Abstract: Objective To investigate the significance of hot filtration in component detection of licorice-containing traditional Chinese medicine preparations by taking the herb pair of Genkwa Flos and Radix et Rhizoma Glycyrrhizae as an example in order to elucidate the supramolecular interactions between these herbs and provide chemical-level evidence for the “eighteen incompatible medicaments” theory in traditional Chinese medicine. Methods The experimental approach involved multiple analytical techniques. High-Performance liquid chromatography (HPLC) was employed to quantitatively analyze the content variations of marker components in decoctions prepared at different herb ratios and at two temperatures (85℃ and 35℃). Scanning electron microscopy (SEM) was used to examine the morphological characteristics and structural changes induced by temperature variations in the co-decoction solutions. Comparative HPLC analyses were conducted between single-herb decoctions and combined decoctions to evaluate the impact of self-assembly formation on the dissolution behavior of the marker components. The standard curve method was applied for precise quantification of all target compounds under different preparation conditions. Results The investigation revealed significant temperature-dependent phenomena. The physical state of the Genkwa Flos-Radix et Rhizoma Glycyrrhizae decoction was thermally responsive in that a solution state was maintained at 85℃, but turned into a semi-gel state at 35℃ and room temperature, and no complete gelation was formed that would enable tube inversion. HPLC analysis demonstrated substantially higher recovery rates for all three marker components (Yuanhuacine, Yuanhuadine, and Genkwanin) in decoctions filtered at 85℃ compared to those processed at 35℃. SEM observations provided visual evidence of temperature-mediated nanostructural evolution, revealing well-defined nanoparticles approximately 400 nm in diameter at 85℃ that progressively aggregated into larger structures around 2 μm when the temperature decreased to 35℃. Comparative analysis established that the self-assembly formation in combined decoctions significantly enhanced the dissolution efficiency of the marker components compared to single-herb preparations. Quantitative determination yielded specific content values: 0.012 0±0.000 1 to 0.045 1±0.001 4 μg for Yuanhuacine, 0.008 2±0.000 06 to 0.028 3±0.000 9 μg for Yuanhuadine, and 0.119 9±0.000 7 to 0.255 0±0.003 9 μg for genkwanin at different preparation ratios. Conclusion This research provides insights into the temperature-responsive self-assembly behavior in incompatible herb pairs via integrated component quantification and morphological characterization. The findings clearly demonstrate that the decoction process facilitates the initial formation of supramolecular nanostructures, while subsequent cooling drives the progressive growth and aggregation of these nanoparticles. This study is expected to advance the theoretical framework of traditional Chinese medicine by establishing a supramolecular foundation for comprehending herbal compatibility and decoction processes.

Key words: Genkwa Flos-Radix et Rhizoma Glycyrhizae, Glycyrrhizinic Acid, Eighteen Incompatibilities, Toxicity, Temperature, Mechanism, High-Performance Liquid Chromatography (HPLC), Scanning Electron Microscopy (SEM)