Antithrombotic activity of Punica granatum, L (Pomegranate): Experimental investigation and exploration of its mechanism of action on primary and secondary hemostasis in vitro and ex vivo

Abstract

Hemostasis is the physiological process that stops bleeding by forming a thrombus (or blood clot). However, the inappropriate formation of a thrombus can constitute a pathophysiological event involving the same steps as those of hemostasis. Despite some adverse effects, antithrombotic therapy remains an effective means in the prevention and treatment of thrombotic diseases. In this regard, scientific research aimed at discovering new remedies of natural origin is particularly active.This study aims to evaluate the antithrombotic activity of the aqueous extract of Punica granatum peels in vivo. To understand the mechanism of action of this activity, we will investigate primary and secondary hemostasis in vitro and ex vivo. Furthermore, we will examine its antioxidant activity, toxicity, and phytochemical composition to identify the molecules responsible for the observed effects.The antithrombotic activity is assessed in vivo using the acute pulmonary thromboembolism model in mouse. Thrombosis is induced by injecting the thrombogenic solution (epinephrine + collagen) into the lateral caudal vein. Platelet aggregation, both in vitro and ex vivo, is performed on washed platelets isolated by centrifugation from rat blood. The platelet suspension is pre-incubated with the extract for 1 min. Aggregation is triggered by adding the following agonists: ADP, thrombin, collagen, and arachidonic acid. The bleeding time is measured in the absence and presence of the extract following an apical section of the mouse tail. This time corresponds to the duration of flow between the first and last drops of blood. To evaluate plasma coagulation, activated partial thromboplastin time (aPTT) and prothrombin time (PT) were measured under conditions both with and without the extract, using appropriate reagents. Acute toxicity is conducted by administering a single oral dose of the extract to mice (5 and 10 g kg-1). Possible changes in animal behavior are observed for fifteen days. The identification of polyphenols and flavonoids is performed by High Performance Liquid Chromatography (HPLC).The results obtained show that P. granatum exerted a significant preventive antithrombotic action (80 % protection) in mice. Furthermore, the extract inhibited platelet aggregation induced in vitro by all agonists in a dose-dependent manner, prolonged bleeding time, and coagulation times in rats. The results of the ex vivo study in rats confirmed these effects. We also noted that the extract did not exhibit toxicity following administration, and HPLC analysis revealed a richness of polyphenols and flavonoids in the extract.P. granatum exhibited an interesting antithrombotic activity, which could be attributed to its antiplatelet and anticoagulant actions. The phytochemical compounds identified in this extract could, at least in part, be responsible for these effects. Therefore, the demonstrated activities of this plant on hemostasis could lead to the development of more effective remedies and represent a means of prevention against thrombotic events.