| Dendrobium huoshanense has better curative effect in clinic,due to the lack of resources has not been a specific varieties to "Pharmacopoeia of the People’s Republic of China",only as a source of Dendrobium belong to similar species,therefore,quality control of Dendrobium huoshanense has not a clear standard.In recent years,large-scale breeding technology and Bionics wild cultivation technology is being continued to develop and promote the sustainable development and utilization of the resources of Dendrobium huoshanense in AnHui Province.However,there have been good and bad quality of raw materials and finished products during the development of the market,common Dendrobium moniliforme,Dendrobium officinale processed into HuoDou to pretend to fight profiteering,is not conducive to clinical medicine prescription medication,it is necessary to establish the quality standard of Dendrobium huoshanense.2010,2015 edition of "Pharmacopoeia of the People’s Republic of China" in the quality standards of Dendrobium officinale focused on polysaccharide and mannose,by phenol-sulfuric acid method for the determination of polysaccharides content of Dendrobium officinale,calculated on the dried basis,containing anhydrous glucose(C6H12O6)meters shall be not less than 25%,Also provides mannose(C6H12O6)content range calculated by drying after polysaccharide hydrolysis,should be 13.0%～38.0%,the ratio of mannose and glucose peak area should be 2.4 to 8.0.but polysaccharide are not specific component in Dendrobium,our previous study of Dendrobium huoshanense found that the flavonoid composition more,content is relatively high,and Dendrobium candidum has obvious difference,but the content of glucose,mannose and mannose and glucose peak area ratios of Dendrobium officinale have certain similarity.So only in accordance with polysaccharide as an index can not accurately evaluate the quality of the merits of Dendrobium officinale,and flavonoids from two species of Dendrobium are of unknown composition.Therefore,this study on the basis of the preliminary study,using mass spectrometry study of Dendrobium huoshanense and Dendrobium officinale flavonoid composition,establish a more scientific and perfect quality control system,have a very important theoretical and practical significance to the quality and development of Dendrobium huoshanense and Dendrobium officinal.Objective1.To establish the UHPLC/UV/MS spectrum characteristics of Dendrobium huoshanense,and the total model will be applied to the identification and evaluation of the merits;2.We will use Electrospray Ionization Mass Spectrometry technology analysis the flavonoids of the Dendrobium huoshanense and Dendrobium officinale,lay the foundation for later study;3.To establish content of flavonoid components in Dendrobium huoshanense by UHPLC-MS/MS method,provide quality reference data for the construction of rare and endangered species of medicinal Dendrobium huoshanense;4.To establish content of flavonoid components in Dendrobium officinale by UHPLC-MS/MS method for perfecting the quality evaluation system of Dendrobium officinale.5.The Dendrobium officinale,Dendrobium moniliforme and Dendrobium huoshanense will be processed under the same conditions,comparing differences with the characteristic fingerprint of Dendrobium huoshanense;Methods1.Liquid-phase mass spectrometry technique was used to establish UHPLC/UV and UHPLC/MS spectrum characteristics of Dendrobium huoshanense,main screen of mobile phase system,temperature,flow rate and flow compared to cases,and methodological evaluation,finalized column was Hypersil GOLD C18(100 × 2.1mm ID,1.9μm,Thermo,USA);column temperature was 35℃;detection wavelength was 335nm;recording time was 35min;injection volume 2μL.2.Used ion trap mass spectrometry(ESI/MSn)analysis the chemical constituents of Dendrobium huoshanense spectrum characteristics comprehensively,mass spectrometry conditions for ESI ion source:scan range 100～1000m/z;negative mode detection;atomizing nitrogen sheath gas(40psi);atomizing auxiliary gas for nitrogen(10psi);capillary temperature 300℃;spray voltage was 3.0KV;collision gas was helium;collision energy was 35%.3.UHPLC-MS/MS method for the determination of flavonoids index components in Dendrobium huoshanense was as follow:Hypersil GOLD C18(100 × 2.1mm ID,1.9μm)column,methanol and lOmM aqueous ammonium acetate as the mobile phase gradient elution with negative ion electrospray ionization mode,select reaction monitoring mode scanning and standard external standard solution.4.Used UHPLC-MS/MS method to quantitative three flavonoids in Dendrobium officinale,and the content of 12 batches Dendrobium officinale was analyzed by Spass statistics.5.The State Pharmacopoeia Commission Specific chromatogram similarity evaluation system software(2004A version)was adopted to generate a total of mode;and used the same method to analysis and compare the different breed source of Dendrobium.Results1.The establishment of 11 batches Dendrobium huoshanense flavonoids UHPLC/UV/MS spectrum characteristics,where the similarities in the UHPLC/UV spectrum characteristic were 0.794 to 0.985,identified 17 common peaks;and,the similarities in the UHPLC/MS spectrum characteristics were 0.81 to 0.954,identified a total of nine peaks.2.Eight flavonoids were identified in Dendrobioum huoshanense,which are Apigenin-6,8-di-c-β-D-glucoside,Apigenin-6-c-β-D-xyloside-8-c-β-D-glucoside,Isoschaf--toside,Schaftoside,Apigenin-6,8-di-c-a-L-arabinoside,vitexin-2"-o-rhamnoside,Naringin,Apigenin-6-c-a-L-arabinoside-8-c-β-D-xyloside.Combined the literature explore the fragmentation patterns preliminarily,so the methods can be used as a qualitative methods of Dendrobium huoshanense and accurate.3.UHPLC-MS/MS method was developed for the content of three index components of flavonoids in Dendrobium huoshanense.Isoschaftoside,schaftoside and Apigenin-6,8-di-c-a-L-arabinoside respectively range a good linear relationship in 7.5～51ng,5.3～53ng,7.5～120ng.and the average recovery(n=6)were 97.6%,98.1%,97.1%,RSD were 2.22%、2.26%、2.80%.4.Nine flavonoids were identified in Dendrobioum officinale,which were Apigenin-6,8-di-c-β-D-glucoside,Apigenin-6-c-β-D-xyloside-8-c-β-D-glucoside,Isoschaf--toside,Schaftoside,Apigenin-6-c-β-D-glucoside-8-c-p-D-xyloside,Rutin,Vitexin-2"--o-rhamnoside,Isovitexin-2"-o-rhamnoside,Naringin.5.UHPLC-MS/MS method was developed for the content of two index components of flavonoids in Dendrobium officinale.Apigenin-6,8-di-c-β-D-glucoside,and Rutin respectively range a good linear relationship in 4.5～18ng,0.15～187.5ng,and the average recovery(n=6)were 99.9%,97.2%,RSD were 2.20%,2.83%.6.The spectrum characteristics of Dendrobium huoshanense,Dendrobium officinale,and Dendrobium moniliforme were quite different,which Dendrobium moniliforme chromatography was mainly for the two characteristic peaks,had significant differences with Dendrobioum huoshanense;The characteristic spectrum of Dendrobioum huoshanense was applied as a mode control to analysis the similarity of 8 batches of Dendrobium officinale and 6 batches of Dendrobium moniliforme,and the similarities r were 0.096～0.406,0.279～0.391espect ively.Conclusion1.The method of UHPLC/UV/MS characteristic spectrum about Dendrobioum huoshanense is acourate,reliable,repeatable.While the characteristic spectrum of Dendrobioum huoshanense is the mutual mode control,Dendrobioum huoshanense,Dendrobium officinale and Dendrobium moniliforme have big differences,which provide a method for authentication of Dendrobium huoshanense.2.We identify eight flavonoids from Dendrobioum huoshanense combining with the analysis of its fragmentation patterns,it is helpful for more component of identification in Dendrobioum.3.The UHPLC-MS/MS method for content determination depend on the flavonoids index component in Dendrobioum huoshanense and Dendrobium officinale is simple and easy to operate,can be used for quality control. |
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