Analytical profiling of phenolic and flavonoid constituents in Ononis angustissima using LC–ESI–MS/MS coupled with multivariate optimization

Ononis angustissima, a member of the Fabaceae family long employed in folk remedies, has remained largely unexplored in terms of its phytochemical composition and bioactivity. In this work, we applied a face-centered central composite design to refine the ethanolic extraction process for phenolic and flavonoid constituents, achieving peak yields of 76.44 ± 0.76 mg GAE/g dry weight (dw) and 66.42 ± 0.83 mg QE/g dw, respectively. Comprehensive LC–ESI–MS/MS analysis unveiled nineteen principal metabolites, among which luteolin (143.55 ± 1.76 μg/g), salicylic acid (45.82 ± 0.24 μg/g dw), and trans-ferulic acid (40.53 ± 0.43 μg/g dw) were most abundant. The chloroform partition demonstrated remarkable butyrylcholinesterase inhibitory activity (IC50 of 20.52 ± 0.58 μg/mL), showing stronger activity than galantamine under the tested conditions. Additionally, the ethanolic extract exhibited pronounced, selective, and dose-dependent antiproliferative effects against the DLD-1 and CAPAN-1 tumor cell lines, with IC50 of 0.82 ± 0.07 and 0.87 ± 0.06 mg/mL, respectively, while showing considerably lower cytotoxicity toward the L929 fibroblasts. Molecular docking studies further predicted strong interactions of flavonoids, particularly luteolin and quercetin, with both acetylcholinesterase and butyrylcholinesterase active sites, underscoring their neuroprotective potential. Moreover, ADME profiling of the identified metabolites indicated generally favorable pharmacokinetic properties, with luteolin, caffeic acid, and trans-ferulic acid emerging as promising lead compounds due to their absorption potential, lipophilicity, and bioavailability. Collectively, these results highlight O. angustissima as a valuable source of bioactive compounds worthy of further investigation for potential anticancer and neuroprotective applications. However, given the in vitro and in silico nature of this study, additional research, including bioassay-guided isolation, mechanistic studies, and in vivo validation, is necessary to confirm its therapeutic relevance.