Two New Triterpenoid Glycosides from the Roots of Rosa cymosa Tratt.

Article abstract of DOI:10.1002/hlca.201500548

Two new triterpenoid glycosides, 3α,19α,23α-trihydroxy-2-oxo-12-ursen-28-O-β-d-glucopyranoside (1) and 3α,19α,23α-trihydroxy-2-oxoolean-12-en-28-O-β-d-glucopyranoside (2) as well as three known compounds, 2α,3α,19α-trihydroxyolean-12-en-28-O-β-d-glucopyranoside (3), 2α,3α,19α,23-tetrahydroxy-12-ursen-28-O-β-d-glucopyranoside (4), and 2α,3β,19α,23-tetrahydroxyurs-12-en-28-oic acid (5) were isolated from 75% EtOH extract of Rosa cymosa. Their structures were elucidated by extensive spectroscopic methods. All the isolated compounds displayed moderate inhibitory activity against LPS-induced NO production in macrophages.

Full text of DOI:10.1002/hlca.201500548

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Helv. Chim. Acta 2016, 99, 482 – 486
FULL PAPER
Two New Triterpenoid Glycosides from the Roots of Rosa cymosa TRATT.
by Guo-Xu Maa)1), Xiao-Yan Huangb)1), Hua-Nian Daib), Xiao-Qing Zhongc), Yan-Lin Zhouc), Zuo-Lin Sud),
Ying-Zi Heb), Jun-Shan Yanga), Jing-Quan Yuan*b)e), and Xu-Dong Xu*a)
a) Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College,
Beijing 100193, P. R. China (phone: + 86 10 57833296; e-mail: xdxu@implad.ac.cn)
b) College of Chemistry and Materials Science, Guangxi Teachers Education University, Nanning 530001, P. R. China
(phone: + 86 0771 5601290; e-mail: yjqgx@163.com)
c) Guilin Sanjin Pharmaceutical Company Limited, Guilin 541004, P. R. China
d) Guangxi Lingfeng Pharmaceutical Company Limited, Hezhou 542800, P. R. China
e) Guangxi Institute of Medicinal Plant, Nanning 530023, P. R. China
Two new triterpenoid glycosides, 3a,19a,23a-trihydroxy-2-oxo-12-ursen-28-O-b-D-glucopyranoside (1) and 3a,19a,
23a-trihydroxy-2-oxoolean-12-en-28-O-b-D-glucopyranoside (2) as well as three known compounds, 2a,3a,19a-trihydroxyolean-
12-en-28-O-b-D-glucopyranoside (3), 2a,3a,19a,23-tetrahydroxy-12-ursen-28-O-b-D-glucopyranoside (4), and 2a,3b,19a,
23-tetrahydroxyurs-12-en-28-oic acid (5) were isolated from 75% EtOH extract of Rosa cymosa. Their structures were
elucidated by extensive spectroscopic methods. All the isolated compounds displayed moderate inhibitory activity against
LPS-induced NO production in macrophages.
Keywords: Rosa cymosa TRATT., Triterpenoid glycosides, Anti-inflammatory activity, Ursane triterpenes, Oleanane triterpenes.
Results and Discussion
Introduction
Compound 1 was isolated as a white amorphous powder
20
Rosa cymosa TRATT. is a member of the genus Rosa in
the Rosaceae family, and is mainly distributed in the
south of China [1]. The roots of R. cymosa were tradition-
ally used as folk medicine for the treatment of rheuma-
toid arthritis, contusions, diarrhea, and descensus uteri [2]
[3]. Modern pharmacological studies indicated that the
roots of R. cymosa displayed significant biological activi-
ties, such as anticoagulation [4][5], antibiosis [3], anti-
inflammatory [6], and antioxidant [7]. Recent phytochemi-
cal studies suggested that the triterpenoid constituents
were mainly responsible for the biological activities of this
plant. With the aim of finding new bioactive triterpenoid
agents, the 75% EtOH extraction of this plant were
examined, and two new triterpenoid glycosides, named as
3a,19a,23a-trihydroxy-2-oxo-12-ursen-28-O-b-D-glucopyra-
noside (1) and 3a,19a,23a-trihydroxy-2-oxoolean-12-en-
28-O-b-D-glucopyranoside (2) as well as three known
compounds (Fig. 1) were obtained. In this article, we
describe the isolation and structure determination of the
isolated compounds, and their inhibitory activity against
lipopolysaccharide (LPS)-induced NO production in
macrophages.
with ½aꢀD = +27.4 (c = 0.12, MeOH), exhibiting a quasi-
molecular ion peak at m/z 687.3810 [M + Na]+ (calc. for
687.3792) in the positive-ion mode. In conjunction with
1
the analysis of H- and 13C-NMR (APT) (Table 1) spec-
tra, the formula of compound
1 was deduced as
C36H56O11. Its IR spectrum showed the presence of OH
group absorptions at 3573 – 3392 cmꢁ1, CO signal at 1689
1
cmꢁ1, and C=C signal at 1653 cmꢁ1. The H-NMR spec-
trum 1 displayed the presence of six Me signals at d(H)
0.72 (s), 0.90 (s), 1.00 (d, J = 6.6), 1.07 (s), 1.27 (s), 1.52
(s); one olefinic H-atom at d(H) 5.41 (br. s, H–C(12));
one O-bearing CH signal at d(H) 5.00 (s), two HO–CH2
groups at d(H) 3.93 (d, J = 10.8), 3.64 (d, J = 10.8), 4.30
(br. d, J = 12.0), 4.38 (br. d, J = 12.0); and one anomeric
H-atom signal at d(H) 6.18 (d, J = 8.4). The above data
suggested that 1 was an ursane triterpenoid saponin
derivative with a sugar moiety [8 – 11]. The 13C-APT
spectrum displayed 36 C-atom signals (Table 1) including
six Me C-atom signals at d(C) 14.2, 17.0, 17.4, 17.4, 24.8,
27.3; two olefinic C-atom signals at d(C) 124.2 (C(12)),
139.8 (C(13)); two CO signals at d(C) 177.3 (C(28)), 213.1
(C(2)); one sugar moiety C-atom signals at d(C) 96.2,
74.4, 79.6, 71.7, 79.7, 62.8; and other three oxygenated
C-atom signals at d(C) 78.0, 65.3, 73.0. The assignment of
1
)
These authors contributed equally to this work.
€
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DOI: 10.1002/hlca.201500548

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Helv. Chim. Acta 2016, 99, 482 – 486
483
Fig. 1. The structures of compounds 1 – 5
1H-NMR and 13C-APT spectroscopic data of 1 were hydrolysis of 2 afforded sugar moiety of D-glucose and
1
based on the HSQC, HMBC, and H,1H-COSY spectrum.
identified by TLC and HPLC analysis. In the NOESY
In fact, the above data of compound 1 were similar to spectrum, the enhancement from H–C(3) (d(H) 5.00) to
those of the known compound 2a,3a,19a,23-tetrahydroxy- Me(24) (d(H) 0.81) and Me(24) to Me(25) (d(H) 0.97)
12-ursen-28-O-b-D-glucopyranoside (4) [12][13], except for
the OH group at C(2) in compound 4 wherein it was O-
bearing to CO group in compound 1. In the HMBC spec-
trum (Fig. 2), the correlations from H–C(3) (d(H) 5.00)
to C(2) (d(C) 213.1) and C(4) (d(C) 49.0) confirmed the
difference. The sugar moiety was located at C(28) on the
basis of the correlation from the anomeric H-atom signal
indicated that the OH group at C(3) was a-oriented.
Thus, the structure of 2 was identified as 3a,19a,23a-trihy-
droxy-2-oxoolean-12-en-28-O-b-D-glucopyranoside.
The three known compounds, 2a,3a,19a-trihydroxyolean-
12-en-28-O-b-D-glucopyranoside (3) [14][15], 2a,3a,19a,23-
tetrahydroxy-12-ursen-28-O-b-D-glucopyranoside (4) [5], and
2a,3b,19a,23-tetrahydroxyurs-12-en-28-oic acid (5) [13] were
1
at d(H) 6.18 (d, J = 8.4) to the CO signal at d(C) 177.3 (C identified by comparing their H- and 13C-NMR data with
(28)). The type and absolute configuration of the sugar
was identified as D-glucose on the basis of TLC method
the reported literatures.
All five compounds were studied for their anti-inflam-
comparison with authentic monosaccharide (CHCl3/ matory activities on LPS-induced nitric oxide (NO) pro-
MeOH/H2O 3:2:0.2, visualization with EtOH/5%. H2SO4 duction in RAW 264.7. The results are presented in
spraying), followed by HPLC analysis. The NOESY spec- Table 2. It may be said that all the tested compounds
trum was also employed to confirm the configuration of
showed moderate inhibitory activities against the produc-
compound 1. The NOE correlations of H–C(3) (d(H) tion of NO with IC50 values between 3.24 and 9.28 lg/ml
5.00, s) with Me(24) (d(H) 0.72, s) indicated the a-orienta- compared to aminoguanidine used as the positive control.
tion of the OH group. Therefore, the structure of 1 was
elucidated as 3a,19a,23a-trihydroxy-2-oxo-12-ursen-28-O- The work was financially supported by National Natural
b-D-glucopyranoside.
Compound 2 was isolated as a white amorphous pow-
der with ½aꢀD = +19.1 (c = 0.12, MeOH). Its molecular (No. 1140001-38) and Guangxi Natural Science Founda-
formula was assigned as C36H56O11 on the basis of HR- tion Program (2010GXNSFD013052) and the Technologi-
ESI-MS at m/z 687.3802 [M + Na]+ (calc. for 687.3790) in cal Large Platform for Comprehensive Research and
Science Foundation of China (No. 81360683) and Guangxi
Scientific Research and Technology Development Projects
20
the positive-ion mode. Its IR spectrum showed the pres-
Development of New Drugs in the Twelfth Five-Year
ence of OH group absorptions at 3623 – 3452 cmꢁ1, CO ‘Significant New Drugs Created’ Science and Technology
1
signal at 1699 cmꢁ1, and C=C bond at 1643 cmꢁ1. The H Major Projects (No. 2012ZX09301-002-001). The authors
and 13C-NMR (APT) (Table 1) spectroscopic data were also thank Dr. Dev Sooranna, Imperial College London,
quite similar to those of 1, except for the Me group at C
(19) in compound 1 wherein it was transferred to C(20) in
compound 2. This difference was fully supported by the
2D-NMR spectra. In the HMBC spectrum, the correla-
tions from the H-atom signal of an O-bearing group at d
(H) 3.55 (d, J = 4.8, H–C(19)) to d(C) 45.0 (C(18)), 35.9
(C(20)), and Me signals at d(H) 1.12 (s, Me(29)), 0.96
(s, Me(30)) to C(20) (d(C) 35.9) indicated that Me(29)
and Me(30) were both attached to C(20). Therefore, com-
pound 2 was an oleanane triterpenoid saponin [6][9]. Acid
for editing the manuscript.
Experimental Part
General
Optical rotations: PerkinElmer 341 digital polarimeter
(PerkinElmer, Norwalk, CT, USA). UV and IR spectra:
Shimadzu UV2550 and FTIR-8400S spectrometers (Shi-
madzu, Kyoto, Japan), respectively. NMR Spectra:
€
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484
Helv. Chim. Acta 2016, 99, 482 – 486
Table 1. 1H- and 13C-NMR Data (600 MHz and 150 MHz, resp., in (D5)pyridine) for Compounds 1 – 2
Position
1
1
2
d(H) (J in Hz)
d(C), type
d(H) (J in Hz)
d(C), type
2.42 (d, J = 12.0)
2.11 (d, J = 12.0)
54.1, CH2
2.46 (d, J = 12.0)
2.19 (d, J = 12.0)
53.9, CH2
2
3
4
5
6
213.1, C
78.0, CH
49.0, C
213.1, C
78.0, CH
42.6, C
5.00 (s)
5.00 (s)
2.07 – 2.10 (m)
1.24 – 1.27 (m)
1.47 – 1.49 (m)
1.36 – 1.38 (m)
1.57 – 1.61 (m)
47.7, CH
19.2, CH2
2.79 – 2.81 (m)
1.33 – 1.36 (m)
1.59 – 1.62 (m)
1.36 – 1.38 (m)
1.95 – 1.97 (m)
48.4, CH
19.3, CH2
7
33.2, CH2
33.4, CH2
8
9
42.6, C
46.9, CH
38.0, C
41.0, C
47.0, CH
36.8, C
1.99 – 2.01 (m)
1.99 – 2.02 (m)
5.47 (t, J = 2.4)
10
11
12
13
14
15
1.86 – 1.89 (m)
5.41 (t, J = 2.4)
24.4, CH2
124.2, CH
139.8, C
43.9, C
24.5, CH2
123.0, CH
144.9, C
44.1, C
2.30 – 2.34 (m)
1.08 – 1.12 (m)
2.94 – 2.97 (m)
1.92 – 1.95 (m)
29.6, CH2
2.26 – 2.29 (m)
1.03 – 1.05 (m)
2.64 – 2.66 (m)
1.99 – 2.01 (m)
29.4, CH2
16
26.4, CH2
28.3, CH2
17
18
19
20
21
50.4, C
54.8, CH
73.0, C
50.4, C
2.81 (s)
3.56 (br. s)
3.55 (d, J = 4.8)
45.0, CH
81.4, CH
35.9, C
1.18 – 1.21 (m)
1.31 – 1.34 (m)
1.91 – 1.94 (m)
0.99 – 1.01 (m)
1.24 – 1.26 (m)
3.93 (d, J = 10.8)
3.64 (d, J = 10.8)
0.72 (s)
42.5, CH
27.0, CH2
1.33 – 1.35 (m)
2.21 – 2.24 (m)
1.00 – 1.02 (m)
1.33 – 1.36 (m)
4.02 (d, J = 10.8)
3.73 (d, J = 10.8)
0.81 (s)
29.3, CH2
22
23
41.3, CH2
65.3, CH2
32.9, CH2
65.3, CH2
24
25
26
27
28
29
30
10
14.2, CH3
17.4, CH3
17.0, CH3
24.8, CH3
177.3, C
14.1, CH3
17.2, CH3
17.6, CH3
25.0, CH3
177.6, C
1.07 (s)
0.90 (s)
1.52 (s)
0.97 (s)
1.12 (s)
1.55 (s)
1.27 (s)
27.3, CH3
17.4, CH3
96.2, CH
74.4, CH
79.6, CH
71.7, CH
79.7, CH
62.8, CH2
1.12 (s)
0.96 (s)
29.1, CH3
25.2, CH3
96.3, CH
74.5, CH
79.3, CH
71.5, CH
79.6, CH
62.6, CH2
1.00 (d, J = 6.6)
6.18 (d, J = 8.4)
4.11 – 4.14 (m)
4.20 – 4.22 (m)
4.24 – 4.27 (m)
4.09 – 4.12 (m)
4.30 (br. d, J = 12.0)
4.38 (br. d, J = 12.0)
6.36 (d, J = 7.8)
4.19 – 4.22 (m)
4.28 – 4.30 (m)
4.36 – 4.38 (m)
4.18 – 4.20 (m)
4.39 (br. d, J = 12.0)
4.44 (br. d, J = 12.0)
20
30
40
50
60
Bruker AV 600 NMR spectrometer (chemical shift values
TLC analysis. All solvents used were of analytical grade
are presented as d values with TMS as the internal stan- (Beijing Chemical Works, Beijing, P. R. China).
dard; Bruker, Billerica, MA, USA). HR-ESI-MS: LTQ-
Obitrap XL spectrometer (Thermo Fisher Scientific, Bos-
Plant Material
ton, MA, USA). Silica gel (SiO2; 100 – 200 and 300 – 400
mesh, Qingdao Marine Chemical Plant, Qingdao, P. R. The roots of Rosa cymosa were provided by Guilin Sanjin
China) was used for column chromatography. Precoated
SiO2 GF254 plates (Zhi Fu Huang Wu Pilot Plant of Sil-
ica Gel Development, Yantai, P. R. China) were used for
Pharmaceutical Company Limited, collected from Guilin,
Guangxi Province, P. R. China and identified by Prof. Bin
Dai, Guangxi Research Institute of Minority Medicine. A
€
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Helv. Chim. Acta 2016, 99, 482 – 486
485
and yielded compounds 2 (10 mg, tR = 19.7 min) and 4
(10 mg, tR = 14.2 min).
3a,19a,23a-Trihydroxy-2-oxo-12-ursen-28-O-b-D-glucopyra-
noside (= 1-O-[(3a)-3,19,23-trihydroxy-2,28-dioxours-12-
en-28-yl]-b-D-glucopyranose; 1). White amorphous powder.
20
½aꢀD = +27.4 (c = 0.12, MeOH); UV (MeOH): 208 (4.01). IR
(KBr): 3573-3392, 1712, 1689, 1653. 1H- and 13C-NMR
((D5)pyridine): see Table 1. HR-ESI-MS: 687.3810
([M + Na]+, C36H56NaO1þ1; calc. 687.3720).
3a,19a,23a-Trihydroxy-2-oxoolean-12-en-28-O-b-D-gluco-
pyranoside (= 1-O-[(3a,19a)-3,19,23-Trihydroxy-2,28-dioxo-
olean-12-en-28-yl]-b-D-glucopyranose; 2). White amorphous
Fig. 2. Key HMBC correlations of compound 1
20
powder. ½aꢀD = +19.1 (c = 0.12, MeOH). UV: (MeOH): 208
1
(4.01). IR (KBr): 3623-3452, 1723, 1699, 1643. H- and 13C-
Table 2. Inhibitory Activity of Compounds 1 – 5 on LPS-Induced
NO Production in Raw 264.7 Macrophages
NMR ((D5)pyridine): see Table 1. HR-ESI-MS: 687.3802
([M + Na]+, C36H56NaO1þ1; calc. 687.3720).
Compounds
IC50 [lg/ml]a)
Acid Hydrolysis of 1 – 2
1
9.28 ꢂ 0.43
7.96 ꢂ 0.32
3.42 ꢂ 0.61
2.21 ꢂ 0.55
3.24 ꢂ 0.63
0.94 ꢂ 0.20
2
Compounds 1 – 2 (each 2.0 mg) were treated with 1M HCl
(1 ml) and heated under reflux for 3 h, respectively. After
extraction with CH2Cl2 (3 ml 9 3), the H2O-soluble layer
was evaporated to dryness and subjected to the HPLC
analysis under the following conditions, respectively:
HPLC column, Purospher STAR NH2, 5 mm 9 250 mm
(Merck, Darmstadt, Germany); detection, optical rotation
(Chiralyser-MP (IBZ Messtechnik GMBH, Hannover,
Germany)); mobile phase, MeCN/H2O (68:32, v/v); and
flow rate 1.0 ml/min. D-Glucose from 1 – 2 presented in the
aq. phase was carried out by comparison of its retention
time and optical rotation with that of authentic samples, tR
24.7 min (positive).
3
4
5
Aminoguanidineb)
a) Value present mean ꢂ SD of triplicate experiments. b) Positive
control substance.
voucher specimen (NO. 02132181) was deposited with the
Guangxi Botanical Garden of Medical Plant.
Extraction and Isolation
The dried root of Rosa cymosa (10.0 kg) were extracted
three times with 75% EtOH. The 75% EtOH extraction
was concentrated under reduced pressure and the residue
was dissolved in H2O. The mixture was extracted using
petroleum ether (PE) and AcOEt, respectively. The H2O-
soluble fraction was subjected to D-101 macroreticular
resin eluted with H2O, EtOH/H2O (35:65), EtOH/H2O
(55:45), EtOH/H2O (75:25), and EtOH/H2O (95:5),
respectively. The EtOH/H2O (75:25) fraction (40.0 g) was
subject to column chromatography on SiO2 eluting with
CH2Cl2/MeOH gradient (40:1; 20:1; 10:1; 5:1, 0:1 v/v), to
yield five fractions (Frs. I-V). The Fr. II (2.1 g) was
separated using SiO2 CC eluting with CH2Cl2/MeOH
(80:1; 60:1; 40:1; 20:1; 10:1; 5:1, v/v) to give six subfrac-
tions, II1 – 6. Subfraction II3 was prepared by HPLC using
MeOH/H2O (65:35, v/v) on YMC-Pack ODS-A column to
give compounds 3 (20.0 mg, tR = 20.5 min) and 5
(23.0 mg, tR = 33.1 min). Subfraction II4 was prepared by
HPLC using MeOH/H2O (65:35, v/v) on YMC-Pack
Assay for Inhibitory Ability Against LPS-Induced
NO Production in RAW 264.7 Macrophages
RAW 264.7 macrophages were seeded in 24-well plates
(105 cells/well). The cells were coincubated with drugs
and LPS 1 lg/ml) for 24 h. The amount of NO was
assessed by determining the nitrite concentration in the
cultured RAW 264.7 macrophage supernates with Griess
reagent. Aliquots of supernates (100 ll) were incubated,
in-sequence, with 50 ll of 1% sulfanilamide and 50 ll of
0.1% naphthylethylenediamine in 2.5% phosphoric acid
soln. The absorbance was recorded on a microplate
reader at a wavelength of 570 nm.
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[2] Editorial Committee of Flora of China, ‘Flora of China’,
Science Press, Beijing, 1985, p. 763.
ODS-A column to give compound 1 (10.0 mg, tR
=
21.6 min). The Fr. III (3.4 g) was subjected to SiO2 CC
eluting with CH2Cl2/MeOH (60:1; 40:1; 20:1; 10:1, v/v) to
afford four subfractions III1 – 4. Subfractions III2 and III3
were separated by prep. HPLC using MeOH/H2O (58:42)
and (65:35) on YMC-Pack ODS-A column, respectively,
[3] W. Z. Xie, ‘Collection of Chinese Herbal Medicines’, People’s
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€
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