Characterization of Three Oleane-Type Saponins from Panax ginseng

Full text of DOI:10.1007/s10600-015-1530-2

DOI 10.1007/s10600-015-1530-2
Chemistry of Natural Compounds, Vol. 51, No. 6, November, 2015
CHARACTERIZATION OF THREE OLEANE-TYPE SAPONINS
FROM Panax ginseng
1
2
1
1*
Yin-Ping Jin, Wei Li, Shi Yan, and Ying-Ping Wang
Ginseng, the root and rhizome of Panax ginseng C. A. Mey. (Araliaceae), has been used as a very old Chinese
medicine by the people in the Eastern Asia regions for over 2000 years. P. ginseng has very extensive pharmacological
activities, including antifatigue and antidiabetes, as well as activities in the prevention of cancer and slowing the aging process
[1–3]. The pharmacological properties of ginseng are generally attributed to ginsenosides. Based on the structure of aglycone,
the ginsenosides can be divided into protopanaxadiol-type saponins (PPD), protopanaxatriol-type saponins (PPT), and oleanane-
type saponins. More than 150 ginsenosides have been isolated from various parts of ginseng and processed ginseng so far
[4–11], which belong mainly to dammarane-type saponins. However, to date, only four oleanane-type saponins have been
isolated from P. ginseng, including ginsenoside-Ro, ginsenoside-Ro methyl, polyacetylene ginsenoside-Ro [12], and ginsenoside-
Ri [13]. In this study, three oleanane-type saponins, identified as chikusetsusaponin IVa (1), zingibroside R (2), and oleanolic
1
acid 28-O-ꢀ-D-glucopyranoside (3), were further isolated from the plant. Numerous activity studies have been performed on
the dammarane-type ginsenosides, but little attention has been focused on oleanane-type ginsenosides. To further investigate
their antitumor activities, cytotoxicity assays against HepG2, B16, and Lewis tumor cell lines in vitro have been performed
using MTT methods.
Compound 1 was obtained as a white amorphous powder.Acid hydrolysis of compound 1 afforded glucose, glucuronic
1
acid, and oleanolic acid, which were separately identified by direct comparison with authentic samples using TLC. The H NMR
spectrum of compound 1 showed seven singlet methyl signals (ꢁ 0.81, 0.86, 0.92, 0.94, 0.96, 1.06, 1.17) together with
an olefinic proton at ꢁ 5.26 (1H, m) and two anomeric proton signals at ꢁ 5.39 (1H, d, J = 8.2 Hz) and 4.39 (1H, d, J = 7.8 Hz).
The presence of olefinic carbon signals (ꢁ 122.4 and 143.4) suggested that compound 1 could be an oleanane-type saponin.
–
–
–
The negative ion ESI-MS of compound 1 exhibited ions at m/z 793.7 [M – H] , 631.4 [M – H – Glc] , 613.4 [M – H – Glc – H O] ,
2
–
–
–
569.3 [M – H – Glc – H O – CO ] , 497.2 [M – H – Glc – CO – C H O ] , and 455.2 [M – H – Glc – GluA] . In comparison
2
2
2
3 6 3
with the negative ion ESI-MS of ginsenoside-Ro [14], it had closely similar fragmentation pathways, except for a loss of
162 Da (one glucose unit). The physicochemical properties and spectral data (Table 1) of compound 1 were identical to
chikusetsusaponin IVa [15].
30 29
21
19
13
11
28
C
25
5
17
15
26
1
OR
2
9
10
O
3
27
7
R O
1
24
23
1 - 4
2
1: R = GlcUA, R = Glc; 2: R = GlcUA ꢂGlc, R = H
1
2
1
2
2
3: R = H, R = Glc; 4: R = GlcUA ꢂGlc, R = Glc
1
2
1
2
1) Institute of Special Wild Economic Animals and Plant, Chinese Academy of Agricultural Sciences, No. 4899 Juye
Street, Jingyue Economic Development District, Changchun City, 130112, Jilin Province, P. R. China, e-mail:
yingpingw1967@126.com; 2) College of Chinese Medicinal Materials, Jilin Agricultural University, No. 2888 Xincheng
Street, Jingyue Economic Development District, Changchun City, 130118, Jilin Province, P. R. China. Published in Khimiya
Prirodnykh Soedinenii, No. 6, November–December, 2015, pp. 1025–1027. Original article submitted December 13, 2013.
0009-3130/15/5106-1193 02015 Springer Science+Business Media New York
1193

13
TABLE 1. C NMR Data of Compounds 1–3 (150 MHz, TMS, ꢁ, ppm)
1*
2**
C atom
1*
2**
3**
C atom
3**
Glc UA
1
2
3
4
5
6
7
8
38.4
25.6
89.7
39.3
55.6
17.9
31.7
38.8
47.6
36.5
23.1
122.4
143.4
41.5
27.5
22.6
46.6
41.2
45.8
30.1
33.5
32.6
27.1
14.6
15.5
16.3
24.9
176.6
32.1
22.5
38.4
26.4
88.9
39.3
55.5
18.2
33.0
39.5
47.7
36.7
23.5
122.3
144.6
41.9
28.1
23.5
46.4
41.7
46.2
30.7
34.0
33.0
27.9
16.5
15.2
17.1
25.9
179.9
33.0
23.4
40.6
29.7
78.6
41.0
56.3
20.4
34.8
41.5
49.8
39.0
25.0
123.4
144.6
43.8
29.9
25.4
48.6
43.4
47.8
32.4
35.6
34.1
30.4
18.1
17.2
19.1
27.7
176.9
34.7
25.3
3- -Sugar
O
105.6
71.8
76.3
73.9
75.1
179.8
105.2
82.7
76.9
72.9
77.7
172.3
Glc (1–2)
105.8
77.5
78.1
71.4
77.2
62.4
1ꢃ
2ꢃ
3ꢃ
4ꢃ
5ꢃ
6ꢃ
9
1ꢃꢃ
2ꢃꢃ
3ꢃꢃ
4ꢃꢃ
5ꢃꢃ
6ꢃꢃ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Glc
94.3
72.5
77.3
69.7
76.9
61.0
Glc
96.2
74.6
79.8
71.6
79.4
62.7
28- -Sugar
O
1ꢃꢃꢃ
2ꢃꢃꢃ
3ꢃꢃꢃ
4ꢃꢃꢃ
5ꢃꢃꢃ
6ꢃꢃꢃ
______
*13
**13
C NMR data were measured in CD OD;
C NMR data were measured in pyridine-d , Glc UA – ꢀ-D-glucuronic acid.
5
3
Compound 2 was obtained as a white amorphous powder.Acid hydrolysis of compound 2 afforded glucose, glucuronic
acid, and oleanolic acid, which were separately identified by direct comparison with authentic samples using TLC.
1
The H NMR spectrum of compound 2 showed seven singlet methyls (ꢁ 0.77, 0.92, 0.95, 0.97, 1.07, 1.25, 1.27) together with
an olefinic proton at ꢁ 5.42 (1H, m) and two anomeric proton signals at ꢁ 5.39 (1H, d, J = 7.2 Hz), 5.00 (1H, d, J = 7.2 Hz).
The presence of olefinic carbon signals (ꢁ 122.3 and 144.6) suggested that compound 2 could be an oleanane-type saponin.
C
–
–
–
The negative ion ESI-MS of compound 2 exhibited ions at m/z 793.6 [M – H] , 731.5 [M – H – CO – H O] , 631.4 [M – H –Glc] ,
613.4 [M – H – Glc – H O] , 569.3 [M – H – Glc – H O – CO ] , 551.5 [M – H – Glc – 2H O – CO ] , and 455.2 [M – H – Glc – GluA] .
2
2
–
–
–
–
2
2
2
2
2
In comparison with compound 1, it had closely similar fragmentation pathways, except for the characteristic ion moiety
m/z 731.5 (CO and H O unit) [12]. The physicochemical properties and spectral data (Table 1) of compound 2 were identical
2
2
to zingibroside R [16].
1
Compound 3 was obtained as a white amorphous powder. Acid hydrolysis of compound 3 afforded glucose and
1
oleanolic acid, which were separately identified by direct comparison with authentic samples using TLC. The H NMR spectrum
of compound 3 showed seven singlet methyls (ꢁ 0.90, 0.92, 0.94, 1.04, 1.15, 1.24, 1.26) and an olefinic proton at ꢁ 5.47 (1H, m)
and 6.36 (1H, d, J = 8.4 Hz) for the anomeric proton signal. The presence of olefinic carbon signals (ꢁ 123.4 and 144.6)
suggested that compound 3 could be an oleanane-type saponin. The configuration of the anomeric position was determined to
1
be ꢀ on the basis of the coupling constant of the anomeric proton signal in the H NMR spectrum of compound 3 (1H, d,
13
J = 8.4 Hz). The signals due to the ꢀ-D-glucopyranosyl moiety were observed in the C NMR spectrum at ꢁ 96.2, 74.6, 79.8,
71.6, 79.4, and 62.7. The physicochemical properties and spectral data (Table 1) of compound 3 were identical to oleanolic
acid 28-O-ꢀ-D-glucopyranoside [16].
1194

The isolated oleanane-type saponins were all tested for their cytotoxicities against HepG2, B16, and Lewis tumor cell
lines using the MTT method with compound K as positive control. Among them, compounds 1, 3, and 4 showed no cytotoxicity
effect, while compound 2 showed potent cytotoxic activities against HepG2, B16, and Lewis cell lines with IC values of
50
35.39, 35.44, and 47.30 ꢄM, respectively.
Plant Material. The roots of P. ginseng C. A. Mey. were collected from Jilin Province, China, in August 2010.
Red ginseng, obtained by steaming fresh P. ginseng roots at 120ꢅC for 8 h, is manufactured by the National R & D Center for
Ginseng and Pilose Antler Product Processing. The botanical origin of the material was identified by Dr. Jing-hui Zhao,
Institute of Special Wild Economic Animals and Plants, CAAS, Jilin Province. A voucher specimen (No. 2010127) was
deposited at the Institute of Special Wild Economic Animals and Plants, CAAS, Jilin, China.
Extraction and Isolation. The air-dried roots of red ginseng (12.8 kg) was extracted with methanol, sonified (4 ꢆ 60 L;
each 1 h), and concentrated in vacuo to give the MeOH extract. The MeOH extract was suspended in H O and extracted with
2
n-BuOH. The concentrated butanol fraction was suspended with excess water and then subjected to D macroporous absorption
101
resin eluting with H O, 20% EtOH, 70% EtOH, and 95% EtOH in sequence. The 70% EtOH fraction (294.0 g) was subjected
2
to silica gel column chromatography eluted with CH Cl –MeOH (100:1ꢂ0:1) to yield seven fractions (Fr.1–7). Fractions 1–7
2
2
were subjected to silica gel and RP-18 column chromatography and preparative HPLC, affording 19 saponins (1–19).
On the basis of spectroscopic and chemical methods, the structures of compounds 1–19 were elucidated as chikusetsusaponin
IVa (1), zingibroside R (2), oleanolic acid 28-O-ꢀ-D-glucopyranoside (3), and ginsenoside – Ro (4), 20 (S)-Rh (5), 20 (R)-Rh
1
1
1
(6), 20 (S)-Rg (7), 20 (R)-Rg (8), 20 (S)-Rg (9), 20 (R)-Rg (10), Rg (11), Rk (12), Rs (13), Re (14), Rg (15), Rb (16), Rb
2
2
3
3
5
1
3
1
1
2
(17), Rc (18), and Rd (19) [12]. Compounds 1–3 were oleanane-type saponins isolated for the first time from P. ginseng.
Cytotoxicity Assays. The cytotoxic activities of compounds 1–4 against HepG2, B16, and Lewis tumor cell lines
were evaluated using the MTT assay. The assay was performed in the Cell Culture Laboratory, Pharmaceutical College, Jilin
University. Compound K was used as the positive control with IC of 8.1, 6.1, and 8.9 ꢄM for HepG2, B16, and Lewis tumor
50
cells, respectively.
ACKNOWLEDGMENT
The authors gratefully acknowledge financial support from the Key Project in the National Science and Technology
Pillar Program (2011ZX09401-305-10-02).
REFERENCES
1.
2.
3.
Y. Z. Xiang, H. C. Shang, X. M. Gao, and B. L. Zhang, Phytother. Res., 22, 851 (2008).
C. Bachran, S. Bachran, M. Sutherland, D. Bachran, and H. Fuchs, Mini-Rev. Med. Chem., 8, 575 (2008).
P. Y. Yue, N. K. Mak, Y. K. Cheng, K. W. Leung, T. B. Ng, D. T. Fan, H. W. Yeung, and R. N. Wong, Chin. Med.,
2, 6 (2007).
4.
5.
L. W. Qi, C. Z. Wang, and C. S. Yuan, Nat. Prod. Rep., 28, 467 (2011).
T. S. Wang, W. C. Chang, and P. G. Xiao, China Ginseng, Liaoning Science and Technology Publishing House,
Shenyang, 2001, 671 pp.
6.
7.
D. Q. Dou and Y. J. Chen, Chin. J. Med. Chem., 7, 202 (1997).
J. D. Park, Y. H. Lee, and S. I. Kim, Arch. Pharm. Res., 21, 615 (1998).
8.
9.
10.
11.
F. Qiu, Z. Z. Ma, Y. P. Pei, S. X. Xu, X. S. Yao, and Y. J. Chen, Chin. J. Med. Chem., 8, 205 (1998).
J. M. Zhao, N. Li, H. Zhang, C. F. Wu, H. R. Piao, and Y. Q. Zhao, Bioorg. Med. Chem. Lett., 21, 1027 (2011).
G. V. Malinovskaya, V. V. Makhanꢃkov, V. A. Denisenko, and N. I. Uvarova, Chem. Nat. Compd., 27, 253 (1991).
J. G. Cho, D. Y. Lee, S. Shrestha, S. K. Lee, H. M. Kang, S. H. Son, D. C. Yang, and N. I. Baek, Chem. Nat. Compd.,
49, 882 (2013).
12.
13.
14.
15.
16.
H. Hikino, Y. Kiso, J. Kinouchi, S. Sanada, and J. Shoji, Planta Med., 51, 62 (1985).
L. Fu, X. G. Li, and S. R. Yang, J. Jilin Agric. Univ., 20, 30 (1998).
Y. J. Li, H. L. Wei, L. W. Qi, J. Chen, M. T. Ren, and P. Li, Rapid Commun. Mass Spectrom., 24, 2975 (2010).
V. S. Pires, D. Guillaum, G. Grace, and E. P. Schenkel, J. Agric. Food Chem., 45, 1027 (1997).
R. L. Nie, T. Morita, R. Kasai, J. Zhou, C. Y. Wu, and O. Tanaka, Planta Med., 4, 322 (1984).
1195