LC-MS/MS-based phytochemical characterization and biological activities of the endemic species Stachys milasensis

Stachys milasensis Ö. Güner is a recently described endemic species of Türkiye for which phytochemical and biological data are currently unavailable. In this study, aqueous and methanolic extracts obtained from the aerial parts of S. milasensis were comprehensively evaluated for their phenolic composition, antioxidant capacity, and enzyme inhibitory activities. Total phenolic and flavonoid contents were determined spectrophotometrically, while individual phytochemicals were characterized using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Antioxidant properties were assessed by DPPH, ferric reducing antioxidant power (FRAP), cupric reducing antioxidant capacity (CUPRAC), phosphomolybdenum, and metal-chelating assays, whereas enzyme inhibitory activities against cholinesterases, tyrosinase, α-amylase, and α-glucosidase were investigated. The methanolic extract exhibited higher total phenolic and flavonoid contents, reaching 77.69 mg gallic acid equivalents (GAE) per g extract and 71.53 mg rutin equivalents (RE) per g extract, respectively. LC-MS/MS analysis identified chlorogenic acid as the predominant constituent in both extracts, with concentrations of 10 613 and 11 160 µg g^-1 extract in the aqueous and methanolic extracts, respectively. The methanolic extract also contained considerable amounts of verbascoside (730 µg g^-1 extract), hyperoside (95.5 µg g^-1 extract), and luteolin-7-glucoside (83.4 µg g^-1 extract). Among the antioxidant assays, the methanolic extract demonstrated stronger DPPH radical scavenging (96.80 mg trolox equivalents (TE) per g extract), CUPRAC (297.89 mg TE per g extract), and FRAP (157.56 mg TE per g extract) activities. Furthermore, among the tested extracts, the methanolic extract exhibited the highest inhibitory activities against α-glucosidase (1009.48 mg acarbose equivalents (ACE) per g extract), α-amylase (247.54 mg ACE per g extract), and tyrosinase (67.79 mg kojic acid equivalents (KAE) per g extract). This study provides the first comprehensive phytochemical and biological characterization of S. milasensis, contributes the chemotaxonomic knowledge of the genus Stachys, and establishes a foundation for future comparative phytochemical studies.