panax ginseng C. A. Meyer was collected in the Jilin Province, P. R.
China, in October 2007. Specimens were verified by Prof. Zhong-Zhen Zhao, School of Chinese
Medicine, Hong Kong Baptist University. A voucher specimen (PG-0710) was deposited with the
Research and Development Division, School of Chinese Medicine, Hong Kong Baptist University.
Extraction and Isolation. Air-dried, milled roots of Panax ginseng C. A. Meyer (7.0 kg) were
extracted with 70% EtOH (3ꢁ21 l) under reflux, and the extracts were evaporated to obtain a brown
residue (ca. 1.8 kg). The residue was dissolved in H2O (7 l) and partitioned successively with petroleum
ether (3ꢁ7 l), AcOEt (3ꢁ7 l), and BuOH (3ꢁ7 l) to give a petroleum ether-soluble fraction (110 g),
an AcOEt-soluble fraction (120 g), and BuOH-soluble fraction (185 g), resp. The BuOH extract (180 g)
was separated by CC (SiO2; CHCl3/MeOH 10 :1!3 :7). Various fractions were combined according to
their TLC behavior to obtain nine fractions, Frs. A!I. Fr. D (17 g) was subjected to CC (SiO2; AcOEt/
MeOH/H2O 50 :8 :1; and RP-18; MeCN/H2O 20 :80!40 :60) and finally purified by HPLC (MeCN/
H2O 35 :65!38 :62) to obtain compounds 5 (4.5 mg), 6 (39 mg), ginsenoside Rd (100 mg), ginsenoside
Rs2 (20 mg), pseudoginsenoside RC1 (6 mg), and vinaginsenoside R16 (5.5 mg). Fr. E (10 g) was
repeatedly purified by CC (SiO2; AcOEt/MeOH/H2O 50 :8 :1; and RP-18; MeCN/H2O 20 :80!40 :60),
and HPLC (MeCN/H2O 35 :65) to yield 4 (15 mg), ginsenoside Rs1 (25 mg), and gypenoside XVII
(15 mg). Fr. F (14 g) was subjected to CC (SiO2; AcOEt/MeOH/H2O 50 :10 :1; and RP-18; MeCN/H2O
35 :65) and finally purified by HPLC (MeCN/H2O 30 :70!32 :68) to afford 3 (25 mg), ginsenoside Rc
(350 mg), and quinquenoside R1 (8 mg). Fr. G (15 g) was subjected to CC (D101 macroporous resin;
H2O, 20% EtOH, and 50% EtOH). The 50% EtOH fraction was purified by CC (RP-18; MeCN/H2O
35 :65) and HPLC (MeCN/H2O 3 :7) to give 2 (6.5 mg), ginsenosides Rb2 and Rb3 (90 and 20 mg, resp.).
Fr. H (45 g) was separated by CC (D101 macroporous resin; H2O, 20% EtOH, and 50% EtOH). The
50% EtOH fraction was then purified by CC (RP-18; MeCN/H2O 25 :75!35 :65, MeOH/H2O 65 :35),
and finally by HPLC (MeCN/H2O 27:73 and 38 :62) to afford 1 (8 mg), and ginsenosides Ra1, Ra2, and
Rb1 (45, 40, and 110 mg, resp.). Fr. I (13 g) was separated by CC (D101 macroporous resin; H2O, 20%
EtOH, and 50% EtOH). The 50% EtOH fraction was then purified by CC (ODS; MeCN/H2O 25 :75!
35 :65) and then by HPLC (MeCN/0.1% AcOH 3 :7) to obtain malonylginsenoside Rb1 (10 mg).
Alkaline Hydrolysis of Compounds 1–6. MeOH Solns. of 1–6 (ca. 0.2 mg/ml each) were hydrolyzed
with MeONa (20 mm) for 12 h at r.t. The reaction mixtures of 1a–6a were neutralized with HCOOH

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
(20 mm) and then subjected to UPLC/HR-ESI-MS analysis under the following conditions: column,
Acquity UPLC BEH C18 (2.1ꢁ100 mm, 1.7 mm); solvent, 0.1% HCOOH in MeCN (A) and 0.1%
HCOOH in H2O (B); gradient elution from 10 to 40% A over 6 min at a flow rate of 0.35 ml/min; HR-
ESI-MS (neg.; Nebulizer, 2.0 bar; dry heater, 1808; dry gas, 8.0 l/min; cap., 4000 V; end plate offset,
ꢀ500 V; and collision cell RF, 550.0 Vpp). The deacyl saponins were identified by comparing their
UPLC retention times and HR-MS data with those of authentic samples. Compounds 1a (tR 3.954 min;
HR-ESI-MS: 1209.6266 ([MꢀH]ꢀ )) and 2a (tR 3.948 min; HR-ESI-MS: 1209.6236 ([MꢀH]ꢀ )) were
identical to ginsenoside Ra1 (tR 3.955 min; HR-ESI-MS: 1209.6274 ([MꢀH]ꢀ , C58H97O2ꢀ6 ; calc.
1209.6274). Compound 3a (tR 3.862 min; HR-ESI-MS: 1107.5977 ([MꢀH]ꢀ )) was identified as
ginsenoside Rb1 (tR 3.866 min; HR-ESI-MS: 1107.5956 ([MꢀH]ꢀ , C54H91O2ꢀ3 ; calc. 1107.5957).
Compound 4a (tR 4.078 min; HR-ESI-MS: 1077.5878 ([MꢀH]ꢀ ), 1123.5865 ([MþHCOO]ꢀ )) was
determined as ginsenoside Rb2 (tR 4.072 min; HR-ESI-MS: 1077.5867 ([MꢀH]ꢀ , C53H89O2ꢀ2 ; calc.
1077.5851), 1123.5877 ([MþHCOO]ꢀ , C54H91O2ꢀ4 ; calc. 1123.5906)). Compounds 5a (tR 3.972 min; HR-
ESI-MS: 1077.5873 ([MꢀH]ꢀ ), 1123.5883 ([MþHCOO]ꢀ )) and 6a (tR 3.963 min; HR-ESI-MS:
1077.5854 ([MꢀH]ꢀ ), 1123.5863 ([MþHCOO]ꢀ )) were determined as ginsenoside Rc (tR 3.971 min;
HR-ESI-MS: 1077.5870 ([MꢀH]ꢀ , C53H89O2ꢀ2 ; calc. 1077.5851; and 1123.5876 ([MþHCOO]ꢀ
,
C54H91Oꢀ24 ; calc. 1123.5906).
Ginsenoside Ra4 (¼(20S)-3-O-{b-d-6-O-[(E)-But-2-enoyl]glucopyranosyl-(1!2)-b-d-glucopyra-
nosyl}-20-O-[b-d-xylopyranosyl-(1!4)-a-l-arabinopyranosyl-(1!6)-b-d-glucopyranosyl]protopanaxa-
diol¼(3b,12b)-12-Hydroxy-20-{[b-d-xylopyranosyl-(1!4)-a-l-arabinopyranosyl-(1!6)-b-d-glucopyr-
anosyl]oxy}dammar-24-en-3-yl 2-O-{6-O-[(2E)-But-2-enoyl]-b-d-glucopyranosyl}-b-d-glucopyranoside;
1). Awhite amorphous powder. [a]2D5 ¼ þ15.3 (c¼0.3, MeOH). UV (MeOH): 210 (3.84), 252 (3.43). IR
(KBr): 3392, 2944, 2878, 1716, 1654, 1552, 1445, 1417, 1385, 1312, 1265, 1165, 1078, 1043, 921, 894, 839,
785, 647, 578, 535. 1H- and 13C-NMR: Tables 1 and 2, resp. HR-ESI-MS: 1277.6519 ([MꢀH]ꢀ
,
C62H101Oꢀ27 ; calc. 1277.6536).
Ginsenoside Ra5 (¼(20S)-3-O-[b-d-6-O-Acetylglucopyranosyl-(1!2)-b-d-glucopyranosyl]-20-O-
[b-d-xylopyranosyl-(1 !4)-a-l-arabinopyranosyl-(1 !6)-b-d-glucopyranosyl]protopanaxadiol ¼
(3b,12b)-12-Hydroxy-20-{[b-d-xylopyranosyl-(1!4)-a-l-arabinopyranosyl-(1!6)-b-d-glucopyrano-
syl]oxy}dammar-24-en-3-yl 2-O-(6-O-Acetyl-b-d-glucopyranosyl)-b-d-glucopyranoside; 2). A white
amorphous powder. [a]2D5 ¼ þ9.5 (c¼0.4, MeOH). IR (KBr): 3392, 2943, 2878, 1739, 1637, 1551, 1453,
1
1385, 1369, 1307, 1250, 1202, 1164, 1079, 1041, 920, 892, 841, 786, 648, 624, 575, 535. H- and 13C-NMR:
Tables 1 and 2, resp. HR-ESI-MS: 1251.6356 ([MꢀH]ꢀ , C60H99O2ꢀ7 ; calc. 1251.6379).
Ginsenoside Ra6 (¼(20S)-3-O-{b-d-6-O-[(E)-But-2-enoyl]glucopyranosyl-(1!2)-b-d-glucopyra-
nosyl}-20-O-[b-d-glucopyranosyl-(1!6)-b-d-glucopyranosyl]protopanaxadiol¼(3b,12b)-20-{[6-O-(b-
d-Glucopyranosyl)-b-d-glucopyranosyl]oxy}-12-hydroxydammar-24-en-3-yl 2-O-{6-O-[(2E)-But-2-eno-
yl]-b-d-glucopyranosyl}-b-d-glucopyranoside; 3). A white amorphous powder. [a]2D5 ¼ þ17.6 (c¼0.6,
MeOH). UV (MeOH): 212 (3.83), 252 (2.87). IR (KBr): 3400, 2944, 2879, 1715, 1656, 1553, 1445, 1384,
1314, 1231, 1168, 1076, 923, 892, 838, 627, 579, 535. 1H- and 13C-NMR: Tables 1 and 2, resp. HR-ESI-MS:
1175.6223 ([MꢀH]ꢀ , C58H95O2ꢀ4 ; calc. 1175.6219), 1221.6277 ([MþHCOO]ꢀ , C59H97O2ꢀ6 ; calc.
1221.6274).
Ginsenoside Ra7 (¼ 3-O-{b-d-6-O-[(E)-But-2-enoyl]glucopyranosyl-(1!2)-b-d-glucopyranosyl}-
20-O-[a-l-arabinopyranosyl-(1!6)-b-d-glucopyranosyl]protopanaxadiol¼(3b,12b)-20-{[6-O-(a-l-
Arabinopyranosyl)-b-d-glucopyranosyl]oxy}-12-hydroxydammar-24-en-3-yl 2-O-{6-O-[(2E)-But-2-eno-
yl]-b-d-glucopyranosyl}-b-d-glucopyranoside; 4). A white amorphous powder. [a]2D5 ¼ þ14.0 (c¼0.6,
MeOH). UV (MeOH): 214 (3.83), 252 (3.18). IR (KBr): 3400, 2944, 2878, 1717, 1655, 1552, 1542, 1445,
1384, 1314, 1174, 1078, 921, 889, 863, 839, 780, 647, 579, 536. 1H- and 13C-NMR: Tables 1 and 2, resp. HR-
ESI-MS: 1145.6084 ([MꢀH]ꢀ , C57H93O2ꢀ3 ; calc. 1145.6113), 1191.6142 ([MþHCOO]ꢀ , C58H95O2ꢀ5 ; calc.
1191.6168).
Ginsenoside Ra8 (¼(20S)-3-O-{b-d-4-O-[(E)-But-2-enoyl]glucopyranosyl-(1!2)-b-d-glucopyra-
nosyl}-20-O-[a-l-arabinofuranosyl-(1!6)-b-d-glucopyranosyl]protopanaxadiol¼(3b,12b)-20-{[6-O-
(a-l-Arabinofuranosyl)-b-d-glucopyranosyl]oxy}-12-hydroxydammar-24-en-3-yl 2-O-{4-O-[(2E)-But-2-
enoyl]-b-d-glucopyranosyl}-b-d-glucopyranoside; 5). A white amorphous powder. [a]2D5 ¼ ꢀ19.3 (c¼0.2,
MeOH). UV (MeOH): 211 (3.98), 252 (3.03). IR (KBr): 3392, 2944, 2879, 1717, 1651, 1445, 1384, 1309,

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1863
1267, 1231, 1172, 1078, 1043, 890, 839, 810, 647, 575. 1H- and 13C-NMR: Tables 1 and 2, resp. HR-ESI-MS:
1145.6095 ([MꢀH]ꢀ , C57H93O2ꢀ3 ; calc. 1145.6113), 1191.6127 ([MþHCOO]ꢀ , C58H95O2ꢀ5 ; calc.
1191.6168).
Ginsenoside Ra9 (¼(20S)-3-O-{b-d-6-O-[(E)-But-2-enoyl]glucopyranosyl-(1!2)-b-d-glucopyra-
nosyl}-20-O-[a-l-arabinofuranosyl-(1!6)-b-d-glucopyranosyl]protopanaxadiol¼(3b,12b)-20-{[6-O-
(a-l-Arabinofuranosyl)-b-d-glucopyranosyl]oxy}-12-hydroxydammar-24-en-3-yl 2-O-{6-O-[(2E)-But-2-
enoyl]-b-d-glucopyranosyl}-b-d-glucopyranoside; 6). A white amorphous powder. [a]2D5 ¼ þ0.7 (c¼1.2,
MeOH). UV (MeOH): 217 (3.65), 252 (2.99). IR (KBr): 3392, 2944, 2879, 1710, 1655, 1542, 1445, 1384,
1314, 1233, 1197, 1076, 924, 889, 839, 808, 647, 576, 535. 1H- and 13C-NMR: Tables 1 and 2, resp. HR-ESI-
MS: 1145.6092 ([MꢀH]ꢀ , C57H93O2ꢀ3 ; calc. 1145.6113), 1191.6152 ([MþHCOO]ꢀ , C58H95O2ꢀ5 ; calc.
1191.6168).
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Received July 13, 2010