Classiflication And Chemical Diversity Of Fallopia Multiflora And Its Infraspecific Taxa

[Objective] Fallopia multiflora(Thunberg)Haraldson was first recorded in “Kai Bao Ben Cao”,which is one of the traditional Chinese medicine.It has a long history of medicinal use.In recent years,numerous studies have been conducted on the relationships among F.multiflora,F.multiflora var.angulata(S.Y.Liu)H.J.Yan,Z.J.Fang & Shi Xiao Yu,and F.multiflora var.ciliinervis(Nakai)Yonekura & H.Ohashi.The content of specific chemical constituents and index stilbene glycosides of F.multiflora in different habitats was obviously different.However,Whether this difference is still unclear related to morphological characteristics and gene mutation.This paper aims to solve the following problems:(1)In this study,we clarified the taxonomic relationship among F.multiflora,F.multiflora var.angulata,and F.multiflora var.ciliinervis,with the aim providing a scientific basis for rational evaluation of the resources and quality of F.multiflora.(2)To establish a rapid and effective method to identify F.multiflora and its related species on the market.(3)This paper elucidated the relationship between stilbene glycoside and chemical diversity in tuber root of F.multiflora,with the aim providing a scientific basis for quality evaluation of the germplasm resources and clinical application of F.multiflora.[Methods](1)The macroscopic and microscopic morphology of root tubers or rhizomes,stems,and leaves among Fallopia multiflora,F.multiflora var.angulata,and F.multiflora var.ciliinervis were compared.The content of eleven chemical components(catechin,polydatin,stilbene glycoside,emodin,emodin-8-O-?-Dglucopyranoside,rhein,chrysophanol,aloe emodin,quercetin,physcion and resveratrol)in the three taxa were determined by using HPLC-DAD,and chemical diversity was further evaluated by using principal component analysis and hierarchical cluster analysis.Molecular phylogenies were mapped using two chloroplast markers(mat K and psb A-trn H region)and a ribosomal nuclear markers(ITS2 region)to construct phylogenetic trees of NJ,ML and BI systems.(2)According to the SNP loci on the psb A-trn H sequence,specific primers of F.multiflora and its related species were designed and amplified by PCR.Using allele specificity PCR to identifying F.multiflora,F.multiflora var.angulata,F.multiflora var.ciliinervis,and F.denticulata(Huang)A.J.Li from different regions of China.(3)Based on the classification of F.multiflora,F.multiflora var.angulata,F.multiflora var.ciliinervis,the contents of twelve chemical constituents(proanthocyanidin B1,catechin,cis-stilbene glycoside,polydatin,trans-stilbene glycoside,emodin-8-O-D-glucoside,aloe emodin,emodin,chrysophanol,resveratrol,and physcione)in F.multiflora were simultaneously determined by using HPLC-DAD-VWD,and chemical diversity was further evaluated by using hierarchical cluster analysis.[Results](1)The results showed that the subterranean organs of Fallopia multiflora and F.multiflora var.angulata were root tubers,whereas those of F.multiflora var.ciliinervis are rhizomes,according to an analysis of macroscopic and microscopic morphological characteristics.In the phylogenetic tree,F.multiflora and F.multiflora var.angulata clustered into a clade with bootstrap support rates(mat K + psb A-trn H: NJ-BS/ML-BS/BI-PP = 99/85/0.99).F.multiflora var.ciliinervis was clustered into a clade.In addition,F.multiflora,F.multiflora var.angulata and F.multiflora var.ciliinervis had obvious differences in their chemical compositions.The root tuber of F.multiflora contained high levels of stilbene glucoside and catechin compounds,but lower contents of polydatin and anthraquinone compounds.In contrast to F.multiflora,rhizomes of F.multiflora var.ciliinervis contained high levels of polydatin and anthraquinone compounds,lacked stilbene glucoside.The contents of eleven components of F.multiflora var.angulata is lower than others.Based on principal component analysis and hierarchical cluster analysis,the individuals of F.multiflora and F.multiflora var.angulata were clustered into a clade.However,the individuals of F.multiflora var.ciliinervis clustered into a single clade.(2)By optimizing the conditions of PCR reaction of F.multiflora and its related species from different habitats,we established a rapid AS-PCR method.When the annealing temperature was raised to 48 ? with 30 cycle number,the result of F.multiflora and its related species identification is the best.(3)The content of stilbene glycoside ranged from 0.2% to 4.9%,and the content of free anthraquinones ranged from 0.01% to 1.2%.The contents of chrysophanol and aloe-emodin was very low,and not be detected.According to HCA analysis,F.multiflora was divided into five types.The content of stilbene glycoside <1% for A,1%~2.5% for B,2.5%~3% for C,3%~4.5% for D,>4.5% for E.A type doesn't comply with the provisions of the 2015 edition of the Chinese Pharmacopoeia.[Conclusion](1)In this study,we compared Fallopia multiflora,F.multiflora var.angulata,and F.multiflora var.ciliinervis based on macroscopic and microscopic morphology,molecular phylogeny,and chemical analyses.The results provided strong evidence for establishing the classification of F.multiflora,F.multiflora var.angulata,and F.multiflora var.ciliinervis.Our findings indicated that F.multiflora var.ciliinervis should be considered as a separate species due to the substantial morphological,chemical,and molecular differences compared with F.multiflora and F.multiflora var.angulata,whereas F.multiflora var.angulata should be treated as a variant of F.multiflora due to their closer relationship between these two taxa.(2)It's simple and reliable to identify the authenticity of F.multiflora by allele loci specific PCR.(3)F.multiflora can be divided into different chemical types according to the content difference of stilbene glycoside.The content of free anthraquinone was relatively low when the content of stilbene glycoside was higher in F.multiflora.