Perfil metabólico das raízes de Senna spectabilis e exploração de fungos associados à sua microbiota para produção e biotransformação de metabólitos secundários
Data
2018-04-02
Autores
Monteiro, Afif Felix
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Neste trabalho, analisou-se o perfil químico do extrato etanólico das raízes de mudas adultas de S. spectabilis por meio da aplicação da técnica de CLAE-EM e CLAE-EM/EM, permitindo observar a predominância de alcaloides piperidínicos, bem como de metabólitos de outras classes biossintéticas, incluindo triterpenoide pentacíclico (PT), antraquinonas, flavonas e esteroide. O fracionamento do extrato proporcionou o isolamento e a subsequente elucidação estrutural via técnicas de EM e RMN, de cinco metabólitos secundários: os alcaloides espectalina e cassina; o TP ácido betulínico; o ácido tetradecanoico; e um novo dibenzofurano, o qual consiste no primeiro relato de sua respectiva classe biossintética para S. spectabilis, bem como para o gênero Senna. Essas classes de quimiotipos possuem propriedades estruturais e bioatividades de reconhecido interesse para química medicinal. Nesse contexto, dado ao grande potencial de micróbios associados a plantas medicinais, realizou-se o cultivo de fungos endofíticos e da microbiota relacionada à rizosfera de S. spectabilis, em diferentes meios de cultivo (CZAPEK, Malte e PDB). Os extratos obtidos foram analisados por CLAE-DAD e submetidos a triagem em ensaios antibacterianos. Entre os extratos ativos promissores, o extrato de Fusarium solani em Malte (FsM) foi selecionado e fracionado por CLAE semipreparativa, fornecendo a lactona β-resorcílica (-)-(2R*, 4S*, 8R*)-(Z)-pochonina N, a qual encontra-se em fase de avaliação biológica, e o ácido carboxílico β-lactônico hymeglusina, um composto cujo o potencial antibiótico é bem conhecido. O extrato FsM foi, ainda, analisado por CLAE-EM/EM e os dados gerados foram tratados utilizando-se a abordagem de “redes de interações moleculares” através da plataforma GNPS, propiciando a identificação de onze metabólitos adicionais, entre os quais prevalece a classe de dicetopiperazinas. Esses resultados indicam que a microbiota de S. spectabilis constitui uma fonte rica para a produção de metabólitos com uma ampla diversidade estrutural e, consequentemente, capazes de desempenhar uma gama de funções biológicas. Por fim, investigou-se a capacidade de biotransformação de um substrato diterpenoide (esqueleto halimano) pelos fungos Fusarium oxysporum, Myrothecium verrucaria e Rhinocladiella similis, os quais forneceram sete análogos estruturais oxifuncionalizados, promovendo a biocatálise de reações notáveis, como a oxidação de ligações C-H sp3 não ativadas em posições específicas dos anéis da decalina, bem como a hidratação da ligação dupla-13,14 C-C da cadeia lateral. Essas modificações geraram novas funcionalidades (hidroxi, oxo, formil e carboxi), as quais correspondem a novos sítios reativos e, portanto, podem permitir o alcance de um número maior de análogos estruturais de relevância não apenas química, mas também biológica. O substrato e seus produtos de biotransformação foram avaliados quanto a atividade citotóxica contra as linhagens celulares HCT-116 (carcinoma cólon-retal) e MCF-7 (adenocarcinoma mamário), porém apenas o derivado 19-hidroxi desempenhou inibição intensificada frente às linhagens, em 50 μM. Portanto, estes microrganismos se mostraram fontes enzimáticas prolíficas para a oxidação/redução de diterpenoides com baixa reatividade, revelando um potencial praticamente inexplorado de fungos endofíticos e rizosféricos de Senna spectabilis para a transformação microbiana de outros substratos naturais e ou sintéticos.
This work describes the chemical profiling of the ethanolic extract from roots (ER) of Senna spectabilis’ seedlings by HPLC-HR-ESI-MS and HPLC-IT-MS/MS. This approach allowed observing the prevalence of piperidine alkaloids, as well as metabolites belonging to other biosynthetic classes, including pentacyclic triterpene (PT), anthraquinones, flavones, and steroid. The fractionation of ER provided the isolation, and then the structural elucidation through MS and NMR-based techniques, of five secondary metabolites: piperidine alkaloids spectaline and casine, PT betulinic acid, tetradecanoic acid, and a new dibenzofuran. The dibenzofuran compound comprises the first report of its class for S. spectabilis, and therefore for Senna genus. These chemotypes possess outstanding chemical and bioactive properties for application in medicinal chemistry. In this context, the great potential of plantassociated microbes motivated the cultivation of endophytic and rhizosphere fungi belonging to S. spectabilis microbiota to be performed in different cultivation media (CZAPEK, Malt, and PDB). The resulting extracts were analyzed by HPLC-DAD and subjected to screening in antibacterial assays. The Malt extract of Fusarium solani (FsM), one of the active ones, was fractionated by semi-preparative HPLC, affording the β-resorcylic lactone (-)-(2R*, 4S*, 8R*)-(Z)-pochonin N, which is currently being assayed for antibacterial activity, together with the β-lactonic carboxylic acid hymeglusin, a well-known compound for its antibiotic potential. FsM was further analyzed by HPLC-IT-EM/EM and the data were treated applying the molecular networking approach through GNPS platform, allowing the identification of eleven additional metabolites, including diketopiperazines predominantly. These results indicate that S. spectabilisʹ microbiota comprises a rich source for production of metabolites exhibiting a wide range of structural diversity and, consequently, able to display a variety of bioactivities. Finally, it was investigated the fungal capability of Fusarium oxysporum, Myrothecium verrucaria, and Rhinocladiella similis, to biotransform a diterpenoid substrate (halimane skeleton). These fungi provided seven oxifunctionalized analogs by biocatalysing noteworthy reactions: oxidation of nonactivated sp3 C-H bonds, as well as hydration of the C-C 13,14-double bond from the side-chain. The new generated (hydroxyl, oxo, formil and carboxy) functionalities correspond to new active sites and can allow a further number of structural derivatives to be obtained, not only for chemical but also for biological purposes. The substrate and its bioproducts were accessed for cytotoxic activities against the tumor cell lines HCT-116 e MCF-7 however, only compound 19-hydroxi displayed some enhanced inhibition to both cells, in [50 μM]. Therefore, the abovementioned microorganisms proved to be prolific enzymatic sources for the oxidation/reduction towards weakly reactive diterpenoids, revealing, an untapped potential from endophytes and rhizosphere microorganisms for the transformation of natural and or synthetic substrates.
This work describes the chemical profiling of the ethanolic extract from roots (ER) of Senna spectabilis’ seedlings by HPLC-HR-ESI-MS and HPLC-IT-MS/MS. This approach allowed observing the prevalence of piperidine alkaloids, as well as metabolites belonging to other biosynthetic classes, including pentacyclic triterpene (PT), anthraquinones, flavones, and steroid. The fractionation of ER provided the isolation, and then the structural elucidation through MS and NMR-based techniques, of five secondary metabolites: piperidine alkaloids spectaline and casine, PT betulinic acid, tetradecanoic acid, and a new dibenzofuran. The dibenzofuran compound comprises the first report of its class for S. spectabilis, and therefore for Senna genus. These chemotypes possess outstanding chemical and bioactive properties for application in medicinal chemistry. In this context, the great potential of plantassociated microbes motivated the cultivation of endophytic and rhizosphere fungi belonging to S. spectabilis microbiota to be performed in different cultivation media (CZAPEK, Malt, and PDB). The resulting extracts were analyzed by HPLC-DAD and subjected to screening in antibacterial assays. The Malt extract of Fusarium solani (FsM), one of the active ones, was fractionated by semi-preparative HPLC, affording the β-resorcylic lactone (-)-(2R*, 4S*, 8R*)-(Z)-pochonin N, which is currently being assayed for antibacterial activity, together with the β-lactonic carboxylic acid hymeglusin, a well-known compound for its antibiotic potential. FsM was further analyzed by HPLC-IT-EM/EM and the data were treated applying the molecular networking approach through GNPS platform, allowing the identification of eleven additional metabolites, including diketopiperazines predominantly. These results indicate that S. spectabilisʹ microbiota comprises a rich source for production of metabolites exhibiting a wide range of structural diversity and, consequently, able to display a variety of bioactivities. Finally, it was investigated the fungal capability of Fusarium oxysporum, Myrothecium verrucaria, and Rhinocladiella similis, to biotransform a diterpenoid substrate (halimane skeleton). These fungi provided seven oxifunctionalized analogs by biocatalysing noteworthy reactions: oxidation of nonactivated sp3 C-H bonds, as well as hydration of the C-C 13,14-double bond from the side-chain. The new generated (hydroxyl, oxo, formil and carboxy) functionalities correspond to new active sites and can allow a further number of structural derivatives to be obtained, not only for chemical but also for biological purposes. The substrate and its bioproducts were accessed for cytotoxic activities against the tumor cell lines HCT-116 e MCF-7 however, only compound 19-hydroxi displayed some enhanced inhibition to both cells, in [50 μM]. Therefore, the abovementioned microorganisms proved to be prolific enzymatic sources for the oxidation/reduction towards weakly reactive diterpenoids, revealing, an untapped potential from endophytes and rhizosphere microorganisms for the transformation of natural and or synthetic substrates.
Descrição
Palavras-chave
Fungos do solo, Microbiota, Rizosfera, Fungos endofíticos, Biotransformação (metabolismo)