Three New Compounds from Aspergillus terreus PT06-2 Grown in a High Salt Medium

To investigate the structurally novel and bioactive natural compounds from marine-derived microorganisms under high salinity, the fungus Aspergillus terreus PT06-2 was isolated from the sediment of the Putian Sea Saltern, Fujian, China. Three new compounds, terremides A (1) and B (2) and terrelactone A (3), along with twelve known compounds (4–15) were isolated and identified from the fermentation broth of A. terreus PT06-2 at 10% salinity. Among these metabolites, compounds 4 and 15 only produced in the 10% salinity culture, were identified as methyl 3,4,5-trimethoxy-2-(2-(nicotinamido) benzamido) benzoate, and (+)-terrein, respectively. The new compounds 1 and 2 exhibited antibacterial activity against Pseudomonas aeruginosa and Enterobacter aerogenes with MIC values of 63.9 and 33.5 μM, respectively. Compounds 5 showed moderate anti-H1N1 activity and lower cytotoxicity with IC50 and CC50 values of and 143.1 and 976.4 μM, respectively.


The Effects of Salt Stress on Production of Secondary Metabolites from Aspergillus terreus PT06-2
Salt-tolerant fungi belong to extremophiles which can survive under the conditions of zero to high salinity. However, using salt-tolerant fungi to produce new secondary metabolites at high salinity was rarely reported [2][3][4][5][6]. In order to investigate the effect of high salt stress on fungal secondary metabolites, A. terreus PT06-2 was cultured at 0%, 3% and 10% salt. The amount of EtOAc extracts of metabolites in 10% salinity was the largest (222 mg vs. 165 and 200 mg of 0% and 3% salinity). The chemical diversities of the metabolites in 10% salinity were increased (Figure 3). Compounds 4 and 15 were not produced by A. terreus PT06-2 when cultivated in 0% and 3% salinity media. The other sole secondary metabolites were a kind of red pigments (Figure 3) whose structures were not identified. Figure 3. HPLC profiles of secondary metabolites from A. terreus PT06-2 cultured in different salt conditions (0%, 3% and 10%, respectively). Peaks circled were red pigments (HPLC eluent: 0-60 min, 5-100% CH 3 OH; flow rate: 1 mL/min).

General Experimental Procedures
Optical rotations were obtained on a JASCO P-1020 digital polarimeter. UV spectra were recorded on a Beckman DU 640 spectrophotometer. IR spectra were obtained on a Nicolet NEXUS 470 spectrophotometer as KBr disks. 1 H NMR, 13 C NMR, and DEPT spectra and 2D-NMR were recorded on a JEOL JNMECP 600 spectrometer using TMS as internal standard, and chemical shifts were recorded as δ values. ESIMS was measured on a Q-TOF Ultima Global GAA076 LC mass spectrometer. Semipreparative HPLC was performed using an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5 μm, 4 mL/min). HPLC was performed using an ODS column (YMC-pack C18, 4.6 × 250 mm, 5 μm, 2 mL/min). TLC and column chromatography (CC) were performed on plates precoated with silica gel GF254 (10-40 μm) and over silica gel (200-300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 (Amersham Biosciences, Sweden), respectively. All the materials for the culture medium of A. terreus PT06-2 was purchased from Qingdao Marine Chemical Factory, Qingdao, China.

Fungal Material
The fungus A. terreus PT06-2 was isolated from sediment (saline 20%), Putian Saltern of Fujian Province of China. It was identified according to its morphological characteristics and analyses of its 18S rRNA sequence (Genbank JN006059) by Prof. C. X. Fang, China Center for Type Culture Collection. A voucher specimen is deposited in our laboratory at −80 °C. The working strain was prepared on potato dextrose agar slants and stored at 4 °C.

Fermentation and Extraction
The fungus A. terreus PT06-2 was grown under static conditions at 28 °C for 35 days in 100 1000-mL conical flasks containing liquid medium (300 mL/flask, pH 7.0) composed of glucose (10 g/L), maltose (20 g/L), mannitol (20 g/L), monosodium glutamate (10 g/L), yeast extract (3 g/L), corn steep liquor (1 g/L), and 10% salt (NaCl 8%, MgSO 4 0.5%, KH 2 PO 4 0.5%, NH 4 Cl 0.5% and KCl 0.5%). The fermented whole broth (30 L) was filtered through cheesecloth to separate the supernatant from the mycelia. The supernatant was concentrated under reduced pressure to about 5 L and then extracted three times with EtOAc to give an EtOAc solution, while the mycelia were extracted three times with acetone. The acetone was removed under reduced pressure to afford a residual aqueous solution. This aqueous solution was extracted three times with EtOAc to give a further EtOAc crude extract. Both EtOAc solutions were combined and concentrated under reduced pressure to give an extract (31.2 g).
The fungus A. terreus PT06-2 was incubated under the same conditions in 0%, 3% and 10% salt. The chemical diversities of the secondary metabolites of EtOAc extract were investigated with HPLC.

Chemical Transformation of 1 into 2
Compound 1 (2 mg, 5 μmol) was dissolved in MeOH (0.5 mL) and stirred for 24 h at room temperature. The reaction mixture was purified by semi-preparative HPLC eluting with 70% CH 3 OH to give compound 2 (1.8 mg, 96% yield) as colorless crystals.

X-ray Crystal Structure of 2
Compound 2 was obtained as colorless monoclinic crystals with molecular formula C 21

Bioassay
Antimicrobial activity against E. aerogenes, P. aeruginosa, S. aureus and C. albicans was evaluated using an agar dilution method [29]. The tested strains were cultivated in LB agar plates for bacteria and in YPD agar plates for C. albicans, at 37 °C. Compounds and positive controls were dissolved in 5% DMSO-H 2 O at different concentrations from 1000 to 62.5 μg/mL and then from 50 to 0.78 μg/mL, using continuous 2-fold dilution. The test solutions (5 μL) were absorbed onto paper disks (5 mm diameter) and placed on the assay plates. After 24 h incubation, zones of inhibition (mm in diameter) were recorded. The minimum inhibitory concentrations were defined as the lowest concentration at which an inhibition zone could be observed.
Cytotoxicity against HL-60 and BEL-7402 cancer cell lines and confluent MDCK cells was assayed by the MTT method [28]. Cell lines were grown in RPMI-1640 supplemented with 10% FBS under a humidified atmosphere of 5% CO 2 and 95% air at 37 °C. Cell suspensions, 200 μL, at a density of 5 × 10 4 cell mL −1 were plated in 96-well microtiter plates and incubated for 24 h. Then, 2 μL of the test solutions (in MeOH) were added to each well and further incubated for 72 h. The MTT solution (20 μL, 5 mg/mL in IPMI-1640 medium) was then added to each well and incubated for 4 h. Old medium containing MTT (150 μL) was then gently replaced by DMSO and pipetted to dissolve any formazan crystals formed. Absorbance was then determined on a SPECTRA MAX PLUS plate reader at 540 nm. The CC 50 was calculated as the compound concentration necessary to reduce cell viability by 50%. Vp-16 (Etoposide) was used as the positive control with IC 50 values of 0.042 and 0.83 μM, respectively.
The antiviral activity against H1N1 was evaluated by the CPE inhibition assay [30,31]. Confluent MDCK cell monolayers were firstly incubated with influenza virus (A/Puerto Rico/8/34 (H1N1), PR/8) at 37 °C for 1 h. After removing the virus dilution, cells were maintained in infecting media (RPMI 1640, 4 μg/mL of trypsin) containing different concentrations of test compounds at 37 °C. After 48 h incubation at 37 °C, cells were fixed with 100 μL of 4% formaldehyde for 20 min at room temperature. After removal of the formaldehyde, the cells were stained with 0.1% crystal violet for 30 min. The plates were washed and dried, and the intensity of crystal violet staining for each well was measured in a microplate reader (Bio-Rad, USA) at 570 nm. The IC 50 was calculated as the compound concentration required inhibiting influenza virus yield at 48 h post-infection by 50%. Ribavirin was used as the positive control with the IC 50 values of 100.8 μM.

Conclusions
In summary, fifteen metabolites including three new compounds were isolated and identified from the fermentation broth of A. terreus PT06-2 under 10% salinity conditions. High salt stress affected the quantity and profile of secondary metabolites. The new compounds 1 and 2, and the sole metabolite 4 produced under high salt stress conditions showed antibacterial activity. The typical metabolite of A. terreus, butyrolactone I (5), showed anti-H1N1 activity with low cytotoxicity, indicating that 5 might be a promising drug candidate for anti-influenza virus.