A rapid at-line LC-QTOF-MS/MS based targeting and guided isolation approach uncovers 2-methoxy-1,4-naphthoquinone from Impatiens balsamina Linn. as an anti-Malassezia agent

Background: Impatiens balsamina Linn. is an annual plant traditionally used to treat several cutaneous fungal infections, including conditions often associated with Malassezia spp. However, its anti-Malassezia activity, biomarkers, and underlying mechanisms remain insufficiently defined. This study establishes a rapid at-line LC-ESI-QTOF-MS/MS screening platform coupled with a colorimetric antifungal assay to identify anti-Malassezia biomarkers and assess their modes of action. Methods: An at-line LC-ESI-QTOF-MS/MS platform was used to screen I. balsamina extracts and identify bioactive constituents. The most active extract was subjected to preparative HPLC for isolation of biomarkers. The resazurin-based microdilution antifungal assays were performed against M. furfur. The mechanisms of action of the identified compounds were investigated by lipase assay and biofilm formation assay. Results: The ethyl acetate extract exhibited the most potent antifungal activity. The LC-ESI-QTOF-MS/MS and bioactive chromatograms pinpointed 2-methoxy-1,4-naphthoquinone (2MN) as the only active compound. Purified 2MN showed MIC values of 6.510 ± 1.906 µg/mL and MFC values of 8.854 ± 4.543 µg/mL. The compound reduced extracellular lipase activity and effectively inhibited biofilm formation of M. furfur. Conclusions: The at-line LC-ESI-QTOF-MS/MS platform successfully guided and reduced the time and sample scale for purification of the anti-Malassezia compound. 2MN from I. balsamina exerts a significant antifungal effect and reduces fungal biofilm. Our rapid targeting approach and findings here encourage the discovery of antifungal phytochemicals as a candidate for anti-Malassezia therapy.