ITS2 ribotyping, in vitro anti-inflammatory screening, and metabolic profiling of fungal endophytes from the Mexican species Crescentia alata Kunth

Abstract

Endophytic fungi represent a promising biotechnological tool to identify and produce in large scale novel anti-inflammatory bioactive compounds. In this study, Crescentia alata Kunth was selected following the ethnomedical criteria and a total of 219 isolates grouped in 86 morphotypes were obtained. From these, 44 isolates that presented a pigment-producing morphotype were selected as the screening panel. The ITS2 ribotypes of the selected endophytic fungi were annotated and classified phylogenetically based on a sequence-structure analysis. The isolates belonged to 17 genera: Colletotrichum, Fusarium, Lophotrichus, Podospora, Xylaria, Diaporthe, Aspergillus, Periconia, Didymella, Prosthemium, Trematophoma, Cladosporium, Cercospora, Pseudocercosporella, Aureobasidium, Bjerkandera, and Trametes. From the anti-inflammatory screening with the Griess assay only 14.77% were highly active with no significant difference compared to indomethacin, and showed promising in vitro anti-inflammatory effect tested in murine macrophages induced with bacterial lipopolysaccharides (LPS). Interestingly, 11.36% of the extracts increased the production of nitrite on LPS-induced macrophages. None of the extracts at the tested concentrations presented a pro-inflammatory effect on non-induced macrophages, nor a cytotoxic effect (cell viability >85%) in the resazurin bioassay. Metabolic profiling of the endo-metabolome and exo-metabolome extracts using Thin Layer Chromatography (TLC), revealed that the exo-metanolome extracts had a relative higher number and diversity of chemical groups. The 1H NMR metabolomic analysis showed characteristic signals that differentiate the fungal genera with high anti-inflammatory activity from those with the least activity. These signals could be associated with the group of terpenes. This is the first report on the isolation of endophytes from C. alata, from which 13 isolates exhibit pharmacological value as sources of potential anti-inflammatory and immuno-modulatory compounds. These bioactive metabolites are likely to belong to the groups of terpenes.