New Anti-Inflammatory Cembranes from the Cultured Soft Coral Nephthea columnaris

Two new cembranes, columnariols A (1) and B (2), were isolated from the cultured soft coral Nephthea columnaris. The structures of cembranes 1 and 2 were elucidated by spectroscopic methods. In the anti-inflammatory effects test, cembranes 1 and 2 were found to significantly inhibit the accumulation of the pro-inflammatory iNOS and COX-2 protein of the lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Compound 1 exhibited moderate cytotoxicity toward LNCaP cells with an IC50 value of 9.80 μg/mL.


Introduction
Among the natural products isolated from soft corals, the cembranes are major representative compounds. Previous investigations of soft corals belonging to the genus Nephthea (family Nephtheidae), collected off the waters of Taiwan, have yielded several interesting cembranes [1,2]. In our continuing studies, a sample originally collected off the coast of Southern Taiwan, identified as Nephthea columnaris (Studer, 1895), yielded two new cembranes, columnariols A (1) and B (2) (Scheme 1). In this paper, we deal with the isolation, structure determination and bioactivity of cembranes 1 and 2.
In the NOESY experiment of 2 ( Figure 2), H-1 correlated with H-3, but not with H-2, and large coupling constants were recorded between H-1/H-2 (J = 10.0 Hz) and H-2/H-3 (J = 9.2 Hz) and indicating that the dihedral angles between H-1/H-2 and H-2/H-3 are approximately 180° and the configurations of both chiral carbons C-1 and C-2 were assigned as R*. H-11 correlated with H3-18, but not with H2-20 confirming the S*-configuration of C-11 by modeling analysis. Furthermore, no correlation was found between H-3/H3-18 and H-7/H3-19, indicating that C-3/4 and C-7/8 carbon-carbon double bonds had an E-configuration.   In the in vitro anti-inflammatory activity test, the upregulation of the pro-inflammatory iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) protein expression of LPS (lipopolysaccharide)-stimulated RAW264.7 macrophage cells was evaluated using immunoblot analysis. At a concentration of 50 μM, compounds 1 and 2 were found to significantly reduce the levels of iNOS and COX-2, respectively, relative to the control cells stimulated with LPS only (Figure 3). To evaluate the cytotoxic effects of compounds 1 and 2 on the viability of RAW264.7 macrophage cells, we used the alamar blue assay. Both compounds 1 and 2 (20 and 50 μM) did not significantly affect the viability of macrophage cells 16h after treatment. Thus, compounds 1 and 2 might be promising as anti-inflammatory agents, as they do not exhibit cytotoxicity to RAW264.7 macrophage cells.  Cytotoxicity of cembranes 1 and 2 toward MOLT-4 (human acute lymphoblastic leukemia), SUP-T1 (human T-cell lymphoblastic lymphoma), DLD-1 (human colorectal adenocarcinoma), LNCaP (human prostatic carcinoma) and MCF7 (human breast adenocarcinoma) tumor cells were studied, and the results are shown in Table 3. LNCaP cell line was more sensitive to the cytotoxic effects of 1.

General Experimental Procedures
Optical rotation values were measured with a Jasco P-1010 digital polarimeter (Japan Spectroscopic Corporation, Tokyo, Japan). IR spectra were obtained on a Varian Digilab FTS 1000 FT-IR spectrophotometer (Varian Inc., Palo Alto, CA, USA); absorptions are reported in cm −1 . NMR spectra were recorded on a Varian Mercury Plus 400 NMR spectrometer (Varian Inc., Palo Alto, CA, USA) using the residual solvent (CDCl3, δH 7.26 ppm for 1 H NMR and δC 77.1 ppm for 13

Animal Material
Specimens of the octocoral Nephthea columnaris (Studer, 1895) were collected originally by hand using SCUBA equipment off the coast of the Southern Taiwan, and transplanted to five 0.6-ton cultivating tanks equipped with a flow-through sea water system in February 2012. The cultured octocorals for this research work were collected from the tanks in May 2013. Prof. Chang-Feng Dai, Institute of Oceanography, National Taiwan, identified the soft coral. Living reference specimens are being maintained in the authors' marine organism cultivating tank and a voucher specimen (NMMBA-TWSC-12005) was deposited in the National Museum of Marine Biology and Aquarium, Taiwan.

Extraction and Isolation
Sliced bodies of Nephthea columnaris (wet weight 800.0 g, dry weight 76.6 g) were extracted with a mixture of MeOH and DCM (1:1) (1.6 L × 5). The extract was partitioned between EtOAc and H2O. The EtOAc layer (7.4 g) was separated on silica gel and eluted using n-hexane/EtOAc (stepwise, 100:1-pure EtOAc) to yield 17 fractions A-Q. Fraction J was chromatographed on NP-HPLC using a mixture of n-hexane and acetone (2:1) to afford 14 fractions J1-J14. Fraction J3 was separated by NP-HPLC using a mixture of n-hexane and acetone (2:1) as the mobile phase to yield 6 fractions J3A-J3F. Fraction J3B was purified by RP-HPLC using a mixture of acetonitrile and water (1:1) as the mobile phase to afford 1 (2.3 mg, tR = 22 min). Fraction J5 was purified by RP-HPLC using a mixture of MeOH and water (3:2) as the mobile phase to afford 2 (2.0 mg, tR = 22 min).

In Vitro Anti-Inflammatory Assay
According to our previous and other studies for the in vitro anti-inflammatory activity assay, we used LPS induced RAW murine macrophage cell line which was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) [4][5][6][7]. The in vitro anti-inflammatory activity of Compounds 1 and 2 was measured by examining the inhibition of lipopolysaccharide (LPS)-induced upregulation of pro-inflammatory iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) protein expression in macrophage cells using Western blotting analysis [7][8][9]. Briefly, inflammation in macrophages was induced by incubating them for 16 h in a medium containing only LPS (10 ng/mL) without compounds. For the anti-inflammatory activity assay, Compounds 1, 2 and dexamethasone (10 μM) were added to the cells 10 min before the LPS challenge. The cells were then for western blot analysis. The immunoreactivity data were calculated with respect to the average optical density of the corresponding LPS-stimulated group. RAW264.7 macrophage cells viability was determined after treatment with alamar blue (Invitrogen, Carlsbad, CA, USA), the tetrazolium dye that is reduced by living cells to fluorescent products. This assay is similar in principle to the cell viability assay using 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide and has been validated as an accurate measure of the survival of RAW264.7 macrophage cells [10,11]. For statistical analysis, 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.

MTT Antiproliferative Assay
Based on our previous and other studies from the cytotoxicity, there are different tumor cell lines (include MOLT-4, SUP-T1, DLD-1, LNCaP and MCF7 cells) which were obtained from ATCC [12][13][14][15][16]. Cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine and antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin) at 37 °C in a humidified atmosphere of 5% CO2. Cells were seeded at 4 × 10 4 per well in 96-well culture plates before treatment with different concentrations of the tested compounds. In cell culture experiments, compounds 1 and 2 were dissolved in 100% dimethyl sulfoxide (DMSO) (clear). The final concentration of DMSO in the culture medium was 0.02% and the compounds were made concentrations of 1.25, 2.5, 5, 10 and 20 μg/μL prior to the experiments. After treatment for 72 h, the cytotoxicity of the tested compounds was determined using a MTT cell proliferation assay (thiazolyl blue tetrazolium bromide, Sigma-M2128). The MTT is reduced by the mitochondrial dehydrogenases of viable cells to a purple formazan product. The MTT-formazan product was dissolved in DMSO. Light absorbance values (OD = OD570 − OD620) were recorded at wavelengths of 570 and 620 nm using an ELISA reader (Anthos Labtec Instrument, Salzburg, Austria) to calculate the concentration that caused 50% inhibition (IC50), i.e., the cell concentration at which the light absorbance value of the experiment group was half that of the control group. These results were expressed a percentage of the control ± SD established from n = 4 wells per one experiment from three separate experiments.

Conclusions
Octocorals have been well-recognized as an important source of potential bioactive marine natural products. However, because of the octocorals are claimed to be threatened species and most of the compounds from octocorals are difficult to obtain by chemical methods at this stage, bioactive substances from cultured marine invertebrates will play an important role in this field. Our studies on Nephthea columnaris for the extraction of additional natural substances, have led to the isolation of two new cembranes, colummnariols A (1) and B (2). Compounds 1 and 2 are potentially anti-inflammatory and may become lead compounds in future marine anti-inflammation drug development [17,18]. To the best of our knowledge, this is the first time to study the natural products from N. columnaris. These results suggest that continuing investigation of novel secondary metabolites together with the potentially useful bioactivities from this marine organism are worthwhile for future drug development. The octocoral Nephthea columnaris will be transplanted to culturing tanks located in the National Museum of Marine Biology and Aquarium, Taiwan, for extraction of additional natural products to establish a stable supply of bioactive material [19].