| Objective :Based on the accelerated solvent extraction technique and LC-PDA-TOF-MS,we established the qualitative component profiles of Alstonia scholaris leaves from different origins;meanwhile,based on the fingerprint profiles of A.scholaris leaves combined with three chemometric analysis methods of similarity analysis,cluster analysis and discriminant analysis,we selected suitable qualitative and quantitative indicators and established the HPLC-UV multi-component method for A.scholaris leaves(Scholaricine,Akuammidine,19,20-(E)-Vallesa mine,Tubotaiwine and Picrinine),The method was used to quantify 19 batches of A.scholaris leaves.The above method was used to evaluate the quality of 19 batches of A.scholaris leaf samples in a more comprehensive and scientific manner,and also to provide a reference for other quality control studies on Chinese medicine.In addition,this paper also provides a preliminary discussion on the phylogeny of the leaves of A.scholaris,and provides ideas for further research on the association between chemical and genetic levels of A.scholaris.Methods:1.Optimization of the extraction of the active ingredients from A.scholaris leaves:The main active components(Picrinine and Vallesamine)in A.scholaris leaves were used as the main objects of study,and the optimal extraction conditions for accelerated solvent extraction technique were obtained according to the single-factor exa mination and response surface center design(Box-Behnken).2.Identification of the components of A.scholaris leaves: HPLC-PDA-TOF-MS technique was established to qualitatively analyze the components in A.scholaris leaves by high resolution mass spectrometry information,UV information,and combined with the information obtained from literature review.3.Establishment and analysis of HPLC-UV fingerprint profiles of A.scholaris leaves:Based on the established accelerated solvent extraction technique and high performance liquid chromatography(HPLC)method,fingerprint profiles of A.scholaris leaf samples from different origins were established,and the similarities and differences in chemical composition were investigated by cluster analysis,similarity analysis and discri minant analysis,and the samples of different qualities of A.scholaris leaves were distinguished into two categories,and 27 common peaks and 17 distinguished peaks were obtained.were obtained as qualitative and quantitative indicators,respectively.4.Multi-component content deter mination of A.scholaris leaves: Based on the distinguishing peaks(quantitative index components)identified in the above experiments,multi-component content determination of five quantitative index components(Scholaricine,Akuammidine,19,20-(E)-Vallesamine,Tubotaiwine and Picrinine)was carried out with the aid of phase fingerprint similarity analysis and cluster analysis to make a more comprehensive quality evaluation of A.scholaris leaves.5.Preliminary study on the genealogy and geography of Alstonia scholaris leaves:phylogenetic study of Alstonia scholaris leaf based on one cp DNA,one n DNA and one nr DNA,and analysis of genetic relationship among different populations.Results:1.Optimized extraction of the effective active substances from A.scholaris leaves:The experiment was carried out using a fully automated accelerated solvent extraction technique.17 experimental protocols were designed based on the results of single-factor experiments and the principles of Box-Behnken test design,and the extraction conditions were finally established with an extraction temperature of 150°C,an extraction time of 10 min,and two cycles.Under these conditions,the total extraction efficiency of the two alkaloid components,19,20-(E)-Vallesa mine and Picrinine,from the leaves of A.scholaris were maximized.Under these conditions,the total peak area was 288259,which was basically consistent with the predicted value of 289776,confir ming that the equation was plausible.The validity of the model was confirmed using ANOVA analysis.2.Identification of the components of the leaves: HPLC-PDA-TOF-MS technique was used to identify the components of the leaves.16 components of the ASE extract of the leaves were identified,namely,angstilobine B acid,Scholaricine,quercetin,Akuammidine,19,20-(E)-Vallesa mine,16-formyl-5a-methoxystricta mine,Tubotaiwine,nareline,Picrinine,10-methoxyalstiphyllanine H,5,7-dihidroxi-2’,4’-dimetoxiisofiavone isoliquiritigenin,chylieodiscie acid,ursolic acid,oleanolic acid,betulinic acid,chondroitin.3.Establishment and analysis of HPLC-UV fingerprint profiles of A.scholaris leaves: Based on the established accelerated solvent extraction technique and high performance liquid chromatography(HPLC)method,fingerprint profiles of A.scholaris leaf samples from different origins were established,and the similarities and differences in chemical composition were investigated by cluster analysis,similarity analysis and discri minant analysis,and the samples of different qualities of A.scholaris leaves were distinguished into two categories,and 27 common peaks and 17 distinguished peaks were obtained.were obtained as qualitative and quantitative indicators,respectively.4.Multi-component content deter mination of A.scholaris leaves: Combining the results of the above three analytical methods,the five components of A.scholaris leaves,Scholaricine,Akuammidine,Vallesa mine,Tubotaiwine and Picrinine,were selected as quantitative indicators to establish an HPLC multi-component content deter mination method.The proposed method can effectively separate the index components in the samples,and the linearity of the five components was good in the linear range(r>0.999),and the relative standard deviations(RSDs)of the spiked recoveries were less than 2%.The results showed that the content of alkaloids varied widely among the origins.The alkaloid content of A.scholaris leaves from Honghe and Wenshan in Yunnan is high and the composition is basically the same as that of other origins,which can be considered as a resource substitute for the leaves of A.scholaris.5.Preli minary phylogenetic study of Alstonia scholaris leaf: The experiment was conducted to study the phylogeny of five populations of Alstonia scholaris leaf by one cp DNA trn F-trn C,one nr DNA ITS and one n DNA SAP.Sequence characterization of the three gene fragments was carried out using Dna SP software,and the final results were: cp DNA full length 905 bp,with 5 variant loci and GC content 0.358;nr DNA matrix length 560 bp,with 7 variant loci detected,7 parsimony loci and GC content0.656;n DNA detected 8 variant loci.The maximum likelihood plots showed low expenditure rates(less than 30)for each branch,and the results indicated that the three fragments failed to resolve the differentiation relationships among subspecies populations of Alstonia scholaris leaves,and further study by genomic approaches is urgently needed.Conclusions:1.A convenient,fast and reproducible extraction method is the first and most critical step in the study of the compound group of A.scholaris leaf.In this study,we optimized the accelerated solvent extraction method by response surface design to establish the optimal extraction conditions,which ensured the stability of the extraction rate of the active ingredients of A.scholaris leaf and also laid the foundation for the subsequent substance base study.2.In this study,the fingerprint profiles of the compound groups of A.scholaris leaves from different origins were established,and the similarities and differences of their chemical compositions were studied by combining cluster analysis,similarity analysis and discri minant analysis.And the content analysis of five main components of 19 batches of A.scholaris leaf from different origins was carried out to evaluate the quality of A.scholaris leaf samples from multiple dimensions.The fingerprinting method of traditional Chinese medicine based on chemometric identification technology,combined with multi-component content deter mination,can evaluate the quality of traditional Chinese medicine in a global way.3.The study of genetic differentiation revealed that the genetic style of A.scholaris leaves did not agree with the origin distribution style at the compound level.The nucleotide variation rate of the infraspecific populations was low,while the differentiation of different origins of A.scholaris was better at the compound level.Further phylogenetic studies are needed to further investigate the phylogeny of the leaves. |
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