Three new cycloart-7-ene triterpenoid glycosides from Cimicifuga dahurica and their anti-inflammatory effects

Article abstract of DOI:10.1080/14786419.2020.1719487

Ten cycloart-7-ene triterpenoid glycosides, including three new compounds (1–3), were isolated from the roots of Cimicifuga dahurica. Their structures were elucidated on the basis of extensive spectroscopic analyses, chemical methods and comparison with literatures. In addition, the isolates were evaluated for their inhibitory effects on the production of NO, as well as the expressions of iNOS and COX-2 in LPS-stimulated RAW 264.7 macrophages. The results showed that compounds 3, 5, 6, 7 and 8 can reduce the release of NO in a dose-dependent manner. Mechanistically, Western blot analysis indicated that the NO inhibitory effects relied on down-regulating the expression of iNOS, and partially associated with lowering the expression of COX-2.

Full text of DOI:10.1080/14786419.2020.1719487

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Three new cycloart-7-ene triterpenoid glycosides
from Cimicifuga dahurica and their anti-
inflammatory effects
Qian-Qian Pang, Yu-Dan Mei, Yuan-Chu Zhang, Ling-Xian Liu, Dan-Feng Shi,
Da-Bo Pan, Xin-Sheng Yao, Hai-Bo Li & Yang Yu
To cite this article: Qian-Qian Pang, Yu-Dan Mei, Yuan-Chu Zhang, Ling-Xian Liu, Dan-Feng Shi,
Da-Bo Pan, Xin-Sheng Yao, Hai-Bo Li & Yang Yu (2020): Three new cycloart-7-ene triterpenoid
glycosides from Cimicifugaꢀdahurica and their anti-inflammatory effects, Natural Product Research,
DOI: 10.1080/14786419.2020.1719487
To link to this article: https://doi.org/10.1080/14786419.2020.1719487
View supplementary material
Published online: 03 Feb 2020.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=gnpl20

NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1719487
Three new cycloart-7-ene triterpenoid glycosides from
Cimicifuga dahurica and their anti-inflammatory effects
a
ꢀ
b
ꢀ
a
a
Qian-Qian Pang , Yu-Dan Mei , Yuan-Chu Zhang , Ling-Xian Liu ,
a
a
a
b
a
Dan-Feng Shi , Da-Bo Pan , Xin-Sheng Yao , Hai-Bo Li and Yang Yu
a
Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy and Guangdong
Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan
b
University, Guangzhou, People’s Republic of China; State Key Laboratory of New-tech for Chinese
Medicine Pharmaceutical Process, Jiangsu Kanion Pharmaceutical Co. Ltd, Jiangsu, Lianyungang,
People’s Republic of China
ABSTRACT
ARTICLE HISTORY
Ten cycloart-7-ene triterpenoid glycosides, including three new
compounds (1–3), were isolated from the roots of Cimicifuga
dahurica. Their structures were elucidated on the basis of exten-
sive spectroscopic analyses, chemical methods and comparison
with literatures. In addition, the isolates were evaluated for their
inhibitory effects on the production of NO, as well as the expres-
sions of iNOS and COX-2 in LPS-stimulated RAW 264.7 macro-
phages. The results showed that compounds 3, 5, 6, 7 and 8 can
Received 7 November 2019
Accepted 11 January 2020
KEYWORDS
Cimicifuga dahurica;
cycloart-7-ene triterpenoid
glycosides; anti-
inflammatory; NO; iNOS;
COX-2
reduce the release of NO in
a dose-dependent manner.
Mechanistically, Western blot analysis indicated that the NO
inhibitory effects relied on down-regulating the expression of
iNOS, and partially associated with lowering the expression of
COX-2.
1. Introduction
The genus Cimicifuga plants, belonging to the family Ranunculaceae, have an exten-
sive history of use in China, Japan, Europe and North America (Guo et al. 2017).
Cimicifuga Rhizome, originated from C. heracleifolia Kom., Cimicifuga dahurica (Turcz.)
Maxim. and C. foetida L., is officially listed in Chinese Pharmacopeia (Commission NP
CONTACT Yang Yu
The first two authors contributed equally to this work.
1018yuyang@jnu.edu.cn; Hai-Bo Li
lihaibo1985124@sina.com
ꢀ
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1719487.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
Q.-Q. PANG ET AL.
2
015) for the treatment of headache, toothache, aphtha, sore throat and uterine pro-
lapse. Previous phytochemical studies have revealed that it mainly contained 9,19-
cycloartane triterpenes (Liu et al. 2003; Sun et al. 2016; Hao et al. 2019), cinnamic acids
(Qin et al. 2016), chromones (Shi et al. 2018) and nitrogenous compounds (Zhang
et al. 2013). More strikingly, cycloartane triterpenes have been reported to be the
major characteristic components in Cimicifuga showing diverse bioactivities, including
anti-tumour (Liu et al. 2014; Sun et al. 2017), neuro-protection (Lv et al. 2017), anti-
inflammation (Thao et al. 2018) and so on (Su et al. 2016).
Natural compounds in medical plants are always found to play an important role in
inflammatory disease (Maione et al. 2016). In our research for anti-inflamatory agents
from Cimicifuga plants, an investigation of C. dahurica resulted in the isolation of new
bioactive phenolic acids (Lu et al. 2019a, 2019b). As a continuing effort to enrich and
deeply investigate the potent anti-inflamatory agents, ten cycloart-7-ene triterpenoid
glycosides, including three new triterpenes (1–3), were obtained from the extract of
C. dahurica. Their structures were unambiguously elucidated by analysing spectro-
scopic data, using chemical methods, or comparing their NMR data with those in the
literatures. In addition, we examined the in vitro effects of the isolates on the produc-
tion of nitric oxide (NO), as well as the expressions of inducible NO synthase (iNOS)
and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 cells.
2
. Results and discussion
.1. Structure determination
Compound 1 was isolated as a white amorphous powder, m.p. 190–195 C, [a]25 D
23.4. Its molecular formula was established as C H O based on the HRESIMS data
2
ꢁ
–
35 54 10
þ
(
m/z 657.3605 [M þ Na] , calcd. for C H O Na, 657.3615), accounting for nine degrees
35 54 10
1
of unsaturation. The H NMR spectrum (600 MHz, in pyridine-d ) of 1 showed typical
5
signals due to a cyclopropane methylene [d 1.12, 0.55, each, d, J ¼ 4.0 Hz, H -19], seven
H
2
tertiary methyls [d 1.78 (H -21), 1.59 (H -18), 1.54 (H -27), 1.48 (H -26), 1.39 (H -28),
H
3
3
3
3
3
1
.38 (H -29), 1.11 (H -30)], an olefin [d 6.18, dd, J ¼ 7.6, 2.0 Hz, H-7] and an anomeric
3
3
H
0
proton [d 4.89 (1 H, d, J ¼ 7.5 Hz, H-1 )], indicating a cycloartane-type triterpenoid
H
1
3
glycoside. The C NMR (150 MHz, in pyridine-d ) and DEPT 135 spectra of 1 displayed a
5
total of 35 carbon signals. Except for five glycosyl carbons, the remaining 30 aglycone
carbons were resolved as 9 quaternary carbons (a carbonyl group), 7 methines (an ole-
finic, and 5 oxygenated), 7 methylenes and 7 methyls. In addition, an NMR comparison
analysis with a known compound (Nishida and Yoshimitsu 2011), allowed the establish-
ment of a cycloart-7-en rings A–D in 1. This conclusion can be supported by three spin
1
1
systems (H -1/H -2/H-3, H-5/H -6/H-7 and H -11/H -12) in H– H COSY, as well as key
2
2
2
2
2
correlations of H -29(30)/C-3, 4, 5; H -19/C-1, 9, 10, 11; H -18/C-12, 13, 14, 17; H -28/C-8,
3
2
3
3
13, 14, 15 and H-15, 17/C-16 (supplementary material Figure S1) in HMBC spectrum.
Meanwhile, the COSY correlations of H -22/H-23/H-24, together with HMBC correlations
2
of H -21/C-17, 20, 22, H -26(27)/C-24, 25 and H-24/C-20, led to the establishment of a
3
3
five-membered side chain (ring E) with the C20, 24-ether linkage (supplementary mater-
ial Figure S1). The hydrolysis of compound 1 offered a D-xylose, which was determined
by derivatisation and HPLC analysis (Tanaka et al. 2007). A b-D-xylose was deduced

NATURAL PRODUCT RESEARCH
3
Figure 1. Structures of compounds 1–10.
due to the J value (J1 , 2
0
0
¼7.5 Hz). The sugar unit was confirmed to attached to C-3 by
0
0
the HMBC correlation peaks of H-1 (d 4.89)/C-3(d 88.3), and H-3 (d 3.54)/C-1 (107.6).
H
C
H
Thus, the planar structure of 1 was established.
The relative stereochemistry of 1 was disclosed by coupling constants and the
ROESY experiment. The large coupling constants of H-3 (d 3.54, dd, J ¼ 11.6, 4.1 Hz)
H
supported the assignment of 3b-OH. Moreover, ROESY correlations of H -29/H-3/H-5,
3
H -30/H -19/H -18/H-15 and H -28/H-17, indicated that A/B, C/D rings were trans-
3
2
3
3
fused, and H-17 and 15-OH were a-oriented (supplementary material Figure S1). In
addition, based on a reported method of computational chemistry (Gan et al. 2015),
the free rotation of the C-17-C-20 single bond was considered to be restricted to
some extent in 20,24-epoxy-cycloartane triterpenoids. The H-24a and H-23b were
assigned by key correlations of H-17/H -21, H -21/H-24 and H-23/H -26(27) observed
3
3
3
in ROESY spectrum (supplementary material Figure S1). Hence, compound 1 was eluci-
ꢀ
ꢀ
dated as (20 S,24 R)-20,24-epoxy-15a,23b,25-trihydroxy-3b-(b-D-xylopyranosyloxy)-9,19-
cycloart-7-en-16-one (Figure 1).
ꢁ
Compound 2 was isolated as a colourless amorphous powder, m.p. 295–301 C,
[
a]25 D –52.4 and its molecular formula C H O was determined by HRESIMS data
35 54 10
þ
(
m/z 657.3620, [M þ Na] , calcd. for C H O Na, 657.3615), implying nine degrees of
35 54 10
13
unsaturation. The C NMR spectrum of 2 was similar to those of 1 except C-17 side
chain moiety (Table S1). With the aid of 2 D NMR spectra, A–D rings of cyclopropane
structure in 2 were assigned as the same as 1 (supplementary material Figure S2).
Besides, a sequence connected through the correlations of H-17/H-20(H -21)/H -22 in
3
2
1
1
the H– H COSY, combined with the long-range correlations of H -22/C-23, H-24/C-23,
2
2
5, H -26(27)/C-24, 25, led to a C20–C27 side chain. Therefore, the structure of 2 was
3
established as an 11-dehydroxylated derivative of 15a-hydroxyximicidol-3-O-b-D-xylo-
pyranoside (Kadota et al. 1995). By comparing their NMR data, it could be deduced
that 2 possessed the same stereochemistry with this known compound, which was
further confirmed by the ROESY experiment (supplementary material Figure S2).
Except for C-24, the other chiral centres in compound 2 were consistent with 1 for
ROESY correlations of H-3/H -29, H -19/H -18(H-20)/H-15 and H -28/H-17/H -21.
3
2
3
3
3
Further, 24a-OH was determined by ROESY correlations of H -21/H-22a (d 3.24) and
3
H

4
Q.-Q. PANG ET AL.
H-22b (d 4.22)/H-24. As a result, compound 2 was established and named 11-dehy-
H
dro-15a-hydroxyximicidol-3-O-b-D-xylopyranoside (Figure 1).
ꢁ
Compound 3 was obtained as a white amorphous powder, m.p. 266–270 C, [a]25
D –37.6, and a molecular formula determined to be C H O on the basis of
3
5 54 10
þ
HRESIMS data (m/z 635.3795, [M þ H] , calcd. for C H O , 635.3795). Compound 3
3
5 55 10
possessed the same aglycone as bugbanoside F and 7,8-didehydro-12-b-hydroxyl-cimi-
genol-3-O-b-D-galactopyranoside for their similar NMR data (supplementary material
1
1
Table S1), which can be supported by the detailed analysis of key H– H COSY and
HMBC correlations (supplementary material Figure S3). Similarly, the sugar unit in 3
was determined as a b-D-xylose by the same method as compound 1. For the stereo-
chemistry of 3, the large coupling constants of H-3 (d 3.49, dd, J ¼ 11.6, 4.1 Hz) and
H
1
H-12 (d 4.36, dd, J ¼ 11.3, 5.2 Hz) were observed in H NMR spectrum. Observed
H
ROESY correlations were H-5/H-3/H -29, H -30/H -19/H -18/12-OH, H-15/H -18/H-20,
3
3
2
3
3
H -28/H-17/H -21, H-23/H -26(27) and H-24/H-17, indicating the existence of 3b-OH,
3
3
3
12b-OH, 15a-OH, H-17a, H-23a and H-24b (supplementary material Figure S3).
Furthermore, as concluded from those 24-configuration triterpenes (Li et al. 1993), the
absolute configuration of C-24 in 3 can be assigned as 24S for a single or broad single
peak (J < 1.0 Hz) (whereas a double peak, J ꢂ 4.0 Hz for 24 R). Thereafter, compound 3
was established as 12b-hydroxy-7,8-didehydrocimigenol-3-O-b-D-xylopyranoside
(Figure 1).
The seven known compounds (4–10) were identified as (23R,24R)-16b,23;16a,24-die-
poxy-cycloart-7-en-3b,12b,15a,25-tetraol-3-O-b-D-xylopyranoside (4) (Yoshimitsu et al.
006), (23R,24R)-16b,23;16a,24-diepoxy-12b-acetoxy-cycloart-7-en-3b,15a,25-triol-3-O-b-D-
2
xylopyranoside (5) (Yoshimitsu et al. 2006), 25-O-acetyl-7,8-didehydrocimigenol-3-O-b-D-
xylopyaranoside (6) (Kusano et al. 1999), 24-epi-7,8-didehydrocimigenol-3-O-b-D-xylopyar-
anoside (7) (Li et al. 1993), 7,8-didehydrocimigenol-3-O-b-D-xylopyaranoside (8) (Kusano
et al. 1999), 24-O-acetyl-7,8-didehydro-hydroshengmanol 3-O-b-D-xylopyranoside (9)
(Kusano et al. 1999) 24-epi-24-O-acetyl-7,8-didehydro-hydroshengmanol-3-O-b-D-xylopyra-
noside (10) (Kusano et al. 1999), respectively, by comparison of their spectroscopic data
with those reported in the literatures.
2.2. Anti-inflammatory activities
For evaluating the anti-inflammatory effects, compounds were assayed on NO produc-
tion in LPS-stimulated RAW264.7 cells. As shown in supplementary material Figure S6,
the NO production in the model groups (simulated with LPS) increased significantly,
whereas the production of NO decreased in the treatment groups as compared with
that in model cells. The results suggested that compounds inhibited the release of NO
in a dose-dependent manner. In conclusion, some of the isolates can significantly
inhibit NO-production during LPS-stimulated inflammatory response.
In order to further illuminate the anti-inflammatory effects, compounds 3 and 5
were selected as examples to test the impact on protein levels of iNOS and COX-2 by
Western blot. As shown in supplementary material Figure S7, the expressions of iNOS
and COX-2 in non-treated cells simulated with LPS was significantly increased com-
pared with the control groups. The decreases of iNOS protein in the treatment groups

NATURAL PRODUCT RESEARCH
5
were consistent with the trends of NO production, whereas the protein level of COX-2
did not noticeably decrease. The results of Western blot analysis indicated that the
anti-inflammatory effects of 3 and 5 mainly relied on down-regulating the expression
of iNOS, and partially associated with lowering the expression of COX-2.
The cytotoxicity of the tested compounds was evaluated in the presence or
absence of LPS by MTT assay as reported previously (Mei et al. 2019). All the tested
compounds did not decrease the viability of RAW264.7 cells when they were incu-
bated with or without LPS (0.5 lg/mL) at the test concentration (data were present in
supplementary material Figure S4 and S5).
3. Experimental
3.1. Instrumentations and reagents
Melting points were determined on an X-5 micromelting point apparatus (Beijing TECH
Instrument Co. Ltd, Beijing, China) and are reported uncorrected. Optical rotations were
measured on a JASCO P-1020 polarimeter with a 1-cm cell at room temperature. IR spec-
tra were recorded in KBr disc with a JASCO FT/IR-480 Plus. NMR spectra were recorded
on a Bruker Avance 600 spectrometer (Bruker Co. Ltd., Bremen, Germany) in pyridine-d5
(CIL, USA). HRESIMS spectra were performed on a Waters Synapt G2 Q-TOF (Waters,
Manchester, UK) with a Waters RP-18 column (Acquity BEH, 1.7m, / 3.0 ꢃ 150 mm). HPLC
analyses were performed on a Shimadzu LC-20AB (or Waters 2695) coupled with a
Phenomenex Gemini C18 column (5lm, / 4.6 ꢃ 250 mm; FLM Inc., Guangzhou, China),
while preparative HPLC was performed on an LC-20AB (or Waters 1515) using a
Phenomenex Gemini C18 column (5 lm, / 10 ꢃ 250 mm; FLM Inc., Guangzhou, China).
Column chromatography (CC) was carried out over Diaion HP-20 (Mitsubishi Chemical
Co.), silica gel (200–300 mesh, Qingdao Marine Chemical Ltd.), ODS-silica gel (12 nm,
S-50 lm, YMC Ltd.), Sephadex LH-20 (Amersham Pharmacia Biotech), pre-coated silica gel
plate (SGF254, 0.2 mm, Yantai Chemical Industry Research Institute).
The methanol and acetonitrile of HPLC grade (Merck, Shanghai, China) were used in
HPLC. L-Cysteine methyl ester hydrochloride, o-tolyl isothiocyanate and the standard
monosaccharides were purchased from Aladdin Reagent CO., Ltd., Shanghai, China, while
the other solvents were purchased from Baishi Chemical Industry CO., Ltd., Tianjin, China.
3.2. Plant material
The roots of C. dahurica were collected in Heilongjiang Province, China, in June 2015,
and were identified by Prof. Haixue Kuang (Heilongjiang University of Chinese
Medicine). A sample (JNUCD201506) was deposited in Institute of Traditional Chinese
Medicine & Natural Products, college of pharmacy, Jinan University, Guangzhou, China.
3.3. Extraction and isolation
Air-dried rhizomes of C. dahurica (19.5 kg) were pulverised and extracted three times
with 60% EtOH under reflux conditions. The 60% EtOH extract was concentrated under
reduced pressure, yielding a viscous residue (3.6 kg), which was subjected to HP-20

6
Q.-Q. PANG ET AL.
macroporous adsorption resin CC to yield H O fraction, 30% ethanol fraction (384 g)
2
and 95% ethanol fraction (534 g). The 95% ethanol fraction (450 g) was subjected to
silica gel CC using a stepwise gradient elution of CH Cl –CH OH (100:0, 99:1, 98:2,
2
2
3
97:3, 95:5, 90:10, 80:20, 70:30, 60:40, 0:100) to afford 12 subfractions (A–L). Fraction G
(
40 g) was chromatographed over an ODS CC using CH OH–H O as eluent (30:70,
3
2
50:50, 70:30, v/v) to give 7 subfractions (G1–G7). Fraction G4 (7.0 g) was further sepa-
rated on a silica gel column eluted with CH Cl –CH OH (100:1 to 0:100, v/v), affording
2
2
3
subfractions G4A–G4E. By using semi-preparative HPLC, subfraction G4B was purified
with CH CN–H O (35:65) to give 3 (13.8 mg) and 8 [160 mg, [a]25D –15.3 (c 1.0,
3
2
MeOH)], while G4C offer 1 (5.2 mg) and 2 (180 mg) with CH CN–H O (30:67). Fraction
3
2
G5 (21 g) was further separated into five subfractions G5A–G5G on a silica gel CC by
using CH Cl –CH OH gradiently (100:1 to 0:100, v/v). Then subfraction G5C was puri-
2
2
3
fied by Sephadex LH-20 (CH OH) and semi-preparative HPLC with CH CN–H O (40:60),
3
3
2
giving 4 [15.3mg, [a]25D –22.3 (c 1.0, MeOH)], 5 [113 mg, [a]25D –28.5 (c 1.0, MeOH)], 6
46 mg, [a]25D –15.5 (c 1.0, MeOH)], and 9 [14.3 mg, [a]25 D–46.4 (c 1.0, MeOH)].
Subfraction G5D was purified by using semi-preparative HPLC with CH CN–H O (45:55)
[
3
2
to yield 7 [53mg, [a]25D –13.7 (c 1.0, MeOH)] and 10 [67 mg, [a]25D –26.4 (c
.0, MeOH)].
1
3.4. Spectroscopic data of compounds 1–3
ꢀ ꢀ
20 S,24 R)-20,24-epoxy-15a,23b,25-trihydroxy-3b-(b-D-xylopyranosyloxy)-9,19-cycloart-
(
7
ꢁ
-en-16-one (1). Colourless amorphous powder; m.p. 190–195 C, [a]25D –23.4 (c 0.5,
ꢄ1
1
MeOH); IR (KBr) ꢀmax: 3409, 2967, 2937, 2872, 1737, 1457, 1382, 1069, 1046 cm ; H
NMR (pyridine-d , 600 MHz) and C NMR (pyridine-d , 150 MHz) data, seen supplemen-
13
5
5
þ
tary material Table S1 and Figures S9–S17; HR ESI MS: m/z 657.3605 [M þ Na] (calcd.
for C H O , 657.3615).
3
5 55 10
1
1-dehydro-15a-hydroxyximicidol-3-O-b-D-xylopyranoside (2). White amorphous
ꢄ1
powder; IR(KBr) ꢀ
3397, 2969, 2935, 2872, 1735, 1700, 1465, 1383, 1078, 1039 cm
;
max
ꢁ
1
13
m.p. 295–301 C, [a]25D –52.4 (c 1.0, MeOH); H NMR (pyridine-d , 600 MHz) and
C
5
NMR (pyridine-d , 150 MHz) data, seen supplementary material Table S1 and Figures
5
þ
S18–S26; HR ESI MS: m/z 657.3620 [M þ Na] (C H O , calcd. 657.3615).
3
5 55 10
1
2b-hydroxy-7,8-didehydrocimigenol-3-O-b-D-xylopyranoside (3). White amorph-
ꢄ1
13
ꢁ
ous powder; IR(KBr) mmax 3452, 2976, 2950, 1169, 1066, 1032 cm ; m.p. 266–270 C,
a]25D –37.6 (c 1.0, MeOH); H NMR (pyridine-d , 600 MHz) and C NMR (pyridine-d5,
1
[
5
1
50 MHz) data, seen supplementary material Table S1 and Figures S27–S35; HR ESI MS:
þ
m/z 635.3795 [M þ H] (calcd. for C H O , 635.3795).
35 55 10
3.5. Acid hydrolysis of compounds 1–3
The acid hydrolysis of compounds 1–3 was performed as the literature (Tanaka et al.
2
007). A solution of each compound (1.0 mg) was hydrolysed in 2 mL HCl (2 mol/L)
ꢁ
under 80–90 C for 1 h. The reaction mixture was partitioned between EtOAc and H O
2
twice. The H O layer was concentrated under reduced pressure, giving a saccharide
2
residue. The residue was dissolved in pyridine (1.0 mL) containing L-cysteine methyl

NATURAL PRODUCT RESEARCH
7
ꢁ
ester hydrochloride (2.5 mg) and heated at 60 C. 1 h later, o-tolyl isothiocyanate
ꢁ
(
10 lL) was added and further reacted at 60 C for 1 h. Then, the reaction mixture
was directly analysed by the HPLC system and detected with a UV detector at the
ꢁ
wavelength of 250 nm on a C18 column at 35 C. The mobile phase was
CH CN ꢄ H O ꢄ HCOOH (25:75:0.1, v/v/v) at a flow rate of 0.8 mL/min. The standard
3
2
monosaccharides, D-xylose and L-xylose, were subjected to the same method. As a
result, the monosaccharides of 1–3 were determined to be D-xylose by comparing
retention time with derivatisation of standard monosaccharides in HPLC chromato-
gram (supplementary material Figure S8).
3.6. Anti-inflammation activity assay
The assay was carried out as previously described (Zhao et al. 2012) with minor
modifications.
3.6.1. Cell cultures
Mouse RAW264.7 macrophage cells were obtained from ATCC and cultured in
Dullbecco’s modified Eage’s medium (DMEM) containing 10% foetal bovine serum
(
1
Gibco: C11995500BT), 100 U/mL penicillin and 100 lg/mL streptomycin (Gibco:15140-
ꢁ
22) with 5% CO at 37 C.
2
3.6.2. Measurement of cell viability
The viability of mouse RAW264.7 macrophage cells was determined by MTT
(
Methylcyclopentadienyl Manganese Tricarbonyl, Aladdin: T100896-5g). The RAW264.7 cells
4
were seeded into 96-well plate (Nest, Biotech, China) with a density of 2.5 ꢃ 10 cells/mL.
After treatment with series concentrations of compounds for 24 h, the absorbance
(490 nm) was recorded on a microplate reader (BioTek Instruments: SN258282).
3.6.3. Bioassay for NO production
Nitrite, as an indicator of NO production, was measured for its concentration in
medium according to Griess method (Beyotime Biotechnology, S0021). Hydrocortisone
(
Aladdin: C1710078) and curcumin (Meilunbio: N0223A) were used as positive controls.
4
RAW264.7 cells were seeded into 96-well plates at 2.5 ꢃ 10 cells/well and cultured
overnight. Subsequently, the cells were incubated in new medium which included
ꢁ
0
.5 lg/mL LPS with or without the compounds for additional 24 h at 37 C. The cell-
free supernatant and Griess reagents (Griess Reagent I and Griess Reagent II) were
completely mixed with the same amount of 50 lL. Absorbance of the final product
was measured at 540 nm on the microplate reader. The nitrite concentration and
inhibitory rate were calculated according to the standard calibration curve. The
residual cell-free supernatant was removed and added 100 lL fresh DMEM, and then
the viability of the RAW264.7 cells was determined by MTT.
3.6.4. Western blot assay
Western blot analysis was performed to evaluate the expression of inflammation-
associated proteins, such as iNOS and COX-2 proteins. Cells were collected, then

8
Q.-Q. PANG ET AL.
resuspended with radio immunoprecipitation assay buffer (Fdbio: FD008) supple-
mented with 0.1 mM PMSF protease inhibitor (Fdbio: FD1001). The cell suspension was
vortexed and lysed by ultrasound and then centrifuged at 12,000 rpm, then the super-
natant was taken and reserved. The total protein concentrations were determined by
BCA protein assay kit (Beytime: P0010). The same amount of proteins for each sample
was loaded to 8% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-
PAGE), and the proteins separated on the gel were transferred onto a polyvinylidene
difluoride membrane (Immobilon: IPVH00010). After blocking with 5% skim milk (BD:
2
32100), the membranes were then incubated with specific primary antibodies (iNOS:
ꢁ
CST13120s; COX-2: 12282S) at 4 C overnight, followed by incubation with correspond-
ing secondary antibodies (Fdbio: FDR007). The protein blots were finally detected by
enhanced chemiluminsecence system (ImmunoCruz: sc-2048).
3.6.5. Statistical analysis
At least triplicate experiments were conducted to obtain all results, and the data were
displayed as mean ± SEM. Statistical analysis was performed on GraphPad, version 4.00,
One-way ANOVA followed by Tukey’s test were used in the statistical teats. A value of
p < 0.05 was considered statistically significant.
4. Conclusions
In this study, ten cycloart-7-ene triterpenoid glycosides, including three new com-
pounds (1–3), were isolated from the roots of C. dahurica (Figure 1). Their structures
were elucidated on the basis of extensive spectroscopic analyses, chemical methods
and comparison with literatures. In addition, the isolates were evaluated for their
inhibitory effects on the production of NO, as well as the expressions of iNOS and
COX-2 in LPS-stimulated RAW 264.7 macrophages.
Acknowledgement
The authors are grateful to State Key Laboratory of New-Tech for Chinese Medicine
Pharmaceutical Process, Jiangsu Kanion Pharmaceutical Co. Ltd. for their assistance of activity test.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was supported by grants from the National Natural Science Foundation of China
(Grant Nos. 81630097, 81602984, 81803347 and 81903426).
References
Commission NP. 2015. The pharmacopeia of the People’s Republic of China. Beijing: China
Medical Science Press, 1, p. 73–74.

NATURAL PRODUCT RESEARCH
9
Gan LS, Zheng DJ, Liu Q, Zhou J, Zhang MZ, Yao W, Shao BH, Mo JX, Zhou CX. 2015. Eight new
cycloartane triterpenoids from Beesia calthifolia with hepatoprotective effects against D-gal-
actosamine induced L02 cell damage. Bioorg Med Chem Lett. 25(18):3845–3849.
Guo Y, Yin T, Wang X, Zhang F, Pan G, Lv H, Wang X, Owoicho Orgah J, Zhu Y, Wu H. 2017.
Traditional uses, phytochemistry, pharmacology and toxicology of the genus Cimicifuga: a
review. J Ethnopharmacol. 209(1):264–282.
Hao YM, Luo W, Jiang GZ, Lv CN, Lu JC. 2019. One new and seven known triterpene glycosides
from the aerial parts of Cimicifuga dahurica. J Asian Nat Prod Res. 10.1080/10286020.2019.
1638370
Kadota S, Li JX, Tanaka K, Namba T. 1995. Constituents of Cimicifugae Rhizoma. II. Isolation and
structures of new cycloartenol triterpenoids and related compounds from Cimicifuga foetida
L. Tetrahedron. 51(4):1143–1166.
Kusano A, Takahira M, Shibano M, Miyase T, Kusano G. 1999. Studies on the constituents of
Cimicifuga Species. XXVI. Twelve new cycloart-7-enol glycosides from the underground parts
of Cimicifuga simplex WORMSK. Chem Pharm Bull. 47(4):511–516.
Li JX, Kadota S, Hattori M, Yoshimachi S, Shiro M, Oogami N, Mizuno H, Namba T. 1993.
Constituents of Cimicifugae Rhizoma. I. Isolation and characterization of ten new cycloartenol
triterpenes from Cmicifuga heracleifolia KOMAROV. Chem Pharm Bull. 41(5):832–841.
Liu DL, Li YJ, Yao N, Xu J, Chen ZS, Yiu A, Zhang CX, Ye WC, Zhang DM. 2014. Acerinol, a cyclo-
lanstane triterpenoid from Cimicifuga acerina, reverses ABCB1-mediated multidrug resistance
in HepG2/ADM and MCF-7/ADR cells. Eur J Pharmacol. 733:34–44.
Liu Y, Chen D, Si J, Tu G, An D. 2003. Cimidahuside 1 and 2, two new cyclolanostanol xylosides
from the aerial parts of Cimicifuga dahurica. Nat Prod Res. 17(4):243–246.
Lu Q, Li HB, Pang QQ, Zhang WY, Su ZZ, Pan DB, Yao XS, Yu Y. 2019. New phenylpropanoid allo-
pyranosides from the rhizomes of Cimicifuga dahurica. Bioorg Med Chem Lett. 29(14):
1774–1778.
Lu Q, Zhang WY, Pan DB, Shi DF, Pang QQ, Li HB, Yao XJ, Yao ZH, Yu Y, Yao XS. 2019. Phenolic
acids and their glycosides from the rhizomes of Cimicifuga dahurica. Fitoterapia. 134:485–492.
Lv C, Yang F, Qin R, Qi Z, Zhou W, Lu J. 2017. Bioactivity-guided isolation of chemical constitu-
ents against H O -induced neurotoxicity on PC12 from Cimicifuga dahurica (Turcz.) Maxim.
2
2
Bioorg Med Chem Lett. 27(15):3305–3309.
Maione F, Russo R, Khan H, Mascolo N. 2016. Medicinal plants with anti-inflammatory activities.
Nat Prod Res. 30(12):1343–1352.
Mei YD, Zhang N, Zhang WY, Tang JS, Zhou H, Yu Y, Yao XS. 2019. Two new ursane-type nortri-
terpenes from Lonicera macranthoides and their iNOS-inhibitory activities. Chin J Nat Med.
17(1):27–32.
Nishida M, Yoshimitsu H. 2011. Six new cycloartane glycosides from Cimicifuga Rhizome. Chem
Pharm Bull. 59(10):1243–1249.
Qin R, Zhao Y, Zhao Y, Zhou W, Lv C, Lu J. 2016. Polyphenolic compounds with antioxidant
potential and neuro-protective effect from Cimicifuga dahurica (Turcz.) Maxim. Fitoterapia.
115:52–56.
Shi QQ, Lu J, Peng XR, Li DS, Zhou L, Qiu MH. 2018. Cimitriteromone A-G, macromolecular triter-
penoid-chromone hybrids from the Rhizomes of Cimicifuga foetida. J Org Chem. 83(17):
10359–10369.
Su Y, Chi WC, Wu L, Wang QH, Kuang HX. 2016. Photochemistry and pharmacology of 9,19-
cyclolanostane glycosides isolated from genus Cimicifuga. Chin J Nat Med. 14(10):721–731.
Sun HY, Huang MH, Yao N, Hu JY, Li YI, Chen LP, Hu N, Ye WC, Tai WCS, Zhang DM et al. 2017.
The cycloartane triterpenoid ADCX impairs autophagic degradation through Akt overactiva-
tion and promotes apoptotic cell death in multidrug-resistant HepG2/ADM cells. Biochem
Pharmacol. 146:87–100.
Sun HY, Liu BB, Hu JY, Xu LJ, Chan SW, Chan CO, Mok DKW, Zhang DM, Ye WC, Chen SB. 2016.
Novel cycloartane triterpenoid from Cimicifuga foetida (Sheng ma) induces mitochondrial
apoptosis via inhibiting Raf/MEK/ERK pathway and Akt phosphorylation in human breast car-
cinoma MCF-7 cells. Chin Med. 11:1

1
0
Q.-Q. PANG ET AL.
Tanaka T, Nakashima T, Ueda T, Tomii K, Kouno I. 2007. Facile discrimination of aldose enan-
tiomers by reversed-phase HPLC. Chem Pharm Bull. 55(6):899–901.
Thao NP, Lee YS, Luyen BTT, Oanh HV, Ali I, Arooj M, Koh YS, Yang SY, Kim YH. 2018. Chemicals
from Cimicifuga dahurica and their inhibitory effects on pro-inflammatory cytokine production
by LPS-stimulated bone marrow-derived dendritic cells. Nat Prod Sci. 24(3):194.
Yoshimitsu H, Nishida M, Sakaguchi M, Nohara T. 2006. Two new 15-deoxycimigenol-type and
three new 24-epi-cimigenol-type glycosides from Cimicifuga Rhizome. Chem Pharm Bull.
54(9):1322–1325.
Zhang F, Han LF, Pan G-X, Peng S, Andre N. 2013. A new phenolic amide glycoside from
Cimicifuga dahurica. Yao Xue Xue Baol. 48(8):1281–1285.
Zhao F, Gao Z, Jiao W, Chen L, Chen L, Yao X. 2012. In vitro anti-inflammatory effects of beta-
carboline alkaloids, isolated from Picrasma quassioides, through inhibition of the iNOS path-
way. Planta Med. 78(18):1906–1911.