Decoction Slices Confirmation,chemical Composition Analysis And Quality Standard Research Of The Famous Classical Formulae "Houpo Wen Zhong Tang"

The formulae have a wide range of clinical applications and the characteristics of traditional Chinese medicine are called famous classical formulae.All of them were born in Qing Dynasty or before it(before 1911 AD).In recent years,the national administration of traditional Chinese medicine has promulgated a series of guiding principles for the study of famous classical formulae.The decoction slices confirmation(inclouding medicinal materials processing),chemical composition analysis and quality standard research are the key technical difficulties in the study of famous classical formulae."Houpo wen zhong tang" is from “Nei wai shang bian huo lun” written by Lee dong-yuan in Jin Dynasty.It has been used for a century to treat diseases of spleen and stomach."Houpo wen zhong tang" has definite clinical effect and high development value.However,the existing research on "Houpo wen zhong tang" can not meet the requirements of the development of famous classical formulae.The key technical difficulties and the weakness in the research of "Houpo wen zhong tang" are taken as the entry point in this research.Firstly,modern instrumental analysis techniques,such as liquid chromatography and liquid chromatography-mass spectrometry,combined with multivariate statistical analysis and fingerprint analysis were used to confirm slices of "Houpo wen zhong tang".Secondly,by comparing the changes of chemical composition in licorice with different processing methods,the processing method of decoction slices was determined.Thirdly,base on the analysis of chemical constituents in vitro and in vivo of "Houpo wen zhong tang",the potential medicinal ingredients were determined.Finally,base on the potential medicinal ingredients,the fingerprint and quality evaluation method of "Houpo wen zhong tang" were established.The main research contents include the following aspects:1.Determination of Citri Reticulatae Pericarpium processing method in "Houpo wen zhong tang".Firstly,the fingerprint of Citri Exocarpium Rubrum and Citri Reticulatae Pericarpium was established by ultra-high performance liquid chromatography(UHPLC).It was found that there was little difference between water extracts from Citri Exocarpium Rubrum and Citri Reticulatae Pericarpium in the fingerprint.Therefore,15 batches of water extracts from Citri Exocarpium Rubrum and Citri Reticulatae Pericarpium were analyzed by UHPLC-Q-TOF-MS.Then the raw data were imported into SIMCA-P software for multivariate statistical analysis.Principal component analysis(PCA)and orthogonal partial least squares discrimination analysis(OPLS-DA)in unsupervised mode were selected to select the compounds with VIP > 1 and p < 0.05 in Citri Exocarpium Rubrum and Citri Reticulatae Pericarpium as potential markers.Then,UNIFI software,ChemBank,PubChem and other websites were used to identify these markers,and a total of 44 different compounds in the water extracts of Citri Exocarpium Rubrum and Citri Reticulatae Pericarpium were identified.The differences of these compounds were directly analyzed by heat map.By comparing the standard and retention time,15 of the 44 different compounds were successfully identified in the fingerprint.Finally,the quantitative analysis of 12 compounds with high peak purity and good separation degree was carried out.It was found that the contents of organic acids and PMFs in the water extract of Citri Exocarpium Rubrum were higher than those in the water extract of Citri Reticulatae Pericarpium.While the contents of oxyside flavonoids in Citri Exocarpium Rubrum were lower than those in the Citri Reticulatae Pericarpium.Experimental results show that the chemical composition of Citri Exocarpium Rubrum and Citri Reticulatae Pericarpium is different.Citri Reticulatae Pericarpium can not substitute Citri Exocarpium Rubrum in "Houpo wen zhong tang".2.Study on processing methods of licorice in "Houpo wen zhong tang".The differences of different processing methods of licorice in "Houpo wen zhong tang" were studied from two aspects: the differences of contents in vitro and the differences of pharmacokinetics in vivo.First of all,the processing methods and their evolution were summarized from literature.The processing method of licorice in "Houpo wen zhong tang" was determined to be roasting.It was different with the processing method of licorice in pharmacopoeia.For that the processing technology of processed licorice was studied.Single factor experiment was carried out with the wetting water and the color change of licorice during processing.The processing technology established was as follows: licorice wetting with 0.4 times or 0.6 times water for the slices,and then the slices were put them on the wire mesh and baked until the color changed in yellow or dark yellow.In order to make the processing technology capable of large industrial production,two processing methods,frying and baking,were selected to optimize the modern processing technology.Based on the content of liquiritin and glycyrrhizic acid in licorice,the technological parameters of frying were optimized.The established processing technology is: the raw licorice slices wetting by0.6 times of water,until all the water suction into slices,then put them into induction cooker at 250 ? to fry them until the color changes into yellow.The method has good stability and reproducibility.The differences about licorice,roasted licorice,fried licorice and honey-roastd licorice were studied based on pharmacokinetics.The main pharmacodynamics where the main pharmacodynamics components in licorice: glycyrrhizic acid(GL),glycyrrhetinic acid(GA),liquiritin(LQ),isiquiriritin(LQ),isiquiriritin(ILQ),liquiritin apioside(LA),glycyrrhizic acid(GL)and glycyrrhetinic acid(GA)as the investigation index.Base on the analysis of the c-t curves and the main pharmacokinetic parameters of the seven main pharmacodynamic components in licorice,roasted licorice,fried licorice and honey-roastd licorice.It was found that the trend of roasted licorice and fried licorice was similar.However,it is significantly different from licorice and honey-roastd licorice,It was found that compared with licorice group,LG decreased and ILG increased in the roasted licorice group,fried licorice group and honey-roastd licorice group.In addition,the LQ increased in the roasted licorice group and fried licorice group,decreased in the honey-processed licorice group.It shows that heating can promote the conversion between LG and ILG,LQ and ILQ.Compared with the licorice group,GL in the roasted licorice group fried and licorice group were increased,GL in the honey-roasted group were decreased,GA in the roasted licorice group and fried licorice group were decreased but GA in the honey-roastd group were increased.It was meant that honey could promote GLmetabolism into GA.The results showed that there were significant differences in the content of the main pharmacodynamics components of licorice,roasted licorice and honey-roastd licorice.And the prosessing method of licorice in "Houpo wen zhong tang" can not substitute by honey-roastd licorice.3.Establishment of the quality evaluation standard of Magnolia officinalis in "Houpo wen zhong tang".Magnolia officinalis is the most important medicine in "Houpo wen zhong tang".The quality of Magnolia officinalis is meaning for the quality standard of "Houpo wen zhong tang".In the pharmacopoeia,magnolol and honokiol were selected as the indexes to measure the quality of Magnolia officinalis.However,both of them are lipid-soluble components with low content in decoction.Therefore,Magnoloside A and syringaresinol are added as the quality evaluation indexes of water-soluble components in the quality standard of Magnolia officinalis.On the basis of pharmacopoeia,the detection wavelength,mobile phase composition,additives,gradient,chromatographic column,column temperature and flow rate were optimized.Moreover,the extraction solvent,extraction method,amount of solvent,extraction time and extraction times of Magnolia officinalis were investigated.Finally,after the verification of linearity,precision,repeatability,stability,reproducibility and recovery rate,a method for the determination of water solubility and lipid solubility in Magnolia officinalis was established.Furthermore,the content of Magnolia officinalis from different habitats was determined.The evaluation method of effect component index was introduced,and the antioxidant capacity related to the pharmacodynamic effect was selected as the evaluation index.The antioxidant capacity of magnolol,honokiol,syringaresinol and Magnoloside A were tested.According to its antioxidant capacity,the weights of four index components and the effect component index were calculated.Finally,the correlation between the effect component index and the antioxidant capacity of 15 batches of Magnolia officinalis was analyzed.A new method for the quality evaluation of Magnolia officinalis was established.The results laid a foundation for establishing the quality evaluation standard of "Houpo wen zhong tang".4.Chemical composition and blood composition identification in "Houpo wen zhong tang".According to the preparation method,the extraction of "Houpo wen zhong tang" was prepared.The chemical components in the extraction were analyzed by UHPLC-Q-TOF-MS.The preliminary identification results of the chemical components of "Houpo wen zhong tang" were obtained through UNIFI database screening.On the basis of the ultraviolet absorption,retention time,peaks information of these compounds,109 compounds were identified,including alkaloids,flavonoids,lignins,phenylpropanoid glycosides,triterpenes,organic acids.These compounds mainly come from Magnolia officinalis,licorice and Citri Exocarpium Rubrum.Then the compounds of "Houpo wen zhong tang" in plasma were analyzed.The identification results of these components laid a foundation for the screening of the pharmacodynamic components of "Houpo wen zhong tang" and its in vivo study.5.Establishment of fingerprint and content determination method of "Houpo wen zhong tang".Firstly,the HPLC condition of "Houpo wen zhong tang" was optimized by liquid chromatography.And the fingerprint of "Houpo wen zhong tang" was established.Furthermore,the characteristics peaks were assigned by comparing the difference among the liquid phase spectra at different wavelengthsof "Houpo wen zhong tang",single herbs and the negative control.By means of methodology,the determination of soluble content of syringaresinol,Magnoloside A,liquiritin and glycyrrhizic acid in the spectra was realized.Since it was difficult to detect the fat-soluble components in water,methanol was used to redissolve the extraction after freeze-drying.A method was established for the determination of hesperidin,alpinetin,pinocembrin,cardamonin,alnustone,magnolol,honokiol,xylanolactone and dehydroxylanolactone.The contents of 13 components in "Houpo wen zhong tang" were determined.Finally,the fluctuation range of the index components in "Houpo wen zhong tang" was determined.Above all,liquid chromatography and liquid chromatography-mass spectrometry were used to analyze whether Citri Exocarpium Rubrum or Citri Reticulatae Pericarpium should be used in "Houpo wen zhong tang".The composition of "Houpo wen zhong tang" wasconfirmed.Base on the different content of the main medicinal ingredients in licorice,the processing method of "Houpo wen zhong tang" was determined.A new method for quality evaluation of "Houpo wen zhong tang" was established by effect-constituent index.The chemical components in "Houpo wen zhong tang" were identified.It was provided to further elucidate the pharmacodynamic components in "Houpo wen zhong tang".The fingerprint and the content determination method of the pharmacodynamic index components of "Houpo wen zhong tang" were established,which provided the theoretical basis for further development.