Anti-Inflammatory Polyoxygenated Steroids
from the Soft Coral Sinularia sp.
Jui-Hsin Su,1;2 Ching-Li Lo,1 Yi Lu,1 Zhi-Hong Wen,1 Chiung-Yao Huang,1 Chang-Feng Dai,3 and Jyh-Horng Sheu1;2
1Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan 2Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan
3Institute of Oceanography, National Taiwan University, Taipei 112, Taiwan
Received June 11, 2008; E-mail: [email protected]
Four new polyhydroxylated steroids (1–4) were isolated from a Formosan soft coral Sinularia sp. The structures of these metabolites were determined on the basis of spectroscopic (IR, MS, and 1D and 2D NMR) analyses. Among these metabolites, 3 and 4 are rarely found marine steroids with a C-9/C-10 double bond. Compounds 1 and 2 have shown significant inhibition of the accumulation of the pro-inflammatory COX-2 protein of LPS-stimulated RAW264.7 macro-phage cells at 10mM.
Previous chemical investigations on the Formosan soft cor-als of the genus Sinularia have afforded several polyoxygenat-ed steroids.1–5 Recently, we have investigated the chemical
constituents of a Taiwanese soft coral Sinularia sp. and have isolated six new sesquiterpenoids.6,7Our continuing study on
the chemical content of this soft coral also has resulted in the isolation of four new polyhydroxylated steroids (1–4) (Chart 1). The structures of the new metabolites were deter-mined on the basis of extensive spectroscopic analysis, includ-ing 2D NMR (1H–1H COSY, HMQC, HMBC, and NOESY)
spectroscopy. Furthermore, at a concentration of 10mM both compounds 1 and 2 demonstrated an ability to inhibit the ac-cumulation of two pro-inflammatory proteins, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells.
The soft coral Sinularia sp. was kept at 20C immediately after collection. The frozen animals were extracted exhaustive-ly with EtOH, and then the concentrated EtOH extract was par-titioned between EtOAc and H2O. The combined
EtOAc-solu-ble fraction was concentrated under reduced pressure and the residue was repeatedly chromatographed to yield metabolites 1–4.
The HR-ESI-MS of 1 revealed a pseudomolecular ion peak at m=z 439.3554 [M + Na]þ, corresponding to the molecular
formula C28H48O2 (calcd for C28H48O2Na m=z 439.3552).
The IR spectrum displayed absorption bands at 3437 and 1655 cm1 for hydroxy group and carbon–carbon double-bond, respectively. The1H NMR spectrum (Table 1) revealed
six methyl signals [
1.04 (s), 0.92 (d, J ¼ 7:0 Hz), 0.86 (d, J ¼ 7:0 Hz), 0.79 (d, J ¼ 7:0 Hz), 0.77 (d, J ¼ 7:0 Hz), and 0.68 (s)], two oxymethine signals [ 3.99 (m) and 3.85 (s)], and an olefinic proton signal at5.60 (d, J ¼ 5:5 Hz). In the13C NMR spectrum (Table 2), compound 1 showed 28 carbon
resonances, with multiplicities determined by DEPT 90 and 135 experiments. The olefinic carbon signals appearing at
CHO H H OH 3 1 HO H H 1 3 5 7 9 10 11 13 14 18 19 17 OH 20 21 22 25 26 27 24 28 4 HO H H H OH 2 HO H H H OH Chart 1.
Table 1. 1H NMR Data for Sterols 1–4a) No. 1 2 3 4 1 3.85 s 3.85 s 4.20 s 4.20 br s 2
: 2.10 m;: 1.74 m : 2.09 m;: 1.74 m : 2.22 m;: 1.77 m : 2.23 m;: 1.78 m 3 3.99 m 3.99 m 4.03 m 4.04 m 4 : 2.38 m;: 2.33 m : 2.39 m;: 2.30 m : 2.40 m;: 2.30 m : 2.40 m;: 2.30 m 6 5.60 br d (5.5)b) 5.60 d (5.5) 5.72 d (5.5) 5.72 d (6.0) 7 2.00 m; 1.60 m 2.01 m; 1.62 m 2.20 m; 1.62 m 2.22 m; 1.62 m 8 1.47 m 1.48 m 2.05 m 2.06 m 9 1.58 m 1.60 m 11 1.48 m 1.49 m 5.60 d (6.0) 5.61 d (6.0) 12 : 1.19 m;: 2.02 m : 1.20 m;: 2.01 m : 2.12 m;: 2.25 m : 2.14 m;: 2.25 m 14 1.06 m 1.06 m 1.31 m 1.32 m 15 1.60 m; 1.06 m 1.62 m; 1.08 m 1.78 m 1.76 m 16 1.87 m; 1.29 m 1.88 m; 1.30 m 1.89 m; 1.32 m 1.92 m; 1.32 m 17 1.11 m 1.14 m 1.21 m 1.23 m 18 0.68 s 0.69 s 0.64 s 0.65 s 19 1.04 s 1.04 s 1.21 s 1.21 s 20 1.39 m 1.41 m 1.42 m 1.46 m 21 0.92 d (7.0) 0.95 d (7.0) 0.92 d (7.0) 0.92 d (6.5) 22 1.41 m; 0.96 m 1.56 m; 1.17 m 1.41 m; 0.95 m 1.57 m; 1.17 m 23 1.39 m; 0.96 m 2.12 m; 1.88 m 1.39 m; 0.96 m 2.14 m; 1.90 m 24 1.21 m 1.22 m 25 1.58 m 2.24 m 1.57 m 2.24 m 26 0.86 d (7.0) 1.02 d (7.0) 0.86 d (6.5) 1.03 d (7.0) 27 0.79 d (7.0) 1.03 d (7.0) 0.79 d (6.5) 1.04 d (7.0) 28 0.77 d (7.0) 4.71 s; 4.66 s 0.78 d (7.0) 4.73 s; 4.67 sa) Spectra recorded at 500 MHz in CDCl3. b) J values (in Hz) parenthese.
Table 2. 13C NMR Data for Sterols 1–4a)
No. 1 2 3 4 1 73.0 (CH)b) 73.0 (CH) 72.8 (CH) 72.8 (CH) 2 38.2 (CH2) 38.2 (CH2) 36.5 (CH2) 36.5 (CH2) 3 66.5 (CH) 66.5 (CH) 67.4 (CH) 67.4 (CH) 4 41.4 (CH2) 41.6 (CH2) 41.5 (CH2) 41.5 (CH2) 5 137.2 (C) 137.2 (C) 135.9 (C) 135.9 (C) 6 125.7 (CH) 125.7 (CH) 124.5 (CH) 124.5 (CH) 7 31.8 (CH2) 31.8 (CH2) 31.2 (CH2) 32.2 (CH2) 8 31.8 (CH) 31.9 (CH) 34.6 (CH) 34.6 (CH) 9 41.7 (CH) 41.7 (CH) 142.2 (C) 142.2 (C) 10 41.6 (C) 41.7 (C) 45.2 (C) 45.2 (C) 11 20.3 (CH2) 20.3 (CH2) 119.3 (CH) 119.3 (CH) 12 39.4 (CH2) 39.5 (CH2) 41.7 (CH2) 41.7 (CH2) 13 42.3 (C) 42.3 (C) 41.1 (C) 41.1 (C) 14 56.6 (CH) 56.6 (CH) 52.8 (CH) 52.8 (CH) 15 24.3 (CH2) 24.3 (CH2) 25.2 (CH2) 25.2 (CH2) 16 28.1 (CH2) 28.2 (CH2) 28.3 (CH2) 28.3 (CH2) 17 56.0 (CH) 56.0 (CH) 56.2 (CH) 56.0 (CH) 18 11.8 (CH3) 11.8 (CH3) 11.4 (CH3) 11.4 (CH3) 19 19.4 (CH3) 19.4 (CH3) 26.7 (CH3) 26.7 (CH3) 20 36.2 (CH) 35.7 (CH) 35.6 (CH) 35.6 (CH) 21 18.9 (CH3) 18.7 (CH3) 18.4 (CH3) 18.4 (CH3) 22 33.7 (CH2) 34.6 (CH2) 33.6 (CH2) 34.5 (CH2) 23 30.6 (CH2) 31.0 (CH2) 30.5 (CH2) 30.9 (CH2) 24 39.0 (CH) 156.9 (C) 39.0 (CH) 156.8 (C) 25 31.4 (CH) 33.8 (CH) 31.5 (CH) 33.8 (CH) 26 20.5 (CH3) 21.9 (CH3) 20.5 (CH3) 21.9 (CH3) 27 17.6 (CH3) 22.0 (CH3) 17.6 (CH3) 22.0 (CH3) 28 15.4 (CH3) 106.0 (CH2) 15.4 (CH3) 106.0 (CH2)
137.2 (C) and 125.7 (CH) corresponded to one trisubstituted double bond. The resonances appearing at
73.0 (CH) and 66.5 (CH) confirmed the presence of two oxymethine carbons. The1H–1H COSY correlations revealed that 1 has three sepa-rated proton sequences. Interpretation of the COSY and HMQC spectra led to the placement of two hydroxy groups at C-1 (73.0) and C-3 (66.5). This assignment was also sup-ported by HMBC cross-peaks of H2-2/C-3 and H3-19/C-1.Detailed analyses of the1H–1H COSY and HMBC correlations (Figure 1 and Table 3) further established the planar structure of 1.
In the NOESY spectrum of 1 (Figure 2), the NOE correla-tions between H-8 and H3-18 and H3-19 as well as between
H3-18 and H-20 indicated that these protons adapt a
-orien-tation. The hydroxy groups at C-1 and C-3 were determined to have the
and orientations, respectively, on the basis of strong NOE interactions of H3-19 ( 1.04, s) with H-1 (3.85, s), H3-19 with H-4
(2.33, m) and H-4 ( 2.38, m)with H-3 (
3.99, m) (Figure 2). Furthermore, the 24S config-uration of 1 was determined by comparison of NMR data with those of yonarasterol B which was isolated from the soft coral Clavularia viridis8as the proton shift of H3-28,
H 0.77, wasfound to be identical with that of yonarasterol B. Also, the carbon shifts of C24–C28are in excellent agreement with those
of yonarasterol B and (24S)-methylcholestanol (vs. those of (24R)-24-methylcholestanol).9On the basis of the above find-ings and other detailed NOE correlations (Figure 2), the struc-ture of 1 was fully established as (24S)-24-methylcholest-5-ene-1
,3-diol.Compound 2 also obtained as a white powder. The HR-ESI-MS (m=z 437.3394, [M + Na]þ) and NMR data of 2 indicated
the molecular formula, C28H46O2. Both the1H and13C NMR
signals of 2 were found to be very closely related to those of compound 1, suggesting the very similar steroidal skeleton. By comparison of NMR data of 2 with those of 1 (Tables 1 and 2), it was found that a methyl proton signal (
H 0.77 d,J ¼ 7:0 Hz) in 1 was replaced by two exomethylene proton signals (
H 4.71 and 4.66, each s) in 2. This was furtherconfirmed by the HMBC correlations (Table 3) from H2-28
to C-23, C-24, and C-25. Thus, the structure of steroid 2 was established as 24-methylenecholest-5-ene-1
,3-diol.Metabolite 3 was obtained as a white powder and exhibited a pseudomolecular ion peak at m=z 437.3399 [M + Na]þ in
the HR-ESI-MS, appropriate for a molecular formula of
Table 3. Protons to Which Long-Range Correlations Were Observed in the HMBC Experiments on Sterols 1–4
Carbon 1 2 3 4 1 H3-19 H3-19 H3-19 H3-19 2 H-4
3 H-2 H-2, H-4 4 H-6 5 H-4, H3-19 H-4, H3-19 H3-19 H-1, H3-19 6 H-4 H-4 8 H-6 H-6 9 H3-19 H3-19 H-12, H3-19 H-12, H3-19 10 H-6, H3-19 H-6, H3-19 H3-19 H3-19 11 H-12 H-12 12 H3-18 H3-18 H3-18 H3-18 13 H3-18 H3-18 H3-18 H3-18 14 H3-18 H-12, H3-18 H3-18 H3-18 17 H3-18, H3-21 H3-18, H3-21 H3-18, H3-21 H3-18, H3-21 20 H3-21 H3-21 H3-21 H3-21 22 H3-21 H3-21, H2-23 H3-21 H3-21 23 H3-28 H2-28 H3-28 H2-28 24 H3-26, H3-27, H3-28 H2-23, H3-26, H3-27 H3-26, H3-27, H3-28 H2-23, H3-26, H3-27 25 H3-26, H3-27, H3-28 H3-26, H3-27, H2-28 H3-26, H3-27, H3-28 H3-26, H3-27, H2-28 26 H3-27 H3-27 H3-27 H3-27 27 H3-26 H3-26 H3-26 H3-26 : 1H−1H COSY : HMBC HO OH 1 3 6 8 9 11 17 18 19 21 25 26 27 4 13 28 14 7Figure 1. Key1H–1H COSY and HMBC correlations for 1.
20 18 19 8 14 17 HO H H H H OH H H H H H H H H 1 3 11 9
C28H46O2. The IR spectrum of 3 was found to exhibit
absorp-tions of a hydroxy (3384 cm1) group. The spectroscopic data
of 3 (IR,1H and13C NMR) were similar to those of 1, except
that the presence of a C-9/C-10 double bond in 3 could be ob-served. This was confirmed by HMBC correlations (Table 3) from H-12
(2.25), H3-19 (1.21), and C-9 (142.2). Onthe basis of above analysis, the structure of 3 was established as (24S)-24-methylcholest-5,9-diene-1
,3-diol.The new metabolite 4 was isolated as a white powder. It’s molecular formula C28H44O2 was established by HR-ESI-MS
(m=z 435.3242, [M + Na]þ). The 13C NMR spectrum of 4
(Table 2) showed the presence of 28 carbons and the chemical shifts (
HandC) of the tetracyclic system of 4 were close tothose of 3 (Tables 1 and 2). The chemical shifts of the side chain from C-20 to C-28 in 4 are nearly identical with those of 2. Thus, the structure of steroid 4 was established as 24-methylenecholest-5,9-diene-1
,3-diol.It is noteworthy to mention that metabolites 3 and 4 are rare-ly found marine steroids with a double bond at C-9/C-10.10,11
The in vitro anti-inflammatory effect of the steroids 1 and 2 was tested as they were isolated in larger quantities relative to 3 and 4. In this assay, the up-regulation of the pro-inflamma-tory iNOS and COX-2 proteins of the LPS-stimulated RAW 264.7 macrophage cells was evaluated using the immunoblot analysis. At a concentration of 10mM, compounds 1 and 2 could reduce the levels of the iNOS to 53:9 4:6% and 45:7 5:6%, respectively, and COX-2 to 35:8 6:0% and 26:4 5:0%, respectively, relative to the control cells stimu-lated with LPS only (Figure 3). The housekeeping protein,
-actin was not changed notably by the presence of 1 and 2 at the concentration of 10mM. Both compounds 1 and 2 at 10mM significantly inhibited the expression of LPS-induced pro-inflammatory proteins, iNOS and COX-2, in macrophage cells. Thus, 1 and 2 were found to be active anti-inflammatory steroids.Experimental
General Experimental Procedures. Melting points were de-termined using a Fisher-Johns melting point apparatus. Optical ro-tations were measured on a Jasco P-1020 polarimeter. IR spectra were recorded on a Jasco FT/IR-4100 infrared spectrophotometer. NMR spectra were recorded on a Varian Unity INOVA 500 FT-NMR at 500 MHz for1H and 125 MHz for13C, respectively,
in CDCl3. LRMS and HRMS were obtained by ESI on a Bruker
APEX II mass spectrometer. Silica gel (Merck, 230–400 mesh) was used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F-254, 0.2 mm) were used for analytical TLC. High-performance liquid chromatography (HPLC) was per-formed on a Hitachi L-7100 apparatus equipped with a Bischoff refractive index detector, or a Hitachi L-7400 UV detector and with a Merck Hibar Si-60 column (250 21 mm2, 7mm).
Animal Material. Sinularia sp. was collected by hand via scuba off the northern east coast of Taiwan, in May 2004, at depths of 15 to 20 m, and stored in a freezer until extraction. A voucher sample (20040516-6) was deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.
Extraction and Isolation. The sliced bodies of the soft coral Sinularia sp. (1.0 kg, wet wt) were exhaustively extracted with EtOH (1 L 5). The organic layer was filtered and concentrated
under vacuum, and the residue of aqueous suspension was parti-tioned between EtOAc and H2O. The solvent-free EtOAc extract
(9.8 g) was subjected to CC on silica gel and eluted with EtOAc in hexane (0–100%, gradient) to yield 14 fractions. Fraction 9, elut-ing with hexane–EtOAc (4:1), was purified on a Sephadex LH-20 column using acetone as the mobile phase to afford two subfrac-tions. Subfraction 2 was separated by normal phase HPLC using hexane–acetone (8:1) to yield 1 (3.0 mg), 2 (3.6 mg), 3 (0.7 mg), and 4 (0.6 mg), respectively.
24S-24-Methylcholest-5-ene-1
,3
-diol (1): White powder; mp 140–142C; ½
25
D ¼ 58 (c 0.3, CHCl3); IR (neat)
max3437, 1655 cm1; 1H NMR (CDCl
3, 500 MHz) and 13C NMR
(CDCl3, 125 MHz), see Tables 1 and 2; ESIMS m=z 439 (80,
[M + Na]þ); HRESIMS m=z 439.3554 (calcd for C
28H48O2Na,
439.3552).
iNOS Protein Expression (%) 0 20 40 60 80 100
*
*
COX-2 Protein Expression (%) 0 20 40 60 80 100
*
*
iNOS
β
-actin
B
A
-a 2b 1 cCOX-2
β
-actin
-a 2b 1 cFigure 3. Effect of compounds 1 and 2 on iNOS and COX-2 protein expression of RAWCOX-264.7 macrophage cells by immunoblot analysis. (A) Immunoblots of iNOS and
-ac-tin; (B) Immunoblots of COX-2 and -actin. The values are mean SEM. (n ¼ 6). Relative intensity of the LPS alone stimulated group was taken as 100%. Under the same experimental condition CAPE (caffeic acid phenyl-ethyl ester, 10mM) reduced the levels of the iNOS and COX-2 to 2:5 3:7% and 67:2 13:4%, respectively. Significantly different from LPS alone stimulated group (P < 0:05). aStimulated with LPS alone, bstimulatedwith LPS in the presence of 2 (10mM), cstimulated with
24-Methylenecholest-5-ene-1
,3
-diol (2): White powder; mp 141–142C; ½
25
D ¼ 66 (c 0.4, CHCl3); IR (neat)
max3437 cm1;1H NMR (CDCl
3, 500 MHz) and 13C NMR (CDCl3,
125 MHz), see Tables 1 and 2; ESIMS m=z 437 (80, [M + Na]þ); HRESIMS m=z 437.3394 (calcd for C
28H46O2Na,
437.3395).
(24S)-24-Methylcholest-5,9-diene-1
,3
-diol (3): White powder; mp 126–127C; ½
25
D ¼ 40 (c 0.5, CHCl3); IR (neat)
max3384, 1653 cm1;1H NMR (CDCl3, 500 MHz) and13C NMR(CDCl3, 125 MHz), see Tables 1 and 2; ESIMS m=z 437 (50,
[M + Na]þ); HRESIMS m=z 437.3393 (calcd for C
28H46O2Na,
437.3393).
24-Methylenecholest-5,9-diene-1
,3
-diol (4): White pow-der; mp 126–128C; ½
25
D ¼ 30 (c 0.3, CHCl3); IR (neat)
max3437, 1653 cm1;1H NMR (CDCl3, 500 MHz) and13C NMR(CDCl3, 125 MHz), see Tables 1 and 2; ESIMS m=z 435 (90,
[M + Na]þ); HRESIMS m=z 435.3242 (calcd for C
28H44O2Na,
435.3239).
In Vitro Anti-Inflammatory Assay. The anti-inflammatory assay was modified from Ho et al.12 and Park et al.13 Murine
RAW 264.7 macrophages were obtained from the American Type Culture Collection (ATCC, No. TIB-71) and cultured in Dulbecco’s modified essential medium (DMEM) containing 10% heat-inactivated fetal bovine serum, at 37C in a humidified
5% CO2–95% air incubator under standard conditions.
Inflamma-tion in macrophages was induced by incubating them for 16 h in a medium containing only LPS (0.01mg mL1; Sigma, USA)
with-out the presence of test compounds. For the anti-inflammatory activity assay, compounds 1 and 2 were added to the cells 5 min before LPS challenge, respectively. Then, cells were washed with ice-cold PBS, lysed in ice-cold lysis buffer, and then centrifuged at 20000g for 30 min at 4C. The supernatant was decanted from
the pellet and retained for Western blot analysis. Protein concen-trations were determined by a DC protein assay kit (Bio-Rad) modified by the method of Lowry et al.14 Samples containing
equal quantities of protein were subjected to SDS-polyacrylamide gel electrophoresis, and the separated proteins were electrophoret-ically transferred to polyvinylidene difluoride membranes (PVDF; Immobilon-P, Millipore, 0.45mm pore size). The resultant PVDF membranes were incubated with blocking solution, and then incu-bated for 180 min at room temperature with antibodies against in-ducible nitric oxide synthase (iNOS; 1:1000 dilution; Transduc-tion Laboratories) and cyclooxygenase-2 (COX-2; 1:1000 dilu-tion; Cayman Chemical) proteins. The blots were detected using ECL detection reagents (Perkin-Elmer, Western Blot Chemilumi-nescence Reagent Plus) according to the manufacturer instructions
and finally exposed to X-ray film (Kodak X-OMAT LS, Kodah, U.S.A.). The membranes were reprobed with a monoclonal mouse anti-
-actin antibody (1:2500, Sigma) as the loading control. For the immunoreactivity data, the intensity of each drug-treated band is expressed as the integrated optical density (IOD) calculated with respect to the average optical density of the corresponding control (treated with LPS only) band. For statistical analysis, all the data were analyzed by a one-way analysis of variance (ANOVA), followed by the Student–Newman–Keuls post hoc test for multiple comparisons. A significant difference was defined as a P value of < 0:05.Financial support was provided by Ministry of Education (No. 96C031702) and National Science Council of Taiwan (No. NSC 95-2113-M-110-011-MY3) awarded to J.-H. Sheu.
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