Secondary metabolites from the fungus Emericella nidulans.

NIH Public Access Author Manuscript Nat Prod Commun. Author manuscript; available in PMC 2014 July 25.

NIH-PA Author Manuscript

Published in final edited form as: Nat Prod Commun. 2013 September ; 8(9): 1285–1288.

Secondary Metabolites from the Fungus Emericella nidulans Amer H. Tarawneha, Francisco Leóna, Mohamed M. Radwanb, Luiz H. Rosac, and Stephen J. Cutlera Stephen J. Cutler: [email protected] aDepartment

of Medicinal Chemistry, School of Pharmacy, The University of Mississippi, University, MS 38677, USA

bNational

Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS 38677, USA

cDepartamento

de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil


Abstract A new polyketide derivative koninginin H (1), has been isolated from the fungus Emericella nidulans, together with koninginin E (2), koninginin A (3), trichodermatide B (4), citrantifidiol (5), (4S,5R)-4-hydroxy-5-methylfuran-2-one (6), the glycerol derivatives gingerglycolipid B (7), (2S)-bis[9Z,12Z]-1-O, 2-O-dilinoleoyl-3-O-[α-D-galactopyranosyl-(1″→6′)β-Dgalactopyranosyl]glycerol (8), (2S)-bis[9Z,12Z]-1-O, 2-O-dilinoleoyl-3-O-β-Dgalactopyranosylglycerol (9), the cerebroside flavuside B (10), and the known sterols β-sitosterol glucoside and ergosta-5,7,22-trien-3-ol. Their structures were established by extensive NMR studies (1H NMR, 13C NMR, DEPT, 1H–1H COSY, HSQC, HMBC) and mass spectrometry. The antibacterial, antimalarial, antifungal and antileishmanial activities of compounds 1-10 were examined and the results indicated that compound 4 showed good antifungal activity against Cryptococcus neoformans with an IC50 value of 4.9 μg /mL.


Keywords Emericella nidulans; Koninginin; Cerebroside; Galactolipids In our continual search for bioactive compounds from filamentous fungi [1] a series of fungal extracts were screened for various biological activities, and the ethyl acetate extract of Emericella nidulans (Eidam) Vuill. showed the most promising activities and was selected for further studies. The genus Emericella (Ascomycota) is one of the sexual stages associated with Aspergillus [2]. E. nidulans is employed as a model organism in studies of cell biology and gene regulation, as well as in biological modeling [3,4]. Previous phytochemical studies revealed

Correspondence to: Stephen J. Cutler, [email protected].

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the presence of various polyketides with a benzophenone nucleus [5], as well as austin derivatives [6].


The present work describes the isolation and structural elucidation of the constituents of the ethyl acetate extracts of E. nidulans accessed from orange peel. A new polyketide, koninginin H (1), as well as eleven known compounds were isolated, including koninginin E (2) [7], koninginin A (3) [8], trichodermatide B (4) [9], citrantifidiol (5) [10], (4S,5R)-4hydroxy-5-methylfuran-2-one (6) [11], the glycerol derivatives [12]: gingerglycolipid B (7), (2S)-bis[9Z,12Z]-1-O, 2-O-dilinoleoyl-3-O-[α-D-galactorpyranosyl-(1″→6′) β-Dgalactopyranosyl]glycerol (8), (2S)-bis[9Z,12Z]-1-O,2-O-dilinoleoyl-3-O-β-Dgalactopyranosyl glycerol (9), the cerebroside flavuside B 10 [13], and the sterols βsitosterol glucoside and ergosta-5,7,22-trien-3-ol. The structures of the known compounds were confirmed by comparison of their spectroscopic properties with published data. Additionally, the identification of the fatty acids and sugars presented in the glycerol derivatives were confirmed by transesterification and analyzed by GC/MS. To assess whether isolated compounds 1-10 display biological activities, we examined their in vitro antibacterial, antimalarial, antifungal and antileishmanial activities.

NIH-PA Author Manuscript NIH-PA Author Manuscript

Compound 1 was obtained as an amorphous solid. HRESIMS experiments indicated the molecular formula C16H27O5 (calc. for [M + H]+ 299.1858; found 299.1848). The IR spectrum exhibited absorption bands for OH (3252 cm-1) and for a carbonyl group (1615 cm-1). The presence of these groups was confirmed by the 1H and 13C NMR spectra (Table 1). The 13C NMR spectrum exhibited signals for 16 carbons, including one methyl group, eight aliphatic methylenes, four geminal to a heteroatom at δC 66.0, 67.4 72.6, and 81.7, as well as one α,-β-unsaturated carbonyl group at δC 197.5, and substituted olefinic carbons at δC 111.8 and 171.5. These findings, as well as the mass spectral data, suggested a koninginin type polyketide [7, 14]. Both the 1H and 13C NMR spectral data of compound 1 were close to those of koninginin E [7] 2, with the exception of an extra hydroxyl group in compound 1; a detailed comparison of the 13C NMR data are shown in Table 1. Correlation between the signal of the methyl group at δH 1.33 and a proton belonging to an oxygenated carbon at δH 4.02 was obtained in the 1H-1H COSY experiment. Also, in the HMBC experiment, correlation was observed between the methyl group at δH 1.33 and the carbons at δC 40.6 and 67.4 corresponding to C-14, and C-15, respectively. These data confirmed the position of the hydroxyl group at C-15. The relative configuration at C-4, C-9 and C-10 was deduced by comparison of the NMR chemical shifts with those exhibited by similar known compounds [7], and by NOESY experiment. However, the stereochemistry at C-15 of the side-chain could not be confirmed since 1 is a minor secondary metabolite and could not be isolated in enough quantity to perform this study. Due to the relationship of structure 1 to the koninginins it is named koninginin H, the next consecutive designation in this nomenclature. Interestingly, the polyketides of koninginin-type were all isolated from the genus Trichoderma, with the exception of compound (2S)-8-hydroxy-2-((S)-1hydroxyheptyl)-2,6,7,8-tetrahydro-5H-chromen -5-one and analog, which were isolated from Aspergillus espii [15]. This is the first time that this kind of polyketide has been isolated from the Emericella genus. The chemical diversity reported here suggests the high

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probability that different secondary metabolites can be obtained using various stress conditions such as fermentation methods and media.


The isolated compounds 1–10 (Figure 1) were evaluated for their antibacterial, antifungal, antimalarial and antileishmanial activities. The antibacterial activities were tested against Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Escherichia coli, Pseudomonas aeruginosa, and Mycobacterium intracellulare. None of these compounds showed in vitro antibacterial activity. The antifungal activities were evaluated against a panel of pathogenic fungi (Candida albicans, C. glabrata, C. krusei, Cryptococcus neoformans, and Aspergillus fumigatus) associated with opportunistic infections. Amphotericin B was included as a standard antifungal drug for comparison. Only compound 4 exhibited good antifungal activity against Cryptococcus neoformans with an IC50 value of 4.9 μg /mL. The compounds tested were inactive in the antimalarial and antileishmanial bioassays.

Experimental General


Optical rotations were recorded using a Rudolph Research Analytical Autopol V Polarimeter. UV was obtained using a Perkin-Elmer Lambda 3B UV/visspectrophotomer. 1H and 13C NMR spectra were measured on a Bruker model AMX 500 NMR spectrometer with standard pulse sequences, operating at 500 MHz for 1H and 125 MHz for 13C. The chemical shift values were reported in parts per million (ppm) using the residual solvent signal as internal standard. Coupling constants were recorded in Hertz (Hz). Standard pulse sequences were used for COSY, HMQC, HMBC, TOCSY, NOESY and DEPT. High-resolution mass spectra (HRMS) were measured on a Micromass Q-Tof Micro mass spectrometer with a lock spray source. CC was carried out on silica gel (70-230 mesh, Merck), C18 and SPE columns (500 mg Bed, Thermo scientific), and TLC (silica gel 60 F254) was used to monitor fractions from CC. Preparative TLC was carried out on silica gel 60 PF254+366 plates (20 × 20 cm, 1 mm thick). Visualization of the plates was achieved with a UV lamp (λ=254 and 365 nm) and anisaldehyde/acid spray reagent (MeOH-acetic acid-anisaldehyde-sulfuric acid, 85:9:1:5). All HPLC analyses were performed on a Waters LC Module I equipped with a UV detector 486 utilizing the Millenium 32 Chromatography Manager software (Waters). An ODS column (Phenomenex Luna C18, 10 × 250 mm, 5 μm) was used. All HPLC solvents were HPLC grade, filtered through appropriate filters (water through 0.45 μm and organic solvents through 0.22 μm filters) and purged prior to and during analysis with nitrogen at a flow rate of 5 mL/min. GC/MS analyses were carried out on a ThermoQuest Trace 2000 GC, equipped with a single split/splitless capillary injector, a ThermoQuest AS2000 autosampler and a Phenomenex ZB-5 column (30 m × 0.25 mm × 0.25 μm), interfaced to a Thermo Quest-Finnigan Trace MS quadrupole ion trap detector. The injector temperature was 250°C and 1 μL injections were added in splitless mode, with the splitless time set at 60 s, the split flow at 50 mL/min and the septum purge valve set to close 60 s after the injection occurred. The oven temperature was raised from 70 to 270°C (hold 20 min) at a rate of 5°C/min, for a total run time of 60 min; the transfer line temperature was 250°C. Helium was used as the carrier gas at a constant pressure of 20 psi.


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The mass spectrometer was operated in the electron impact mode (EI+) and scanned from 40 to 800 amu at 1 scan/s, with an ionizing voltage of 70 eV and an emission current of 350 μA. Data were recorded using an IBM Netfinity 3000 Workstation with Microsoft Windows NT 4.0 operating system (Build 1381, Service pack 6) and Xcalibur data acquisition and analysis software (Version 1.2). The NIST Mass Spectral Search Program (Version 1.7, Build 11/05/1999) was used for the NIST/EPA/NIH. Linoleic acid standard was purchased from Sigma-Aldrich Chemical Co. (Germany). Fungal material


The strain of Emericella nidulans (Eidam) Vuill. used in this study was collected from a piece of orange peel in Tifton, Georgia in 1978 and its identification was confirmed through phylogenetic, physiological and morphological analysis. A voucher specimen (UM-032009) has been deposited in the culture collection of the Medicinal Chemistry Department, University of Mississippi. The sequence of the fungal strain displayed 99% of identity with different sequences of Aspergillus and Emericella species deposited in GenBank database. However, according to Gazis et al. and Ko et al. [16], sequencing of the ITS region may fail to recognize some Ascomycota taxa and for this reason there are several erroneous fungal sequences deposited in GenBank. To increase the taxonomic accuracy and avoid mistakes on the phylogenetic inferences, the ITS1-5.8S-ITS2 nuclear ribosomal gene sequence of the strain was compared with sequences of type species of Aspergillus and Emericella. The nucleotides difference among the strain and the sequence of the type species E. nidulans NRRL 2395 (AY373888) was of the 10 nucleotide (0.2%). In addition, the strain displayed in CYA after 7 days at 25 ± 2°C, under cool-white fluorescent lights (55 ± 5 μmol/m2/s) with a 12 h photoperiod, colonies spreading rapidly, velvety, in green shades with a whitish margin; anamorph state dominating, with scattered green ascomata within and upon the conidial layer. Ascomata globose, green, solitary, 200 μm diam., maturing in 10 days, softwalled. Asci with 10 μm diam. Ascospores lenticular, 4.0 × 3.0 μm, green, maturing within 7 days, smooth-walled. Using physiological, morphological characteristics and molecular techniques, the strain was identified as Emericella nidulans (Eidam) Vuill. (Ascomycota). Fermentation, extraction and isolation


The broth extract (24.0 g) was obtained as a dark brown gum using the same procedure previously reported [1]. The EtOAc extract (23.0 g) was chromatographed by Si gel Vacuum liquid chromatography (VLC) with fractions stepwise from hexanes to methanol, yielding 8 fractions, each of 250 mL (hexanes; 3:1 hexanes-EtOAc; 2:2 hexanes-EtOAc; 1:3 hexanesEtOAc; EtOAc; 3:1 EtOAc-MeOH; 2:2 EtOAc-MeOH; MeOH). The fraction 1:1 hexanesEtOAc (1.9 g) was purified by VLC with a gradient of MeOH in EtOAc to yield 9 subfractions. Subfraction 2 was purified by crystallization from hexanes/EtOAc to yield the known compound ergosta-5,7,22-trien-3-ol (20 mg). Subfraction 4 was rechromatographed on a C18 solid-phase extraction (SPE) column using MeOH-H2O (4:1) to yield β-sitosterol glucoside (20 mg). Subfractions 7 and 8 were combined and purified over VLC with a gradient of MeOH in DCM and by reverse phase HPLC to afford koninginin H 1 (3.5 mg) and compound 4 (10 mg). The 1:3 hexanes-EtOAc fraction (1.4 g) was purified by VLC with a gradient of MeOH in EtOAc to yield 8 subfractions. Subfraction 3 was rechromatographed by CC over silica gel in dichloromethane (DCM) to give 2 (18.7 mg). Nat Prod Commun. Author manuscript; available in PMC 2014 July 25.

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The fraction eluted by EtOAc (4 g) was subjected to VLC with a gradient of MeOH in EtOAc to yield 4 subfractions, each of 500 mL. The subfractions eluted with EtOAc-MeOH (1:1) (2 g) were purified by C-18 SPE CC using MeOH-H2O (65:35) to yield subfractions A-F. Subfraction B (200 mg) afforded compound 10 (15 mg). Subfraction C (45 mg) was purified by HPLC to give compounds 6 (5 mg) and 7 (15 mg). From subfraction D (80 mg) compound 8 (22 mg) was isolated after C-18 SPE CC using MeOH-H2O (65:35) as eluent. The fraction 1:3 MeOH-EtOAc (1.8 g) was purified by VLC with a gradient of MeOH in EtOAc to yield 10 subfractions, each of 500 mL. Subfraction 4 was purified by HPLC to give compound 5 (12 mg). Subfraction 5 was subjected to Si-CC using DCM-MeOH (19:1) as mobile phase and crystallization from hexanes/EtOAc to give compound 3 (25 mg). Subfraction 9 was chromatographed by C-18 SPE CC using MeOH-H2O (90:10) as eluent to yield compound 9 (20 mg).

Koninginin H (1) [α]25D: + 80 (c 0.01, MeOH) IR (KBr) νmax: 3252, 2928, 2854, 1716, 1615, 1558, 1396, 1245, 1194, 1088 cm-1


UV/Vis λmax (MeOH) nm (log ε): 274 (3.57), 255 (3.77). 1H

NMR (Pyridine-d5, 500 MHz): Table 1.

13C

NMR (Pyridine-d5, 125 MHz): Table 1.

neg HRESIMS m/z: [M + Cl]− 333.1502 (Calcd. for C16H26Cl35O5, 333.1469), 335.1485 (Calcd. for C16H26Cl37O5, 335.1439); pos HRESIMS m/z [M + H]+ 299.1848 (Calcd. for C16H27O5, 299.1858), [M + Na]+ 321.1629 (Calcd. for C16H26NaO5, 321.1678). Transesterification of galactolipids 7-9 Compounds (4 mg/each) were subject to direct transesterification by heating at 80°C for 3 h with 2.5 mL of 2% sulfuric acid in methanol. The reaction mixture was partitioned between MeOH and n-hexanes. The n-hexanes layer containing the fatty acid methyl esters was analyzed by GC/MS.


In vitro antimicrobial assay Compounds 1-10 were tested for antimicrobial activity against a panel of microorganism obtained from the American Type Culture Collection (Manassas, VA) and included the fungi Candida albicans ATCC 90028, C. glabrata ATCC 90030, C. krusei ATCC 6258, Cryptococcus neoformans ATCC 90113, and Aspergillus fumigatus ATCC 204305, and the bacteria Staphylococcus aureus ATCC 29213, methicillin-resistant S. aureus ATCC 33591 (MRSA), Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, and Mycobacterium intracellular ATCC 23068. The bioassays were performed as described previously [17].


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In vitro antimalarial and antileishmanial assays


Antimalarial activity was determined in vitro against chloroquine sensitive (D6, Sierra Leone) and resistant (W2, Indo China) strains of Plasmodium falciparum by measuring plasmodial LDH activity, as described earlier [18]. The antileishmanial activity of the compounds was tested in vitro against a culture of Leishmania donovani promastigotes [19].

Acknowledgments The project described was supported by Grant Number 5P20RR021929 from the National Center for Research Resources (NCRR) and 9P20GM104932 from the National Institute of General Medical Sciences, components of the National Institutes of Health (NIH). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Furthermore, this investigation was conducted in a facility constructed with support from research facilities improvement program C06 RR14503 from the NIH National Center for Research Resources. Dr Luiz H. Rosa was supported by the Conselho Nacional of Desenvolvimento Científico and Tecnológico (CNPq).

References


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Figure 1.

Compounds 1-10 isolated from Emericella nidulas.


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Table 1


NMR spectroscopic data for 1 in (C5D5N)a and 2. 1 δC

Position. 1

197.5

2

33.6

3 4 5 6 7

19.0

8

23.3

2

δH (J in Hz)

δCa

δCb

197.4

197.3

3.00 m, 2.43 m

33.6

33.8

30.9

2.18 m

30.9

29.3

66.0

4.58 br s

66.0

66.6

171.5

171.4

169.3

111.8

111.8

111.9

2.45 m

18.9

17.3

1.93 m, 1.84 m

23.3

22.9


9

81.7

1.02 m

81.6

80.6

10

72.6

3.9 m

72.7

72.9

11

33.70

1.86 m, 1.71 m

32.4

32.8

12

26.8

1.64 m

26.6

25.3

13

27.0

1.64 m

30.0

29.3

14

40.6

1.72 m, 1.56 m

30.9

31.8

15

67.4

4.02 m

23.2

22.6

16

24.7

1.33 d (6.0 )

14.5

14.1

a1

H NMR (500 MHz) and 13C NMR (125 MHz).

b

CDCl3 ref. 7


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