Antipyretic and Antinociceptive Activities of Tri-Phol-That Extract and Its Flavonoids:

Despite the common use of herbal medicines rich in flavonoids to treat fever and pain, the pharmacological mechanisms involved in these effects are poorly understood. Therefore, this study assessed the antipyretic and analgesic effects of Tri-Phol-That extract, characterized its major flavonoids, and investigated its possible molecular interactions with key inflammatory mediators. The antipyretic activity was evaluated against a turpentine-induced pyrexia model in mice. Analgesic activity was evaluated using the acetic acid-induced writhing, tail immersion, and hot plate tests. The concentration of rutin, quercetin, and kaempferol was determined by high-performance liquid chromatography. Molecular docking was performed using GNINA against COX-2 (mouse: 1CX2, human: 5KIR), COX-1 (ovine: 3N8X), and mPGES-1 (human: 4YL1). Interaction profile studies were also performed in the Discovery Studio 2021. The hydroethanolic extract lowered rectal temperature in a dose-dependent manner, with maximum inhibition occurring at 100-200 mg/kg, although the effect was less potent than that of tolfenamic acid. The acetic acid-induced writhing test also showed strong dose-dependent peripheral antinociceptive activity with 84.64% inhibition at 200 mg/kg of extract, comparable to tolfenamic acid. The central analgesic effects of extracts were observed in hot plate tests. Weak central analgesic effects were also observed in tail immersion tests. HPLC analysis indicated rutin (4.01 μg/g), kaempferol (2.13 μg/g), and quercetin (0.96 μg/g) were the most abundant flavonoid compounds. Based on docking results, quercetin had the strongest binding affinity toward COX-2 (-8.44 kcal/mol). Rutin also exhibited the strongest binding affinity toward COX-1 (-7.55 kcal/mol) among the flavonoids tested. On the other hand, kaempferol exhibited the strongest binding affinity toward mPGES-1 (-9.97 kcal/mol). Results also showed that rutin, quercetin, and kaempferol interacted with catalytic residues of ARG120, TYR355, TYR385, SER530, and SER127. Thus, it can be concluded that the extract of Tri-Phol-That had strong antipyretic and analgesic effects via multi-target inhibition of COX-1, COX-2, and mPGES-1, possibly due to the combined effects of rutin, quercetin, and kaempferol. These results support the traditional medicinal use of this extract and provide justification for further investigation as a phytotherapeutic agent.