Kokusaginine metabolism in rats: A comparative in Vitro-In vivo analysis revealing sex-dependent differences

Kokusaginine, derived from Ruta graveolens L., exhibits diverse biological activities. Prior studies confirmed its anti-renal fibrosis properties and conducted pharmacokinetic analyses, but its specific in vivo metabolites, metabolic enzyme phenotypes, and sex-specific differences remain unclear. To elucidate kokusaginine's metabolic enzyme phenotype in rat liver microsomes and delineate its sex-specific metabolic pathways and metabolites both in vitro and in vivo. Using Ultra-Performance Liquid Chromatography with competitive chemical inhibition and Ultra-High Performance Liquid Chromatography - Quadrupole/Time-of-Flight Mass Spectrometry, we assessed the influence of Cytochrome P450 (CYP) enzymes on kokusaginine metabolism and identified its metabolites. Kokusaginine metabolism involves multiple enzymes, primarily CYP2E1 and CYP2C8. Metabolic pathways include demethylation, oxygenation (primary), and glucuronidation. Twelve metabolites were identified in rat plasma, urine, and excrement, with six in liver microsomes. Sex-specific differences were observed. This study systematically investigated kokusaginine's metabolic characteristics in rats of different sexes, providing insights into its in vivo metabolic behavior. By comparing metabolites and elucidating CYP phenotypes, we gained a comprehensive understanding of kokusaginine's pharmacokinetic profile, guiding clinical medication usage and new pharmaceutical development.