Unveiling the Bioactive Potential of Medicago lupulina Flowers: LC-ESI-MS/MS Phytochemical Profiling, Potentiometric Biosensor-Based Antioxidant Evaluation, and In Silico COX-2 Inhibition

Abstract

Plants exhibit significant biological activity and are widely used in the pharmaceutical and food industries due to their bioactive compounds. Medicago lupulina is a plant species belonging to the Fabaceae family. In this study, dried flowers of M. lupulina were extracted with methanol. The total phenolic content and DPPH˙ radical scavenging activity of the obtained extract were determined using a potentiometric biosensor method. In addition, the phenolic content of the extract was determined by LC-ESI-MS/MS. The inhibitory properties of the main components against cyclooxygenase-2 (COX2) were theoretically investigated using molecular docking. Moreover, the pharmacokinetic properties of the main molecules were determined by SwissADME. The methanol extract exhibited a DPPH radical scavenging activity of 39.43% and contained 117.2 mg GAE g⁻¹ extract of total phenolics at 500 ppm. In the LC-ESI-MS/MS analysis, coumarin (23.93), kaempferol (15.79), syringic acid (9.44), isoquercitrin (8.31), salicylic acid (7.49), kaempferol-3-glucoside (6.03), quercetin (4.88) and hesperidin (4.15) were the major constituents (mg g⁻¹ extract). Molecular docking results showed that isoquercitrin (−10.40 kcal/mol), kaempferol-3-glucoside (−10.10 kcal/mol) and hesperidin (−9.90 kcal/mol) exhibited the strongest binding affinities toward the COX-2 enzyme. The BOILED-Egg model indicated that coumarin and salicylic acid possess high gastrointestinal absorption and favorable brain permeability. Therefore, M. lupulina is rich in biologically active secondary metabolites, highlighting its promising potential for applications in the food, pharmaceutical and cosmetic industries.