Bioactive Saponin Ardisiacrispin B Suppresses Colorectal Cancer Progression through Regulation of Aberrant RAS/RAF and PI3K/AKT Signaling Pathways: An Integrated Network Pharmacology and Experimental Validation Study

Abstract Colorectal cancer remains a leading cause of cancer-related morbidity and mortality worldwide, despite advances in current therapeutic strategies. However, the clinical management of CRC is still hindered by limited treatment efficacy, the emergence of drug resistance, and severe systemic toxicity associated with conventional therapies. In this context, natural products have emerged as promising sources for the development of novel anticancer agents due to their diverse bioactive properties, multitarget mechanisms of action, and comparatively lower cytotoxic effects. Here, we investigated the anti-CRC potential and underlying mechanisms of Ardisiacrispin B (AB), a bioactive saponin derived from Ardisia crispa. To this end, we employed an integrated strategy combining network pharmacology, molecular docking, and molecular dynamics simulations to predict candidate targets and pathways, followed by in vitro and in vivo experimental validation using CRC cell lines and a xenograft mouse model. Network pharmacology identified 240 shared targets between AB and CRC, highlighting SRC, AKT1, EGFR, STAT3, and other oncogenic proteins as core nodes. Gene Ontology and KEGG pathway analyses indicated that AB may potentially influence key signaling cascades, including the PI3K–AKT, MAPK, and RAS pathways, which were subsequently investigated experimentally. Molecular docking and 100 ns molecular dynamics simulations predicted stable binding of AB to key targets, characterized by significant hydrogen bonding and favorable free energy profiles. In vitro, AB inhibited proliferation, colony formation, and migration of HCT-116 and SW480 cells in a concentration-dependent manner, while inducing apoptosis and cell cycle arrest via modulation of cleaved caspase-3, Bax, and Bcl-2 levels. Mechanistically, AB suppressed PI3K/AKT signaling and reduced RAS expression while increasing the phosphorylation of the three major MAPK subfamilies, p38, JNK1/2, and ERK1/2, highlighting a complex regulatory network. In vivo, AB significantly reduced tumor growth in HCT-116 xenografts and was well tolerated under the experimental conditions, as indicated by the absence of significant changes in body weight during the treatment period, as evidenced by decreased Ki67 expression and increased cleaved caspase-3 in tumor tissues. Collectively, these findings provide a comprehensive mechanistic framework demonstrating that AB exerts potent anti-CRC effects through multitarget modulation of key oncogenic pathways, offering a promising candidate for further preclinical development and potential therapeutic translation.