| Summary: | (1) Background: <i>Ranunculus bulumei</i> is a flowering plant that belongs to the <i>Ranunculus</i> species. Several <i>Ranunculus</i> species, such as <i>R. aquatilis</i> and <i>R. muricatus</i>, have traditionally been used to treat fever and rheumatism throughout Asia, suggesting that plants belonging to the Ranunculus species may have anti-inflammatory effects. To our knowledge, the pharmacological activity of <i>R. bulumei</i> has not been reported. Therefore, in this study, we aim to assess the anti-inflammatory activity of a methanol extract that was derived from <i>R. bulumei</i> (Rb-ME) in macrophage-mediated inflammatory responses and to identify the molecular mechanism that underlies any anti-inflammatory action. (2) Methods: The anti-inflammatory efficacy of Rb-ME was evaluated while using in vitro and in vivo experiments. The RAW264.7 cells and peritoneal macrophages were stimulated by lipopolysaccharide (LPS). In addition, LPS-induced peritonitis and HCl/EtOH-triggered gastritis models were produced. A nitric oxide (NO) assay, real-time PCR, luciferase reporter gene assay, western blot analysis, plasmid overexpression strategy, and in vitro kinase assay were used to determine the molecular mechanisms and target molecules of Rb-ME. The phytochemical active ingredients of Rb-ME were also identified by high performance liquid chromatograph (HPLC). (3) Results: Rb-ME reduced the production of NO and mRNA expression of iNOS, COX-2, IL-1β, and IL-6 without cytotoxicity. The protein secretion of TNF-α and IL-6 was also decreased by Rb-ME. HPLC analysis indicates that quercetin, luteolin, and kaempferol are the main active ingredients in the anti-inflammatory efficacy of Rb-ME. Rb-ME also blocked MyD88-induced NF-κB promoter activity and nuclear translocation of NF-κB subunits (p65 and p50). Moreover, Rb-ME reduced the phosphorylation of IκBα, Akt, p85, Src, and Syk, which are NF-κB upstream signaling molecules in LPS-activated RAW264.7 cells. According to the in vitro kinase assay, Rb-ME directly inhibits Syk kinase activity. The oral administration of Rb-ME alleviated inflammatory responses and the levels of p-IκBα in mice with LPS-induced peritonitis and HCl/EtOH-induced gastritis. (4) Conclusions Rb-ME has anti-inflammatory capacity by suppressing NF-κB signaling and it has been found to target Src and Syk in the NF-κB pathway. Based on this efficacy, Rb-ME could be developed as an anti-inflammatory herbal medicine.
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