| Foreign medicinal materials are an important way to discover new sources of medicinal plants to use in Chinese medicine.There have been many successful cases since the introduction of foreign medicinal materials in China more than 2000 years ago.Typical examples are American ginseng,saffron,and turmeric.In both traditional and modern times,European motherwort(Leonurus cardiaca)and Chinese motherwort(Leonurus japonicus)have similarities in medical application.They have similar efficacy in gynecological diseases,but European motherwort has broader treatment indications and extends to neurological and cardiovascular diseases.Both herbs are known to have outstanding advantages for postpartum disease,in fact this is the most famous use for the Chinese species.A comparative study of the two species'textual history has not been attempted before.By identifying as many texts as possible through history of use of the two species,a understanding of the trajectory of the uses.preparations,and formulations can offer an unique lens that can be used to view the two species side by side.Due to their botanical relationship and the historical similarities in the use of these two medicinal plants,it can be speculated that they may have some similar chemical components.However,the current research of the chemical composition of these two species is difficult to clarify mainly due to the following limitations:1)the quality of Chinese medicinal materials;the control standard is the content of a single(or several)components,and it is difficult to focus on the evaluation of complex components;2)the analysis techniques used are simple thin-layer chromatography.HPLC-UV,etc.,thus it is difficult to perform accurate qualitative and quantitative analysis of complex components;3)the source of the analysis sample is often unclear,there is a lack of a direct comparison of these two speices using accurate morphological classification and identification of the medicinal materials;4)the chemical analysis of the two has mostly been carried out separately,lacking a systematic comparative analysis.Therefore,in order to clarify the differences between western(L.cardiaca)and eastern(L.japonicus),we must use modern analysis techniques to systematically study the similarities and differences in chemical composition of the two based on comprehensive sample collection and accurate identification.In this study,there were three sets of data obtained from three different methods,together these data establish the foundation of a systematic method for integration of foreign medicinal plants into Chinese medicine.The first data set comes from a systematic literature study of each plant accessing over 200 titles in 7 languages from over 2000 years.Second,a systematic population sampling of European motherwort and Chinese motherwort(L.cardiaca:3 populations in Poland in Europe,17 populations in 4 states in the United States,1 population in China;L japonicus:7 populations in China,2 populations in the United States)was performed based on morphological classification and microscopic identification of the samples,a genetic analysis of the chloroplast genome populations.Finally,a study of the metabolomics based on UPLC-Q-TOF-MS and UPLC-MS/MS technology.A comparative chemical analysis aims to clarify the material basis of the differences between western(L.cardiaca)and eastern(L.japonicus)motherwort species.The main findings are as follows:1.Historical Textual Analysis of L.cardiaca and L.japonicusThe Leonurus genus has a long history of use in both Chinese medicine and Western medicine.However,textual investigation revealed that although there have been some erroneous claims for L.cardica in Dioscorides' De Materia Medica(65 AD),a text with over 600 medicinal plants,the description of the plant some authors claim to be motherwort does not match any plant in the Leonurus genus.The plant is also absent in the famous texts by the Persian physician Aviceanna(980-1037).The earliest date for a textual appearance of L.cardiaca is 1485 in a German text(Gartder Gesundheit).However,the most frequently used Asian relative to L.cardiaca,L.japonicus,has a textual history that spans nearly two thousand years with its first appearance in the Shen Nong Bencao Jing(circa 1 st century AD).Textual analysis revealed a significant difference in the history of the two plants with regards to plant parts used,applications,and even preparation methods.While L.cardiaca has been prepared and used in a consistant manner over its 500 plus years of textual history,L japonicus has had significant changes over its history.For example,the author of the Shen Nong Bencao Jing focuses on the application of the seed of L.japonicus.Although the herbaceous material is mentioned in this text and the single usage for that plant part is repeated by later authors,there was never a development of this application and it was eventually dropped completely.However,starting in the Tang dynasty,the emphasis on the seed slowly shifted toward the herbaceous parts of the plant.By the the time Li Shizhen writes the Bencao Gengmu(1596),the two parts are clearly separated and the emphasis is on the herbaceous plant part.Even by the Song dynasty,we can see that formularies such as the Taiping Sheng Hui Fang(992)favor the herb over the seed in formulas by about a 3:1 ratio.Moreover,we see a change in usage of the herbaceous parts,from the exclusive application in postpartum diseases to a significant broadening that encompasses most diseases in gynecology.By the Qing dynasty the use of L.japonicus and L.cardiaca are nearly the same with respect to their application in gynecological diseases.However,although there is some textual support for the use of L.japonicus for some neurological diseases in postpartum woman,this use had fallen out of favor by the Qing dynasty.In contrast,L.cardiaca is heavily favored for conditions such as anxiety,insomnia,heart diseases,and many related conditions.Therefore,based on textual study,we can say that L.cardiaca can be applied in all the same clinical situations where L.japonicus is applied,but expands on those applications in gynecology to encompass a wide array of neurological and heart diseases.2.Population genetics analysis of L.cardiaca and L.japonicus chloroplast genomesThe genetic diversity and genetic structure of L.cardiaca and L.japonicus were analyzed through chloroplast genome population genetics.This data helped to establish a DNA molecular identification system for identification of L.cardiaca and L.japonicus origins and individual identification.It also was used as an evaluation of germplasm resources of the two species using conservation and utilization strategy analysis,while inferring genotypes or haplotypes that can be used for future selection of high-quality chemical types to make a resource assessment for the development and utilization of European motherwort(L.cardiaca)in China.Using high-throughput sequencing,the chloroplast genomes of 22 European motherwort samples and 61 Chinese motherwort samples were sequenced and assembled.The results show that the chloroplast genomes of European and Chinese motherwort are basically the same,both of which are typical double-stranded circular structure of the angiosperm chloroplast genome.They include four parts:two inverted repeat regions,a large single copy region,and a small single copy region.Gene annotation found that the species and number of coding genes of the two species were the same,encoding 114 unique genes,including 80 genes encoding proteins,4 rRNA genes,and 30 tRNA genes.The overall length of the European motherwort genome varies from 151,236-151,831 bp,while Chinese motherwort is smaller,151,395-151,733 bp.Intraspecific variation in the genome of European Motherwort is small,with 22 sequences containing 238 mutation sites,with a mutation rate of 0.16%,forming 8 haplotypes with a haplotype polymorphism of 0.732;62 Motherwort sequences include 312 Mutation loci,the mutation rate was 0.21%,the number of haplotypes was 52,and the number of haplotypes was 0.99.The base substitution and transversion ratios are similar in both species,with the smallest AT/TA transversion,but the CT/GA replacement and AC/TG transversion of European Motherwort occupies a significantly larger proportion,indicating that European Motherwort and Chinese motherwort have a different evolution history.The results of phylogenetic and haploid reticulation tree construction show that European Motherwort has a clear pedigree and geographic structure.European Motherwort from Poland(Europe);Illinois,California,Oregon(United States);and Xinjiang(China)are gathered into four distinct groups.The branches indicate that there is no genetic infiltration and human-induced germplasm hybrids.The haplotypes corresponding to each branch can be used as molecular geographic identification of origin.Different haplotypes may correspond to different chemical types,which can be selected as high-quality chemical types and molecular markers for breeding.If this species is introduced and cultivated in China in the future,they should be introduced according to different haplotypes and high-quality chemical types,and blind and disorderly introductions should be avoided because this could cause germplasm hybridization and lead to germplasm degradation.The Chinese motherwort phylogenetic tree and haploid reticulated evolution tree also form 4 distinct branches,but do not have a pedigree geographic structure.Individuals of the same origin are not clustered together,but form a multi-line evolutionary relationship with individuals of other origins.The haplotype corresponding to each branch cannot be used as a molecular geographical indication for identification of the origin.The reason for this result may be the result of the artificial exchange of germplasm between origins or populations during the long-term cultivation of this species.Therefore,the key to the protection and utilization of germplasm resources is germplasm purification.3.Comparative Analysis of Chemical Composition of European Motherwort and MotherwortUPLC-Q-TOF-MS and UPLC-MS/MS technology combined with plant metabolome technology were used for the first time to qualitatively and quantitatively analyze European and Chinese motherwort from different sources to determine the main compounds,including the main differentiating marker compounds,as well as the key similarities and differences.Based on the literature review,a database of molecular and structural formulas of 164 compounds in the Leonurus genus was established and the characteristics of the compounds were analyzed.Common substituents and substitution positions were determined and possible biosynthetic pathways of metabolites were constructed using 23 different chemical standards to analyze samples by UPLC-Q-TOF-MS.This analysis,combined with the literature,allowed for mass spectrometry cleavage pathways to be developed and chromatographic retention of flavonoids,phenylethanoid glycosides,chlorogenic acids,alkaloids,phenolic acids and other compounds were studied and analyzed,and the diagnostic product ion(DPIs),characteristic product ion abundance ratio(DPIR),neutral loss was determined.Based on the above the data produced for these two species was analyzed.First,structural analysis and identification of compounds with high content and known structure in the two species was established,next the combination of mass spectrometry and chromatographic characteristics of identified compounds,the ClogP value,intramolecular hydrogen bonding and other information such as compound content was analyzed and estimated structures for all compounds in both species were determined.Results:A total of 330 compounds were identified or inferred from European and Chinese motherwort,including 38 potentially new compounds,and more than 200 compounds were found for the first time from these species.The identified compounds included 83 flavonoids,70 phenylethanoid glycosides.81 glucuronic acids,10 alkaloids,11 iridoids,13 chlorogenic acids,10 phenols acids and their glycosides,10 sugars and their glycosides,28 fatty acid derivatives.10 caffeic acid glucosides,and 5 other compounds,including 70 groups of isomers.This can be used as the foundation for the composition of metabolites in L.cardiaca and L.japonicus in any future research.UPLC-Q-TOF analysis was performed on different European and Chinese motherwort samples using plant metabolome analysis strategies.On the whole,the metabolites of the two are similar.The content of phenylethanoid glycosides in European motherwort is generally higher.Although the content of flavonoids and glucaric acid are different in the two,the total content of European motherwort is higher.Stachydrine is also significantly higher in European motherwort but leonurine is,essentially,not present.In order to further obtain the key markers of the difference between the two,the liquid quality data was imported into Progenesis QI for summing,peak alignment,peak extraction,grouping,and other processing to obtain data sets for different samples.Furthermore,the Sumproduct function of Excel was used to automatically screen European motherwort and Chinese motherwort with a compound peak area difference of more than 3 times.Ezinfo software was also used to analyze the two species with composition analysis,partial least squares analysis,orthogonal partial least squares analysis,S-Plot diagrams,and VIP values to search for and identify compounds with significant differences.12 key differential markers in European Motherwort and Motherwort were identified(verbascoside,lavandulifolioside,leonuronide A,isolavandulifolioside,leonuronide B,leonurine,syringic acid,2-cis-caffeoyl glucaric acid,phaseolic acid,genkwanin,leonurusoide F,apigenin 7-(6"-coumaroyl)-galactopyranoside),and pass The data validated that 12 differentiating marker compounds are well clustered and can be used to identify European motherwort and Chinese motherwort samples.The UPLC-Q-TOF-MS method and the LC-MS/MS method were used to analyze the content of 37 major compounds in European and Chinese motherwort,and 11 of the compounds were accurately and quantitatively analyzed,and the results showed that the average content of European motherwort was more than three times the average content of motherwort.The compounds are mainly stachydrine,verbascoside,lavandulifolioside,leonuronide A and B,lindenin,and glucaric acid and fatty acids.In Chinese motherwort the average content of leonurine and astragalin is more than twice that of European Motherwort.The quantitative analysis results are consistent with the qualitative analysis results. |
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