The Anthraquinone Derivatives from the Fungus Alternaria sp. XZSBG-1 from the Saline Lake in Bange, Tibet, China

Four new anthraquinone derivatives 1–4 were obtained along with seven known compounds 5–11 from the extracts of the fungal strain Alternaria sp. XZSBG-1 which was isolated from the sediments of the carbonate saline lake in Bange, Tibet, China. Their structures were determined by spectroscopic methods, mainly by 2D NMR spectra. Compound 1 is a novel tetrahydroanthraquinone with an epoxy ether bond between C-4a and C-9a. In the primary bioassays, compound 3 (alterporriol T) exhibited inhibition of a-glucosidase with a IC50 value 7.2 μM, and compound 9 showed good inhibitory activity against the HCT-116 and HeLa cell lines, with IC50 values of 3.03 and 8.09 μM, respectively.


Introduction
In 2002, we reported research on the metabolites of a fungal strain from a saline lake locates in the Bahamas [1]. Since that time, it seems that that little research on fungal metabolites from saline lakes has been published. Recently, we have been interested in the microorganisms from Tibetan saline lakes, which live under the special plateau habitat conditions, including low temperatures and high salt levels that are similar to those of the ocean, and we have thus obtained some unique and significant compounds.
Compounds 3 and 4 also contain a chiral axis. Comparing the CD spectra ( Figure 6) with that of the the known compound alterporriol N, because the CD spectra were less sensitive to the configuration of the four chiral centres [10,16], according to the trends of CD between compound 3 and alternporriol N, we suggest that the absolute configuration of the chirality axis for compound 3 can be assigned as aS.
The compounds 5-11 were identified by comparing their spectroscopic data with those of the corresponding known compounds.

General Procedures
Column chromatography (CC) was performed using silica gel (200-300 mesh, Qingdao Marine Chemical, Qingdao, China). The HPLC system consisted of a Waters 2010 series (Waters, Milford, MA, USA). A mini ODS column (250 × 10 mm, 10 μm particle size) was used. Melting points were determined on an X-4 micro-melting point apparatus and were uncorrected. Circular dichroism was measured on a Schmidt Haensch Polartronic HH W5 polarimeter (Schmidt, Germany) and was uncorrected. UV spectra were measured on a Shimadzu UV-3501 PC spectrophotometer (Shimadzu, Japan). 1 H (400 MHz) and 13 C-NMR (100 MHz) data were recorded on a Bruker AVANCE 400 spectrometer (Bruker, Switzerland) with TMS as internal standard. LC/MS data were acquired using an Applied Biosystems/MDS Sciex (Applied Biosystems, Grand Island, NY, USA) and ana ESI source. HR-ESIMS were measured on a Shimadzu LCMS-IT-TOF.

Fungal Material
The fungal strain Alternaria sp. XZSBG-1 was isolated from a piece of sediment involving rotten branches and leaves, which was collected from the saline carbonate lake of Bamucuo, in Bange county, Tibetan Autonomous Region, China in July 2007. The strain was deposited in the Guangdong Province Key Laboratory of Functional Molecules in Oceanic Microorganism, School of Chemistry and Chemical Engineering, Sun Yat-sen University of China, Guangzhou, China.

Identification of Fungal Cultures
The fungal strain was identified as an Alternaria sp. according to morphologic traits and a molecular biological protocol by DNA amplification and sequencing as described [12,20]. The sequence data have been submitted to GenBank, accession number HM622756, and the strain was identified as Alternaria sp. Its 591 base pair ITS sequence had 99% sequence identity to that of Alternaria sp. IA249 (AY154699).

Fermentation, Extraction, and Isolation of Alternaria sp. XZSBG-1
The fungal strain was cultivated in potato glucose liquid medium (15 g of glucose and 3 g of crude sea salt in 1 L of potato infusion) in 1 L Erlenmeyer flasks each containing 400 mL of culture broth at 25 °C without shaking for 4 weeks. The fermentation broth (80 L) was filtered. The culture broth was extracted three times with an equal volume of EtOAc. The combined EtOAc layers were evaporated to dryness under reduced pressure to give an EtOAc extract (25.2 g). The mycelia was extracted with MeOH three times. The MeOH layer was evaporated under vacuum; then the combined residue was suspended in H2O and partitioned with EtOAc to provide the EtOAc extract (28.0 g), all the crude extract combined together (53.2 g) was subjected to silica gel column chromatography (CC, petroleum ether, EtOAc v/v, gradient) to generate eight fractions (Fraction 1−8).  Table 3; 13 C-NMR (DMSO-d6), see Table 3 Table 3; 13 C-NMR (DMSO-d6), see Table 3 Table 2; 13 C-NMR (DMSO-d6), see Table 2 Table 3; 13 C-NMR (DMSO-d6), see Table 3