Research On The Serum Pharmaceutical Chemistry Of Calculus Bovis Cultivated By Glucuronidase And Natural Calculus Bovis

The first partMETHOD FOR DETERMINATION OF THE CHOLIC ACIDCOMPONENTS IN SERUM.ObjectiveEstablish a method of serum cholic acid HPLC-ELSD determination for the Calculusbovis serum pharmaceutical chemical and pharmacokinetic study.Methods（1）column:Agela Venusil MP C18（4.6mm×250mm,5μm）column; mobile phase:acetonitrile （A）,0.2%formic acid in water （B）, gradient elution,0min30%A,70%B,15min55%A,45%B;30min90%A,10%B;flow rate:1.0ml/min; column temperature:25°C; Detector:evaporative light scattering detector. Settings of ELSD detector: drift tube temperature85°C; nitrogen flow2.0L·min^-1; Impact OFF （2） serum sample processing method:Takeserum100μl added methanol900μl, vortex mixing,12000rpm for centrifuge5min;supernatant,0.22μm filter membrane; the filtrate900μl,37°C water bath evaporated todryness, the solid residue at-20°C cryogenic preservation, remove the spare before thetest. The solid residue is dissolved with100μl flow, vortex mixing,12000rpm forcentrifuge5min, take20μl injection for analysis.ResultsThe analytical method detection limit was1μg·ml^-1, limit of quantification was5μg·ml^-1. The RSD%values of Repeatability and Stability were less than3.91; extractionrecoveries RSD%values less than3.67. The method of analysis biological samplesconform the limit requirements.ConclusionEstablished a mathod to detect the content of cholic acid in serum by HPLC-ELSD determination, the method is specificity, the sample preparation method is simple and fast,provide a method for pharmacokinetics and serum pharmaceutical chemistry study. The second partTRANSITIONAL COMPOSITION IN THE SERUM OFCALCULUS BOVIS SERUMObjectiveUse HPLC-ELSD and HPLC-MS analysis to confirm the transitional composition intothe blood after intragastric administration calculus bovis and calculus bovis cultivated byglucuronidase.Methods（1） column: Agela Venusil MP C18（4.6mm×250mm,5μm） column; mobile phase:acetonitrile （A）,0.2%formic acid in water （B）, gradient elution,0min30%A,70%B,15min55%A,45%of B%A30min90,10%B; flow rate:1.0ml/min; column temperature:25°C; Detector: evaporative light scattering detector. Settings of ELSD detector: drift tubetemperature85°C; nitrogen flow2.0L·min^-1; Impact OFF （2） mass spectrometryconditions. Ion source: ESI source; detection methods: negative ion detection; scan mode:full ion scan （SCAN）; scan range:100^-1500amu The; capillary voltage:4.0kV （positiveand negative ions）, fragmentation voltage:150V; source temperature:99℃; to solvent gas:N₂; drying temperature:350°C; dry gas flow rate:9L·min^-1; dry gas pressure:35psi.（3）pretreatment of serum samples: Take serum100μl added methanol900μl, vortex mixing,12000rpm for centrifuge5min; supernatant,0.22μm filter membrane; take filtrate900μl,37°C water bath evaporated to dryness The solid residue stored at-80℃cryopreservationbefore the test, remove the spare. The solid residue is dissolved with100μl mobile phase,vortex mixing,12000rpm for centrifuge5min, take20μl injection for analysis.ResultsThe steady-state serum of rats after ig. calculus bovis and calculus bovis cultivated byglucuronidase compared to the blank serum ten transitional composition confirmed, whichcan determine the composition of chenodeoxycholic acid and deoxycholic acid. Massspectrometry confirmed that it is the same substance.ConclusionThe steady-state serum of rats after ig.calculus bovis were detected ten kinds ofcompounds in the transitional composition.The steady-state serum of rats after ig.calculusbovis cultivated by glucuronidase were detected siven kinds of compounds in thetransitional composition. suggesting that the calculus bovis cultivated by glucuronidasehave the different material basis for the efficacy of the calculus bovis. The third partPHARMACOKINETIC OF CHENODEOXY CHOLIC ACID ANDDEOXYCHOLIC ACIDObjective:Determine the cholic acid composition in rats surem after intragastric administrationcalculus bovis and calculus bovis cultivated by glucuronidase.Use3P97software tocalculate the pharmacokinetic parameters, provide a theoretical basis for the calculus bovisand calculus bovis cultivated by glucuronidase.Methods（1） Wistar rats40, were randomly divided into4groups （n=10）. Intragastricadministration per group: single dose of calculus bovis4.0g·kg^-1,2.0g·kg^-1and the calculusbovis cultivated by glucuronidase3.8g·kg^-1,2.0g·kg^-1. After intragastric administration of5min,10min,20min,30min,1h,2h,3h,4h,5h,6h,7h,9h in the tail tip blood wascollected about0.5ml.（2） take the serum100μl, added methanol900μl, vortex mixing,12000rpm for centrifuge5min; supernatant,0.22μm filter membrane; the filtrate900μl,37°C water bath evaporated to dryness, solid residue100μl mobile phase, vortex mixing,12000rpm for centrifuge5min, take20μl injection analysis, the calculus bovis cultivatedby glucuronidase group determination of the content of chenodeoxycholic acid, calculusbovis group determination of ursodeoxycholic acid content.（3）Using the3P97pharmaco-kinetic software carried out automatically fitting the best-compartment model as a standardto calculate the main pharmacokinetic parameters of each rat and calculated the mean, theuse of statistical moments be calculated by AUC and MRT value and calculating the doseof the absolute bioavailability.Results（1） Rats after ig. calculus bovis, T（peak）of deoxycholic acid was0.72h, T1/2Ka0.16h,T1/2αdistribution phase half-life under conditions of two doses of about1.4h, eliminationhalf-life T1/2βapproximately6h; apparent distribution coefficient of the V/F were smaller.Two dose groups of deoxycholic acid in the body of the average residence time MRT（0^∞）and MRT（0^t）, no significant differences in AUC（0^∞）and AUC（0^t）, a significant differencebetween the AUC and the dose was positively correlated.（2） Rats after ig. calculus boviscultivated by glucuronidase chenodeoxycholic acid T（peak）is about0.78h, T1/2Kaapproximately0.35h, the distribution phase half-life under conditions of two doses of T1/2αabout0.9h, elimination half-life T1/2βabout6h, the apparent distribution coefficient V/F high-dose was significantly greater than the low dose group, the peak concentration C（max）,area under the concentration-time curve the AUC, the clearance rate significantly different,high two dose group of chenodeoxycholic acid in the body of the average residence timeMRT（0^∞）and AUC（0^∞）and AUC（0^t）, significant differences in dose are positivelycorrelated.ConclusionThe pharmacokinetic characteristics of Chenodeoxycholic acid and deoxycholic acidconform the two-compartment model.