Angiotensin-converting enzyme (ACE) plays a key role in blood pressure regulation. This study elucidated the inhibitory mechanisms of matrine (MT) and oxymatrine (OMT), with particular emphasis on how N-oxidation

influences their interactions with ACE. Enzyme kinetics revealed that OMT exhibited better inhibitory efficacy, with a lower IC₅₀ value than MT. Fluorescence spectroscopy and isothermal titration calorimetry confirmed that both alkaloids formed static, enthalpy-driven complexes with ACE, dominated by hydrogen bonding and electrostatic interactions. Circular dichroism spectroscopy demonstrated that OMT induced more pronounced reductions in the α-helical content of ACE, reflecting stronger conformational alterations upon complexation. Molecular docking speculated that N-oxidation endowed OMT with additional hydrogen bonds and hydrophobic contacts within the catalytic pocket and the flexible lid region (α₁–α₃ helices). Molecular dynamics simulations

further demonstrated that OMT stabilized the lid region and reduced residue fluctuations, which is consistent with enhanced binding stability. Mechanistically, the addition of N-oxidation increased the molecular polarity and rigidity of OMT, thereby improving shape and electrostatic complementarity at the ACE-ligand interface, which enhanced interfacial noncovalent interactions and promoted tighter complex formation. Overall, these findings provide structural insight into how N-oxidation modulates the bioactivity of quinolizidine alkaloids, offering a theoretical basis for the rational design of functional food ingredients targeting blood pressure regulation.