| Dendrobium nobile Lindl is grown in Guizhou,Yunnan,Guangxi and Sichuan province and it is one of genuine species recorded in Chinese Pharmacopoeia of every versions.The plant Dendrobium nobile Lindl has the function of nourishing yin,clearing heat,tonifying stomach and promoting fluid.Along with Dendrobium officinale as well as Dendrobium huoshanense,these three Dendrobium species are recognized as high value traditional Chinese medicine.In ancient times,the classical writings such as Shen Nong' s Herbal Classic and Compendium of Materia Medica had recorded Dendrobium nobile Lindl.Amount these Dendrobium species,Dendrobium nobile Lindl was specialized in clearing heat,nourishing Yin and tonifying stomach.These functions are closely related to the constituents in Dendrobium nobile Lindl.Dendroine is the main component in Dendrobium nobile Lindl.In the epast versions of the Chinese Pharmacopoeia,the content of the Dendroine is an index of quality evaluation.There exist several research paper about the polysaccharide content in Dendrobium genues,however,the content of flavonoid glucoside in Dendrobium nobile Lindl has not been report.This study is based on the chromatographic fingerprint which has been studied thoroughly by our research group previously.There are huge differences between the chromatographic fingerprints of phenols in Dendrobium officinale,Dendrobium huoshanense and several other species.Also the chromatographic fingerprints of flavonoid glucoside in different Dendrobium species are different,so it can be applied to distinguished Dendrobium species with each other.There is a flavonoid glucoside with the highest content in the chromatographic fingerprint in Dendrobium nobile Lindl.Therefore,in this study,we aim to extract and purified this flavonoid glucoside,quantitative determination as well as anti-oxidant activities will also be investigate in order to provide some references in the establishment of the quality control.Objective1.This study is to extract,separate and purified the flavonoid glucoside in the stem of Dendrobium nobile Lindl which was grown in Chishui city,Guizhou province.2.10 batches of Dendrobium nobile Lindl was used to establish the quantitative determination method by HPLC.3.The flavonoid glucoside separated from the Dendrobium nobile Lindl would be used as an indicative components in order to check its existence in Dendrobium nobile of different origin,Dendrobium officinale,Dendrobium huoshanense,Dendrobium devoninum and Dendrobium moniliforme.The specificity of this flavonoid glucoside in Dendrobium nobile Lindl was explored.4.The oxidative damage model of MSC was established,and the protective function of flavonoid glucoside against oxidative damage in different concentration was test by MTT assay.The anti-oxidant ability of the flavonoid glucoside in Dendrobium nobile Lindl was studied to provide the basic data for further pharmaceutical research.MethodsIn this experiments,solvent extraction method was performed to extract the target component in Dendrobium nobile Lindl.Macroporous resin,sephadex LH-20 and silica gel column was used for the separation process to obtain pure flavonoid glucoside.And the chemical structure of it will be elucidated by physic-chemical characters and modern spectroscopy analysis.The flavonoid glucoside separated from Dendrobium nobile Lindl was used as a standard reference.Its content in the 10 batches of Dendrobium nobile Lindl was tested through RP-HPLC equipped with an Agilent SB-Aq column.The mobile phase was Acetonitrile?A?-0.2%phosphoric acid,and the elution gradient was shown below:0?10min 5%A?16%A,10?30min 16%A?25%A.The detection wavelength was 270 nm and the flow rate was 1.OmL/mL-1.The flavonoid glucoside separated from Dendrobium nobile Lindl was used as a standard reference.This study is to identificate Dendrobium nobile in different producing areas and different species of Dendrobium by high performance liquid phase method.Its content was tested through RP-HPLC equipped with an Agilent SB-Aq column.The mobile phase was Methanol?A?-0.2%phosphoric acid,and the elution gradient was shown below:0?10min 25%A?30%A,10?15min 30%A?33%A,15?25min 33%A?35%A;25?40min 35%A;40?60min 35%A?50%A;The detection wavelength was 340 nm and the flow rate was 1.0mL/mL-1.The MSC were separated and the oxidative damage model was build.The protective effect of flavonoid glucoside against oxidative damage in different concentration was test through MTT assay.Results1.In this study,we separated the highest content flavonoid glucoside in Dendrobium nobile Lindl and its chemical structure was characterized as 6-C-?-rhamnose-8-C-?-glucose-apigenin.2.The linear regression equation of the flavonoid glucoside in Dendrobium nobile Lindl is y=1640.1x-7881.2?r=1?.The linearity is good in the concentration range of 0.58?g?14.5?g.The average returning rate?n=6?is range of 98.38%?103.33%,and the RSD is 1.69%.It is certain that the detection method has good accuracy,repeatability and stability.I can be employed for the quality control of Dendrobium nobile Lindl.According to the determination results of the 10 batches Dendrobium nobile Lindl samples from Chishui city,Guizhou province,the flavonoid glucoside content isin the range of 0.103%?0.170%.3.In this part,we compared Dendrobium nobile with other origins in Chishui,Yunnan,Sichuan,Guangxi and other species.The corresponding peaks of Dendrobium flavonoids in Chishui were not related to the peaks of Dendrobium nobile in Yunnan and Dendrobium nobile in Sichuan.Dendrobium in Guangxi might contain this component in the corresponding position.Dendrobium officinale might contain Chishui Dendrobium nobile flavonoid glycosides,Dendrobium huoshanense,Dendrobium devonianum Paxt,Dendrobium moniliforme?L.?Sw.,are not included in this component.4.The cell viability was measured by MTT assay.Flavonoid glycosides from Chishui Dendrobium nobile had no toxicity on MSC with the concentration of 1-100?g/mLcompared with the control group.When pretreated with 600?M H202 for 2 h,the cell viability reduced significantly by 54%.Therefore,600?M H202 for 2 h was chosen for the following experiments.The protective effect of flavonoid glycosides against H2O2-induced cell inhibition was assessed by MTT test.While pretreated with 1,10,25,50,100?g/mL,the protective effect of flavonoid glycosides on H202-induced cell inhibition displayed dose-dependence.Conclusion1.The flavonoid glucoside content in Dendrobium nobile Lindle is in the rage of 0.103%?0.170%,when compare with the regulated concentration in Chinese pharmacopoeia the concentration?no less than 0.4%?in Dendrobium nobile Lindle is higher.2.We established a method for determination the content of flavonoid glucoside in Dendrobium nobile Lindle,and this would provide certain basis for the development of quality control of this remedy.3.The specificity of the flavonoid glycosides of Dendrobium nobile was proved by HPLC compared with that of Dendrobium nobile with other origins in Chishui,Yunnan,Sichuan,Guangxi and other species.4.Different concentration of flavonoid glucoside in Dendrobium nobile Lindle has restore effect on MSC oxidative injured by H202 and showed dose-effect relationship.It can be concluded that flavonoid glucoside in Dendrobium nobile Lindle has certain protective effect on MSC. |
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