Four New Citrinin Derivatives from a Marine-Derived Penicillium sp. Fungal Strain

Four new citrinin derivatives, including two citrinin dimers and two citrinin monomer derivatives, were isolated and identified from a marine-derived fungal strain Penicillium sp. ML226 along with six known related compounds. Their structures were elucidated by spectroscopic and chemical methods. The new compounds showed modest cytotoxic activity against HepG-2 cell line and weak antimicrobial activity against Staphylococcus aureus.


Introduction
The search for new bioactive natural products is still the main way of discovering new drugs. Investigating the secondary metabolites of microorganisms isolated from specific ecological environments may increase the chance of finding compounds with novel skeletons and varied and unique bioactivities. It was reported that the specific situations that microorganisms live in might activate some silent genes and induce some unique biosynthetic pathways [1]. Marine microorganisms have attracted extensive attention in this context. Marine fungi are an important resource to find OPEN ACCESS chemically and biologically diverse compounds due to their special living environment [2,3]. In order to search for new bioactive natural products, a marine-derived fungal strain, ML226, authenticated as Penicillium sp., was isolated from the Taiwan Strait, China. The EtOAc extract of Penicillium sp. ML226 exhibited cytotoxic and antimicrobial activity. Chemical investigation of the EtOAc extract of Penicillium sp. ML226 led to the isolation of two new citrinin dimers-penicitrinone E (1) and penicitrinol J (2)-two new citrinin monomer derivatives-penicitrinol K (3) and citrinolactone D (4)-together with six known compounds-penicitrinone A [4] (5), penicitrinone B [4] (6), citrinolactone B [5] (7), citrinin [6] (8), 2,3,4-trimethyl-5,7-dihydroxy-2,3-dihydrobenzofuran [7] (9) and phenol A [8] (10) (Figure 1). In this paper, we report the isolation and structural elucidation of compounds 1-10 and the cytotoxic and antimicrobial activity of 1-4. They all showed weak cytotoxicity against HepG-2 cell line in the concentration of 10 μg/mL with inhibition rate from 6% to 30%. Compounds 2 and 3 showed weak antimicrobial activity against Staphylococcus aureus.  H-NMR data of 1 showed four tertiary methyl signals, two aromatic methyl signals, four sp 3 methine protons (two oxygenated), and one hydroxyl proton ( Table 1). The 13 C-NMR and DEPT spectra for 1 displayed 24 carbon signals comprising four tertiary methyls, two aromatic methyls, four sp 3 methines (two oxygenated), two carbonyl carbons, and 12 sp 2 quaternary carbons (Table 1). Except for those of the benzopyran moiety, the NMR data were quite similar to those of 5 [4], indicating that they shared the same molecular skeleton. Compared with those of 5, the NMR spectra of 1 exhibited an additional carboxyl group (δ c 165.4), two downfield shifts effect of C-1 (+4.6 ppm) and C-8 (+2.7 ppm) because of the inductive effect of the additional carboxyl group. The C-7 of 5 is a sp 2 methine carbon but the C-7 of 1 is a sp 2 quaternary carbon, indicating that the carboxyl group was linked to C-7. This deduction was further supported by analyses of the 2D (HMQC, 1 H-1 H COSY and HMBC) NMR spectra ( Figure 2). The relative configuration of the two methyl residues in the benzopyran moiety was determined as trans based on the NOESY correlation of 4-CH 3 with 3-H and J 3,4 (<0.5 Hz); and the relative configuration of the two methyl residues in the benzofuran moiety was determined as trans based on the NOESY correlation of 3′-CH 3 with 2′-H and J 2′,3′ (=4.3 Hz) ( Figure 3).   427.1757). The 1 H-NMR data of 2 showed four tertiary methyl signals, two aromatic methyl signals, five sp 3 methine protons (three oxygenated), and two hydroxyl protons ( Table 1). The 13 C-NMR and DEPT spectra for 2 displayed 24 carbon signals including four tertiary methyls, two aromatic methyls, five sp 3 methines (three oxygenated), one carbonyl carbon, and 12 sp 2 quaternary carbons ( Table 1). The NMR data were quite similar to those of 1 except for those of the benzopyran moiety. Compared with those of 1, the NMR spectra of 2 exhibited an additional oxygenated sp 3 methine proton (δ H 5.71) and an additional oxygenated sp 3 methine carbon (δ c 66.3), but missed one carbonyl carbon (δ c 183.8 in 1). These indicated one of the two additional protons was linked to C-1, the other was the hydroxyl proton of 6-OH, which was further supported by the downfield shift effect of C-4a (+12 ppm) and the highfield shifts effect of H-3 (−1.07 ppm) and H-4 (−0.26 ppm) as a result of the missing of the double bond between C-1 and C-8a, and the 2D (HMQC, 1 H-1 H COSY and HMBC) NMR spectra ( Figure 2). The NOESY correlation of 3-H with 4-CH 3 and J 3,4 (=6.1 Hz) established the trans of the two methyl residues in the benzopyran moiety; The NOESY correlation of 2′-H with 3′-CH 3 Table 2). The 13 C-NMR and DEPT spectra of 3 displayed signals for three tertiary methyls, one aromatic methyl, one sp 3 methylene, three sp 3 methines (two being oxygenated) and eight quaternary carbons (Table 2). Compared to those of 2, compound 3 shared the same benzopyran moiety with 2 which was further supported by the 2D (HMQC, 1 H-1 H COSY and HMBC) spectra ( Figure 2), and the NMR spectra of 3 exhibited a high-field shift effect of C-1 (−1.01 ppm). The 1 H-1 H COSY correlations between 1-H and 11-Ha, 1-H and 11-Hb, and key HMBC correlations from 1-H, 11-H and 10-CH 3 to corresponding carbons indicated that C-11 was linked to C-1, C-10 was linked to C-11, and 10-CH 3 was linked to C-10 ( Figure 2). Finally C-10 was linked to C-8 via O and 10-OH was linked to C-10, which established by the molecular formula of 3. The NOESY correlation of 3-H with 4-CH 3    269.0790). The 1 H-NMR data of 4 showed one aromatic methyl signal, one methoxyl group, two sp 3 methylene signals, two aromatic protons, one oxygenated sp 3 methine proton, and one hydroxyl proton ( Table 3). The 13 C-NMR spectrum of 4 displayed signals for two methyls (one of them oxygenated), two sp 3 methylenes, one oxygenated sp 3 methine, two sp 2 methines, one carbonyl carbon, and 6 sp 2 quaternary carbons ( Table 3). The NMR data were quite similar to those of 7 [5]. By comparison with those of 7, the NMR spectra of 4 exhibited an additional methoxyl group (δ H 3.50, δ c 57.3) attached C-9, and which was further supported by HMBC correlations from 9-OCH 3 to C-9 ( Figure 2).

Cytotoxic and Antimicrobial Activity
The new compounds 1-4 were tested for cytotoxic effects against the HeLa and HepG-2 cell lines using the MTT method [15]. However, they exhibited no remarkable cytotoxic activity against any of the cell lines in the concentration of 10 μg/mL (100% of cis-platinum as positive control). The results of cytotoxic tests of compounds 1-4 are shown in Table 4. The antimicrobial activity of compounds 1-4 against Staphylococcus aureus (CMCC26003), Escherichia coli (CMCC44103), Candida albicans (AS2.538), and Aspergillus niger (ACCC30005) were also evaluated by paper diffusion method with concentration of 20 μg/6 mm paper disk. Only compounds 2 and 3 showed weak antimicrobial activity against Staphylococcus aureus CMCC26003 with inhibition zones of 10 and 9 mm diameter, respectively (18 mm of gentamicin as positive control).

General Procedures
Optical rotations were obtained on a PerkinElmer 341 automatic polarimeter. UV spectra were recorded on a Persee TU-1901 spectrophotometer. IR spectra were recorded on a Nicolet Avatar 330FT spectrometer. 1 H-NMR, 13 C-NMR, and DEPT spectra and 2D NMR spectra were recorded on a Bruker Avance Ⅲ-600 NMR spectrometer using TMS as internal standard, and chemical shifts were recorded as values. HRESIMS data were measured on a Bruker FT-ICR-MS mass spectrometer. ESIMS was measured on a Finnigan mass spectrometer. TLC was carried out using glass-precoated silica gel GF 254

Fungal Material
The fungal strain Penicillium sp. ML226 was isolated from the sediment of Fu Gong mangrove region, Long Hai, Taiwan Strait, China. It was identified according to its morphological characteristics and ITS sequence. It was identified as a sporulating fungus by traditional morphology. A BLAST search result showed that the internal transcribed spaces (ITS) sequence of ML226 was highly homologous (96% percent similarity) to that of a Penicillium species (JX192960), indicating that ML226 belongs to this genus. The voucher specimen is deposited in our laboratory at−80 °C. The producing strain was prepared on potato dextrose agar slants and stored at 4 °C.

Fermentation and Extraction
The fungus Penicillium sp. ML226 was inoculated on slope of YMG media (glucose 4.0, malt extract 10.0, yeast extract 4.0, pH 7.2) in a 250 mL solanad type flask containing solid media (25 mL/flask) at 28 °C for 4 days to afford spores. Then the spores were obtained by scraping and agitating from the slope of YMG media using 120 mL ddH 2 O. Solid media fermentation was performed with YMG media (12 L) at 28 °C for 7 days, and the spores was inoculated with inoculating loop. The cultured agar was chopped, diced and extracted with EtOAc-MeOH-AcOH (80:15:5, 3.5 liters) at room temperature overnight. The organic solution was collected through filtration, and the remaining agar residue was extracted several times more as described above until the filtrate was colourless. The combined filtrates were concentrated under vacuum to remove organic solvents. The aqueous solution was extracted five times with EtOAc to give an EtOAc solution, which was concentrated under vacuum to give a crude EtOAc extract. Then the EtOAc extract was dissolved with MeOH to give a MeOH solution. The MeOH solution was concentrated under vacuum to give a crude extract (5.00 g).

Biological Assays
Cancer cell lines were derived from the cell bank of the Chinese Academy of Sciences. The cytotoxicities of the compounds 1-5 were measured by the MTT (Sigma) assay [15]. The cells in 100 μL of culture medium were plated in each well of 96-well plates (Falcon, CA). After 24 h of incubation for a density of 5 × 10 3 /100 μL medium, the cells were treated in triplicate with the concentration of 10 μg/mL of every compound for 72 h at 37 °C. A 20 μL aliquot of MTT solution (5 mg/mL) was added directly to all wells and incubated for 4 h at 37 °C. To quench the reaction, 100 μL of triplex solution (10% SDS, 5% isobutanol, 12 mM HCl) was added to each well and incubated overnight at 37 °C. The optical density of each well was measured with a microplate reader (M-3350, Bio-Rad) at 595nm (excitation). Growth inhibition rates were calculated with the following equation: