Molecular Identification Of Medicinal Plants Of Bupleurum And Investigation Of The Quality Of Bupleurum Medicinal Materials

Different species Bupleurum usually have different pharmacological activities,for example,Bupleurum yinchowense Shan et Y.Li has better neuroregulation effect,B.chinense has better antipyretic effect than B.scorzonerifolium.Thus,the abuse of different Bupleurum species will lead to the quality of Bupleuri Radix uncontrollable and corresponding drug safety.And it is very difficult to identify these Bupleurum species using traditional morphological identification method,establishing a fast and effective method to identify these Bupleurum species is very necessary.In this paper,DNA barcoding technology is used to establish molecular identification method,and HPLC is used in detecting saikosaponins content to evaluate the quality of Bupleuri Radix.First,we selected 4 kinds of DNA barcodes,ITS,psbA-trnH,matK and rbcL,amplifying them in different Bupleurum species and obtaining identification efficiency to selecting the best DNA barcode.Second,Amplifying this barcode in large sample to establish sequence data,the data was analyzed by Single Nucleotide Polymorphism(SNP),Kimura 2-parameter(K2P)distance,cluster tree,"haplotype-species" method.Third,the molecular identification method based on "haplotype-species" was established.In quality evaluation part,the content of saikosaponins,saikosaponin a,d,c,was detected by HPLC.Based on saikosaponins content,the quality of different species Bupleurum plants was evaluated through statistical analysis.Finally,the molecular identification and quality evaluation methods are used in investigating market Bupleuri Radix to give reasonable suggestion.The results in this paper are as follow:1.We described and summarized morphological and powder microscopic characteristics of 7 kinds of Bupleurum medical plants and summarized the defect of morphological identification method.2.We analyzed 4 kinds of DNA barcodes identification efficiency and selected the best barcode,ITS sequence,to identify different Bupleurum species.3.The molecular identification method was established to identify common Bupleurum medical plants.Through variation points,K2P distance,cluster analysis and "haplotypes-species" analysis,19 ITS haplotypes and 86 variation points were obtained.4.We evaluated the quality of Bupeluri Radix based on saikosaponins content.The content of SSa,SSd and SSc has positive correlation relationship.According to saikosaponins content,the quality order of different Bupleurum species are as follow:B.marginatum var.stenophyllum>B.yinchowense>B.chinense>B.bicaule>B.scorzonerifolium.And Bupleuri Radix which comes from Shanxi and Gansu has better quality than which comes from Ningxia.5.We investigated the quality of market Bupleuri Radix.The market Bupleuri Radix was identified by our molecular identification method,and we found that the main species in market were marginatum var.stenophyllum and B.chinense.The species in 5 Chinese herbal medicine market,Anguo,Yuzhou,Bozhou,Guangzhou and Hehuachi,were more than one species.The content of SSa and SSd in Bupleuri Radix should not be lower than 0.3%according to Chinese Pharmacopoeia,and the market Bupleuri Radix 's percent of pass was 100%.However,there is significant difference of saikosaponins content in different species,the content of B.marginatum var.stenophyllum was over 4%but B.bicaule was only 0.4%.