Anti-inflammatory components of Chrysanthemum indicum flowers

Article abstract of DOI:10.1016/j.bmcl.2014.11.054

One new octulosonic acid derivative, chrysannol A (1), along with 17 known compounds (2-18), were isolated from Chrysanthemum indicum flowers. Their structures were determined from 1D NMR, 2D NMR, HR-ESI-MS spectral data, and comparisons with previous reports. The effects of these compounds on lipopolysaccharide (LPS)-induced nitric oxide (NO) and tumor necrosis factor alpha (TNF-α) production by RAW 264.7 cells were investigated. Compound 8 showed the highest inhibition of NO production of 46.09% at a concentration of 10.0 μM. Compounds 7, 10, 11, and 16 inhibited TNF-α secretion at all concentration tested (0.4, 2.0, and 10.0 μM), with inhibition values ranging from 22.27% to 33.13%. In addition, compound 8 and 9 decrease COX-2 and iNOS protein on Western blot analysis in dose dependent manner.

Full text of DOI:10.1016/j.bmcl.2014.11.054

Bioorganic & Medicinal Chemistry Letters 25 (2015) 266–269
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Anti-inflammatory components of Chrysanthemum indicum flowers
Bui Thi Thuy Luyen a, Bui Huu Tai a,b, Nguyen Phuong Thao a,b, Ji Yun Cha c, Hoon Yeon Lee c,
a,
Young Mi Lee c, , Young Ho Kim
⇑
⇑
a College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea
b Institute of Marine Biochemistry (IMBC), Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Caugiay, Hanoi, Viet Nam
c Department of Oriental Pharmacy, College of Pharmacy, Wonkwang University and Wonkwang Oriental Medicines Research Institute, Iksan, Jeonbuk 570-749, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 July 2014
Revised 5 November 2014
Accepted 20 November 2014
Available online 27 November 2014
One new octulosonic acid derivative, chrysannol A (1), along with 17 known compounds (2–18), were
isolated from Chrysanthemum indicum flowers. Their structures were determined from 1D NMR, 2D
NMR, HR-ESI-MS spectral data, and comparisons with previous reports. The effects of these compounds
on lipopolysaccharide (LPS)-induced nitric oxide (NO) and tumor necrosis factor alpha (TNF-
tion by RAW 264.7 cells were investigated. Compound 8 showed the highest inhibition of NO production
of 46.09% at a concentration of 10.0 M. Compounds 7, 10, 11, and 16 inhibited TNF- secretion at all
concentration tested (0.4, 2.0, and 10.0 M), with inhibition values ranging from 22.27% to 33.13%. In
a) produc-
l
a
Keywords:
l
Chrysanthemum indicum
Chrysannol A
NO
addition, compound 8 and 9 decrease COX-2 and iNOS protein on Western blot analysis in dose
dependent manner.
Ó 2014 Elsevier Ltd. All rights reserved.
TNF-a
Anti-inflammation
Chrysanthemum indicum L. (Compositae), which is widespread
in Korea, is a well-known herb and medicinal plant with small yel-
low flowers. The flowers of C. indicum have been used in mixed
spices, as a food additives for masking flavors, and used in teas
and alcoholic beverages in Korea since ancient times.1 C. indicum
has a long history of use in traditional Korea and Chinese medi-
cines for the treatment of infectious diseases, including pneumonia
and pertussis, and in the treatment of colitis, stomatitis, cancer,
fever, sores, vertigo, inflammation, and hypertension. In terms of
the chemical constituents of this plant, several sesquiterpenes,
flavonoids, and phenolic compounds have been isolated and found
to exhibit inhibitory activity against rat lens aldose reductase.2–4
Additionally, its extracts have been reported to have central and
peripheral analgesic properties, to reduce blood pressure, have
anti-inflammatory and immunomodulatory activities, acetylcho-
linesterase inhibitory activity, and inhibitory activity against
various bacteria and viruses.5–7
Compound 18 was obtained as a colorless oil. The molecular for-
mula of 1 was determined to be C14H22O8 by HR-ESI-MS at m/z
353.1008 [M+Cl]ꢀ (Calcd C14H22O8Cl for 353.1003). The 1H NMR
spectrum of 1 showed signals assignable to four oxygenated
methine protons H-1 (dH 4.43, t, J = 4.8 Hz), H-2 (dH 4.00, t,
J = 4.8 Hz), H-3 (dH 4.12, br t, J = 4.8 Hz), and H-7 (dH 4.31, m),
one oxygenated methylene group H-9a (dH 4.68, dd, J = 3.6,
12.3 Hz) and H-9b (dH 4.85, dd, J = 8.4, 12.3 Hz), one methoxy at
dH 3.78 (s), two methylenes H-4ax (dH 2.21, dd, J = 4.8, 15.0 Hz),
H-4eq (dH 2.26, dd, J = 1.2, 15.0 Hz), and H-30a (dH 1.47, m) and
H-30b (dH 1.67, m), one methine H-20 (dH 2.40, m), and two methyl
groups H-40 (dH 0.91, t, J = 7.2 Hz) and H-50 (dH 1.13, d, J = 7.2 Hz)
(see Table 1). The 13C NMR, DEPT and HMQC spectra of 1 showed
the presence of 14 carbon resonances, consisting of three methyls,
three methylenes, four oxygenated methines, and four quaternary
carbons. Two carbon resonance signals at dC 178.3 (C-10), 169.6 (C-
10) were assigned to two carbonyl groups. Eight carbon resonance
signals at dC 104.8, 40.3, 65.8, 69.5, 78.4, 81.1, 64.3, and 169.6 and
the absence of an anomeric proton indicated the presence of 3-
deoxy-2-octulosonic acid.9 Examination of the COSY spectra of 1
showed two spin systems, H9/H7/H1/H2/H3/H4 and H50/H20/H30/
H40 (see Fig. 2). HMBC correlation were observed between
methylene proton H-4eq/H-4ax (dH 2.26/2.21) and C-2 (dC 69.5),
C-3 (dC 65.8), between H-1 (dH 4.43) and C-5 (dC 104.8), and
between H-3 (dH 4.12) and C-5 (dC 104.8). The quaternary carbon
C-10 (dC 169.6) and the exclusive methoxy group 10-OCH3 (dC
53.2) were assigned to a methyl carboxylate on the basis of the
In this study, we report a new compound (1) and 17 known com-
pounds (2–18) isolated from the flowers of C. indicum (see Fig. 1).
All compounds were assessed for inhibitory activity against LPS-
induced NO and TNF-a production in RAW 264.7 cells. In addition,
the effects on LPS-induced COX-2 and iNOS in RAW 264.7 cells of
compound 8 and 9 were evaluated using Western blot analysis.
⇑
Corresponding authors. Tel.: +82 63 850 6807; fax: +82 63 850 7055 (Y.M.L.);
tel.: +82 42 821 5933; fax: +82 42 823 6566 (Y.H.K.).
E-mail addresses: ymlee@wku.ac.kr (Y.M. Lee), yhk@cnu.ac.kr (Y.H. Kim).
http://dx.doi.org/10.1016/j.bmcl.2014.11.054
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

B. T. T. Luyen et al. / Bioorg. Med. Chem. Lett. 25 (2015) 266–269
267
O
3'
1'
O
H3CO
GlcO
H3CO
GlcO
OH
OH
9
5'
6
7
O
O
OH
3
1
5
3
OCH3
OCH3
8
H
2
O
HO
10
H
n
OGlc
OCH3
H
H
H
1
R3
O
9'
R1O
COOR2
4
5
n = 1
n = 2
R4
HO
HO
3'
1'
7'
1
8'
5
3
R1
O
R5O
OR3
R5
Caff
5'
OR4
OGlc
R2
O
18
R1
OGlc
OH
R2
R3
R4
R5
R1
H
R2
R3
R4
R5
H
OH
O
12
13
14
15
16
17
6
7
8
9
H
H
OH
OH
H
H
Caff
Caff
H
H
H
HO
HO
H
CH3
CH3
H
H
OCH3 OH OCH3
O
H
Caff
H
H
H
OH OH OH
OGlc OH OH
OH
OH
H
H
H
H
HO
Caff
H
Caff
Caff
Caff
OH
CH3
CH3
H
Caff
H
10 OH OGlc OCH3 OH
Glc
11
H
Caff
OGlc
H
H
OH
Figure 1. The structure of isolated compounds from the flowers of Chrysanthemum indicum.
Table 1
The NMR spectroscopic data for compound 1 (in CD3OD)
at C-1 (dC 78.4) and C-7 (dC 81.1) suggested the presence of a bicy-
clo-ketal group in the methyl octulosonate moiety. These findings
suggested a methyl 2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]octane-
5-carboxylate moiety.10 In addition, analysis of COSY spectra
together with HMBC correlations of the methyl protons H-50
(dH 1.13, d, J = 7.2 Hz) and C-10 (dC 178.3), C-20 (dC 42.3), and C-30
(dC 27.8) confirmed the presence of a 20-methyl butanoic acid moi-
ety. The configuration of the 20-methyl butanoic acid substituent
was identified as the S-isomer by analyzing the ester formed with
(R)-(+)-1-phenylethanol by GC (see Supporting information). The
HMBC correlation of the methylene protons H2-9 (dH 4.85, and
4.68) and carboxylic group C-10 (dC 178.3) indicated that the
20S-methyl butanoyl moiety was attached to the methyl octuloson-
ate moiety at C-9 via an ester linkage. The absolute configuration of
1 was determined based on the NOESY spectrum, the three-bond
(3JH–H) 1H–1H spin coupling, and circular dichroism (CD) spectrum.
The coupling constant between protons H-3 (dH 4.12) and H-4ax
(dH 2.21) was 4.8 Hz, indicating an equatorial configuration of
H-3. The observation of a NOESY correlation between proton H-2
(dH 4.00) and H-4ax (dH 2.21) suggested an axial orientation of H-
2; hence the 4.8 Hz coupling constant between H-2 and H-3 also
indicated equatorial configuration of H-1. The NOESY interaction
Pos.
1
dCa
78.4
69.5
65.8
40.3
dbH (mult., J in Hz)
1
2
3
4
4.43 (t, 4.8)
4.00 (t, 4.8)
4.12 (br t, 4.8)
2.21 (dd, 4.8, 15.0)
2.26 (dd, 1.2, 15.0)
—
5
7
9
104.8
81.1
64.3
4.31 (m)
4.68 (dd, 3.6, 12.3)
4.85 (dd, 8.4, 12.3)
—
3.78 (s)
—
2.40 (m)
1.47 (m)
1.67 (m)
0.91 (t, 7.2)
1.13 (d, 7.2)
10
OCH3
10
169.6
53.2
178.3
42.3
20
30
27.8
40
50
11.9
17.0
Assignments were done by DEPT, HMQC, HMBC, COSY, and NOESY experiments.
Measured at a150 MHz, b600 MHz.
HMBC correlation of the methoxy protons at dH 3.78 and C-10. The
downfield shift of the signals of two oxygenated methine carbons
of H-1 (dH 4.43)/H-9 (dH 4.68) supported the same
of H-1 and oxygenated methylene C-9 (see Fig. 2). Moreover, a
a-configuration
O
O
O
O
OH
COSY
HMBC
NOESY
H
O
HO
H
OCH3
H
H
H
Figure 2. The keys COSY, HMBC, and NOESY correlations for compound 1.

268
B. T. T. Luyen et al. / Bioorg. Med. Chem. Lett. 25 (2015) 266–269
0.4 μM
2 μM
10 μM
40
35
30
25
20
15
10
5
0
e
L
1
2
3
4
5
6
7
8
9
0
1
2
3
1
4
5
7
8
1
b
i
l
1
1
1
1
1
16
1
PS
c
i
x
o
h
e
ec
l
V
e
Compounds
C
Figure 3. Effect of compounds 1–18 on the LPS-induced NO production on RAW 264.7 cells. Data were represented as mean SD of at least three independent experiments
performed in triplicates. Celecoxib (1.0 M) was used as a positive control.
l
positive Cotton effect was observed at 230 nm and indicated the
‘S’-configuration of carbon C-5.11 Therefore, the structure of
compound 1 were determined to be methyl (1S,2R,3R,5S,7R)-7-
(20S-methyl butanoyloxymethyl)-2,3-dihydroxy-6,8-dioxabicyclo
[3.2.1] octane-5-carboxylate, a new compound named chrysannol A.
Other known compounds were identified by comparison of
their NMR data to previous reports, including dihydrosyringin
7, 10, 11, and 16 showed potent inhibitory activities at all of the
test concentration (0.4, 2.0, and 10.0 M) with inhibition values
of 22.27% to 33.13%. Compounds 1, 12, and 18 displayed significant
inhibitory activities of 21.96%, 24.73%, and 23.02% at 10.0 M, and
14.42%, 16.13%, and 11.78% when diluted to 2.0 M, respectively.
Other compounds showed moderate inhibitory activities with inhi-
bition values of 13.02% to 19.45% at a concentration of 10.0 M. At
0.4 and 2.0 M, the remaining compounds were weak or inactive,
with the exception of compounds 7, 10, 11, and 16. Flavonoids
(6–11) significantly inhibited TNF- production. The glycoside
compounds and 9–11 showed higher inhibitory activities
than the aglycones, 7 and 8. These implied that the sugar
moiety in the flavonoids enhanced the inhibitory activity of these
compounds. In addition, the location and number of caffeoyl moi-
eties in structures 12–17 are important for the activities of quinic
acid derivatives.
l
l
l
l
(2),12 syringin (3),13 benzyl b-
D
-glucopyranoside (4),14 b-pheny-
l
lethoxy-b-
D
-glucopyranoside (5),15 luteoloside (6),16 tricin (7),17
quercetin (8),18 quercimetrin (9),19 isorhamnetin 3-O-b-
D-gluco-
a
side (10),20 apigetrin (11),21 chlorogenic acid (12),22 chlorogenic
acid methyl ester (13),23 cryptochlorogenic acid methyl ester
(14),23 cynarin (15),24 methyl 3,5-di-O-caffeoyl quinate (16),25
methyl 3,4-di-O-caffeoyl quinate (17),26 (Z)-50-hydroxyjasmone
6
50-O-b- -glucopyranoside (18).27 Eight compounds (3, 6, 7, 8, 9,
D
11, 12, and 15) were determined previously and other compounds
(2, 4, 5, 10, 13, 14, and 16–18) were isolated for the first time from
this plant.
In conclusion, our results indicated that the flavonoids and qui-
nic acid derivatives showed marked inhibitory effects on the pro-
The anti-inflammatory effects of the isolated compounds in
RAW 264.7 cells were investigated (see Supporting information).
duction of NO and TNF-a secretion. Compounds 8 and 9 also
At a concentration of 50.0 lM, the MTT assay results showed that
compounds 1–18 did not affect cell viability (data not shown).
Thus, the effects of compounds 1–18 on LPS-induced production
COX-2
(A)
iNOS
Actin
of the inflammatory mediators NO and TNF-
were evaluated at concentrations lower than 50
tion was measured using Griess reaction assays. Each compound
was screened at concentrations of 0.4, 2.0, and 10.0 M (see
Fig. 3). The results showed that compound 8 exhibited the highest
inhibitory activity of 46.09% at 10.0 M. Compound 9 exhibited
a
in RAW 264.7 cells
lM. NO produc-
LPS (1μg/ml)
Compound 8 (μM)
Celecoxib (1μM)
-
-
-
+
-
+
0.4
-
+
2
-
+
10
-
+
-
l
l
-
+
potent inhibitory activity of 36.55% at the same concentration.
The remaining compounds were weak or inactive. Additionally,
aglycone 8 (inhibition value: 46.09%) displayed inhibitory activity
that was higher than glycoside, compounds 6 and 9–11 (inhibition
values decreased from 36.55% to inactive). The lack of a hydroxyl
group in compound 7 also caused decreased inhibitory activity.
The effect of compounds 8 and 9 on LPS-induced COX-2 and iNOS
in RAW 264.7 cells were also investigated using Western blot
analysis. The results showed that compounds 8 and 9 decreased
COX-2 and iNOS protein expression in dose dependent manner
(see Fig. 4).
(B)
COX-2
iNOS
Actin
LPS (1µg/ml)
Compound 9 (µM)
Celecoxib (1µM)
-
-
-
+
-
+
+
+
10
-
+
-
0.4
-
2
-
-
+
Similarly, TNF-a secretion levels by cells stimulated with or
Figure 4. Effects of compounds 8 (A) and 9 (B) on LPS-induced COX-2 and iNOS in
without compounds were also evaluated (see Fig. 5). Compounds
RAW 264.7 cells using Western blot analysis.

B. T. T. Luyen et al. / Bioorg. Med. Chem. Lett. 25 (2015) 266–269
269
2 μM
10 μM
0.4 μM
10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
S
1
2
3
4
5
6
7
8
9
2
5
7
8
1
b
i
le
C
10
11
1
13
14
1
16
1
P
c
i
x
L
h
e
eco
l
V
e
Compounds
Figure 5. Effects of compounds 1–18 on the LPS-induced TNF-
a production on RAW 264.7 cells. Data were represented as mean SD of at least three independent
experiments performed in triplicates. Celecoxib (1.0 M) was used as a positive control.
l
4. Matsuda, H.; Morikawa, T.; Toguchida, I.; Harima, S.; Yoshikawa, M. Chem.
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decreased COX-2 and iNOS protein expression based on Western
blot analysis. Thus, the extract of C. indicum was rich in phenolic
compounds, which may in part explain the use of this plant in
the treatment of inflammatory conditions. These result are impor-
tant for the development of C. indicum as an anti-inflammatory
functional food. Further studies should determine the efficacy of
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26
8. Chrysannol A (1): Colorless oil; [
(log ): 208 (2.10) nm; IR (KBr)
CD [k nm,
] (MeOH): 230 (+18.58) HR-ESI-MS m/z: 353.1008 [M+Cl]ꢀ (Calcd
14H24O9Cl for 353.1003); 1H NMR (600 MHz, CD3OD) and 13C NMR (150 MHz,
a
]
ꢀ5.76 (c 0.08, MeOH); UV (MeOH) kmax
D
e
m ;
max: 3444, 2968, 1732, 1463, 1178, 1084 cmꢀ1
D
e
C
CD3OD) are given in Table 1.
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Acknowledgments
This work was supported by the Priority Research Centers Pro-
gram through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education, Science and Technology
(2009-0093815), Republic of Korea and by a Grant from the Korea
Food and Drug Administration for Studies on Standardization of
Herbal Medicine (2012). This research was also financially sup-
ported by the Ministry of Knowledge Economy (MKE), Korea Insti-
tute for Advancement of Technology (KIAT) through the Inter-ER
Cooperation Projects (R0002019).
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.
2014.11.054.
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