| The Scorzonera austriaca Willd.is perennial herb of Compositae. As a tradionalmedicine herb, Scorzonera austriaca Willd. is distributed in the north and part of northwestof China. Pharmacological experiments proved hepatoprotective effect of flavonoids extractfrom Scorzonera austriaca Willd. Therefore it is necessary to study on flavonoids inScorzonera austriaca Willd. Scorzonera austriaca Willd. is used as a raw material in thisthesis, including four aspects: the extraction and isolation of total flavonoids fromScorzonera austriaca Willd., purification of total flavonoids by macroporous resin, HPLCfingerprint of flavonoids purified and quantitative determination of three flavonoids byHPLC-UV. It can provide strong security for quality control of Scorzonera austriaca, andlay foundations for pharmacological activity study on anti-HBV.Research results are as follow:Firstly, study on the chemical constituents, the extraction and separation ofScorzonera austriaca Willd.10kg of Scorzonera austriaca Willd. was extracted with70%ethanol three times, a week each time. The extract was filtered, concentrated and extractedwith ethyl ether.The water solution was subjected to D-101macroporous resin column,concentrating60%ethanol elution part and267g total flavonoids was collected. Threeflavonoids were isolated with silica gel column and ODS columns:5,7,3′,4′-tetrahydroxyl-6-C-β-D-glucopyranosyl flavone;5,7,3′,4′-tetrahydroxyl-8-C-β-D-glucopyranosyl flavone;5,7,4′-trihydroxy-flavonoid-8-C-β-D-glucopyranosyl.Secondly, establish a method for examining the content of flavonoids compounds inScorzonera austriaca Willd.5,7,4′-trihydroxy-flavonoid-8-C-β-D-glucopyranosyl was usedas reference substance, AlCl3-MeOH was chosen as the color-developing agent, PH ofHAc-NaAc buffer solution was5, and the ultraviolet-visible spectrophotometry wasadopted to establish the calibration curve, with the detection wavelength at390nm.Thirdly, purification of flavonoids extracted from Scorzonera austriaca Willd bymacroporous resin was studied. By static adsorption and desorption tests, D4020resin waschosen for the separation of flavonoids due to its higher adsorption and desorptioncapacity.The content of flavonoids in the extract purified by D4020macroporous resin isthe highest. The saturated resin was first washed with5%ethanol to remove impurities,collecting the purified component washed with10-30%ethanol.The content of flavonoidswas improve to90%at least.Establish HPLC fingerprint of purified flavonoids in Scorzonera austriaca Willd. TheHPLC fingerprint of purified flavonoids in Scorzonera austriaca Willd. consisted of14 mutual peaks and3mutual peaks were confirmed by3reference substances. The resultsthat the similarity of12samples of purified flavonoids in Scorzonera austriaca Willd.wasmore than0.9indicated the correlation of HPLC fingerprint and helped to obtain themedicinal pattern.Getting purified flavonoids extracted from12batches of Scorzonera austriaca Willd.with the method above. The chromatogram peak of the separated flavonoid(5,7,3′,4′-tetrahydroxyl-6-C-β-D-glucopyranosyl flavones) was chosen as the referencesubstance, to establish the HPLC fingerprint chromatographic conditions. Chromatographiccondition was as follow: Phenomenex Gemini5μm C18110A chromatographic column（250mm×4.6mm，5μm）,the UV detection wavelength is337nm, column temperature is30℃, injection volume is10μl. Mobile phase A is water-0.1%methane acid,mobile phaseB is acetonitrile. Gradient elution:0.01~7.00min, B is97~86%;7.01~30.00min, B is86~83%;30.01~60.00min, B is83~65%,60.01~65.00min, B is65~0%. The flow rate is0.8ml·min-1.At last, establish the method of determining the content of5,7,3′,4′-tetrahydroxyl-6-C-β-D-glucopyranosyl flavone;5,7,3′,4′-tetrahydroxyl-8-C-β-D-glucopyranosyl flavonesand5,7,4′-trihydroxy-flavonoid-8-C-β-D-glucopyranosyl by HPLC-UV. The methodologyvalidation proved good linearity, precision and stability, while the recovery was in98%~99%. The difference of the content of three flavones from12batches of Scorzoneraaustriaca Willd. determinated by the method above showed individual characteristics. |
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