Elucidating the Pharmacological Basis of Xianling Cifang Granules Against Breast Cancer. A Metabolomic Profiling Study

Objective: To analyze the constituents and metabolic products of Xianling Cifang Granules (XLCF) in the serum of mice. The potential targets of XLCF in the treatment of breast cancer (BC) were explored by combining network pharmacology and molecular docking technology. Methods: Serum was collected from mice following oral administration of XLCF and analyzed using UHPLC-Q Exactive Orbitrap-MS. Absorbed prototype constituents and metabolites were identified by comparing retention times, accurate masses, MS/MS fragments, and isotopic patterns. Network pharmacology predicted potential therapeutic targets, and molecular docking (Autodock/Pymol) validated interactions between key constituents and targets. Results: Our comprehensive metabolomic profiling elucidates the pharmacological basis of XLCF against BC by identifying its absorbed constituents and their potential therapeutic links. It identified 122 prototype constituents of XLCF entering the systemic circulation. Icaritin (a metabolite derived from Epimedium brevicornu Maxim ) was identified as a pivotal constituent due to its high bioavailability and established anti-BC activity, specifically inducing redox-mediated apoptosis via the SIRT6/NF-κB pathway and modulating the immunosuppressive microenvironment in triple-negative breast cancer (TNBC). Additionally, 62 serum metabolites exhibited significant alterations post-XLCF treatment, indicative of metabolic reprogramming involving carboxylation, hydroxylation, glucuronidation, and sulfation. Network pharmacology implicated inflammation and cellular metabolism pathways in the therapeutic effects of XLCF. Molecular docking confirmed that Icaritin, as the principal bioactive component, formed stable interactions with core targets ADORA1, AKR1B1, and ADORA3. Conclusion: This integrated approach delineates the anti-BC mechanism of XLCF, 122 absorbed constituents (with Icaritin as key) and 62 altered metabolites drive systemic metabolic reprogramming, acting through ADORA1, AKR1B1 and ADORA3 targets to modulate critical pathways. These findings provide robust pharmacological evidence supporting the clinical application of XLCF against BC and demonstrate the value of combining metabolomics with target prediction for Traditional Chinese Medicine (TCM) research. Experimental validation of the identified targets is warranted. Keywords: Xianling Cifang granules, UHPLC-Q exactive Orbitrap-MS, prototypes, metabolites, network pharmacology