Estudo de modificadores químicos epigenéticos no cultivo de fungos endofíticos associados à Paepalanthus chiquitensis (Eriocaulaceae) e avaliação da atividade antimicrobiana
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2020-03-27
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Universidade Estadual Paulista (Unesp)
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Este trabalho apresenta o estudo dos efeitos de modificadores epigenéticos e eliciadores químicos na diversificação metabólica de Curvularia lunata e Fusarium fujikuroi, fungos isolados de Paepalanthus chiquitensis (Eriocaulaceae). Os fungos foram cultivados em PDB (caldo batata dextrose), e as seguintes condições foram investigadas: (PDB + SAHA) ácido hidroxâmico suberoilanilida, (PDB + AZA) 5-azacitidina, (PDB + AA) ácido anacárdico, PDB + Cu2+ e PDB + Mg2+. Essas condições foram comparadas com o controle PDB. O estudo químico de C. lunata, resultou no isolamento das novas γ-lactamas espirocíclicas C3 e C4, e outros cinco compostos conhecidos: triticonas E (C1) e F (C2), ácido 5-O-metilcurvulínico (C5), ácido curvulínico (C6) e curvulina (C7). As estruturas foram elucidadas por análises espectroscópicas e pela comparação com dados da literatura. Além disso, um estudo computacional foi realizado para determinar a configuração absoluta do C‒3' de C3 e C4. As triticonas C1 e C2 apresentaram boa atividade antibacteriana para Escherichia coli, com CIM de 62,5 μg/mL. Dos eliciadores químicos utilizados, a suplementação com Mg2+ foi a condição que apresentou as diferenças mais significativas no perfil metabólico de F. fujikuroi. O extrato PDB + Mg2+ preparado do cultivo em escala ampliada foi purificado por métodos cromatográficos e resultou no isolamento e identificação das substâncias: ácido indolacético (F3) e seus derivados F4, F8 e F17, assim como a auxina ácido fenilacético (F2) e seus derivados F6, F11, F12, F15 e F18. Os compostos F2, F3 e F4 apresentaram os melhores valores de CIM para todas as leveduras do gênero Cândida avaliadas. As análises quantitativas por CLAE-DAD dos metabólitos isolados ácido fusárico, AIA, ácido acético-2-(4-butilpicolinamida) e terpestacina foram quantificadas nos extratos obtidos dos cultivos, e o método desenvolvido foi validado. Paralelamente, em colaboração com a Universidade de Oklahoma, foram estudados os seguintes fungos de solo: Aspergillus arcoverdensis que resultou no isolamento da verruculogena, fumitremorgina e, veriditoxina, apresentou potente atividade contra Trichomonas vaginalis (1,0 μM). O estudo bioguiado de Cylindrocarpon pauciseptatum possibilitou o isolamento da fujikurina B, responsável pela atividade antituberculose, não apresentando citotoxicidade em células J774. Por fim, os estudos de Talaromyces verruculosis, resultou em frações e substâncias que exibiram atividades biológicas frente as cepas de T. vaginalis, M. tuberculosis e C. albicans na formação de biofilme, sendo não citotóxicas em células J774 e HepG2. O estudo bioguiado de T. verruculosis resultou no isolamento de metabolitos com potente atividade inibitória na formação do biofilme de C. albicans: duclauxina, bacillisporina F e 1-epi-bacillisporin F.
Eriocaulaceae, Paepalanthus chiquitensis, Fusarium fujikuroi, Curvularia lunata, fungo endofítico, epigenética
This work presents the studies of the effects of epigenetic modifiers and chemical elicitors on the metabolic diversification of Fusarium fujikuroi and Curvularia lunata, isolated of Paepalanthus chiquitensis (Eriocaulaceae). The fungi were grown in PDB (potato dextrose broth) and the following conditions were investigated: (PDB +SAHA) suberoylanilide hydroxamic acid, (PDB +AZA) 5-azacitidine, (PDB +AA) anacardic acid, PDB +Cu2+ and PDB + Mg2+. These conditions were compared with the PDB control. The chemical study of C. lunata, resulted in the isolation of new spirocyclic-γ-lactams C3 and C4, and five known compounds: triticones E (C1) and F (C2),5-O-methylcurvulinic acid (C5), curvulinic acid (C6) and curvulin (C7). The structures were elucidated by spectroscopic analysis and by comparison with data from the literature. In addition, a computational study was carried out to determine the absolute configuration in the C-3' (C3 and C4). The extract and compounds were tested against bacteria and yeasts. The C1 and C2 showed good antibacterial activity for E. coli, with a minimum inhibitory concentration of 62.5 μg/mL. The supplementation with Mg2+ into PDB medium was the condition that showed the most significant differences in the metabolic profile of F. fujikuroi. The chemical study of extract PDB + Mg2+ resulted in the isolation and identification of the metabolites: indoleacetic acid (F3) and its derivatives F4, F8 and F17, as well of the phenylacetic acid class (F18) the derivatives F2, F6, F11, F12 and F15 and F18. The compounds F2, F3 e F4 have showed interesting MIC values against all yeast strains tested. The quantitative analysis by HPLC-DAD of the metabolites fusaric acid, indole acetic acid, acetic acid-2-(4-butylpicolinamide) and terpestacin were quantified in the extracts prepared of the cultured studied, besides the method was validated. In collaboration with Oklohama University, others activities were studied the following soil fungi: Aspergillus arcoverdensis, resulted in isolation of verruculogen, fumitremorgin and veriditoxin that demonstrated potential activity against Trichomonas vaginalis (1.0 μM). The bioguided study of Cylindrocarpon pauciseptatum made it possible to isolate fujikurin B, responsible for anti-Mycobacterium tuberculosis activity and non-cytotoxicity in the J774 cell line. Finally, the studies of Talaromyces verruculosis, result in fractions and substances that exhibit several biological activities against T. vaginalis, M. tuberculosis and C. albicans in the biofilm formation, besides showed non cytotoxic in J774 and HepG2 cells. The bioguided study of T. verruculosis resulted in the isolation of potent metabolites able to inhibit the formation of C. albicans biofilm, named: duclauxin, bacillisporin F and 1-epi-bacillisporin F.
Eriocaulaceae, Paepalanthus chiquitensis, Fusarium fujikuroi, Curvularia lunata, fungo endofítico, epigenética
This work presents the studies of the effects of epigenetic modifiers and chemical elicitors on the metabolic diversification of Fusarium fujikuroi and Curvularia lunata, isolated of Paepalanthus chiquitensis (Eriocaulaceae). The fungi were grown in PDB (potato dextrose broth) and the following conditions were investigated: (PDB +SAHA) suberoylanilide hydroxamic acid, (PDB +AZA) 5-azacitidine, (PDB +AA) anacardic acid, PDB +Cu2+ and PDB + Mg2+. These conditions were compared with the PDB control. The chemical study of C. lunata, resulted in the isolation of new spirocyclic-γ-lactams C3 and C4, and five known compounds: triticones E (C1) and F (C2),5-O-methylcurvulinic acid (C5), curvulinic acid (C6) and curvulin (C7). The structures were elucidated by spectroscopic analysis and by comparison with data from the literature. In addition, a computational study was carried out to determine the absolute configuration in the C-3' (C3 and C4). The extract and compounds were tested against bacteria and yeasts. The C1 and C2 showed good antibacterial activity for E. coli, with a minimum inhibitory concentration of 62.5 μg/mL. The supplementation with Mg2+ into PDB medium was the condition that showed the most significant differences in the metabolic profile of F. fujikuroi. The chemical study of extract PDB + Mg2+ resulted in the isolation and identification of the metabolites: indoleacetic acid (F3) and its derivatives F4, F8 and F17, as well of the phenylacetic acid class (F18) the derivatives F2, F6, F11, F12 and F15 and F18. The compounds F2, F3 e F4 have showed interesting MIC values against all yeast strains tested. The quantitative analysis by HPLC-DAD of the metabolites fusaric acid, indole acetic acid, acetic acid-2-(4-butylpicolinamide) and terpestacin were quantified in the extracts prepared of the cultured studied, besides the method was validated. In collaboration with Oklohama University, others activities were studied the following soil fungi: Aspergillus arcoverdensis, resulted in isolation of verruculogen, fumitremorgin and veriditoxin that demonstrated potential activity against Trichomonas vaginalis (1.0 μM). The bioguided study of Cylindrocarpon pauciseptatum made it possible to isolate fujikurin B, responsible for anti-Mycobacterium tuberculosis activity and non-cytotoxicity in the J774 cell line. Finally, the studies of Talaromyces verruculosis, result in fractions and substances that exhibit several biological activities against T. vaginalis, M. tuberculosis and C. albicans in the biofilm formation, besides showed non cytotoxic in J774 and HepG2 cells. The bioguided study of T. verruculosis resulted in the isolation of potent metabolites able to inhibit the formation of C. albicans biofilm, named: duclauxin, bacillisporin F and 1-epi-bacillisporin F.
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Palavras-chave
Eriocaulaceae, Paepalanthus chiquitensis, Fusarium fujikuroi, Curvularia lunata, fungo endofítico, epigenética, Paepalanthus chiquitensis, endophytic fungi, epigenetics