Effect of timosaponin E1 and E2 on superoxide generation induced by various stimuli in human neutrophils and on platelet aggregation in human blood.

We have reported that six steroidal saponins isolated from Anemarrhenae rhizoma had various effects on stimulus-induced superoxide generation in human neutrophils. In this paper, two novel steroidal saponins, timosaponins E1 and E2 were isolated from Anemarrhenae rhizoma, and the effects of these steroidal saponins on superoxide generation in human neutrophils were investigated. Timosaponins E1 and E2 significantly inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide generation in a concentration-dependent manner, but not that induced by arachidonic acid (AA). On the other hand, both compounds enhanced superoxide generation induced by phorbol 12-myristate 13-acetate (PMA) in a concentration-dependent manner. The superoxide generation induced by PMA with timosaponins E1 and E2 was suppressed by staurosporine, an inhibitor of protein kinase C, but was not suppressed by genistein, an inhibitor of protein tyrosine kinase. Tyrosyl phosphorylation of a 58 kDa protein, which was increased by fMLP, was inhibited by timosaponins E1 and E2. Timosaponins E1 and E2 also inhibited the generation of a 47 kDa protein and platelet aggregation in human blood. The results suggest that protein tyrosine kinase participates in fMLP-mediated superoxide generation by timosaponin E1- and E2-treated human neutrophils.