Mass spectrometry-guided isolation of naphthalenoid pulvinic acids from the Ectomycorrhizal Fungus Pisolithus arhizus

Pisolithus arhizus is a worldwide distributed ectomycorrhizal fungus used in traditional medicine for wound healing and antimicrobial properties. It is also known for natural dye production, ecological significance, and ectomycorrhizal associations that detoxify polluted soils and support plant growth. In this study, an integrated dereplication strategy combining liquid chromatography-tandem mass spectrometry (LC-MS 2 ) with molecular networking was employed to investigate the phytochemical profile of the dichloromethane-methanol crude extract from the fruiting bodies of Pisolithus arhizus (Sclerodermataceae). The molecular networking analysis revealed distinct clusters of naphthalenoid pulvinic acid derivatives and enabled the rapid targeting, isolation, structure elucidation, and identification of a previously undescribed pisoquinone close analogue, named methylpisoquinone ( 1 ), alongside the known norbadione A. The isolated compounds were evaluated for their antimicrobial activities against Staphylococcus aureus , Escherichia coli , Candida albicans , and Mycobacterium bovis BCG . Both compounds were inactive at the tested concentrations. • Molecular networking enabled the targeted isolation of naphthalenoid pigments. • Structures were determined based on the NMR and HRESIMS data. • The study revealed the chemical diversity of naphthalenoid pigments in P. arhizus.