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Master's Dissertation
DOI
https://doi.org/10.11606/D.87.2022.tde-20122023-152813
Document
Author
Full name
Rafael Conrado
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2022
Supervisor
Committee
Souza, Ana Olívia de (President)
Maldonado, Gabriel Padilla
Oliveira, Rodrigo Cardoso de
Vasconcellos, Suzan Pantaroto de
Title in Portuguese
Avaliação da Ação Biológica de Metabólitos Secundários Produzidos pelo Fungo Penicillium sp.
Keywords in Portuguese
Penicillium maximae
Ação antifúngica
Citotoxicidade
Metabólitos secundários
Abstract in Portuguese
Neste estudo, uma linhagem de fungo isolada do solo do bioma Caatinga foi identificada por análise da região espaçadora transcrita interna (ITS) do DNA ribossômico (rDNA) e da morfologia por microscopia óptica, microscopia eletrônica de varredura (MEV) e de transmissão (MET), como Penicillium maximae. Por ensaio de antagonismo em relação a diversos fitopatógenos, o P. maximae inibiu o crescimento de Curvularia lunata, Fusarium sacchari, Fusarium verticillioides, e de Fusarium vasinfectum, Fusarium subglutinans, e de Fusarium phaseoli. Para a biossíntese de metabólitos secundários, o fungo P. maximae foi cultivado em meio líquido batata dextrose por 15 dias a 28 ºC e 150 rpm, e do processamento do sobrenadante da cultura por extração em fase sólida com metanol obteve-se uma fração bruta (SPE100) que no fracionamento por cromatografia líquida de alta eficiência (CLAE/HPLC) resultou em sete frações (F1-F7). No ensaio de ação antifúngica por determinação da concentração inibitória mínima (CIM) contra os fitopatógenos acima descritos, as frações F1-F7 não foram efetivas até 500 µg/mL. No teste de citotoxicidade por MTT [3-(4,5-dimetiltiazol-2il)-2,5-difenil brometo de tetrazol] em células tumorais de mama humana (MCF-7 e MDA-MB-231), de câncer de pâncreas (MIA-PaCa-2) e de melanoma humano (SKMELL-28), a fração F6 a 250 e 500 µg/mL inibiu o crescimento celular da linhagem MCF-7 em 66,6% e 72,2%, e a fração F7 a 125, 250 e 500 µg/mL em 64,1%; 81,4% e 70,5%, respectivamente. Nas células MIAPaCa-2, a fração F6 inibiu 42,7% do crescimento celular, e a fração F7 64,3%, ambas as frações a 500 µg/mL. Até 500 µg/mL, essas frações não foram citotóxicas para fibroblastos normais HDFα, e a 1.000 µg/mL não foram tóxicas para larvas de Galleria mellonella, por até 120 horas. Os metabólitos secundários presentes nas frações F6 e F7 apresentam especificidade pelas células tumorais, e os dados são promissores para continuidade do estudo e purificação dos metabólitos secundários produzidos por esta espécie.
Title in English
Evaluation of the Biological Action of Secondary Metabolites Produced by the Fungus Penicillium sp
Keywords in English
Penicillium maximae
Antifungal action
Cytotoxicity
Secondary metabolites
Abstract in English
In this study, a fungal strain isolated from the soil of the Caatinga biome was identified by the internal transcribed spacer (ITS) analysis of ribosomal DNA (rDNA) and morphology by optical microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) as Penicillium maximae. Through antagonism assay against several phytopathogens the P. maximae inhibited the growth of Curvularia lunata, Fusarium sacchari, Fusarium verticillioides, and Fusarium vasinfectum, Fusarium subglutinans, and Fusarium. phaseoli. For the biosynthesis of secondary metabolites, the fungus P. maximae was cultivated in potato dextrose liquid medium for 15 days at 28 ºC and 150 rpm. The processing of the culture supernatant by solid phase extraction with methanol, resulted in a crude fraction (SPE100) which through high performance liquid chromatography (HPLC/HPLC) fractionation resulted in seven fractions (F1-F7). By the minimum inhibitory concentration assay against the above-described phytopathogens, until 500 µg/mL the F1-F7 fractions did not showed antifungal activity . The cytotoxicity of the fractions F1- F7 was evaluated by using the MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazole bromide] test in human breast tumor cells (MCF-7 and MDA-MB -231), pancreatic cancer (MIA-PaCa-2) and human melanoma (SKMELL-28). The F6 fraction at 250 µg/mL inhibited the cell growth of MCF-7 by 66.6% and at 500 µg/mL 72.2%, and the F7 fraction at 125, 250 and 500 µg/mL in 64.1%; 81.4% and 70.5%, respectively. In MIA-PaCa-2 cell, the F6 fraction inhibited 42.7% of cell growth and the F7 fraction 64.3%, both fractions at 500 µg/mL. These fractions up to 500 µg/mL were not cytotoxic to normal HDFα fibroblasts, and at 1,000 µg/mL were not toxic to Galleria mellonella larvae until 120 hours. The secondary metabolites present in the F6 and F7 fractions present specificity for the tumor cells, and the results are promising for the continuity of the study and purification of the secondary metabolites produced by this species.
 
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Release Date
2026-01-14
Publishing Date
2024-01-16
 
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