Isolation of a new cytotoxic polyhydroxysterol from the South China Sea soft coral Sinularia sp.

Abstract A new polyhydroxysterol, (22E)-24-methylenecholestane-22-ene-3β,5α,6β-triol (1), together with four known analogues (2–5) were isolated from the South China Sea soft coral Sinularia sp. Their structures were elucidated by spectroscopic analyses and comparison with previously reported data. All these compounds exhibited cytotoxic effect against both HepG2 and HeLa cell lines with IC50 values ranging from 8.36 to 37.30 μM. Preliminary structure–activity relationship study identified that the presence of double bonds in the side chains of these polyhydroxysterols significantly reduced the biological effect obtained.


Introduction
Soft coral of the genus Sinularia, belonging to the family Alcyoniidae, involves over 100 described species (Jia et al. 2006;Zhang et al. 2015). Previous chemical investigations on 50 species of this genus have led to the discovery of over 500 structurally and biologically interesting compounds, most of which fall into the classes of terpenoids, sterols and polyamines (Lakshmi & Kumar 2009;Liang et al. 2013;Shaaban et al. 2013), demonstrating a broad spectrum of biological activities, such as cytotoxicity (Wang et al. 2009;Sun et al. 2012;Aboutabl et al. 2013;Pejin et al. 2014), antimicrobal (Dmitrenok et al. 2003;Kumar & Lakshmi 2006;Hunt et al. 2012) and anti-inflammatory (Shindo et al. 1992;Radhika et al. 2005) effects. During our course of searching for structurally unique and biologically interesting natural products from the invertebrates inhabited the South China Sea Wang et al. 2010;Zhang et al. 2013;Zhang et al. 2014;Liu et al. 2015), one new (1) and four known polyhydroxysterols (2-5) have been isolated from the soft coral Sinularia sp. which was collected from the Leizhou Island ( Figure 1). Structurally, compound 1, bearing an unusual Δ 22,24 -diene system in the side chain, is a kind of steroid rarely discovered from the soft coral, to the best of our knowledge, only four examples involving sinugrandisterols B, D (Ahmed et al. 2007), nebrosteroids D, e (Huang et al. 2008), have been reported until now. Herein, we report the isolation, and structure elucidation of these metabolites, as well as their cytotoxic effect against both HepG2 and HeLa cell lines.
The isolates were evaluated for cytotoxic effect against both HepG2 and HeLa cell lines. Pleasingly, all polyhydroxysterols tested exhibited cytotoxicity against these two cell lines in spite of having a different level of efficacy (Table 1). Interestingly, compounds 4 and 5, none of them having double bond in the side chain, demonstrated significant potential against both HepG2 and HeLa cell lines, with IC 50 being 12.40/8.82 and 8.36/16.48 μM, respectively. In contrast, compound 1, containing two pairs of double bonds in the side chain, was the least effective in terms of the cytotoxicity against both two cell lines involved (IC 50 37.30/19.32 μM for HepG2/HeLa, respectively), clearly suggesting that the double bond in the side chain significantly influenced the biological effect tested. Compared to 1, both compounds 2 and 3 exhibited almost threefold more cytotoxic against HepG2 cell line and comparable effect against HeLa cell line, revealing that more double bonds in the side chain resulted in less cytotoxic effect. Moreover, 2 and 3 displayed almost the same potential against both two cell lines, as shown by their very close IC 50 values (Table 1), suggesting the position of the double bond in the side chain was not an essential element influencing the bioactivity studied here.

General experimental procedures
Optical rotation was measured on Anton Par MCP-500 polarimeter (AntonPaar, Graz, Austria). uV spectrum was recorded on a Tu-1900 spectrophotometer (Beijing, China). IR spectra were recorded on a nicolet-6700 spectrometer (Thermo, Waltham, American). nMR experiments were measured on a Bruker Avance 300 spectrometer (Bruker, Switzerland) operating at 300 and 75 MHz for 1 H and 13 C, respectively, with TMS as internal standard and chemical shifts reported in ppm. High-resolution mass spectra (HRMS) were performed on a Finnigan-MAT-95XL mass spectrometer (Thermo Quest Finnigan, Bremen, Germany). The semipreparative HPLC was performed on Aglient1200 system (Agilent, PaloAlto, American) equipped with a reversed-phase C18 column (4 μM, 250 × 10 mm, Phenomenex). CC was performed with silica gel (230-400 mesh, Merck). TLC was carried out with aluminium sheet precoated silica gel G 60 . Spots were visualised under uV light or by spraying with 5% H 2 SO 4 in etOH followed by heating.

Animal material
The soft coral Sinularia sp. was collected at Leizhou Peninsula in the South China Sea, Zhanjiang, Guangdong Province, China, in July 2013, and was identified by Dr Rob van Soest (university of Amsterdam, the netherlands). A voucher specimen (2013-02) has been deposited in the Laboratory of Marine natural Products, Department of Chemistry, Jinan university, Guangzhou, P.R. China.

Extraction and isolation
The fresh material of Soft Coral Sinularia sp. was crushed mechanically and extracted with 95% ethanol three times (each time for 15 days) at room temperature. The ethanol extract was filtered and evaporated under reduced pressure to get a black brown residue. The residue was suspended in water and partitioned between ethyl acetate (etOAc) and water. The etOAc extract was concentrated to afford 159 g residue, which was subject to silica gel CC eluted with the gradient solution of Pe (petroleum ether)-etOAc (5-100%) and etOAc-MeOH (50-100%) to give six fractions (Frs.1-6). Fr.3 (23 g) was further purified by silica gel CC using the gradient solution of Pe-etOAc (50-100%) as eluent to obtain three sub-fractions (Frs.3a-3c). Finally, Fr.3b was further purified by semipreparation reversed phase HPLC (82% acetonitrile-H 2 O, flow rate: 2.0 mL/min) to yield 1 (9.5 mg), 2 (88 mg), 3 (6 mg), 4 (48.2 mg) and 5 (5.4 mg).

Cytotoxic assay
The isolated compounds 1-5 were tested for cytotoxicity against human liver carcinoma HepG2 and human cervical carcinoma HeLa cell lines using MTT method (Alley et al. 1988;Chao et al. 2012). In briefly, fresh HepG2 or HeLa cells (1.0 × 10 4 cells/well) were cultured in a 96-well plate and incubated for 12 h. The cells were then treated with the tested compounds (1-5) at various concentrations and then incubated at 37 °C in a 5% CO 2 atmosphere for 48 h. The supernatant was removed, followed by the addition of 25 μL of MTT [2.5 mg. mL −1 in phosphate-buffered saline (PBS)] to each well. After incubation at 37°C for 4 h, 100 μL of dimethyl sulfoxide (DMSO) was added to each well and incubated for another 20 min. The absorbance of each well was then measured at 570 nm using a Thermo scientific Multiskan FC multiplate photometer (Waltham, MA, uSA). The IC 50 values were calculated from the compound concentration-response curves.

Conclusion
In summary, in the course of our searching for bioactive metabolites from marine organisms, one new polyhydroxysterol (1) featuring an unusual Δ 22,24 -diene system in the side chain, along with four known analogues (2-5) were isolated from the South China Sea Sinularia sp. The cytotoxic effect of these polyhydroxysterols (1-5) against the proliferation of HepG2 and HeLa cell lines was evaluated. Interestingly, all these isolates exhibited moderate to significant cytotoxicity against both of the two cell lines employed. Preliminary structureactivity relationship study was discussed, suggesting the presence of double bonds in the side chains of these polyhydroxysterols significantly influenced the biological effect studied. More double bonds in the side chain resulted in less cytotoxicity regardless of the position of the double bond.

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