# 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

## TL;DR

This study explores how a Thai traditional herbal remedy, Benjalokawichian, reduces fever by identifying its active compounds and mechanisms of action.

## Contribution

The study integrates network pharmacology, molecular docking, and in vitro testing to uncover the antipyretic mechanisms of a traditional polyherbal remedy.

## Key findings

- BLW extract significantly inhibits nitric oxide production in macrophages with an IC50 of 69.10 μg/mL.
- Key compounds like perforatic acid and peucenin-7-methyl ether reduce NO production in a dose-dependent manner.
- Molecular docking shows strong binding affinities of BLW compounds to targets like TNF and NFKB1.

## Abstract

Benjalokawichian (BLW) is a classic antipyretic polyherbal remedy used in Thai traditional medicine (TTM) to reduce toxic fever (TF). This study aimed to shed light on the mechanisms of action and identify bioactive components of BLW responsible for TF treatment. The methods that combine network pharmacology, molecular docking, and the inhibition of nitric oxide (NO) production in LPS-induced RAW 264.7 were employed for these objectives. Network pharmacology served as a means to identify 15 potential bioactive compounds, 88 possible therapeutic targets, and 4 hub genes related to BLW. Among the significant targets, TNF, PTGS2, STAT3, and NFKB1 were closely linked to the metabolic pathways of phenylalanine, arachidonic acid, and tyrosine, which are vital in managing infections, inflammation, proliferation, and apoptosis in the TF microenvironment. Additionally, molecular docking analysis indicated that core compounds displayed strong binding affinities for the key targets, with binding energies ranging between −4.5 and −11.1 kcal/mol. The in vitro assay demonstrated that BLW extract significantly inhibited NO production in LPS-activated RAW 264.7 macrophages, presenting an IC50 value of 69.10 μg/mL, and no cytotoxic effects on RAW 264.7 macrophages. 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. In summary, this research indicates that BLW provides therapeutic benefits for TF via a complex interplay of different 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.