Comprehensive phytochemical profiling and bioactivity assessment of the endemic Hypericum heterophyllum: Chemotypic variation revealed through validated LC–MS/MS analysis

Abstract

Hypericum heterophyllum Vent., an endemic species of Türkiye, was investigated for its phytochemical compo sition and biological activities using two populations (HHA: Ankara population and HHB: Afyonkarahisar pop ulation) collected from distinct geographical regions. The 80% ethanolic extracts were evaluated for total phenolic and flavonoid content, antioxidant capacity, antimicrobial activity, enzyme inhibitory effects, and detailed metabolite profiling through a fully validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. Among the two populations, HHA exhibited higher total phenolic and flavonoid contents, which were associated with significantly stronger antioxidant activities across multiple assays. Furthermore, HHA showed more pronounced α-glucosidase inhibitory activity compared to HHB, whereas α-amylase and cholinesterase inhibition remained weak to moderate and population-dependent. LC–MS/MS analysis identified neochlorogenic acid, hyperoside, quercitrin, catechin, and procyanidin B2 as the principal metabolites driving these activities. Both extracts exhibited complex and phenolic-rich chemical profiles; however, HHA consistently contained higher levels of major caffeoylquinic acids, flavonoids, and flavan-3-ols, which was reflected in su perior antioxidant performance (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6- sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), and cupric reducing antioxidant capacity (CUPRAC)), stronger tyrosinase and α-glucosidase inhibition, and enhanced antimicrobial activity—particularly against Gram-positive bacteria—compared to HHB. Overall, H. heterophyllum, especially the HHA chemotype, emerges as a promising natural source of bioactive compounds with potential applications in nutraceutical and pharmaceutical fields.