| Dendrobium is the second largest genus in the orchid,there are 76 species discovered in China,and nearly 40 kinds are used of medicine.Dendrobium was set out in the Eastern Han Chinese "Shen Nong Ben Cao Jing" firstly.It has been widely used in the treatment of weakness after febrile diseases,thirsty,fireexcess from yindeficiency,asthenia-syndrome and other diseases.Dendrobium contains several chemical constituents such as polysaccharide,flavonoids,dibenzyls,phenanthrene,alkaloidsand amino acids.Modern experiments have showed that they havea number of pharmacological effects like enhancing the immunologic function,anti-tumor,promoting the secretion of digestive juices,inhibiting platelet aggregation,decreasing high blood lipid and blood sugar,antioxidant,anti-aging,anti-pyreticanalgesics and so on.The records of sources of Dendrobium were the fresh or dry stems of Dendrobium nobile,Dendrobium chrysotoxum,Dendrobium fimbriatum and its allied species of the same genera in the 2015 editions of "Chinesepharmacopoeia",with Dendrobium offinale single listed separately.According to the ’ Chinese Herbal Medicine Quality Standards in GuangdongProvince’(I),Flickingeria Fimbriata(Bl.)Hawkes,the whole dry grass with pseudobulbs also named daigua dendrobuim,as one of the medical dendrobiums,is distributed in southern China,including Guangdong,Guangxi,Hainan,Yunnanand Guizhou Provinces.It has the advantage ofbenefiting stomach,nourishingyinand clearing heat,clinical using at deficiency Yin-fluid、dry mouth polydipsia、eat less and retching,besides deficiency-heat and blurring after illness.Thus it is used as one of the substitution products of Dendrobiumin Guangdong province.The chemical research about Herba flickingeriae was few,and thus no quality standard has been recorded.In this research,we investigated in chemical constituents systematically,and also establishedits quality standard,which might provide scientific basis for its clinical application and further research.Traditional Chinese medicine fingerprint technology is recognized as the most effective method of traditional Chinese medicine quality controlathome and abroad.Herba flickingeriae was studied qualitatively and quantitatively by characteristic fingerprint technology combined with contentdetermination methods and thethin-Layer Chromatography,which can provide the methodologybasis for quality control.Objective:1.To establish HPLC characteristic spectrum of Herba flickingeriae and to analysis the correlation between Herba flickingeriaeand its water solution,evaluating the effect of extract by decoction.2.To establish a method for determination of schaftoside,isoschaftoside and protocatechuic acid by HPLC-MS/MS.3.To establish the method of the studies of Herba flickingeriaeby thethin-Layer Chromatography(TLC).Methods:1.The characteristic spectrum mainly includes the screening of chromatographic conditions,the methodological evaluation,the alignment of characteristic peaks,and specificchromatogram similarity evaluation system software writed by the State Pharmacopoeia Commission Specific chromatogram similarity evaluation system software(2004A version)was adopted to generate a total of mode;and use the same method to analysis and compare the different breed and different provenance source of Herba flickingeriae with water decoction,and the correlation analysis between it and the medicinal material.The characteristic spectrum from Herba flickingeriae and its water solutionwas established.HPLC was employed with ZORBAX SB Cis column,the acetonitrile-0.1%phosphoric acid solutionwas employed as a mobile phase by gradient elution,detection wavelength was 220nm and 340nm,column temperature was35℃，and flowrate was 1.0 mL/min.2.The schaftoside,isoschaftoside and protocatechuic acid was identified by HPLC-MS combining with retention time and UV of reference substances.Besides,a method for determination of schaftoside and isoschaftoside was set up.HPLC method was used with the Zorbax SB-Aqcolumn,the acetonitrile-0.1%phosphoric acid(15:85)was employed as a mobile phase by gradient elution,detection wavelength was 340 nm.Column temperature was 35 ℃,and flow rate was 1.0 mL/min.In the same way,HPLC method of protocatechuic acid was used with the Zorbax SB-Aqcolumn,the acetonitrile-0.1%phosphoric acid(2:98)was employed as a mobile phase,detection wavelength was 220 nm.Column temperature was 30℃,and flow rate was 1.0 mL/min.3.Two thin-Layer Chromatography method were adopted to identify flavonoidsand protocatechuic acid separately from Herbaflickingeriae.Identification of flavonoids:using polyamidefilm,itsmobile phase system was water-alcohol-aceticacid-butanone-acetone(6.5:2.2:1:1.3:0.5)and color-developingagent was 0.3%AICl3 solution;Identification of protocatechuic acid:using silica gel G plate,its mobile phase system was trichloromethane-methyl alcohol-formic acid(9:1.8:0.5)and color-developingagent was 5%FeCl3 ethanol solution.Results:1.The HPLC characteristic spectrum analysis methodof medium polarity part from Herbaflickingeriae was established.(1)The 12 batches of Herba flickingeriae were divided into 4 groups in the detection wavelength of 220nm,with 21-26 common peaks were separated and 19 of which belong to all 4 groups.The similarities of the first group of 5 batches were 0.950～0.988 with 21 common peaks,the second group of 3 batches were 0.943～0.987 with 26 common peaks,and the third group of 3 batches were 0.977～0.988 with 23 common peaks.The fourth group of 1 batch had one more common peak than the third group,which differed from the latter.The 12 batches were divided into 2 groups in the detection wavelength of 340nm,with 7 to 8 common peaks of flavonoids.The similarities of the first group of 11 batches were 0.966-0.988,while the second group had one more common peak with obvious differerence.The similarity between the common pattern of the first group and the second group was only 0.671.(2)The method as same as the characteristic spectrum of Herba flickingeriae was adopted to establish the characteristic spectrum for Herba flickingeriae water decoction by HPLC.The 12 batches of Herba flickingeriae were divided into 4 groups in the detection wavelength of 220nm,with 21～25 common peaks were separated and 17 of which belong to all 4 groups.The similarities of the first group of 5 batches were 0.906～0.972 with 16 common peaks,the second group of 3 batches were 0.955～0.986 with 26 common peaks,and the third group of 3 batches were 0.979～0.989with23 common peaks.The fourth group of 1 batch had one more common peak than the third group,which differed from the latter.The 12 batches were divided into 2 groups in the detection wavelength of 340nm,with 7 to 8 common peaks of flavonoids.The similarities of the first group of 11 batches were 0.909-0.985,while the second group had one more common peak with obvious difference.The similarity between the common pattern of the first group and the second group was only 0.659.The similarities of each batch sample betweenHerba flickingeriaeand and its water solution of Herba flickingeriae were 0.733～0.919,which prompted conventional water decoction can basicallykeep the main characteristic component ofHerbaflickingeriae.2.Three contents(schaftoside,isoschaftoside and protocatechuic)was identified by HPLC-MS/MS and the method of contentdetermination was established.(1)The regression equation of schaftoside and isoschaftoside were y=1074.6x-22.302(r=0.9999),y=959.0x-30.767(r=0.9999),which the linear ranges were0.188μg～2.261μg,r=0.9999,0.220μg～2.635μg,r=0.9999,respectively.The average recovery of schaftosidewas 100.2%and RSD was 0.40%(n=6),and the average recovery of isochaftoside was 101.5%and RSD was 1.18%(n=6).The content of Schaftoside and Isoschaftoside from 12 batches of Herba flickingeriae were 0.244～1.469mg/g,0.307～1.630mg/g,respectively.(2)A method for determination of protocatechuic acid in Herba flickingeriae was established by HPLC.Baseline separation was obtained for protocatechuic acid content.There gression equation of protocatechuic acid was y = 5567.4x-11.717,r=1.0000,which the linear ranges was 0.055～1.100 μg,respectively.The average recovery was 101.0%and RSD was 2.54%(n=6).The content of protocatechuic acid in the 12 samples of commercially available Herba flickingeriae were among0.0374-0.203 mg/g,with distinct difference.3.The thin-Layer Chromatography was adopted to identify flavonoids and protocatechuic acid from Herba flickingeriae.Identification of Schaftoside and Isoschaftoside,there were six main spots;Identification of protocatechuic acid,the colour was also obvious.Conclusion:1.The analysis means of HPLC characteristic spectrum of Herba flickingeriae are accurate and reliable,also has a good repetitiveness,which provide the methodology basis for identification and quality control for Herba flickingeriae.In the detection wavelength of 340nm,the common peaks of flavonoids are steady,with low difference among samples,while in the detection wavelength of 220nm,the information of common peaks is abundant and the methods take a better advantage to distinguish the quality of Herbaflickingeriae.A great quantity of main characteristicingredients is remained by water cooking generally in the characteristic spectrum of Herba flickingeriaewater decoction,which provides the methodologybasis for the studies of the material basis of Herba flickingeriae.2.Two Flavone C-glycosides(Schaftoside,Isoschaftoside)and protocatechuic acidare identified from Herba flickingeriae.Their methods of content determination are accurate and reliable withagood resolution and repeatability,providing effective content componentsas the indicators for Herba flickingeriae.3.The method of TLC of Herba flickingeriaeis simple.The identified flavonoids and protocatechuic acid of chromatography are distinct and the spots are clear,which provides the evidence of identification for the quality control of Herba flickingeriae. |
PDF Full Text Request