Integrated Metabolomics and Network Pharmacology to Explore Potential Mechanisms of Bubali Cornu (Water Buffalo Horn) Water Extract on Hemostasis

A BSTRACT Background: Bubali Cornu (Water Buffalo Horn [WBH]) is a traditional Chinese medicinal material widely used for its hemostatic and blood-cooling properties. While its clinical application in managing bleeding has been documented, the underlying mechanisms of action remain poorly understood. Materials and Methods: To elucidate the molecular basis of WBH’s hemostatic effect, we employed an integrative approach combining network pharmacology and untargeted metabolomics. Bleeding and clotting time assays were conducted in mice to assess hemostatic efficacy. Key active compounds and target genes were identified through bioinformatic databases, and metabolomic profiling was performed using liquid chromatography mass spectrometry/mass spectrometry followed by multivariate statistical analysis. Results: WBH administration significantly reduced both bleeding and clotting times compared to the wild-type group. Network pharmacology analysis identified several active compounds – such as arginine, aspartic acid, alanine, 4-guanidino-1-butanol, guanidine, and cholesterol – that were enriched in amino acid biosynthesis pathways. Metabolomic analysis further revealed upregulation of histidine metabolism, a pathway closely associated with coagulation regulation. Conclusions: These findings suggest that WBH enhances hemostasis primarily through modulation of amino acid metabolism, particularly histidine-related pathways. This study provides novel mechanistic insights into WBH’s pharmacological action and demonstrates the utility of integrating network pharmacology with metabolomics in the study of traditional Chinese medicines.