New 20(S)-protopanaxadiol type saponins from the leaves of Panax notoginseng and their potential anti-inflammatory activities

Article abstract of DOI:10.1016/j.steroids.2020.108696

Through the combination of various chromatographies, 11 new 20(S)-protopanaxadiol (PPD) type saponins, named as notoginsenosides NL-E₁ ? NL-E₄ (1–4), NL-F₁ (5), NL-F₂ (6), NL-G₁ (7), NL-G₂ (8), NL-H₁ ? NL-H₃ (9–11) were obtained from the leaves of Panax notoginseng. Their structures were ascertained based on the extensive spectroscopic methods and chemical reactions. Meanwhile, the 20(S)-PPD type saponins with aglycone, (20S,24ζ)-3β,12β,20,24,25-pentahydroxy dammarane, was only found from the leaves of P. notoginseng. The characteristic could be used to distinguish the extracts of P. notoginseng leaves from its other medicinal parts such as roots, rhizomes, flowers or seeds. Furthermore, the nitric oxide (NO) inhibitory activities of all compounds were examined in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. As a result, compounds 2–7, 10 could exert NO inhibitory activity at 25 μM without cytotoxicity. Moreover, the inhibitory activities of them were in dose-dependent manner at 1, 10, and 25 μM. Especially, notoginsenoside NL-F₂ (6) still possessed strong biological activity at 1 μM.

Full text of DOI:10.1016/j.steroids.2020.108696

Journal Pre-proofs
New 20(S)-Protopanaxadiol Type Saponins from the Leaves of Panax noto‐
ginseng and Their Potential Anti-Inflammatory Activities
Jingya Ruan, Fan Sun, Ying Zhang, Dandan Zheng, Guilin Xiang, Wei Zhao,
Yi Zhang, Tao Wang
PII:
S0039-128X(20)30122-7
DOI:
Reference:
https://doi.org/10.1016/j.steroids.2020.108696
STE 108696
To appear in:
Steroids
Received Date:
Revised Date:
Accepted Date:
15 May 2020
24 June 2020
30 June 2020
Please cite this article as: Ruan, J., Sun, F., Zhang, Y., Zheng, D., Xiang, G., Zhao, W., Zhang, Y., Wang, T.,
New 20(S)-Protopanaxadiol Type Saponins from the Leaves of Panax notoginseng and Their Potential Anti-
Inflammatory Activities, Steroids (2020), doi: https://doi.org/10.1016/j.steroids.2020.108696
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Article
New 20(S)-Protopanaxadiol Type Saponins from the
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Leaves of Panax notoginseng and Their Potential
Anti-Inflammatory Activities
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Jingya Ruan 1,¶, Fan Sun 1,¶, Ying Zhang 2, Dandan Zheng 2, Guilin Xiang 3, Wei Zhao
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1, Yi Zhang 1,2,* and Tao Wang 1,2,
*
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Tianjin Key Laboratory of TCM Chemistry and Analysis, Tianjin University of
Traditional Chinese Medicine, 10 Poyanghu Road, West Area, Tuanbo New Town,
Jinghai District, 301617, Tianjin, China
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Institute of TCM, Tianjin University of Traditional Chinese Medicine, 10 Poyanghu
Road, West Area, Tuanbo New Town, Jinghai District, 301617, Tianjin, China
Wenshan Miaoxiang Sanqi Limited Company, South Kaihua Road, 663000, Wenshan
City, Yunnan Province, China
J. Ruan and F. Sun contributed equally to this work.
* Correspondence:zhwwxzh@tjutcm.edu.cn (Y.Z.); wangtao@tjutcm.edu.cn (T.W.);
Tel./Fax: +86-22-5959-6168 (T.W.)
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Abstract: Through the combination of various chromatographies, 11 new
20(S)-protopanaxadiol (PPD) type saponins, named as notoginsenosides NL-E1NL-E4
(14), NL-F1 (5), NL-F2 (6), NL-G1 (7), NL-G2 (8), NL-H1NL-H3 (911) were obtained
from the leaves of Panax notoginseng. Their structures were ascertained based on the
extensive spectroscopic methods and chemical reactions. Meanwhile, the 20(S)-PPD type
saponins with aglycone, (20S,24ζ)-3β,12β,20,24,25-pentahydroxy dammarane, was only
found from the leaves of P. notoginseng. The characteristic could be used to distinguish the
extracts of P. notoginseng leaves from its other medicinal parts such as roots, rhizomes,
flowers or seeds. Furthermore, the nitric oxide (NO) inhibitory activities of all compounds
were examined in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. As a result,
compounds 27, 10 could exert NO inhibitory activity at 25 μM without cytotoxicity.
Moreover, the inhibitory activities of them were in dose-dependent manner at 1, 10, and 25
μM. Especially, notoginsenoside NL-F2 (6) still possessed strong biological activity at 1
μM.
Keywords: the leaves of Panax notoginseng; 20(S)-protopanaxadiol type saponins; RAW
246.7 cell; anti-inflammatory activity
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1. Introduction
Panax notoginseng (Burk.) F. H. Chen (Panax genus, Acanthopanax family), whose
rhizomes and roots have a wide application in traditional Chinese medicine (TCM) for the
treatment of inflammation-related diseases, is rich in saponins [1]. In clinical, many of the
pharmaceutical preparations which possess the ability to treat cardiovascular and
cerebrovascular diseases are made by the total saponins of P. notoginseng [2]. While
because of the over-exploitation and harsh growing conditions of it, the suitability of P.
notoginseng to the cultivated soil is reducing constantly, its production decreases and prices
rise year by year [3]. In the light of literatures, the leaves of P. notoginseng, which own rich
saponins as well, have drawing more and more attentions [48]. As a result, this study is
focusing on saponins from the leaves of P. notoginseng which exhibit anti-inflammatory
activity.
In this article, the investigation of saponins from P. notoginseng leaves was
accomplished by multiple chromatographic and spectrophotometric methods, as well as
chemical reactions. And similar to the reference [9], the in vitro anti-inflammation
screening model was established by RAW264.7 cells, then the inhibitory ability of saponins
on the NO release induced by lipopolysaccharide (LPS) was evaluated. Moreover, a
summary of structure-activity relationships were carried out as well.
2. Experimental
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2.1. Experimental Procedures for Phytochemistry Study
2.1.1. General Experimental Procedures
NMR spectra were determined on Bruker ascend 600 MHz and/or Bruker ascend 500 MHz
NMR spectrometer (Bruker BioSpin AG Industriestrasse 26 CH-8117, Fällanden,
Switzerland) with tetramethylsilane as an internal standard. Negative-ion mode
ESI-Q-Orbitrap MS were measured on a Thermo ESI-Q-Orbitrap MS mass spectrometer
connected to the UltiMate 3000 UHPLC instrument via ESI interface (Thermo, Waltham,
MA). Optical rotations, UV and IR spectra were run on a Rudolph Autopol® IV automatic
polarimeter(l = 50 mm) (Rudolph Research Analytical, Hackettstown NJ, USA), Varian
Cary 50 UV-Vis (Varian, Inc., Hubbardsdon, MA, USA) and Varian 640-IR FT-IR
spectrophotometer (Varian Australia Pty Ltd., Mulgrave, Australia), respectively.
CC were performed on macroporous resin D101 (Haiguang Chemical Co., Ltd.,
Tianjin, China), silica gel (48–75 μm, Qingdao Haiyang Chemical Co., Ltd., Qingdao,
China), ODS (50 μm, YMC Co., Ltd., Tokyo, Japan), MCI gel CHP 20P (Mitsubishi
Chemical Corporation, Osaka, Japan) and Sephadex LH-20 (Ge Healthcare Bio-Sciences,
Uppsala, Sweden). High performance liquid chromatography (HPLC) column: Cosmosil
5C18-MS-II (4.6 mm i.d. × 250 mm, 5 µm, Nakalai Tesque, Inc., Tokyo, Japan) was used to
analysis, and Cosmosil 5C18-MS-II (20 mm i.d. × 250 mm, 5 µm, Nakalai Tesque, Inc.,
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Tokyo, Japan) as well as Venusil PrepG C18 column (25 mm i.d. × 50 mm, 10 µm, Agela
Technonologies, Inc., Tianjin, China) were used to separate the constituents.
2.1.2. Plant Material
The leaves of Panax notoginseng (Burk.) F. H. Chen were collected from Shilin country,
Kunming city, Yunnan province, China, and identified by Dr. Wang Tao (Institute of
Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine). The
voucher specimen was deposited at the Academy of Traditional Chinese Medicine of
Tianjin University of TCM.
2.1.3. Extraction and Isolation
The dried leaves of P. notoginseng (8 kg) was extracted three times with 50% EtOH under
reflux for 3 h, 2 h, and 2 h, successively. Evaporation of the solvent under reduced pressure
provided the 50% EtOH extract (2.67 kg). Then an aliquot (2.1 kg) of the 50% EtOH
extract was subjected to D101 resin CC (H2O → 95% EtOH) to give H2O (760.0 g) and
95% EtOH eluted fraction (695.0 g), respectively.
The 95% EtOH eluate (150.0 g) was subjected to silica gel CC [CH2Cl2-MeOH (1:0 →
100:3 → 100:7 → 10:1 → 8:1 → 3:1 → 2:1 → 1:1 → 0:1, v/v)] to yield 12 fractions (Fr.
1Fr. 12). Fraction 7 (30.0 g) was separated by MCI gel CHP 20P CC [MeOH-H2O (65:35
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→ 70:30 → 75:25 → 80:20 → 100:0, v/v)], and 12 fractions (Fr. 7-1Fr. 7-12) were given.
Fraction 7-4 (447.3 mg) was isolated by pHPLC [CH3CN-1% HAc (26:74, v/v), Cosmosil
5C18-MS-II column] to produce notoginsenoside NL-E1 (1, 10.4 mg). Fraction 7-5 (480.0
mg) was purified by pHPLC [CH3CN-1% HAc (30:70, v/v), Cosmosil 5C18-MS-II column]
to gain notoginsenosides NL-H1 (9, 23.1 mg) and NL-H2 (10, 22.8 mg). Fraction 7-6 (800.0
mg) was separated by pHPLC [MeOH-1% HAc (70:30, v/v), Cosmosil 5C18-MS-II column],
and seven fractions (Fr. 7-6-1Fr. 7-6-7) were obtained. Fraction 7-6-4 (179.5 mg) was
purified by pHPLC [CH3CN-1% HAc (32:68, v/v), Cosmosil 5C18-MS-II column] to give
notoginsenoside NL-G1 (7, 32.6 mg). Fraction 8 (25.0 g) was subjected to MCI gel CHP
20P CC [MeOH-H2O (40:60 → 60:40 → 70:30 → 80:20 → 100:0, v/v)] to gain 11 fractions
(Fr. 8-1Fr. 8-11). Fraction 8-6 (1.6 g) was separated by pHPLC [MeOH-1% HAc (60:40,
v/v), Venusil PrepG C18 column], and 13 fractions (Fr. 8-6-1Fr. 8-6-13) were yielded.
Fraction 8-6-6 (135.3 mg) was purified by pHPLC [CH3CN-1% HAc (26:74, v/v),
Cosmosil 5C18-MS-II column] to produce notoginsenoside NL-H3 (11, 18.4 mg). Fraction 9
(15.0 g) was isoalted by MCI gel CHP 20P CC [MeOH-H2O (60:40 → 70:30 → 80:20 →
100:0, v/v)], and 10 fractions (Fr. 9-1Fr. 9-10) were given. Among them, Fraction 9-3
(254.2 mg) was further separated by pHPLC [CH3CN-1% HAc (22:78, v/v), Cosmosil
5C18-MS-II column] to yield notoginsenosides NL-E2 (2, 5.1 mg), NL-E3 (3, 6.4 mg),
NL-E4 (4, 3.4 mg), NL-F1 (5 13.0 mg), and NL-F2 (6, 5.1 mg). And Fraction 9-4 (500.0 mg)
was isolated by pHPLC [CH3CN-1% HAc (26:74, v/v), Cosmosil 5C18-MS-II column] to
give 11 fractions (Fr. 9-4-1Fr. 9-4-10). Fraction 9-4-6 (86.0 mg) was further purified by
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pHPLC [MeOH-1% HAc (60:40, v/v), Cosmosil 5C18-MS-II column] to obtain
notoginsenoside NL-G2 (8, 16.5 mg).
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Notoginsenoside NL-E1 (1): White powder; []D 4.8 (conc 0.50, MeOH); IR max
(KBr) 3351, 2940, 2877, 1570, 1411, 1160, 1074, 1033 cm1; 1H NMR (C5D5N, 600 MHz)
spectroscopic data:  0.77, 1.53 (1H each, both m, H2-1), 1.82, 2.22 (1H each, both m,
overlapped, H2-2), 3.37 (1H, dd, J = 3.6, 11.4 Hz, H-3), 0.73 (1H, br. d, ca. J = 12 Hz, H-5),
[1.35 (1H, m), 1.49 (1H, m, overlapped), H2-6], [1.20 (1H, m), 1.48 (1H, m, overlapped),
H2-7], 1.38 (1H, m, overlapped, H-9), 1.49, 1.96 (1H each, both m, overlapped, H2-11),
4.06 (1H, m, H-12), 2.07 (1H, dd, J = 10.2, 10.2 Hz, H-13), [0.97 (1H, m), 1.57 (1H, m,
overlapped), H2-15], 1.45, 1.82 (1H each, both m, overlapped, H2-16), 2.50 (1H, q like, ca.
J = 10 Hz, H-17), 0.96 (3H, s, H3-18), 0.80 (3H, s, H3-19), 1.65 (3H,s, H3-21), [2.10 (1H, m,
overlapped), 2.90 (1H, m), H2-22], 2.07, 2.20 (1H each, both m, overlapped, H2-23), 3.83
(1H, br. d, ca. J = 8 Hz, H-24), 1.57 (6H, s, H3-26 and 27), 1.32 (3H, s, H3-28), 1.00 (3H, s,
H3-29), 0.91 (3H, s, H3-30), 4.96 (1H, d, J = 7.2 Hz, H-1'), 4.05 (1H, dd, J = 7.2, 9.0 Hz,
H-2'), 4.26 (1H, dd, J = 8.4, 9.0 Hz, H-3'), 4.23 (1H, dd, J = 9.0, 9.6 Hz, H-4'), 4.03 (1H, m,
overlapped, H-5'), [4.42 (1H, dd, J = 4.8, 10.2 Hz), 4.62 (1H, br. d, ca. J = 11 Hz), H2-6'],
5.14 (1H, d, J = 7.8 Hz, H-1''), 3.91 (1H, dd, J = 7.8, 7.8 Hz, H-2''), 4.15 (1H, dd, J = 7.8,
8.4 Hz, H-3''), 3.94 (1H, dd, J = 8.4, 9.0 Hz, H-4''), 4.03 (1H, m, H-5''), [4.10 (1H, dd, J =
6.0, 10.2 Hz), 4.71 (1H, br. d, ca. J = 11 Hz), H2-6''], 5.69 (1H, br. s, H-1'''), 4.90 (1H, br. s,
H-2'''), 4.78 (1H, m, overlapped, H-3'''), 4.78 (1H, m, overlapped, H-4'''), [4.19 (1H, dd, J =
6.0, 12.0 Hz), 4.32 (1H, br. d, ca. J = 12 Hz), H2-5''']; 13C NMR (C5D5N, 150 MHz)
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spectroscopic data: see Tables 1a and 1b; ESI-Q-Orbitrap MS m/z 949.53735 [MH]
(calcd for C47H81O19, 949.53666).
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Notoginsenoside NL-E2 (2): White powder; []D 3.1 (conc 0.26, MeOH); IR max
(KBr) 3355, 2939, 2877, 1569, 1413, 1157, 1074, 1033 cm1; 1H NMR (C5D5N, 600 MHz)
spectroscopic data:  0.73, 1.54 (1H each, both m, H2-1), 1.82, 2.22 (1H each, both m,
overlapped, H2-2), 3.27 (1H, dd, J = 4.2, 12.0 Hz, H-3), 0.66 (1H, br. d, ca. J = 11 Hz, H-5),
1.35, 1.45 (1H each, both m, overlapped, H2-6), [1.19 (1H, m), 1.43 (1H, m, overlapped),
H2-7], 1.35 (1H, m, overlapped, H-9), 1.49, 1.96 (1H each, both m, overlapped, H2-11),
4.06 (1H, m, H-12), 2.06 (1H, dd, J = 10.8, 10.8 Hz, H-13), [0.96 (1H, m), 1.58 (1H, m,
overlapped), H2-15], 1.43, 1.82 (1H each, both m, overlapped, H2-16), 2.50 (1H, q like, ca.
J = 11 Hz, H-17), 0.96 (3H, s, H3-18), 0.80 (3H, s, H3-19), 1.65 (3H, s, H3-21), [2.08 (1H,
m, overlapped), 2.90 (1H, m), H2-22], 2.06, 2.20 (1H each, both m, overlapped, H2-23),
3.84 (1H, br. d, ca. J = 9 Hz, H-24), 1.58 (3H, s, H3-26), 1.58 (3H, s, H3-27), 1.29 (3H, s,
H3-28), 1.11 (3H, s, H3-29), 0.91 (3H, s, H3-30), 4.94 (1H, d, J = 7.8 Hz, H-1'), 4.26 (1H,
dd, J = 7.8, 9.0 Hz, H-2'), 4.33 (1H, m, overlapped, H-3'), 4.34 (1H, m, overlapped, H-4'),
3.94 (1H, m, overlapped, H-5'), [4.37 (1H, dd, J = 5.4, 12.0 Hz), 4.59 (1H, dd, J = 1.8, 12.0
Hz), H2-6'], 5.39 (1H, d, J = 7.8 Hz, H-1''), 4.14 (1H, m, overlapped, H-2''), 4.28 (1H, dd, J
= 9.0, 9.0 Hz, H-3''), 4.15 (1H, m, overlapped, H-4''), 3.93 (1H, m, overlapped, H-5''), [4.48
(1H, dd, J = 3.6, 11.4 Hz), 4.51 (1H, dd, J = 2.4, 11.4 Hz), H2-6''], 5.15 (1H, d, J = 7.8 Hz,
H-1'''), 3.91 (1H, dd, J = 7.8, 8.4 Hz, H-2'''), 4.17 (1H, dd, J = 8.4, 9.0 Hz, H-3'''), 3.93 (1H,
dd, J = 9.0, 9.5 Hz, H-4'''), 4.03 (1H, m, H-5'''), [4.11 (1H, dd, J = 6.6, 10.8 Hz), 4.71 (1H,
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dd, J = 1.2, 10.8 Hz), H2-6'''], 5.71 (1H, d, J = 1.8 Hz, H-1''''), 4.91 (1H, br. s, H-2''''), 4.79
(1H, m, overlapped, H-3''''), 4.78 (1H, m, overlapped, H-4''''), [4.21 (1H, dd, J = 4.8, 11.4
13
Hz), 4.32 (1H, m, overlapped), H2-5'''']; C NMR (C5D5N, 150 MHz) spectroscopic data:
see Tables 1a and 1b; ESI-Q-Orbitrap MS m/z 1111.59045 [MH] (calcd for C53H91O24,
1111.58948).
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Notoginsenoside NL-E3 (3): White powder; []D 5.6 (conc 0.32, MeOH); IR max
(KBr) 3358, 2939, 2877, 1565, 1417, 1157, 1072, 1043 cm1; 1H NMR (C5D5N, 600 MHz)
spectroscopic data:  0.74, 1.52 (1H each, both m, H2-1), [1.83 (1H, m, overlapped), 2.19
(1H, m), H2-2], 3.29 (1H, dd, J = 4.2, 12.0 Hz, H-3), 0.68 (1H, br. d, ca. J = 11 Hz, H-5),
1.36, 1.47 (1H each, both m, overlapped, H2-6), 1.20, 1.46 (1H each, both m, H2-7), 1.36
(1H, m, overlapped, H-9), 1.48, 1.94 (1H each, both m, H2-11), 4.06 (1H, m, H-12), 2.06
(1H, m, overlapped, H-13), [1.00 (1H, m), 1.58 (1H, m, overlapped), H2-15], [1.44 (1H, m),
1.83 (1H, m, overlapped), H2-16], 2.51 (1H, q like, ca. J = 10 Hz, H-17), 0.95 (3H, s,
H3-18), 0.78 (3H, s, H3-19), 1.65 (3H, s, H3-21), [2.08 (1H, m), 2.89 (1H, m), H2-22], 2.07,
2.22 (1H each, both m, H2-23), 3.84 (1H, br. d, ca. J = 9 Hz, H-24), 1.58 (3H, s, H3-26),
1.59 (3H, s, H3-27), 1.28 (3H, s, H3-28), 1.11 (3H, s, H3-29), 0.90 (3H, s, H3-30), 4.94 (1H,
d, J = 7.8 Hz, H-1'), 4.12 (1H, dd, J = 7.8, 9.0 Hz, H-2'), 4.35 (1H, m, overlapped, H-3'),
4.10 (1H, m, overlapped, H-4'), 3.97 (1H, m, H-5'), [4.36 (1H, m, overlapped), 4.59 (1H, dd,
J = 1.8, 10.8 Hz), H2-6'], 5.54 (1H, d, J = 7.8 Hz, H-1''), 4.20 (1H, dd, J = 7.8, 9.0 Hz,
H-2''), 4.29 (1H, dd, J = 9.0, 9.0 Hz, H-3''), 4.22 (1H, dd, J = 9.0, 9.0 Hz, H-4''), 3.88 (1H,
m, H-5''), [4.36 (1H, m, overlapped), 4.51 (1H, dd, J = 2.5, 11.0 Hz), H2-6''], 5.40 (1H, d, J
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= 7.2 Hz, H-1'''), 4.11 (1H, m, overlapped, H-2'''), 4.14 (1H, m, overlapped, H-3'''), 4.15 (1H,
m, overlapped, H-4'''), [3.70 (1H, dd, J = 9.6, 9.6 Hz), 4.31 (1H, m, overlapped), H2-5'''],
5.13 (1H, d, J = 7.2 Hz, H-1''''), 3.92 (1H, dd, J = 7.2, 9.0 Hz, H-2''''), 4.17 (1H, m,
overlapped, H-3''''), 3.93 (1H, dd, J = 9.0, 9.0 Hz, H-4''''), 4.03 (1H, m, H-5''''), [4.09 (1H, m,
overlapped), 4.70 (1H, br. d, ca. J = 10 Hz), H2-6''''], 5.71 (1H, br. s, H-1'''''), 4.91 (1H, br. s,
H-2'''''), 4.79 (1H, m, overlapped, H-3'''''), 4.78 (1H, m, overlapped, H-4'''''), [4.21 (1H, dd, J
= 5.4, 12.6 Hz), 4.31 (1H, m, overlapped), H2-5''''']; 13C NMR (C5D5N, 150 MHz)
spectroscopic data: see Tables 1a and 1b; ESI-Q-Orbitrap MS m/z 1243.63171 [MH]
(calcd for C58H99O28, 1243.63174).
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Notoginsenoside NL-E4 (4): White powder; []D 3.5 (conc 0.17, MeOH); IR max
1
(KBr) 3354, 2933, 2873, 1571, 1414, 1072, 1043 cm1; H NMR (C5D5N, 600 MHz)
spectroscopic data:  0.75, 1.53 (1H each, both m, H2-1), [1.83 (1H, m, overlapped), 2.20
(1H, m), H2-2], 3.30 (1H, dd, J = 3.6, 12.0 Hz, H-3), 0.69 (1H, br. d, ca. J = 12 Hz, H-5),
1.36, 1.47 (1H each, both m, overlapped, H2-6), [1.20 (1H, m), 1.44 (1H, m, overlapped),
H2-7], 1.36 (1H, m, overlapped, H-9), [1.48 (1H, m, overlapped), 1.95 (1H, m), H2-11],
4.08 (1H, m, H-12), 2.06 (1H, dd, J = 10.2, 10.2 Hz, H-13), [0.98 (1H, m), 1.57 (1H, m,
overlapped), H2-15], 1.42, 1.83 (1H each, both m, overlapped, H2-16), 2.52 (1H, q like, ca.
J = 11 Hz, H-17), 0.94 (3H, s, H3-18), 0.80 (3H, s, H3-19), 1.64 (3H, s, H3-21), 2.08, 2.90
(1H each, both m, H2-22), [2.11 (1H, m), 2.22 (1H, m, overlapped), H2-23], 3.84 (1H, br. d,
ca. J = 10 Hz, H-24), 1.57 (3H, s, H3-26), 1.56 (3H, s, H3-27), 1.28 (3H, s, H3-28), 1.12 (3H,
s, H3-29), 0.93 (3H, s, H3-30), 4.94 (1H, d, J = 7.8 Hz, H-1'), 4.13 (1H, dd, J = 7.8, 9.0 Hz,
10

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H-2'), 4.37 (1H, m, overlapped, H-3'), 4.10 (1H, dd, J = 9.0, 9.0 Hz, H-4'), 3.97 (1H, m,
H-5'), [4.35 (1H, m, overlapped), 4.59 (1H, br. d, ca. J = 12 Hz), H2-6'], 5.53 (1H, d, J = 7.8
Hz, H-1''), 4.21 (1H, dd, J = 7.8, 9.0 Hz, H-2''), 4.30 (1H, dd, J = 9.0, 9.0 Hz, H-3''), 4.21
(1H, m, overlapped, H-4''), 3.87 (1H, m, H-5''), [4.37 (1H, m, overlapped), 4.50 (1H, br. d,
ca. J = 10 Hz), H2-6''], 5.41 (1H, d, J = 7.2 Hz, H-1'''), 4.12 (1H, dd, J = 7.2, 9.0 Hz, H-2'''),
4.15 (1H, m, overlapped, H-3'''), 4.15 (1H, m, overlapped, H-4'''), [3.70 (1H, dd, J = 10.2,
10.2 Hz), 4.33 (1H, m, overlapped), H2-5'''], 5.13 (1H, m, overlapped, H-1''''), 3.87 (1H, dd,
J = 7.2, 9.0 Hz, H-2''''), 4.15 (1H, m, overlapped, H-3''''), 4.04 (1H, m, overlapped, H-4''''),
4.04 (1H, m, overlapped, H-5''''), [4.25 (1H, dd, J = 3.6, 12.0 Hz), 4.78 (1H, br. d, ca. J = 12
Hz), H2-6''''], 4.99 (1H, d, J = 7.2 Hz, H-1'''''), 4.06 (1H, dd, J = 7.2, 8.4 Hz, H-2'''''), 4.16
(1H, m, overlapped, H-3'''''), 4.21 (1H, m, overlapped, H-4'''''), [3.74 (1H, dd, J = 10.2, 10.2
13
Hz), 4.32 (1H, m, overlapped), H2-5''''']; C NMR (C5D5N, 150 MHz) spectroscopic data:
see Tables 1a and 1b; ESI-Q-Orbitrap MS m/z 1243.63049 [MH] (calcd for C58H99O28,
1243.63174).
25
Notoginsenoside NL-F1 (5): White powder; []D +2.8 (conc 0.65, MeOH); IR max
1
(KBr) 3358, 2940, 2877, 1568, 1387, 1076, 1035 cm1; H NMR (C5D5N, 600 MHz)
spectroscopic data:  [0.72 (1H, m), 1.52 (1H, m, overlapped), H2-1], [1.82 (1H, m,
overlapped), 2.20 (1H, m), H2-2], 3.27 (1H, dd, J = 4.2, 12.0 Hz, H-3), 0.66 (1H, br. d, ca. J
= 11 Hz, H-5), [1.34 (1H, m, overlapped), 1.47 (1H, m), H2-6], 1.17, 1.42 (1H each, both m,
H2-7), 1.35 (1H, m, overlapped, H-9), [1.51 (1H, m, overlapped), 1.93 (1H, m), H2-11],
4.14 (1H, m, overlapped, H-12), 2.11 (1H, dd, J = 10.8, 10.8 Hz, H-13), [0.95 (1H, m), 1.52
11

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(1H, m, overlapped), H2-15], 1.35, 1.82 (1H each, both m, overlapped, H2-16), 2.55 (1H, q
like, ca. J = 10 Hz, H-17), 0.93 (3H, s, H3-18), 0.80 (3H, s, H3-19), 1.63 (3H, s, H3-21),
2.47 (2H, m, H2-22), 1.91, 2.42 (1H each, both m, H2-23), 3.85 (1H, br. d, ca. J = 10 Hz,
H-24), 1.51 (3H, s, H3-26), 1.55 (3H, s, H3-27), 1.27 (3H, s, H3-28), 1.10 (3H, s, H3-29),
0.94 (3H, s, H3-30), 4.92 (1H, d, J = 7.8 Hz, H-1'), 4.26 (1H, dd, J = 7.8, 8.4 Hz, H-2'), 4.32
(1H, m, overlapped, H-3'), 4.33 (1H, m, overlapped, H-4'), 3.95 (1H, m, overlapped, H-5'),
[4.35 (1H, dd, J = 5.4, 10.5 Hz), 4.58 (1H, br. d, ca. J = 11 Hz), H2-6'], 5.39 (1H, d, J = 7.8
Hz, H-1''), 4.13 (1H, dd, J = 7.8, 8.4 Hz, H-2''), 4.26 (1H, dd, J = 7.8, 8.4 Hz, H-3''), 4.15
(1H, m, overlapped, H-4''), 3.95 (1H, m, overlapped, H-5''), [4.46 (1H, dd, J = 3.6, 11.4 Hz),
4.52 (1H, dd, J = 3.0, 11.4 Hz), H2-6''], 5.14 (1H, d, J = 7.8 Hz, H-1'''), 3.92 (1H, dd, J = 7.8,
7.8 Hz, H-2'''), 4.17 (1H, m, overlapped, H-3'''), 4.08 (1H, dd, J = 7.8, 7.8 Hz, H-4'''), 4.06
(1H, m, overlapped, H-5'''), [4.30 (1H, m, overlapped), 4.74 (1H, br. d, ca. J = 11 Hz),
H2-6'''], 5.00 (1H, d, J = 7.2 Hz, H-1''''), 4.04 (1H, dd, J = 7.2, 7.8 Hz, H-2''''), 4.17 (1H, m,
overlapped, H-3''''), 4.21 (1H, m, H-4''''), [3.74 (1H, dd, J = 9.6, 9.6 Hz), 4.32 (1H, m,
13
overlapped), H2-5'''']; C NMR (C5D5N, 150 MHz) spectroscopic data: see Tables 1a and
1b; ESI-Q-Orbitrap MS m/z 1111.58997 [MH] (calcd for C53H91O24, 1111.58948).
25
Notoginsenoside NL-F2 (6): White powder; []D +0.8 (conc 0.26, MeOH); IR max
(KBr) 3356, 2937, 2877, 1570, 1415, 1164, 1072, 1042 cm1; 1H NMR (C5D5N, 600 MHz)
spectroscopic data:  [0.75 (1H, m), 1.56 (1H, m, overlapped), H2-1], [1.83 (1H, m,
overlapped), 2.20 (1H, m), H2-2], 3.30 (1H, dd, J = 3.0, 12.0 Hz, H-3), 0.68 (1H, br. d, ca. J
= 12 Hz, H-5), 1.35, 1.45 (1H each, both m, overlapped, H2-6), [1.19 (1H, m), 1.44 (1H, m,
12

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overlapped), H2-7], 1.37 (1H, m, overlapped, H-9), [1.51 (1H, m, overlapped), 1.96 (1H, m),
H2-11], 4.15 (1H, m, overlapped, H-12), 2.11 (1H, dd, J = 10.2, 10.2 Hz, H-13), [0.97 (1H,
m), 1.55 (1H, m, overlapped), H2-15], [1.37 (1H, m, overlapped), 1.79 (1H, m), H2-16],
2.55 (1H, q like, ca. J = 10 Hz, H-17), 0.93 (3H, s, H3-18), 0.80 (3H, s, H3-19), 1.64 (3H, s,
H3-21), [2.47 (2H, m), H2-22], [1.91 (1H, m), 2.44 (1H, m, overlapped), H2-23], 3.86 (1H,
br. d, ca. J = 10 Hz, H-24), 1.51 (3H, s, H3-26), 1.55 (3H, s, H3-27), 1.28 (3H, s, H3-28),
1.11 (3H, s, H3-29), 0.95 (3H, s, H3-30), 4.94 (1H, d, J = 7.8 Hz, H-1'), 4.13 (1H, dd, J =
7.8, 9.0 Hz, H-2'), 4.36 (1H, m, overlapped, H-3'), 4.10 (1H, dd, J = 8.5, 8.5 Hz, H-4'), 3.98
(1H, m, H-5'), [4.35 (1H, m, overlapped), 4.59 (1H, br. d, ca. J = 11 Hz), H2-6'], 5.53 (1H, d,
J = 7.2 Hz, H-1''), 4.21 (1H, m, overlapped, H-2''), 4.29 (1H, dd, J = 9.6, 9.6 Hz, H-3''),
4.21 (1H, m, overlapped, H-4''), 3.89 (1H, m, H-5''), [4.37 (1H, m, overlapped), 4.51 (1H,
br. d, ca. J = 11 Hz), H2-6''], 5.40 (1H, d, J = 7.2 Hz, H-1'''), 4.11 (1H, dd, J = 7.2, 9.0 Hz,
H-2'''), 4.14 (1H, m, overlapped, H-3'''), 4.15 (1H, m, overlapped, H-4'''), [3.70 (1H, dd, J =
9.6, 9.6 Hz), 4.33 (1H, m, overlapped), H2-5'''], 5.13 (1H, m, overlapped, H-1''''), 3.92 (1H,
dd, J = 7.2, 9.0 Hz, H-2''''), 4.17 (1H, m, overlapped, H-3''''), 4.06 (1H, m, overlapped,
H-4''''), 4.05 (1H, m, overlapped, H-5''''), [4.28 (1H, dd, J = 2.4, 10.8 Hz), 4.75 (1H, br. d,
ca. J = 11 Hz), H2-6''''], 5.00 (1H, d, J = 7.2 Hz, H-1'''''), 4.05 (1H, dd, J = 7.2, 8.4 Hz,
H-2'''''), 4.16 (1H, m, overlapped, H-3'''''), 4.22 (1H, m, overlapped, H-4'''''), [3.76 (1H, dd, J
= 9.0, 9.0 Hz), 4.33 (1H, m, overlapped), H2-5''''']; 13C NMR (C5D5N, 150 MHz)
spectroscopic data: see Tables 1a and 1b; ESI-Q-Orbitrap MS m/z 1243.63147 [MH]
(calcd for C58H99O28, 1243.63174).
13

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Notoginsenoside NL-G1 (7): White powder; []D +5.5 (conc 1.01, MeOH); IR max
(KBr) 3362, 2943, 2877, 1648, 1452, 1387, 1162, 1075, 1038 cm1; 1H NMR (C5D5N, 500
MHz) spectroscopic data:  [0.77 (1H, m), 1.54 (1H, m, overlapped), H2-1], [1.83 (1H, m,
overlapped), 2.22 (1H, m), H2-2], 3.36 (1H, dd, J = 4.5, 12.0 Hz, H-3), 0.73 (1H, br. d, ca. J
= 12 Hz, H-5), 1.37, 1.51 (1H each, both m, overlapped, H2-6), [1.20 (1H, m), 1.48 (1H, m,
overlapped), H2-7], 1.39 (1H, m, overlapped, H-9), [1.54 (1H, m, overlapped), 1.96 (1H, m),
H2-11], 4.12 (1H, m, H-12), 2.04 (1H, m, overlapped, H-13), 0.98, 1.54 (1H each, both m,
overlapped, H2-15), 1.39, 1.83 (1H each, both m, overlapped, H2-16), 2.55 (1H, q like, ca. J
= 11 Hz, H-17), 0.98 (3H, s, H3-18), 0.81 (3H, s, H3-19), 1.65 (3H, s, H3-21), [1.95 (1H, m,
overlapped), 2.62 (1H, dt like, ca. J = 5, 14 Hz), H2-22], [2.04 (1H, m, overlapped), 2.33
(1H, m), H2-23], 4.45 (1H, t like, ca. J = 7 Hz, H-24), 4.93, 5.25 (1H each, both br. s,
H2-26), 1.95 (3H, s, H3-27), 1.31 (3H, s, H3-28), 0.99 (3H, s, H3-29), 0.96 (3H, s, H3-30),
4.94 (1H, d, J = 7.5 Hz, H-1'), 4.05 (1H, dd, J = 7.5, 9.0 Hz, H-2'), 4.27 (1H, dd, J = 9.0,
9.0 Hz, H-3'), 4.19 (1H, m, overlapped, H-4'), 4.00 (1H, m, H-5'), [4.40 (1H, dd, J = 5.5,
11.5 Hz), 4.60 (1H, dd, J = 2.0, 11.5 Hz), H2-6'], 5.12 (1H, d, J = 7.5 Hz, H-1''), 3.88 (1H,
dd, J = 7.5, 8.0 Hz, H-2''), 4.15 (1H, m, overlapped, H-3''), 4.03 (1H, m, overlapped, H-4''),
4.03 (1H, m, overlapped, H-5''), [4.25 (1H, m, overlapped), 4.74 (1H, br. d, ca. J = 11 Hz),
H2-6''], 4.99 (1H, d, J = 8.0 Hz, H-1'''), 4.03 (1H, m, overlapped, H-2'''), 4.15 (1H, m,
overlapped, H-3'''), 4.17 (1H, m, overlapped, H-4'''), [3.73 (1H, dd, J = 10.0, 11.5 Hz), 4.32
13
(1H, dd, J = 5.0, 11.5 Hz), H2-5''']; C NMR (C5D5N, 125 MHz) spectroscopic data: see
Tables 1a and 1b; ESI-Q-Orbitrap MS m/z 931.52795 [MH] (calcd for C47H79O18,
14

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931.52609).
25
Notoginsenoside NL-G2 (8): White powder; []D 0.6 (conc 0.72, MeOH); IR max
(KBr) 3358, 2941, 2877, 1646, 1456, 1386, 1166, 1073, 1040 cm1; 1H NMR (C5D5N, 500
MHz) spectroscopic data:  [0.74 (1H, m), 1.49 (1H, m, overlapped), H2-1], [1.83 (1H, m,
overlapped), 2.19 (1H, m), H2-2], 3.29 (1H, dd, J = 3.5, 11.0 Hz, H-3), 0.67 (1H, br. d, ca. J
= 12 Hz, H-5), [1.37 (1H, m, overlapped), 1.50 (1H, m), H2-6], 1.19, 1.45 (1H each, both m,
H2-7), 1.37 (1H, m, overlapped, H-9), 1.55, 1.95 (1H each, both m, overlapped, H2-11),
4.14 (1H, m, overlapped, H-12), 2.06 (1H, m, overlapped, H-13), 0.99, 1.55 (1H each, both
m, overlapped, H2-15), [1.39 (1H, m), 1.83 (1H, m, overlapped), H2-16], 2.57 (1H, q like,
ca. J = 10 Hz, H-17), 0.97 (3H, s, H3-18), 0.79 (3H, s, H3-19), 1.66 (3H, s, H3-21), [1.95
(1H, m, overlapped), 2.66 (1H, t like, ca. J = 5 Hz), H2-22], [2.06 (1H, m, overlapped), 2.36
(1H, m), H2-23], 4.48 (1H, t like, ca. J = 6 Hz, H-24), 4.95, 5.27 (1H each, both br. s,
H2-26), 1.97 (3H, s, H3-27), 1.28 (3H, s, H3-28), 1.11 (3H, s, H3-29), 0.96 (3H, s, H3-30),
4.96 (1H, d, J = 8.5 Hz, H-1'), 4.17 (1H, m, overlapped, H-2'), 4.38 (1H, m, overlapped,
H-3'), 4.11 (1H, dd, J = 9.0, 9.0 Hz, H-4'), 4.00 (1H, m, H-5'), [4.38 (1H, m, overlapped),
4.62 (1H, br. d, ca. J = 11 Hz), H2-6'], 5.56 (1H, d, J = 7.5 Hz, H-1''), 4.23 (1H, m,
overlapped, H-2''), 4.32 (1H, m, overlapped, H-3''), 4.23 (1H, m, overlapped, H-4''), 3.91
(1H, m, H-5''), [4.38 (1H, m, overlapped), 4.54 (1H, br. d, ca. J = 10 Hz), H2-6''], 5.44 (1H,
d, J = 7.0 Hz, H-1'''), 4.14 (1H, m, overlapped, H-2'''), 4.17 (1H, m, overlapped, H-3'''), 4.17
(1H, m, overlapped, H-4'''), [3.71 (1H, dd, J = 10.0, 10.0 Hz), 4.35 (1H, m, overlapped),
H2-5'''], 5.15 (1H, d, J = 8.0 Hz, H-1''''), 3.93 (1H, dd, J = 8.0, 8.0 Hz, H-2''''), 4.18 (1H, dd,
15

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J = 7.0, 8.0 Hz, H-3''''), 4.08 (1H, m, overlapped, H-4''''), 4.07 (1H, m, overlapped, H-5''''),
[4.29 (1H, m), 4.78 (1H, br. d, ca. J = 11 Hz), H2-6''''], 5.02 (1H, d, J = 7.5 Hz, H-1'''''), 4.07
(1H, m, overlapped, H-2'''''), 4.20 (1H, dd, J = 8.5, 8.5 Hz, H-3'''''), 4.23 (1H, m, overlapped,
13
H-4'''''), [3.76 (1H, dd, J = 11.0, 11.0 Hz), 4.35 (1H, m, overlapped), H2-5''''']; C NMR
(C5D5N, 125 MHz) spectroscopic data: see Tables 1a and 1b; ESI-Q-Orbitrap MS m/z
1225.62292 [MH] (calcd for C58H97O27, 1225.62117).
25
Notoginsenoside NL-H1 (9): White powder; []D 4.5 (conc 1.15, MeOH); IR max
(KBr) 3366, 2943, 2877, 1649, 1455, 1388, 1313, 1075, 1033 cm1; 1H NMR (C5D5N, 500
MHz) spectroscopic data:  [0.76 (1H, m), 1.53 (1H, m, overlapped), H2-1], 1.80, 2.22 (1H
each, both m, H2-2), 3.36 (1H, dd, J = 4.5, 12.0 Hz, H-3), 0.72 (1H, br. d, ca. J = 12 Hz,
H-5), [1.36 (1H, m), 1.48 (1H, m, overlapped), H2-6], [1.19 (1H, m), 1.48 (1H, m,
overlapped), H2-7], 1.39 (1H, m, H-9), [1.52 (1H, m, overlapped), 1.98 (1H, m), H2-11],
4.14 (1H, m, H-12), 2.07 (1H, dd, J = 10.5, 10.5 Hz, H-13), [0.97 (1H, m, overlapped), 1.55
(1H, m), H2-15], 1.36, 1.82 (1H each, both m, H2-16), 2.55 (1H, q like, ca. J = 10 Hz,
H-17), 0.94 (3H, s, H3-18), 0.78 (3H, s, H3-19), 1.64 (3H, s, H3-21), [2.24 (1H, dd like, ca.
J = 3, 14 Hz), 2.46 (1H, dt like, ca. J = 3, 14 Hz), H2-22], [2.05 (1H, m, overlapped), 2.32
(1H, tt like, ca. J = 3, 14 Hz), H2-23], 4.46 (1H, dd, J = 4.5, 8.0 Hz, H-24), 4.91, 5.25 (1H
each, both br. s, H2-26), 1.95 (3H, s, H3-27), 1.31 (3H, s, H3-28), 0.99 (3H, s, H3-29), 0.95
(3H, s, H3-30), 4.96 (1H, d, J = 8.0 Hz, H-1'), 4.06 (1H, dd, J = 8.0, 8.5 Hz, H-2'), 4.28 (1H,
dd, J = 8.5, 9.0 Hz, H-3'), 4.22 (1H, dd, J = 9.0, 9.0 Hz, H-4'), 4.03 (1H, m, overlapped,
H-5'), [4.42 (1H, dd, J = 5.5, 11.5 Hz), 4.63 (1H, dd, J = 2.0, 11.5 Hz), H2-6'], 5.16 (1H, d,
16

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J = 7.5 Hz, H-1''), 3.98 (1H, dd, J = 7.5, 8.0 Hz, H-2''), 4.20 (1H, dd, J = 8.0, 8.5 Hz, H-3''),
4.02 (1H, dd, J = 8.5, 8.5 Hz, H-4''), 4.03 (1H, m, overlapped, H-5''), [4.11 (1H, dd, J = 6.0,
10.5 Hz), 4.68 (1H, dd, J = 1.5, 10.5 Hz), H2-6''], 5.68 (1H, d, J = 1.5 Hz, H-1'''), 4.89 (1H,
dd, J = 1.5, 3.5 Hz, H-2'''), 4.81 (1H, dd, J = 3.5, 6.0 Hz, H-3'''), 4.78 (1H, m, H-4'''), [4.24
(1H, dd, J = 4.0, 12.0 Hz), 4.35 (1H, dd, J = 3.0, 12.0 Hz), H2-5''']; 13C NMR (C5D5N, 125
MHz) spectroscopic data: see Tables 1a and 1b; ESI-Q-Orbitrap MS m/z 931.52588 [MH]
(calcd for C47H79O18, 931.52609).
25
Notoginsenoside NL-H2 (10): White powder; []D +2.6 (conc 1.14, MeOH); IR max
(KBr) 3391, 2943, 2877, 1648, 1454, 1387, 1163, 1077, 1039 cm1; 1H NMR (C5D5N, 500
MHz) spectroscopic data:  0.78, 1.55 (1H each, both m, overlapped, H2-1), [1.83 (1H, m,
overlapped), 2.23 (1H, m), H2-2], 3.37 (1H, dd, J = 4.5, 11.5 Hz, H-3), 0.72 (1H, br. d, ca. J
= 12 Hz, H-5), [1.37 (1H, m), 1.48 (1H, m, overlapped), H2-6], [1.19 (1H, m), 1.48 (1H, m,
overlapped), H2-7], 1.38 (1H, m, H-9), [1.55 (1H, m, overlapped), 1.98 (1H, m), H2-11],
4.19 (1H, m, overlapped, H-12), 2.09 (1H, dd, J = 10.5, 10.5 Hz, H-13), 0.97, 1.55 (1H
each, both m, overlapped, H2-15), 1.37, 1.83 (1H each, both m, overlapped, H2-16), 2.55
(1H, q like, ca. J = 11 Hz, H-17), 0.93 (3H, s, H3-18), 0.79 (3H, s, H3-19), 1.65 (3H, s,
H3-21), [2.28 (1H, dd like, ca. J = 4, 13 Hz), 2.50 (1H, dt like, ca. J = 3, 13 Hz), H2-22],
[2.07 (1H, m), 2.38 (1H, tt like, ca. J = 4, 13 Hz), H2-23], 4.46 (1H, dd, J = 4.0, 8.0 Hz,
H-24), 4.90, 5.25 (1H each, both br. s, H2-26), 1.93 (3H, s, H3-27), 1.31 (3H, s, H3-28), 0.99
(3H, s, H3-29), 0.97 (3H, s, H3-30), 4.95 (1H, d, J = 7.5 Hz, H-1'), 4.06 (1H, dd, J = 7.5, 8.0
Hz, H-2'), 4.28 (1H, dd, J = 8.0, 9.5 Hz, H-3'), 4.23 (1H, dd, J = 9.0, 9.5 Hz, H-4'), 4.03
17

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(1H, m, overlapped, H-5'), [4.42 (1H, dd, J = 5.5, 11.5 Hz), 4.62 (1H, dd, J = 2.0, 11.5 Hz),
H2-6'], 5.14 (1H, d, J = 7.5 Hz, H-1''), 3.93 (1H, dd, J = 7.5, 8.5 Hz, H-2''), 4.19 (1H, dd, J
= 8.5, 9.0 Hz, H-3''), 4.09 (1H, dd, J = 9.0, 9.5 Hz, H-4''), 4.06 (1H, m, overlapped, H-5''),
[4.29 (1H, dd, J = 6.0, 11.5 Hz), 4.75 (1H, dd, J = 2.0, 11.5 Hz), H2-6''], 5.00 (1H, d, J = 7.5
Hz, H-1'''), 4.04 (1H, dd, J = 7.5, 8.0 Hz, H-2'''), 4.17 (1H, dd, J = 8.0, 8.5 Hz, H-3'''), 4.22
(1H, m, overlapped, H-4'''), [3.73 (1H, dd, J = 10.0, 11.0 Hz), 4.34 (1H, dd, J = 5.0, 11.0
Hz), H2-5''']; 13C NMR (C5D5N, 125 MHz) spectroscopic data: see Tables 1a and 1b;
ESI-Q-Orbitrap MS m/z 931.52771 [MH] (calcd for C47H79O18, 931.52609).
25
Notoginsenoside NL-H3 (11): White powder; []D 0.9 (conc 0.92, MeOH); IR max
(KBr) 3378, 2941, 2877, 1649, 1456, 1366, 1167, 1075, 1042 cm1; 1H NMR (C5D5N, 500
MHz) spectroscopic data:  [0.76 (1H, m), 1.54 (1H, m, overlapped), H2-1], [1.81 (1H, m,
overlapped), 2.16 (1H, m), H2-2], 3.28 (1H, dd, J = 4.5, 12.0 Hz, H-3), 0.68 (1H, br. d, ca. J
= 12 Hz, H-5), 1.35, 1.48 (1H each, both m, H2-6), 1.18, 1.46 (1H each, both m, H2-7), 1.36
(1H, m, overlapped, H-9), [1.50 (1H, m, overlapped), 1.96 (1H, m), H2-11], 4.16 (1H, m,
overlapped, H-12), 2.07 (1H, dd, J = 10.5, 10.5 Hz, H-13), [0.96 (1H, m), 1.53 (1H, m,
overlapped), H2-15], 1.36, 1.81 (1H each, both m, overlapped, H2-16), 2.57 (1H, q like, ca.
J = 10 Hz, H-17), 0.93 (3H, s, H3-18), 0.79 (3H, s, H3-19), 1.64 (3H, s, H3-21), [2.25 (1H,
dd like, ca. J = 4, 14 Hz), 2.47 (1H, dt like, ca. J = 4, 14 Hz), H2-22], [2.04 (1H, m), 2.34
(1H, tt like, ca. J = 4, 14 Hz), H2-23], 4.44 (1H, dd, J = 4.0, 9.5 Hz, H-24), 4.89, 5.25 (1H
each, both br. s, H2-26), 1.93 (3H, s, H3-27), 1.27 (3H, s, H3-28), 1.10 (3H, s, H3-29), 0.95
(3H, s, H3-30), 4.91 (1H, d, J = 7.5 Hz, H-1'), 4.07 (1H, dd, J = 7.5, 9.5 Hz, H-2'), 4.33 (1H,
18

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m, overlapped, H-3'), 4.08 (1H, m, overlapped, H-4'), 3.94 (1H, m, H-5'), [4.33 (1H, m,
overlapped), 4.56 (1H, dd, J = 2.0, 12.0 Hz), H2-6'], 5.49 (1H, d, J = 7.5 Hz, H-1''), 4.18
(1H, m, overlapped, H-2''), 4.27 (1H, m, overlapped, H-3''), 4.20 (1H, dd, J = 8.5, 8.5 Hz,
H-4''), 3.85 (1H, m, H-5''), [4.36 (1H, dd, J = 5.0, 12.0 Hz), 4.46 (1H, dd, J = 2.5, 12.0 Hz),
H2-6''], 5.39 (1H, d, J = 6.5 Hz, H-1'''), 4.09 (1H, m, overlapped, H-2'''), 4.12 (1H, m,
overlapped, H-3'''), 4.13 (1H, m, overlapped, H-4'''), [3.68 (1H, dd, J = 11.0, 11.0 Hz), 4.31
(1H, dd, J = 3.0, 11.0 Hz), H2-5'''], 5.12 (1H, d, J = 7.5 Hz, H-1''''), 3.92 (1H, dd, J = 7.5,
8.5 Hz, H-2''''), 4.15 (1H, dd, J = 8.5, 8.5 Hz, H-3''''), 4.04 (1H, m, overlapped, H-4''''), 4.04
(1H, m, overlapped, H-5''''), [4.26 (1H, m, overlapped), 4.73 (1H, br. d, ca. J = 11 Hz),
H2-6''''], 4.98 (1H, d, J = 7.5 Hz, H-1'''''), 4.02 (1H, dd, J = 7.5, 8.0 Hz, H-2'''''), 4.19 (1H, m,
overlapped, H-3'''''), 4.20 (1H, m, overlapped, H-4'''''), [3.72 (1H, dd, J = 9.5, 11.0 Hz), 4.34
(1H, m, overlapped), H2-5''''']; 13C NMR (C5D5N, 125 MHz) spectroscopic data: see Tables
1a and 1b; ESI-Q-Orbitrap MS m/z 1225.62134 [MH] (calcd for C58H97O27, 1225.62117).
Acid Hydrolysis of 1–11: Compounds 1–11 were hydrolyzed by 1M HCl and aqueous
layer were obtained by using the reported method [9]. As results, D-glucose and D-xylose
were detected from the acid hydrolysate of 48, 10 and 11; D-glucose and L-arabinose were
found in 1, 2, 9; while D-glucose, D-xylose, along with L-arabinose displayed in the aqueous
layer of acid hydrolysate of 3. All of the sugar were confirmed by comparison of tR with the
relative authentic samples [tR: 9.4 min (D-xylose), 10.3 min (L-arabinose), and 12.4 min
(D-glucose), all of them showed positive optical rotations].
19

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2.2. Experimental Procedures for Bioassay
The MTT and nitrite levels measurements of 1–11, as well as statistical analysis of were
conducted by using the method reported previously [9,10].
3. Results and Discussion
Various chromatographic methods including D101 macroporous resin, silica gel, ODS,
Sephadex LH-20 column chromatorgraphies (CC) and preparative HPLC chromatorgraphy
1
13
(pHPLC), kinds of spectrophotometric methods including UV, IR, H NMR, C NMR,
1H-1H COSY, HSQC, HMBC, NOESY, MS, and []D, along with chemical reactions were
used to research saponins from the 50% ethanol extract of P. notoginseng leaves. And 11
new 20(S)-protopanaxadiol (PPD) type saponins, named as notoginsenosides NL-E1NL-E4
(14), NL-F1 (5), NL-F2 (6), NL-G1 (7), NL-G2 (8), NL-H1NL-H3 (911) were obtained
from it (Figure 1).
-Figure 1-
25
Notoginsenoside NL-E1 (1) was obtained as an optically active ([]D 4.8, MeOH)
white powder. The molecular formula, C47H82O19 (m/z 949.53735 [MH]; calcd for
C47H81O19, 949.53666) of it was established on negative-ion ESI-Q-Orbitrap MS analysis.
20

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436
437
Its IR spectrum exhibited absorption maxima at 3351 and 1074 cm–1, accounting for
hydroxyl and ether function, respectively. The presence of D-glucose and L-arabinose in 1
was confirmed by acid hydrolysis and HPLC analysis [9]. The 1H and 13C NMR (Tables 1a
and 1b) spectra suggested the presence of two β-D-glucopyranosyl [δ 4.96 (1H, d, J = 7.2
Hz, H-1'), 5.14 (1H, d, J = 7.8 Hz, H-1'')] and one -L-arabinofuranosyl [δ 5.69 (1H, br. s,
13
H-1''')]. 47 signals showed in its C NMR spectrum. Except for the 17 signals assigned to
the above-mentioned sugar moieties, the other 30 resonances displayed in up field, which
indicated notoginsenoside NL-E1 (1) was a triterpene saponin. Eight methyl signals
appeared at δ 0.80, 0.91, 0.96, 1.00, 1.32, 1.65 (3H each, all s, H3-19, 30, 18, 29, 28, and 21)
and 1.57 (6H, s, H3-26 and 27), together with three oxygenated methine signals showed at δ
3.37 (1H, dd, J = 3.6, 11.4 Hz, H-3), and 3.83 (1H, br. d, ca. J = 8 Hz, H-24), 4.06 (1H, m,
H-12) suggested it was a dammarane type saponin. Seven moieties written in bold lines
were denoted according to the proton and proton correlations observed in the 1H-1H COSY
spectrum (Figure 2). And in its HMBC experiment, the long-range correlations were found
from H3-18 to C-79, C-14；H3-19 to C-1, C-5, C-9, C-10; H3-21 to C-17, C-20, C-22;
H3-26, H3-27 to C-24, C-25; H3-28 to C-35, C-29; H3-29 to C-35, C-28; H3-30 to C-8,
C-1315. Then, the planar structure of the aglycone of notoginsenoside NL-E1 (1) was
confirmed to be 3,12,20,24,25-pentahydroxy dammarane. Meanwhile, the coupling
1
constants of J2,3 (3.6, 11.4 Hz) and J12,13 (10 Hz) showed in the H NMR specrtum of 1
suggested both H-3 and H-13 were in an axial bond. In combination with the NOE
correlations between δH 0.73 (H-5) and δH 1.32 (H3-28), 1.38 (H-9), 3.37 (H-3); δH 0.91
21

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(H3-30) and δH 1.38 (H-9), 2.50 (H-17), 4.06 (H-12); δH 1.00 (H3-29) and δH 0.80 (H3-19);
δH 0.96 (H3-18) and δH 0.80 (H3-19), 2.07 (H3-13) observed in its NOESY spectrum (Figure
3), the relative configuration of its aglycone was confirmed. Moreover, according to the
cross peaks from δH 4.96 (H-1') to δC 89.0 (C-3); δH 5.14 (H-1'') to δC 83.5 (C-20); δH 5.69
(H-1''') to δC 68.4 (C-6'') (Figure 2), the connectivities between sugar and aglycone, as well
as sugar moieties each other were established.
-Figure 2-
-Figure 3-
-Table 1a-
- Table 1b-
Notoginsenosides NL-E2 (2), NL-E3 (3), as well as NL-E4 (4) were white powder. The
molecular formula of them were determined to be C53H92O24 (m/z 1111.59045 [MH];
calcd for C53H91O24, 1111.58948), C58H100O28 (m/z 1243.63171 [MH]; calcd for
C58H99O28, 1243.63174), and C58H100O28 (m/z 1243.63049 [MH]; calcd for C58H99O28,
1243.63174) on the basis of negative-ion ESI-Q-Orbitrap MS analysis, respectively. The
1
13
comparison of the H, C NMR (Tables 1a and 1b) data of compounds 24 with those of
compound 1 suggested they owned the same aglycone as 1. And the main difference
between compounds 2, 3, and 4 was the different types of substitution glycosyl. The 1H, 13C
1
NMR spectra along with 2D NMR spectra including H-1H COSY, HSQC, HMBC
22

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indicated the glycosyl groups in 24 were β-D-glucopyranosyl(1→2)-β-D-glucopyranosyl
and -L-arabinofuranosyl(1→6)-β-D-glucopyranosyl;
β-D-xylopyranosyl(1→2)-β-D-glucopyranosyl(1→2)-β-D-glucopyranosyl
-L-arabinofuranosyl(1→6)-β-D-glucopyranosyl;
and
β-D-xylopyranosy(1→2)-β-D-glucopyranosyl(1→2)-β-D-glucopyranosyl
and
β-D-xylopyranosyl(1→6)-β-D-glucopyranosyl, respectively. Moreover, the long-range
correlations were displayed from δH 4.94 (H-1') to δC 89.0 (C-3); δH 5.39 (H-1'') to δC 83.5
(C-2'); δH 5.15 (H-1''') to δC 83.4 (C-20); δH 5.71 (H-1'''') to δC 68.4 (C-6''') in
notoginsenoside NL-E2 (2); from δH 4.94 (H-1') to δC 89.0 (C-3); δH 5.54 (H-1'') to δC 82.9
(C-2'); δH 5.40 (H-1''') to δC 84.6 (C-2''); δH 5.13 (H-1'''') to δC 83.8 (C-20); δH 5.71 (H-1''''')
to δC 68.4 (C-6'''') in notoginsenoside NL-E3 (3); and from δH 4.94 (H-1') to δC 89.0 (C-3)；
δH 5.53 (H-1'') to δC 82.8 (C-2'); δH 5.41 (H-1''') to δC 84.6 (C-2''); δH 5.13 (H-1'''') to δC
83.8 (C-20); δH 4.99 (H-1''''') to δC 69.9 (C-6'''') in notoginsenoside NL-E4 (4) (Figure 2).
Consequently, the planar structure of notoginsenosides NL-E2NL-E4 (24) were
identified.
1
13
1
The H, C NMR (Tables 1a and 1b), H-1H COSY, HSQC, and HMBC spectra of
notoginsenosides NL-F1 (5) and NL-F2 (6) indicated the aglycones of them were identical
to each other. And the planar structure of them was also 3,12,20,24,25-pentahydroxy
dammarane like compounds 14. D-glucose and D-xylose were detected from their acid
hydrolysis product [9]. The long-range correlations were observed from δH 4.92 (H-1') to δC
89.1 (C-3); δH 5.39 (H-1'') to δC 83.3 (C-2'); δH 5.14 (H-1''') to δC 84.1 (C-20); δH 5.00
23

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(H-1'''') to δC 69.8 (C-6''') in the HMBC spectrum of compound 5; While, which could be
found from δH 4.94 (H-1') to δC 89.0 (C-3); δH 5.53 (H-1'') to δC 82.8 (C-2'); δH 5.40 (H-1''')
to δC 84.6 (C-2''); δH 5.13 (H-1'''') to δC 84.1 (C-20); δH 5.00 (H-1''''') to δC 69.8 (C-6'''') in
that of compound 6 (Figure 2). Then, the planar structures of notoginsenosides NL-F1 (5)
and NL-F2 (6) were denoted.
According to the above-mentioned identification, we knew that notoginsenosides
NL-E1NL-E4 (14) and notoginsenosides NL-F1 (5), NL-F2 (6) had the same planar
structure, 3,12,20,24,25-pentahydroxy dammarane. Since their chemical shifts of H-121
and
C-121
were
identical
to
those
of
known
compound,
3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-20-O-β-D-xylopyranosyl(1→6)-β-D-gl
ucopyranosyl-darmmar-25-ene-3β,12β,20S,24S-tetraol [11], the aglycone of them was
identified as (20S,24ζ)-3β,12β,20,24,25-pentahydroxy dammarane. Meanwhile, the
difference of ∆δC26 C27
notoginsenosides NL-F1 (5), NL-F2 (6) (14: 0.4 ≤ ∆δC26 C27
were displayed, which might be caused by the difference configuration of C-24. Comparing
their ∆δC26 C27 with that of
-
between notoginsenosides NL-E1NL-E4 (14) and
-
≤ 0.6; 5, 6: ∆δC26 C27 = 0.1)
-
-
3-O-[β-D-glucopyranosyl(1→2)-β-D-glucopyranosyl]-(20S,24S)-9β,19-cyc1o1anostan-3β,1
6β,20,24,25-pentao1 (∆δC26-C27 = 0.1) [12], the aglycone of notoginsenosides NL-F1 (5) and
NL-F2 (6) was denoted as (20S,24S)-3β,12β,20,24,25-pentahydroxy dammarane; and that of
notoginsenosides
NL-E1NL-E4
(14)
was
identified
to
be
(20S,24R)-3β,12β,20,24,25-pentahydroxy dammarane. On the other hand, the HPLC
24

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519
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521
retention time (tR) of notoginsenosides NL-E4 (4) and NL-F2 (6) owned the same planar
structure was 15.3 min and 11.1 min (Figure 4), repectively. Comparing with the similar
phenomenon of C-24 epimeric 20(S)-PPD type saponins (tR of 24R configuration was
longer than that of 24S configuration) [13,14], the C-24 configuration of compounds 16
was furtherly confirmed. Consequently, the structures of notoginsenosides NL-E1NL-E4
(14), NL-F1 (5), and NL-F2 (6) were elucidated. Among them, the planar structure of
notoginsenoside NL-F1 (5) had been identified [15], but the absolute configuration of it was
reported here firstly.
-Figure 4-
1
Moreover, comparising the H NMR chemical shifts of 22, 23, 26, and 27 positions of
notoginsenosides NL-E1NL-E4 (14) with those of notoginsenosides NL-F2 (5), NL-F2 (6),
it was found that the difference of C-24 configuration had great influence on the chemical
shifts of H2-22 and H2-23. And ∆δH22b H22a
while ∆δH22b H22a ⁓ 0, ∆δH23b H23a > 0.5 for 24S configuration (Table 2). Meanwhile,
∆δH26 H27 could not be used to determined the configuration of C-24.
-
> 0.8, ∆δH23b-H23a < 0.2 for 24R configuration;
-
-
-
-Table 2-
The molecular formula of notoginsenoside NL-G1 (7) was determined to be C47H80O18
25

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524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
(m/z 931.52795 [MH]; calcd for C47H79O18, 931.52609) on the basis of ESI-Q-Orbitrap
MS analysis. D-glucose and D-xylose were yielded after acid hydrolyzing [9]. Its 1H and 13C
NMR (Tables 1a and 1b) spectra displayed the signals assignable to two
β-D-glucopyranosyl [δ 4.94 (1H, d, J = 7.5 Hz, H-1'), 5.12 (1H, d, J = 7.5 Hz, H-1'')] and
one β-D-xylopyranosyl [δ 4.99 (1H, d, J = 8.0 Hz, H-1''')], seven methyl groups [δ 0.81,
0.96, 0.98, 0.99, 1.31, 1.65, 1.95 (3H each, all s, H3-19, 30, 18, 29, 28, 21 and 27)], three
oxygenate methine [δ 3.36 (1H, dd, J = 4.5, 12.0 Hz, H-3), 4.12 (1H, m, H-12), 4.45 (1H, t
like, ca. J = 7 Hz, H-24)], and one terminal ethylene [δ 4.93, 5.25 (1H each, both br. s,
H2-26)]. The NMR data for aglycone of 7 were remarkably reminiscent of those of
compounds 16 besides C-2227. One terminal ethylene appeared, but one methyl and one
oxygenate quaternary carbon disappeared in the aglycone of 7 comparing with 16.
Furthermore, according to the cross peaks from δH 4.93, 5.25 (H2-26) to δC 18.2 (C-27),
76.1 (C-24), 149.5 (C-25); δH 1.95 (H3-27) to δC 76.1 (C-24), 110.4 (C-26), 149.5 (C-25)
observed in its HMBC experiment (Figure 5), the aglycone of notoginsenoside NL-G1 (7)
was clarified to be (20S,24ζ)-3β,12β,20,24-tetrahydroxy-dammar-25-ene.
-Figure 5-
1
13
Comparing the H, C NMR (Tables 1a and 1b) data of notoginsenoside NL-G2 (8)
with those of 7, it could be revealed that they had exactly the same aglycone. In
combination with 2D NMR (1H-1H COSY, HSQC, HMBC) determination, one more
26

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551
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557
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561
562
563
β-D-xylopyranosyl(1→2)-β-D-glucopyranosyl [δ 5.44 (1H, d, J = 7.0 Hz, H-1'''), 5.56 (1H,
d, J = 7.5 Hz, H-1'')] was found to appear in 8. The resonance of C-2' was shifted down
field by 6.8 ppm comparing with 7, which suggested the linkage position of
β-D-xylopyranosyl(1→2)-β-D-glucopyranosyl was C-2'. The correlation from δH 5.56 (H-1'')
to δC 82.6 (C-2') observed in its HMBC spectrum (Figure 4) clarified the above description.
The 1H and 13C NMR (Tables 1a and 1b) and 2D NMR spectra including 1H-1H COSY,
HSQC, HSQC-TOCSY, and HMBC analysis indicated the aglycone of notoginsenosides
NL-H1NL-H3 (911) was idenentical to each other, whose planar structure was the same
as that of notoginsenosides NL-G1 (7) and NL-G2 (8). Meanwhile, the substituted glycosyl
in notoginsenoside NL-H2 (10) was in accordance with that of notoginsenoside NL-G1 (7);
and the similar situation was found between notoginsenoside NL-H3 (11) and
1
13
notoginsenoside NL-G2 (8). However, the H and C NMR spectra of notoginsenoside
NL-H1 (9) revealed the presence of two β-D-glucopyranosyl [δ 4.96 (1H, d, J = 8.0 Hz,
H-1'), 5.16 (1H, d, J = 7.5 Hz, H-1'')] and one -L-arabinofuranosyl [δ 5.68 (1H, d, J = 1.5
Hz, H-1''')]. Moreover, the cross peaks from δH 4.96 (H-1') to δC 88.8 (C-3), δH 5.16 (H-1'')
to δC 83.7 (C-20) and δH 5.68 (H-1''') to δC 68.3(C-6'') were displayed for 9; δH 4.95 (H-1')
to δC 88.8 (C-3); δH 5.14 (H-1'') to δC 83.7 (C-20); δH 5.00 (H-1''') to δC 69.9(C-6'') were
observed for 10; δH 4.91 (H-1') to δC 89.0 (C-3); δH 5.49 (H-1'') to δC 83.1(C-2'); δH 5.39
(H-1''') to δC 84.6 (C-2''); δH 5.12 (H-1'''') to δC 83.7 (C-20); δH 4.98 (H-1''''') to δC 69.9
(C-6'''') were found for 11 in their HMBC experiments (Figure 4), respectively. Then, the
planar structures of notoginsenosides NL-H1NL-H3 (911) were determined.
27

564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
Comparing the NMR data and chromatographic retention behavior of 7 with 10, and 8
with 11, the following results: (1) ΔδH22b-H22a > 0.6 for 7 and 8, < 0.3 for 10 and 11 (Table 3);
(2) δC-26: 7 > 10, 8 > 11 (Table 3); (3) tR: 7 > 10, 8 > 11 (Figures 6 and 7) were obtained.
These differences were similar to those of C-24 epimeric ginsenosides, 24R-ginsenoside
M7cd and 24S-ginsenoside M7cd (Table 3) [13]. Then, the aglycone of notoginsenosides
NL-G1
(20S,24R)-3β,12β,20,24-tetrahydroxydammar-25-ene; while that of notoginsenosides
NL-H1NL-H3 (911) was elucidated as
(7)
and
NL-G2
(8)
was
denoted
to
be
(20S,24S)-3β,12β,20,24-tetrahydroxydammar-25-ene.
-Figure 6-
-Figure 7-
-Table 3-
Moreover, the NO inhibitory effects of compounds 111 were examined in
LPS-stimulated RAW264.7 cells. Pretreatment of compounds 27, 10 at noncytotoxic
concentration (25 µM) (Figure S72) decreased the NO production significantly (Table 4)
comparing with control group, which indicated that they had the anti-inflammatory activity.
The activity comparison of compounds 17 and 10 indicated that the inhibition of
triterpenoids on NO production increased with the increase of the number of substituted
sugars (4 > 2 > 1; 6 > 5); comparing the activities of 4 and 6, it was found that the
28

585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
configuration of 24-OH in the structure of 24,25-dihydroxy-substituted saponins had a
strong influence on the activity (24S > 24R).
-Table 4-
Moreover, the inhibitory activities of compounds 27 and 10 were in dose-dependent
at 1, 10, and 25 μm. Especially, compound 6 still possessed strong biological activity at 1
μM (Figure 8).
-Figure 8-
4. Conclusion
In conclusion, 11 new 20(S)-PPD type saponins, notoginsenosides NL-E1NL-E4 (14),
NL-F1 (5), NL-F2 (6), NL-G1 (7), NL-G2 (8), NL-H1NL-H3 (911) were obtained from the
50% ethanol extract of P. notoginseng leaves.
Among them, the saponins with aglycone, (20S,24ζ)-3β,12β,20,24,25-pentahydroxy
dammarane, were only found from the leaves of P. notoginseng [5,7,16]. The characteristics
could be used to distinguish the extracts of P. notoginseng leaves from its other medicinal
parts such as roots, rhizomes, flowers or seeds.
Moreover,
the
rules
of
determining
C-24
configuration
of
29