Study On The Anti-inflammatory Consititituents From Two Marine Sponge In The South China Sea

The sea, the world’s largest life habitat, covers about 71% of earth surface area.There are great amount of secondary metabolites with unique chemical structure,various potent biological activities and special physiological mechanism in the vast ocean. The structures of the metabolites are different fom those of the compounds extracted from land plants due to the particularity of marine environment, for example, high salinity, high pressure, hypoxia and light, and the close correlation between biological chains. Thus, marine natural products have become one of the most important resources. Soft coral and Marine sponge have increaingly captured the attention of scientists in recent years. In order to find the diversity of structure, potent biological activities and special physiological mechanism in marine secondary metabolites, the chemical consitituents and ant-inflammatory activity of extractives from Sinularia flexibilis had been studied; and the anti-RA mechanism of Stellettin B,which is separated from Jaspis stellifera had also been studied.Repeated column chromatography, including silica gel, TLC and preparative HPLC, was used to investigate the chemical constituents of Sinularia flexibilis and 12 compounds were isolated from Me OH extract of Sinularia flexibilis. By analysis of IR, MS, 1D and 2D NMR spectra, the structures of isolates were identified as follows:sinulaflexiolide L(1), isosinuflexibilin D(2), thioflexibilolide A(3),(-)14-deoxycrassin(4),(1R,13 S,12S,9S,8R,5S,4R)-9-acetoxy-5,8:12,13-diepoxycembr-15(17)-en-16,4-olide(5),Sinulariolide(6),dihydrosinularin(7),sinularin(8),11-dehydrosinulariolide(9),epoxycembrane A(10),diepoxyceane A(11),flexilarin B(12). Compound 1 and 2 are new compounds.We examined anti-inflammatory activities of the several isolates from Sinularia flexibili. In the non-cytotoxic concentrations, Griess method was used to test the suppression effects of LPS-induced NO production in RAW264.7 cells. The results showed that compounds5, 6, 7and 9 had strong suppression effects of NO and their IC50 values were 24.0 μM, 42.2 μM, 6.1 μM and 8.5 μM, respectively.To explore the role of Stellettin B in the pathogenesis of RA, FLS cells from patients were adopted as the object of study. The MTT-based assay was used to evaluate the cytotoxicity of Stellettin B. Flow cytometric was used to determine the effects of Stellettin B on the cell cycle of FLS cells. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of IL-1β, TNF-α, IL-17, MMP-3 andTIMP-1 in the supernatant of LPS-induced FLS cells. The results were shown as below:1. Stellettin B exhibited significant inhibitory effect on FLS cells with an IC50 value of 5.86 μM.2. Cell apoptosis rate was greatly increased after the cells were treated with Stellettin B(1, 5, 10 μM). However, when the concentration reached 50 μM,the necrosis rate was obviously increased. Flow cytometric analysis showed that treatment with Stellettin B(1, 5 μM) against FLS cells induced G2/M-phase arrest.3. After the cells were treated with Stellettin B, the levels of IL-1β and TNF-αexpressed in FLS cells were significantly decreased. However, IL-17 was weakly suppreseed. MMP-3 was markedly reduced. TIMP-1 was significantly increased. MMP-3/TIMP-1 showed the trend of increase with the decease of the concentration.The results of anti-inflammatory activities test showed that Sinularia flexibili contained anti-inflammatory ingredients and compounds5, 6, 7and 9 had strong suppression effects of NO. The anti-inflammatory mechanism of Stellettin B was proved to be related to inhibition of synovial cell proliferation, induction of cell G2/M arrest, inhibition of proinflammatory cytokines such as IL-1β, TNF-α and IL-17 and regulation of MMP-3/TIMP-1 ratio.