Anticoagulative Efficacy Of Blood-quickening And Stasis-transforming Medicinal

ObjectivesTo summarize the clinical efficacy of blood-quickening and stasis-transforming medicinal on anticoagulative therapy of heartvascular disease. By experiment, to confirm and assess the anticoagulative efficacy of them, and provide some evidence for clinical therapy.MethodsExperimental study1. Antithrombin bioactivity experimentFirst, eight varieties of Traditional Chinese Medicinal, included leech, silkworm, earthworm, wingless cockroach, salvia, ligusticum, red peony and saffron had been distilled as liquid test sample medicine which oncentration is equivalent to lg/L crude drug. Then respectively, after 0.2mL Tris-HCl buffer contained 0.5% fibrinogen was dripped on microtitration plate and 50μL test sample was mixed into it, thrombin which concentration is 40μ/mL would be titrated into the test sample step by step. Simultaneously, it was timed. The consumed dosage of thrombin would be recorded ultimatily when some filiform coagulum had been seeked out by needle, and converted into the antithrombin unit per gram (AT-U) of the tset sample as distilled water was the control.Formula:AT-U=C1V1/C2V2WU:thrombin activity unit per gram, U/g; C1:the concentration of thrombin solution, U/mL, it is 40U/mL in this experiment; C2:the concentration of test sample, g/mL, it is lg/mL in this experiment; V1:the consumed thrombin solution volume, mL; V2:the added volume of test sample, mL, it is 0.05mL in this experiment; W:the added Tris-HCl buffer volume, it is 0.2mL in this experiment.It is an antithrombin unit when the consumed thrombin solution volume counteract a unit thrombin 2.Anticoagulative experiment about the formula composition of Trditional Chinese Medicine in rat modelThere were three varieties of Traditional Chinese Medicine selected from the former eight varieties that had potent AT-U. They were labled A, B and C, and combined each other as four groups, A+B,A+C,B+C,and A+B+C. Walfarin was the positive controlled drug.By randomised controlled method, 60 Wistar rices were divided into 6 groups: placebo group, positive drug controlled group, A+B,A+C,B+C,and A+B+C four experimental groups. Every group had 10 rices. Distilled water was fed in placebo group. In positive group, Warfarin was dissolved in distilled water and fed to rices with fourfold adult dosage,0.4 mg/kg. In every experimental group, the dosage was eightfold adult's dosage,2g/kg/d. All test drug had been fed to rices by intragastric administration once every day for 7 days. Then,3mL blood was taken from abdominal aorta of rice after the last feeding 1 hour. Prothrombin time(PT), active partial thromboplastin time(APTT), thrombin time(TT) and fibrinogen(FIB) were tested.Statistics:By SPSS13.0 software, the difference between groups would be tested through analysis of variance(ANOVA) method, and the difference between the dealed groups and the blank group, the dealded groups each other would be tested through multiple comparison method.Clinical studyBetween Janury 2009 to December 2009 in Guangdong Province Tradtional Chinese Medicine(TCM) Hospital,60 cases patients of Coronary Heart Disease(CHD) diagnosised on 1979 WHO standard were randomizedly divided into two group:treatment group and control group. Complex Thrombolysis Capsual was adminstrated to patients in treatment group and asprin was dosed in control group for the following 30 days. Blood sample were taken from peripheral blood of patients to test PT, TT, APTT, FIB and a symptom questionnaire was filled at first day and at 31th day respectively.Result Experimental studyAll drug groups'thrombin activity unit (AT-U) are significantly higher than the placebo group. They are wingless cockroach, salvia, red peony, silkworm, leech, earthworm, ligusticum, and saffron respectively based on the gradation of their AT-U value from high to low. Be compared to leech, the AT-U value of wingless cockroach, salvia, red peony and ligusticum are higher significantly than its; saffron's AT-U value is lower and silkworm earthworm's are no difference. Be compared to silkwom respectively, wingless cockroach, salvia and red peony's AT-U value are higher; earthworm, ligusticum, and saffron's AT-U value are lower; leech's is no difference. Be compared to earthworm respectively, beech, silkworm, wingless cockroach, salvia and red peony's AT-U value are higher; ligusticum and saffron's are no difference. Be compared to wingless cockroach, the other herbs'AT-U value are lower. Be compared to salvia, wingless cockroach's AT-U value is higher; beech, silkworm, earthworm, ligusticum and saffron's AT-U value are lower; and red peony's is no difference. Be compared to ligusticum, beech, silkworm, wingless cockroach, salvia and red peony's AT-U value are higher; and earthworm and saffron's are no difference. Be compared to red peony, the AT-U value of wingless cockroach is higher; beech, silkworm, earthworm, ligusticum and saffron's AT-U value of are lower; and salvia's is no difference. Be compared to saffron, the AT-U value of beech, silkworm, wingless cockroach, salvia and red peony are higher; earthworm and ligusticum's are no difference. As a whole, the potent three herbs of AT-U value are wingless cockroach, slvia and red peony.The difffrent formula composition of wingless cockroach, slvia and red peony had inordinately influence to TT, PT, APTT of rice. The difference has statistical significance, P<0.05. Warfarin and four types of composition of herbs all can prolong the PT and TT of rice significantly, p<0.05. Be compared to Warfarin positive group, the potency of four herbs composition group have no difference in prolonging PT and TT, p>0.05. There are no difference about PT and TT of rice between the four herbs composition groups while compared each other one by one. Be compared to placebo group, warfarin positive group and the other three herbs group, the wingless cochroach A + salvia B + red peony C group prolonged APTT of rice significantly, p<0.05. There are no significant difference in FIB value of rice between the whole test group each other.Clinical studyThere are no stastic difference in general state between two groups p>0.05. In other words, there are comparability between them. No difference was in PT, TT, APTT and FIB befor treatment, p>0.05; PT and TT in two groups was obviously higher after treatment, p<0.05, but the value was in the normal range. Compared the two groups, there was no significant difference in PT, TT, APTT, FIB between them after treatment, p>0.05. there was no differene in score of TCM symptom questionaire between two groups befor therapy, p>0.05. But after therapy, the score was lower than befor in two groups and the score of treatment group was significant lower than which of control group, p<0.05.ConclusionPresently, much emphasis has been placed on the development of direct thrombin inhibitors (DTIs) in anticoagulation therapy field. It is proved that hirudin is the most potent direct thrombin inhibitor. Based on our study, we find that besides beech, there are wingless cockroach, salvia, red peony, silkworm, earthworm, ligusticum and saffron that have some element directly inhibiting thrombin. Among which, wingless cockroach, salvia and red peony have potent thrombin activity significantly.In vitro animal experiment, the blood-quickening and stasis-transforming medicinal including wingless cockroach, salvia, red peony, silkworm, beech, earthworm, ligusticum and saffron all have exactly anticoagulative efficacy. In vivo rice model anticoagulation experiment, different four formula compositions, wingless cockroach A + salvia B, wingless cockroach A + red peony C, salvia B + red peony C and wingless cockroach A + salvia B + red peony C, have the identical anticoagulative efficacy compared to wrfarin. The possible anticoagulation mechanism of blood-quickening and stasis-transforming medicinal may contain intrinsic, extrinsic and common pathway. In conclusion, blood-quickening and stasis-transforming medicinal, whether single herb or formula composotion, all have eaxctly anticoagulative efficacy. They should be effective, safe, and convenient anticoagulant agents in clinical anticoagulative therapy. In clinical study, it was proved that blood-quickening and stasis-transforming medicinal fomula, Complex Thrombolysis Capsual had siminar efficacy as asprin in anticoagulant therapy to the CHD patients. Complex Thrombolysis Capsual was superior to the asprin to relieve the symptom of CHD patients. This may be relevant to its anticoagulation although its efficacy was mild. Therefor Complex Thrombolysis Capsual may be as a alternative to asprin for patients who can not tolerate it. It will point out that blood-quickening and stasis-transforming medicinal have safe anticoagulative efficacy, and may be as the substitude of the western anticoagulative medicinal to some extent.