Study And Application Of Chinese Herbal Medicine Fingerprints In Quality Control And In Vitro Absorption And Metabolism

There is a long history of herbal medicine in far Eastern countries;in particular Chinese people have utilized herbs and plants to treat various diseases for more than 8000 years. Traditional Chinese medicine (TCM) has been modified to some extend in other countries, such as Korea and Japan and has attracted considerable attention even in European and North American countries. These drugs are complex mixtures, containing usually hundreds of chemically different constituents but only a few, if not one, compounds are responsible for the beneficial and/or hazardous effects. Furthermore, biologically active compounds form just a minute part of herbs being diluted with a large amount of proteins, sugars or tannins, which, in some cases, does not contribute to the pharmaceutical effect but they make the quality control of crude drugs and their medical preparations extremely difficult. Therefore, very efficient and selective methods, including the extraction techniques are required for identification and quantitative analysis of the active compounds or drug standardization. Chromatography and electromigration methods represent main techniques applied in this field due to their powerful separation efficiency combined with sensitive detection as indicated by several recent reviews covering the TCM preparations or selective active compounds in a variety of sample matrices (Cinchona alkaloids, indole alkaloids or tropane alkaloids).Traditionally, chromatography and relative techniques are also used to evaluatethe quality of plant raw materials, as the diversity of components and their content vary with not only the species but also with the growing conditions, the season when plants are harvested, the process methods and storage duration. In the medicine plant production, it is not always necessary to produce very pure preparations. Herbal drugs are then standardized to selected component to assure comparability of different preparations of plant origin. In this respect, fingerprint analysis has been accepted by WHO, as a methodology for the assessment of herbal medicines. Fingerprint is also used to control quality of TCM and their raw materials as stated by the Chinese Pharmacopoeia. Conventional research focuses mainly on the determination of the most active or hazardous components, while fingerprinting can offer integral characterization of a complex system with a quantitative degree of reliability. The identification of crude herbs based on geographical origins is crucial in order to Fingerprints can ensure authenticity, quality, safety and efficacy of the raw material before it is converted to the final product.In this paper, the fingerprints of Ginkgo biloba L. and Elschotzia blanda Benth are established for further research. One is used for the determination of Ginkgo biloba L. and Elschotzia blanda Benth extract and preparation;one is for the uptake and transport of Elschotzia blanda Benth extract in Caco-2 cell model;another is for in vitro metabolism of Elschotzia blanda Benth extract;the last is for the inductive effect of Elschotzia blanda Benth extract on rat cytochrome P450.Study and application of fingerprints in the quality of Chineseherbal medicine1. Study and application on fingerprints of the Elschotzia blanda Benth extractsand tabletsAIM: To establish a method for determination of Elschotzia blanda Benth, its extractsand tablets with HPLC fingerprints, so as to control the quality of the tablets.METHODS: HPLC-DAD method was used to determine the constituents in tablets.TMDiamonsil Clg (200mmx4.6mm, 5um) was used as analysis column, and acetonitrile/KH2PO4 was used as mobile phase with gradient elution. The columntemperature was at 20 °C. The HPLC profile of chemical constituents of extracts and tablets were analyzed using similarity software. RESULTS: The fingerprints of the extract and the tablet from the same batch were slightly different because of preparing procedure. While the fingerprints of different batches of tablets made from the same extract were similar to each other. CONCLUSION: This method is accurate, reproducible, simple and can be used as analytical method for the routine quality control of the Elschotzia blanda Benth tablets.2. Study and application on fingerprints of Ginkgo biloba leaves preparations AIM: To establish a method for determination of Ginkgo biloba L, its extract and preparations with HPLC fingerprints, so as to control the quality of the preparations. METHODS: HPLC-DAD method was used to determine the constituents inTMpreparations. Diamonsil C]g (200mmx4.6mm, 5um) was used as analysis column,and acetonitrile/KH2PC>4 was used as mobile phase with gradient elution. The column temperature was at 24 °C. The HPLC profile of chemical constituents of control sample and preparations were analyzed using similarity software. RESULTS: The fingerprints of three preparations from different companies were slightly different because of the different preparing procedure. But the fingerprints of different batches of the same preparation from the same company were similar to each other. CONCLUSION: This method is accurate, reproducible, simple and can be used as analytical method for the routine quality control of Ginkgo biloba preparations.3. Application of HPLC retention index in Chinese herbal medicine fingerprints AIM: To establish a stable and reproducible fingerprint method for the indentification of Ginkgo biloba L. and Elschotzia blanda Benth by using chromatography retention index. METHODS: HPLC was used to determine the constituents in herbal medicines. DiamonsilTMc^ (200*4.6mm,5um) was used as analysis column, acetonitrile/KH2PO4 was used as mobile phase with gradient elution. The fingerprints of Ginkgo biloba L.and Elschotzia blanda Benth extract were analyzed by retention index using a series of alkane-2- one. The relative standard deviation (RSD) of retention time, relative retention time and retention index of the common peaks from the chromatograms under the different conditions was calculated and compared to eachother. RESULTS: The retention indexes of the common peaks in the fingerprints of Ginkgo biloba L. and Elschotzia blanda Benth under the different chromatographic conditions showed no significant difference (P>0.05). And the RSD of the retention indexes was the least compared to those of retention time and relative retention time. CONCLUSION: Above results showed that the developed method was accurate, reproducible, simple, and overcome the drawback that the slight variety of chromatographic conditions resulted in poor reproducibity in usual fingerprints method.In vitro uptake and tranport of the Elsholtzia blanda benth extract in Caco-2 cell model1. In vitro uptake of the Elsholtzia blanda benth extract in Caco-2 cell modelAIM: To investigate the uptake characteristic of the Elsholtzia blanda benth extract on human epithelial cell line (Caco-2). METHODS: A human intestinal epithelial cell model Caco-2 cell in vitro cultured was applied to study the uptake of the Elsholtzia blanda benth extract at small intestine. The effects of time, pH, concentration, temperature and P-glycoprotein inhibitors on the uptake of Elsholtzia blanda benth extract were investigated. The determination of Elsholtzia blanda benth extract was performed by HPLC. RESULTS: The uptake rate of the Elsholtzia blanda benth extract by Caco-2 cell depended on the extracelluar temperature and pH. The uptake rate was 2.7 fold at 4°C as that at 37°C, and was 1.6 fold at pH 6.0 as that at pH 8.0. The uptake rates of the Elsholtzia blanda benth extract were linear with the total flavones concentration increasing from 0.415 to 6.64mg.mL"1 and within 4hours at 3.32mg/ml total flavones. Verapamil, cyclosporin A, cimetidine and ouabain can all significantly increase the uptake rates of the Elsholtzia blanda benth extract, and especially in the lower cincentration of the Elsholtzia blanda benth extract, except cimetidine. CONCLUSION: The uptake characteristic of the Elsholtzia blanda benth extract in Caco-2 cell was primarily rapid passive diffusion and depended on temperature, pH, time and concentration, and partly mediated by p-glycoprotein.2. In vitro transport of the Elsholtzia blanda benth extract in Caco-2 cell modelAIM: To investigate the transport mechanism of the Elsholtzia blanda benth extract on human epithelial cell line (Caco-2). METHODS: A human intestinal epithelial cell model Caco-2 cell in vitro cultured was applied to study the transport mechanism of the Elsholtzia blanda benth extract at small intestine. The transport characteristic of the Elsholtzia blanda benth extract was investigated under the conditions of different incubate time, different incubate temperature, different pH of incubate medium, different extract concentration and P-glycoprotein inhibitors present. The contents of the Elsholtzia blanda benth extract in apical chamber (AP) and basolateral chamber (BL) were determined by HPLC. RESULTS: The transport ability of the Elsholtzia blanda benth extract significantly depended on the extracelluar pH and temperature. The apparent permeability coefficient (Pm) value of the Elsholtzia blanda benth extract from AP to BL at pH 6.0 was 4.8 fold greater than that at pH 7.4, while Pm value of BL-AP at pH 7.4 was 1.4 fold greater than that at pH6.0;At the same Papp value of AP-BL at 37°C was 3.9 fold greater than that at 4°C. The flux of the Elsholtzia blanda benth extract across the Caco-2 cell layer (apical to basolateral side or basolateral to apical side) was linear with time for up to 10 hours and concentration for up to 6.64mgmL"1. The Pm from the basolateral to the apical side was 0.25-0.44 times as that from the apical to the basolateral side at pH 6.0. Verapamil, cyclosporine A, cimetidine, ouabain and indomethacin can all increase the transport rate of the Elsholtzia blanda benth extract from apical to basolateral side, but they affected the transport rate of the Elsholtzia blanda benth extract from basolateral to apical side differently. Verapamil, cyclosporine A and ouabain can reduce the transport rate from basolateral to apical side and increase the transport rate from apical to basolateral side. But the increasing degree of the transport rate from apical to basolateral side was weakened with the increasing concentration of the Elsholtzia blanda benth extract. Cimetidine can raise the transport from basolateral to apical side. While indomethacin reduced the transport from basolateral to apical side at low concentration and raised it at high concentration. CONCLUSION: The transport of the Elsholtzia blanda benth extract in Caco-2 cell layer was pH, temperature, time and concentration dependent. And rapid passive diffusion and active transport were the major pathways for theElsholtzia blanda benth extract absorption, and partly mediated by p-glycoprotein. In vitro metabolism and inductive effect of the Elsholtzia blanda benth extract on rat cytochrome P450 enzyme1. In vitro metabolism of the Elsholtzia blanda benth extract in rat liver microsomesAIM: To obtain the information about metabolic mechanism of the Elsholtzia blanda benth extract by liver microsomal enzymes. METHODS: An HPLC fingerprints method was established for determination of the Elsholtzia blanda benth extract in rat liver microsomes. The metabolism of the Elsholtzia blanda benth extract was investigated in five kinds of rat liver microsomal incubates pretreated with Phenobarbital (PB), dexamethasone (Dex), P-naphthoflavone (BNF), diphenytriazol and control. The Elsholtzia blanda benth extract was incubated in different rat liver microsomes. The concentration of the major constituents of remaining extract in microsomal incubates was determined by HPLC fingerprints. RESULTS: The assay was linear from 0.020016 to 0.5004mg.mL"1(calculated on the content of total flavones against the major constituent peak area) for the Elsholtzia blanda benth extract in rat liver microsomal incubates. The average extraction recovery and method recovery was 75.39% and 98.67% (?=4), intra-day and inter-day variation coefficients were less than <1.5% (n =4) . The method allowed study of the metabolism of the Elsholtzia blanda benth extract in rat liver microsomes in vitro. The metabolism phase I of the Elsholtzia blanda benth extract was weak, and the concentration of the Elsholtzia blanda benth extract after 120 min was only reduced for 8.8%, 9.0% and 17.1% respectively, in the microsomes pretreateded with BNF, Dex and PB, calculated on the major constituent. The metabolism phase II of the Elsholtzia blanda benth extract was mainly mediated by the microsome treated with BNF or diphenytriazol. After 120 min incubation, in the microsomes treateded with BNF and diphenytriazol, the concentration of the Elsholtzia blanda benth extract was reduced for 31.2% and 66.5% respectively, calculated on the major constituent. And they were significantly different from control microsome (p<0.05). CONCLUSION: These results revealed that the metabolism ability of the Elsholtzia blanda benthextract was weaker in phase I reaction mediated by P450 than in phase II reaction mediated by UDP-glucuronosyltransferase, and the metabolism phase II of the Elsholtzia blanda benth extract was primarily mediated by the microsome induced by BNF or diphenytriazol.2. In vivo inductive effect of the Elsholtzia blanda benth extract on cytochrome P450AIM: To study the induction of the Elsholtzia blanda benth extract on cytochrome P450. METHODS: Sprague-Dawley rats were pretreated with the different daily dose of the Elsholtzia blanda benth extract for different days. The selected typical substrates for CYP subfamilies were incubated with the rat liver microsome pretreated with the Elsholtzia blanda benth extract, respectively. The catalytic activities of rat liver microsomes were determined by measuring the product formation or by monitoring the substrate loss, and compared with that in the control microsome and that in the microsome induced by BNF, PB or Dex, respectively. The typical substrates for CYP subfamilies were phenacetin and ethoxyresorufin for CYP1A;aminopyrine and pentoxyresorufin for CYP2B;diazepam and testosterone for CYP3A;chlorzoxazone for CYP2E1;omeprazole for CYP2C19;diclofenac for CYP2C9. RESULTS: 1) The activity of ethoxyresorufm-0-deethylase (EROD) and the metabolic ability of aminopyrine, phenacetin, chlorzoxazone and diclofenac in microsomes pretreated with the Elsholtzia blanda benth extract showed no significant difference comparing with control group (/?>0.05), and there was no correlation between the metabolic degree and the treatment day of the Elsholtzia blanda benth extract. While the pentoxyresorufin-Odealkylase (PROD) activity in the microsomal incubates pretreated for three-days or five-days showed significant difference comparing with control group (p<0.05), but also significant difference comparing with that pretreated with PB. 2) The metabolic ability of testosterone, omeprazole and diazepam in microsomes pretreated with the Elsholtzia blanda benth extract showed significant difference comparing with control group (p<0.05), and there was the correlation between the metabolic degree and the treatment day of the Elsholtzia blanda benth extract. The specific inhibitor of CYP3A, ketoconazole, inhibited significantly the metabolism of diazepam and testosterone.CONCLUSION: These results revealed that the Elsholtzia blanda benth extract might induce CYP3A.