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Sesquiterpenoids from Inula racemosa Hook. f. Inhibit Nitric Oxide Production

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A novel trinorsesquiterpene (1), three new (2-4), and 10 known sesquiterpenes were isolated from the roots of Inula racemosa Hook. f. The structures and absolute configurations of the new sesquiterpenes were elucidated by extensive spectroscopic and computational methods. All compounds were evaluated for their inhibition of LPS-induced nitric oxide production in RAW264.7 macrophages, and the results indicated that compounds 9, 12, and 13 moderately inhibited nitric oxide production with IC₅₀ values of 7.39 ± 0.36, 6.35 ± 0.26, and 5.39 ± 0.18 µM, respectively.
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Introduction
!
The genus Inula, from the Compositae family,
comprises more than 100 species and is mainly
found in Asia, Europe, and Africa [1]. These plants
are used for the treatment of digestive disorders
and inflammation, and their extracts are reported
to have anti-inflammatory, antibacterial, antihep-
atitic, antidiabetes, and antitumor activities [26].
In previous phytochemical studies, various ses-
quiterpene lactones (SLs) have been isolated from
Inula species, including I. britannica, I. salsoloides,
I. japonica,andI. lineariifolia, and these SLs have
shown diverse pharmacological activities [710].
I. racemosa is distributed in the Qinghai-Xizang
plateau and is used in the traditional Chinese
medicine Zangmuxiang[11]. As part of our on-
going research program on bioactive secondary
metabolites from the Inula genus, I. racemosa
was screened for its anti-inflammatory effects,
which led to the isolation and characterization of
a novel trinorsesquiterpene (1), three new (24),
and 10 known sesquiterpenes (l
"Fig. 1). Addi-
tionally, we determined the absolute configura-
tion of compounds 24using the density func-
tional theory (DFT) method for the calculation of
electronic circular dichroism (ECD). Their inhibi-
tory activities against lipopolysaccharide (LPS)-
induced nitric oxide (NO) production in
RAW264.7 macrophages were determined in this
study.
Materials and Methods
!
Instruments and chemicals
Optical rotations were measured on a Perkin-El-
mer 341 digital polarimeter (Perkin-Elmer). CD
spectra were recorded using a JACSO J-815 spec-
trometer. UV spectra were obtained on a Shimad-
zu UV-2550 spectrometer. IR spectra were re-
corded on a Bruker Vector 22 spectrometer using
KBr pellets. ESIMS was performed using an Agi-
lent 1100 LC/MSD-Trap (ESIMS), and HRESIMS
was performed using a Waters QTOF micromass
spectrometer. NMR spectra were obtained on a
Bruker Avance 400 MHZ NMR spectrometer in
CDCl3with TMS as an internal standard. Column
chromatography (CC) was performed using silica
gel (SiO2, 100300 mesh; Qingdao Haiyang Chem-
ical & Special Silica Gel Co, Ltd.) and Sephadex LH-
20 (GE Healthcare Bio-Sciences AB). TLC was per-
formed using precoated SiO2GF254 plates (Qing-
dao Haiyang Chemical & Special Silica Gel Co,
Ltd.). Zones were visualized under UV light
(254 nm) or by spraying with 10 % H2SO4followed
Abstract
!
A novel trinorsesquiterpene (1), three new (24),
and 10 known sesquiterpenes were isolated from
the roots of Inula racemosa Hook. f. The struct ures
and absolute configurations of the new sesquiter-
penes were elucidated by extensive spectroscopic
and computational methods. All compounds were
evaluated for their inhibition of LPS-induced ni-
tric oxide production in RAW264.7 macrophages,
and the results indicated that compounds 9,12,
and 13 moderately inhibited nitric oxide produc-
tion with IC50 values of 7.39 ± 0.36, 6.35 ± 0.26,
and 5.39 ± 0.18 µM, respectively.
Abbreviation
!
iNOS: inductible nitric oxide synthase
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/
plantamedica
Sesquiterpenoids from Inula racemosa
Hook. f. Inhibit Nitric Oxide Production
Authors Shou-De Zhang1, Jiang-Jiang Qin1, Hui-Zi Jin1, Yin-Hua Yin2, Hong-Lin Li3, Xian-Wen Yang4, Xia Li2, Lei Shan2,
Wei-Dong Zhang1, 2
Affiliations The affiliations are listed at the end of the article
Key words
l
"Inula racemosa Hook. f.
l
"Compositae
l
"sesquiterpenes
l
"density functional theory
l
"nitric oxide
received June 30, 2011
revised August 2, 2011
accepted Sept. 19, 2011
Bibliography
DOI http://dx.doi.org/
10.1055/s-0031-1280294
Published online October 14,
2011
Planta Med 2012; 78: 166171
© Georg Thieme Verlag KG
Stuttgart · New York ·
ISSN 00320943
Correspondence
Lei Shan
Department of Natural Product
Chemistry
School of Pharmacy
Second Military Medical
University
325 Guohe Road
Shanghai 200433
China
Phone: + 86 21 34 2059 89
Fax:+862134205989
shanleish@yahoo.com.cn
Correspondence
Wei-Dong Zhang
Department of Natural Product
Chemistry
School of Pharmacy
Second Military Medical
University
325 Guohe Road
Shanghai 200433
China
Phone: + 86 21 34 2059 89
Fax: +862134205989
wdzhangy@hotmail.com
166
Zhang S-D et al. Sesquiterpenoids from Inula Planta Med 2012; 78: 166171
Original Papers
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by heating. The chemical aminoguanidine (purity 98.0%) was
purchased from Sigma.
Plant material
The roots of I. racemosa were collected in Lasa, Xizang, China in
August 2008. The species identity was confirmed by Professor
Han-Ming Zhang in the Department of Pharmacognosy, Second
Military Medical University. A voucher specimen (No.
ZMX2008829) was deposited at the School of Pharmacy, Second
Military Medical University.
Extraction and isolation
The powdered roots (10.0 kg) of I. racemosa were extracted at
room temperature with 95 % EtOH (3 × 10 L, 24 h each), affording
a dark residue (724 g) after evaporation under reduced pressure.
The residue was suspended in distilled water (1500 mL) and ex-
tracted successively with petroleum ether (PE)(5 × 6 L) and CHCl3
(5 × 6 L). The CHCl3extract (52.5 g) was chromatographed on a
silica gel column (1000 g, 100200 mesh, 10 × 70 cm) by eluting
with a stepped gradient of PE/Acetone (20 : 1, 10 : 1, 5 : 1, 2: 1,
1 : 1, each 10 L) giving five fractions (Fr. AE). Fr. A (5.0 g) was sub-
jected to CC over silica gel [100 g, 200300 mesh, 4.5 × 40 cm, PE/
EtOAc (8: 1), 8 L] providing 6(12.0 mg), 7(9.0 mg), 8(5.1 mg), 9
(202.0 mg), and 11 (185.0 mg). Fr. B (3.2 g) was subjected to CC
over silica gel [60 g, 200300 mesh, 4.5 × 40 cm, PE/EtOAc (5 : 1),
5 L] and Sephadex LH-20 [4.0 × 150 cm, CHCl3/MeOH (1: 1),
1500 mL] giving 1(12.2 mg), 5(6.3 mg), 10 (6.6 mg), and 12
(4.2 mg). Following similar procedures, 2(9.4 mg), 4(7.7 mg),
and 14 (6.5 mg) were obtained from Fr. C (2.5 g), while 3
(11.3 mg) and 13 (11.7 mg) were obtained from Fr. D (2.7 g). The
purities of these compounds were > 98%, as determined by HPLC.
Racemosin A (1): white powder; [α]
D
20
12 (c0.075, MeOH); UV
(MeOH) λmax (log ε) 195 (3.12) nm; IR (KBr) νma x 3420, 2924,
2851, 1716, 1647, 1383, 1218, 1062, 529 cm1;1Hand13C NMR
spectroscopic data, see l
"Table 1; ESIMS (positive) m/z 219 [M +
Na]+, ESIMS (negative) m/z 195 [M H]; HRESIMS (positive) m/z
219.1370 [M + Na]+(calcd. for C12H20O2Na, 219.1361).
(7R,8R,10R)-8-Hydroxyeudesma-4(5),11(13)-dien-12-oic acid (2):
white powder; [α]
D
20
+69 (c0.05, MeOH); UV (MeOH) λmax (log
ε) 230 (3.12), 272 (2.21) nm; CD (MeOH), λmax (Δε) 231 nm
(1.81), 270 nm (+ 0.35); IR (KBr) νmax 3446, 2930, 2867, 1705,
1624, 1658, 1383, 1014, 946, 528 cm1;1Hand13C NMR spectro-
scopic data, see l
"Table 1; ESIMS (positive) m/z 273 [M + Na]+,
ESIMS (negative) m/z 249[M H]; HRESIMS (positive) m/z
249.1498 [M H](calcd. for C15H21O3, 249.1490).
(4S,8R,10R)-13-Dimethoxyeudesma-5(6),7(11)-dien-12,8-olide
(3): white powder; [α]
D
20
+67 (c0.09, MeOH); UV (MeOH) λmax
(log ε) 240 (3.84), 277 (4.11) nm; CD (MeOH), λmax (Δε) 239 nm
(4.75), 278 nm (+ 13.52); IR (KBr) νmax 3502, 3004, 2969, 2921,
1713, 1420, 1364, 1223, 1092, 530 cm1;1Hand13C NMR spectro-
scopic data, see l
"Table 1; ESIMS (positive) m/z 315 [M + Na]+,
ESIMS (negative) m/z 291 [M H]; HRESIMS (positive) m/z
315.1588 [M + Na]+(calcd. for C17H24O4Na, 315.1572).
Fig. 1 Chemical structures of compounds 114.
167
Zhang S-D et al. Sesquiterpenoids from Inula Planta Med 2012; 78: 166171
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(4S,8S,10R)-12-Hydroxyeudesma-5(6),7(11)-dien-12,8-olide (4):
white powder; [α]
D
20
180 (c0.21, MeOH); UV (MeOH) λmax (log
ε): 239 (2.14) 279 (3.24) nm; CD (MeOH), λmax (Δε) 237 nm
(+ 1.80), 281 nm (4.02); IR (KBr) νmax 3447, 2930, 2867, 1741,
1656, 1459, 1383, 1216, 1010, 527 cm1;1Hand13C NMR spectro-
scopic data, see l
"Table 1; ESIMS (positive) m/z 271 [M + Na]+,
ESIMS (negative) m/z 247 [M H]; HRESIMS (positive) m/z
271.1335 [M + Na]+(calcd. for C17H24O4Na, 271.1372).
Computational methods
The calculations were performed by the Maestro 7.5 program
and the Gaussion 03 program package. MMFF94 forcefield was
employed to search the low-energy conformations of the com-
pounds. Ground-state geometries were optimized at the B3LYP/
6-31G** level at 298K; total energy of each conformer was ob-
tained, and harmonic frequency analysis was calculated to con-
firm the minima. Excitation energy (in nm) and rotatory strength
R (velocity form Rvel and length form Rlen in 1040 erg-esu-cm/
Gauss) between different states were caculated using the TDDFT
methodology at B3LYP-SCRF/6-31G**//B3LYP/6-31G**, B3PW91-
SCRF/6-31G**//B3LYP/6-31G**, and B3LYP-SCRF/6-311++G**//
B3LYP/6-31G** levels with the COSMO model in methanol solu-
tion. The ECD spectra were then simulated by overlapping Gaus-
sian functions for each transition according to
"ðEÞ¼ 1
2:297 1039 1
ffiffiffiffiffiffiffiffi
2
pX
A
i
EiRie½ðEEiÞ=ð2Þ2
where σis the width of the band at 1/e height and ΔEiand Riare
the excitation energies and rotatory strengths for transition i, re-
spectively; σ=0.20eVandRve l have been used in this work.
Assay for inhibitory activities against
LPS-induced NO production
This assay was conducted as previously described [12]. Briefly,
RAW264.7 cells grown on a 100-mm culture dish were harvested
and seeded in 96-well plates at 2 × 105cells/well. The plates were
pretreated with various concentrations of samples for 30 min and
were then incubated for 24 h with or without 1 µg/mL of LPS. The
nitrite concentration in the culture supernatant was measured
by the Griess reaction [13]. Cell viability was measured using
the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] assay (Sigma-Aldrich) [14]. Statistical calculations were
carried out with the Microsoft Excel software. Results are ex-
pressed as the mean ± SD of 4 independent experiments.
Supporting information
1D and 2D NMR spectra of compounds 14and detailed data of
calculated ECDs are available as Supporting Information.
Results and Discussion
!
Compound 1was obtained as a white powder, and its HRESIMS
showed an [M + Na]+ion at m/z 219.1370, corresponding to the
molecular formula C12H20O2, with three degrees of unsaturation.
The IR spectrum suggested the presence of hydroxy (3420 cm1)
and olefinic (1624 cm1) groups. These observations were consis-
tent with the 1Hand13C NMR signals appearing at ΔH3.99 (1H,
dd, J= 8.0, 1.0 Hz, H-7), 3.55 (1H, ddd, J= 8.0, 4.0, 2.0 Hz, H-8),
and 5.27 (1H, d, J= 1.0 Hz, H-6), as well as ΔC149.7 (C-5), 122.2
(C-6), 73.6 (C-7), and 71.6 (C-8), which were similar to the signals
of alantolactone (9) [15]. Comparison of the NMR data of 1(l
"Ta-
ble 1)and9indicated the absence of the α-methylene-γ-lactone
ring and the presence of an additional hydroxy substituent at C-7
in 1, which was further confirmed by the molecular formula and
the downfield chemical shift of C-7 (ΔC73.6). This assignment
was further verified by the HMBC correlations of H-6/C4 , C- 5 , C-
8, and C-10; H-7/C-5, C-6, C-8, C-9, and C-10; and H-8/C-8 (Fig. 1S).
The relative configuration of 1was confirmed on the basis of NO-
ESY correlations of H-8/H9α(ΔH1.50), H3-14/H9β(ΔH1.62), H-
7/H3-14, and H-7/H3-15 (Fig. 1S). Therefore, the structure of 1
was determined and named racemosin A.
Compound 2gave a molecular formula of C15H22O3from its HRE-
SIMS at m/z 249.1498 [M H], accounting for five degrees of un-
saturation. The IR spectrum showed the presence of hydroxy
(3446 cm1), olefinic (1624, 1658 cm1), and carbonyl (1705 cm1)
Table 1 1H- and 13CNMR data for compounds 14(CDCl3).
No. 1 2 3 4
δCaδHbδCaδHbδCaδHbδCaδHb
1 40.6 t 1.45 m 31.6 t 1.70 m 34.1 t 1.52 m; 1.73 m 32.4 t 1.55 m; 1.72 m
2 18.0 t 1.82 m; 1.42 m 27.5 t 2.00 m; 2.16 m 17.9 t 1.60 m; 1.95 m 16.5 t 1.60 m; 1.87 m
3 33.3 t 1.53 m 31.9 t 1.52 m; 1.76 m 39.5 t 1.63 m; 1.70 m 41.8 t 1.63 m; 1.70 m
4 38.7 d 2.56 m 124.9 s 40.6 d 2.79 m 38.1 d 2.69 m
5 149.7 s 132.9 s 164.2 s 163.9 s
6 122.2 d 5.27 d (1.0) 27.1 t 1.80 m; 2.65 m 114.2 d 6.56 s 114.0 d 6.33 s
7 73.6 d 3.99 dd (8.0, 1.0) 39.6 d 2.40 m 161.2 s 159.2 s
8 71.6 d 3.55 ddd (8.0, 4.0, 2.0) 78.3 d 3.51 dd (7.0, 3.0) 76.0 d 4.78 dd (10.0, 4.0) 76.5 d 5.05 dd (12.0, 5.0)
9 45.3 t 1.62 m; 1.50 m 38.7 t 1.28 m; 2.05 m 43.2 t 1.54 m; 2.16 m 46.5 t 1.54 m; 2.12 m
10 37.7 s 39.4 s 38.4 s 38.3 s
11 144.7 s 116.0 s 118.5 s
12 171.5 s 173.0 s 174.5 s
13 124.7 t 5.68 s; 6.34 s 99.2 d 5.22 s 55.0 t 4.42 s
14 28.6 q 1.18 s 17.3 q 1.04 s 29.5q 1.26 s 26.0 q 1.36 s
15 22.8 q 1.17 d (7.0) 19.0 q 1.60 s 20.6q 1.26 d (7.0) 22.2 q 1.23 d (7.0)
OCH353.9 q
54.1 q
3.37 s
3.40 s
aRecorded at 100 MHz. bRecorded at 400 MHz
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Zhang S-D et al. Sesquiterpenoids from Inula Planta Med 2012; 78: 166171
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groups. A comparison of the 1Hand13C NMR spectra of 2with
those of alloalantolactone suggested that their structures were
related [16]. Analysis of the HMBC spectrum (Fig. 1S) revealed
that the only difference between the two compounds was that
the lactone ring between C-8 and C-12 had been hydrolyzed in
compound 2, which was consistent with its molecular formula.
In the NOESY experiment of 2(Fig. 1S), the cross-peaks of H-8/
H-9α(ΔH1.28) and H-9β(ΔH2.05)/H3-14 suggested that OH-8
and Me-14 were β-oriented. The NOESY correlations between
H-7 and H-8 suggested that H-7 was α-oriented. Thus, the rela-
tive configuration of 2was determined.
Theoretical calculations of the electronic circular dichroism
(ECD) spectrum have been demonstrated as a powerful approach
for determining the absolute configurations of natural products
[1721]. To elucidate the absolute configuration of 2, we calcu-
lated its ECD using time dependent DFT (TDDFT) with the Gaus-
sian 03 program [22]. Firstly, a systematic conformational search
was carried out for compound 2via the Maestro 7.5 program us-
ing MMFF94 force-field with a window of 10 kcal/mol [23]. Three
relatively stable conformations, 2a, 2b,and2c (l
"Fig. 2), were
identified and optimized with ab initio DFT at the B3LYP/6-
31G**level. Apparently, the three different conformers of 2can
be ascribed to the rotation of the C7C11 bond (2a and 2c), as
well as the conformational interconvertion of the A-ring (2a and
2b). The key dihedral angles are listed in l
"Table 2. Conforma-
tional analysis indicated that conformers 2a and 2b are predom-
inantly populated with the distributions of 86.96 % and 12.91 %,
respectively, accounting for more than 99 % of all conformers both
in the gas phase and methanol solution (l
"Table 3), and the dif-
ference between 2a and 2b is observed with the conformational
interconversion of the A-ring. According to this conformational
analysis, the ECD spectra of conformers 2a and 2b were calcu-
lated at the B3LYP-SCRF/6-31G**//B3LYP/6-31G**, B3PW91-
SCRF/6-31G**//B3LYP/6-31G**, and B3LYP-SCRF/6-311++G**//
B3LYP/6-31G** levels with the COSMO [24, 25] model in metha-
nol solution (see spectra in Fig. 2S). The weighted ECD spectra of
the above two conformers are shown in l
"Fig. 3 A, and the calcu-
lated ECDs showed diagnostic negative and positive CEs around
230 and 280 nm, respectively, which corresponded with the ex-
perimental CEs. To further clarify the origin of the experimentally
observed ECD of 2at molecular levels, molecular orbital (MO)
analysis at the B3LYP/6-311++G level with the COMSO model in
methanol solution was also carried out on conformers 2a
(Fig. 3S). The major negative rotatory strength around 230 nm is
contributed by the electronic transition from MO68 (HOMO) in-
volving the π-electrons of the double bond in the A-ring to its cor-
responding unoccupied MO70. In addition, the wave peak around
280 nm derives from the transition from MO68 to MO69 (LUMO).
All the results are consistent with the experimental ECD of 2.On
the basis of the above evidence, the structure of compound 2was
determined to be (7R,8R,10R)-8-hydroxyeudesma-4(5),11(13)-
dien-12-oic acid.
Compound 3was obtained as a white powder and exhibited a
molecular ion [M + Na]+at m/z 315.1588 in the HRESIMS, which
corresponded to the molecular formula of C17H24O4.The1Hand
13C NMR spectroscopic data were similar to those of 13-hy-
droxy-5,7(11)-eudesmadien-8,12-olide (5) [26], except for the
absence of a CH2OH group [ΔC55.4 (t, C-13); ΔH4.44 (2H, s, H-
13)] and the presence of an additional dimethyl acetal group [ΔC
99.2 (d, C-13), 53.9 (q, 13-OMe), 54.1(q, 13-OMe); ΔH5.22 (1H, s,
H-13), 3.37 (3H, s, 13-OMe), 3.40 (3H, s, 13-OMe)] in compound
3, which was further confirmed by HMBC correlations of H-13
with the carbonyl group and the two acetal methoxy groups at
ΔC53.9 and 54.1 (Fig. 1S). We also calculated the ECD spectrum
of 3using the same computational method described above and
Table 2 Key dihedral angles in optimized conformers of 2at the B3LYP/6-
31G* level in the gas phase (degree).
Dihedral angles 2a 2b 2c
C1C2-C3C4 44 52 45
C10C5-C4-C3 253
C6-C5-C4- C3 178 169 176
C7-C6-C5- C4 124 113 125
C9-C8-C7- C6 54 51 48
C14-C10-C5- C4 107 119 109
C12-C11-C7- C6 44 43 171
Table 3 Conformational analysis of compound 2in the gas phase and in MeOH solution.
Species In the gas phase In MeOH
ΔEaPE%bΔGaPG%bΔEscPΔEs%bΔEscPΔEs%bΔEs′′cPΔEs′′%b
2a 0.00 82.80 0.00 86.96 0.00 84.15 0.00 84.06 0.00 84.50
2b 0.94 16.90 1.13 12.91 1.00 15.62 1.00 15.59 1.02 15.23
2c 3.33 0.30 3.87 0.13 3.50 0.23 3.24 0.35 3.40 0.27
aRelative zero point energy and relative Gibbs free energy at the B3LYP/6-31G** level, respectively (kcal/mol). bConformational distribution calculated by using the respective
parameters. cRelative energy at the B3LYP-SCRF/6-31G**//B3LYP/6-31G**, B3PW91-SCRF/6-31G**//B3LYP/6-31G**, B3LYP-SCRF/6-311++G**//B3LYP/6-31G** levels with the
COSMO model, respectively (kcal/mol)
Fig. 2 Optimized geometries of individual con-
formers of compound 2at the B3LYP/6-31G** level
in the gas phase.
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found that the results are similar and consistent with the experi-
mental spectrum (l
"Fig. 3 B). Molecular orbitals analysis of 3
(Fig. 4S) indicated that the electronic transitions from MO79
(HOMO) to MO80 (LUMO) involving the electrons of the α,β-un-
saturated ketones in 3afford positive rotatory strengths around
285 nm, which are consistent with the strong positive CE at
277 nm in the experimental ECD of 3. The negative rotatory
strengthen at 250 nm derives from the transition from MO77 to
MO80, which are associated with another negative CE at 240 nm
in the experimental ECD of 3(Fig. 4S). Therefore, the structure of
3was determined to be (4S,8R,10R)-13-dimethoxyeudesma-5
(6),7(11)-dien-12,8-olide.
Compound 4has the molecular formula C15H20O3based on its
HRESIMS at m/z 271.1335 [M + Na]+, which was the same as 13-
hydroxy-5,7(11)-eudesmadien-8,12-olide (5) [26]. The 1Hand
13C NMR data of 5were identical with those of 4, suggesting the
same skeleton. Detailed analysis of the 1Hand13C NMR data of 4
and 5revealed that 4was the C-8 epimer of 5, which was further
confirmed by a crucial NOESY correlation between H-8 and H3-
14 (Fig. 1S). A theoretical calculation of the ECD spectrum of 4
was also carried out, and the results showed a high similarity to
the experimental values (l
"Fig. 3 C). Analysis of the molecular or-
bitals of 4(Fig. 5S) revealed that the negative rotatory strength at
290 nm derives from the transition from MO67 (HOMO) to MO68
(LUMO) involving the electrons of the α,β-unsaturated ketones in
4, and the negative rotatory strength at 230 nm and the positive
rotatory strength at 250 nm are generated by the transitions
from MO64 to MO68 and MO65 to MO68, respectively. Thus,
compound 4was determined to be (4S,8S,10R)-12-hydroxyeu-
desma-5(6),7(11)-dien-12,8-olide.
By comparing physical and spectroscopic data with literature val-
ues, the ten known sesquiterpenes (514) were identified as 13-
hydroxy-5,7(11)-eudesmadiendien-8,12-olide (5) [26], 4α-H-eu-
desma-11(13)-en-4,12-diol (6) [27], 3-oxo-eudesma-4,11-dien-
12,8β-olide (7) [28], 11α,13-dihydroalantolactone (8) [15], alan-
tolactone (9) [15], isoalantodiene (10) [29], isoalantolactone (11)
[15], (1(10)E)-5β-hydroxygermacra-1(10),4(15),11-trien-8,12-
olide (12) [30], 2α-hydroxyeudesma-4,11(13)-dien-12,8β-olide
(13) [31], and dehydroivangustin (14) [32].
On the basis of a previous research in which sesquiterpene lac-
tones (SLs) have shown to be a rich natural source of potential
anti-inflammatory compounds [33], all isolates were tested for
inhibitory activities against lipopolysaccharide-induced NO pro-
duction in RAW264.7 macrophages under the concentration
range from 2.5 to 50 µM. The IC50 values obtained (l
"Table 4)sug-
gested that all compounds exhibited inhibitory activities against
NO production (IC50 5.3952.06 µM). These data are comparable
to that of aminoguanidine, an iNOSinhibitor with an IC50 value of
9.12 µM. The cell viability measured by the MTT assay showed
that all the compounds had no significant cytotoxicity to the
RAW264.7 cells at their effective concentration for the inhibition
of NO production.
Acknowledgements
!
This work was supported by NSFC (30725045), the Special Pro-
gram for New Drug Innovation of the Ministry of Science and
Technology, China (2009ZX09311-001, 2009ZX09103-319) and
in part by the Scientific Foundation of Shanghai China
(08DZ1971700, 09dZ1975700, and 10DZ1971400).
Table 4 Inhibitory effects of compounds isolated from I. racemosa against
LPS-induced NO production in RAW264.7 macrophages (n = 4, mean ± SD).
Compounds IC50 (µM)a
140.16 ± 2.31
252.06 ± 2.66
345.99 ± 1.69
412.03 ± 1.12
511.07 ± 1.24
620.39 ± 2.01
718.90 ± 1.33
810.13 ± 1.24
97.39 ± 0.36
10 14.06 ± 1.25
11 12.05 ± 1.45
12 6.35 ± 0.26
13 5.39 ± 0.18
14 15.97 ± 1.26
AGb9.12 ± 0.35
aInhibitory effects against LPS-induced NO production in RAW264.7 macrophages.
bPositive control (98.0%, Sigma) aminoguanidine (AG)
Fig. 3 Calculated and experimental ECD spectra of compounds 2,3,and4
(experimental in MeOH; .... at B3LYP-SCRF/6-31G**//B3LYP/6-31G** level
with the COSMO model in MeOH; –––at B3LYP-SCRF/6-311++G**//B3LYP/6-
31G** level with the COSMO model in MeOH).
170
Zhang S-D et al. Sesquiterpenoids from Inula Planta Med 2012; 78: 166171
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Conflict of Interest
!
All authors stated no conflict of interest and agreed to publish
this paper.
Affiliations
1School of Pharmac y, Shanghai Jiao Tong University, Shanghai, China
2Department of Natural Product Chemistry, School of Pharmacy, Second
Military Medical University,Shanghai, China
3Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, EastChina
University of Science and Technology, Shanghai, China
4Key Laboratory of Marine Bio-Resources Sustainable Utilization, South China
Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou,
China
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... GC investigations on a capillary column with the cyclodextrin derivative proved that the natural olefin 10a was the (+)-enantiomer ( Figure 3). Tri-noreudesmanes 11a-11c were isolated from Inula racemosa [107][108][109]. Compound 11d was isolated from the roots of Inula helenium [110]. ...
... Racemosin A (11a) was identified in Inula racemosa Hook. f [107], and it is an ingredient in several patented drugs to treat rhinitis [111], to treat or prevent myocardial ischemia [112], to treat epidemic haemorrhagic fever [113] and to treat or prevent acute heart failure ( Figure 3) [114]. ...
... Racemosin A (11a) was identified in Inula racemosa Hook. f [107], and it is an ingredient in several patented drugs to treat rhinitis [111], to treat or prevent myocardial ischemia [112], to treat epidemic haemorrhagic fever [113] and to treat or prevent acute heart failure ( Figure 3) [114]. The diastereomers 12a and 12b ( Figure 3) were isolated from the essential oils of Vetiveria zizanioides [115,116], and, therefore, they are components of Haitian vetiver oil [116]. ...
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... A large number of secondary metabolites have been isolated from different extracts/fractions of I. racemosa using chromatography. These secondary metabolites include eudesmulolide esters , isoalantolactone, dihydroisoalantolactone, alantodiene, isoalantodiene (Sharma et al. 2016), sesquiterpenoids (Zhang et al. 2012), and sesquiterpene lactones (Bohlmann et al. 1978). ...
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