Chemical constituents from cimicifuga dahurica and their anti-proliferative effects on MCF-7 breast cancer cells

Article abstract of DOI:10.3390/molecules23051083

This study was designed to search for novel anti-cancer compounds from natural plants. The 70% ethanolic extract from the rizhomes of Cimicifuga dahurica (Turcz.) Maxim. (Ranunculaceae) was found to possess significant in vitro anti-proliferative effects

Full text of DOI:10.3390/molecules23051083

molecules
Article
Chemical Constituents from Cimicifuga dahurica and
Their Anti-Proliferative Effects on MCF-7 Breast
Cancer Cells
Chu Thi Thanh Huyen 1,2, Bui Thi Thuy Luyen 2 ID , Ghulam Jilany Khan 3 ID , Ha Van Oanh 2,
Ta Manh Hung 4, Hui-Jun Li 1 and Ping Li 1,
*
1
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China;
thanhhuyen0187@hotmail.com (C.T.T.H.); cpuli@163.com (H.-J.L.)
2
3
4
Hanoi Univerisity of Pharmacy, 13–15 Le Thanh Tong, Hanoi, Vietnam;
luyenbthoaduoc@gmail.com (B.T.T.L.); havanoanh@gmail.com (H.V.O.)
State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering (BME),
Southeast University, Nanjing 210096, China; U4574904@hotmail.com
National Institute of Drug Quality Control (NIDQC), 48 Hai Ba Trung, Hoan Kiem, Hanoi, Vietnam;
tahungvkn@gmail.com
*
Correspondence: liping@cpu.edu.cn; Tel.: +86-025-8327-1382
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 21 February 2018; Accepted: 25 April 2018; Published: 4 May 2018
Abstract: This study was designed to search for novel anti-cancer compounds from natural plants.
The 70% ethanolic extract from the rizhomes of Cimicifuga dahurica (Turcz.) Maxim. (Ranunculaceae)
was found to possess significant in vitro anti-proliferative effects on MCF-7 breast cancer cells.
A phytochemical investigation using assay-guided fractionation of the ethanolic extract of C. dahurica
resulted in the isolation of one new phenolic amide glycoside
one new 9,19-cycloartane triterpenoid glycoside , and thirteen known constituents
The structures of , and were established using contemporary NMR methods and from their
3, two new lignan glycosides
4
and
7,
6
1,
2,
5, and
8–17.
3,
4,
6
7
HRESIMS data. The anti-proliferative effects of isolated compounds were evaluated using the
BrdU-proliferation kit. Five among the 17 isolated compounds showed significant anti-proliferative
effects (p
cycle arresting effect (p
expression analysis showed a down expression of c-Myc and cyclin D1 which further elucidated
the anti-proliferation mechanism of compound while apoptotic effects were found in association
≤
0.05), wherein compound
7 showed the most significant anti-proliferative and cell
≤
0.05) which followed a dose dependent manner. Western blot protein
7
with Bcl-2 family protein expression variations. Conclusively this study reports the isolation and
identification of 17 compounds from C. dahurica, including four novel molecules, in addition to the
fact that compound 7 possesses significant anti-proliferative and apoptotic effects in vitro that may
require further exploration.
Keywords: Cimicifuga dahurica; lignan; phenolic amide; triterpenoid glycoside; anti-proliferation
1. Introduction
The genus Cimicifuga (now known as Actaea) has been widely used all over the world and since
ancient times has been a traditional Chinese herbal medicine. This genus consists of 28 species
worldwide [
C. dahurica, C. foetida, and C. heracleifolia, commonly known as “Shengma” in Chinese, are officially
listed in the Chinese Pharmacopoeia [ ]. In traditional Chinese medicine, Shengma is widely
used for treating aphtha, sore throat, toothache, and wind-heat headache. It also has been used
in archoptosis, non-erupting measles, spot poison, uterine prolapse, and other diseases [ ].
1], among which nine species are indigenous to China. Three Cimicifuga species, namely,
2
1–4
Molecules 2018, 23, 1083; doi:10.3390/molecules23051083
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1083
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Pharmacological studies have revealed that Cimicifuga possesses anti-menopause symptoms [
antitumor [ ], anti-inflammatory [ ], anti-osteoporosis [ ] and some other effects. In addition to a lot
of researchs about biological activities, the safety of Cimicifuga rhizomes were also conducted [ ].
To date, more than 450 compounds including 9,19-cycloartane triterpenoids, phenylpropanoids,
chromones, lignans, amides and other compounds have been isolated from Cimicifuga spp. plants [ ].
Among them, many compounds such as triterpenoids [10 13] and phenolic compounds [14 15] show
5],
6
7
8
9
1
–
,
potent anti-cancer activities. In continuation of our studies seeking novel anti-cancer agents from
natural products, we focused on assessing the effects of compounds from the ethanolic extract of
C. dahurica.
2. Results and Discussion
2.1. Characterization of Compounds 1–17
Compound
3 was obtained as white powder. The HR-ESI-MS of 3 exhibited the ion peak at
m/z 506.2020 [M + H]+, which was in good agreement with a molecular formula of C25H31NO10
(M = 505.19480). The low-field region of the 1H-NMR spectrum showed the benzene ring signals of
six protons at δH [7.12 (br s, H-6), 7.17 (overlapped, H-2 and H-5)], and [6.83 (d, J = 1.5 Hz, H-20),
6.75 (d, J = 8.0 Hz, H-50), 6.68 (dd, J = 1.5, 8.0 Hz, H-60)] which coupling features suggested that the
structure contained two ABX spin systems (Table 1). The other two olefinic protons at δH 7.47 (d,
J = 15.5 Hz, H-7) and 6.50 (d, J = 15.5 Hz, H-8) were a typical pair of trans-double bonds, while peaks
at δH 3.84 (s) and 3.88 (s) indicative of the presence of two methoxy and two methylene groups at δH
2.79 (t, J = 7.5 Hz, H-70) and 3.51 (t, J = 7.5 Hz, H-80) were also observed. The carbinol protons signal
(
δH 3.70 to 5.32) and 13C-NMR spectra (δC 62.8 to 100.2) suggested the presence of an allose moiety in
the structure of 3 (Table 1).
1
Table 1. H- and 13C-NMR data of 3 in CD3OD.
a
a
b
b
Position
δC
Position
δC
δH (Mult., J in Hz)
δH (Mult., J in Hz)
0
1
2
3
4
5
6
7
8
9
130.9
112.4
150.9
149.7
117.3
122.7
141.4
120.4
168.8
132.0
113.5
148.9
146.0
-
5
6
7
8
116.2
122.2
36.1
42.4
56.7
56.4
100.2
72.0
72.9
68.6
75.9
62.8
6.75 (d, 8.0)
6.68 (dd, 1.5, 8.0)
2.79 (t, 7.5)
3.51 (t, 7.5)
3.88 (s)
0
7.17 *
-
-
0
0
7.17 *
7.12 (br s)
7.47 (d, 15.5)
6.50 (d, 15.5)
3-OCH3
0
3 -OCH3
3.84 (s)
5.32 (d, 8.0)
3.70 *
00
1
2
3
4
5
6
00
00
-
-
4.18 (t, 3.0)
3.63 (dd, 3.0, 10.0)
3.87 (m)
3.71 *
3.88 (dd, 6.0, 12.0)
0
00
1
2
3
0
00
6.83 (d, 1.5)
0
00
-
-
0
4
a
125 MHz, b 500 MHz. * Overlapped signals. Assignments were made using the HMQC and HMBC spectra.
Complete assignments of all carbons and protons were based on HSQC, COSY, and HMBC
experiments. After acid hydrolysis of
confirmed using TLC, based on a sugar standard and analysis of the coupling constant of H-100
(J = 8.0 Hz). Thus, the sugar moiety was identified as -D-allopyranoside. The position of an
allose sugar and two methoxy groups were determined by HMBC experiments. The HMBC
3, the absolute configuration of the sugar unit was
β
correlations between H-100
(δH 5.32) and C-4 (δC 149.7), H-2 (δH 7.17) and H-6 (δH 7.12) with
C-4 suggested the location of the allose at C-4. Two methoxy groups attached at C-3 and C-30
were determined based on the HMBC correlations of the methoxy protons (δH 3.88) with C-3
(
δC 150.9) and H-5 (δH 7.17) with C-3, the methoxy protons (δH 3.84) with C-30 (δC 148.9) and
H-50 (δH 6.75) with C-30, respectively. Using the COSY and HMBC spectra, the structure of
3

Molecules 2018, 23, 1083
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could be divided two parts, where part A was a (2E)-3-[4-(
β
-D-allopyranosyl)-3-methoxyphenyl
unit and part B was a (4-hydroxy-3-methoxyphenyl)ethyl moiety (Figure 1). These parts
were connected through an amide bridge based on the HMBC correlation of H-80 (δH 3.51)
with C-9 (δC 168.8) and the downfield-shift of the nitrogen-bearing methylene group at C-80
(
δC 42.4). From the evidence above, compound
with the (2E)-3-[4-( -D-allopyranosyl)-3-methoxy-phenyl]-N-[2-(4–hydroxy-3-methoxyphenyl)
ethyl]-2-propenamide structure.
3 was determined as a new compound
β
1 R = β-D-glucopyranosyl
2 R = β-D-glucopyranosyl
3 R = β-D-allopyranosyl
8 R = β-D-glucopyranosyl
9
4 R1 = R4 = β-D-allopyranosyl, R2 = R3 = OMe
7 R1 = R4 = β-D-allopyranosyl, R2 = R3 = H
5 R1 = β-D-xylopyranosyl, R2 = β-D-glucopyranosyl
6 R1 = β-D-xylopyranosyl, R2 = β-D-glucopyranosyl
13 R1 = H, R2 = H
10 24α
11 24β
17 R1 = β-D-xylopyranosyl, R2 = H
12 R = H
14 R = β-D-xylopyranosyl, ∆7
15 R = β-D-xylopyranosyl
16 R = β-D-xylopyranosyl
Figure 1. Structures of compounds 1–17 isolated from C. dahurica.

Molecules 2018, 23, 1083
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Compound
4
was obtained as a white powder. The HR-ESI-MS spectra displayed the presence of
an ion peak at m/z 765.2573 [M + Na]+, indicating a molecular formula of C34H46O18 (M = 742.26841).
The 1H-NMR spectrum of
4 showed the resonance signal of methine protons at δH 3.14 (br s, H-1 and
H-5), 4.79 (d, J = 7.5 Hz, H-2 and H-6), methylene protons [δH 3.96 (dd, J = 3.0, 9.0 Hz, H-4) and 4.32
(dd, J = 6.5, 9.0 Hz, H-8)], aromatic protons [δH 6.74 (s, H-20, H-200, H-60, and H-600)], and methoxy
protons [δH 3.88 (s)]. The presence of a sugar moiety was recorded by the chemical signal of the
anomeric proton [δH 5.17 (d, J = 7.5 Hz, H-1000)] (Table 2). The 13C-NMR spectrum of
4 exhibited the
resonance signals of 16 carbons including the aglycone and sugar unit. The carbon chemical shifts of
the aglycone at δC 55.7 (C-1 and C-5), 87.2 (C-2 and C-6), 73.0 (C-4 and C-8), and aromatic carbons
ranging from 104.8 to 154.4 suggested that
4
is a lignan skeleton with a tetrasubstituted aromatic ring.
with those of a previously reported lignan
Comparing the 1H- and 13C-NMR spectroscopic data of
4
named liriodendrin [16]00r0evealed the similarity of their chemical shifts. However, the downfield-shift of
000
the proton signal at H-1 , H-3 to 5.17 (d, J = 7.5 Hz), 4.15 (t, J = 2.5 Hz), respectively, and upfield-shift
0
000 000
of C-2 ”, 3 , 4 in the sugar unit (δC 73.1, 72.2, and 68.7) indicated the presence of an allose moiety in
the structure of instead of glucose as in liriodendrin. These findings, together with the correlations
of H-2000, H-3000, and H-4000 and the absence of correlation between H-1000 and H-3000 in the ROESY
spectrum indicated that the protons at C-1000 were in a -configuration and C-20”, C-30”, C-4000 were
the -configuration. In addition, acid hydrolysis of and comparison of the TLC, specific rotation,
4
β
α
4
and properties of sugar unit with a sugar standard, in combination with the analysis of the coupling
constant of H-1000 (J = 7.5 Hz) confirmed the absolute the sugar to be a β-D-allopyranoside (Table 2).
1
Table 2. H- and 13C-NMR Data of 4 and 7 in CD3OD.
4
7
Position
a
a
b
b
δC
δC
δH (Mult., J in Hz)
δH (Mult., J in Hz)
1, 5
2, 6
4, 8
55.7
87.2
73.0
3.14 (br s)
4.79 (d, 7.5)
3.96 (dd, 3.0, 9.0)
55.4
87.0
72.7
3.09 (br s)
4.74 (br s)
3.86 *
4.32 (dd, 6.5, 9.0)
4.22 (d, 6.0)
-
7.01 (br s)
-
0
00
00
00
00
00
00
1 , 1
139.4
104.8
154.4
136.0
154.4
104.8
57.1
103.9
73.1
72.2
-
6.74 (s)
-
137.1
111.6
150.8
147.6
117.7
119.8
56.8
100.6
72.0
72.8
0
2 , 2
0
3 , 3
0
4 , 4
-
-
-
0
5 , 5
7.16 (d, 8.5)
6.90 (br d, 8.0)
3.88 (s)
5.26 (d, 7.5)
3.63 *
4.17 (br s)
3.60 *
3.82 m
0
6 , 6
6.74 (s)
3.88 (s)
5.17 (d, 7.5)
3.62 *
4.15 (t, 2.5)
3.65 *
OCH3
000 0000
Allo-1 , 1
000 0000
2
3
4
5
6
, 2
, 3
, 4
, 5
000 0000
000 0000
68.7
76.3
63.0
68.6
75.7
62.8
000 0000
3.64 (m)
3.78 (dd, 6.0, 12.0)
3.68 *
000 0000
, 6
3.65 *
3.85 (dd, 6.0, 12.0)
a
125 MHz, b 500 MHz. * Overlapped signals. Assignments were made using the HMQC and HMBC.
000 0 00
(δH 5.17) and C-4 , C-4 (δC 136.0), as well
In the HMBC spectrum, the correlations between H-1
as H-20, H-200, H-60, H-6” (δH 6.74), with C-40, C-400 determined the allose moiety to be linked at C-40
and C-400. These evidences indicated that compound
4 was a new compound which was named
syringaresinol di-O-β-D-allopyranoside.
Compound
C32H42O16 (M = 682.24729), as determined by sodium adduct ion at m/z 705.2362 [M + Na]+ in
the HR-ESI-MS. The structure of showed similar lignan-skeleton chemical shifts with those of
including carbon signals at δC 55.4, 87.0, 72.7, aromatic carbons from 111.6 to 150.8, and an allose
7 was also obtained as a white amorphous powder with the molecular formula
7
4,

Molecules 2018, 23, 1083
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0
00
moiety. However, the resonance signals of aromatic proton at δH 7.01 (br s, H-2 , 2 ), 7.16 (d, J = 8.5 Hz
,
H-50, 500), 6.90 (br d, J = 8.0 Hz, H-60, 600), as well as the HMBC correlation of H-20 (δH 7.01) and
C-40 (δC 147.6), C-60 (δC 119.8); H-50 (δH 7.16) and C-10 (δC 137.1), C-30 (δC 150.8); H-60 (δH 6.90) and
C-20 (δC 111.6), C-40 (δC 147.6) suggested the presence of an aromatic ring ABX spin system in the
structure of
7 7 with
(Table 2). In addition, comparing the 1H- and 13C-NMR spectroscopic data of
those of a previously reported lignan named (+)-pinoresinol di-O-β-D-glucopyranoside [16] revealed
the similarity of their chemical shifts. However, the downfield-shift of the proton signal at H-1000,
H-3000 to 5.26 (d, J = 7.5 Hz), 4.17 (brs), respectively, and upfield-shift of C-2000, 3000, 4000 to the sugar
unit (δC 72.0, 72.8, and 68.6) indicated the presence of an allose moiety in the structure of
glucose as is (+)-pinoresinol di-O- -D-glucopyranoside. Thus, compound was identified as a new
compound which was named (+)-pinoresinol di-O-β-D-allopyranoside.
7 instead of
β
7
Compound
6 was obtained as a white amorphous powder. The HR-ESI-MS of 6 displayed a
quasimolecular ion [M + Na]+ at m/z 805.4344, consistent with a molecular formula of C41H66O14
1
(M = 782.44526). The H-NMR spectrum of
6 (Table 3) indicated the presence of characteristic
cyclopropane methylene signals [δH 0.43 and 0.68 (each 1H, d, J = 4.0 Hz, H-19)], one sec- and
six tert-methyl groups [δH 1.17 (s, H-18), 0.93 (d, J = 6.5 Hz, H-21), 1.20 (s, H-26), 1.13 (s, H-270),
1.16 (s, H-28), 1.06 (s, H-29), and 0.91 (s, H-30)], and two anomeric protons [δH 4.30 (d, J = 8.0 Hz, H-1 )
and 4.28 (d, J = 7.5 Hz, H-100)]. In the 13C-NMR spectrum, the signals of a cyclopropane methylene
carbon at δC 32.0 (C-19), four methine carbons bearing oxygen at δC 90.1 (C-3), 89.0 (C-15), 72.2 (C-23),
and 90.1 (C-24), a ketone carbon at δC 112.4 (C-16), and two anomeric carbons at δC 107.3 (C-10)
and 105.2 (C-100) were observed. The evidences above suggested that
triterpenoid diglycoside. After careful comparision, it was found that the NMR spectroscopic data
of and 3-O- -L-arabinopyranosyl cimigenol 15-O- -D-glucopyranoside were similar, except for the
presence of a xylose unit in instead of an arabinose unit [17]. The difference in the carbon chemical
shift of C-20, C-30 and C-40 of xylose unit [δC 75.6, 78.1, and 71.3, respectively] and those of arabinose
6 was a 9,19-cimigenol-type
6
α
β
6
unit [δC 73.1, 74.9, and 69.7] was recorded. Acid hydrolysis of
6 yielded an aglycone—cimigenol—and
two sugar units.
1
Table 3. H- and 13C-NMR Data of 6 in CD3OD.
a
a
b
b
Position
δC
Position
δC
δH (Mult., J in Hz)
δH (Mult., J in Hz)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
a
33.5
30.5
90.1
42.0
49.8
22.1
27.3
48.8
21.2
27.7
27.0
34.8
42.5
48.3
89.0
112.4
60.3
19.9
32.0
25.0
20.2
38.8
72.2
1.29 (m)/1.57 (m)
1.95 (m)/1.70 (m)
3.21 (dd, 4.5, 11.5)
24
25
26
27
28
90.1
71.9
27.3
25.3
12.6
26.0
15.5
3.45 (s)
-
1.20 (s)
1.13 (s)
1.16 (s)
1.06 (s)
0.91 (s)
-
1.72 (m)
0.81 (m)/1.62 (m)
1.96 (m)/1.21 (m)
29
30
1.36 (m)
3-Xylose
0
-
-
1
2
3
4
107.3
75.6
78.1
71.3
66.7
4.30 (d, 8.0)
3.28 (dd, 7.5, 8.0)
3.34 (dd, 8.0, 8.0)
3.58 *
3.19 (dd, 10.0, 11.0)
3.84 (dd, 5.0, 11.0)
0
0
1.22 (m)/2.15 (m)
1.66 (m)/1.74 (m)
0
0
-
-
5
3.97 (s)
-
1.46 (d, 11.0)
15-Glucose
00
1
2
3
4
5
6
105.2
75.4
78.0
72.4
77.5
63.0
4.28 (d, 7.5)
3.21 (dd, 8.0, 9.0)
3.37 *
3.26 *
3.31 (m)
3.62 (dd, 5.5, 11.5)
3.79 (dd, 2.5, 11.5)
00
00
1.17 (s)
0.43 (d, 4.0)/0.68 (d, 4.0)
1.68 (m)
00
00
00
0.93 (d, 6.5)
1.03 (m)/2.35 (ddd, 2.5, 6.0, 13.0)
4.42 (br d, 9.0)
125 MHz, b 500 MHz. * Overlapped signals. Assignments were made using the HMQC and HMBC.

Molecules 2018, 23, 1083
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1
The coupling constant of the anomeric protons in the H-NMR spectrum of
8.0 Hz) indicated the
6
(J = 7.5 and
β
-configuration. In addition, the absolute configuration of the sugar moiety
was confirmed as a D-xylopyranoside and D-glucopyranoside by comparing their TLC and specific
rotations. The positions of the xylose and glucose units were determined based on HMBC analysis.
In the HMBC spectrum, correlations were observed between the anomeric proton of the xylose
unit [δH 4.30 (d, J = 8.0 Hz, H-10)] and the methine signal [δC 90.1 (C-3)], the anomeric proton
of glucose [δH 4.28 (d, J = 7.5 Hz, H-100)] and the methine signal [δC 89.0 (C-15)]. Furthermore,
the downfield-shift of C-3 and C-15 suggested that the xylose moiety was attached to C-3 and glucose
was linked at C-15, respectively. Thus, compound
6 was elucidated to be 3-O-β-D-xylopyranosyl
cimigenol-15-O-β-D-glucopyranoside.
The spectrums of compound
The structures of known compounds were identified as geniposide (
24-O-acetylhydroshengmanol 3-O- -d-xylopyranosyl-15-O- -d-glucopyranoside
prim-O-glucosylcimifugin ( ) [21], isoferulic acid ( ) [22], acerinol (10) [23], 24-epi-acerinol
11) [24], 25-O-acetyl-cimigenol (12) [25], cimigenol (13) [26], 25-O-acetyl-7,8-didehydrocimigenol
3-O- -D-xylopyranoside 14 27], 25-O-acetylcimigenol 3-O- -D-xylopyranoside (23R,24S)
15) [28], 24-epi-7,8-didehydrocimigenol 3-O- -D-xylopyranoside (16) [24], (23R,24S) cimigenol
-D-xylo-pyranoside (17) [29], based on comparison of their spectroscopic data with those in the
3,
4,
6
,
7
can be found in supplementary materials (Figure S1–S26).
) [18], paeoniflorin ( ) [19],
20],
1
2
β
β
(5)
[
8
9
(
β
(
)
[
β
(
β
3-O-
β
literature. Compounds 1, 2, 10 were isolated from this plant for the first time.
2.2. Cytotoxicity Activity
2.2.1. Cell Proliferation Activity
MCF-7 breast cancer cells were exposed to epidermal growth factor (EGF) which was known
as a stimulator of proliferation (positive control). The anti-proliferative effects of the extract of
C. dahurica and five purified compounds (compounds
with 50 ng/mL EGF had statistically significant differences compared to control vehicle (Figure 2A).
Among these five purified compounds, compound had the most potent anti-proliferative effect at
1, 2, 7, 8, 9) under co-incubation of MCF-7 cells
7
the same concentration. In addition, the similar anti-proliferative effect of 7 was demonstrable under
co-incubation of MCF-7 cells with 50 ng/mL EGF (Figure 2B).
Figure 2. Proliferation of MCF-7 cells exposed to the extraction of Cimicifuga dahurica Maxim. at the
concentration of 30
µg/mL or 10
µ
M of purified compounds (
1–
17) (A) and the increasing doses of
compound under simultaneous application of 50 mg/mL EGF (
7
B
). Means
±
SD, n = 5 of three
#
independent experiments: **** p < 0.0001 for the EGF group versus the control group; p < 0.05,
##
p < 0.01, ### p < 0.001, #### p < 0.0001 versus the EGF group.

Molecules 2018, 23, 1083
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To date, only two lignans have been found in the plants of Cimicifuga genus, actaealactone,
a neolignan derived from C. racemosa [30] and (+)-isolariciresinol-3-O- -D-glucopyranoside from
C. dahurica [31]. Actaealactone exhibited a small stimulating effect on the growth of MCF-7 breast cancer
cells about 1.24-fold (14 M) compared to untreated cells, while compound exhibited the significantly
M), showing more potential
β
µ
7
anti-proliferative effect on MCF-7 cells even at the lower concentration (3
anti-proliferative effect.
µ
2.2.2. Cell Cycle Distribution and Western Blot Analyses
For exploration of the anti-proliferation mechanism of compound
7, western blotting was
performed to determine the effect of compound to the levels of cyclin D1 and c-Myc. The cyclin D1
7
proto-oncogene exercises powerful control over the mechanisms that regulate the mitotic cell cycle,
and overexpression of cyclin D1 is common in human cancers [32]. C-Myc encodes for a transcription
factor that is a key regulator of a wide variety of cellular processes. In most non-malignant cells,
Myc levels are low, its constitutive expression is linked to the pathogenesis of many human cancers [33].
As shown in Figure 3A–C, the downregulation of cyclin D1 protein level and c-Myc level were observed
in the cells treated with compound
we further assessed the cell cycle phases to explore the variations. Our results showed that compound
caused cell cycle transition arrest in the G0/G1 phase that was markedly increased in dose dependent
7. After observing the expression variation of Cyclin D1 and c-Myc,
7
manner (Figure 3D,E) in breast cancer MCF-7 cells. The increased arrest of cell cycle at G0/G1 phase
was contrastingly reduced in subsequent S and G2/M phase indicting that the cell cycle was arrested
within G0/G1 to S phase while a very small proportion was arrested in G2/M phase. These results
seconded our earlier results of Cyclin D1 and c-Myc (Figure 3A–C).
We examined the effect of the caspase inhibitors and the nucleoside addition on compound
7-induced cell cycle arrest in MCF-7 cells by adding a caspase 3 inhibitor (50
caspase inhibitor (50 M, Z-VAD-FMK) or a nucleoside mixed solution of dATP, dCTP, dGTP and
dTTP (125 M of each) (dNTP Mixture) to the cultured media with compound (30 M). As shown in
µM, Z-DEVD-FMK), a pan
µ
µ
7
µ
Figure 4, the cell cycle arrest at G0/G1 phase was reversed by a pan caspase inhibitor or a caspase 3
inhibitor but not the nucleoside addition. These results demonstrated that pan caspase and caspase 3
maybe had major roles in the mechanism of cell cycle arrest effects of compound 7 on MCF-7 cells.

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Figure 3. The effect of compound
and then treated with compound
was performed using antibody against cyclin D1 and c-Myc.
western blot analysis. ( ) The results of the western blotting in cyclin D1 and c-Myc were evaluated
in three independent experiments and normalized with -actin. ( ) After treating the cells with
varying concentrations of compound , the cells were collected and fixed with 75% ethanol and then
7
on breast cancer MCF-7 cell cycle. The cells were plated overnight
at the indicated concentrations for 48 h. ( ) The western blot
-actin was used as internal control for
7
A
β
B,C
β
D,E
7
re-incubated in PBS with RNase and then stained with propidium iodide and then subjected to flow
cytometry for DNA content was determination percentage at various cell cycle phases. * p < 0.1,
** p < 0.01, *** p < 0.001, **** p < 0.0001 versus the control group, ns: no significant difference.

Molecules 2018, 23, 1083
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Figure 4. The effects of a caspase 3 inhibitor, a pan caspase inhibitor and nucleoside on compound
7-induced cell cycle arrest in MCF-7 cells. *** p < 0.001 versus the control group.
2.2.3. Induction of Apoptosis by Compound 7 in MCF-7 Cells
After MCF-7 cells were treated with compound 7 (0, 1, 3, 10, 30 µM) for 48 h, cells were stained
with annexin V/PI and examined using a BD flow cytometer. Early and late apoptosis and necrotic
cells were discriminated. The corresponding quantities of total cell apoptosis and early apoptosis
were 4.8% and 2.1%; 8.2% and 2.3%; 13.8% and 4.0%; 17.8% and 3.3%; 27.0% and 6.9%; respectively.
The data demonstrated that apoptotic cells were found to be increased in a dose-dependent manner
(Figure 5A). We further assessed the expression variations of Bcl-2 family proteins including Bcl-2,
Bcl-XL (anti-apoptotic proteins) and Bax (pro-apoptotic protein). Our results showed that after 48 h
treatment of compound 7, the expression of anti-apoptotic proteins (Bcl-2, Bcl-XL) were decreased in
dose dependent manner alternatively the expression of pro-apoptotic protein (Bax) was increased in
breast cancer MCF-7 cells (Figure 5B).

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Figure 5. Apoptosis induced by compound
using Annexin V/PI double staining assay after treatment with compound
were harvested and stained with PI and Annexin V-FITC in darkness for 15 min, followed by flow
cytometry analysis. Lower right: Annexin V+/PI (early apoptosis); upper right: Annexin V+/PI+
(late apoptosis and necrosis). ( ) The expression of Bcl-2, Bcl-XL and Bax was assessed through western
7
in MCF-7 cells.
(
A) Apoptosis quantification,
7
for 48 h. MCF-7 cells
−
B
blotting analyses of MCF-7 (treated for 48 h with varying concentrations of the compound),
used as internal control.
β-actin was
To determine whether cell apoptosis was dependent upon caspase activation and the influence
of nucleoside in the cultured media on the cells apoptosis produced by compound , we examined
the effects of the caspase inhibitors and the nucleoside addition on compound -induced apoptosis
7
7
in MCF-7 cells using Annexin V-FITC/PI kit. As shown in Figure 6, apoptosis was reduced by a pan
caspase inhibitor or a caspase 3 inhibitor but not the nucleoside addition. These results demonstrate
that compound
7 induces apoptosis in MCF-7 cells by activating a pan caspase and caspase 3-dependent
apoptotic pathway.

Molecules 2018, 23, 1083
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Figure 6. The effects of a caspase 3 inhibitor, a pan caspase and nucleoside on compound
apoptosis in MCF-7 cells.
7-induced
Collectively in our present study we conclusively found four new compounds including one
new phenolic amide glycoside , two new lignan glycosides and , and one new 9,19-cycloartane
triterpenoid glycoside . In addition to these four novel compounds we also isolated and purified
thirteen other previously known compounds , and 17 from the ethanol extract of C. dahurica.
3
4
7
6
1,
2,
5
8–
In continuation of our studies searching for novel anti-cancer agents from natural products, we found
that the ethanol extract from the rhizomes of C. dahurica exhibited considerable inhibitory effects on the
proliferation of MCF-7 cells through the BrdU-proliferation test. Moreover we found that compound
had significant anti-proliferatory and apoptotic effects against breast cancer MCF-7 cells. In additions,
we also found that compound had significant cell cycle arresting (at G0/G1-S) effects following
graded dose response effectiveness. The apoptotic effects and cell cycle arresting (at G0/G1-S) effects
of compound maybe through caspase 3 and pan caspase-dependent pathways. Although compounds
and were two chemically similar lignans with the same sugar moieties, the higher anti-proliferative
activity in MCF-7 cells of compared to suggested that two methoxy units in the benzene ring (rather
7
7
7
7
4
7
4
than four methoxy units) may be associated with increased biological potency.
3. Materials and Methods
3.1. General Experimental Proceduces
1
The H and 13C, DEPT, HSQC, HMBC, ROESY, and COSY NMR spectra were recorded on a
Avance 500 MHz instrument (Bruker, Switzerland). The high-resolution electrospray ionization mass
spectra (HR-ESI-MS) were obtained from an Agilent 6530 Accurate-Mass Q-TOF LC/MS system
(Agilent Technologies Co., Ltd., Waldbronn, Germany). Semi-preparative HPLC was performed
on a Agilent 1100 series system,)using an ODS column (Zorbax SB-C18, 9.4
Agilent Technologies Inc., Santa Clara, CA, USA). Column chromatography was performed on silica gel
(200–300 mesh and 300–400 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), SephadexTM
×
250 mm, 5 µm,
LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), and octadecyl silica (ODS, 50 µm, YMC Co.,
Ltd., Tokyo, Japan). Thin-layer chromatography was performed on a precoated GF254 plate (Qingdao
Haiyang Chemical Co., Ltd., Qingdao, China), RP-18 F254S plates (Merck, Kenilworth, NJ, USA) and
compounds were visualized by spraying with aqueous 10% H2SO4 and then heating for 3–5 min.

Molecules 2018, 23, 1083
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3.2. Plant Material
The rizhomes of Cimicifuga dahurica were collected from Panshi City, Jilin Province, China in
November 2015. The plant materials were authenticated by Prof. Li Hui-Jun of the Department
of Pharmacognosy, China Pharmaceutical University. A voucher specimen (No. XASM201511)
was deposited in the State Key Laboratory of Natural Medicines, China Pharmaceutical University,
Nanjing, China.
3.3. Extraction and Isolation
The air-dried rizhomes (45 kg) of Cimicifuga dahurica were successively extracted three times with
70% EtOH (3
pressure to afford a dark brown residue extract (3.0 kg), which was suspended in H2O and partitioned
successively with petroleum ether (3 3 L), EtOAc (3 3 L), and n-BuOH (3 3 L) to afford petroleum
×
400 L) for 2 h under reflux. The combined extracts were concentrated under reduced
×
×
×
ether (70 g, CP), EtOAc (1200 g, CE), n-BuOH (368 g, CB) fractions, and a H2O layer (W).
The n-butanol fraction (368 g) was fractionated on a silica gel column chromatography (CC)
eluting with gradient solvent systems of CH2Cl2/MeOH (0–100% MeOH, step-wise) to obtain five
fractions including: CB-1 (35 g), CB-2 (78 g), CB-3 (42 g), CB-4 (12 g), and CB-5 (175 g). Fraction CB-4
(12 g) was isolated by CC over silica gel, eluting with solvent systems of CH2Cl2/MeOH (10/1, v/v)
to obtain three subfractions (CB-4.1 through CB-4.3). Compounds 4 (10 mg), 7 (11 mg) and 8 (20 mg)
were afforded from fraction CB-4.1 (7.5 g) by silica gel CC eluting with CH2Cl2/MeOH (1/1, v/v),
and purified by RP-C18 silica gel using acetone/H2O (3/2, v/v) as eluent. Fraction CB-4.2 (2.5 g) was
fractioned on RP-C18 silica gel eluting with acetone/H2O (2.5/1, v/v), and further isolated by silica gel
CC using solvent system CH2Cl2/acetone/MeOH (5/1/0.1, v/v/v) to give compounds 5 (8 mg), and 6
(11 mg). Fraction CB-4.3 (1.1 g) was subjected by SephadexTM LH-20 CC eluting with MeOH/H2O
(1/1, v/v) and further isolated by RP-C18 silica gel using solvent system MeOH/H2O (2/1, v/v) to
give compounds 1 (42 mg), 2 (20 mg), and 3 (24 mg).
A portion of the ethylacetat fraction (700 g) was applied to a silica gel column chromatography
(CC) using a stepwise gradient elution of CH2Cl2/MeOH (0–100% MeOH) to afford seven fractions
including CE-1 (32 g), CE-2 (50 g), CE-3 (55 g), CE-4 (98 g), CE-5 (250 g), CE-6 (100 g) and
CE-7 (55 g). Fraction CE-4 (98 g) was chromatographed on a silica gel column by eluting with
petroleum ether/aceton (100/0
Fraction CE-4.1 (10 g) give compound 11 (80 mg) on crystallization from EtOAc. Repeated silica
gel column chromatography of fraction CE-4.2 (30 g) with petroleum ether/EtOAc (20/1 1/1)
→
4/1, v/v) to give three subfractions (CE-4.1 through CE-4.3).
→
and recrystallization to yield compounds 10 (70 mg), 12 (100 mg) and 13 (200 mg). Fraction CE-5
(250 g) was subjected to a silica gel column chromatography with a gradient mixture of petroleum
ether/aceton (5/1
was crystallized from MeOH to give an addition crop of compound
further separated into three subfractions (CE-5.4.1 to CE-5.4.3) on a silica gel column (CH2Cl2/acetone,
4/1 3/2, v/v). A part of fraction CE-5.4.1 (4.0 g) was purified by RP-C18 silica gel using MeOH/ H2O
→
1/1, v/v) as eluent to give 5 subfractions (CE-5.1 to CE-5.5). Fraction CE-5.2 (80 g)
9
(10 g). Fraction CE-5.4 (53 g) was
→
(4/1, v/v) as eluent, followed by purification using semi-preparative HPLC eluted with CH3CN/H2O
(60:40), to yield compounds 14 (30 mg) and 15 (60 mg). A portion of fraction CE-5.4.2 (5 g) was applied
on RP-C18 silica gel using MeOH/ H2O (3/1, v/v) as eluent to give compound 16 (50 mg). Repeated
silica gel column chromatography of fraction CE-5.5 (40 g) using CH2Cl2/acetone (2/1, v/v) and
recrystallization furnished compound 17 (1.5 g).
3.4. Acid hydrolysis and Sugar Identification
The new compounds 3, 4, 6 and 7 (2 mg of each) were dissolved in MeOH (2 mL) and refluxed
with 1.0 N HCl (2 mL) for 3 h and following by extracting with ethyl acetate. The water layer was
then neutralized by Ag2CO3, and filtered to give a monosaccharide. Each monosaccharide of those
compounds had an Rf (n-BuOH–AcOH–H2O, 4:1:1) and specific rotation [α
]D + 52.7◦ (c 0.10, H2O);

Molecules 2018, 23, 1083
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◦
◦
[
α
]D + 24.3 (c 0.10, H2O); [
α
]D + 14.4 (c 0.05, H2O) corresponding to those of D-glucose, D-xylose and
D-allose (Sigma-Aldrich, St. Louis, MO, USA), respectively.
3.5. Biological Evaluations
3.5.1. Cell Culture
MCF-7 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD,
USA). Cells were cultured and growth in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco,
Grand Island, NY, USA) medium containing 10% fetal bovine serum (FBS, Gibco, Grand Island, NY,
USA), 100 units/mL penicillin, and 100
in 5% CO2—95% air.
µ
g/mL streptomycin (Gibco, Grand Island, NY, USA) at 37◦C
3.5.2. Anti-Proliferative Activity
After treatment with C. dahurica extract at the concentration of 30 mg/mL and 10
compounds ( 17) under co-incubation with 50 ng/mL EGF, MCF-7 cells were incubated with BrdU
labelling solution (10 M) for 2 h and fixed with fixation solution for 30 min at room temperature.
L anti-BrdU peroxidase-labelled antibody was added for 90 min and washed three times.
L/well of substrate solution for colorimetric quantification was added at room temperature
µM of purified
1–
µ
100
100
µ
µ
for 5–30 min until colour development was sufficient for photometric detection. The absorbance was
measured at 405 nm using a Synergy 2 multi-detection microplate reader (Bioteck, Winooski, VT, USA).
3.5.3. Cell Cycle Distribution Analyses
We cultured breast cancer MCF-7 cells in small dishes and after 24 h of incubation the cells were
treated with varying concentrations (1, 3, 10 and 30 µM) of compound 7. The cells were then further
incubated for 48 h and then collected. The harvested cells were centrifuged at 1000 rpm for 5 min,
washed thoroughly with ice cold PBS and were then subjected to fixation in 75% ethanol over night
at 4 ◦C. After fixation the ethanol was removed and cells (about 1
×
106) were re-suspended in PBS
having RNase (25
µg/mL◦), then the cells were further incubated for another half hour in propidium
iodide (50
µg/mL) at 37 C and subjected to flow cytometer analyses. Cell cycle distribution was
analyzed by ModFitLT (Verity, Software House, Topsham, ME, USA).
To determine whether cell cycle distribution is dependent upon caspase activation and the
influence of the nucleoside addition to apoptotic activity of compound
Z-DEVD-FMK, Medchem Express, Monmouth Junction, NJ, USA), a pan caspase inhibitor (50
Z-VAD-FMK, Beyotime Biotechnology, Shanghai, China) or a nucleoside mixed solution of dATP,
dCTP, dGTP and dTTP (125 M of each) (dNTP Mixture, Beyotime Biotechnology) were added to the
M) to incubation for 48 h and followed by flow cytometer analyses as
7, a caspase 3 inhibitor (50
µM,
µM,
µ
media with compound
described above.
7 (30 µ
3.5.4. Annexin V/PI Analyses
The Annexin V-FITC/PI apoptosis detection kit (Keygen Biotechnology, Nanjing, China) was
used to detect the effects of compound . MCF-7 cells (3
105 cells/well) were seeded in 6-well plates
and cultured for 24 h. After treatment of cells with compound (0, 3, 10, 30 M) for 48 h, adherent and
floating cells were collected and washed with ice cold PBS, then cells were centrifuged. The pellet was
suspended with binding buffer (500 L), stained with Annexin V-FITC (5 L) and PI (5 L), and then
7
×
7
µ
µ
µ
µ
incubated for 15 min at room temperature in the dark. After incubation, cells were analyzed with a
BD Accuri C6 Flow Cytometer (BD Biosciences, San Jose, CA, USA) to determine the percentage of
apoptotic cells.
To determine whether cell apoptosis is dependent upon caspase activation and the influence of the
nucleoside addition on apoptotic activity of compound
7
, a caspase 3 inhibitor (50
µM Z-DEVD-FMK,
Medchem Express, Monmouth Junction, NJ, USA), a pan caspase inhibitor (50
µM Z-VAD-FMK,

Molecules 2018, 23, 1083
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Beyotime Biotechnology, Shanghai, China) or a nucleoside mixed solution of dATP, dCTP, dGTP and
dTTP (125 M of each) (dNTP Mixture, Beyotime Biotechnology) were added to the media with
µ
compound 7 (30 µM) and incubated for 48 h.
3.5.5. Western Blotting
MCF-7 cells were seeded in 6-well plates and treated with compound 7. Cells were collected and
washed with cold phosphate-buffered saline (PBS). The collected cells were lysed on ice for 30 min
in 100 L lysis buffer (120 mM NaCl, 40 mM Tris (pH 8), 1% Nonidet P-40 and protease inhibitors)
µ
and centrifuged at 12,000 rpm for 30 min. BCA protein assay kit (Beyotime, Shanghai, China) was
used to measure protein concentrations as described in a previous report [34]. Whole-cell extracts
were subjected to electrophoresis on polyacrylamide gels and then transferred onto nitrocellulose
membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween-20
(TBST). Then, anti-cyclin D1, anti-Bcl-2, anti-Bcl-XL, anti-Bax antibodies (Cell Signaling Technology,
Danvers, MA, USA), anti c-Myc antibody (Abcam, Cambridge, MA, USA) or anti-actin antibody
(Santa Cruz Biotechnology, Dallas, TX, USA) was bound overnight at 4 ◦C. After washing with TBST,
HRP conjugated secondary antibody (Keygen, Nanjing, China) was bound for 1 h at room temperature.
Finally, protein bands were detected using a Tanon 5200 Multi Chemiluminescent Imaging System
(Tanon, Shanghai, China). The ratio of cyclin D1/
Image J.
β-actin and c-Myc/β-actin were analyze using NIH
3.5.6. Statistics
Values are presented as mean
followed by Bonferroni post-hoc testing using GraphPad Prism 6.01 (GraphPad Software Inc.,
±
SD. p-values were calculated by one-way analysis of variance
San Diego, CA, USA). p < 0.05 was considered statistically significant. * p < 0.05; ** p < 0.01; *** p < 0.001
**** p < 0.0001.
;
Supplementary Materials: Supplementary materials are available on line.
Author Contributions: Ping Li and Hui-Jun Li conceived and designed the experiments; Chu Thi Thanh
Huyen, Bui Thi Thuy Luyen, Ta Manh Hung performed the experiments; Chu Thi Thanh Huyen, Ha Van Oanh
Ghulam Jilany Khan analyzed the data; Ghulam Jilany Khan contributed reagents/materials/analysis tools;
and Chu Thi Thanh Huyen wrote the manuscript. All authors read and approved the final manuscript.
,
Acknowledgments: This study was supported by the 111 projiect (no. B16046).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–17 are available from the authors.
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