A New Sesquiterpenoid from Chrysanthemum indicum

Article abstract of DOI:10.1007/s10600-019-02898-y

A novel glucoside sesquiterpenoid (1) was isolated from Chrysanthemum indicum L. flowers, together with 14 known compounds (2–15), among which eight compounds (3, 4, 8, and 10–14) were isolated from the plant for the first time. Their structures were determined on the basis of the interpretation of spectroscopic data, viz., ESI-MS, HR-TOF-MS, IR, and NMR. The effects of these compounds onlipopolysaccharide-induced nitric oxide production by RAW 264.7 cells were investigated. Compounds 2, 3, and 7 showed strong or moderate inhibitory effects at 100 μM, and the IC₅₀ values of 3 and 7 were 36.09 and 9.59 μM, respectively.

Full text of DOI:10.1007/s10600-019-02898-y

DOI 10.1007/s10600-019-02898-y
Chemistry of Natural Compounds, Vol. 55, No. 6, November, 2019
A NEW SESQUITERPENOID FROM Chrysanthemum indicum
1,2
3
4
3
Jia Chen, Xu Yang, Bo Li, Kun Yang,
2*
1
4*
Yiqin Wang, Kexing Sun, Yong Zhang,
and Weiliang Zhu
4
A novel glucoside sesquiterpenoid (1) was isolated from Chrysanthemum indicum L. flowers, together
with 14 known compounds (2–15), among which eight compounds (3, 4, 8, and 10–14) were isolated
from the plant for the first time. Their structures were determined on the basis of the interpretation of
spectroscopic data, viz., ESI-MS, HR-TOF-MS, IR, and NMR. The effects of these compounds on
lipopolysaccharide-induced nitric oxide production by RAW 264.7 cells were investigated. Compounds 2, 3,
and 7 showed strong or moderate inhibitory effects at 100 μM, and the IC values of 3 and 7 were
50
36.09 and 9.59 μM, respectively.
Keywords: Chrysanthemum indicum, sesquiterpenoid, glucoside, nitric oxide production.
Dendranthema indicum is the dried flowers and inflorescences of Chrysanthemum indicum L.As a traditional Chinese
medicine, it has been used for heat-clearing and detoxifying, detumescence, and removing intensive heat from the liver
and improving acuity of sight [1]. C. indicum has many pharmacological effects such as anti-inflammatory [2–4], antibacterial
[5], and immunomodulatory properties [6]. The flowers of this herb are also commonly used historically as tea to treat some
eye diseases [7]. Phytochemical investigation on the plant has shown the presence of sesquiterpenoids, flavonoids, and phenolic
compounds [1–3].
So far, there are only few studies focused on water-soluble chemical constituents of C. indicum. In our previous
study, the water extract of C. indicum showed stronger anti-inflammatory activity than the other extractions [3]. Herein, we
present the isolation and structure elucidation of one novel glucoside sesquiterpenoid (1) and eight compounds (3, 4, 8, and
10–14) from C. indicum for the first time. In addition, all compounds were tested for their abilities to inhibit nitric oxide
production, which is one of the indicators of inflammatory activity.
Compound 1 was obtained as a white amorphous powder. The molecular formula was determined to be
–
C
H
O from the quasimolecular ion peak at m/z 427.1979 [M – H] (calcd 427.1974) in the HR-ESI-MS. Two
21 32
9
–
fragment ion peaks of the negative ion ESI-MS at m/z 265.1 and 247.0 were attributed to [M – C H O – H] and
[M – C H O – H O – H] , respectively, which suggested the presence of one glucose moiety (C H O ) in the
molecule. The IR spectrum (KBr) exhibited absorptions for hydroxyl (3425 cm ), ester carbonyl (1767 and 1735 cm ), and
olefinic functionalities (1654 cm ). The H NMR spectrum of 1 (Table 1) showed the proton signals of three methyl
protons at δ 1.83 (3H, s), 1.40 (3H, d, J = 6.7 Hz), and 1.17 (3H, s), an anomeric proton signal at δ 4.43 (1H, d, J = 7.8 Hz)
and signals of one methylene group at δ 3.86 (1H, dd, J = 11.9, 2.1 Hz) and 3.72 (1H, dd, J = 11.9, 4.8 Hz)
6
10 5
–
6
10
5
2
6 12 6
–1
–1
–1
1
13
corresponding to a β-configuration of the glucose. The C NMR spectrum (broadband and DEPT 135°) showed 21
signals comprising three methyl groups, three methylenes, 12 methines, and three quaternary carbons in an HSQC experiment.
Five tertiary C-atoms (δ 105.2, 78.4, 77.9, 75.4, 71.3) and one methylene signal (δ 62.5) showed the presence of a glucose unit.
1) Shanghai Children′s Medical Center, Shanghai Jiao Tong University, 200127, Shanghai, P. R. China; 2) Shanghai
University of Traditional Chinese Medicine, 201203, Shanghai, P. R. China; 3) Chengdu University of Traditional
Chinese Medicine, 611137, Chengdu, P. R. China; 4) Drug Discovery and Design Centre & CAS Key Laboratory of
Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 201203, Shanghai, P. R. China,
e-mail: zhangyong109@simm.ac.cn. Published in Khimiya Prirodnykh Soedinenii, No. 6, November–December, 2019,
pp. 926–929. Original article submitted November 22, 2018.
1076
0009-3130/19/5506-1076 ©2019 Springer Science+Business Media, LLC

1
13
TABLE 1. H and C NMR Data for 1 (CD OD, δ, ppm, J/Hz)
3
C atom
C atom
δH
δC
δH
δC
1
2
2.57 (1H, dd, J = 16.1, 8.0)
2.32 (1H, overlapped)
2.24 (1H, m)
54.6
34.4
11
12
13
14
15
Glc-1′
2′
3′
4′
2.66 (1H, dd, J = 13.6, 6.8)
–
43.0
181.9
16.5
30.2
17.6
105.2
75.4
78.4
71.3
77.9
62.5
1.40 (3H, d, J = 6.7)
3
4
5
6
7
8
9
5.48 (1H, br.s)
127.0
144.0
56.0
82.5
54.5
83.2
44.3
1.17 (3H, s)
1.83 (3H, s)
–
2.70 (1H, t, J = 8.9)
4.18 (1H, t, J = 10.1)
2.61 (1H, m)
4.43 (1H, d, J = 7.8)
3.22 (1H, m)
3.29–3.39 (1H, m, overlapped)
3.29–3.39 (1H, m, overlapped)
3.29–3.39 (1H, m, overlapped)
3.86 (1H, dd, J = 11.9, 2.1)
3.72 (1H, dd, J = 11.9, 4.8)
3.94 (1H, m)
2.32 (1H, dd, J = 15.2, 5.0)
2.12 (1H, dd, J = 15.2, 4.3)
–
5′
6′
10
74.2
O
O
12
15
O
O
13
OH
OH
11
5
6
O
O
O
3
O
HO
1'
HO
8
10
HO
HO
1
OH
OH
HO
HO
14
1
1
Fig. 1. Structure and H– H COSY correlations of 1.
–
These characteristic signals, in combination with the ESI-MS data (m/z 265.1 [M – C H O – H] ), implied that 1 is a
6
10 5
glucoside guaianolide-type sesquiterpenoid. The NMR data of compound 1 were comparable to the known compound
chrysanolide B [8]. The differences in NMR data for 1 and chrysanolide B were at C-8 and the signals of glucose (Table 1).
The signals of the glucose unit and low-frequency shift of the 8-position by 83.2 ppm further revealed the presence of the
sugar moiety at C-8, which was also substantiated by an HMBC experiment. The entire sequence of protons attached to the
1
1
guaianolide skeleton was established by an H– H COSY experiment (Fig. 1). The absolute configuration of the sugar was
determined by acid hydrolysis followed by GC analysis. In the HMBC spectrum, the long-range correlations of H-1/C-2, C-5,
and C-10; H-2/C-1 and C-3; H-3/C-2, C-4, and C-15; H-5/C-1, C-4, C-6, and C-10; H-6/C-8; H-7/C-6, C-8, and C-11;
H-8/C-7 and C-1′; H-9/C-8 and C-10; H-11/C-7, C-12, and C-13; H-13/C-7, C-11, and C-12; H-14/C-1 and C-10;
H-15/C-3, C-4, and C-5 were used to establish the molecular skeleton of 1 as being the same as the typical guaianolide
sesquiterpene [8, 9]. The relative configuration of 1 was determined from its ROESY spectrum and J-based configurational
analysis. The ROESY correlations of H-6/H-11, H-8/H-6, H-14, and H-1′ suggest that CH-6, CH-8, CH-11, CH -14, and
3
CH-1′ are in the β-orientation. The correlations of H-1/H-5 and H-7/H-13 suggest that CH-1, CH-5, CH-7, and
CH -13 are in the α-orientation. Thus, compound 1 was elucidated as 10α-hydroxy-8α-O-(β-D-glucopyranosyl)-1αH,5αH,
3
6βH,8βH,7αH,11βH,11α-methylguaia-3-enolide.
The 14 known compounds (2–15) were identified as eriodictyol-7-O-β-D-glucopyranosiduronic acid (2) [7],
luteolin-7-O-β-D-glucopyranosiduronic acid (3) [10], diosmin (4) [11], apigenin-7-O-β-D-rutinoside (5) [12], buddleoside (6)
[10], luteolin-7-O-β-D-glucoside (7) [10], 5-hydroxy-4′-methoxyflavone-7-O-α-L-rhamnopyranosyl(1→6) [2-O-acetyl-β-D-
glucopyranosyl(1→2)]-β-D-glucopyranoside (8) [13], 4-O-caffeoylquinic acid (9) [14], guanosine (10) [15], adenosine (11)
[15], thymidine (12) [16], uridine (13) [15], bis(2-ethylhexyl)phthalate (14) [17], and uracil (15) [18] by comparison of their
spectroscopic data with those reported in the literature.
To investigate whether compounds 1–15 have anti-inflammatory effects, inhibitory activities of the isolates on
lipopolysaccharide induced nitric oxide production in mouse macrophage RAW264.7 cells were evaluated. Before the assay,
the cytotoxicity of each isolate was detected, and no isolate was found that showed cytotoxicity to RAW264.7 cells
(data not shown). As shown in Fig. 2, compounds 2, 3, and 7 showed strong or moderate inhibitory effects at 100 μM.
In particular, the IC value of 7 was 9.59 μM, which was better than the positive control aminoguanidine hydrochloride
50
(IC = 15.03 μM), and that of 3 was 36.09 μM.
50
1077

1.25
1.00
0.75
0.50
0.25
0
C
P
1
2
3
4
5
6
7
8
9
10
11 12
13
14 15
Compounds
Fig. 2. Effect of compounds 1–15 on the LPS-induced nitric oxide production on RAW 264.7 cells. Data are
the means s.d. of three wells. C – control; P – positive control (50 mM aminoguanidine hydrochloride).
EXPERIMENTAL
General Experimental Procedures. Macroporous resin Diaion HP20 (Mitsubishi Chemical Corporation, Japan),
MCI (GEL CHP 20P, Mitsubishi Chemical Corporation, Japan), Sephadex LH-20 (Pharmazia, Uppsala, Sweden), and ODS
(50 μm, YMC Co., Ltd, Kyoto, Japan) were used for column chromatography. Optical rotations were determined on a Rudolph
Autopol VI 90079 polarimeter (Rudolph ResearchAnalytical, Hackettstown, NJ, USA). IR spectra were recorded on a Thermo
Nicolet 6700 spectrophotometer (Thermo Fisher Scientific, MA, USA). NMR spectra were obtained on a Bruker AV-400
spectrometer (Bruker Corporation, Rheinstetten, Switzerland). Chemical shifts are given in δ values (ppm) relative to
tetramethylsilane as an internal standard. 2D NMR spectra were recorded on a BrukerAV-500 spectrometer (Bruker Corporation,
Rheinstetten, Switzerland). ESI-MS were measured on a Finnigan LCQ-DECA spectrometer (Thermo Fisher, Pittsburgh, PA, USA).
HR-ESI-MS were measured on a Waters Q-TOF Ultima Premier instrument (Waters, Milford, MA, USA).
Plant Material. The dried flowers and inflorescences of Chrysanthemum indicum L. were purchased from the market
of Lotus Pond Medicinal Materials from Chengdu, China in 2016. The plants were identified by Dr. Yan-Hong Shi, Institute of
Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine. A voucher specimen (No. YJ-20160923) was
deposited in the Drug Discovery and Design Centre, Shanghai Institute of Materia Medica, Chinese Academy of Sciences.
Extraction and Isolation. Dried flowers and inflorescences of Chrysanthemum indicum L. (5 kg) were crushed and
reflux-extracted with water (40 L × 2 times, 1 h for each). The solvent was evaporated under reduced pressure and concentrated
to yield 8 L of concentration liquid, which was extracted with EtOAc and n-BuOH. The n-BuOH extract (240 g) was subjected
to HP20 (Diaion) column chromatography, with EtOH–H O (0% to 100%) to yield five fractions (Frs. 1–5), all fractions being
2
monitored by TLC (n-BuOH–acetic acid–water, 4:1:5).
Fraction 2 (3.3 g, 20% EtOH) was subjected to repeated CC on ODS and eluted with MeOH–H O (0% to 60%) to
2
yield six subfractions. Subfraction 3 was further separated by Sephadex LH-20 and eluted with 30% MeOH to afford
compound 1 (7.6 mg). Subfraction 4 was also purified by Sephadex LH-20 (30% MeOH) to provide two subfractions
(Subfrs. A and B). Subfraction B was separated by ODS with MeOH–H O (10%, 20%, and 30%) to give compounds 2 (14.3 mg)
2
and 3 (48.2 mg). Then Subfr. Awas separated by ODS with MeOH–H O (0% and 10%) to obtain compounds 12 (15.3 mg) and
2
15 (4.6 mg). In addition, Fr. 1 was separated by ODS with MeOH–H O (0% and 10%) to afford compounds 10 (21.4 mg),
2
11 (7.1 mg), and 13 (9.6 mg).
Fraction 3 (4.0 g, 40% EtOH) was subjected to MCI and eluted with MeOH–H O (20% to 100%) to yield five
2
subfractions. Subfraction 2 (0.9 g, 40% MeOH) was further purified by ODS and eluted with MeOH–H O (20% to 60%) to
2
obtain compounds 4 (8.0 mg), 5 (26.6 mg), and 6 (74.2 mg).
Fraction 4 (8.2 g, 60% EtOH) was also subjected to MCI and eluted with MeOH–H O (20% to 100%) to yield four subfractions.
2
Subfraction 3 (1.2 g) was purified by ODS with MeOH–H O (20% to 80%) to give compounds 7 (122.1 mg) and 9 (14.8 mg).
2
Subfraction 4 was further separated by Sephadex LH-20 with 60% MeOH to afford compounds 8 (9.7 mg) and 14 (15.4 mg).
10α-Hydroxy-8α-O-(β-D-glucopyranosyl)-1αH,5αH,6βH,8βH,7αH,11βH,11α-methylguaia-3-enolide (1). White
25
–1
amorphous powder; [α] –3.8° (c 0.5, MeOH). IR (KBr, ν , cm ): 3425, 1767, 1735, 1654, 1384, 1077, 1048, 1500,
D
max
1
13
–
1462, 1365, 1201, 1072. For H and C NMR (CD OD) spectral data, see Table 1. HR-ESI-MS m/z 427.1979 [M – H]
3
(calcd for C H O , 427.1974).
21 31
9
Determination of Sugar Components. The acid hydrolysis and detection of sugars were conducted according to the
method described in [19]. Briefly, compound 1 (1 mg) was refluxed in 2 mL 10% HCl–dioxane (1:1) for 2 h, and the solution
1078

was evaporated under N . The residue was dissolved in anhydrous pyridine (100 μL), 0.1 mLcysteine methyl ester hydrochloride
2
(200 μL) was added, and the mixture was warmed at 60°C for 1 h. Next, 2.6 mL of the trimethylsilylation reagent
HMDS–TMCS (hexamethyldisilazane–Me SiCl–pyridine, 2:1:10) was added, with warming at 60°C for another 30 min.
3
The mixture was partitioned between cyclohexane and H O (each 2 mL). The cyclohexane extract was subjected to GC
2
analysis to identify the sugars. GC conditions: cap. column, DB-5 (30 m × 0.25 mm × 0.25 μm); detection, FID; detector temp.,
280°C; injection temp., 280°C; initial temp. maintained at 100°C for 5 min and then raised to 280°C at the rate of 35°C/min,
final temp. maintained for 9 min; carrier, N gas. The absolute configurations of the sugars were determined by comparing the
2
retention times (t ) of derivatives of the sugars with those of authentic sugars prepared in a similar way. The t values of the
R
R
derivatives were 14.16 min (D-glucose).
Anti-inflammatory Effects Assay. Cell Culture. RAW 246.7 cells were maintained in Dulbecco′s modified Eagle′s
medium (high-glucose condition) supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin
at 37°C in 5% CO .
2
Measurement of Nitric Oxide Production. RAW 246.7 cells were seeded in 96-well plates at a density of
50.000 cells/well. After incubation for 24 h, cells were treated with the isolates (100 μM) for 30 min and then stimulated with
LPS (100 ng/mL) for 24 h. The culture supernatant (50 μL) was transferred to another 96-well plate and reacted with Griess
reagent (50 μL) for 10 min. Then, the absorbance at 540 nm was measured using a microplate spectrophotometer. The compounds
were dissolved in Me SO, and the final concentration of Me SO was < 0.1%. Aminoguanidine hydrochloride, which inhibits
2
2
nitric oxide synthase activity, was used as the positive control.
ACKNOWLEDGMENT
This work was supported by the Natural Science Foundation of Young Scholars of China (81603270), the China
Postdoctoral Science Foundation (2017M611608), and the Shanghai Sailing Program (16YF1414100), which are gratefully
acknowledged. In addition, Jia Chen and Xu Yang contributed equally to this work.
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