Integrated LC–ESI–MS/MS and Chemometric Profiling of <i>Carduus pycnocephalus</i> subsp. <i>albidus</i> : Linking Phenolic Diversity to Antioxidant and Enzyme Inhibitory Potentials

This study presents the first molecular-level characterization of Carduus pycnocephalus subsp. albidus using LC-ESI-MS/MS and chemometric analysis to elucidate its phenolic diversity and biofunctional significance. Methanolic extracts prepared by maceration (MAC-ME), ultrasound-assisted extraction (UAE-ME), and Soxhlet extraction (SOE-ME) were compared in terms of phytochemical composition and biological activities. Twenty-three phenolic compounds were quantified, with chlorogenic acid (9452-9840 µg/g), hesperidin (3678-4069 µg/g), and hyperoside (1028-1145 µg/g) as major constituents. SOE-ME and UAE-ME exhibited the highest total phenolic content (36.38 and 36.14 mg GAEs/g) and flavonoid content (46.78 and 46.51 mg REs/g). Antioxidant assays revealed significant extraction method-dependent variation. SOE-ME showed the strongest reducing and radical-scavenging capacities (CUPRAC = 0.96 mg/mL; FRAP = 0.74 mg/mL; ABTS•⁺ = 1.45 mg/mL), while UAE-ME had the best metal-chelating ability (IC₅₀ = 2.63 mg/mL). The relative antioxidant capacity index (RACI) ranked SOE-ME highest (0.458). Enzyme inhibition was moderate: SOE-ME and UAE-ME were most active against acetylcholinesterase (IC₅₀ = 1.27-1.31 mg/mL), whereas UAE-ME showed the strongest inhibition against α-amylase (2.10 mg/mL); tyrosinase inhibition was similar across extracts (1.37-1.38 mg/mL). Strong correlations were observed between total phenolics/flavonoids and antioxidant activity (r > 0.87) as well as between these contents and acetylcholinesterase inhibition (r ≈ 0.93). These results identify C. pycnocephalus subsp. albidus as a phenolic-rich thistle exhibiting extraction-dependent chemical and biofunctional diversity.