Oligossacarídeos obtidos por hidrólise química de polissacarídeos fúngicos: purificação e atividade anticoagulante
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Data
2017-03-03
Autores
Ferreira, Jéssica Moreli [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Polissacarídeos e oligossacarídeos de plantas, algas, bactérias e fungos representam um grupo de moléculas com potencial para aplicação em terapia medicamentosa e em biotecnologia. Os oligossacarídeos podem ser obtidos, a partir dos respectivos polissacarídeos, por degradação química, física ou bioquímica. β-glucanas secretadas pelo fungo ascomiceto Lasiodiplodia theobromae (EPSgraviola, EPSlaranja e EPSmanga), cujas soluções apresentam elevado grau de viscosidade, dificultando os testes de atividade biológica, foram hidrolisadas parcialmente com TFA 0,2 M, 100 °C, em tempos variáveis, para a obtenção dos respectivos oligossacarídeos, que foram analisados por cromatografia líquida de íons. O EPSmanga mostrou-se, nas condições estudadas, o substrato ideal para a produção de oligossacarídeos, especialmente aos 45 minutos de hidrólise. As frações solúveis, contendo o material hidrolisado, foram submetidas, inicialmente, à cromatografia de filtração em gel Sephadex G-15, que forneceu dois picos, um composto predominantemente por mono e dissacarídeos e outro contendo oligossacarídeos maiores, de onde foi separada a Fração I, a qual foi submetida à cromatografia de adsorção em carvão-Celite, utilizando um gradiente descontínuo de etanol-água 0 a 60% (v/v). A fração etanol-água a 50%, denominada Fração II, contendo a maior proporção de oligossacarídeos com maior DP foi submetida a uma segunda condição de eluição, utilizando um gradiente contínuo de etanol-água 42-49%, gerando frações ainda compostas por mais de um oligossacarídeo. A Fração II contendo oligossacarídeos com DP > 5 foi sulfatada, e os oligossacarídeos com DS de 0,80 foram submetidos à cromatografia de filtração em gel Sephadex G-15, para remoção de sal. Após análises por FT-IR para acompanhar o aparecimento de novas bandas, provenientes dos grupos sulfatos, os oligossacarídeos derivatizados foram utilizados em testes de atividade anticoagulante. O aumento do tempo para a formação do coágulo, nos testes do aPTT e TT, foram concentração-dependente, indicando que os oligossacarídeos sulfatados apresentam ação anticoagulante.
Polysaccharides and oligosaccharides of plants, algae, bacteria and fungi represent a group of molecules that have potential to application in drug therapy and in biotechnology. The oligosaccharides can be obtained, from their respective polysaccharides, by chemical, physical or biochemical degradation. β-glucans secreted by the fungus Lasiodiplodia theobromae (EPSgraviola, EPSorange e EPSmango), whose solutions have high viscosity, what makes the tests of biological activity difficult, were partially hydrolyzed with TFA 0,2 M, 100 °C, in variable times, in order to obtain their respective oligosaccharides, that were analyzed by liquid chromatography of ions. The EPSmango was, in the studied conditions, the ideal substrate to the production of oligosaccharides, especially at 45 minutes of hydrolysis. The soluble fractions, that contain the hydrolyzed material, were initially submitted to gel filtration chromatography on Sephadex G-15, that provided two peaks, one peak with mono and disaccharides and the other one containing bigger oligosaccharides, named Fraction I. That fraction I was submitted to charcoal-Celite adsorption chromatography using a discontinuous gradient ethanol-water 0 to 60% (v/v). The fraction containing oligosaccharides with higher DP (ethanol-water 50%) was named Fraction II, that was submitted to charcoal-Celite adsorption chromatography using a continuous gradient ethanol-water 42-49%, generating fractions still composed of a mixture of oligosaccharides. So, the Fraction II was sulfated, and the produced oligosaccharides with DS = 0,80 were submitted to gel filtration chromatography on Sephadex G-15, in order to remove the salt. After FT-IR analysis, to accompany the appearance of new bands from the sulfate groups, the oligosaccharides were used in anticoagulant activity tests. The increases of time to clot formation, in the aPTT and TT tests, were concentration-dependent, indicating that the sulfated oligosaccharides have anticoagulant action.
Polysaccharides and oligosaccharides of plants, algae, bacteria and fungi represent a group of molecules that have potential to application in drug therapy and in biotechnology. The oligosaccharides can be obtained, from their respective polysaccharides, by chemical, physical or biochemical degradation. β-glucans secreted by the fungus Lasiodiplodia theobromae (EPSgraviola, EPSorange e EPSmango), whose solutions have high viscosity, what makes the tests of biological activity difficult, were partially hydrolyzed with TFA 0,2 M, 100 °C, in variable times, in order to obtain their respective oligosaccharides, that were analyzed by liquid chromatography of ions. The EPSmango was, in the studied conditions, the ideal substrate to the production of oligosaccharides, especially at 45 minutes of hydrolysis. The soluble fractions, that contain the hydrolyzed material, were initially submitted to gel filtration chromatography on Sephadex G-15, that provided two peaks, one peak with mono and disaccharides and the other one containing bigger oligosaccharides, named Fraction I. That fraction I was submitted to charcoal-Celite adsorption chromatography using a discontinuous gradient ethanol-water 0 to 60% (v/v). The fraction containing oligosaccharides with higher DP (ethanol-water 50%) was named Fraction II, that was submitted to charcoal-Celite adsorption chromatography using a continuous gradient ethanol-water 42-49%, generating fractions still composed of a mixture of oligosaccharides. So, the Fraction II was sulfated, and the produced oligosaccharides with DS = 0,80 were submitted to gel filtration chromatography on Sephadex G-15, in order to remove the salt. After FT-IR analysis, to accompany the appearance of new bands from the sulfate groups, the oligosaccharides were used in anticoagulant activity tests. The increases of time to clot formation, in the aPTT and TT tests, were concentration-dependent, indicating that the sulfated oligosaccharides have anticoagulant action.
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Palavras-chave
β-glucanas fúngicas, Hidrólise ácida parcial, HPAEC/PAD, Cromatografia, Oligossacarídeos sulfatados, Atividade anticoagulante, Fungal β-glucans, Partial acid hydrolysis, Chromatography, Sulfated oligosaccharides, Anticoagulant activity