Evaluate The Integrated Chemical Quality,Choleretic Validity And Developed Bar Code Recognition System For Dida

OBJECTIVE: To establish a method for the determination of 7 effective constituents in the Swertia mileensis simultaneously by using HPLC-DAD spectrum technique.Optimizing the processes of extracting 7 effective constituents in Swertia mileensis for determination.To analyze and compare the content of 7 effective constituents in 11 species which are used as Dida.Integrated evaluation of them.To supply a basis for the resources exploitation.To determine four natural iridoid compounds from Swertia by quantitative analysis of multi-components with single marker(QAMS),and examine the feasibility of using the relative correction factors(RCFs)between the different compounds.To carry out comparative study on Tibetan medicine Dida’s heredity and genetic relationships and develope its bar code recognition system based on ITS.To compare the cholagogic effect of four plants used as the Tibetan medicine Dida(Swertia mileensis,Swertia mussotii,Swertia chirayita,Saxifraga tangutica).METHODS: Most appropriate absorption wavelengths of different iridoids,xanthenones and oleanolic acid have been performed by using 3D spectrum technique.According to the results,the wavelength 210 nm,240 nm and 265 nm had been turned on during the detection of determination 7 effective constituents.The L9(34)orthogonal test was used for optimization.7 effective constituents in the extract was the index,and the concentration was determined by HPLC,the effects of every liquid to solid ratio,extract time,temperature of extraction,and the number of extraction considered were investigated.The date of determination 7 effective construction of 11 Didas was normalized and integrated by three-dimension.The RCFs between the four natural iridoid compounds were determined by HPLC.With any of the four compounds as reference,a QAMS method was established for detect the quantitation of the other four compounds.Subsequently,the new QAMS method was used to determine the contents of four compounds contained in nine Swertia,and compare with external standard methods,in order to evaluate the accuracy of the QAMS method.Use DNA method,to extract DNA from 14 Didas plant tissues,get ITS barcode’s information by PCR amplification and sequencing.Calculate the genetic distance of Kimura 2-parameter(K2P)by MEGA 6.06,and constructe the Neighbor-Joining(NJ)tree.100 male rats were randomly divided into 10 groups,respectively as normal control group,the positive control group(Ursodeoxycholic Acid Tablets)0.05 g/kg,the Tibetan medicine Dida high dose groups 1.5 g/kg,low dose groups 0.75 g/kg.All groups were administered orally on rats,once every day,continuous administration of 7 days.To observe the choleretic action reagent by common bile duct intubation experiment and the analysis of the amount of bile secretion and bile composition.RESULTS: Determination of 7 effective constituents indicated good linearity over the linear range with coefficients(r)of 0.9999 respectively.The recoveries were found in the average range of 98 % to 102 % with RSD being less than 3 %.The optimum ultrasonic extraction condition was confirmed that every liquid to solid ratio of 10,30 min for each time,50 ℃ and ultrasonic extraction 3 times.RCFs of four natural iridoid compounds is stable in a certain linear range,and repeatability was good in different experimental conditions.The quantitative results of both external standard method and QAMS had no significant difference.The ITS barcode’s number of variable sites and indels are higher,and its aligned length is suitable.The Tibetan medicine Dida could enhance the amount of bile secretion and affect the secretion of components in rat bile.Such as,decrease the contents of total cholesterol and total bilirubin,increase the bile acid.CONCLUSION: HPLC-DAD spectrum technique is a simple,efficient and highly reproducible way to control the quality of Swertia mileensis.The optimized process is simple,reasonable and feasible.This experiment shows that these plants used as Dida in Tibet medicine mostly contain these 7 effective constituents,but the difference is notable.The integrated evaluation by the integrated chemical quality index(ICQI)has reference value for quality control.The method established in this research is accurate and feasible,so as to provide reliable basis for the application of the QAMS method in quality control of traditional Chinese medicines.Developed Tibetan medicine Dida’s bar code recognition system based on ITS barcode,which can identify the species.They all have a part as choleretic effect.The studies provide basic date for identification,pharmaceutical analysis,evaluating the quality,the equality and difference on pharmacodynamics,molecular pharmacognosy of multi-origin Tibetan medicine Dida.And provide reference for the study on identification of multi-origin medicinal plants.