Biological Activities and LC–QTOF–MS-Based Phytochemical Characterization of Onosma alboroseum Fisch. et Mey. subsp. alboresum var. alboroseum Extracts and Extract-Loaded Nanoparticles

<b>Background/Objectives:</b> This study provides the first comprehensive evaluation of the antioxidant, antimicrobial, and enzyme inhibitory activities of <i>Onosma alboroseum</i> subsp. <i>alboroseum</i> var. <i>alboroseum</i>, including a novel nanoformulation-based comparative assessment of its most active extract. The study further aimed to investigate whether nanoparticles modulate the biological performance of the extract. <b>Methods:</b> Antioxidant activity was assessed using DPPH, FRAP, and CUPRAC assays, and total phenolic content was determined by the Folin-Ciocalteu method. Antimicrobial activity was evaluated using agar well diffusion and microdilution assays, while enzyme inhibitory activities were assessed through anticholinesterase and anti-urease assays. The most biologically active extract was subjected to LC-QTOF-MS-based tentative metabolite profiling and subsequently formulated into nanoparticles for comparative biological evaluation. <b>Results:</b> Among the extracts studied, the methanol extract had the highest total phenolic content and demonstrated superior antioxidant, antimicrobial, and enzyme inhibitor activities. LC-QTOF-MS profiling indicated a phenolic-rich composition, with rosmarinic acid as the predominant compound based on relative peak area. The methanol extract was encapsulated within alginate nanoparticles for subsequent comparative biological assessment. While the crude extract showed superior activity in antioxidant assays, nanoparticles enhanced cholinesterase and urease inhibition (28.03% and 12.11%, respectively) and improved antibacterial efficacy in microdilution assays (MIC range: 3.13-12.5 µg/mL), although no inhibition was observed in agar diffusion tests. <b>Conclusions:</b> These findings indicate the first time that the methanol extract of <i>Onosma alboroseum</i> subsp. <i>alboroseum</i> var. <i>alboroseum</i> represents a phenolic-rich source of bioactive constituents and a nanoparticle formulation that can modulate specific biological activities depending on the assay system, highlighting the relevance of formulation strategy in phytochemical-based pharmaceutical applications.