Study On The Genetic Relationship And Quality Evaluation Of The Source Plants Of The Chinese Medicine Vitex Officinalis

Purpose:Viticis Fructus is a dry,ripe fruit of Vitex trifolia L var.simplicifolia Cham?Vitex.rotundifolia L?and V.trifolia Linn.It has the effect of dispelling wind and cooling and clearing the head.It is a commonly used Chinese medicinal material,and it has been listed as a national grade 3 key protected wild medicinal species.It is commonly used clinically for the treatment of wind-heat fever,dizziness,headache,red eyes and swelling,and tinnitus and deafness.There is much controversy over whether the Vitex.rotundifolia L should be used as an independent species in the vitex or a variant under the trifoliate.In addition,his research on taxonomy has been confined to morphology,subjectivity is relatively large,and there is no research on molecular biology.At present,in the Chinese Pharmacopoeia,V.rotundifolia L is used as a variant of Viticis Fructus.Its fruit and the fruit of V.trifolia are collected at the same time as Viticis Fructus.However,the results of the previous study showed that the differences in the chemical composition and pharmacological effects of the fruits of these two species were different.Different sources of traditional Chinese medicines of the same variety have different chemical compositions and pharmacological effects,which may directly affect the efficacy of clinical medications.Therefore,it may be unfavorable to use both fruits of V.rotundifolia L and fruit of V.trifolia as the medicinal materials for Viticis Fructus.Clinical drug safety and quality control.Other studies have shown that the major components in the fruit of V.rotundifolia L vary greatly among different populations in China,and molecular markers have revealed that the genetic diversity and chemical diversity are closely related,but for V.rotundifolia L the genetic variation background and variation pattern of V.rotundifolia L and V.trifolia are not clear.Therefore,in this study,chloroplast?cpDNA?gene and nuclear gene?ITS?variation and microsatellite molecular marker?SSR?analysis were used to study the phylogeography of the populations of V.rotundifolia L and V.trifolia.The study of population genetics has explored the genetic structure and genetic diversity of the V.rotundifolia L and V.trifolia populations,and initially revealed the genetic background,variation patterns,and evolutionary history of V.rotundifolia L and V.trifolia.In order to solve the problems of taxonomy of the two species,the species of the V.rotundifolia L and V.trifolia provide some molecular evidence and put forward their own views.The chemical fingerprinting and content determination of the fruits of V.rotundifolia L and V.trifolia,which were used to provide the theoretical basis for the clinical application and quality evaluation of Viticis Fructus and to provide reference for the revision of the Fructus Viticis collection method.The genetic diversity and chemical diversity analysis of the genetic diversity of the two species were combined to provide a corresponding protection strategy for the germplasm resources of the source plants of the fruits of V.trifolia,so as to ensure the stability of the germplasm resources of the the fruits of V.rotundifolia L and optimize the selection.Good quality and stable populations provide some help for the effectiveness and safety of clinical medications and provide a basis for quality control.Materials and methods:Materials:The leaves and dried fruits of V.rotundifolia and V.trifolia were identified by Professor Luping Qin from Zhejiang University of Traditional Chinese Medicine.Orientin,isoquercetin,vitexin,casticin and artemisetin were purchased from Chengdu Purifa Technology Development Co.,Ltd.Methods:1.Phylogenetic study of the original plants of Viticis FructusTwo cpDNA sequences?trnG-trnS,trnH-psbA?of 25 V.rotundifolia and V.trifolia populations with 158 individuals and a nrDNA?ITS?sequences of 27 V.rotundifolia and V.trifolia populations with 179 individuals have been studied by using Phylogeography methods.The results of sequencing were used DNAsp 5.1,PERMUT,ARLEQUIN 3.5,MrBayes 3.1.2,TCS 1.2,BEAST and other software to detect the genetic diversity and genetic differentiation,and carried out phylogenetic analysis and pedigree analysis,and population genetic structure analysis can also be performed for each population and the time of differentiation of the two species can be estimated.This reveals the relationship between the V.rotundifolia and V.trifolia and determines the classification level of V.rotundifolia.2.Study on genetic diversity of the original plants of Viticis Fructus based on SSR markersThe genetic diversity and genetic structure of the 245 individuals of 25 populations of V.rotundifolia were analyzed by using 9 pairs of SSR markers.Popgene32,Creuvus2.0,GenAlex 6.502,STRUCTURE,BOTTLENECK1.2.02 and other software were used to analyze the genetic diversity and genetic structure of V.rotundifolia and V.trifolia,and the bottleneck effect was detected for the population with more than 10 individuals.According to the results of genetic diversity,the protection of wild germplasm resources of Viticis Fructus provides corresponding strategies and protection of populations,which provides a theoretical basis for the sustainable use of Viticis Fructus.3.Study on Chemical Diversity of Viticis Fructus from Different SourcesThe HPLC fingerprint of Viticis Fructus was established by gradient elution method.The fingerprints of Viticis Fructus from different sources were studied.Secondly,HPLC method was used to determine the content of five components of Orientin,isoquercetin,vitexin,casticin and artemisetin in different sources,which provided a theoretical basis for the quality evaluation of Viticis Fructus.4.The relationship between genetic diversity and chemical diversity of Viticis Fructus from Different SourcesThe clustering map constructed based on the genetic distance of SSR molecular markers and the clustering map constructed based on the genetic distance of cpDNA sequences were compared with the clustering map constructed based on chemical distance,so as to inherit the genetic diversity and chemical diversity of Viticis Fructus.The relationship between them is explored.Results:1.Based on the sequence of cpDNA two-step spacers?trnG-trnS,trnH-psbA?from 158individuals of 25 V.rotundifolia L and V.trifolia populations and 179 individuals of 27 V.rotundifolia L and V.trifolia populations.A total of 9 haplotypes were found in the phylogeography of nrDNA?ITS?sequences.The overall level of haplotype was(h_cp=0.562,h_nr=0.465),nucleotide polymorphism(?_cp=0.0012,?_nr=0.00139),and the overall level of genetic diversity was low.The haplotype diversity and nucleotide polymorphism of V.trifolia population was higher than that of V.rotundifolia L.Molecular variance analysis?AMOVA?showed that the genetic variation among species was 76.44%at the species level,indicating that obvious differentiation has been observed between the V.rotundifolia L and V.trifolia and the genetic variation?80.34%?in the single leaf Manjing population is higher than that of its population.Genetic variation between groups?19.64%?.Haplotype phylogenetic tree based on haplotype distribution and Bayesian analysis revealed that V.rotundifolia L and V.trifolia had different haplotypes,and haplotypes were not shared,and an insignificant phylogeographical structure(N_ST>G_ST,P<0.05).Similar topology to phylogeographical relationships indicated a single phylogenetic split.The haplotypes could be roughly clustered into two lineages:V.rotundifolia and V.trifolia with high bootstrap support.The statistical parsimony haplotypes network of all samples revealed a single network.Divergence times were estimated using the program of BEAST.The divergence times estimated at approximately 0.18 million years ago?mya;95%HPD:0.08-0.37?between V.rotundifolia and V.trifolia.V.rotundifolia and V.trifolia,appeared to have diverged from each other during the late Pleistocene.In the figure,it is presumed th V.rotundifolia evolved from V.trifolia.Based on cpDNA data,the population of V.rotundifolia and V.trifolia both showed a relatively smooth,unimodal distribution and closely matched the distribution under an exponential growth rate indicating a rapid demographic expansion.2.For the first time,9 pairs of SSR markers were used to analyze the genetic diversity and genetic structure of the V.rotundifolia and V.trifolia.Populations from 25 populations of 245individuals of the V.rotundifolia and V.trifolia.A total of 48 alleles were detected.The average expected heterozygosity was 0.4507,revealing that the genetic diversity of V.rotundifolia and V.trifolia.was lower,and the results were consistent with the cpDNA sequence results.The variance analysis of the molecular data revealed that the genetic variation of the V.rotundifolia and V.trifolia.populations mainly existed in the population?78.02%,77.37%?.The Hardy-Weinberg equilibrium test found that 9 out of 9 SSR loci deviated significantly from the Harvard equilibrium,suggesting that the population experienced a bottleneck effect.Subsequent BOTTLENECK bottleneck testing revealed that some groups experienced bottleneck effects or founder effects.According to the test results of Mantel test,there was no significant correlation between genetic distance and geographical distance on the overall level.R²=0.0172,P=0.21?>0.05?,indicating the genetic differentiation and geography Isolation was irrelevant,suggesting that this may be due to the rapid expansion events of V.rotundifolia and V.trifolia.,and the STRUCTURE analysis showed that the introgression of genes among various populations was more evident.According to the research results of genetic diversity,the corresponding in situ conservation strategies and ex situ conservation strategies were formulated to protect the germplasm resources of the original plants of Viticis Fructus.3.In the study of the chemical diversity of Viticis Fructus medicinal materials from different sources,the chemical fingerprints of 13 populations of the fruits of V.rotundifolia and V.trifolia were analyzed by high performance liquid chromatography.The results of the analysis showed that the similarity calculation results between the fruits V.rotundifolia and V.trifolia were lower,indicating that the chemical composition difference was more obvious.The UPGMA system cluster diagram of the fingerprint showed that when the chemical distance was 0.05,the V.rotundifolia and V.trifolia could be divided into two groups,further illustrating that the chemical composition of the V.rotundifolia and V.trifolia were different.In studies on the determination of chemical constituents of Viticis Fructus medicinal materials from different sources,the isoorientin,orientin,vitexin,casticin and artemitin were been measured.The results showed that the contents of orientin and isoorientin in the fruit of V.trifolia were significantly higher than those of V.rotundifolia,and the content of orientin and isoorientin in the population of 3_GX?Guangxi?was the highest;The content of casticin in the fruit of V.rotundifolia was significantly higher than that V.trifolia,with the highest content in DC?Duchang?.Therefore,in clinical application,the fruits of V.rotundifolia and V.trifolia should be used separately to treat different diseases using different sources of Viticis Fructus medicinal materials.It is recommended that the Chinese Pharmacopoeia should separate and collect the V.rotundifolia and V.trifolia.And establish different quality standards so as to correctly and effectively guide clinical medication.4.In phylogeography based on cpDNA sequences,the haplotype tree clustering results showed that the haplotypes of V.rotundifolia and V.trifolia can be clearly divided into two groups,consistent with the clustering results of chemical fingerprinting.However,the genetic distance UPGMA clustering map based on SSR molecular markers is inconsistent with the UPGMA clustering results of chemical fingerprinting.Comprehensive analysis of the results of the two markers and the analysis of the growth environment of the V.rotundifolia and V.trifolia.It was speculated that the chemical composition difference between the fruits of V.rotundifolia and V.trifolia is due to genetic factors and environmental factors.In order to effectively utilize,develop and protect the germplasm resources of the original plant of Viticis Fructus,combined with the results of genetic diversity and chemical diversity research,DL?Dalian?and 3_GX?Guangxi?should be selected for the V.rotundifolia and V.trifolia,respectively.Two populations established a GAP base to ensure the genetic diversity and chemical diversity of their populations.Conclusion:1.Molecular Phylogeography show that V.rotundifolia and V.trifolia a close genetic relationship,and V.rotundifolia is evolved from V.trifolia;two species have significant differentiation,differentiation time was 0.18 mya.2.The results of genetic diversity showed that V.rotundifolia and V.trifolia had lower genetic diversity,and the conservation of wild resources of Viticis Fructus should be strengthened to ensure the sustainable use of their germplasm resources.3.Chemical composition studies showed that there were significant differences in the chemical constituents between V.rotundifolia and V.trifolia,and the quality of DC,HT and DL's Viticis Fructus were the best.4.According to the morphological differences between V.rotundifolia and V.trifolia and the combination of their pedigree geography,genetic diversity,and chemical composition,it is recommended to use Phytophthora as an independent species under the genus Vitex.5.It is recommended that the Chinese Pharmacopoeia separate or contain different quality standards for V.rotundifolia and V.trifolia.