The rapid discovery, target preparation, and bioactivity evaluation of 5-lipoxygenase and acetylcholinesterase inhibitors from the flowers of Astragalus membranaceus (Fisch) Bge

This study investigated the bioactive components of Astragalus membranaceus flowers that target 5-lipoxygenase (5-LOX) and acetylcholinesterase (AChE), enzymes linked to inflammation and Alzheimer’s disease. Using receptor-ligand affinity ultrafiltration combined with HPLC-MS, seven active compounds were identified with purities exceeding 98 %. Among them, 9,10-dimethoxy-pterocarpan-3-O-β-D-glucoside (DMPTG) exhibited the strongest dual inhibition of 5-LOX and AChE, with IC50 values of 5.69 × 10⁻⁵ mol/L and 3.97 × 10⁻⁵ mol/L, respectively. Enzyme-catalyzed reaction kinetics confirmed the reversible inhibition of both enzymes. Extraction conditions were optimized using response surface methodology, achieving a target compound yield of 5.96 % under simplified parameters. Efficient isolation was accomplished using semi-preparative liquid chromatography and high-speed countercurrent chromatography, overcoming the limitations of single techniques. Molecular docking and dynamics simulations revealed stable interactions between DMPTG and the target enzymes, supported by strong binding energies and minimal structural fluctuations. Biological assays demonstrated that DMPTG significantly protected glutamate-injured PC12 cells by modulating the expression of apoptosis-related proteins, restoring Bcl-2 levels, and reducing Bax and Bad expression. This regulatory effect underscores the neuroprotective potential of the compound. This comprehensive strategy integrates advanced screening, extraction, and bioactivity evaluation, providing a robust foundation for developing dual-target inhibitors. The findings highlight the industrial and pharmacological value of A. membranaceus flowers in treating inflammation and neurodegenerative disorders. [Display omitted] •A binary target was employed to screen for highly bioactive components.•A “two in one” extraction and targeted preparation methodology was established.•A complex separation protocol was optimized for high yield and purity.•Compounds with potent molecular and cellular level bioactivities were identified.•A quaternary bioactivity evaluation process helps avoid false-positive results.