African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 5233

Full Length Research Paper

Diketopiperazine alkaloids produced by the endophytic fungus Penicillium citrinum and evaluation of their antileishmanial activity

Luiza Guimarães Tunes
  • Luiza Guimarães Tunes
  • Genômica Funcional e Proteômica de Leishmania ssp. e Trypanosoma cruzi, Instituto René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, MG, Brazil.
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Vívian Nicolau Gonçalves
  • Vívian Nicolau Gonçalves
  • Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
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Daniela Nabak Bueno
  • Daniela Nabak Bueno
  • Laboratório de Química de Produtos Naturais Bioativos, Instituto René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, MG, Brazil.
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Carlos Leomar Zani
  • Carlos Leomar Zani
  • Laboratório de Química de Produtos Naturais Bioativos, Instituto René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, MG, Brazil.
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Luiz Henrique Rosa
  • Luiz Henrique Rosa
  • Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
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Betania Barros Cota
  • Betania Barros Cota
  • Laboratório de Química de Produtos Naturais Bioativos, Instituto René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, MG, Brazil.
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  •  Received: 26 June 2019
  •  Accepted: 26 August 2019
  •  Published: 31 October 2019

 ABSTRACT

Chromatographic fractionation of the antileishmanial extract obtained from fermentation of the endophytic fungus Penicillium citrinum, isolated from leaves of Ageratum myriadenia, yielded three diketopiperazine alkaloids; cyclo(L-Pro-L-Leu) (1), cyclo-(L-Pro-L-Phe) (2) and tryprostatin B (3). The structures of these compounds were established on the basis of spectroscopic methods and comparison with the literature. Compounds 1 and 2 were active against both amastigote-like forms of Leishmania (Leishmania) amazonensis and intracelular amastigotes of L. (Leishmania) infantum with approximately 50% of parasite growth inhibition at 100 μM. None of the compounds were considered toxic against human leukemia monocyte cell line (THP-1) at 100 μM. It is the first report about isolation of these diketopiperazines from P. citrinum and their antileishmanial potential against L. (L.) infantum.

 

Key words: Microfungi, natural products, in vitro, Leishmania, amphotericin B, cytotoxic activity.


 INTRODUCTION

Penicillium is cosmopolitan genera and comprises pathogens, saprobes, opportunists or endophytes taxa, which are able to produce a large number of secondary metabolites being reported 1338 exometabolites (Frisvad, 2015). Penicillium citrinum is assigned to section Citrina with a worldwide distribution, occurring commonly in soils; they  are  known  as  producers  of  the  mycotoxins citrinin and citreoviridin (Houbraken et al., 2011).
 
Penicitrinine A has been claimed as antitumor agents in patents (Zheng et al., 2015; Ying et al., 2015). Other secondary metabolites isolated from these species such as penicillanthranin A and chrysophanol, which are anthraquinone-citrinin derivatives, have exhibited moderate  antibacterial  activity   against  Staphylococcus
 
aureus, both with MIC values of 16 mg/ml (Khamthong et al., 2012).
 
In previous study, it was shown that the crude extract from the fungus Penicillium citrinum UFMGCB 579, isolated from leaves of bioactive plant species Ageratum myriadenia, had antileishmanial activity (Rosa et al., 2010) in the assays using amastigotes-like forms of Leishmania (Leishmania) amazonensis. Chemical investigation of the ethyl acetate extracts from the broth and mycelia of P. citrinum UFMGCB 579 led to the isolation of three metabolites. In this study, the occurrence of three diketopiperazine alkaloids in addition to the evaluation of their antileishmanial activity is reported.

 


 MATERIALS AND METHODS

General experimental procedures
 
Thin-layer chromatography (TLC) analyses were conducted on silica gel G-60/F254 (0.25 mm, Merck) and the spots were visualized under visible light after heating the plate sprayed with a mixture (1:1) of ethanolic solutions of vanillin (1% w/v) and sulfuric acid (10% v/v). Gel Permeation Chromatography (GPC) was carried out by using two coupled glass columns (Büchi column n° 17980) filled with Sephadex LH-20 TM (GE Healthcare, U.S.A.) gel. Semi-preparative HPLC purifications were carried out by a Shimadzu chromatographic system (Shimadzu, Kyoto, Japan), equipped with a LC6AD pump and a dual wavelength detector (SPD10A). The mass spectra were acquired in a maXis ETD high-resolution ESI-QTOF mass spectrometer (Bruker Daltonics, Bremen, Germany). The optical rotations were determined on a Modular Circular Polarimeter MCP 300 (Anton Paar, Ashland, Virginia, USA). 1D and 2D nuclear magnetic resonance (NMR) experiments were run on a Bruker Avance 400 MHz spectrometer (Bruker Biospin, Rheinstetten, Germany).
 
Fungal isolation
 
The endophytic fungus P. citrinum was isolated from the plant Ageratum myriadenia (DC) R.M. King & H. Rob (Asteraceae) that was deposited in the herbarium of the “Departamento de Botânica da Universidade Federal de Minas Gerais” under the code BHCB 5816. A fungal sample was deposited at “Coleção de Microrganismos e Células da Universidade Federal de Minas Gerais” under the code UFMGCB 579 and at GenBank by accession number FJ466725 (Rosa et al., 2010).
 
Fermentation and extraction
 
The fungus was cultured in a bioreactor containing 10 L of MEC medium (2% malt extract, 0.1% peptone, 1.5% glucose) for fourteen days at 28°C and 150 rpm. Both filtrate and biomass were extracted with ethyl acetate (EtOAc). The organic solvent was removed using a rotary evaporator to afforded 3.3 g of EtOAc extract.
 
Isolation of secondary metabolites from the endophytic fungi
 
All extract was subjected to GPC using EtOH as mobile phase at flow rate of 120 ml/h to  produce  16  fractions  after  TLC  analyses.
 
Fraction 6 (210 mg) was purified on semi-preparative HPLC, using a Shim-pack® C18 column (5 μm, 250 x 20 mm i.d.), detection at λ 220 and 254 nm, flow rate of 7 ml/min and eluted with mixture of MeOH:H2O (MeOH, 10-100% in 60 min and 100% for 10 min) to obtain 1 (12 mg). Fraction 8 (220 mg) was also purified as same conditions and yielded compounds 2 (5 mg) and 3 (2.8 mg), respectively.
 
Cyclo(L-Pro-L-Leu) ((3S,8aS)-3-(2-methylpropyl)-2,3,6,7,8,8a-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione)(1) is a colorless amorphous solid (8.0 mg); [a]D25 -117.0 (c 0.40, MeOH). The molecular formula is C11H18N2O2, as determined by HR-ESI-MS (m/z 211.1419, [M+H]+; calc. for C11H19N2O2: 211.1441). 1H NMR (400 MHz, CDCl3) δ ppm 0.96 (d, J= 6.58 Hz, H-12b), 1.0 (d, J= 6.58 Hz, H-12a), 1.76 (m, H-11), 2.02 and 1.90 (m, H-4b and a), 2.35 and 2.13 (m, H-5b and a), 2.07 and 1.53 (m, H-10b and a), 3.57 (m, H-3), 4.02 (dd, J=9.38, 3.47 Hz, H-9), 4.12 (t, J=8.14 Hz, H-6), 6.07 (br s, NH).13C NMR (100 MHz, CDCl3) δ ppm 21.2 (CH3, C-12b), 22.8 (CH2, C-4), 23.3 (CH3, C-12a), 24.7 (CH, C-11), 28.1 (CH2, C-5), 38.7 (CH2, C-10), 45.5 (CH2, C-3), 53.4 (CH, C-9), 59.0 (CH, C-6), 166.2 (C=O, C-1), 170.3 (C=O, C-7).
 
Cyclo-(L-Pro-L-Phe) ((3S,8aS)-3-benzyl-2,3,6,7,8,8a-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione) (2) is a colorless amorphous solid (5.0 mg); [a]D25 -71.5 (c 0.25, MeOH). The molecular formula is C14H16N2O2, as determined by HR-ESI-MS (m/z 245.1255, [M+H]+; calc. for C14H17N2O2: 245.1285).1H NMR (400 MHz, CDCl3) δ ppm 1.80 (m, H-4), 2.09 and 1.24 (m, H-5), 3.16 (d, J=5.01 Hz, H-10), 3.36 and 3.54 (m, H-3), 4.06 (ddd, J= 10.75, 6.44, 1.67 Hz, H-6), 4.43 (tt, J= 5.01, 5.01, 0.99 Hz, H-9), 7.23 (br m., H-2’/6’ and H-4’), 7.27 (br m, H-3’/5’).13C NMR (100 MHz, CDCl3) δ ppm 21.4 (CH2, C-4), 28.0 (CH2, C-5), 36.8 (CH2, C-10), 44.6 (CH2, C-3), 56.3 (CH, C-9), 58.7 (CH, C-6), 126.7 (CH, C-4’), 129.6 (CH, C-2’/-6’), 128.1 (CH, C-3’/-5’), 136.0 (C, C-1’), 165.5 (C=O, C-1), 169.5 (C=O, C-7).
 
Tryprostatin B ((3S,8aS)-3-[[2-(3-methylbut-2-enyl)-1H-indol-3-yl]methyl]-2,3,6,7,8,8a-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione) (3) is a pale yellow amorphous solid (2.8 mg); [a]D25 -29.5 (c 0.14, MeOH). The molecular formula was C21H25N3O2, as determined by HR-ESI-MS (m/z 352.2019, [M+H]+; calc. for C21H26N3O2: 352.2020). 1H NMR (400 MHz, CDCl3) δ ppm 1.65 and 1.38 (m, H-14), 1.75 (br s, H-21), 1.76 (br s, H-22), 1.95 and 0.93 (m, H-13), 3.20 (dd, J= 14.80, 6.90 Hz H-8b), 3.24 (dd, J= 11.3, 3.9 Hz, H-15b), 3.34 (d J= 4.60 Hz, overlapped, H-8a), 3.49 (m, H-15a)  3.50 (m, H-18), 3.97 (ddd, J=10.87, 6.51, 1.44 Hz, H-12), 4.37 (ddd, J=6.80, 4.6, 1.8 Hz, H-9), 5.35 (tt, J= 7.21, 7.21, 1.34 Hz, H-19), 6.96 (td, J= 7.52, 7.52, 1.15 Hz, H-5), 7.02 (td, J= 7.52, 7.52, 1.15 Hz, H-6), 7.27 (d, J= 7.94 Hz, H-7), 7.46 (d, J= 7.81 Hz, H-4), 8.52 (s, NH-1). 13C NMR (101 MHz, CD3OD-d4) δ ppm 18.0 (CH3, C-22), 22.5 (CH2, C-14), 25.9 (CH3, C-21), 26.2 (CH2, C-18), 28.6 (CH2, C-8), 29.0 (CH2, C-13), 46.0 (CH2, C-15), 57.2 (CH, C-9), 60.1 (CH, C-12), 104.5 (C, C-3), 111.7 (CH, C-7), 119.1 (CH, C-4), 119.8 (CH, C-5), 121.9 (CH, C-6), 122.1 (CH, C-19), 129.5 (C, C-3a and C-2), 134.7 (C, C-20), 137.3 (C, C-7a), 167.5 (C, C-17), 170.4 (C, C-11).
 
Assays of anti-leishmanial activity
 
Stationary phase promastigotes of L. (Leishmania) amazonensis (strain IFLA/BR/67/PH-8) were stimulated to differentiate into amastigote-like forms by rising the incubation temperature to 32°C and lowering the pH of the medium to 6.0. The parasites were seeded in 96-well plates using 90 μl of parasite suspension at 16 × 108 parasites per milliliter, followed by 10 μl of the sample-tests to give a final concentration of 100 μM. Amphotericin B at 0.5 μM was used as positive control. The plates were incubated at 32°C for 72 h and the number of parasites was estimated using the methyl thiazolyl tetrazolium (MTT). The results were calculated from the absorbance  measurements  using the percentage of parasite death in relation to the controls without drug (Teixeira et al., 2002).
 
 
THP-1, maintained in RPMI 1640 medium supplemented with 10 % FBS cells and differentiated in the presence of 20 ng/ml phorbol myristate acetate (PMA) for 72 h at 37°C, were infected at a parasite/macrophage ratio of 10:1 for 3 h with L. (Leishmania) infantum (strain MHOM/MA/67/ITMAP-263) promastigotes expressing the firefly luciferase as reporter gene. Non-internalized parasites were removed by five washes with HEPES/NaCl buffer (20 mM HEPES, 0.15 M NaCl, 10 mM glucose, pH 7.2). The infected cells were then treated with 100 μM of the sample-tests and of amphotericin B at 0.5 μM. After 72 h, RPMI was aspirated and the luciferase activity was assessed by adding 20 μl of reconstituted One-Glo™ Luciferase Assay System solution as enzyme substrate (Promega, Madison, WI, USA). Luciferase activity was measured in a luminometer SpectraMax M5 (Molecular Devices, Sunnyvale, CA, USA) using 1 s integration/well (Roy et al., 2000).
 
Cell viability assays
 
Non-infected THP-1 macrophages were used as signal background while non-treated infected THP-1 cells were used as control for growth comparison (Garcia et al., 2013). THP-1 macrophages were seeded in 96-well plates at a density of 2 × 105 cells per well. After 72 h treatment with compounds at a final concentration of 100 μM, the cell death was estimated using the MTT. The results were calculated from the absorbance measurements using the percentage of cell death in relation to the controls of untreated cells.
 
Statistical analysis
 
Statistical analyses were performed using Graph Pad Prism software 5.03 (GraphPad Software, Inc., San Diego, CA, USA). Differences were assessed by analysis of variance (ANOVA). Differences were considered statistically significant when p< 0.05.


 RESULTS AND DISCUSSION

In an ongoing survey of the bioactive potential of microorganisms present in the Brazilian ecosystems, the EtOAc extract of an endophytic fungus P. citrinum UFMGCB 579 was able to inhibit the growth of amastigotes-like forms of L. (L.) amazonensis by 86% at 20 mg/ml and showed an IC50 value of 4.6 mg/ml (Rosa et al., 2010). The crude EtOAc extract was subjected to preparative GPC by medium pressure liquid chromatography to afford a series of fractions. HPLC purification yielded the known compounds cyclo-(L-Pro-D-Leu) (1), cyclo-(L-Pro-L-Phe) (Campbell et al., 2009) (2) and tryprostatin B (3) (Cui et al., 1996) (Figure 1).
 
These compounds were elucidated by comparing their spectral data (MS, and 1D and 2D NMR data) and specific rotation values with those in the literature values.
 
 
The cyclo-(L-Pro-Leu)  (1)  was  reported  to be isolated  from the EtOAc extract of the culture broth of the marine fungus Penicillium chrysogenum, which was obtained from the North China Sea (Wang et al., 2014) and it was obtained together with cyclo-(L-Pro-L-Phe) (2) from a neomycin resistant mutant of the marine-derived fungus Penicillium purpurogenum G59 (Wang et al., 2016).
 
Compounds 1 and 2 have antibacterial activity against gram positive and gram negative bacteria (Kumar et al., 2012; Mangamuri et al., 2016) and tryprostatin B (3) is a microtubule inhibitor on the cell cycle progression in the M phase of mouse tsFT210 cells (Cui et al., 1996). Compound 3 has also immunosuppressive activity against mouse splenic lymphocytes stimulated with lipopolysaccharide (IC50 3 mg/ml) (Fujimoto et al., 2000) and cytotoxic activity against human leukemia cancer cell line K562 (IC50 21.1 μM) (Wollinsky et al., 2012).
 
Compounds 1 and 2 were active against both intracellular amastigotes and amastigote-like forms of Leishmania parasites, showing approximately 50% of parasite growth inhibition at 100 μM (Figure 2). Compound 3 was more active in the amastigote-like model than in the intracellular amastigote form of the parasite (p < 0.001), showing a reduction of parasite growth of 55 and 19%, respectively. In addition, treatment with amphotericin B reduced more intracellular amastigote form of the parasite than amastigote-like form (p < 0.05). This could be explained by the different susceptibility of different species of Leishmania or by an activation of the compound by the macrophage. The variation of susceptibility to drugs in different species and strains of Leishmania is described in clinical isolates and reference strains, reinforcing the difficulty to find a single drug that can treat all forms of leishmaniasis (Fernández et al., 2012). The intracellular amastigote model is the best to represent human infection and it is important in the drug discovery process because it is the only in vitro model that allows the discovery of pro-drugs that need to be metabolized by macrophages in order to become active, such as antimony, and of drugs that activates the immune response of the macrophages in order to eliminate the parasites (Siqueira-Neto et al., 2010).
 
 
None of the compounds (1-3) were toxic for THP-1 cells when they were tested up to 100 mM (p < 0.0001, Figure 3). They reduced the viability of cells less than 10% at 100 mM (1 = 1%; 2 = 0%; 3 = 8%). Amphotericin B, although more active than the compounds against the parasites, is also more toxic, showing 95% of cell death at the same concentration.
 

 


 CONCLUSION

This study demonstrated that the endophytic fungus P. citrinum is a source of the diketopiperazine alkaloids. To the researchers’ knowledge, this is the first report on the occurrence of tryprostatin B in Penicillium species and of the diketopiperazines (1-2) in the P. citrinum. The biological evaluation of these compounds showed that they are not toxic for THP-1 although they have shown weak antiparasitic potential against two Leishmania species in comparison with amphotericin B.

 


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.


 ACKNOWLEDGEMENTS

The authors are grateful to the FAPEMIG (APQ-00193-08), CAPES and to the Program of Technological Development in Tools for Health (PDTIS-FIOCRUZ), RPT13A platform for the use of its facilities. They are especially grateful to Rubens Lima do Monte Neto for helping with bioassays and Silvia Reni Bortolin  Uliana for providing the Leishmania strain.



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