Neuroprotective triterpene saponins from the leaves of Panax notoginseng

Article abstract of DOI:10.1080/14786419.2019.1677657

Two new triterpene saponins, namely notoginsenoside Ng5 (1) and notoginsenoside Ng6 (2) were isolated from the leaves of Panax notoginseng, along with five known ones. Their structures were determined by chemical methods, NMR and X-ray experiments. The absolute configuration of compound 3 with four sugar units was confirmed by single crystal X-ray analysis. Compounds 2–4 and 6 inhibited PC12 cell damage induced by serum deprivation, and increased cell viability from 58.7 ± 6.7% to 66.7 ± 4.5%, 76.1 ± 6.1%, 64.7 ± 5.2% and 67.2 ± 5.0% at 10 μM, respectively.

Full text of DOI:10.1080/14786419.2019.1677657

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Neuroprotective triterpene saponins from the
leaves of Panax notoginseng
Ji-Wu Huang, Yi-Qian Du, Chuang-Jun Li, Jing-Zhi Yang, Jie Ma, Ying-Da Zang,
Nai-Hong Chen & Dong-Ming Zhang
To cite this article: Ji-Wu Huang, Yi-Qian Du, Chuang-Jun Li, Jing-Zhi Yang, Jie Ma, Ying-Da
Zang, Nai-Hong Chen & Dong-Ming Zhang (2019): Neuroprotective triterpene saponins from the
leaves of Panaxꢀnotoginseng, Natural Product Research, DOI: 10.1080/14786419.2019.1677657
To link to this article: https://doi.org/10.1080/14786419.2019.1677657
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1677657
Neuroprotective triterpene saponins from the leaves of
Panax notoginseng
Ji-Wu Huang, Yi-Qian Du, Chuang-Jun Li, Jing-Zhi Yang, Jie Ma, Ying-Da Zang,
Nai-Hong 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, China
ABSTRACT
ARTICLE HISTORY
Received 8 September 2019
Accepted 21 September 2019
Two new triterpene saponins, namely notoginsenoside Ng5 (1)
and notoginsenoside Ng6 (2) were isolated from the leaves of
Panax notoginseng, along with five known ones. Their structures
were determined by chemical methods, NMR and X-ray experi-
ments. The absolute configuration of compound 3 with four sugar
units was confirmed by single crystal X-ray analysis. Compounds
2–4 and 6 inhibited PC12 cell damage induced by serum depriv-
ation, and increased cell viability from 58.7 6.7% to 66.7 4.5%,
76.1 6.1%, 64.7 5.2% and 67.2 5.0% at 10 lM, respectively.
KEYWORDS
Panax notoginseng leaves;
triterpene saponins;
neuroprotective
CONTACT Dong-Ming Zhang
zhangdm@imm.ac.cn
Ji-Wu Huang and Yi-Qian Du contributed equally to the work.
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1677657.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

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J.-W. HUANG ET AL.
1. Introduction
Panax notoginseng, also referred to as ‘Chinese ginseng’, is a perennial herb with dark
green leaves widely cultivated in Southwestern China. To date, more than 200 chem-
ical compounds have been isolated from this plant and its endophytic fungi, most of
them are the triterpene saponins (Wang et al. 2016; Jin et al. 2017). Recent studies
have shown that Panax notoginseng (P. notoginseng) saponins possessed various bio-
logical effects, including neuroprotection (Liu et al. 2017), anti-cancer (Gu et al. 2017),
anti-depression (Zhang et al. 2016), anti-inflammation (Li et al. 2019) and inhibiting
vascular smooth muscle cell proliferation (Fang et al. 2018), etc. After firstly reporting
the isolation of three novel tricyclic tetranordammarane saponins from the P. notogin-
seng leaves (Liu et al. 2018), we continue with our investigation to further enrich the
diversity of bioactive saponins of this plant. Herein, we present the discovery of seven
dammarane-type saponins, including two new saponins, termed notoginsenoside Ng5
(1) and notoginsenoside Ng6 (2), and five known saponins (3–7) from the ethanolic
and water extracts of this herb. As an important source of neuroprotective constitu-
ents, plants are receiving much attention from science world (Carito et al. 2014; Les
et al. 2017; Venditti and Bianco 2019). In this study, we evaluated, in the serum depriv-
ation-induced PC12 cell damage model, the neuroprotective effects of compounds
1–7 at 10 lM. Owing to the difficulty obtaining high quality crystal, single crystal of
saponins were seldom reported. Here, for the first time, a single crystal of compound
3
was reported to confirm the absolute configuration of 12(R),23(R)-epoxy
dammarane-type saponin.
2. Results and discussion
Compound 1 was isolated as a white powder, [a]2D5 –8.8 (c 0.10, CH3OH). Its formula
was established as C57H94O23 by HR-ESI-MS data: m/z 1169.6059 [M þ Na]þ; calcd for
C57H94O23Na, 1169.6078. The presence of hydroxyl, ester and olefin groups were
deduced by the adsorption bands at 3360, 1712 and 1657 cmꢀ1 in its IR spectrum. In
13C NMR spectrum (Table S1, supplementary material), 57 carbon signals were
observed, 30 of them were assigned to the aglycone, including eight methyl carbons
(dC 16.0, 16.2, 16.5, 17.4, 17.9, 22.2, 25.8 and 28.0), three oxygen substituted carbons
(dC 70.1, 83.5 and 89.2) and a pair of olefinic carbons (dC 125.9 and 131.0). According
to the HSQC spectrum, the proton signals could be assigned to the above 30 carbons,
indicating the sapogenin of 1 was 20(S)-protopanaxadiol (He et al. 2005). Four sugar
units were suggested from four pair anomeric protons/carbons at dH 4.90 (1H, d,
J ¼ 7.2 Hz, H-10)/dC 104.9, dH 5.32 (1H, d, J ¼ 7.8 Hz, H-100)/dC 106.2, dH 5.12 (1H, d,
1
J ¼ 7.2 Hz, H-1000)/dC 98.0 and dH 4.97 (1H, d, J ¼ 7.2 Hz, H-10000)/dC 105.7 in H and 13C
NMR spectra (Table S1, supplementary material). Their absolute configurations were
proved to be D-glucose and D-xylose by the acid hydrolysis and GC analysis of 1.
Besides, the positions and sequences of the sugar moieties were confirmed by HMBC
spectrum according to the correlations (Figure S6, supplementary material) from H-10
to C-3 (dC 89.2), H-100 to C-20 (dC 84.2), H-1000 to C-20 (dC 83.5), and H-10000 to C-6000 (dC
69.9). Furthermore, apart from 53 carbons due to the skeleton and sugars, the remain-
ing signals of 1 were assigned to a crotonic group (dC 17.8, 123.2, 144.7 and 166.6) (Li

NATURAL PRODUCT RESEARCH
3
Figure 1. Structures of compounds 1 and 2.
et al. 2018), and it was placed at C-600 position of glucose according to the HMBC cor-
relations from H-600 [dH 4.99 (d, 11.4), 4.88 (m)] to C-100000 (dC 166.6). Consequently, the
structure of compound 1 was deduced as 3-O-f6-O-[(E)-but-2-enoyl]-b-D-glucopyrano-
syl-(1!2)-b-D-glucopyranosylg-3b,12b,20(S)-trihydroxy-dammar-24-ene 20-O-b-D-xylo-
pyranosyl-(1!6)-b-D-glucopyranoside (notoginsenoside Ng5) (Figure 1).
Compound 2 was isolated as a white powder, [a]2D5þ 27.8 (c 0.10, CH3OH). Its for-
mula was established as C58H98O27 by HR-ESI-MS data: m/z 1249.6182 [M þ Na]þ; calcd
for C58H98O27Na, 1249.6188. The presence of hydroxyl and olefin groups were deduced
1
by the adsorption bands at 3385 and 1647 cmꢀ1 in its IR spectrum. Its H NMR spec-
trum (Table S1, supplementary material) presented eight aglycone methyl singlets at
dH 0.83, 0.89, 1.00, 1.11, 1.29, 1.57, 1.57 and 1.61, and two olefinic proton signals at dH
6.10 (1H, d, J ¼ 15.6 Hz) and 6.22 (1H, m). Two olefinic carbons (dC 122.7 and 142.4)
and five anomeric carbons (dC 98.3, 103.2, 104.3, 104.8 and 106.4) were observed from
the 13C NMR spectrum (Table S1, supplementary material) of 2. By comparing the 13C-
1
and H-NMR data with references, the core of compound 2 was identical to the agly-
cone of notoginsenoside Fh5 (Liu et al. 2017). The absolute configurations of sugars
were proved to be D-glucose, D-xylose and L-arabinose by the acid hydrolysis and GC
experiments of 2. Furthermore, the positions and sequences of the sugar moieties
were established by the correlations from H-10 (1H, 4.95, d, J ¼ 6.3 Hz) to C-3 (dC 88.9),
H-100 (1H, 5.54, d, J ¼ 7.8 Hz) to C-20 (dC 83.0), H-1000 (1H, 5.44, d, J ¼ 6.3 Hz) to C-200 (dC
84.5), H-10000 (1H, 5.19, d, J ¼ 7.8 Hz) to C-20 (dC 83.4), and H-100000 (1H, 5.01, d,
J ¼ 6.0 Hz) to C-60000 (dC 69.1) in HMBC spectrum (Figure S15, supplementary material).
Finally, in the view of the above evidences, compound 2 was established as 3-O-b-D-
xylopyranosyl-(1!2)-b-D-glucopyranosyl-(1!2)-b-D-glucopyranosyl-3b,12b,20(S),
25-
tetrahydroxydammar-23-ene
20-O-a-L-arabinopyranosyl-(1!6)-b-D-glucopyranoside
(notoginsenoside Ng6) (Figure 1).
Compound 3 was isolated as a colourless crystal (MeOH/H2O, 1:1). Its formula was
established as C53H88O22 by HR-ESI-MS data: m/z 1075.5673 [M ꢀ H]–; calcd for C53H87O22,
1
1075.5694. Its H NMR spectrum presented eight aglycone methyls from corresponding
singlets at dH 0.82, 0.93, 1.08, 1.11, 1.28, 1.50, 1.68 and 1.82 and an olefinic proton from

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J.-W. HUANG ET AL.
Figure 2. ORTEP drawings of compound 3.
singlet at dH 5.54 (1H, d, J ¼ 7.2 Hz). Its 13C NMR spectrum presented two olefinic carbons
(dC 129.2 and 131.1) and four anomeric carbons (dC 99.3, 105.1, 106.0 and 106.3). By com-
paring their NMR data with those of literature, compound 3 was determined as quinquefo-
loside-Lb (Jiang et al. 2008). Finally, according to the X-ray crystallography analysis [Cu Ka,
Flack parameter: 0.03 (8)] of 3, its absolute configuration was unquestionably confirmed,
as depicted in Figure 2. Crystallographic data of 3 can be accessed via the Cambridge
Crystallographic Data Centre (CCDC: 1897315).
The remaining saponins were established as notoginsenoside LX (4) (Li et al. 2014),
ginsenoside F3 (5) (Li et al. 2016), notoginsenoside LK1 (6) (Li et al. 2019) and notogin-
senoside Fc (7) (Yang et al. 1983). All the isolated compounds were tested for their
neuroprotective effect on serum free induced PC12 cell. Compounds 2–4 and 6 were
active against serum deficiency induced PC12 cell damage (Figure S1, supplementary
material), and increased cell viability at 10 lM from 58.7 6.7% to 66.7 4.5%,
76.1 6.1%, 64.7 5.2% and 67.2 5.0%, respectively.
3. Experimental section
3.1. Plant materials
Leaves of P. notoginseng were acquired in Wenshan, Yunnan province of China, in
2015, and authenticated by associate Prof. Lin Ma of our school. A sample (ID-22816)
of P. notoginseng leaves has been deposited at our herbarium.

NATURAL PRODUCT RESEARCH
5
3.2. Instruments and chemicals
See SI-1 in supplementary material.
3.3. Extraction and isolation
See SI-2 in supplementary material. The air-dried leaves of P. notoginseng (25 kg) were
successively extracted with ethyl alcohol and water. The ethanolic extract was applied
to a diatomite column, a D101 column, silica gel column, MPLC system and p-HPLC to
give compounds 3 (25 mg), 4 (18 mg), 5 (6 mg), 6 (10 mg), 7 (8 mg). And the water
extract was subjected to a PRP-512B column, Sephadex LH-20 gel CC, MPLC system
and p-HPLC to obtain compounds 1 (20 mg) and 2 (8 mg).
3.3.1. Notoginsenoside Ng5 (1)
White powder, [a]2D5–8.8 (c 0.10, CH3OH); UV (CH3OH) kmax (log e): 203 (3.22) nm; IR
(microscope) ꢀmax: 3360, 2936, 1712, 1657, 1384, 1074 cmꢀ1; HRESIMS m/z 1169.6059
1
[M þ Na]þ (calcd for C57H94O23Na, 1169.6078); 13C NMR (150 MHz, C5D5N) and H NMR
(600 MHz, C5D5N) spectral data are listed in Table S1, supplementary material.
3.3.2. Notoginsenoside Ng6 (2)
White powder, [a]2D5þ 27.8 (c 0.10, CH3OH); UV (CH3OH) kmax (log e): 203 (3.25) nm; IR
(microscope) ꢀmax: 3385, 2970, 2938, 1647, 1388, 1079, 1047 cmꢀ1; HRESIMS m/z
1249.6182 [M þ Na]þ (calcd for C58H98O27Na, 1249.6188); 13C NMR (150 MHz, C5D5N)
and 1H NMR (600 MHz, C5D5N) spectral data are listed in Table S1, supplemen-
tary material.
3.4. Acid hydrolysis of new saponins (1 and 2)
See SI-3 in supplementary material.
3.5. Absolute configuration of sugars
See SI-4 in supplementary material.
3.6. Neuroprotection bioassays
Neuroprotection bioassays were carried out as described by Li, Chen, et al (Li et al.
2011), and compounds 1–7 were tested for neuroprotective activity against serum
deprivation induced PC12 cell by using MTT method. Results were expressed as
the means SD.
4. Conclusions
In this chemical investigation of P. notoginseng leaves led to the discovery of seven tri-
terpene saponins, including two new saponins (1 and 2), together with five known
ones (3 2 7). The single crystal data of 12(R),23(R)-epoxy dammarane-type saponin (3)

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J.-W. HUANG ET AL.
was reported for the first time. Bioactive experiment results revealed that compounds
2–4 and 6 showed moderate neuroprotective effects on serum deficiency treated
PC12 cell at 10 lM, but their structure–activity relationship still need to be explored.
Disclosure statement
All authors declare no conflicts of interest.
Funding
This work was supported by grants from the National Natural Science Foundation of China (No.
81730093), CAMS Innovation Fund for Medical Sciences (No. 2016-I2M-2-003 and 2017-I2M-3-
010), and independent project of State Key Laboratory of Bioactive Substance and Function of
Natural Medicines (No. GTZA201803).
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