【作者】 陈晓伟；

【导师】 阮新民；

【作者基本信息】 广州中医药大学 ， 中西医结合临床， 2010， 博士

【摘要】 背景抗凝治疗对于心血管病的防治有着重要的意义。现有抗凝治疗主要以西药为主,包括肝素、华法林、水蛭素等。其中肝素为注射剂,不便长期抗凝；华法林作为主要的口服剂广泛应用于心血管抗凝治疗中,但其药物安全窗窄,需要临床检测的特点直接带来了临床依从性差。而临床中,活血化瘀中药在抗凝治疗方面已经广为应用,大多为口服,具有服用方便、相对安全、副作用较小、病人依从性好的特点,但缺乏系统评价这类中药的抗凝效果。目的总结活血化瘀中药在心血管疾病抗凝治疗中的作用,结合实验确认其抗凝作用,以及抗凝血酶活性；评价不同药物配伍的抗凝效果,为临床抗凝治疗提供依据。方法实验研究1.抗凝血酶效价实验分别取含有0.5%纤维蛋白原的Tris-HCl缓冲液0.2mL,于室温下滴于滴定板上,加入50μL待测液充分混匀,滴加5μL凝血酶(40U／mL)迅速匀散,同时计时,用针尖拨动溶液,当有丝状凝结物被针挑起时,记下时间,其所需凝血酶,用量即为V1,若lmin内未凝结,则以分钟为间隔继续滴加,每次2μL,记下针尖能挑起丝状凝结时的消耗凝血酶的体积。用同法以蒸馏水作对照。按下式计算每克供试品抗凝血酶活性单位AT-U。式中：U—每1g药材抗凝血酶活性效价,单位U／g；C1—凝血酶溶液的浓度,U／mL；该实验中为40U／mL；C2—供试品溶液的浓度,g／mL；该实验中为lg／mLV1—消耗凝血酶溶液的体积,mL；V2—供试品溶液的加入量,mL；该实验中为0.05mL；W—取样量。该实验中为0.2mL。中和一个单位的凝血酶的量,为一个抗凝血酶活性单位。2.药物配伍抗凝血实验选取抗凝血酶效价实验中抗凝血酶活性最强的三味中药,A、B、C,组成A+B、A+C、B+C、A+B+C四种配伍；阳性对照药为华法林钠片,2.5mg／片。采用完全随机对照设计,将60只wistar大鼠分为空白组、阳性对照组,A+B、A+C、B+C、A+B+C四个配伍组,共6组中,每组10只。空白组给予蒸馏水；阳性对照华法林组给予成人剂量0.1mg/kg的4倍(0.4 mg/kg)的华法林,溶于蒸馏水；试验药物各组按成人常用量8倍折合成小鼠用量2g／kg／d给药。各组均灌胃给药,每日1次,连续7天,末次给药后1h,自腹主动脉采血测定凝血酶时间(TT)、凝血酶原时间(PT)、活化部分凝血酶原时间(APTT)、纤维蛋白原含量(FIB)。统计处理：采用SPSS13.0软件,运用多样本均数比较的方差分析(analysis of variance, ANOVA)的方法,比较各处理组之间的差异性；多个样本均数间的多重比较(multiple comparison)的方法,比较各处理组与空白组、各处理组两两之间的差异性。临床研究本研究选取2009年1月至2009年12月,在广东省中医院心脏中心就诊,根据WHO1979年关于冠心病诊断标准,确诊为冠心病,并且属于慢性心肌缺血综合症者共60例。完全随机对照将他们分为治疗组,对照组,每组30例。治疗组给予中成药复方血栓通胶囊3粒,tid,连续应用30天；对照组：给予拜阿司匹林100mg,qd,连续应用30天；两组均给予扩冠等常规治疗。各组分别于纳入试验当天(服药前)、服药后第31天分别采外周抗凝血送检PT、TT、APTT、FIB；并记录症状疗效评分表。结果实验研究所有药物组抗凝血酶活性单位AT-U均显著高于空白组；按AT-U从大到小顺序排列,依次为土鳖、丹参、赤芍、僵蚕、水蛭、地龙、川芎、红花。其中,分别与水蛭比较,土鳖、丹参、赤芍、川芎的抗凝血酶活性单位AT-U大于水蛭,红花小于水蛭,而僵蚕、地龙则无显著差异;分别与僵蚕比较,土鳖、丹参、赤芍大于僵蚕,地龙、川芎、红花小于僵蚕,水蛭无显著差异；分别与地龙比较,水蛭、僵蚕、土鳖、丹参、赤芍大,川芎、红花无差异；与土鳖比较,各组均显著小于它；与丹参比较,土鳖大于它,水蛭、僵蚕、地龙、川芎、红花小,赤芍无显著差异；与川芎比较,水蛭、僵蚕、土鳖、丹参、赤芍均大于它,地龙、红花无显著差异；与赤芍比较,土鳖大于它,水蛭、僵蚕、地龙、川芎、红花小于它,丹参无显著差异；与红花比较,水蛭、僵蚕、土鳖、丹参、赤芍均大于它,地龙、川芎无显著差异。抗凝作用最强的前三个药物为土鳖、丹参、赤芍。土鳖、赤芍、丹参三者不同的配伍用药,对大鼠TT、PT、APTT均有不同的影响,差异有显著性意义(P<0.05)；其中华法林与四种中药配伍方式均有延长大鼠的PT、TT效果(P<0.05)；与华法林阳性对照组比较,四种中药配伍延长PT、TT的作用差异无显著性意义(P>0.05)；四种中药配伍彼此之间两两比较,对PT、TT的影响无显著差异(P<0.05)；与空白组、华法林、以及其它三种配伍比较,土鳖A+丹参B+赤芍C明显延长APTT(P<0.05)。空白组、华法林组、土鳖A+丹参B、土鳖A+赤芍C、丹参B+赤芍C、土鳖A+丹参B+赤芍C对FIB的影响无显著性差异。临床研究治疗组、对照组两组患者一般情况差异无统计学意义(>0.05),具有可比性。治疗前两组间PT、TT、APTT、FIB比较无显著差异(p>0.05);治疗后和治疗前同组比较,治疗组与对照组的PT、TT均较治疗前有所升高(p<0.05),但都尚在正常值范围内；治疗后两组间PT、TT、APTT、FIB比较无显著差异(p>0.05),均值均在正常范围内。治疗前两组间中医症状评分值无显著差异(p>0.05)；治疗前后比较,治疗组、对照组症状评分均有下降,差异有显著性(p<0.05);治疗后,两组间比较,治疗组症状评分值下降更显著(p<0.05)。结论现在及今后抗凝治疗的热点在于发现直接的凝血酶抑制剂,水蛭素已经被证实具有强大的直接抑制凝血酶作用。在本研究中,我们发现活血化瘀中药中,除水蛭外,土鳖、丹参、赤芍、僵蚕、地龙、川芎、红花也含有凝血酶的直接抑制成分,其中以土鳖、丹参、赤芍抗凝血酶活性最强。体外实验中,活血化瘀药物土鳖、丹参、赤芍、僵蚕、水蛭、地龙、川芎、红花均有确切抗凝作用；在大鼠体内抗凝实验中,不同配伍土鳖A+丹参B、土鳖A+赤芍C、丹参B+赤芍C、土鳖A+丹参B+赤芍C,四种方式具有与华法林同样的抗凝效果,并且抗凝机制涉及环节更多,涵盖了凝血过程的内源性、外源性以及共同途径。所以认为活血化瘀中药单味药、配伍组合均有确切抗凝效果,活血化瘀中药可以作为临床抗凝治疗中有效、安全、方便的制剂。活血化瘀中成药复方血栓通胶囊在治疗“冠心病”属于慢性心肌缺血综合症者时,与拜阿司匹林一样具有改善临床症状的功效,且比拜阿司匹林效果更为显著。其具有一定抗凝作用,这种抗凝作用是温和的,仅引起PT、TT轻微升高,并未引起凝血指标超出正常值；这种抗凝作用与拜阿司匹林的抗凝作用相比,无显著差异,所以对于属于慢性心肌缺血综合症的冠心病患者,如果不能耐受阿司匹林,其二级预防可以选用复方血栓通胶囊。更多还原

【Abstract】 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.更多还原