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Master's Dissertation
DOI
10.11606/D.60.2006.tde-09052007-103936
Document
Author
Full name
Denise Oliveira Guimarães
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2006
Supervisor
Committee
Pupo, Monica Tallarico (President)
Araujo, Angela Regina
Lopes, Joao Luis Callegari
Title in Portuguese
Prospecção química e biológica em fungos endofíticos associados a ´Viguiera arenaria´ (Asteraceae)
Keywords in Portuguese
atividade biológica
fungos endofíticos
metabólitos secundários
Abstract in Portuguese
Foram isolados 37 fungos endofíticos de Viguiera arenaria (VA1 a VA37), sendo 32 oriundos de folhas e 5 de raízes. Os fungos foram classificados por métodos de biologia molecular, sendo Glomerella cingulata a espécie predominante. Os fungos foram cultivados em culturas fermentativas em duas etapas, em pequena escala, para obtenção dos extratos em AcOEt, BuOH e MeOH. Os extratos em AcOEt foram avaliados em ensaios antimicrobianos, citotóxicos frente a células leucemia T humana (JURKAT) e frente às enzimas GAPDH de Trypanosoma cruzi e APRT de Leishmania tarentolae. Diversos extratos apresentaram atividades biológicas significativas. Os perfis químicos dos extratos em AcOEt foram avaliados através de CLAE-UV e RMN 1H. Os fungos VA1 (Glomerella cingulata) e VA17 (Fusarium sp.), selecionados após as triagens química e biológica, foram cultivados em escala ampliada. A partir do extrato AcOEt do fungo VA1 foram isoladas duas substâncias, nectriapirona (I) e tirosol (II). A nectriapirona apresentou atividade citotóxica significativa contra células de leucemia T humana (linhagens JURKAT) e melanoma (linhagens B16F10). Ergosterol (III) foi isolado do extrato micelial metanólico do fungo VA1. A partir do extrato micelial metanólico do fungo VA5 (G. cingulata), cultivado em pequena escala, foi isolado o manitol (IV). Após do cultivo em escala ampliada do fungo VA17 (Fusarium sp.) foram isolados três derivados dicetopiperazinícos, um oriundo do extrato AcOEt, substância V, ainda não relatada na literatura, e dois do extrato micelial MeOH, fusaperazina B (VI) e substância VII, também inédita na literatura. O isolamento das substâncias foi realizado através de técnicas cromatográficas, como CC, CCDP e CLAE. As estruturas químicas foram elucidadas com auxílio de técnicas espectroscópicas (RMN 1H e 13C, HMQC, HMBC, NOE-diff) e espectrométricas (ESI-MS). Experimentos de modelagem molecular foram realizados com a fusaperazina B (VI), que corroboraram com os dados de NOE-diff, indicando que a conformação dobrada do anel dicetopiperazínico é a mais predominante. Foi ainda realizada avaliação do perfil químico dos extratos obtidos em pequena escala dos fungos classificados como G. cingulata, via CLAE e RMN 1H, verificando-se diferenças químicas significativas, o que pode sugerir variabilidade genética entre as linhagens. O tirosol (II) foi detectado na maioria dos extratos de G. cingulata.
Title in English
Chemical and biological prospection in endophytic fungi found in association with Viguiera arenaria (Asteraceae)
Keywords in English
biological activity
endophytic fungi
secondary metabolites
Abstract in English
A total of 37 endophytic fungi were isolated from Viguiera arenaria (VA1 to VA37), 32 from the leaves and 5 from the roots. Endophytes were classified by means of molecular biology methods, and Glomerella cingulata was the predominant species. The endophytes were cultured in a two step fermentative process in small scale to give the EtOAc, BuOH and MeOH extracts. The EtOAc extracts were submitted to antimicrobial assays, citotoxic assays against human leukemia T cells (JURKAT) and assays against two enzymes, GAPDH from Trypanosoma cruzi and APRT from Leishmania tarentolae. Several extracts showed promising activities in the bioassays. The chemical profiles of the EtOAc extracts were obtained through HPLC and 1H NMR. Endophytes VA1 (Glomerella cingulata) e VA17 (Fusarium sp.) were cultured in large scale after chemical and biological screenings. Nectriapyrone (I) and tirosol (II) were isolated from the EtOAc extract from VA1. Nectriapyrone showed high citotoxicity against leukemia T human cells (JURKAT) and melanoma cells (B16F10). Ergosterol (III) was isolated from the micelial MeOH extract of VA1. Mannitol (IV) was isolated from the micelial MeOH extract from the fungus VA5 (G. cingulata). Extracts from VA17 (Fusarium sp.), obtained in large scale, yielded three diketopiperazine derivatives. A novel derivative was isolated from the EtOAc extract (compound V). Two derivatives were obtained from the micelial MeOH extract, fusaperazine B (VI) and a new diketopiperazine (VII). The isolation of the compounds was carried out using chromatography techniques (column, prep. TLC, and HPLC) and the identification was achieved by spectroscopic (1D and 2D NMR) and spectrometric methods (ESI-MS). Molecular modeling experiments were carried out with fusaperazine B (VI), showing that the diketopiperazine ring is predominantly in the folded conformation, in agreement with NOE-diff experiments. Significant differences were observed in the HPLC profiles and 1H NMR spectra of the extracts from the G. cingulata strains, which might be related to genetic variability among the strains. Tirosol (II) was detected in the majority of G. cingulata extracts
 
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Publishing Date
2007-05-21
 
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