Antifungal activity of liriodenine on agents of systemic mycoses, with emphasis on the genus Paracoccidioides

Adriele Dandara Levorato Vinche Iván de- la-Cruz-Chacón Alma Rosa González-Esquinca Julhiany de Fátima da Silva Gisela Ferreira Daniela Carvalho dos Santos Hans Garcia Garces Daniela Vanessa Moris de Oliveira Camila Marçon Ricardo de Souza Cavalcante Rinaldo Poncio Mendes About the authors

Abstract

Background:

Endemic systemic mycoses remain a health challenge, since these opportunistic diseases are increasingly infecting immunosuppressed patients. The simultaneous use of antifungal compounds and other drugs to treat infectious or non-infectious diseases has led to several interactions and undesirable effects. Thus, new antifungal compounds should be investigated. The present study aimed to evaluate the activity of liriodenine extracted from Annona macroprophyllata on agents of systemic mycoses, with emphasis on the genus Paracoccidioides.

Methods:

The minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were determined by the microdilution method. The cellular alterations caused by liriodenine on a standard P. brasiliensis (Pb18) strain were evaluated by transmission and scanning electron microscopy.

Results:

Liriodenine was effective only in 3 of the 8 strains of the genus Paracoccidioides and in the Histoplasma capsulatum strain, in a very low concentration (MIC of 1.95 μg.mL-1); on yeasts of Candida spp. (MIC of 125 to 250 μg.mL-1), including C. krusei (250 μg.mL-1), which has intrinsic resistance to fluconazole; and in Cryptococcus neoformans and Cryptococcus gattii (MIC of 62.5 μg.mL-1). However, liriodenine was not effective against Aspergillus fumigatus at the studied concentrations. Liriodenine exhibited fungicidal activity against all standard strains and clinical isolates that showed to be susceptible by in vitro tests. Electron microscopy revealed cytoplasmic alterations and damage to the cell wall of P. brasiliensis (Pb18).

Conclusion:

Our results indicate that liriodenine is a promising fungicidal compound that should undergo further investigation with some chemical modifications.

Keywords:
Liriodenine; Paracoccidioidomycosis; Systemic mycosis; Antifungal compounds; Antimicrobial compounds; Medicinal plants

Background

Endemic systemic mycoses remain a health challenge, especially because these opportunistic affections are continuously infecting immunosuppressed patients. The simultaneous use of antifungal compounds and other drugs to treat infectious or non-infectious diseases has led to several interactions and undesirable effects. Therefore, alternative antifungal compounds should be found. The present study aimed to evaluate the activity of liriodenine extracted from Annona macroprophyllata on agents of systemic mycoses, particularly the genus Paracoccidioides.

Paracoccidioidomycosis (PCM) is a systemic mycosis limited to Latin America and is caused by different species of the genus Paracoccidioides - P. brasiliensis complex, P. americana, P. restrepiensis, P. venezuelensis, and P. lutzii [11. Mendes RP, Cavalcante RS, Marques SA, Marques MEA, Venturini J, Sylvestre TF, et al. Paracoccidioidomycosis: current perspectives from Brazil. Open Microbiol J. 2017 Oct 31;11:224-82.

2. Teixeira MM, Theodoro RC, de Carvalho MJ, Fernandes L, Paes HC, Hahn RC, et al. Phylogenetic analysis reveals a high level of speciation in the Paracoccidioides genus. Mol Phylogenet Evol. 2009 Aug;52(2):273-83.

3.Matute DR, McEwen JG, Puccia R, Montes BA, San-Blas G, Bagagli E, et al. Cryptic speciation and recombination in the fungus Paracoccidioides brasiliensis as revealed by gene genealogies. Mol Biol Evol. 2006 Sep 8;23(1):65-73.

4. Muñoz JF, Farrer RA, Desjardins CA, Gallo JE, Sykes S, Sakthikumar S, et al. Genome diversity, recombination, and virulence across the major lineages of Paracoccidioides. mSphere. 2016;1.
-55. Turissini DA, Gomez OM, Teixeira MM, McEwen JG, Matute DR. Species boundaries in the human pathogen Paracoccidioides. Fungal Genet Biol. 2017 Sep;106:9-25.]. These fungi are the main causative agents of endemic mycoses in this region [11. Mendes RP, Cavalcante RS, Marques SA, Marques MEA, Venturini J, Sylvestre TF, et al. Paracoccidioidomycosis: current perspectives from Brazil. Open Microbiol J. 2017 Oct 31;11:224-82.]. PCM affects mainly rural workers, leads to severe pulmonary sequelae in most cases, is considered a neglected disease, and is the 8th cause of death among the chronic diseases in Brazil [66. Mendes RP. Paracoccidioidomycosis surveillance and control. J Venom Anim Toxins incl Trop Dis. 2010;16(2):194-7. https://doi.org/10.1590/S1678-91992010000200003.
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8. Kamikawa CM, Mendes RP, Vicentini AP. Standardization and validation of Dot-ELISA assay for Paracoccidioides brasiliensis antibody detection. J Venom Anim Toxins incl Trop Dis. 2017 Feb 15;23:11. https://doi.org/10.1186/s40409-017-0101-3.
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9. Ribeiro SM, Nunes TF, Cavalcante RS, Paniago AMM, Mendes RP. A scoping study of pulmonary paracoccidioidomycosis: severity classification on radiographic and tomographic evaluation. J Venom Anim Toxins incl Trop Dis. 2020;26:e20200107. doi: 10.1590/1678-9199.
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The etiologic agents of other systemic mycoses were also studied due to their prevalence and importance, namely:

  • histoplasmosis, an endemic disease with the highest incidence in AIDS patients [1111. Adenis AA, Aznar C, Couppié P. Histoplasmosis in HIV- infected patients: a review of new developments and remaining gaps. Curr Trop Med Rep. 2014 Mar 28;1(2):119-28.];

  • candidiasis, for its high incidence in neutropenic patients and as the main cause of nosocomial infections [1212. Uzun O, Ascioglu S, Anaissie EJ, Rex JH. Risk factors and predictors of outcome in patients with cancer and breakthrought candidemia. Clin Infect Dis. 2001 Jun 15;32(12):1713-7.];

  • cryptococcosis, because of the high incidence in AIDS patients and the difficulties in its treatment [1313. Sfadar A, Chatuvedi V, Cross EW, Park S, Bernard EM, Armstrong D, et al. Prospective study of Candida species in patients at a comprehensive cancer center. Antimicrob Agents Chemother. 2001 Jul;45(7):2129-33.,1414. Van der Horst CM, Saag MS, Cloud GA, Hamill RJ, Graybill JR, Sobel JD. Treatment of cryptococcal meningites associated with the acquired immune deficiency syndrome. N Engl J Med. 1997 Jul 3;337(1):15-21.];

  • aspergillosis, for its incidence in tuberculosis patients with pulmonary cavities, and in neutropenic patients developing invasive disease [1515. Denning DW, Pleurery A, Cole DC. Global burden of chronic pulmonary aspergilosis as a sequel to pulmonary tuberculosis. Bull World Health Organ. 2011 Dec 1;89(12):864-72.].

The incidence of fungal infections has increased dramatically in recent decades, posing serious problems for the management of immunosuppressed patients with systemic mycoses [1616. Limper AH, Adenis A, Le T, Harrison TS. Fungal infections in HIV/AIDS. Lancet Infect Dis. 2017 Nov;17(11):e334-43.

17. Putot A, Perrin S, Jolivet A, Vantilcke V. HIV - associated disseminated histoplasmosis in western French Guiana, 2002-2012. Mycoses. 2015 Mar;58(3):160-6.

18. Tang JL, Kung HC, Lei WC, Yao M, Wu UI, Hsu SC, et al. High incidences of invasive fungal infections in acute myeloid leukemia patients receiving inductin chemotherapy without systemic antifugal prophylaxis: a prospective observational study in Taiwan. PLoS One. 2015 Jun 10;10(6):e0128410.
-1919. Harrison N, Mitterbauer M, Tobudic S, Kalhs P, Rabitsch W, Greinix H, et al. Incidence and characteristics of invasive fungal diseases in allogeneic hematopoietic stem cell transplant recipients: a retrospective cohort study. BMC Infect Dis. 2015;15:584.]. During the same period, a wide gap has occurred in the development of antimicrobial compounds: few antifungal and many antibacterial compounds [2020. Spellberg B, Powers JH, Brass EP, Miller LG, Edwards JE Jr. Trends in antimicrobial drug development: implications for the future. Clin Infect Dis. 2004 May 1;38(9):1279-86.,2121. Walsh CT, Wencewicz TA. Prospects for new antibiotics: a molecule-centered perspective. J Antibiot (Tokyo). 2014 Jan;67(1):7-22.].

The development of new drugs derived from plant extracts have increased in the last few years [2222. Sharifi-Rad J, Salehi B, Varoni EM, Sharopov F, Yousaf Z, Ayatollahi SA, et al. Plants of the Melaleuca Genus as antimicrobial agents: from farm to pharmacy. Phytother Res. 2017 Oct;31(10):1475-94.

23. Ghaderi L, Moghimi R, Aliahmadi A, McClements DJ, Rafati H. Development of antimicrobial nanoemulsion-based delivery systems against selected pathogenic bacteria using a thymol-rich Thymus daenensis essential oil. J Appl Microbiol. 2017 Oct;123(4):832-40.
-2424. Van Vuuren S, Holl D. Antimicrobial natural product research: A review from a South African perspective for the years 2009-2016. J Ethnopharmacol. 2017 Aug 17;208:236-52.]. Plants produce different compounds with biological properties and activity against diseases of different etiologies, such as inflammatory diseases, infectious diseases, and cancer. Liriodenine (Fig. 1) is an oxoaporphine alkaloid, yellow needle, fluorescent, plain, with an oxo group in position 7. Liriodenine has antibacterial, antifungal, antiprotozoal and cytotoxic properties, is isolated from plants of several families, including the Annonaceae [2525. Hufford CD, Funderburk MJ, Morgan JM, Robertson LW. Two antimicrobial alkaloids from heartwood of Liriodendron tulipifera L. J Pharm Sci. 1975 May; 64(5):789-92.

26. Hufford CD, Sharma AS, Oguntimein BO. Antibacterial and antifungal activity of liriodenine and related oxoaporphine alkaloids. J Pharm Sci.1980 Oct;69(10):1180-3.

27. Graziose R, Rathinasabapathy T, Lategan C, Poulev A, Smith PJ, Grace M, et al. Antiplasmodial activity of aporphine alkaloids and sesquiterpene lactones from Liriodendron tulipifera. J Ethnopharmacol. 2011 Jan 7;133(1):26-30.

28. Costa EV, da Cruz PE, de Lourenço CC, de Souza Moraes VR, de Lima Nogueira PC, Salvador MJ. Antioxidant and antimicrobial activities of aporphinoids and other alkaloids from the bark of Annona salzmannii A. DC. (Annonaceae). Nat Prod Res. 2013 May 15;27(11):1002-06.

29. De La Cruz Chacón I, González-Esquinca AR. Liriodenine alkaloid in Annona diversifolia during early development. Nat Prod Res. 2012 Jul;26(1):42-9.

30. De La Cruz Chacón I, González-Esquinca AR, Feferb PG, Jímenez Garcia LFJ. Liriodenine, early antimicrobial defence in Annona diversifolia. Z Naturforsch C J Biosci. 2011 Jul-Aug;66(7-8):377-84.
-3131. Clark AM, Watson ES, Asfaq MK, Hufford CD. In vivo efficacy of antifungal oxoaporphine alkaloids in experimental disseminated candidiasis. Pharm Res. 1987 Dec;4(6):495-8.]. Its antifungal activity was observed on Candida albicans (ATCC 10231), Aspergillus niger (ATCC 16888), Trichophyton mentagrophytes (ATCC 9972), and the phytopathogenic fungi Aspergillus glaucus and Rhizopus stolonifer [2525. Hufford CD, Funderburk MJ, Morgan JM, Robertson LW. Two antimicrobial alkaloids from heartwood of Liriodendron tulipifera L. J Pharm Sci. 1975 May; 64(5):789-92.,2626. Hufford CD, Sharma AS, Oguntimein BO. Antibacterial and antifungal activity of liriodenine and related oxoaporphine alkaloids. J Pharm Sci.1980 Oct;69(10):1180-3. ,2828. Costa EV, da Cruz PE, de Lourenço CC, de Souza Moraes VR, de Lima Nogueira PC, Salvador MJ. Antioxidant and antimicrobial activities of aporphinoids and other alkaloids from the bark of Annona salzmannii A. DC. (Annonaceae). Nat Prod Res. 2013 May 15;27(11):1002-06.,3030. De La Cruz Chacón I, González-Esquinca AR, Feferb PG, Jímenez Garcia LFJ. Liriodenine, early antimicrobial defence in Annona diversifolia. Z Naturforsch C J Biosci. 2011 Jul-Aug;66(7-8):377-84.].

Figure 1.
Structure of liriodenine.

The objective of the present study was to evaluate the antimicrobial activity of liriodenine against fungi of the genus Paracoccidioides and other causative agents of systemic mycoses in vitro, as well as observe the morphological alterations caused by this compound in a standard P. brasiliensis (Pb18) strain.

Materials and Methods

Ethics statement

This study was approved by the Research Ethics Committee of Botucatu Medical School (FMB) of São Paulo State University (UNESP), Botucatu, SP, Brazil, under protocol number 2.257.787.

Liriodenine

Liriodenine was obtained from the Laboratory of Plant Physiology and Chemistry, Institute of Biological Sciences, Chiapas University of Arts and Sciences. Two of the authors, ICC and ARGE, are researchers from the Service of Plant Physiology and Chemistry and were responsible for supplying the sample. Liriodenine was extracted from Annona macroprophyllata, which is planted for commercial purposes. Moreover, the present research did not involve any endangered or threatened species.

Plant materials

Root bark (300 g) of Annona macroprophyllata (≡ Annona diversifolia) located at the geographical coordinates 16º46’34” N 93º07’14” W were collected in September 2011 in Tuxtla Gutiérrez, Chiapas, Mexico. The taxonomic sample was under reference voucher #352 of the Eizi Matuda Herbarium (HEM) of the Chiapas University of Arts and Sciences, Mexico.

Liriodenine extraction

Root barks were dried at room temperature (at 25-30 ºC for one week). After thorough grinding, the plant material was moistened with a saturated sodium carbonate (Na2CO3) solution and left to dry for 48 hours at room temperature. Alkaloids were extracted with chloroform (CHCl3) by constant stirring for 1 hour and then filtered and washed with distilled water. The CHCl3 phases were extracted into a 1 M hydrochloric acid (HCl) solution before being alkalinized to pH 9.5 with a saturated solution of Na2CO3. The alkaline solution was then reextracted with CHCl3, dried with anhydrous sodium sulfate (Na2SO4), filtered and evaporated at approximately 25 °C to obtain total alkaloids.

From 300 g of dried and powdered roots, 1200 mg of total alkaloids were extracted and a solid yellow precipitate was obtained. This was purified by fractionated crystallizations with a CHCl3-MeOH system to obtain 310 mg of solid yellow needles, which indicated positive results in both the Dragendorff’s and the Mayer’s tests.

Liriodenine standard was obtained as yellow needles from CHCl3, with a melting point (m.p.) of 280-282 °C. The product was identified as liriodenine by Dr. Miguel Angel Pérez Farrera from the herbarium of the National Autonomous University of Mexico. Spectrometric identification data were reported in De La-Cruz-Chacón and González-Esquinca [2929. De La Cruz Chacón I, González-Esquinca AR. Liriodenine alkaloid in Annona diversifolia during early development. Nat Prod Res. 2012 Jul;26(1):42-9.].

Chemicals

Hydrochloric acid, methanol, chloroform, water, sodium carbonate and anhydrous sodium sulfate were purchased from J.T. Baker, USA. The methanol and water used were HPLC grade.

Microorganisms

Strains and clinical isolates of agents of systemic mycoses were evaluated in this study: 8 from the genus Paracoccidioides, 5 of the genus Candida, 2 of the genus Cryptococcus - 1 of the Cryptococcus neoformans complex and 1 of the Cryptococcus gattii complex, 1 of Histoplasma capsulatum, and 1 Aspergillus fumigatus.

Strains obtained from the American Type Culture Collection (ATCC) and clinical isolates of the Laboratory of Research on Tropical Diseases, Botucatu Medical School (FMB), São Paulo State University (UNESP), SP, Brazil, were used in this study and include the following fungi of the genus Paracoccidioides: P. brasiliensis - Pb18 (fungal collection of FMB/UNESP; isolated from a PCM patient with the chronic form); Pb192 (fungal collection of FMB/UNESP; unknown clinical form); Pb339 (ATCC 3206; chronic clinical form); P. lutzii - PL01 (ATCC MYA-826; unknown clinical form); and PL8334 (unknown clinical form). Isolates number 234, 326 and 531 were obtained from PCM patients admitted in the Infectious Diseases Ward of Botucatu Medical School, UNESP, cultivated in the Research Laboratory on Tropical Diseases and identified at the Research Laboratory of Microbiology, Botucatu Biosciences Institute, UNESP. Isolate number 234 was obtained from the sputum of a PCM patient with the chronic form (August 8th, 2009), isolate number 326 from the oral mucosa of a PCM patient with the acute/subacute form (February 8th, 2012), and isolate number 531 from the scrotum of a PCM patient with the chronic form (January 9th, 2017). The following strains were used as representatives of fungi that can cause other systemic mycoses: Candida albicans (ATCC 90028), Candida tropicalis (ATCC 750), Candida glabrata (ATCC 90030), Candida parapsilosis (ATCC 22019), Candida krusei (ATCC 6258), Cryptococcus neoformans (ATCC 28957), Cryptococcus gattii (AFLP4), Histoplasma capsulatum (ATCC 26029), and Aspergillus fumigatus (ATCC 7100).

Molecular characterization and phylogenetic analysis of clinical isolates of fungi of the genus Paracoccidioides

The clinical isolates from PCM patients were identified by sequencing the exon 2 region of gp43 glycoprotein, a highly variable genetic region which allows species differentiation [33.Matute DR, McEwen JG, Puccia R, Montes BA, San-Blas G, Bagagli E, et al. Cryptic speciation and recombination in the fungus Paracoccidioides brasiliensis as revealed by gene genealogies. Mol Biol Evol. 2006 Sep 8;23(1):65-73.]. DNA extraction was performed following the "Quick CTAB" protocol [3232. Zhang Y, Hagen F, Stielow B, Rodrigues AM, Samerpitak K, Zhou X, et al. Phylogeography and evolutionary patterns in Sporothrix spanning more than 14 000 human and animal case reports. Persoonia. 2015 Dec;35:1-20.], and exon 2 of gp43 was amplified by PCR in a Veriti Thermocycler (Applied Biosystems, Foster City, CA, USA) using primers gp43-E2F (CCAGGAGGCGTGCAGGTGTCCC-3') and gp43-E2R (GCCCCCTCCGTCTTCCATGTCC-3') [3333. Cisalpino PS, Puccia R, Yamauchi LM, Cano MI, da Silveira JF, Travassos LR. Cloning, characterisation, and epitope expression of the major diagnostic antigen of Paracoccidioides brasiliensis. J Biol Chem. 1996 Feb 23;271(8):4553-60.]. To perform the amplification, GoTaq® Green Master Mix (Promega, Madison, WI, USA) was used with a 25 μL reaction volume containing 3 μL of genomic DNA (300 ng/μL) and 0.5 μM of each primer. Cycling was performed as follows: initial DNA denaturation at 94 °C for 5 minutes; 35 cycles of denaturation at 94 °C for 1 minute, annealing at 58 °C for 1 minute, extension at 72 °C for 1 minute and a final extension at 72 °C for 10 minutes.

After confirming the amplification by visualization on a 1.5% agarose gel, the PCR product was purified using ExoProStar (GE Healthcare, Milwaukee, WI, USA) and sequenced on an ABI 3500 DNA analyzer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. The quality of the sequences was verified by sequencing analysis software (Applied Biosystems), and only nucleotides with Phred ≥ 20 were included. Sequencing editing and phylogenetic analysis were performed using MEGA v7.0 software [3434. Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016 Jul;33(7):1870-4.]. Molecular identification was performed by the Basic Local Alignment Search Tool (BLAST) of the National Biotechnology Information Center (NCBI). Sequence alignments were performed using the Clustal W algorithm. Phylogenetic construction was performed by the maximum likelihood (ML) method [3535. Kimura M. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980 Dec;16(2): 111-20.] with 1000 bootstrap replications and using P. lutzii as an external group.

The sequences reported in this paper have been deposited in the GenBank database under the following accession numbers: MH367527, MH 367528, MH367529 and MH367531.

Susceptibility tests

Susceptibility testing was performed by determining the minimum inhibitory concentration (MIC) by the microdilution method. The MIC of liriodenine was determined for all microorganisms and compared to that of standard antifungal agents (fluconazole - FLC and amphotericin B - AmB) for each genus. RPMI 1640 medium (Sigma) was used without compounds or solvents as a control for growth and sterility. Dimethyl sulfoxide (DMSO) solvent was used as a control for toxicity at the same volume used in the assay. The microdilution test for fungi of the genus Paracoccidioides was performed using the protocol proposed by de Paula and Silva et al. [3636. de Paula e Silva AC, Oliveira HC, Silva JF, Sangalli-Leite F, Scorzoni L, Fusco-Almeida AM, et al. Microplate alamarBlue Assay for Paracoccidioides susceptibility testing. J Clin Microbiol. 2013 Apr;51(4):1250-2.]. For yeasts, the microdilution method was used according to the specifications of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [3737. Arendrup MC, Meletiadis J, Mounton JW, Lagrou K, Hamal P. European Comittee on Antimicrobial Susceptibility Testing (EUCAST). Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeast. EDEF 7.3.1.2017. http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/AFST/Files/EUCAST_E_Def_7_3_1_Yeast_testing__definitive.pdf.
http://www.eucast.org/fileadmin/src/medi...
]. Susceptibility testing of filamentous fungi was performed according to document M38-A2 of the Clinical and Laboratory Standards Institute (CLSI, 2008) [3838. Clinical Laboratory Standard Institute (CLSI). Reference method for broth dilution antifungal susceptibility testing of filamentous fungi, second ed. Approved standard, M38- A2. CLSI, Wayne, PA. 2008.]. The susceptibility of Histoplasma capsulatum was evaluated by the microdilution method according to document M27-A2 of the CLSI (2002), modified by Brilhante et al. [3939. Brilhante RS, Fechine MA, Cordeiro Rde A, Rocha MF, Ribeiro JF, Monteiro AJ, et al. In vitro effect of sulfamethoxazole-trimethoprim against Histoplasma capsulatum var. capsulatum. Antimicrob Agents Chemother. 2010 Sep;54(9):3978-9.].

Liriodenine was dissolved in dimethyl sulfoxide. Serial dilutions were prepared in RPMI 1640 medium, maintaining a constant volume of 1 mL per tube. Liriodenine was tested at 10 concentrations ranging from 500 to 0.97 μg.mL-1. Volumes of 100 μL of each dilution were distributed in 96-well microplates. The commercial antifungals amphotericin B (Sigma) and fluconazole (Sigma) were included as controls. The final concentrations ranged from 16 to 0.03 μg.mL-1 for amphotericin B and from 128 to 0.25 μg.mL-1 for fluconazole. The suspensions of yeast-phase cells of the genus Paracoccidioides were adjusted to a final concentration of 0.5x103 to 2.5 x 103 cells/mL in RPMI 1640 medium supplemented with 2% glucose. The plates were incubated for 72 hours at 37 ºC under shaking at 150 rpm. After 48 hours of incubation, 20 μL Alamar Blue dye (Invitrogen) was added to all wells, and the plates were incubated for an additional 24 hours. The tests were carried out in triplicate for all fungi. The MIC was determined by the observation of staining after 72 hours of incubation. The cutoff point was the lowest concentration of liriodenine that caused a significant reduction (≥ 50% inhibition) in growth when compared to the control. For fluconazole, the MIC endpoint was defined as the lowest drug concentration resulting in at least 50% growth reduction compared with the control, while that of amphotericin B was the lowest concentration that resulted in at least 90% qrowth reduction compared with the positive control. The plates were read from the highest to the lowest concentration.

Minimum fungicide concentration (MFC)

After the determination of MICs, subcultures were prepared from all microplate wells containing the concentrations of liriodenine ≥ to that of the MICs. For fungi of the genus Paracoccidioides, aliquots (100 µL) of each well were removed and plated on brain heart infusion broth (Difco) containing 0.5% gentamicin, 4% horse serum, and culture filtrate of strain Pb339. Sabouraud agar (Difco) was used for the other fungi. The plates were incubated according to the specific time of each microorganism. The MFC was defined as the lowest concentration of liriodenine resulting in ≤ 1 visible colony on the culture medium after the incubation period.

Transmission electron microscopy

Transmission electron microscopy (TEM) was performed at the Center of Electron Microscopy of São Paulo State University (UNESP), Botucatu Biosciences Institute, Botucatu, SP, Brazil.

Strain Pb18 was treated with 31.2 μg.mL-1 of liriodenine in a 24-well plate. The final volumes of liriodenine and inoculum were adjusted to 1 mL, and Karnovsky’s fixative was added after 48 hours of incubation at 37 ºC. After this period, the material was removed from the fixative and washed three times for 5 minutes each in 0.1 M phosphate buffer, pH 7.3. The material was immersed in osmium tetroxide 0.1 M, pH 7.3, for 2 hours. Next, the material was washed three times for 10 minutes each in distilled water and immersed in 0.5% uranyl acetate for approximately 2 hours. After dehydration in an increasing acetone series, a mixture of Araldite® resin + 100% acetone (1:1) was added, and the material was left to stand for 12 hours at room temperature. Pure resin was added for approximately one hour at 37 ºC, and the material was embedded. Ultrathin (90 nm) sections were cut from the blocks and counterstained with uranyl acetate in 50% alcohol for 20 minutes, followed by counterstaining with lead citrate for 10 minutes. The sections were observed under a Tecnai Spirit transmission electron microscope (FEI Company).

Scanning electron microscopy

For scanning electron microscopy (SEM) performed at the same center, strain Pb18 was cultured in RPMI 1640 medium with 2% glucose and treated with the MIC of liriodenine on coverslips coated with poly-L-lysin for 48 hours at 37 ºC. After this period, the medium was discarded, and the material was fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3, for 30 minutes. The fixative was removed from the wells, and the coverslips were washed three times for 5 minutes each in distilled water. The coverslips were immersed in 0.5% osmium tetroxide 0.1 M, pH 7.3, in distilled water for 40 minutes. The osmium tetroxide was then discarded, and the coverslips were washed again three times for 10 minutes in distilled water. After dehydration in an increasing alcohol series, the coverslips were critical-point dried, mounted on stubs, and sputter coated. The images were obtained with a Zeiss EVO LS 15 microscope.

Results

Phylogenetic analysis of clinical isolates

Figure 2 shows the phylogenetic analysis of the exon 2 region of gp43. Clinical isolate 326 was grouped together with strains deposited as P. brasiliensis S1a, isolate 531 was grouped together with strains deposited as P. brasiliensis S1b, and isolate 234 was grouped together with strains deposited as PS3 (presently called P. restrepiensis). Strain 192, also used as a standard strain, was grouped with PS3.

Figure 2.
Molecular phylogenetic analysis by the maximum likelihood method using exon 2 of the gp 43 region. The evolutionary history was inferred using the maximum likelihood method based on the Kimura 2-parameter model. The tree with the highest log likelihood is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 11 nucleotide sequences. Evolutionary analyses were conducted in MEGA7. The species names were concordant with the latest accepted taxonomies for the species (old and recent species names).

Minimum inhibitory concentration

Table 1 shows the results of the antifungal activity of liriodenine isolated from Annona macroprophyllata.

Table 1.
Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of liriodenine isolated from Annona macroprophyllata, evaluated in 17 fungal isolates.

Among the eight fungal strains of the genus Paracoccidioides, only strains Pb18, Pb192 and Pb531 were susceptible to liriodenine, with MICs of 31.2, 62.5 and 250 μg.mL-1, respectively. The remaining standard strains and clinical isolates were resistant to the highest concentration evaluated.

Regarding the yeasts used, liriodenine exhibited antifungal activity against most of the strains tested when compared to the amphotericin B and fluconazole controls, except for Candida tropicalis, which was found to be resistant to the highest concentration evaluated.

Liriodenine did not exhibit antifungal activity against the Aspergillus fumigatus strain. However, the Histoplasma capsulatum strain was susceptible to liriodenine at a concentration of 1.95 μg.mL-1. The solvent DMSO did not show activity for all strains and clinical isolates evaluated.

Minimum fungicide concentration

Liriodenine exhibited fungicidal activity against all standard strains and clinical isolates that were sensitive in in vitro tests. The MFC was the same as the MIC in all fungi evaluated, with the exception of Histoplasma capsulatum and Candida krusei (Table 1).

Scanning electron microscopy

Ultrastructural analysis of yeast-phase cells of P. brasiliensis revealed single cells or clusters with several buds (Figs. 3 and 4). SEM of yeast-phase cells not treated with liriodenine showed slightly irregular surfaces and prolongations that attached the cells to the contact surface, which in this case was the culture plate. The prolongations were short, had a regular and delicate morphology, and were visualized close to the cell body, resembling a “halo” around the cell. The single cells exhibited discrete irregularities on their surface that resembled “ribbings”. In addition, a filamentous region (ligament) was detected, which was interpreted as the onset of a bud (Figs. 3A and 3B). Clusters of cells with several ligaments to their buds were noted in other images (Figs. 3C and 3D).

Changes in ultrastructural morphology were observed after treatment with liriodenine at a concentration of 31.2 μg.mL-1 (Figs. 3E, 3F, 3G, and 3H). The adhesion “halo” to the culture medium was rarely observed in single cells, although the morphology was similar to that observed in untreated cells in terms of the round shape of the cell and discrete surface irregularities (“ribbings”) (Fig. 3E). However, the most evident ultrastructural alterations were observed in cell clusters, which contained very deformed cells with irregular ligaments, apparently without a lining membrane; plates of material were juxtaposed with these clusters (Figs. 3F, 3G, and 3H).

Figure 3.
Scanning electron microscopy of yeast-phase cells of standard Paracoccidioides brasiliensis (PB18) strain. (A-D) Cells not treated with liriodenine. (E-H) Cells treated with liriodenine at the MIC of 31.2 μg.mL-1. li: ligament; p: prolongation; jm: juxtaposed material.

Transmission electron microscopy

TEM of cells not treated with liriodenine revealed a round shape, regular contours, and cell walls with uniform thickness. The nucleus and organelles were found to be preserved and intact, and mitochondria, discrete endoplasmic reticula and vacuoles with variable sizes and contents were observed. Some smaller vacuoles were electron-lucent and did not contain visible material; however, most vacuoles observed in the cytoplasm of cells were dense, circular and filled with finely flocculent, homogenous and mildly dense material. Ligaments could be detected between cells and their buds (Figs. 4A, 4B, and 4C).

In contrast, marked changes were found in the morphology of cells treated with liriodenine, as demonstrated by SEM. However, TEM showed details of the cytoplasmic and nuclear alterations. The shape of the cells was altered, and there were only irregular-shaped cells. The cells generally had depressions in their walls, and the plasma membrane exhibited variable shapes, with a sickle shape predominant. Cytoplasmic alterations were very evident and included large cytoplasmic areas without a defined contour filled with finely flocculent, homogenous and mildly dense material, similar to the material observed inside the dense circular vacuoles of the control group. These images suggest the fusion of these various vacuoles in treated cells. In addition, large vacuoles with an irregular, or even triangular, shape containing the same material were noted (Figs. 4D, 4E, 4F, 4G, 4H, 4I, 4J, and 4K).

Figure 4.
Transmission electron microscopy of yeast-phase cells of the standard Paracoccidioides brasiliensis (Pb18) strain. (A-C) Cells not treated with liriodenine. (D-K) Cells treated with liriodenine at the MIC of 31.2 μg.mL-1. dv: dense vacuole; v: electron-lucent; nu: nucleus; mi: mitochondrion; cw: cell wall; fo: sickle shape.

Discussion

Nowadays, despite the discovery of new antifungal compounds, some problems persist including low urinary excretion of active drugs, impaired treatment of urinary tract infections, and reduced diffusion into the cerebrospinal fluid regardless of the degree of meningeal inflammation. Moreover, interactions with different drugs and restrictions concerning treatment of pregnant women also constitute limitations [4040. Kauffman CA. Candiduria. Clin Infect Dis. 2005 Sep 15;41(Suppl 6):371-6.

41. Saag MS, Cloud GA, Graybill JR, Sobel JD, Tuazon CU, Johnson PC, et al. A comparison of itraconazole versus fluconazole as maintenance therapy for AIDS-associated cryptococcal meningitis. National Institute of Allergy and Infectious Diseases Mycoses Study Group. Clin Infect Dis. 1999 Feb;28(2):291-6.

42. Piscitelli SC, Gallicano KD. Interactions among drugs for HIV and opportunistic infections. N Engl J Med. 2001 Mar 29;344(13):984-96.
-4343. Lee BE, Feinberg M, Abraham JJ, Murthy AR. Congenital malformations in a infant born to a woman treated with fluconazole. Pediatric Infect Dis J. 1992 Dec;11(12):1062-4.]. These difficulties also justify continuous research to identify new antifungal compounds, including those of plant origin.

The use of plant extracts for the treatment and prophylaxis of diseases is one of the oldest practices of humans. The search for new plant-derived drugs has developed substantially in recent years due to increasing resistance to antimicrobial compounds, undesirable effects of existing compounds and drug interactions [4444. Oz T, Guvenek A, Yildiz S, Karaboga E, Tamer YT, Mumcuyan N, et al. Strength of selection pressure is an important parameter contributing to the complexity of antibiotic resistance evolution. Mol Biol Evol. 2014 Sep;31(9):2387-401.,4545. Gibbons S. Anti-staphylococcal plant natural products. Nat Prod Rep. 2004 Apr;21(2):263-77.]. The main advantage of this approach over modifications in already existing compounds is the chance of identifying new prototypes of drugs with clearly different chemical structures, thus avoiding cross-resistance and currently observed drug interactions [3131. Clark AM, Watson ES, Asfaq MK, Hufford CD. In vivo efficacy of antifungal oxoaporphine alkaloids in experimental disseminated candidiasis. Pharm Res. 1987 Dec;4(6):495-8.].

Few studies have evaluated the activity of plant-derived compounds against pathogenic fungi. The present study demonstrated the antifungal activity of liriodenine against three fungal strains of the genus Paracoccidioides; however, greater activity was observed against yeasts of the genus Cryptococcus and against the thermally dimorphic fungus Histoplasma capsulatum. Among the studies focusing on the genus Paracoccidioides, two extracts derived from the genus Annona [4646. Lima LAR, Johann S, Cisalpino PS, Pimenta LPP. In vitro antifungal activity of fatty acid methyl ester of the seeds of Annona cornifolia A.St.-Hil (Annonaceae) against pathogenic fungus Paracoccidioides brasiliensis. Rev Soc Bras Med Trop. 2011;44(6):777-80.,4747. Lima LARS , Alves TMA, Zani CL, Sales Júnior PA, Romanha AJ, Johann S, et al. In vitro cytotoxic, antifungal, trypanocidal and leishmanicidal activities of acetogenins isolated from Annona cornifolia A. St. -Hil. (Annonaceae). An Acad Bras Ciênc. 2014 Jun;86(2):829-39.] and three evaluated compounds extracted from other plant species [4848. San-Blas G, San-Blas F, Gil F, Apitz-Castro R. Inhibition of growth of the dimorphic fungus paracoccidioides brasiliensis by ajoene. Antimicrob Agents Chemother. 1989 Sep;33(9):1641-4.

49. Johann S, Cisalpino PS, Watanabe GA, Cota BB. Antifungal activity of extracts of some plants used in Brazilian traditional medicine against the pathogenic fungus Paracoccidioides brasiliensis. Pharma Biol. 2010;48(4):388-96.
-5050. Gullo FP, Sardi JC, Santos VA, Sangalli-Leite F, Pitangui NS, Rossi SA, et al. Antifungal activity of maytenin and pristimerin. Evid Based Complement Alternat Med. 2012 May 22;2012:340787.] were used. In this respect, Lima et al. [4646. Lima LAR, Johann S, Cisalpino PS, Pimenta LPP. In vitro antifungal activity of fatty acid methyl ester of the seeds of Annona cornifolia A.St.-Hil (Annonaceae) against pathogenic fungus Paracoccidioides brasiliensis. Rev Soc Bras Med Trop. 2011;44(6):777-80.] evaluated the activity of fatty acid methyl esters (FAMEs) against 12 clinical isolates of P. brasiliensis by the broth microdilution method and observed inhibition of all isolates, with MIC values ranging from 3.4 to 55.5 μg.mL-1. This MIC range was similar to that observed in the present study. In another study, Lima et al. [4747. Lima LARS , Alves TMA, Zani CL, Sales Júnior PA, Romanha AJ, Johann S, et al. In vitro cytotoxic, antifungal, trypanocidal and leishmanicidal activities of acetogenins isolated from Annona cornifolia A. St. -Hil. (Annonaceae). An Acad Bras Ciênc. 2014 Jun;86(2):829-39.] investigated the activity of acetogenins extracted from Annona cornifolia against Pb01 and Pb18 by the broth microdilution method. Concentrations of 1.17 to 150 μg.mL-1 were tested. Acetogenins exhibited activity against the two strains at concentrations ≤ 150 μg.mL-1 and were found to be more active than the combination of sulfamethoxazole and trimethoprim, but less active than amphotericin B.

Using the dry weight method, San-Blas et al. [4848. San-Blas G, San-Blas F, Gil F, Apitz-Castro R. Inhibition of growth of the dimorphic fungus paracoccidioides brasiliensis by ajoene. Antimicrob Agents Chemother. 1989 Sep;33(9):1641-4.] demonstrated that ajoene, a sulfur compound found in garlic (Allium sativum), inhibited the growth of P. brasiliensis by 90% in the yeast phase and by 60% in the mycelial phase at a concentration of 11.7 μg.mL-1. Electron microscopy revealed that 11.7 μg.mL-1 of ajoene affected the integrity of the fungal cytoplasmic membrane, but cell lysis was only observed at a concentration of 46.8 μg.mL-1.

The hexane fractions of hydroalcoholic extracts of Baccharis dracunculifolia and Piper regnellii exhibited antifungal activity against three strains of the genus Paracoccidioides (Pb18, Pb01 and Pb339). Compounds from Baccharis dracunculifolia DC were active against Pb18, Pb01 and Pb339, with MICs of 7.8, 7.8 and 30 μg.mL-1, respectively, while the MIC of compounds from Piper regnellii was 7.8 μg.mL-1 for the three strains evaluated [4949. Johann S, Cisalpino PS, Watanabe GA, Cota BB. Antifungal activity of extracts of some plants used in Brazilian traditional medicine against the pathogenic fungus Paracoccidioides brasiliensis. Pharma Biol. 2010;48(4):388-96.]. Comparison of these results with those of the present study shows that strain Pb339 is resistant to liriodenine but susceptible to the hexane fractions, while strain Pb18 is more sensitive to liriodenine. Furthermore, the compounds maytenin and pristimerin derived from Maytenus ilicifolia exhibited antifungal activity against yeasts and filamentous fungi. Strain Pb18 was sensitive to both compounds at a concentration of < 0.12 mg/L. Maytenin provided better results than pristimerin. No cytotoxic effects of these compounds on oral mucosa keratinocytes were observed, suggesting an adequate safety level [5050. Gullo FP, Sardi JC, Santos VA, Sangalli-Leite F, Pitangui NS, Rossi SA, et al. Antifungal activity of maytenin and pristimerin. Evid Based Complement Alternat Med. 2012 May 22;2012:340787.].

The ultrastructural data of yeast-phase cells of P. brasiliensis treated with liriodenine revealed alterations mainly in the cell wall and, to a lesser extent, alterations in the cytoplasm. Similar findings have been reported for ajoene, including cell wall damage, cell lysis and deterioration of the structures in yeast-phase cells of P. brasiliensis [4848. San-Blas G, San-Blas F, Gil F, Apitz-Castro R. Inhibition of growth of the dimorphic fungus paracoccidioides brasiliensis by ajoene. Antimicrob Agents Chemother. 1989 Sep;33(9):1641-4.].

This study revealed a MIC of liriodenine against Histoplasma capsulatum ATTC 26029 of 1.95 μg.mL-1. Alanís-Garza et al. [5151. Alanís-Garza BA, González-González GM, Salazar-Aranda R, Waksman de Torres N, Rivas-Galindo VM. Screening of antifungal activity of plants from the northeast of Mexico. J Ethnopharmacol. 2007 Dec 3;114(3):468-71.] demonstrated the antifungal activity of hexane, ethyl acetate and butanol extracts isolated from 15 plants in the Northeastern region of Mexico against Histoplasma capsulatum had MICs ranging from 16 to 1.000 μg.mL-1. Even lower MIC values were observed for maytenin (0.97 mg/L) and pristimerin (0.48 mg/L) [5050. Gullo FP, Sardi JC, Santos VA, Sangalli-Leite F, Pitangui NS, Rossi SA, et al. Antifungal activity of maytenin and pristimerin. Evid Based Complement Alternat Med. 2012 May 22;2012:340787.].

In recent years, an increase in invasive infections caused by yeasts of the genus Candida has been observed, especially among immunosuppressed patients [5252. Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev. 2007 Jan;20(1):133-63.]. Candida albicans is the main causative agent of candidemia, although the prevalence of non-Candida albicans etiology has been increasing [5353. Nucci M, Queiroz-Telles F, Alvarado-Matute T, Tiraboschi IN, Cortes J, Zurita J, et al. Epidemiology of candidemia in Latin-America: a laboratory-based survey. PLoS One. 2013 Mar 19;8(3):e59374.]. The susceptibility of Candida species to commonly used antifungal agents is variable; C. krusei exhibits intrinsic resistance to fluconazole [5454. Kaiyar SK, Edlind TD. Identification and expression of multidrug resistance-related ABC transporter genes in Candida krusei. Med Mycol. 2001 Feb;39(1):109-16.], while C. glabrata shows acquired resistance [5555. Pfaller MA, Diekema DJ, Gibbs DL, Newell VA, Ellis D, Tullio V, et al. Results from the ARTEMIS DISK Global Antifungal Surveillance Study, 1997 to 2007: a 10.5-Year Analysis of Susceptibilities of Candida Species to Fluconazole and Voriconazole as Determined by CLSI Standardized Disk Diffusion. J Clin Microbiol. 2010 Apr;48(4):1366-77.].

In the present study, liriodenine exhibited antifungal activity against most Candida strains, with MICs of 125 to 250 μg.mL-1, except for C. tropicalis, which was resistant to the highest concentration evaluated. These values are similar to those reported for pristimerin [5050. Gullo FP, Sardi JC, Santos VA, Sangalli-Leite F, Pitangui NS, Rossi SA, et al. Antifungal activity of maytenin and pristimerin. Evid Based Complement Alternat Med. 2012 May 22;2012:340787.], with MICs ranging from 7.81 to 250 mg/L, and for the hexane extract of Annona glabra [5656. Padmaja V, Thankamany V, Hara N, Fujimoto Y, Hisham A. Biological activities of Annona glabra. J Ethnopharmacol. 1995 Aug 11;48(1):21-4.], which inhibited the growth of C. albicans at a concentration of 100 μg.mL-1. The present study also showed that liriodenine has lower MIC values than the methanol and ethyl acetate extracts of Duguetia furfuracea against C. albicans and C. krusei, with MICs ≥ 1024 μg.mL-1, although acting against C. tropicalis [5757. Pinho FVS de A, da Cruz LC, Rodrigues NR, Waczuk EP, Souza CES, Coutinho HDM, et al. Phytochemical Composition, Antifungal and Antioxidant Activity of Duguetia furfuracea A. St.-Hill. Oxid Med Cell Longev. 2016;2016:7821051.]. Liriodenine was also more active than FAMEs of Annona cornifolia, which exhibited MICs of 500 μg.mL-1 or higher against C. albicans and C. parapsilosis strains [4646. Lima LAR, Johann S, Cisalpino PS, Pimenta LPP. In vitro antifungal activity of fatty acid methyl ester of the seeds of Annona cornifolia A.St.-Hil (Annonaceae) against pathogenic fungus Paracoccidioides brasiliensis. Rev Soc Bras Med Trop. 2011;44(6):777-80.]. Finally, the MIC values presented by liriodenine against Candida species ranged between 125 and 250 μg.mL-1 while these values were much lower, 3.12 μg.mL1, for the same substance, but extracted from Liriodendron tulipifera, Magnoliaceae [3131. Clark AM, Watson ES, Asfaq MK, Hufford CD. In vivo efficacy of antifungal oxoaporphine alkaloids in experimental disseminated candidiasis. Pharm Res. 1987 Dec;4(6):495-8.]. This difference can be explained by the plant liriodenine was extracted as well as phase of its development.

Cryptococcosis is one of the main mycoses that affects immunosuppressed patients, especially those infected with human immunodeficiency virus, because of the severity of clinical manifestations, the incidence and intensity of sequelae, and the high mortality rate [5858. Chastain DB, Henao-Martínez AF, Franco-Paredes C. Opportunistic invasive mycoses in AIDS: cryptococcosis, histoplasmosis, coccidiodomycosis, and talaromycosis. Curr Infect Dis Rep. 2017 Aug 22;19(10):36. ]. Cryptococcal meningitis should be treated with a combination of amphotericin B or its lipid derivatives with flucytosine. However, classical amphotericin B requires intravenous administration and careful control of side effects. The lipid derivatives of amphotericin B are less toxic but are expensive and require intravenous administration. Finally, flucytosine is not available in many countries, including Brazil. These difficulties have led to the use of high doses of fluconazole, which is fungistatic and provides slow clearance of infection. This scenario encouraged the investigation of other therapeutic regimens, including available antifungal compounds, the combination of antifungals with immunomodulators, and the search for other drugs with anticryptococcal activity [5959. Perfect JR, Dismukes WE, Dromer F, Goldman DL, Graybill JR, Hamill RJ, et al. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the infectious diseases society of America. Clin Infect Dis. 2010 Feb 1;50(3):291-322.

60. Kon AS, Grumach AS, Colombo AL, Penalva ACO, Wanke B, Telles FQ, et al. Consenso em criptococose - 2008. Rev Soc Bras Med Trop. 2008 Sep 1;41(5):524-44.

61. Armstrong-James D, Brown GD, Netea MG, Zelante T, Gresnigt MS, Van de Veerdonk FL, et al. Immunotherapeutic approaches to treatment of fungal disease. Lancet Infect Dis. 2017 Dec;17(12):e393-402.
-6262. Jarvis JN, Meintjes G, Rebe K, Williams GN, Bicanic T, Williams A, et al. Adjunctive interferon-γ immunotherapy for the treatment of HIV-associated cryptococcal meningitis: a randomized controlled trial. AIDS. 2012 Jun 1;26(9):1105-13.].

In the present study, liriodenine exhibited activity against isolates of two complexes, Cryptococcus neoformans complex and C. gattii complex, with an MIC of 62.5 μg.mL-1 for both species. The MIC observed in this study was much lower than the MICs for FAMEs of Annona cornifolia [5454. Kaiyar SK, Edlind TD. Identification and expression of multidrug resistance-related ABC transporter genes in Candida krusei. Med Mycol. 2001 Feb;39(1):109-16.] (≥ 500 μg.mL-1) and lower than three compounds from Ocimum basilicum - essential oil, crude ethanol extract and hexane fraction (MICs of 156 to 2.500 μg.mL-1) [6363. Cardoso NN, Alviano CS, Blank AF, Romanos MT, Fonseca BB, Rozental S, et al. Synergism effect of the essential oil from Ocimum basilicum var. Maria Bonita and its major components with fluconazole and its influence on ergosterol biosynthesis. Evid Based Complement Alternat Med. 2016;2016:5647182.]. However, the MICs of maytenin (0.48 to 3.9 mg/L) and pristimerin (0.97 to 7.8 mg/L) were even lower [5050. Gullo FP, Sardi JC, Santos VA, Sangalli-Leite F, Pitangui NS, Rossi SA, et al. Antifungal activity of maytenin and pristimerin. Evid Based Complement Alternat Med. 2012 May 22;2012:340787.].

The MICs of amphotericin B and fluconazole on Candida species and Cryptococcus species, used as controls, were very similar to those that had been published by other authors [6464. Pfaller MA, Messer SA, Boyken L, Hollis R, Rice C, Tendolkar S, et al. In vitro activities of voriconazole, posaconazole, and fluconazole against 4,169 clinical isolates of Candida spp and Cryptococcus neoformans collected during 2001 and 2002 in the ARTEMIS global antifungal surveillance program. Diagn Microbiol Infect Dis. 2004 Mar;48(3):201-5.

65. Pfaller MA, Diekema DJ, Messer SA, Boyken L, Hollis RJ. Activities of fluconazole and voriconazole against 1,586 recent clinical isolates of Candida species determined by broth microdilution, disk diffusion, and Etest methods: report from the ARTEMIS Global Antifungal Susceptibility Program, 2001. J Clin Microbiol. 2003 Apr;41(4):1440-6.

66. Yang YL, Li SY, Cheng HH, Lo HJ, TSARY Hospitals. The trend of susceptibilities to amphotericin B and fluconazole of Candida species from 1999 to 2002 in Taiwan. BMC Infect Dis. 2005 Nov 3;5:99.
-6767. Swinne D, Watelle M, Nolard N. In vitro activities of voriconazole, fluconazole, itraconazole and amphotericin B against non Candida albicans yeast isolates. Rev Iberoam Micol. 2005 Mar;22(1):24-8.].

Aspergillus fumigatus is the main etiological agent of invasive aspergillosis that affects neutropenic patients [6868. Kontoyiannis DP, Marr KA, Park BJ, Alexander BD, Anaissie EJ, Walsh TJ, et al. Prospective surveillance for invasive fungal infections in hematopoietic stem cell transplant recipients, 2001-2006: overview of the Transplant-Associated Infection Surveillance Network (TRANSNET) Database. Clin Infect Dis. 2010 Apr 15;50(8):1091-100.,6969. Neofytos D, Fishman JA, Horn D, Anaissie E, Chang CH, Olyaei A, et al. Epidemiology and outcome of invasive fungal infections in solid organ transplant recipients. Transpl Infect Dis. 2010 Jun;12(3):220-9.]. Treatment is performed mainly with triazole compounds such as voriconazole and posaconazole [7070. Patterson TF, Thompson GR, Denning DW, Fishman JA, Hadley S, Herbrecht R, et al. Practice Guidelines for the Diagnosis and Management of Aspergillosis: 2016 Update by the Infectious Diseases Society of America. Clin Infect Dis. 2016 Aug 15;63(4):433-42.]. However, surveillance studies have revealed the emergence of resistant clinical and environmental isolates worldwide, [7171. Verweij PE, Lestrade PP, Melchers WJ, Meis JF. Azole resistance surveillance in Aspergillus fumigatus: beneficial or biased? J Antimicrob Chemother. 2016 Aug;71(8):2079-82.

72. Nabili M, Shokohi T, Moazeni M, Khodavaisy S, Aliyali M, Badiee P, et al. High prevalence of clinical and environmental triazole-resistant Aspergillus fumigatus in Iran: is it a challenging issue? J Med Microbiol. 2016 Jun;65(6):468-75.

73. Verweij PE, Chowdhary A, Melchers WJ, Meis JF. Azole Resistance in Aspergillus fumigatus: Can We Retain the Clinical Use of Mold-Active Antifungal Azoles? Clin Infect Dis. 2016 Feb 1;62(3):362-8.

74. Meis JF, Chowdhary A, Rhodes JL, Fisher MC, Verweij PE. Clinical implications of globally emerging azole resistance in Aspergillus fumigatus. Philos Trans R Soc Lond B Biol Sci. 2016 Dec 5;371(1709):20150460.

75. Chowdhary A, Sharma C, Meis JF. Azole-resistant aspergillosis: Epidemiology, molecular mechanisms, and treatment. J Infect Dis. 2017 Aug 15;216(Suppl 3):436-44.
-7676. Gonçalves SS, Souza ACR, Chowdhary A, Meis JF, Colombo AL. Epidemiology and molecular mechanisms of antifungal resistance in Candida and Aspergillus. Mycoses. 2016 Apr;59(4):198-219.] particularly in patients previously using azole compounds or infected with fungi exposed to azole derivatives used in agriculture [7777. Chowdhary A, Kathuria S, Xu J, Meis JF. Emergence of azole-resistant Aspergillus fumigatus strains due to agricultural azole use creates an increasing threat to human health. PLoS Pathog. 2013 Oct;9(10):e1003633.,7878. Snelders E, Huis In 't Veld RA, Rijs AJ, Kema GH, Melchers WJ, Verweij PE. Possible environmental origin of resistance of Aspergillus fumigatus to medical triazoles. Appl Environ Microbiol. 2009;75:4053-7.]. In the present study, liriodenine did not exert antifungal activity against the Aspergillus fumigatus strain. On the other hand, maytenin at a concentration of 125 mg.L and pristimerin at a concentration of 250 mg/L were found to be active against this fungus, with maytenin exhibiting the best fungicidal activity [5050. Gullo FP, Sardi JC, Santos VA, Sangalli-Leite F, Pitangui NS, Rossi SA, et al. Antifungal activity of maytenin and pristimerin. Evid Based Complement Alternat Med. 2012 May 22;2012:340787.].

The mechanism of the antifungal action of liriodenine has not yet been clarified. In vitro and in vivo studies demonstrated that liriodenine inhibits topoisomerase II, an enzyme that induces DNA double strand breaks and is necessary to relieve torsional stress during replication. The antifungal action of liriodenine maybe similar to that of antibiotics that inhibit this enzyme, leading to defective synthesis of messenger RNA and proteins and consequent death of the microorganism [7979. Ho Woo S, Nan-Jun Sun NJ, Cassady JM, Snapka RM. Inhibition of Topoisomerase II by Liriodenine. Biochem Pharmacol. 1997 Aug 15;54(4):467-73.].

Liriodenine inhibits the biosynthesis of dopamine and dopamine content induced by L-DOPA in PC12 cells [8080. Jin CM, Lee JJ, Yang YJ, Kim YM, Kim YK, Ryu SY, et al. Liriodenine inhibits dopamine biosynthesis and L-DOPA-induced dopamine content in PC12 cells. Arch Pharm Res. 2007;30(8):984-90.]. Since melanin, an important virulence factor of Cryptococcus species, is also synthesized from L-DOPA, it would be interesting to investigate the possible antimelanogenic activity of liriodenine.

However, a study using Saccharomyces cerevisiae suggested another mechanism of action of liriodenine on yeasts. That study showed that liriodenine interrupts mitochondrial iron-sulfur synthesis based on three findings:

  • liriodenine caused cellular iron imbalance through the induction of genes that are important for iron uptake and cellular iron homeostasis;

  • genome analysis demonstrated that yeast mutants with deletions in the genes responsible for iron homeostasis are hypersensitive to liriodenine;

  • treatment of yeasts with liriodenine produced cellular defects that resembled deficiencies in mitochondrial iron-sulfur synthesis, including an increase in mitochondrial iron levels, a decrease in the activity of sulfur enzymes, and an increase in oxidative stress [8181. Tripathi SK, Xu T, Feng Q, Avula B, Shi X, Pan X, et al. Two plant-derived aporphinoid alkaloids exert their antifungal activity by disrupting mitochondrial iron-sulfur cluster biosynthesis. J Biol Chem. 2017 Oct 6;292(40):16578-93.].

Biochemical models designed to determine the mechanism of action of antifungal compounds should be used for the evaluation of liriodenine [8282. Visbal G, San-Blas G, Murgich J, Franco H. Paracoccidioides brasiliensis, paracoccidioidomycosis, and antifungal antibiotics. Curr Drug Targets Infect Dis. 2005 Sep;5(3):211-26.,8383. San-Blas G, San-Blas F. Immune and chemical responses to paracoccidioidomycosis. In: Jacobs PH, Nall L, editor’s. Fungal disease: biology, immunology and diagnosis. New York: Marcel Dekker. Pp. 219-35. 1997.].

Conclusion

The present in vitro study demonstrated the fungicidal activity of liriodenine against yeasts and some dimorphic fungi. Therefore, it is believed additional evaluation of this compound in animal models of infection should be carried out, followed by the estimation of its safety and efficacy for the treatment of human infections. Our initial findings also encouraged the modification of liriodenine chemical structure, in order to turn it into a more effective product.

Acknowledgments

The authors would like to thank Professor Eduardo Bagagli for supporting the molecular characterization and phylogenetic analysis of the clinical isolates of the genus Paracoccidioides.

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  • Availability of data and materials

    All data generated or analyzed during this study are included in this article.
  • Funding

    ADL was the recipient of a doctoral scholarship from the Coordination for the Improvement of Higher Education Personnel (CAPES).
  • Ethics approval

    The present study was approved by the Research Ethics Committee of Botucatu Medical School (FMB/UNESP) under protocol number 2.257.787.
  • Consent for publication

    Not applicable.

Publication Dates

  • Publication in this collection
    28 Oct 2020
  • Date of issue
    2020

History

  • Received
    01 Mar 2020
  • Accepted
    06 Oct 2020
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