ABSTRACT: Different therapeutic approaches, particularly those involving plant-derived compounds, have been explored for treating inflammatory diseases. This study evaluated the antioxidant and anti-inflammatory properties of eugenol (EU) in its pure form, bis-eugenol (BIS), and clove essential oil (OE) at different concentrations (5, 10, and 25 μg/mL). Chemical analysis confirmed that OE contains a high proportion of eugenol (45-90%), with the presence of eugenol acetate differentiating it from pure eugenol, while bis-eugenol was successfully synthesized and characterized with a 97% yield. Antioxidant activity assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays showed that 25 μg/mL of each compound effectively neutralized free radicals and reduced ferric ions, with DPPH radical scavenging of about 80% and FRAP values ranging from 200 to 300 μM Fe2+ equivalents. In RAW 264.7 macrophages, the extracts were noncytotoxic and maintained above 70% cell viability under H2O2-induced oxidative stress, with BIS25 showing the highest protective effect. Regarding cellular mechanisms, OE25 preserved superoxide dismutase (SOD) and catalase activity, while BIS10 and BIS25 significantly reduced catalase activity. EU10, EU25, and OE25 led to notable glutathione activity depletion. BIS25 was the only compound to significantly reduce nitric oxide production. All extracts downregulated toll-like receptor 4 (TLR-4) expression. BIS10 induced NRF2, and IL-10 increased with BIS10 and OE10. Tumor necrosis factor alpha (TNF-α) levels decreased across all BIS and OE concentrations. Our findings indicate that bis-eugenol exhibited the most pronounced antioxidant and anti-inflammatory effects among the compounds tested. Its chemical structure appears to confer greater stability and reduce the generation of phenoxy radicals, which may account for its superior efficacy in cellular protection and inflammatory modulation, especially through activation of the NRF2 pathway. Notably, bis-eugenol was the only compound that simultaneously suppressed TLR4/nuclear factor kappa B (NF-κB) pathways while upregulating NRF2 and IL-10, suggesting that its mechanism of action involves both direct inflammatory inhibition and the activation of endogenous protective pathways. However, further studies are needed to elucidate the influence of its molecular structure on these mechanisms and to confirm its primary role in mediating these effects.
