br Materials and methods br Results br Discussion

Materials and methods
Results
Discussion Since early times, herbs and their extracted substances have been used as foods and medicinal resources worldwide (Petrovska, 2012). A Ginger is one of the most important and oldest spices, consisting of the prepared and sun-dried rhizomes of Zingiber officinale and it 839 is widely consumed almost all over the world and it is more popular in tropical countries or warm regions (Katiyar et al., 1996). Bioactive compounds from ginger have been extensively characterized over several decades of study, and earlier studies demonstrated that ZGR, found in Zingiber officinale, had a variety of pharmacological activities such as anti-inflammatory, anti-apoptotic, and protecting myocardial infarction and irritable bowel disorder (Banji et al., 2014, Hemalatha and Prince, 2015, Kim et al., 2010, Rao et al., 2011). However, the anti-FXa and anti-platelet activity and FXa inhibitory activity of ZGR have not yet been reported. In this study, ZGR showed anti-FXa and anti-platelet aggregation activities and potent antithrombotic activity using ex vivo models of arterial and pulmonary thrombosis. Interestingly, ZGR did not prolong bleeding times in mice at effective antithrombotic doses, indicating a favorable efficacy to bleeding ratio. FXa is a trypsin-like serine protease that plays a pivotal role in the blood coagulation cascade (Dahlback, 2000). Noting that direct thrombin inhibitors have a narrow therapeutic index, we hypothesized that selective inhibition of FXa is a potential and promising strategy for the design of new antithrombotic agents, especially since FXa is positioned at the beginning of the common extrinsic and intrinsic coagulation pathways (Bauer, 2006, Loffredo et al., 2015). Numerous FXa inhibitors have been isolated from animals and fall within a molecular mass range of 1–29 kDa (Chen et al., 2015, Jiang et al., 2011, Thakur et al., 2014, Waxman et al., 1990). However, ZGR (with molecular weights of 194.23) is relatively smaller than any other known FXa inhibitors are, providing a substantial advantage over larger anticoagulant proteins or peptides, including low immunogenicity and low production costs. Clotting time and platelet aggregation assays are the most commonly used methods for determining the efficacy of novel anti-thrombotic drugs (Jauch et al., 2013). In our experiments, the anticoagulant effects of ZGR were verified using aPTT in vitro and ex vivo assays. We observed that the aPTTs were longer in mice treated with ZGR than they were in the vehicle-treated mice, with no obvious increases in PT. The ex vivo clotting time in mice treated with ZGR was also prolonged in a dose-dependent manner. ZGR is also selective platelet aggregation inhibitors, as shown by inhibition of ADP, collagen and U46619-induced platelet aggregation, but not thrombin-induced platelet aggregation. These data confirm that ZGR did not inhibit the activity or production of thrombin under these conditions (data not shown). ZGR showed similar anti-thrombotic efficacy to rivaroxaban (a direct FXa inhibitor) such as increased blood clotting time, delayed thrombogenesis and thrombogenic time, and inhibition of the production and activity of FXa. Although rivaroxaban did not affect platelet aggregation induced by ADP, collagen, or U46619, ZGR effectively and concentration-dependently inhibited ADP-, collagen- or U46619-induced platelet aggregation. Platelet aggregation plays a pathophysiological role in a variety of thromboembolic disorders and many kinds of anti-platelet aggregation drugs have been developed and are routinely applied to treat these diseases, either alone or in combination with other platelet inhibitors. However, these drugs have various side effects after prolonged use (Eikelboom et al., 2012, Ferraris et al., 2011). Based on the results that ZGR effectively inhibited ADP-, collagen- or U46619-induced platelet aggregation, it is desirable to develop more clinically acceptable drugs derived from natural resources, as these often seem to display fewer adverse effects.