Assessing Digestive Transformations of Withania somnifera Extracts via LC–MS/MS Profiling with a Focus on Bioactive Compounds Withaferin A, Withanolide A, Withanoside IV, and Untargeted Metabolomics

Botanical extracts are widely employed as health care agents but lack the rigorous vetting required for FDA-approved pharmaceuticals. Research on phytochemical bioavailability and transformation has mainly focused on liver metabolism and plasma binding, while gastrointestinal and microbiome metabolism studies remain limited. This study combined digestive in vitro assays with mass spectrometry-based metabolomics and molecular networking to analyze metabolites in Withania somnifera leaf and root extracts, along with three known bioactive reference standards: withaferin A, withanolide A, and withanoside IV. Both withaferin A and withanoside IV underwent significant in vitro transformation, while withanolide A remained stable across conditions. Molecular networking revealed that withanolides in the root extract were largely stable, whereas many in the leaf extract were more labile under the assay conditions. Detailed network analysis also enabled the identification of specific metabolite transformations. These findings support the refinement of in vitro models to better predict in vivo behavior in complex botanical mixtures.