Enhanced anti-inflammatory effects of (-)-linalool, (-)-borneol and β-caryophyllene via NF-κB/Nrf2 and arachidonic acid pathways

Background: Chronic immune-mediated inflammation remains difficult to treat with single-target approaches. Terpenoids are known to modulate immune responses, but whether specific combinations can produce enhanced effects and through which mechanisms is not well understood. Objectives: This study investigates whether a fixed-ratio combination (5:23:26) of (-)-Linalool, (-)-Borneol and β-Caryophyllene (Formula 1) exerts enhanced anti-inflammatory effects and explores the underlying molecular mechanisms. Methods: Two complementary models were used: release of slow-reacting substance of anaphylaxis (SRS-A) in isolated guinea pig ileum and LPS-activated RAW264.7 macrophages. PGE2, LTB4, NO and iNOS levels were measured. Network pharmacology, followed by experimental validation, identified key targets and pathways. Cellular morphology, apoptosis and NF-κB p65 nuclear accumulation were assessed by Hoechst 33342/PI staining and fluorescent plasmid transfection. Results: Formula 1 achieved 79.88% inhibition of SRS-A release, significantly surpassing every single component (P < 0.001) and the positive control quercetin. It also reduced PGE2, LTB4, NO and iNOS levels more effectively than any single compound. In LPS-stimulated macrophages, Formula 1 reduced cell deformation and apoptosis and markedly reduced p65 nuclear translocation. Mechanistically, it increased the Nrf2/HO-1 axis while down-regulating NF-κB and the arachidonic acid pathway. Notably, even when (-)-Linalool was reduced to one-tenth of its dose, Formula 1 maintained comparable efficacy. Conclusions: The combination of (-)-Linalool, (-)-Borneol and β-Caryophyllene produces enhanced anti-inflammatory effects through coordinated regulation of multiple pathways, as supported by the Highest Single Agent (HSA) model. These findings provide a basis for developing multi-target therapies for immune-mediated inflammatory diseases