Research On Biological Assessment Of Anti-hemolysis Compound Puerarin Injection

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.