Bioprospecção dos fungos endofíticos associados as variedades morfoanatômicas de Casearia sylvestris e estudo químico de Preussia sp. por abordagens OSMAC
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Data
2016-12-02
Autores
Somensi, Andressa [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Micro-organismos associados a vegetais desempenham um papel importante na descoberta e desenvolvimento de novos fármacos. Com isso, a busca por novas fontes de substâncias bioativas tem se intensificado, destacando os estudos realizados com fungos endofíticos, os quais apresentam uma elevada diversidade taxonômica e estrutural dos metabólitos secundários, sendo estes com potencial antitumoral, antimicrobiano e antifúngico. Estas observações motivaram o desenvolvimento do presente trabalho, o qual visa a bioprospecção dos fungos endofíticos associados as variedades morfoanatômicas da espécie vegetal Casearia sylvestris e o estudo do perfil metabolômico de Preussia sp por abordagens OSMAC (One Strain Many Comounds). Para tanto, o isolamento dos fungos endofíticos conduziu a vinte e quatro linhagens puras, sendo onze linhagens de C. sylvestris var. lingua e treze de C. sylvestris var. sylvestris, resultando em extratos brutos promissores na produção de substâncias bioativas. A bioprospecção inicial conduziu a seleção do fungo endofítico Preussia sp., afim de realizar um estudo químico da espécie por meio de abordagens OSMAC. O cultivo em laboratório, nem sempre mimetiza o habitat natural dos micro-organismos e não exploram de maneira completa toda diversidade das vias biossintéticas. Pequenas alterações nos parâmetros físicos e químicos de fermentação são capazes de modificar completamente o perfil metabolômico desses micro-organismos. Essas estratégias buscam elicitar novas rotas biossintéticas, aumentando a diversidade dos metabólitos secundários produzidos a partir de uma única cepa fúngica. Com isso, foram estabelecidas três estratégias de estudo, variando a composição nutricional do meio cultivo (MDB, Milho e MDB+Cu2+). O estudo de Preussia sp., cultivado em meio líquido de MDB, levou ao isolamento de seis dicetopiperazinas, uma isoflavona (Daidzeína) e o ácido 2-(4´-hidroxifenil) propiônico. Do cultivo da linhagem em meio sólido de Milho foram isoladas duas xantonas, sendo uma isolada pela primeira vez como produto natural, três oxepinocromanonas e três furocromanonas, sendo uma furocromanona descrita pela primeira vez na literatura. O cultivo da linhagem em meio líquido de MDB com adição de íons cobre, conduziu ao isolamento de duas oxepinocromanonas, sendo uma inédita, além de uma pirriolida e a 2,4-diidroxi-3,6-dimetilbenzaldeído. Varredura no espectro de UV do extrato bruto do cultivo em MDB, na ausência de cobre, não indicou a presença de cromonas, sugerindo que este metal esteja elicitando a produção desta classe de metabólitos. Esse resultado fortalece a abordagens de novas ferramentas eficazes na elicitação de novas vias biossintética por meio de alterações nos parâmetros de cultivos. Algumas substâncias puras foram submetidas à avaliação da atividade fitotóxica e erradicação de biofilme formado pela bactéria S. epidermidis ATCC 34984 e apresentaram bioatividade. Portanto, esses resultados ressaltam a importância de estudos da diversidade metabólica de fungos endofíticos, uma vez que estes possam ser uma fonte promissora de novos metabólitos secundários bioativos por meio de uma única cepa fúngica.
Microorganisms associated with plants perform an important role for discovery and development of new drugs. Hence, the search for new sources of natural bioactive compounds has been intensified, emphasizing the studies carried out with endophytic fungi, that present a high taxonomic and structural diversity of secondary metabolites, with antitumoral, antimicrobial and antifungal potential. These observations led to the purpose of the present work, that aimed the bioprospection of endophytic fungi associated with two morpho-anatomical varieties of the vegetal species of Casearia sylvestris and the metabolomic profile of Preussia sp. by the OSMAC (One Strain Many Comounds) approach. Therefore, the isolation of endophytic fungi conducted to twenty four pure strains, eleven strains from C. sylvestris var. lingua and thirteen from C. sylvestris var. sylvestris, resulting in promissing crude extracts for production of bioactive compounds. The initial bioprospecting led to the selection of the endophytic fungus Preussia sp. and a chemical study by OSMAC approach were accomplished. Cultivation in the laboratory doesn’t always mimics the natural habitat of the microorganisms and because of that the diversity of biosynthetic pathways aren’t fully exploit. Small changes in the physical and chemical parameters of fermentation are capable to modify completely the metabolomic profile of these microorganisms. These strategies are used to elicit new biosynthetic pathways and increase the production of secondary metabolites using only one fungal strain. Thereby, three strategies varying the nutritional composition of the culture medium (PDB, Corn and PDB+Cu2+) were selected. In the study of Preussia sp., cultivated in liquid medium of PDB, six diketopiperazines, one isoflavone (daidzein) and the 2-(4-Hydroxyphenyl) propanoic acid were isolated. With the same strain, cultivated in solid medium of corn, two xantones, one of them isolated for the first time as a natural product, three oxepinochromenones and three furochromanones, one of them described for the first time in the literature, were isolated. The cultivation in liquid medium of PDB with addition of copper ions, afforded to the isolation of two oxepinochromenones, one already known and the other a novel molecule, besides the pirriolide and the 2,4-dihydroxy-3,6-dimethylbenzaldehyde. It wasn’t found, with a closer look in the UV spectrum of the crude extracts obtained from the liquid medium of PDB without copper addition, the presence of chromones, suggesting that the metal elicited the production of this class of metabolite. This result strengthens that the new approaches for eliciting new biosynthetic pathways through changes in cultivation parameters are effective tools. Some of the isolated compounds were submitted to evaluation of the phytotoxic activity and eradication of biofilm formed by the bacterium S. epidermidis ATCC 34984 and presented bioactivity. Finally, these results highlights the importance of studies of the metabolic diversity of endophytic fungi, since it may be a promising source in the discovery of new bioactive fungal secondary metabolites through a single fungal strain.
Microorganisms associated with plants perform an important role for discovery and development of new drugs. Hence, the search for new sources of natural bioactive compounds has been intensified, emphasizing the studies carried out with endophytic fungi, that present a high taxonomic and structural diversity of secondary metabolites, with antitumoral, antimicrobial and antifungal potential. These observations led to the purpose of the present work, that aimed the bioprospection of endophytic fungi associated with two morpho-anatomical varieties of the vegetal species of Casearia sylvestris and the metabolomic profile of Preussia sp. by the OSMAC (One Strain Many Comounds) approach. Therefore, the isolation of endophytic fungi conducted to twenty four pure strains, eleven strains from C. sylvestris var. lingua and thirteen from C. sylvestris var. sylvestris, resulting in promissing crude extracts for production of bioactive compounds. The initial bioprospecting led to the selection of the endophytic fungus Preussia sp. and a chemical study by OSMAC approach were accomplished. Cultivation in the laboratory doesn’t always mimics the natural habitat of the microorganisms and because of that the diversity of biosynthetic pathways aren’t fully exploit. Small changes in the physical and chemical parameters of fermentation are capable to modify completely the metabolomic profile of these microorganisms. These strategies are used to elicit new biosynthetic pathways and increase the production of secondary metabolites using only one fungal strain. Thereby, three strategies varying the nutritional composition of the culture medium (PDB, Corn and PDB+Cu2+) were selected. In the study of Preussia sp., cultivated in liquid medium of PDB, six diketopiperazines, one isoflavone (daidzein) and the 2-(4-Hydroxyphenyl) propanoic acid were isolated. With the same strain, cultivated in solid medium of corn, two xantones, one of them isolated for the first time as a natural product, three oxepinochromenones and three furochromanones, one of them described for the first time in the literature, were isolated. The cultivation in liquid medium of PDB with addition of copper ions, afforded to the isolation of two oxepinochromenones, one already known and the other a novel molecule, besides the pirriolide and the 2,4-dihydroxy-3,6-dimethylbenzaldehyde. It wasn’t found, with a closer look in the UV spectrum of the crude extracts obtained from the liquid medium of PDB without copper addition, the presence of chromones, suggesting that the metal elicited the production of this class of metabolite. This result strengthens that the new approaches for eliciting new biosynthetic pathways through changes in cultivation parameters are effective tools. Some of the isolated compounds were submitted to evaluation of the phytotoxic activity and eradication of biofilm formed by the bacterium S. epidermidis ATCC 34984 and presented bioactivity. Finally, these results highlights the importance of studies of the metabolic diversity of endophytic fungi, since it may be a promising source in the discovery of new bioactive fungal secondary metabolites through a single fungal strain.
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Palavras-chave
Casearia sylvestris, Fungos endofíticos, Preussia sp., OSMAC, Endophytic fungi