| This work was supported by National Natural Science Foundation of China and Natural Science Foundation of Jiangsu Province,China—Part experiment content of "Material basis and correlated mechanism research of efficacy changes of the herb pair constituented by Angelica sinensis and Ligusticum chuanxiong with different proportions and preparation methods".This dissertation is divided into two major parts.In the first part,this paper reviewed the system in effect relations,drug drug interactions,berganottin chemical and pharmacological scattered research progress.There are six chapters in second part,specific research content and correlated conclusion as follows:There are three segments in the first chapter about the sample preparation of danggui and chuanxiong.First segment,Angelica-river xiong for different ratio(1:0,1:0.176,1:0.364,1:0.577,1:0.839,1:1.192,1:1.732,1:2.747,1:5.67,0:1)mixed samples of the medicinal material thermal circumfluence water extraction(simulation water frying),thermal circumfluence 50%aqueous extract(simulation wine boiled)and first water extraction and 95%ethanol-extraction(simulation powder),extract,reduced enrichment and dry for various sample preparation after 30.In the second segment,According to aromatherapy powder angelica and the proportion of river xiong(3:2)says take medicine,a total of 1.5 kg,with contain different ethanol concentration(0%,5%,10%,15%,20%,30%,40%,50%,70%,95%)eight times the amount alcohol thermal water reflux extraction two times,each time 2 h,natural cooling,pour out aqueous,filtration liquid,merger extract by vacuum reduced pressure concentration dry get 10 samples preparation.In the third segment,According to a certain proportion(0:1,1:5,2:5,2:3,1:1,3:2,5:2,5:1,1:0)says take angelica and wine river xiong,mixed,500 g,with eight times the amount of water on hot reflux extraction 2 h,natural cooling,pour out water solution,then add in the sameamount of water again hot reflux extraction 2 h,filtering,liquid with 95%ethanol to the thermalreflux extraction 2 h,natural cooling,pour out aqueous,filtration liquid,and water extractionliquid combination on the vacuum reduced pressure concentration,dry to extract,samplepreparation nine.There are four segments in the second chapter,to the rightness blood deficiency mice are medicine blood enriching ffunction concentration-response relation and interaction.In the first segment,The blood deficient mice model was induced by injection of different dosage of phenylhydrazine and cyclophosphamide.Changes of orbitblood routine,thymus index and spleen index of model mice were tested,to found the best model to study the concentration-response relation and interaction of the herb pair constituted by Angelica sinensis and Ligusticum chuanxiong.The fifth model suited to study the concentration-response relation and interaction.In the second segment,to study the enriching blood effect of Fo-shou-san(FSS)extracted with different ethanol concentration on blood deficiency animal.The blood deficient mice model was induced by injection of phenylhydrazine and cyclophosphamide.The body weight of mice was recorded during the induction of model.After the model was established,blood was collected from the retroorbital plexus,and the blood routine,thymus index,spleen index,and liver index were determined.Total antioxidant capacity(T-AOC)and superoxide dismutase(SOD)in liver were detected by colorimetric method and xanthine oxidase method,respectively.Those indexes were calculated to a single index by the weight coefficient method for exploring the total enriching blood effect.FSS extracted with different ethanol concentration could increase the number of white blood cell,red blood cell,platelet and hematocrit in model mice;increase weight,thymus index,and spleen index of model mice,while decrease liver index of model mice;and increase the activity of T-AOC and SOD in liver.The order of FSS extracted with different concentration of ethanol on total enriching blood effect was:40%>30%>20%>50%>15%>70%>10%>5%>95%>0%.FSS extracted with different ethanol concentration had the activity of enriching blood on blood deficiency model mice,and 40%ethanol extraction showed the best effect.In the third segment,study on nourishing and tonifying blood effects of the herb pair constituted by danggui and chuanxiong by drug interaction.The blood deficiency mice were induced by injecting N-acetylphenylhydrazine and cyclophosphamide.,the effects of Gui-Xiong with different proportions(0:1,1:5,2:5,2:3,1:1,3:2,5:2,5:1,1:0)and different concentrations on the peripheral blood index and organ index were observed.Then all indexes were integrated to the total nourishing effect value by comprehensive index method.The interaction was analyzed by response surface method.Model parameters were estimated with nonlinear regression.The three-dimensional response surfaces were constructed with Matlab software.In the response surface,most compatibility of Gui-Xiong showed synergistic action,some showed addition action,and few of them showed the antagonist action.The proportion from 4:1 to 2:1 of Gui-Xiong had the addition action;the other proportions had the obvious addition action at low dose,and synergistic action at high dose.In the fourth segment,The blood deficient mice model was induced by injection of phenylhydrazine and cyclophosphamide,and mice were divided into 3 groups,control group,model group,treatment group(FSS).Endogenous metabolites of three groups were determined with UPLC-Q-TOF/MS.Pattern recognition methods including PLS-DA,OPLS-DA were applied to analyze multivariate data,and T-test was used in significant statistical analysis.In pattern recognition,model group could be distinguished from other groups.24 potential metabolic biomarkers(L-Cysteine;Citric acid;Arachidonic acid;Xanthine;Glucose 6-phosphate;Leukotriene B4;Gentisic acid;L-Dopa)were identified qualitatively.These endogenous substances of the treatment group mice in vivo had varying degrees of recovery,and many metabolic pathways(Arachidonic acid metabolism;Histidine metabolism;Cysteine and methionine metabolism;Citrate cycle(TCA cycle);Tryptophan metabolism;Tyrosine metabolism;Thiamine metabolism;Glycolysis or Gluconeogenesis)were identified using ingenuity pathway analysis(IPA).A metabolomic feature network of FSS to protect against the blood deficient mice was constructed.Changed metabolities can be certainly recovered by FSS,and the treatment effect of FSS can be connected with the regulation of 7 related metabolic pathways.There are three segments in the third chapter about the rightness rat medicine are acute blood stasis model the amount of blood rheology effect relation and interaction.In the first segment,study on interaction of activating blood effects of the herb pair constituted by danggui and chuanxiong by response surface method.Based on the acute blood stasis rats model,the effects of Gui-Xiong with different proportions(0:1,1:5,2:5,2:3,1:1,3:2,5:2,5:1,1:0)and different concentrations on hemorheology were observed.Then all indexes were integrated to the total activating blood circulation effect value by comprehensive index method.The interaction was analyzed by response surface method.Model parameters were estimated with nonlinear regression.The three-dimensional response surfaces were constructed with Matlab software.The proportion from 2:1 to 1.3:1 of Gui-Xiong had the synergistic action(the value is-0.9);the dose of danggui ranged from 2 to 3.5 g,and chuanxiong from 7 to 9 g showed antagonist action(the value is 0.2),and the other proportions hadn't the obvious addition and antagonism action.In the second segment,Aromatherapy scattered to rat acute blood stasis model of the amount of blood rheology effect relations.Observation of acute blood stasis citron scattered the influence of rat models,this paper discusses the relationship between blood live scattered citron effect.The acute blood stasis in the rat model,through the determination of whole blood viscosity,plasma viscosity,blood sedimentation,blood pressure product,whole blood viscosity and high cutting reduction of the whole blood viscosity reduction low shear,a visit to citron scattered blood stasis rats on hemorheology of.Finally the weight coefficient method will all index for total blood effect value integration,to evaluate the total citron scattered for activating blood circulation and eliminating stasis effect.Results show that,compared with the normal group,of the indexes in model group,all have significant differences,tip made model success.Compared with model group,give berganottin after scattered,can reduce made from blood rheology model of each index value.Show that scattered on acute citron blood stasis model rats have blood role,six times the equivalent of the optimal clinical effect,0.25 times the worst effect,present a clear concentration-response relationship.In the third segment,Urine metabonomics study of FSS on acute blood stasis rats model.To observe urinary metabolic changes of acute blood stasis rats and the regulating action of FSS on abnormal metabolism,and to find the potential biomarkers.To explore mechanisms of FSS in treatment of acute blood stasis rats.Based the acute blood stasis rats model,and rats were divided into 3 groups,control group,model group,treatment group(FSS).Endogenous metabolites of three groups were determined with UPLC-Q-TOF/MS.Pattern recognition methods including PLS-DA,OPLS-DA were applied to analyze multivariate data,and T-test was used in significant statistical analysis.In pattern recognition,model group could be distinguished from other groups.43 ions which had a significant contribution to the classification from rat urine metabolic profiles of model group were screened further,11 potential metabolic biomarkers were identified qualitatively.These endogenous substances of the treatment group rats in vivo had varying degrees of recovery,and 3 metabolic pathways were identified using ingenuity pathway analysis(IPA).Ametabolomic feature network of FSS to protect against acute blood stasis rats about urine was constructed.Changed metabolities can be certainly recovered by FSS,and the treatment effect of FSS can be connected with the regulation of 3 related metabolic pathways.Fourth chapter,to describe the effects of FSS on the peripheral blood index and hemorheology and blood cogulation function in acute blood deficiency-blood stasis rats.Based acute blood deficiency-blood stasis rats model,orbitblood routine,Na+-K+-ATP,Ca2+-Mg2+-ATP,thymus index and spleen index of model mice were tested to observe the nourishing blood effect;Whole blood viscosity,plasma viscosity,erythrocyte sedimentation rate and haematocrit were tested to observe the activating blood effect of FSS on hemorheology;TT,prothrombin time(PT),activated partial thromboplastin time(APTT),and plasma fibrinogen(FIB)were tested to observe the effect on blood cogulation function.In comparison with normal control group,indexes in model group changed significantly.In comparison with model group,FSS could protect against acute blood stasis rats.FSS could improve obviously the abnormality of hemorheology and blood cogulation function in acute blood deficiency-blood stasis rats;the results provided the pharmacological data for the theory can enhance the effect of nourishing-activating blood circulation and dissipating blood stasis,and established the basis to reveal the scientific innovation of FSS.Fifth chapter,Study on interaction of antioxidant effects of danggui and chuanxiong with different preparation methods and proportions.Anti-oxidative activity of Gui-Xiong with different proportions and extracted by water,ethanol,alcohol-water were evaluated by means of DPPH.The pharmacodynamic interaction was analyzed by response surface method.Model parameters were estimated with nonlinear regression.The three-dimensional response surfaces were constructed with Matlab software.Different preparation methods and different proportions of Gui-Xiong had anti-oxidative effects at different degrees with dosage dependant.The water extract of Gui-Xiong had narrow synergistic range with only spot distribution;it had six-band distribution of antagonist range.Synergistic ranges of ethanol extract were band shapes,while antagonist action was spot distribution.Synergistic ranges of alcohol-water extract were three-band shapes,while antagonist action was spot distribution.In short,the alcohol-water extract of Gui-Xiong showed a wider synergistic action range than the ethanol extract.And most of range showed no obvious synergistic and antagonist action.This dissertation established the basis for revealing the composition law and scientific connotation of Gui-Xiong by studying the bio-effect changes of Gui-Xiong herb pair on nourishing and activating blood,anti-oxidation,and the effects of the blood deficiency model in mice plasma and the acute blood-stasis model rats urine by Metabolomics studies.Conclusion:This paper,by establish the effect of raise blood and promoting blood circulation evaluation system,the more comprehensive index method for the effect of the indicators integration,response surface method used in the quantitative evaluation of gui-xiong compatibility interactions,are to be found with the quantity medicine with different than the method of interaction with nature significantly different,and show concentration-response relationship.Compared to see,blood compatibility to the synergy effect coveraged than blood effect,antioxidation synergy minimum range that gui-xiong compatibility,the overall efficacy prefer to keep blood enriching blood.This paper also used the metabolomic research ideas,from blood deficiency model in plasma,acute blood stasis model of rat in urine,to find after drug compatibility of related metabolic pathways,to reveal the composition of medicine,to lay the foundation for the law and scientific connotation.This paper characteristics and innovations:1 The index synthetic index method of traditional Chinese medicine reflect the efficacy of the drug indexes all standardized and integration,so as to achieve the integration of suitable for comparative evaluation effect.2 The response surface method design and research gui-xiong compatibility of the interaction of the nature and degree of Chinese crude drug compatibility for modern research provides the ideas and methods for reference.3 Based on the metabolomic research idea to explore the effect of compatibility are mechanism,which reveals the foundation of rule. |
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