Cycloartane triterpenoids from Actaea vaginata with anti-inflammatory effects in LPS-stimulated RAW264.7 macrophages

Article abstract of DOI:10.1016/j.phytochem.2019.01.003

Five undescribed cycloartane triterpenoids, including two cycloartane trinor-triterpenoids, were isolated from a 70% ethanol extract of the whole plant of Actaea vaginata (Ranunculaceae), together with thirteen known cycloartane triterpenoids. Their structures were determined by spectroscopic techniques and quantum chemical calculations for intramolecular noncovalent interactions with reduced density gradient method. All compounds were evaluated for their anti-inflammatory effects by a lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production model in RAW264.7 macrophage cells, and some showed potent inhibitory effects with IC₅₀ values ranging from 5.0 to 24.4 μM. Further mechanism studies showed that one compound dose-dependently suppressed LPS-induced NO production and pro-inflammatory cytokines secretion, and decreased the expression of iNOS, through inhibiting NF-κB activation.

Full text of DOI:10.1016/j.phytochem.2019.01.003

Phytochemistry160(2019)1–10
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Cycloartane triterpenoids from Actaea vaginata with anti-inflammatory
effects in LPS-stimulated RAW264.7 macrophages
Zhu-Jun Fanga,1, Tian Zhangb,1, Shi-Xin Chena, Yan-Lan Wanga, Chang-Xin Zhoua, Jian-Xia Moa,
Yong-Jiang Wua, You-Kai Xuc, Li-Gen Linb,∗∗, Li-She Gana,d,∗
a College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, PR China
b State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao 999078, PR China
c Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun 666303, PR China
d Hangzhou Institute of Innovative Medicine, Zhejiang University, 291 Fucheng Road, Hangzhou 310018, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Five undescribed cycloartane triterpenoids, including two cycloartane trinor-triterpenoids, were isolated from a
70% ethanol extract of the whole plant of Actaea vaginata (Ranunculaceae), together with thirteen known cy-
cloartane triterpenoids. Their structures were determined by spectroscopic techniques and quantum chemical
calculations for intramolecular noncovalent interactions with reduced density gradient method. All compounds
were evaluated for their anti-inflammatory effects by a lipopolysaccharide (LPS)-stimulated nitric oxide (NO)
production model in RAW264.7 macrophage cells, and some showed potent inhibitory effects with IC50 values
ranging from 5.0 to 24.4 μM. Further mechanism studies showed that one compound dose-dependently sup-
pressed LPS-induced NO production and pro-inflammatory cytokines secretion, and decreased the expression of
iNOS, through inhibiting NF-κB activation.
Actaea vaginata (Ranunculaceae)
Cycloartane trinortriterpenoids
Reduced density gradient
Inducible NO synthase
NF-κB pathway
1. Introduction
products have been well known for their large diversities in structure
and an important source for drug candidates with fewer adverse effects
Inflammation is a natural defense response to noxious stimuli, pa-
thogens, and immune reactions, and a key player in the pathogenesis of
many acute and chronic diseases, including cardiovascular diseases,
neurodegeneration, diabetes, and even aging (Escárcega et al., 2018;
Ryan and Kelly, 2016). The early phase of inflammation involves the
release of nitric oxide (NO) and prostaglandin produced by inducible
nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), respec-
tively, associated with increased expression levels of pro-inflammatory
cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-6
(IL-6), and interleukin-1 beta (IL-1β) (Lawrence et al., 2001). Nuclear
factor-kappa B (NF-κB) plays a pivotal role in inflammation through
controlling the transcription of pro-inflammatory cytokines, chemo-
kines, adhesion molecules, inducible enzymes, and growth factors
(Taniguchi and Karin, 2018).
(Newman and Cragg, 2016). Extensive research focused on natural
products with significant anti-inflammatory activities such as iridoids
(Lee et al., 2018), sesquiterpenoids (Xue et al., 2018), diterpenoids
(Zhou et al., 2018), flavonoids (Park et al., 2018), and lignans (Hwang
et al., 2003) has been carried out. Notably, recent studies revealed that
some cycloartane triterpenoids exhibited promising anti-inflammatory
activities (Youn et al., 2016).
As a part of our continuous research on cycloartane triterpenoids
from medicinal plants (Zhang et al., 2018; Gan et al., 2015; Mo et al.,
2014), the whole plants of Actaea vaginata (Maxim.) J.Compton (Ra-
nunculaceae) (synonym Souliea vaginata (Maxim.) Franch.) collected
from Taibai mountain in Shaanxi province of China were studied phy-
tochemically. A. vaginata is widely distributed in the highlands of
southwest and northwest China (Editorial Committee of Flora of China,
1979), and its rhizomes are commonly used as a traditional Tibetan
medicine for the treatment of conjunctivitis, stomatitis, pharyngitis,
and enteritis (Jia and Li, 2005). Previous phytochemical work have
Although non-steroidal anti-inflammatory drugs (NSAIDs) have
been widely used to relieve pain and inflammation, their gastro-
intestinal and cardiovascular toxicity are always criticized. Natural
∗ Corresponding author. College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, PR China.
∗∗ Corresponding author. State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao 999078,
PR China.
E-mail addresses: ligenl@umac.mo (L.-G. Lin), lsgan@zju.edu.cn (L.-S. Gan).
1 These authors contributed equally.
https://doi.org/10.1016/j.phytochem.2019.01.003
Received 10 September 2018; Received in revised form 17 November 2018; Accepted 7 January 2019
0031-9422/©2019ElsevierLtd.Allrightsreserved.

Z.-J. Fang et al.
Phytochemistry160(2019)1–10
Fig. 1. Chemical structures of compounds 1–5.
demonstrated that A. vaginata are rich in cycloartane triterpenoids
(Zhou et al., 2006; Wu et al., 2017a,b). However, the anti-inflammatory
activities and related mechanisms of these cycloartane triterpenoids
from A. vaginata have not been reported. In the current study, five
undescribed cycloartane triterpenoids and nortriterpenoids (1–5)
(Fig. 1), including two rare trinortriterpenoids (1, 2), together with
thirteen known cycloartane triterpenoids (6–18), were systematically
isolated. The structures were elucidated via spectroscopic techniques
and quantum chemical calculations for intramolecular noncovalent
interactions with reduced density gradient method. All compounds
hydroxylmethyl structure on the side chain. In the HMBC spectrum
(Fig. 2), the cycloartane skeleton was confirmed by correlations from
H3-29 and H3-30 to C-3, C-4, and C-5, from H-12 to C-9 and C-13, from
H2-19 to C-1, C-5, C-8, C-9, C-10, and C-11, from H3-18 to C-12, C-13,
and C-17, from H3-28 to C-13, C-14, and C-15, and from H3-21 to C-17,
C-20, and C-22. HMBC correlations from H-16 to C-23, from H2-22 to C-
17 and C-23, and from H2-24 to C-22 and C-23 verified connections on
the side chain. The configuration was determined by NOESY correla-
tions of H-3/H3-29, H3-29/H-5, H-5/H-7α, H-7α/H3-28, H3-28/H-12, H-
12/H-17, H-17/H3-21, H-17/H-16, and H-16/H-24a for the α-oriented
hydrogens. Meantime, the key β-oriented hydrogens were identified by
NOESY correlations of H3-30/H-19β, H-19β/H-8, and H-8/H3-18
(Fig. 2). As the absolute configuration on the main skeleton of these
cycloartane triterpenoids have already been clarified by techniques
such as single-crystal X-ray diffraction analysis with Cu Kα irradiation
(Nian et al., 2013), consequently, compound 1 was identified as
(16S,20R,23R)-12β-acetoxy-16,23-epoxy-3β,23,24-trihydroxy-9,19-cy-
clolanostane and represents the first example of a cycloartane trinor-
triterpenoid. Compound 2 showed almost the same NMR data as that of
1 except for the additional signals of a sugar moiety. An anomeric hy-
drogen at δH 4.88 in the 1H NMR spectrum and five carbon signals at δC
108.0, 76.1, 79.1, 71.7, and 67.6, as well as a strongly deshielded
oxygen-bearing methine at δC 88.6, indicated a xylosyl moiety at C-3,
similar to that of cimilactone A (6) (Liu et al., 2002). Furthermore, a
key HMBC correlation between H-1′ and C-3 also confirmed that
compound 2 is a 3-O-xylosyl derivative of 1. The closely comparable
NMR spectra for the sugar moiety of 2 (Tables 1 and 2) to those of co-
isolated known compounds 6, 7, 9, 11–13, 15–18, and the previously
reported 9,19-cycloartane-type glycoside derivatives from this plant
(Wu et al., 2017a,b), together with experiments of acid hydrolysis and
co-TLC with an authentic D-xylose sample (Ju et al., 2002a), suggested
that the same D-xylopyranoside unit in 2. The structure of 2 was finally
defined as (16S,20R,23R)-12β-acetoxy-16,23-epoxy-23,24-dihydroxy-
3β-(β-D-xylopyranosyloxy)-9,19-cyclolanostane.
were assayed for their anti-inflammatory effects on
a lipopoly-
saccharide (LPS)-stimulated NO production model in RAW264.7 mac-
rophage cells. The potential action mechanism of one of the most potent
compound 3 was further investigated on pro-inflammatory cytokines,
chemokines, and inducible enzymes by immunostaining, ELISA, and
western blotting methods.
2. Results and discussion
Compound 1 was isolated as white amorphous powder. The mole-
cular formula was determined as C29H46O6 by a HRESIMS pseudo ion
peak at 513.3190 [M + Na]+ (calcd for C29H46O6Na, 513.3192). The
IR spectrum of 1 showed a vibrational absorption peak for ester group
at 1733 cm−1. The 1H NMR spectrum (Table 1) exhibited two shielded
geminal hydrogen signals at δH 0.35 (1H, d, J = 4.2 Hz, H-19α) and
0.67 (1H, d, J = 4.2 Hz, H-19β) assignable to the CH2-19 group of a
cycloartane type triterpenoid, as well as four methyl singlets at δH 0.87
(s, Me-28), 0.79 (s, Me-30), 0.97 (s, Me-29), and 1.13 (s, Me-18), one
methyl doublet at δH 0.91 (d, J = 6.5 Hz, Me-21), and one acetyl methyl
singlet at δH 2.01 (3H, s, OAc). The 13C NMR (Table 2) and DEPT (135)
spectra revealed an acetyl group at δC 171.0 and 21.9 (eOAc), and
exhibited four oxygen-bearing carbons at δC 78.7 (C-3), 77.3 (C-12),
71.9 (C-16), and 64.7 (C-24). The above data indicated a unique cy-
cloartane trinor-triterpenoid structure for 1, other than those reported
tetranor-triterpenoids cimilactones A-C (6–8) from this plant (Liu et al.,
2002; Nian et al., 2013). Compared to the NMR spectra of cimilactones
A-C, a deshielded hemiacetal carbon signal at δC 97.5 and a terminal
oxygen-bearing methylene at δC 64.7 indicated a 23-hemiacetal, 24-
The molecular formula of compound 3, C37H58O10, suggested one
less unsaturated degree than that of asiaticoside A (9) (Gao et al.,
2006). The 1H NMR spectrum of 3 (Table 1) also exhibited typical cy-
clopropyl methylene hydrogen signals at δH 0.21 (1H, d, J = 4.0 Hz, H-
2

Z.-J. Fang et al.
Phytochemistry160(2019)1–10
Table 1
1H NMR data for compounds 1–5 (δ in ppm, J in Hz, 500 MHz).
No.
1a
2b
3b
4a
5b
1
α 1.61, m
α 1.56, m
α 1.53, m
α 1.60, m
α 1.54, m
β 1.22, m
β 1.15, m
β 1.12, m
β 1.21, m
β 1.12, m
2
α 1.76, m
β 1.59, m
3.28, dd (10.6, 4.3)
1.28, m
α 2.31, m
α 1.91, m
β 2.33, m
3.49, dd (11.7, 4.2)
1.25, m
α 1.59, m
β 1.79, m
3.30, dd (10.9, 4.3)
1.30, m
α 1.87, m
β 2.29, m
3.48, dd (11.5, 4.1)
1.28, m
β 1.86, m
3
5
6
3.48, m
1.29, m
α 1.57, m
α 1.50, m
α 1.50, m
α 1.62, m
α 1.51, m
β 0.85, m
β 0.70, m
β 0.73, m
β 0.85, m
β 0.78, m
7
α 0.99, m
α 0.93, m
α 0.96, m
α 1.42, m
α 1.00, m
β 1.37, m
β 1.29, m
β 1.25, m
β 1.11, m
β 1.34, m
8
11
1.70, m
1.63, dd (11.7, 5.4)
α 2.75, dd (15.9, 8.9)
β 1.20, m
1.66, m
1.74, dd (12.4, 5.3)
α 2.65, dd (16.0, 9.4)
β 1.01, dd (16.0, 3.8)
5.04, dd (9.4, 3.8)
α 2.10, dd (14.3, 7.7)
β 1.90, dd (14.3, 3.0)
4.23, ddd (7.7, 6.7, 3.0)
2.03, br d (6.7)
1.10, s
1.60, m
α 2.65, dd (16.2, 9.1)
β 1.03, m
α 2.68, dd (15.9, 9.3)
β 1.18, dd (15.9, 4.0)
5.34, dd (9.3, 4.0)
α 1.98, m
β 1.87, m
4.22, m
2.11, br d (6.7)
1.35, s
α 0.21, d (4.0)
β 0.61, d (4.0)
2.48, m
α 2.75, dd (16.0, 8.9)
β 1.17, dd (16.0, 3.5)
5.11, dd (8.9, 3.5)
α 1.94, m
β 1.72, m
4.42, m
1.77, dd (15.9, 7.4)
1.32, s
α 0.25, d (4.2)
β 0.62, d (4.2)
1.62, m
0.95, d (6.4)
α 1.47, m
β 2.25, dd (14.1, 2.8)
12
15
4.84, dd (9.1, 3.1)
α 2.09, m
5.14, m
α 1.92, dd (12.7, 8.1)
β 1.68, dd (12.7, 6.7)
4.46, dd (14.7, 7.3)
1.80, m
β 1.88, m
16
17
18
19
4.32, dd (15.0, 7.8)
1.65, m
1.13, s
1.34, s
α 0.35, d (4.2)
β 0.67, d (4.2)
1.56, m
α 0.24, d (4.1)
β 0.59, d (4.1)
1.84, m
α 0.38, d (4.5)
β 0.73, d (4.5)
2.36, m
1.12, d (7.3)
6.01, d (4.0)
20
21
22
0.91, d (6.5)
α 1.89, m
0.98, d (5.9)
α 2.54, m
1.16, d (7.2)
5.22, d (3.3)
β 1.26, m
β 1.47, m
24
a 3.41, d (12.0)
b 3.65, d (12.0)
a 3.87, d (11.8)
b 4.14, d (11.8)
4.34, d (4.3)
25
26
27
28
29
30
OAc
OCH3
1′
2′
3′
4′
5′
3.20, dt (13.7, 6.9)
1.09, d (6.8)
1.08, d (6.8)
0.97, s
0.99, s
0.81, s
3.44, m
1.62, s
1.68, s
0.89, s
1.35, s
1.05, s
2.14, s
1.21, d (3.0)
1.20, d (3.0)
0.88, s
1.34, s
1.05, s
0.87, s
0.97, s
0.79, s
2.01, s
0.89, s
1.34, s
1.03, s
2.14, s
2.05, s
2.11, s
3.26, s
4.88, d (7.5)
4.06, m
4.19, t (8.7)
4.26, m
3.77, m
4.39, dd (11.3, 5.2)
4.88, d (7.5)
4.06, m
4.19, m
4.26, m
3.77, dd (11.2, 10.3)
4.39, dd (11.2, 5.2)
4.86, d (7.5)
4.04, dd (8.4, 7.5)
4.15, dd (8.7, 8.4)
4.23, ddd (9.0, 8.7, 5.1)
3.74, dd (10.9, 9.0)
4.36, dd (10.9, 5.1)
a
Recorded in chloroform-d.
Recorded in pyridine-d5.
b
19α) and δH 0.61 (1H, d, J = 4.0 Hz, H-19β), as well as one acetyl
methyl singlet at δH 2.14 (3H, s, OAc). Compared to the 13C NMR
spectrum of 9, the absence of the ketone carbon C-24 in 9 and the
presence of an oxygen-bearing carbon at δC 80.0 in 3 indicated that the
ketone carbonyl group is saturated to form a hydroxyl. The doublets for
CH3-26 and CH3-27 in 9 were deformed into two singlets in 3, and C-25
was deshielded from δC 35.4 in 9 to δC 73.4 in 3, which suggested an
additional hydroxyl group linked to C-25 (Table 2). In the HMBC
spectrum (Fig. 3), besides the signals verifying the cycloartane skeleton,
correlations from H3-27 and H3-26 to C-25 and C-24, and from H-24 to
C-22, C-23, and C-25 showed the 24,25-dihydroxyl side chain linked to
C-23. The 16,23-epoxy and 3-O-xylose linkage were respectively con-
firmed by HMBC correlations from H-16 to C-23 and from the anomeric
hydrogen H-1′ to C-3. The β-D-xylosyl moiety was again confirmed by
the above mentioned protocol. NOESY correlations (Fig. 3) of H-3/H3-
29, H3-29/H-5, H-8/H3-18, H3-18/H-20, H-12/H-17, H-17/H-16, and
H-17/H3-21 indicated the same configurations of 3 on the cycloartane
skeleton as that of 9, while the configuration at C-24 was unclear. The
configuration at C-24 on the relatively relaxed side chain was firstly
analyzed by conformational searching. To avoid the flexible hydroxyl
groups on the sugar moiety, Monte Carlo searching using molecular
mechanism with MMFF force field in the Spartan 14 program was
carried out for two model compounds, the C-24 epimer pairs of the 3-
methylated aglycone of 3 (supplementary data S51, compounds 3a and
3b). In a relative energy window of 0–2 kcal/mol, the result showed 3
lowest energy conformers for the 24R-3a and 5 ones for the 24S-3b,
respectively. These conformers were further optimized using DFT-D3 at
the M06-2X-D3/6-311G(d) level in vacuum using the Gaussian 09
program (Frisch et al., 2009). The results showed two predominating
conformers (over 94% Boltzmann distribution) for each compound.
Inspection of the 3D lowest energy structures indicated that in-
tramolecular hydrogen bonds might play important role in reducing
molecular energy and stabilizing conformation of the side chain by
forming of envelop-like five-membered ring or chair-like six-membered
ring. The existence of intramolecular hydrogen bonds was further
confirmed by computation using reduced density gradient (RDG)
method for noncovalent interactions (Fig. 4, supplementary data S51).
RDG calculation results for the dominant conformers of the 24R-3a and
the 24S-3b were shown in Fig. 4, which were generated by using
Multifwn program (Lu and Chen, 2012). For 24R-3a, a deep green color
of gradient isosurface, corresponding to a X-axis spike value around
−0.02, suggested the existence of weak hydrogen bonds at C(25)
eOeH…OeC(23) and C(24)eOeH…OeC(25). For 24S-3b, gradient
isosurface with dark green color indicated that weak hydrogen bonds
also existed at C(25)eOeH…OeC(23) and C(24)eOeH…OeC(25)
while directions of the adjacent groups are totally different. The above
calculations showed a relatively fixed side chain for 3, and the R con-
figuration at C-24 on the intramolecular hydrogen-bonded side chain
3

Z.-J. Fang et al.
Phytochemistry160(2019)1–10
Table 2
and 13C NMR spectra (Tables 1 and 2) very close to those of 4 and
asiaticoside A (9). Instead of the trisubstituted double bond in 9,
compound 5 exhibited an additional methoxy group as indicated by a
1H NMR methyl singlet at δH 3.26 (3H, s, OMe) and one 13C NMR
carbon signal at δC 51.1 (OMe). The methoxy group was determined as
23-OMe by a key HMBC correlation from OCH3 to C-23 (δC 103.5).
Strong NOESY correlations of H3-18/H3-26, and H-16/H3-OMe clearly
showed the α-configuration of 23-OMe. The β-D-xylosyl moiety was also
confirmed by acid hydrolysis and co-TLC with an authentic D-xylose
sample. Thus, the structure of 5 was determined as (16S,20R,23S)-12β-
acetoxy-16,23-epoxy-23-methoxy-3β-(β-D-xylopyranosyloxy)-9,19-cy-
clolanostan-24-one.
13C NMR data for compounds 1–5 (δ in ppm, 125 MHz).
No.
1a
2b
3b
4a
5b
1
2
3
4
5
6
7
8
32.0
30.3
78.7
40.5
46.8
20.7
25.8
46.1
20.0
26.7
36.7
77.3
47.7
48.5
43.4
71.9
56.6
13.2
30.2
25.0
20.9
40.2
97.5
64.7
32.4
30.4
88.6
41.7
47.5
20.9
26.2
46.3
20.6
27.2
37.2
77.6
48.9
48.3
43.8
71.9
57.4
14.0
30.0
26.2
21.5
40.5
101.8
61.9
32.5
30.4
88.6
41.7
47.5
20.8
26.3
47.1
21.0
27.6
37.0
77.4
49.4
48.3
46.2
75.2
52.9
13.5
30.7
25.1
26.3
106.3
154.2
80.0
73.4
28.0
26.4
21.3
26.2
15.8
171.2
21.7
32.1
30.2
78.6
40.6
46.8
20.6
26.0
47.0
20.5
27.2
36.6
77.2
48.7
48.0
45.7
75.2
51.6
12.5
31.0
25.3
24.6
116.0
149.0
201.3
34.8
18.5
18.5
21.2
25.6
14.1
171.0
21.5
32.4
30.3
88.5
41.7
47.5
20.9
26.2
46.2
20.5
27.2
37.1
77.4
49.2
48.3
43.8
73.0
56.3
14.0
30.1
25.9
21.3
39.3
103.5
213.0
35.7
20.0
20.0
19.4
26.1
15.7
170.9
22.0
51.1
107.9
76.0
79.0
71.6
67.5
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
OAc
By comparing their NMR data with those reported in the literature,
structures of the known compounds (Table S50) were identified as ci-
milactone A (6) (Liu et al., 2002), cimilactone B (7) (Liu et al., 2002),
cimilactone C (8) (Nian et al., 2013), asiaticoside A (9) (Gao et al.,
2006), 25-O-acetylcimigenol (10) (Li et al., 1993), 25-O-acet-
ylcimigenol-3-O-β-D-xylopyranoside (11) (Kusano et al., 1994), cimi-
genol-3-O-β-D-xyloside (12) (Chen et al., 2007), neocimicigenoside B
(13) (Mimaki et al., 2006), 27-deoxyacetylacteol (14) (Kadota et al.,
1995), 23-epi-26-deoxyactein (15) (Chen et al., 2002), 12-deacetyloxy-
15α-hydroxy-23-epi-26-deoxyactein (16) (Zhou et al., 2006), acteol-3-
O-β-D-xylopyranosyl-3′‐O-β-D-xylopyranoside (17) (Zhu et al., 2016),
and beesioside P (18) (Ju et al., 2002b), respectively.
The anti-inflammatory effects of all compounds (1–18) were eval-
uated by an LPS-stimulated NO production model on RAW264.7 mac-
rophage cells. The results showed that compounds 3, 5, 9, 14, and 15
exhibited strong NO production inhibitory activity (< 20 μΜ) with IC50
values ranging from 5.0 to 17.8 μΜ, and compounds 1 and 4 showed
moderate NO production inhibitory activity (Liu et al., 2016), while the
19.8
25.6
14.1
171.0
21.9
20.1
26.0
15.8
171.0
22.1
positive control indomethacin exhibited an IC50 value of 3.9
0.3 μM
OCH3
1′
2′
3′
4′
108.0
76.1
79.1
71.7
67.6
108.1
76.1
79.1
71.7
67.6
(Table 3). After careful analysis of the structural features of these cy-
cloartane triterpenoids and nortriterpenoids as well as their NO pro-
duction inhibitory potency, some regular structure-activity relationship
patterns were summarized. (1) Cycloartane compounds with a 16,23-
epoxy, 22 (23)-en side chain (3–5, 9) and a 16,23; 23,26-diepoxy spiro-
side chain (14, 15) showed NO production inhibitory activity, com-
pared to the inactive compounds with a 16,23; 16,24-diepoxy side
chain (10–12) and the tetranortriterpenoid lactones (6–8). (2) An
acetoxy group at C-12 is necessary for the NO production inhibitory
activity of cycloartane triterpenoids and nortriterpenoids in LPS-sti-
mulated RAW264.7 cells, such as compounds 1, 3–5, 9, 14, and 15. (3)
For cycloartane triterpenoids and nortriterpenoids containing a hy-
droxyl or acetoxy group at C-15, the NO production inhibitory activity
was totally disappeared, such as compounds 10–13, 16, and 18. Taken
together, the ring system type of the side chain, the existence of an
acetoxy group at C-12, and the absence of an oxygen-bearing group at
C-15 are essential for the NO production inhibitory activity of cy-
cloartane triterpenoids and nortriterpenoids in LPS-stimulated
RAW264.7 cells.
5′
a
Recorded in chloroform-d.
Recorded in pyridine-d5.
b
was then determined by key NOESY correlations between H3-18 and
H3-26/H3-27. Therefore, the structure of compound 3 was elucidated as
(16S,20S,24R)-12β-acetoxy-16,23-epoxy-24,25-dihydroxy-3β-(β-D-xy-
lopyranosyloxy)-9,19-cyclolanost-22(23)-ene.
Compound 4 possessed a molecular formula of C32H48O5 and
showed similar 1D NMR data (Tables 1 and 2) to those of asiaticoside A
(9). Compared to the 13C NMR spectrum of 9, the absence of a xylosyl
moiety indicated that 4 might be the aglycone of 9. 2D NMR experi-
ments further confirmed the structure of 4 as (16S,20S)-12β-acetoxy-
16,23-epoxy-3β-hydroxy-9,19-cyclolanost-22(23)-en-24-one.
Compound 5 gave a molecular formula of C38H60O10 and showed 1H
Fig. 2. Key HMBC (blue arrows) and NOESY (red dotted double arrows) correlations of 1. (For interpretation of the references to color in this figure legend, the
reader is referred to the Web version of this article.)
4

Z.-J. Fang et al.
Phytochemistry160(2019)1–10
Fig. 3. Key HMBC (blue arrows) and NOESY (red dotted double arrows) correlations of 3. (For interpretation of the references to color in this figure legend, the
reader is referred to the Web version of this article.)
Fig. 4. Gradient isosurfaces (RDG = 0.5 a.u.) for the dominant conformers of 24R-3a and 24S-3b and the corresponding plots of the reduced density gradient versus
the electron density multiplied by the sign of the second Hessian eigenvalue.
As the most potent one among the undescribed compounds, com-
pound 3 was chosen in the following studies. Compound 3 inhibited
LPS-induced NO production in a dose dependent manner (Fig. 5A),
while didn't obviously affect cell viability up to 40 μM as determined by
an MTT assay (Fig. 5B). The Western blotting results suggested com-
pound 3 reversed LPS-induced increase of iNOS protein in a dose de-
pendent manner (Fig. 5C), which was further supported by the im-
munostaining results (Fig. 5D). Next, the inhibitory effects of compound
(IKKα/β) and degraded subsequently through the 26S proteasome-
mediated pathway, allowing NF-κB to translocate into the nucleus to
induce transcription of pro-inflammatory genes (Tak and Firestein,
2001). As shown in Fig. 6A, the phosphorylation of IKKα/β, IκBα and
p65 were increased remarkably by LPS stimulation, which were sup-
pressed by treatment of compound 3. Since the nuclear translocation of
NF-κB transcriptional subunit p65 is a critical step for the activation of
NF-κB signaling pathway, immunofluorescent staining was performed
to determine the translocation of p65. The immunostaining results re-
vealed greater proportion of p65 translocated to the nucleus in LPS
stimulated RAW264.7 cells, while pre-treatment of 3 significantly re-
duced the p65 nuclear accumulation (Fig. 6B). Thus, treatment of 3
prevented degradation of IκBα and translocation to the nucleus of NF-κB
in LPS-induced RAW264.7 cells.
3
on LPS stimulated elevation of pro-inflammatory cytokines in
RAW264.7 cells were evaluated. The ELISA results showed that 3 sig-
nificantly suppressed the levels of TNF-α, IL-6, and MCP-1 in LPS sti-
mulated macrophages (Fig. 5E). Taken together, the above data in-
dicates that compound 3 suppresses LPS stimulated inflammatory
responses in macrophages.
NF-κB is a heterodimer consisting of two subunits (p50 and p65),
which induces transcription of related genes (Gomez et al., 2005).
Under unstimulated conditions, NF-κB dimers are inactive in the cyto-
plasm combining with its inhibitor protein κBα (IκBα). Upon in-
flammatory stimuli, such as LPS, IκBα is phosphorylated by IκB kinase
The anti-inflammatory effect of cycloartane triterpenoids and nor-
triterpenoids was only reported in few studies and one report showed
that 3,4; 9,10-seco-cycloartane type triterpenoids activate mitogen-ac-
tivated protein kinase kinase 1 (MPKK1), resulting in TNF-α production
and activation of NF-κB (Li et al., 2013). The current study showed that
5

Z.-J. Fang et al.
Phytochemistry160(2019)1–10
Table 3
Haiyang Chemical Co. Ltd.), C18 reverse-phased silica gel (40–75 μm,
FujiChromatorex), MCI gel (CHP20P, 75–150 μm, Mitsubishi Chemical
Industries Ltd.), D-101 macroporous resin (Chemical Plant of Nankai
University, Tianjin, People's Republic of China), and Sephadex LH-20
gel (Amersham Pharmacia Biotech.) were used for column chromato-
graphy. An EX-1600 HPLC system (Shanghai Wufeng Scientific
Inhibitory effects of compounds 1–18 against LPS-induced NO production in
RAW264.7 cells.
Compounds
IC50 (μM)a
Cell viability (%)b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
24.4
‒
2.0
93.1
84.7
90.6
96.8
96.5
93.8
92.0
97.7
90.8
77.2
95.0
90.4
99.5
99.5
91.3
90.9
98.2
97.2
97.5
3.3
3.6
1.8
1.7
0.6
0.9
1.5
0.4
1.0
2.6
0.8
1.3
3.6
2.6
1.5
0.5
0.9
0.6
0.6
c
Instruments Co. Ltd.) with
a
semi-preparative C18 column
6.1
0.2
0.6
1.4
(250 mm × 10 mm, 10 μM, Phenomenex) was also applied for the iso-
lation. All solvents were of analytical grade (Tianjin Yongda Chemical
Reagent Co. Ltd., Tianjin, People's Republic of China). Dulbecco's
Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were
purchased from Life Technologies/Gibco Laboratories (Grand Island,
NY, USA). LPS (Escherichia coli, serotype 0111: B4), 4′,6-diamidino-2-
phenylindole (DAPI), and Griess reagent were purchased from Sigma-
Aldrich (St. Louis, MO, USA). Antibodies against iNOS, p65, p-p65, p-
IKK-α/β, IKK-α, IKK-β, p-IκB-α, and IκB-α were purchased from Cell
Signaling Technologies (Beverly, MA, USA). Antibody against GAPDH
was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
SuperSignal West Femto Maximum Sensitivity Substrate was obtained
from Life Technologies/Thermo Fisher Scientific (Grand Island, NY,
USA).
20.4
17.8
> 30
> 30
> 30
5.7
0.2
c
‒
> 30
> 30
> 30
5.0
11.9
> 30
> 30
> 30
3.9
0.2
2.4
17
18
Indomethacind
0.3
a
IC50 values were expressed as mean
Cell viability at 30 μM.
The samples showed cytotoxic effects to cells at 30 μM (more than 10% cell
SD (n = 6).
b
c
4.2. Plant material
death).
Whole plants of Actaea vaginata (Maxim.) J.Compton
(Ranunculaceae) (synonym Souliea vaginata (Maxim.) Franch.) were
collected from the Taibai mountain, Baoji, Shaanxi, China, in
September 2014 (33°58′56.496″ N 107°45′43.7688″ E), and authenti-
cated by one of the authors Jian-Xia Mo. A voucher specimen has been
deposited in the Institute of Modern Chinese Medicine, Zhejiang
University (accession number AV-2014-I).
d
Indomethacin (5 μM) was used as a positive control.
compound 3 suppressed the phosphorylation of IKKα/β and IκBα, and
inhibited the nuclear translocation of p65. The anti-inflammatory effect
of cycloartane triterpenoids and nortriterpenoids might be mediated
through the following pathways: (1) Inhibit the dimerization of Toll-
like receptor 4 (TLR4). (2) Block the MyD88 (myeloid differentiation
primary response gene 88)-and/or TRIF (TIR-domain-containing
adaptor molecule 1)-dependent pathway. (3) Prevent the phosphor-
ylation of specific serine residues of IκB proteins by a multiprotein
complex termed the IKK complex. (4) Hinder the nuclear translocation
of p65/p50 complex (Napetschnig and Wu, 2013). Further studies are
needed to fully determine the biochemical mechanisms involved in the
anti-inflammatory effect of cycloartane triterpenoids and nor-
triterpenoids.
4.3. Extraction and isolation
The air-dried whole plant of A. vaginata (5 kg) was powdered and
extracted with 70% EtOH (3 × 25 L) at room temperature. After re-
moval of the solvent under reduced pressure, a crude extract (585.6 g)
was obtained. The extract was then suspended in H2O (2 L) and parti-
tioned successively with petroleum ether, EtOAc, and n-BuOH (volume
ratio of 1:1) to give three fractions, SVP (35.2 g), SVE (300.7 g), and
SVB (203.5 g), respectively. The EtOAc fraction SVE was subjected to a
D-101 macroporous resin column eluted with aqueous EtOH
(30%–95%, stepwise) to afford eight subfractions of SVEA–SVEH. SVEC
(29.0 g) was separated on a silica gel column (CH2Cl2‒MeOH, 25:1 to
20:1) to afford four subfractions of SVEC1–SVEC4. SVEC1 (2.4 g) was
further chromatographed on a Sephadex LH-20 column (MeOH), fol-
lowed by a C18 reversed-phase column (80% aqueous MeOH) to obtain
compounds 9 (13.3 mg), 11 (24.8 mg), and 5 (30.5 mg). Compounds 6
(19.5 mg, tR = 16.8 min) and 15 (42.1 mg, tR = 22.4 min) were ob-
tained by semipreparative HPLC (65% aqueous MeOH) of SVEC2
(2.5 g). SVEC3 (6.2 g) was fractionated by a silica gel column (cyclo-
hexane–acetone, 3:1) to yield three sub-fractions of SVEC3a–SVEC3c.
SVEC3a (2.2 g) was further chromatographed on a C18 reversed-phase
silica gel column (60% aqueous MeOH) to obtain compounds 3
(62.5 mg), 16 (120.2 mg), and 7 (22.3 mg). SVEC4 (1.9 g) was subjected
to a silica gel column (cyclohexane–acetone, 7:2) to afford compounds
18 (22.0 mg) and 13 (90.4 mg). SVED (32.8 g) was separated by a silica
gel column (CH2Cl2–acetone, 10:1 to 1:1) to give fourteen subfractions
of SVED1–SVED14. SVED1 (4.2 g) was further separated by a silica gel
column (petroleum ether‒acetone, 5:1 to 1:1), followed by a Sephadex
LH-20 column (MeOH) to afford compounds 10 (76.5 mg) and 14
(47.9 mg). Next, SVED5 (2.5 g) was subjected to a silica gel column
(petroleum ether–acetone, 7:1 to 1:1) to afford four subfractions of
SVED5a–SVED5d. SVED5b (0.4 g) was further chromatographed on a
3. Conclusions
Two unprecedented cycloartane trinor-triterpenoids (1, 2), together
with other three undescribed (3–5) and thirteen known (6–18) ones,
were discovered from the traditional Tibetan medicine Actaea vaginata.
A reduced density gradient calculation for intramolecular noncovalent
interactions allowed the determination of relative configuration of C-24
secondary hydroxyl group on the rotatable side chain of compound 3.
As one of the most potent compound in an LPS-stimulated NO pro-
duction assay, compound 3 dose-dependently suppressed LPS-induced
NO production and pro-inflammatory cytokines secretion, and de-
creased the expression of iNOS, through inhibiting NF-κB activation.
4. Experimental
4.1. General experimental procedures
Optical rotations were acquired on a Jasco p-1010 polarimeter. IR
spectra were recorded on a Jasco FT/IR-4100 spectrometer. NMR
spectra were obtained on a Bruker AVANCE III 500 MHz spectrometer
(Bruker Co., Switzerland) with TMS as the internal standard. ESIMS and
HRESIMS were measured on a Finnigan LCQDECA XP instrument and an
Agilent Q-TOF 1290 LC/6224 MS system. Precoated silica gel GF254
plates (Qingdao Haiyang Chemical Co. Ltd., Qingdao, People's Republic
of China) were used for TLC. Silica gel (200–300 mesh, Qingdao
silica gel column (CH2Cl2‒MeOH, 70:1 to 30:1), followed by
a
Sephadex LH-20 column (MeOH) to afford compound 8 (10.5 mg).
6

Z.-J. Fang et al.
Phytochemistry160(2019)1–10
Fig. 5. Cytotoxic and anti-inflammatory effects of compound 3. (A) NO production in LPS-induced RAW264.7 cells treated with different concentrations of 3. (B) The
cell viability of RAW264.7 cells treated with different concentrations of 3. (C) 3 suppressed iNOS expression in a concentration-dependent manner. GAPDH was used
as an internal loading control. (D) The immunostaining of iNOS. Nuclei were stained with DAPI. Scale bar = 25 μM. (E) Effect of compound 3 on the production of
TNF-α, IL-6, and MCP-1 in LPS-induced RAW264.7 cells. The levels of TNF-α, IL-6 and MCP-1 were determined by ELISA kits. N = 6–9. *p < 0.05, **p < 0.01,
***p < 0.001, 3 vs LPS. ###p < 0.001, LPS vs vehicle control.
Compound 1 (22.0 mg) was obtained from SVED5d (0.3 g) via silica gel
column eluted with CH2Cl2–MeOH (40:1). SVED14 (3.3 g) was sub-
jected to a C18 reversed-phase silica gel column (from 30% to 100%
aqueous MeOH) to afford five subfractions of SVED14a‒SVED14e.
SVED14c (0.3 g) was further chromatographed on a silica gel column
(CH2Cl2‒acetone, 6:1 to 3:1) to afford compound 2 (15.7 mg). SVED14d
(0.5 g) was subjected to a silica gel column (CH2Cl2‒MeOH, 10:1 to 4:1)
and then was further purified by a Sephadex LH-20 column (MeOH) to
afford compound 17 (31.2 mg). SVEG (14.5 g) was subjected to a silica
gel column (petroleum ether‒EtOAc, 5:1 to 0:1) to give two subfrac-
tions of SVEG1–SVEG2. SVEG1 (0.9 g) was further chromatographed on
a Sephadex LH-20 column (MeOH), followed by a silica gel column
(CH2Cl2‒MeOH, 100:1) and preparative TLC (CH2Cl2‒MeOH, 50:1) to
afford compound 4 (5.8 mg). The n-BuOH fraction SVB was subjected to
4.3.1. (16S,20R,23R)-12β-acetoxy-16,23-epoxy-3β,23,24-trihydroxy-
9,19-cyclolanostane (1)
White amorphous powder; [α]3D0− 19.4 (c 0.15, MeOH); IR (KBr)
νmax 2961, 2929, 2869, 2338, 1733, 1456, 1376, 1261, 1023, 873,
801 cm−1 1H and 13C NMR data, see Tables 1 and 2; ESIMS (positive)
;
m/z 513 [M + Na]+; ESIMS (negative) m/z 489 [M − H]−; HRESIMS
(positive) m/z 513.3190 [M + Na]+ (calcd for C29H46O6Na, 513.3192).
4.3.2. (16S,20R,23R)-12β-acetoxy-16,23-epoxy-23,24-dihydroxy-3β-(β-
D-xylopyranosyloxy)-9,19-cyclolanostane (2)
[α]30
White amorphous powder;
D − 27.9 (c 0.11, MeOH); IR (KBr)
νmax 2934, 1734, 1653, 1457, 1364, 1243, 1164, 1044, 976, 795 cm−1
;
1H and 13C NMR data, see Tables 1 and 2; ESIMS (positive) m/z 605
[M − H2O + H]+; HRESIMS (positive) m/z 645.3623 [M + Na]+
(calcd for C34H54O10Na, 645.3615).
a
D-101 macroporous resin column eluted with aqueous EtOH
(10%–95%, stepwise) to afford seven subfractions of SVBA–SVBG.
SVBG (50.0 g) was further chromatographed by a silica gel column
(CH2Cl2‒MeOH, 25:1 to 4:1) to afford six subfractions of
SVBG1–SVBG6. SVBG3 (0.8 g) was purified on a Sephadex LH-20
column (MeOH) to afford compound 12 (65.0 mg).
4.3.3. (16S,20S,24R)-12β-acetoxy-16,23-epoxy-24,25-dihydroxy-3β-(β-
D-xylopyranosyloxy)-9,19-cyclolanost-22(23)-ene (3)
White amorphous powder; [α]1D3− 62.5 (c 0.10, MeOH); IR (KBr)
νmax 2936, 2865, 2358, 2338, 1735, 1456, 1371, 1254, 1163, 1032,
7

Z.-J. Fang et al.
Phytochemistry160(2019)1–10
Fig. 6. Compound 3 inhibited NF-κB activation in LPS-stimulated RAW264.7 cells. (A) The protein levels of phospho-IKKα/β, IKKα, IKKβ, phospho-IκBα, IκBα,
phospho-p65 and p65 were detected by Western blot analysis. GAPDH was used as an internal loading control. (B) The nuclear translocation of p65 was detected by
immunostaining. Nuclei were stained with DAPI. Scale bar = 10 μM.
981, 889 cm−1
;
1H and 13C NMR data, see Tables 1 and 2; ESIMS
(sign(λ2)ρ) was different colored and projected to the RDG isosurfaces
and the area, the strength, and the type of NCI in molecular will be
clearly exhibited. The different colored 3D gradient isosurfaces and 2D
plots of the reduced density gradient versus of sign(λ2)ρ were carried
out via Multifwn 3.6 (Lu and Chen, 2012) and VMD 1.9.3 program
(Humphrey et al., 1996). The data was obtained by evaluating M06-2X-
D3/6-311G(d) density and gradient values on cuboid grids. In order to
get a proper size of isosurface, the value of RDG function was set as 0.5
a.u., and the surfaces of gradient isosurfaces were colored on a blue-
green-red scale according to values of sign(λ2)ρ, ranging from −0.035
to 0.02 a.u. The strength of NCI was increasing gradually from the
middle to the both sides, and the blue region means strong attractive
interactions, e.g. hydrogen-bonds. The red region implies repulsive in-
teractions and the green region represents van der Waals interactions.
(positive) m/z 663 [M + H]+, 685 [M + Na]+; ESIMS (negative) m/z
697 [M + Cl]−; HRESIMS (positive) m/z 663.4116 [M + H]+ (calcd
for C37H59O10, 663.4108).
4.3.4. (16S,20S)-12β-acetoxy-16,23-epoxy-3β-hydroxy-9,19-cyclolanost-
22(23)-en-24-one (4)
Brown oil; [α]1D3− 61.0 (c 0.09, MeOH); IR (KBr) νmax 2961, 2927,
2857, 2362, 2339, 1720, 1636, 1460, 1380, 1241, 1203, 1108, 1026,
928 cm−1 1H and 13C NMR data, see Tables 1 and 2; ESIMS (positive)
;
m/z 513 [M + H]+, 535 [M + Na]+; HRESIMS (positive) m/z
513.3584 [M + H]+ (calcd for C32H49O5, 513.3580).
4.3.5. (16S,20R,23S)-12β-acetoxy-16,23-epoxy-23-methoxy-3β-(β-D-
xylopyranosyloxy)-9,19-cyclolanostan-24-one (5)
White amorphous powder; [α]1D3− 65.0 (c 0.11, MeOH); IR (KBr)
νmax 2936, 2865, 2363, 2339, 1721, 1639, 1462, 1371, 1249, 1163,
4.6. Cell culture
1051, 986 cm−1 1H and 13C NMR data, see Tables 1 and 2; ESIMS
;
Mouse derived RAW264.7 macrophages (American Type Culture
Collection, ATCC, Manassas, VA) were cultured in DMEM with 10%
FBS. Cells were maintained in a humidified incubator with 5% CO2 at
37 °C.
(positive) m/z 699 [M + Na]+; ESIMS (negative) m/z 711 [M + Cl]−
;
HRESIMS (positive) m/z 699.4083 [M + Na]+ (calcd for C38H60O10Na,
699.4084).
4.7. MTT assay
4.4. Acid hydrolysis and TLC analysis of sugar moieties of compounds 2, 3,
and 5
RAW264.7 cells were seeded into 96-well plates at a density of
1 × 104 cells per well and incubated overnight. Cells were then treated
with different concentrations of 3 for 24 h. The cell viability was de-
termined by the MTT assay, as described previously (Feng et al., 2017).
The configuration of sugar moieties was established according to the
published method with modifications (Ju et al., 2002a; Gan et al.,
2015). Compounds 2, 3, and 5 (5.0 mg for each) were refluxed with
10% HCl in EtOH (10 mL) for 6 h, respectively. Each reaction mixture
was diluted with H2O and extracted with EtOAc (3 × 15 mL). After
neutralizing with Na2CO3, the presence of D-xylose in the aqueous layer
was detected by co-TLC (n-BuOH-HAce-H2O, 4:1:1, Rf = 0.48) with an
authentic sample.
4.8. Measurement of NO production
RAW264.7 cells were seeded into 24-well plates (1 × 105 cells per
well) and allowed to adhere for 24 h. Cells were then treated with
different concentrations of isolates for 1 h, followed by stimulation with
1 μg/mL LPS. DMSO (Sigma-Aldrich) was used as vehicle, with the final
concentration of DMSO being maintained at 0.1% of all cultures.
Indomethacin (5 μM) was used as a positive control. After 18 h in-
cubation, the supernatant was collected to determine NO content using
Griess reagent (Liu et al., 2016; Li et al., 2018).
4.5. Reduced density gradient calculation for noncovalent interactions in
compound 3
The reduced density gradient (RDG) function was applied to analyze
intramolecular non-covalent interactions (NCI) in a simply, clear, and
visual way (Johnson et al., 2010). RDG can show the area of NCI, and ρ
implied the strength of NCI, and the sign of the second Hessian eigen-
value (sign(λ2)) was introduced to give the type of NCI. Therefore, the
electron density multiplied by the sign of the second Hessian eigenvalue
4.9. Western blot analysis
Western blot analysis was performed as described previously (Li
8

Z.-J. Fang et al.
Phytochemistry160(2019)1–10
et al., 2018). In brief, protein concentration was measured using a BCA
protein assay kit (Thermo Scientific). Equal amount of proteins
(15–30 μg) were separated by SDS-PAGE and transferred to PVDF
membranes (Bio-Rad, Hercules, CA, USA). After blocked with 5% non-
fatted milk for 1 h, the blots were probed with specific primary anti-
bodies overnight at 4 °C, and then incubated with horseradish perox-
idase conjugated secondary antibody for 1 h at room temperature. The
immune-blotting signals were detected using SuperSignal West Femto
Maximum Sensitivity Substrate under visualization in ChemiDoc™ MP
Imaging System (Bio-Rad).
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The cell media were centrifuged at 4000 rpm for 10 min at 4 °C and
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were determined using commercial ELISA kits (Neobiosciences,
Shenzhen, China), according to the manufacturer's instructions.
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4.11. NF-κB immunofluorescence
After incubation on chamber slides overnight, RAW264.7 cells were
pretreated with 3 (10 μM) for 1 h, and then stimulated with LPS (1 μg/
mL) for 1 h. The cells were fixed immediately in 4% formaldehyde for
20 min after washing with PBS (pH 7.4), and then permeabilized with
0.5% Triton X-100 (Bio-Rad) for 20 min at room temperature. The slides
were then incubated with a primary antibody against NF-κB p65 (1:100
dilution) at 4 °C overnight. The slides were washed with PBS and in-
cubated with Texas Red-conjugated anti-rabbit IgG secondary antibody
at 1:1000 dilution (Thermo Scientific) at room temperature for 1 h.
Finally, the slides were washed and stained with DAPI for nucleus. The
nuclear translocation of p65 was observed by confocal microscopy
(Olympus, Tokyo, Japan).
4.12. Statistical analysis
All data were expressed as mean
SD based on at least three in-
dependent experiments and analyzed by Graphpad Prism 6 (GraphPad
Software, San Diego, CA, USA). One-way ANOVA was used for statis-
tical comparison, and p-values less than 0.05 were considered statisti-
cally significant.
Acknowledgements
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This work was financially supported by National Key R&D Program
of China (No. 2017YFE0102200), National Natural Science Foundation
of China (21372198, 81473103, 81872756), Key Technology R&D
Program of Zhejiang Province (2018C02034), Zhejiang Provincial
Natural Science Foundation of China (LR17H300001), and Science and
Technology Development Fund, Macao S.A.R (FDCT 102/2017/A).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.phytochem.2019.01.003.
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Cimicifuga racemosa and their effects on CRF-stimulated ACTH secretion from AtT-20
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