| Xiang-Sha-Liu-Jun-Zi-Jia-Jian(XSLJZJJ)granules,is conducted on the basis of Xiang-Sha-Liu-Jun-Zi-Tang with the adding and subtracting of the herbs,consisting of nine herbal medicines including Radix Astragali preparata(Zhihuangqi),Radix Codonopsis(Dangshen),Rhizoma Corydalis(Yanhusuo)and so on,which has been used for the treatment of spleen-deficiency and qi-stagnation syndrome of functional dyspepsia(FD).There were many studies about the chemical constituents of each single herb in the formula,and these herbs contained various structure types of compounds,including flavonoid aglycones,flavonoid glycosides,triterpene saponins,triterpenoid acid,alkaloids,lactones,polyacetylene glycosides,amino acids,organic acids and so on.However few studies reported the chemical components,the compounds in vivo and their metabolic pathway of the global formula,so do as the objective evaluation of its effect and the effective substances on the treatment of spleen-deficiency and qi-stagnation syndrome of FD.In this study,the Chemome of XSLJZJJ granules was analyzed qualitatively firstly,and based on the profiling of chemical compounds,the metabolism of this preparation in normal rats was studied.Then,taking XSLJZJJ granules as the drug carrier,spleen-deficiency and qi-stagnation syndrome of FD as the disease model,the potential effective substances of this formula were explored starting from serum medicinal chemistry and pathogenic organ-intestinal tract two main ways combining biological evaluation system.This study provided a reference for the illumination of the effective substances of TCM or formula.The main research contents are as follows:1.Compounds analysis of Xiang-Sha-Liu-Jun-Zi-Jia-Jian granules and its representative single herbHigh resolution liquid chromatography-mass spectrometry technology has become a powerful tool to study the chemical composition of traditional Chinese medicine.But the various chemical compositions and the content in large range of compound in traditional Chinese medicine make it not easy to identify peaks clearly and accurately when collecting or analyzing data.Based on the identification strategy of component in formula from the the high dose of herb to the equivalent dose of the formula combining with characteristic ions,multistage mass fragmentation pattern,retention behavior,reference substance and literature at the same time,we applied high performance liquid chromatography combined with linear ion trap-electrostatic field Fourier transform mass spectrometry(HPLC-LTQ-Orbitrap/MS)to identify components of Xiang-Sha-Liu-Jun-Zi-Jia-Jian granules simultaneously.A total of 169 compounds were identified or tentatively characterized,including 30 flavonoid aglycones,29 flavonoid glycosides,26 alkaloids,23 triterpenic acids,19 triterpene saponins,16 lactones,5 sesquiterpenoids,4 amino acids,3 hexanol glycosides,2 hexenol glycosides,2 polyacetylene glycosides,2 phenylpropanoid glycosides,2 phenolic acids,2 nucleosides,2 saccharides,1 iridoid glycoside and one other compound(5-hydroxymethylfuraldehyde).The results showed that the HPLC-LTQ-Orbitrap/MS method combining with the herbal affiliation of the components in TCM formula was a rapid and reliable method which could be used to identify the chemical constituents from the extract of Xiang-Sha-Liu-Jun-Zi-Jia-Jian granules.This study provide reference for revealing the effective substances and clinical effect of this formula,and provide a strategy for multi-component qualitative analysis of TCM formula.In addition,the global compounds of Ligusticum chuanxiong Hort.(Chuanxiong)which was the representative herb of Xiang-Sha-Liu-Jun-Zi-Jia-Jian granules were characterized comprehensively by UPLC-Q-TOF/MS~E.A total of 81 phthalides including 26 potential new ones from Chuanxiong were characterized finally using the complementary information of"structure-mass spectrometry" and "structure-retention time" aimed at the isomerism of phthalides in Chuanxiong.This part of the study provided a reference for the quality control and pharmacological research of Chuanxiong herbs,and also lay the material and method foundation for the study on chemical compounds of Xiang-Sha-Liu-Jun-Zi-Jia-Jian granules.2.The metabolism study of Xiang-Sha-Liu-Jun-Zi-Jia-Jian granules in spleen-deficiency and qi-stagnation syndrome of FD model ratsIn this study,Xiang-Sha-Liu-Jun-Zi-Jia-Jian granules for the treatment of spleen-deficiency and qi-stagnation syndrome of FD as the medicine carrier,on the base of the chemical compositions of the formula and the mass fragmentation rules,and the metabolism of "representative standards-single herbal medicine-formula" as the main way,a research strategy based on UPLC-Q-TOF/MS~E technology combining the screening method of medicine metabolites was proposed to characterize drug-induced constituents of the formula in normal rats’plasma and feces samples.A total of 75 drug-induced constituents were identified in rat plasma samples including 27 prototype components and 48 metabolites.And a total of 74 drug-induced constituents were identified in the rat feces samples,including 32 prototype components and 42 metabolites.This study could help to reveal the potential effective substances of this formula,which provided a reference for the study method of metabolism of TCM formula,further pharmacodynamics research and the application in clinical.In order to reveal the effective substances on the treatment of spleen deficiency and qi stagnation syndrome of FD furtherly,The rat model was established by Iodoacetamide and small platform fatigue factors,and was also evaluated.To reveal effective substances of this formula in vivo,we analyzed and characterized the compositions with migration elimination rules in model rat blood after given XSLJZJJ granules and identified drug-induced constituents in pathogenic organ-intestinal tract(feces).After setting the candidate ion pair list based on the previous analysis of the normal blood component analysis,blood samples were analyzed at the time of administration of each rat before and after administration in MRM scanning mode of UPLC-Q-TOF/MS~E technique.The chromatographic peak areas of each ion channel corresponds to the retention time were calculated,then the trend figure of components with migration elimination in blood was descripted.The results showed that.eight compounds including tetrahydropalmatine,glycyrrhizic acid,tetramethoxy flavone and dehydroeburicoic acid et al.had better migration and elimination behavior,indicating that these compounds were more likely to be effective.Besides,stool samples were also analyzed with UPLC-Q-TOF/MS~E technique.A total of 73 drug-induced constituents were identified,including 32 prototype components and 41 metabolites,of which 69 components compared with those detected in normal rats were common.Tetrahydrocannabinol、tetrahydrofuran Tetrahydrohexane Tetrandrine and or sulfated products four metabolites only were found in the model group,which indicated that these metabolites and their prototypes may be associated with pharmacological effects.In short,this study explained the potential effective substances of Xiang-Sha-Liu-Jun-Zi-Jia-Jian granules to treat spleen-deficiency and qi-stagnation syndrome of functional dyspepsia,which laid the foundation for the further pharmacological study and pharmacodynamics evaluation of the formula.3.The study on the regulation of Xiang-Sha-Liu-Jun-Zi-Jia-Jian granules to intestinal microecology in model rats based on the diversity of gut microbiota16S rDNA sequencing technique was used to study the regulation effect of Xiang-Sha-Liu-Jun-Zi-Jia-Jian granules on gut microbiota of model rats.Differences of diversity of gut microbiotain for the characterization of disease model by a statistical analysis of bioinformatics between the normal group and the model group,which mainly related to the genus of Prevotella,Alloprevotella,Ruminococcus and Enterococcus.After the intervention of the medicine,the diversity of the gut microbiota was mainly related to the genus of Ruminococcus,Prevotella and Alloprevotella.It was reported that Ruminococcus P01-3 could metabolize glycyrrhizic acid.And another research showed that there was a stable interaction between alkaloids of Yanhusuo and gut microbiota,a variety of metabolites of tetrahydropalmatine and a number of protoberberine type alkaloid were detected in the feces samples.The results provided an objective reference for evaluating the efficacy of the formula on the treatment of spleen-deficiency and qi-stagnation syndrome of functional dyspepsia,and further revealed the potential effective substances of the formula by the retrospect of chemical composition of this formula.In this paper,we analyzed and characterized chemical compositions of Xiang-Sha-Liu-Jun-Zi-Jia-Jian granules systematically,also proposed a identification strategy of TCM formula which was carried out from the the high dose of herbs to the equivalent dose of the formula.Taking the chemical compositions of the formula and their mass fragmentation rules as the foundation and the metabolism of "representative standards-single herbal medicine-formula" in vivo as the main route,a research strategy based on UPLC-Q-TOF/MS~E combining with the screening method of medicine metabolites was proposed to characterize drug-induced constituents in normal rats’ blood and feces samples.After a comprehensive qualitative analysis of components of this formula in vitro and in vivo,to reveal effective substances of this formula preliminarily,we analysed and characterized the compositions with migration elimination behavior in model rat blood after given XSLJZJJ granules and identified drug-induced constituents in feces sample.Combining with the study of the diversity of gut microbiota,previous reports and the evaluation of efficacy,this study reveals the potential effective substances which had a reverse regulation effect on spleen-deficiency and qi-stagnation syndrome of FD in Xiang-Sha-Liu-Jun-Zi-Jia-Jian granules.The current study provided a reference for the research of efficacy material basis and offered theoretical basis for the quality evaluation and clinical application of TCM formula. |
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