Study On Resource Of Chrysanthemum Indicum L. And Quality Evaluation Of Chrysanthemi Indici Flos

Chrysanthemi Indici Flos is the capitulum of Chrysanthemum indicum L.in the compositae family.Flowers are gathered and dried or dried after steaming in the sun when the flowers just bloom in autumn or winter,It has the efficacy of heat-clearing and detoxifying,reducing fire and clearing liver.It is used for the disease of furuncle and phyma,sore red swollen eyes,and headache and vertigo.Chrysanthemi Indici Flos is extensively applied in the sphere of medicines,health products and daily chemical products,because of its curative effect,C.indicum L.is widely distributed in the physical environment such as lopes,grasslands,fields and roadside in northeast China,north China,central China,south China,east China and southwest China.At present,Chrysanthemi Indici Flos as the merchandise in the market is mainly wild resource.Because of the increased demand and destructive harvesting of medicinal herb collector,the distribution area and resources of C.indicum L.were decreased significantly.The widely variation of the conetent of linarin exists due to the affection of genetic and physical environment.The price of Chrysanthemi Indici Flos has increased by 325% in the past five years to 68 yuan/kg.Therefore,it is of great significance to carry out the research on germplasm resource of C.indicum L.and quality evaluation of Chrysanthemi Indici Flos.In order to achieve the job of resources protection,tender breeding and improving quality standards,the theories and methods of pharmaceutical botany,medicinal plant resources and medicinal plant ecology were combined with modern analytical techniques and molecular biology methods in this topic.The research of resource investigation,sample collection,germplasm resource nursery,chemical composition analysis,genetic diversity identification analysis and germplasm difference evaluation of C.indicum L were conducted.The main results were as follows: Ⅰ.THE RESEARCH OF RESOURCE INVESTIGATION OF C.indicum L.The data of the C.indicum L.resources of 50 counties or cities in Anhui,Hubei,Henan,Shaanxi,Shanxi,Guangdong and Guangxi provinces were collected.The results showed that the growth environment was different,the adaptability was strong,and the distribution area was not obvious.In the east longitude E107.54° ~ E118.10°,N23.18° ~ N35.16°latitude,18 m ~ 1083 m above sea level,have grown to the Dabie Mountains,Qinling Mountains,Zhongtiao Mountains,Funiu Mountains,Taihang Mountains and plains and plains for the transfer zone to plain areas for less.In recent years,price rose sharply,only China Resources Sanjiu Pharmaceutical Co.,Ltd.in Yangxin County,Hubei Province has built wild species of planting base,and were certified with GAP in 2015.In the existing survey area,the amount of Chrysanthemi Indici Flos was 5441.4 tons,but the actual recoverable amount of Chrysanthemi Indici Flos was lower in the area of investigation,and most of the Chrysanthemi Indici Flos did not reach the "Chinese Pharmacopoeia".The provisions can not be used as a supply of medicinal herbs.It has been far from being able to meet the annual demand of Chrysanthemi Indici Flos.Ⅱ.THE GERMPLASM RESOURCE COLLECTION OF C.indicum L.32 representative germplasms of C.indicum L.were selected and cultivated in the germplasm resources nursery which were established in Yangxin County,Huangshi City,Hubei Province based on the investigation of resource,genetic diversity,and the content of mulberry glycoside to build C.indicum L.natural research platform for germplasm preservation,germplasm analysis,comparison,following-up fine germplasm screening,applying for new varieties and other works to provide experimental material.The results showed that C.indicum L.had great diversity in leaf shape,leaf tip,leaf base,leaf margin,leaf crack,auricle,plant height,stem color,flowering period,flower diameter size and so on.Ⅲ.THE SCREENING AND EVALUATION OF GERMPLASM OF C.indicum L.After the establishment of the germplasm resources,the germplasm of C.indicum L.was continuously expanded and purified.And the phenotypic characteristics of most germplasm were realized after four consecutive years.The germplasms of Hubei Xinzhou,Hubei Luotian,Henan Huangchuan,Anhui Huoqiu,Anhui Shucheng and Anhui Tongcheng were selected according to the origin distribution,plant morphology,flowering difference and blossom content,and the difference was the most obvious.We had a germplasm evaluation of the six different types.The research content included the comparison of the agronomic traits,the comparison of the leaf epidermis electron microscopy,the ultrastructural comparison of the leaves,the comparison of the morphological structure,the comparison of the pollen morphology,and the comparison of the chemical composition.The results were as follows:The highest yield appeared in Anhui Tongcheng germplasm because of its advantage in the plant height,canopy,inflorescence diameter,100 flower dry weight and single plant flower yield and other aspects.The glands of germplasm in Hubei Xinzhou were the most widely distributed.So,the germplasm in Hubei Xinzhou can be used as insect-resistant germplasm cultivation.The furrow arms of Hubei Luotian were the longest.So,the germplasm in Hubei Luotian can be used as a low-temperature germplasm material for breeding.Anhui huoqiu county germplasm has the advantage in tolerance of drought,strong wind and other natural conditions because of low density of lenticel.Anhui shucheng germplasm is more suitable for growing in warm and humid climate because of high density of lenticel.The content of linarin in Hubei Xinzhou germplasm,Hubei Luotian germplasm,Anhui Huoqiu germplasm reached the qualified standard according to Chinese Pharmacopoeia.In order to avoid excessive concentration of waste time,the shortage of labor caused the waste of wild chrysanthemum in time to be picked,and the chrysanthemum germplasm variety,rich wild chrysanthemum resources.These germplasms were chosen to conduct standardization and scale GAP planting.Ⅳ.THE QUALITY EVALUATION OF CHRYSANTHEMI INDICI FLOS BASED ON CHEMICAL COMPOSITION118 Chrysanthemi Indici Flos from different regions were selected and quantitative analysised of linarin were conducted.The highest acceptability of Chrysanthemi Indici Flos appears in Hubei province(55.6%),and the highest content of linarin(2.02%)appears in Xinzhou district,Hubei province.The above results show that Hubei is more appropriate for enterprises to promote the wild chrysanthemum medicinal materials procurement source and GAP planting.Only 25 samples in 118 were eligible for pharmacopoeia,and the eligible rate was only 21.2%.It is suggested that the determination of chrysanthemum in the pharmacopoeia of linarin is worth further investigation.Therefore,in order to establish a more reasonable evaluation system of the wild chrysanthemum flowers,this study established the UPLC method and quantitative analysis method for determination of wild chrysanthemum flower six kinds of chemical composition and UPLC-Q-TOF/MS technology online identification of wild chrysanthemum flower of the chemical composition of the qualitative analysis method.The samples were extracted for 30 minutes by 70% methanol in the ratio of samples to methanol is 1:250.The mobile phases were acetonitrile and 1% aqueous-water(phase A: 50:50,v/v;phase B: 2:98,v/v).The temperature of column was maintained at 40 ℃,and UV-monitoring was performed at 326 nm.The content of chlorogenic acid,linarin,3,5-dicapryrylcinic acid,luteolin,apigenin and aconitine of chrysanthemi indici flos were quantitatively analyzed in the above conditions.In this study,UPLC-Q-TOF / MS was used to qualitatively analyze the chemical constituents of the root,stem and leave of C.indicum L.The 8 components were identified,including 4 phenolic acids,1 flavonoids,2 flavonoid glycosides and 1 Chrysanthemum lactone.UPLC-Q-TOF / MS was used to qualitatively analyze the chemical constituents of the flowers of Chrysanthemi indici flos.The 14 components were identified,including 4 phenolic acids,3 flavonoids,7 flavonoid glycosides,indicating that the chemical composition of Chrysanthemi indici flos varied.The eight major chemical components of the parts(roots,stems and leaves)at different times(May 15,September 15 and November 15)were semi-quantitatively analyzed for the first time.The results showed that the content of phenolic acids was higher in the roots.The contents of chlorogenic acid and 3,5-dicaffeoylquinic acid were higher in stem and leaves,and the contents of other phenolic compounds were lower.The content of the flavonoids was the highest in the leaves and the lowest in the roots.The content of the Chrysanthemum lactone was the highest in the leaves.From the chronological point of view,in addition to acacia and 4,5-dicaffeoylquinic acid,the content of other composition was the highest in November.Indicating that at the end of October,early November after the flowers,and then harvest other parts of the C.indicum L.,achieving the comprehensive development and utilization.The results showed that the content of phenolic acids was the lowest in flowers,while the contents of flavonoids(except acacin)were the highest in the flowers.And the content of aconin was the highest in the leaves,and the lowest in the roots and flowers.The content of Chrysanthemum lactone was the highest in the leaves,but not in the roots and flowers.The luteolin and apigenin were the main components of the flowers,and the roots,stems and leaves of the C.indicum L.were not detected.C.indicum L.flower is a medicinal site,in order to prevent the lawless elements of the leaves mixed with flowers.So it can be added in the quality standards of Chrysanthemum lactone detection items,to prevent non-medicinal parts of the pseudo-medicinal parts of the behavior.Roots,stems,leaves of C.indicum L.Luteolin and apigenin are the unique components of the flower,and the roots,stems and leaves of the C.indicum L.are not detected and can be used as the TLC identification of Chrysanthemi indici flos To improve the identification of Chrysanthemi indici flos,check the quality standards to provide a scientific basis.Ⅴ.THE GENETIC DIVERSITY OF C.indicum L.BY SSR MAKERSequencing of transcriptome was carried out using the mixed tissue samples of Luotian,Hubei Province.70 samples with genotype uniqueness were identified by 20 pairs of primers with good polymorphism.Using the four pairs of primers with good polymorphism,the success rate of 88.6% was the highest,which was the best method to develop the fingerprints of wild chrysanthemum germplasm.The homology of the two germplasm groups was high compared with the two transcripts in Luotian,Anhui Province and Shucheng,Anhui Province,but the two genes had different homologous genes.From the perspective of evolutionary biology,the germplasm has a homologous gene,and the two species differ greatly in terms of "positive selection of genes" and similar in "positive selection of genes".