Comprehensive Chemical Characterization And Comparison Of Ziziphi Spinosae Semen And Ziziphi Mauritianae Semen

RATIONALES: Both Ziziphi Spinosae Semen(ZSS)and Ziziphi Mauritianae Semen(ZMS)were derived from the dried seeds of the genus Ziziphus.The classical application of ZSS and ZMS is supplementing the liver,quieting the heart,arresting the sweat,and promoting the production of the body fluids.Alone or in combination with other herbs,ZSS is the most frequently used herb for treating insomnia.In addition,ZMS was used widely in Yunnan as local used medicine.Recently,the reduction of the wildlife resources of ZSS and shortage of market supply resulted in the adulteration of ZSS by substituting with ZMS.Because the material basis and chemical difference between ZSS and ZMS are not clear,the combination of the two seeds can't guarantee the clinical safety of traditional Chinese medicine.Traditional Chinese medicine is the essence of Chinese culture and it has a history of thousands of years.The material basis of traditional Chinese medicine is complex,including primary metabolites,like amino acids,sugars,fatty acids and other as well as secondary metabolites such as flavonoids,saponins and alkaloids.Therefore,it is particularly important to find and establish a Chinese medicine analysis method that is suitable for the overall characteristics and complex substance system of Chinese medicine.In recent years,nuclear magnetic resonance(NMR),liquid chromatography mass spectrometry(LC-MS),gas chromatography mass spectrometry(GC-MS)analysis technology have been developing rapidly.However,various instruments are suitable for different substances,and a single technique cannot satisfy the complex substance analysis of Traditional Chinese medicine.Therefore,the innovative development and integration of multiple platform technologies to systematically characterize the chemical components of TCM can promote the development of the material basis research of TCM.At present,mass spectrometer can be used in combination with a variety of chromatographic techniques.With its characteristics of high sensitivity,high resolution and abundant information,high resolution mass spectrometry has been favored by more and more researchers of Traditional Chinese medicine and applied to the analysis of various traditional Chinese medicine.Plant metabolomics is an important branch of metabolomics.1H NMR,LC-MS and GC-MS are commonly used tools in plant metabolomics,with advantages of short detection time,good reproducibility and batch analysis.Metabolomics technology will obtain high-throughput data,while multivariate statistical analysis can extract information from complex data to the maximumextent and reflect the overall difference of sample grouping.In conclusion,a rapid method for identifying the chemical constituents of ZSS was established by high resolution mass spectrometry.The chemical profiles and differences of ZSS and ZMS were comprehensively analyzed by using a variety of analytical technology platforms and based on untargeted plant metabolomics.The difference metabolites were found and analyzed quantitatively.Objective: To establish a rapid,efficient and accurate method for the characterization of the chemical constituents of ZSS;The primary and secondary metabolites were compared and difference metabolites were found by untargeted metabolomics;A targeted quantitative method for simultaneous analysis of 9 components by LC-MS/MS was established and applied to the quality research for ZSS and ZMS.Methods:1.UHPLC-Q-Orbitrap MS analysis technology was used to obtain the mass spectrometry data of ZSS by using Full MS/dd ms2 data acquisition model.Establish a local database of chemical constituents and a theoretical molecular network of different types of compounds.The characteristics ions obtained by flavonoids,saponins and alkaloids under different normalized collision energies were imported into the Compound Class Scoring module of CD software to run.The running results were matched with the theoretical molecular network,and the structure of the compound was identified quickly by combining with MS/MS fragmentations of the compounds.2.Based on 1H NMR,GC-MS and LC-MS untargeted metabolomics techniques,amino acids,sugars,fatty acids and secondary metabolites in ZSS and ZMS were identified by combining with references,HMDB data,NIST database and rapid identification strategy of chemical constituents.Then the preprocessed data were imported into SIMCA-P software for multivariate statistical analysis.According to principal component analysis,orthogonal partial least squares discriminant analysis and s-plot,16 batches of ZSS and 13 batches of ZMS were analyzed and the differential metabolites were screened by VIP> 1 and p <0.05.The relative peak area of different metabolites was used to analyze the relative content.3.The standard analytical methods using UHPLC-MS/MS have been developed for the simultaneous quantification of 9 components.Various validation parameters such as specificity,linearity,detection limit,quantitation limit,accuracy and precision were successfully obtained and individually validated.The method was used to determine the content of 29 batches of ZSS and ZMS,and the results were analyzed by principal component analysis and partial least squares discriminant analysis.Results:1.By using UHPLC-Q-Orbitrap MS,a rapid identification strategy for the chemical constituents in ZSS was established.Firstly,the in-house library of ZSS was established.There were 114 compounds,including 50 flavonoids,36 saponins and 28 alkaloids.Secondly,based on the structural characteristics of the compounds,a theoretical molecular network was established to predict the chemical constituents of ZSS,thus 144 flavonoids,240 alkaloids and 32 saponins were predicted.Analyzing fragmentation pathways of reference substance,the characteristics ions of flavonoids,alkaloids and saponins were determined.Through bridging with CD software,a total of169 chemical components were matched.Among which 90 compounds were preliminarily identified including 64 flavonoids,21 alkaloids and 5 saponins compounds in ZSS.2.The 1H NMR technology was first time to be employed for the analysis of ZSS and ZMS.Peaks from ZSS and ZMS were assigned based on comparison with chemical shifts of standard references,HMDB as well as reported literature data.A total of 30 compounds were assigned to amino acids,organic acids and sugars.Then different metabolites were selected based on two criterions: VIP> 1 and p < 0.05.Resultantly,12 compounds,including leucine,isoleucine,alanine,lysine,glutamine,asparaginate,methionine,choline,citric acid,acetic acid,saccharose and raffinose,were considered as marker compounds differentiating ZSS and ZMS based on the results of 1H NMR.Moreover,the results indicated that leucine,isoleucine,alanine,lysine,glutamine,asparaginate,methionine,choline,citric acid,and acetic acid were higher in ZSS while saccharose and raffinose were higher in ZMS.A total of 28 compounds were tentatively identified from ZSS and ZMS by comparison with NIST library by the GC-MS metabolomics technique.those data from GC-MS analysis demonstrated that the major metabolic differences between two seeds were involved in stearic acid,palmitic acid,arachidic acid,oleic acid,behenic acid,linoleic acid and vitamin E.the contents of MUFA(stearic acid,palmitic acid,arachidic acid,oleic acid and behenic acid)in ZMS were significantly higher than those in ZSS while the contents such as linoleic acid and vitamin E were much higher in ZSS than those in ZMS.Based on UHPLC-Q-Orbitrap MS technology,in total 90 secondary metabolites including 64 flavonoids,21 alkaloids,and 5 saponins were tentatively characterized in ZSS and 69 secondary metabolites in ZMS.29 secondary metabolites were identified as differential compounds from ZSS and ZMS.9 genkwanin C-glycosides were presented higher levels in ZMS than in ZSS.In addition,the content of 3 cyclopeptide alkaloids in ZMS was much higher than that in ZSS.These saponin compounds,such as Ju A,Ju B,pro Ju A and pro Ju B,obviously occurred at higher levels in ZSS than in ZMS.3.A simultaneous determination method of UPLC-MS/MS was developed for the deter mination of 9 components of ZSS and ZMS: coclaurine,magnoflorine,swertisin,spinosin,6' '-feruloylspinosin,vicenin II,kaempferol-3-O-rutinoside,jujuboside A and jujuboside B.The method was proved to be reasonable and feasible by the Methodological investigation,and the contents of 29 batches of ZSS and ZMS were determined.It was found that the conte nts of coclaurine,magnoflorine,swertisin,kaempferol-3-O-rutinoside,jujuboside A and jujub oside B in ZSS were much higher than those of coclaurine,magnoflorine,swertisin,kaempf erol-3-O-rutinoside,and jujuboside B in ZMS,in the meanwhile jujuboside A could not be detected in ZMS.On the other hand,the contents of Vicenin II,spinosin and 6'''-feruloyls pinosin in ZSS were much lower than Vicenin II,spinosin and 6'''-feruloylspinosin in ZMS.The results of PCA and PLS-DA of multivariate statistical analysis showed that the nine c omponents could distinguish ZSS from ZMS,which indicated that the 9 components could be effectively distinguished ZSS from the ZMS.All of these have hindered the effective qu ality control of ZSS.Conclusion: Based on the above research,a rapid identification strategy for the chemical constituents in ZSS was established by using UHPLC-Q-Orbitrap MS in this paper and systematically characterized the secondary metabolites in ZSS.1H NMR,GC-MS and LC-MS untargeted metabolomics were used to comprehensively characterize the primary and secondary metabolites in ZSS and ZMS and the difference metabolites were identified combined with multivariate statistical analysis.A method for simultaneous determination of 9 chemical constituents in ZSS and ZMS by UPLC-MS/MS was established,which provides a scientific basis for establishing a scientific and reasonable quality standard of ZSS.