• JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
 
  Bookmark and Share
 
 
Master's Dissertation
DOI
https://doi.org/10.11606/D.41.2019.tde-05032020-094343
Document
Author
Full name
Eunizinis dos Santos Kawafune
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2019
Supervisor
Committee
Ferreira, Marcelo José Pena (President)
Maldonado, Gabriel Padilla
Sartorelli, Patrícia
Title in Portuguese
Metabolômica de fungos endofíticos associados à Baccharis oxyodonta DC.(Asteraceae): da quimiodiversidade à citotoxicidade
Keywords in Portuguese
Baccharis
Citotoxicidade
Fungos endofíticos
Metabolômica
Produtos naturais
Redes moleculares
Abstract in Portuguese
Os microrganismos representam uma espetacular fonte de metabólitos bioativos que podem apresentar aplicações como agentes agrotóxicos, antibióticos, imunossupressores, antiparasitários, antitumorais, entre outros. Nesse grupo, os fungos endofíticos apresentam grande potencial na caracterização de substâncias bioativas, especialmente componentes citotóxicos. O objetivo geral deste trabalho foi contribuir com o conhecimento a respeito da diversidade química e biológica de fungos endofíticos associados aos indivíduos masculino e feminino de Baccharis oxyodonta DC. (Asteraceae). Para tal, os fungos endofíticos dos indivíduos feminino e masculino de B. oxyodonta foram isolados e identificados por sequenciamento da região ITS e construção de árvores filogenéticas. No indivíduo feminino foram isolados 28 fungos pertencentes aos gêneros Alternaria, Annulohypoxylon, Cercospora, Colletotrichum, Phomopsis e Preusia, e as famílias Dydimellaceae e Strophariaceae. Já do indivíduo masculino foram obtidos 20 fungos distribuídos entre os gêneros Alternaria, Aspergillus, Cladosporium, Penicillium, Periconia e Phomopsis. Para cultivo das cepas utilizou-se três meios de cultivos diferentes (arroz, BDA e Czapek). Após extração, as 136 amostras resultantes foram analisadas por CLAE/EM-EM e, a partir dos dados obtidos, foi realizada uma análise metabolômica que possibilitou a visualização do impacto das diferentes condições de cultivo no perfil metabólico das cepas. Dessa análise pode-se verificar que o meio de cultivo BDA gerou a maior diversidade metabólica e, também, foram identificadas diferentes classes de substâncias tais como, antraquinonas, xantonas, amidas, lactonas sesquiterpenicas, ácidos graxos, isocumarinas, dicetopiperazinas e policetídeos. Os extratos foram avaliados em relação ao potencial citotóxico, utilizando o ensaio do MTT, sendo 11 extratos considerados ativos frente a linhagem tumoral HCT-116 (carcinoma colorretal), quando testados na concentração de 50 μg.mL-1. Com base nos resultados de citotoxicidade e do perfil metabólico, duas cepas, Alternaria sp. 1 ePhomopsis sp. 4, foram selecionadas para o cultivo em larga escala. Após procedimentos cromatográficos e análise espectral dos isolados, quatro componentes do extrato bruto de Alternaria sp. 1 foram identificados como alternariol, alternariol-metiléter, altenusina e 5'-metoxi-6-metil-bifenil-3,4,3'-triol e, a partir do extrato bruto de Phomopsis sp. 4 foram identificadas a sidowinina B, o ácido 8-hidroxi-6-metil-xantona carboxílico, inéditos no gênero, e phomopsixantona, inédita em literatura. As substâncias foram avaliadas em relação a citotoxicidade entretanto apenas o alternariol exibiu atividade de inibição moderada frente a linhagem tumoral HCT-116
Title in English
Metabolomics of endophytic fungi associated with Baccharis oxyodonta DC. (Asteraceae): from chemiodiversity to cytotoxicity
Keywords in English
Baccharis
Cytotoxicity
Endophytic fungi
Metabolomics
Molecular networking
Natural products
Abstract in English
Microorganisms represent a spectacular source of bioactive metabolites and they may have applications such as pesticides, antibiotics, immunosuppressants, antiparasitics, antitumor, among others. Endophytic fungi are a rich source of bioactive compounds, especially cytotoxic components. The objective of this work was to contribute to the knowledge about the chemical and biological diversity of endophytic fungi from male and female Baccharis oxyodonta DC. (Asteraceae). Thereby, the endophytic fungi of male and female individuals from B. oxyodonta were isolated and identified by sequencing of ITS region and using phylogenetic tree construction. Twenty-eight fungi belonging to the Alternaria, Annulohypoxylon, Cercospora, Colletotrichum, Phomopsis and Preusia genera, and the families Dydimellaceae and Strophariaceae were isolated from females. From the male, 20 fungi distributed among the genera Alternaria, Aspergillus, Cladosporium, Penicillium, Periconia and Phomopsis were obtained. Strains were cultivated using three different media (rice, BDA and Czapek). After extraction procedures, the resulting 136 samples were analyzed by HPLC/MS-MS and a metabolomic approach was performed to allow the visualization of the impact of different culture conditions on the metabolic profile of strains. From this analysis could be verified that BDA medium furnished the highest metabolic diversity and were identified different classes of compounds such as anthraquinones, xanthones, amides, sesquiterpene lactones, fatty acids, isocoumarins, diketopiperazines and polyketides. The extracts were evaluated for cytotoxic potential using the MTT assay. Eleven extracts tested at a concentration of 50 μg.mL-1 were considered active against the HCT-116 tumor cell line (colorectal carcinoma). Based on the cytotoxicity and metabolic profile results, two strains, Alternaria sp. 1 and Phomopsis sp. 4, were selected for large-scale cultivation. After chromatographic procedures and spectral analysis of the isolated compounds, four components from Alternaria sp. 1 were identified as alternariol, alternariol methylether, altenusine and 5'-methoxy-6-methyl-biphenyl-3,4,3'-triol and from the crude extract of Phomopsis sp. 4 were identified sydowinin B, 8-hydroxy-6-methyl-xanthone carboxylic acid, unpublished in the genus, and a new compound phomopsixanthone. Isolated compounds were evaluated for cytotoxicity, however only alternariol showed moderate inhibition activity against the HCT-116 tumor cell line.
 
WARNING - Viewing this document is conditioned on your acceptance of the following terms of use:
This document is only for private use for research and teaching activities. Reproduction for commercial use is forbidden. This rights cover the whole data about this document as well as its contents. Any uses or copies of this document in whole or in part must include the author's name.
There are withheld file due to requirements (data publishing, patents or rights).
Release Date
2023-12-18
Publishing Date
2020-12-01
 
WARNING: Learn what derived works are clicking here.
All rights of the thesis/dissertation are from the authors
CeTI-SC/STI
Digital Library of Theses and Dissertations of USP. Copyright © 2001-2022. All rights reserved.