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Yataprasen is a topical Thai herbal remedy for the treatment of musculoskeletal pain and is included in Kumpe Thart Phra Narai, the first Thai textbook of traditional medicine. The herbal preparation is made from a hydroethanolic extract of a mixture of 13 medicinal plants, of which Putranjiva roxburghii Wall. leaves are the major ingredient. In this study, we investigated the underlying mechanism of action for the anti-inflammatory effects of the Yataprasen remedy, its main ingredients, and the phytochemicals isolated from P. roxburghii leaves. The anti-inflammatory effects of the Yataprasen remedy, along with its main ingredients, including the leaves of Baliospermum solanifolium (Burm.) Suresh, Melia azedarach L., P. roxburghii , Senna siamea (Lam.) Irwin & Barneby, and Tamarindus indica L. were determined by measuring prostaglandin E 2 (PGE 2) secretion, nitric oxide (NO) production, and the synthesis of inflammatory biomarkers in lipopolysaccharide (LPS)-treated RAW264.7 macrophage cells. The active ingredients of the P. roxburghii leaves were separated by chromatography and spectroscopic measurements were used to identify their chemical structures. Ethanol extracts of the Yataprasen remedy and some of its ingredients significantly suppressed LPS-induced PGE 2 secretion and NO production in a dose-dependent manner. Treatment of RAW264.7 cells with ethanolic extracts of the Yataprasen remedy (50 μg/mL) significantly inhibited LPS-induced mRNA expression of TNF-α, COX-2, iNOS, and NF-κB. Among the plant ingredient extracts, P. roxburghii leaf extract exhibited the highest inhibitory effects on LPS-induced TNF-α and iNOS expression. Moreover, T. indica leaf extract showed the highest activity on the inhibition of LPS-induced COX-2 and NF-κB expression. Putraflavone, podocarpusflavone A, and amentoflavone were isolated biflavonoids from P. roxburghii leaf extract and showed the inhibitory effects on LPS-induced PGE 2 secretion and NO synthesis in RAW264.7 cells. Of the isolated biflavonoids, amentoflavone exhibited the strongest anti-inflammatory activity by inhibiting the expression of TNF-α, COX-2, and iNOS. The results support reported the anti-inflammatory effects of the Yataprasen remedy, which are associated with the downregulation of proinflammatory mediators. P. roxburghii , along with its biflavonoids, are the impact components that contribute to the anti-inflammatory effects of the herbal remedy. [Display omitted] • Yataprasen suppressed COX-2 expression, resulting in a reduction of PGE 2 secretion. • Yataprasen suppressed iNOS expression, resulting in a reduction of NO production. • Yataprasen inhibited TNF-α synthesis in RAW264.7 macrophages. • Among ingredients, P. roxburghii extract showed the highest anti-inflammatory effect. • Among bioflavonoids, amentoflavone showed the strongest anti-inflammatory effect. [ABSTRACT FROM AUTHOR]