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Flavonoids from the flowers of Chrysanthemum morifolium Ramat.

Authors:
  • Henan University of Chinese Medicine,zhengzhou,China

Abstract and Figures

OBJECTIVE: To study the chemical constituents in the flowers of Chrysanthemum morifolium Ramat. METHODS: The compounds were isolated with Diaion HP-20, Toyopearl HW-40, Sephadex LH-20, silica gel column chromatography and preparative HPLC. The structures of the compounds were identified by physiochemical properties and spectral analysis. RESULTS: Twenty compounds were obtained, and their structures were identified as luteolin (1), apigenin (2), acacetin (3), diosmetin (4), acacetin 7-O-β-D-glucoside (5), acacetin 7-O-β-D-glucoside (6), acacetin7-O-(6″-O-acetyl)-β-D-glucoside (7), eriodictyol (8), naringenin (9), artemetin (10), 5-hydroxy-6, 7, 3', 4'-tetramethoxyflavone (11), 5, 7-dihydroxy-3', 4'-dimethoxyflavone (12), 4'-methoxyctricin (13), 3', 5'-dimethoxy-4', 5, 7-trihydroxyflavone (14), 5, 6-dihydroxy-3, 7, 3', 4'-tetramethoxyflavone (15), luteolin 7-O-β-D-glucuronide methyle ester (16), dihydroquercetin-7-β-D-glucoside (17), quercetin 3-O-β-D-glucoside(18), quercetin 3-O-β-D-glucoside (19), and acacetin 7-O-β-(6″-(E)-crotonylglucopyranoside) (20). CONCLUSION: Compounds 9-20 were isolated for the first time from this plant.
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Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Four new sesquiterpenoids from Dendranthema morifolium (Ramat.) kitam
owers
Wenjing Chen
a,b
, Mengnan Zeng
a,b
, Meng Li
a,b
, Fang Li
a,b
, Xuan Zhao
a,b
, Hui Fan
a,b
,
Xiaoke Zheng
a,b
, Weisheng Feng
a,b,
a
School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou 450046, China
b
Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment & Chinese Medicine Development of Henan Province, Zhengzhou 450046, China
ARTICLE INFO
Keywords:
Dendranthema morifolium (Ramat.) kitam
Sesquiterpenoids
Structural elucidation
Anti-inammatory activity
ABSTRACT
Four new sesquiterpenoids, chrysanthguaianolactones C-F (14), together with four known sesquiterpenoids
(58), including 3α,4α,10β-trihydroxy- 8α-acetoxyguai-1,11(13)-dien-6α,12-olide (5), 3α,4α,10β-trihydroxy-
8α-acetoxy 11βH-guai-1-en-12,6α-olide (6), 8α-(angelyloxy)-3β,4β-dihydroxy-5αH,6βH,7αH, 11αH-guai-
1(10)-en-12,6-olide (7) and 3β,4α-dihydroxy-8α-angelyloxy-1(10),11(13) dien-6α,12-olide (8) were obtained
from an ethyl acetate fraction, which was yielded from an acetone extract of Dendranthema morifolium (Ramat.)
kitam. Their structures were determined with extensive spectroscopic (UV, IR, HR-ESIMS, and 1D and 2D NMR)
analyses. In addition, compounds 18 were evaluated for their anti-inammatory eects on H9c2 cardiocytes
impaired by lipopolysaccharide (LPS). Among them, compound 2 and compounds 58 exhibited anti-in-
ammatory eects against LPS-induced inammation.
1. Introduction
Dendranthema morifolium (Ramat.) kitam (Flos chrysanthemum) is a
traditional Chinese medicine that, has been widely used as a daily
beverage for thousands of years. It was recorded in the Chinese medical
classics Shennongs Herbaand is thought to be a top-gradeherb in
China. Pharmacological investigations have shown that Flos chry-
santhemum exhibits antibacterial (He et al., 2013), antioxidant (Lin
et al., 2010), anti-inammatory, and heart-protective (Lii et al., 2010)
characteristics. Previous phytochemical studies on caeic acid deriva-
tives, avonoids, triterpenoids, glycosides and alkaloids have been
isolated from Flos chrysanthemum (Yuan et al., 2015; Qu et al., 2017).
Based on a bioassay-guided isolation, further phytochemical study was
undertaken to investigate the chemical constituents of a 50% acetone
extract from Flos chrysanthemum, which led to the isolation of four
new sesquiterpenoids, chrysanthguaianolactones C-F (14), along with
four known sesquiterpenoids (58), 3α,4α,10β-trihydroxy-8α-acetox-
yguai-1,11(13)- dien-6α,12-olide (5), 3α,4α,10β-trihydroxy-8α-
acetoxy-11βH-guai-1-en-6α,12-olide (6), 8α-(angelyloxy)-3β,4β-dihy-
droxy-5αH,6βH,7αH,11αH-guai-1(10)-en-12,6-olide (7) and 3β,4α-di-
hydroxy-8α-angelyloxy-1(10),11(13)-dien-6β,12-olide (8). Their struc-
tures were determined in detail through extensive spectroscopic
analysis (1D and 2D NMR spectroscopy and mass spectrometry) and
compared with previous reports in the literature.
2. Results and discussion
The structures of the sesquiterpenoid compounds (Fig. 1) were de-
termined through analysis using HR-ESIMS, 1D and 2D NMR spec-
troscopy, including
1
H
1
H COSY, HMBC and HSQC experiments.
Compound 1 was obtained as colorless crystals.The molecular for-
mula was determined to be C
20
H
28
O
7
by analysis of HR-ESIMS (m/z
403.1725 [M + Na]
+
, calcd. for C
20
H
28
O
7
Na, 403.1727) with seven
degrees of unsaturation. The IR spectrum showed the presence of OH
groups (3393 cm
1
), CO groups (1766, 1706 cm
1
) and C]C bonds
(1456 cm
1
). The
1
H NMR spectrum of 1 consisted of ve methyl
groups at δ
H
1.51 (s, H-14), 1.48 (s, H-15), 1.18 (d, J= 6.9 Hz, H-13),
1.90 (s, H-5) and 2.01 (d, J= 7.2 Hz, H-4); one methylene group at δ
2.13 (dd, J= 15.2, 4.8 Hz, H-9a) and 2.00 (brd, J= 15.2 Hz, H-9b);
three oxygenated methane groups at δ5.42 (m, H-8), 4.51 (t,
J= 10.8 Hz, H-6) and 4.08 (d, J= 2.6 Hz, H-3); and two olenic
protons at δ6.19 (m, J= 7.2 Hz, H-3) and 5.98 (t, J= 2.6 Hz, H-2).
The
13
C NMR spectrum indicated four olenic carbon signals at δ
C
153.8
(C-1), 128.2 (C-2), 128.6 (C-2) and 140.5 (C-3); two carbonyl carbon
signals at δ180.5 (C-12) and 168.0 (C-1); ve O-bearing carbon signals
at δ83.4 (C-3), 82.6 (C-4), 78.2 (C-6), 73.2 (C-8) and 71.7 (C-10); ve
methyl carbon signals at δ29.3 (C-14), 23.2 (C-15), 20.7 (C-5), 16.1 (C-
4) and 15.1 (C-13); one CH
2
carbon signals at δ46.0 (C-9); and three
CH carbon signals at 59.1 (C-5), 54.6 (C-7) and 42.8 (C-11). Four
https://doi.org/10.1016/j.phytol.2017.11.009
Received 12 July 2017; Received in revised form 3 November 2017; Accepted 10 November 2017
Corresponding author at: School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou, 450046, China.
E-mail address: fwsh@hactcm.edu.cn (W. Feng).
Phytochemistry Letters 23 (2018) 52–56
Available online 20 November 2017
1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
T
degrees of unsaturation were attributed to two C]O groups and two
pairs of C]C bonds; the remaining three degrees of unsaturation in-
dicated that 1 had a tricyclic ring skeleton. Comparison of the NMR
data of 1 with indicumolide A and known sesquiterpenoids 6 (Feng
et al., 2009; Tan et al., 1998), which was also isolated from this genus,
suggested that 1 was a tricyclic guaiane-type sesquiterpenoid.
The
1
H
1
H COSY correlations were observed for the H-3/H-2/H-5/
H-6/H-7/H-8/H-9 spin system. The planar structure of 1 was outlined
mainly by HMBC experiment (Fig. 2). The HMBC spectrum clearly de-
monstrated correlations from Me-13 to C-7, C-11 and C-12; from Me-15
to C-4, and C-5; from Me-14 to C-1, C-9 and C-10; from H-2 to C-5, C-10,
C-3 and C-1; from H-6 to C-11, C-7, C-5, C-4, C-1and C-8; from H-3 to C-
5, C-4, C-2 and C-1; from H-5 to C-6, C-7, C-2 and C-1; and other cor-
relations shown in Fig. 2. A hydroxyl group was further determined to
be located at C-3 in 1 by the detailed analysis of HMBC correlations
between H-2 and C-3. The remaining hydroxyls were respectively
placed on C-4 and C-10 judging from the HMBC correlations with the
nearby proton signals shown in Fig. 2. Additionally, the HMBC corre-
lations from Me-5to C-1, C-2and C-3; from Me-4to C-1, C-2, and C-
3; and from H-3to C-1, C-4, and C-5revealed the presence of a 2-
methylbut-2-enoyl moiety in 1, and its (Z)-conguration was de-
termined by the correlation of H-5with H-3in the NOESY experiment.
Moreover, the HMBC correlation from H-8 to C-1indicated that the 2-
methylbut-2-enoyl moiety was attached to C-8.
The relative conguration of 1 was deduced from the NOESY
spectrum. The NOESY correlations were observed between H-2 and H-
3, Me-14, between Me-15 and H-3, H-6, and between H-7 and Me-13.
since the H-5 of the guaiane-type sesquiterpenoid was dened in the α-
conguration (Tan et al., 1998), the structure of 1 was unambiguously
elucidated as 3α,4α,10β-trihydroxy-8α-angelyloxy-11βH-guai-1-en-
6α,12-olide, named chrysanthguaianolactone C.
Compound 2was obtained as colorless crystals. The molecular
formula was determined to be C
20
H
26
O
7
by analysis of HR-ESIMS (m/z
401.1567 [M + Na]
+
, calcd. for C
20
H
26
O
7
Na, 401.1570) with eight
degrees of unsaturation. The IR spectrum showed the presence of OH
groups (3416 cm
1
), CO groups (1772, 1700 cm
1
) and C]C bonds
(1456 cm
1
). The NMR spectroscopic data from 2(Table 1) resembled
those of 1 except for the missing resonances assigned to CHeCH
3
in
1, showing an eC]CH group instead, which showed the resonance
signals at C-11 (δ140.4) and C-13 (δ120.7). the HMBC correlations are
shown in Fig. 2. Furthermore, the stereochemistry was established by a
NOESY experiment, in which H-6/H-15 and H-3/H-15 correlated with
each other. This established a β-orientation for the protons at C-3, C-6,
and C-15. Additionally, H-5/H-14 showed an NOESY correlation con-
rming the α-orientation of the protons at C-5 and C-14. Thus, com-
pound 2was elucidated as 3α,4α,10β-trihydroxy-8α-angelyloxyguai-
1,11(13)-dien-6α,12-olide, and named chrysanthguaianolactone D.
Compound 3was obtained as colorless crystals.The molecular for-
mula was determined to be C
21
H
23
O
10
by analysis of HR-ESIMS (m/z
467.1888 [M + Na]
+
, calcd. for C
21
H
23
O
10
Na, 467.1887) with six
degrees of unsaturation. The IR spectrum showed the presence of OH
groups (3381 cm
1
), CO groups (1647 cm
1
) and C]C bonds
(1373 cm
1
). The NMR spectroscopic data of 3(Table 1) resembled
those of 7 except for the missing resonances assigned to a 2-methylbut-
2-enoyl moiety in 1, showing an a glucopyranoside group instead,
which showed the resonance signals at C-1(δ105.4), C-2(δ75.5), C-3
(δ78.7), C-4(δ71.5), C-5(δ78.0), and C-6(δ62.8). In the acid
hydrolysis of 3, D-glucose was respectively aorded and conrmed by
TLC and optical rotations compared with the reference substances,
(Zhang et al., 2008, 2016). The conguration was determined by
measuring the optical rotation value and the large
3
J
H1,H2
coupling
constant. Based on the results described above, the structure of the
carbohydrate fragment of 3 was determined to be (1 6)-β-D-gluco-
pyranoside. Moreover, the HMBC correlation from H-8 to C-1indicated
Fig. 1. Structure of compounds 18.
Fig. 2. Key HMBC and
1
H
1
H COSY correlations of 14.
W. Chen et al. Phytochemistry Letters 23 (2018) 52–56
53
that the β-D-glucopyranoside moiety was attached to C-8. Furthermore,
the similar HMBC correlations suggested that 3 had the same relative
conguration as 7 (Fig. 2). The relative conguration of 3 was deduced
from the NOESY spectrum. The NOESY correlations were observed
between H-5 and H-7, H-11, Me-15, between H-3 and Me-15, and be-
tween H-6 and H-8. Thus, compound 3 was elucidated as 3α,4α-dihy-
droxy-8α-O-β-D-glucopyranoside-11βH-guai-1(10)-en-6α,12-olide, and
named chrysanthguaianolactone E.
Compound 4 was obtained as colorless crystals. The molecular
formula was determined to be C
17
H
24
O
7
by analysis of HR-ESIMS (m/z
363.1415 [M + Na]
+
, calcd. for C
17
H
24
O
7
Na, 363.1414) with six de-
grees of unsaturation. The IR spectrum showed the presence of OH
groups (3503, 3372, 2985 cm
1
), CO groups (1763 cm
1
) and C]C
bonds (1370 cm
1
). The
1
H NMR spectrum of 4 consisted of four me-
thyl groups at δ
H
2.05 (s, H-2), 1.90 (s, H-14), 1.36 (s, H-15) and 1.23
(d, J= 7.1 Hz, H-13), one methylene group at δ2.70 (dd, J= 14.3,
4.9 Hz, H-2a) and 1.74 (dd, J= 14.3, 4.9 Hz, H-2b), three oxygenated
methane groups at δ5.33 (m, H-8), 4.42 (t, J= 10.0 Hz, H-6) and 3.67
(t, J= 5.3 Hz, H-3), one olenic protons at δ5.44 (d, J= 4.5 Hz, H-9).
The
13
C NMR spectrum indicated two olenic carbon signals at δ
C
143.4
(C-10) and 124.8 (C-9), two carbonyl carbon signals at δ181.1 (C-12)
and 172.2 (C-1), ve O-bearing carbon signals at δ81.5 (C-4), 80.6 (C-
1), 79.5 (C-3), 77.2 (C-6), and 75.9 (C-8), four methyl carbon signals at
δ24.9 (C-14), 23.5 (C-15), 21.0 (C-1) and 15.6 (C-13), a CH
2
carbon
signals at δ47.4 (C-2), and three CH carbon signals at 63.5 (C-5), 48.0
(C-7) and 42.6 (C-11). Three degrees of unsaturation were attributed to
two C]O groups and one pair of C]C bonds; the remaining three
degrees of unsaturation indicated that 4 had a tricyclic ring skeleton.
We compared the NMR data of 4 with those of tricyclic guaiane-type
sesquiterpenoids (Ahmed et al., 2004.), to which it was similar. The
NMR spectroscopic data of 4 (Table 1) resembled those previously re-
ported in the literature (Ahmed et al., 2004), except for the missing
resonances assigned to a eC]CH group, showing an CHeCH
3
group
instead, which showed the resonance signals at C-11 (δ42.6), C-12 (δ
181.1) and C-13 (δ15.6). the HMBC correlations are shown in Fig. 2.
The stereochemistry of compound 4 was established by a NOESY ex-
periment, in which H-5/H-15 and H-5/H-7 were correlated with each
other. The presence of H-5 downeld at δ2.34 supported the α-or-
ientation of the hydroxyl group at C-1 (Ahmed et al., 2004). Thus,
compound 4 was elucidated as 1α,3α,4β-trihydroxy-8α-acetoxy-9-en-
6α,12-olide, and named chrysanthguaianolactone E.
The known compounds were identied as 3α,4α,10β-trihydroxy-8α-
acetoxyguai-1,11(13)-dien-6α,12-olide (Ahmed et al., 2004)(5),
3α,4α,10β-trihydroxy-8α-acetoxy-11βH-guai-1-en-6α,12-olide (Tan
et al., 1998) (6), 8α-(angelyloxy)-3β,4β-dihydroxy-5αH,6βH,7αH,
11αH-guai-1(10)-en-12,6-olide (Feng et al., 2009)(7), and 3β,4α-di-
hydroxy-8α-angelyloxy-1(10),11(13) dien-6β,12-olide (Wang et al.,
2014) (8) by comparing their physical and spectroscopic data with
values reported in the literature.
To investigate whether compounds 18 protect H9c2 cells from LPS-
induced inammation, H9c2 cells were treated with 20 μM LPS in the
presence or absence of compounds 18 (10 μM), and the absorbance
was assessed by MTT assay. Among them, compound 2and compounds
58 exhibited anti-inammatory eects against LPS-induced in-
ammatory (Fig. 3).
3. Experimental
3.1. General experimental procedures
NMR spectra were recorded at room temperature in CD
3
OD using a
Bruker Avance III 500 NMR spectrometer with TMS as an internal
standard (500 MHz for
1
H NMR and 125 MHz for
13
C NMR). Optical
rotation was measured with an AP-IV (Rudolph Research Analytical,
USA). The IR spectrum was determined on a Nicolet iS10 Microscope
Table 1
1
H NMR Spectroscopic Data of Compounds 14 (500 MHz).
No. 1 (CD
3
OD) 2 (CD
3
OD) 3 (CD
3
OD) 4 (CD
3
OD)
2 5.98 (t, 2.6) 5.98 (brd, 2.6) 2.73 (brd,
16.5)
2.70 (dd, 14.3,
4.9)
2.26 (brd,
16.5)
1.74 (dd, 14.3,
4.9)
3 4.08 (d, 2.6) 4.09 (d, 2.6) 3.65 (d, 4.5) 3.67 (t, 5.3)
5 2.93 (dd, 10.8,
2.3)
3.04 (dd, 11.0,
2.3)
2.60 (d, 10.2) 2.34 (d, 9.5)
6 4.51 (t, 10.8) 4.48 (t, 11.0) 4.02 (t, 10.2) 4.42 (t, 10.0)
7 2.50 (m) 3.57 (m) 2.13 (m) 3.16 (m)
8 5.42 (m) 5.36 (m) 3.60 (m) 5.33 (m)
9 2.13 (dd, 15.2,
4.8)
2.18 (dd, 15.8,
5.2)
2.73 (brd,
12.6)
5.44 (d, 4.5)
2.00 (brd, 15.2) 2.02 (dd, 15.8,
5.2)
2.37 (brd,
12.6)
11 2.64 (m) 2.73 (brq,
11.6)
2.61 (m)
13 1.18 (d, 6.9) 6.10 (d, 3.4) 1.43 (d, 6.9) 1.23 (d, 7.1)
5.48 (d, 3.4)
14 1.51 (s) 1.51 (s) 1.75 (s) 1.90 (s)
15 1.48 (s) 1.48 (s) 1.48 (s) 1.36 (s)
14.42 (d, 7.8)
23.18 (m) 2.05 (s)
36.19 (m, 7.2) 6.23 (m, 7.2) 3.30 (m)
42.01 (d, 7.2) 2.02 (d, 7.2) 3.31 (m)
51.90 (s) 1.90 (s) 3.32 (m)
63.85 (d, 10.7)
3.65 (m)
Data assignment was based on HSQC and HMBC experiments.
Fig. 3. The anti-inammatory activity of compounds 18 against
LPS- induced inammatory in H9c2 cells (n = 3); ** P< 0.01
compared with the Control; ## P< 0.01 compared with the
Model.
W. Chen et al. Phytochemistry Letters 23 (2018) 52–56
54
Spectrometer (Thermo Scientic, USA). HR-ESIMS spectra were re-
corded on a Bruker maxis HD mass spectrometer. UV spectra were re-
corded on a Shimadzu UV-2401PC apparatus. Preparative HPLC was
conducted using a Saipuruisi LC-50 instrument with an UV200 detector
(Beijing, China) and a YMC-Pack ODS-A column (250 × 20 mm, 5 μm
and 250 × 10 mm, 5 μm). Column chromatography was performed
with a Diaion HP-20 (Mitsubishi Chemical Corporation, Tokyo, Japan),
Toyopearl HW-40, MCI gel CHP-20 (TOSOH Corp., Tokyo, Japan),
Sephadex LH-20 (4070 μm, Amersham Pharmacia Biotech AB,
Uppsala, Sweden), Lichroprep RP-18 gel (4063 μm, Merck, Darmstadt,
Germany), and silica gel (160200 mesh, Marine Chemical Industry,
Qingdao, China). TLC was performed on self-made silica gel G (Qingdao
Marine Chemical Industry) plates, CH
2
Cl
2
:MeOH:H
2
O (10:1:0.1, v/v),
and CH
2
Cl
2
:MeOH:H
2
O (4:1:0.1, v/v) as the eluent, and spots were
visualized by spraying with 10% H
2
SO
4
in ethanol (v/v) followed by
heating. The chemical reagents were supplied by Beijing Chemical Plant
(Beijing, China) and Tianjin NO. 3 Reagent Plant (Tianjin, China).
3.2. Plant materials
Dendranthema morifolium (Ramat.) kitam were collected from
Jiaozuo, Henan Province, China, in 2015. Identied by Prof Sui-qing
Chen (Henan University of Traditional Chinese Medicine). A voucher
specimen (No. 20150715A) was deposited in the Research Department
of Natural Medicinal Chemistry, School of Pharmacy, Henan University
of Traditional Chinese Medicine.
3.3. Extraction and isolation
The Chrysanthemum morifolium Ramat (11.2 kg) were fractured in
50% acetone (25 L × 3) thrice in a ash extractor at room temperature
(1829 °C). The ltrated solution was condensed under reduced pres-
sure to produce an extract (2.5 kg). The extract was dissolved in water
to a total volume of 9 L and then partitioned with petroleum ether
(boiling point 6090 °C, 5 L × 3) for depigmentation. The aqueous
layer was sequentially partitioned with ethyl acetate (5 L × 4) and n-
butanol (5 L × 4) to give ethyl acetate-soluble (352.0 g) and n-butanol-
soluble (410.0 g) fractions. The ethyl acetate-soluble fraction were
subjected to silica gel (1.0 kg) column (30 cm × 16 cm i.d.) chroma-
tography which was eluted with dichloromethane (CH
2
Cl
2
)/methanol
(MeOH) (v/v, 50:1, 30 L 30:1, 30 L 20:1, 30 L 5:1, 30 L) to
provide fractions AD according to their TLC proles. After removing
the solvents, 64.2 g, 58.6 g, 31.0 g, and 72.2 g were obtained from the
extracts, respectively.
Fraction B (58.6 g) was separated by Toyopearl HW-40 column
chromatography which was eluted with MeOH-H
2
O (v/v, 1:10
3:7 1:1 7:3 10:0, 3 L each) to furnish fractions B1B32.
Fraction B6 (210 mg) was separated by Sephadex LH-20 column chro-
matography (CC), which was eluted with MeOH and puried by HPLC
using methanol (MeOH)/H
2
O (v/v, 55:45) as a mobile phase at the ow
rate of 5 mL/min to yield compounds 1 [retention time (t
R
) 39.3 min,
11.4 mg] and 2 (t
R
41.0 min, 4.5 mg).
Fraction C (31.0 g) was separated on a silica gel column and eluted
with petroleum CH
2
Cl
2
-MeOH (80:1, 60:1, 40:1, 20:1, 10:1, and 5:1) to
aord fractions C1C6. Fraction C2 (518.5 mg) was subjected to
Toyopearl HW-40 and eluted with MeOH to yield Fraction C2.1-C2.5.
Fraction C2.3 was separated on MCI gel CHP-20 and eluted with 40%
MeOH to aord Fraction C2.3.1- C2.3.4. Fraction C2.3.1.2 was puried
by preparative PHPLC (42% MeOH/H
2
O, 5 mL/min) to yield compound
3(t
R
52.6 min, 3.0 mg) and 4 (t
R
64.5 min, 15.0 mg).
Fraction D (72.2 g) was isolated by column chromatography in-
cluding a Toyopearl HW-40, RP-18, MCI gel CHP-20 and Sephadex LH-
20 to yield compounds 5 (2.5 mg), 6 (56.8 mg) 7 (6.2 mg) and 8
(2.5 mg).
3.3.1. Chrysanthguaianolactone C (1)
Colorless crystals: [α]20 D +62.8 (c 0.23, CH
3
OH); UV (CH
3
OH)
λ
max
(logε): 203 (0.72) nm, 217 (0.74) nm; IR (KBr) ν
max
cm
1
: 3393,
2933, 1766, 1706, 1456, 1379, 1239, 1141, 1007 and 963 cm
1
. HR-
ESIMS m/z: 403.1725 [M + Na]
+
(calcd. for C
20
H
28
O
7
Na 403.1727).
1
H NMR (500 MHz, CD
3
OD) spectral data (Table 1);
13
C NMR
(125 MHz, CD
3
OD) spectral data (Table 2).
3.3.2. Chrysanthguaianolactone d (2)
Colorless crystals: [α]20 D + 45.6 (c 0.12, CH
3
OH); UV (CH
3
OH)
λ
max
(logε): 204 (1.38) nm, 216 (1.20) nm; IR (KBr) ν
max
cm
1
: 3416,
2982, 2935, 1772, 1700, 1641, 1456, 1236, 1004 and 975 cm
1
. HR-
ESIMS m/z: 401.1567 [M + Na]
+
(calcd. for C
20
H
26
O
7
Na 401.1570).
1
H NMR (500 MHz, CD
3
OD) spectral data (Table 1);
13
C NMR
(125 MHz, CD
3
OD) spectral data (Table 2).
3.3.3. Chrysanthguaianolactone e (3)
Colorless crystals: [α]20 D +46.7 (c 0.13, CH
3
OH); UV (CH
3
OH)
λ
max
(logε): 205 (1.92) nm; IR (KBr) ν
max
cm
1
: 3381, 2973, 2872,
1647, 1373, 1311, 1079, 1025 and 984 cm
1
. HR-ESIMS m/z:
467.1888 [M + Na]
+
(calcd. for C
21
H
32
O
10
Na 467.1887).
1
H NMR
(500 MHz, CD
3
OD) spectral data (Table 1);
13
C NMR (125 MHz,
CD
3
OD) spectral data (Table 2).
3.3.4. Chrysanthguaianolactone F (4)
Colorless crystals: [α]20 D +53.2 (c 0.19, CH
3
OH); UV (CH
3
OH)
λ
max
(logε): 207 (2.45) nm; IR (KBr) ν
max
cm
1
: 3503, 3372, 2985,
2941, 1763, 1718, 1370, 1251, 1177, 1010 and 972 cm
1
. HR-ESIMS
m/z: 363.1415 [M + Na]
+
(calcd. for C
17
H
24
O
7
Na 363.1414).
1
H NMR
(500 MHz, CD
3
OD) spectral data (Table 1);
13
C NMR (125 MHz,
CD
3
OD) spectral data (Table 2).
3.4. Activity assay
The rat cardiac H9c2 myocardial cells were spontaneously im-
mortalized ventricular rat embryo myoblasts that were purchased from
Biowit Technologies (Shenzhen, China). The cells were maintained in
Dulbeccos modied Eagles medium (DMEM) supplemented with 10%
fetal bovine serum at 37 °C in a water-saturated 5.0% CO
2
incubator.
The cells were split upon reaching a conuency of 80% using trypsin-
EDTA, and then seeded onto 96-well plates at a density of 2.0 × 10
4
Table 2
13
C NMR Spectroscopic Data of Compounds 14 (125 MHz).
No. 1 (CD
3
OD) 2 (CD
3
OD) 3 (CD
3
OD) 4 (CD
3
OD)
1 153.8 154.1 137.2 80.6
2 128.2 128.1 39.9 47.4
3 83.4 83.4 79.9 79.5
4 82.6 82.3 83.9 81.5
5 59.1 59.2 54.1 63.5
6 78.2 78.2 80.9 77.2
7 54.6 49.8 60.9 48.0
8 73.2 72.5 80.8 75.9
9 46.0 45.1 44.8 124.8
10 71.7 72.4 127.9 143.4
11 42.8 140.4 41.9 42.6
12 180.5 171.2 181.8 181.1
13 15.1 120.7 16.6 15.6
14 29.3 28.7 24.2 24.9
15 23.2 23.2 23.4 23.5
1168.0 168.1 105.4 172.2
2128.6 128.6 75.5 21.0
3140.5 140.7 78.7
416.1 16.1 71.5
520.7 20.7 78.0
662.8
Data assignment was based on HSQC and HMBC experiments.
W. Chen et al. Phytochemistry Letters 23 (2018) 52–56
55
cells L
1
(200 μL/well) and incubated for 24 h before treatment.
Thereafter, the cells were exposed to LPS (20 ug/ml) for 24 h and then
incubated in fresh medium with compounds 18 (10 μM) for an addi-
tional 24 h. The eects of compounds 18 on LPS induced sepsis in
H9c2 cells were assessed using the MTT assay, as previously described
(Han et al., 2008). The absorbance of each well was then measured on a
microplate spectrophotometer at a wavelength of 490 nm. Experiments
were performed in triplicate and the values are the averages of three
(n = 3) independent experiments. Individual data are expressed as the
mean ± standard deviation (SD). A post hoc Dunnetts test was used to
obtain corrected p-values in the group comparisons. Statistical analyses
were performed with one-way ANOVA (SPSS version 17.0). A pvalue
less than or equal to 0.05 was considered statistically signicant.
Acknowledgements
Our work was supported by the central government guide local
science and technology development funds (14104349) and the Key
Technology and Quality Characteristics of Quality Control of Authentic
Medicinal Materials Rehmannia glutinosa,Dioscoreae rhizome,
Achyranthes bidentata Blume in Henan (171100310500).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.phytol.2017.11.009.
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