LC–ESI–MS/MS Phenolic Profiling and In Vitro Biological Activities of Astragalus lusitanicus subsp. orientalis Extracts

ABSTRACT This study reports the phytochemical composition and multifunctional biological activities of Astragalus lusitanicus Lam. subsp. orientalis . Ethanol and water extracts were prepared using ultrasonic‐assisted extraction and evaluated for total phenolic and flavonoid contents, as well as their antioxidant, antimicrobial, and enzyme inhibitory properties. LC–ESI–MS/MS analysis revealed major phenolic constituents, including hesperidin, caffeic acid, protocatechuic acid, and quercetin. The water extract exhibited higher total phenolic (40.36 mg GAE/g) and flavonoid (12.39 mg RE/g) levels, correlating with superior antioxidant performance in 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), and cupric ion reducing antioxidant capacity (CUPRAC) assays. In contrast, the ethanol extract demonstrated stronger inhibitory effects against acetylcholinesterase, tyrosinase, and α‐amylase. Antimicrobial assays (disc diffusion and broth microdilution) showed that the ethanol extract exhibited significant activity against Pseudomonas aeruginosa , Klebsiella pneumoniae , and Listeria monocytogenes , while the water extract displayed moderate inhibition. These findings show that the extracts display measurable in vitro antioxidant, antimicrobial, and enzyme inhibitory activities. The combined presence of various phytochemicals may contribute to the observed activities; however, potential applications would require additional mechanistic, toxicity, and formulation studies. This work provides new chemical and biological insights into an underexplored Astragalus taxon, contributing to the discovery of natural bioactive agents relevant to medicinal chemistry.