The Study On Mass Spectrometry Identification And Multi-component Quantitative Studies Of Huangqi Herb Pair And Its Compound Preparation

The compatibility of traditional Chinese medicine(TCM)prescriptions is the key to the effectiveness of prescriptions.TCM syndrome differentiation is achieved by the changes in compatibility of prescription.The law of TCM compatibility and studies of pharmacodynamic material basis is the breakthrough to solve the key problem of prescription,and is one of the national key research field of modern Chinese medicine(CM)composition content.To explore the material basis for efficacy of CM and its combination has become a key problem in today's CM circles.Huangqi(Astragalus Membranaceus)-Sanqi(Panax Notoginseng)herb pair,on the compatibility of the herb(Huangqi)on invigorating body and the herb(Sanqi)on promoting blood circulation and removing blood stasis drugs,is widely used in clinical for angina,myocardial infarction,chronic nephritis and other diseases.However few systematic study has been reported on the compatibility law and the pharmacodynamic material of Huangqi-Sanqi.The UPLC/ESI-LTQ-Orbitrap MS' combined mass spectrometry has been employed for the research on of chemical components dentification and their mass fragmentation rules of Huangqi-Sanqi herb pair,including isoflavonids and saponins.We also apply this analytical technique and method for the research of material basis of another Chinese combination involving Huangqi-the Bupiyishen decoction,of which the major chemical structure categories(including phenylpropanoid,lobetyolins,stilbene glycosides and anthraquinones)have been investigated by mass spectrometry.This would reveal the application prospect of UPLC/ESI-LTQ-Orbitrap MSn in the field of TCM herb pair or compound recipe.In addition,concerning the complexity of chemical compounds and the difference of their contents,a quantitative study of multi components on these two kinds of prescriptions has been developed to discuss the influence of compatibility and concentration-drying technics on dynamic change process of effective components at the medicinal substances level.The method for multi-components quantitativity based on LC-MS technology has been adhibited to observe the dissolution changes of flavonoids and saponins before and after compatibility,as well as under different ratio condition.Through the quantitative detection method by HPLC-MS,the dissolution influence of chemical instituents caused by different concentration and drying processes of Bupiyishen decoction was investigated.Objective1.Ultra-high-pressure liquid chromatography with linear iontrap-Orbitrap mass spectrometry(HPLC-LTQ-Orbitrap-MSn)is applied to separately illustrate the chemical composition of Radix Astragali and Panax Notoginseng,and the mass fragmentation rule of different chemical structure types,including astragalosides,soyasaponins and notoginsenosides is also investigated.2.To establish a HPLC-LTQ-Orbitrap-MSn analytical method,compre-hensively reflect and explain chemical compositions of Huangqi-Sanqi herb pair.To accurately classify and identify the main chemical component peaks,and initially establish the Huangqi-Sanqi fingerprint based on combined mass spectrometry technology.3.To establish a HPLC-MS method for quantitative determination of six isoflavones and eight saponins,to observe the dissolution changes of flavonoids and saponins before and after compatibility,as well as under different ratio condition;To discuss the dynamic change process of components,which influenced by the compatibility of the herb pair at the medicinal substances level.4.To establish a HPLC-LTQ-Orbitrap-MSn analytical method,compre-hensively and fast explain the mass fragmentation rule of different chemical structure types of major herbs,including Astragalus,Codonopsis,Radix Polygoni Multiflori,Radix Salviae Miltiorrhizae.5.To overcome the restriction of single component quantitation of effective substance,quantitative detection method of seven representative chemical composition by HPLC-MS is established to investigate the dissolution change of chemical instituents caused by different concentration and drying processes of Bupiyishen decoction.Methods1.HPLC-LTQ-Orbitrap-MS" analysis was employed and both positive and negative modes were used to analyzed the sample solution of Radix Astragali and Panax Notoginseng,separately.A Phenomenex Kinelex C18(2.1 mm × 100 nm,1.7 ?m)reversed-phase column equipped with a Phenomenex ultra-efficient C18 guard column was used for chromatography.The mobile phase consisted of 0.1%formic acid in water(A)and acetonitrile(B);flow rate,300 ?L/min;injection volume,5 ? L.Data-dependent MSn scanning was performed to minimize total analysis time as it can trigger fragmentation spectra of target ions and prevent repetition by dynamic exclusion settings.2.Chemical fingerprint of Huangqi-Sanqi herb pair was established by HPLC-LTQ-Orbitrap-MS'.By comparison with experimental data of single herb,as well as literature statistics and contrast by reference compounds,the main chromatographic peaks of the prescription were quickly identifed.3.To establish a HPLC-MS method for quantitative determination of six isoflavones and eight saponins,A ACQUITY BEH C18(2.1 mm × 50 mm,1.8 ?m)reversed-phase column equipped with a BEH C18 Vanguard column was used for chromatography.The mobile phase consisted of 0.1%formic acid in water(A)and acetonitrile(B);flow rate,300 ?L/min;injection volume,5 ?L.Multiple-reaction monitoring mode was selected and targeted parent ion pair was quantitative detected.The amount of dissolved chemical composition of different proportion of Huangqi-Sanqi herb pair was determined.The dissolution change of Huangqi compounds was observed by comparing four proportion groups,including 3:0?3:0.5?3:1 and 3:2.Meanwhile,The dissolution change of Sanqi compounds was inpected by comparing proportion groups,namely,3:0?3:0.5?3:1 and 3:2.4.HPLC-LTQ-Orbitrap-MSn analysis was applied and both positive and negative modes were used to detect the sample solution of Bupiyishen decoction.A Phenomenex Kinelex C18(2.1 mm × 100 mm,1.7?m)reversed-phase column equipped with a Phenomenex ultra-efficient C18 guard column was used for chromatography.The mobile phase consisted of 0.1%formic acid in water(A)and acetonitrile(B);flow rate,200 ?L/min;injection volume,5 ?L.Data-dependent MSn scanning was performed to minimize total analysis time as it can trigger fragmentation spectra of target ions and prevent repetition by dynamic exclusion settings.Meanwhile,relative peak area of main components before and after compatibility was compared,in order to investigate the influence of compatibility on the dissolution of chemical composition.5.To establish a HPLC-MS method for quantitative determination of seven components,A ACQUITY BEH C18(2.1 mm × 50 mm,1.8 ?m)reversed-phase column equipped with a BEH C18 Vanguard column was used for chromatography.The mobile phase consisted of 0.1%formic acid in water(A)and acetonitrile(B);flow rate,250 ?L/min;injection volume,5 ?L.Multiple-reaction monitoring mode was selected and targeted parent ion pair was quantitative detected.The amount of dissolved chemical composition by different concentration and drying processes of Bupiyishen decoction was detectedd and compared.Results1.The chemical constituents of Radix Astragali and Panax Notoginseng could be rapidly identified through the respective establishment of mass data processing strategy of two herbs.By comparing precise mass ratio,multi-stage mass fragment ions,product mass fragmentation rule of reference compounds,48 flavonoids of Astragalus were totally identified,including 18 isoflavones,10 pterocarpans,8isoflavans,7 flavones,5isoflavanone.Besides,31 saponins in Astragalus were characterized,including astragalosides and soyasaponins;49 notoginsenosides was detected in Panax Notoginseng.2.Comparing the acquired mass informations of Huangqi-Sanqi herb pair with accurate relative molecular weight information and multi-stage mass spectrometry fragmentation informations of their single herbs,39 different types of chemical constituents in the prescription were identified fastly by the mass spectra.According to the exact molecular weight informations from HR-MS and the characteristic fragmentation informations obtained by CID scanning,each compound peak of flavonoids and saponins were determined.And through comparison of the retention time and mass behavior with reference compounds,provides the evidence for the compounds identification.3.The method for multi-components quantitativity based on LC-MS technology has been adhibited to observe the dissolution changes of flavonoids and saponins before and after compatibility.The dissolution of main components of Radix Astragali and Panax Notoginseng has somehow changed under different ratio condition,which caused by different compatibility ratios.Compared with the water extracts of Huangqi,the contents of 6 isoflavones and 3 saponins of Huangqi first increased and then decreased with the amount of compatibility of Sanqi increased.One the other hand,compared with the Sanqi water extracts,the contents of the instituents of Sanqi increased after the compatibility of the two berbs.But the contents of panax notoginseng saponins,namely ginsenoside Rg1,Rd and Rb1 have been gradually decreased with the rising amount of compatibility of Huangqi.4.Due to the better response of compounds of Bupiyishen decoction in the negative ion mode,the negative ion was used for detection and analysis and most of the compounds were preliminaryly structurelly confirmed.The LC-MS spectrum of the decoction mainly contains 80 chromatographic peaks,and better separation achieved between these peaks.The retention time and precise molecular weight of each peak,as well as their tandem mass spectrometry fragmentation information were obtained by detecting,the structure of each chemical compound was confirmed by compared data with those of the reference compound and related literature.The comparison of relative peak area showed that the contents of chemical compounds has been changed before and after the compatibility of decoction.The contents of Astragalus isoflavones and saponins slightly increased while contents of salvianolic acids subtle decreased.5.Through the quantitative detection method by HPLC-MS,the content of chemical instituents were influenced by different concentration and drying processes of Bupiyishen decoction.Among its drying products,the main components of frozen drying and spray drying ones incurred smaller losses after Spray drying or frozen drying,the content of each main component were higher and more stable,so the spray drying method could shorten the drying time,reduce the loss of effective components,and the process condition was reasonable.ConclusionThe herb pair and combination which contain Astragalus,namely Huangqi-Sanqi herb pair and Bupiyishen decoction,were studied in this topic.The UPLC/ESI-LTQ-Orbitrap MSn combination of mass spectrometry was applied to identifying the chemical material basis of these prescriptions and elucidate mass fragmentation rules and pathways of different structure types.In addition,the multi-component quantitative analysis of these two kinds of prescriptions carried out separately,the dynamic changing process of their instituents affected by their compatibility proportion,concentration or drying process was discussed.Aiming at the difficult problems of identification of chemical constituents of TCM material foundations in the research,the thesis adopted liquid chromatography-mass spectrometry methods and exploratorily studied variety chemical components of traditional herbs,including Astragalus,Codonopsis,Radix Polygoni Multiflori,Panax Notoginseng,Salvia miltiorrhiza and Cuscuta Chinensis.The study summarized the mass fragmentation law defferent chemical structure types(including Astragalus saponins,Astragalus isoflavones,notoginsenosides,phenylpropanoid glycosides,stilbene glycosides and anthraquinones and et al),and the characteristic fragmentation ions and pathways were concluded as well.The results show a good prospect of application of UPLC/ESI-LTQ-Orbitrap MSn combination of mass spectrometry in the complex system of traditional Chinese medicine.According to complication and content difference of chemical compounds of TCM pescriptions,the compounds dissolution changes of Huangqi-Sanqi herb pair(including isoflavones and saponins)under different ratio conditions of compatibility.The dissolution of main components of Radix Astragali and Panax Notoginseng has somehow changed under different ratio condition,which caused by different compatibility.Quantitative detection method of representative chemical compounds by HPLC-MS is established to investigate the dissolution change of chemical instituents caused by different concentration and drying processes of Bupiyishen decoction.the main components of frozen drying and spray drying ones incurred smaller losses after Spray drying or frozen drying,the content of each main component were higher and more stable,so the spray drying method could shorten the drying time,reduce the loss of effective components,and the process condition was reasonable.