Vukovic_2026_Plants.(Basel)_15_

Alchemilla subcrenata Buser is a montane Alchemilla (Rosaceae) species with established traditional medicinal use but only limited prior phytochemical characterization, restricted to preliminary observations on phenolic content and peroxidase activity. This study reports the first systematic optimisation of microwave-assisted extraction (MAE) conditions for this species using a Box-Behnken design within a Response Surface Methodology (RSM) framework. Three extraction variables, ethanol concentration (30-70%, v/v), solid-to-liquid ratio (1:10-1:20, w/v), and extraction time (10-30 min), were simultaneously optimised against twelve response variables encompassing total phenolic and flavonoid contents, six antioxidant assays, and four enzyme inhibitory activities (acetylcholinesterase, butyrylcholinesterase, tyrosinase, and alpha-amylase). Second-order polynomial models demonstrated good predictive ability for most responses (R(2) = 0.878-0.971 for eleven of the twelve responses), with the exception of metal chelation, which showed a lower fit (R(2) = 0.671) and was excluded from optimisation; the multiobjective optimization identified optimal conditions of 60% ethanol, 1:12.5 (w/v) solid-to-liquid ratio, and 10 min extraction time. Under optimal conditions, MAE delivered exceptional in vitro antioxidant and enzyme inhibitory activities from A. subcrenata. LC-MS/MS characterisation, representing the first comprehensive phenolic profiling of this species, identified 28 phenolic compounds (1599.7 microg/g extract), dominated by quercetin-3-O-hexoside, gallic acid, catechin, and kaempferol glycosides; naringenin and aesculetin represent novel phytochemical records for A. subcrenata. The abundant presence of quercetin hexosides and hydroxycinnamic acids offers a mechanistic explanation for the broad, multi-target bioactivity observed, positioning A. subcrenata as a promising source of bioactive phenolics that warrants further investigation for potential nutraceutical, pharmaceutical, and cosmeceutical applications.