Flexibilisquinone, a New Anti-Inflammatory Quinone from the Cultured Soft Coral Sinularia flexibilis

A new quinone derivative, flexibilisquinone (1), was isolated from the cultured soft coral Sinularia flexibilis, originally distributed in the waters of Taiwan. The structure of quinone 1 was established by extensive spectroscopic methods, particularly 1D and 2D NMR experiments. In the in vitro anti-inflammatory effects test, quinone 1 was found to significantly inhibit the accumulation of the pro-inflammatory iNOS and COX-2 proteins of the LPS-stimulated RAW264.7 macrophage cells.

The spectral data of 1 were in full agreement with those of a known quinone analogue, sarcophytonone, which was isolated from a Chinese soft coral Sarcophyton crassocaule [24].  (C) relative density of COX-2 immunoblot. The relative intensity of the LPS-stimulated group was taken to be 100%. Band intensities were quantified by densitometry and are indicated as the percent change relative to that of the LPS-stimulated group. Compound 1 and dexamethasone (Dex) significantly inhibited LPS-induced iNOS and COX-2 protein expression in macrophage. The experiment was repeated three times. (* p < 0.05, significantly different from the LPS-stimulated group).

General
Optical rotations were measured with a JascoP1010 digital polarimeter (Japan Spectroscopic Corporation, Tokyo, Japan). Infrared spectra were obtained on a Varian Diglab FTS 1000 FT-IR spectrophotometer (Varian Inc., Palo Alto, CA, USA). UV spectra were recorded on a Hitachi U-3210 UV spectrophotometer (Hitachi Ltd. Tokyo, Japan). NMR spectra were recorded on a Varian Mercury Plus 400 NMR spectrometer (Varian Inc.) at 400 MHz for 1 H and 100 MHz for 13 C in CDCl 3 at 25 °C. ESIMS and HRESIMS data were recorded on Bruker APEX II mass spectrometer (Bruker, Bremen, Germany). Column chromatography was performed on silica gel (230-400 mesh, Merck, Darmstadt, Germany). TLC was carried out on precoated Kieselgel 60 F 254 (0.25 mm, Merck) and spots were visualized by spraying with 10% H 2 SO 4 solution followed by heating. Normal phase HPLC (NP-HPLC) was performed using a system comprised of a Hitachi L-7110 pump (Hitachi Ltd. Tokyo, Japan) and a Rheodyne 7725 injection port (Rheodyne LLC. Rohnert Park, CA, USA). A normal phase column (Supelco Ascentis ® Si Cat #:581515-U, 25 cm × 21.2 mm, 5 µm, Sigma-Aldrich, St. Louis, MO, USA) was used for NP-HPLC.

Animal Material
Specimens of the cultured soft coral Sinularia flexibilis (specimen no. CSC-1) were collected by hand in a 80 ton cultivation tank located in the National Museum of Marine Biology and Aquarium (NMMBA), Taiwan, in July 2006 and stored in a freezer (−20 °C) until extraction. A voucher specimen was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan.

In Vitro Anti-Inflammatory Assay
Murine macrophage (RAW264.7) cell line was purchased from ATCC. In vitro anti-inflammatory activity of compound 1 was measured by examining the inhibition of lipopolysaccharide (LPS)-induced up-regulation of pro-inflammatory iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) proteins expression in macrophage cells using western blotting analysis [26][27][28]. Briefly, inflammation in macrophages was induced by incubating them for 16 h in a medium containing only LPS (10 ng/mL) without compounds. For anti-inflammatory activity assay, compound 1 (1, 5, 10 or 20 µM) or dexamethasone (Dex; 1 µM) were added the cells 10 min before LPS challenge. The cells were then for western blot analysis. The immunoreactivity data are calculated with respect to the average optical density of the corresponding LPS-stimulated group. 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.

Conclusions
Octocorals have been well-recognized as an important source of potential medicinal-use agents. However, because of the corals are claimed to be threatened species and most of the compounds from octocorals are difficult to obtain by chemical methods, bioactive compounds from cultured soft corals will play an important role in this field. Our further studies on the chemical constituents of a cultured soft coral Sinularia flexibilis grown in the culture tanks with a flow-through sea water system located in the National Museum of Marine Biology and Aquarium, Taiwan for the extraction of additional natural products in order to establish a stable supply of bioactive material, have led to the isolation of a new quinone derivative, flexibilisquinone (1), and this compound was found to significantly inhibit the accumulation of the pro-inflammatory iNOS protein of the LPS-stimulated RAW264.7 macrophage cells, suggesting that quinone 1 is worthy of further biomedical investigation.