Iceberg pharmacokinetic strategy for simultaneous evaluation of five flavonoids from Viticis fructus extract in rats using UHPLC–ESI–MS/MS

Viticis fructus (VF) is the dry and ripe fruit of Vitex trifolia L. var. simplicifolia Cham or Vitex trifolia L., which contains several flavonoids exhibiting promising pharmacological effects. However, the pharmacokinetic behaviors of these flavonoids and their metabolites remain unclear. This study aimed to comprehensively evaluate the pharmacokinetics of five flavonoids from VF in rats using ultra-high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UHPLC–ESI–MS/MS) and the Iceberg pharmacokinetic strategy. After gavage administration of VF extract to rats, only small amounts of free vitexicarpin, chrysosplenol D and luteolin were detected in rat plasma, while the contents of free quercetin and kaempferol were both lower than their corresponding lower limits of quantification. In contrast, enzymatic hydrolysis with β-glucosidase revealed significant amounts of quercetin, kaempferol, vitexicarpin, chrysosplenol D, and luteolin. The maximum plasma concentration (C_max) and the area under the curve from time zero to the last measurable concentration (AUC_0–t) values of total vitexicarpin, chrysosplenol D and luteolin after enzymatic hydrolysis were all significantly higher than those free values before enzymatic hydrolysis, respectively (P < 0.05). These findings indicate that most flavonoids from VF primarily exist as phase II metabolites in rat plasma, which can prolong their residence time in the body through intestinal hepatic circulation and other pathways. These phase II metabolites with higher concentrations may be an essential material basis for their pharmacological effects.