| Dai medicine is one of the four major minority medicines in China,which is also an important part of the national minority medicine.Due to the influence of natural and historical conditions and the incomplete document of Dai medicine herbal literature,there exists a common phenomenon that different Dai medicines are under the same name,or the same Dai medicines has different names.This phenomenon causes confusion in the use of Dai medicine.This study aims to clarify the varieties of Dai medicine resources,to establish the identification system of,to provide a scientific reference to the promotion the industrialization process of Dai medicine.Literature analysis,ethno-botany and mathematical statistical analysis have been used for the systematic study of Dai medicine.The DNA barcode method has been applied to establish the Dai medicine DNA barcode database.The combination of DNA barcode and high resolution melting(HRM)curve technique has also been applied in the identification and market supervision of Dai medicine.The main results are as follows:1.Based on the past 40 years research studies,the use of professional database query verification and the review of 35 monographs and 143 periodical literature,a total of 8590 Dai medical drug information has been collected.Apart 97 information of medicinal material which has not document the Latin name or the Latin name cannot be confirmed,8493 medicinal information are available.The 1654 kinds of drugs used in the current Dai medical have been recorded in the Dai drug resources catalog information set.All these drugs are classified according to the plant/animal/mineral name,Dai name,family,medicinal name,plant part,Latin name,function,literature source and the standard collection.2.On the basis of the above research,this study analysis and sort out the types,traditional utilization characteristics and quality standards of Dai medicinal resources systematically,using the ethnobotany and mathematical statistics.The results are as follows:2.1 Dai medicine contains a wide range of drugs,and there are currently recorded a total of 1654 kinds of drugs.Among them are 1491 kinds of herbs,belonging to 191 families,836 genera,including 2 families 2 genera 4 species of lichens,7 families 9 genera 9 species of fungi,1 family 1 genera of mosses,2 families 2 genera 2 species of algae,19 families 26 genera 42 species of ferns,7 families 7 genera 13 species of gymnosperms,153 families 789 genera 1420 species of angiosperms;141 kinds of animal drugs,belonging to90 families 123 genera;22 kinds of mineral drugs.1491 kinds of Dai medicinal plants,159 kinds of medicinal plants are endemic to China and 152 kinds of plants have been listed as endangered and rare species,belonging to70 families and 133 genera and 62 families and 133 genera,respectively.There are 68 species of the Dai animal medicine endangered and rare species,accounting for 48.22% of the total number.2.2 From the usage characteristics,the highest frequency of medication Dai medicinal parts are root and rhizome(Radix et Rhizoma),followed by the whole grass and leaves.In terms of the treatment of diseases,most of the plant drugs have been used to treat the digestive system diseases,followed by other diseases such as injuries,circulatory system,skin,respiratory and urinary system diseases;most of the animal drugs have been used to treat the skin and circulatory diseases,followed by the digestive system and infectious diseases;while mineral medicine are mainly used to treat the skin and circulatorydiseases.In terms of the usage methods,most of the plant drugs are used in the form of soup,followed by patches;most of the animal drugs are used in the form of powder,followed by patches;while mineral drugs are mainly for external use.2.3 According to the review results of the Dai medicine quality standard,785 Dai medicinal materials have been documented from all levels,accounting for 47.46% of the total number of medicinal herbs.Among them,there were 327 kinds of medicinal herbs accepted by the Pharmacopoeia of the People’s Republic of China.Taking the Legume and Euphorbiaceae as an example,this study analyzes the name,variety and base,parts,function and quality standard and so on.The results show that it is a common phenomenon that "variety-name-base" of Dai medicine was not standardized,and the descriptions of medicinal parts and efficacy were still to be standardized.At the same time,the quality standard of Dai medicine was extremely imperfect,which hindered industrialization process of Dai medicine.So the research of Materia Medica,resource and the use status of Dai medicine should be strengthened,and a systematic research with modern drug research technology should also be carried out to promote the variety classification and the establishment of quality standard system of Dai medicine.3.The total DNA of the Dai medicine was extracted by the DNA kit.The ITS2 or psb A-trn H sequence was amplified by PCR and sequenced in two directions.The obtained sequences were then assembled by Codon Code Aligner 5.1.5,and the low-quality sequences and primer regions were removed to get the target ITS2 or psb A-trn H sequences.The standard DNA sequences of 300 common Dai medicines were established.On the basis of the standard DNA sequence,the preliminary work of our group and the database technology,we combine the Gen Bank sequence,BLAST analysis of error prevention and phylogenetic tree analysis to verify the reliability of thesequence,and establish the Dai medicine DNA barcode identification system.The system consists of a sample database and a sequence database,containing more than 1,000 kinds of Dai medicine and a large number of adulterate products and their closely related species.4.Four kinds of medicinal materials and their counterfeit goods,including‘Habinhao’,‘Yaganzhuang’,‘Yalingwu’,were studied by DNA barcode technology,and the HRM technique was also applied in the identification of Dai medicine.The results show that DNA barcode and HRM can be applied for the identification of Dai medicinal herbs and their adulterate products effectively.The method proved to be easy to handle and standardize,which provides a powerful means for the safety of clinical medication and market supervision of Dai medicine.4.1 48 samples from 12 Clerodendrum species and ITS2,psb A-trn H sequences downloaded from Genebank were used to construct the DNA barcode database of Clerodendrum.We used this database to identify 27 commercial samples “Ha-bin-hao” purchased in drug stores and hospitals.Results indicated that the intra-specific genetic distance were smaller than inter-specific ones in ITS2 regions of Clerodendrum species.While in psb A-trn H regions between the species,it is the opposite.Therefore,ITS2 regions as DNA barcodes can stably and accurately distinguish Clerodendrum species from its adulterants.According to the market research of medicinal herbs,the commercial samples of “Ha-bin-hao” on the market were complex sources,and only one in 27 samples have been identified as C.japonicum(Thunb.)Sweet.Terefore,we present the ITS2 barcode as a powerful tool for the market supervision of “Ha-bin-hao”.4.2 30 samples from 9 Paris species and its adulterants were used to construct the HRM curve model.We used this model to identify 10 crude drug of ‘Yaganzhuang’ purchased from the market.All the samples can be assignedto the corresponding species group.Further DNA sequencing results are consistent with the HRM curve.It’s indicated that the HRM curve is feasible as a means of market supervision.4.3 Based on the 16 species of genus Hedyotis and a total of 70 ITS2 sequences,we establish a DNA barcode database of ‘Yallingwu’ and its close relatives.The results showed that the phylogenetic tree constructed by ITS2 sequence and the method of comparison and the minimum distance can significantly distinguish between 16 species of Hedyotis.We then applied the data base to identify 15 batches of H.diffusa collected from the market.7 batches of them gather toghter as one cluster,which had the highest similarity with H.diffusa.3 bathes of them gather as another cluster,which showed a high similarity with the H.tenelliflora Bl..The other5 batches are some other species.Then,based on the results of DNA barcode,the study applied the HRM to identify the known ‘Yalingwu’(H.diffusa)and its adulterate products.The results show that HRM can identify ‘Yalingwu’(H.diffusa)and its adulterate products effectively.4.4 Based on the ITS2 sequence,42 batches of ‘Hepajulei’(Radix Saposhnikoviae Divaricatae)collected from market were studied.The results showed that 12 bathes of medicinal had the highest similarity with Radix Saposhnikoviae Divaricatae;15 batches of them had the highest similarity with Seseli mairei;5 batches of them had the highest similarity with Pimpinella candolleana;8 batches of them had the highest similarity with Peucedanum ledebourielloides;1 batch of them had the similarity with Carum carvi;1 batch of them had the similarity with Peucedanum praeruptorum.Based on the ITS2 sequence,the NJ tree,the BLASTl and nearest distance can distinguish the above six species.HRM was used to identify the four kinds of known ‘Hepajulei’(S.divaricata)and its adulterate products.The results showed that HRM could effectively identify the ‘Hepajulei’ and its counterfeitproducts.This study shows that the origin of ‘Hepajulei’ in market place is complex.By the molecular identification methods,including DNA barcode and HRM,we can regulate it effectively.To sum up,this study has sorted out the Dai medicine resource systematically,and clarifies the varieties of Dai medicine resource for the first time.On this basis,300 kinds of commonly used Dai medicine standard DNA sequences together with a unified database of Dai medicine were established.At the same time,the DNA barcode and its combination of HRM method were successfully applied for the identification of several common Dai medicinal materials and their adulterate products,which provides a model for the molecular identification of other plant medicines.This study is of great importance for the identification,the safety of clinical usage,the protection of endangered species and the sustainable use of Dai medicine. |
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