Natural products from endophytic fungi found in association with Asteraceae species and antibiotic assay in the \Caenorhabditis elegans\ infection model

Author(s)

Denise Oliveira Guimarães

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2010-02-05

Examining board members

Monica Tallarico Pupo; Ilana Lopes Baratella da Cunha Camargo; Fernando Batista da Costa; Edson Rodrigues Filho; Dulce Helena Siqueira Silva

• Advisor: Monica Tallarico Pupo

Abstract

The search for small bioactive molecules from under-explored natural sources that possess specific ecological interaction in their respective habitat has been emphasized as strategic for the discovery of bioactive compounds. Endophytic microorganisms are still relatively under-explored sources of natural products. These microorganisms live inside the host plant tissues without causing symptoms of disease to the host. The specific ecological interaction between the endophytes and their host plants may trigger the biosynthesis of new small bioactive molecules. The endophytic fungi \"Glomerella cingulata\" and \"Guignardia mangiferae\" were isolated from \"Viguiera arenaria\" leaves. G. mangiferae was cultured using different conditions: Czapek, malt extract and potato-dextrose broth liquid media and rice solid medium. Crude extracts and fractions obtained after \"G. mangiferae\" cultivation were evaluated in antimicrobial, cytotoxic and enzymatic assays. Chromatographic procedures were carried out with crude extracts and led to the isolation of 13 compounds, five of them new in the literature. From the novel compounds four are tricycloalternarene derivatives. The crude ethanolic extract obtained from malt extract cultivation was the most promising extract in the biological assays. Chaetoglobosin D, a cytotoxic compound against cancer cell lines previously described in the literature, was isolated from this bioactive extract. The other extracts, fractions and compounds assayed showed weak or moderate activity in the performed assays. The production of tyrosol by \"G. cingulata\" was investigated, since several endophytic fungal strains from Asteraceae species had already produced this compound. Tyrosol may act as a signaling molecule for the production of other metabolites in endophytic fungi. In order to quantify the production of tyrosol by \"G. cingulata\", an analytical method was validated under Guidance of Industry recommendations. Fluid culture analyses were performed after the cultivation of \"G. cingulata\" under different conditions using Czapek liquid medium. The best condition for tyrosol production occurred at pH 5.0, 120 rpm, 30 ºC, six days of incubation. Cultivation of \"G. cingulata\" with and without tyrosol addition in the Czapek liquid medium did not allow the conclusion that tyrosol act as a signaling molecule for metabolite production by endophytic fungi. During sandwich training at Department of Molecular Biology of Massachusetts General Hospital (Harvard Medical School), under Dr. Frederick M. Ausubel supervision, 170 samples from Brazil, the most part obtained from endophytic fungi, were assayed using the nematode \"Caenorhabditis elegans\" infected with the pathogen \"Enterococcus faecalis\". About 29% of pure compounds and 2% of extracts assayed were able to promote the survival of the infected nematode. The polyketides chaetoviridins were found to be the most active compounds in this \"in vivo\" antibacterial assay. (AU)