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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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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
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
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.
iNOS: inductible nitric oxide synthase
Supporting information available online at
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
"Inula racemosa Hook. f.
"density functional theory
"nitric oxide
received June 30, 2011
revised August 2, 2011
accepted Sept. 19, 2011
Published online October 14,
Planta Med 2012; 78: 166171
© Georg Thieme Verlag KG
Stuttgart · New York ·
ISSN 00320943
Lei Shan
Department of Natural Product
School of Pharmacy
Second Military Medical
325 Guohe Road
Shanghai 200433
Phone: + 86 21 34 2059 89
Wei-Dong Zhang
Department of Natural Product
School of Pharmacy
Second Military Medical
325 Guohe Road
Shanghai 200433
Phone: + 86 21 34 2059 89
Fax: +862134205989
Zhang S-D et al. Sesquiterpenoids from Inula Planta Med 2012; 78: 166171
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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]
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]
+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).
(3): white powder; [α]
+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.
Zhang S-D et al. Sesquiterpenoids from Inula Planta Med 2012; 78: 166171
Original Papers
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(4S,8S,10R)-12-Hydroxyeudesma-5(6),7(11)-dien-12,8-olide (4):
white powder; [α]
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
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
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
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
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
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.
Zhang S-D et al. Sesquiterpenoids from Inula Planta Med 2012; 78: 166171
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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
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-
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.
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).
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.
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,
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Original Papers
Downloaded by: University of Science & Technology of China. Copyrighted material.
... 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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The compounds 11,12,13-tri-nor-sesquiterpenes are degraded sesquiterpenoids which have lost the C3 unit of isopropyl or isopropenyl at C-7 of the sesquiterpene skeleton. The irregular C-backbone originates from the oxidative removal of a C3 side chain from the C15 sesquiterpene, which arises from farnesyl diphosphate (FDP). The C12-framework is generated, generally, in all families of sesquiterpenes by oxidative cleavage of the C3 substituent, with the simultaneous introduction of a double bond. This article reviews the isolation, biosynthesis and biological activity of this special class of sesquiterpenes, the 11,12,13-tri-nor-sesquiterpenes.
... 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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Context Traditionally, Inula racemosa Hook. f. (Asteraceae) has been reported to be effective in cancer treatment which motivated the authors to explore the plant for novel anticancer compounds. Objective To isolate and characterize new cytotoxic phytoconstituents from I. racemosa roots. Materials and methods The column chromatography of I. racemosa ethyl acetate extract furnished a novel sesquiterpene lactone whose structure was established by NMR (1D/2D), ES-MS and its cytotoxic properties were assessed on HeLa, MDAMB-231, and A549 cell lines using MTT and LDH (lactate dehydrogenase) assays. Further, morphological changes were analyzed by flow cytometry, mitochondrial membrane potential, AO-EtBr dual staining, and comet assay. Molecular docking and simulation were performed using Glide and Desmond softwares, respectively, to validate the mechanism of action. Results The isolated compound was identified as racemolactone I (compound 1). Amongst the cell lines tested, considerable changes were observed in HeLa cells. Compound 1 (IC50 = 0.9 µg/mL) significantly decreased cell viability (82%) concomitantly with high LDH release (76%) at 15 µg/mL. Diverse morphological alterations along with significant increase (9.23%) in apoptotic cells and decrease in viable cells were observed. AO-EtBr dual staining also confirmed the presence of 20% apoptotic cells. A gradual decrease in mitochondrial membrane potential was observed. HeLa cells showed significantly increased comet tail length (48.4 µm), indicating broken DNA strands. In silico studies exhibited that compound 1 binds to the active site of Polo-like kinase-1 and forms a stable complex. Conclusions Racemolactone I was identified as potential anticancer agent, which can further be confirmed by in vivo investigations.
... To the best of our knowledge, this is the first report of the isolation of HEDO from I. britannica. With the exception of its nitric oxide production inhibitory effect in LPS-induced RAW 264.7 cells [32], little is known about the biological activity of this compound to date. ...
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2α-Hydroxyeudesma-4,11(13)-dien-8β,12-olide (HEDO), a eudesmane-type sesquiterpene lactone belonging to large group of plant terpenoids isolated from Inula britannica, displays cytotoxic activity against diffuse large B cell lymphoma cells in vitro. However, the molecular mechanism of the anticancer effect remains unclear. In this study, we showed that HEDO inhibits cell growth by inducing apoptosis in lymphoma cell lines through its antiproliferative activity. HEDO increases the depolarization of mitochondrial membrane potential and upregulated intracellular reactive oxygen species (ROS). Furthermore, we examined the cell cycle effect, and our results provided evidence that the arrest of the cell cycle at the SubG0/G1 phase plays an important role in the ability of HEDO to inhibit cell growth in Ontario Cancer Institute (OCI)-LY3 lymphoma cells by preventing nuclear factor-kappa B (NF-κB) signaling. In addition, HEDO induced apoptosis by instigating the activation of Bcl-2-associated X (BAX) and cleaved caspase-3, decreasing B-cell lymphoma 2 (BCL2), B-cell lymphoma-extra large (BCL-XL), and procaspase 3 expression levels. Based on these findings, we suggest that HEDO has potential as an anticancer drug of lymphoma by inducing ROS-dependent accumulation of SubG0/G1 arrest and apoptosis in OCI-LY3 cells.
From ancient times, therapeutic herbal plants are extensively utilized in various traditional medicine practices. One such plant is Inula racemosa Hook. f., which is also known as Puskarmool. I. racemosa is mainly found in India’s temperate and sub-alpine regions particularly in Kashmir, Himachal Pradesh, and Uttarakhand. Different ethnic communities use this plant to treat Asthma, respiratory illness, skin diseases, heart disorders, and other ailments. The main phytochemical compounds identified in different parts of the plant are sesquiterpene lactones-alantolactone and isoalantolactone. The plant is well recognized for its diverge range of biological potential such as anti-inflammatory, analgesic, antifungal, hepatoprotective, anti-allergic, adrenal beta-blocking, and cardioprotective effects. So, the present review aims to offer useful data related to phytochemistry, pharmacology, toxicity and conservation of I. racemosa. in a systematic form which will be useful for its further exploration.
Sesquiterpene lactones (SL) are widely distributed in nature (formed biosynthetically in plants from farnesyl pyrophosphate) and are a structurally diverse class of terpenoids with 15 carbon atoms in the skeleton and, in addition to the lactone cycle, can contain various functional groups. Some of them exhibit biological activity both in a rather wide range and in relation to a specific target. An increase in the number of undescribed natural plant compounds of this class, as well as detection in various plant species, opens up new possibilities for their use for the purposes of medical chemistry, phytochemistry, pharmacognosy, chemotaxonomy, and related fields. Using the example of SL of the eudesmane structural type found in plants of the genus Inula, this review attempts to show the relevance of studies of such compounds that investigate the mechanism of action on various biological models, including the goal of developing new effective antitumor agents.
Nitric oxide (NO), an endogenous important signaling molecule, is being placed inside the cell and also between cells of the plants, animals, and bacteria cells. It participates in almost all cellular and bodily functions; it has a significant role in homeostasis in the regulation of the central nervous system, gastrointestinal, respiratory, genital, and urinary systems such as micro- and macrovascularization, inhibition of platelet aggregation, and neurotransmission. However, the overproduction of NO is implicated in some diseases such as arthritis, asthma, cerebral ischemia, and Parkinson's diseases besides neurodegeneration. Therefore, to have a proper understanding of the molecular mechanisms on the induction of NO on these diseases, the interest in studying therapeutic nitric oxide synthase (NOS) inhibitors has increased. This chapter provides an overview of the main advances in design strategies, the mechanism of action at the molecular level, and the search for selective NOS inhibitors from plants and plant secondary metabolites.
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Ten undescribed noreudesmane-type sesquiterpenoids, including eight 12,13-dinoreudesmanes and a pair of 11,12,13-trinoreudesmane epimers were isolated from the whole plant of Artemisia hedinii. Their structures were elucidated by extensive analysis of spectroscopic data, including MS, 1D and 2D NMR, and their absolute configurations were confirmed by X-ray diffraction experiments and DFT calculations. Compounds 1–5, 7–10 were evaluated for their anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated murine macrophages RAW264.7 cells, and all of them could significantly inhibit the LPS induced CCL2 mRNA expression in a dose-dependent manner.
Twenty-one eudesmane-type sesquiterpenes, including five new compounds, were isolated from the roots of Inula helenium. The structures of the new compounds (1-5) were determined by extensive spectroscopic data interpretation, single-crystal X-ray diffraction analysis and ECD calculations. Six compounds can synergistically enhance cisplatin effect against ovarian cancer cells, the structure−activity relationship for the synergistic effect of these compounds with cisplatin was revealed for the first time, which provides useful clues to develop novel sensitizers to overcome drug resistance in cancer. In addition, fifteen sesquiterpenes exhibited significant anti-inflammatory activity, which provided promising candidates for development of anti-inflammatory agent.
As a genus of the Asteraceae, Inula is widely distributed all over the world, and several of them are being used in traditional medicines. A number of metabolites were isolated from Inula species, and some of these have shown to possess ranges of pharmacological activities. The genus Inula contains abundant sesquiterpenoids, such as eudesmanes, xanthanes, and sesquiterpenoid dimers and trimers. In addition, other types of terpenoids, flavonoids, and lignins also exist in the genus Inula. Since 2010, more than 300 new secondary metabolites, including several known natural products that were isolated for the first time from the genus Inula. Most of them exhibited potential bioactivities in various diseases. The review aimed to summarize the advance of recent researches (2010–2020) on phytochemical constituents, biosynthesis, and pharmacological properties of the genus Inula for providing a scientific basis and supporting its application and exploitation for new drug development.
The mutations and deregulation of Wnt signaling pathway occur commonly in human cancer and cause the aberrant activation of β-catenin and β-catenin-dependent transcription, thus contributing to cancer development and progression. Therefore, β-catenin has been demonstrated as a promising target for cancer prevention and therapy. Many natural products have been characterized as inhibitors of the β-catenin signaling through down-regulating β-catenin expression, modulating its phosphorylation, promoting its ubiquitination and proteasomal degradation, inhibiting its nuclear translocation, or other molecular mechanisms. These natural product inhibitors have shown preventive and therapeutic efficacy in various cancer models in vitro and in vivo. In the present review, we comprehensively discuss the natural product β-catenin inhibitors, their in vitro and in vivo anticancer activities, and underlying molecular mechanisms. We also discuss the current β-catenin-targeting strategies and other potential strategies that may be examined for identifying new β-catenin inhibitors as cancer preventive and therapeutic drugs.
A new eudesmanolide and a new aromatic derivative were isolated from the roots of Carpesium cernuun. Their structures were elucidated as 13-hydroxy-5,7(11)-eudesmadien-12,8-olide and 3-methyl-8-acetoxy-9, 10-diisobutyryloxy-p-cymene by spectral methods (EIMS, FAB-MS, ID and 2D NMR).
The structure of two new sesquiterpene lactones from Iva angustifolia Nutt. has been elucidated. One of these, ivangustin, is the double bond isomer 2a of asperilin. The second, ivangulin (6), represents a new structure type and is the methyl ester of a ring A seco-eudesmanolide.
A new eudesmanolide and a new aromatic derivative were isolated from the roots of Carpesium cernuun. Their structures were elucidated as 13-hydroxy-5,7(11)-eudesmadien-12,8-olide and 3-methyl-8-acetoxy-9,10-diisobutyryloxy-p-cymene by spectral methods (EIMS, FAB-MS, 1D and 2D NMR). Carpesium cernuun L. has long been used as chinese folk medicine for its anti-inflammatory, pain-relief, and detoxication properties 1 . Up to now, no phytochemical studies of Carpesium cernuun has been carried out. Here we report the structure elucidation of a new eudesmanolide 1 and a new aromatic derivative 2, which were obtained from this plant.
Two new sesquiterpene lactones, alantodiene and isoalantodiene, have been isolated from Inula racemosa. Both lactones display biological activity as plant growth regulators. Their structures have been elucidated using spectral data and chemical correlation. The most active plant growth regulator yet isolated from this source is isoalantodiene which is a potent root initiator with hypocotyl cuttings of Vigna radiata and it also increases the nitrate reductase activity in this plant.
An investigation of Inula helenium, I. royleana, I. salicina and I. bifrons afforded in addition to known sesquiterpene lactones 20 new lactones, the eudesmanolides 3, 6 and 8–12, the germacranolides 14–18 and 20–22, the guaianolides 23 and 25, the pseudoguaianolide 26, the xanthanolides 28 and 32 and the cyclopropane analogue 30. Structures and configurations of these new compounds were established by extensive PMR studies and by some chemical transformations. Some of the investigated species contain besides the widely distributed pentaynene 33 several known thymol derivatives together with a new one (40).
The aerial parts of Pulicaria undulata afforded two new eudesmanolides, one being of a glaucolide type, and a nor-guaianolide. The structures were elucidated by high field 1H NMR techniques.
Three new sesquiterpenes (1-3), together with four known sesquiterpene lactones, were isolated from the flowers of Inula britannica var. chinensis. Structures were established on the basis of high-field ID and 2D NMR methods supported by HRMS. All sesquiterpene lactones were tested for cytotoxicity as well as apoptotic ratio in human COLO 205, HT 29, HL-60, and AGS cancer cells. Compounds 3 and 4, two α-methylene γ-lactone-bearing sesquiterpenes, were modestly active in these assays. © 2006 American Chemical Society and American Society of Pharmacognosy.
Starting from the screening in conductors, an algorithm for the accurate calculation of dielectric screening effects in solvents is presented, which leads to rather simple explicit expressions for the screening energy and its analytic gradient with respect to the solute coordinates. Thus geometry optimization of a solute within a realistic dielectric continuum model becomes practicable for the first time. The algorithm is suited for molecular mechanics as well as for any molecular orbital algorithm. The implementation into MOPAC and some example applications are reported.