Comprehensive characterization of the in vitro and in vivo metabolites of xylocarpin H using UHPLC-Q-TOF-MS/MS

Xylocarpin H, a limonoid from the Xylocarpin granatum, exhibits significant biological activities, including antioxidant, antiviral, analgesic, hypnotic, antimalarial, and antidepressant effects, demonstrating considerable potential for drug development. However, few studies have been reported to identify and classify active metabolites responsible for such excellent biological activities. In this study, we utilized ultra high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC–Q–TOF–MS/MS) coupled with multiple data postprocessing techniques to rapidly identify in vivo and in vitro metabolites of xylocarpin H. Consequently, a total of 32 metabolites (23 in phase I and 9 in phase II) of xylocarpin H were detected both in rat feces, urine, bile, blood, intestinal flora, and rat and human liver microsomes. The metabolic pathways of xylocarpin H mainly involve hydrolysis, oxidation, reduction, addition, glucose conjugation, glucuronide conjugation, and sulfate conjugation. The metabolite M15 of xylocarpin H was synthesized by ester hydrolysis and its structure was determined by nuclear magnetic resonance spectroscopy. This study elucidates the metabolic pathways of xylocarpin H and provides insights into the origin of its pharmacological activity.