THE NORPURPUREINE ALKALOID FROM ANNONA PURPUREA INHIBITS HUMAN PLATELET ACTIVATION IN VITRO

摘要

Background: The leaves of Annona purpurea have yielded several alkaloids with anti-aggregation activities against rabbit platelets. This is promising in the search for agents that might act against platelets and reduce the incidence of cardiovascular diseases. Since significant differences in platelet function have been reported between human and animal platelets, a study focusing on the effect of A. purpurea extracts against human platelet activation is necessary. Methods: The compounds in an A. purpurea ethanolic extract underwent bio-guided fractionation and were used for in vitro human platelet aggregation assays to isolate the compounds with anti-platelet activity. The bioactive compounds were identified by spectroscopic analysis. Additional platelet studies were performed to characterize their action as inhibitors of human platelet activation. Results: The benzylisoquinoline alkaloid norpurpureine was identified as the major anti-platelet compound. The IC50 for norpurpureine was 80 μM against platelets when stimulated with adenosine 5′-diphosphate (ADP), collagen and thrombin. It was pharmacologically effective from 20 to 220 μM. Norpurpureine (220 μM) exhibited its in vitro effectiveness in samples from 30 healthy human donors who did not take any drugs during the 2 weeks prior to the collection. Norpurpureine also gradually inhibited granule secretion and adhesion of activated platelets to immobilized fibrinogen. At the intra-platelet level, norpurpureine prevented agonist-stimulated calcium mobilization and cAMP reduction. Structure–activity relationship analysis indicates that the lack of a methyl group at the nitrogen seems to be key in the ability of the compound to interact with its molecular target. Conclusion: Norpurpureine displays a promising in vitro pharmacological profile as an inhibitor of human platelet activation. Its molecular target could be a common effector between Ca2+ and cAMP signaling, such as the PLC-PKC-Ca2+ pathway and PDEs. This needs further evaluation at the protein isoform level.