| The genus Fritillaria(Liliaceae)comprises approximately 140 species of bulbous perennials,which are widely distributed in the North Temperate Zone.In China,there are 24 species and four varieties of this genus and 15 of them are endemic to China.Fritillariae cirrhosae bulbus,known by the Chinese name "Beimu" and derived from the bulbs of several Fritillaria species,has been used as an antitussive,expectorant and antisthmatic herb in traditional Chinese medicine(TCM)for many years.In the Pharmacopoeia of the People’s Republic of China(Vol.1)(2015),Zhe Beimu(in the middle and lower reaches of the Yangtze River),Chuan Beimu(in Qinghai Tibet Plateau and its adjacent areas),Yi Beimu(in Xinjiang),Ping Beimu(in the Northeast of China)and Hu Bei Beimu(in Hubei)were officially recorded and listed,involving up to ten species and one variety of Fritillaria genus.Among them,there are five species and one variety in the source plants of Chuan Beimu,namely F.cirrhosa,F.unibracteata,F.przewalskii,F.delavayi,F.taipaiensis,and F.wabuensis.Wild resources of Fritillariae cirrhosae bulbus are mainly distributed in the Qinghai Tibet Plateau and its adjacent areas,such as Gansu,Qinghai,Sichuan,Tibet,and Yunnan.A few species also extend to Shaanxi,Chongqing and Shanxi.Fritillariae cirrhosae bulbus has high development value and great market demand.In addition to the source plants of Fritillariae cirrhosae bulbus recorded in the Pharmacopoeia of the People’s Republic of China(Vol.1)(2015),there are many other Fritillaria species in the Qinghai Tibet Plateau and its adjacent areas and their bulbus are not collected separately and used as Fritillariae cirrhosae bulbus in the drug market.Because of the complexity of morphological variation,overlapping of geographical distribution,difficulty of classification and identification,the application of Fritillaria medicinal species in this area is confused.Therefore,in this study,the resources of Fritillaria in the Qinghai-Tibet Plateau and its adjacent areas were investigate to obtain the information about their distribution,individual morphological variation,habitat characteristics,cultivation status and so on.Then the collected materials were used to explore on the chloroplast genomics of Fritillaria.The phylogeny of Fritillaria species was inferred by using chloroplast genome to discuss species relationship of Fritillaria.At the same time,UPLC-ELSD was established to simultaneously determine the contents of six alkaloids in Fritillaria:peimisine,imperialine,sipeimine-3-D-glucoside,verticine,verticinone and hupehenine.This method also laid a foundation for the quality evaluation of Fritillariae cirrhosae bulbus.Investigation on Fritillaria medicinal resources included plantation base investigation and field investigation.According to the plantation base survey,there are five Fritillaria species mainly cultivated in this area,namely,F.cirrhosa,F.unibracteata,F.taipaiensis,F.wabuensis and F.mellea.According to the field survey,there are ten wild Fritillaria species,namely,F.delavayi,F.unibracteata,F.przewalskii,F.sichuanica,F cirrhosa,F.yuzhongensis and F.cirrhosa var.ecirrhosa.The information on their habitats and resources were summarized.There are abundant wild Fritillaria species in Qinghai-Tibet Plateau and adjacent areas,but the wild resources are severely damaged.The scale of cultivated Fritillariae cirrhosae bulbus is not enough to meet the market demand.Fritillariae cirrhosae bulbus are mainly collected from wild and complex in source plants.The resources of wild Fritillaria species decreased sharply due to environmental damage and over excavation.The investigation of Fritillaria resources in this area can provide scientific reference for the utilization and development of Fritillaria resources and the planting of Fritillariae cirrhosae bulbus in this area.A total of 18 samples including 13 species(variaties involved)of Fritillaria in Liliaceae were sequenced by using the Illumina sequencing platform,and their assembly was completed.There are 112 different genes in the chloroplast genome of Fritillaria in Liliaceae,including 78 protein coding genes,30 tRNA genes and 4 rRNA genes.Repeat sequence analysis showed that the chloroplast genome of Fritillaria in Liliaceae contains an average of 71 SSRs,including 54 mono SSRs,13 Di SSRs,7 tetra SSRs and 1 penta SSR.The average forward repeat(F),palindrome repeat(P)and tandem repeat(T)sequences of the chloroplast genome of Fritillaria in Liliaceae were 18,21 and 24,respectively.The phylogenetic tree was constructed by the sequence of protein coding region of chloroplast genome.The results showed that the close species could be well separated,and the support rate was high.By combining with morphological analysis,the status of some species such as F.wabuensis,F.mellea and F.cirrhosa var.ecirrhosa should be recognized;F.yuzhongensis is closely related to F.przewalskii and F.taipaiensis on the molecular data.It should be evaluated whether F.yuzhongensis can be used as the source plant of Fritillariae cirrhosae bulbus from their chemical components and pharmacological activity;F.roylei is not supported to be merged into F.cirrhosa,but whether it can be used as Fritillariae cirrhosae bulbus needs further chemical and pharmacological research to confirm.It also indicated that the whole chloroplast genome in molecular identification of Fritillaria species in Qinghai Tibet Plateau and was a reliable and promising method.Using the method of UPLC-ELSD and chemometrics,the four index components of five kinds of Fritillariae Bulbus,namely imperialine,sipeimine-3-D-glucoside,verticine,verticinone and the other two alkaloids,peimisine and hupehenine,were detected simultaneously.The chemical components of five kinds of Fritillariae Bulbus were compared by the methods of cluster analysis(HCA)and principal component analysis(PCA).Compared with HPLC-ELSD,this method not only greatly saves the analysis time,but also can simultaneously detect the content of 6 alkaloids in five kinds of Fritillaria Bulbus.Compared with the detection of a single alkaloid,this method can reflect the chemical composition and content of five kinds of Fritillariae Bulbus more comprehensively,so as to provide scientific basis for the quality control and comprehensive utilization of Fritillaria species. |
