Integrating HPLC Fingerprinting, Gray Relational Analysis, and In Vitro–In Vivo Evaluation of Xiaochaihu Decoction and Its Nano‐Phase

BACKGROUND: Self-assembled nano-systems (SANS) formed in traditional Chinese medicine (TCM) decoctions have recently emerged as promising carriers for enhancing the oral bioavailability of poorly soluble components. However, the pharmacokinetic characteristics of SANS in Xiaochaihu (XCH) decoction remain insufficiently investigated. OBJECTIVE: This study aimed to clarify the component distribution and pharmacodynamic relevance of the nanoemulsion phase of XCH decoction (N-XCH) and to assess its potential to improve pharmacokinetics and tissue targeting. METHODS: N-XCH was characterized by particle size analysis, zeta-potential measurement, TEM, UV-Vis, and FTIR spectroscopy. HPLC fingerprinting was used to compare the distribution of active components among the four phase states of XCH decoction. Gray relational analysis (GRA) was applied to relate index components to hepatoprotective and antipyretic endpoints. In vitro release, in vivo pharmacokinetic, and tissue distribution studies were performed to evaluate the release behavior and in vivo disposition of representative components. RESULTS: Fingerprint and GRA analyses indicated that N-XCH is the optimal dissolution phase for co-loading active constituents, and identified saikosaponin A as a key component associated with both hepatoprotective and antipyretic effects. In vitro, N-XCH displayed sustained-release behavior obeying Weibull kinetics with Fickian diffusion. In rats, N-XCH significantly increased the AUC of baicalin, wogonoside, liquiritin, and glycyrrhetinic acid compared with the decoction, while maintaining similar or slightly reduced Cmax values and altered tissue exposure profiles with enhanced hepatic uptake (RUE > 1) for multiple analytes. CONCLUSION: N-XCH, as a self-assembled nanoemulsion phase in XCH decoction, markedly improves the systemic exposure and liver targeting of representative actives without excessive peak concentrations. These findings highlight the potential of SANS in TCM decoctions to optimize oral bioavailability and organ targeting, and provide an integrated analytical-pharmacokinetic framework for linking spectrum-effect relationships with nano-phase behavior.