Abstract

Colorectal cancer (CRC) remains one of the most prevalent malignancies worldwide, emphasizing the need for safer and more effective natural therapeutics. The ethanolic extract of the Five-Flower Remedy (FFE), a traditional Thai polyherbal formulation used for detoxification and inflammation, was investigated for its cytotoxic and mechanistic effects against human colorectal adenocarcinoma (HT-29) cells through an integrated experimental and computational approach. FFE significantly inhibited HT-29 cell proliferation in a dose- and time-dependent manner, reducing cell viability to 20 %, 65 %, and 90 % at 500 μg/mL after 24, 48, and 72 h, respectively ( p < 0.01 and 0.001 respectively). In the soft-agar assay, FFE at 250 μg/mL markedly suppressed colony formation, decreasing both colony area and number by approximately 55–60 % compared with the untreated control. LC-MS/MS QTOF profiling identified 312 metabolites, predominantly lipid amides such as oleamide and 1-hexadecanoylpyrrolidine, together with phenolic and alkaloid constituents. Network pharmacology and enrichment analyses revealed 40 overlapping targets between FFE compounds and 3039 colorectal cancer-related genes, highlighting the VEGF, MAPK, PKC, and inflammatory signaling pathways mediated by key targets MAPK14, PLA2G4A, PRKCA, and HDAC family proteins. Molecular docking demonstrated that 1-hexadecanoylpyrrolidine exhibited plausible binding affinity toward cPLA2 and PKC (−5.5 kcal/mol), comparable to the chemotherapeutic drug 5-fluorouracil (−5.4 to −5.5 kcal/mol) at p38. These findings suggest that FFE speculates multi-targeted anticancer effects by modulating angiogenic, inflammatory, and metastatic signaling pathways, providing the first mechanistic evidence supporting its traditional use and suggesting its potential as a safe alternative therapy for colorectal cancer management.