2.9. In vitro anticoagulant activity

The anticoagulant activity was determined through prothrombin time (PT) test and activated partial thromboplastin time (aPTT) test using methods described previously with slight modification [35].

In this study, prothrombin time (PT) was measured according to Thromboplastin LI kit by Agappe diagnostics Switzerland GmbH and activated partial thromboplastin time (aPTT) was measured according to aPTT kit by Analyticon Biotechnologies AG, Germany. Nine parts of blood and one part of 3.2% trisodium citrate (Na₃C₆H₅O₇) solution (0.109 M) were mixed gently in sterile 15 mL falcon tubes. The tubes were centrifuged immediately for 15 min at 3000 rpm to obtain platelet-poor plasma. 100 μL of plasma was placed into the test Eppendorf tubes at 37 °C and incubated for 3 min. Then 100 μL extracts of different concentrations were added to the tubes. 200 μL pre-warmed (37 °C) PT reagent was added into each of the test Eppendorf tubes. A timer was started simultaneously and the clotting time in seconds was recorded. For aPTT, 50 μL plasma was taken into each of the test Eppendorf tubes. 50 μL extracts of different concentration was added to the test Eppendorf tubes. The test Eppendorf tubes were subjected to incubation at 37 °C for 2 min. Then pre-warmed (37 °C) 50 μL Calcium Chloride (CaCl₂) (0.025 M) was added to each of the test Eppendorf tubes. A timer was started simultaneously and the clotting time in seconds was recorded.