| Alpine gentian herb is a staple Tibetan medicine which is used commonly.It is clinically used to cure virus disease and laryngitis.It is an important ingredient in the product of three flavors gentian flower pill and other classical Tibetan medicines.It is a complex source and its mainstream species are mainly Gentiana szecheyii Kanitz,G.algida Pall,and G.purdomii Marq..As an National Class One Endangered Tibetan,G.szecheyii Kanitz can not achieve sustainable use of resources and there are significant differences in chemical composition between G.szecheyii and G.algida Pall.,G.purdomii Marq..G.szecheyii is short of gentiopicroside and swertiamarin which are the represents of iridoid constituents.In addition,its pharmacodynamic material is unclear;G.algida Pall,and G.purdomii Marq.have similar original plant Imorphology,and between the two types presence a transition,leading to confusion classification.In order to solve these problems,we used ISSR molecular marker and ITS2 sequence to analysis the inter-species relationships of G.algida Pall,and G.purdomii Marq.and explore the genetic diversity of G.szecheyii Kanitz in this paper,while using acute pharyngitis model rats to investigate what is the active component of alcoholic extract of G.szecheyii Kanitz to treat acute pharyngitis model in mice.The results are as follows:(1)Twelve ISSR primers were used for PCR to analysis seventy-two individuals from five populations,of which two from G.algida Pall,and three from G.purdomii Marq.They produced 161 band loci,the percentage of polymorphic loci was 92.54%.the Nei's gene diversity(H)was 0.1818?0.1976 and Shannon's information index(I)was 0.2634?0.2847.Genetic differentiation coefficient(Gst)was 0.4193.Gene flow(Nm)was 0.6926.The results showed that G.algida Pall,and G.purdomii Marq.had abundant genetic diversity,genetic diversity mainly comes from the within group variation,because of the lack of gene exchange among populations,the genetic differentiation among populations was large.(2)ISSR clustering results of G.algida Pall,and G.purdomii Marq.showed that when the genetic similarity coefficient of 63%,five populations could be divided into three categories,which population GSW and population GSY are gathered for a class,the group of MXB and the group of MXH get together for a class,the group of MXR alone.While interspecifics which between population MXR and population MXH and interspecifics which between group MXR and group GSW genetic distance were the same,and interspecifics genetic distance between population GSW and population MXH was less than the genetic distance within species of G.algida Pall.,interspecifics genetic distance between population MXR and population GSW was less than the genetic distance within species of G.purdomii Marq..In view of this,the"plant height,flower number,peduncle length" was unable to distinguish clearly between G.algida Pall,and G.purdomii Marq..If according to the "higher plants in China," in "corolla spots in the presence " to identify G.algida Pall,and G.purdomii Marq.,the ISSR clustering results showed that portion of G.algida Pall,and G.purdomii Marq.gather together,also cannot distinguish between G.algida Pall,and G.purdomii Marq..(3)ITS2 sequence comparison between G.algida Pall,and G.purdomii Marq.showed that ITS2 sequence of seventy-two individuals from five populations was mainly divided into two types,and each category contains individuals of different populations of G.algida Pall,and G.purdomii Marq.,genetic distance range from 0.0000?0.9984.Based on ITS2 sequences and ISSR analysis results,it is recommended that should merge G.algida Pall,and G.purdomii Marq.(4)ISSR markers were used to analyze the genetic diversity of G.szecheyii Kanitz.Twelve ISSR primers were used for PCR to analysis sixty individuals from four populations,of which come fromm G.szecheyii Kanitz,percentage of polymorphic loci was 89.80%.Nei's gene diversity(H)range from 0.1364?0.2070,Shannon's information index(I)range from 0.2140?0.3168,genetic differentiation(Gst)was 0.3434,gene flow(Nm)was 0.9560.Compared with other endangered plants,G.szecheyii Kanitz have abundant genetic diversity,genetic diversity mainly comes from the within group variation.G.szecheyii Kanitz.in the endangered state is not caused by lack of genetic diversity,mostly from the environmental pressure caused by human factors and their biological characteristics.(5)ITS2 sequence of G.szecheyii Kanitz comparison showed that ITS2 sequences of all individual which from G.szecheyii Kanitz showed high consistency.The ITS2 sequences in different populations were relatively stable and were not affected by environment,geographical distribution and so on.(6)ITS2 sequences of mainstream species of Alpine gentian herb that were collected in our laboratory were comparing analyzed with that of confusable varieties of Alpine gentian herb which were obtained from GenBank together,and phylogenetic tree was constructed by MEGA6.06.The results showed that the phylogenetic tree and the traditional classification of cultivars showed consistency.G.algida Pall.,G.purdomii Marq.and G.frigid Haenke were indistinguishable through the ITS2,while other species could be distinguished ITS2 sequences could be used as a DNA barcode of G.szecheyii Kanitz to identify G.szecheyii Kanitz and its adulterants effectively.(7)Acute pharyngitis model was induced by 25%ammonia chemical stimulation.Different polar parts of G.szecheyii Kanitz on the rat model of pharyngeal pathological morphology and serum IL-10,TNF-? expression were observed.The results showed that extract,ethyl acetate and n-butanol part of G.szecheyii Kanitz could quickly repair the pathological tissue of the rat's throat.They had statistically significant difference compared with model control group(P<0.05).Extract and n-butanol part of G.szecheyii Kanitz could significantly reduce the level of IL-10 and TNF-? in serum of rats,and had highly significant difference compared with model control group(P<0.01).The influence of alcoholic extract of G.szecheyii Kanitz on the throat and inflammatory factors in ratsvery site on rat showed that total extract and n-butanol part had a good curative effect on acute pharyngitis. |
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