New Terpenes from the Egyptian Soft Coral Sarcophyton ehrenbergi

Chemical investigations of the Egyptian soft coral Sarcophyton ehrenbergi have led to the isolation of compounds 1–3 as well as the previously reported marine cembranoid diterpene sarcophine (4). Structures were elucidated by comprehensive NMR and HRMS experimentation. Isolated compounds were in vitro assayed for cytotoxic activity against human hepatocarcinoma (HepG2) and breast adenocarcinoma (MCF-7) cell lines.


Introduction
The Red Sea serves as an epicenter for marine bio-diversity, with a high endemic biota. Indeed, of the 180 soft coral species identified world-wide, approximately 40% are native to the Red Sea [1]. Soft coral (Cnidaria: Anthozoa: Octocorallia) are an important structural component of coral reef communities contributing significantly to coral-reef biomass [2,3]. Soft coral have also been the subject of biological studies since the 19th century with coral belonging to the genus Sarcophyton (Alcyoniidae) well recognized as a rich source of terpenoids [4]. For cembranoid-type diterpenes, a wide range of biological activities have been reported including antitumor, ichthyotoxic, anti-inflammatory, neuroprotective, antibacterial, antiangiogenic, antimetastatic, anti-osteoporotic, and cytotoxic properties [5]. Diterpenes have been isolated previously from Sarcophyton ehrenbergi [6][7][8][9][10]; however, to the best of our knowledge, this is the first chemical investigation of the Red Sea soft coral Sarcophyton ehrenbergi (Figure 1). In the course of our research for bioactive substances from marine sources [11,12], chromatographic separation of an ethyl acetate extract of the alcyonacean soft coral, S. ehrenbergi has led to the isolation of three compounds 1-3 as well as the previously reported marine cembranoid diterpene sarcophine (4) (Figure 2). Structures of these isolated metabolites were elucidated by comprehensive NMR and HRMS techniques. Identified compounds were in vitro assayed for cytotoxic activity in two human cancer cell lines.

Results and Discussion
The EtOAc extract of freshly collected S. ehrenbergi was subjected to normal and reverse phase chromatography to afford the isolated metabolites as reported.

Animal Material
Soft coral S. ehrenbergi was collected from the Egyptian Red Sea off the coast of Hurghada in March 2012. The soft coral was identified by co-author Alhammady with a voucher specimen (03RS27) deposited in the National Institute of Oceanography and Fisheries, Marine Biological Station, Hurghada, Egypt.

Extraction and Separation
Frozen soft coral (4 kg, total wet weight) was chopped into small pieces and extracted with ethyl acetate at room temperature (4 L × 5). The combined ethyl acetate extracts were concentrated in vacuo to a brown gum. The dried EtOAc-soluble material (250 g) was subjected to gravity chromatography in a silica gel column (6 × 120 cm) using n-hexane-EtOAc gradient separated into eight fractions.

Cell Culture
Cell-culture material was purchased from Cambrex BioScience (Copenhagen, Denmark) and chemicals from Sigma-Aldrich (St. Louis, MO, USA), except where noted otherwise. Human hepatocarcinoma and breast adenocarcinoma cell lines, HepG2 and MCF-7 respectively, purchased from the American type culture collection (ATCC, Manassas, VA, USA) were used to evaluate the cytotoxic effect of the isolated metabolites. Cells are routinely cultured in Dulbecco's modified eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/mL penicillin G sodium, 100 units/mL streptomycin sulphate and 250 ng/mL amphotericin B. Cells are maintained at sub-confluency in humidified air at 37 °C containing 5% CO 2 . For sub-culturing, monolayer cells are harvested after trypsin/EDTA treatment. Cells for bioassays were assayed at 75% confluence. Test compounds were dissolved in dimethyl sulphoxide (DMSO) and diluted to begin with a concentration of 100 μg/mL and serially diluted to a final concentration of 12.5 μg/mL. Assays were run in triplicate unless noted otherwise.

Anti-Tumor Activity
Compound cytotoxicity against HepG2 and MCF-7 cell lines was assayed via a 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) spectrophotometric cell-viability assay. The MTT assay is based on the ability of active mitochondrial dehydrogenase enzyme of living cells to cleave the tetrazolium ring of the yellow MTT to form dark-blue insoluble formazan crystals that are largely impermeable to cell membranes, resulting in its accumulation within healthy cells. With loss of cell viability, formazan crystals leak into the media. Cell viability is monitored and quantified spectrophotometrically from formazan crystals extracted from the cells [16]. Briefly, cells are diluted in serum-free media to 5.0 × 10 4 cells/well based on turbidity measurements, plated into flat-bottom 96-well microplates, and incubated for 48 h with 20 μL of test sample. After incubation, media is removed and 40 μL MTT (0.5 mg MTT/ ml 0.9% aqueous NaCl) is added into each well and then incubated for an additional 4 h. MTT crystals are solubilized by adding 180 µL of acidified isopropanol to each well and plates are agitated at room temperature, followed by absorbance measurements at 570 nm using a microplate ELISA reader (BMG LABTECH, Ortenberg, Germany). Triplicate repeats were performed for each concentration and the average was calculated. Data were expressed as the percentage of relative viability compared with the untreated cells and with the vehicle control. Cytotoxicity was reported as a relative viability compared with the untreated control. Percent relative viability was determined based cell viability: (Absorbance of treated cells/absorbance of control cells) × 100. Half maximal inhibitory concentrations (IC 50 ) were calculated based on dose-response curves.

Conclusions
Three new (1-3) and one previously reported (4) terpenes were isolated and chemically characterized from the Red Sea soft coral S.ehrenbergi. Identified compounds 1-3 exhibited in vitro cytotoxic activity against a breast adenocarcinoma cell line (MCF-7) with IC 50 values of 192.87, 68.57, and 114.41 µm/mL, respectively.