The Basic Research Of Xinshenning Capsules

XinSuNing Capsule is a modified recipe of an ancient prescription named Huanglian Wendan Decoction("Itemized Discrimination of Exogenous Diseases Caused by Six Factors")and contains 11 traditional Chinese medicines including Rhizoma coptidis,Pinellia ternate,Poria cocos,Immature Bitter Orange,Antifeverile dichroa root,Lotus plumula,Sophora flavescens,Sweet wormwood herb,Ginseng,Radix ophiopogonis,Glycyrrhiza uralensis.It has been a proved recipe clinically used in treating the phlegm heat disturbing mind type arrhythmia(premature heartbeat)and in 2005 it was approved as a new drug by the State Food and Drug Administration and came to the market.With the prominent limitations of anti-arrhythmic chemical drugs(those chemical drugs may have proarrhythmic effects and antiarrhythmic effects at the same time),traditional Chinese medicines represented by XinSuNing Capsule is receiving more clinical acceptance for their remarkable efficacy in antiarrhythmic treatment by blocking multiple ion channels and regulating the non-ion channels.The newly conducted evidence-based medicine study has verified the definite clinical effects of XinSuNing capsules in treating the phlegm-heat disturbing mind type arrhythmia(premature heartbeat),but its chemical material basis,mechanism of action and quality control have not been studied.This paper taking XinSuNing capsule as the study object,conducts a study on the main chemical compositions of XinSuNing capsule,represents its overall chemical outline,speculates its molecular mechanism and based on this establishes a qualify control method concerning the efficacy and combining the qualitative and quantitative analyses.First,the main chemical compositions contained in XinSuNing capsule were studied.With the combination of the silica gel column chromatography,ODS column chromatography,Sephadex LH-20 column chromatography,RT-HPLC and other chromatographic separation methods,22 monomer compositions were separated and obtained.Taking advantage of their physicochemical properties and modern spectroscopy techniques(1H-NMR,13C-NMR,etc.),structures of the 22 compounds were identified.These 22 compounds are berberine,daucosterol,sophora flavescens chalcone,coptisine,dihydroberberine,dihydrocoptisine,dihydrocoptisine,hesperidin,jateorhizine,isoliquiritigenin,Kushenol O,sophocarpidine,sophocarpine,coumarin,N-trans-feruloyltyramine,liquiritigenin,nobiletin,hesperetin,8-oxyberberrubine,4-(2-hydroxy-vinyl)-benzene-1,2-diol,5-demethenyl nobiletin,scopoletin and P-sitosterol.Among them,five chemical compositions are from Rhizoma coptidis,four from Immature Bitter Orange,four from Sophora flavescens,two from Glycyrrhiza uralensis and the rest seven from other medicinal materials.With the technique of HPLC-LTQ-Orbitrap-MS/MS,the overall chemical profile of XinSuNing capsule was represented and its compositions were speculated.Welch Xtimate C18 Column(4.6 × 250 mm,5 ?m)was adopted and acetonitrile-0.2%ammonium hydroxide and 0.1%aqueous solution of formic acid were used as the mobile phase for the gradient elution.ESI-LTQ-Orbitrap-MS/MS was employed for the pattern detection of cations and anions.Taking the advantage of information about the quasi-molecular ion peak and fragment ion and the comparison with the documentation,109 constituents in total were recognized in XinSuNing capsule,including 38 alkaloids,42 flavonoids,12 triterpenes,six coumarins,one organic acids,and five undefined constituents.Among them,the structures of 16 constituents were precisely determined through the direct comparison with the standard substance.This verified the feasibility and reliability of HPLC-LTQ-Orbitrap-MS/MS in the chemical composition inference.Based on the study of chemical composition and the chemical outline of XinSuNing capsule represented with HPLC-LTQ-Orbitrap-MS/MS technique,a database of the chemical constituents of XinSuNing capsule was established.After the target prediction based on the chemical similarity,similar drugs with a similar score greater than 0.6 and their corresponding targets were extracted as the potential targets of XinSuNing capsule.In the meantime,arrhythmia and related symptoms(palpitation,dizziness and xerostomia)in databases DrugBank and OMIM constitute a target bank relating the disease to the symptoms.Then with the PPI database,four PPI meshes concerning XinSuNing capsule were built:Potential target of action-Arrhythmia-related targets,Potential target of action-Palpitation-related targets,Potential target of action-Dizziness-related targets and Potential target of action-Xerostomia-related targets.Finally,analysis was conducted based on features of the network topology(degree,closeness,betweenness and coreness)and 35 key targets that can have effects on arrhythmia,related palpitation,dizziness and xerostomia were selected,such as CHRM1,CYSLTR1,NFKB1,TNF,DRD2,ESR1,HTR1A,OPRK1,OXTR,PPARA,PTGER3,SSTR2,SSTR3,ANXA1,AR,BCL2,AGTR1,DRD4,HTR2A,PTGS2,HTR2B,MAPK14,EDNRA,TP53 and so on.The key targets mainly participate in the following pathways:(1)They act on the biological pathways related with arrhythmia,such as the metabolic pathway of multiple nutrients,and then play a role in regulating the weak constitution,heart failure and other symptoms of patients with cardiovascular and cerebrovascular diseases.(2)They participate in the pathways for oxidative phosphorylation,anaerobic glycolysis,synthesis and degradation of fatty acids and other purposes,provide energy to the organism and alleviate the ischemia and anoxia in the progression of cardiovascular and cerebrovascular diseases;they participate in angiogenesis and the contraction of vascular smooth muscles and play a role in alleviate the symptoms of ischemia.(3)They participate in pathways related with the nervous system regulation,regulate several immune pathways,thus enhancing the immunologic function of the organism to cure the arrhythmia.On this basis,emphasis was put on the 22 compounds.14 compositions among them can be combined with the key targets and are considered as the key active ingredients.They are 5-demethenyl Nobiletin,palmatine,N-trans-feruloyltyramine,?-sitosterol,nobiletin,scopoletin,liquiritigenin,daucosterol,sophocarpine,hesperetin,sophora falvescens chalcone,coumarin(Comp.13),berberine and isoliquiritigenin.HPLC was employed with Welch Xtimate C18 chromatographic columns(4.6 x 250 mm,5?m)as the analytical columns,and acetonitrile-aqueous solution of formic acid(0.2%ammonium hydroxide,0.2%formic acid)as the mobile phase for gradient elution.The flow velocity was 1.0 ml/min;the wavelength was detected to be 254nm and the HPLC finger-print of XinSuNing capsule was established.This method has good specificity,accuracy,repeatability and stability.When referring to the relevant technical requirements and the practical situation of HPLC finger-print of XinSuNing capsule,29 common peaks were assigned.And through the comparison with the reference substances,12 chromatographic peaks among the 29 peaks were identified.The similarity of fingerprints of ten batches of commercially available preparations was calculated by the Similarity Evaluation System for Chromatographic Fingerprint of Chinese Traditional Medicine(Version 2004A)and the values of similarity between the finger-print of the commercially available preparation and the reference finger-print are in the range of 0.98 to 0.997.After the similarity analyses of finger-prints of single medicine and combination drug,it is found that Rhizoma coptidis and Immature Bitter Orange make the biggest contribution to the full chromatogram with a rate of contribution of 0.21 respectively.Pinellia ternate,Lotus plumule and Glycyrrhiza uralensis make the smallest contribution,with the rates of contribution being 0.02,0.04 and 0.06 respectively.referring to the selection method of Quality Marker of Traditional Chinse Medicine and taking into the actual situation of mass production,this paper took berberine,palmatine and hesperidin as the index components and established a method based on the chemical finger-print to determine the content of index components of XinSuNing capsule.The Chromatographic condition as aforesaid proved stable and reliable after the linear relation test and the experimental verifications in terms of accuracy,repeatability and stability.The recoveries of the three index components were100.59%,100.41%and99.71%respectively.This paper based the chemical composition study of XinSuNing capsule,represented the overall chemical outline of XinSuNing capsule and inferred its compositions with liquid chromatography.As for structures to be inferred,the paper took advantage of target spot prediction and network pharmacological methods and speculated the possible molecular mechanism and active ingredients that make XinSuNing capsule work.Integrating the results of chemical compositions of XinSuNing and the finger-print study,the paper tentatively established a quality control method concerning the efficacy and combining the qualitative and quantitative analyses.The paper lays a foundation for comprehensively revealing the effective substance and mechanism of action of XinSuNing capsule.The newly established quality control method is more scientific than the existing standard,lifts the scientific content of the product and can be used as reference for the study of pharmacodynamic material basis and the elevated standard of other compound Chinese patent medicines.