UHPLC‐DAD‐HRMS 2 Characterization of Novel Dihydrochalcone Conjugates From the Antiviral Mansoa hirsuta Extracts: Diagnostic Fragmentation Pathways and Dereplication of Oligomeric Polyphenols

High‐resolution mass spectrometry (HRMS) coupled with ultra‐high‐performance liquid chromatography (UHPLC) enables rapid characterization of complex polyphenolic constituents in medicinal plants. Mansoa hirsuta (Bignoniaceae), a neotropical liana traditionally used to treat infectious diseases, remains poorly characterized chemically. This study reports the comprehensive UHPLC–DAD–HRMS/MS profiling of ethanolic extracts from leaves, stems, and fruits, leading to the identification of 57 phenolic compounds, including 40 previously unreported flavonoid conjugates. Accurate mass measurements and diagnostic MS/MS fragmentation patterns enabled the structural characterization of five classes of dihydrochalcone‐derived metabolites: dihydrochalcone‐tyramine, ‐flavanone (mansoin‐type), ‐flavan‐3‐ol, and ‐flavonol conjugates. Characteristic fragment ions, particularly those corresponding to the dihydrochalcone core and tyramine moiety, supported the annotation of novel oligomeric flavonoids. Organ‐specific metabolite distribution revealed leaves enriched in Mansoin A–type heterotrimers, stems containing phenylethanoid glycosides and cinnamoyl‐mangiferins, and fruits predominated by simpler dihydrochalcone‐tyramine conjugates. All extracts exhibited low cytotoxicity (CC 50 > 400 μg mL −1 ) and selective antiviral activity against Zika (ZIKV), chikungunya (CHIKV), Mayaro (MAYV), and dengue virus serotype 2 (DENV‐2). The stem extract showed the highest selectivity against ZIKV (SI > 25.5), whereas the fruit extract displayed the greatest potency against DENV‐2 (EC 50 = 25.4 ± 1.2 μg mL −1 ; SI > 19.7). These findings expand the phytochemical knowledge of M. hirsuta , demonstrate the utility of HRMS‐based metabolite profiling for dereplication of complex flavonoids, and provide diagnostic MS/MS fragmentation patterns applicable to the characterization of related oligomeric flavonoids in other plant species.