Identification and Bioactivity of Compounds from the Mangrove Endophytic Fungus Alternaria sp.

Racemic new cyclohexenone and cyclopentenone derivatives, (±)-(4R*,5S*,6S*)-3-amino-4,5,6-trihydroxy-2-methoxy-5-methyl-2-cyclohexen-1-one (1) and (±)-(4S*,5S*)-2,4,5-trihydroxy-3-methoxy-4-methoxycarbonyl-5-methyl-2-cyclopenten-1-one (2), and two new xanthone derivatives 4-chloro-1,5-dihydroxy-3-hydroxymethyl-6-methoxycarbonyl-xanthen-9-one (3) and 2,8-dimethoxy-1,6-dimethoxycarbonyl-xanthen-9-one (4), along with one known compound, fischexanthone (5), were isolated from the culture of the mangrove endophytic fungus Alternaria sp. R6. The structures of these compounds were elucidated by analysis of their MS (Mass), one and two dimensional NMR (nuclear magnetic resonance) spectroscopic data. Compounds 1 and 2 exhibited potent ABTS [2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)] scavenging activities with EC50 values of 8.19 ± 0.15 and 16.09 ± 0.01 μM, respectively. In comparison to Triadimefon, compounds 2 and 3 exhibited inhibitory activities against Fusarium graminearum with minimal inhibitory concentration (MIC) values of 215.52 and 107.14 μM, respectively, and compound 3 exhibited antifungal activity against Calletotrichum musae with MIC value of 214.29 μM.


Introduction
Marine microbes are recognized as an abundant source of biologically active natural products with structural diversity [1]. Among them, mangrove fungi constitute the second largest ecological group of marine fungi and produce a large number of chemicals with novel functions and structures [2]. Alternaria is a genus of ascomycete fungi with a widespread distribution, several species of which are known as major plant pathogens that may cause severe agricultural spoilage [3]. Chemical and bioactive investigations to Alternaria sp. from marine environments have led to several novel compounds with antimicrobial [4][5][6], cytotoxic [7][8][9], and MptpB (Mycobacterium tuberculosis protein tyrosine phosphatase B) inhibitory activities [10].
More detailed experimental information for Chiral HPLC analysis of Compounds 1 and 2 are shown in Figures S37 and S38 at the Supplementary Information. Natural products are usually biosynthesized in one enantiomer form. Enantiomerically opposite products are less than 1% relative to the overall abundance of natural products, which often result from the action of stereochemically distinct enzymes that can give single and opposite enantiomeric products from achiral substrates [20]. For examples, (±)-tylopilusin A and (±)-tylopilusin B were two naturalfully substituted 2-cyclopenten-1-one racemates produced by one kind of microorganism, with similar chiral centers as compound 2.
The structures, key HMBC and NOESY correlations of compounds 1-4 are shown in Figures 1 and 2, respectively. More detailed spectra are available at the Supplementary Information (Figures S1-S34).

Biological Activity
All of the new compounds were tested for ABTS radical scavenging activity. The results are showed in Table 3. Compounds 1 and 2 exhibited higher ABTS scavenging activities with EC50 values of 8.19 ± 0.15 and 16.09 ± 0.01 μM, respectively, than ascorbic acid (EC50 17.14 ± 0.11 μM), which was used as the positive control, while compounds 3 and 4 did not exhibit antioxidant activities (EC50 > 500 μM) in this test. According to the data, compounds 1 and 2 both have marked ABTS radical scavenging activities. The ABTS radical scavenging activity curves of compounds 1 and 2 are available at the Supplementary Information (Figure S39). The antimicrobial activity of compounds 1-3 and 5 were evaluated in vitro. The results of their antibacterial and antifungal activities are summarized in Table 4. All the tested compounds exhibited antifungal activities against Fusarium graminearum with MIC values of 107.14-862.07 μM, in comparison to the positive control-Triadimefon (MIC 510.64 μM). Among them, the activities of compounds 2, 3, and 5 were higher than the positive control with MIC values of 215.52, 107.14 and 474.68 μM, respectively. Compound 3 also exhibited more potent antifungal activity against Calletotrichum musae with MIC value of 214.29 μM than Triadimefon (MIC 340.43 μM), while compounds 2 and 5 only showed moderate activities with MIC values of 862.07 and 474.68 μM, respectively. Compound 1 was inactive towards Calletotrichum musae (MIC > 1970.44 μM). Compound 3 bears a -Cl group at C-4 and exhibits greater antifungal activity against Fusarium graminearum and Calletotrichum musae than compound 5 with no substitute at C-4, suggesting that the -Cl group at C-4 is the key reason to increase the antifungal activity.
Additionally, compounds 1, 2 and 5 displayed no activities neither against Gram positive bacterium Staphyloccocus aureus nor against Gram negative bacterium Escherichia coli with MIC value ≥1265.82 μM.

General Experimental Procedures
IR spectra were obtained on a Nicolet 5DX-Fourier transform infrared spectrophotometer (Thermo Electron Corporation, Madison, WI, USA). All NMR experiments were recorded on a Bruker AVIII 600 MHz NMR spectrometer (Bruker BioSpin GmbH company, Rheinstetten, Germany), using deuterated dimethyl sulfoxide or chloroform as solvent and the residual solvent resonance as internal standard, and coupling constants (J) are in Hz. HR-ESI-MS were performed on LCMS-IT-TOF (Shimadzu, Janpan) mass spectrometer. Chromatography was carried out on a silica gel column (200-300 mesh; Qingdao haiyang chemicals Co., Ltd., Qingdao, China). CD data were measured on a Chirascan™ CD spectrometer (Applied Photophysics, London, UK). Optical rotations were measured on a Jasco P-1020 digital polarimeter. Chiral HPLC analyses were carried out using an Agilent 1100 liquid chromatograph equipped with a single pump, a UV-detector and a normal phase S-Chiral B cellulose-based column (Acchrom Technologies Co., Ltd., CHN, 5 μm, 150 × 4.6 mm) at a flow rate of 1.0 mL/min. All other reagents used were analytical grade.

Fungal Material and Fermentation
The strain of Alternaria sp. (collection No. R6) was isolated from the root of a marine semi-mangrove plant Myoporum bontioides A. Gray collected from the mangrove in Leizhou peninsula, Guangdong Province, China. Its species was identified by compared with the morphological characteristics with that of Alternaria sp. [9]. This fungus is stored in College of Materials and Energy (Formerly College of Science), South China Agricultural University, Guangzhou, China. The small agar slices bearing mycelia were cut from stock cultures and transferred aseptically to a 500 mL Erlenmeyer flask containing 250 mL of GYT medium (1% glucose, 0.1% yeast extract, 0.2% peptone, 0.2% crude sea salt), and incubated at 28 °C, 120 rpm for 6 days as seed culture. The fermentation was performed using 1 L Erlenmeyer flasks, each containing a rice medium (100 mL water, 100 g rice, 0.3 g crude sea salt). One milliliter of seed culture was added into each flask and incubated at room temperature for 30 days under static conditions.

Extraction and Isolation
The fermented rice substrate (75 flasks) was extracted repeatedly with methanol (4 × 100 mL for each flask) and solvent was combined and filtered through cheesecloth. The filtrate was concentrated to 5 L in vacuo below 50 °C and extracted five times using an equal volume of ethyl acetate and n-butyl alcohol in sequence, respectively. The combined ethyl acetate extract (48.

The ABTS Radical Scavenging Activity Assay
The scavenging activity of ABTS radical was measured as described in previous literature [22] with minor modifications. The ABTS radical stock solution was preparred by reacting ABTS diammonium salt solution (7 mM) and potassium persulphate solution (2.45 mM) at room temperature in the dark for 12-16 h before use. The solution was diluted with ethanol until the absorbance at 734 nm reaching 0.7 ± 0.02 units. Three milliliters of diluted ABTS radical working solution was added to 1 mL of ethanolic solution of the test compound whose concentrations ranged from 3.13 to 200 μg/mL, and the final concentrations of the test compound in the mixed solution were 0.78-50 μg/mL in capped test tubes. The mixture was shaken vigorously and incubated at 30 °C in a water bath for 60 min in the dark. The absorbance was measured at 734 nm. Ascorbic acid was taken as a positive control. The scavenging ability was calculated as: ABTS radical scavenging activity (%) = [1 − (A1 − A2)/A0] × 100, where A0 is the absorbance in the lack of the test compound, A1 is the absorbance in the presence of the test compound and ABTS, and A2 is the absorbance in the lack of ABTS. The EC50 values (the concentration of the antioxidant required to scavenge 50% of ABTS present in the mixed solution) were obtained from liner regression analysis of the concentration-response curve plotted for each tested compound. In case the scavenging rate was found below 50% at the maximum tested concentration (50 μg/mL in the mixed solution), the test concentration would be directly increased to 500 μM to check whether the scavenging rate was still below 50%. If the scavenging rate was below 50% at 500 μM, it came to conclusion that EC50 was higher than 500 μM. Otherwise, further dilution would be made to acquire accurate value of the EC50.

Antimicrobial Activity Assay
Testing of antimicrobial activities of compounds was performed through determination of minimal inhibitory concentration (MIC) by the broth tube dilution method [23] in relation to fungi (Fusarium graminearum, Calletotrichum musae), and bacteria (Escherichia coil, Staphyloccocus aureus). Stock solution of compound was prepared in 5% (v/v) DMSO aqueous solution. Half a milliliter of the solution was further diluted with 0.5 mL of broth medium, and the final sample concentrations in the mixtures were 400, 200, 100, 50, 25, 12.5 and 6.25 μg/mL in capped test tubes. The tested microorganism taken from the culture medium was immediately diluted with sterilized water, and the final concentration of the microorganism was approximately 1 × 10 5 c.f.u/mL in water. Ten microliters of microorganic suspension was added into each tube, and then bacteria were incubated at 37 °C for 24 h, fungi were incubated at 28 °C for 48 h. The MIC was defined as the lower concentration at which no microbial growth could be observed. As a positive control of growth inhibition, Cefradine and Kanamycin was used in the case of bacteria, Triadimefon in the case of fungi. Negative control of solvent influence was detected in parallel.

Conclusions
This investigation led to the isolation of one new cyclohexenone derivative, one new cyclopentenone derivative, and two new xanthone derivatives, which enriched the library of natural compounds. This was the first report showing that these compounds were produced by the fungus Alternaria sp. Compounds 1 and 2 exhibited higher ABTS radical scavenging activities with EC50 values of 8.19 ± 0.15 and 16.09 ± 0.01 μM, respectively, than ascorbic acid (EC50 17.14 ± 0.11 μM), which revealed that they could be potential antioxidant drugs and/or lead compounds for constructing an antioxidant compound library. In comparison to Triadimefon, compounds 2 and 5 exhibited more potent inhibitory activity against Fusarium graminearum, and compound 3 exhibited more potent antifungal activity against both Calletotrichum musae and Fusarium graminearum, which suggested that the three compounds were worthy of consideration for the development and research of antifungal agents.