Two new saponins from the leaves of Panax notoginseng

Article abstract of DOI:10.1080/10286020.2017.1403421

Two new saponins, notoginsenosides Ng1 (1) and Ng2 (2), together with seven known compounds (3–9), were isolated from the leaves of Panax notoginseng. Their structures were elucidated by UV, IR, HRESIMS, and NMR experiments. Compounds 6 and 7 showed moder

Full text of DOI:10.1080/10286020.2017.1403421

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Two new saponins from the leaves of Panax
notoginseng
Ji-Wu Huang, Fang-You Chen, Chuang-Jun Li, Jing-Zhi Yang, Jie Ma, Xiao-
Guang Chen & Dong-Ming Zhang
To cite this article: Ji-Wu Huang, Fang-You Chen, Chuang-Jun Li, Jing-Zhi Yang, Jie Ma,
Xiao-Guang Chen & Dong-Ming Zhang (2017): Two new saponins from the leaves of Panax
notoginseng, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2017.1403421
To link to this article: https://doi.org/10.1080/10286020.2017.1403421
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Date: 01 December 2017, At: 05:43

Journal of asian natural Products research, 2017
https://doi.org/10.1080/10286020.2017.1403421
Two new saponins from the leaves of Panax notoginseng
Ji-Wu Huang, Fang-You Chen, Chuang-Jun Li, Jing-Zhi Yang, Jie Ma, Xiao-Guang Chen
and Dong-Ming Zhang
state Key laboratory of Bioactive substance and function of natural Medicines, institute of Materia Medica,
chinese academy of Medical sciences and Peking union Medical college, Beijing 100050, china
ABSTRACT
ARTICLE HISTORY
received 12 september 2017
accepted 7 november 2017
Two new saponins, notoginsenosides Ng1 (1) and Ng2 (2), together
with seven known compounds (3–9), were isolated from the leaves
of Panax notoginseng. Their structures were elucidated by UV, IR,
HRESIMS, and NMR experiments. Compounds 6 and 7 showed
moderate cytotoxic activities against HCT-116, with IC50 values of
4.98 and 0.64 μmol/L, respectively.
KEYWORDS
araliaceae; Panax
notoginseng; saponins;
anticancer
O
O
OH
O
OH
O
O
OH
OH
O
O
H
HO
OH
OH
HO
O
O
O
HO
OH
OH
O
O
O
OH
OH
O
OH
OH
HO
OH
O
OH
O
OH
OH
OH
OH
2
1
OH
OH
1. Introduction
Panax notoginseng (Burk.) F.H.Chen (Araliaceae), a traditional precious herb, is mainly
cultivated in Yunnan Province and Guangxi Zhuang Autonomous Region [1]. Due to its
excellent pharmacological effects on the immune system, cardiovascular system as well
as anti-inflammatory and anti-tumor activities, chemical investigations have been made
since decades ago and various bioactive components including saponins, flavonoids, and
cyclopeptides have been isolated from this plant [2]. Modern pharmacological researches
have revealed that the dammarane-type saponins are the main bioactive constituents in P.
notoginseng. Over the past years, most studies were based on the root of this plant, and few
phytochemical experiments have been performed on the leaves. For the further develop-
ment and utilization of this valuable Chinese plant, we collected the air-dried leaves and
conducted an extensive research on P. notoginseng. In this paper, we report the isolation
and structure elucidation of two new saponins, notoginsenosides Ng1 (1) and Ng2 (2),
CONTACT dong-Ming Zhang
zhangdm@imm.ac.cn
© 2017 informa uK limited, trading as taylor & francis Group

2
J.-W. HUANG ET AL.
along with seven known compounds notoginsenoside-LX (3), notoginsenoside FP2 (4),
gypenoside-XVII (5), notoginsenoside Fe (6), ginsenoside Rd (7), ginsenoside Rb3 (8),
and notoginsenoside E (9) (Figure 1). Furthermore, the anticancer assay was performed
against five human cancer cell lines.
2. Results and discussion
Notoginsenoside Ng1 (1) was isolated as white powder. Its molecular formula was estab-
lished as C47H78O17 by the HRESIMS ion peak at m/z 937.5150 [M + Na]+. 1H NMR spec-
trum of 1 (Table 1) exhibited eight methyl signals (δH 0.82, 0.94, 1.01, 1.10, 1.31, 1.51, 1.68,
1.83) and an olefinic proton signal at δH 5.54 (1H, d, J = 7.2 Hz). e 13C NMR spectrum
of 1 presented 47 carbon signals, of which the anomeric carbons of three sugar moieties
(δC 99.3, 106.4, and 106.9) were observed. e signals at δC 129.2 (C-24) and 131.2 (C-25)
showed the existence of two olefinic carbon signals. e 13C NMR data (Table 1) were similar
to those of notoginsenoside-LX (3) [3], except for the appearance of a xylopyranosyl unit.
Acid hydrolysis of compound 1 provided D-glucose (Glc) and D-xylose (Xyl) [tR (min):
20.33 and 14.76] by GC. e standard monosaccharide derivatives were made and analyzed
by GC under the same condition. Retention times were consistent. According to HSQC,
HMBC, and ROESY experiments, two β-D-glucopyranoses (δH 4.99 and 5.11) and one
β-D-xylopyranose (δH 4.99) were identified. In the HMBC spectrum of 1 (Figure 2), the
location and sequence of the sugar moieties were observed, with key long-range correlations
from δH 4.99 (H-1′ of Glc I) to δC 88.7 (C-3), from 5.11 (H-1′′ of Glc II) to 82.0 (C-20), and
from δH 4.99 (H-1′′′ of Xyl) to 70.8 (C-6′′ of Glc II). e correlations between H-23 (δH 4.85,
1H, t, J = 8.4 Hz) and C-12 (δC 79.6) in HMBC spectrum indicated the presence of the ether
linkage between C-23 and C-12. e α-orientation of H-23 and H-12 was demonstrated
by the ROESY correlations between H-12 (δH 3.68, 1H, overlapped) and H-9 (δH 1.48, 1H,
overlapped)/H-30 (δH 1.10, 3H, s)/H-17 (δH, 3.21, 1H, td, J = 10.8, 4.2 Hz), as well as corre-
lations between H-23 (δH 4.85, 1H, t, J = 8.4 Hz) and H-12 (δH 3.68, 1H, overlapped)/H-17
(δH, 3.21, 1H, td, J = 10.8, 4.2 Hz). e ROESY correlations between H-21 (δH 1.51, 3H, s)
and H-13 (δH 1.59, 1H, t, J = 10.2 Hz), as well as between H-13 and H-19 (δH 0.82, 3H, s),
indicated the configuration of C-21 was β (Figure 2).
26
Glc III
Ara(f)
24
25
27
21
21
O
O
OH
22
22
27
O
O
O
23
O
OH
20
24
OH
O
25
20
17
23
12
12
18
17
16
H
13
11
11
19
OH
HO
OH
18
14
19
26
OH1
13
1
4
16
9
9
14
2
2
3
10
8
7
15
8
7
15
10
5
3
5
30
4
30
HO
HO
O
O
6
6
O
O
O
O
OH
O
Glc I
OH
OH
29
28
29
28
O
OH
OH
OH
OH
HO
OH
Glc II
Glc I
O
OH
OH
OH
Xyl
Glc II
2
OH
OH
OH
1
Figure 1. structures of compounds 1 and 2.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Table 1. 1h and 13c nMr (600 Mhz, 150 Mhz in pyridine-d5) spectral data for compounds 1 and 2.
Position
1 δC
1 δH (J in Hz)
0.85 *, 1.52 *
1.83 *, 2.27 *
3.37 dd (11.4, 2.4)
2 δC
2 δH (J in Hz)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
39.4
26.8
88.7
39.7
56.4
18.5
35.2
39.8
50.6
37.1
30.1
79.6
49.8
51.3
32.6
25.6
46.6
15.5
16.5
82.0
24.6
52.1
39.2
26.8
89.0
39.7
56.4
18.4
35.1
40.0
50.2
36.9
30.9
70.1
49.5
51.5
30.7
26.7
51.9
16.0
16.3
83.2
21.9
32.7
0.73 t (13.2), 1.55 *
1.82 *, 2.20–2.22 m
3.28 dd (11.4, 4.2)
0.73 d (11.4)
1.37 *, 1.48 *
1.19–1.21 m, 1.37 *
0.65–0.68 m
1.36 *, 1.47 *
1.16–1.19 m, 1.47 *
1.48 *
1.36 *
1.37 *, 1.92 *
3.68 *
1.59 t (10.2)
1.47 *, 1.97 dd (10.8, 4.8)
4.16 *
2.03 t (10.8)
1.07 dd (11.4, 7.8), 1.48 *
2.10–2.14 m, 2.27 *
3.21 td (10.8, 4.2)
0.94 s
1.01 *, 1.55 *
1.36 *, 1.82 *
2.61 q (10.2)
0.93 s
17
18
19
20
21
22
0.82 s
0.82 s
1.51 s
2.27 *, 2.84 *
1.59 s
3.17 ddd (16.8, 10.2, 6.0),
3.43 ddd (14.4, 9.6, 4.2)
23
24
25
26
27
28
29
30
72.6
129.2
131.2
26.7
18.9
28.1
4.85 t (8.4)
5.54 d (7.2) 202.5
29.8 2.10 ddd (15.0, 9.6, 6.0), 2.73–2.77 m
144.4
17.8
125.4
28.1
16.6
17.4
1.68 s
1.83 s
1.31 s
1.01 s
1.10 s
1.86 s
5.78 s, 6.37 s
1.29 s
1.12 s
0.97 s
16.8
17.0
3-O-sugar Glc i
3-O-sugar Glc i
1′
2′
3′
4′
5′
6′
106.9
75.8
78.9
71.9
4.99 * 105.1
4.06 *
4.25 *
4.25 *
4.94 d (7.8)
4.26 *
4.16 *
3.96 *
4.34 *
83.5
78.1
72.0
78.0
62.9
78.4
63.1
20-O-sugar Glc ii
99.3
4.06 *
4.42–4.43 m, 4.62 d (11.4)
Glc ii
4.34 *, 4.58 d (10.8)
1′′
2′′
3′′
4′′
5′′
6′′
5.11 d (7.8) 106.1
3.93 td (8.4, 3.0)
5.39 d (7.2)
4.16 *
4.16 *
4.34 *
3.96 *
75.3
78.8
71.8
76.9
70.8
Xyl
106.4
74.9
78.3
77.2
79.4
71.7
78.3
62.7
4.25 *
4.16 *
4.16 *
4.38 *, 4.76 d (10.2)
20-O-sugar Glc iii
4.99 *
4.26 *, 4.50 s
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
98.0
74.9
78.4
71.7
76.7
68.8
ara(f)
110.3
83.5
78.8
85.9
62.7
5.13 d (7.8)
3.96 *
4.16 *
4.16 *
4.09 *
4.06 *
4.16 *
4.25 *
71.1
67.2
3.68 *, 4.38 *
4.16 *, 4.72 q (9.6)
1′′′′
2′′′′
3′′′′
4′′′′
5′′′′
5.71 d (1.2)
4.89 br s
4.83 q (6.0)
4.76 dt (7.2, 4.2)
4.26 *, 4.34 *
*overlapped.

4
J.-W. HUANG ET AL.
O
O
OH
O
O
O
OH
OH
OH
H
O
O
H
HO
H
OH
OH
H
H
HO
O
O
O
HO
OH
OH
O
O
O
OH
OH
O
OH
OH
HO
OH
HMBC
O
OH
O
OH
OH
OH
OH
ROESY
2
1
OH
OH
Figure 2. Key hMBc and roesY correlations of compounds 1 and 2.
From the above evidence, the structure of compound 1 was elucidated as (20S)-3-
O-β-D-glucopyranosyl-20-O-β-D-xylopyranosyl(1→6)-β-D-glucopyranosyl-12β,23R-
epoxydammar-24-ene (Figure 1), named notoginsenoside Ng1.
Notoginsenoside Ng2 (2) was also obtained as white powder. Its molecular formula was
established as C53H88O23 by the HRESIMS ion peak at m/z 1115.5613 [M + Na]+. e enone
absorption was showed at 219 nm in its UV spectrum. 1H NMR spectrum of 2 (Table 1)
showed seven methyl signals (δH 0.82, 0.93, 0.97, 1.12, 1.29, 1.59, 1.86), two olefinic proton
signals at δH 5.78 (1H, s) and 6.37 (1H, s), and four anomeric proton signals at δH 4.94 (1H,
d, J = 7.8 Hz), 5.13 (1H, d, J = 7.8 Hz), 5.39 (1H, d, J = 7.2 Hz), and 5.71 (1H, d, J = 1.2 Hz).
e 13C NMR spectrum of 2 showed four anomeric carbon signals (δC 98.0, 105.1, 106.1,
and 110.3) and two olefinic carbon signals at δC 144.4 (C-25) and 125.4 (C-27). e signal
at δC 202.5 (C-24) showed the existence of a carbonyl group. e carbon signals in the 13C
NMR spectrum (Table 1) of 2 were similar to those of notoginsenoside-B [4], except the
five signals in 2 due to an arabinofuranosyl unit.
Acid hydrolysis of compound 2 provided D-glucose and L-arabinose (Ara) [tR (min):
20.33 and 14.79] by GC. e standard monosaccharide derivatives were made and analyzed
by GC under the same condition. Retention times were consistent. According to HSQC,
HMBC, and ROESY experiments, three β-D-glucopyranoses and one α-L-arabinofuranose
were identified. In the HMBC experiment of 2 (Figure 2), long-range correlations were
observed between δH 4.94 (H-1′ of Glc I) and δC 89.0 (C-3), 5.39 (H-1′′ of Glc II) and 83.5
(C-2′ of Glc I), 5.13 (H-1′′′ of Glc III) and 83.2 (C-20), 5.71 (H-1′′′′ of Ara) and 68.8 (C-6′′′
of Glc III).
From the above evidence, the structure of compound 2 was characterized as (20S)-3-
O-[β-D-glucopyranosyl(1→2)β-D-glucopyranosyl]-20-O-[α-L-arabinofuranosyl(1→6)β-D-
glucopyranosyl]-12β-hydroxydammar-25-en-24-one, named notoginsenoside-Ng2.
By comparison with literatural data, compounds 3–9 were identified as notoginseno-
side-LX (3) [3], notoginsenoside FP2 (4) [5], gypenoside-XVII (5) [6], notoginsenoside
Fe (6) [7], ginsenoside Rd (7) [8], ginsenoside Rb3 (8) [8], and notoginsenoside E (9) [9],
respectively.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Cytotoxic assay of saponins 1–9 was carried out by MTT (3-(4,5-dimethylthi-
azolyl)-2,5-diphenyltetrazolium bromide) method as represented in the reference [10].
3. Experimental
3.1. General experimental procedures
Optical rotations were performed on an Autopol V automatic polarimeter (RUDOLPH,
Hackettstown, NJ, U.S.A.). UV spectra were taken with a JASCO V-650 spectrophotom-
eter (JASCO, Tokyo, Japan). IR spectra were recorded on a Nicolet 5700 spectrometer
using an FT-IR microscope transmission method (ermo Electron, Madison, WI, U.S.A.).
NMR spectra were acquired with VNS-600 (VARIAN, Palo Alto, CA, U.S.A.). HRESIMS
were measured using an Agilent 1100 series LC/MS ion trap mass spectrometer (Agilent
Technologies, Santa Clara, CA, U.S.A.). GC was performed on the Agilent technologies
7890A apparatus (Agilent Technologies, Inc, Germany) with flame ionization detector and
a HP-5 column (60 m × 0.25 mm). e column temperature was programmed from 200 to
280 °C at a rate of 10 °C/min. Nitrogen was used as the carrier gas at 1.5 ml/min. e injector
and detector temperature was at 300 °C and the injection volume was 1.0 μl. MPLC system
was composed of two C-605 pumps, a C-635 UV detector (Büchi, Flawil, Switzerland),
and an ODS column (YMC, 600 × 60 mm, 50 μm, Kyoto, Japan). Column chromatography
was performed with MCI gel (CHP20/P120, Mitsubishi Chemical, Tokyo, Japan), Celite
(Sinopharm Chemical Reagent Co., Ltd, Shanghai, China), macroporous resin (HPD-100,
Hebei Cangzhou Baoen Chemical Co. Ltd, Cangzhou, China). Preparative HPLC was per-
formed on a Shimadzu LC-6AD instrument with a SPD-20A detector (Shimadzu, Kyoto,
Japan) and a reversed-phase C18 column (YMC-Pack ODS-A, 250 × 20 mm, 5 μm, Kyoto,
Japan) at 25 °C at a flow rate of 10.0 ml/min using the following eluent MeOH/H2O 72:28
(HPLC system I); CH3CN/H2O 24:76 (HPLC system II), 25:75 (HPLC system III), 35:65
(HPLC system IV), detection at 203 nm.
3.2. Plant material
Panax notoginseng leaves were collected from Wen-Shan, Yunnan, China, in June 2015, and
identified by associate Professor Ma Lin of Institute of Materia Medica, Chinese Academy
of Medical Sciences and Peking Union Medical College (IMM CAMS&PUMC). A voucher
specimen has been deposited at the Herbarium of IMM CAMS&PUMC (ID-22816).
3.3. Extraction and isolation
e air-dried P. notoginseng leaves (25 kg) were extracted with 95% ethanol (150 L × 2) and
then concentrated under vacuum at 50 °C to give a residue (5.1 kg). e ethanol extract
(3.6 kg) was subjected to Celite column (petroleum ether; chloroform; acetone; acetone/
EtOH 4:1; acetone/EtOH 1:1; 95% EtOH; MeOH) to afford seven fractions: Fr.A-Fr.G.
Fr.E (379 g) was applied to macroporous resin column (HPD-100, 4.0 kg) using EtOH/
H2O (0, 30%, 60%, 95%) to yield 4 fractions, E0-E3. E2 (113 g) was subjected to a MCI gel
column (H2O/EtOH 9:1-95:5) to obtain five fractions, E2A-E2E. E2B (30 g) was applied
to a Sephadex LH-20 gel column to afford ten subfractions, E2Ba-E2Bj. E2Bc (10 g) was

6
J.-W. HUANG ET AL.
loaded onto a MPLC system and eluted with 45–95% MeOH/H2O (9 h) to yield 25 fractions
(B1-B25). e following four compounds were isolated by preparative HPLC: compounds
3 (19.1 mg; tR 46.0 min), 4 (7.6 mg; tR 95.0 min), and 2 (8.7 mg; tR 113.0 min) were purified
from fraction B16 by HPLC system II; compound 1 (10.0 mg; tR 45.0 min) was purified from
fraction B18 by HPLC system III. E2D (15 g) was applied to MPLC system and eluted with
60–90% MeOH/H2O (9 h) to yield 25 fractions (D1-D25). e following five compounds
were isolated by preparative HPLC: compounds 9 (4.7 mg; tR 13.0 min), 8 (19.8 mg; tR
43.0 min), and 7 (33.3 mg; tR 57.0 min) were purified from fraction D18 by HPLC system I;
compounds 5 (3.0 mg; tR 35.0 min) and 6 (28.0 mg; tR 57.0 min) were purified from fraction
D20 by HPLC system IV.
3.3.1. Notoginsenoside Ng1 (1)
White powder; [ꢀ]2D0 –13.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 203 (2.58), 254 (2.01)
nm; IR (microscope) νmax: 3404, 2942, 1639, 1451, 1380, 1079 cm−1; 1H NMR (600 MHz,
pyridine-d5) and 13C NMR (150 MHz, pyridine-d5) spectral data, see Table 1; HRESIMS:
m/z 937.5150 [M + Na]+ (calcd for C47H78O17Na, 937.5131).
3.3.2. Notoginsenoside Ng2 (2)
White powder; [ꢀ]20 +3.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 203 (2.84), 219 (2.84),
254 (2.69) nm; IR D(microscope) νmax: 3361, 2932, 1732, 1662, 1388, 1074 cm−1; H NMR
1
(600 MHz, pyridine-d5) and 13C NMR (150 MHz, pyridine-d5) spectral data, see Table 1;
HRESIMS: m/z 1115.5613 [M + Na]+ (calcd for C53H88O23Na, 1115.5609).
3.4. Acidic hydrolysis
Solutions of compounds 1 and 2 (2.0 mg each) in 2.5 N HCl (4.0 ml) were stirred at 90 °C
for 12 h. Afer cooling, each reaction mixture was diluted to 4.0 ml with water and then
extracted by EtOAC (8.0 ml × 3). e aqueous layer was repeatedly evaporated to dryness
with water until neutral. e aqueous layer residues were dissolved in anhydrous pyridine
(2.0 ml) and stirred with L-cysteine methyl (1.0 mg) for 2 h at 60 °C and then dried by a
stream of N2. Furthermore, 0.5 ml of N-trimethylsilylimidazole was added and the mixture
was stirred at 60 °C for another 2 h. Afer cooling, each reaction mixture was diluted to
2.0 ml with water and then extracted by hexane (4.0 ml × 3). e hexane layer was dried
under N2 steam at room temperature. e residue was dissolved in hexane (1.0 ml), and
the solution was analyzed by GC.
3.5. Cytotoxic activities
As the data shown in Table 2, compounds 6 and 7 exhibited moderate cytotoxic activities
against HCT-116, with IC50 values 4.98 and 0.64 μmol/L, respectively.
Table 2. cytotoxicities of compounds 6 and 7.
IC50 (μmol/L)
Compound
HCT-116
HepG2
BGC-823
A549
A2780
6
4.98
0.64
<0.01
>10
>10
<0.01
>10
>10
<0.01
>10
>10
<0.01
>10
>10
0.735
7
taxol

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
Acknowledgments
We are grateful to the Department of Instrumental Analysis of IMM CAMS&PUMC for the meas-
urement of the UV, IR, HRESIMS, and NMR.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
is research program was financially supported by the National Natural Science Foundation of
China [grant number 81630094] and CAMS Innovation Fund for Medical Sciences (CIFMS) [grant
number 2016-I2M-1-010] and [grant number 2016-I2M-2-003].
References
[1] H.Z. Fu, R.J. Zhong, D.M. Zhang, and D. Wang, J. Asian. Nat. Prod. Res. 15, 1139 (2013).
[2] T. Wang, R.X. Guo, G.H. Zhou, X.D. Zhou, Z.Z. Kou, F. Sui, C. Li, L.Y. Tang, and Z.J. Wang,
J. Ethnopharmacol. 188, 234 (2016).
[3] D.W. Li, J.Q. Cao, X.L. Bi, X.C. Xia, W. Li, and Y.Q. Zhao, J. Gins. Res. 38, 28 (2014).
[4] M. Yoshikawa, T. Murakami, T. Ueno, K. Yashiro, N. Hirokawa, N. Murakami, J. Yamahara,
H. Matsuda, R. Saijoh, and O. Tanaka, Chem. Pharm. Bull. 45, 1093 (1997).
[5] X.Y. Wang, D. Wang, X.X. Ma, Y.J. Zhang, and C.R. Yang, Helv. Chim. Acta. 91, 60 (2008).
[6] R.W. Teng, H.Z. Li, D.Z. Wang, Y.N. He, and C.R. Yang, Chin. J. Magn. Reson. 19, 25 (2002).
[7] T.R. Yang, R. Kasai, J. Zhou, and O. Tanaka, Phytochemistry. 22, 1473 (1983).
[8] H.Z. Li, Y.J. Zhang, and C.R. Yang, Nat. Prod. Res. Dev. 18, 549 (2006).
[9] M. Yoshikawa, T. Murakami, T. Ueno, N. Hirokawa, K. Yashiro, N. Murakami, J. Yamahara,
H. Matsuda, R. Saijoh, and O. Tanaka, Chem. Pharm. Bull. 45, 1056 (1997).
[10] N. Ganot, S. Meker, L. Reytman, A. Tzubery, and E.Y. Tshuva, Jove-J. Vis. Exp. 81, e50767 (2013).