| This work was supported by National Natural Science Foundation of China(81274058)—Part experiment content of "Study on dose-effect relationship and synergistic interation of nourishing and activating blood effect for the compatibility of Danggui and Honghua" and the open project program of jiangsu key laboratory for high technology research of TCM formulae and jiangsu collaborative innovation center of Chinese medicinal resources industrialization(No.FJGJS-2015-11).This graduation thesis is made up of two major parts.In the first part,this paper reviewed the methods and progress of herb pairs and compound Xin-Sheng-Hua granules.There are three chapters in second part,specific research content and correlated conclusion as follows:Chapter 2.The research of pharmacodynamics of Xin-Sheng-Hua granules based on herb pairs containing Danggui(1)Comparatively evaluating the effect contribution of nourishing blood of herb pairs containing Angelicae Sinensis Radix on Xin-Sheng-Hua Granule by withdrawal analysisFor Xin-Sheng-Hua Granule(XSHG),herb pairs could be formed when Angelicae Sinensis Radix(Danggui,DG)is combined with the other single herbs,respectively.Our aim was to investigate the contribution of herb pairs containing Danggui to XSHG on hemolytic and aplastic anemia(HAA)mice by withdraw analysis.Subsequently,the combination of acetyl phenylhydrazine(APH)and cyclophosphamide(CP)were used to induce the HAA model.100 mice were divided into 10 groups,the dose groups were orally administrated with the XSHG and six different samples of decomposed recipes(DY,DC,DT,DH,DJ and DZ)once per day for continuous 7 days from the experiment began.The test indicators included peripheral blood routine,organ index and ATPase activities,then principal component analysis(PCA)model were developed to evaluate the integrate effects of hematinic function of XSHG and its decomposed recipes.And the relative distance calculation method between dose groups and control group from the PCA score plot,was adopted to evaluate the integrated effects of hematinic function of different samples.And the orders of hematinic effects of various groups were as follows,XHSG>DJ>DT>DZ>DH>DC>DY.These findings showed that withdraw analysis method is a valuable tool to analyze the impacts of nourishing blood of herb pairs containing Danggui on XSHG,which might provide a good reference for illustrating the drug interactions from the aspect of herb pair.(2)Comparatively evaluating the effect contribution of promoting blood circulation of herb pairs containing Angelicae Sinensis Radix on Xin-Sheng-Hua Granule by withdrawal analysisXin-Sheng-Hua-Granule(XSHG)is a popular remedy commonly used in clinic for the treatment of lochiostasis after delivery.To comparatively investigate the roles of herb pairs containing Angelicae Sinensis Radix(Danggui)upon the formula by evaluating the blood coagulation and hemorheology function in acute blood stasis rats.Acute blood stasis rat model was established by ice water bath and subcutaneous injection of adrenaline.And the blood stasis mice were administrated intragastrically with different samples of the formula minus herb pairs containing Danggui and the whole formulae.Afterwards,multi-attribute comprehensive index methods and principal component analysis were both applied to comprehensively assess the total effects of the formula minus herb pairs containing Danggui on promoting blood circulation and dissipating blood stasis.Compared with model group,different samples(XSHG,SHD,DY,DC,DT,DH,DJ and DZ)could improve the blood hemorheology indexes,coagulation parameters and platelet aggregation in acute blood stasis rats.And the effects of promoting blood circulation by removing blood stasis became poor when excluding herb pairs containing Danggui from the formula,the sample DY and DC had the weakest effect of activating blood circulation and dissipating blood stasis,and the effect of sample DY was slightly poorer than DC.In conclusion,various herb pairs containing Danggui played different roles on the effects of improving the abnormality of hemorheology and coagulation function.And the herb pairs Danggui-Yimucao were particularly important for the formula,which was consistent with the characteristics of XSHG and the traditional effect of Yimucao.Chapter 3.Study of material basis of Xin-Sheng-Hua granules in vivo and in vitro(1)Comparative analysis of main bio-active components of Xin-Sheng-Hua Granule and its single herbs by ultra-high-performance liquid chromatography coupled with a triple quadrupole electrospray tandem mass spectrometryXin-Sheng-Hua Granule,a representative formula for postpartum hemorrhage,has been used clinically to treat postpartum diseases.Its main bio-active components were comprised of aromatic acids,phthalides,alkaloids,flavonoids,gingerols,and etc.To investigate the changes of main bio-active constituents in its seven single herbs before and after compatibility,a rapid,simple and sensitive method was developed for comparative analysis of 27 main bio-active components by using ultra-high-performance liquid chromatography coupled with a triple quadrupole electrospray tandem mass spectrometry for the first time.Sufficient separation of 27 target constituents were achieved on a Thermo Scientific Hypersil GOLD column(100 mm×3 mm,1.9?m)within 20 min under the optimized chromatographic conditions.Compared with the theorized content,the observed content of each analyte showed remarkable differences in Xin-Sheng-Hua Granule except thymine,p-coumaric acid,senkyunolide I,senkyunolide H and ligustilide;the total contents of 27 components increased significantly.And the content variation degrees for the different components were gingerols>flavonoids>aromatic acids>alkaloids>phthalides.The results could provide a good reference for the quality control of Xin-Sheng-Hua Granule,and might be helpful to interpret the drug interactions based on variation of bio-active components.(2)The material basis of herb pairs containing Danggui on Xin-Sheng-Hua granulesAmong the seven herbs of XSHG,herb pairs could be formed when Angelicae Sinensis Radix(Danggui,DG)is combined with the other single herbs,respectively.Based on the previous studies,the results indicated that herb pairs Danggui-Yimucao and Danggui-Chuanxiong played significant roles in the formulae.Our aim was to discover the potential reasons of their pharmacodynamics effects based on the content variations of effective components in these samples.A total of 21 effective components of these different samples were analyzed for discovering the potential reasons of their curative effects by ultra-high-performance liquid chromatography coupled with a triple quadrupole electrospray tandem mass spectrometry(UHPLC-TQ-MS/MS).Further analysis of these samples by UHPLC-TQ-MS/MS revealed that XSHG underwent complicate changes when herb pairs containing Danggui were excluded from XSHG respectively.Compared with the amounts of the analytes in XSHG,vast majority of active compounds in the discomposed samples decreased to some extent.Especially for the sample DY,except liquiritigenin,the contents of other constituents reduced compared with the formulae XSHG,thus herb pair Danggui-Yimucao might be particularly important for this formulae.These findings showed that withdraw analysis method is a valuable tool to analyze the impacts of herb pairs containing Danggui on XSHG,which could lay foundation to reveal the compatibility rules of the formulae.Further study about the metabolic changes of the formulae XSHG in the rat should be conducted to discover the action mechanism of pharmacodynamics.(3)Pharmacokinetic comparison of fifteen major bio-active components in normal and blood deficiency rats after oral administration of Xin-Sheng-Hua granules by UHPLC-TQ/MSA sensitive UHPLC-MS/MS method for the simultaneous quantification of fifteen constituents(trigonelline,stachydrine,hydroxysafflor yellow A,chlorogenic acid,amygdalin,leonurine,liquiritin,ferulic acid,senkyunolide I,senkyunolide H,senkyunolide A,glycyrrhizic acid,ligustilide,n-butylphthalide and glycyrrhetic acid)in rat plasma has been developed and validated.Chloramphenicol and clarithromycin was used as the internal standard(IS).The analytes were separated using a C18 column with a 20 min gradient elution,using both positive(ESI+)and negative electrospray ionization(ESI-)in multiple reaction monitoring(MRM)mode.The correlation coefficient(r)were no less than 0.9925 for the 15 analytes.The precision of inter-day and intra-day was less than 11.87%.The relative error of accuracy was in the range of ±12.38%.The mean recovery of 15 analytes were higher than 92.28%.The vadilated method was successfully applied to investigate the pharmacokinetic study after oral dosing of XSHG(9.72 g·kg-1)in normal and blood deficiency rats,respectively.Except alkaloids,glycyrrhizic acid and glycyrrhetic acid,the Cmax and t1/2 of other analytes were relatively short,and basically eliminated within 6 h.Compared with control group,the AUC0-t and AUC0-? of aromatic acids,butylphthalides and alkaloids were all significantly higher in blood deficiency rats(P<0.05).Especially for alkaloids,their Cmax,ti/2,AUC0-t and AUC0-? were higher than other constituents(P<0.01),indicating that alkaloids could be the significant potential active compounds in XSHG.Chapter 4.The tonifying and promoting blood mechanism of Xin-Sheng-Hua granules on basis of metabolomics and network pharmacology(1)Metabolomic strategy to investigate nourishing blood action mechanism of formulae Xin-Sheng-Hua granule in blood deficiency ratsIn this investigation,global metabolic strategy by UHPLC-QTOF/MS along with pattern recognition was originally developed to indentify differentiating potential metabolites of hemorrhagic anemia.Finally,altogether 18 potential endogenous metabolites(3 downregulated and 15 upregulated)were confirmed,and 10 metabolic pathways about blood deficiency were discovered.XSHG,SHD,DY,DG and YMC had different pharmacological effects by adjusting certain perturbed pathways near to normal condition.Overall,the metabolomic results of different groups were in accordance with the peripheral blood parameters.Different dose groups had individual effects on blood deficiency rats,and the orders of nourishing blood were as follows:XSHG>SHD>DY>DG>YMC.These results indicated that the formulae XSHG had the best treatment effects of nourishing blood by up-regulating or down-regulating the perturbed biomarkers.The strategy successfully suggested that the applicability of metabolomics in revealing nourishing blood effects of XSHG;the results above can supply assistance for better clarifying the action mechanisms of hemorrhagic anemia;and intergrative metabolomic research by UHPLC-QTOF/MS could be beneficial for investigating the mechanisms of the classic formulae XSHG and SHD.Futhermore,a representative example was displayed that close correlation were obtained between endogenous metabolites and pharmacological indexes.Different groups could regulate pantothenic acid and coenzyme a biosynthesis,cysteine-methionine metabolism and sphingolipid metabolism.(2)Network pharmacology research on major active components of Xin-Sheng-Hua granulesFormulae XSHG has been clinically used for the treatment of postpartum hemorrhage syndrome(PHS)through tonifying blood for years.In the present study,a feasible system pharmacology model based on chemical,pharmacokinetic and pharmacological data was developed via network construction approach to clarify the mechanisms of nourishing blood of Xin-Sheng-Hua granules.Fifty-eight active ingredients of XSHG possessing favorable pharmacokinetic profiles and biological activities were selected,interacting with 46 PHS-related targets to provide potential synergistic therapeutic actions.Systematic analysis of the constructed networks revealed that these targets such as TNF-a,HIF-1?,IL1? IL6,MAPK1,MAPK10 and PTGS2 were mainly involved in HIF-1 signaling pathway,MAPK sinaling pathway,TNF signaling pathway and sphingolipid signaling pathway.The contribution index of each active constituent also indicated nine compounds,including stachydrine,Z-ligustilide,ferulic acid,senkyunolide I,amygdalin,leonurine,hydroxysafflor yellow A,glycyrrhizic acid and senkyunolide A.PHS could be associated with the decreased erythrocyte synthesis,disorders of energy metabolism,microcirculation disturbance and elevated inflammatory factor.(3)Experimental verification of key targets from potential signaling pathway of blood deficiency syndromeAccording to metabolomics and network pharmacology analysis,the key targets(EPOR,TNF-?,IL-6,F2,HIF-1?,COASY,ACSS2,SPHK1,S1PR1,CBS and AHCY)of potential signaling pathway were verficated experimentally.The levels of EPOR,TNF,IL-6 and F2 from rat plasma were analyzed by ELISA method,and western blot was applied to investigate the expression of other target protein in liver.ELISA and western results shown that formulae XSHG could obviously increase the levels of EPOR,TNF-?,IL-6,F2,COASY,ACSS2,CBS and AHCY(P<0.05),while the expression of HIF-1?,SPHK1 and S1PR1 were decreased(P<0.05)significantly after administration of extract XSHG.As a result,formulae XHSG could have obvious promotion effects of hematopoietic function through increasing erythrocyte function,enhancing immune function,improving energy metabolism and microcirculation.These results successfully explained the polypharmcological mechanisms underlying nourishing blood action of formulae XSHG for the treatment of hemorrhage syndrome,and also probed into the potential novel therapeutic strategies for hemorrhage syndrome in the clinical application. |
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