| Radix Asteris is a famous traditional Chinese medicine (TCM), which has been used for more than one thousand years. It has many effects, such as moistening lung, dispersing phlegm and relieving cough, and so on. Triterpeniods are usually considered as the main pharmacological effective compounds in it. Many water soluble compounds from the medicinal herb Aster tataricus had remarkable inhibition of development of tumor, and antioxidant activity such as quercetin and kaemferol.With the help of dynamic and kinetic mechanisms and mathematics method, pharmacokinetics of traditional Chinese medicine (TCM),under the guide of chinese medical science theory, studied absorption, distribution, biotransformation and elimination of active constituents or parts, simple or compound recipes of TCM, and relations of pharmacokinetics and pharmacodynamics of TCM. As one of important contents of modernization of TCM, pharmacokinetics of TCM might combine the studies of phytochemistry and pharmacodynamics and were beneficial for illuminating efficacy of TCM, identifying therapeutical basis and mechanism of action of TCM.In the present study, a reversed phase high performance liquid chromatography method for determination of the six chemical components in the Radix Asteris was developed using different detectors and extraction methods according to their polarities and structures. Quantification of the six components is important for the quality control of Radix Asteris. The developed method was precise and accurate and the method can be effectively applied for the quanlity control and evaluation of Radix Asteris at different samples. This chemical evidence based analysis can also provide valuable information for better understanding about the relationship between the chemical characteristic and the therapeutic efficacy of the Radix Asteris. Absorption, distribution, metabolism and excretion of shionone in rats were investigated for illuminating mechanism of action of theirs. The research provided a significant exploration for pharmacokinetics and therapeutic basis of TCM.Part one Simultaneous quantification of 3 lipid soluble active ingredients in Radix Asteris by HPLC-ELSDObjective: To establish a high-performance liquid chromatography method with evaporative light scattering detection for simultaneous determination of three major triterpeniods, i.e. shionone, friedelin, epi-friedelinol, in Radix Asteris.Methods: The optimal chromatographic conditions were achieved on a RP18 column (150 mm×3.9 mm, 5μm) with gradient elution of acetonitrile and 0.05% acetic acid in 22 min; the column temperature was 30 ?C. The ELSD was set at an evaporating temperature of 40 ?C and nitrogen gas pressure of 15 PSI. The validation of the method included tests of linearity, sensitivity, precision, repeatability, stability and accuracy.Results: All calibration curves showed good linear regression (r 2 > 0.9991) within test ranges. The established method showed good precision and accuracy with overall intra-day and inter-day variations of 1.6%–2.9% and 1.7%–2.4%, respectively, and overall recoveries of 97.35%–101.13% for the three compounds analyzed. Conclusion: The method developed was successfully applied to quantify the main triterpeniods in 14 Radix Asteris samples.Part two Simultaneous determination of 3 water soluble active ingredients in Radix Asteris by HPLCObjective: To establish HPLC method for the determination of contents of ferulic acid, quercetin and kaemferol in Radix Asteris.Methods: The samples were extracted with 80% methanol. A HPLC method detected by 3 different wavelengths (310 nm for quercetin, 360 nm for kaemferol and 365 nm for ferulic acid) had been developed. The chromatographic procedure for determination was carried out using DiamonsilTM C18 (250 mm×4.6 mm, 5μm) as an analytic column gradient eluted with a mixture consisting of acetonitrile and 0.05 % H3PO4.Results: The ranges of calibration curve of ferulic acid, quercetin, and kaemferol were 0.011～0.277 mg/mL, 0.004～0.106 mg/mL and 0.006～0.126 mg/mL, respectively. The established HPLC method was applied to determine 15 samples of Radix Asteris. The results showed that the ranges of ferulic acid, quercetin and kaemferol were 0.15～1.43 mg/g, 0.09～0.36 mg/g and 0.03～1.12 mg/g, respectively.Conclusion: A simple, effective and feasible HPLC method to determine the contents of quercetin, kaemferol and ferulic acid was established for the quality control of Radix Asteris.Part three Studies on pharmacokinetics and tissue distribution of shionone in the ratsObjective: 1 A HPLC method for determination of shionone in rat plasma was developed for studying the pharmacokinetics of in rats after after oral administration of shionone. 2 A HPLC method was developed and validated for determination of shionone in rat tissues.Methods: 1. After oral administration of shionone, blood samples were obtained from fossa orbitalis vein according to the specific schedule, 0, 10, 20, 30, 60, 90, 120, 180, 240, 360 and 580 min and collected in heparinized centrifuge tube, respectively. Sample was pretreated by being extracted with ethyl ether using friedelin as internal standar (IS) , the supernatant was collected and evaporated to dryness under a gentle stream of nitrogen. The residue was then reconstituted with 50μL acetonitrile, and an aliquot (20μL) of the supernatant was injected into the HPLC system. The separation was achieved by HPLC on a RP18 column (150 mm×3.9 mm, 5μm) with a mobile phase composed of acetonitrile-0.05% phosphoric acid water(98:2, v/v) at a flow rate of 1.0 mL/min. UV detection was set at 200 nm and friedelin was chosen as the internal standard (IS). 2 Rats were randomLy assigned to six groups. After administration of shionone, heart, liver, lung, spleen, kidney, brain, stomach and small intestine samples were obtained at 0.5, 1, 2, 3 and 5 h, respectively. Tissue samples were weighed rapidly and put into normal saline solution to remove the blood or content, blotted on filter paper, and then were weighed for wet weight and homogenized in saline solution. Preparation of tissues samples and HPLC analysis conditon were same with the plasma samples. The method was applied to study tissues distribution of shionone in rats after oral administration of shionone at a dose of 270 mg/kg.Results: 1 The calibration curve in plasma was linear over the range of 0.04295~1.719μg/mL and the linear regression equation of analytes is Y=0.1039X?0.2654; Precision and accuracy of the present method for determination of shionone are acceptable in rat plasma; the recovery of shionone in rat plasma was 76.93% ~ 81.27%. The study of stability demonstrated samples were stable. The main pharmacokinetic parameters were as follows: T1/2(ka) 40.61±9.0 min,T1/2(ke) 72.41±9.7 min,Cmax 0.39±0.35μg/mL,Tmax 76.08±10 min, AUC(0-∞) 93.32±75μg﹒min/mL, MRT(0-t) 171.97±16 min, AUC(0-t) 86.29±72μg﹒min/mL. 2 The LOD of current assay based on S/N = 3 was 10 and 13, 22 and 24, 20 ng/mL in heart, liver, spleen, lung, kidney, stomach, small intestine and brain. The intra-day and inter-day presion were less than 10%, and the extraction recoveries of different tissues were between 63%~73%.Conclusion: 1 The developed method was successfully applied to the pharmacokinetics and tissue distribution research after oral administration of shionone to healthy Sprague–Dawley rats. 2 A one-compartment open model gave the best fit to the plasma concentration time curves obtained in rats, and the weight was 1. It revealed that shionone was absorbed and eliminated rapidly in rats. 3 Stomach,intestine and lung were the main distribution tissues of shionone in rats. It was also found there was no accumulation of shionone in rat tissues.Part four Studies on the excretion of shionone in ratsObjective: A RP-HPLC method was developed for the determination of shionone in excretion of rats and studying its excretion. Methods: The urine, feces and bile samples were collected at predetermined time after shionone was orally administered to 6 rats. The concentrations of shionone were determined by HPLC-UV method, the samples were separated with a mobile phase consisting of acetonitrile and 0.05%H3PO4 and the detection wavelength was at 200 nm.Results: Shionone recovered mostly from feces (45.35%) in 72 h, 0.0001% was found from urine in 72 h and nothing was deteced from bile.Conclusion: A reliable HPLC method is developed and proved to be approporiate for the determination of shionone in excretion of rats. The major amount of shionone was recovered from feces after shionone was orally administrated to rats and the main time of excretion was between 6～12 h.Part five Metabolism of shionone in vivo and in vitroObjective: To study the metabolites and metabolic pathway of shionone in vivo and in vitro.Methods: Culture solution of intestinal bacteria produced from rat feces and hepatic microsomal enzyme were used to research the metabolism of shionone in vitro. After taking orally shionone, blood, bile, urine, feces were pretreated and analyzed by HPLC-PDA.Results: Only shionone was present in both gastric content and culture solution of intestinal bacteria. Shiononeand its metabolites were not detected in the bile samples. In addition to free shionone, metabolite M1 and M2 were detected. In the feces, free shionone was the major form of shionone. In the liver microsome incubation mixture, gastric content and culture solution of intestinal bacteria, no metabolite was found.Conclusion: The results of this work demonstrated that shionone is not high in blood, and can be more easily excreted through feces. But it can make an effect. Two following reasons might be helpful to explain this fact. The one was shionone might be converted to its metabolites in rat after ingestion and its metabolites can make an effect. The other was that shionone had made an effect before excretion, however, exact cause needed to be investigated further.Part six Drug-protein binding determination of shiononeObjective: To study the binding of shionone to rat plasma, human plasma and bovine serum albumin (BSA).Methods: A HPLC method was established for the determination of shionone in rat plasma, human plasma and bovine serum albumin (BSA). The protein binding rates of shionone with rat plasma, human plasma and BSA proteins were determined.Results: Its in vitro protein binding rates with the three mediums were high. Conclusion: The binding to rat plasma increased firstly, and then decreased with increasing drug concentration, but the results increased with increasing drug concentration for human plasma and bovine serum albumin (BSA). |
