New 1,4-Dienonesteroids from the Octocoral Dendronephthya sp.

Two new steroids, dendronesterones D (1) and E (2), featuring with 1,4-dienone moiety, along with three known steroids, methyl 3-oxochola-4,22-diene-24-oate (3), 5α,8α-epidioxy-24(S)- methylcholesta-6,22-dien-3β-ol (4), and 5α,8α-epidioxy-24(S)-methylcholesta-6,9(11),22-trien-3β-ol (5), were isolated from an octocoral Dendronephthya sp. The structures of steroids 1 and 2 were elucidated by using spectroscopic methods and steroid 1 was found to exhibit significant in vitro anti-inflammatory activity in lipopolysaccharides (LPS)-induced RAW264.7 macrophage cells by inhibiting the expression of the iNOS protein.

Using an in vitro pro-inflammatory suppression assay, the effects of steroids 1-5 on the release of iNOS and COX-2 protein from LPS-stimulated RAW264.7 macrophage cells were assessed. The results of the in vitro pro-inflammatory suppression assay showed that steroid 1 at 10 µM suppressed the expression of iNOS/β-actin and COX-2/β-actin to 24.2 ± 10.6 and 70.4 ± 11.9%, as compared with LPS alone group ( Figure 5). Compounds 1-5 did not significantly affect the viability of macrophage cells 16 h after treatments.
Using an in vitro pro-inflammatory suppression assay, the effects of steroids 1-5 on the release of iNOS and COX-2 protein from LPS-stimulated RAW264.7 macrophage cells were assessed. The results of the in vitro pro-inflammatory suppression assay showed that steroid 1 at 10 μM suppressed the expression of iNOS/β-actin and COX-2/β-actin to 24. 2 ± 10.6 and 70.4 ± 11.9%, as compared with LPS alone group ( Figure 5). Compounds 1-5 did not significantly affect the viability of macrophage cells 16 h after treatments.  Figure S21). The relative intensity of iNOS/ COX-2 to β-actin bands was normalized to LPS-stimulated group, and cells treated with dexamethasone were used as a positive control. (* p < 0.05, significantly different from the LPSstimulated group). Data are expressed as the mean ± SEM (n = 3 or 4).

Discussion
Dendronephthya spp. have been demonstrated to have a wide structural diversity of interesting steroids that possess various pharmacological properties, specifically in anti-inflammatory activities  Figure S21). The relative intensity of iNOS/ COX-2 to β-actin bands was normalized to LPS-stimulated group, and cells treated with dexamethasone were used as a positive control. (* p < 0.05, significantly different from the LPS-stimulated group). Data are expressed as the mean ± SEM (n = 3 or 4).

Discussion
Dendronephthya spp. have been demonstrated to have a wide structural diversity of interesting steroids that possess various pharmacological properties, specifically in anti-inflammatory activities [13,14]. In our study of Dendronephthya sp., two previously unreported steroids, dendronesterones D (1) and E (2), were isolated together with the previously described marine steroids, methyl 3-oxochola-4,22-dien-24-oate (3), 5α,8α-epidioxy-24(S)-methylcholesta-6,22-dien-3β-ol (4), and 5α, 8α-epidioxy-24(S)-methylcholesta- 6,9(11),22-trien-3β-ol (5). In the present study, the structures of new metabolites 1 and 2 were elucidated by spectroscopic methods and anti-inflammatory activities of steroids 1-5 were assessed using inhibition of pro-inflammatory iNOS and COX-2 release from macrophages. The results indicated that dendronesterone D (1) showed the most potent suppressive effects on iNOS release and steroids 2 and 3 showed more weak suppressive effects on iNOS/β-actin and COX-2/β-actin expression than those of 1. The results suggested that the anti-inflammatory activities of steroids 1-3 were mainly reliant on the functional group at C-11. Furthermore, steroid 5 was found to be inactive in terms of reducing the expression of iNOS/β-actin, indicating that the anti-inflammatory activities of steroids 4 and 5 are dependent on the existence of the carbon-carbon double bond between C-9/11. bromophenol blue, 10% glycerol, and 50 mM Tris-HCl (pH 7.2)) was added to the samples, and the protein lysates (50 µg) loaded onto tricine SDS-polyacrylamide (7% or 10%) gel. After electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; pore size, 0.45 µM; Millipore, Bedford, MA, USA) at 135 mA overnight at 4 • C in transfer buffer (50 mM Tris-HCl, 380 mM glycine, 1% SDS, 20% methanol). The PVDF was incubated overnight at 4 • C with the anti-iNOS, anti-COX-2, or anti-β-actin antibodies. A horseradish peroxidase-conjugated secondary antibody was used for detection. Anti-iNOS (catalog no. 160862) and anti-COX-2 (catalog no. 160106) antibodies were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). The β-actin antibody (catalog no. Actin sigma A5441) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Immunoreactive bands were visualized by enhanced chemiluminescence (ECL kit; Millipore) and the BioChemi Imaging System and relative densitometric quantification was performed using LabWorks v6.2 (UVP, Upland, CA, USA). Bands for iNOS, COX-2, and β-actin antibodies were recognized at~135, 72, and~45 kDa, respectively. The experiment was repeated 3−4 times and data presented as the mean ± standard error of the mean (SEM). For statistical analysis of immunoblot, the integrated optical density of the LPS group was set to 100%, and β-actin was used to verify that equivalent amounts of protein were loaded in each lane. The data was analyzed by analysis of variance (ANOVA) with the Student-Newman-Keuls post hoc test for multiple comparisons. The difference was significant when p was less than 0.05.

Cell Viability
The RAW264.7 macrophage cell viability was determined after treatment with alamar blue (invitrogen, Carlsbad, CA, USA) [18], a 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-2yl)-2,5-diphenyltetrazolium bromide and has been validated as an accurate measure of the survival of RAW264.7 macrophage cells.