Integrated Computational and Experimental Study Reveals Isorhoifolin as a Potential Therapeutic Agent Against Hepato-cellular Carcinoma Through Putative EGFR and CYP1B1 Interactions

<title>Abstract</title> <p> Chaihu Shugan San (CSS), a well-established traditional Chinese medicine formula, has demonstrated anticancer effects on hepatocellular carcinoma (HCC). However, the main active components and the mechanism of action of CSS are not clear. We aimed to de-termine the main anti-HCC ingredient of CSS and clarify its therapeutic mechanisms us-ing computational and experimental approaches. A network pharmacology approach was used to find therapeutic targets of CSS for HCC and perform GO and KEGG analyses of its related pathways and functions. CSS-derived compounds and HCC-related targets were evaluated via molecular docking. The optimal compound-target interactions were characterized via 500 ns molecular dynamics (MD) simulations, free energy landscape (FEL) analysis, and MM/PBSA calculations. Finally, we evaluated the <italic>In vitro</italic> anti-HCC efficacy of the lead compound in HepG2 and Huh7 cells through MTT assay, cell proliferation and Annexin V-FITC/PI assays. Network pharmacology predicted many targets associated with oxidative stress, chemical carcin-ogenesis, nitrogen metabolism and receptor signaling pathways for CSS in the regulation of HCC. Molecular docking screened two top compounds namely <italic>Isorhoifolin</italic> and 3,5,6,7,8,3,4-heptamethylflavone. In MD simulation analysis, the docked complexes formed by <italic>Isorhoifolin</italic> and its targets were relatively stable compared with corresponding controls. According to MM/PBSA, <italic>Isorhoifolin</italic> had relatively strong binding ability with EGFR (-274.20 kJ/mol) compared to Erlotinib (-234.21 kJ/mol) and with CYP1B1 (-383.03 kJ/mol) compared to 2-Phenylquinazoline-4-amine (-142.81 kJ/mol). <italic>In vitro</italic> experiments showed that <italic>Isorhoifolin</italic> inhibit HCC viability (IC ₅₀ value of 20.48 µM in HepG2 cells and 26.93 µM in Huh7 cells), suppressed HCC cell proliferation in dose-dependent manner and induced apoptosis of HCC cells. The study identified <italic>Isorhoifolin</italic> as a key anti-HCC bioactive ingredient in CSS and demonstrated potent anti-HCC activity and exhibited predicted interactions with EGFR and CYP1B1 in computational analyses, based on integrating multi-scale approaches. </p>