Diterpenos tetranorlabdanos e isocumarinas produzidos por Botryosphaeria parva, um fungo endofítico em Eugenia jambolana Lam. (Myrtaceae)
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Data
2016-03-10
Autores
Monfardini, Júlia Dietsche [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Os micro-organismos têm demonstrado serem valiosas fontes de princípios ativos de uso clínico, no qual a penicilina é o exemplo mais conhecido de metabólito secundário, de importância medicinal, produzido por fungos. A espécie vegetal Eugenia jambolana Lam., popularmente conhecida como Jambolão, é utilizada na medicina popular brasileira, principalmente no tratamento de diabetes. O fungo Botryosphaeria parva, isolado das folhas da E. jambolana (L.) e codificado como Ej_F01, foi cultivado em escala ampliada em três meios de cultivo: Czapek®, PDB e Milho. Após o período de fermentação do endófito no meio de milho, foi feita uma extração direta com MeOH, seguida de filtração e evaporação do solvente. O extrato obtido foi solubilizado em AcOEt e fez-se três partições líquido-líquido com H2O, após a evaporação do solvente orgânico, o extrato foi dissolvido em CH3CN e foi feita uma nova partição líquido-líquido com Hexano, após a evaporação da CH3CN, foi obtido o extrato de interesse. Após o período de fermentação do endófito nos meios líquidos (PDB e Czapek®), a suspensão micelar foi filtrada a pressão reduzida, separando os micélios dos meios fermentados. Em seguida, os filtrados aquosos foram submetidos a três partições líquido-líquido com AcOEt, e após a evaporação do solvente orgânico, foram obtidos os extratos brutos de interesse. Todos os extratos foram fracionados utilizando técnicas cromatográficas, como Cromatografia em Coluna e/ou Cromatografia Líquida de Alta Eficiência preparativa. Após o fracionamento dos extratos brutos, foi possível obter sete substâncias oriundas do extrato do milho, seis substâncias do extrato do Czapek®, e uma do extrato de PDB, que foram submetidas a análises espectrométricas (EM, RMN de 1H, 13C, 1D e 2D) para a determinação/identificação estrutural. As substâncias 05, 08, 09 e 11 foram identificadas como 5-hidroximeleina, rel. (3S, 4S)-4-hidroximeleina, meleina e rel. (3S, 4R)-4-hidroximeleina, respectivamente, pertencentes à classe das isocumarinas; as substâncias 01, 03, 06 e 07 foram identificadas como botryosphaerin A, 13,14,15,16-tetranorlabd-7-eno-19,6β:12,17-diolídeo, CJ-14445 e oidiolactona E, respectivamente, pertencentes à classe dos diterpenos tetranorlabdanos; a substância 12 foi identificada como botryosphaerona D, pertencente à classe das naftalenonas. A substância 02, inédita, foi nomeada como rel. (4S, 5R, 6R, 10S)-18-hidroxi-13,14,15,16-tetranorlabd-7,9-dieno-19,6β:12,17-diolídeo, codificada como botryosphaerin I. Os estudos relacionados ao B. parva têm como finalidade verificar sua produção metabólica em diferentes meios de cultivo, bem como a avaliação do potencial biológico dos extratos brutos, frente aos ensaios antioxidante, antifúngico e anticolinesterásico.
Microorganisms have proven to be valuable sources of active principles for clinical use, wherein the penicillin is the best known example of a secondary metabolite of medicinal importance, produced by fungi. The plant species Eugenia jambolana Lam., commonly called Black Plum, is used in traditional Brazilian medicine, especially in the treatment of diabetes. The Botryosphaeria parva fungus, isolated from leaves of E. jambolana (L.) encoded as Ej_F01, was grown on an enlarged scale in three different culture media: Czapek®, PDB and corn. After the fermentation period in the corn media, a direct extraction was done with MeOH followed by filtration and evaporation of the solvent. The extract was disolved in AcOEt and made three liquid-liquid partition with H2O, after evaporation of the organic solvent, the extract was dissolved in CH3CN and was made a new liquid-liquid partition with Hexane, after evaporation of the CH3CN, it was obtained extract of interest. After the fermentation period in liquid media (PDB and Czapek®), the micellar suspension was filtered under reduced pressure, separating the mycelium from the fermented broth. Then, the aqueous filtrates were subjected to three liquid-liquid partition with AcOEt, and after evaporation of the organic solvent, there were obtained the crude extracts of interest. All extracts were fractionated using chromatographic techniques such as column chromatography and/or preparative High Performance Liquid Chromatography efficiency. After the fractioning of crude extracts, it was possible to obtain seven substances derived from corn extract, six substances obtained from Czapek extract, and a substance obtained from the PDB extract, which were submitted to spectrometric analysis (MS-ESI, NMR 1H, 13C, 1D and 2D) for determining and structural identification. The substances 05, 08, 09 and 11 were identified as 5-hydroxymelein, rel. (3S, 4S)-4-hydroxymelein, Melein and rel. (3S, 4R)-4-hydroxymelein, respectively, belonging to the class of isocoumarins; the substances 01, 03, 06 and 07 were identified as botryosphaerin A, 13,14,15,16-tetranorlabd-7-ene-19,6β:12,17-diolide, CJ-14445 and oidiolactone E, respectively, belonging to the class of tetranorlabdanes diterpenoids, and substance 12 was identified as botryosphaerone D, belonging to the class of naftalenones. A new tetranorlabdane diterpenoid was obtained, substance 02, named rel. (4S, 5R, 6R,10S)-18-hydroxy-13,14,15,16-tetranorlabd-7,9-dien-19,6β:12,17-diolide and encoded botryosphaerin I. These studies related to the B. parva are intended to verify your metabolic production in different culture media as well as the evaluation of the biological potential of extracts compared to antioxidant, antifungal and anticholinesterase trials.
Microorganisms have proven to be valuable sources of active principles for clinical use, wherein the penicillin is the best known example of a secondary metabolite of medicinal importance, produced by fungi. The plant species Eugenia jambolana Lam., commonly called Black Plum, is used in traditional Brazilian medicine, especially in the treatment of diabetes. The Botryosphaeria parva fungus, isolated from leaves of E. jambolana (L.) encoded as Ej_F01, was grown on an enlarged scale in three different culture media: Czapek®, PDB and corn. After the fermentation period in the corn media, a direct extraction was done with MeOH followed by filtration and evaporation of the solvent. The extract was disolved in AcOEt and made three liquid-liquid partition with H2O, after evaporation of the organic solvent, the extract was dissolved in CH3CN and was made a new liquid-liquid partition with Hexane, after evaporation of the CH3CN, it was obtained extract of interest. After the fermentation period in liquid media (PDB and Czapek®), the micellar suspension was filtered under reduced pressure, separating the mycelium from the fermented broth. Then, the aqueous filtrates were subjected to three liquid-liquid partition with AcOEt, and after evaporation of the organic solvent, there were obtained the crude extracts of interest. All extracts were fractionated using chromatographic techniques such as column chromatography and/or preparative High Performance Liquid Chromatography efficiency. After the fractioning of crude extracts, it was possible to obtain seven substances derived from corn extract, six substances obtained from Czapek extract, and a substance obtained from the PDB extract, which were submitted to spectrometric analysis (MS-ESI, NMR 1H, 13C, 1D and 2D) for determining and structural identification. The substances 05, 08, 09 and 11 were identified as 5-hydroxymelein, rel. (3S, 4S)-4-hydroxymelein, Melein and rel. (3S, 4R)-4-hydroxymelein, respectively, belonging to the class of isocoumarins; the substances 01, 03, 06 and 07 were identified as botryosphaerin A, 13,14,15,16-tetranorlabd-7-ene-19,6β:12,17-diolide, CJ-14445 and oidiolactone E, respectively, belonging to the class of tetranorlabdanes diterpenoids, and substance 12 was identified as botryosphaerone D, belonging to the class of naftalenones. A new tetranorlabdane diterpenoid was obtained, substance 02, named rel. (4S, 5R, 6R,10S)-18-hydroxy-13,14,15,16-tetranorlabd-7,9-dien-19,6β:12,17-diolide and encoded botryosphaerin I. These studies related to the B. parva are intended to verify your metabolic production in different culture media as well as the evaluation of the biological potential of extracts compared to antioxidant, antifungal and anticholinesterase trials.
Descrição
Palavras-chave
Botryosphaerin I, Botryosphaerona D, Atividade antioxidante, Atividade antifúngica, Atividade anticolinesterásica, Antioxidant activity, Antifungal activity, Acetylcholinesterase activity