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1446
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Secondary Metabolites from Saussurea deltoidea and Their Inhibitory
Activity on Lipopolysaccharide-Induced Tumor Necrosis Factor Production
by Yu-Lin Tung a ) 1), Ming-Jen Cheng b )1), Nai-Yun Hu c ), Ying-Chu Shih c ), Shu-Jiau Chiou c ),
and Ih-Sheng Chen* a )
a
) Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University,
Kaohsiung, Taiwan 807, R.O.C. (phone: þ 886-7-3121101 ext 2191; fax: þ 886-7-3210683;
e-mail: [email protected])
b
) Bioresource Collection and Research Center (BCRC), Food Industry Research and Development
Institute (FIRDI), Hsinchu, Taiwan 300, R.O.C. (e-mail: [email protected])
c
) Biomedical Engineering Research Laboratories, Industrial Technology Research Institute (ITRI),
Hsinchu, Taiwan 300, R.O.C.
Bioassay-guided fractionation of the whole plants of Saussurea deltoidea led to the isolation and
structure elucidation of one new sesquiterpene, deltoidealactone (1), together with 23 known compounds
2 – 24. Their structures were established on the basis of extensive 1D- and 2D-NMR analyses in
combination with HR-MS experiments. Deltoidealactone (1) inhibits tumor necrosis factor (TNF-a)
production in U937 cells in vitro with an IC50 value of 1.47 mg/ml.
Introduction. – The genus Saussurea (Compositae) consists of ca. 400 species
distributed throughout Asia and Europe, with five species occurring in Taiwan [1].
Many Saussurea plants are commonly used as traditional Chinese medicines for curing
bleeding, gynecological diseases, and rheumatism [2], and are well-known to contain
sesquiterpenoids [3 – 6].
S. deltoidea (DC.) C. B. Clarke is a perennial herb, and distributed in China,
Himalaya and Taiwan [1]. Eight known compounds, mainly triterpenoids and
flavonoids, have been isolated from the same species in Mainland China previously
[7]. However, the bioactivity of this species has not been investigated. The MeOH
extract of Formosan species showed inhibitory activity on lipopolysaccharide (LPS)induced tumor necrosis factor (TNF)-a release in a human myeloid leukemic cell line,
U937, and the aim of this study was the isolation of its chemical constituents and to
study their bioactivitiy. Bioassay-guided fractionation led to the isolation of one new
germacrane sesquiterpene analog, deltoidealactone (1), together with the 23 known
compounds, 2 – 24, from the AcOEt-soluble fraction of the whole plants of S. deltoidea.
The structure elucidation of 1 and the inhibitory activity on LPS-induced TNF-a
production of the isolates are described herein.
Results and Discussion. – 1. Chemistry. The AcOEt-soluble fraction of the MeOH
extract was fractionated by a combination of silica-gel and RP-18 column chromatog1)
Y.-L. T. and M.-J. C. contributed equally to the work in this paper.
2011 Verlag Helvetica Chimica Acta AG, Zrich
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1447
raphy, and preparative TLC to yield 24 compounds, 1 – 24, the structures of which
were elucidated by 1D- and 2D-NMR spectral analysis and comparison with literature
data.
Compound 1 was obtained as colorless needles with [a] 25
D ¼ 13.7 (c ¼ 0.038,
CHCl3 ). The molecular formula was determined as C19H22O6 on the basis of the [M þ
Na] þ peak at m/z 369.1314 (calc. 369.1314 for C19H22NaO6 ) in the HR-ESI-MS. The
UV absorption (lmax 220 nm) confirmed the presence of a conjugated C¼C bond. The
bands at 3395, 1773, 1707, and 1658 cm 1 in the IR spectrum revealed the presence of
OH, g-lactone, aldehyde, and ester CO groups, respectively. Nine indices of hydrogen
deficiency (IHD) were determined from the molecular formula, and 13C-NMR
(Table 1), and DEPT spectra. Comparison of the 1H- and 13C-NMR spectra (Table 1)
with those of known sesquiterpenoids [8] indicated that 1 is a sesquiterpene with a
germacrane skeleton containing an a-methylidene g-lactone group, two olefinic CH2
groups, and one (hydroxymethyl)acrylate group. The 1H,1H-COSY, HMBC (Fig. 1),
1448
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Table 1. 1H- and 13C-NMR Data (CDCl3 , 600 and 150 MHz, resp.) of Compound 1. d in ppm, J in Hz. CAtom numbering as indicated in formula 1.
1
CH2(1)
CH2(2)
HC(3)
C(4)
CH2(5)
HC(6)
HC(7)
HC(8)
HC(9)
C(10)
C(11)
CO(12)
CH2(13)
HCO(14)
Me(15)
CO(1’)
C(2’)
CH2(3’)
CH2(4’)
HOC(3’)
d(C )
d( H )
22.7
24.6
128.7
131.8
46.4
69.6
49.8
75.6
146.8
142.4
133.4
168.7
128.4
193.8
17.0
164.9
139.3
62.3
126.0
–
2.18 (td, J ¼ 12.6, 6.2), 2.77 (dt, J ¼ 12.6, 6.2)
1.93 – 1.96 (m), 2.47 – 2.50 (m)
5.28 (t, J ¼ 8.1)
–
2.35 (t, J ¼ 11.4), 2.65 (br. d, J ¼ 11.4)
5.13 (td, J ¼ 11.4, 2.1)
3.09 (dd, J ¼ 10.3, 2.1)
4.94 (br. d, J ¼ 10.3)
6.24 (dd, J ¼ 10.3, 1.2)
–
–
–
5.78 (d, J ¼ 1.5), 6.41 (d, J ¼ 1.5)
9.45 (d, J ¼ 1.2)
1.87 (s)
–
–
4.31 (d, J ¼ 5.4)
5.90 (d, J ¼ 1.2), 6.24 (d, J ¼ 1.2)
2.13 (br. t, J ¼ 6.6)
and NOESY data (Fig. 1) established the structure of 1 as rel-(3aS,4S,5E,9Z,11aR)-6formyl-2,3,3a,4,7,8,11,11a-octahydro-10-methyl-3-methylene-2-oxocyclodeca[b]furan4-yl 2-(hydroxymethyl)acrylate, and it was named deltoidealactone.
The 1H-NMR spectrum of 1 (Table 1) showed signals of one aldehyde group at
d(H) 9.45 (d, J ¼ 1.2, HC(14)), one vinyl Me group at d(H) 1.87 (s, Me(15)), one
exocyclic olefinic CH2 group at d(H) 5.78, 6.41 (2d, J ¼ 1.5, CH2(13)), one terminal CH2
group at d(H) 5.90, 6.24 (2d, J ¼ 1.2, CH2(4’)), O-bearing CH2 group at d(H) 4.31 (d, J ¼
5.4, CH2(3’)), two O-bearing CH groups at d(H) 4.94 (br. d, J ¼ 10.3, HC(8)) and 5.13
(td, J ¼ 11.4, 2.1, HC(6)), two olefinic H-atoms at d(H) 5.28 (t, J ¼ 8.1, HC(3)) and
Fig. 1. Significant COSY (—
—), NOESY ( $ ), and HMBC ( ! ) correlations of 1
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1449
6.24 (dd, J ¼ 10.3, 1.2, HC(9)), two CH2 groups at d(H) 2.18 (td, J ¼ 12.6, 6.2, 1 H of
CH2(1)), 2.77 (dt, J ¼ 12.6, 6.2, 1 H of CH2(1)), 1.93 – 1.96, 2.47 – 2.50 (2m, CH2(2)), and
one CH H-atom at d(H) 3.09 (dd, J ¼ 10.3, 2.1, HC(7)), which pointed to a
germacrane type sesquiterpene [8].
The 13C- and DEPT-NMR spectra indicated (Table 1) that compound 1 is a
sesquiterpene derivative with signals for 19 C-atoms, which were classified as seven
quaternary C-atoms comprising four olefinic C-atoms (d(C) 131.8, 133.4, 139.3, and
142.4), two ester CO C-atoms (d(C) 164.9 and 168.7), and one CO C-atom (d(C)
193.8), five CH groups, including two sp2 olefinic C-atoms (d(C) 128.7 and 146.8), two
O-bearing C-atoms (d(C) 69.6 and 75.6), and one sp3 C-atom (d(C) 49.8), six CH2
groups, containing one O-bearing CH2 group (d(C) 62.3), two exocyclic olefinic Catoms (d(C) 126.0 and 128.4), and three aliphatic CH2 groups (d(C) 22.7, 24.6 and
46.4), and one vinyl Me group (d(C) 17.0). Seven degrees of unsaturation were
attributed to three CO groups, two terminal C¼C bonds, and two pairs of C¼C bonds;
the remaining two degrees of unsaturation indicated that 1 had a bicyclic ring skeleton.
Based on the above evidences and the fact that some sesquiterpenoids [3 – 6] and its
analogs tomenphantins A and B [8] were isolated from Elephantopus tomentosus
(Compositae) [8], compound 1 was suggested to be a bicyclic sesquiterpenoid with
germacrane-type skeleton.
By the 1H,1H-COSY (Fig. 1) and HSQC data, two contiguous structural sequences
were derived from correlations from HC(1) (d(H) 2.18, 2.77; d(C) 22.7) to HC(2)
(d(H) 1.93 – 1.96, 2.47 – 2.50; d(C) 24.6), from HC(2) to HC(3) (d(H) 5.28; d(C)
128.7), and from HC(5) (d(H) 2.35, 2.65; d(C) 46.4) to HC(6) (d(H) 5.13; d(C)
69.6), from HC(6) to HC(7) (d(H) 3.09; d(C) 49.8), from HC(7) to HC(8) (d(H)
4.94; d(C) 75.6), from HC(8) to HC(9) (d(H) 6.24; d(C) 146.8), in accord with the
presence of three spin systems corresponding to the following two fragments:
CH2(1)CH2(2)CH(3) and CH2(5)CH(6)CH(7)CH(8)CH(9) (Fig. 1).
HMBC Data (Fig. 1) enabled to establish the full connectivity within the molecule.
Correlations between the H-atom signal at d(H) 2.18, 2.77 (HC(1)) and the C-atom
signals at d(C) 146.8 (C(9)) and 142.4 (C(10)) revealed that C(1) (d(C) 22.7) was
connected with C(10) (d(C) 142.4), and the HMBC cross-peaks HC(2)/C(1), C(3),
and C(4), HC(3)/C(2), Me(15), and C(5), HC(5)/C(4), C(6), C(7), and Me(15),
HC(7)/C(9), and HC(9)/C(1), established a ten-membered ring of
CH2(1)CH2(2)CH(3)C(4)CH2(5)CH(6)CH(7)CH(8)CH(9)C(10).
The other five-membered ring (a-methylidene g-lactone) was composed of
C(6)C(7)C(11)C(12)O, ascertained by the 1H,13C-NMR long-range correlations
between the H-atom signal at d(H) 3.09 (HC(7)) and the C-atom signals at d(C) 133.4
(C(11)), and 168.7 (C(12)), and between the H-atom signal at d(H) 5.78/6.41
(HC(13)) and the C-atom signals at d(C) 49.8 (C(7)), 133.4 (C(11)), and 168.7
(C(12)) in HMBC spectrum. Finally, the correlations between the H-atom signal at
d(H) 4.31 (HC(3’)) and the C-atom signals at d(C) 164.9 (C(1’)), 139.3 (C(2’)), and
126.0 (C(4’)), and between the H-atom signals at d(H) 5.90 and 6.24 (HC(4’)), and the
C-atom signals at d(C) 164.9 (C(1’)), 126.0 (C(2’)), and 62.3 (C(3’)) confirmed the
presence of a 2-(hydroxymethyl)acrylate sequence ((O)COC(¼CH2 )CH2OH).
An aldehyde group in the molecule was indicated by the band at 1707 cm 1 in the IR
spectrum and confirmed by the signal at d(C) 193.8 in the 13C-NMR spectrum. HMBCs
1450
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
between the CHO group (d(C) 193.8) and both HC(1) (d(H) 2.18, 2.77) and HC(9)
(d(H) 6.24) established the position of the aldehyde group as C(10)HO. Furthermore,
the attachment of one Me group (d(H) 1.87) to C(4) was disclosed according to the
HMBC cross-peak Me(15)/C(4).
Finally, the above assignments were similar to those of the known germacranolide
tomenphantin A [8], while the low-field resonance of the C(8) (d(H) 4.94; d(C) 75.6)
located the 2-(hydroxymethyl)acrylate ((O)COC(¼CH2 )CH2OH) side chain
(C(1’)C(2’)C(3’)C(4’)) at this C-atom. Complete 1H- and 13C-NMR assignments
(Table 1) were achieved through a combination of COSY, HSQC, HMBC, and NOESY
experiments. The full assignments of the C-atom resonances based on HSQC and
HMBC techniques were compiled in Table 1.
The relative configuration of 1 was deduced from a NOESY spectrum (Fig. 1) in
combination with biogenetic considerations [8] and comparison with epitulipinolide
[9], the relative configuration of which was based on a CD spectrum analysis.
According to the NOESY spectrum, the HC(6) is b-oriented, which was confirmed by
the NOE HC(6)/HC(8), indicating that HC(6) and HC(8) are on the same side of
the molecular plane, tentatively assumed as b-orientation. On the other hand, no
detectable NOESY effect could be observed between HC(6) and HC(7), as in
tomenphantin A [8], and thus the a-orientation of the HC(7) was confirmed. Based
on the information from the 1H-NMR, COSY, and NOESY spectra, a computergenerated 3D structure was obtained by using the above-mentioned molecularmodeling program with MM2 force-field calculations for energy minimization (Fig. 2).
The calculated distances between HC(6) and HC(8) (2.356 ) is less than 4.00 ,
and between HC(6) and HC(7) (4.486 ) is more than 4.00 ; this is consistent with
the well-defined NOESY (Fig. 1) observed for each of the H-atom pairs. Consequently,
the relative configuration of compound 1 was established to be rel-(6R,7S,8S) (Fig. 1).
The other known isolates, i.e., lupeol (2) [10], betulonic acid (3) [11], taraxasterol
(4) [12], taraxasteryl acetate (5) [13], oleanolic acid (6) [14], b-amyrin (7) [15], a
mixture of b-sitosterol (8) and stigmasterol (9) [16], a mixture of b-sitostenone (10)
and stigmasta-4,22-dien-3-one (11) [17], vanillin (12) [18], p-hydroxybenzoic acid (13)
[19], vanillic acid (14) [20], methyl 4-hydroxybenzoate (15) [20], a-tocopherylquinone
(16) [21], methyl indole-3-carboxylate (17) [22], balansenate II (18) [23], stearic acid
(19) [24], a mixture of tetradecanoic acid (20), pentadecanoic acid (21), and palmitic
acid (22) [25], and a mixture of methyl palmitate (23) and methyl stearate (24) [26],
were identified by comparison with literature data.
2. Biological Studies. Tumor necrosis factor (TNF-a), also known as cachectin, is a
cytokine produced by cells of the mononuclear system, and is recognized as one of the
key mediators of inflammatory responses [27]. Through its interaction with cells like
macrophages, fibroblasts, and endothelial cells, TNF-a promotes the immune response
and local inflammatory processes [27 – 29]. Lipopolysaccharide (LPS) stimulation
effects severe biological responses within host cells, including fever, procoagulant
activity, septic shock, and death. However, the stimulation of LPS involved in several
pro-inflammatory mediators, including nitric oxide (NO), interleukins (IL-6 and IL-1),
and tumor necrosis factor (TNF-a), both in vivo and in vitro, is mediated through
activation of the host immune and inflammatory cells. TNF-a is a mediator in the
inflammatory response involved in host defense. In this study, we investigated the
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1451
Fig. 2. Most stable conformation for compound 1 as predicted by molecular-mechanics (MM2)
calculations
inhibitory activity of isolated compounds using LPS-induced macrophage, an in vitro
model.
The inhibitory activity on LPS-induced TNF-a production were conducted for
eleven compounds, 1, 4 – 9, 12, 16, 18, and 19, isolated from the whole plants of S.
deltoidea, while the amount of other compounds available was too low to allow testing
(Table 2). Compound 1 inhibited significantly human TNF-a with an IC50 value of
1.47 mg/ml, in comparison to those of the positive control 4-[4-(4-fluorophenyl)-2-[4(methylsulfinyl)phenyl]-1H-imidazol-5-yl]pyridine (SB 203580) with an IC50 value of
0.19 mg/ml for TNF-a inhibition, respectively. The high cell viability ( > 80% of control
group) indicated that the inhibitory activity of LPS-induced human myeloid leukemic
cell line by compound 1 did not result from its cytotoxicity.
Conclusions. – This is the first report regarding the secondary metabolites from the
whole plants of S. deltoidea except eight common known compounds which have been
isolated from aerial part of the same plant in Mainland China previously [7]. Through a
comprehensive investigation, 24 pure natural products with different skeletons were
obtained. The metabolite 1, found in this study, was a new, naturally occurring
compound. Interestingly, this is the first report of a sesquiterpene with a germacrane
skeleton compared to other guaiane- or eudesmane-type sesquiterpenes isolated from
1452
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Table 2. Inhibitory Effects of Test Compounds on the TNF-a Production Stimulated by LPS in Human
Myeloid Leukemic Cell Line U93
No.
Compounds
IC50 [mg/ml]
1
4
5
6
7
8/9
12
16
18
19
Deltoidealactone
Taraxasterol
Taraxasteryl acetate
Oleanoic acid
b-Amyrin
b-Sitosterol/stigmasterol
Vanillin
a-Tocopheryl quinone
Balansenate II
Stearic acid
4-[4-(4-Fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-1H-imidazol-5-yl]pyridine
( SB 203580) a )
1.47
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
0.19
a
) Positive control.
this plant previously [3 – 6]. This new type could stimulate future phytochemical
studies.
This work was kindly supported by the National Science Council of the Republic of China (NSC 962628-B-037-003-MY3).
Experimental Part
General. TLC: Silica gel 60 F254 precoated plates (Merck). Column chromatography (CC): silica gel
60 (SiO2 ; 70 – 230 or 230 – 400 mesh; Merck) or Spherical C18 (20 – 40 mm; Silicycle). HPLC: Spherical
C18 column (250 10 mm, 5 mm; Waters), LDC-Analytical-III apparatus, UV/VIS detector (SPD10A,
Shimadzu); MeCN/H2O 10 : 1 as mobile phase, flow rate 1.0 ml/min. M.p.: Yanaco micro-melting point
apparatus; uncorrected. UV Spectra: Jasco UV-240 spectrophotometer; lmax (log e) in nm. Optical
rotation: Jasco DIP-370 polarimeter; in CHCl3 . IR Spectra: Perkin-Elmer-2000 FT-IR spectrophotometer; ñ in cm 1. 1H-, 13C- and 2D-NMR spectra: Varian-Gemini-200, Varian-Unity-Plus-400, and VarianMercury-400 spectrometers; d in ppm rel. to Me4Si, J in Hz. GC/MS: Trace GC/POLARIS Q Thermo
Finnigan; in m/z (rel. %). EI-MS: VG-Biotech Quatro-5022 mass spectrometer; in m/z (rel. %). ESI- and
HR-ESI-MS: Bruker APEX-II mass spectrometer; in m/z.
Plant Material. The whole plants of S. deltoidea were collected from Wutai, Pingtung County, Taiwan,
in July 2007, and identified by I.-S. C., College of Pharmacy, Kaohsiung Medical University. A voucher
specimen (Chen 1187) has been deposited with the Herbarium of the School of Pharmacy, College of
Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan, R.O.C.
Extraction and Isolation. Whole plants of S. deltoidea (2.7 kg) were sliced and extracted with cold
MeOH (3 30 l, 3 d each) at r.t. The extract was concentrated under reduced pressure and partitioned
with AcOEt/H2O 1 : 1 (v/v) to afford an AcOEt-soluble fraction (270 g) and a H2O-soluble fraction
(100 g).
The AcOEt fraction (100 g) was subjected to CC (SiO2 (2 kg, 230 – 400 mesh); hexane/Me2CO
gradient) to give 14 fractions: Frs. 1 – 14. Fr. 2 (5.7 g) was applied to CC (SiO2 ) (250 g, 230 – 400 mesh);
hexane/CH2Cl2 gradient) to furnish twelve fractions: Frs. 2-1 – 2-12. Fr. 2-4 (742 mg) was subjected to CC
(RP-C18 (10 g); with acetone/MeOH 60 : 1) to yield 18 (5.6 g) and four fractions: Frs. 2-4-1 – 2-4-4. Fr. 24-4 (5 mg) was purified by prep. TLC (SiO2 ; hexane/CHCl3 10 : 1): 19 (2.8 mg). Fr. 2-6 (537 mg) was
applied to CC (SiO2 (250 g, 230 – 400 mesh); hexane/AcOEt gradient) to obtain 13 fractions: Frs. 2-6-1 –
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1453
2-6-13. Fr. 2-6-2 (25.6 mg) was purified by prep. TLC (SiO2 ; hexane/CHCl3/AcOEt 100 : 1 : 1): 5 (6.3 mg),
20 (2.8 mg), a mixture of 20, 21, and 22 (2.5 mg), and a mixture of 23 and 24 (4.6 mg). Fr. 3 (1.4 g) was
applied to CC (SiO2 (100 g, 230 – 400 mesh); hexane/Me2CO gradient) to obtain ten fractions: Frs. 3-1 – 310. Fr. 3-1 (13 mg) was purified by CC (RP-C18 (20 g); acetone/H2O 10 : 1), to obtain 14 fractions: Frs. 31-1 – Fr. 3-1-14. Fr. 3-1-14 (1.2 g) was applied to CC (SiO2 (100 g, 230 – 400 mesh); CH2Cl2/Me2CO
gradient) to obtain 14 fractions: Frs. 3-1-14-1 – 3-1-14-14. Fr. 3-1-14-3 (25 mg) was purified by prep. TLC
(SiO2 ; CH2Cl2/AcOEt 1 : 5): 3 (3.5 mg) and 6 (2.3 mg). Fr. 3-5 (13 mg) was subjected to CC (RP-C18
(10 g); acetone/H2O 10 : 1), to obtain 2 (1.8 mg). Fr. 3-5-3 (13 mg) was subjected to CC (RP-C18 (10 g);
acetone/H2O 10 : 1) to obtain 7 (1.8 mg). Fr. 3-7 (14 mg) was applied to CC (RP-C18; MeOH/H2O 10 : 1)
to obtain five fractions: Frs. 3-7-1 – 3-7-5. Fr. 3-7-2 (3.2 mg) was applied to CC (SiO2 (230 – 400 mesh);
CH2Cl2/Me2CO gradient) to obtain 12. Fr. 4 (4.9 g) was subjected to CC (SiO2 (100 g, 230 – 400 mesh);
hexane/Me2CO gradient) to provide eleven fractions: Frs. 4-1 – 4-11. Fr. 4-4 (51 mg) was subjected to CC
(RP-C18; Me2CO/MeOH 20 : 1) to give eight fractions: Frs. 4-4-1 – 4-4-8. Compound 16 (12.9 mg) was
obtained from Fr. 4-4-4. Fr. 4-4-7 (13 mg) was submitted to CC (SiO2 (230 – 400 mesh); hexane/AcOEt
3 : 1) to afford seven fractions: Frs. 4-4-7-1 – 4-4-7-7. A mixture of 8 and 9 (16.8 mg) was obtained from
Fr. 4-4-7-5. Fr. 5 (675 mg) was subjected to CC (SiO2 (200 g, 230 – 400 mesh); hexane/Me2CO 3 : 1) to
yield eight fractions: Frs. 5-1 – 5-8. Fr. 5-4 (51 mg) was subjected to CC (SiO2 (230 – 400 mesh); hexane/
Me2CO 10 : 1) to afford eight fractions: Frs. 5-4-1 – 5-4-8. A mixture of 10 and 11 (5.6 mg) was obtained
from Fr. 5-4-5. Fr. 9 (743 mg) was subjected to CC (Sephadex LH-20 (50 g); MeOH): Frs. 9-1 – 9-5. Fr. 9-1
(20 mg) was purified by prep. TLC (SiO2 ; hexane/Me2CO 20 : 1): 17 (1.0 mg). Fr. 13 (30.6 g) was
subjected to CC (SiO2 (2000 g, 230 – 400 mesh); CH2Cl2/Me2CO gradient) to afford eight fractions:
Frs. 13-1 – 13-6. Fr. 13-3 (50 mg) was submitted to CC (SiO2 (230 – 400 mesh); hexane/Me2CO 2 : 1) to
obtain seven fractions: Frs. 13-3-1 – 13-3-7. Fr. 13-3-5 was recrystallized from MeOH to furnish 1 (1.9 mg).
Fr. 13 – 9 (1.7 g) was subjected to CC (RP-C18; Me2CO/H2O 1 : 1) to yield eight fractions: Frs. 13-9-1 – 139-8. Fr. 13-9-1 (6 mg) was purified by prep. TLC (SiO2 ; CHCl3/MeOH 10 : 1): 14 (1.4 mg) and 15 (0.8 mg).
Fr. 13-9-2 (149 mg) was subjected to CC (Sephadex LH-20 (50 g); MeOH): Frs. 13-9-2-1 – 13-9-2-9.
Fr. 13-9-2-5 (40 mg) was purified by prep. TLC (SiO2 ; CHCl3/MeOH 10 : 1): 13 (0.4 mg).
Deltoidealactone ( ¼ rel-(3aR,4R,5E,9Z,11aS)-6-Formyl-2,3,3a,4,7,8,11,11a-octahydro-10-methyl-3methylidene-2-oxocyclodeca[b]furan-4-yl 2-(Hydroxymethyl)prop-2-enoate; 1). Colorless needles. M.p.
182 – 1858. [a] 25
D ¼ þ 13.7 (c ¼ 0.038, CHCl3 ). UV (MeOH): 220 (3.35). IR (KBr): 3395 (COOH), 1773
(g-lactone), 1707 (CHO), 1658 (a,b-unsaturated ester C¼O). 1H- and 13C-NMR: see Table 1. ESI-MS:
369 ([M þ Na] þ ). HR-ESI-MS: 403.1882 ([M þ Na] þ , C19H22NaO þ6 ; calc. 369.1314).
Cell-Based Assay for Cytokine Release. The human myeloid leukemic cell line U937 obtained from
the American Type Culture Collection (Rockville, MD) was used in this study. The cells were cultured in
RPMI 1640 medium (Invitrogen) containing 10% fetal calf serum (FCS) at 378 and 5% CO2 , and
maintained in an exponential growth status. For differentiation induction, the cells were incubated in
T150 flask at an initial concentration of 4 l05 cells/ml with 50 ng/ml phorbol 12-myristate 13-acetate
(PMA; Sigma, MO) for 24 h and then transferred to the same medium without PMA for further 48 h.
The cells were collected by gently scraping the flask with a rubber policeman (Bellco Glass, Vineland,
NJ) for the test article assay on inhibition of cytokine secretion. The PMA treated U937 cell suspensions
were plated 1.6 105 cell/well onto 96-well culture plates. The cells were mixed with test article, and
incubated for 30 min at 378, 5% CO2. Lipopolysaccharide (LPS; 200 ng/ml, Sigma, MO) was added into
the assay medium and incubated for another 4 h. The culture media were collected for TNF-a analysis
with commercial assay kit from R&D systems (Minneapolis, MN). Results were calculated with GraFit
analysis and expressed as IC50 (mg/ml).
Cytotoxicity. The cytotoxicity assay was performed by the MTT ( ¼ 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-2H-tetrazolium bromide; Sigma) method; 100 ml of 1 mg/ml MTT was incubated with cells for
1 h at 378. One hundred ml DMSO was added to dissolve the crystals, and OD560 was measured with
ELISA reader (Tecan Spectrafluor plus, Switzerland). Results were expressed as percentage of cell
viability of LPS only stimulated cell.
1454
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
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Received June 18, 2010
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