Carotenoids and flavonoids synergistically attenuate inflammation in endothelial cells induced by TNF-α

Objective: Carotenoids and flavonoids are both plants origin and have a well described anti-oxidant activity. Recently we demonstrated the protective effect of carotenoids on endothelial cells. In the current study we aimed to evaluate the possible synergistic activity of these substances as anti-oxidant and anti inflammatory mediators on endothelial cells. Methods: Endothelial cells (EA.hy926) were pre-incubated with vehicle, lycopen, dissolved chocolate, epicatechin solution, combination of lycopen with chocolate and combination of lycopen with epicatechin solution for 18-24h. After incubation, stable oxidized products of NO examined. In another assay pre incubated cells with the above substances were induced with TNF-a for 6h. The activated cells were examined for protein abundance of: eNOs, adhesion molecules ICAM-1, VCAM-1 and osteopontin (OPN). Results: There was a significant augmentation of NOx production after pre incubation with each substance separately. Moreover, the combination of lycopen with either chocolate or epicatechin resulted in synergistic effect. Lycopen with chocolate 8.1 5.1 (OD) and lycopen with epicatechin 6.9 2.3 (OD), versus control 1.4 1.2 (OD) and lycopen 2.3 0.9 (OD) respectively (p<0.005). Pre incubation with lycopen, chocolate or epicatechin didn’t have significantly effect on the activation of VCAM-1 and ICAM-1by TNF-a. However, the combination of either lycopen and chocolate or lycopen with epicatechin inhibited significantly the expression of both VCAM-1 and ICAM-1by TNF-a (25.5 2.6 and 12.5 2.1 versus 46.6 11.6; 31.3 15.3 and 19.0 5.3 versus 45.3 11.9 respectively p<0.001). The expression of the inflammatory mediator osteopontin, was also inhibited to a great extent by combination between lycopen with chocolate or epicatechin. (2.0 0.3 and 3.6 0.5 versus 12.2 1.1 respectively p<0.001). Regarding the expression of eNOs, both lycopen, chocolate and epicatechin increased significantly the expression of eNOs. However, the combination of these sustenance didn’t reach synergistic effect. Conclusion: in the current study we demonstrated the superiority of the combination carotenoids and flavonoids compared to each substance alone, as oxidative stress and inflammation inhibitor.