Isolation and Structure Elucidation of Cembranoids from a Dongsha Atoll Soft Coral Sarcophyton stellatum

Six new polyoxygenated cembrane-based diterpenoids, stellatumolides A–C (1–3), stellatumonins A and B (4 and 5), and stellatumonone (6), were isolated together with ten known related compounds (7–16) from the ethyl acetate (EtOAc) extract of soft coral Sarcophyton stellatum. The structures of the new compounds were established by extensive spectroscopic analyses, including 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and data comparison with related structures. Compounds 8 and 14 were isolated from a natural source for the first time. The isolated metabolites were shown to be not cytotoxic against a limited panel of cancer cells. Compound 9 showed anti-inflammatory activity by reducing the expression of proinflammatory cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) proteins in lipopolysaccharide (LPS)-stimulated mouse leukaemic monocyte macrophage (RAW 264.7) cells.

Stellatumolide C (3) was found to have the same molecular formula (C20H28O4) as compound 2 and the same hydroxyl and unsaturated γ-lactone functionalities on the basis of its HREIMS and IR spectra. The NMR data of 3 (Tables 1 and 2) were found to be mostly identical to those of 2, with the exception that the chemical signal of C-7 was found to be shifted upfield (ΔδC − 2.7 ppm) in comparison with that of 2, and the variational J values of 9.5 and 5.0 Hz at H-7 of 3 relative to that of 2 (7.0 and 3.0 Hz). After interpretation of the 2D NMR spectra, in particular the NOE correlations including that of H3-18/H3-19, compound 3 was established as the 7-epimer of compound 2.   Further detailed examination of 2D NMR correlations (Figure 2) established the gross structure of 2 as 4,8-epoxy-7β-hydroxy-cembra-1(15),2,11-trien-16,2-olide. Careful investigation of NOESY correlations in combination with measuring distances between relevant protons in the MM2 energyminimized model enabled resolution of the relative configuration of compound 2 ( Figure 4). The NOE interactions of H3-18 with H3-19 and H3-20, H3-20 with one of the methylene protons at C-10 (δH 1.82, dd, J = 14.5), H2-10 with H3-19, and H3-19 with H-7 (δH 3.44 dd, J = 7.0, 5.0) revealed that the methyl groups at C-4 and C-8 were on the same face as H-7, and assuming that H3-18 was α-oriented, both C-7 and C-8 were therefore of the S* configuration. Moreover, the NOE correlations displayed by H3-17/H-14, H3-18/H-3, and H3-20/H-10 assigned the geometry of the double bonds at C-1/C-15, C-2/C-3, and C-11/C-12 as Z, Z, and E, respectively. On the basis of the above findings, the structure of stellatumolide B (2) was elucidated.
Cytotoxicities of metabolites 1−16 against the growth of human hepatocellular liver carcinoma (HepG2), human breast cancer (MDA-MB231), and human lung adenocarcinoma (A549) cell lines were screened. None of the metabolites exhibited inhibitory activity against the growth of the tested cancer cells (IC50 > 20 μg/mL).
Furthermore, the in vitro anti-inflammatory activity of (+)-sarcophine (9) on inhibition of the expression of COX-2 and iNOS proteins in the lipopolysaccharide (LPS)-stimulated mouse leukaemic monocyte macrophage cell line (RAW 264.7) was further evaluated, due to the sufficient quantity of 9 (104.3 mg) isolated from this investigation. The results showed that 9 could effectively inhibit the LPS-induced expression of iNOS protein at 50 and 100 μM. Compound 9 also could significantly inhibit the expression of COX-2 at 25−100 μM (Figure 8).
Cytotoxicities of metabolites 1-16 against the growth of human hepatocellular liver carcinoma (HepG2), human breast cancer (MDA-MB231), and human lung adenocarcinoma (A549) cell lines were screened. None of the metabolites exhibited inhibitory activity against the growth of the tested cancer cells (IC 50 > 20 µg/mL).
Furthermore, the in vitro anti-inflammatory activity of (+)-sarcophine (9) on inhibition of the expression of COX-2 and iNOS proteins in the lipopolysaccharide (LPS)-stimulated mouse leukaemic monocyte macrophage cell line (RAW 264.7) was further evaluated, due to the sufficient quantity of 9 (104.3 mg) isolated from this investigation. The results showed that 9 could effectively inhibit the LPS-induced expression of iNOS protein at 50 and 100 µM. Compound 9 also could significantly inhibit the expression of COX-2 at 25-100 µM (Figure 8).

Animal Material
The soft coral Sarcophyton stellatum Kukenthal (Alcyoniidea) was collected by hand via self-contained underwater breathing apparatus (SCUBA) at a depth of 10-15 m along the coast of Dongsha Atoll, Taiwan, and stored in at −20 •

In Vitro Anti-Inflammatory Assay
RAW264.7 cells were purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cell were cultured in DMEM, supplemented with RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin G, and 100 µg/mL streptomycin (Gibco/BRL, Gran Island, NY, USA), at 37 • C in an incubator with 5% CO 2 . RAW 264.7 cells were seeded onto a 6-well plate with 2 × 10 6 cell per well and cultured for 24 h. The cells were pretreated with the compound 9 for 1 h and treated with lipopolysaccharide (LPS, 1 µg/mL) from Escherichia coli 055:B5 (Sigma-Aldrich, St. Louis, MO, USA) in the presence or absence of the compound 9 (25, 50, and 100 µM). After 24 h LPS treatment, the cell lysates were prepared using Cell Lysis Buffer (Cell Signalling Technology, Beverly, MA, USA). The expression of COX-2 and iNOS proteins was measured using Western blotting analysis.

Western Blotting Analysis
Protein concentrations were measured using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). Protein extracts were boiled, loaded into sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and electrotransferred to polyvinylidene fluoride (PVDF) membranes. After blocking in 5% nonfat milk in TBST buffer (20 mM Tris-HCl, 120 mM NaCl, and 0.1% Tween 20) for 1 h, the membranes were incubated with antibodies for COX-2 (#4842), iNOS (#13120), and β-actin (#3700) (all purchased from Cell Signaling Technology, Danvers, MA, USA) at 4 • C with gentle agitation overnight. The membranes were then washed and incubated with horseradish peroxidase-labelled rabbit and mouse secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) for 2 h at room temperature. After successive washes, the membranes were developed with an enhanced chemoluminescence (ECL) kit (Amersham Biosciences, Buckinghamshire, UK), and blots were visualized using a LAS3000 system (Fujifilm, Tokyo, Japan). Densitometric analysis was performed with ImageJ software (National Institute of Health, Bethesda, MD, USA).

Statistical Analysis
The data are expressed as the mean ± SD. One-way ANOVA followed by Tukey's post-hoc test (Graphpad Prism 5.0, GraphPad Software, San Diego, CA, USA) was used to compare multiple groups according to the experiments. p values < 0.05 were considered statistically significant.