Studies On Classification And Active Components Of Chinese Traditional Medicine Zanthoxylum Nitidum

Objective: Verifying the original plant and Latin name of Zanthoxylum nitidum,correcting the wrong Latin name of the Z.nitidum var.fastuosum,and pointing out that there is a difference between the type-3 of Z.nitidum var.nitidum and the Z.nitidum var.tomentosum;RP-HPLC was used to detect five active components and tested if there was significant sinal of toddaloactone in Z.nitidum,which provided a basis for the development and utilization of the germplasm resources and their quality control,and to resolve the controversy about the toddaloactone in Z.nitidum;Chloroplast gene sequences were used to estimate the phylogenetic relationship and genetic diversity,which provided a molecular basis for the conservation and utilization of the germplasm resources,the identification of the three types and variety in Z.nitidum and the evaluation of rationality for original classification.Methods:(1)The textual research of normaltive Latin name of type-3 in Z.nitidum var.nitidum was conducted,and we compared the morphological diffference of three types and variety of Z.nitidum through the comprehensive evidence from literature review,specimens and field investigation;(2)Thin layer chromatography(TLC)and High Performance Liquid Chromatography(HPLC)was used to detect the signal of toddaloactone in the original variety and variety of Z.nitidum;(3)HPLC was used to examine the five components in both three types of Z.nitidum var.nitidum and Z.nitidum var.tomentosum;(4)Four primer pairs was selected to amplify and sequence the chloroplast fragments based on 339 indibiduals from 30 populations of Z.nitidum covered the whole distribution in China.After the manual alignment,multiple analyses were used to clarify their phylogenetic relationship and genetic diversity.Result:(1)Type-3 named as Z.nitidum var.nitidum had only two synonyms with Z.nitidum var.negelect How mss and Z.nitidum f.fastuosum How ex Huang based on all collected publications,hence the Latin name Z.nitidum var.fastuosum was irregular.(2)Toddaloactone was not detected in both Z.nitidum var.nitidum and Z.nitidum var.tomentosum.(3)RP-HPLC showed that the content of nitidine chloride was the highest level in type-3 of Z.nitidum var.nitidum and Z.nitidum var.tomentosum,but was the lowest in type-1;there was no significant difference in the content of sanguinarium chloride among the three types of Z.nitidum var.nitidum and Z.nitidum var.tomentosum;the content of allocryptopine was the highest level in Z.nitidum var.tomentosum,and with the lowest in type-1,however,type-1contains the highest level in L-sesamin and L-asarinin.Clustering analysis was performed based on tested 9 samples according to the content of five components sum,which matched well with the results of the morphological classification for three types of Z.nitidum var.nitidum and Z.nitidum var.tomentosum.(4)Totally,9 haplotypes were obtained based on 4 chloroplast fragments.Phylogenetic analyses suggested Z.nitidum var.tomentosum and three types of Z.nitidum var.nitidum corresponded to different haplotypes and clades,and the genetic differentiation and specific haplotypes were examined,although several shared chroloplast haplotypes were found between type-1and type-3,probabily produced by introgression.Additionally,some specific haplotypes were distributed across narrow or special regions,which provided insight into the geographic tracing of germplasm resources for Z.nitidum.Conclusions: We clarified the confusion that no significant signal of toddaloactone is detected in both varities and all types,and corrected the error Latin name,which would provide a basis for subsequent researchers.Comparison analysis of the five chemical constituents suggested the significant difference was found in three types of Z.nitidum var.nitidum and Z.nitidum var.tomentosum,which was mostly supported by the molecular evidence in spite of partly shared clades with distinct haplotypes between type-1 and type-3 probably produced by introgression.Therefore,the classification of Chinese traditional medicine Z.nitidum matched well based on three different lines of evidence from morphological traits,chemical constituents and genetic differentiation,which was of great significance to guide development,geographic tracing and utilization of germplasm resources for Z.nitidum.