Clovamide and clovamide-rich extracts of three Trifolium species as antioxidants and moderate antiplatelet agents in vitro.

This in vitro study provides a new insight into the phytochemical, antioxidant, antiplatelet and cytotoxic profiles of commercial trans-clovamide and clovamide-rich extracts, isolated from aerial parts of three Trifolium species (i.e. T. clypeatum L., T. obscurum Savi and T. squarrosum L.). Under inorganic experimental conditions, the EC for the investigated substances varied between 4.87 (clovamide) and 7.91 μg/ml (T. clypeatum) according to the DPPH assay. The most effective 1 mM ONOO scavenger was clovamide (IC = 19.29 μg/ml), and the weakest was the T. obsucurum extract (IC = 41.79 μg/ml). The antioxidant action of the examined substances (1-50 μg/ml) was also evaluated in blood plasma (under 100 μM ONOO-induced oxidative stress) using 3-nitrotyrosine, protein thiol groups, lipid hydroperoxides and thiobarbituric acid-reactive substances as biomarkers. All examined substances were more effective in limiting the oxidative damage to blood plasma components than the nitrative damage. Their anti-nitrative action was statistically significant only at the highest concentration (50 μg/ml). Measurements of platelet adhesion and aggregation found the compounds to have moderate antiplatelet properties of up to 20% inhibition of platelet adhesion at concentrations of 1-5 μg/ml. Additionally, clovamide and Trifolium extracts demonstrated no cytotoxicity towards blood platelets or peripheral blood mononuclear cells.