Secondary Metabolites with Nitric Oxide Inhibition from Marine-Derived Fungus Alternaria sp. 5102

Two new benzofurans, alternabenzofurans A and B (1 and 2) and two new sesquiterpenoids, alternaterpenoids A and B (3 and 4), along with 18 known polyketides (5−22), were isolated from the marine-derived fungus Alternaria sp. 5102. Their structures were elucidated on the basis of extensive spectroscopic analyses (1D and 2D NMR, HR-ESIMS, and ECD) and X-ray crystallography, as well as the modified Mosher’s method. Compounds 2, 3, 5, 7, 9–18, and 20–22 exhibited potent anti-inflammatory activity by inhibiting the production of NO in RAW264.7 cells activated by lipopolysaccharide with IC50 values in the range from 1.3 to 41.1 μM. Structure-activity relationships of the secondary metabolites were discussed.


Results and Discussion
Alternabenzofuran A (1) was obtained as a colorless crystal. The molecular formula of 1 was established as C 14 H 16 O 6 based on the HR-ESIMS data ( Figure S1) Figure S3) and HSQC data ( Figure S4)   A 7-hydroxybenzofuran was assigned by 1 H-1 H COSY correlations ( Figure S5) between H-4 and H-5, H-5 and H-6, H-6 and H-7, and HMBC correlations ( Figure S6) from H-4 to C-3a and C-7a, and H-3 to ester carbonyl C-1, as well as four-bond W-type correlation from H-6 to ester carbonyl C-1 ( Figure 2). The HMBC correlations from H-8 to C-3 and H-3 to C-9 indicated that C-8 of acetoxy was linked to C-3 of 7-hydroxy-benzofuran group. At the same time, 1 H-1 H COSY correlations between H-12 and H-10, H-10 and H-11, H-11, and HMBC correlations from a H-12 to C-10 and C-11, and H-13 to C-10 constructed a 2,3-butanediol group. The key HMBC correlations from H-10 to another ester carbonyl C-9 suggested that 2,3-butanediol group was connected to benzofuran moiety by ester bond. Finally, compound 1 was crystallized upon slow evaporation of chloroform solvent to give a crystal of the monoclinic space group P2 1 , which was detected by X-ray crystallography. The refinement of the Cu Kα data resulted in a Flack parameter [18] of 0.04 (17) and a Hooft parameter [19] of 0.07 (5), which assigned the absolute configuration of 1 as 3S, 10S, and 11S ( Figure 3). The absolute configuration of secondary alcohol was further resolved by a modified Mosher's method [20,21]. The chemical shifts for H-10, H-11, H-12, H-13 of 1a and 1b were measured as δ H 5.19, 5.32, 1.20, 1.17 for 1a, and δ H 5.18, 5.31, 1.31, 1.07 for 1b, respectively. The observed differences of chemical shifts (∆δ = δ S − δ R ) ( Figure 4) indicated that the C-11 absolute configuration is S in agreement with the X-ray crystallography analysis.
In this work, all isolated compounds  were evaluated for their inhibition of nitric oxide (NO) production in RAW264.7 cells activated by lipopolysaccharide (LPS) using the Griess assay with indomethacin as a positive control (Table 3) . Compounds 2, 3, 7, 9,10, 12-15, 17,18 and 20-22 showed stronger anti-inflammatory activity compared to the positive control indomethacin whose IC 50 was 35.8 ± 5.7 µM. Among them, compounds 3, 7, 9 and 14 displayed significant inhibitory effects on the production of NO with IC 50 values below 10 µM, while compounds 5 and 11 exhibited moderate anti-inflammatory activity with IC 50 values of 41.1 and 39.0 µM, respectively. To investigate whether the inhibitory activities of the active compounds were due to their cytotoxicity, the effects of the tested compounds on cell proliferation/viability were evaluated using the MTT method. Meanwhile, compounds 2, 9, 11, 14, and 17 (up to 100 µM) did not show any significant cytotoxicity with LPS treatment for 24 h. The pro-inflammatory enzymes, inducible nitric oxide synthase (iNOS) for nitric oxide (NO) production and cyclooxygenase-2 (COX-2) for prostaglandin production, have been shown to play key roles in inflammatory processes. Therefore, further studies are required to clarify the underlying mechanism of the active compounds. In comparison of anti-inflammatory activity of two sesquiterpene thujopsene, 3 showed much stronger activity than that of 4, indicating that the hydroxymethyl group (C-14) played an important role in anti-inflammatory action. For isocoumarins with 6/6/5 system (8 and 9), the keto carbonyl group at C-9 made no difference to the anti-inflammatory activity, while the hydroxyl group at Mar. Drugs 2020, 18, 426 8 of 14 C-9 made a more positive contribution to the anti-inflammatory activity. For another isocoumarins with 6/6/6 system (11)(12)(13)(14)(15), the substitution with the hydroxyl group at C-10 made a more positive contribution to the anti-inflammatory activity, while other substitution made no difference to the anti-inflammatory activity.
There has been a tremendous increase in pharmacological research on anti-inflammatory of marine-derived molecules, and more than 150 anti-inflammatory compounds derived from marine fungi have been reported past two decades [37,38]. The anti-inflammatory compounds are classified into different chemical classes, such as terpenes [39,40], steroids [41], polyketides [15], alkaloids [42], and peptides [43]. For example, tanzawaic acid Q (isolated from a marine-derived fungus, Penicillium steckii 108YD142) inhibited the lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins and mRNA expressions in RAW 264.7 macrophages. Amestolkolides B (obtained from the mangrove endophytic fungus Talaromyces amestolkiae YX1) showed strong anti-inflammatory activity by inhibiting nitric oxide (NO) production in lipopolysaccharide activated in RAW264.7 cells with IC 50 values of 1.6 ± 0.1 Mm in vitro [40]. A serial of mono-and dimeric sorbicillinoids (isolated from the marine-derived fungus Trichoderma reesei 4670) exhibited potent anti-inflammatory activity by inhibiting the production of NO in RAW264.7 cells activated by lipopolysaccharide with IC 50 values in the range from 0.94 to 38 µM, whose structure-activity relationships were discussed [15]. Oxepinamide A (isolated from a marine-derived fungus Acremonium sp. from the surface of the Caribbean tunicate Ecteinascidia turbinata.) showed potent anti-inflammatory effect with the inhibition rate of 82% at the standard testing dose of 50 µg per ear by a topical resiniferatoxin (RTX)-induced mouse ear edema assay [42]. Alternaramide (isolated from a marine Alternaria sp. SF-5016) showed the inhibition of the production of PGE2 and NO correlated with down-regulation of iNOS and COX-2 expression in LPS-induced RAW264.7 and BV2 macroglia cells with IC 50 values ranging from 27.63 to 40.52 µM, and suppressed the NF-κB and MAPK signaling pathway, as well as reduced the Toll-like receptor 4 (TLR4) and myeloid differentiation primary response gene 88 (MyD88) at the mRNA and protein levels [43]. Though there is no marine-derived anti-inflammatory agent currently on the market, the chemical diversity and biological activities of marine-derived molecules will provide medical and chemical researchers with a plenty variety of promising lead compounds for the development of anti-inflammatory marine drugs.

Biological Material
The fungal strain 5102 was isolated from an actiniae collected in the Laishizhou island (22 • 27 49.7" N 114 • 32 21.4" E), Shenzhen City, Guangdong Province, China, in April 2016. The fungus was identified as Alternaria by an ITS sequence with 99% query coverage and 99% similarity to which has been deposited in GenBank under accession number EFJ809940.1. The fungal strain 5102 was deposited in GenBank with accession number MT742159.

Extraction, Isolation, and Characterization
The fungus was activated and purified on PDA plate, then implanted in a conical flask with PDB liquid (four 500 mL Erlenmeyer flasks; each containing 12 g of PDB powder and 15 g of artificial sea salt and 500 mL distilled H 2 O) and cultured in a shaker chamber for three days (150 RPM, 28 • C) to obtained seed liquid. The fungus's seed liquid cultured on a rice medium (150 bottles 500 mL conical flask; each bottle with 50 g of rice, 15 g of sea salt and 60 mL sterile water) with room temperature under daylight and stilling culture for one month. The fermented material was extracted with EtOAc three times and concentrated under reduced pressure. The EtOAc extract (45.9 g) was subjected to CC on silica gel (100-200 mesh)   model for MeOH was used. ECD spectra were generated using the program SpecDis 3.0 (University of Würzburg, Würzburg, Germany) and OriginPro 8.5 (OriginLab, Ltd., Northampton, MA, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.30 ev. All calculations were performed by Tianhe-2 in the National Super Computer Center in Guangzhou.

Cell Viability Assay and Anti-Inflammatory Activity
Cell viability was measured using the conventional MTT assay. RAW 264.7 cells were seeded in 96-well plates at a density of 1.5 × 10 5 cells/mL. After 12 h, the cells were treated with LPS (1 µg/mL) and samples, followed by additional incubation for 24 h at 37 • C. MTT stock solution (2 mg/mL) was added to wells for a total reaction volume of 110 µL. After 4 h incubation, the supernatants were aspirated. The formazan crystals in each well were dissolved in 50 µL of DMSO, and the absorbance was measured using a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA) at the wavelength of 490 nm. Relative cell viability was evaluated based on the quantity of MTT converted to the insoluble formazan salt. The optical density of formazan generated in the control cells represented 100% viability. The data were expressed as mean percentages of the viable cells compared to the respective control.
After pre-incubation of RAW 264.7 cells (1.5 × 10 5 cells/mL) with LPS (1 µg/mL) and samples at 37 • C for 24 h, the quantity of nitrite accumulated in the culture medium was measured as an indicator of NO production. Briefly, 50 µL of cell culture medium were mixed with 100 µL Griess reagent, and incubated at room temperature for 10 min. The absorbance was determined at 540 nm wavelength with a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA).

Statistical Analysis
Each experiment was performed at least three times independently, and the resulting data are presented as the mean ± standard deviation. The comparison of three or more groups used one-way analysis of variance, followed by Tukey's multiple comparison tests. Statistical analysis was performed using GraphPad Prism software, version 3.03 (GraphPad Software Inc, GraphPad Software Inc., San Diego, CA, USA).