A Preliminary Study On The Quality Evaluation And Mechanism Of The Microscopic Characteristic Index Method Of Arctium Chinensis Medicinal Materials

Purpose:This research is on the National Natural Science Foundation of China:the research on the accumulation of arctiin in the development of burdock seed based on the combination of frozen section,micro cutting and liquid quality(81773852)And National Natural Science Foundation of China:with the support of the project of construction and mechanism of quality marker system of Chinese herbal medicine(81874338),the author visited and investigated the various origins of Arctium in the early stage,and through the literature investigation and Research on Arctii,and combined with the market investigation and the origin investigation of Arctii,the distribution of resources in different production areas of burdock seed was clarified,The aim is to standardize the market order of burdock seed medicine and realize the standardization and scientificization of the development of Chinese medicine industry.The microscopical characteristic index of 30 batches of burdock seeds from different regions and30 batches of burdock seeds from the protection nursery of key Chinese herbal medicines in Liaoning Province,Dalian campus of Liaoning University of traditional Chinese medicine were measured.The chemical components and secondary metabolites of burdock in the skin samples of burdock seeds were analyzed and compared by using uplc-q-tof-ms technology The results of sequencing the whole genome of Arctium lappa in the early stage of the project group,selected the whole genome of burdock and four other species of Compositae,and compared and analyzed the Arctium genome.The purpose of this study is to explain the formation mechanism of this microscale of Arctium lappa,and to explore the biological formation mechanism of this microscale marker,which is the biological formation mechanism of Fructus cells in Arctium,which is the biological formation mechanism of Fructus cells in burdock,which is a biomarker of traditional Chinese medicine.The new ideas and methods for molecular identification of materials are provided.Materials and Methods:1.Materials:In the study of resource distribution of Arctium lappa L.in different producing areas,members of the research group collected 40 samples of Arctium lappa L.from 22 shops in Bozhou Chinese herbal medicine market of Anhui,Anguo Chinese herbal medicine market of Hebei,Hehuachi Chinese herbal medicine market of Chengdu and Yulin Chinese herbal medicine market of Guangxi.We visited Kuandian County of Liaoning Province,Xiuyan County of Jilin Province,Wuchang County of Heilongjiang Province,Pucheng County of Gansu Province and Weiyuan County of Shaanxi Province,and collected 20 samples of Arctium lappa L.from farmers in the producing areas.30 batches of Arctium lappa L.from July to October in 2019 were collected from the protection garden of key varieties of traditional Chinese medicine in Liaoning Province,Dalian campus of Liaoning University of traditional Chinese medicine(E 121°52?38.06?,N 39°03?46.59?)and 30 batches of Arctium lappa L.from different places were collected from different provinces and cities in China.They were identified as the dry and mature fruits of Arctium lappa L.by Professor Kang Tingguo and Professor Xu Liang of Liaoning University of traditional Chinese medicine.The Arctium lappa L.for gene expression analysis were collected by laser microdissection and liquid chromatography-mass spectrometry(LC-MS)in the key variety protection garden of Liaoning Province(E 121°52?38.06?,N 39°03?46.59?),including bud stage,early flowering stage,full flowering stage,late flowering stage and mature stage.2.Methods:2.1 The research on the distribution of Arctium lappa L.resources in different producing areas includes the summary and arrangement of herbal literature,modern monographs,Chinese Pharmacopoeia of various editions,and the standards of Chinese herbal medicine of various provinces.Based on the field investigation on the market of large and medium-sized medicinal materials in China,40 samples of Arctium lappa L.from 22 shops were collected for statistics of 1000 grain weight,withered rate and doping rate.The main producing areas of Arctium lappa L.were investigated,and 20 samples of Arctium lappa L.were collected for statistics of 1000 grain weight,shrinkage rate and doping rate.2.2 Considering that the factors affecting the suspension effect of the prepared samples are mainly sample weight and comminution size,etc.and the investigation level of each factor is different,so the optimization conditions of the micro quantitative method are investigated by uniform design method in this experiment.The optimized conditions are sample weight 450 mg,glycerin quantity 5 ml.using this condition,take 3 samples of Arctium lappa L.seeds,and operate according to the above conditions of uniform optimization.Record the number and area of stone cells(10000?m²)under each loading.According to the formula P=(X×V)/(V'×W),calculate the number of characteristic index and area characteristic index of stone cells according to the formula,and calculate the average and RSD.The content of arctiin in 30 batches of burdock seeds from the same origin and 30batches of different regions in China was analyzed by HPLC,and the data were introduced into SPSS software for correlation analysis.2.3 The chemical components and secondary metabolites of arctiin in the pericarp of Arctium lappa L.at different growth stages were quantitatively analyzed and compared by liquid chromatography-mass spectrometry(UPLC-Q-TOF-MS),so as to reveal the accumulation of arctiin in the growth stage and the material accumulation rule of the stone cells in the pericarp of Arctium lappa L.The dynamic sampling of Arctium lappa L.planted in the garden,the selection of five different periods of fresh Arctium lappa L.medicinal materials,making frozen sections,through laser micro cutting technology to capture each period of stone cells,in order to achieve the purpose of accurately capture stone cells as a micro characteristic marker samples;using the reference protein sequence of Arctium lappa L.and other Compositae plant genome analysis Blast comparison of white files(https://www.ncbi.nlm.nih.gov/),parameter E?Value is 1e-5;extract identity>=50,align?Ratio>=50%protein;Pfam was used?Scan was used to search whether the protein contained the corresponding domain.The genes with complete domain were selected as candidate genes,and the selected candidate genes were extracted for expression analysis.The purpose of this series of experiments is to preliminarily explore the mechanism of the microscopic characteristic structure of stone cells.Results:1.Through the investigation of Arctium lappa L.in five provinces,combined with the statistics of 20 pieces of Arctium lappa L.in terms of 1000 grain weight,withering rate and doping rate,the members of the research group found that in the process of collecting and processing Arctium lappa L.by air selection,the impurities of Arctium lappa L.were removed,and the screening of impurities by farmers was not thorough enough,or there was no withered and dried Arctium lappa L.and Arctium lappa L.by air selection There are more impurities and shrunken Fructus Arctii,which lead to the lower 1000 grain weight of Fructus Arctii.2.In the study of the correlation between the microscopic characteristic index and arctiin content of 30 batches of Arctium lappa L.from the same origin,Y:arctiin content;X1:microscopic characteristic index of stone cell number;x2:microscopic characteristic index of stone cell area.Y=-0.365X₁+27.196,r=-0.9843 P=0.000,Y=-0.3514X₂+26.707,r=-0.9902P=0.000,both of them had significant statistical significance.The two groups of data not only had significant correlation,but also showed negative correlation,that is,the greater the number of stone cells and area microscopic characteristic index of Arctium lappa L.,the lower the content of arctiin.3.In the study of the correlation between the microscopical characteristic index of burdock seed and arctiin content in 30 batches of different producing areas,Y=-0.2307X₁+17.582,r=-0.909 P=0.000,Y=-0.2225X₂+17.157,r=-0.899 P=0.000,both of them have significant statistical significance.The data of the two groups are not only significant correlation,but also negative correlation.That is,the larger the number of pericarp cells and the area micro characteristic index,the lower the content of arctiin.4.In the UPLC-Q-TOF-MS method,33 main chromatographic peaks were observed in the analysis of 5 different stages of development of burdock peel.32 of them were identified,including amino acids,phenolic acids(caffeic acid derivatives such as chlorogenic acid),lignans and fatty acids.The identified components include amino acids(1),phenolic acids(8),lignans(19),fatty acids(4);29 compounds were identified in the first stage,32 compounds were identified in the second stage,32 compounds were identified in the third stage,33compounds in the fourth period,with the most species.The fifth stage burdock contains 32compounds.5.In the bioinformatics analysis of genes related to the formation of stone cells in the endocarp of Arctium lappa L.,18 gene families,4CL,C4H,CCo AOMT,CCR,PAL,ACT,C3H,CAD,COMT,CSE,DIR,F5H,HCT,LAC,PLR,POD,PPO and SIRD,were selected and compared by correlation analysis Four genes,4CL,HCT,DIR and LAC,which were highly related to lignin accumulation and stone cell formation,were screened out by P-value and correlation coefficient.Conclusion:1.According to the statistics of Arctium lappa L.from different producing areas,it is found that the Arctium lappa L.can be divided into two specifications:selection and control according to the doping rate,1000 grain weight and shrinkage rate.2.In the study of the correlation between the microscopic characteristic index of Arctium lappa L.and the content of arctiin in 30 batches of Arctium lappa L.from the same origin and different origins,there was a highly negative correlation between the microscopic characteristic index of the number of stone cells and the area of stone cells and the content of arctiin.According to the experimental results,the microscopic characteristic index of the number of Arctium lappa L.meeting the national standard should not be higher than 60.81pieces/mg,and the area characteristic index should not be higher than 617700?m²/mg.3.In the experiment of 5 different stages of development of burdock by UPLC-Q-TOF-MS method:in the analysis and detection,the samples of different batches of burdock were obtained a better total ion flow chromatography,and the separation degree of each peak was better;in terms of mass spectrometry data,the response value of negative ion scanning mode was better than that of positive ion mode,and the detection was performed The quality measurement accuracy is good,and it can meet the requirements of identifying molecular formula and fragment element composition by accurate mass number.Finally,33main chromatographic peaks were observed from the peel of burdock seeds from 5 different batches,and 32 of them were identified.Then,the selected ion response intensity of the main compounds(including unknown)was compared with Unifi software.The results were amino acids,phenolic acids(caffeic acid derivatives such as chlorogenic acid),lignans and fatty acids component.4.In the experiment of bioinformatics analysis of genes related to stone cell formation in the endocarp of Arctium lappa L.,the reference protein sequences were used to blast the genomic protein files of Arctium lappa L.and other Compositae plants,and the genes with complete domains were used as candidate genes.After expression analysis and correlation analysis,four genes with cor greater than 0.7 and P value less than 0.05 were selected:4CL,DIR,HCT and LAC.These genes can provide an important research direction for the molecular formation mechanism of stone cells.The negative correlation between the formation of stone cells and the content of arctiin is also preliminarily clarified.