Beyond chemical composition: solvent pretreatment modulates supramolecular assembly and interactions with MRSA to enhance antibacterial efficacy of Scutellariae Radix-Coptidis Rhizoma

Background Scutellariae Radix - Coptidis Rhizoma herb pair is a classic traditional Chinese medicine (TCM) combination with heat-clearing, dampness-drying and detoxifying effects, extensively applied clinically against bacterial infections. Traditional water decoction and ethanol pretreatment are mainstream extraction approaches, whereas existing comparisons merely emphasize quantitative variations of bioactive compounds, neglecting the underlying supramolecular assembly differences of effective substances. Comparative analysis of solvent-induced supramolecular variations provides a novel perspective to interpret differential pharmacodynamic material bases of TCM formulas. Methods Macroscopic and microscopic morphologies of HH-0 and HH-50 were firstly characterized by visual inspection, SEM and DLS. Their phytochemical profiles were subsequently analyzed by UHPLC-Q-Orbitrap HRMS and HPLC. Antibacterial activities against MRSA were further quantified via turbidimetry and plate assays. UV, FT-IR and MD simulations revealed divergent supramolecular assembly behaviors. Finally, untargeted metabolomics and assembly-peptidoglycan interaction simulations elucidated the underlying anti-MRSA mechanisms. Results The solvent pretreatment endowed HH-0 and HH-50 supramolecules with distinct characteristics. HH-50 featured rougher supramolecular surfaces and higher Zeta potential with favorable dispersibility. The two supramolecules shared consistent active constituents, and HH-50 showed elevated berberine abundance and enhanced anti-MRSA activity. Notably, HH-50 maintained superior antibacterial efficacy and interference against peptidoglycan synthesis and energy metabolism even at identical berberine doses. This confirmed that divergent supramolecular conformations determined antibacterial differences, as diminished hydrogen bonds and weakened van der Waals and electrostatic interactions enhanced the binding affinity between assemblies and MRSA. Conclusion Beyond chemical composition, solvent pretreatment (water versus 50% ethanol) markedly modulates supramolecular assembly behaviors and subsequent assembly-bacteria interactions, thereby regulating the anti-MRSA efficacy of the Scutellariae Radix - Coptidis Rhizoma herb pair. This work emphasizes that supramolecular configurations, rather than simple differences in chemical contents, dominate the pharmacological activities of traditional Chinese medicine preparations.