LAMA-1: A Cerebroside Isolated from the Deep-Sea-Derived Fungus Penicillium chrysogenum †

Chemical investigation of the ethyl acetate extract of Penicillium chrysogenum strain S003, a fungus isolated from Red Sea deep sediment, led to the isolation of a cerebroside molecular species LAMA (1) along with three other known compounds, ergosterol (2), epidioxyergosterol (3), and kojic acid (4). The structures of the isolated compounds were elucidated by interpretation of spectral data, including detailed 1D and 2D NMR (One and two dimensional Nuclear Magnetic Resonance) and mass spectrometry. The cytotoxic activities of isolated compounds 1–4 against five human carcinoma cells were evaluated using sulforhodamine B (SRB) assay. Compounds 2 and 3 displayed promising cytotoxic profiles against lung cancer (A-549), prostate (DU-145), breast adenocarcinoma (MCF-7), and hepatocellular (HepG2) cell lines, with IC50 values of 21.26, 19.3; 1.50, 6.10; 16.95, 13.6; and 2.89, 3.07 µM, respectively, while they were inactive against HeLa cells. Compounds 1 and 4 showed weak cytotoxic profiles against all cell lines under investigation.


Introduction
Marine-derived fungi are recognized as a great source of potentially useful natural products [1,2]. Since the late 1990s, hundreds of novel compounds have been isolated and characterized from marine habitats annually [3], in stark contrast to their terrestrial counterparts. The most explored fungal isolates by the natural products chemists are the Penicillium species, which are considered a major source for drug discovery [4][5][6][7][8]. Currently, as reported in many recent reviews [9][10][11][12][13], the Penicillium genus still represents a major producer of diverse biologically active metabolites.
The Penicillium genus and its different spp. are fast-growing, salt-tolerant, and can easily be obtained and subcultured from various substrates, so the high number of reported metabolites could be justified by this fact. This has encouraged many researchers to study variable Penicillium species isolated from different habitats [6]. Their extensive investigation is concerned with the isolation, characterization, and bioactivity assessment of the obtained secondary metabolites. Among the significant reported activities are antibacterial [14][15][16][17], cytotoxic, and anticancer [16][17][18][19][20][21] activities.
In the course of our ongoing efforts to isolate and identify drug leads from marine metabolites [22][23][24][25][26][27], we have investigated the extract of the Red Sea deep-sediment isolated fungus Penicillium chrysogenum strain S003.

Isolation and Purification of Compounds 1-4
The chromatographic fractionation of extracts from the broth and mycelia of fungus Penicillium chrysogenum strain S003 using silica gel and Sephadex LH-20 column chromatography resulted in the isolation of four separate compounds (1)(2)(3)(4). Compound 1 was assigned as a cerebroside molecular species, and the name LAMA was assigned to this compound.
The sugar moiety and length of the fatty acid residues were identified via methanolysis with methanolic hydrochloric acid of compound 1 followed by partition with n-hexane. After separating the two layers, the hexane layer gave a mixture of fatty acid methyl esters (FAMEs), while the aqueous layer afforded methylated sugar moiety. HPLC comparison between the methylated sugar against standard methylated sugars (glucose and galactose) confirmed that the sugar moiety was glucose (glucose t R = 14.11 min, galactose t R = 13.27). The anomeric proton-coupling constant at δ H 4.91 (1H, d, J = 7.8 Hz) and the chemical shift of the anomeric carbon δ C (105.6) confirmed the β-configuration of the glucopyranoside moiety (α-glucopyranoside: J = 3.7 Hz; δ C 98.5) [36]. Analysis of the negative FAB-MS spectrum of the FAME mixture exhibited the presence of four components at m/z: 269, 283, 297, and 453 [M − H] − , which were considered as FAME-1, FAME-2, FAME-3, and FAME-4, indicating C-16, C-17, C-18, and C-22 FAMEs, respectively ( Figure S8). Compound 1 (Figure 1) was first hydrolyzed, then the reaction mixture was extracted with hexane, and the hexane layer was concentrated to afford a mixture of the methyl esters of the α-hydroxy configuration fatty acids [α] 19 D = −5.0 (c 0.12, CHCl 3 ) [39][40][41][42]. The method of Tanaka et al. [43] was used for determination of the absolute configuration of sugar moiety. Direct HPLC analysis of the sugar moiety reaction mixture exhibited a peak at t R =18.68 min, which was matched with derivatives of D-glucose, confirming the absolute configuration of the sugar moiety (L-glucose t R = 19.22 min). From previous data, the structure of compound 1 was identified as molecular species cerebroside, as shown in Figure 1. The name LAMA was assigned to compound 1.
Due to the considerable importance of determining the molecular species composition of sphingolipids, isolation and structure elucidation of the cerebroside components in the molecular species of LAMA were conducted. By using reversed phase column, LAMA was fractionated to give LAMA-1.  Figures S7 and S9), the structure of this compound was considered resolved, as shown in Figure 2.
Metabolites 2020, 10, x FOR PEER REVIEW 4 of 9 The negative-ion FAB-MS spectrum [M − H] − showed a succession of molecular ion peaks ( Figure S7) at m/z: 710, 724, 738, and 752, with characteristic fragments at m/z: 310 and 268 indicating C-16 and C-19 long-chain bases. Therefore, the previously mentioned spectral data showed that compound 1 was assumed to be a molecular species of sphingosine-type cerebrosides with 2-hydroxy fatty acid.
The sugar moiety and length of the fatty acid residues were identified via methanolysis with methanolic hydrochloric acid of compound 1 followed by partition with n-hexane. After separating the two layers, the hexane layer gave a mixture of fatty acid methyl esters (FAMEs), while the aqueous layer afforded methylated sugar moiety. HPLC comparison between the methylated sugar against standard methylated sugars (glucose and galactose) confirmed that the sugar moiety was glucose (glucose tR = 14.11 min, galactose tR = 13.27). The anomeric proton-coupling constant at δH 4.91 (1H, d, J = 7.8 Hz) and the chemical shift of the anomeric carbon δC (105.6) confirmed the βconfiguration of the glucopyranoside moiety (α-glucopyranoside: J = 3.7 Hz; δC 98.5) [36]. Analysis of the negative FAB-MS spectrum of the FAME mixture exhibited the presence of four components at m/z: 269, 283, 297, and 453 [M − H] − , which were considered as FAME-1, FAME-2, FAME-3, and FAME-4, indicating C-16, C-17, C-18, and C-22 FAMEs, respectively ( Figure S8). Compound 1 ( Figure  1) was first hydrolyzed, then the reaction mixture was extracted with hexane, and the hexane layer was concentrated to afford a mixture of the methyl esters of the α-hydroxy configuration fatty acids [α] = −5.0 (c 0.12, CHCl3) [39][40][41][42].
The method of Tanaka et al. [43] was used for determination of the absolute configuration of sugar moiety. Direct HPLC analysis of the sugar moiety reaction mixture exhibited a peak at tR =18.68 min, which was matched with derivatives of D-glucose, confirming the absolute configuration of the sugar moiety (L-glucose tR = 19.22 min). From previous data, the structure of compound 1 was identified as molecular species cerebroside, as shown in Figure 1. The name LAMA was assigned to compound 1.
Due to the considerable importance of determining the molecular species composition of sphingolipids, isolation and structure elucidation of the cerebroside components in the molecular species of LAMA were conducted. By using reversed phase column, LAMA was fractionated to give LAMA-1.  In addition to LAMA-1, the structures of other known compounds 2-4 ( Figure 1) were elucidated by comparing their detailed NMR spectral data with those in the literature. These compounds were identified as ergosterol (2) [28][29][30], epidioxyergosterol (3), [30,31], and kojic acid (4) [32-35].

Cytotoxic Activity of Isolated Compounds 1-4
The isolated compounds 1-4 were evaluated for their cytotoxic activity against five selected human cancer cell lines (

Biological Materials
The fungus Penicillium chrysogenum strain S003 (Figure 3) was cultured from deep-sea sediment from the Red Sea, and the fungal strain was identified based on a previously described method [26].

Biological Materials
The fungus Penicillium chrysogenum strain S003 (Figure 3) was cultured from deep-sea sediment from the Red Sea, and the fungal strain was identified based on a previously described method [26].

Fermentation and Extraction of Fungus Penicillium chrysogenum Strain S003
Fungus Penicillium chrysogenum strain S003 was cultured at 25 ℃ in 2 L Erlenmeyer flasks containing 500 mL of Czapek-Dox yeast liquid culture medium, composed of (NaNO3 3.0 g/L, KCl 0.5 g/L, K2HPO4 0.1 g/L, MgSO4·7H2O 0.5 g/L, FeSO4 0.01 g/L, sucrose 30.0 g/L, yeast extract 5.0 g/L and NaCl 20.0 g/L). After cultivation for 30 days under static conditions, 15 L of the whole broth was filtrated using cheesecloth. Extraction of the broth with ethyl acetate was performed three times, and was further dried under vacuum to afford an ethyl acetate extract (1.4 g), while the mycelia were extracted three times with MeOH. The MeOH solution was partitioned with n-hexane, followed by evaporation to give a MeOH extract and an n-hexane extract (5 g). The MeOH extract was dissolved in water and partitioned with CHCl3, followed by evaporation under reduced pressure to produce

Fermentation and Extraction of Fungus Penicillium chrysogenum Strain S003
Fungus Penicillium chrysogenum strain S003 was cultured at 25°C in 2 L Erlenmeyer flasks containing 500 mL of Czapek-Dox yeast liquid culture medium, composed of (NaNO 3 3.0 g/L, KCl 0.5 g/L, K 2 HPO 4 0.1 g/L, MgSO 4 ·7H 2 O 0.5 g/L, FeSO 4 0.01 g/L, sucrose 30.0 g/L, yeast extract 5.0 g/L and NaCl 20.0 g/L). After cultivation for 30 days under static conditions, 15 L of the whole broth was filtrated using cheesecloth. Extraction of the broth with ethyl acetate was performed three times, and was further dried under vacuum to afford an ethyl acetate extract (1.4 g), while the mycelia were extracted three times with MeOH. The MeOH solution was partitioned with n-hexane, followed by evaporation to give a MeOH extract and an n-hexane extract (5 g). The MeOH extract was dissolved in water and partitioned with CHCl 3 , followed by evaporation under reduced pressure to produce an CHCl 3 extract (2.8 g). The resulting extracts were subjected to further fractionation and separation to obtain the pure compounds.

Conclusions
Chemical investigation of deep-sediment-derived Penicillium chrysogenum S003 yielded a cerebroside molecular species (1), and three known compounds: ergosterol (2), epidioxyergosterol (3), and kojic acid (4). The chemical structures of purified compounds 1-4 were characterized using spectroscopic studies and by comparison with available data in the literature. The cytotoxic activities of isolated compounds 1-4 against five human carcinoma cells were evaluated using an SRB assay. Compounds 2 and 3 displayed a promising cytotoxic profiles against lung cancer (A-549), prostate cancer (DU-145), breast adenocarcinoma (MCF-7), and hepatocellular carcinoma (HepG2) cell lines with IC 50 values of 21.26, 19.3; 1.50, 6.10; 16.95, 13.6; and 2.89, 3.07 µM, respectively, while they were inactive against HeLa cells. Compounds 1 and 4 showed weak cytotoxic profiles against all cell lines under investigation.

Conflicts of Interest:
All contributing authors declare no conflicts of interest to disclose, whether financial or of any other nature.