Anti-Food Allergic Compounds from Penicillium griseofulvum MCCC 3A00225, a Deep-Sea-Derived Fungus

Ten new (1–10) and 26 known (11–36) compounds were isolated from Penicillium griseofulvum MCCC 3A00225, a deep sea-derived fungus. The structures of the new compounds were determined by detailed analysis of the NMR and HRESIMS spectroscopic data. The absolute configurations were established by X-ray crystallography, Marfey’s method, and the ICD method. All isolates were tested for in vitro anti-food allergic bioactivities in immunoglobulin (Ig) E-mediated rat basophilic leukemia (RBL)-2H3 cells. Compound 13 significantly decreased the degranulation release with an IC50 value of 60.3 μM, compared to that of 91.6 μM of the positive control, loratadine.


Introduction
For the past decade, the trend to discover new compounds from marine microorganisms continues to rise [1], especially from marine fungi [2,3], which accounted for 68% of the reported new marine natural products in 2019 [4]. Of particular importance is the Penicillium species, which are recognized as the richest source for the discovery of biologically important and structurally unique secondary metabolites [5][6][7][8].
As our ongoing research for novel and bioactive secondary metabolites from the deep sea-derived microorganisms [8][9][10][11], the fungal strain Penicillium griseofulvum isolated from the Indian Ocean sediment was selected for a systematic chemical examination. As a result, five carotanes, four naphthalenes, and three viridicatol derivates were obtained [12,13]. A continuous study, however, led to the isolation of 10 new ( Figure 1) and 26 known compounds. Herein, we report the isolation, structure elucidation, and biological activity of these compounds.

Results and Discussion
Compound 1 was isolated as a white powder. Its molecular formula was established as C 17  3 Hz) confirmed the two benzene units and deduced another fragment of C-7/C-8. By the HMBC (Heteronuclear Multiple-bond Correlation) correlations of H-7 (δ H 5.16) to C-1/C-2/C-6/C-9 and H-6 (δ H 7.60)/7 -NMe (δ H 2.89) to C-7 , 1 was then assigned a phenylpropionyl moiety and a benzamide groups ( Figure 2). However, the limited HMBC correlations hindered the connection of these two fragments. Fortunately, crystals of 1 were obtained. By the single X-ray crystallography (Figure 3), the absolute configuration of 1 was then unambiguously assigned as 2-(2R,3Sdihydroxy-3-phenyl-propionylamino)-N-methyl-benzamide, and named penigrisamide.     Figure 4). On the basis of the above evidences, 3 was then assigned as N,N-pyroglutamylleucinmethylester.
Compound 4 was obtained as a colorless oil. Its molecular formula was established    Compound 3 was obtained as a colorless oil. Its molecular formula was established as C17H27N3O5 on the basis of the protonated molecule peak at m/z 376.1841 [M + Na] + in its (+)-HRESIMS spectrum, requiring six degrees of unsaturation. Diagnostic NMR data for 3 suggested the presence of a pyroglutamylleucinmethylester (20) [15]. Moreover, the 1 Figure 4). On the basis of the above evidences, 3 was then assigned as N,N-pyroglutamylleucinmethylester.
Compound 4 was obtained as a colorless oil. Its molecular formula was established   Table 2) exhibited 19 carbons, including three methyl singlets (one oxygenated), two methylenes, seven methines (five olefinic), and seven nonprotonated carbons (one carbonyl and two ketone groups). These signals were closely similar to those of aurantiomide C (11) [14], except that the terminal amino group in 11 was replaced by the methoxy unit (δ C 52.2) in 2. The assumption was confirmed by the HMBC correlation of 17-OMe (δ H 3.46) to C-17 (δ C 173.9). Accordingly, the structure of 2 was determined as 17-deamino-17-methoxylaurantiomide C, and named aurantiomoate C.  Compound 3 was obtained as a colorless oil. Its molecular formula was established as C 17  The molecular formula of 5 was established as C 24 H 32 O 7 by the ion peak at m/z 455.2040 [M + Na] + in its positive HRESIMS. The 1 H and 13 C NMR spectra exhibited 24 carbons, including three doublets and five singlet methyls, one methoxyl, four methines (two oxygenated and two olefinic), and eleven quaternary carbons (six olefinic and two carbonyl carbons). These signals were closely similar to those of penicyrone A [16] except that the hydroxy (δ C 82.6) at the C-9 position in penicyrone A was replaced by the carbonyl (δ C 202. 8       and H 3 -18 (δ H 1.28) to C-5/C-6/C-7. Therefore, 6 was established as 4,5-dihydro-4,5epoxyverrucosidinol, and named verrucosidinol B.
Compound 7 had a molecular formula C17H14O6 as assigned by its positive HRESIMS at m/z 337.0690 [M + Na] + . Its 1 H and 13 C NMR spectroscopic data greatly resembled those of helvafuranone [18] except for an additional hydroxy substituent at the C-8 position. By detailed analysis of its 1D and 2D NMR spectroscopic data, 7 was then established as 8hydroxyhelvafuranone.
Compound 10 was obtained as a colorless oil. Its molecular formula was established as C 10  In the HMBC spectrum, H-3 (δ H 3.76) was correlated to C-7 and C-1, which constructed a hexacyclic ring via an ether bond between C-1 and C-7. Accordingly, 10 was established as 9-hydroxy-3,7-epoxydecanoic acid.

General Experimental Procedures and Fungal Fermentation
Penicillium griseofulvum, isolated from a sediment sample of the Indian Ocean at a depth of 1420 m, was deposited at the Marine Culture Collection of China (MCCC) with the accession number MCCC 3A00225. It was cultivated on corn medium in 100 × 1 L Erlenmeyer flasks for 62 days. The detailed general experimental procedures, fungal fermentation, and extraction were reported previously [12].

Maryer's Method
As reported [40], compounds 3 and 4 (each for 1 mg) were separately dissolved in HCl (1 mL) and incubated for 24 h. The hydrolysate was dried and dissolved in acetone. Then NaHCO 3 and FDAA were added to incubate for 1 h. After being cooled, the mixture was dissolved in 50% aqueous CH 3 CN to yield FDDA derivatives. The corresponding standard amino acids were treated with the same procedures. The FDAA derivates were analyzed by HPLC at 254 and 340 nm by comparing the retention times with those of standards.

Induced CD (ICD) Experiment
Compound 8 and dimolybdenum tetracetate [Mo 2 (OAc) 4 ] were resolved in dried DMSO. Their CD spectra were recorded immediately. Then the ICD spectra were measured every 3 min until they were stationary. The inherent CD data of compound 8 was subtracted to provide its induced CD spectrum as described previously [41,42].

Anti-Food Allergic Experiment
The in vitro anti-food allergic experiment was conducted according to the reported method [43]. Briefly, IgE-sensitized RBL-2H3 cells were treated with tested compounds for 1 h. Then cells were stimulated with dinitrophenyl-bovine serum albumin. The bioactivities were quantified by measuring the fluorescence intensity of the hydrolyzed substrate in an Infinite M200PRO fluorometer (Tecan, Zurich, Switzerland). Phosphate-buffered saline (PBS) buffer and loratadine were used as negative and positive controls, respectively.

Conclusions
From the deep sea-derived fungus Penicillium griseofulvum MCCC 3A00225, 10 new and 26 known compounds were obtained. The structures of the new compounds were determined by extensive analysis of their NMR and HRESIMS spectra, the absolute configurations were confirmed by different methods including the single X-ray crystallography, Marfey's method, and ICD experiment etc. (−)-Cyclopenol (13) showed the strongest in vitro anti-food allergic activity with an IC 50 value of 60.3 µM in IgE-mediated RBL-2H3 cells. Funding: The work was supported by grants from the National Natural Science Foundation of China (21877022), the COMRA program (DY135-B2-08), and the Xiamen Southern Oceanographic Center (17GYY002NF02).

Conflicts of Interest:
The authors declare no conflict of interest.