The Purification, Characterization, and Biological Activity of New Polyketides from Mangrove-Derived Endophytic Fungus Epicoccum nigrum SCNU-F0002

Six new polyketides, including one coumarin (1), two isocoumarins (2 and 3), dihydroradicinin (4), and two benzofuranone derivatives (7 and 8), together with seven known analogues (5–6 and 9–13) were isolated from the culture of the mangrove endophytic fungus Epicoccum nigrum SCNU-F0002. The structures were elucidated on the interpretation of spectroscopic data. The absolute configuration of Compounds 2 and 3 were determined by comparison of their ECD spectra with the data of their analogue dihydroisocoumarins described in the literature. The absolute configuration of 4 was determined by single-crystal X-ray diffraction. All the compounds were screened for their antioxidant, antibacterial, anti-phytopathogenic fungi and cytotoxic activities. Using a DPPH radical-scavenging assay, Compounds 10–13 showed potent antioxidant activity with IC50 values of 13.6, 12.1, 18.1, and 11.7 μg/mL, respectively. In addition, Compounds 6 and 7 showed antibacterial effects against Bacillus subtilis (ATCC 6538), Escherichia coli (ATCC 8739), and Staphylococcus aureus (ATCC 6538), with MIC values in the range of 25–50 μg/mL.


Introduction
Marine natural products play a significant role in the drug discovery and development process [1]. Marine-derived fungi are widely recognized as an emerging source for the production of novel and bioactive secondary metabolites [2]. Marine fungi associated with mangroves live in extreme ecosystems characterized by high salinity, which may determine the production of unique metabolites [3,4]. Exploring the secondary metabolites with excellent biological activity and pharmacy value from mangrove-derived fungi has attracted great attention of both pharmaceutical and natural product chemists [1][2][3][4]. Different species belonging to the genus Epicoccum have been reported to produce many bioactive secondary metabolites with antiviral [5], antibacterial [6], antifungal [7], anti-inflammatory [8], and cytotoxic activities [9]. As part of our ongoing research on bioactive compounds from mangrove endophytic fungi [10,11], the chemical investigation of the ethyl acetate extract of the endophytic fungus Epicoccum nigrum SCNU-F0002, which was isolated from the fresh fruit of the mangrove plant Acanthus ilicifolius L., yielded five new polyketide compounds (1, 2, 3, 7, and 8) and one fungus-derived polyketide (4), which was previously semi-synthesized from radicinin [12], together with seven known compounds (5,6, and 9-13) ( Figure 1). In this paper, we report the purification, structure elucidation, and bioactivities of these compounds.

General Experimental Procedures
HR-ESI-MS data were measured on a Thermo Fisher Scientific Q-TOF mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). NMR spectra were performed on a Bruker AVANCE NEO 600 MHz spectrometer (Bruker BioSpin, Switzerland), using TMS as an internal standard. IR spectra were carried out on a Nicolet Nexus 670 spectrophotometer in KBr discs. CD spectra were measured on a ChirascanTM CD spectrometer (Applied Photophysics, London, UK). UV spectra were measured on a PERSEE TU-1990 spectrophotometer. Single-crystal data were recorded on an Oxford Gemini S Ultra diffractometer (Oxford Instrument, Oxfordshire, UK). Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Stockholm, Sweden) column chromatography (CC) was carried out on silica gel (200-300 mesh, Qingdao Marine Chemical Factory, Qingdao, China). Thin layer chromatography (TLC) was detected on a silica gel GF254 plate (Qingdao Marine Chemical Ltd, Qingdao, China). A Phenomenex Luna (Phenomenex, Torrance, CA, USA) C18 column (250 × 10 mm, 5 µm, 5 mL/min) was used for semipreparative HPLC. All solvents were of analytical grade, except for those used for HPLC, which were of chromatographic grade. DPPH was purchased from Sigma (Steinheim, Germany).

Fungal Materials
The fungus SCNU-F0002 used in the study was isolated from fresh fruit of the mangrove plant Acanthus ilicifolius L., which was collected in January 2018 from the Qi'ao island Mangrove Nature Reserve in Guangdong province, China. It was obtained using the standard protocol for isolation [30]. Initially, the plant fruit was washed with sterile water and surface-sterilized in a 100 mL beaker with 75% ethanol for 1 min. This was followed by dipping the sample into a 5% sodium hypochlorite for 1 min, and the plant parts were then rinsed with sterile water, cut into 3 mm sections, and plated on potato dextrose agar (PDA) (potatoes: 300 mg/mL; dextrose: 20 mg/mL; agar: 15 mg/mL; chloramphenicol: 1 mg/mL) with penicillin (100 units/mL) and streptomycin (0.8 mg/mL). The plates were incubated at 25 ± 1 • C. The endophytic fungal strains were isolated by routine microbiology. The fungal isolates were numbered and stored at 4 • C in triplicate on PDA slants. Fungal identification was carried out using a molecular biological protocol by DNA amplification and sequencing of the ITS region [31]. The sequence data of the fungal strain have been deposited at Gen Bank with accession no. MN096740. A BLAST search result showed that the sequence was most similar (100%) to the sequence of Epicoccum nigrum. A voucher strain was deposited at the School of Chemistry and Environment, South China Normal University, Guangzhou, China, with the access code SCNU-F0002.

Extraction and Isolation
The fungus Epicoccum nigrum SCNU-F0002 was grown under static conditions at 25 • C for 28 days in a solid autoclaved rice substrate medium containing 50 g of rice and 50 mL of 3% saline water. After incubation, the mycelia and solid rice were extracted with EtOAc, and the extracts were concentrated to yield 22.5 g of residue under reduced pressure. The residue was then subjected to a silica gel column (80 × 10 cm) and eluted with a gradient of petroleum ether/EtOAc from 1:0 to 0:1 and divided into 36 fractions. Fraction 10 (115 mg) was chromatographed on Sephadex LH-20 CC (CH 2 Cl 2 /MeOH v/v, 1:1) and silica gel CC (petroleum ether/EtOAc, v/v, 1:5) gave Compounds 4 (7 mg), 5 (5 mg), and 7 (10 mg). Fraction 18 (600 mg) was further eluted on silica gel CC (CH 2 Cl 2 /MeOH v/v, 100:2) to yield Compounds 1 (4 mg), 6 (5.1 mg), and 8 (6.2 mg). Fraction 28 (240 mg) was purified via the Sephadex LH-20 CC (CH 2 Cl 2 /MeOH v/v, 3:1) to yield Subfraction 28.5 (30 mg), which was purified by silica gel CC (CH 2 Cl 2 /MeOH v/v, 96:4) to afford Compounds 2 (3.8 mg), 9 (6 mg), and 10 (5.3 mg).   Table 2 To maximize the likelihood of success, a full sphere of data was collected using Cu Kα radiation. A total of 3935 reflections were collected, yielding a Flack parameter x and standard uncertainty u for this structure of −0.06 (13) based on 860 Friedel pairs. The value of u is slightly beyond the limit of enantiopure-sufficient distinguishing power. However, further confirmation of the absolute configuration was obtained from the examination of Bayesian statistics on Bijvoet pairs [32] implemented using the program PLATON [33]. The calculated Hooft y parameter was −0.06 (10) with G = 1.1 (1). The calculated probability values P3 (true), P3 (twin), and P3 (wrong) were 1.000, 0.000, and 0.000, respectively. This confirmed the absolute configuration of the two stereocenters as 2S and 3S. Moreover, these results are consistent with the relative configuration of radicinin that was proposed in [12,34] on the basis of NMR data and X-ray crystal data.

DPPH Radical Scavenging Activity Assay
The DPPH free radical scavenging assay is based on previously reported methods [35,36], but with minor modifications. The assay was performed on a 96-well microplate. A total of 200 µL of the reaction mixture consists of a series of 100 µL of different concentrations (2, 25, 50, 100, 200 µg/mL) of the tested compound (in ethanol) and 100 µL of 0.16 mM DPPH (in ethanol). The reaction mixture was incubated for 30 min at room temperature in the dark. The absorbance at 517 nm was recorded on a microplate reader, and the inhibition rate was calculated. Vitamin C was used as a positive control.

Antimicrobial Activity Assay
The antimicrobial activities against five bacteria (S. aureus (ATCC 6538), B. subtilis (ATCC 6633), E. coli (ATCC 8739), P. aeruginosa (ATCC 9027), and S. enteritidis (ATCC 14028)) along with three phytopathogenic fungi (P. italicum (BNCC 118157), C. musae (BNCC 226680), and G. zeae (BNCC 116158)) were evaluated on 96-well microtiter plates using a modification of the broth microdilution method [37,38]. The microbial test strain was incubated, and the microbial strain suspension was diluted to a seeding density of 5 × 10 5 cfu compared to the MacFarland standard. The fungi was cultured in PDB (potato dextrose broth) medium (6.75 g of potatoes, 0.45 g of dextrose, and 300 mL of distilled H 2 O) at 28 • C (160 rpm) for 48 h, and the bacteria was cultured in LB medium (3 g of peptone, 1.5 g of sodium chloride, 0.3 g of dextrose, 1.5 g of yeast extract, and 300 mL of distilled H 2 O) at 37 • C (160 rpm) for 24 h. Under the sterile environment, microorganism suspensions (100 µL) of each strain were poured into the wells containing 100 µL of 2-fold serially diluted single compounds in the corresponding culture medium for a final volume of 200 µL. The fungi and bacteria were then incubated at 28 • C for 48 h and at 37 • C for 24 h, respectively. The antimicrobial effect was evaluated by optical density measurement at 595 nm. The amount of growth in each well was compared with a blank control, which consisted only of the medium (an agent in DMSO and PDB), and the MIC was recorded as the lowest concentration of the agent that completely inhibits growth. All experiments were performed at least three times. Triadimefon and ciprofloxacin were used as positive controls for fungi and bacteria, respectively.
Author Contributions: Z.Y. performed the experiments for the isolation, structure elucidation, and biological evaluation and prepared the manuscript; S.W., M.D., H.G., C.H., X.Z., J.H., and Z.S. contributed to the fermentation, extraction, and structure characterization of all compounds; Y.L. supervised the research work and revised the manuscript.
Funding: This research received no external funding.