Mechanistic Insights Into the Anti‐Constipation Potential of Xiaogan Jianwei Formula: Integration of HPLC‐Q‐TOF MS/MS, Network Pharmacology, In Vivo Validation, and Gut Microbiota

This study aims to explore the mechanism of action and potential bioactive constituents of Xiaogan Jianwei Formula (XG) in the treatment of constipation. The chemical constituents of XG and its absorbable components were characterized using HPLC-Q-TOF MS/MS, and network pharmacology was used to predict key targets and pathways, identifying the cAMP/PKA signaling pathway as a target pathway. In vivo experiments were performed to evaluate anti-constipation effects, including measurements of fecal water content, intestinal transit rate, MUC2 expression, cAMP/PKA/AQP3 signaling, and gut microbiota composition via 16S rRNA sequencing. In total, 138 chemical constituents were identified from XG, of which 32 components were absorbable; flavonoids were the main active ingredients. Pharmacological studies showed that XG significantly increased fecal water content and intestinal transit rate in loperamide-induced constipated rats without affecting physiological parameters. XG upregulated the expression of cAMP, PKA, p-PKA, and AQP3 and restored MUC2 expression. Moreover, XG improved gut microbiota diversity, enriched beneficial bacteria such as Akkermansia, and reduced pathogenic taxa like Helicobacter and Campylobacterota. XG alleviates constipation by regulating the cAMP/PKA/AQP3 signaling axis and modulating the gut microbiota. This study provides a scientific basis for the clinical application of XG and supports its development as an effective medicine for constipation.