Sesquiterpenoids from Chrysanthemum indicum with Inhibitory Effects on NO Production

Article abstract of DOI:10.1021/acs.jnatprod.0c01121

Three new guaianolide lactones (1-3) and four new 9-oxonerolidol glucosides (5-8) together with 20 known compounds were isolated from the MeOH extract of the flowers of Chrysanthemum indicum. Their structures were elucidated based on the interpretation of NMR, HRESIMS, and electronic circular dichroism (ECD) data along with acid hydrolysis. Of the isolates, sesquiterpenoids 1-4 and 15 and flavones 17 and 18 exhibited inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide production in RAW 264.7 cells with IC50 values in the range 0.2-27.0 μM.

Full text of DOI:10.1021/acs.jnatprod.0c01121

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Article
Sesquiterpenoids from Chrysanthemum indicum with Inhibitory
Effects on NO Production
Jun Gu Kim, Jin Woo Lee, Thi Phuong Linh Le, Jae Sang Han, Yong Beom Cho, Haeun Kwon,
Dongho Lee, Mi Kyeong Lee, and Bang Yeon Hwang*
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sı Supporting Information
ABSTRACT: Three new guaianolide lactones (1−3) and four new 9-oxonerolidol glucosides (5−8) together with 20 known
compounds were isolated from the MeOH extract of the flowers of Chrysanthemum indicum. Their structures were elucidated based
on the interpretation of NMR, HRESIMS, and electronic circular dichroism (ECD) data along with acid hydrolysis. Of the isolates,
sesquiterpenoids 1−4 and 15 and flavones 17 and 18 exhibited inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide
production in RAW 264.7 cells with IC50 values in the range 0.2−27.0 μM.
he dried flowers of Chrysanthemum indicum L. (Aster-
aceae) have been used as a traditional herbal medicine in
RESULTS AND DISCUSSION
■
T
Compound 1 was isolated as a white, amorphous powder. Its
molecular formula was assigned as C H O from the
HRESIMS data (m/z 367.1517 [M + Na] ; calcd 367.1516)
South Korea for the treatment of inflammation, hypertension,
1
20 24
5
headache, vertigo, and respiratory diseases. Previous phyto-
+
chemical investigations on this plant reported the presence of
and NMR data (Tables 1 and 2), indicating nine indices of
2
−4
various types of sesquiterpenoids and flavonoids,
some of
1
hydrogen deficiency. The H NMR spectrum showed the
which exhibited cytotoxic, antiviral, antioxidant, antiosteopo-
presence of an olefinic proton at δ 6.08 (s), a pair of exocyclic
5
−9
H
rosis, and anti-inflammatory activities.
It has recently been
methylene protons at δ 6.31 (d, J = 3.0 Hz) and 5.79 (d, J =
H
reported that highly oxidized guaianolide sesquiterpenoids
from C. indicum showed anti-inflammatory activity via
inhibition of LPS-induced NF-κB pathway and down-
3
.0 Hz), six methines at δ 5.24 (dt, J = 4.3, 9.9 Hz), 4.38 (dd,
H
J = 9.4, 10.9 Hz), 3.21 (m), 3.16 (m), 2.78 (dd, J = 3.9, 7.1
Hz), and 2.60 (m), and two methyl groups at δ 2.33 (s) and
.96 (d, J = 7.5 Hz), which are characteristic signals for α-
10
H
regulation of MAPK activation. Several 1,10-seco-guaianolide
0
sesquiterpenoids isolated from C. indicum were found to have
10
methylene-γ-lactone-bearing guaianolide sesquiterpenoids.
11
inhibitory effects on nitric oxide production. Owing to these
interesting biological activities, extensive phytochemical studies
have been pursed. As part of an ongoing research program for
the discovery of potential anti-inflammatory agents from
medicinal plants, three new guaianolide-type sesquiterpenoids
1
The remaining H NMR signals for an olefinic proton at δH
6
=
1
.17 (dq, J = 1.5, 7.3 Hz), two methyl groups at δ 2.01 (dd, J
1.6, 7.2 Hz) and 1.93 (m), and the C NMR signals at δC
66.5 (C-1′), 135.6 (C-3′), 127.0 (C-2′), 20.6 (C-5′), and
H
13
1
−3 and four new 9-oxonerolidol glucosides 5−8 together
Special Issue: Special Issue in Honor of A. Douglas
Kinghorn
with 20 known compounds were isolated from the CH Cl -
2
2
and n-BuOH fractions of the dried flowers of C. indicum.
Herein, the isolation, structure determination of new
compounds, and the inhibitory effects of all 27 compounds
on nitric oxide (NO) production in LPS-stimulated RAW
Received: October 15, 2020
Published: March 5, 2021
2
64.7 macrophages are described.
©
2021 American Chemical Society and
American Society of Pharmacognosy
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62

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1
5.9 (C-4′) revealed the presence of an angeloyloxy group. In
Compound 2 was obtained as a white, amorphous powder.
Its molecular formula was assigned as identical to that of 1
(C H O ) from the HRESIMS data (m/z 367.1518 [M +
Na] ; calcd 367.1516). The H and C NMR data (Tables 1
and 2) were closely similar to those of 1, with the main
1
3
the C NMR spectrum, apart from the above angeloyloxy
group, 15 carbon signals were observed and classified as two
methyls, two methylenes, seven methines, two carbonyl
carbons, and two quaternary carbons with the aid of HSQC
data. The position of the angeloyloxy group at C-8 was
confirmed by the HMBC correlation from H-8 to C-1′ (Figure
2
0
24
5
+
1
13
difference in the chemical shifts of H-1 and H-5 (δ 1.96 and
H
2.79 for compound 2; δH 2.78 and 3.21 for compound 1).
1
1
). The relative configuration of 1 was assigned by the NOESY
These data were consistent with the reported H NMR data
experiment, in which the correlations of H-1/H-5, H-1/H-10,
H-7/H-10, and H-5/H-10 indicated that H-1, H-5, H-7, and
H-10 were arbitrarily assigned α-orientations. NOESY
correlations of H-6/H-8, H-6/CH -14, and H-8/CH -14
(δ 2.75 and 3.12 for epi-8α-angeloxycichoralexin; δ 1.97 and
2.87 for 8α-angeloxycichoralexin), suggesting the β- and α-
H
H
orientation of H-1 and H-5, respectively, in compound
12,14,15
2.
The small difference in the coupling constant between
3
3
revealed the β-orientations of H-6, H-8, and CH -14 (Figure
H-1 and H-5 (J ) for compounds 1 and 2 (7.1 and 4.3 Hz,
3
1,5
1
). In addition, the vicinal coupling constants of H-6/H-7 and
respectively) supported the above inference. The NOESY
correlations of H-1/H-6, H-1/H-8, H-8/CH -14, H-1/CH -
H-7/H-8 (J = 10.9 and 9.9 Hz, respectively) support the trans-
3
3
13
orientation of both H-6/H-7 and H-7/H-8. The calculated
and experimental ECD spectrum matched well, which
confirmed the absolute configuration of 1 (Figure 3).
Consequently, the structure of chrysaindilide A (1) was
determined as (+)-(1S,5R,6R,7R,8S,10S)-8-angeloyloxy-10-
methyl-2-oxoguai-3,11(13)-dien-6,12-olide.
14, and H-5/H-10 unambiguously revealed the β-orientation
of H-1 in compound 2 (Figure 2). A strong negative Cotton
effect observed at 200−223 nm in compound 2, in contrast to
the positive Cotton effect at 226 nm (Δε +18.0) in compound
1, implied the inversed (1R) absolute configuration compared
with (1S) of compound 1. The experimental ECD spectrum
5
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1
Table 1. H NMR (400 and 500 MHz) Data for Compounds
1−4 in CDCl3
no.
1
2
3
4
1
2.78, dd
1.96, dd
(4.3, 11.6)
(
3.9, 7.1)
2
2.49, m
.70, m
3.41, br s
2.50, m
2.71, d (17.6)
3.42, br s
2
3
4
5
6.08, s
5.91, s
Figure 1. Key HMBC and NOESY correlations of compound 1.
3.21, m
4.38, dd
2.79, dd
3.08, d (11.1) 3.11, d (10.6)
(
4.3, 11.5)
6
7
8
9
3.79, dd
(10.5, 10.5)
2.92, m
3.71, t (10.4)
3.74, t (10.4)
(
9.4, 10.9)
3.16, m
3.14, tt
3.20, tt
(3.0, 10.2)
4.89, dt
(2.1, 10.6)
(
3.0, 10.2)
5.24, dt (4.3,
5.32, t (10.6)
1.67, m
4.81, dt (2.1,
10.5)
2.16, m
9
.9)
1.78, m
2.26, dd (2.3,
1
3.5)
2
.26, m
2.19, m
1.98, m
2.45, m
2.51, m
1
1
1
1
0
1
2
3
2.60, m
Figure 2. Key HMBC and NOESY correlations of compound 2.
was determined as (+)-(1R,5R,6R,7R,8S,10S)-8-angeloyloxy-
5.79, d (3.0)
5.74, br s
6.33, br s
1.50, d (6.5)
2.28, s
5.57, d (3.0)
6.16, d (3.0)
1.74, s
5.55, d (3.0)
6.16, d (3.0)
1.76, s
6
.31, d (3.0)
1
0-methyl-2-oxoguai-3,11(13)-dien-6,12-olide.
Compound 3 was isolated as a white, amorphous powder. Its
1
1
1
2
3
4
5
′
′
′
0.96, d (7.5)
2.33, s
1.68, s
1.69, s
molecular formula of C H O was determined from the
2
0
26
5
+
HRESIMS data (m/z 369.1672 [M + Na] ; calcd 369.1672)
2.40, m
1.72, m
and NMR data (Tables 1 and 2), indicating eight indices of
6.17, dq (1.5,
6.01, dq (1.4,
7.2)
6.21, dq (1.4,
7.3)
1
13
hydrogen deficiency. A detailed analysis of H and C NMR
spectra indicated that the structure of compound 3 was close
to that of angeloylajadin (4), previously isolated from C.
7
.3)
1
.50, m
4
′
′
2.01, dd (1.6,
.2)
1.91, dd (1.5,
7.3)
1.78, br s
0.94, t (7.4)
2.03, dd (1.5,
7.3)
1.90, d (7.3)
7
16
indicum, only differing by the presence of a 2-methylbuta-
5
1.93, m
1.20, d (7.0)
noyloxy group [δ 2.40 (H-2′), 1.72 (H-3′), 1.50 (H-3′), 1.20
H
Assignments were based on COSY, HSQC, and HMBC experiments.
(
H-5′), 0.94 (H-4′); δ 175.7 (C-1′), 41.2 (C-2′), 26.3 (C-3′),
C
1
6.8 (C-5′), 11.8 (C-4′)] instead of an angeloyloxy group. The
Table 2. 13C NMR (100 and 200 MHz) Data for
Compounds 1−4 in CDCl and Pyridine-d5
position of the 2-methylbutanoyloxy group was determined by
3
the HMBC correlation between H-8 (δ 4.81) and C-1′ (δ
H
C
no.
1
2
3
4
175.7) (Figure 4). The relative configuration of the trans-fused
α-methylene-γ-unsaturated lactone moiety was assigned by the
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
2
3
4
5
53.4
208.1
132.5
178.5
52.5
78.4
49.2
71.0
40.5
53.3
208.0
131.8
178.2
53.0
76.8
49.4
70.1
42.3
136.5
33.2
63.5
66.8
51.3
77.9
56.5
69.9
41.4
128.5
137.0
168.8
120.6
22.0
18.9
175.7
41.2
26.3
11.8
16.8
136.4
33.2
63.5
66.9
51.4
78.0
56.6
69.7
41.5
127.0
137.0
168.9
120.8
22.2
NOESY correlation between H-6 (δ 3.71) and H-8 (δ 4.81)
H
H
and the absence of correlation between H-6 and H-7, along
with the large coupling constants of H-5/H-6 (J = 11.1 Hz),
H-6/H-7 (J = 10.4 Hz), and H-7/H-8 (J = 10.5 Hz) (Figure
4
). In addition, the β-orientation of the epoxide moiety was
deduced by the NOESY correlation among H-3 (δ 3.41), H -
H
3
1
5 (δH 1.68), and H-5 (δH 3.08) (Figure 4). The absolute
configuration was determined by the analysis of the
experimental ECD data. In a guaianolide with a double bond
in the seven-membered B-ring, the inversion of the CE at
0
1
2
3
4
5
′
′
′
′
′
30.7
29.7
140.1
169.1
124.9
16.8
141.9
167.3
127.0
20.3
2
50−270 nm was reported by Moiseeva et al., while a negative
CE was observed in compounds with a saturated B-ring such as
17
1
and 2. The positive CE at 207 nm (Δε + 13.0) and 260 nm
(
Δε + 0.24), and the negative CE at 232 nm (Δε −1.60),
19.8
21.0
19.0
17
matched well with that of ajadin and confirmed the (6S, 7R)
configuration of compound 3 (Figure S21, Supporting
Information). Consequently, the structure of 8-O-methylbuta-
noylajadin (3) was determined as (+)-(3S,4R,5S,6S,7R,8S)-8-
methylbutanoyloxy-10-methylguai-1(10),11(13)-dien-6,12-
olide.
166.5
127.0
135.6
15.9
166.2
128.2
137.5
15.5
166.6
128.7
140.3
15.7
20.6
20.3
20.5
Assignments were based on COSY, HSQC, and HMBC experiments.
Compound 5 was obtained as a colorless oil. The molecular
formula was assigned as C H O from the HRESIMS data
2
1
34
7
+
showed good agreement with the calculated ECD spectrum
Figure 3). Consequently, the structure of chrysaindilide B (2)
(m/z 421.2200 [M + Na] ; calcd 421.2197), with five indices
1
(
of hydrogen deficiency. The H NMR data (Table 3) exhibited
5
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Figure 3. Experimental and calculated ECD spectra of compounds 1 and 2.
1
Table 3. H NMR (900 MHz) Data for Compounds 5−8 in
a
CDCl3
no.
1
5
6
7
8
5.20, d (17.7)
.24, d (11.2)
5.21, d (17.3)
5.24, d (11.2)
5.19, d (17.6)
5.26, d (11.0)
5.21, d (17.6)
5.22, d (11.0)
5
2
5.87, dd
5.87, dd
(11.0, 17.6)
5.81, dd
(11.0, 17.6)
5.97, dd
(11.0, 17.6)
(
11.0, 17.6)
3
4
Figure 4. Key HMBC and NOESY correlations of compound 3.
1.65, m
1.64, m
1.57, m
1.65, m
1.73, m
5
6
2.05, m
5.21, t (7.0)
2.04, m
5.21, m
1.51, m
2.11, t (7.4)
1.57, m
2.44, m
signals for three vinylic protons at δ 5.87 (dd, J = 11.0, 17.6
Hz, H-2), 5.24 (d, J = 11.2 Hz, H-1), and 5.20 (d, J = 17.7 Hz,
H
2.76, m
H-1); two olefinic protons at δ 6.09 (s, H-10) and 5.21 (t, J =
H
7
8
9
7
2
.0 Hz, H-6); three methylene groups at δ 3.03 (s, H -8),
H 2
3.03, s
6.09, s
3.01, s
6.02, s
6.07, s
6.03, s
6.05, s
.05 (m, H -5), and 1.65 (m, H -4); and four methyl groups at
2
2
δ 2.13 (s, CH -13), 1.88 (s, CH -12), 1.59 (s, CH -14), and
H
3
3
3
1
1
1
1
1
1
1
2
3
4
5
6
0
1
2
3
4
5
′
′
′
′
′
2.29, d (6.9)
2.11, h (6.8)
0.90, d (6.7)
0.90, d (6.7)
1.59, s
1
.35 (s, CH -15), which are characteristic signals for the linear
3
sesquiterpenoid, 9-oxonerolidol. In addition, six proton signals
1.88, s
2.13, s
1.59, s
1.35, s
4.39, d (7.7)
3.38, t (8.3)
3.51, t (9.1)
3.58, t (9.1)
3.25, d (9.4)
3.79, m
1.89, s
2.17, s
2.13, s
1.35, s
4.41, d (7.7)
3.35, t (8.3)
3.54, t (8.9)
3.56, t (9.0)
3.32, m
1.89, s
2.15, s
1.87, s
1.36, s
4.45, d (7.7)
3.39, t (7.9)
3.56, m
3.55, m
3.36, m
at δ 3.2−4.5 implied the presence of a monosaccharide group.
H
1
In the H NMR data, the resonance at δ 4.39 (J = 7.7 Hz)
H
correlated to the anomeric carbon at δ 97.9 in the HSQC
C
1.35, s
spectrum and suggested a β-glycosidic linkage. The D-
glucopyranose moieties of the new compounds 5−8 were
defined by comparison with a standard sample in the RP-
HPLC analysis after acid hydrolysis and subsequent derivatiza-
4.40, d (7.7)
3.38, t (8.5)
3.52, t (9.1)
3.59, t (9.2)
3.25, m
13
tion (Figure S50, Supporting Information). The C NMR and
HSQC spectra of 5 revealed 21 carbon signals corresponding
to four methyls, five methylenes, eight methines (including
three olefinic carbons at δC 142.3, 129.2, and 123.0), an
′
3.78, m
3.78, dd
(4.9, 11.5)
3.78, dd
(5.5, 11.7)
3.89, dd (2.7,
11.8)
3.86, dd (2.9,
1
1.7)
oxygenated tertiary carbon (δ 80.8), two olefinic quaternary
C
a
carbons (δC 156.0 and 129.7), and a carbonyl carbon (δC
Assignments were based on COSY, HSQC, and HMBC experiments.
1
99.5), of which 15 signals were ascribable to the 9-
oxonerolidol and 6 signals for the glucosyl moiety (Table 4).
The location of the β-glucosyl unit was determined at C-3 of
the 9-oxonerolidol moiety by the HMBC correlation of H-1′
([α]25
+13.0).
19,20
Consequently, the structure of compound
D
5 was defined as (3S)-9-oxo-(E)-nerolidol-3-O-β-D-glucopyr-
anoside.
Compound 6 was obtained as a colorless oil. The HRESIMS
data (m/z 423.2356 [M + Na] ; calcd 423.2353) indicated a
molecular formula of C21
more than 5, implying the saturation of one double bond. The
C NMR spectrum showed two pairs of double bonds at δ
142.2 (C-2) 129.4 (C-6), 129.1 (C-7), and 116.1 (C-1)
(
δ 4.39) to C-3 (δ 80.8) (Figure 5). The trans geometry of
H
C
6
(7)
the Δ
double bond was determined by the NOESY
+
correlation of H-6 (δ 5.21) and H -8 (δ 3.03) along with the
H
2
H
chemical shift of CH -14 (δ 16.5) (Figure S29, Supporting
H O , with two hydrogen atoms
36 7
3
C
1
8
Information). The absolute configuration of the agylcone
was determined by comparing its specific rotation value
1
3
C
2
5
(
[α]
+14.4) with (3S)-9-oxonerolidol of natural origin
D
,
5
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1
3
7(8)
Table 4. C NMR (225 MHz) Data for Compounds 5−8 in
the location of the Δ
double bond (Figure 5). The (E)
a
CDCl3
configuration of the double bond at C-7/C-8 was assigned by
the NOESY correlation between H -6 (δ 2.11) and H-8 (δ
H
.02) together with the chemical shift of C-14 (δ 19.1) in the
C NMR spectrum (Figure 6). Therefore, the structure of
compound 7 was determined as (E)-3,7,11-trimethyldodeca-
,7,10-trien-9-one-3-O-β-D-glucopyranoside.
2
H
no.
5
6
7
8
6
C
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
2
3
4
5
6
116.1
142.3
80.8
40.9
22.8
129.2
129.7
55.2
199.5
123.0
156.0
27.8
20.8
16.5
22.8
97.9
73.4
76.5
69.8
75.3
61.9
116.1
142.2
80.7
40.9
22.8
129.4
129.1
54.2
209.7
50.9
24.5
22.6
22.6
16.5
22.8
97.9
73.4
76.5
69.8
75.3
61.9
116.4
142.0
80.6
40.9
21.7
41.4
157.2
126.0
191.8
126.2
154.8
27.8
20.6
19.1
22.8
97.7
73.8
76.5
70.6
75.2
62.6
115.7
142.4
80.6
41.0
22.4
34.5
159.4
126.1
191.4
126.3
155.2
27.9
20.7
25.8
24.2
98.1
73.9
76.6
70.8
75.2
62.8
13
1
0
1
2
3
4
5
′
′
′
′
′
Figure 6. Key NOESY correlations of compounds 7 and 8.
Compound 8, obtained as a colorless oil, was assigned the
same molecular formula as that of 7, as determined from the
HRESIMS data (m/z 421.2193 [M + Na] ; calcd 421.2197).
The H and C NMR data of 8 (Tables 3 and 4) were highly
similar to those of 7, except for the C NMR chemical shift of
C-6 (δ 34.5) and C-14 (δ 25.8). The H− H COSY and
HMBC data of 8 revealed the same correlations as those of 7,
suggesting that these compounds have the same 2D structure
Figure 5). The deshielded signal for CH -14 (δ 25.8) as well
+
1
13
1
3
1
1
C
C
′
a
Assignments were based on COSY, HSQC, and HMBC experiments.
(
3 C
as the NOESY correlation between H-8 (δ 6.03) and CH -14
H
3
7
(8)
(
δ 1.87) clearly supported the Z-configuration of the Δ
double bond (Figure 6). Therefore, the structure of
H
18
compound 8 was assigned as (Z)-3,7,11-trimethyldodeca-
1,7,10-trien-9-one-3-O-β-D-glucopyranoside, which is the geo-
metric isomer of 7. However, whether compound 8 is a natural
product or an artifact derived from compound 7 by trans-cis
isomerization remains unresolved at this time due to sample
limitations.
The 20 known compounds were identified as angeloylajadin
16
21
(
4), 5-hydroxy-9-oxonerolidol (9), 8-hydroxycarvotanace-
22 23
tone (10), loliolidie (11), 3,5,6,-trihydroxy-7-megastimen-
2
4
25
26
9
-one (12), indicumenone (13), chrysetunone (14), 8-
27 28
angeloyloxycostunolide (15), (2S)-hesperetin (16), acace-
tin (17), apigenin (18), diosmetin (19), luteolin-7-O-
glucoside (20), luteolin-7-O-glucuronide (21), luteolin-7-
O-rutinoside (22), apigenin-7-O-glucoside (23), apigenin-7-
29
30
31
1
1
30
30
Figure 5. H− H COSY and HMBC correlations of compounds 5−8.
32
4
1
33
34
(
Table 4). In the H NMR spectrum (Table 3), two methyl
O-glucuronide (24), linarin (25), acacetin-7-O-glucoside
35 36
doublets (δ 0.90, J = 6.7 Hz), a methylene doublet (δ 2.29, J
(26), and acacetin-7-O-glucuronide (27), respectively, by
comparing their physicochemical and spectroscopic data with
those of published values.
H
H
=
6.9 Hz), and the methine heptet (δH 2.11, J = 6.8 Hz)
indicated the 10(11)-dihydro derivative of 5. These results
were supported by the HMBC correlations from H -10 and H-
All compounds were evaluated for their inhibitory effects on
LPS-induced NO production in RAW 264.7 macrophage cells.
Aminoguanidine was used as positive controls (IC50 = 20.7
μM) (Table 5). Cell viability was evaluated using an MTT
assay, indicating that none of the test compounds showed any
significant cytotoxicity at their effective concentration for the
inhibition of NO production (Table 5). Among the isolates,
sesquiterpenoids with an α-methylene-γ-unsaturated lactone
moiety (compounds 1, 3, 4, and 15) showed significant
inhibitory effects on NO production with IC50 values ranging
from 0.2 to 9.9 μM. Comparison of the inhibitory effects of
compounds 1 (IC = 1.8 μM) and 2 (IC = 27.0 μM), which
2
1
1
1
1 to C-9 as well as the H− H COSY correlations of CH -10/
2
H-11/CH -12/CH -13 (Figure 5). The (E) configuration of
the Δ double bond was assigned by the NOESY correlation
of H-6 (δ 5.21) and H -8 (δ 3.01) along with the C NMR
3
3
6
(7)
1
3
H
2
H
chemical shift of CH -14 (δ 16.5) (Figure S35, Supporting
3
C
Information). Consequently, the structure of compound 6 was
determined as (3S)-3-hydroxy-3,7,11-trimethyldodeca-1,6-
dien-9-one-3-O-β-D-glucopyranoside.
Compound 7, obtained as a colorless oil, was assigned the
same molecular formula as 5, as determined from the
+
HRESIMS data (m/z 421.2197 [M + Na] ; calcd 421.2197).
5
0
50
1
13
The H and C NMR data of 7 (Tables 3 and 4) resembled
have the opposite configuration at C-1, suggested that the
conformation of the guaianolide might be important for the
NO inhibition. In the linear sesquiterpenoids, 9-oxonerolidol
glucoside derivatives 5−8 (IC values from 97.8 to >100 μM)
7(8)
those of 5, except for the Δ
double bond in 7 instead of
in 5. The HMBC correlation from H-6 to C-8 and CH3-
4 and the correlation from H-8 and H-10 to C-9 supported
6
(7)
Δ
1
5
0
5
66
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Article
Preparative HPLC was performed using Waters HPLC system
equipped with two Waters 515 pumps with a 2996 photodiode-
array detector using a YMC J’sphere ODS-A column (5 μm, 250 × 20
mm, i.d., flow rate 6.0 mL/min). TLC was performed using precoated
silica gel 60 F254 (0.25 mm, Merck) plates, and spots were detected by
a 10% vanillin−H SO in water spray reagent.
Table 5. Inhibitory Effects of Compounds 1−27 on LPS-
a
Induced NO Production in Macrophage RAW 264.7 Cells
IC
CC
IC
CC
5
0
50
50
(μM)
50
b
c
b
c
compound
(μM)
(μM)
22.5
>100
compound
(μM)
27.0
>100
1
2
3
4
5
6
7
8
9
1.8
27.0
3.0
0.2
>100
>100
>100
97.8
48.4
48.8
56.7
29.9
>100
>100
15
16
17
18
19
20
21
22
23
24
25
26
27
9.9
31.2
5.9
18.4
34.5
87.5
>100
>100
53.7
>100
>100
2
4
Plant Material. The dried flowers of C. indicum were purchased
from Kyungdong herbal market (Seoul, Korea) in April 2019 and
identified by one of the authors (B.Y.H.). A voucher specimen
21.5
21.7
>100
>100
>100
>100
>100
60.9
65.1
>100
(CBNU-2019-04-CI) has been deposited at the Herbarium of the
College of Pharmacy, Chungbuk National University, Korea.
>100
>100
>100
>100
>100
>100
>100
>100
Extraction and Isolation. The dried flowers of C. indicum (3.0
kg) were extracted with MeOH (3 × 18 L) by maceration for 3 days
at room temperature, filtered, and evaporated under reduced pressure
to obtain a MeOH extract. This extract (110 g) was suspended in
distilled water and partitioned sequentially with n-hexane (2 × 2 L),
CH Cl (2 × 2 L), EtOAc (2 × 2 L), and n-BuOH (2 × 2 L). The
1
1
1
1
1
0
1
2
3
4
98.0
>100
>100
>100
>100
2
2
45.4
92.4
CH Cl -soluble fraction (20.0 g) was chromatographed on a silica gel
2 2
column and eluted with CH Cl −MeOH gradient system (100:1 to
2
2
0:1) to give 12 fractions (CIC1−CIC12). CIC6 (1.8 g) was separated
by RP-MPLC with MeOH−H O (10:90 to 100:0) to obtain 12
2
Aminoguanidine was used as the positive control (IC = 20.7 μM).
5
0
a
fractions (CIC6-1−CIC6-12). CIC6-5 (77 mg) was further purified
Results are expressed as the mean IC50 values in μM from triplicate
experiments. IC : 50% inhibitory concentration. CC : 50%
b
c
by preparative HPLC (MeCN−H O, 23:77, isocratic) to yield 8-
2
50
50
hydroxycarvotanacetone (10) (t = 21.2 min, 4.3 mg), loliolide (11)
R
cytotoxic concentration.
(
(
tR = 23.8 min, 4.2 mg), and 3,5,6-trihydroxy-7-megastimen-9-one
12) (t = 26.6 min, 16.1 mg). CIC6-7 (57.3 mg) was purified by
R
exhibited weaker inhibitory effects than those of aglycone 9
IC50 = 48.4 μM), suggesting that the glucosylation of 9-
preparative HPLC (MeCN−H O, 40:60, isocratic) to give (2S)-
2
(
hesperetin (16) (t = 19.8 min, 4.0 mg). CIC6-10 was recrystallized
R
oxonerolidol derivatives decreased the inhibition of NO
production. In the flavonoids, the aglycone of the flavone
compounds (17−19) also showed moderate inhibitory effects
with IC values ranging from 5.9 to 34.5 μM. However, the
with MeOH and afforded acacetin (17) (15.1 mg). CIC7 (5.0 g) was
separated by RP-MPLC and eluted with MeOH−H
00:0) gradient system to afford 15 fractions (CIC7-1−CIC7-15).
CIC7-7 (424 mg) was purified by preparative HPLC (MeCN−H O,
O (5:95 to
2
1
2
5
0
6
0:40, isocratic) to give indicumenone (13) (t = 15.5 min, 2.0 mg)
R
flavone glycosides (compounds 20−27) showed weaker
inhibitory effects on NO production (IC50 values from 45.4
to >100 μM). The α-methylene-γ-unsaturated lactone moiety
is a well-known pharmacophore, which showed various
bioactivities such as antitumor, antimicrobial, and anti-
and chrysetunone (14) (t = 17.3 min, 5.9 mg). CIC7-9 (248 mg)
R
was separated by preparative HPLC (MeCN−H O,45:55, isocratic)
2
to yield apigenin (18) (t = 19.5 min, 1.6 mg), diosmetin (19) (t =
R
R
2
0.9 min, 1.8 mg), and 5-hydroxy-9-oxonerolidol (9) (t = 27.8 min,
R
1
.5 mg). CIC9 (1.0 g) was separated by RP-MPLC with MeOH−
2
37−39
inflammatory activities.
Our results also demonstrated
H O (10:90 to 100:0) to give 14 fractions (CIC9-1−9-14). CIC9-10
that sesquiterpenoids 1, 3, 4, and 15 containing the α-
methylene-γ-unsaturated lactone moiety showed the strongest
inhibitory effects on the NO production.
(100 mg) was subjected to Sephadex LH-20 column chromatography
(MeOH, isocratic) to afford 3 fractions (CIC9-10-1−CIC9-10-3).
CIC9-10-2 (90 mg) was further purified by preparative HPLC
(
1
MeCN−H O, 35:65, isocratic) to give compound 5 (t = 31.2 min,
In conclusion, guaianolides 1−4 and germacrane 15 type
sesquiterpenoid lactones as well as flavones 17 and 18 from the
flowers of C. indicum showed inhibitory effects on NO
production in LPS-induced RAW 264.7 macrophages.
Nerolidol derivatives have been reported from the same
plant and many plants in the genus Chrysanthemum. However,
this is the first report of the isolation of 9-oxonerolidiol
glucosides from C. indicum. The flowers of C. indicum may be
worthy of potential for the study of anti-inflammatory agents.
2
R
0.6 mg), 6 (t = 44.9 min, 11.2 mg), 7 (t = 32.4 min, 2.8 mg), and 8
R
R
(
tR = 40.8 min, 2.6 mg). CIC11 (2.0 g) was separated by RP-MPLC
with MeOH−H O (10:90 to 100:0) to afford 11 fractions (CIC11-
2
1
−CIC11-11). CIC11-8 (67 mg) was separated by Sephadex LH-20
(MeOH, isocratic) to give compound 1 (17.1 mg) and the other three
fractions (CIC11-8-1−CIC11-8-3). CIC11-8-2 (40 mg) was further
purified by preparative HPLC (MeCN−H O, 52:48, isocratic) to give
2
compound 2 (t = 23.9 min, 3.3 mg). CIC11-10 (67 mg) was purified
R
by preparative HPLC (MeCN−H O, 60:40, isocratic) to afford
2
compound 3 (t = 46.9 min, 3.5 mg) and angeloylajadin (4) (t =
R
R
EXPERIMENTAL SECTION
43.8 min, 3.5 mg). CIC11-11 (61 mg) was further separated by
■
preparative HPLC (MeCN−H O, 77:23, isocratic) to give 8-
General Experimental Procedures. Optical rotations, UV, and
IR spectra were obtained on a JASCO DIP-1000 polarimeter, a
JASCO UV-550 spectrophotometer, and a JASCO FT-IR 4100
spectrometer, respectively. Experimental ECD spectra were measured
using a JASCO J-715 spectrometer. Briefly, the dissolved solutions of
each compound in MeOH were transferred into a 1 mm path cell, and
the spectra were recorded from 400 to 200 nm with 0.5 nm of data
pitch and a 100 nm/min scanning speed at 25 °C. NMR spectra were
recorded on a Bruker AVANCE 400, 500, and 900 MHz
spectrometers using CDCl and pyridine-d as solvents. ESIMS and
2
angeloyloxyconstunolide (15) (t = 30.2 min, 1.6 mg).
R
The n-BuOH fraction (14.0 g) was chromatographed on a silica gel
column and eluted with CH Cl −MeOH gradient system (20:1 to
2
2
0
:1) to give 9 fractions (CIB1−CIB9). CIB3, CIB4, and CIB5 were
recrystallized in MeOH and afforded acacetin-7-O-glucoside (26)
10.4 mg), apigenin-7-O-glucoside (23) (12.2 mg), and luteolin-7-O-
glucoside (20) (6.2 mg), respectively. CIB8 (2.0 g) was subjected to
RP-MPLC and eluted with a H O−MeOH gradient system (80:20 to
60:40) to give 6 fractions (CIB8-1−CIB8-6). CIB8-4 (274 mg) was
further separated by preparative HPLC (MeCN−H O, 30:70,
isocratic) to yield luteolin-7-O-rutinoside (22) (t = 7.2 min, 8.9
mg), luteolin-7-O-glucuronide (21) (t = 12.1 min, 2.5 mg), and
(
2
3
5
HRESIMS data were obtained with LCQ Fleet and maXis 4G mass
spectrometers, respectively. Column chromatography was performed
on silica gel (Merck, 70−230 mesh) and Sephadex LH-20 (25−100
μm, Pharmacia). MPLC was performed on a Biotage Isolera Prime
chromatography system with Lichroprep RP-18 (Merck, 40−63 μm).
2
R
R
apigenin-7-O-glucuronide (24) (t = 17.5 min, 5.0 mg). CIB8-6 (222
R
mg) was further purified by preparative HPLC (MeCN−H O, 40:60,
2
5
67
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J. Nat. Prod. 2021, 84, 562−569

Journal of Natural Products
pubs.acs.org/jnp
Article
isocratic) to give linarin (25) (t = 7.8 min, 1.0 mg) and acacetin-7-O-
was added, and the mixture was heated for one more hour at 60 °C.
The reaction mixture was directly analyzed by Waters 2695 Alliance
HPLC system equipped with a 2996 photodiode-array detector and a
R
glucuronide (27) (t = 15.4 min, 4.5 mg).
R
Chrysaindilide A (1). This compound is a white amorphous
powder; [α]25 +26 (c 0.7, MeOH); UV (MeOH) λmax (log ε) 220
Phenomenex luna 5u C (2) column (5 μm, 250 × 20 mm, i.d., flow
D
18
(
2.47) nm; ECD (MeOH, c 0.25) λmax (Δε) 267 (−0.15), 226
rate 0.8 mL/min) at 25 °C with isocratic elution of 25% CH CN in
3
−
1
(
+18.0) nm; IR νmax (film) 1770, 1700, 1261, 1145, 1027, 676 cm ;
0.1% formic acid solution for 40 min. The injection volume was 10
μL, and peaks were detected at 250 nm. The standards of D-glucose
and L-glucose (Sigma-Aldrich, St. Louis, MO, USA) were converted
to phenylthiocarbamate derivative by the same method and analyzed
under the same chromatographic conditions. The D-glucose and L-
1
13
H NMR (400 MHz, CDCl ) and C NMR (100 MHz, CDCl ), see
3
3
+
Tables 1 and 2; HRESIMS m/z 367.1517 [M + Na] (calcd for
C H NaO , 367.1516).
20
24
5
Chrysaindilide B (2). This compound is a white amorphous
powder; [α]25 +16 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 225
glucose showed t values of 20.02 and 18.61 min, respectively. The
R
D
hydrolysates of 5−8 showed similar retention time with standard D-
glucose derivative (19.99−20.03 min) (Figure S50, Supporting
Information).
(
2
6
2.20) nm; ECD (MeOH, c 0.5) λmax (Δε) 348 (+1.79), 267 (+0.43),
34 (+14.1), 214 (−18.6) nm; IR ν (film) 1770, 1706, 1238, 1073,
max
−1 1 13
59 cm ; H NMR (500 MHz, CDCl ) and C NMR (200 MHz,
3
+
Measurement of LPS-Induced NO Production and Cell
Pyridine-d ), see Tables 1 and 2; HRESIMS m/z 367.1518 [M + Na]
5
Viability. RAW 264.7 cells were seeded into 96-well tissue culture
(
calcd for C H NaO , 367.1516).
20 24 5
6
plates at 2 × 10 cells/mL and stimulated with 1 μg/mL of LPS in the
8
-O-Methylbutanoylajadin (3). This compound is a colorless oil;
2
5
presence or absence of compounds. Aminoguanidine was used as
positive controls. After incubation at 37 °C for 24 h, 100 μL of cell-
free supernatant was mixed with 100 μL of Griess reagent containing
equal volumes of 2% (w/v) sulfanilamide in 5% (w/v) phosphoric
acid and 0.2% (w/v) N-(1-naphthyl)ethylenediamine solution to
determine nitrite production. Absorbance was measured at 550 nm
against a calibration curve with sodium nitrite standards. Cell viability
of the remaining cells was determined by MTT (Sigma Chemical Co.,
St. Louis, MO, USA) based colorimetric assay.
[α] +14 (c 0.3, CHCl ); UV (MeOH) λ (log ε) 210 (1.78) nm;
D 3 max
ECD (MeOH, c 1.0) λmax (Δε) 260 (+0.24), 232 (−1.60), 207
−
1
(
+13.0) nm; IR νmax (film) 1774, 1710, 1261, 1151, 1031, 680 cm ;
1
13
H NMR (400 MHz, CDCl ) and C NMR (100 MHz, CDCl ), see
Tables 1 and 2; HRESIMS m/z 369.1672 [M + Na] (calcd for
3
3
+
C H NaO , 369.1672).
20
26
5
(
3S)-9-Oxo-(E)-nerolidol-3-O-β-D-glucopyranoside (5). This com-
2
5
pound is a colorless oil; [α]
+14 (c 0.1, MeOH, aglycone); UV
D
(
1
MeOH) λmax (log ε) 239 (1.60) nm; IR ν (film) 3650, 1620,
max
−1
1
13
444, 1031, 663 cm ; H NMR (900 MHz, CDCl ) and C NMR
3
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c01121.
(
+
225 MHz, CDCl ), see Tables 3 and 4; HRESIMS m/z 421.2200 [M
■
3
+
Na] (calcd for C H NaO , 421.2197).
*
2
1
34
7
(
3S)-3-Hydroxy-3,7,11-trimethyldodeca-1,6-dien-9-one-3-O-β-D-
2
5
glucopyranoside (6). This compound is a colorless oil; [α] +11 (c
D
.1, MeOH, agylcone); UV (MeOH) λmax (log ε) 210 (2.30), 292
−
1
1
1D NMR, 2D NMR, and HRESIMS of compounds 1−3
and 5−8 and detailed ECD spectrum of compounds 1−
1
3
3
3
+
1
13
3. H and C NMR and HPLC chromatogram of 20
C H NaO , 423.2353).
known compounds (PDF)
21
36
7
(
E)-3,7,11-Trimethyldodeca-1,7,10-trien-9-one-3-O-β-D-gluco-
2
5
pyranoside (7). This compound is a colorless oil; [α] +5 (c 0.1,
D
AUTHOR INFORMATION
Corresponding Author
MeOH, aglycone); UV (MeOH) λmax (log ε) 272 (1.34) nm; IR ν
■
max
−
1
1
(
film) 3652, 1626, 1437, 1031, 663 cm ; H NMR (900 MHz,
13
CDCl ) and C NMR (225 MHz, CDCl ), see Tables 3 and 4;
Bang Yeon Hwang − College of Pharmacy, Chungbuk
National University, Cheongju 28160, South Korea;
orcid.org/0000-0002-4148-6751; Phone: +82-43-261-
3
3
+
HRESIMS m/z 421.2197 [M + Na] (calcd for C H NaO ,
2
1
34
7
4
21.2197).
(
Z)-3,7,11-Trimethyldodeca-1,7,10-trien-9-one-3-O-β-D-gluco-
2814; Email: byhwang@chungbuk.ac.kr
2
5
D
pyranoside (8). This compound is a colorless oil; [α] +5 (c 0.1,
MeOH, aglycone); UV (MeOH) λmax (log ε) 272 (1.04) nm; IR ν
max
Authors
−
1
1
(
film) 3380, 1643, 1116, 1018, 698 cm ; H NMR (900 MHz,
Jun Gu Kim − College of Pharmacy, Chungbuk National
University, Cheongju 28160, South Korea
Jin Woo Lee − College of Pharmacy, Chungbuk National
University, Cheongju 28160, South Korea
13
CDCl ) and C NMR (225 MHz, CDCl ), see Tables 3 and 4;
HRESIMS m/z 421.2193 [M + Na] (calcd for C H NaO ,
3
3
+
2
1
34
7
4
21.2197).
ECD Computational Method. The preliminary geometry for
compounds 1 and 2 were obtained by the Chem3D software, and
corresponding conformers were searched by the procedure
implemented in Spartan ′14 software under the MMFF force
Thi Phuong Linh Le − College of Pharmacy, Chungbuk
National University, Cheongju 28160, South Korea
Jae Sang Han − College of Pharmacy, Chungbuk National
University, Cheongju 28160, South Korea
Yong Beom Cho − College of Pharmacy, Chungbuk National
University, Cheongju 28160, South Korea
40
field. Selected conformers were further optimized with density
function theory (DFT) calculations at the B3LYP/6-31+G(d,p) level
41
using the Gaussian 09W package. Time-dependent DFT ECD
calculation of optimized conformers were performed at the CAM-
B3LYP/TZVP level with a CPCM solvent model in MeOH. The
overall calculated curves were obtained as sum of Gaussian functions
in accordance with the Boltzmann weighting after UV correction.
Acid Hydrolysis of Compounds 5−8 and Determination of
Sugar Configuration. Compounds 5−8 (each 1.0 mg) were treated
with 1 N HCl (1.0 mL) at 80 °C for 1 h and evaporated in vacuo. The
resultant mixture of sugar and aglycone were partitioned with CH Cl
Haeun Kwon − Department of Plant Biotechnology, College of
Life Sciences and Biotechnology, Korea University, Seoul
02841, South Korea
Dongho Lee − Department of Plant Biotechnology, College of
Life Sciences and Biotechnology, Korea University, Seoul
02841, South Korea; orcid.org/0000-0003-4379-814X
Mi Kyeong Lee − College of Pharmacy, Chungbuk National
University, Cheongju 28160, South Korea; orcid.org/
2
2
and water. The water fraction (sugar portion) was evaporated and
dissolved in anhydrous pyridine (1 mL) followed by addition of L-
cysteine methyl ester hydrochloride (4.0 mg, Sigma-Aldrich, St. Louis,
MO, USA). After heating the mixture at 60 °C for 1 h, 10 μL of
phenylisothiocyante solution (Sigma-Aldrich, St. Louis, MO, USA)
0000-0001-5814-2720
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c01121
5
68
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J. Nat. Prod. 2021, 84, 562−569

Journal of Natural Products
pubs.acs.org/jnp
Article
Notes
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Shwarzt, B.; Prokscht, P. Phytochemistry 1994, 36, 389−392.
28) Chen, H. J.; Chung, C. P.; Chiang, W.; Lin, Y. L. Food Chem.
5
(
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(
This work was supported by the National Research
Foundation of Korea (NRF) grant funded by the Korea
government (MIST) (No. 2020R1A2C1008406) and by the
Korea Basic Science Institute under the R&D program
2
011, 126, 1741−1748.
(29) Yim, S. H.; Kim, H. J.; Lee, I. S. Arch. Pharmacal Res. 2003, 26,
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(30) Han, X. H.; Hong, S. S.; Hwang, J. S.; Lee, M. K.; Hwang, B. Y.;
Ro, J. S. Arch. Pharmacal Res. 2007, 30, 13−17.
(Project No. C140440) supervised by the Ministry of Science
(
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and ICT.
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(32) Wang, M.; Simon, J. E.; Aviles, I. F.; He, K.; Zheng, Q. Y.;
DEDICATION
■
Tadmor, Y. J. Agric. Food Chem. 2003, 51, 601−608.
(33) Lee, M. H.; Son, Y. K.; Han, Y. N. Arch. Pharmacal Res. 2002,
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Dedicated to Dr. A. Douglas Kinghorn, The Ohio State
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products.
(34) Quintin, J.; Lewin, G. J. Nat. Prod. 2004, 67, 1624−1627.
(35) Li, Y. L.; Li, J.; Wang, N. L.; Yao, X. S. Molecules 2008, 13,
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Biosci. 2011, 66, 251−259.
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