Identification Of 5 Begonia Medicinal Plants Based On DNA Barcoding And Fingerprint

ObjectiveThis study focused on the identification and quality evaluation of five Begonia medicinal plants through the study of DNA barcoding,chromatographic fingerprints and chemical components,in order to provide basis for authentication and quality evaluation of Begonia medicinal plants and the foundation for the establishment of quality standard of Begonia medicinal plants.Methods1.Materia medica of the five Begonia medicinal plants was investigated through looking up literatures and ancient books,sorting the specimens,field investigation,and collecting samples.2.The five candidate DNA barcodes(ITS2 and ITS,psb A-trn H,mat K and rbc L)recommended in the world were applied to test 151 samples from 31 medicinal plants of the genus Begonia.Suitable DNA barcod was screened by comparing the results of success rates of PCR amplification and sequencing test,intra-specific and inter-specific genetic distances,"barcoding gap",the identification reliability and neighbor-joining tree(NJ)tree of the five loci.Addtionally,the genetic relationship of the Begonia medicinal plants were analyzed.3.Ultra-performance liquid chromatography-photodiode array(UPLC-PDA)was applied to establish chromatographic fingerprints of the five Begonia medicinal plants(B.evasiana,B.sinensis,B.pedatifida,B.smithiana and B.henryi),and ultra-high performance liquid chromatography-quantitative time of flight-tandem mass spectrometry(UPLC-QTOF-MS/MS)was used to get the fragment information of the common peaks so as to assign some components.Chemometrics methods such as similarity evaluation,cluster analysis,principal component analysis and orthogonal partial least-squares discriminant analysis were used to analyze the differences of the five Begonia medicinal plants.Results1.The originate,resources distribution,conventional name,medicinal herb alias,morphological characteristics and functional indications of the five Begonia medicinal plants(B.evasiana,B.sinensis,B.pedatifida,and B.henryi)were confirmed.2.The results of success rates of PCR amplification and sequencingtest,intra-specific and inter-specific genetic distances,"barcoding gap",the identification reliability and neighbor-joining(NJ)tree of the five loci suggested that ITS2 could distinguish B.pedatifida,B.smithiana and B.henryi and other the genus Begonia medicinal plants in the species level except Begonia evasiana(B.grandis subsp.grandis)and Begonia sinensis(B.grandis subsp.sinensis).Therefore,ITS2 was the ideal DNA barcode for the identification of the genus Begonia medicinal plants.3.The UPLC fingerprints were firstly established for the medicinal plants from five species of the genus Begonia including B.evasiana,B.sinensis,B.pedatifida,B.smithiana and B.henryi.The chemical constituents and their content of the five medicinal plants Begonia were revealed comprehensively.The chemical components in B.evasiana,B.sinensis and B.henryi were similar including phenolic acid,flavonoid glycoside,flavone and triterpenes.Similar chemical components existed in B.pedatifida and B.smithiana,such as phenolic acid,flavonoid glycoside,flavone,without triterpenes.4.The accurate molecular weight of compound and fragment ions information were obtained by UPLC-QTOF-MS/MS.Their respective common peaks of the five medicinal plants were assigned by comparing their fragment pathways with those of standard compounds and literatures.29 compounds,from B.evansiana,B.sinensis,B.pedatifida,B.smithiana and B.henryi were identified,including 3 organic acid compounds,21 flavonoids and flavonoid glycoside,5 triterpene and their saponin components.Meantime,the differences of components among the five medicinal plants were compared with each other.5.The CA and PCA method were applied to discriminate the five Begonia medicinal plants.The results of CA were better than the results of PCA.The results of CA showed that B.evansiana,B.pedatifida and B.smithiana were clustered into one group,respectively.However,B.henryi and B.sinensis were gathered in one group,Variables,namely chemical marker,that have the ability for discrimianation of the five species were found based on PLS-DA.Thus,the distinction of chemical component and characteristic ingredients of the 5 medicinal plants were definited.ConclusionThe resource identification and quality assessment of the five Begonia medicinal plants were researched systematically in this study.The originate and resources distribution of the genus Begonia were confirmed through research on materia medica,wild resource investigation and sample collecting.The results of DAN barcoding indicated that the ITS2 sequence was the ideal sequence to discriminate the genus Begonia medicinal plants and its relative adulterants in the species level.The chromatographic fingerprints of the five Begonia medicinal plants were firstly established by UPLC-MS so as to reflect the differences of category and contents of chemical components from the five Begonia medicinal plants,and the chemical compositions and characteristics of the five medicinal plants of the genus Begonia were confirmed.Thus,these provided accurate,systematic and efficient methods and basis for the authentication,quality standard and quality assessment of the genus Begonia medicinal plants.