Integration of Network Pharmacology, Molecular Docking, and In Vitro Nitric Oxide Inhibition Assay to Explore the Mechanism of Action of Thai Traditional Polyherbal Remedy, Mo-Ha-Rak, in the Treatment of Prolonged Fever

Background: Prolonged fever (PF) is one of the most challenging clinical conditions due to its complex molecular mechanisms and limited effective treatments. Objective: The current study aimed to explore the mechanism of action of Mo-Ha-Rak (MHR), a Thai traditional polyherbal remedy, in PF treatment. Methods: Integration of network pharmacology, molecular docking, and inhibition of nitric oxide (NO) production in LPS-induced RAW264.7 macrophages approaches were used. Results: The study identified 86 potential active compounds, 131 potential therapeutic targets, and 9 hub genes for MHR. Key targets with the highest degree of connectivity in PF, including TNF, IL6, IL1B, PTGS2, STAT3, and NFKB1, are closely associated with arachidonic acid metabolism pathways, which play critical roles in infections, inflammation, cell proliferation, and apoptosis in the PF microenvironment. Molecular docking analysis suggested that core compounds exhibited strong binding affinities for four key targets, viz. TNF, IL6, IL1B, and PTGS2, with binding energies ranging from −4.1 to −9.8 kJ/mol. MHR exhibited dose-dependent reduction of NO production at concentrations of 10–100 µg/mL. Among the biomarkers of MHR tested, ellagic acid, loureirin A, resveratrol, and rhein showed potential to inhibit NO production. Conclusions: This study demonstrates that MHR exerts its therapeutic effects on PF through a complex network of multiple compounds, targets, and pathways. These findings highlight the mechanisms of PF and the role of MHR in modulating the arachidonic acid metabolism pathway, which underlies the development of fever.