Objectives: Fraxinus excelsior L. (FE) is traditionally used to treat inflammatory and pain disorders. This study aimed to identify the constituents of FE leaves and evaluate the effects of its n-hexane (FEH), ethyl acetate (FEE), methanol (FEM) extracts and constituents on the viability of THP-1 cells and their ability to release pro-inflammatory cytokines. Methods: THP-1 cell viability was assessed using an MTT assay. The immunomodulatory activity was evaluated by measuring TNF-α and IL-12 released by LPS-stimulated THP-1 cells using ELISA. Key findings: Triterpenes, tyrosol esters, alkanes, phytyl and steryl esters, pinocembrin and bis(2-ethylhexyl)phthalate were isolated from FE. The tyrosol esters showed no significant effect on THP-1 cell viability. FEH, FEE, FEM, and pinocembrin, ursolic acid, oleanolic acid had IC50 values of 56.9, 39.9, 124.7 μg/mL and 178.6, 61.5 and 199.8 μM, respectively. FE extracts, ursolic acid, oleanolic acid, and pinocembrin significantly reduced TNF-α/IL-12 levels. The tyrosol esters did not significantly affect TNF-α/IL-12 production. Conclusions: FE was able to reduce pro-inflammatory cytokine production indicating a mechanistic focus in its use for inflammation and pain. Further investigations are warranted to unravel the mode of action of the tested constituents and discover other potentially active compounds in FE extracts
