Optimization of culture conditions of Beauveria bassiana and Mycoleptodiscus indicus aiming the production of metabolites with antimicrobial, antitumor and antiparasitic activity

Author(s)

Willian Jonis Andrioli

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2012-03-16

Examining board members

Jairo Kenupp Bastos; Roberto Gomes de Souza Berlinck; João Batista Fernandes; Ian Castro Gamboa

• Advisor: Jairo Kenupp Bastos

Abstract

It is known that microorganisms are considered as promising source of bioactive molecules and endophytic and entomopathogenic fungi are included in this group. Therefore, we selected the entomopathogenic fungus Beauveria bassiana and the endophytic fungus Mycoleptodiscus indicus to initiate the prospection aiming to determine the favorable growing conditions to obtain secondary metabolites with biological activity. This work was guided by biological assays and chromatographic analysis. The fractions obtained in the cultivation in a large scale were subjected to biological tests (leishmanicidal, antimalarial, antimicrobial and cytotoxic). The dichloromethane and ethyl acetate fractions of both fungi showed promising results in antitumor and leishmanicidal assays. Then, these fractions were subjected to chromatographic processes that allowed the isolation of compounds whose chemical structures were elucidated through the use of NMR techniques, mass spectrometry, UV, IR, ?D, and X-ray diffraction. From cultures of B. bassiana were obtained compounds from the pyridone class: pyridovericin, pyridovericin-N-O-(4-O-methyl-?-D-glucopyranoside) and pyridovericin-15-deoxy, being the latter two novel compounds; and alloxazine class: 7,8-dimethylalloxazine (lumichrome) and 1-methyl-11-hydroxylumichrome, being this last one unprecedented. From M. indicus cultures were obtained compounds from two classes: azaphilone and chromene. In this thesis we report the derivatives eugenitin, 6-methoxyeugenin and 9-hydroxyeugenin, which have been isolated from a wide variety of plants and fungi. Azaphilones are a class of structurally diverse fungal metabolites, known pigments with pyran-quinone structure and contains a highly oxygenated bicyclic core and a chiral quaternary center. Then, we obtained two known azaphilones: austdiol and austdiol diacetate (product of acetylation), and three unprecedent azaphilones: austdiol dimer, austdiol dihydroisocoumarin and austdiol chromen. The three new azaphilones from M. indicus have an unusual pattern of connection between two aromatic polyketides, a methylene bridge. The isolation of these molecules reinforces the hypothesis that the biosynthesis occurs with the participation of one formaldehyde molecule. The purified compounds were submitted to biological assays. In the cytotoxic assay the azaphilones austdiol and austdiol diacetate, and the pyridone pyridovericin displayed activity against tumor cell lines, although at higher concentrations than the standard compounds used. Moreover, there was some degree of selectivity, since the assayed compounds were not toxic to human lymphocytes. Also in leishmanicidal assays, austdiol, austdiol diacetate and pyridovericin displayed activity, although at levels lower than leishmanicidal standards. Thereby, it can be considered that azaphilone and pyridone compounds are good leader compounds to search for cytotoxic and leishmanicidal agents, since these classes of compounds have biological dynamicity. Finally, the isolation and elucidation of new metabolites belonging to azaphilone and pyridone classes contribute to the enrichment of these important fungal metabolites and, likewise, this study demonstrates the importance of endophytic and entomopathogenic fungi in natural products chemistry. (AU)