Pharmacokinetic Study On The Combination Of Total Alkaloids Of Rhizoma Coptidis And Panax Notoginseng Saponins

Rhizoma Coptidis and Panax Notoginseng are traditional Chinese medicines.Preliminary studies have found that when the combination of total alkaloids extracted from Rhizoma Coptidis and total saponins from Panax Notoginseng,the effect of treating diabetic complications is well.Among them,berberine,epiberberine,coptisine and palmatine in the total alkaloids of Rhizoma Coptidis are the main active components;in the total saponins of Panax Notoginseng,notoginsenoside R1,ginsenoside Rb1,Rg1,Re,and Rd is the main active ingredient.In order to further explore the safety and efficacy of the composition of total alkaloids of Rhizoma Coptidis and total saponins of Panax Notoginseng.To study the drug absorption,distribution,metabolism and excretion of the main active ingredients in rats and inhibition of human CYP450 enzymes in vitro.1.Development and validation simultaneous determination method of nine active componentsAn LC-MS/MS method for the simultaneous quantitation nine active components in rat plasma has been developed and validated.ESI positive ion and MRM mode was used for detection.The detection transitions are:m/z 336.1→m/z 320.2（berberine）,m/z 336.1→m/z 320.1（epiberberine）,m/z 320.0→m/z 292.0（coptisine）,m/z 352.0→m/z 337.3（palmatine）,m/z 955.4→m/z 775.7（notoginsenoside R1）,m/z 823.4→m/z 643.7（ginsenoside Rgl）,m/z 1131.5→m/z 365.4（ginsenoside Rb1）,m/z 969.5→m/z 789.7（ginsenoside Re）,m/z 969.6→m/z 789.8（ginsenoside Rd）,m/z 285.2→m/z 193.1（IS-diazepam）,m/z 803.6→m/z 283.1（IS-digoxin）.The linear range of berberine,epiberberine and coptisine was 0.12520 ng/mL;palmatine was 0.1-16 ng/mL;R1,ginsenoside Rg1,and ginsenoside Re was 0.580ng/mL;Rb1 was 2.5-400 ng/mL,and ginsenoside Rd was 0.75-120 ng/mL.Analytical method validation was performed according to the Chinese Pharmacopoeia guideline.The results showed that this method is suitable for pharmacokinetic study of the combination.2.PharmacokineticAfter single dose,parameter Tmax of eight active ingredients（except ginsenoside Re）was between 2 h and 6 h,and Vd values of eight active ingredients were larger,indicating that their widely distributed in rats.Except for ginsenoside Rb1,t1/2 of the other seven compounds were between 10 h and 12 h,indicating that the elimination in rats was slow.Tmax was slightly shortened with multiple dose compared to single dose.Except for ginsenosides Rg1,Rb1 and Panax notoginseng saponins R1,AUC0-t of other active ingredients were all increased.In addition,except for ginsenoside Rd and Panax notoginseng saponin R1,t1/2 of the other active ingredients did not change significantly.No accumulation of eight active ingredients in rats.The PK data provide a useful platform on which to base future clinical studies of combination.3.Tissue distributionThe results showed that seven active ingredients have been detected after oral administration 1h,except Panax notoginseng saponin R1 and ginsenoside Re.The results indicating that the seven active ingredients were distributed rapidly and widely in rats.Except for coptisine,the concentrations of the eight active ingredients were higher in stomach and small intestine,which indicated that the active ingredients were mainly absorbed through the gastrointestinal tract after the composition was administered by gavage.After 24 hours of oral administration,the concentration of active ingredients in the stomach and small intestine was still higher than that in the rest of tissues.However,compared with the concentrations of active ingredients in the stomach and small intestine after 1 h and 3 h of oral administration,it showed a downward trend,indicating that composition is slower to digest in the gastrointestinal tract of rats.4.Metabolism and ExcretionTriple TOF 6600 plus combined with IDA technology was used to identify metabolites in rat urine and feces.In these samples,three probable saponins and twenty probable alkaloid metabolites were found.They are protopanaxadiol PPD,ginsenoside CK and ginsenoside Rg3;berberine,coptisine and palmatine through hydroxylation,demethylation,glucuronidation and sulfation etc.An LC-MS/MS method has been developed for the determination nine active ingredients in rat urine and feces.The maj ority of the administered dose was excreted in urine and feces.But,the results indicating that gastrointestinal excretion is the primary route of elimination for all nine active ingredients.After 96 hours of oral administration,part of the active ingredients still be detected in rat feces,and the composition takes a long time to digest and excrete in vivo.5.Inhibitory effect on human CYP450 EnzymeThe inhibition of the main six human CYP450 enzyme isoforms was investigated using a mixed probe-substrate method.The study found that the composition has inhibitory effect on the four enzyme subtypes of CYP1A2,CYP2D6,CYP2C9,and CYP3A4（with testosterone as a substrate）.