| Background and Purpose: In recent years, our research team has committed to the chemical and pharmacological studies of veratrum nigrum L.Var. ussurience Nakai alkaloids (VnA), which was first found to have a strong anti-arterial and anti-venous thrombosis role; so far, we have isolated 11 different ester-type isosteroidal alkaloids, one of which was identified as neogermbudine (NG) by using modern analytical techniques. This paper aims to study the antiarterial and antivenous thrombosis effect and hemorheological effects of NG, bleeding time and coagulation parameters; and investigate the antithrombotically active components of VnA so as to provide experimental basis for the development and clinical application of VnA as a new high efficacious antithrombotic drug.Methods:1. Electrical stimulation induced rat carotid artery thrombosis model Rats was anesthetized by urethane, then the unilateral carotid artery was isolated surgically, a direct current stimulating electrode and a temperature electrode were placed at heart-proximal and heart-remote ends of carotid artery, respectively. The continual electric stimulation (1.6mA,7min) was started on the carotid artery and cause endometrial vascular injury, thus thrombosis. The time elapsing from the beginning of stimulation to abrupt fall in temperature of carotid artery surface was recorded by means of the temperature electrode of the instrument. This time represented the occlusion time (OT) of carotid artery injured electrically, namely, thrombus formation time. Then, the percent increase of OT were calculated and compared with control group.2. Blood stasis induced inferior vena cava thrombosis model Rats were anesthetized by urethane, the midline abdominal wall was incised, then inferior vena cava was separated surgically, the left renal veins was ligated to induce stasis. Then, abdominial cavity was closed,and reopened four hours after ligation. The blood vessel of inferior vena cava was clamped at 2cm below the ligation .The thrombus clot in the blood vessel was removed (if any) and weighted afer drying at 60℃for 20 min. The animal numbers of thrombus formation and thrombus drying weight, which were used to evaluate the antivenous thrombotic effects, were recorded so as to calculate thrombus formation rate (animal number of thrombosis/test animal number) and percent inhibition of thrombus weight respectively.3. The determination of platelet aggregationThe turbidimetry developed by Dr. Born was used to determine the platelet aggregation. The blood was collected from rat heart, with sodium citrate as anticoagulant agent. Then, the platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared by centrifugation, after that, the platelet aggregation rate at 5 min was determined in an aggregometer using ADP as an aggregation inducer.4. The determination of hemorheology parametersThe blood was collected from rat heart with heparin as an anticoagulant and tested for hemorheological parameters including whole blood viscosity at low and high shear rate and plasma viscosity measured in a viscometer.5. The determination of bleeding timeThe hemorrhage transection of mouse tail model was employed to determine the bleeding time (BT). Mice was placed on the fixator, the vertical tail was cut at 1.5 mm from the caudal end every 30 seconds, blood drops were collected onto filter paper placed tangentially to animal's tail. The time between the cutting of the mouse tail and the end of hemorrhage was considered as the bleeding time.6. The determination of whole blood clotting timeThe capillary glass tube method was used to measure the whole blood clotting time (CT). The capillary glass tube of 1 mm diameter was insert into mouse's canthus venous plexus until the blood column to 5 mm, then the capillary tube was broken manually at a short segment at intervals of 30 seconds until a blood coagulation filament appears, the time from the beginning to blood coagulation filament appearing is recorded and considered as the blood clotting time.7. The determination of thrombin time and recalcification timeRabbit blood was taken from heart, 50ul plasm obtained after centrifugation of the citrated blood, 50ul each of 0.1mol?L-1Tris-HCL buffer(PH=7.4) and thrombin test solution were consecutively added into the small cup of the coagulometer. The thrombin time (TT) was given automatically by the instrument. Similarly, the recalcification time (RT) was given automatically after addition of 100ul each of plasma and 0.025mol/L CaCl2 solution.Results:1. The effect of NG on artery thrombosis of rats With the doses increased, OT values dose-dependently extended. When NG are 5.0μg?kg-1,10.0μg?kg-1,20.0μg?kg-1,40.0μg?kg-1,the OT values are 857.7s,930.60s,1040.40s,1171.60s.The OT percent prolongation at high dose of NG(40.0μg?kg-1)was basically identical to that of LAS group at 18.0mg?kg-1.2. The effect of NG on venous thrombosis of ratsCompared with the NS group, with gradual increase in doses, the thrombosis rate (the number of animals thrombosis / number of animals tested) and the weight of formed thrombus gradually decreased. When NG are 5.0μg?kg-1,10.0μg?kg-1,20.0μg?kg-1,40.0μg?kg-1, the thrombosis rate are 9/10,8/10,6/10,5/10,and the weights of formed thrombus are 0.0499g,0.0386g,0.0292g and 0.0164g. The effect of high dose of NG(40.0μg?kg-1)on the venous thrombosisis is close to that of heparin(400.0μg?kg-1)as a positive drug.3. The effect of NG on platelet aggregation of ratsThe decreased rate of platelet aggregation was observed during in vitro and ex vivo experiment with NG. The inhibition rates were 17.02%, 34.89%, 46.76%, 60.96% at dose of 5.0μg?kg-1,10.0μg?kg-1,20.0μg?kg-1,40.0μg?kg-1 of NG in ex vivo experiment and 17.58%, 30.82%, 44.33%, 55.16% at dose of 25.0μg?ml-1,50.0μg?ml-1,100.0μg?ml-1,200.0μg?ml-1 of NG in in vitro experiment, respectively. The above results showed a good dose-effect relationship.4. The effect of NG on hemorheology of rabbitsAfter administration of drug, the whole blood viscosity at both low and high shear rate decreased significantly and dose-dependently. The decrease percentage of low-dose NG(5.0μg?kg-1) at 20 min, 1h, 3h post dosing are 10.75%, 24.95%, 35.70% at low shear rate and 9.97%, 23.45%, 32.61% at high shear rate, respectively. And the other doses of NG were showed the similar viscosity lowering fashion. And with the dose increasing, the decreasing trend is more evident, showing good dose-effect relationship. After administration, a dose-dependently decrease in plasma viscosity was also observed in the same way as the whole blood viscosity. Compared with the pre-dosing, the decrease percentage caused by NG at 20 min, 1h, 3h post dosing were 12.35%, 19.14%, 30.25% at low dose(5.0μg?kg-1) and 23.72%, 36.54%, 48.72% at high dose(40.0μg?kg-1).5. The effect of NG on BT and CT of ratsCompared with the NS , the NG showed the significant prolongation in BT and CT with the percentage of prolongation being 12.30%, 33.12%, 52.90%, 71.33% for BT and 23.44%, 53.97%, 82.60%, 93.10% for CT at doses of 5.0μg?kg-1,10.0μg?kg-1,20.0μg?kg-1,40.0μg?kg-1 , respectively. 6. The effect of NG on TT and RT of rabbits The NG could significantly prolong the TT and RT in a dose- and time-dependent manner. In the low-dose(5.0μg?kg-1), the TT prolongation rates at various time points after administration were 33.16%, 46.53%, 60.42%; RT prolongation rates were 15.10%, 33.39%, 49.57%, and the others showed the same trend with the low dose group.Conclusions:Neogermbudine (NG), as a single component isolated from herb veratrum nigrum L.Var. ussurience Nakai (VnA), has significant inhibiting effect against arterial thrombosis and venous thrombosis; and it can also significantly reduce whole blood viscosity and plasma viscosity; significantly inhibit platelet aggregation in ex vivo and in vitro experiment; and prolong the BT,CT,TT and RT; and all the above effects caused by NG showed a good dose-effect relationship. The above mentioned findings suggest that NG may be an important antithrombotic active ingredient of VnA.
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