NMR Analysis of Complex Formation Underlying the Precipitation of Rosmarinic Acid with Protoberberine Derivatives

Rosmarinic acid (RA), a representative polyphenol, forms insoluble complexes with protoberberine alkaloids (PBAs), leading to precipitate formation. In this study, the precipitation behavior and complex formation between RA and three PBAs, coptisine (CO), palmatine (PA), and jatrorrhizine (JA), were investigated using NMR analysis. The addition of RA to aqueous PBA solutions produced precipitates composed of RA and PBAs at a 1 : 1 M ratio, and the amounts of precipitate followed the order CO > PA > JA. Job’s plot and NMR titration analyses revealed that all RA–PBA complexes formed in aqueous solution had a 1 : 1 stoichiometry, and the association constants (K) followed the order RA–CO > RA–PA > RA–JA. Substantial upfield shifts in the proton signals of PBAs suggested that π–π and CH–π interactions are involved in complex formation. Interestingly, the order of the K values was consistent with that of the precipitate amounts, suggesting an association between complex-forming ability and precipitation tendency. However, the relatively small differences in the K values suggest that precipitation is also influenced by other physicochemical factors, including complex solubility. Thermodynamic analyses suggested that the rigid, planar structure of CO may contribute to its high binding affinity by minimizing the entropy penalty during complex formation, whereas the two methoxy groups in PA and the methoxy and hydroxy groups in JA may increase it and reduce binding affinity. These findings provide physicochemical insights into polyphenol–alkaloid interactions and support the rational design and stability assessment of natural-product-based formulations and herbal medicines.