The anti-adhesive and anti-aggregatory effects of phenolics from Trifolium species in vitro

The present in vitro study includes a comparative evaluation of anti-platelet (anti-thrombotic) properties of plant phenolics, isolated from nine different clover (Trifolium) species. The analysis covered phenolic fractions isolated from T. alexandrinum L., T. fragiferum L., T. hybridum L., T. incarnatum L., T. pallidum Waldst et Kit., T. resupinatum L. var. majus Boiss, T. resupinatum L. var. resupinatum, T. scabrum L., and T. pratense L. (red clover). The inhibitory effects of plant preparations (1-50 µg/ml) on hemostatic functions of blood platelets were assessed by measurements of thrombin- or ADP-induced platelet adhesion to fibrinogen, platelet aggregation in platelet-rich plasma (activated with ADP or collagen), and by the determination of PF-4 secretion from platelet α-granules. The influence of T. phenolics on arachidonic cascade in blood platelets was also determined. T. resupinatum var. majus, T. resupinatum var. resupinatum, and T. scabrum had the strongest anti-platelet effects. These preparations displayed the most evident anti-adhesive and anti-aggregatory effects in response to all of the used agonists: thrombin (0.2 U/ml), ADP (10 µM), and collagen (2 µg/ml), and their inhibitory properties were also confirmed by an analysis of PF-4 secretion. T. scabrum and some of other examined clover species possess significantly higher concentrations of both isoflavones and other bioactive phenolics, when compared to red clover. The obtained results suggest that these clovers contain substances with potent anti-platelet properties.