Melampodium divaricatum (Rich.) DC. extracts exert anti-inflammatory effects via inhibition of the NLRP3 inflammasome

Ethnopharmacological relevance: Melampodium divaricatum (Rich.) DC. (MelD), commonly known as butter daisy, is a medicinal plant widely distributed in Central and South America. In traditional medicine, particularly in Mexico and other parts of Central America, decoctions prepared from the aerial parts of MelD have been widely used to treat inflammation-associated ailments, including dysentery, fever, infections, and pain. Despite its long-standing ethnomedicinal use, the molecular and immunological mechanisms underlying its anti-inflammatory effects, particularly in the context of innate immune regulation, remain largely unexplored. Aim of the study: Based on the ethnomedicinal use of MelD in Nicaragua for the treatment of inflammation-related conditions such as fever and pain, this study aimed to investigate whether extracts of MelD modulate the activation of the NLRP3 inflammasome, a central regulator of innate immune responses. In addition, we sought to elucidate the underlying molecular mechanisms and to characterize the major bioactive constituents associated with these anti-inflammatory effects. Materials and methods: Methanolic extracts were prepared from the aerial parts of MelD collected in Nicaragua. Cytotoxicity was evaluated using the EZ-CYTOX cell viability assay. The effects of the extract on NLRP3 inflammasome activation were examined in lipopolysaccharide (LPS)-primed human THP-1 macrophages and murine macrophage cell lines. Inflammasome activation was induced by ATP or nigericin stimulation. Interleukin-1β (IL-1β) secretion was quantified by enzyme-linked immunosorbent assay (ELISA), while ASC speck formation and oligomerization were assessed by immunofluorescence microscopy and chemical cross-linking, respectively. NLRP3 ATPase activity was measured using the ADP-Glo™ Max assay. The in vivo anti-inflammatory activity of the extract was further evaluated using chemical- and tail-fin injury–induced zebrafish inflammation model. Chemical profiling of the extract was performed by ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC–QTOF/MS). Results: The MelD extract significantly inhibited NLRP3 inflammasome activation and subsequent IL-1β secretion in a concentration-dependent manner without inducing cytotoxicity. Mechanistic analyses revealed that the extract suppressed ASC speck formation and ASC oligomerization, indicating an impairment of inflammasome assembly. Notably, treatment with the extract did not affect the expression levels of NLRP3 or pro–IL-1β during the priming phase, suggesting preferential interference with the activation step of the inflammasome. In agreement with the in vitro findings, the extract markedly reduced inflammatory responses, including neutrophil and macrophage recruitment, in the zebrafish inflammation model. UPLC–QTOF/MS analysis identified flavonoid C-glycosides as major constituents of the extract, implicating these compounds as potential contributors to the observed anti-inflammatory activity. Conclusions: This study provides experimental evidence that MelD extract exerts anti-inflammatory effects by targeting the assembly and activation of the NLRP3 inflammasome. These findings offer a pharmacological rationale for the traditional use of MelD in the management of fever and pain and highlight its potential as a natural source of bioactive compounds for the development of therapeutics against NLRP3-driven inflammatory disorders.