| The basal species of Atractylodes chinensis(DC.),Koidz and Atractylodes lancea(Thunb.)DC.are recorded in Chinese Pharmacopoeia 2015.Atractylodes chinensis(DC.),Koidz,Atractylodes lancea(Thunb.)DC.and Atractylodes japonica Koidz.ex Kitam.,collectively known as Atractylodes lancea in the latest Chinese botany,are perennial herbs of the genus Atractylodes in the Compositae family.In recent years,there are many adulterants of Atractylodis Rhizoma in the market due to the shortage of market supply and other reasons,which makes the traditional Chinese medicine containing Atractylodis Rhizoma has a huge potential safety hazard.The identification of traditional Chinese medicine has always been the focus of attention.Compared with the traditional method,the DNA metabarcoding technology can be used to sequence the mixed samples with uniform identification standard and high sensitivity.It is more accurate and convenient to identify the origin of raw materials of Chinese patent medicine.Objective:In this study,Atractylodis Rhizoma and its adulterants were selected as the research object,and Atractylodis Rhizoma and its adulterants were collected from the origin.By constructing the standard reference DNA barcoding database of Atractylodis Rhizoma and its adulterants,the DNA extraction method of the Chinese patent medicine containing Atractylodis Rhizoma was optimized.The molecular identification of Atractylodis Rhizoma in Ermiao Wan was carried out by using the DNA metabarcoding technology based on the genetic information of species,This paper discusses the feasibility of the identification of the raw materials of Chinese patent medicine by the DNA metabarcoding technology,which provides a new method for the identification of the raw materials of Chinese patent medicine.Methods:1.Atractylodis Rhizoma and its adulterants were collected for DNA extraction,obtain ITS2,mat K and rbc L sequences.Species were identified based on BLAST alignment and NJ phylogenetic tree.Another 29 sequences were downloaded from Genbank to supplement the database,which was used together with the obtained standard sequences to construct the standard reference DNA barcoding database of Atractylodis Rhizoma and its adulterants.2.To explore the influence of different cleavage times,different purification methods and different lysates on DNA quality.The Chinese patent medicines containing Atractylodis Rhizoma collected in the Pharmacopoeia were systematically sorted out,and the Chinese patent medicines containing Atractylodis Rhizoma were screened according to the factors such as DNA quality,dosage form,and mode of drug administration of the Chinese patent medicines containing Atractylodis Rhizoma,and representative experimental subjects were selected for the follow-up identification study of this study.3.The raw materials of Ermiao Wan in Chinese patent medicine were purchased from offline pharmacies,including Atractylodis Rhizoma,fried Cortex Phellodendri and American ginseng,which were used to verify the sensitivity of DNA metabarcoding technology.DNA barcoding technology was used to identify it to ensure the accuracy of the original.4.Referring to the method prescribed in the Pharmacopoeia,two control samples were prepared,one of which was added with American ginseng.After that,DNA was extracted from 3 boxes of Ermiao Wan commercial samples and 2 Ermiao Wan experimental control samples,and high-throughput sequencing was performed on the extracted quality DNA.Sequence obtained by data analysis,the assembled sequences were compared by the standard DNA database of Atractylodis Rhizoma and its adulterants constructed in the first part to analyze whether there are adulterants or common near-source species of Atractylodis Rhizoma in Chinese patent medicine.Results:1.The DNA quality of Atractylodis Rhizoma and its adulterants is good and stable.The gel electrophoresis of PCR products amplified by ITS2,rbc L and mat K universal primers can obtain bright and single bands,indicating that the amplification success rate is 100 %.A total of 9 PCR products with bright and single bands were sequenced in two directions,and 9 sequences were obtained by sequence splicing.The sequencing success rate was 100 %.Three ITS2 sequences,three rbc L sequences and three mat K sequences were obtained by data analysis of the obtained sequences.The NJ phylogenetic tree was constructed together with 29 sequences downloaded from Genbank.Atractylodes lancea(Thunb.)DC.and Atractylodes macrocephala Koidz.showed good monophylety in the NJ phylogenetic tree based on ITS2 sequence,which could be effectively distinguished.However,the branch of NJ phylogenetic tree based on rbc L sequence and mat K sequence is disordered,and the sequence difference is very small,which cannot distinguish Atractylodes lancea(Thunb.)DC.and Atractylodes macrocephala Koidz..2.It is concluded from the experiment that the GP1 pyrolysis solution is used for one time,and the column purification of GD and PW is the optimal experimental step.DNA extraction experiments on 12 kinds of Chinese patent medicines containing Atractylodis Rhizoma.Among them,Ermiao Wan has the highest DNA concentration and the best quality.Ermiao Wan is selected as a representative research object for subsequent macro barcode technology identification study.3.The sequence of Phellodendri Chinensis Cortex and Panacis quinquefolii Radix ITS2 were input into the DNA barcode identification system of Chinese medicinal materials for BLAST comparison(website: http://www.tcmbarcode.cn).The results showed that Phellodendri Chinensis Cortex was Phellodendron chinense Schneid.and the Panacis quinquefolii Radix was Panax quinquefolium L..The ITS2 sequence of Atractylodis Rhizoma was compared with the standard DNA barcoding database of Atractylodis Rhizoma and its adulterants obtained in the first part,and the NJ phylogenetic tree was constructed.The results showed that the ITS2 sequence of Atractylodis Rhizoma was clustered with Atractylodes lancea(Thunb.)DC.,indicating that the rhizome of Atractylodis Rhizoma was Atractylodes lancea(Thunb.)DC..The above three kinds of medicinal materials are authentic.4.The authentic Atractylodes lancea(Thunb.)DC.could be detected in all 3 commercial samples and 2 self-made control samples of Ermiao Wan,but A36 and EMW8 have found adulterants of Atractylodes macrocephala Koidz.;A60 has found unlabeled traditional Chinese medicines in the prescriptions of Glycyrrhiza uralensis Fisch.,Scutellaria baicalensis Georgi,Paeonia lactiflora Pall.,and Sanguisorba officinalis L.;EMW8 has found Fallopia multiflora(Thunb.)Harald.,and five samples have found molds,fungi and a small amount of Triticum aestivum L.,Secale cereale L.,Vigna unguiculata(L.)Walp.Valeriana officinalis L.and other non-pharmacopoeial species sequence.Conclusion:By collecting samples of Atractylodis Rhizoma and its adulterants,DNA extraction and sequence analysis,DNA barcoding technology can be used to accurately identify samples of Atractylodis Rhizoma and its adulterants,and a reference DNA barcoding database of Atractylodis Rhizoma and its adulterants is successfully constructed.The identification of Ermiao Wan,a representative Chinese patent medicine containing Atractylodes pallidus,by using the DNA metabarcoding technology based on PCR-free can accurately and effectively identify the basal materials of Atractylodis Rhizoma in Chinese patent medicine,and provide a new method for the basal materials identification of Chinese patent medicine. |
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