Objective To explore the self-assembly behavior and molecular association patterns of glycyrrhizic acid (GA) and genkwanin (Gen) after combination, and to analyze the correlation between the incompatibility and the molecular state of Gancao (Glycyrrhizae Radix et Rhizoma)-Yuanhua (Genkwa Flos) from the microscopic perspective. Methods Employ dynamic light scattering (DLS), scanning electron microscopy (SEM), and ultrafiltration to characterize the particle size, morphology and ultrafiltration behavior of GA-Gen mixed solutions. Results When GA and Gen were combined at mass ratios of 55:1 and 45:1, the particle size decreased significantly from 100—150 nm (GA monomer solutions) to approximately 50 nm. After combination, DLS showed a light intensity signal that was not present in the monomer solution at 10—100 nm. SEM images showed that the GA-Gen compatible solutions formed well-defined cubic particles, distinctly different from the globular and long needle-like morphologies of the individual components. When ultrafiltration membranes with molecular weight cut-off (M_w) of 3 000, 10 000, 30 000, 50 000 and 100 000 were used for separation, it was found that with the increase of GA concentration, the transmittance of GA gradually decreased, and the molecular weight at 10% transmittance (T₁₀) and the association coefficient (N) also gradually increased. At the same concentration, the transmittance of GA in the compatibility system was higher than that in the GA monomer system, and T₁₀ and N were lower than those in the monomer system. In addition, the transmittance of Gen in GA-containing system was significantly improved. The decoction system of medicinal materials is consistent with the trends of monomer system. Comparing the SEM of single decoction and co-decoction, the particle size of Glycyrrhizae Radix et Rhizoma single decoction was regular and the size was relatively uniform, and the particle size of Genkwa Flos single decoction was greatly different. The co-decoction failed to reproduce the regular cube of monomer compatibility, but the particle uniformity was between the two single decoctions. Conclusion GA and Gen do not simply coexist in aqueous solution. Instead, Gen is incorporated into GA micelles via molecular substitution, leading to the formation of new self-assembled associates. These findings provide a novel perspective for understanding the mechanism of “reverse” of Glycyrrhizae Radix et Rhizoma-Genkwa Flos herb pair.

glycyrrhizic acid  /  genkwanin  /  self-assembly  /  micelle  /  ultrafiltration separation  /  microscopic analysis  /  permeability  /  association coefficient  /  Glycyrrhizae Radix et Rhizoma  /  Genkwa Flos  /  incompatibility  /  dynamic light scattering