Deciphering the Material Basis of an Herbal Formula via Integrated Two-dimensional Chromatography, In Silico Screening, and Comparative Pharmacokinetics: A Case Study of Kaiyu Jiangzhuo Formula

Abstract Kaiyu Jiangzhuo (KYJZ) is a Chinese medicine formula composed of nine herbs. Its efficacy in alleviating glycolipid metabolic disorders and reducing the incidence of diabetes mellitus in overweight patients with prediabetes has been widely validated. Although our previous studies separately characterized the chemical constituents of the KYJZ decoction and the components detected in plasma, the relationship between chemical profiling and systemic exposure remains undefined. In particular, it is unclear which decoction constituents are absorbed into systemic circulation and how obesity modulates their pharmacokinetic profiles. This study aimed to comprehensively profile the components of KYJZ and identify potential bioactive constituents by integrating in silico screening, comparative pharmacokinetic, and tissue distribution studies. Two-dimensional liquid chromatography–mass spectrometry (2D-LC–MS) and a hybrid method (data-dependent acquisition/high-resolution multiple reaction monitoring) were employed to decipher the chemical profile and circulating components of KYJZ, respectively. Components exhibiting high binding affinity for antiobesity targets (composite Z-score >0.4) were selected for further pharmacokinetic study. In summary, 316 compounds were identified in KYJZ using 2D-LC–MS/MS. Among these, 99 compounds were recognized as circulating constituents, and 16 with a composite Z-score >0.4 were specifically selected for comparative pharmacokinetic evaluation in mice fed a normal-chow diet (NCD) or a high-fat diet (HFD). Comparative pharmacokinetics revealed that long-term HFD feeding significantly changed the pharmacokinetic behavior of circulating components in mice, especially protoberberine-type alkaloids. Despite their low plasma exposure, they were detected at their highest concentrations in the liver relative to all other tissues examined, suggesting that the liver may represent a relevant organ contributing to the antiobesity effects of KYJZ. Our findings highlight the capability of two-dimensional chromatography to characterize complex herbal medicines and establish a novel analysis pipeline integrating chemical profiling, in silico screening, and comparative pharmacokinetic analysis for identifying bioactive components in traditional Chinese medicine formulas.