Ajuga vestita Boiss: a “natural gold mine” for the design of novel nutraceuticals and phytopharmaceuticals

Members of the Ajuga genus are of significant interest due to their traditional applications in various countries, and have therefore been the subject of phytochemical research investigating their natural bioactive compounds. The aim of this study was to determine the chemical composition and biological activities of various Ajuga vestita extracts (ethyl acetate, ethanol, ethanol/water and water). The biological effects assessed included antioxidant, enzyme inhibition and cytotoxic activities. The extracts were chemically profiled using ultra-high-performance liquid chromatography – electrospray ionization – quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS), which identified 121 compounds, including iridoids, flavonoids, and phenolic acids. The ethanol/water extract exhibited superior radical scavenging activity (2,2-Diphenyl-1-picrylhydrazyl (DPPH): 43.13 mg Trolox equivalent (TE)/g; 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS): 45.37 mg TE/g) and reducing power (cupric reducing antioxidant capacity (CUPRAC): 103.43 mg TE/g; ferric reducing antioxidant power (FRAP): 45.29 mg TE/g). Regarding enzyme inhibition, the ethanol extract exhibited the greatest inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), glucosidase, and tyrosinase, whereas the ethyl acetate extract was most effective against amylase. Regarding cytotoxicity, the ethyl acetate extract exhibited the strongest effect, significantly reducing cell viability in all cell lines and having a particularly strong impact on murine bone marrow stromal (S17) cells, with viability at 30.8 ± 2.7% at a concentration of 100 µg/mL. By contrast, the aqueous extract exhibited low cytotoxicity, maintaining high cell viability (over 94% at 50 µg/mL and over 74% at 100 µg/mL). Additionally, network pharmacology, molecular docking and molecular dynamics simulations were employed to investigate the multi-target mechanisms and binding stability of A. vestita phytochemicals. These findings provide new, detailed insights into A. vestita and suggest its potential as a raw material for developing health-promoting nutraceutical and pharmaceutical products.