Isolation of caffeic acid derivatives and their anti-inflammatory effects in raw 264.7 cells from Parasenecio firmus

It is well known that the pro-inflammatory mediators such as nitric oxide (NO) and prostaglandin E₂ (PGE₂) are involved in several inflammatory diseases and lipopolysaccharide (LPS) can stimulate these inflammatory responses. Caffeic acid derivatives (CAD) are treated as important anti-inflammatory chemicals. The aim of this study is to investigate isolation and anti-inflammatory effects of CAD which are the major compounds of Parasenecio firmus. Bioassay-guided separation on methanol extract of P. firmus using the multiple chromatography steps resulted in the isolation of three CAD that are identified as 3,5-caffeoylquinic acid, 4,5-dicaffeoylquinic acid and 5-caffeoylquinic acid (5-CQA). The anti-inflammatory effects of CAD were investigated via study on the molecular mechanisms in lipopolysaccaride (LPS)-stimulated RAW 264.7 cells. CAD dose-dependently reduced the productions of NO and PGE₂ induced by LPS. It was found that 5-CQA showed the highest inhibition on NO production in RAW 264.7 cells. In addition, 5-CQA significantly suppressed the LPS induced expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNF-α) at the mRNA and protein levels. These results were strongly supported by decrease in DNA binding of nuclear factor-kappa B (NF-κB) and nuclear translocation of p65 and p50 subunits of NF-κB. in addition, 5-CQA dose-dependently reduced the production of IL-6, IL-1β and TNF-α in LPS-stimulated RAW 264.7 macrophage cells. Thus, these results suggest that the anti-inflammatory activity of 5-CQA is associated with the down-regulation of iNOS, COX-2 and TNF-α through the negative regulation of the NF-κB pathway in RAW 264.7 cells.