Constipation affects more than 14% of the global population with complex pathogenesis. Compound Traditional Chinese medicines (TCM), such as CDD-2101, a manufactured version of a time-tested TCM, hold great promise for constipation treatment due to the multiple-component-multiple-target strategy. Here, a novel multiple network analysis based on metabolomics and network pharmacology was developed to decipher the complex interplays between CDD-2101 and constipation. First, CDD-2101 alleviated constipation in a loperamide-induced acute constipation mice model and no adverse effect was observed. Then, fecal metabolomics and phytochemistry profiles were conducted by liquid chromatography-mass spectrometry analysis. The untargeted metabolomics built a metabolomic network related to significantly modulated pathways during the constipation alleviation, including bile acid secretion, steroid hormone biosynthesis, glycerophospholipid metabolism, and linoleic acid metabolism. Based on the phytochemistry, network pharmacology predicted similar modulated pathways and built a hub-target network including retinoid X receptor alpha (RXRA), cytochrome P450 1A1 (CYP1A1), cytochrome P450 1A2 (CYP1A2), and phospholipase A2-IVA (PLA2G4). Third, integrated networks were built to select the key pathways and targets, as well as the key ingredients, including rhein, hesperetin, albiflorin, and magnolol from the TCM, which were corroborated by molecular docking, in vitro enzyme assays, and targeted metabolomics results. Finally, an integrated network was built to elucidate the multi-ingredient-multi-symptom linkage during the treatment. In conclusion, the novel multiple-network analysis revealed the complex “multi-component-multi-target-multi-pathway” mechanisms of CDD-2101 against constipation. Our work provides insight into the understanding of active compounds and pharmacological mechanisms, contributing to the development of natural medicines.
