Avaliação química e biológica de fungos endofíticos associados as algas marinhas Acanthophora spicifera, Dichotomaria marginata e Sargassum vulgare
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Data
2018-09-24
Autores
Honório, Alana Evangelista
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Universidade Estadual Paulista (Unesp)
Resumo
A química de produtos naturais é uma estratégia de grande sucesso na descoberta de compostos bioativos para o desenvolvimento de novos fármacos. Neste contexto destacam-se os estudos com plantas, micro-organismos, e mais recentemente organismos marinhos. Avanços recentes nos estudos com algas marinhas e de seus fungos associados, com destaque para os fungos endofíticos, revelaram bioatividades relevantes, como anti-inflamatória, antioxidante antimicrobiana e antitumoral, além de estruturas químicas diversificadas, incluindo compostos halogenados. Este trabalho explorou o potencial químico e biológico dos endófitos associados às algas A. spicifera, D. marginata e S. vulgare, permitindo o isolamento de 40 linhagens fúngicas e a identificação de doze gêneros e espécies por taxonomia molecular e morfológica. Após isolados, preservados e cultivados no meio líquido extrato de Malte preparado com água do mar ou água ultra pura, foram obtidos os extratos brutos em acetato de etila, que foram avaliados quanto ao perfil químico por RMN de 1H, CLAEDAD, CG-EM e CL-EM, além de triagem biológica em ensaios antimicrobianos contra linhagens de bactérias super-resistentes e formadoras de biofilme, Doença de Chagas, Leishmaniose, bem como em ensaio anticolinesterásico, de inibição da atividade de protease de veneno de serpente e citotóxico frente as linhagens tumorais HCT-116 e MTT. A análise química permitiu a identificação de 78 compostos por CG-EM e 26 por CL-EM. A avaliação biológica dos extratos brutos e frações semipurificadas mostrou alto porcentual de amostras bioativas, incluindo algumas com potenciais acima dos padrões utilizados. Após a prospecção inicial, linhagens de Hypoxylon monticulosum isoladas de A. spicifera (AS 09 MQ) e de D. marginata (DM 02 MQ), foram selecionadas para investigação de sua produção metabólica para verificação de possíveis interações entre hospedeiro e micro-organismos associados, bem como a descoberta de novos compostos. O cultivo das duas linhagens de H. monticulosum, em água ultrapura, levou ao isolamento de dois compostos majoritários para cada extrato, sendo Diidroesporotriolídeo (S02) comum aos dois extratos. Essa classe de compostos pertence à família das furofurandionas e são comumente relatadas para o gênero Avaliação química e biológica de fungos associados às algas marinhas Acanthophora spicifera, Dichotomaria marginata e Sargassum vulgare. Hypoxylon. Do extrato de DM 02 MQ foi isolada uma Ciclohexadiona (S03) inédita na literatura. Essas substâncias apresentaram atividade antimicrobiana contra diferentes linhagens de bactérias super-resistentes. A linhagem de Annulohypoxylon stygium (AS 01 MQ) isolada da alga A. spicifera foi cultivada nos meios líquido Czapek e Malte utilizando planejamento fatorial 2^3 com 5 réplicas no ponto central, afim de avaliar como as condições de cultivo, variação de pH, tempo de crescimento e tipo de água afetam sinergicamente a produção em massa e a diversidade química de seus componentes. Os resultados obtidos para o cultivo em Malte mostrou que a variável água é o fator determinante para melhora do cultivo do endófito, enquanto para Czapek, a variável tempo representou o fator determinante. A análise química permitiu identificar 20 compostos por CG-EM. Esses dados confirmaram a expectativa de que as condições de cultivo podem influenciar a produção de metabólitos secundários, permitindo planejar e direcionar os experimentos de acordo com as respostas desejadas. Os dados gerados nesse trabalho demonstraram a importância do estudo dos organismos marinhos, como os fungos endofíticos, contribuindo para o conhecimento da quimiodiversidade desses micro-organismos associados às algas A. spicifera, D. marginata e S. vulgare, ainda sub-explorada do ponto de vista químico, confirmando que estes são uma fonte prolífica de metabólitos novos, bioativos e de elevada quimiodiversidade.
The chemistry of natural products is a highly successful strategy in the discovery of bioactive compounds for the development of new drugs. In this context studies with plants, microorganisms and more recently marine organisms stand out. Recent advances in marine algae studies, along with studies on their associated fungi, with emphasis on endophytic fungi, disclosed relevant bioactivities such as anti-inflammatory, antimicrobial,antitumor and antioxidant, as well as strong chemodiversity, including halogenated compounds. This work explored the chemical and biological potential of endophytes associated to marine algae A. spicifera, D. marginata and S. vulgare andled to the identification of twelve endophytic fungal strains by molecular and morphological taxonomy. After isolation, preservation and cultivation in Malte liquid medium prepared with sea water or ultra pure water, crude extracts were obtained in ethyl acetate, and were evaluated for their chemical profile by 1H NMR, HPLC-DAD, GC-MS and LC-MS, in addition to biological screening against multi-resistant bacterial strains and biofilm-forming bacteria, against Chagas disease, Leishmaniasis, as well as anti-cholinesterase assay, inhibition of snake venom protease activity and cytotoxic activity against human colon adenocarcinoma (HCT-116). Chemical analyses led to the identification of 78 compounds by GC-MS and 26 by LC-MS. Biological evaluation of crude extracts and fractions disclosed a high rate of bioactive samples, including some higher potentials than standard compounds. After preliminary prospection, Hypoxylon monticulosum strains isolated from A. spicifera (AS 09 MQ) and D. marginata (DM 02 MQ) were selected for their metabolic production investigation and detection of possible host-microorganisms interactions as well as the discovery of new compounds. Cultivation of H. monticulosum, in ultrapure water, led to the isolation of two major compounds from each extract, with dihydroesporotriolide (SO2) as a common constituent in both extracts. This class of compounds belongs to the furofurandione family and are widely reported in genus Hypoxylon. From DM 02 MQ extract, a novel Cyclohexadione (SO3) was isolated. These substances showed antimicrobial activity against multi-resistant bacterial strains. In addition, Annulohypoxylon stygium (AS 01 MQ) isolated from A. spicifera was selected for chemometric studies and was grown in Czapek and Malte liquid media using 2 ^ 3 factorial design with 5 replicates at the central point in order to evaluate how the Avaliação química e biológica de fungos associados às algas marinhas Acanthophora spicifera, Dichotomaria marginata e Sargassum vulgare. culture conditions, pH variation, growth time and type of water might synergistically affect mass production yield and chemical diversity of its components. The results obtained for the Malte cultivation evidenced that the variable water is the determining factor for the improvement of the endophyte culture, whereas for Czapek, time was the determining factor variable . Their chemical analysis allowed the identification of 20 compounds by GC-MS. These data confirm the expectation that the cultivation conditions can modify, increase or direct the production of secondary metabolites, and it is possible to plan and direct the experiments according to the desired responses. The data generated in this study demonstrated the importance of marine organisms studies, such as endophytic fungi, and contributed to the knowledge of the chemodiversity of microorganisms associated with marine algae A. spicifera, D. marginata and S. vulgare, which are still underexplored, and confirm that they represent a prolific source of novel, bioactive and highly chemodiverse metabolites.
The chemistry of natural products is a highly successful strategy in the discovery of bioactive compounds for the development of new drugs. In this context studies with plants, microorganisms and more recently marine organisms stand out. Recent advances in marine algae studies, along with studies on their associated fungi, with emphasis on endophytic fungi, disclosed relevant bioactivities such as anti-inflammatory, antimicrobial,antitumor and antioxidant, as well as strong chemodiversity, including halogenated compounds. This work explored the chemical and biological potential of endophytes associated to marine algae A. spicifera, D. marginata and S. vulgare andled to the identification of twelve endophytic fungal strains by molecular and morphological taxonomy. After isolation, preservation and cultivation in Malte liquid medium prepared with sea water or ultra pure water, crude extracts were obtained in ethyl acetate, and were evaluated for their chemical profile by 1H NMR, HPLC-DAD, GC-MS and LC-MS, in addition to biological screening against multi-resistant bacterial strains and biofilm-forming bacteria, against Chagas disease, Leishmaniasis, as well as anti-cholinesterase assay, inhibition of snake venom protease activity and cytotoxic activity against human colon adenocarcinoma (HCT-116). Chemical analyses led to the identification of 78 compounds by GC-MS and 26 by LC-MS. Biological evaluation of crude extracts and fractions disclosed a high rate of bioactive samples, including some higher potentials than standard compounds. After preliminary prospection, Hypoxylon monticulosum strains isolated from A. spicifera (AS 09 MQ) and D. marginata (DM 02 MQ) were selected for their metabolic production investigation and detection of possible host-microorganisms interactions as well as the discovery of new compounds. Cultivation of H. monticulosum, in ultrapure water, led to the isolation of two major compounds from each extract, with dihydroesporotriolide (SO2) as a common constituent in both extracts. This class of compounds belongs to the furofurandione family and are widely reported in genus Hypoxylon. From DM 02 MQ extract, a novel Cyclohexadione (SO3) was isolated. These substances showed antimicrobial activity against multi-resistant bacterial strains. In addition, Annulohypoxylon stygium (AS 01 MQ) isolated from A. spicifera was selected for chemometric studies and was grown in Czapek and Malte liquid media using 2 ^ 3 factorial design with 5 replicates at the central point in order to evaluate how the Avaliação química e biológica de fungos associados às algas marinhas Acanthophora spicifera, Dichotomaria marginata e Sargassum vulgare. culture conditions, pH variation, growth time and type of water might synergistically affect mass production yield and chemical diversity of its components. The results obtained for the Malte cultivation evidenced that the variable water is the determining factor for the improvement of the endophyte culture, whereas for Czapek, time was the determining factor variable . Their chemical analysis allowed the identification of 20 compounds by GC-MS. These data confirm the expectation that the cultivation conditions can modify, increase or direct the production of secondary metabolites, and it is possible to plan and direct the experiments according to the desired responses. The data generated in this study demonstrated the importance of marine organisms studies, such as endophytic fungi, and contributed to the knowledge of the chemodiversity of microorganisms associated with marine algae A. spicifera, D. marginata and S. vulgare, which are still underexplored, and confirm that they represent a prolific source of novel, bioactive and highly chemodiverse metabolites.
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Palavras-chave
Hypoxylon monticulosum, Annulohypoxylon stygium, Marine microorganisms, Endophytic fungi, Marine algae, Chemometrics, Micro-organismos marinhos, Fungos endofíticos, Algas marinhas, Quimiometria