| Arnebia Radix is a major traditional Chinese medicine which commonly used in China.The sources in "Chinese Pharmacopoeia"(2020 edition)include Arnebia euchroma and Arnebia guttata.However,in circulation,the plants are mixed for Arnebia Radix because the roots of many Arnebia Radix plants contain naphthoquinone.In addition to the shortage of genuine Arnebia Radix resources,a large amount of imported Arnebia Radix with unknown origin has flowed into the market and mixed with Arnebia Radix intentionally or inadvertently.As a result,the quality of Arnebia Radix materials cannot be effectively guaranteed,which affects the clinical efficacy.The medicinal Arnebia Radix is usually divided into two types:hard Arnebia Radix and soft Arnebia Radix.The genuine source of Arnebia Radix is soft Arnebia Radix and belongs to the soft Arnebia Radix genus.Xinjiang as an authentic production area of Arnebia Radix,has a vast territory,which leads to the in the content of index components in Arnebia Radix in different counties and cities of Xinjiang.Is no longer desirable to vaguely consider that the entire Xinjiang is genuine production of Arnebia Radix.Due to the different sources and origins of medicinal materials,the influence of genes or external factors,the medicinal materials are also quite different in terms of chemical compositions,which leads to uneven efficacy in the clinical medication process,and brings great difficulty to the quality control of the medicinal materials.Arnebia plants are concentrated in Xinjiang.At present,Arnebia Radix products have the problems of very few domestic resources and severe protection situation.The severe shortage of Arnebia Radix resources is far from being able to meet the needs of the modern Chinese medicine industry.The resources and quality of Arnebia Radix have become the current problem of traditional Chinese medicine resources to be solved.Carrying out the investigation of Arnebia Radix germplasm resources,studying the genetic diversity of Arnebia Radix,and improving the quality evaluation system of medicinal materials are the basis for effective protection and rational use of Arnebia Radix.In this study,combined with the fourth national survey of traditional Chinese medicine resources,combining visits and field surveys,route sampling and the estimation,vegetation with soil type and area,the wild resources of the genus Arnebia in 39 counties and cities in Xinjiang were investigated.Using the "Traditional Chinese Medicinal Materials Biological Resources Regionalization Analysis System" developed by the Chinese Academy of Surveying and Mapping to explore the areas where Arnebia euchroma,Arnebia guttata,and Arnebia decumbens materials can grow in Xinjiang,by constructing the ecological environment database of traditional Chinese medicinal materials,the regional analysis of Arnebia euchroma,Arnebia guttata,and Arnebia decumbens in Xinjiang has been completed.On this basis,traditional source,traits,microscopy,and physical and chemical identification of medicinal materials derived from three species of Arnebia in Xinjiang were carried out,and DNA barcode technology based on ITS2,content determination by HPLC,and chemical fingerprinting technology were used to identify and evaluate the quality of three species of Arnebia in different counties and cities in Xinjiang to improve the quality evaluation system of Arnebia species.At the same time,using ISSR molecular marker technology to explore the genetic diversity of the three species of Arnebia in Xinjiang and to explore the genetics of Arnebia.Discuss the genetic diversity characteristics of Arnebia and its relationship with the quality of medicinal materials,and provide theoretical basis for effective protection and rational development and utilization of Arnebia germplasm resources.The research content and results are as follows:(1)The results of the investigation of 3 kinds of Arnebia resources showed that the 3 kinds of Arnebia Radix resources in different counties and cities in Xinjiang are all wild resources,and there are significant differences during them.Arnebia euchroma is mainly distributed in grassland and meadow.It is the most widely distributed and the most abundant Arnebia Radix in this survey,and it is also the main source of commercially available Arnebia Radix;Arnebia guttata is mainly distributed on gravel Gobi and hillsides;Arnebia decumbens is mainly distributed on the edge of the Gurbantong Desert.The exploring of the areas where three species of Arnebia plants are suitable for growth in Xinjiang by using the "Chinese Medicinal Materials Biological Resources Zoning and Analysis System" which was developed by the Chinese Academy of Surveying and Mapping.The ecological environment database of Chinese medicinal materials was constructed through the "Chinese Medicinal Materials Biological Resources Regionalization Analysis System",and the regionalization analysis of three species of Arnebia in Xinjiang was completed.(2)Analyze the genetic diversity of three species of Arnebia Radix using ISSR molecular marker technology.Six pairs of primers detected a total of 48 allele loci in 120 individual samples.The total number of effective alleles was 9.3455,and the value range was 1.3813(860)-1.6403(814).The average number of effective alleles per locus was 1.5770.The higher Ne in 39 populations were A.decumbens population in Wusu,A.euchroma population in Bole,A.decumbens population in Tecos,A.decumbens population in Karamay,and A.guttata population in Hejing.The lowest was A.guttata population in Hami(1.000).The Nei's genetic diversity index(He)and Shannom Information Index(?)indicated that A.decumbens population in Wusu,A.decumbens population in Tecos,A.decumbens population in Karamay,A.euchroma population in Bole,A.Euchroma population in Tashkurgan had rich genetic diversity,but A.guttata population in Hami,A.guttata population in Tuoli,A.guttata population in Mulei had low genetic diversity.The genetic distance between populations is the smallest between Arnebia euchroma and Arnebia guttata,which is 0.0627;the genetic distance between Arnebia guttata and Arnebia decumbens is the largest,which is 0.1640.The individual-based UPGMA chart shows that there are more cross-mixing of individuals between groups and groups,indicating that there is more genetic variation in the group.Using NTSYSpc2.1 principal component analysis,120 samples were divided into 3 parts.The first part is composed of Arnebia decumbens,the second part is composed of most of Arnebia guttata and a small amount of Arnebia euchroma,and the third part is composed of large part of Arnebia euchroma and several and Arnebia guttata.(3)The comprehensive evaluation and analysis of 3 kinds of soft purple herbal materials in different counties and cities in Xinjiang was completed by using plant source identification method,microscopic identification,TLC,Content determination according "Chinese Pharmacopoeia".Three kinds of Arnebia Forsk.have certain differences in plant morphology,medicinal properties,microstructure of vegetative organs,and powder characteristics of medicinal materials.In particular,Arnebia euchroma is quite different from Arnebia guttata and Arnebia decumbens.But the difference between Arnebia guttata and Arnebia decumbens is small.The control medicinal materials of Arnebia Radix and L-shikonin,?,?'-dimethylacryloyl akanin were used as the control respectively,and cyclohexane:xylene:ethyl acetate:formic acid(6:4:0.5:0.4)is a developing agent,the spots of the test product are concentrated,clear and bright,with good separation and moderate Rf value.The moisture content of the 3 medicinal materials of the genus Arnebia from 46 origins is 4.25%-14.09%,which are in compliance with the pharmacopoeia.The total ash content is 9.73%-20.52%,and the acid-insoluble ash content is 1.562%-9.177%.It is recommended that the total ash content of this product does not exceed 20.0%,and the acid-insoluble ash content does not exceed 10.0%.The water content and ash content of different producing areas are quite different,indicating that in Xinjiang,the main producing area of Arnebia euchroma,the quality of medicinal materials in each distribution area also has great differences.(4)Based on the DNA barcode research of ITS2 sequence.The sequence length of the ITS2 region is 399 bp,and the number of variable positions is 71.The intraspecies distance is 0.0025-0.006,and the interspecies distance is 0.0745-0.0915.The samples are grouped according to the region,the genetic distance within the group is 0-0.0494,and the genetic distance between the groups is 0-0.0941.Analyzing the distribution of genetic distance,there are obvious barcoding gaps between the samples.Through the analysis of the constructed NJ cluster tree,a separate evolutionary branches can be formed,which indicates that the ITS2 sequence can distinguish three species of Arnebia from different producing areas.(5)The roots of three species of Arnebia genus all contain naphthoquinones.There are differences in species and content among different species.There are also large differences in different producing areas within the same species.The results of content determination by HPLC showed the total naphthoquinones in Arnebia euchroma is the highest,with an average of 42.8914 mg/g,followed Arnebia guttata,with an average of 12.3323 mg/g,and Arnebia decumbens has the lowest content,with an average The value is 6.8404 mg/g.The cluster analysis and principal component analysis of the chemical components of 7 naphthoquinones in 46 batches of samples can distinguish Arnebia decumbens from Arnebia euchroma and Arnebia guttata,while the Arnebia euchroma and Arnebia guttata have a small overlap.Orthogonal least squares discriminant analysis showed that ?-acetoxyisovaleryl arkanine and?,?'-dimethylacryloyl arkanine contributed the most among the three kinds of Arnebia,which can be used as a difference marker to distinguish the three kinds of Arnebia.(6)HPLC fingerprint analysis was performed on 46 batches of 3 kinds of Arnebia samples.The "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System(2012A Edition)" was used to calibrate 19 common chromatographic peaks and evaluate the similarity with the common pattern map as the reference map.Except for the similarity of Ae-5 and Ae-6 of Arnebia euchroma,which is lower than 0.7,the similarities of the other 16 samples are all higher than 0.9;the similarity of the 18 batches of Arnebia guttata samples was between 0.546-0.99.Compared with the control map,the similarity of the other samples except Ag-7 and Ag-13 samples is higher than 0.8;the comparison of Arnebia euchroma and Arnebia guttata shows that the similarity between Arnebia guttata and Arnebia euchroma is not high.The results of OPLS-DA showed that 7 compounds such as isobutyryl shikonin,?,?'-dimethylacryloylakkanin,levorotatory shikonin,etc can be used as specific markers to distinguish the two kinds of Arnebia Radix.Moreover,the 7 compounds are generally higher than those of Arnebia guttata in Arnebia euchroma. |
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