Systematic Characterization of the In Vitro and In Vivo Chemical Constituents of Shenmai Formula by UHPLC-Orbitrap MS

Shenmai Formula (SMF) is a classic traditional Chinese medicine for cardiovascular diseases. Its multicomponent complexity and obscure in vivo metabolic fate hinder understanding of its therapeutic material basis. Because many constituents undergo extensive gastrointestinal and hepatic biotransformation, in vitro profiling alone cannot reveal the actual circulating entities; systematic in vitro-in vivo mapping is therefore essential to define SMF's true bioactive material basis. An in-house constituent database was built from literature (PubMed/CNKI). SMF extract and rat plasma (blank and drug-containing) were analyzed by UHPLC-Orbitrap MS, and constituents were identified by integrating database matching with published literature. Fifty-eight in vitro constituents (mainly saponins) were characterized. Integrating in vitro and in vivo data, 19 circulating prototypes and 14 biotransformed metabolites were mapped in rat plasma. Their biotransformation was driven mainly by deglycosylation, oxidation, methylation, desaturation, and sulfation. The 19 prototypes and 14 metabolites define SMF's in vivo profile and represent the primary potential bioactive candidates for its efficacy. Mapping these active forms and pathways provides a targeted material basis for future pharmacological, pharmacokinetic, and mechanistic studies.