Caracterização química e avaliação citotóxica de heteropolissacarídeos dos fungos Colletotrichum gloeosporiodes e Fusarium sp.
Carregando...
Data
2019-08-14
Autores
Alexandre-Polido, Samara Marrye Aguiar
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
Muitas pesquisas apontam que moléculas naturais, como polissacarídeos extraídos de fungos, podem ser aplicados em diferentes ensaios biológicos. Embora existam trabalhos com os fungos Colletotrichum gloeosporioides e Fusarium sp, relacionados com produção de metabólitos ou com fitopatogenicidade, a lacuna sobre a composição química da biomassa desses organismos ainda existe. Assim, este trabalho teve como objetivo principal realizar estudos inéditos para esclarecer essa composição, focando nos polissacarídeos. Isso irá fornecer dados adicionais sobre os micro-organismos, além de disponibilizar novas moléculas para ensaios biológicos. Para isso, os micro-organismos foram cultivados e suas biomassas submetidas a procedimentos de extrações etanólica e aquosa. Os extratos aquosos foram tratados com ciclos de congelamento e descongelamento, diálise e precipitação com etanol, resultando nos precipitados etanólicos do C. gloeosporioides (PEH2O-C) e do Fusarium sp (PEH2O-F). Ambos foram purificados por cromatografia de filtração em gel, onde o PEH2O-C foi separado em cinco frações, com a PIV-C selecionada para os demais estudos. As análises de caracterização química (por HPAEC/PAD, CG-EM e RMN) revelaram que PIV-C possui galactose e glucose como componentes majoritários, sendo a cadeia principal formada por unidades β-D-galactofuranosídicas ligadas em (1→5) e (1→6), com algumas dessas últimas unidades substituídas em O-3 por resíduos α-D-glucopiranosídicos. A cromatografia de filtração em gel separou o PEH2O-F em três frações, sendo a PII-F selecionada para os estudos de caracterização. As análises mostraram que PII-F possui manose e galactose como monossacarídeos principais e que se trata de uma β-D-galactofuranana 1→6 com substituição em O-2, principalmente, por resíduos α-D-manopiranosídicos 2-O- ligados. Testes de citotoxicidade realizados com PIV-C e PII-F mostraram que a viabilidade das células CHO-K1 não apresentaram diferenças significativas quando comparadas com a viabilidade do controle negativo, nos tempos experimentais de 24 e 48 horas. Além disso, o teste de genotoxicidade com a fração PII-F mostrou que o heteropolissacarídeo não causou danos significativos ao DNA das células CHO-K1. Esses resultados apontam para novas perspectivas de estudos com os polissacarídeos presentes nas frações PIV-C e PII-F, onde poderão ser investigadas aplicações para essas moléculas.
Many researches suggest that natural molecules as fungal polysaccharides can be applied in different biological assays. Although there are works with Colletotrichum gloeosporioides and Fusarium sp related to metabolite production or phytopathogenicity, there still is a gap on the chemical composition of the biomass of these organisms. Thus, the main goal of this work was to performed unpublished studies to clarify this composition as for polysaccharides. This will provide additional data on microorganisms and new molecules for biological assays. For this, the fungi were cultivated and their biomass submitted to ethanolic and aqueous extraction procedures. The aqueous extracts were submitted to freeze-thaw cycles, dialysis and precipitation with ethanol, resulting in ethanolic precipitate from C. gloeosporioides and Fusarium sp named, respectively, PEH20-C and PEH2O-F. These materials were purified by gel filtration chromatography and the procedure separated the PEH20-C fraction into five peaks being the PIV-C selected for research. Chemical characterization analyses (HPAEC/PAD, GC-MS and NMR) revealed that galactose and glucose were the main components and the polysaccharide is a (1→5, 1→6) linked-β-D-Galf main chain partially substituted in O-3 by α-D-glucopyranosyl residues. The gel permeation chromatography separated the PEH2O-F in three peaks and PII-F was selected for characterization studies. The analysis from PII-F showed mannose and galactose as the main monosaccharides and the polysaccharide is (1→6) linked-β-D-Galf main chain partially substituted in O-2 by α-(1→2) linked mannopyranosyl residues. Cytotoxicity assays performed with PIV-C and PII-F, showed that there was no significant difference in the viability of CHO-K1 cells, when compared to the negative control, in the experimental time of 24 and 48 hours. The genotoxicity assay showed that PII-F did not cause significant damage to the DNA of CHO-K1 cells. These results showed that new perspectives of studies with polysaccharides from fractions PIV-C and PII-F where applications for these molecules could be investigated.
Many researches suggest that natural molecules as fungal polysaccharides can be applied in different biological assays. Although there are works with Colletotrichum gloeosporioides and Fusarium sp related to metabolite production or phytopathogenicity, there still is a gap on the chemical composition of the biomass of these organisms. Thus, the main goal of this work was to performed unpublished studies to clarify this composition as for polysaccharides. This will provide additional data on microorganisms and new molecules for biological assays. For this, the fungi were cultivated and their biomass submitted to ethanolic and aqueous extraction procedures. The aqueous extracts were submitted to freeze-thaw cycles, dialysis and precipitation with ethanol, resulting in ethanolic precipitate from C. gloeosporioides and Fusarium sp named, respectively, PEH20-C and PEH2O-F. These materials were purified by gel filtration chromatography and the procedure separated the PEH20-C fraction into five peaks being the PIV-C selected for research. Chemical characterization analyses (HPAEC/PAD, GC-MS and NMR) revealed that galactose and glucose were the main components and the polysaccharide is a (1→5, 1→6) linked-β-D-Galf main chain partially substituted in O-3 by α-D-glucopyranosyl residues. The gel permeation chromatography separated the PEH2O-F in three peaks and PII-F was selected for characterization studies. The analysis from PII-F showed mannose and galactose as the main monosaccharides and the polysaccharide is (1→6) linked-β-D-Galf main chain partially substituted in O-2 by α-(1→2) linked mannopyranosyl residues. Cytotoxicity assays performed with PIV-C and PII-F, showed that there was no significant difference in the viability of CHO-K1 cells, when compared to the negative control, in the experimental time of 24 and 48 hours. The genotoxicity assay showed that PII-F did not cause significant damage to the DNA of CHO-K1 cells. These results showed that new perspectives of studies with polysaccharides from fractions PIV-C and PII-F where applications for these molecules could be investigated.
Descrição
Palavras-chave
Colletotrichum gloeosporioides, Fusarium sp, Galactofuranana, Espectroscopia de RMN de 1H e 13C, Ensaio do MTT, Galactofuranan, 1H e 13C NMR spectroscopy, MTT assay