Design Studies Of The Fractional Optimization Of TCM Formula (MXST)

Design Studies of the Fractional Optimization of TCM Formula(MXST)A Traditional Chinese Medical formula(TCM formula) is one of the most important parts of the Chinese medical science.Studies on composing prescriptions and drug compatibility are the core of TCM formulas.With the changes of medical mode,more and more new TCM formulas are required.It is the key question to design the prescriptions.This paper puts forward and verifies a new design method of new TCM formulas.Orthogonal experiment design is used during the formulation design of a new TCM formula which is composed of a classical prescription and effective TCM.Two key links in a new TCM formula are the best dose design and drug compatibility,which is obtained by using classical animal model and special pharmacology index; The best chemical preparation is obtained by using the chemical and analysis techniques.System studies on the pharmacology embody the whole view of chemistry of compound prescriptions.System researches on the pharmacology and Pharmacokinetics and the correlation with TCM theory promote the subject development of the TCM prescription.By taking MXSTF as a research object,exploratory researches on TCM formula prove the feasibility and scientific.1 The base of prescription formulation and dose screening on the basis of pharmacodynamics of MXSTFFormula screening by the research of pharmacodynamics was studied firstly.The basic recipe is drafted based on the clinical efficacy and PRC codex.Analgesic effect was the observation index by the orthogonal design.Efficacy impact was studied by TCM formulas with different proportions and doses on three animal models of migraine,then the best drug compatibility and dose ratio were screened.1.1 Influences on the model rats of migraine induced by nitroglycerin by TCM formulas with different proportions and dosesThe influences on the model rats of migraine induced by nitroglycerin by TCM formulas with different proportions and doses were observed.Results:The tenth sample(MXSTF) has the best analgesic effect on the model rats of migraine induced by nitroglycerin.Compared to the model control group,it is showed statistical difference(P<0.05).1.2 Studies on the pain time of model mouse induced by hot plate by TCM formulas with different proportions and dosesThe studies on the pain time of model mouse induced by hot plate by TCM formulas with different proportions and doses were observed.Results:Pain Time was obviously postponed on the model mouse induced by hot plate because of the second sample,the eighth sample and the tenth sample.Compared to the model control group,it is showed statistical difference(P<0.05).1.3 Influences on the delay time of model mouse of induced by 2%formaldehyde by TCM formulas with different proportions and dosesThe influences on the delay time of model mouse of induced by 2%formaldehyde by TCM formulas with different proportions and doses were observed.Results:Delay time was obviously longer then other samples on the model mouse ofâ…¡-phase-pain induced by 2%formaldehyde because of the sixth sample, the seventh sample and the tenth sample.It is showed statistical difference when compared to the model control group(P<0.05).In a word,the base of prescription formulation and dose screening of the TCM formula curing migraine were set up according to these pharmacological effects,which include the famous drug pair and classical drug team.2 Studies on the pharmacology of TCM formulas with different therapeutic principle compatibilityThe influence on the contents of index components was observed by TCM formulas with different therapeutic principle compatibility in rat's brain tissue and serum.The model rats of migraine induced by nitroglycerin and special pharmacology index were used during the experiment in order to verify the pharmacological action of MXSTF.The contents of 5-HT,CGRP and ET-1 in rat's serum were statistically analyzed among normal control group,positive drug control group and TCM formulas groups with different therapeutic principle compatibility.Compared with model group,there were significant differences in 5-HT level from the group of Rhizoma Gastrodiae and Rhizoma Chuanxiong(P<0.05),in 5-HT and ET-1 levels from MXSTF water extract(P<0.05),and in 5-HT,ET-1å’ŒCGRP levels from MXSTF alcohol extraction(P<0.05).The levels of 5-HT in rat's brain tissue were statistically analyzed among normal control group,positive drug control group and TCM formulas groups with different therapeutic principle compatibility. The results show that there were obvious differences among Rhizoma Gastrodiae and Rhizoma Chuanxiong group,MXSTF water extract and MXSTF alcohol extraction when compared with model group(P<0.05 ).The pharmacological actions show that MXSTF is superior to Rhizoma Gastrodiae and Rhizoma Chuanxiong group and MXSTF alcohol extraction is better than MXSTF water extract.3 Studies on the pharmacy of MXSTFThe pharmacy studies is the base of the study of TCM formula.In order to improve clinical effect,the main material basis of drug must be extracted as much as possible.Technology is a key link to the study on the pharmacy of TCM formula.It is one of the issues that how to correctly evaluate the preparation process of Chinese medicine.3.1 Effects on the pharmacodynamic of different ingredients extracted from ChuanxiongPharmacodynamic experiments were as indexes when the route of drug extraction is difficult to determine.Many ingredients were extracted from Chuanxiong.All of them were compared with the pharmacodynamics of migraine.The mode with the combination of Chinese Medicine Chemistry and Pharmacology of TCM is used to determine the extraction route of Chuanxiong.The content of 5-HT was determined by electrochemical method during migraine.Then chuanxiong is extracted by ethanol;Other drugs were also extracted by ethanol.3.2 Determination of the extraction route and the extraction wayUsing effective components of monarch drug and ministerial drugs as a parameter,the effects of mixed extracting and separate extracting were observed.The ratio of mixed extracting/separate extracting for Gastrodin is 102.42%,the ratio of mixed extracting/separate extracting for Puerarin is 108.54%.The dates showed that mixed extracting is more beneficial to the extraction of Tianma and Gegen.The ratio of mixed extracting/separate extracting for Ferulic acid is 99.46%.The mixed extracting is equal to separate extracting for Ferulic acid,so the mixed extracting was chosen for Chuanxiong. 3.3 Studies on the technology of MXSTFThe optimum mixture extraction process by L₉(3⁴) is as follows:80%alcohol extracting third,adding 8times amount of alcohol into Chinese medical materials,once 2 hours.The method has features of low cost,simple process and effectiveness,which will be advantageous to extend and use the formula.4 Quality control of TCM formula(MXSTF)4.1 Instrument and testing drugHPLC:Waters 600 pump,Waters 996 detertor,Waters 717 auto sampler,Millennium³² Login;TLC scanner,TRANSILLUMINATOR 2020D;air compressor pump,TOSHIBA Corporation.Reference substance:Ferulic acid,Gastrodin,Puerarin;Reference drugs:Rhizoma Chuanxiong,Radix Angelicae Dahuricae.4.2 TLC chromatographic identificationn-Hexane-ethyl acetate(9:1) was used for Rhizoma Chuanxiong with crude standard as reference; Trichloromethane- methanol-water(7:2.5:0.25) was used for Radix puerariae lobatae;Petroleum ether-Ether(1:1) was used for Radix Angelicae Dahuricae with crude standard as reference.4.3 Determination4.3.1 Determination of Ferulic acid in sampleColumn:SymmetryC₁₈(3.9×150 mm,5μm);The mobile phase A was acetonitrile-0.05%acetic water, the mobile phase B was methanol.0-45 min,A from 100%to 60%,B from 0%to 40%;45 - 50 min,A from 60%to 0%,B from 40%to 100%;The detection wavelength was 323 nm;Column temperature was 30℃;The flow rate was 1.0 ml·min^-1;A good linearity of Ferulic acid presented over the range of 0.02715-0.4887μg(γ=0.9998,n=7);The average recovery rate for the content determination was up to 98.22%,RSD=1.30%(n=5);Determination of Ferulic acid in the preparation was not less than 0.18 mg per one gram.4.3.2 Determination of Gastrodin in sampleColumn:SymmetryC₁₈(3.9×150 mm,5μm);The mobile phase was acetonitrile-0.05%acetic water (2:98);The detection wavelength was 220 nm;The flow rate was 0.6 ml·min^-1;Column temperature was room temperature;A good linearity of Gastrodin presented over the range of 0.1032-1.0320μg(γ=1.0000, n=7);The average recovery rate for the content determination was up to 97.81%,RSD=1.49%(n=6); Determination of Gastrodin in the preparation was not less than 1.92 mg per one gram.4.3.3 Determination of Puerarin in sampleColumn:SymmetryC₁₈(3.9×150mm,5μm);The mobile phase A was acetonitrile-0.05%acetic water, the mobile phase B was methanol.0-45 min,A from 100%to 60%,B from 0%to 40%;45 - 69 min,A from 60%to 0%,B from 40%to 100%;The detection wavelength was 250 nm;Column temperature was 30℃;The flow rate was 1.0 ml·min^-1;A good linearity of Puerarin presented over the range of 0.164-2.05μg(γ=0.9998,n=9);The average recovery rate for the content determination was up to 97.87%, RSD=1.57%(n=6);Determination of Puerarin in the preparation was not less than 13.18 mg per one gram.5 Studies on Absorption Kinetics of Multi-composition by Intestinal Backflow in vivoIntestinal absorption behaviors of Ferulic acid,Gastrodin and Puerarin were observed in this thesis. Compared to single herb,the uptakes amount per unit area of Ferulic acid increase in the TCM formula, while the uptake amounts of Gastrodin and Puerarin per unit area decrease.Synergistic or antagonistic effect was shown between compatibility in TCM formula.With drug concentration increasing,the uptake amounts of Gastrodin and Puerarin were increased.The absorption way fits the passive transport mechanism;Based on the great decrease of Gastrodin in TCM formula,the studies of compatibility of Tianma with different drugs were carried out.The absorption rate of Chuanxiong and Tianma,Baizhi and Tianma were all for 90%,while the absorption rate of Gengen and Tianma was 46%.Therefore Gengen is the herb source which restrains the absorption of Gastrodin in intestinal.6 Qualitative Study on Multi-composition in TCM Formula by LC-MS-MSThis paper established gradient elution system which can include multi-composition in the same chromatogram.Eight kinds of elements of Chuanxiong and Gengen dissolved in Krebes -Ringer's solution were determined with LC-MS-MS negative ion scan mode,which are Ferulic acid,daidzein 4'-7-diglucoside,3'-OH Puerarin,Puerarin,3'-OCH₃ Puerarin,Puerarin xyloside,Daidzin and Daidzein respectively.It needs to further research on two unknown materials and Scoparone.The source of Scoparone needs to be further determined because the TCM formula do not include the plants which Scoparone coming from,namely Artemisia scoparia Waldst.et Kit.,A.capillaris Thunb.,Aegle marmelos Corr.and Bupleurum fruticescens L.Qualitative study by LC-MS-MS is as a basis for further studies on the material base and metabolism in vivo.