| There are three main varieties of Codonopsis Radix(CR),namely Codonopsis pilosula(Franch.)Nannf.,Codonopsis pilosula var.modesta(Nannf.)L.T.Shen and Codonopsis tangshen Oliv.had been widely used in Chinese herbal medicine.Previous studies on CR mainly focused on polysaccharides,and natural products isolation of the weakly polar components.However,there is little research on the content determination and activity of the weakly polar components in CR.In order to gain a deeper understanding of the compound structural types,contents,and related activities of the weakly polar components in CR,this thesis first carried out the identification of the chemical components of the weakly polar parts of CR by using ultra performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS).Further,UPLC-MS fingerprints of the weakly polar portion were constructed,and spectral-efficiency correlation analysis of anti-inflammatory activity was carried out.A UPLC-Q-TOF-MS method was developed for the determination of 14components with possible anti-inflammatory activity in 23 batches of CR samples.And the identification of 3 varieties of CR was achieved by combining the content of the 14components with chemometric analysis.The specific research contents were as follows:1.Analysis of the chemical composition of the weakly polar components of CRUPLC-Q-TOF-MS/MS technique was used to optimize the mobile phase,ionization source and mass spectrum parameters.By comparing the retention time,relative molecular weight,primary and secondary mass spectrum information of the compounds,and combining with references,a total of 36 compounds(11 triterpenoids,5 sterols,8 alkaloids and nitrogenous compounds,1 hexoside and hexenol glycosides,2 alkynes and alkenes,5 phenolic and organic acids,3 sesquiterpenes and 1 other compound)were identified in the weakly polar components of CR.Triterpenoids and sterols are a group of natural products with a unique skeleton and good physiological activity,but there are few reports on the mass spectrometric analysis of triterpenoids and sterols in CR.Therefore,the mass spectrometric patterns of the triterpenes and sterols of CR were investigated.The effects of two ionisation sources,including electrospray ionisation ionization(ESI)and atmospheric pressure chemical ionisation(APCI),on the ionisation values of triterpenoids and sterols by mass spectrometry were compared.The results indicated that the APCI source was more suitable than the ESI source for the ionisation of triterpenoids and sterols in CR.The primary and secondary mass spectral information of triterpenoids and sterols in CR were concluded,the possible cleavage pathways of triterpenoids and sterols were speculated,and the identification patterns of two types of pentacyclic triterpenoid isomers in CR were summarized,which provides a reference for the analysis and identification of similar substances.2.Study on the spectrum-effect relationship of the anti-inflammatory of the weakly polar fraction of CRThe fingerprints of the weakly polar fraction of 18 batches of CR were constructed by using UPLC-MS and 20 shared peaks were labelled.The similarity of the fingerprints of the 18 batches of CR samples ranged from 0.850 to 0.973 indicating that the weakly polar fraction components of CR samples from different varieties,bases and growth years were different.An in vitro inflammation model was constructed by mouse mononuclear macrophages RAW264.7 and the inhibition rate of 18 batches of CR extracts on NO was determined.Grey correlation method was used to analyze the relationship between shared peak areas in the fingerprints and NO inhibition rates.It was found that peak 7,10,12,17 and 20 contributed the most to the anti-inflammatory effect,among them,shared peak 12 was presumed to be a sterol compound,shared peak17 wasβ-sitosterol and shared peak 20 was friedelin.Triterpenoids and sterols(glut-5-en-3β-ol,stigmasta-4,22-dien-3-one,stigmasterol,taraxerol,codopitirol A,24-methylenecycloartanol,codonopliate C were associated with anti-inflammatory,and the correlations of activity were all greater than 0.8.These results show that triterpenoids and sterols in the weakly polar components of CR play an important role in the anti-inflammatory effect.3.Determination of the content of 14 terpenoids and sterols of CR by UPLC-Q-TOF-MSDue to the low content,complex types of components,similar structure,no UV absorption,difficulty of ionizing,and hard to obtain comparison products,the content determination of triterpenes and sterols in CR has rarely been reported so far.Therefore,based on the 1 sesquiterpene lactone,10 triterpenoids and 3 sterols isolated from CR in our previous experiments,a UPLC-Q-TOF-MS method for the determination of 14components was constructed.All 14 compounds showed good linearity in their respective ranges with R~2≥0.9925,mean spiked recoveries in the range of81.25%～118.05%(RSD<4.00%),intraday precision RSD<4.54%and interday precision RSD<6.82%.The methodological investigation confirmed the simplicity and feasibility of the method.The method was further used to determine the content of 14components in 23 batches of 3 varieties of CR samples.It was found that the content of friedelin,taraxerol,β-sitosterol and stigmasterol in all batches of CR samples was greater than 2/10,000,which met the minimum requirements of the pharmacopoeia for the index content components.The experiment results illustrated that the content of 14chemical components in the 3 varieties of CR samples was different.Furthermore,with the combination of linear discriminant analysis,the 3 varieties of CR samples can be successfully distinguished. |
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