Steroid constituents from the soft coral Sinularia microspiculata

Abstract A methanol extract of the soft coral Sinularia microspiculata revealed five sterols, including two new compounds. Using combined chromatographic and spectroscopic experiments, the new compounds were found to be 7-oxogorgosterol (1) and 16α-hydroxysarcosterol (2). Their structures were determined on the basis of spectroscopic data (1H and 13C NMR, HSQC, HMBC, 1H-1H COSY, NOESY, and FT-ICR-MS) and by comparing obtained results to the values indicated in previous studies. Among the isolated compounds, 3 showed weak cytotoxic effects against HL-60 (IC50  =  89.02  ±  9.93 μM) cell line, whereas 5 was weakly active against HL-60 (IC50  =  82.80  ±  13.65 μM) and SK-Mel2 (IC50  =  72.32  ±  1.30 μM) cell lines.


Introduction
Sinularia soft corals (phylum Cnidaria, class Anthozoa, subclass Octocorallia, order Alcyonacea) are a rich source of steroids and terpenoids [1][2][3]. Steroids are a highly diverse group of metabolically active compounds. The typical sterols have a 3β-hydroxy-∆ 5 -(or ∆ 0 -) cholestane nucleus and a C 8 -C 10 side chain. Marine organisms are of particular interest for research due to their high content of oxysterols, which are involved in a variety of biological activities [3].
In continuation of our ongoing research on the steroid constituents of Vietnamese corals [4,5], we report the isolation and structure elucidation of five sterols (Figure 1), including two new compounds, from the soft coral Sinularia microspiculata.

Results and discussion
Using combined chromatographic separations, two new and three known steroids were isolated from the methanol extract of the soft coral S. microspiculata spectroscopic data (1D, 2D NMR, and MS) and comparison with previously reported values, the known compounds were identified as sarcophytosterol (3) [6], 3β-hydroxypregna-5, 16-dien-20-one (4) [7], and 3β,7α-dihydroxyergosta-5,24(28)-diene (5) [8]. This is the first report of compounds 3-5 from S. microspiculata. Compound 1 was isolated as a white powder. Its molecular formula, C 30 H 48 O 2 , was determined by a quasi-molecular ion peak at m/z 463.3539 [M + Na] + on Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The 13 C NMR spectrum showed 30 carbon signals including 7 methyls, 8 methylenes, 10 methines, and 5 quaternary carbons. The seven methyl groups were identified by signals at δ C 12.  bond at C-5/C-6 and the ketone at C-7. Detailed analysis of other HMBC correlations clearly identified the planar structure of compound 1 ( Figure 2). The proton signals of H-3 at δ H 3.65-3.69 (1H, m, J 1/2 = 11.0 Hz) is indicative for its α-orientation [11] (versus t, J = 2.5 or 3.0 Hz for β-orientation of H-3 without a large J value attributable to an axial-axial coupling [12,13]). This was further confirmed by an agreement of the 13 C NMR chemical shift for C-3 (δ C 70.5) of 1 with that of 3β-hydroxycholest-5-en-7-one at δ C 70.61 [11] which is quite different from that of aragusterol G at δ C 66.4 [14]. Moreover, the 13 C-NMR data for the side chain of 1 were essentially identical to those of crassumsterol [15] and 7β-hydroxygorgosterol [16], suggesting the same configurations in the side chain of these three compounds, which was also assigned by nuclear overhauser effect spectroscopy (NOESY, see Figure 3). Thus, the structure of 1 was determined to be 7-oxogorgosterol.

Biological material
The sample of the soft coral Sinularia microspiculata Tixier-Durivault was collected at Da Den, Quangninh, Vietnam, in April 2014, and identified by Prof. Do Cong Thung. A voucher specimen (No. SM09) was deposited at the Institute of Marine Biochemistry and Institute of Marine Environment and Resources, VAST, Vietnam.

Extraction and isolation
The samples of S. microspiculata were washed, cut into small pieces, dried at 50 °C, and then powdered. The dried powder (2 kg) was extracted three times with methanol under ultrasonic condition. The resulting solutions were filtered, combined, and concentrated under reduced pressure to obtain the methanol residue (M, 200 g). The methanol residue was suspended in water and extracted in turn with n-hexane and dichloromethane resulting in extracts of n-hexane (H, 42 g), dichloromethane (D, 3 g), and an aqueous layer.

Cytotoxic assays
Cytotoxic evaluations were performed by following the previously described protocols [21,22].

Acknowledgments
The authors are grateful to the Institute of Chemistry, VAST, for the provision of the spectroscopic instrument.

Disclosure statement
No potential conflict of interest was reported by the authors.