Citrinin Monomer and Dimer Derivatives with Antibacterial and Cytotoxic Activities Isolated from the Deep Sea-Derived Fungus Penicillium citrinum NLG-S01-P1

Two previously unreported citrinin dimer derivatives, penicitol D (1) and 1-epi-citrinin H1 (2), were isolated from the culture of a deep sea-derived fungus Penicillium citrinum NLG-S01-P1, together with 11 biogenetic related compounds (3–13). A plausible biogenetic pathway for compounds 2–4 was proposed. Their structures, including absolute configurations, were established through analysis of extensive spectroscopic data and time-dependent density functional theory (TD-DFT) ECD calculations. Compounds 1 and 2 showed antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA). Compounds 5 and 10 displayed relatively stronger activities than the other compounds against Vibrio vulnificus and Vibrio campbellii. Compound 1 showed the most potent cytotoxic activity towards the HeLa cell.

The antibacterial and cytotoxic activity of compounds 1-13 were evaluated using strains of methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 43300, CGMCC 1.12409), Vibrio vulnificus MCCC E1758, Vibrio campbellii MCCC E333, Vibrio rotiferianus MCCC E385, and A549 and HeLa cell lines ( Table 2). For strains of MRSA, compounds 1 and 2 showed similar activities in comparison to the positive control chloramphenicol with MIC values ranging from 7 to 8 µg/mL. For strains of V. vulnificus, compounds 1-13 showed weaker activities than the positive control erythromycin. Compounds 5 and 10 exhibited relatively stronger activities than the other compounds against V. vulnificus and V. campbellii, with MIC values ranging from 15 to 17 µg/mL, respectively.
For cytotoxicity, compounds 1-13 displayed weaker activities than the positive control doxorubicin. Compound 1 exhibited the most potent cytotoxic activities towards HeLa cells, with the IC 50 value of 4.1 µM.

Fungal Material
The fungal strain was identified based on ITS sequence homology (99% similarity with P. citrinum strain SFC101144 with Genbank Accession No. MF185963.1 (max score 900, e value 0.0, query cover 100%)). The ITS1-5.8S-ITS2 rDNA sequence of the fungus has been submitted to GenBank with the accession number MK266987 (Text S1). The strain was deposited at the Third Institute of Oceanography, Ministry of Natural Resources, China.

Fermentation, Extraction, and Isolation
Strain NLG-S01-P1 was cultured on PDA plates at 28 • C for 3 days. Then, six plugs (5 mm diameter) were transferred to 12 Erlenmeyer flasks (1 L), each containing 500 mL PD medium in sterile conditions. Erlenmeyer flasks were shaken on a rotary shaker at 28 • C and 120 rpm for 3 days to form seed cultures (1 × 10 8 spores/mL). Next, seed cultures (40 × 100 mL) were transferred to flasks (40 × 1 L) containing 70 g of rice, 0.1 g of corn flour, 0.3 g of peptone, 0.1 g of sodium glutamate, and 100 mL of naturally sourced and filtered seawater. After 28 days, the fermented broth was dried, smashed, and extracted with EtOAc. The organic solvent was evaporated under reduced pressure to afford the EtOAc extract (40 g), which was subjected to silica gel column chromatography using PE-acetone (9.8:0.

ECD Calculation
Conformational searches were carried out by means of a Merck Molecular Force Field (MMFF). A restricted number of low-energy conformations were obtained falling in an energy window with the threshold of 5 kcal/mol, and then conformers were initially optimized at B3LYP/6-31G(d) level in MeOH using the conductor-like polarizable continuum model (CPCM). The theoretical calculations were carried out using Gaussian 09 (Wallingford, CT, USA). The optimized-conformers with Boltzmann-based population lower than 1% were filtered out, and the remaining chosen for ECD calculation were conducted using TD-DFT at wB97XD/def2-SVP [16]. Rotatory strengths for a total of 30 excited states were calculated. The Boltzmann-averaged ECD spectrum was obtained with the aid of Multiwfn 3.6 software. Electronic transitions were expanded as Gaussian curves, with a full width at half maximum (FWHM) for each peak set to 0.3 eV, which gives a good fitting with the experimental width of the bands [17].

Antibacterial Assay
Antibacterial activities against MRSA (ATCC 43300, CGMCC 1.12409), V. vulnificus (MCCC E1758), V. campbellii (MCCC E333), and V. rotiferianus (MCCC E385) were tested by continuous dilution in 96-well plates using resazurin as a surrogate indicator. The MIC value was determined to be the lowest concentration that did not induce the color change by observing the blue-to-pink color change [18][19][20].

Cytotoxicity Assay
Cytotoxic activities of compounds 1-13 were evaluated using A549 (adenocarcinomic human alveolar basal epithelial cell) and HeLa (cervical cancer cell) cells by Cell Counting Kit-8 (CCK-8) (DOJINDO) assay at 48 h post-treatment with doxorubicin as a positive control, following the manufacturer's instructions [21]. The cell lines were purchased from the cell bank of Chinese Academy of Sciences, Shanghai, China.

Conclusions
In the current research, we have isolated four citrinin dimer and nine monomer derivatives, including two novel dimers, penicitol D (1) and 1-epi-citrinin H1 (2). The absolute configuration of 1-2 was elucidated using the TD-DFT ECD method and a plausible biogenetic pathway for 2-4 was proposed. Compounds 1 and 2 showed similar anti-MRSA activities in comparison to chloramphenicol, and compound 1 displayed the most potent cytotoxic activity towards HeLa cells, with an IC 50 below 5 µM. Both 1-epi-citrinin H1 (2)-as an epimer of citrinin H1-and the anti-vibiro activities of compounds 1-13 are reported here for the first time. Penicitol D (1) is the second citrinin dimer with a single oxygen-bridging center with a similar structure skeleton to the previously reported penicitol B [22]. The previously unreported compounds in the current study enrich the structural diversity of citrinin dimers.
Author Contributions: W.W. carried out ITS sequencing, the isolation and structural elucidation of compounds, ECD calculation, and wrote this paper. Y.L. performed the antibacterial activity evaluation. W.K. and M.G. contributed to the cytotoxic activity assay. B.Z. carried out the fermentation of fungi. F.L. designed the experiments of the bioactivity assay. Z.S. contributed to the revision of the paper.