Identification of bioactive natural products from endophytic fungi isolated from Globularia alypum

Abstract

The endophytic mycobiota constitute a prolific source of bioactive natural products that has evolved during the symbiotic endophyte−plant relationship. The diversity of fungal endophytes isolated from the roots of the plant Globularia alypum (Plantaginaceae, Scrophulariales) collected in Batna (Algeria) were studied in the present thesis. In total, seventeen fungal strains were isolated and identified to species level by means of morphological and molecular phylogenetic methods. The phylogenetic analysis of these fungal endophytes revealed their affinities to three classes of the phylum of Ascomycota: Eurotiomycetes, Dothideomycetes and Sordariomycetes representing nine genera: Alternaria, Aspergillus, Chaetomium, Dendrothyrium, Diaporthe, Fusarium, Macrophomina, Penicillium and Preussia. Among the isolated fungal endophytes, eight were selected for small scale fermentation in three different liquid media. Their extracts showed prominent antimicrobial activity in a screening for novel antibiotics by the serial dilution assay. A scale-up of fermentation of selected five fungi including three strains of Chaetomium madrasense, Dendrothyrium variisporum and Preussia similis, yielded twenty eight secondary metabolites, twelve of which turned out to be novel natural products. The structures of the isolated metabolites were elucidated using a combination of spectral methods, including 1D and 2D NMR spectroscopy, high-resolution mass spectrometry, and CD spectroscopy. The biological activities of both, the known and new compounds were extensively studied. Thirteen metabolites were isolated from the Preussia similis complex including six new bicyclic polyketides 1-6 (for which the trivial names preussilides A−F are proposed) and one new dimer of 2 amino benzoid acid moieties, along with several known cytochalasins and xanthones derivatives. The preussilides were tested for antimicrobial and antiproliferative effects, and, in particular, preussilides A and C showed selective activities against eukaryotes. Subsequent studies on their influence on the morphology of human osteosarcoma cells (U2OS) suggest that these polyketides might target an enzyme involved in coordination of the cell division cycle. Hence, they might, for instance, affect timing or spindle assembly mechanisms, leading to defects in chromosome segregation and/or spindle geometry. Studies on the secondary metabolite production of Dendrothyrium variisporum in various culture media led to the isolation of twelve compounds. Massarilactones D and H were isolated as the major components as well as two new furanone derivatives for which we propose the trivial names (5S)-cis-gregatin B and graminin D, three new anthranilic acid derivatives, two known anthranilic acid analogues and three cyclopeptides.