Construction Of A Method For The Identification Of Biological Components Of The Chinese Patent Medicine Tiedi Wan Based On The Full Length Of Chloroplast And Mitochondrial Genomes

The identification of biological components of Chinese patent medicines is a major difficulty in the identification of Chinese medicines.The misfeeding or false feeding of raw materials of Chinese patent medicines has seriously affected the safety and efficacy of clinical use of Chinese patent medicines and their development in the international market,and there is an urgent need for more effective identification methods to solve the problem of species identification of prescription ingredients of Chinese patent medicines.Tiedi Wan is a famous prescription in history.It has good clinical effect on chronic pharyngitis.In the 2020 edition of the Chinese Pharmacopoeia,Tiedi Wan is a large honey pill made from 10 Chinese herbs,including Ophiopogonis Radix,Platycodonis Radix,Canarii Fructus,Scrophulariae Radix,Fritilariae Thunbergia Bulbus,Trichosanthis Pericarpium,Poria,Glycyrrhiza Radix et Rhizoma,Membrana Follicularis Ovi,and Chebulae Fructus,crushed into a fine powder with condensed honey.The current 2020 edition of the Chinese Pharmacopoeia only has microscopic identification of seven species for Tiedi Wan,thin-layer chromatography with gallic acid and bezoar A as controls,and HPLC for the determination of glycyrrhetinic acid content.The traditional methods do not correspond to the species and the components,and only some of the species can be identified,not all of the prescription components.Molecular identification techniques have been used in the identification of Chinese herbal medicines,and DNA barcoding is highly accurate,regardless of the morphological structure and chemical composition of the herbs.DNA metabarcoding and shotgun metabarcoding techniques based on DNA barcode identification are available for the detection of biological components of Chinese patent medicines.As research entered the genomic era,“super barcode” was introduced for species identification studies,enabling the differentiation of hard-to-identify populations and more accurate identification down to the subspecies level of species.Therefore,this thesis takes the Chinese patent medicine Tiedi Wan as an example,establishes the chloroplast genome and mitochondrial genome databases of10 raw herbs and their close relatives,removes low-quality data by shotgun sequencing,mapping reads to the chloroplast and mitochondrial genome databases,and uses python scripts for species composition analysis,in an attempt to construct a biological composition identification method for the Chinese patent medicine Tiedi Wan based on the full length of the chloroplast and mitochondrial genomes,providing a new method for the identification of Chinese patent medicines and a new means of regulation for the safe circulation of Chinese patent medicines in the market.ObjectiveIn this study,the Tiedi Wan,which contains 10 types of botanical,animal and fungal herbs,was used as an example to establish an analytical procedure for the identification of proprietary Chinese medicines based on the full length of chloroplasts and mitochondria,and to systematically and comprehensively identify the Tiedi Wan’s basal species in conjunction with traditional identification methods,thus providing a more comprehensive method for the identification of Chinese patent medicines.Methods1.Each herbal drink and basal species of Ophiopogonis Radix,Platycodonis Radix,Canarii Fructus,Scrophulariae Radix,Fritilariae Thunbergia Bulbus,Trichosanthis Pericarpium,Poria,Glycyrrhiza Radix et Rhizoma,Membrana Follicularis Ovi,and Chebulae Fructus were collected from pharmacies for morphological identification and DNA barcoding to determine their authenticity according to the Flora of China and the Chinese Pharmacopoeia.Morphological identification was carried out according to the Chinese Pharmacopoeia,after which the DNA of each raw material was extracted.The four DNA barcodes of ITS2,psb A-trn H,mat K and rbc L were used for botanicals,and the COI barcode was amplified and sequenced for animal drugs,and the primers were removed and compared with the Gen Bank database of NCBI to identify the accuracy of the raw materials and control herbs.2.Two mock samples were prepared in the laboratory according to the Tiedi Wan formula in the 2020 edition of the Chinese Pharmacopoeia and one of them was added with the same amount of American ginseng powder as the minimum dosage for a positive control.Three samples of different batches of commercial Tiedi Wan were collected from pharmacies and tested using traditional identification methods.Seven microscopic features of the commercial Tiedi Wan powder were observed under a biological microscope.Seven microscopic features of the commercial Tiedi Wan powder were observed under a biological microscope.Thin layer chromatography(TLC)was used to observe the spot positions and colours of the commercial samples and the control by chromatography using gallic acid and bezoar A as controls.High performance liquid chromatography(HPLC)was used to determine the content of glycyrrhetinic acid in commercial sample solutions and glycyrrhetinic acid control solutions under the same liquid phase system.3.In this study,DNA of acceptable quality was extracted using the extraction steps in the DNA extraction kit method,and the DNA of these five samples was sequenced in high throughput using the Illumina Nova Seq platform.Four types of DNA barcodes were generated from the high-throughput dataset using the Shotgun metabarcoding method.The Taxa of each barcode is assigned to NCBI’s Gen Bank library by blast for accurate identification of authentic species in Chinese patent medicines.Finally,a statistical and taxonomic visualisation of the species composition of Chinese patent medicines.4.Integration of existing databases of chloroplast and mitochondrial genomes of the constituent species of the Iron Flute Pill formula through public databases.A mapping-based strategy was used to analyse high-throughput data from the chloroplast and mitochondrial genomes to identify the biological components of the iron flute pills.Sequences from the database were used as the reference genome for mapping using bowtie2v2.3.4.3,and sequencing depth and coverage were calculated using samtools v1.10.The reads mapped to the genome were extracted using seqtk v1.3 and statistics were calculated.Backtracking of mapped reads to genes and gene spacer regions by python script.Screening of genes and gene spacer regions with high confidence for species composition analysis.Data from each sample was visualised and analysed by Megan v6.21 to compare the differences between samples.Results1.DNA barcoding results showed that ITS2,psb A-trn H,mat K,rbc L and COI sequences were successfully sequenced with 7,8,4,6 and 1 sequences,respectively.The mat K sequences of Canarii Fructus,Scrophulariae Radix,Glycyrrhiza Radix et Rhizoma and the rbc L sequence of Scrophulariae Radix were not successfully sequenced.Species for which sequences were not obtained downloaded the corresponding sequences in the Genbank database for use as reference sequences.Microscopic features of seven species,including Poria,Fritilariae Thunbergia Bulbus,Ophiopogonis Radix,Scrophulariae Radix,Chebulae Fructus,Platycodonis Radix and Glycyrrhiza Radix et Rhizom were observed by analysis of sample powders.The points in the chromatograms obtained for the test solutions of the three commercial samples of Tiedi Wan correspond to the position and colour of the spots obtained for the control solution.The three commercial samples were detected by HPLC at 3.06 mg/g,3.09 mg/g and 3.05 mg/g of the active ingredient glycyrrhetinic acid,respectively.2.An average of 6.8 Gb of shotgun reads were sequenced in the genomic DNA of mock and commercial samples.97,11,10,14 and 1operational taxonomic unit(OTUs)were generated for ITS2,psb A-trn H,rbc L,mat K and COI,respectively.All marker components,including eight plants,one fungus and one animal,were successfully detected in both mock and commercial samples,with Chebulae Fructus,poria and Fritilariae Thunbergia Bulbus identified by sequencing of the plastid genome.In addition,four unlabelled plants were detected in the drug samples and 30 fungi,such as Schwanniomyces,Diaporthe and Fusarium,were detected in the mock and commercial samples.3.The chloroplast genomes of the constituent species of Tiedi Wan ranged from 127,952 bp to 171,818 bp in length,with a difference of 43,866 bp in length between species.The chloroplast genome and mitochondrial genome sequences were mapped to an average of 129,466.8 reads in all samples,with a total of647,334 reads mapped.The coverage of the two mock samples was above95% for all species except Poria and Chebulae Fructus,which had less than90% coverage,and Phoenix coat,which had 100% coverage.The highest number of reads was obtained in the commercial samples HSZY056,with most species having coverage above 95% and Phoenix coat reaching 100%coverage in all three commercial samples.Among the mapping distribution results for all plant species,four regions obtained high numbers of reads,namely the psb K-rps2,rpo C2-psa B,ycf2-ndh F,and ycf1-ycf2 regions.All the ingredients of Tiedi Pills were successfully detected,with Poria being better identified in the commercial samples.ConclusionThe traditional identification methods of microscopic identification,TLC and HPLC of commercial Chinese patent medicines indicated that the quality of commercial samples Tiedi Wan met the requirements of the Chinese Pharmacopoeia.The microscopic identification,however,only examined the microscopic characteristics of seven species and could not form a specific test.The thin-layer chromatography and high performance liquid chromatography detection of one or two of its chemical components did not correspond to one another,and there were problems of incomplete detection.The Shotgun metabarcoding method is a useful addition to the traditional methods for identifying plant,animal and fungal components of herbal products in both homemade and commercial samples of Tiedi Wan.However,in some samples not all species were obtained and for difficult to detect species could not be successfully detected.The chloroplast genome and mitochondrial genome established in this thesis as a super barcode analysis method can identify all the biological components of Tiedi Wan.This identification method can be used as a powerful tool for the simultaneous identification of plant,fungal and animal components in Tiedi Wan,which can be combined with traditional methods to ensure the quality of Tiedi Wan.