Untargeted LC-QTOF-MS/MS metabolomics and integrative pharmacology identify candidate triterpenoids associated with the gastrointestinal activity of Baliospermum montanum root extract.

Baliospermum montanum (Willd.) has been investigated for its gastrointestinal effects, but its metabolite composition and potential mechanisms underlying these effects remain insufficiently characterized. This study employed an integrative framework combining untargeted metabolomics, computational pharmacology, and in vivo evaluation to characterize candidate constituents and potential mechanisms associated with the gastrointestinal effects of B. montanum root extract. Methanolic root extract was profiled using untargeted LC-QTOF-MS/MS in positive and negative electrospray ionization modes. Facility-assisted database/library annotation and manual curation identified 15 prioritized putative metabolite annotations, including the triterpenoid candidates cucurbitacin C, ganoderic acid F, ursolic acid, and 24-hydroxyglycyrrhetic acid. In accordance with the Metabolomics Standards Initiative framework, these compounds were classified as Level 2 putative annotations because authentic reference standards were not analyzed under identical experimental conditions. Network pharmacology, molecular docking, and molecular dynamics simulations were performed against gastrointestinal regulatory targets, including dopamine receptor D2 (DRD2), 5-hydroxytryptamine receptor 1A (5-HT1A), and cystic fibrosis transmembrane conductance regulator (CFTR). Cucurbitacin C showed a comparatively favorable predicted interaction profile among the prioritized candidates, while the corresponding DRD2-associated complex maintained a root-mean-square deviation below 2 Å during molecular dynamics simulation, supporting stability of the simulated complex. In vivo, oral administration of the root extract to Wistar rats for five consecutive days at 300-900 mg/kg produced significant dose-related changes in fecal characteristics, stool output, and defecation frequency, with the greatest responses observed at 900 mg/kg (p < 0.001). No significant changes in body weight or serum AST, ALT, and ALP were observed under the tested conditions, providing no biochemical evidence of hepatocellular injury. Histopathological examination of the colon demonstrated alterations in goblet-cell mucin, crypt morphology, epithelial appearance, edema, and inflammatory features, consistent with changes in colonic secretory activity and mucosal architecture. Collectively, these findings indicate that B. montanum root extract produces measurable gastrointestinal effects accompanied by alterations in intestinal secretory and mucosal parameters. Integrative computational analysis identified potential interactions of prioritized putative metabolites with gastrointestinal regulatory targets, with cucurbitacin C emerging as a candidate constituent for further investigation. These findings provide a basis for future experimental validation of the metabolite-target relationships underlying the gastrointestinal activity of B. montanum root extract.Keywords: Baliospermum montanum; Untargeted LC-MS/MS metabolomics; Molecular dynamics simulation; Cucurbitacin C; Gastrointestinal motility.