| This research work was supported by the Shaanxi Provincial Key R&D Project Key Industrial Innovation Chain（Group）-Social Development Field Funding Project-“Demonstrative Research on the Compatibility Theory and Application Technology of Chinese Medicine Compounds Based on the Distribution of Effective Substances”.The research object of this thesis is the compound Chinese patent medicine Qixuehe capsule,which has the functions of soothing the liver and regulating qi,promoting blood circulation and removing blood stasis,and relieving pain.It is mainly used clinically for various symptoms of qi stagnation and blood stasis type menstrual disorders.However,at present,its effective substances and mechanism of action are unclear,and the quality control indicators are single,which greatly limits its clinical rational application and product upgrade and optimization.Therefore,based on integration effects and quality markers,this study carried out the efficacy evaluation,drug-effective material basis and mechanism of action,quality markers,prescription simplification,and components compatibility on Qixuehe capsule,which not only provides scientific evidence for the products upgrading and optimization of Qixuehe capsule,but provides methodological references for the optimization research of the compatibility of traditional Chinese medicine formulae.The thesis is divided into 5 chapters,the specific research contents are as follows:Chapter 1.Literature researchSection 1.Advances in computational network pharmacology for traditional Chinese medicine（TCM）researchTraditional Chinese medicine（TCM）,as a complementary and alternative medicine,has been developed and practiced in China and surrounding countries for its remarkable clinical efficacy.Chinese medicine is the main material means of TCM prevention and treatment of diseases.Hence,systematic analysis and elucidation of the complex chemical basis and action mechanisms of TCM will be highly beneficial,and promote the modernization of TCM.Nowadays,the widespread application of network pharmacology has unveiled the mystery of TCM to some extent by constructing the relationship of“herb-compound-target-disease-syndrome”.Moreover,it can promote the development of drug discovery,medical guidance and the dissection of the syndrome in TCM.With the integration of computational techniques into network pharmacology,the efficiency of data mining and the accuracy of active compounds identification and target fishing have been improved unprecedentedly,and the“herb-compound-target-disease-syndrome”network has been more systematically and comprehensively explained to reflect the holistic mechanisms of TCM.Therefore,a comprehensive overview of each aspect of the use of computational techniques in TCM network pharmacology is urgent.This chapter systematically dissects the core contents involved in TCM computational network pharmacology and severs the cutting-edge study examples to compare and analyze the advantages and limitations of different computational techniques,which provide the insights and methods for computational network pharmacology on TCM research.Section 2.The research idea and design of this paperThis section aims to explain the overall framework of research ideas,paper design and research content.By replicating the pathological model of qi stagnation and blood stasis syndrome in rats around Qixuehe capsule,the following key problems are intended to be solved:（1）Systematically elucidate the pharmacodynamic substance basis and dose-effect relationship of Qixuehe capsule;（2）The quantitative identification of quality markers of Qixuehe capsule;（3）The optimization and discovery the simplified prescriptions and compatibility components of Qixuehe capsule;（4）To preliminarily explore the mechanism of Qixuehe capsule in treating qi stagnation and blood stasis syndrome.Chapter 2.Research on the material basis and mechanism of the action of Qixuehe capsule based on qi stagnation and blood stasis syndromeSection 1.Evaluation of pharmacodynamics of Qixuehe capsule on qi stagnation and blood stasis syndromeLong-term subcutaneous injection of low-dose epinephrine and chronic unpredictable stimulation were used to replicate the qi stagnation and blood stasis syndrome model in rats.The rats were divided into normal control group,model group,Qixuehe capsule clinical equivalent dose,clinical 3 times dose,clinical 6 times dose administration groups,compound danshen dripping pills（CDDP,positive control）group,respectively,through hemorheology（WBV,PV）and coagulation function（PT,APTT,TT,FIB）indexes,combined with principal component analysis（PCA）and orthogonal/partial least squares discriminant analysis（OPLS-DA/PLS-DA）analysis,the effect of Qixuehe capsule in the treatment of qi stagnation and blood stasis syndrome was evaluated.The results showed that each dose group of Qixuehe capsule has significant effect on promoting blood circulation and removing blood stasis,especially the clinical equivalent dose of Qixuehe capsule is the best.This study provides a basis for the rational clinical application of Qixuehe capsule.Section 2.A network pharmacology approach to uncover the mechanisms of Qixuehe capsule for the treatment of qi stagnation and blood stasis syndromeTo explore the active components and mechanisms of Qixuehe capsule in treating qi stagnation and blood stasis syndrome.By the combination of TCMSP,TCM-MESH databases and literature mining,taking OB≥30%,DL≥0.18 and Caco-2≥-0.4 as criteria,the active components of Qixuehe capsule were screened.The target proteins of Qixuehe capsules were predicted based on TCMSP,TCM-MESH,CTD and other databases,and all target proteins were assigned to the corresponding pathological processes of qi stagnation and blood stasis syndrome according to the molecular function of target proteins by Uniprot and Gene Cards database.The“drug-component-target-pathological process/manifestation”network module of Qixuehe capsule was constructed by Cytoscape.Then,GO enrichment and pathway analysis were carried out on the targets of each network module.Through network pharmacology,Qixuehe capsule may improve hemorheological abnormality mainly via 185 compounds-51 targets;184 compounds-68 targets associated with Qixuehe capsule in improving coagulation disorders;174 compounds-24 targets associated with Qixuehe capsule in relieving pain;191 compounds-24 targets associated with Qixuehe capsule in anti-inflammation;105 compounds-27 targets associated with Qixuehe capsule in improving immunity.Conclusion Qixuehe capsule may regulate and improve the 5 pathological processes such as hemorheological abnormality,coagulation disorders,pain,inflammation and immunity to treat qi stagnation and blood stasis syndrome.This study systematically revealed the potential active components and possible mechanisms of Qixuehe capsule in the treatment of qi stagnation and blood stasis syndrome,which provided a theoretical basis for further experimental research on the chemical material basis and mechanism of action.Section 3.Study on the dose-effect relationship and mechanism of action of Qixuehe capsule for treating qi stagnation and blood stasis syndrome based on metabonomicsLong-term subcutaneous injection of epinephrine hydrochloride and chronic unpredictable stimulation were used to replicate the qi stagnation and blood stasis syndrome model in rats.The rats were divided into model group,normal control group,and Qixuehe capsule administration groups（0.5 times dose group,clinical equivalent dose group,2 times the dose group,3 times the dose group,4 times the dose group,5 times dose group）,through hemorheology（WBV and PV）and coagulation function（PT,APTT,TT,FIB）related indicators to evaluate the effect of Qixuehe capsule for promoting blood circulation and removing blood stasis,combined with multi-attribute comprehensive index methods to determine the dose-effect relationship of Qixuehe capsule in treating qi stagnation and blood stasis syndrome:Qixuehe capsule clinical equivalent 5 times dose>Qixuehe capsule clinical equivalent dose>Qixuehe capsule clinical equivalent 4 times dose>Qixuehe capsule clinical equivalent 1/2 times dose>Qixuehe capsule clinical equivalent 2 times dose>Qixuehe capsule clinical equivalent 3 times dose.UPLC-Q-TOF-MS technology was used to determine the endogenous metabolites in the plasma of each group of rats.Principal component analysis（PCA）and orthogonal/partial least-squares discriminant analysis（OPLS-DA/PLS-DA）were used for multivariate statistical analysis of endogenous markers in plasma,and P test for potential differential metabolites was performed based on SPSS19.0.The results showed that the metabolic profile of the model group rats in the PCA score chart can be better separated from other groups.Furthermore,metabolites such as Lyso PC（18:0）,Lyso PC（16:0）,PC（16:0/22:6（4Z,7Z,10Z,13Z,16Z,19Z））,Lyso PC（18:1（9Z））,Lyso PC（20:4（5Z,8Z,11Z,14Z））,etc.are qualitatively identified as potential biomarkers from the plasma metabolic profiles of model group rats,and these endogenous substances were recovered to varying degrees in other administration groups.Through the Met PA pathway analysis,the qi stagnation and blood stasis syndrome model is mainly related to 7 metabolic pathways,including Glycerophospholipid metabolism,Linoleic acid metabolism,Alpha-Linolenic acid metabolism,Glycosylphosphatidylinositol（GPI）-anchor biosynthesis,Ether lipid metabolism,Arachidonic acid metabolism,and Primary bile acid biosynthesis.Qixuehe capsule can call back the abnormally changed endogenous differential metabolites of rats with qi stagnation and blood stasis syndrome,and regulate the above-mentioned metabolic pathways to treat qi stagnation and blood stasis syndrome.Chapter 3.Study on the quality markers of Qixuehe capsuleSection 1.Identification of quality markers of Qixuehe capsule based on analytic hierarchy process and entropy weight methodsQixuehe capsule is a Chinese patent compound medicine developed for treating the disorder of qi and blood（the common etiology of gynecological disease）for its significant effect on smoothing liver and regulating qi,activating blood circulation and relieving pain.However,the quality control of Qixuehe capsule has always been a bottleneck problem limiting sustainable development due to its complex formulae（15 herbs）and multiple effects.Therefore,the TCM quality markers quantitative identification system based on the combination of analytic hierarchy process and entropy weight methods established previously by our research group was employed in this study.Taking the different effects of Qixuehe capsule as the entry point,the comprehensive scores of chemical substances in Qixuehe capsule under the items of effectiveness（smoothing liver and regulating qi,activating blood circulation and relieving pain）,testability and specificity was calculated and integrated respectively.Subsequently,by analyzing the compatibility relationship of Qixuehe capsule,15 active ingredients with high comprehensive scores including Ferulic acid,Quercetin,Caffeic acid,Kaempferol,Rutin,Z-Ligustilide,Senkyunolide I,Vanillic acid,Protocatechuic acid,Chlorogenic acid,Rosmarinic acid,Senkyunolide A,Gallic acid,Tetrahydropalmatine and Eugenol have been found to be the quality markers of Qixuehe capsule.Collectively,this study not only provides scientific evidence for further research on the improvement and standardization of quality standards of Qixuehe capsule but also provides methodological references for the quantitative identification of quality markers for multi-effect TCM formulae.Section 2.Study on the quantitative method of main components of Qixuehe capsule based on quality markersIn this study,high performance liquid chromatography was used to establish a quantitative research method for the simultaneous determination of 5 main components in Qixuehe capsule.Cluster analysis was used to analyze the rules of 5 components in 10batches of Qixuehe capsules and established the fingerprints of Qixuehe capsule.Using Waters Sun Fire C18 chromatographic column（4.6 mm×150 mm,5μm）;using methanol（A）-0.2%formic acid water（B）as mobile phase,gradient elution;flow rate 1.0 m L·min-1;detection wavelength 280nm（salvianolic acid B,（Z）-ligustilide,naringin,neohesperidin）,235nm（paeoniflorin）;column temperature 30℃;sample volume 20μL;The chromatographic peaks all reached the baseline separation of 5 components in 10 batches of Qixuehe capsule from the market,The linear equations of paeoniflorin,naringin,neohesperidin,salvianolic acid B,and ligustilide are Y=24784X-59263（r=0.9999）,Y=30425X-13282,（R=0.9999）,Y=39424X-36032（r=0.9998）,Y=7868.5X-16625（r=0.9999）,Y=21701X-36214（r=0.9999）,respectively;the average sample recovery rate was 100.53%（RSD=0.68%）,100.09%（RSD=2.01%）,98.82%（RSD=1.72%）,103.19%（RSD=1.03%）,92.43%（RSD=2.08%）;among 10 batches of blood and capsule samples The content range of the above 5 components is 2.42～4.93 mg·g-1,2.80～3.16 mg·g-1,1.28～1.44 mg·g-1,2.82～4.54 mg·g-1,2.21～3.62 mg·g-1;Then based on cluster analysis to evaluate the difference in the component content of 10 batches of Qixuehe capsule samples,the results show that the difference in the content of the above 5 components of Qixuehe capsule is small and the quality is uniform.The method is simple,sensitive,reliable and accurate,and provides a reference for the quality improvement research of Qixuehe capsule.Chapter 4.Prescription simplification study of Qixuehe capsuleSection 1.Comparative study of Qixuehe capsule prescription simplification effects based on quality marker analysisBased on the relevant data of the research on quality markers of Qixuehe capsule,the scores of the chemical components of Qixuehe capsule in the functions of soothing the liver and regulating qi,promoting blood circulation and removing blood stasis,and relieving pain were obtained,and the weight of each functional item was obtained according to the analytic hierarchy process and entropy weight method,further the comprehensive score of active components of Qixuehe capsule was obtained by comprehensive analysis,and then assign each active component to Qixuehe capsule corresponding herbs according to the source of medicinal materials,that is,to obtain the contribution of each herb in Qixuehe capsule prescription to the effect score,delete the herbs that contributes less to the efficacy,Qixuehe capsule simplification prescription B（removing Persicae Semen,Platycodonis Radix,and Linderae Radix）and Qixuehe capsule simplification prescription C（removing peach Persicae Semen,Platycodonis Radix,Linderae Radix,Bupleuri Radix,and Cimicifugae Rhizoma）were obtained.Based on the rat model of qi stagnation and blood stasis syndrome,the hemorheology（WBV and PV）and coagulation function（PT,APTT,TT,FIB）related indicators to evaluate the effect of Qixuehe capsule on promoting blood circulation and removing blood stasis.Comprehensive analysis by multi-attribute comprehensive index methods shows that the self-made Qixuehe capsule original prescription group,Qixuehe capsule simplified prescription group B high-dose and Qixuehe capsule simplified prescription C low-dose group have significant effect on promoting blood circulation and removing blood stasis,and can obviously improve the abnormalities of hemorheology and coagulation functionare in rats with qi stagnation and blood stasis syndrome,but its effect of promoting blood circulation and removing blood stasis is not as good as that of the clinically equivalent of Qixuehe capsule group.This study provides a reference for the in-depth study of Qixuehe capsule prescription simplification.Section 2.Study on the mechanism of Qixuehe capsule simplified prescription based on metabolomicsLong-term subcutaneous injection of low-dose epinephrine hydrochloride and chronic unpredictable stimulation were used to replicate the qi stagnation and blood stasis model in rats.The rats were divided into normal control group,model group,clinical equivalent dose group,self-made Qixuehe capsule original prescription group,streamline the prescription B low doses of Qixuehe,streamline the prescription dose of B middle dose of Qixuehe,streamline the prescription B high dose of Qixuehe,streamline the prescription C low doses of Qixuehe,streamline prescription C middle dose of Qixuehe,and streamline the prescription C high dose of of Qixuehe,compound danshen dripping pills（CDDP,positive control）group.UPLC-Q-TOF-MS technology was used to determine the endogenous metabolites in the plasma of each group of rats.Principal component analysis（PCA）and orthogonal/partial least-squares discriminant analysis（OPLS-DA/PLS-DA）were used for multivariate statistical analysis of endogenous markers in plasma,and P test for potential differential metabolites was performed based on SPSS 19.0.The results showed that the metabolic profile of the model group rats in the PCA score chart can be better separated from other groups.Furthermore,metabolites such as,Palmitic acid,Retinyl ester,PE（P-18:0/22:4（7Z,10Z,13Z,16Z））,PC（20:4（8Z,11Z,14Z,17Z）/18:4（6Z,9Z,12Z,15Z））,PC（22:6（4Z,7Z,10Z,13Z,16Z,19Z）/16:1（9Z））,8,11,14-Eicosatrienoic acid,Lyso PC（20:5（5Z,8Z,11Z,14Z,17Z））,etc.,are qualitatively identified as potential biomarkers from the plasma metabolic profiles of model group rats,and these endogenous substances were recovered to varying degrees in other administration groups.Through the Met PA pathway analysis,the qi stagnation and blood stasis syndrome model is mainly related to 9 metabolic pathways,including Biosynthesis of unsaturated fatty acids,Linoleic acid metabolism,Glycerophospholipid metabolism,alpha-Linolenic acid metabolism,Fatty acid biosynthesis,Glycosylphosphatidylinos-itol（GPI）-anchor biosynthesis,Retinol metabolism,Arachidonic acid metabolism,Primary bile acid biosynthesis,etc.Qixuehe capsule simplified prescription can call back the abnormally changed endogenous differential metabolites of rats with qi stagnation and blood stasis syndrome,and regulate the above-mentioned metabolic pathways to treat qi stagnation and blood stasis syndrome.Chapter 5.Study on the components compatibility of Qixuehe capsuleSection 1.Comparative study on effect between components compatibility of Qixuehe capsule based on quality marker analysisBased on the relevant data of the research on quality markers of Qixuehe capsule,the scores of the chemical components of Qixuehe capsule in the functions of soothing the liver and regulating qi,promoting blood circulation and removing blood stasis,and relieving pain were obtained,and the weight of each functional item was obtained according to the analytic hierarchy process and entropy weight method,futher the comprehensive scores of the Qixuehe capsule active ingredients in soothing liver and regulating qi,promoting blood circulation and removing blood stasis,and relieving pain effects were obtained by comprehensive analysis,and finally determined that ferulic acid was the representative component of soothing liver and regulating qi,and salvianolic acid B was the representative component of promoting blood circulation and removing blood stasis,tetrahydropalmatine was the representative component of relieving pain,respectivelly.Set up three dosage groups of ferulic acid-salvianolic acid B-tetrahydropalmatine composition（WBV and PV）with high,medium and low doses.Based on the aforementioned rat model of qi stagnation and blood stasis syndrome,through hemorheology（WBV and PV）and coagulation function（PT,APTT,TT,FIB）related indicators to evaluate the effect of Qixuehe capsule on promoting blood circulation and removing blood stasis,and the results showed that the composition of ferulic acid-salvianolic acid B-Tetrahydropalmatine had a dose-dependent:high-dose>medium-dose>Qixuehe capsule clinical equivalent dose>low-dose.Section 2.Study on the mechanism between components compatibility of Qixuehe capsule based on metabolomicsLong-term subcutaneous injection of low-dose epinephrine hydrochloride and chronic unpredictable stimulation were used to replicate the qi stagnation and blood stasis model in rats.The rats were divided into normal control group,model group,Qixuehe capsule clinically equivalent administration group,ferulic acid-salvianolic acid B-tetrahydropalmatine compatibility group low dose（TSF-L）,ferulic acid-salvianolic acid B-tetrahydropalmatine compatibility group medium dose（TSF-M）,ferulic acid-Salvianolic acid B-Tetrahydropalmatine compatibility group high-dose（TSF-H）,compound danshen dripping pills（CDDP,positive control）administration group.UPLC-Q-TOF-MS technology was used to determine the endogenous metabolites in the plasma of each group of rats.Principal component analysis（PCA）and orthogonal/partial least-squares discriminant analysis（OPLS-DA/PLS-DA）were used for multivariate statistical analysis of endogenous markers in plasma,and P test for potential differential metabolites was performed based on SPSS 19.0.The results showed that the metabolic profile of the model group rats in the PCA score chart can be better separated from other groups.Furthermore,metabolites such as Lyso PC（18:0）,PC（18:0/16:1（9Z））,Lyso PC（18:2（9Z,12Z））,PC（20:4（5Z,8Z,11Z,14Z）/18:0）,Lyso PC（20:3（5Z,8Z,11Z））,etc.,are qualitatively identified as potential biomarkers from the plasma metabolic profiles of model group rats,and these endogenous substances were recovered to varying degrees in other administration groups.Through the Met PA pathway analysis,the qi stagnation and blood stasis syndrome model is mainly related to 9 metabolic pathways,including Linoleic acid metabolism,Glycerophospholipid metabolism,Taurine and hypotaurine metabolism,alpha-Linolenic acid metabolism,Glycosylphos-phatidylinositol（GPI）-anchor biosynthesis,Arachidonic acid metabolism,Biosynthesis of unsaturated fatty acids,Tyrosine metabolism,Primary bile acid biosynthesis,etc.Ferulic acid-salvianolic acid B-tetrahydropalmatine compatibility group can call back the abnormally changed endogenous differential metabolites of rats with qi stagnation and blood stasis syndrome,and regulate the above-mentioned metabolic pathways to treat qi stagnation and blood stasis syndrome. |
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