| Dendrobium(D.)is originally recorded in the "Shen Nong's Herbal Classic" and the traditional valuable Chinese medicine,which have been widely used in the treatment of weakness after ferbrile diseases,thirsty,fire excess from yin deficiency and so on.There are 74 species and 2 variant species in China.D.moniliforme(L.)Sw.of this study is also one of the important species.The history of Dendrobium in Lu'an has a long history.The case of"Fan Ziji Ran" cloud,Dendrobium,out of Lu'an."Ming Yi Bie Lu" in the "Dendrobium Sheng Liuan Valley water near the stone".Lu'an is located in today's Anhui province.And it is including D.huoshanense C.Z.Tang et S.J.Cheng?D.officinale Kimura et Migo and D.moniliforme.There apear the phenomenon that D.huoshanense?D.officinale are posing by D.moniliforme.With Little research of D.officinale was done,it was difficult to identify different kinds of Dendrobiums.And itwas difficult to identify the quality of D.moniliforme good or bad in the market.The recent study focused on polysaccharides,alkaloids and small molecule compounds,but there was no research report on phenolic compounds.About the characteristics of fingerprints of D.officinale,did many studies such as the Characteristic fingerprints of D.Huoshanense,D.officinale,D.nobile Lindl,D.loddigesii.To the identification of the authenticity of the quality of Dendrobium,it provides a great basis.Studing the determination of Characteristic components in order to provide the basis for the quality control.Objective:l?To extract,separate and purified the flavonoid glucoside in the stem of D.moniliforme.The structure of flavonoid glycosides was obtained by structural analysis,which provide the foundation for studing on the determination of characteristic flavonoid glycosides in D.monilforme.2?To establish a method to determine vitexin-2 "-O-rhamnoside and apigenin-6-C-[?-rhamnopyranosyl-(1?2)]-?-glucopyransyl,which were separated.And ten batches of D.moniliforme were determined.3?To establish the TLC methods of vitexin-2 "-O-rhamnoside and apigenin-6-C-[?-rhamnopyranosyl-(1?2)]-p-glucopyransyl in D.moniliforme.4?Based on liquid chromatography-mass spectrometry(LC-MS),the flavonoids were analyzed qualitatively,and the UHPLC/UV/MS spectrum characteristics were established.The common peaks were marked and their structures were deduced.The study will be applied to the identification and evaluation of the merits,and will be provided the foundation for studing on D.moniliforme5?To study the protective effect of flavonoid glycoside on rat bone marrow mesenchymal stem cells(MSC)oxidative damage by H2O2.Explore its antioxidant capacity in order to provided the foundation for further pharmacological studies.Methods:1?The solvent extraction method,AB-8 macroporous resin method,hydroxypropyl dextran gel chromatography and high performance liquid chromatography,were used to isolate flavonoid glycosides of D.moniliforme.UV spectrum,hydrogen spectrum(1H-MR),carbon spectrum(13C-NMR)and mass spectrometry(MS)were to identify the structure of the monomer.2?The method of determination of vitexin-2"-O-rhannoside and apigenin-6-C-[?-rhamnopyranosyl-(1?2)]-?-glucopyransyl of D.moniliforme:Chromatographic condi-tions:Agilent SB-Aq column for the column,methanol-10mM ammonium acet-ate as the mobile phase,gradient elution,0?10min:25%30%;10?15min:30%?33%;15?25min:33%35%;25?40min:35%;The flowrate was 1.0mLˇmin-1 and detection wavelength was set at 340nm.3?Using TLC methods to identify respectively three batches of D.moniliforme,which also were studied in different temperature and humidity conditions.4?Based on liquid chromatography-mass spectrometry(LC-MS),the flavonoids were analyzed qualitatively,and the UHPLC/UV/MS spectrum characteristics were established.The characteristic pattern common pattern was analyzed by using the software of the similarity evaluation of the chromatographic fingerprints of the Chinese Pharmaco-poeia.And the structure of the common peaks were deduced by UHPLC-ESI-MSn analysis5?To establish the oxidative damage model of MSC,and to study the protective effect of flavonoid glycoside on rat bone marrow mesenchymal stem cells(MSC)oxidative damage by MTTˇResults:1?Two Flavonoid glycosides,vitexin-2 "-O-rhamnoside and apigenin-6-C-[?-rhannopyranosyl-(1?2)]-?-glucopyransyl,were seperated and ldentificated from D.moniliforme.2?The calibration curves of vitexin-2 "-O-rhamnoside showed a good linearity in the rage of 0.344?5.16 ?g(r=0.9999).The average recoverie was 102.8%and RSD was 1.59%(n=6).The calibration curves of apigenin-6-C-[arhamnopyranosyl-(1?2)]-?-glucopyransyl showed a good linearity in the rage of 0.268?4.02 ?g(r=0.9998).The average recoverie was 99.4%and RSD was 1.62%(n=6).The content of vitexin-2 "-O-rhamnoside is in the range of 0.055%?0.136%with 10 bacths.The content of apigen-in-6-C-[?-rhamnopyranosyl-(1?2)]-?-glucopyran syl was in the rage of 0.050%?0.117%?3?In the reference substance chromatograms of vitexin-2 "-O-rhamnoside and apigenin-6-C-[?-rhamnopyranosyl-(1?2)]-?-glucopyransyl,same colours were appeared respectively in the same location with reference.When the plates were under the condition of normal or low temperature or humidity,those chromatographs unfolded ideally whose characteristie spots were clear and round similarly and the application are nice.4?Based on liquid chromatography-mass spectrometry(LC-MS),the UHPLC/UV/MS spectrum characteristics were established.There are five common peaks in the fingerprints of D.moniliforme,four flavonoid glycosides were separated and identificated,Vitexin(1),vitexin-2-O-rhannoside(2),Isovitexin(3),apigenin-6-C-[?-rhamnop-yranosyl-(1?2)]-?-glucopyransyl(4).5?Vitexin-2"-O-rhamnoside did not significantly promote the growth of MSC cells.Compared with the blank control group,H2O2 group the proliferation ability of MSC cells decreased significantly indicating(P<0.01)which description modeling success.Compared with H2O2 groud,the different concentration of vitexin-2 "-O-rhainoside has restore effect on MSC oxidative injured by H2O2 and showed dose-effect relationship(P<0.01).Conclusion:1?Vitexin-2 "-O-rhamnoside and apigenin-6-C-[?-rham-nopyranosyl-(1?2)]-?-glucopyransyl were separated firstly from D.moniliforme.;2?The method by HPLC to determine the vitexin-2"-O-rhamnosideand apigenin-6-C-[?-rham-nopyranosyl-(1?2)]-?-glucopyransy were established.The quantitative deter-mination method with good accuracy,repeatability and stability is suitable for the quality control of D.moniliforme;3?These methods were high reliability,strong separating force,with the advantages of short on time,high sensitivity,coloring conveniently,which can be used as the TLC methods of vitexin-2 "-O-rhamnoside and apigenin-6-C-[?-rhamnopyranosyl-(1?2)]-?-glucopyransyl in D.moniliforme.4?The UHPLC/UV/MS spectrum characteristics of D.moniliforme were established,which will be applied to the identification and evaluation of the merits,and will be provided the foundation for studing on D.moniliforme;5?The different concentrations of vitexin-2"-O-rhamnoside had restore effect on MSC oxidative injured by H2O2 and showed dose-effect relationship,which Reveal vitexin-2"-O-rhamnoside have antioxidant effect. |
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