Pharmacokinetics Study On Multi-Components Of Radix Salviae Miltiorrhizae And The Quality Assessment Of Its Preparation

OBJECTIVE:(1)To develop a new reversed phase high performance liquid chromatographic method(RP-HPLC)for determining the concentration of Tanshinoneâ… in rat plasma following intravenous injection of Tanshinoneâ… solution, and the pharmacokinetic parameters of Tanshinoneâ… was caculated for the first time by Drug and Statistics 1.0 program.To develop A for simultaneous determinination of four tanshinones in rat plasma was developed for application in the pharmacokinetics study.(2)To investigate the effects of Fufang Danshen tablets on cytochrome P450 in rats.(3)To establish a high-performance liquid chromatography coupled with diode array detection and evaporative light scattering detection method for simultaneously determining eleven components of three different structural types in Fufang Danshen Tablet.And to assess the quality of Fufang Danshen Tablet basing on Chemometrics.METHODS:(1)The HPLC assay was carried out using a Cosmosil C₁₈column. The mobile phase was acetonitrile-0.05 mol l^-1ammonium acetate buffer with 1% acetic acid(66:34,v/v).The flow rate was 1.0 ml min^-1.The detection wavelength was set at 263 nm.The plasma concentration of Tanshinoneâ… after bolus injecting was determined using RP-HPLC developed.Moreover,their pharmacokinetics parameters were estimated by DAS1.0.The assay was conducted on a VP-ODS C₁₈column(4.6 min×250 mm;5μm),and the mobile phase was consisted of methanol and 0.05 mol·L^-1ammonium acetate buffers in a volume ratio of 70:30.The flow rate was 1.0 ml min^-1.The detection wavelength was set at 263 nm.Testosterone propionate was used for internal standard.The plasma concentrations of four tanshinones were determined after oral administration of extraction of Salvia Mitiorrhiza Bunge.And pharmacokinetics parameters were estimated by DAS1.0.(2)The metabolic change of phenacetin being the probe drug was studied in vivo. After given isotonic Na chloride,Cimetidine,phenobarbital sodium and Fufang Danshen tablets by oral administration respectively,the rats were all given phenacetin solution by intra-peritoneal injection.Then the blood samples were collected at different time.An HPLC method developed was used to determine the concentrations of the probe drug in rat plasma.And pharmacokinetics parameters were estimated by DAS1.0.(3)Using the developed HPLC-DAD-ELSD method,five different wavelengths were chosen as the monitoring wavelength to determine salvianolic acid B, dihydrotanshinone,crytotanshinone,tanshinoneâ… ,methylenetanshinone and tanshinoneâ…¡A,and an evaporative light scattering detector combined was employed to determine five saponins(notoginsenoside R1,ginsenoside Rg1,Re,Rb1 and Rd).RESULTS:(1)The assay accuracy was better than 92%,and the precision of Tanshinoneâ… at low to high concentrations was better than 9 and 11%for intra-day and inter-day assays,respectively.The recovery of the method exceeded 88.3%for Tanshinoneâ… .The assay showed good linearity(r=0.9998)over a relatively wide concentration range from 0.05 to 10.0μg ml^-1.The average concentration-time profiles of TS I after intravenous injection at a dose of 3 mg kg^-1in rats were well described with two-compartment model.After intravenous injection of TS I supernatant solution,TS I was eliminated rapidly with a short value of t_1/2β.The response was linear in the concentration range of 0.01-12.75μg·mL^-1for cryptotanshinone,0.01-16.25μg·mL^-1for tanshinoneâ… ,0.02-13.75μg·mL^-1for methltanshinone,0.02-13.25μg·mL^-1for tanshinoneâ…¡A,respectively.The recovery of assay was high than 88.7%.The pharmacokinetic paramenters showed that cryptotanshinone and tanshinoneâ…¡A after oral administration were fitted with two-compartment open models while the methltanshinone and tanshinoneâ… were fitted with one -compartment open models.(2)The t_1/2of probe drug in groups of isotonic Na chloride,Cimetidine,phenobarbital sodium and Fufang Danshen tablets was 93.51±9.21,161.67±10.95,85.36±8.98, 77.93±6.54 min,respectively.And Fufang Danshen Tablets has certain induction to the rats CYP450.(3)The developed method was successfully applied to quantify the eleven components in fifteen batches Fufang Danshen Tablet samples from four different factories.Moreover,the quality was assessed by principal component analysis and hierarchical clustering analysis;it demonstrated that the preparation procedure and the growth area of raw materials were more important factor influencing the quality of Fufang Danshen Tablet.CONCLUSION:(1)The HPLC method we have developed,was simple, sensitive and specific,and could be used for the analysis of large numbers of plasma samples.The assay was validated to meet the requirements of pharmacokinetic studies, and the results of validation have been showed that this method is sensitive,accurate and reproducible.Therefore,this method could be used to assay multi-components of Radix Salviae Miltiorrhizae in clinical samples and other biological fluid samples following appropriate adjustments.The method developed was simple and reliable and could be used to simultaneously determine the four kinds of tanshinones in rat plasma.(2)Fufang Danshen Tablets has certain induction to the rats CYT450.(3)The results indicated that the method could be used as a convenient and reliable method in the multi-component determination and quality control of traditional Chinese medicine.NEW IDEAS:(1)The pharmacokinetics studies of multi-components in the extraction of Radix Salviae Miltiorrhizae were investigated for the first time.And the developed method was applied to the pharmacokinetic studies of tanshinoneâ… and methltanshinone for the first time.(2)Effects of Fufang Danshen Tablets on liver Cytochrome P450 in Rats was investigated for the first time.(3)A simple quantitative method based on HPLC-DAD-ELSD was developed for the routine analysis of eleven active components in Fufang Danshen Tablet.