New Anti-inflammatory Cyclopeptides From a Sponge-Derived Fungus Aspergillus violaceofuscus

Three new cyclic peptides including a cyclic tetrapeptide (1), an aspochracin-type cyclic tripeptide sclerotiotide L (2) and a diketopiperazine dimer (3), have been isolated from the ethyl acetate extract of a marine sponge-derived fungus Aspergillus violaceofuscus. The structures of all compounds were unambiguously elucidated on the basis of HRESIMS, 1D and 2D NMR spectroscopic data, MS/MS experiments and chemical methods. Compounds 1 and 3 showed anti-inflammatory activity against IL-10 expression of the LPS-induced THP-1 cells with inhibitory rates of 84.3 and 78.1% respectively at concentration of 10 μM.

The sponge-derived fungi have been proven to be a prolific source of cyclic peptides (Amagata et al., 2006;Yu et al., 2008). In previous search for structurally unique cyclic peptides from marine sponge-derived fungus Nigrospora oryzae PF18 collected off the Xisha Islands in the South China Sea, we have identified a series of new cyclohexadepsipeptides oryzamides A-C (Ding et al., 2016). As part of our continuing quest for new bioactive molecules, chemical investigation of secondary metabolites of the fungus Aspergillus violaceofuscus from the marine sponge Reniochalina sp. resulted in the identification of three new cyclopeptides, including a cyclic tetrapeptide violaceotide A (1), an aspochracin-type cyclic tripeptide sclerotiotide L (2), and a new diketopiperazine dimer (3) (Figure 1). Herein, the isolation, structure elucidation and anti-inflammatory studies of the three new cyclic peptides were described.

General Experimental Procedures
Optical rotations were determined on a Rudolph research analytical autopol VI polarimeter with a 1 dm length cell at room temperature. UV spectra were performed on a Persee TU-1950 UV-VIS spectrophotometer. The NMR spectra were recorded on a Bruker AMX-600 instrument. HRESIMS data were obtained on a Waters Xevo G2-XS Q-Tof mass spectrometer. Reversedphase HPLC was performed on Waters X-Bridge C18 (5 µm) columns with a Waters 1525 separation module equipped with a Waters 2998 photodiode array detector. MPLC was accomplished using a Interchim PuriFlash 450 chromatography system. Silica gel 60 (200-300 mesh; Yantai, China), Sephadex LH-20 (18-110 µm, Pharmacia Co.) and ODS (50 µm, YMC Co.) were used for column chromatography.

Fungal Strain and Fermentation
The fungus Aspergillus violaceofuscus was isolated from the inner part of the marine sponge Reniochalina sp. collected from the Xisha Islands in the South China Sea. The sample was deposited at the Research Center for Marine Drugs, School of Medicine, Shanghai Jiao Tong University. This strain was identified based on the morphology analyze and ITS gene sequencing (GenBank accession No. FJ491681).
The strain was cultivated on potato dextrose agar at 28 • C for 7 days. Large scale fermentation was carried out in 50 erlenmeyer flasks (2 L) each containing 80 g of rice and 120 mL of distilled H 2 O with 0.3% (m/v) peptone. Each flask was inoculated with 20 mL of cultured broth and incubated under static conditions at room temperature for 40 days.

Extraction and Isolation
The fermented substrate was exhaustively extracted with ethyl acetate to provide the residue (26.0 g) after removal of the organic solvent under reduced pressure.

Advanced Marfey's Analysis of Compound 1
Compound 1 (1 mg) were hydrolyzed in HCl (6 M; 1 mL) for 18 h at 110 • C. The solutions were then evaporated to dryness and redissolved in H 2 O (200 µL). The aqueous hydrolysate was added with 1% (w/v) solution of 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA, 100 µL) in acetone and 1 M NaHCO 3 (40 µL). After treating at 45 • C for 90 min, the reactions were quenched by the addition of HCl (1 M, 40 µL). Appropriate amino acid standards were treated with L-FDLA and D-FDLA as described above and yielded the L-FDLA and D-FDLA standards. Marfey's derivatives of 1 was subjected to UPLC-MS selected ion chromatography on a reversed-phase column (Waters ACQUITY HS T3 column; 1.8 µm, 2.1 × 100 mm) with a linear gradient from 10 to 60% aqueous CH 3 CN containing 0.1% formic acids over 18 min and their retention times were compared with those from the authentic standard derivatives.

Marfey's Analysis of Compound 3
Compound 3 (0.4 mg) was dissolved in 6 N HCl (1 mL) and heated at 110 • C for 24 h. Then, the solvent was evaporated under reduced pressure and resuspended in 50 µL of H 2 O. The hydrolysates were treated with 200 µL of 1% (w/v) 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (FDLA) in acetone and 40 µL of 1.0 N NaHCO 3 . The reaction mixtures were heated at 45 • C for 2 h, cooled to room temperature, and then neutralized with 40 µL of 1 N HCl. Standard D-Leu and D/L-Leu were derivatised in a similar fashion separately. The derivatives of the hydrolysates and the standard amino acids were analyzed by LC-MS selected ion chromatography on a reversed-phase column (Waters XBridge C18 column; 5 µm, 4.6 × 250 mm; 1.0 mL/min) with a linear gradient from 10 to 100% aqueous CH 3 CN containing 0.1% formic acids over 30 min. The retention times for FDLA derivatives of standard D-Leu and L-Leu were 21.8 and 18.7 min, respectively, while this for FDLA derivatives of compound 3 were 21.8 min.

Anti-inflammatory Assay
THP-1 (a human acute monocytic leukemia cell line) cells (CCTCC) were maintained in RPMI-1640 supplemented with 10% (v/v) FBS and 0.05 mmol/L 2-mercaptoethanol at 37 • C in a 5% CO 2 and humidified environment. THP-1 cells (5 × 10 5 /mL) were differentiated using 160 nmol/L PMA for 36 h. Differentiation of PMA-treated cells was enhanced by removing the PMA-containing media and the cells were incubated in FBS free, fresh RPMI 1640 for a further 12 h, and then stimulated with compounds or/and LPS at the indicated concentrations and time periods.
Cytokines IL-6, IL-10, MCP-1, and TNF-α in the culture media of THP-1 cells treated with 10 µM compounds or/and 0.1 µg/mL LPS were determined by flow cytometry using the Human Inflammation Cytometric Bead Array (CBA) according to the instruction of the manufacturer (BD Biosciences, San Jose, CA, USA). Cytokine levels were measured on a FACSCalibur flow cytometer (BD Biosciences Pharmingen). The concentrations were assessed by using FCAP Array software.
The absolute configurations of the amino acids were determined by the advanced Marfey's method after acid hydrolysis (Fujii et al., 1997). Compound 1 was hydrolyzed and then derivatized with L-FDLA. The UPLC-MS comparison between Marfey's derivatives of the hydrolysate of 1 and appropriate amino acid standards assigned the L configurations for Thr, Tyr, N-Me-Ala, and Ile (Supplementary Figure 23). The final structure of 1 was elucidated as cyclo-[L-Thr-L-O-Me-Tyr-L-N-Me-Ala-L-Ile] and named as violaceotide A.
The relative configurations of 3 were deduced from the observed NOESY correlations (Figure 6). The key NOESY correlations of H-11/H-17 and H-11/ H-2, indicated that these protons were on the same face. The NOESY correlations of H-5 ′ with H-11 and H-11 with H-2 suggested cis-fused ring junction at C-2 and C-3. Marfey's method (Cho et al., 2018) was employed to determine the absolute configuration at C-15. Compound 3 was hydrolyzed and derivatized with L-FDLA and analyzed by LC-MS to the establishment of absolute configuration of Leu residues.  By comparing the retention times of authentic standards of Land D-forms of Leu, the hydrolysate was identified to contain a unit of D-Leu (Supplementary Figure 25). Therefore, the absolute configurations of 3 were assigned as 2R, 3R, 11S, 15R (Figure 1).
To the best of our knowledge, compound 3 has the same planar structure as a unnamed and ambiguous compound without any spectroscopic data or optical rotation published on a patent (Masashi et al., 1995). Our compound possesses the different stereochemistry with it. Therefore, 3 was reported as a new diketopiperazine dimer herein.
Compounds 1-3 were evaluated their inhibitory activities against the production of four cytokines levels in the serum of human acute monocytic leukemia cell line THP-1 by using the human inflammation cytometric bead array (CBA) assay ( Table 4). The cytokines, including IL-6, IL-10, MCP-1, TNF-α in this study, are pivotal mediators that contribute to inflammation and various related diseases (Liu et al., 2017;Wu et al., 2017). Notably, treatment of THP-1 cells by LPS showed a significant elevation in the secretion of the cytokines (P < 0.01). Results showed that the THP-1 cells pretreated with compounds 1 and 3 showed a significant decrease in the LPS-induced expression of IL-10 with inhibitory rates of 84.3 and 78.1% (P < 0.01), respectively. These compounds did not show cytotoxicity against THP-1 cells after 24 h treatment.

CONCLUSIONS
From the marine sponge-derived fungus Aspergillus violaceofuscus, three new cyclic peptides were obtained.
Aspochracin-type cyclic tripeptide sclerotiotide L (2) and a diketopiperazine dimer (3) showed anti-inflammatory activity against IL-10 expression of the LPS-induced THP-1 cells, which indicated that the marine sponge-derived microorganism are a fertile source of compounds with novel structures and significant bioactivities.

AUTHOR CONTRIBUTIONS
JL, BG, and LY: performed the experiments; JL: identified the structures and analyzed the data; HL and FY: conceived and designed the experiments; JL and FY: wrote the paper. All authors listed have approved the work for publication.