Two new dammarane-type triterpenoid saponins from notoginseng medicinal fungal substance

Article abstract of DOI:10.1080/10286020.2016.1189907

Two new dammarane-type triterpenoid saponins, namely ginsenoside Rk₆(1) and ginsenoside-Rh₂₂(2), were isolated from notoginseng medicinal fungal substance. The structures of 1 and 2 were established as 3β,6α,12β,26-tetrahydroxydammar

Full text of DOI:10.1080/10286020.2016.1189907

Journal of Asian Natural Products Research
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Two new dammarane-type triterpenoid saponins
from notoginseng medicinal fungal substance
Wei Xu, Xin Liu, Xiao-Li Liu, Ai-Ling Jia, Xin-Wen Wang & Zhi-dong Qiu
To cite this article: Wei Xu, Xin Liu, Xiao-Li Liu, Ai-Ling Jia, Xin-Wen Wang & Zhi-dong Qiu
(2016): Two new dammarane-type triterpenoid saponins from notoginseng medicinal fungal
substance, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2016.1189907
To link to this article: http://dx.doi.org/10.1080/10286020.2016.1189907
Published online: 07 Jun 2016.
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Date: 19 June 2016, At: 21:01

Journal of asian natural Products research, 2016
http://dx.doi.org/10.1080/10286020.2016.1189907
Two new dammarane-type triterpenoid saponins from
notoginseng medicinal fungal substance
Wei Xu1, Xin Liu1, Xiao-Li Liu, Ai-Ling Jia, Xin-Wen Wang and Zhi-dong Qiu
college of Pharmacy, changchun university of chinese Medicine, changchun 130017, china
ABSTRACT
ARTICLE HISTORY
received 21 March 2016
accepted 11 May 2016
Two new dammarane-type triterpenoid saponins, namely ginsenoside
Rk6 (1) and ginsenoside-Rh22 (2), were isolated from notoginseng
medicinal fungal substance. The structures of 1 and 2 were established
as 3β,6α,12β,26-tetrahydroxydammar-20(21),24(25)(E)-diene-6-O-β-
D-glucopyranoside and 3β,6α, 20(S)-trihydroxy-12(R),23(R)-expoxy-
13(S),17(S)-dammar-24-ene-6-O-β-D-glucopyranoside on the basis
of spectroscopic analysis and chemical analysis, respectively.
KEYWORDS
araliaceae; notoginseng;
triterpenoid saponin;
ginsenoside rk6;
ginsenoside-rh22
1. Introduction
e main roots and rhizomes of Panax notoginseng (Burk.) F. H. Chen (Araliaceae) (San Qi)
are widely used as a renowned traditional Chinese medicine prescribed for the treatment
of hemoptysis, traumatic bleeding, vomiting, and blood in the stool [1]. To date, over 80
triterpenoid saponins have been isolated and reported from P. notoginseng [2]. It is well
known that rare ginsenosides have better performances in bioactivity and bioavailability
than others. Biotransformation was considered to be a highly efficient and regiospecific
method to preparation of rare saponins [3]. In our previous study, the notoginseng medicinal
fungal substance (NMFS) was obtained by the incubation of the main roots and rhizomes
of P. notoginseng with the complex enzyme system in natural microorganisms, by which
the saponins can be transformed into rare saponins, and some of them are new active
ingredients [4]. In the course of exploring biologically active components from NMFS,
triterpenoid saponins and phytosterol had been obtained [5,6]. As a continuous study of
this project, two new dammarane-type triterpenoid saponins, namely ginsenoside Rk6 (1)
and ginsenoside-Rh22 (2) (Figure 1), were isolated. We herein report the isolation and
structural elucidation of these two new saponins.
2. Results and discussion
Compound 1 was obtained as a white amorphous powder. Its molecular formula was deter-
mined as C36H60O9 based on the negative precursor ion observed at m/z 671.3929 [M + Cl]− in
the HR-ESI-MS, indicating seven degrees of unsaturation. e 1H and 13C NMR spectroscopic
CONTACT Zhi-dong Qiu
13321549939@163.com
1these authors contributed to the work equally and should be regarded as co-first authors.
© 2016 informa uK limited, trading as taylor & francis Group

2
W. Xu eT AL.
24
22
26
21
24
20
17
OH
21
O
OH
27
OH
20
H
25
11
O
13
19
18
7
H
1
10
15
3
HO
HO
5
O
OH
OH
O
O
HO
HO
HO
HO
OH
OH
Figure 1. structures of compounds 1 and 2.
Table 1. 1h (500 Mhz) and 13c nMr (125 Mhz) spectral data of 1 and 2 (measured in cd3od).
1
2
Position
δC
δH (J = Hz)
δC
δH (J = Hz)
1
2
3
4
5
6
7
8
40.0
27.2
79.5
40.2
61.5
80.6
45.2
41.8
51.1
40.1
32.4
73.5
52.4
51.8
32.9
31.3
48.5
17.2
17.6
155.6
108.4
34.1
27.0
126.6
135.6
68.7
13.5
31.1
15.8
16.7
105.2
75.2
78.8
71.4
77.4
62.6
1.03–1.09 (m), 1.70–1.78 (m)
1.55–1.63 (m), 1.63–1.71 (m)
39.9
27.3
79.5
40.2
61.6
80.6
45.2
41.5
51.1
40.2
30.1
81.4
50.0
51.8
31.0
24.8
48.5
17.1
17.7
74.8
25.8
54.4
73.1
128.2
134.0
25.4
18.2
31.0
17.7
16.7
105.2
75.2
78.8
71.4
77.4
62.6
1.04–1.10 (m), 1.68–1.74 (m)
1.55–1.63 (m), 1.60–1.68 (m)
3.11 (dd, 10.0, 5.0)
3.10 (dd, 10.0, 5.0)
–
1.11 (d, 10.0)
4.10 (td, 10.0, 5.0)
1.60–1.68 (m), 2.03–2.11 (m)
–
1.48 (br. d, 10.0)
–
1.16–1.24 (m), 1.75–1.81 (m)
3.53–3.63 (m)
1.84 (t, 10.0)
–
1.14–1.20 (m), 1.66–1.74 (m)
1.40–1.46 (m), 1.98–2.04 (m)
2.54–2.62 (m)
1.13 (s)
0.99 (s)
–
4.70 (s), 4.89 (s)
2.05–2.11 (m), 2.15–2.21 (m)
2.14–2.22 (m), 2.24–2.32 (m)
5.45 (t, 5.0)
–
1.11–1.15 (m)
4.09 (td, 10.0, 5.0)
1.59–1.67 (m), 2.03–2.11 (m)
–
1.49–1.55 (m)
–
1.10–1.18 (m), 1.78–1.84 (m)
3.44 (td, 10.0, 5.0)
1.41 (t, 10.0)
–
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1′
1.30–1.36 (m), 1.80–1.88 (m)
1.72–1.78 (m), 1.80–1.86 (m)
2.48–2.56 (m)
1.07 (s)
0.98 (s)
–
1.18 (s)
1.84–1.90 (m), 1.90–1.96 (m)
4.44 (br. t, 10.0)
5.12 (d, 10.0)
–
–
3.92 (s)
1.67 (s)
1.33 (s)
1.00 (s)
0.94 (s)
4.35 (d, 7.5)
3.21 (t, 5.0)
1.70 (s)
1.67 (s)
1.33 (s)
0.98 (s)
0.99 (s)
4.35 (d, 7.5)
3.21 (t, 5.0)
3.31–3.37 (m)
3.23–3.31 (m)
3.23–3.31 (m)
3.62–3.68 (m),
3.81(br. d, 10.0)
2′
3′
4′
5′
3.31–3.37 (m)
3.23–3.31 (m)
3.23–3.31 (m)
3.63–3.69 (m),
3.83 (dd, 10.0, 5.0)
6′
data (Table 1) of 1 indicated the presence of 36 C-atom signals, corresponding to six singlet
methyls, two double bonds, eight sp3 methylenes, four aliphatic sp3 methines, three oxy-
gen-bearing methines, four aliphatic quaternary carbons, and one oxygen-bearing methylene,
which were confirmed by HMQC and DEPT experiments. ese data revealed compound 1

JouRnAL of AsiAn nATuRAL PRoduCTs ReseARCH
3
OH
OH
O
OH
H
HO
OH
O
O
OH
OH
O
O
OH
OH
HO
HO
OH
OH
Figure 2. Key hMBc correlations of compounds 1 and 2.
to be a dammarane-type triterpenoid, similar to ginsenoside Rk3 [7]. Six carbon signals at
δC 105.2, 75.2, 78.8, 71.4, 77.4, and 62.6 in the 13C NMR spectrum and one anomeric proton
signal at δH 4.35 (1H, d, J = 7.5 Hz) in the 1H NMR spectrum were detected, which suggested
the presence of one β-glucopyranosyl group. is assumption was confirmed by a detailed
analysis of the 1H NMR, HMBC, and HMQC data. Furthermore, acid hydrolysis of 1 fur-
nished d-glucose, identified through GC analysis [8]. e glycosylation site was defined by
the HMBC correlation H-1′/C-6 (δC 80.6) (Figure 2). e spectral evidence from HMQC and
HMBC indicated 1 to be very similar to ginsenoside Rk3. e only difference between the two
compounds was the appearance of an additional oxygen atom in 1. In comparison with the
corresponding resonance C-26 (δC 25.7) in ginsenoside Rk3, C-26 in 1 shifed downfield to δC
68.7 indicating the oxygen atom was attached to C-26, forming a hydroxyl group. is inference
was also confirmed in change of chemical shif of C-27 (ginsenoside Rk3: δC 17.3, 1: δC 13.5).
In the 13C NMR spectrum of 1, the signals of C-26 and C-27 were observed at δC 68.7 and
13.5, respectively, suggesting that the C-24 and C-25 double bond in 1 have an E-configuration
[9]. Ultimately, the chemical structure of 1 was established as 3β,6α,12β,26-tetrahydroxy-
dammar-20(21),24(25)(E)-diene-6-O-β-D-glucopyranoside, designated as ginsenoside Rk6.
Compound 2 was obtained as a white amorphous powder. Its molecular formula was
determined as C36H60O9 based on the negative precursor ion observed at m/z 671.3929
1
[M + Cl]− in the HR-ESI-MS, indicating seven degrees of unsaturation. e H and 13C
NMR spectroscopic data (Table 1) of 2 suggested the presence of 36 carbon signals, corre-
sponding to eight singlet methyls, one double bond, seven sp3 methylenes, four aliphatic sp3
methines, four oxygen-bearing methines, four aliphatic quaternary carbons, and one oxy-
gen-bearing quaternary carbons, which were further confirmed by the HMQC and DEPT
experiments. ese data revealed compound 2 to be a dammarane-type triterpenoid similar
to ginsenoside-Rh9 [10]. e monosaccharide group in 2 was determined as β-D-glucosyl
substituent using the same method as that employed in 1. e NOESY correlations between
H-30 with H-17 and H-12, but not with H-13, indicated the configurations of C-12, C-17,
and C-13 were R, S, and S, respectively. e NOESY correlation of H-23 with H-12, but
not with H-21, indicated the configurations of C-23 and C-20 were R and S, respectively.
e only difference is the position of the glucose unit between 2 and ginsenoside-Rh9. e
glycosylation site was affirmed by the HMBC correlation of H-1′/C-6 (Figure 2). erefore,
the chemical structure of 2 was established as 3β,6α, 20(S)-trihydroxy-12(R),23(R)-expoxy-
13(S),17(S)-dammar-24-ene-6-O-β-D-glucopyranoside, designated as ginsenoside-Rh22.

4
W. Xu eT AL.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured on a Jasco DIP-140 digital polarimeter (Jasco, Tokyo, Japan)
at 20 °C. e 1D and 2D NMR spectra were recorded on a Bruker AV 500 spectrometer
(Bruker, Fallanden, Switzerland) in CD3OD. Chemical shifs were referenced to the mul-
tiplet signals of CD3OD residual (δH 3.31 and δC 48.7). e HR-ESI-MS data were meas-
ured on a ACQUITY I-Class UPLC®/Xevo® G2-S QTOF LC-MS system (Waters, Milford,
MA, USA) mass spectrometer. GC analysis for the determination of the sugar moiety was
performed on an Agilent GC-7820 A coupled with a FID detector with a HP-5 capillary
column (0.32 mm × 30 m, 0.25 μm, Agilent, Santa Clara, USA). Semi-preparative HPLC
was performed on a Hitachi instrument (pump LC-2130, UV detector LC-2030, Hitachi,
Tokyo, Japan) equipped with a YMC-Pack ODS-AM column (10 mm × 300 mm, 5 μm,
YMC, Tokyo, Japan) at a flow rate of 1.2 ml/min. Column chromatography was performed
on silica gel (100–200 mesh, Qingdao Marine Chemical Co., Ltd, Qingdao, China). in
layer chromatography (TLC) was conducted on silica gel plates (GF254, Qingdao Marine
Chemical Co., Ltd, Qingdao, China) and RP-18 F254 (Merck, Darmstadt, Germany).
3.2. Plant material
San Qi was purchased from Ji Lin An Pharmaceutical Co., Ltd. (Jilin Province, China)
in March, 2013. Authentication of San Qi was performed by professor Li-Li Weng from
Changchun University of Chinese Medicine. A voucher specimen (No. SQ03222013) was
deposited at College of Pharmacy, Changchun University of Chinese Medicine. NMFS was
obtained by biotransformation of notoginseng.
3.3. Extraction and isolation
e dried NMFS (5.0 kg) was powdered and extracted with 70% EtOH (40 L × 4, 2 h for
each time) at 80 °C. Afer removal of EtOH under the reduced pressure, the obtained
EtOH extract (980 g) was suspended in H2O (4 L) and then successively partitioned with
petroleum ether, EtOAc, and n-BuOH. e n-BuOH fraction (240 g) was separated into
50% EtOH fraction and 70% EtOH fraction by D101 macroporous resin. About 70% EtOH
fraction (93 g) was loaded onto silica gel column (2100 g, 100 mm × 650 mm) and eluted
with step gradient solvent system of CH2Cl2-MeOH (100:0 → 0:100) to yield 14 fractions
(Frs. A–N). Fr. J (3.5 g, CH2Cl2-MeOH 2:1) was chromatographed on open ODS (100 g,
28 mm × 230 mm), using a step gradient of MeOH-H2O (0:100 → 100:0) to yield 6 frac-
tions (Frs. JI–JVI). Fr. JIII (320 mg, MeOH-H2O 100:0) was purified by semi-prep. HPLC
(MeOH: H2O 70:30, flow rate of 1.2 ml/min) to afford 1 (3.6 mg, tR 33.6 min) and 2 (6.1 mg,
tR 37.0 min).
3.3.1. Ginsenoside Rk6 (1)
White amorphous powder; [ꢀ]D20 +20.2 (c 0.40, CH3OH). IR (KBr) νmax cm−1: 3390, 2926,
1650, 1455, and 1020. 1H NMR (500 MHz, CD3OD) and 13C NMR (125 MHz, CD3OD) spec-
tral data see Table 1; HR-ESI-MS: m/z 671.3929 [M + Cl]− (calcd for C36H60O9Cl, 671.3926).

JouRnAL of AsiAn nATuRAL PRoduCTs ReseARCH
5
3.3.2. Ginsenoside-Rh22 (2)
White amorphous powder; [ꢀ]D20 −15.4 (c 0.20, CH3OH). IR (KBr) νmax cm−1: 3500, 1380,
and 1070. 1H NMR (500 MHz, CD3OD) and 13C NMR (125 MHz, CD3OD) spectral data
see Table 1; HR-ESI-MS: m/z 671.3929 [M + Cl]− (calcd for C36H60O9Cl, 671.3926).
3.4. Determination of the sugar configuration in compounds 1 and 2
A solution of each compound (3.0 mg) in 1.0 N HCl (dioxane/H2O 1:1; 1 ml) was heated at
100 °C in a water bath for 10 h. Afer removal of dioxane under vacuum, the solution was
extracted with EtOAc (1 ml × 3). e H2O-soluble fraction was concentrated to dryness, and
the obtained residue was dissolved in pyridine (each 2 ml). L-cysteine methyl ester hydro-
chloride (2.0 mg each) was added to the pyridine solution. e mixture was kept at 60 °C
for 1.5 h, dried under vacuum, and trimethylsilylated with 1-(trimethylsilyl)-1H-imidazole
(0.1 ml) at 60 °C for 1 h. e mixture was suspended in H2O (1 ml) and then extracted
with hexane (1 ml × 3) [8]. e supernatant was analyzed by GC under the following con-
ditions: capacity column, HP-5 (0.32 mm × 30 m, 0.25 μm); column temperature, 160 °C
(4 °C/min), 200 °C (kept for 5 min, 10 °C/min), 240 °C (kept for 10 min); carrier gas, He at
1.0 ml/min, split ratio 1:10; injection temperature, 270 °C; detection temperature, 280 °C;
and injection volume, 1.0 μl. Compounds 1 and 2 both gave a peak corresponding to the
authentic standard of d-glucose at 18.42 min.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
is work was financially supported by National Science and Technology Supporting Plan, China
[Number 2012BAI29B05].
References
[1] Editorial Board of Chinese Pharmacopoeia, Chinese Pharmacopoeia, 1 vol. (Chinese Medical
Science and Technology Press, Beijing, 2015), p. 11.
[2] C.Z. Gu, J.J. Lv, X.X. Zhang, Y.J. Qiao, H. Yan, Y. Li, D. Wang, H.T. Zhu, H.R. Luo, C.R. Yang,
M. Xu, and Y.J. Zhang, J. Nat. Prod. 78, 1829 (2015).
[3] W.N. Wang, B.X. Yan, W.D. Xu, Y. Qiu, Y.L. Guo, and Z.D. Qiu, Molecules. 20, 19291 (2015).
[4] X.H. Wang, Q.D. Li, and Q.E. Cao, Chin. Tradit. Herb. Drugs. 32, 267 (2001).
[5] C.F. Ding, M.J. Wang, Z.D. Qiu, and W. Xu, Chin. J. Exp. Tradit. Med. Form. 21, 43 (2015).
[6] W. Xu, C.F. Ding, X.W. Wang, X.L. Liu, and Z.D. Qiu, Chin. Chem. Lett. 26, 1298 (2015).
[7] I.H. Park, N.Y. Kim, S.B. Han, J.M. Kim, S.W. Kwon, H.J. Kim, M.K. Park, and J.H. Park, Arch.
Pharm. Res. 25, 428 (2002).
[8] W. Xu, J.M. Wu, Z. Zhu, Y. Sha, J. Fang, and Y.S. Li, HeIv. Chim. Acta. 93, 2007 (2010).
[9] H.P. Wang, X.B. Yang, X.W. Yang, J.X. Liu, W. Xu, Y.B. Zhang, L.X. Zhang, and Y.P. Wang, J.
Asian Nat. Prod. Res. 15, 579 (2013).
[10] D.Q. Dou, Y.J. Chen, L.H. Liang, F.G. Pang, N. Shimizu, and T. Takeda, Chem. Pharm. Bull.
49, 442 (2001).