Prospecção química em Pseudofusicoccum stromaticum, um fungo Endofítico em Eugenia jambolana (Myrtaceae)
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Data
2017-02-10
Autores
Rahmé, Gustavo Morandi [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O fungo endofítico P. stromaticum isolado dos frutos maduros da espécie vegetal E. jambolana foi cultivado em escala ampliada no meio líquido de Malte por 28 dias a 25 oC no modo estático e, no meio sólido de milho tipo canjica por 21 dias a 25 oC no modo estático. Após 28 dias de crescimento no meio líquido, este foi submetido a sucessivas extrações com acetato de etila (AcOEt), que após evaporado forneceu o extrato bruto de Malte AcOEt. O meio sólido, após 21 dias de cultivo, foi extraído com metanol, seguido de filtração e evaporação fornecendo o extrato bruto metanólico. Este foi submetido à partição líquido/líquido com acetato de etila e água, sendo que a fração AcOEt foi evaporada fornecendo o extrato bruto AcOEt. Este foi particionado com acetonitrila e hexano, e após evaporação dos solventes forneceu os extratos acetonitrila (ACN) e hexânico do meio sólido. O extrato ACN do meio sólido passou por novas partições com acetonitrila e hexano, por apresentar ácidos graxos ainda, fornecendo o extrato bruto de milho NP (novas partições). O extrato bruto de Malte AcOEt, foi fracionado em coluna de bancada, sendo coletadas 11 frações (Fr-1 Ps Ma a Fr-11 Ps Ma). Já o extrato bruto de milho NP sofreu partição utilizando-se cartucho de C18, com base em seu cromatograma, resultando em 6 frações (Fr-1 Ps Milho a Fr-6 Ps Milho). Das 6 frações obtidas do extrato de milho NP, escolheu-se trabalhar com a primeira, a qual sofreu partição no CLAE-DAD preparativo, resultando em 8 frações (Fr-1.1 Ps Milho a Fr-1.8 Ps Milho). O extrato bruto de Malte AcOEt e suas frações, além do extrato bruto de milho NP, foram submetidos à avaliação química por RMN de 1H e biólogica contra o fungo fitopatogêno Cladosporium sphaerospermum (e no caso do milho ao C. cladosporioides também) e de inibição da enzima acetilcolinesterase, apresentando atividade, enquanto o extrato bruto de milho NP se mostrou inativo. Foram identificadas duas substâncias inéditas, sendo uma da Fr-6 Ps Malte, um ftalato (4,5-dimetil-3,4,5,6-tetrahidrobenzo[c][1,6]dioxecina-1,8-diona) e outra da Fr-1.3 Ps Milho, uma cumarina (2-(7-hidroxi-4-metil-2-oxo-2H-cromen-5-il) ácido acético) e foram propostas as estruturas de outras duas substâncias.
The endophytic fungus P. stromaticum isolated from the ripe fruits of the E. jambolana was cultivated in large scale in the malt liquid medium for 28 days at 25 oC in the static mode and in the solid medium of hominy corn for 21 days at 25 oC in static mode. After 28 days of growth in the liquid medium, it was made successive extractions with ethyl acetate (EtOAc). The organic layer was evaporated affording the crude extract of Malt EtOAc. The solid medium, after 21 days of cultivation, was extracted with methanol, followed by filtration and evaporation to give the crude methanolic extract. This was subjected to the liquid/liquid partition with EtOAc and H2O, being this EtOAc fraction evaporated giving the crude EtOAc extract. After that, the extract was partitioned with acetonitrile and hexane, and after the evaporation of the solvents provided the acetonitrile (ACN) and hexane extracts from the solid medium. The ACN extract of the solid medium was partitioned again with ACN and hexane, due the remaining fatty acid, providing the corn crude extract NP (new partitions). The crude extract of Malt EtOAct was fractionated in a chromatographic column, resulting in11 fractions (Fr-1 Ps Ma to Fr-11 Ps Ma). The crude extract of corn NP was fractionated by solid phase extraction in reverse phase (C18), based on its chromatogram, resulting in 6 fractions (Fr-1 Ps Corn to Fr-6 Ps Corn). Among the corn fractions collected, it was chosen the fraction Fr-1 Ps Corn to the chemical studies. This fraction was fractionated by preparative HPLC-DAD resulting in 8 fractions. The crude extract of Malt EtOAc and its fraction and the crude extract of corn NP, were submitted to the chemical evaluation by 1H NMR and biological against the phytopathogenic fungus Cladosporium sphaerospermum (and in the case of corn to C. cladosporioides as well). Besides, anticolinesterasic activity was made, the crude extract of Malt EtOAc and its fractions showed activity. The fractionating of the crude extracts and then of the fractions gotten afforded the isolation of 2 new substances, one of the Fr-6 Ps Malt, a phthalate (4,5-dimethyl-3,4,5,6-tetrahydrobenzo [c] [1,6] dioxecyn-1,8-dione) and another from Fr-1.3 Ps Corn, a coumarin (2- (7-hydroxy-4-methyl-2-oxo-2H-chromen-5-yl) acetic acid) and was possible to propose two structures for another two substances.
The endophytic fungus P. stromaticum isolated from the ripe fruits of the E. jambolana was cultivated in large scale in the malt liquid medium for 28 days at 25 oC in the static mode and in the solid medium of hominy corn for 21 days at 25 oC in static mode. After 28 days of growth in the liquid medium, it was made successive extractions with ethyl acetate (EtOAc). The organic layer was evaporated affording the crude extract of Malt EtOAc. The solid medium, after 21 days of cultivation, was extracted with methanol, followed by filtration and evaporation to give the crude methanolic extract. This was subjected to the liquid/liquid partition with EtOAc and H2O, being this EtOAc fraction evaporated giving the crude EtOAc extract. After that, the extract was partitioned with acetonitrile and hexane, and after the evaporation of the solvents provided the acetonitrile (ACN) and hexane extracts from the solid medium. The ACN extract of the solid medium was partitioned again with ACN and hexane, due the remaining fatty acid, providing the corn crude extract NP (new partitions). The crude extract of Malt EtOAct was fractionated in a chromatographic column, resulting in11 fractions (Fr-1 Ps Ma to Fr-11 Ps Ma). The crude extract of corn NP was fractionated by solid phase extraction in reverse phase (C18), based on its chromatogram, resulting in 6 fractions (Fr-1 Ps Corn to Fr-6 Ps Corn). Among the corn fractions collected, it was chosen the fraction Fr-1 Ps Corn to the chemical studies. This fraction was fractionated by preparative HPLC-DAD resulting in 8 fractions. The crude extract of Malt EtOAc and its fraction and the crude extract of corn NP, were submitted to the chemical evaluation by 1H NMR and biological against the phytopathogenic fungus Cladosporium sphaerospermum (and in the case of corn to C. cladosporioides as well). Besides, anticolinesterasic activity was made, the crude extract of Malt EtOAc and its fractions showed activity. The fractionating of the crude extracts and then of the fractions gotten afforded the isolation of 2 new substances, one of the Fr-6 Ps Malt, a phthalate (4,5-dimethyl-3,4,5,6-tetrahydrobenzo [c] [1,6] dioxecyn-1,8-dione) and another from Fr-1.3 Ps Corn, a coumarin (2- (7-hydroxy-4-methyl-2-oxo-2H-chromen-5-yl) acetic acid) and was possible to propose two structures for another two substances.
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Palavras-chave
Fungos endofíticos, Pseudofusicoccum stromaticum, Eugenia jambolana, Química orgânica, Produtos naturais, Metabólitos, Micro-organismos, Fungal endophyte