克服溶血的复方葛根素注射制剂生物评价研究
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摘要
葛根素(Puerarin)为中药葛根(Radix Puerariae)的主要有效成分之一,为异黄酮类化合物,现代药理实验证明葛根素具有扩张冠脉血管、降低血压和心肌耗氧、抗心律失常和明显限制急性心肌梗塞等药理作用。由于其自身化学结构的特点,葛根素在水中的溶解度仅为0.46%,而且据有关葛根素口服在动物或人体的药动学研究表明,葛根素口服自胃肠道吸收较快,但吸收程度差,生物利用度低,口服给药一般要2-3周甚至2-3个月方能起效,从而影响口服给药临床的疗效发挥。因此,药学研究人员提取葛根素单体,制成葛根素注射制剂,临床用于辅助治疗冠心病、心绞痛、心肌梗死、视网膜动、静脉阻塞、突发性耳聋及缺血性脑血管病、小儿病毒性心肌炎、糖尿病等。葛根素注射液有效成分清楚,疗效确切,质量稳定,在临床应用广泛,是中药西制的代表。
葛根素注射液在临床应用广泛,但可导致严重不良反应,急性血管内溶血是葛根素严重不良反应的主要临床症状,具有发病急、进展快、病情危重的特点,严重时引起病人死亡。据统计,2003年1月1日至2005年6月30日间,有关葛根素注射剂的不良反应病例报告共1006例,其中,严重不良反应30例,死亡11例。严重不良反应以急性血管内溶血为主,共18例,死亡8例,占死亡病例的73%。溶血不良反应致死的病例占葛根素不良反应死亡病例的73%。
目前,有研究报道葛根素注射剂引起的血管内溶血为Ⅱ型变态反应,但实验数据为临床个别病例的研究结果,无法说明葛根素口服给药不发生血管内溶血的事实,亦未能制订防止过敏反应的临床合理用药指南,引发了管理部门和医务工作者对其安全性的全面怀疑,临床医生慎用或不应用葛根素注射液,导致葛根素生产急剧萎缩,整个葛根素的种植、加工、提取、制剂产业链停滞。这一事件,也对中药注射液的安全性带来极大的负面影响。
因此,深入探讨葛根素注射液溶血不良反应的发生机制,建立阐明葛根素注射液诱发急性血管内溶血的原因,探寻解决葛根素注射液溶血不良反应的方法,具有重要的现实意义。
本文参照《中国药典》2005年版二部溶血检查项下方法,以葛根素注射液为研究对象,进行24只Beagle犬的红细胞的体外偶发性溶血实验,结果发现:丙二醇浓度为3.3-16.7 mg/ml、葛根素浓度为0.33-1.67mg/ml、观察时间为3小时,未发现葛根素注射液溶血。提示临床前研究中现有的溶血评价实验方法,无法预测临床中的葛根素注射液偶发性溶血不良反应的实际情况,凸显了现有临床前溶血安全性评价方法的不足,因此有进一步进行深人研究、改良方法的必要。
本文通过系统研究葛根素注射液溶血不良反应研究的相关文献,参考《中国药典》2005年版二部溶血检查法,通过提高药物浓度到丙二醇浓度为100mg/ml、葛根素浓度为10mg/ml,可提高溶血发生率,在24个动物个体的情况下可观测到丙二醇和葛根素注射液的溶血现象,并依此建立中药注射剂偶发性溶血评价方法,对葛根素注射液引致急性血管内溶血不良反应的原因进行探讨。实验结果显示:丙二醇浓度为100mg/ml时发现4例溶血,溶血发生率为16.6%,与生理盐水组存在显著性差异(0.05＞P＞0.01),但溶血率与丙二醇浓度不存在相关性(P＞0.05);葛根素注射液组丙二醇浓度为100 mg/ml、葛根素浓度为10mg/ml时,出现了16例溶血,溶血发生率为66.7%,与生理盐水组存在极显著性差异(P＜0.01),溶血率与葛根素浓度不存在极显著相关性(P＜0.01),相关系数r=0.9523。对葛根素注射液和丙二醇液的偶发性溶血评价实验结果进行析因分析,结果显示:葛根素注射液组溶血发生概率,显著高于丙二醇组溶血发生概率(P＜0.01)。提示:丙二醇作为辅料,虽有引起溶血的风险,但不是主要因素。引起溶血不良反应的主要因素是药物成分葛根素本身。其引起溶血的因素可能与与葛根素嵌入红细胞的双层磷脂膜,引起红细胞膜流动性和红细胞变形能力下降有关。
文献研究发现,牛磺酸具有增强红细胞膜流动性,维持细胞膜的稳定性,保护红细胞膜,防止溶血的作用。本实验通过中药注射液偶发性溶血评价方法,分别取注射蒸馏水、生理盐水和已上市葛根素注射液为对照,考察在相同的葛根素浓度下,加入不同浓度牛磺酸,观察24小时内红细胞的状态,评价牛磺酸抗葛根素溶血的效果。实验结果显示:与葛根素注射液高剂量组比较,复方葛根素注射液Ⅰ、Ⅱ、Ⅲ各组均未出现溶血,复方葛根素注射液Ⅰ、Ⅱ、Ⅲ各组溶血发生率均与葛根素注射液高剂量组存在极显著差异(P＜0.01);与生理盐水组比较,复方葛根素注射液Ⅰ、Ⅱ、Ⅲ各组溶血发生率均与生理盐水组无差异(P=1.00)。复方葛根素注射液Ⅰ、Ⅱ、Ⅲ各组与葛根素注射液高剂量组均含有10mg/ml葛根素;复方葛根素注射液Ⅰ、Ⅱ、Ⅲ各组分别含5mg/ml、10mg/ml、20mg/ml牛磺酸,葛根素注射液高剂量组含有100mg/ml丙二醇,而葛根素注射液高剂量组溶血率为79.2%,显著高于复方葛根素注射液Ⅰ、Ⅱ、Ⅲ各组。实验结果显示:不同浓度的牛磺酸与葛根素配伍应用,均可拮抗由葛根素诱发的溶血不良反应,并可使发生率降低至生理盐水水平。提示可应用中药方剂配伍理论,将中药单体成分葛根素与牛磺酸配伍应用,制成无溶血不良反应的复方葛根素注射液,以消除葛根素单独应用的不良反应。
本文对复方葛根素制剂成型工艺中辅料选择、用量、pH值以及冻干工艺进行考察,确定注射用复方葛根素粉针的制备工艺为:取葛根素5g、牛磺酸25g,加适量注射用水,搅拌使溶解;加入注射用甘露醇,用碳酸钠调pH值至6-7之间,加注射用水至400ml,0.2μm微孔滤膜过,每支西林瓶装溶液2ml。置冷冻干燥箱内,预冻温度为-40℃,冷肼温度为-60℃,保温1h。然后抽真空,当真空度达到10Pa以下后,开始加温升华,并保证升华时样比产品的共晶点温度低5℃,应低于28℃,并在此状况下维持10小时,直到产品中的冻结冰升华完毕为止;主干燥完成后迅速升温,样品终点温度28℃,保温1h后出箱。上述工艺小试各批次产品的成品率均在90%以上,各批产品的外观和再分散性均符合规定。
虽然牛磺酸可消除葛根素所引发的急性血管内溶血不良反应,大大提高了原葛根素注射液的安全性,但葛根素与牛磺酸配伍组成的新复方制剂属化学药品注册分类1,按照《药品注册管理办法》规定,应进行复方制剂中多种成分的药代动力学相互影响研究。为了比较葛根素与牛磺酸配伍复方制剂与各组分单独应用时的动力学差异,对牛磺酸与葛根素配伍的合理性进行探讨,本文建立了血浆中葛根素、牛磺酸的HPLC测定法,进行复方葛根素注射制剂中两组分在犬体内的药动学相互影响实验。
本实验采用6%高氯酸沉淀蛋白预处理方法,用甲醇—0.2%磷酸为流动相,在250nm处测定了血清中葛根素的浓度。在此色谱条件下,葛根素在1.01-60.8μg/ml范围内线性关系良好,该方法专属性强,灵敏度高,准确度及精密度均满足低血药浓度葛根素测定的要求。
本实验采用6%高氯酸沉淀蛋白,2,4—二硝基氟苯(DNFB)柱前衍生化预处理方法,用甲醇—pH5.5乙酸钠、乙酸缓冲液为流动相,在360nm处测定了血清中牛磺酸的浓度。进行24小时内Beagle犬血清中牛磺酸含量动态变化考察,发现不同Beagle犬个体血清中牛磺酸浓度差异较大,同一Beagle犬个体血清中牛磺酸浓度在24h内较稳定,因此,本实验以给药前空白血清中牛磺酸的浓度作为Beagle犬体内牛磺酸基粗?进行牛磺酸在Beagle犬体内的药物动力学研究。在此色谱条件下,牛磺酸在18.4-368.0μg/ml范围内线性关系良好,该方法专属性强,灵敏度高,准确度及精密度均满足低血药浓度牛磺酸测定的要求。
应用所建立方法,进行了6只Beagle犬单剂量静脉注射用复方葛根素粉针、葛根素注射液、牛磺酸注射液后的药物动力学研究。实验结果表明,静脉注射葛根素注射液、牛磺酸注射液和注射用复方葛根素粉针后,葛根素和牛磺酸在犬体内的处置均符合二室模型。单剂量静脉注射葛根素注射液后,犬体内葛根素的t_(1/2α)=3.25±1.76min,t_(1/2β)=55.5±4.78min,AUC_(0-t)=2364.93±158.45μg·min/ml;单剂量静脉注射葛根素牛磺酸注射液后,犬体内牛磺酸的t_(1/2α)=11.31±4.15min,t_(1/2β)=177.8±228.73min,AUC_(0-t)=10361.69±1868.41μg·min/ml;单剂量静脉注射复方葛根素制剂后,犬体内葛根素的t_(1/2α)=3.73±0.55min,t_(1/2β)=56.0±2.70min,AUC_(0-t)=2489.57±115.83μg·min/ml,犬体内牛磺酸的t_(1/2α)=12.34±8.28min,t_(1/2β)=201.0±166.46min,AUC_(0-t)=11811.21±3771.71μg·min/ml。
应用配对t检验法对单剂量静脉注射葛根素注射液和复方葛根素粉针中葛根素的t_(1/2α)、t_(1/2β)、V(c)、Cl(s)、AUC、AUC_(0-∞)、AUC_(0-t)、MRT_(0-∞)、MRT_(0-t)进行检验,结果显示:复方葛根素中牛磺酸与葛根素配伍后牛磺酸的药物动力学过程与牛磺酸单独给药的药物动力学过程没有显著性差异(P＞0.05)。应用配对t检验法对单剂量静脉注射葛根素注射液和复方葛根素粉针中牛磺酸的t_(1/2α)、t_(1/2β)、V(c)、Cl(s)、AUC、AUC_(0-∞)、AUC_(0-t)、MRT_(0-∞)、MRT_(0-t)进行检验,结果显示:复方葛根素中葛根素与牛磺酸配伍后葛根素的药物动力学过程与葛根素单独给药的药物动力学过程没有显著性差异(P＞0.05)。提示将葛根素和牛磺酸组成注射用复方制剂后,葛根素和牛磺酸的药物动力学特征未发生改变,两组分配伍应用无药物动力学的相互作用,为组方的合理性与安全性提供依据。
通过对氯仿诱发小鼠心率失常的影响实验对牛磺酸、葛根素配伍前后药效相互影响进行探讨。发现与生理盐水对照组比较,心得安、复方葛根素注射高剂量均能显著延缓小鼠出现心律失常(P＜0.01);与心得安阳性对照药比较,复方葛根素注射液低、中、高剂量给药组小鼠出现心率失常的时间与心得安组接近,无统计学差异(P＞0.05),提示复方葛根素注射液抗氯仿诱发的小鼠心率失常的效果与心得安相似;与复方葛根素注射液中剂量给药组比较,拆方成分葛根素、牛磺酸抗氯仿诱发的小鼠心率失常的效果弱于复方,但无统计学差异(P＞0.05)。
通过对垂体后叶素所致大鼠心肌缺血的影响实验对牛磺酸、葛根素配伍前后药效相互影响进行探讨。发现垂体后叶素所致的急性心肌缺血模型空白对照组的大鼠,用生物机能系统记录分析心电图,大都表现为S-T段抬高、心率减慢,T波高耸,个别T波低平或倒置,并大多数出现了严重的心搏失常(房早或室早所致的心搏间歇或二联律、三联律等,个别甚至出现室性心动过速)。给予一定剂量的复方葛根素注射液后,与模型空白对照组比较,能显著改善垂体后叶素所致大鼠急性心肌缺血的表现,如st段变化幅度减少,T波倒置、严重心律失常发生的例数均减少,血清心肌酶CK、LDH-L、AST的释放亦明显减少。以上结果显示复方葛根素注射液具有一定抗心肌缺血、心律失常的作用。结果显示:牛磺酸消除葛根素溶血不良反应的同时,葛根素、牛磺酸两个成分配伍有一定的协同增效作用。为牛磺酸、葛根素配伍的合理性提供了药效学依据。
本研究应用中药注射液偶发性溶血评价方法,发现牛磺酸可拮抗葛根素溶血不良反应。提示可通过有效成分组方来消除葛根素的溶血不良反应,将牛磺酸与葛根素配伍,制成无溶血不良反应的复方葛根素注射液。通过复方葛根素制剂成型工艺研究,解决葛根素和牛磺酸的相容性问题和产品的稳定性问题,制成注射用复方葛根素粉针。应用药物动力学和药效学研究方法,对牛磺酸、葛根素配伍前后牛磺酸对葛根素药物动力学、药效的影响进行评价,发现葛根素与牛磺酸配伍不会影响各自的药物动力学过程,并且两个组方成分有一定的协同增效作用,提示二者可联合用药,为组方的合理性提供科学依据。
Radix Puerariae is origin in roots of leguminous plant Pueraria lobata(Willd.) Ohwi or Pueraria thomsonii Benth.Puerarin,one kind of the flavonoids,is the effective ingredient of Radix Puerariae.Modern pharmacological experiments prove that Puerarin can expanse coronary blood vessels,lower blood pressure and myocardial oxygen consumption,and cure arrhythmic and significantly pharmacological effects such as reliefing acute myocardial infarction.Chemical structure as a result of its own characteristics,puerarin solubility in water is only 0.46%,and oral Puerarin,according to the animal or human pharmacokinetics studies,have shown that oral administration of Puerarin is rapidly absorbed from the gastrointestinal tract,but the rate of absorption is poor,and bioavailability is low,oral administration of a general 2-3 to 2-3 weeks or even months to become effective,thus affecting the clinical efficacy of its oral administration.Therefore, pharmaceutical researchers extracted Puerarin monomer,made of puerarin injection preparations,for the adjuvant treatment of clinical coronary heart disease,angina, myocardial infarction,retinal artery and vein occlusion,sudden deafness and ischemic cerebrovascular disease,viral myocarditis,diabetes among children and so on.Active ingredient of Puerarin injection that is effective,quality,stability,a wide range of clinical application,and as a representive Chinese medicines prepared in the Western system.
Puerarin injection,applied widely in clinic,lead to serious adverse reactions such as acute intravascular hemolysis,which is the main clinical symptom.What is worse,it is acute symptom,and fatal in some serious cases as it progresses quickly.According to statistics,during the days from 1~(st) January,2003 to 30~(th) June,2005,the puerarin injection adverse reaction cases report a total of 1006 cases,of which 30 cases is serious adverse reactions,11 cases of death.Serious adverse reactions to the main acute intravascular hemolysis,a total of 18 cases,eight cases of death,accounting for 73%.Deaths caused by Puerarin adverse reactions accounted for 73%in all fatal cases.
At present,it is reported that puerarin injection intravascular hemolysis is caused by typeⅡallergy,but the experimental data are only individual cases of clinical research findings,which could not show whether oral administration of puerarin is the reason of intravascular hemolysis,and fail to find clinical rational drug use guidelines to prevent an allergic reaction.Therefore,its security is comprehensively suspected by concerned departments and medical workers,clinicians should keep a serious eye on puerarin injection, resulting in a dramatic shrinkage of puerarin production,the of the whole Puerarin, including its cultivation,processing,extraction,preparation stagnant industry chain.As the result to this section,the incident negatively affects the safety of traditional Chinese medicine injection of a great.
Therefore,it is of great sense to engage in further study of mechanism of puerarin injection adverse reaction,the establishment of expounded puerarin injection-induced acute causes of intravascular hemolysis,finding solution of its adverse reactions.
In this paper,reference to the method of "Chinese Pharmacopoeia" version 2,2005 Hemolytic inspection,puerarin was studied in puerarin injection applied for 24 Beagle dogs occasional red blood cell hemolysis in vitro experiments,results were as follows:there is no hemolytic with propylene glycol concentration of 3.3-16.7 mg/ml and the concentration of puerarin 0.33-1.67mg/ml for 3 hours' observation.The experiment suggests that pre-clinical studies prompted current experimental methods of evaluation of hemolysis can not predict the clinical sporadic hemolytic as puerarin injection adverse reactions, highlighting disadvantage of the current pre-clinical safety evaluation of hemolytic,it need to be put under further studies,and to improved methods as well.
In this paper,through a systematic study of puerarin injection adverse reactions hemolytic research papers,reference to "Chinese Pharmacopoeia" version 2,2005 Hemolytic test,by increasing the drug concentration to the propylene glycol concentration of 100mg/ml,puerarin concentration of 10mg/ml,puerarin can increase the incidence of hemolysis,and in the 24 individual animals experiment hemolysis can be observed in cases of puerarin injection with addition of propylene glycol.Therefore the establishment of sporadic hemolytic evaluation of puerarin in traditional Chinese medicine injection after that,and applied to explore the mechanism acute intravascular haemolytic adverse reactions due to puerarin injection.The results showed that:propylene glycol concentration of 100mg/ml and then four cases of hemolysis were found,and hemolytic rate was 16.6%. there was a significant difference(0.05＞P＞0.01) with the saline group,but the hemolysis rate and the concentration of propylene glycol do not exist any correlation(P＞0.05); puerarin injection group including propylene glycol concentration of 100 mg/ml,and concentration of 10mg/ml Puerarin,the emergence of 16 cases of hemolysis led to 66.7%as hemolytic rate,there are significant differences(P＜0.01)with the saline group,hemolysis rate and the concentration of Puerarin does not have significant correlation(P＜0.01), correlation coefficient r=0.9523.Factorial analysis in Puerarin injection of propylene glycol solution and sporadic hemolytic evaluation of test results showed that:puerarin injection group hemolytic probability is significantly higher than the probability of occurrence hemolytic propanediol group(P＜0.01).It suggests:propylene glycol as the accessories,although the risk of causing hemolysis,is not a major factor.Adverse reactions caused by hemolysis is Puerarin itself as major factor.The mechanism of hemolytic may be that Puerarin embedded with the double red blood cell membrane phospholipids, erythrocyte membrane caused by decreasing liquidity and a decline in red blood cell deformability.
Literature study found that taurine enhanced the mobility of erythrocyte membrane and maintain its stability,as a result for protectiing of red cell membrane and preventing the role of hemolysis.In this study,through sporadic hemolytic evaluation methods in the injection of Chinese medicine.Distilled water for injection,saline and puerarin injection as a control study,and while concentration of puerarin is in the same,anti-hemolytic effect of puerarin with taurine was evaluated by adding different concentrations of taurine,which was observed within 24 hours for red blood cells status.The results showed that:puerarin injection with high-dose group,compound puerarin injectionⅠ,Ⅱ,Ⅲin each group had no hemolysis,compound puerarin injection ofⅠ,Ⅱ,Ⅲin each group,compared with hemolytic Puerarin high-dose injection group,were significant difference(P＜0.01); Compared with the saline group,compound puerarin injection ofⅠ,Ⅱ,Ⅲthe incidence of hemolysis in each group with no difference(P=1.00).Compound puerarin injectionⅠ,Ⅱ,Ⅲpuerarin injection group and the high-dose group contained 10mg/ml Puerarin; compound puerarin injection ofⅠ,Ⅱ,Ⅲ,respectively,each group contains 5mg/ml, 10mg/ml,20mg/ml taurine,and high-dose group of puerarin injection containing 100mg/ml propylene glycol,and puerarin injection hemolytic rate of high-dose group and 79.2%, significantly higher than that of compound puerarin injection ofⅠ,Ⅱ,Ⅲ.The results showed that:different concentrations of taurine existing in Puerarin,can be antagonistic by puerarin-induced hemolysis and the incidence can be reduced to the level of normal as saline group.It suggested that through combination theory under the direction of traditional Chinese medicine,Chinese medicine Puerarin monomer component,which can produce adverse reactions combined with taurine,compound puerarin injection to eliminate a separate application Puerarin adverse reactions.
In this paper,preparation of compound Puerarin including accessories molding process selection,dosage,pH value and freeze-drying process is tested to determine the compound injection of puerarin injection for preparation:Puerarin 5g,Taurine 25g,plus adequate water for injection,stirring allows the dissolved;adding mannitol for injection with sodium carbonate adjusted pH values to be 6-7,plus water for injection to 400ml, filter through 0.2μm microporous membrane,and each bottle of solution-resistant 2ml.Buy frozen oven,the pre-freezing temperature of -40℃,hydrazine cold temperature -60℃, heat 1h.Vacuum and then,when the vacuum reaches the following 10Pa after sublimation temperature and then to ensure that when the sample sublimation products lower eutectic temperature by 5℃,as be lower than 28℃,and in this case for 10 hours,until it product freeze until ice sublimation;shortly after the completion of the main drying-up,sample the end in the temperature 28℃,the thermal insulation box maintain for 1h.The above-mentioned process in small test of the batch yield of 90%in all batches,the appearance and re-dispersion are consistent with the provisions.
Although intravascular hemolysis acute adverse reactions caused by Puerarin can be the eliminated with co-exist with taurine,and then the safety of puerarin injection the original can be greatly enhanced,Puerarin combined with taurine,compound preparation of the new chemicals registered Category 1,in accordance with the "management of drug registration",it shall be carried out in a variety of components of compound preparation of the pharmacokinetics study of mutual influence.In order to compare the kinetic differences of Puerarin compound preparation combined with taurine and a separate application of each component.In order to explore the the rationality of combination of taurine and Puerarin.In this paper,the establishment of determination of puerarin,Taurine in plasma by HPLC,the compound of puerarin injection of two formulations in dogs Pharmacokinetics of interaction tests will be designed and confirmed.
In this study,the use of 6%perchloric acid pretreatment method of protein precipitation,with methanol -0.2%phosphoric acid as mobile phase,in 250nm,the concentration of puerarin were determined.Under such conditions of chromatography, puerarin in the scope of 1.01-60.8μg/ml has a good linear relationship,the method is of specificity,high sensitivity,accuracy and precision,which meet the demand determination of lowest plasma concentration of puerarin.
In this study,concentrations of taurine in serum is determinated by pre-treatment of pre-column derivatization method,using of 6%perchloric acid precipitated protein,2,4-dinitrofluorobenzene(DNFB),using methanol-pH5.5 sodium acetate,acetic acid buffer as the mobile phase,in 360nm measured serum.Carried out within 24 hours of Beagle dog serum study dynamic changes of taurine content and found that concentration of different taurine varied greatly different individuals Beagle dog serum,serum concentrations of taurine in the same Beagle dogs individual in 24h is a more stable.Therefore,the test blank serum is to justified as the taurine in the Beagle dog pharmacokinetics by reducing the basis value of taurine in Beagle dogs before administration of the concentration of taurine,.In the chromatographic conditions,the scope of taurine in 18.4-368.0μg/ml has a good linear relationship,the method is of specificity,high sensitivity,accuracy and precision,which meets the determination of plasma concentration of taurine requirements.
With application of established methods,pharmacokinetic study is carried out in six Beagle dogs after injection with a single dose of intravenous injection of compound puerarin,puerarin injection,of taurine.Test results show that intravenous injection of puerarin injection,taurine injection and the injection of compound after administration, puerarin and taurine in the disposal of dogs are in line with the two-compartment model.A single dose of intravenous injection of puerarin injection,the dogs Puerarin t1/2α=3.25±1.76min,t1/2β=55.5±4.78min,AUC0-t=2364.93±158.45μg·min/ml;a single dose of intravenous injection of puerarin Su-taurine injection,the dogs taurine t1/2α=11.31±4.15min,t1/2β=177.8±228.73h,AUC0-t=10361.69±1868.41μg·min/ml;a single dose of intravenous injection of compound Puerarin preparations,the dogs Puerarin t1/2α=3.73±0.55min,t1/2β=56.0±2.70min,AUC0-t=2489.57±115.83μg·min/ml,taurine t1/2αdogs=12.34±8.28min,t1/2β=201.0±166.46min,AUC0-t=11811.21±3771.71μg·min /ml.
Paired t test is applied to analyses the data a single dose of intravenous injection of puerarin and compound puerarin injection incuding 1/2α,t1/2β,V(c),Cl(s),AUC, AUC0-∞,AUC0-t,MRT0-∞,MRT0-t test.Results showed that:taurine and Puerarin compound Puerarin,combined with taurine were no significant differences with separate taurine pharmacokinetics(P＞0.05).Paired t test the application of a single dose of intravenous injection of puerarin and puerarin injection compound taurine t1/2α,t1/2β,V (c),Cl(s),AUC,AUC0-∞,AUC0-t,MRT0-∞,MRT0-t test.Results showed that: Compound Puerarin,combined with taurine,there is no significant difference(P＞0.05) between puerarin process and separate the process of drug pharmacokinetics after the pharmacokinetics of puerarin.It suggested that Puerarin and taurine compound preparation of the composition after injection and pharmacokinetic characteristics of taurine did not change the application of two non-distribution of Ng interaction pharmacokinetics,which provides the basis for the group's rationality and security.
In order to explore interaction effects on the effects of Abnormal heart rate test in mice induced by Chloroform,both before and after Puerarin combined with taurine.Result shows that:compared with the saline control group,both propranolol group and compound puerarin of high-dose injection could significantly delay the emergence of arrhythmia in mice(P＜0.01);Compared with the control drug propranolol,compared puerarin injection low,middle and high dosing mice appear abnormal heart rate of time and close to propranolol group,no significant statistic difference(P＞0.05),prompted puerarin injection of curing heart disorders compound chloroform-induced effects in mice is similar to propranolol;and Compound dosing of puerarin injection group,the split Puerarin ingredients,taurine against chloroform-induced disorders in mice the effect of heart rate are less than that of compound,but no statistic significant difference(P＞0.05).
By testing effect of pituitrin-induced myocardial ischemia in rats of taurine,to explore mutual influence before and after the combined puerarin.It was found that pituitrin-induced model of acute myocardial ischemia control group of rats,with the biological function of the system analysis of ECG records,mostly manifested as ST-segment elevation,heart rate, T-wave high-rise,individual low-or T-wave inverted,and the majority of a serious cardiac disorders(room or rooms as early as early or intermittent cardiac arrest induced bigeminy, such as triple law,individual or even ventricular tachycardia).Compound to give a certain dose of puerarin injection,the control group and model comparison,can significantly improve the pituitrin-induced myocardial ischemia in rats with acute manifestations,such as reduced st segment,T wave inversion,serious arrhythmia the number of cases that occurred were reduced,serum enzymes CK,LDH-L,AST release was also markedly reduced.These results indicate that a certain compound puerarin injection of anti-myocardial ischemia,arrhythmias.
In this study evaluation methods of sporadic hemolytic in injection made of Chinese medicine and found that taurine could antagonize Puerarin hemolytic adverse reactions.It suggested the active ingredient group Puerarin side to eliminate adverse effects of hemolysis,the combination of taurine and Puerarin,made compound puerarin injection without adverse reactions hemolytic.Through the preparation of compound forming process Puerarin research,resolve compatibility stability of products made of taurine and Puerarin,as preparation compound puerarin injection powder.In pharmacokinetics and pharmacodynamics research,Puerarin,both before and after combined with taurine Puerarin taurine on pharmacokinetics,efficacy evaluation of the impact and found Puerarin compatibility with taurine does not affect their pharmacokinetic process and composition of the two groups to have a certain synergy,suggesting that can provide a reasonable scientific basis for the two can be combined for the group.
葛根素注射液在临床应用广泛,但可导致严重不良反应,急性血管内溶血是葛根素严重不良反应的主要临床症状,具有发病急、进展快、病情危重的特点,严重时引起病人死亡。据统计,2003年1月1日至2005年6月30日间,有关葛根素注射剂的不良反应病例报告共1006例,其中,严重不良反应30例,死亡11例。严重不良反应以急性血管内溶血为主,共18例,死亡8例,占死亡病例的73%。溶血不良反应致死的病例占葛根素不良反应死亡病例的73%。
目前,有研究报道葛根素注射剂引起的血管内溶血为Ⅱ型变态反应,但实验数据为临床个别病例的研究结果,无法说明葛根素口服给药不发生血管内溶血的事实,亦未能制订防止过敏反应的临床合理用药指南,引发了管理部门和医务工作者对其安全性的全面怀疑,临床医生慎用或不应用葛根素注射液,导致葛根素生产急剧萎缩,整个葛根素的种植、加工、提取、制剂产业链停滞。这一事件,也对中药注射液的安全性带来极大的负面影响。
因此,深入探讨葛根素注射液溶血不良反应的发生机制,建立阐明葛根素注射液诱发急性血管内溶血的原因,探寻解决葛根素注射液溶血不良反应的方法,具有重要的现实意义。
本文参照《中国药典》2005年版二部溶血检查项下方法,以葛根素注射液为研究对象,进行24只Beagle犬的红细胞的体外偶发性溶血实验,结果发现:丙二醇浓度为3.3-16.7 mg/ml、葛根素浓度为0.33-1.67mg/ml、观察时间为3小时,未发现葛根素注射液溶血。提示临床前研究中现有的溶血评价实验方法,无法预测临床中的葛根素注射液偶发性溶血不良反应的实际情况,凸显了现有临床前溶血安全性评价方法的不足,因此有进一步进行深人研究、改良方法的必要。
本文通过系统研究葛根素注射液溶血不良反应研究的相关文献,参考《中国药典》2005年版二部溶血检查法,通过提高药物浓度到丙二醇浓度为100mg/ml、葛根素浓度为10mg/ml,可提高溶血发生率,在24个动物个体的情况下可观测到丙二醇和葛根素注射液的溶血现象,并依此建立中药注射剂偶发性溶血评价方法,对葛根素注射液引致急性血管内溶血不良反应的原因进行探讨。实验结果显示:丙二醇浓度为100mg/ml时发现4例溶血,溶血发生率为16.6%,与生理盐水组存在显著性差异(0.05＞P＞0.01),但溶血率与丙二醇浓度不存在相关性(P＞0.05);葛根素注射液组丙二醇浓度为100 mg/ml、葛根素浓度为10mg/ml时,出现了16例溶血,溶血发生率为66.7%,与生理盐水组存在极显著性差异(P＜0.01),溶血率与葛根素浓度不存在极显著相关性(P＜0.01),相关系数r=0.9523。对葛根素注射液和丙二醇液的偶发性溶血评价实验结果进行析因分析,结果显示:葛根素注射液组溶血发生概率,显著高于丙二醇组溶血发生概率(P＜0.01)。提示:丙二醇作为辅料,虽有引起溶血的风险,但不是主要因素。引起溶血不良反应的主要因素是药物成分葛根素本身。其引起溶血的因素可能与与葛根素嵌入红细胞的双层磷脂膜,引起红细胞膜流动性和红细胞变形能力下降有关。
文献研究发现,牛磺酸具有增强红细胞膜流动性,维持细胞膜的稳定性,保护红细胞膜,防止溶血的作用。本实验通过中药注射液偶发性溶血评价方法,分别取注射蒸馏水、生理盐水和已上市葛根素注射液为对照,考察在相同的葛根素浓度下,加入不同浓度牛磺酸,观察24小时内红细胞的状态,评价牛磺酸抗葛根素溶血的效果。实验结果显示:与葛根素注射液高剂量组比较,复方葛根素注射液Ⅰ、Ⅱ、Ⅲ各组均未出现溶血,复方葛根素注射液Ⅰ、Ⅱ、Ⅲ各组溶血发生率均与葛根素注射液高剂量组存在极显著差异(P＜0.01);与生理盐水组比较,复方葛根素注射液Ⅰ、Ⅱ、Ⅲ各组溶血发生率均与生理盐水组无差异(P=1.00)。复方葛根素注射液Ⅰ、Ⅱ、Ⅲ各组与葛根素注射液高剂量组均含有10mg/ml葛根素;复方葛根素注射液Ⅰ、Ⅱ、Ⅲ各组分别含5mg/ml、10mg/ml、20mg/ml牛磺酸,葛根素注射液高剂量组含有100mg/ml丙二醇,而葛根素注射液高剂量组溶血率为79.2%,显著高于复方葛根素注射液Ⅰ、Ⅱ、Ⅲ各组。实验结果显示:不同浓度的牛磺酸与葛根素配伍应用,均可拮抗由葛根素诱发的溶血不良反应,并可使发生率降低至生理盐水水平。提示可应用中药方剂配伍理论,将中药单体成分葛根素与牛磺酸配伍应用,制成无溶血不良反应的复方葛根素注射液,以消除葛根素单独应用的不良反应。
本文对复方葛根素制剂成型工艺中辅料选择、用量、pH值以及冻干工艺进行考察,确定注射用复方葛根素粉针的制备工艺为:取葛根素5g、牛磺酸25g,加适量注射用水,搅拌使溶解;加入注射用甘露醇,用碳酸钠调pH值至6-7之间,加注射用水至400ml,0.2μm微孔滤膜过,每支西林瓶装溶液2ml。置冷冻干燥箱内,预冻温度为-40℃,冷肼温度为-60℃,保温1h。然后抽真空,当真空度达到10Pa以下后,开始加温升华,并保证升华时样比产品的共晶点温度低5℃,应低于28℃,并在此状况下维持10小时,直到产品中的冻结冰升华完毕为止;主干燥完成后迅速升温,样品终点温度28℃,保温1h后出箱。上述工艺小试各批次产品的成品率均在90%以上,各批产品的外观和再分散性均符合规定。
虽然牛磺酸可消除葛根素所引发的急性血管内溶血不良反应,大大提高了原葛根素注射液的安全性,但葛根素与牛磺酸配伍组成的新复方制剂属化学药品注册分类1,按照《药品注册管理办法》规定,应进行复方制剂中多种成分的药代动力学相互影响研究。为了比较葛根素与牛磺酸配伍复方制剂与各组分单独应用时的动力学差异,对牛磺酸与葛根素配伍的合理性进行探讨,本文建立了血浆中葛根素、牛磺酸的HPLC测定法,进行复方葛根素注射制剂中两组分在犬体内的药动学相互影响实验。
本实验采用6%高氯酸沉淀蛋白预处理方法,用甲醇—0.2%磷酸为流动相,在250nm处测定了血清中葛根素的浓度。在此色谱条件下,葛根素在1.01-60.8μg/ml范围内线性关系良好,该方法专属性强,灵敏度高,准确度及精密度均满足低血药浓度葛根素测定的要求。
本实验采用6%高氯酸沉淀蛋白,2,4—二硝基氟苯(DNFB)柱前衍生化预处理方法,用甲醇—pH5.5乙酸钠、乙酸缓冲液为流动相,在360nm处测定了血清中牛磺酸的浓度。进行24小时内Beagle犬血清中牛磺酸含量动态变化考察,发现不同Beagle犬个体血清中牛磺酸浓度差异较大,同一Beagle犬个体血清中牛磺酸浓度在24h内较稳定,因此,本实验以给药前空白血清中牛磺酸的浓度作为Beagle犬体内牛磺酸基粗?进行牛磺酸在Beagle犬体内的药物动力学研究。在此色谱条件下,牛磺酸在18.4-368.0μg/ml范围内线性关系良好,该方法专属性强,灵敏度高,准确度及精密度均满足低血药浓度牛磺酸测定的要求。
应用所建立方法,进行了6只Beagle犬单剂量静脉注射用复方葛根素粉针、葛根素注射液、牛磺酸注射液后的药物动力学研究。实验结果表明,静脉注射葛根素注射液、牛磺酸注射液和注射用复方葛根素粉针后,葛根素和牛磺酸在犬体内的处置均符合二室模型。单剂量静脉注射葛根素注射液后,犬体内葛根素的t_(1/2α)=3.25±1.76min,t_(1/2β)=55.5±4.78min,AUC_(0-t)=2364.93±158.45μg·min/ml;单剂量静脉注射葛根素牛磺酸注射液后,犬体内牛磺酸的t_(1/2α)=11.31±4.15min,t_(1/2β)=177.8±228.73min,AUC_(0-t)=10361.69±1868.41μg·min/ml;单剂量静脉注射复方葛根素制剂后,犬体内葛根素的t_(1/2α)=3.73±0.55min,t_(1/2β)=56.0±2.70min,AUC_(0-t)=2489.57±115.83μg·min/ml,犬体内牛磺酸的t_(1/2α)=12.34±8.28min,t_(1/2β)=201.0±166.46min,AUC_(0-t)=11811.21±3771.71μg·min/ml。
应用配对t检验法对单剂量静脉注射葛根素注射液和复方葛根素粉针中葛根素的t_(1/2α)、t_(1/2β)、V(c)、Cl(s)、AUC、AUC_(0-∞)、AUC_(0-t)、MRT_(0-∞)、MRT_(0-t)进行检验,结果显示:复方葛根素中牛磺酸与葛根素配伍后牛磺酸的药物动力学过程与牛磺酸单独给药的药物动力学过程没有显著性差异(P＞0.05)。应用配对t检验法对单剂量静脉注射葛根素注射液和复方葛根素粉针中牛磺酸的t_(1/2α)、t_(1/2β)、V(c)、Cl(s)、AUC、AUC_(0-∞)、AUC_(0-t)、MRT_(0-∞)、MRT_(0-t)进行检验,结果显示:复方葛根素中葛根素与牛磺酸配伍后葛根素的药物动力学过程与葛根素单独给药的药物动力学过程没有显著性差异(P＞0.05)。提示将葛根素和牛磺酸组成注射用复方制剂后,葛根素和牛磺酸的药物动力学特征未发生改变,两组分配伍应用无药物动力学的相互作用,为组方的合理性与安全性提供依据。
通过对氯仿诱发小鼠心率失常的影响实验对牛磺酸、葛根素配伍前后药效相互影响进行探讨。发现与生理盐水对照组比较,心得安、复方葛根素注射高剂量均能显著延缓小鼠出现心律失常(P＜0.01);与心得安阳性对照药比较,复方葛根素注射液低、中、高剂量给药组小鼠出现心率失常的时间与心得安组接近,无统计学差异(P＞0.05),提示复方葛根素注射液抗氯仿诱发的小鼠心率失常的效果与心得安相似;与复方葛根素注射液中剂量给药组比较,拆方成分葛根素、牛磺酸抗氯仿诱发的小鼠心率失常的效果弱于复方,但无统计学差异(P＞0.05)。
通过对垂体后叶素所致大鼠心肌缺血的影响实验对牛磺酸、葛根素配伍前后药效相互影响进行探讨。发现垂体后叶素所致的急性心肌缺血模型空白对照组的大鼠,用生物机能系统记录分析心电图,大都表现为S-T段抬高、心率减慢,T波高耸,个别T波低平或倒置,并大多数出现了严重的心搏失常(房早或室早所致的心搏间歇或二联律、三联律等,个别甚至出现室性心动过速)。给予一定剂量的复方葛根素注射液后,与模型空白对照组比较,能显著改善垂体后叶素所致大鼠急性心肌缺血的表现,如st段变化幅度减少,T波倒置、严重心律失常发生的例数均减少,血清心肌酶CK、LDH-L、AST的释放亦明显减少。以上结果显示复方葛根素注射液具有一定抗心肌缺血、心律失常的作用。结果显示:牛磺酸消除葛根素溶血不良反应的同时,葛根素、牛磺酸两个成分配伍有一定的协同增效作用。为牛磺酸、葛根素配伍的合理性提供了药效学依据。
本研究应用中药注射液偶发性溶血评价方法,发现牛磺酸可拮抗葛根素溶血不良反应。提示可通过有效成分组方来消除葛根素的溶血不良反应,将牛磺酸与葛根素配伍,制成无溶血不良反应的复方葛根素注射液。通过复方葛根素制剂成型工艺研究,解决葛根素和牛磺酸的相容性问题和产品的稳定性问题,制成注射用复方葛根素粉针。应用药物动力学和药效学研究方法,对牛磺酸、葛根素配伍前后牛磺酸对葛根素药物动力学、药效的影响进行评价,发现葛根素与牛磺酸配伍不会影响各自的药物动力学过程,并且两个组方成分有一定的协同增效作用,提示二者可联合用药,为组方的合理性提供科学依据。
Radix Puerariae is origin in roots of leguminous plant Pueraria lobata(Willd.) Ohwi or Pueraria thomsonii Benth.Puerarin,one kind of the flavonoids,is the effective ingredient of Radix Puerariae.Modern pharmacological experiments prove that Puerarin can expanse coronary blood vessels,lower blood pressure and myocardial oxygen consumption,and cure arrhythmic and significantly pharmacological effects such as reliefing acute myocardial infarction.Chemical structure as a result of its own characteristics,puerarin solubility in water is only 0.46%,and oral Puerarin,according to the animal or human pharmacokinetics studies,have shown that oral administration of Puerarin is rapidly absorbed from the gastrointestinal tract,but the rate of absorption is poor,and bioavailability is low,oral administration of a general 2-3 to 2-3 weeks or even months to become effective,thus affecting the clinical efficacy of its oral administration.Therefore, pharmaceutical researchers extracted Puerarin monomer,made of puerarin injection preparations,for the adjuvant treatment of clinical coronary heart disease,angina, myocardial infarction,retinal artery and vein occlusion,sudden deafness and ischemic cerebrovascular disease,viral myocarditis,diabetes among children and so on.Active ingredient of Puerarin injection that is effective,quality,stability,a wide range of clinical application,and as a representive Chinese medicines prepared in the Western system.
Puerarin injection,applied widely in clinic,lead to serious adverse reactions such as acute intravascular hemolysis,which is the main clinical symptom.What is worse,it is acute symptom,and fatal in some serious cases as it progresses quickly.According to statistics,during the days from 1~(st) January,2003 to 30~(th) June,2005,the puerarin injection adverse reaction cases report a total of 1006 cases,of which 30 cases is serious adverse reactions,11 cases of death.Serious adverse reactions to the main acute intravascular hemolysis,a total of 18 cases,eight cases of death,accounting for 73%.Deaths caused by Puerarin adverse reactions accounted for 73%in all fatal cases.
At present,it is reported that puerarin injection intravascular hemolysis is caused by typeⅡallergy,but the experimental data are only individual cases of clinical research findings,which could not show whether oral administration of puerarin is the reason of intravascular hemolysis,and fail to find clinical rational drug use guidelines to prevent an allergic reaction.Therefore,its security is comprehensively suspected by concerned departments and medical workers,clinicians should keep a serious eye on puerarin injection, resulting in a dramatic shrinkage of puerarin production,the of the whole Puerarin, including its cultivation,processing,extraction,preparation stagnant industry chain.As the result to this section,the incident negatively affects the safety of traditional Chinese medicine injection of a great.
Therefore,it is of great sense to engage in further study of mechanism of puerarin injection adverse reaction,the establishment of expounded puerarin injection-induced acute causes of intravascular hemolysis,finding solution of its adverse reactions.
In this paper,reference to the method of "Chinese Pharmacopoeia" version 2,2005 Hemolytic inspection,puerarin was studied in puerarin injection applied for 24 Beagle dogs occasional red blood cell hemolysis in vitro experiments,results were as follows:there is no hemolytic with propylene glycol concentration of 3.3-16.7 mg/ml and the concentration of puerarin 0.33-1.67mg/ml for 3 hours' observation.The experiment suggests that pre-clinical studies prompted current experimental methods of evaluation of hemolysis can not predict the clinical sporadic hemolytic as puerarin injection adverse reactions, highlighting disadvantage of the current pre-clinical safety evaluation of hemolytic,it need to be put under further studies,and to improved methods as well.
In this paper,through a systematic study of puerarin injection adverse reactions hemolytic research papers,reference to "Chinese Pharmacopoeia" version 2,2005 Hemolytic test,by increasing the drug concentration to the propylene glycol concentration of 100mg/ml,puerarin concentration of 10mg/ml,puerarin can increase the incidence of hemolysis,and in the 24 individual animals experiment hemolysis can be observed in cases of puerarin injection with addition of propylene glycol.Therefore the establishment of sporadic hemolytic evaluation of puerarin in traditional Chinese medicine injection after that,and applied to explore the mechanism acute intravascular haemolytic adverse reactions due to puerarin injection.The results showed that:propylene glycol concentration of 100mg/ml and then four cases of hemolysis were found,and hemolytic rate was 16.6%. there was a significant difference(0.05＞P＞0.01) with the saline group,but the hemolysis rate and the concentration of propylene glycol do not exist any correlation(P＞0.05); puerarin injection group including propylene glycol concentration of 100 mg/ml,and concentration of 10mg/ml Puerarin,the emergence of 16 cases of hemolysis led to 66.7%as hemolytic rate,there are significant differences(P＜0.01)with the saline group,hemolysis rate and the concentration of Puerarin does not have significant correlation(P＜0.01), correlation coefficient r=0.9523.Factorial analysis in Puerarin injection of propylene glycol solution and sporadic hemolytic evaluation of test results showed that:puerarin injection group hemolytic probability is significantly higher than the probability of occurrence hemolytic propanediol group(P＜0.01).It suggests:propylene glycol as the accessories,although the risk of causing hemolysis,is not a major factor.Adverse reactions caused by hemolysis is Puerarin itself as major factor.The mechanism of hemolytic may be that Puerarin embedded with the double red blood cell membrane phospholipids, erythrocyte membrane caused by decreasing liquidity and a decline in red blood cell deformability.
Literature study found that taurine enhanced the mobility of erythrocyte membrane and maintain its stability,as a result for protectiing of red cell membrane and preventing the role of hemolysis.In this study,through sporadic hemolytic evaluation methods in the injection of Chinese medicine.Distilled water for injection,saline and puerarin injection as a control study,and while concentration of puerarin is in the same,anti-hemolytic effect of puerarin with taurine was evaluated by adding different concentrations of taurine,which was observed within 24 hours for red blood cells status.The results showed that:puerarin injection with high-dose group,compound puerarin injectionⅠ,Ⅱ,Ⅲin each group had no hemolysis,compound puerarin injection ofⅠ,Ⅱ,Ⅲin each group,compared with hemolytic Puerarin high-dose injection group,were significant difference(P＜0.01); Compared with the saline group,compound puerarin injection ofⅠ,Ⅱ,Ⅲthe incidence of hemolysis in each group with no difference(P=1.00).Compound puerarin injectionⅠ,Ⅱ,Ⅲpuerarin injection group and the high-dose group contained 10mg/ml Puerarin; compound puerarin injection ofⅠ,Ⅱ,Ⅲ,respectively,each group contains 5mg/ml, 10mg/ml,20mg/ml taurine,and high-dose group of puerarin injection containing 100mg/ml propylene glycol,and puerarin injection hemolytic rate of high-dose group and 79.2%, significantly higher than that of compound puerarin injection ofⅠ,Ⅱ,Ⅲ.The results showed that:different concentrations of taurine existing in Puerarin,can be antagonistic by puerarin-induced hemolysis and the incidence can be reduced to the level of normal as saline group.It suggested that through combination theory under the direction of traditional Chinese medicine,Chinese medicine Puerarin monomer component,which can produce adverse reactions combined with taurine,compound puerarin injection to eliminate a separate application Puerarin adverse reactions.
In this paper,preparation of compound Puerarin including accessories molding process selection,dosage,pH value and freeze-drying process is tested to determine the compound injection of puerarin injection for preparation:Puerarin 5g,Taurine 25g,plus adequate water for injection,stirring allows the dissolved;adding mannitol for injection with sodium carbonate adjusted pH values to be 6-7,plus water for injection to 400ml, filter through 0.2μm microporous membrane,and each bottle of solution-resistant 2ml.Buy frozen oven,the pre-freezing temperature of -40℃,hydrazine cold temperature -60℃, heat 1h.Vacuum and then,when the vacuum reaches the following 10Pa after sublimation temperature and then to ensure that when the sample sublimation products lower eutectic temperature by 5℃,as be lower than 28℃,and in this case for 10 hours,until it product freeze until ice sublimation;shortly after the completion of the main drying-up,sample the end in the temperature 28℃,the thermal insulation box maintain for 1h.The above-mentioned process in small test of the batch yield of 90%in all batches,the appearance and re-dispersion are consistent with the provisions.
Although intravascular hemolysis acute adverse reactions caused by Puerarin can be the eliminated with co-exist with taurine,and then the safety of puerarin injection the original can be greatly enhanced,Puerarin combined with taurine,compound preparation of the new chemicals registered Category 1,in accordance with the "management of drug registration",it shall be carried out in a variety of components of compound preparation of the pharmacokinetics study of mutual influence.In order to compare the kinetic differences of Puerarin compound preparation combined with taurine and a separate application of each component.In order to explore the the rationality of combination of taurine and Puerarin.In this paper,the establishment of determination of puerarin,Taurine in plasma by HPLC,the compound of puerarin injection of two formulations in dogs Pharmacokinetics of interaction tests will be designed and confirmed.
In this study,the use of 6%perchloric acid pretreatment method of protein precipitation,with methanol -0.2%phosphoric acid as mobile phase,in 250nm,the concentration of puerarin were determined.Under such conditions of chromatography, puerarin in the scope of 1.01-60.8μg/ml has a good linear relationship,the method is of specificity,high sensitivity,accuracy and precision,which meet the demand determination of lowest plasma concentration of puerarin.
In this study,concentrations of taurine in serum is determinated by pre-treatment of pre-column derivatization method,using of 6%perchloric acid precipitated protein,2,4-dinitrofluorobenzene(DNFB),using methanol-pH5.5 sodium acetate,acetic acid buffer as the mobile phase,in 360nm measured serum.Carried out within 24 hours of Beagle dog serum study dynamic changes of taurine content and found that concentration of different taurine varied greatly different individuals Beagle dog serum,serum concentrations of taurine in the same Beagle dogs individual in 24h is a more stable.Therefore,the test blank serum is to justified as the taurine in the Beagle dog pharmacokinetics by reducing the basis value of taurine in Beagle dogs before administration of the concentration of taurine,.In the chromatographic conditions,the scope of taurine in 18.4-368.0μg/ml has a good linear relationship,the method is of specificity,high sensitivity,accuracy and precision,which meets the determination of plasma concentration of taurine requirements.
With application of established methods,pharmacokinetic study is carried out in six Beagle dogs after injection with a single dose of intravenous injection of compound puerarin,puerarin injection,of taurine.Test results show that intravenous injection of puerarin injection,taurine injection and the injection of compound after administration, puerarin and taurine in the disposal of dogs are in line with the two-compartment model.A single dose of intravenous injection of puerarin injection,the dogs Puerarin t1/2α=3.25±1.76min,t1/2β=55.5±4.78min,AUC0-t=2364.93±158.45μg·min/ml;a single dose of intravenous injection of puerarin Su-taurine injection,the dogs taurine t1/2α=11.31±4.15min,t1/2β=177.8±228.73h,AUC0-t=10361.69±1868.41μg·min/ml;a single dose of intravenous injection of compound Puerarin preparations,the dogs Puerarin t1/2α=3.73±0.55min,t1/2β=56.0±2.70min,AUC0-t=2489.57±115.83μg·min/ml,taurine t1/2αdogs=12.34±8.28min,t1/2β=201.0±166.46min,AUC0-t=11811.21±3771.71μg·min /ml.
Paired t test is applied to analyses the data a single dose of intravenous injection of puerarin and compound puerarin injection incuding 1/2α,t1/2β,V(c),Cl(s),AUC, AUC0-∞,AUC0-t,MRT0-∞,MRT0-t test.Results showed that:taurine and Puerarin compound Puerarin,combined with taurine were no significant differences with separate taurine pharmacokinetics(P＞0.05).Paired t test the application of a single dose of intravenous injection of puerarin and puerarin injection compound taurine t1/2α,t1/2β,V (c),Cl(s),AUC,AUC0-∞,AUC0-t,MRT0-∞,MRT0-t test.Results showed that: Compound Puerarin,combined with taurine,there is no significant difference(P＞0.05) between puerarin process and separate the process of drug pharmacokinetics after the pharmacokinetics of puerarin.It suggested that Puerarin and taurine compound preparation of the composition after injection and pharmacokinetic characteristics of taurine did not change the application of two non-distribution of Ng interaction pharmacokinetics,which provides the basis for the group's rationality and security.
In order to explore interaction effects on the effects of Abnormal heart rate test in mice induced by Chloroform,both before and after Puerarin combined with taurine.Result shows that:compared with the saline control group,both propranolol group and compound puerarin of high-dose injection could significantly delay the emergence of arrhythmia in mice(P＜0.01);Compared with the control drug propranolol,compared puerarin injection low,middle and high dosing mice appear abnormal heart rate of time and close to propranolol group,no significant statistic difference(P＞0.05),prompted puerarin injection of curing heart disorders compound chloroform-induced effects in mice is similar to propranolol;and Compound dosing of puerarin injection group,the split Puerarin ingredients,taurine against chloroform-induced disorders in mice the effect of heart rate are less than that of compound,but no statistic significant difference(P＞0.05).
By testing effect of pituitrin-induced myocardial ischemia in rats of taurine,to explore mutual influence before and after the combined puerarin.It was found that pituitrin-induced model of acute myocardial ischemia control group of rats,with the biological function of the system analysis of ECG records,mostly manifested as ST-segment elevation,heart rate, T-wave high-rise,individual low-or T-wave inverted,and the majority of a serious cardiac disorders(room or rooms as early as early or intermittent cardiac arrest induced bigeminy, such as triple law,individual or even ventricular tachycardia).Compound to give a certain dose of puerarin injection,the control group and model comparison,can significantly improve the pituitrin-induced myocardial ischemia in rats with acute manifestations,such as reduced st segment,T wave inversion,serious arrhythmia the number of cases that occurred were reduced,serum enzymes CK,LDH-L,AST release was also markedly reduced.These results indicate that a certain compound puerarin injection of anti-myocardial ischemia,arrhythmias.
In this study evaluation methods of sporadic hemolytic in injection made of Chinese medicine and found that taurine could antagonize Puerarin hemolytic adverse reactions.It suggested the active ingredient group Puerarin side to eliminate adverse effects of hemolysis,the combination of taurine and Puerarin,made compound puerarin injection without adverse reactions hemolytic.Through the preparation of compound forming process Puerarin research,resolve compatibility stability of products made of taurine and Puerarin,as preparation compound puerarin injection powder.In pharmacokinetics and pharmacodynamics research,Puerarin,both before and after combined with taurine Puerarin taurine on pharmacokinetics,efficacy evaluation of the impact and found Puerarin compatibility with taurine does not affect their pharmacokinetic process and composition of the two groups to have a certain synergy,suggesting that can provide a reasonable scientific basis for the two can be combined for the group.
引文
[1]国家药典委员会,中华人民共和国药典2005年版(一部)[M].北京:化学工业出版社,2005,第一版:233-234.
[2]Junei K,Junichi F,Junko C.Studies on the constituents of Pueraria lobata Ⅲ.Isoflavonoids and related compounds in the roots and the voluble stems[J].Chem.Pharm Bull,1987;35(12):4846-4850.
[3]kaz0hiro Hirakura,Makoto Morita,Kaoru Nakajima,et al.Phenolic glucosides from the root of Pueraria lobala[J].Phytochemistry,1997;46(5):92.
[4]Koichi Takeya,Hideji Itokawa.Isoflavonoids and the other constituents in callus tissues of pueraria lobala[J].Chem.Pharm Bull,1982;30(4):1496.
[5]Jun Mira.Antioxidative,anticancer and antiinflammatory properties of isoflavones from kudzu(Pueraria lobata) root[D].Rutgers The State University of New Jersey-New Brunswick,2003.
[6]Hirakura Kazuhiro,Morita Makoto,Nakajima Kaoru.Phenolic glucosides from the root of Pueraria lobata[J].Phytochemistry,1997;(5):921-928.
[7]Kinjo J E,Takeshita T,Abe Y.Studies on the constituents of Pueraria lobata Ⅵ.Chemical constituents in the flower and leaves[J].Chem.Pharm Bull,1988;36(3):1174-1179.
[8]徐礼燊,刘爱茹,张秀琴.葛根中总黄酮的导数脉冲极谱测定[J].药学学报,1987;22(3):208.
[9]章育中,杨凡.高效液相色谱法测定葛根及其片剂中异黄酮的含量[J].药物分析杂志,1984;4(2):67.
[10]赵世萍,章育中.葛根中异黄酮含量的薄层光密度法测定[J].药学学报,1985;20(3):203-208.
[11]方起程,林茂,孙庆民,等.葛根黄酮的研究[J].中华医学杂志,1974,54(5):271.章育中,杨凡.高效液相色谱法测定葛根及其片剂中异黄酮的含量[J].药物分析杂
[12]Kinjo.J.et al.:Oleanene sapogenols from puerariae Radix.Chem.Pharm.Bull[J].
[1 3]尺登志雄,他.东洋药物0の学的研究,葛根の成分[J].医学中央杂志,1973:296(5):133.
[14]Kinjo.J.et al."Two novel aromatic glycosides pueroside-A and - B,from puerariae Radix[J].retrahedron hett,1985;26(49):6101.
[1 5]陈妙华,等.葛根化学成分的研究[J].中药通报,1985:10(6):34.
[16]左春旭,等.葛根非黄酮成分[J].中草药,1987:18(11):10.
[17]#12
[18]柴田乘二,村上孝夫.和汉药成分の研究葛根成分の化分的研究[J].药学杂志,1959;79(6):757.
[19]范礼理,赵德化,赵敏崎,等.葛根黄酮抗心律失常作用[J].药学学报,1985;(09)647-651.
[20]柴象枢.葛根素抗实验性心律失常的研究[J].中国药理学报,1985;6(3):166.
[21]吕欣然,陈淑梅,孙塘.葛根对β-肾上腺素能受体阻滞作用的研究[J].药学学报,1980;15(4):218-220.
[22]吕宝璋,高尔,单京瑞,等.葛根素对β肾上腺能受体结合作用和腺苷酸环化酶活性的影响[J].解放军医学杂志,1985;10(2):97-100.
[23]苗维纳,沈映君,曾晓荣,等.葛根素对豚鼠心肌细胞动作电位及有效不应期的影响[J].中国药理学通报.2001;17(5):565-569.
[24]苗维纳,沈映君,曾晓荣,等.葛根素对豚鼠心室肌细胞钾离子通道的影响[J].中国应用生理学杂志.2002;18(2):155-156.
[25]曾贵云.葛根的药理研究:Ⅰ葛根对全血压、血管反应性、脑循环及外周循环作用研究[J].中华医学杂志,1974;54(5):265.
[26]周远鹏.葛根药物作用研究:Ⅲ葛根黄酮对急性心肌梗塞犬正常区和梗塞区心肌血流河代谢的作用[J].中华医学杂志,1974;54(5):550.
[27]范礼理,等葛根的药理研究Ⅱ.葛根黄酮对犬冠脉循环、心脏血流动力学和心肌代谢的作用[J].中华医学杂志 1975:55(10):724.
[28]范礼理,D D O'Keefe,W J Powell.葛根素对急性心肌缺血狗区域性心肌血流 与心脏血流动力学的作用[J].药学学报,1984;19(11):801-807.
[29]刘启功,王琳,陆再英,等.葛根素抗心肌缺血及其机理的实验研究[J].临床心血管病杂志,1998;14(5):292-295.
[30]李小鹰,王培仁,邵建华,等.静脉注射葛根素对犬急性心肌梗塞范围的影响[J].山东医学院学报,1984;22(3):9-17.
[31]董士勤,王杰,于恒锡,等.含葛根素冷钾停搏液对低温全心缺血心肌保护作用的电镜观察[J].首都医学院学报,1992;13(3):190.
[32]吉宏龙,王天佑.葛根素抑制大鼠背根神经节细胞河豚毒素不敏感性钠电流[J].中国药理学报,1996;17(2):115.
[33]张秀芹,侯金玲,朱燕,等.葛根素葡萄糖注射对犬实验性心肌缺血的影响[J].中国新药杂志,2001;10(4):272-274.
[34]俞坚武,屈百鸣,赵仲生,等.葛根素对儿茶酚胺诱导大鼠心肌损伤时凋亡相关蛋白的影响[J].中国中西医结合急救杂志,2002;9(1):30-32.
[35]丁殿勋,等.葛根素对急性心肌梗塞时早期与再灌注时前列腺素类物质释放影响的实验研究[J].中国人民解放军进修学院学报 1987;8(4):351.
[36]曾贵云.葛根的药理研究Ⅳ.葛根黄酮对高血压及冠心病患者血浆儿茶酚胺含量的影响[J].中华医学杂志,1979;8(8):479-482.
[37]姜秀莲,徐理纳.葛根素对小鼠实验性微循环障碍的改善作用[J].药学学报,1989;24(4):251-254.
[38]吕欣然,康白,陈维宁,等.葛根素对肾性高血压和血浆肾素活性(PRA)的影响[J].潍坊医学院学报,1985;16(2):9-14.
[39]孙文才,周和平.葛根素对自发性高血压大鼠肾血管核因子-κB表达的影响[J].中国中医急症,2008;17(7):965-966.
[40]陈沪生,王培仁,邵建华,等.葛根素的降压效果及其机理的研究[J].山东医科大学学报,1987;25(3):28.
[41]刘文奇,常志文,卢玲巧,等.葛根素对心肌缺血再灌注损伤的影响[J].中国药学杂志,2000;35(9):642.
[42]常志文,刘琦.缺血再灌注心肌细胞凋亡及药物干预[J].中国新药杂志,2000;9(1):28-30.
[43]朱智彤,姚智,李会强,等.葛根素对乳鼠心肌细胞缺氧-复氧时脂质过氧化损伤的保护作用[J].中草药,2002;33(4):343-346.
[44]陈江斌,许家俐,李建军,等.葛根素对冠心病患者纤溶功能及过氧环脂质的影响[J].中国中西结合杂志,1999;19(11):649-650.
[45]曾小楠,樊光辉,陈宏.葛根素对陈旧性心肌梗死患者血浆心纳素和超氧化物歧化酶的影响[J].临床心血管病杂志,2000;16(10):470-471.
[46]禹志领,张广钦,赵红旗,等.葛根总黄酮对血液粘度、血栓形成及血小板功能的影响[J].中药材,1997;20(9):468.
[47]罗助荣,郑冰.葛根素对不稳定心绞痛患者血小板颗粒膜蛋白、溶酶体膜蛋白及血浆纤溶酶原激活物抑制物、C反应蛋白的影响[J].中国中西医结合杂志,2001,21(1):31-33.
[48]白智峰,成蓓,李大强.葛根素对心房纤颤患者血小板活化和内皮功能的影响[J].河南诊断与治疗杂志,2003;17(2):96-97.
[49]申竹芳,谢明智.葛根素与阿斯匹林复方的降血糖作用[J].药学学报,1985;(11)863-865.
[50]郑琳颖,李博萍,潘竞锵,等.葛根降低D-gal诱导大鼠血糖和改善糖耐量减退的作用[J]中医药临床杂志,2005;17(04)358-359.
[51]韩瑚.葛根素注射液治疗糖尿病36例[J].广州医学院学报,2000;28(03)75-76.
[52]孙世锡,几种中药解热作用之药理研究[J].中华医学杂志,1956;42:964-968.
[53]丹野与三太.葛根の解热作用に就きこ[J].日本药物学杂志,1941;33(3):263.
[53]黄胜光.葛根治疗血管神经性头痛[J].中医杂志,1999.40;(4):197-198.
[54]黄胜光.葛根治疗血管神经性头痛[J].中医杂志,1999.40;(4):197-198.
[55]陈慧.葛根治疗血管性头痛和突发性耳聋[J].中医药管理杂志,2006;14(1):21.
[56]柴艳霞,杨风敏.葛根治疗视网膜动脉阻塞的疗效观察[J].河北中西医结合杂志,1996;5(2):76-77.
[57]董凤增.葛根合剂治疗突发性耳聋的临床报道[J].中国医药学报,1993;8(3):22-23.
[58]辛军,王建寰.大剂量葛根治疗颈椎病80例体会[J].宁夏医学杂志,2008;30(9):857.
[59]刘德山,柴青,高伟.葛根对糖尿病患者神经电生理的影响[J].实用中医药杂志,1997;13(3):24-25.
[60]段有金,王韶颖,三轮一智,等.五种中药对蛋白质非酶糖基化的抑制作用[J].中国糖尿病杂志,1998;6(4):227-229.
[61]李川云.改变不良生活方式对心血管疾病的防治作用[J].中国医药指南,2009;7(2)33-34.
[62]朱秀媛,苏成业,李振华,等.葛根有效成分的代谢研究Ⅲ-葛根素的代谢及其药代动力学分析[J].药学学报,1979;14(6)349-355.
[63]Yasuda T,et al.Urinart and Biliary Metabolites of Puerarin in Rats[J].Biol Pharm Bull.1995,18(2):300-303.
[64]张志荣,游学均,魏振平,等.愈风宁心胶囊在兔体内的药动学和生物利用度研[J]中国药学杂志,1997;32(4):224.
[65]吴燕红,苏子仁,赖小平,等.愈风宁心片中葛根素在小鼠体内的药动学和生物利用度研究[J]中药新药与临床药理,2004;15(4)259-261).
[66]刘玉兰,王世文,颜鸣,等.葛根黄豆苷元固体分散物对自发性高血压大鼠血压的影响[J].沈阳药学院学报,1991.8(2):105.
[67]田景振,魏凤环.葛根黄酮缓释胶囊家兔体内药代动力学研究[J].山东中医药大学学报,2003;27(6)454-457.
[68]刘振威,等.磷脂对大鼠离体肠管吸收葛根素的影响[J].潍坊医学院学报1998;20(4):253-254.
[69]向大雄,李焕德,罗杰英,等.葛根总黄酮生物黏附性缓释片在家犬体内的药动学及生物利用度研究[J].中国药学杂志,2005;4(7)532-535.
[70]王雪莉,邢东明,盖国胜,等.微细化工艺制备的葛根微粉溶出度和生物利用度的研究[J].中国药学杂志;2004;39(4)283-286.
[71]朱秀媛,苏成业,李振华,等.葛根有效成分的代谢研究Ⅲ-葛根素的代谢及其药代动力学分析[J].药学学报,1979;14(6)349-355.
[72]金昔陆,等.葛根素在大鼠、家兔、犬中的药物动力学[J].中国药理学报1992;13(3):284-288.
[73]金昔陆,等.血浆中葛根素的高效液相色谱测定法及其在狗体内的药代动力学[J].药学学报,1997;32(10):782-785.
[74]金昔陆,朱秀媛,等.葛根素在三种动物的药代动力学参数与体重的相关性.徐州医学院学报,1991;11(1)25-26.
[75]金昔陆,程桂芳,朱秀媛,等.葛根素在健康志愿者的药代动力学[J].中国临床药理学杂志,1991;7(2):115-118.
[76]陶忠华.新药葛根紊的研究[J]中国新药杂志,1993;2(6):1-3.
[77]李妮,路玫,陈晖,等.葛根素在糖尿病肾病病人中的药动学[J].中国新药与临床杂志,2002;21(1):51-53.
[78]炎彬,孙虹,何希辉,等.葛根黄酮及其葛根素在正常和脑缺血再灌大鼠体内药动学比较[J].中国药学杂志,2004;39(3):128-132.
[79]李敏,等.中药有效部位复方CBN药代动力学研究[J].中药新药与临床1999;15(4):38-40.
[80]黄雪梅,等.小儿热感宁注射液在兔体内的药代动力学研究[J].中国药房,2001;12(10):584-585.
[81]吴正红,等.葛根素滴眼液眼内药代动力学的研究.中国药科大学学报1998:29(3):221-224.
[82]王庆端,江金花,孙文欣,等.葛根总黄酮的急性毒性及长期毒性实验[J].河南医科大学学报,1999;34(02):48-51.
[83]中国医学科学院药物研究所·中草药现代研究[M].北京:北京医科大学、中国协和医科大学联合出版社,1995:258-268·
[84]史纪兰,黄权光,商希梅,等.葛根素致突变、致畸作用评价[J].中国药理学与毒理学杂志,1992;6(3):223.
[85]金涛.普乐宁引起药物热7例[J].中国现代应用药学,1996;13(04):62.
[86]胡姣娣.葛根素致药物热的特点及护理[J].浙江临床医学,2002;2(1):71.
[87]邵华,袁高辉.静滴葛根素注射液致迟发高热反应23例分析[J].中国中药杂志,2002;27(6):478.
[88]王健,徐灵,杨茵.葛根素注射液引起发热的临床统计[J].西北药学杂志,2003;18(1):29-30.
[89]张峻.190例葛根素注射液不良反应分析[J].广州医药,2003;34(5):55-56.
[90]杨金玉.葛根素注射液不良反应分析[J].辽宁药物与临床,2002;5(4):212-213.
[91]常景梅.普乐林致一过性血红蛋白尿一例[J].中华流行病学杂志,1998;19(06):360.
[92]关颖卓.葛根素注射液致急性溶血性贫血合并肝、肾损害[J].药物不良反应杂志,2004;6(6):413-414.
[93]徐芳,何可男.葛根素罕见的严重不良反应[J].中国中医结合杂志,2003;23(5):362.
[94]张赞玲,尹桃.葛根素注射液引起死亡3例[J].药物不良反应杂志,2004;6(1):41-42.
[95]李奎运,衣淑华.普乐林致过敏性皮疹1例报告[J].药学实践杂志,1998;16(3):175.
[96]王凤,韩红杰,刘万群,等.葛根素注射液静滴致过敏反应4例[J].药物不良反应杂志,2001;3(4):253-254.
[97]李淑娥,何绥平.普乐林注射液引起过敏反应5例[J].药物流行病学杂志,1997;6(4):251.
[98]王焱,陈炜,王永志.普乐林静滴致休克反应3例[J].中国药学杂志,2000;35(5):353.
[99]丁庆明,徐小华.葛根素粉针剂致阵颤l例[J].中国医院药学杂志,2004;24(3)1 90.
[100]杨凤英.葛根素致静脉炎1例[J].中国社区医师,2005;18(7):59.
[101]董胜英,杨军平.葛根素注射液过敏致溶血性黄疽1例[J].实用中西医结合临床,2004;4(1):65.
[102]黄雪融,郑荣远葛根素注射液十年致热不良事件600例综合分析[J].药物流行病学杂志,2004;13(2)72-75.
[103]邓培媛,李群娜,朱玉珍,等.葛根素注射剂不良反应及其影响因素分析[J].药物流行病学杂志,2005;14(1)14-19.
[104]朱玉珍,汪波.马明信.葛根素中毒的实验室诊断及致病机制(附四例实验报告)[J].北京医学,2003;25(6)389-392.
[105]朱玉珍,汪波,马明信.葛根素引起急性溶血和肾功能衰竭的致病机制探讨[J].中国新药与临床杂志,2003;22(11):699-702.
[106]李梅,杨柳,黄伟强,等.不同厂家葛根素注射液溶血实验比较[J].广西医科大学学报,2001;18(4)520.
[107]Tiedemann F,Gmelin L.Einige neue bestandtheile der galle des ochsen.Ann Physik Chen[J].1827;9:326.
[108]宋春梅,马洪波.牛磺酸的生物学作用[J].吉林医药学院学报,2005;26(04)261-265.
[109]MadsenS,OttersenOP,Storm-Mathisen J,el al.Immunocytochemical localization of taurine:methodological aspects.[J].Prog Clin Biol Res,1990;351:37-44.
[110]Kendler Bs et al.Taurine:An overview of its role in preventive medi-cine[J].preventmcd.1989;18:79.
[111]TrenknerE.The role of taurine and glutamate during early Postnatal cerebellar development of normal and weaver mutant mice[J].Adv Exp Med Bio1,1990;268:239-244.
[112]Pasantes-Morales H,Moran J,Schousboe A.Volume-sensitive release of taurine from cuItured astrocytes:properties and mechanism[J].Glia,1990;3(5):427-432.
[113]Rivas-Arancibia S,Dorado-Matrinez C,Borgonio-Perez G,et al.Eefcts of taurine on ozone-induced memory deifcits and liPid Peroxldation levels in brains of young,mature,and old rats[J].Environ Res,2000,82(1):7-17.
[114]Khan SH,Banigesh A,Baziani A,et al.The role of taurine in neuronal Protection of following transient global forebrain ischemia[J].Neurochem Res,2000,25(2):217-223.
[115]Valeev AE,Garashchuk OA,Dashkin AN.Taurine-activated curents in isolated neurons of the rat cerebellum[J].Neiroifziologiia,1990;22(6):780-286.
[116]Lake N.Loss of cardiac myoifbrlls:mechanism of contractile deifcits induced by taurine deifciency[J].Am J Physiol,1993;264(4):1323-1326.
[117]Chahine R,Feng J.Protective elects of taurine against rePerufsion-induced arrhythmias in isolated ischemic rat heart[J].Arzneimlttelofrschung,1998;48(4):360-364.
[118]SethuPathy S,Elanchezhiyan C,Vasudevan K,et al.Antiatherogenic erfect of taurine in high fat diet fed rats.Indian J Exp Biol,2002;40(10):11691172.
[119]蔡长清,陶秋萍,陈渝军.牛磺酸对小儿呼吸道感染解热作用观察[J].医药导报,2001;20(3):185.
[120]李培峰,关红,白音吉日嘎拉等.鸡胆汁有效成分Tau和Ca的解热镇痛作用 研究[J].中兽医医药杂志,2003;2:3-5.
[121]Nakamori K,et al.Effectiveness of taurine in protectiong biomembrane against oxidant.Chem Pharm Bull(Tokyo),1990;38(11):3116
[122]Lin YY,Wright CE,Zagorski M,et al.13C-NMR study of taurine and chlorotaurine in human cells[J].Biochim Biophys Acta.1988;969(3):242-248.
[123]窦学术,杨军,唐朝枢.牛磺酸对小鼠电离辐射损伤的防护探讨[J]中国辐射卫生,1995,4(01)58-59.
[124]周宝宽,林静,于肯明,等.牛磺酸膜稳定作用的实验研究[J]药理学通报1997;13(3):227-228.
[125]张秀珍,沈芳兰,王建华,等.牛磺酸对细胞膜脂流动性的影响[J].青岛医学院学报1996;32(2)106-108.
[126]门秀丽,张连元,赵利军,等.牛磺酸对肢体缺血再灌注后红细胞的保护作用[J].华北煤炭医学院学报,2002;4(2)137-138.
[127]周莉玲,李锐,苏子仁,等.牛磺酸制剂体内药动学的研究[J].中成药研究,1987;(7)1-3.
[128]郭丽仪.薄层扫描法测定人体血液中牛磺酸的含量[J].中草药;2001,32(3)217-218.
[129]钱广生,苏旭,刘三康,等.柱前衍生化-HPLC测定田参氨基酸胶囊中10种氨基酸的含量[J].华西药学杂志,2005;20(2):145-147.
[130]吴月瑛.中药注射剂的不良反应及预防措施[J].现代中西医结合杂志,2008;17(27):4308.
[131]张雅杰,高敬茹,孙艳春,等.中药注射剂不良反应原因剖析与防范[J].黑龙江医药科学,2008;31(8):63-64.
[133]Ollila F,Halling K,Vuorela P,et al.Charactererization of flaconoid-biomenmbrane interaction[J].Arch Biochem Biophys,2002;339(1) 103-108.
[134]Pawlikowska-Pawlega B,Gruszecki WI,Misiak L,et al.The study of the quercetin action on human erythrocyte membranes[J].Biochem Pharmacol,2003;66(4):605-612.
[135]刘蔚,程丽静,温晓旭,等.葛根及刺五加提取物对正常小鼠血液流变学作用的比较[J],广州中医药大学学报,2007;24(3):138-141.
[136]宋学歧,于庆潭.葛根素对潜艇人员体外红细胞变形性和聚集性的影响[J],中华航海医学与高气压医学杂志,2005;12(3)168.
[137]中华人民共和国药典委员会.中华人民共和国药典2005年版二部[M].北京:化学工业出版社,附录:118.
[138]高天,王文莉.葛根素注射液不良反应分析[J]中国中医药信息杂志,2002;10,9(10):74.
[139]侯惠民等.药用辅料应用技术[M]上海:中国医药科技出版社,2002:32.
[140]刘翎.葛根素注射液不良反应及相关因素分析[J]中国药物与临床,2004;4(2):159.
[141]金昔陆,朱秀媛.葛根素在大鼠、家兔、犬中的药物动力学[J].中国药理学报,1992:13(5):284.
[142]金昔陆,朱秀媛.葛根素在三种动物的药代动力学参数与体重的相关性[J].徐州医学院学报,1991:11(1)25-26.
[2]Junei K,Junichi F,Junko C.Studies on the constituents of Pueraria lobata Ⅲ.Isoflavonoids and related compounds in the roots and the voluble stems[J].Chem.Pharm Bull,1987;35(12):4846-4850.
[3]kaz0hiro Hirakura,Makoto Morita,Kaoru Nakajima,et al.Phenolic glucosides from the root of Pueraria lobala[J].Phytochemistry,1997;46(5):92.
[4]Koichi Takeya,Hideji Itokawa.Isoflavonoids and the other constituents in callus tissues of pueraria lobala[J].Chem.Pharm Bull,1982;30(4):1496.
[5]Jun Mira.Antioxidative,anticancer and antiinflammatory properties of isoflavones from kudzu(Pueraria lobata) root[D].Rutgers The State University of New Jersey-New Brunswick,2003.
[6]Hirakura Kazuhiro,Morita Makoto,Nakajima Kaoru.Phenolic glucosides from the root of Pueraria lobata[J].Phytochemistry,1997;(5):921-928.
[7]Kinjo J E,Takeshita T,Abe Y.Studies on the constituents of Pueraria lobata Ⅵ.Chemical constituents in the flower and leaves[J].Chem.Pharm Bull,1988;36(3):1174-1179.
[8]徐礼燊,刘爱茹,张秀琴.葛根中总黄酮的导数脉冲极谱测定[J].药学学报,1987;22(3):208.
[9]章育中,杨凡.高效液相色谱法测定葛根及其片剂中异黄酮的含量[J].药物分析杂志,1984;4(2):67.
[10]赵世萍,章育中.葛根中异黄酮含量的薄层光密度法测定[J].药学学报,1985;20(3):203-208.
[11]方起程,林茂,孙庆民,等.葛根黄酮的研究[J].中华医学杂志,1974,54(5):271.章育中,杨凡.高效液相色谱法测定葛根及其片剂中异黄酮的含量[J].药物分析杂
[12]Kinjo.J.et al.:Oleanene sapogenols from puerariae Radix.Chem.Pharm.Bull[J].
[1 3]尺登志雄,他.东洋药物0の学的研究,葛根の成分[J].医学中央杂志,1973:296(5):133.
[14]Kinjo.J.et al."Two novel aromatic glycosides pueroside-A and - B,from puerariae Radix[J].retrahedron hett,1985;26(49):6101.
[1 5]陈妙华,等.葛根化学成分的研究[J].中药通报,1985:10(6):34.
[16]左春旭,等.葛根非黄酮成分[J].中草药,1987:18(11):10.
[17]#12
[18]柴田乘二,村上孝夫.和汉药成分の研究葛根成分の化分的研究[J].药学杂志,1959;79(6):757.
[19]范礼理,赵德化,赵敏崎,等.葛根黄酮抗心律失常作用[J].药学学报,1985;(09)647-651.
[20]柴象枢.葛根素抗实验性心律失常的研究[J].中国药理学报,1985;6(3):166.
[21]吕欣然,陈淑梅,孙塘.葛根对β-肾上腺素能受体阻滞作用的研究[J].药学学报,1980;15(4):218-220.
[22]吕宝璋,高尔,单京瑞,等.葛根素对β肾上腺能受体结合作用和腺苷酸环化酶活性的影响[J].解放军医学杂志,1985;10(2):97-100.
[23]苗维纳,沈映君,曾晓荣,等.葛根素对豚鼠心肌细胞动作电位及有效不应期的影响[J].中国药理学通报.2001;17(5):565-569.
[24]苗维纳,沈映君,曾晓荣,等.葛根素对豚鼠心室肌细胞钾离子通道的影响[J].中国应用生理学杂志.2002;18(2):155-156.
[25]曾贵云.葛根的药理研究:Ⅰ葛根对全血压、血管反应性、脑循环及外周循环作用研究[J].中华医学杂志,1974;54(5):265.
[26]周远鹏.葛根药物作用研究:Ⅲ葛根黄酮对急性心肌梗塞犬正常区和梗塞区心肌血流河代谢的作用[J].中华医学杂志,1974;54(5):550.
[27]范礼理,等葛根的药理研究Ⅱ.葛根黄酮对犬冠脉循环、心脏血流动力学和心肌代谢的作用[J].中华医学杂志 1975:55(10):724.
[28]范礼理,D D O'Keefe,W J Powell.葛根素对急性心肌缺血狗区域性心肌血流 与心脏血流动力学的作用[J].药学学报,1984;19(11):801-807.
[29]刘启功,王琳,陆再英,等.葛根素抗心肌缺血及其机理的实验研究[J].临床心血管病杂志,1998;14(5):292-295.
[30]李小鹰,王培仁,邵建华,等.静脉注射葛根素对犬急性心肌梗塞范围的影响[J].山东医学院学报,1984;22(3):9-17.
[31]董士勤,王杰,于恒锡,等.含葛根素冷钾停搏液对低温全心缺血心肌保护作用的电镜观察[J].首都医学院学报,1992;13(3):190.
[32]吉宏龙,王天佑.葛根素抑制大鼠背根神经节细胞河豚毒素不敏感性钠电流[J].中国药理学报,1996;17(2):115.
[33]张秀芹,侯金玲,朱燕,等.葛根素葡萄糖注射对犬实验性心肌缺血的影响[J].中国新药杂志,2001;10(4):272-274.
[34]俞坚武,屈百鸣,赵仲生,等.葛根素对儿茶酚胺诱导大鼠心肌损伤时凋亡相关蛋白的影响[J].中国中西医结合急救杂志,2002;9(1):30-32.
[35]丁殿勋,等.葛根素对急性心肌梗塞时早期与再灌注时前列腺素类物质释放影响的实验研究[J].中国人民解放军进修学院学报 1987;8(4):351.
[36]曾贵云.葛根的药理研究Ⅳ.葛根黄酮对高血压及冠心病患者血浆儿茶酚胺含量的影响[J].中华医学杂志,1979;8(8):479-482.
[37]姜秀莲,徐理纳.葛根素对小鼠实验性微循环障碍的改善作用[J].药学学报,1989;24(4):251-254.
[38]吕欣然,康白,陈维宁,等.葛根素对肾性高血压和血浆肾素活性(PRA)的影响[J].潍坊医学院学报,1985;16(2):9-14.
[39]孙文才,周和平.葛根素对自发性高血压大鼠肾血管核因子-κB表达的影响[J].中国中医急症,2008;17(7):965-966.
[40]陈沪生,王培仁,邵建华,等.葛根素的降压效果及其机理的研究[J].山东医科大学学报,1987;25(3):28.
[41]刘文奇,常志文,卢玲巧,等.葛根素对心肌缺血再灌注损伤的影响[J].中国药学杂志,2000;35(9):642.
[42]常志文,刘琦.缺血再灌注心肌细胞凋亡及药物干预[J].中国新药杂志,2000;9(1):28-30.
[43]朱智彤,姚智,李会强,等.葛根素对乳鼠心肌细胞缺氧-复氧时脂质过氧化损伤的保护作用[J].中草药,2002;33(4):343-346.
[44]陈江斌,许家俐,李建军,等.葛根素对冠心病患者纤溶功能及过氧环脂质的影响[J].中国中西结合杂志,1999;19(11):649-650.
[45]曾小楠,樊光辉,陈宏.葛根素对陈旧性心肌梗死患者血浆心纳素和超氧化物歧化酶的影响[J].临床心血管病杂志,2000;16(10):470-471.
[46]禹志领,张广钦,赵红旗,等.葛根总黄酮对血液粘度、血栓形成及血小板功能的影响[J].中药材,1997;20(9):468.
[47]罗助荣,郑冰.葛根素对不稳定心绞痛患者血小板颗粒膜蛋白、溶酶体膜蛋白及血浆纤溶酶原激活物抑制物、C反应蛋白的影响[J].中国中西医结合杂志,2001,21(1):31-33.
[48]白智峰,成蓓,李大强.葛根素对心房纤颤患者血小板活化和内皮功能的影响[J].河南诊断与治疗杂志,2003;17(2):96-97.
[49]申竹芳,谢明智.葛根素与阿斯匹林复方的降血糖作用[J].药学学报,1985;(11)863-865.
[50]郑琳颖,李博萍,潘竞锵,等.葛根降低D-gal诱导大鼠血糖和改善糖耐量减退的作用[J]中医药临床杂志,2005;17(04)358-359.
[51]韩瑚.葛根素注射液治疗糖尿病36例[J].广州医学院学报,2000;28(03)75-76.
[52]孙世锡,几种中药解热作用之药理研究[J].中华医学杂志,1956;42:964-968.
[53]丹野与三太.葛根の解热作用に就きこ[J].日本药物学杂志,1941;33(3):263.
[53]黄胜光.葛根治疗血管神经性头痛[J].中医杂志,1999.40;(4):197-198.
[54]黄胜光.葛根治疗血管神经性头痛[J].中医杂志,1999.40;(4):197-198.
[55]陈慧.葛根治疗血管性头痛和突发性耳聋[J].中医药管理杂志,2006;14(1):21.
[56]柴艳霞,杨风敏.葛根治疗视网膜动脉阻塞的疗效观察[J].河北中西医结合杂志,1996;5(2):76-77.
[57]董凤增.葛根合剂治疗突发性耳聋的临床报道[J].中国医药学报,1993;8(3):22-23.
[58]辛军,王建寰.大剂量葛根治疗颈椎病80例体会[J].宁夏医学杂志,2008;30(9):857.
[59]刘德山,柴青,高伟.葛根对糖尿病患者神经电生理的影响[J].实用中医药杂志,1997;13(3):24-25.
[60]段有金,王韶颖,三轮一智,等.五种中药对蛋白质非酶糖基化的抑制作用[J].中国糖尿病杂志,1998;6(4):227-229.
[61]李川云.改变不良生活方式对心血管疾病的防治作用[J].中国医药指南,2009;7(2)33-34.
[62]朱秀媛,苏成业,李振华,等.葛根有效成分的代谢研究Ⅲ-葛根素的代谢及其药代动力学分析[J].药学学报,1979;14(6)349-355.
[63]Yasuda T,et al.Urinart and Biliary Metabolites of Puerarin in Rats[J].Biol Pharm Bull.1995,18(2):300-303.
[64]张志荣,游学均,魏振平,等.愈风宁心胶囊在兔体内的药动学和生物利用度研[J]中国药学杂志,1997;32(4):224.
[65]吴燕红,苏子仁,赖小平,等.愈风宁心片中葛根素在小鼠体内的药动学和生物利用度研究[J]中药新药与临床药理,2004;15(4)259-261).
[66]刘玉兰,王世文,颜鸣,等.葛根黄豆苷元固体分散物对自发性高血压大鼠血压的影响[J].沈阳药学院学报,1991.8(2):105.
[67]田景振,魏凤环.葛根黄酮缓释胶囊家兔体内药代动力学研究[J].山东中医药大学学报,2003;27(6)454-457.
[68]刘振威,等.磷脂对大鼠离体肠管吸收葛根素的影响[J].潍坊医学院学报1998;20(4):253-254.
[69]向大雄,李焕德,罗杰英,等.葛根总黄酮生物黏附性缓释片在家犬体内的药动学及生物利用度研究[J].中国药学杂志,2005;4(7)532-535.
[70]王雪莉,邢东明,盖国胜,等.微细化工艺制备的葛根微粉溶出度和生物利用度的研究[J].中国药学杂志;2004;39(4)283-286.
[71]朱秀媛,苏成业,李振华,等.葛根有效成分的代谢研究Ⅲ-葛根素的代谢及其药代动力学分析[J].药学学报,1979;14(6)349-355.
[72]金昔陆,等.葛根素在大鼠、家兔、犬中的药物动力学[J].中国药理学报1992;13(3):284-288.
[73]金昔陆,等.血浆中葛根素的高效液相色谱测定法及其在狗体内的药代动力学[J].药学学报,1997;32(10):782-785.
[74]金昔陆,朱秀媛,等.葛根素在三种动物的药代动力学参数与体重的相关性.徐州医学院学报,1991;11(1)25-26.
[75]金昔陆,程桂芳,朱秀媛,等.葛根素在健康志愿者的药代动力学[J].中国临床药理学杂志,1991;7(2):115-118.
[76]陶忠华.新药葛根紊的研究[J]中国新药杂志,1993;2(6):1-3.
[77]李妮,路玫,陈晖,等.葛根素在糖尿病肾病病人中的药动学[J].中国新药与临床杂志,2002;21(1):51-53.
[78]炎彬,孙虹,何希辉,等.葛根黄酮及其葛根素在正常和脑缺血再灌大鼠体内药动学比较[J].中国药学杂志,2004;39(3):128-132.
[79]李敏,等.中药有效部位复方CBN药代动力学研究[J].中药新药与临床1999;15(4):38-40.
[80]黄雪梅,等.小儿热感宁注射液在兔体内的药代动力学研究[J].中国药房,2001;12(10):584-585.
[81]吴正红,等.葛根素滴眼液眼内药代动力学的研究.中国药科大学学报1998:29(3):221-224.
[82]王庆端,江金花,孙文欣,等.葛根总黄酮的急性毒性及长期毒性实验[J].河南医科大学学报,1999;34(02):48-51.
[83]中国医学科学院药物研究所·中草药现代研究[M].北京:北京医科大学、中国协和医科大学联合出版社,1995:258-268·
[84]史纪兰,黄权光,商希梅,等.葛根素致突变、致畸作用评价[J].中国药理学与毒理学杂志,1992;6(3):223.
[85]金涛.普乐宁引起药物热7例[J].中国现代应用药学,1996;13(04):62.
[86]胡姣娣.葛根素致药物热的特点及护理[J].浙江临床医学,2002;2(1):71.
[87]邵华,袁高辉.静滴葛根素注射液致迟发高热反应23例分析[J].中国中药杂志,2002;27(6):478.
[88]王健,徐灵,杨茵.葛根素注射液引起发热的临床统计[J].西北药学杂志,2003;18(1):29-30.
[89]张峻.190例葛根素注射液不良反应分析[J].广州医药,2003;34(5):55-56.
[90]杨金玉.葛根素注射液不良反应分析[J].辽宁药物与临床,2002;5(4):212-213.
[91]常景梅.普乐林致一过性血红蛋白尿一例[J].中华流行病学杂志,1998;19(06):360.
[92]关颖卓.葛根素注射液致急性溶血性贫血合并肝、肾损害[J].药物不良反应杂志,2004;6(6):413-414.
[93]徐芳,何可男.葛根素罕见的严重不良反应[J].中国中医结合杂志,2003;23(5):362.
[94]张赞玲,尹桃.葛根素注射液引起死亡3例[J].药物不良反应杂志,2004;6(1):41-42.
[95]李奎运,衣淑华.普乐林致过敏性皮疹1例报告[J].药学实践杂志,1998;16(3):175.
[96]王凤,韩红杰,刘万群,等.葛根素注射液静滴致过敏反应4例[J].药物不良反应杂志,2001;3(4):253-254.
[97]李淑娥,何绥平.普乐林注射液引起过敏反应5例[J].药物流行病学杂志,1997;6(4):251.
[98]王焱,陈炜,王永志.普乐林静滴致休克反应3例[J].中国药学杂志,2000;35(5):353.
[99]丁庆明,徐小华.葛根素粉针剂致阵颤l例[J].中国医院药学杂志,2004;24(3)1 90.
[100]杨凤英.葛根素致静脉炎1例[J].中国社区医师,2005;18(7):59.
[101]董胜英,杨军平.葛根素注射液过敏致溶血性黄疽1例[J].实用中西医结合临床,2004;4(1):65.
[102]黄雪融,郑荣远葛根素注射液十年致热不良事件600例综合分析[J].药物流行病学杂志,2004;13(2)72-75.
[103]邓培媛,李群娜,朱玉珍,等.葛根素注射剂不良反应及其影响因素分析[J].药物流行病学杂志,2005;14(1)14-19.
[104]朱玉珍,汪波.马明信.葛根素中毒的实验室诊断及致病机制(附四例实验报告)[J].北京医学,2003;25(6)389-392.
[105]朱玉珍,汪波,马明信.葛根素引起急性溶血和肾功能衰竭的致病机制探讨[J].中国新药与临床杂志,2003;22(11):699-702.
[106]李梅,杨柳,黄伟强,等.不同厂家葛根素注射液溶血实验比较[J].广西医科大学学报,2001;18(4)520.
[107]Tiedemann F,Gmelin L.Einige neue bestandtheile der galle des ochsen.Ann Physik Chen[J].1827;9:326.
[108]宋春梅,马洪波.牛磺酸的生物学作用[J].吉林医药学院学报,2005;26(04)261-265.
[109]MadsenS,OttersenOP,Storm-Mathisen J,el al.Immunocytochemical localization of taurine:methodological aspects.[J].Prog Clin Biol Res,1990;351:37-44.
[110]Kendler Bs et al.Taurine:An overview of its role in preventive medi-cine[J].preventmcd.1989;18:79.
[111]TrenknerE.The role of taurine and glutamate during early Postnatal cerebellar development of normal and weaver mutant mice[J].Adv Exp Med Bio1,1990;268:239-244.
[112]Pasantes-Morales H,Moran J,Schousboe A.Volume-sensitive release of taurine from cuItured astrocytes:properties and mechanism[J].Glia,1990;3(5):427-432.
[113]Rivas-Arancibia S,Dorado-Matrinez C,Borgonio-Perez G,et al.Eefcts of taurine on ozone-induced memory deifcits and liPid Peroxldation levels in brains of young,mature,and old rats[J].Environ Res,2000,82(1):7-17.
[114]Khan SH,Banigesh A,Baziani A,et al.The role of taurine in neuronal Protection of following transient global forebrain ischemia[J].Neurochem Res,2000,25(2):217-223.
[115]Valeev AE,Garashchuk OA,Dashkin AN.Taurine-activated curents in isolated neurons of the rat cerebellum[J].Neiroifziologiia,1990;22(6):780-286.
[116]Lake N.Loss of cardiac myoifbrlls:mechanism of contractile deifcits induced by taurine deifciency[J].Am J Physiol,1993;264(4):1323-1326.
[117]Chahine R,Feng J.Protective elects of taurine against rePerufsion-induced arrhythmias in isolated ischemic rat heart[J].Arzneimlttelofrschung,1998;48(4):360-364.
[118]SethuPathy S,Elanchezhiyan C,Vasudevan K,et al.Antiatherogenic erfect of taurine in high fat diet fed rats.Indian J Exp Biol,2002;40(10):11691172.
[119]蔡长清,陶秋萍,陈渝军.牛磺酸对小儿呼吸道感染解热作用观察[J].医药导报,2001;20(3):185.
[120]李培峰,关红,白音吉日嘎拉等.鸡胆汁有效成分Tau和Ca的解热镇痛作用 研究[J].中兽医医药杂志,2003;2:3-5.
[121]Nakamori K,et al.Effectiveness of taurine in protectiong biomembrane against oxidant.Chem Pharm Bull(Tokyo),1990;38(11):3116
[122]Lin YY,Wright CE,Zagorski M,et al.13C-NMR study of taurine and chlorotaurine in human cells[J].Biochim Biophys Acta.1988;969(3):242-248.
[123]窦学术,杨军,唐朝枢.牛磺酸对小鼠电离辐射损伤的防护探讨[J]中国辐射卫生,1995,4(01)58-59.
[124]周宝宽,林静,于肯明,等.牛磺酸膜稳定作用的实验研究[J]药理学通报1997;13(3):227-228.
[125]张秀珍,沈芳兰,王建华,等.牛磺酸对细胞膜脂流动性的影响[J].青岛医学院学报1996;32(2)106-108.
[126]门秀丽,张连元,赵利军,等.牛磺酸对肢体缺血再灌注后红细胞的保护作用[J].华北煤炭医学院学报,2002;4(2)137-138.
[127]周莉玲,李锐,苏子仁,等.牛磺酸制剂体内药动学的研究[J].中成药研究,1987;(7)1-3.
[128]郭丽仪.薄层扫描法测定人体血液中牛磺酸的含量[J].中草药;2001,32(3)217-218.
[129]钱广生,苏旭,刘三康,等.柱前衍生化-HPLC测定田参氨基酸胶囊中10种氨基酸的含量[J].华西药学杂志,2005;20(2):145-147.
[130]吴月瑛.中药注射剂的不良反应及预防措施[J].现代中西医结合杂志,2008;17(27):4308.
[131]张雅杰,高敬茹,孙艳春,等.中药注射剂不良反应原因剖析与防范[J].黑龙江医药科学,2008;31(8):63-64.
[133]Ollila F,Halling K,Vuorela P,et al.Charactererization of flaconoid-biomenmbrane interaction[J].Arch Biochem Biophys,2002;339(1) 103-108.
[134]Pawlikowska-Pawlega B,Gruszecki WI,Misiak L,et al.The study of the quercetin action on human erythrocyte membranes[J].Biochem Pharmacol,2003;66(4):605-612.
[135]刘蔚,程丽静,温晓旭,等.葛根及刺五加提取物对正常小鼠血液流变学作用的比较[J],广州中医药大学学报,2007;24(3):138-141.
[136]宋学歧,于庆潭.葛根素对潜艇人员体外红细胞变形性和聚集性的影响[J],中华航海医学与高气压医学杂志,2005;12(3)168.
[137]中华人民共和国药典委员会.中华人民共和国药典2005年版二部[M].北京:化学工业出版社,附录:118.
[138]高天,王文莉.葛根素注射液不良反应分析[J]中国中医药信息杂志,2002;10,9(10):74.
[139]侯惠民等.药用辅料应用技术[M]上海:中国医药科技出版社,2002:32.
[140]刘翎.葛根素注射液不良反应及相关因素分析[J]中国药物与临床,2004;4(2):159.
[141]金昔陆,朱秀媛.葛根素在大鼠、家兔、犬中的药物动力学[J].中国药理学报,1992:13(5):284.
[142]金昔陆,朱秀媛.葛根素在三种动物的药代动力学参数与体重的相关性[J].徐州医学院学报,1991:11(1)25-26.