Study Of Separation And Detection Of Chemical Components And Quality Control Indexes Of Tussilago Farfara L.

Kuan Donghua,the dried flower buds of Tussilago farfara L.,was traditionally used in China,Europe,South Africa,and Siberia for the treatment of asthma,bronchitis,cough,and phlegm.Tussilago farfara which recorded in Chinese Pharmacopoeia was a pungent and warm-natured genuine medicinal material of Chongqing.Literature review indicateds that the researches of Tussilago farfara were mainly focused on several common active compounds,such as tussilagone,rutin and quercetin,etc.The current quality control standard only limits the content of tussilagone,which impact its clinical safety and effectiveness.To address the above problems,a quality control system of multiple indexes was presented in this thesis based on the existing research achievements of analysis and quality control of natural plant.In this paper,combining the methods of extraction and separation with technology of analysis,the different chemical components of Tussilago farfara were sorted into four types according to their properties and classifications of multiple indexes.Several efficient analysis methods were established for these four type components.The results have supplemented and improved the current quanlity control indexes.The main investigations in this thesis are as the following.(1)On the basis of a large amount references of chemical component,separation and analysis methods,pharmacological activity of Tussilago farfara,the theory and technological scheme of the study of Tussilago farfara quality control indexes were put forward and formed.(2)According to properties of active components,toxic components and macro components of Tussilago farfara,solvent extraction method,acid water extraction method and water-extraction and alcohol-precipitation method were used to extract active components,total alkaloid and polysaccharides of Tussilago farfara,respectively.8 compounds were isolated from petroleum ether extract and ethyl acetate extract of Tussilago farfara by using column chromatography,prepatrtion thin layer chromatography,recrystallization and other technologies,and their structures were identified by IR,ESI-MS and NMR.Alkaloid contents of acid extract and total alkaloid were analyzed by the acid dye colorimetry method.Three crude Tussilago farfara polysaccharides(TPS-1,TPS-2 and TPS-3)were obtained grading-alcoholic precipitation.The results provide the materials basis for analysis of multiple quality control indexes of Tussilago farfara,(3)A solvent system optimization method of HSCCC was established,combined partition coefficient(K)determination with HSCCC analysis.Based on this approach,a ternary solvent system was selected and applied for the HSCCC,and index compound tussilagone was isolated from petroleum ether extract of Tussilago farfara,along with 14-acetoxy-7 β-(3’-ethylcis-crotonoyloxy)-lα-(2’-methylbutyryloxy)-notonipetranone.(4)Tussilagone of Tussilago farfara was analyzed by micellar electrokinetic capillary electrophoresis,with the content of 1.66 mg·g-1.And a HPCE method has been established for simultaneous determination of muitiple components as emphases,such as terpenoids,flavonoids,organic acids,and so on.The main parameters that affected separation efficiency were investigated in details.Under the optimal conditions,10 peaks(2,2-Dimethyl-6-acetylchromanone,Rutin,Isoferulic acid,Hyperoside,Ferulic Acid,Chlorogenic Acid,Isochlorogenic acid A,Quercetin,Gallic acid and Caffeic acid)were recognized,in which 9 components’ content were determined,except Gallic acid.(5)Due to and traces of pyrrolizidine alkaloids in total alkaloids,the full quality control of Tussilago farfara L has been limited.The total amount of retronecine esters-type pyrrolizidine alkaloids(RET-PAs)was quantitied because of a common colored retronecine marker,which was produced with all the different RET-PAs during a derivatization reaction.In order to solve the difficulty of quantitative determination of multiple pyrrolizidine alkaloids in the total alkaloids,an on-line pre-concentration method has been applied to improve determining sensitivity of pyrrolizidine alkaloids in Tussilago farfara L.in which included field-amplified sample stacking and sweeping in micellar electrokinetic chromatography.The main parameters that affected separation and stacking efficiency were investigated in details.Under the optimal conditions,the sensitivity enhancement factors obtained by the developed method for the analytes were from 15-to 12-fold,the limits of detection of senkirkine and senecionine were 2.8 and 4.2 μg·L-1,respectively.Senkirkine and senecionine have been detected in alkaloids of Tussilago farfara L,along ferulic acid methyl ester and methyl caffeate.The developed method was also applied to the analysis of acid extraction of Tussilago farfara L,and senkirkine could be detected directly.(6)Focusing on the comprehensive utilization of Tussilago farfara polysaccharides,the contents of neutral sugar and uronic acid of series Tussilago farfara L.acidic polysaccharides were determined by phenol-sulfuric acid and hydroxy diphenyl between colorimetric methods,respectively.FT-IR spectroscopy was used to further analyze the structural differences among series acidic polysaccharides.In order to further determinate the monosaccharide composition of series Tussilago farfara L.acidic polysaccharides,a facile and sensitive method based on capillary zone electrophoresis of pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone(PMP)had been established.The main parameters that affect separation were investigated and optimized.The proposed PMP-CZE method was an efficient method for monosaccharides composition analysis and quality control of polysaccharides from Tussilago farfara L.All acidic polysaccharides exhibited antioxidant activities which were evaluated by 1,1-diphenyl-2-picrylhydrazyl(DPPH)and cellular antioxidant activity(CAA)assay,and then the TPS-2 was shown better antioxidant activity than TPS-1 and TPS-3.It was also demonstrated that TPS-2 might be helpful to develop potential antioxidant agents for food and pharmaceutical industries.(7)Based on above research,a preliminary design of quality control indexes of Tussilago farfara L was proposed from 3 perspectives.Compared with the existing standards,more active components,toxic components and macro components were used as important indexes into the quality control system in this study.The results complement and improve the index of current quality control.