A new sesquiterpene from the South China Sea gorgonian coral Subergorgia suberosa

Abstract A new sesquiterpene, namely suberosoid (1), was isolated from the South China Sea gorgonian coral Subergorgia suberosa. The chemical structure of 1 was established as an unusual sesquiterpene containing 4-methylenecyclohex-2-enone system, by extensive analyses of NMR spectroscopy and high-resolution mass spectrometry. Suberosoid (1) exhibited cytotoxic effect against HeLa cell lines with IC50 value being 10.6 μM.


Results and discussion
The gorgonian coral S. suberosa was cut into pieces and extracted with 95% ethanol at room temperature. The EtOAc-soluble portion of the extract was purified by column chromatography (CC) on silica gel, octadecyl silane (ODS) repeatedly, and the final purification of compound 1 was achieved by preparative RP-HPLC. The chemical structure of 1 was elucidated by the extensive analyses of NMR spectroscopy and high-resolution mass spectrometry.
The isolate was evaluated for cytotoxic effect against HeLa cell lines. Pleasingly, compound 1 exhibited cytotoxicity with IC 50 being 10.6 μM. The investigation of the mechanism underlying the cytotoxic effect of 1 is going on in our laboratory.

General experimental procedures
Optical rotation was measured on Anton Par MCP-500 polarimeter (AntonPaar, Graz, Austria). UV spectrum was recorded on TU-1900 spectrophotometer (Beijing, China). IR spectrum was recorded on Nicolet-6700 spectrometer (Thermo, Waltham, American). NMR experiments were measured on Bruker Avance 500 spectrometer (Bruker, Switzerland) with TMS as internal standard and chemical shifts being reported in ppm. High-resolution mass spectrum was performed on Finnigan-MAT-95XL mass spectrometer (Thermo Quest Finnigan, Bremen, Germany). The prep-HPLC equipment was composed of a Waters 2545 Binary Gradient module, a Waters 2996 Photo Diode Array (PDA) Detector, and a MassLynx V4.1 workstation. A reversed-phase C18 column (XBridge RP18 Column, 5 μm, 19 × 300 mm) was employed. 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 gorgonian coral S. suberosa was collected at Naozhou Island, South China Sea, in June 2013, and was identified by professor Hui Huang (the South China Sea Institute of Oceanology, Chinese Academy of Sciences). A voucher specimen (2013-06-nz) has been deposited in the Laboratory of Natural Products, School of Pharmaceutical Science, Sun Yat-Sen University, Guangzhou, P.R. China.

Cytotoxic assay
Compound 1 was tested for cytotoxic effect against human cervical carcinoma HeLa cell lines using MTT method (Alley et al.1988). Briefly, freshly prepared HeLa cells (1.0 × 10 4 cells/ well) were cultured in 96-well plate and incubated for 12 h. Then the cells were treated with compound 1 at various concentrations and then incubated at 37 °C in a 5% CO 2 atmosphere for another 48 h. After removing the supernatant, 25 μL of MTT [2.5 mg mL −1 in PBS (phosphate buffered saline)] was added into each well and the mixture was incubated at 37 °C for 4 h, Then 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 value was calculated based on the dose-response curve.

Conclusions
To conclude, in the course of our search for bioactive metabolites from natural resources, one new unusual sesquiterpene, namely suberosoid (1), containing 4-methylenecyclohex-2-enone moiety, was isolated from the South China Sea gorgonian coral S. suberosa. Pleasingly, compound 1, not only demonstrates a rare chemical structure with only two similar sesquiterpenoids reported in the literature, but also exhibits cytotoxic effect against HeLa cell lines, enabling it to be an interesting target to the synthetic organic chemists and pharmacologists.

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