Polyacetyleneginsenoside-Ro, a novel triterpene saponin from Panax ginseng

Article abstract of DOI:10.1016/S0040-4039(01)02310-3

A new oleanolic acid-derived saponin, polyacetyleneginsenoside-Ro (1), was isolated along with the known ginsenosides-Ro methyl ester (2), -Rf, -Rg1, -Rg2, and gingerglycolipid B from the roots of Panax ginseng C. A. Meyer. The new saponin was identified as a ginsenoside-Ro derivative containing a polyacetylene side chain by spectroscopic means including 1D and 2D NMR, and was found to inhibit the replication of human immunodeficiency virus type 1 (HIV-1) with an IC₅₀ value of 13.4 μg/mL (11.1 μM).

Full text of DOI:10.1016/S0040-4039(01)02310-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 973–977
Polyacetyleneginsenoside-Ro, a novel triterpene saponin from
Panax ginseng
Hongjie Zhang, Zhizhen Lu, Ghee T. Tan, Shengxiang Qiu, Norman R. Farnsworth,
John M. Pezzuto and Harry H. S. Fong*
Program for Collaborative Research in Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago,
833 S. Wood St., Chicago, IL 60612, USA
Received 15 November 2001; accepted 4 December 2001
Abstract—A new oleanolic acid-derived saponin, polyacetyleneginsenoside-Ro (1), was isolated along with the known gin-
senosides-Ro methyl ester (2), -Rf, -Rg1, -Rg2, and gingerglycolipid B from the roots of Panax ginseng C. A. Meyer. The new
saponin was identified as a ginsenoside-Ro derivative containing a polyacetylene side chain by spectroscopic means including 1D
and 2D NMR, and was found to inhibit the replication of human immunodeficiency virus type 1 (HIV-1) with an IC50 value of
13.4 mg/mL (11.1 mM). © 2002 Elsevier Science Ltd. All rights reserved.
The root of Panax ginseng C. A. Meyer (Araliaceae),
commonly known as Asian, Chinese, or Korean gin-
seng, is one of the oldest Chinese herbal medicines,
having been recorded in the Shen Nung Ben Cao Jing in
the 1st century AD.1 Its history, medical uses, biologi-
cal effects and chemistry have been extensively studied,
documented, and reviewed.1–5 In recent years, various
formulations prepared from this plant material have
been marketed, in varying degrees of quality, as dietary
supplements in the USA under the Dietary Supple-
ments Health Education Act (DSHEA) of 1994. A host
of pharmacological properties of Asian ginseng are
generally attributed to triterpene glycosides (saponins),
called ginsenosides. More than 30 ginsenosides have
been isolated and identified. Among them, gin-
senosides-Rb1, -Rb2, -Rc, -Rg1, -Re, and -Rd account
for 90% of the total.6 These compounds, along with the
species specific ginsenoisde Rf, have been employed as
marker compounds for the chemical analysis of P.
ginseng products. As part of our continuing studies on
the chemistry and biology of medicinal plants, we
undertook the development of analytical high perfor-
mance liquid chromatography (HPLC) and liquid chro-
matography-mass spectrometry (LC–MS) methods7,8
for the analysis of ginsenosides, and for post-marketing
quality control (profiling and quantitative analysis) of
commercial ginseng products.9,10 To facilitate these
studies, the commercially unavailable ginsenoside Rf
was isolated from a sample of P. ginseng root.11 During
the course of this phytochemical isolation, a new gin-
senoside, which we named polyacetyleneginsenoside-Ro
(1) (Fig. 1), was obtained, together with the known
ginsenosides Ro methyl ester (2),12 -Rf,13 -Rg1,14
-Rg2,15 and gingerglycolipid B.16 Compound 1 is com-
posed of two sub-structural units: an oleanolic acid-
derived triterpene saponin (ginsenoside) and
a
polyacetylene moiety, both of which had previously
been isolated from P. ginseng as separate entities.17,18
Since polyacetyleneginsenoside-Ro (1) is a new natural
product with an unknown profile of biological activity,
it was subjected to a routine battery of bioassays avail-
able in our laboratory. Selected saponins have demon-
strated anti-HIV activity in cell-based assays,19–21 while
others have been shown to possess inhibitory activity
against the HIV-1 protease.22 Consistent with the
reported antiviral activities of the majority of oleanane-
type saponins, polyacetyleneginsenoside-Ro (1) was
found to inhibit the in vitro replication of HIV-1. In
this paper, we report the isolation and characterization
of this new saponin, as well as its anti-HIV activity in
the HOG.R5 cell line. This reporter cell line was con-
structed specifically for quantitating HIV-1 replication
in the microtiter format, and is based on the transacti-
vation of a stably-integrated HIV-1 LTR-green fluores-
cent protein (GFP) transcription unit by the viral Tat
protein.23 The system was validated and adapted as a
moderately high-throughput procedure for screening
anti-HIV potential natural products.24
Keywords: Panax ginseng; Araliaceae; polyacetyleneginsenoside; HIV.
* Corresponding author. Tel.: +1 (312) 996-5972; fax: +1 (312) 413-
5894; e-mail: hfong@uic.edu
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)02310-3

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H. Zhang et al. / Tetrahedron Letters 43 (2002) 973–977
Figure 1. Structures of compounds 1 and 2.
(d), 70.88 (d), 63.01 (d)], two terminal double-bond
carbons [l 138.73 (d), 115.46 (t)], and two triple-bond
carbons [l 80.03 (s), 77.65 (s), 70.58 (s), 66.64 (s)].
Based on the long-range correlation observed in an
HMBC experiment (Fig. 2), an oxy-methine group was
assigned between the double bond and a triple-bond,
which was coupled to a second triple-bond. The latter
triple-bond was attached to a methylene carbon, and
sequentially connected to two oxy-methine groups. A
polyacetylene unit was, thus, elucidated. Alkaline
hydrolysis of 1 with 3% Na2CO3 at room temperature
for 2 h yielded a polyacetylene which was identified as
the known panaxytriol by comparison with published
spectral data.17,26 The correlation between lH 5.57 (m,
H-10%) and lC 169.53 (s, C-6%%) in the HMBC spectrum
fixed the coupling position of the oleanolic acid triter-
pene saponin and the polyacetylene units of 1. The
assignment of the absolute stereochemical configura-
tions at C-9 and C-10 of panaxytriol had been a subject
of debate, having been assigned as 3R, 9R and 10R27–29
and as 3R, 9S and 10S.30 Recently, Gurjar et al.31
prepared four diasteromers of panaxytriol (3R,9R,10R-;
3S,9R,10R-; 3R,9S,10S- and 3S,9S,10S-) and their
corresponding 9,10-acetonides. All four diasteromers
of panaxytriol showed very similar NMR data to those
of naturally occurring panaxytriol, but only the
3R,9S,10S-panaxytriol exhibited the same optical rota-
tion ([h]D−25.5°)31 as that of the naturally occurr-
ing panaxytriol ([h]D−25.4°).28 The 3S,9R,10R and
Polyacetyleneginsenoside-Ro (1) was isolated as a white
powder from the methanol extract of P. ginseng C. A.
Meyer (10 kg) by sequential column chromatography
on Diaion HP-20 and silica gel, followed by preparative
high performance liquid chromatography (HPLC).25
HRMALDI-TOFMS gave [M+Na]+ at m/z 1239.6670,
corresponding to the molecular formula of
1
C65H100NaO21 (calcd for 1239.6655). Characteristic H
NMR signals for methyl groups [l 1.26 (3H, s), 1.21
(3H, s), 1.10 (3H, s), 1.09 (3H, s), 0.91 (3H, s), 0.87
(6H, s); l 33.18 (q), 28.01 (q), 26.12 (q), 23.69 (q), 17.51
(q), 16.68 (q), 15.61 (q)], in addition to the multi-meth-
1
ylene signals in the upfield region of H and 13C NMR
spectra, revealed 1 to be an oleanolic acid triterpene
derivative (Tables 1 and 2). Three sugar units were
easily discerned from its NMR spectra by observing the
relative lowfield signals of the anomeric protons and
carbons [lH 6.34 (1H, d, J=8.08 Hz), 5.35 (1H, d,
J=7.8 Hz), 4.89 (1H, d, J=7.5 Hz); lC 106. 05 (d),
105.57 (d), 95.80 (d)]. Comparison of the 13C NMR
data of 1 with that of ginsenoside-Ro methyl ester (2)
revealed that the characteristic chemical shifts exhibited
by 2 were also found in 1 (Tables 1 and 2). Thus,
compound 1 was determined to be an oleanolic acid
triterpene saponin with a ginsenoside-Ro unit. The
remaining 13C NMR signals of 1 were found to belong
to a methyl carbon [l 14.31 (q)], seven methylene
carbons [l 32.12 (t), 30.98 (t), 30.00 (t), 29.69 (t), 25.23
(2C, t), 22.98 (t)], three oxy-methine carbons [l 76.94

H. Zhang et al. / Tetrahedron Letters 43 (2002) 973–977
Table 1. 1H NMR data of compound 1 (500 MHz, pyridine-d5, J in Hz)
975
H
1
H
1
H
1
Triterpene aglycone moiety
l
Polyacetylene aglycone moiety
l
Sugar moiety
l
H2-1
H2-2
H-3
1.62 m
1.57 m
3.13 dd (12.1,
4.4)
0.69 brd (11.7)
1.42 m
1.31 m
1.80 m
1.65 overlap
1.97 m
5.48 brs
2.35 m
1.34 m
1.97 m
3.19 brd (13.5)
1.76 m
1.30 m
1.39 m
1.18 m
1.21 s
1.09 s
H-1%a
H-1%b
H-2%
5.64 dq (17.0, 1.5)
5.23 dq (10.2, 1.5)
6.22 ddd
(17.0, 10.2, 5.2)
5.34 m
2.99 dd (17.4, 8.2)
2.90 dd (17.2, 4.8)
4.32 m
5.57 m
2.10 m
1.97 overlap
1.19 m
1.19 m
H-1%%
H-2%%
H-3%%
4.89 d (7.5)
4.19 t (8.0)
4.35 overlap
H-5
H-6a
H-6b
H2-7
H-9
H2-11
H-12
H-15a
H-15b
H2-16
H-18
H-19a
H-19b
H2-21
H2-22
Me-23
Me-24
Me-25
Me-26
Me-27
Me-29
Me-30
H-3%
H-4%%
H-5%%
4.42 overlap
4.53 d (9.7)
5.35 d (7.8)
4.10 t (7.9)
4.24 t (8.8)
4.30 t (8.8)
3.92 m
4.47 overlap
6.34 d (8.08)
4.22 t (7.6)
4.35 overlap
4.32 t (9.2)
4.04 m
H-8%a
H-8%b
H-9%
H-1%%%
H-2%%%
H-3%%%
H-4%%%
H-5%%%
H2-6%%%
H-1%%%%
H-2%%%%
H-3%%%%
H-4%%%%
H-5%%%%
H-6%%%%a
H-6%%%%b
H-10%
H-11%a
H-11%b
H2-12%
H2-13%
H2-14%
H2-15%
H2-16%
Me-17%
1.19 m
1.19 m
1.19 m
0.81 t (7.30)
4.47 overlap
4.43 overlap
0.87 s
1.10 s
1.26 s
0.87 s
0.91 s
3S,9S,10S isomers were dextro-rotatory, while the
3R,9R,10R polyacetylene showed an optical rotation of
−12.3°. In addition, the four corresponding 9,10-ace-
tonides of the panaxytriol diasteromers also showed
very similar NMR data to those of the acetonide
prepared from the naturally occurring panaxytriol, but
only the 3R,9S,10S-panaxytriol acetonide ([h]D−28°)31
exhibited a similar optical rotation to that of the natu-
rally panaxytriol acetonide ([h]D−22.5°).27 Based on
these data, the naturally occurring panaxytriol must
have the 3R,9S,10S-configuration.31 Since the poly-
acetylene unit obtained by the alkaline hydrolysis of
polyacetyleneginsenoside-Ro (1) showed similar optical
rotation ([h]D−20.6°) to that of natural panaxytriol, the
stereochemistry of the isolate must be 3%R,9%S,10%S. The
structure of 1 was thus deduced, and given the trivial
name of polyacetyleneginsenoside-Ro.
potency of this compound relative to that of the other
published HIV-1 inhibitory saponins. Only one other
oleanane-type saponin from Panax, zingibroside R1,
has been reported to demonstrate anti-HIV activity.20
Little is known about the mechanism of action of these
compounds. However, two mechanisms were proposed
for the anti-HIV activity of glycyrrhizin: inhibition of
viral adsorption to target cells by interference with
protein kinase C activity and/or disruption of the initial
stages of viral replication caused by non-specific inter-
actions of the compound with the viral membrane.32–34
None of the anti-HIV saponins reported in the litera-
ture, thus far, was found to affect the enzymatic activity
of HIV-1 reverse transcriptase.35,36 Further investiga-
tion into the mechanism of action of this class of
compounds is warranted.
Polyacetyleneginsenoside-Ro (1) is the first example of
a polyacetylene containing ginsenoside and was found
to inhibit the replication of HIV-1 with an IC50 value of
13.4 mg/mL (11.1 mM). The compound was nontoxic
toward the cultured cells up to a concentration of 20
mg/mL (16.4 mM). The reported role of the unusual
monoterpenyl moieties of Gleditsia and Gymnocladus
saponins21 in modulating the anti-HIV activities of
these compounds suggests that the polyacetylene moi-
ety of 1 may well be responsible for the higher antiviral
Acknowledgements
The authors are grateful to the Research Resources
Center, University of Illinois at Chicago for access to
the Bruker DRX 500 MHz instrument for this study.
Biological evaluation was provided by the Bioassay
Research Facility of the College of Pharmacy, Univer-
sity of Illinois at Chicago.

976
H. Zhang et al. / Tetrahedron Letters 43 (2002) 973–977
Table 2. 13C NMR data of compounds 1 and 2 (125 MHz, pyridine-d5)
C
1
2
C
1
2
C
1
2
Triterpene aglycone
moiety
l
l
Polyacetylene aglycone
moiety
l
l
Sugar moiety
l
l
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
38.80 t
26.04 t
89.28 d
39.56 s
55.73 d
18.52 t
33.18 t
39.94 s
48.07 d
37.01 s
23.42 t
123.03 d
144.12 s
42.16 s
28.28 t
23.85 t
47.03 s
41.78 d
46.27 t
30.82 s
34.01 t
32.58 t
28.01 q
16.68 q
15.61 q
17.51 q
26.12 q
176.47 s
23.69 q
33.18 q
38.63 t
26.57 t
89.32 d
39.54 s
55.76 d
18.48 t
33.15 t
39.89 s
48.01 d
36.91 s
23.40 t
122.88 d
144.15 s
42.12 s
28.25 t
23.76 t
47.00 s
41.75 d
46.20 t
30.79 s
34.00 t
32.43 t
28.12 q
16.70 q
15.54 q
17.47 q
26.10 q
176.44 s
23.65 q
33.15 q
C-1%
C-2%
C-3%
C-4%
C-5%
C-6%
C-7%
C-8%
115.46 t
138.73 d
63.01 d
77.65 s
70.58 s
66.64 s
80.03 s
25.23 t
70.88 d
76.94 d
30.98 t
30.00 t
29.69 t
25.23 t
32.12 t
22.98 t
14.31 q
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
C-1%%
C-2%%
C-3%%
C-4%%
C-5%%
C-6%%
C-1%%%
C-2%%%
C-3%%%
C-4%%%
C-5%%%
C-6%%%
C-1%%%%
C-2%%%%
C-3%%%%
C-4%%%%
C-5%%%%
C-6%%%%
105.57 d
82.55 d
77.65 d
72.45 d
77.24 d
169.53 s
106.05 d
77.08 d
78.01 d
71.71 d
78.41 d
62.75 t
95.80 d
74.19 d
78.96 d
71.11 d
79.40 d
62.21 t
105.36 d
82.59 d
77.55 d
72.89 d
76.80 d
170.51 s
105.97 d
77.10 d
77.99 d
71.68 d
78.36 d
62.71 t
95.78 d
74.18 d
78.94 d
71.09 d
79.39 d
62.20 t
C-9
C-9%
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
C-28
C-29
C-30
C-10%
C-11%
C-12%
C-13%
C-14%
C-15%
C-16%
C-17%
Figure 2. Selected HMBC correlations for polyacetyleneginsenoside-Ro (1) (Bruker DRX 500 MHz, pyridine-d5).

H. Zhang et al. / Tetrahedron Letters 43 (2002) 973–977
977
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