Prospecção de metabólitos especiais com potencial antimicrobiano produzidos por fungos endofíticos associados à gramínea Axonopus Leptostachyus (Flüggé) Hitchc (Poaceae)

• Autor(a) principal: Neponuceno, Ricardo Aparecido Rodrigues
• Data de Publicação: 2020
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Institucional da UFMT
• Texto Completo: http://ri.ufmt.br/handle/1/3906
• topic: Bioprospecção; Metabólitos secundários; Atividade antimicrobiana; CNPQ::CIENCIAS AGRARIAS; Bioprospecting; Secondary metabolites; Antimicrobial activity

Endophytic fungi contribute considerably to the production of bioactive metabolites of great economic value and with several applications, such as antibiotics and agrochemistry. In this context, the present work aimed to perform the isolation, purification and structural elucidation of secondary metabolites produced by endophytic fungi associated with Axonopus leptostachyus, aiming at the search for substances with potential antimicrobial action. In this way, 64 endophytes isolated from A. leptostachyus, were submitted to assays to evaluate antimicrobial activity, against 5 strains of bacteria and 5 of human pathogenic candida, semi-quantified and classified into groups of high (AIA), medium (MAI) and low (BAI) inhibitory activity. Additionally, six selected endophytes were also evaluated for the detection of possible antagonists of the phytopathogen Corynespora cassicola. The results of the bioassays added to the multivariate analysis allowed the selection of the endophytic fungi with the best potential for obtaining the bioactive metabolites, with Microsphaeropsis arundinis (P21) being the only one that showed excellent activity in relation to all tests. Against pathogenic bacteria and candida, the fungi Talaromyces varreculosus (P24), Gongronella butleri (P48), Penicillium javanicum (P70) and the unidentified endophyte (P4) stood out with the best antimicrobial activities. The tests also identified that Cladosporum flabelliforme (P5) and Neocosmospora strita (P28) showed excellent activities against pathogenic candida and phytopathogen, while Fusarium succisae (P59) and Fusarium oxysporum (P61) stood out only with activity for C. cassicola. After the bioassays, the endophytes with the best potentials were submitted to cultivation in PDA (Potato Dextrose Agar) and minimal medium, in a reduced and enlarged scale. In the PDA medium, the endophytes were grown in 100 mL of medium (5 Petri dishes) under incubation at 37 °C for 15 to 23 days. Pieces with PDA and mycelia were randomly cut and macerated for 24 h with 150 mL of ethyl acetate (EtOAc). The EtOAc phase was vacuum filtered, concentrated in a rotary evaporator and dried in an air jet, obtaining the crude extracts EtOAc. In the minimal medium, the fungi grew in duplicate of 200 mL under incubation and orbital shaking for 20 days. Then, the micellar suspension was filtered, partitioned with EtOAc, and dried to obtain the crude extracts. In sequence, the extracts from the screening and growth stages on a larger scale were solubilized and analyzed using HPLC-PDA (High-Performance Liquid Chromatography – Photodiode Array) and 1H NMR (Proton Nuclear Magnetic Resonance). After analysis, the P5, P21 and P28 endophytes were chosen for the chromatographic fractionation of their extracts, aiming at the isolation of the compounds responsible for the detected activities. The isolated substances were analyzed using 1H and 13C NMR spectroscopy, gCOSY, gHMBC and gHSQC for the identification or elucidation of their structures. The fractionation of the EtOAc extract of C. flabelliforme (P5) resulted in the isolation of a compound in Tr 26 min. (FR1.16- 17.fim.3), not yet identified. The study of M. arundinis (P21) allowed the identification of a benzolactonic dimer (1), an unprecedented molecular structure for the genus Microsphaeropsis. For the fungus N. striata (P28), substances extracted from the PDA medium were isolated, such as the monorden (radicicol), a lactonic derivative of resorcylic acid (2), and the compounds in fractions FR4.fim.4, FR6.1, FR6.3, FR6.5 and FR1.precipitate, not yet identified, as well as a substance extracted from the minimal medium, under study for structural identification (3).