Integrated network pharmacology, molecular docking, and experimental validation reveal synergistic inhibition of EGFR and PI3K-Akt/JAK2-STAT3 pathways by Esculin and Esculetin to exert anti-colorectal cancer effects.

Colorectal cancer (CRC) remains a global health challenge with limited efficacy of single-target therapies. Esculin and esculetin, the main coumarins of Cortex Fraxini, have been reported to exhibit anti-tumor activities in various cancer cell lines in preclinical studies. This study systematically elucidated the synergistic anti-CRC mechanisms of esculin and esculetin by integrating network pharmacology, bioinformatics, molecular docking, molecular dynamics simulations, and in vitro validation. Network pharmacology predicted 23 overlapping targets, with 10 core proteins significantly enriched in the PI3K-Akt and JAK2-STAT3 pathways, further supported by bioinformatics analysis. Molecular docking and 100-ns molecular dynamics simulations revealed that the esculin-esculetin complex exhibited stronger binding affinity and stable interaction with EGFR compared with each monomer. In vitro, the natural 5:1 combination synergistically inhibited HCT116 and HT29 cell proliferation, suppressed colony formation, migration, and invasion, downregulated the phosphorylation of PI3K, Akt, JAK2 and STAT3, reduced EGFR, TOP1 and CDK2 mRNA, decreased the Bcl-2/Bax ratio, and increased cleaved caspase-3 levels. Quantitative validation by EdU, TUNEL, and flow cytometry confirmed concentration-dependent inhibition of proliferation and induction of apoptosis. These findings demonstrate that esculin and esculetin co-target EGFR and simultaneously block downstream PI3K-Akt and JAK2-STAT3 dual pathways, providing a multi-component, multi-target, multi-pathway synergistic model for C. fraxini-based combination therapy against CRC.