Integration of Network Pharmacology and Molecular Docking Together with an In Vitro Nitric Oxide Inhibition for the Insight for Antipyretic Effects of Benjalokawichian, the Thai Traditional Polyherbal Remedy

Benjalokawichian (BLW) or Ha-Rak is a classic antipyretic polyherbal remedy used in Thai traditional medicine (TTM) to reduce toxic fever (TF). This study aimed to shed light to the mechanisms of action and identify bioactive components of BLW responsible for TF treatment. The approaches that integrate network pharmacology, molecular docking, and inhibition of nitric oxide (NO) production in LPS-induced RAW264.7 were used for these purposes. Network pharmacology was used as a tool to identify 17 potential bioactive compounds, 88 potential therapeutic targets, and 4 hub genes for BLW. Among the key targets, TNF, PTGS2, STAT3, and NFKB1 were closely associated with the phenylalanine, arachidonic acid and tyrosine metabolic pathways, which play critical roles in infections, inflammation, proliferation and apoptosis in the TF microenvironment. On the other hand, molecular docking analysis suggested that core compounds exhibited strong binding affinities for key targets, with binding energies ranging from -4.5 to -11.1 kJ/mol. In vitro assay showed that BLW extract exhibited strong inhibition of NO production in LPS-induced RAW264.7 macrophages, with an IC50 value of 69.10 μg/mL, and no cytotoxicity against RAW264.7 macrophages was observed. Furthermore, the biomarker compounds of BLW extract, viz. perforatic acid and peucenin-7-methyl ether were found to decrease NO production in a dose-dependent manner. Overall, this study demonstrates that BLW exerts its therapeutic effects on TF through a complex network of various compounds, targets, and pathways. These findings serve as a foundation for further research into the mechanisms of action of a polyherbal remedy toward TF to provide scientific evidences for its clinical use.