Anticholinesterase and Antityrosinase Secondary Metabolites from the Fungus Xylobolus subpileatus

is a widely distributed crust fungus reported from all continents except Antarctica, although considered a rare species in several European countries. Profound mycochemical analysis of the methanol extract of resulted in the isolation of seven compounds ( - ). Among them, (3β,22E)-3-methoxy-ergosta-4,6,8 ,22-tetraene ( ) is a new natural product, while the NMR assignment of its already known epimer ( ) has been revised. In addition to a benzohydrofuran derivative fomannoxin ( ), four ergostane-type triterpenes - were identified. The structure elucidation of the isolated metabolites was performed by one- and two-dimensional NMR and MS analysis. Compounds - as well as the chloroform, -hexane, and methanol extracts of were evaluated for their tyrosinase, acetylcholinesterase, and butyrylcholinesterase inhibitory properties. Among the examined compounds, only fomannoxin ( ) displayed the antityrosinase property with 51% of inhibition, and the fungal steroids proved to be inactive. Regarding the potential acetylcholinesterase (AChE) inhibitory activity of the fungal extracts and metabolites, it was demonstrated that the chloroform extract and compounds - exerted noteworthy inhibitory activity, with 83.86 and 32.99%, respectively. The butyrylcholinesterase (BChE) inhibitory assay revealed that methanol and chloroform extracts, as well as compounds and exerted notable activity, while the rest of the compounds proved to be only weak enzyme inhibitors. Our study represents the first report on the chemical profile of basidiome of the wild-growing , offering a thorough study on the isolation and structure determination of the most characteristic biologically active constituents of this species.