Pharmacokinetic Study Of The Major Active Components Of Escin

Escin is a natural mixture of saponins extracted from the seeds of Aesculuschinensis Bge.var.chekiangensis （Hu et Fang）Fang or Aesculus wilsonii Rehd.Escins A, B, C and D are the major bioactive constituents of escin. Escin hasantioedematous, antiinflammatory and venotonic effects, and is predominantly usedin the therapy of chronic venous insufficiency, haemorrhoids and post-operativeoedema. To date, only a few reports have focused on the pharmacokinetics of theindividual saponins of escin in human and more information is needed to ensure thesafety of the drug in clinical use. In this study, we developed a liquidchromatography-mass spectrometric （HPLC-MS/MS） method for the simultaneousquantification of escins A, B, C and D in human plasma and urine. And applied it toa pharmacokinetic study of the four isomers in human.The metabolism of the fourisomers in rat and their plasma protein binding was also studied. The results are asfollowed:1. The development of an HPLC-MS/MS assay for escin in humanbiosamplesA sensitive, selective and rapid method for the simultaneous quantification ofescins A, B, C and D in human plasma and urine was developed. Escin andtelemisartan （internal standard） were extracted from plasma or urine using solidphase extraction on C18columns and separated on a Zorbax Extend C18columnusing methanol-acetonitrile-10mM ammonium acetate （30:30:40，v/v/v） as mobilephase. Detection was carried out by multiple reaction monitoring （MRM） on anAPI4000TMHPLC-MS/MS system with an ESI interface in the positive ion mode.MRM was performed at unit resolution using the mass transition ion-pairs at m/z1131.8â†'m/z807.6for escin and m/z515.2â†'m/z276.1for telemisartan. Linearity,specificity, precision, accuracy, recovery, matrix effects and stability were estimatedaccording to the guidance for method validation of the FDA. The results show thatall validation parameters met the requirements of the guidance criteria. 2. Pharmacokinetic study of escin in humanUsing the established LC-MS/MS method, the pharmacokinetics of escin inhealthy male volunteers were studied after three intravenous （i.v.） infusions of5.77,11.54and23.08mg of sodium aescinate solution and a single oral60mg dose ofsodium aescinate tablets.After i.v. infusion of5.77,11.54and23.08mg of sodium aescinate, respectivevalues of pharmacokinetic parameters for escin A were: t1/23.75,9.32and9.67h;C_max46.3,70.5and138.8ng/mL; and AUC0-∞331,444and658μg/L*h. Linearpharmacokinetics was established based on high correlation coefficients （r>0.83） ofpharmacokinetic parameters (e.g. AUC0-∞and C_max).Corresponding values for escin B were: t1/23.30,3.72and7.75h; C_max76.5,150.7and241.2ng/mL; and AUC_0-∞217,442and843μg/L*h. Pharmacokineticparameters were linear with dose （r>0.99）.Corresponding values for escin C were: t1/217.5,15.0and18.2h; C_max65.0,163.2and230.3ng/mL; and AUC_0-∞861,1813and3962μg/L*h with linearpharmacokinetics （r>0.99）.Corresponding values for escin D were:17.7,9.3and9.7h; C_max19.8,46.3and70.5ng/mL; and AUC0-∞138,331and658μg/L*h with linear pharmacokinetics（r>0.98）.After i.v. infusion of sodium aescinate, escins A, B, C and D were extensivelymetabolized with only about5,2,6and4%of escins A, B, C and D, respectivelyexcreted in urine in the form of unchanged drugs. There was no significant differencein urine excretion for the three doses.After oral administration of sodium aescinate tablets, the C_maxof escins A, B, Cand D in human plasma were <6ng/mL. Corresponding AUC_0-∞values were0.9,0.2,0.5and0.8%of those after i.v. infusion after correction for dose. These resultsindicated the bioavailability of escin is extremely low in human.3. Plasma protein binding in humanProtein binding in human plasma was determined by equilibrium dialysis.Relatively high protein binding （>90%） was found for escins A, B, C and D with no significant differences between the four components.4. Mass-spectrometry fragment of escins A, B, C and DUsing the molecular-weight precision, high-resolution and informationdependent acquisition （IDA） triple quadrupole time-of-flight mass spectrometry, wedetermined the relative fragmentation pattern of each of the four isomers of escin.The four isomers were completely resolved using gradient elution and the methodwas highly reproducible and stable. Using ESI-Triple TOF, the exact molecularweight of each isomer was determined after which IDA technology was used to scanthe exact molecular weight. Combining the software with information in theliterature, the fragmentation patterns of escin isomers were established. The resultsshowed that escins C and D have the same fragmentation patterns as do escins A andB. Because of different R₂and R₃groups, escins have different fragmentation patternsfrom isoescins. Furthermore, we found escine and isoescin show specific ion peak,which could be used to discriminate them. This finding improves our currentknowledge of saponins.5. The metabolism study of escin A., B, C and D in ratsMetabolism of escin was studied in rats using triple time-of-flight massspectrometry. Urine and feces were sampled after intravenous injection of sodiumaescinate. After solid phase extraction, the metabolites were completely separated bygradient elution and by triple TOF using Mass Defect technology. The results showthat new isomers were produced in rats possibly by rearrangement of the ester bond.We also found glycohydrolase metabolites in urine but not in feces samplessuggesting their formation is due to bacteria in gastrointestinal tract.