The Species Identification Of Yangyin Qingfei Wan,Based On Shotgun Metabarcoding

The composition of traditional patent medicine is complex,and the identification of its species composition is a big difficulty in the identification of Chinese medicine.The safety,effectiveness and development of traditional patent medicine in the international market are seriously affected by the error or false feeding of medicinal materials of traditional patent medicine.The quality control model of traditional identification methods based on single or several components has the problem of poor specificity,which is difficult to reflect the overall quality of traditional patent medicine.More effective identification methods are still needed to solve the problem of species identification of traditional patent medicine.Yangyin Qingfei Wan(YYQFW)contain eight medicinal materials of Fritillariae Cirrhosae Bulbus(Chuanbeimu),Paeoniae Radix Alba(Baishao),Scrophulariae Radix(Xuanshen),Ophiopogonis Radix(Maidong),Moutan Cortex(Mudanpi),Rehmanniae Radix(Dihuang),Menthae Haplocalycis Herba(Bohe)and Glycyrrhizae Radix et Rhizoma(Gancao),too many medicinal materials bring difficulty in identification.In addition,YYQFW contains starch and honey,which increase the difficulty of DNA extraction.Moreover,DNA degradation problems exist in the process of drug,which brings difficulties to molecular identification technology.Therefore,on the basis of completing the traditional identification methods of YYQFW stipulated in the 2020 edition of Chinese Pharmacopoeia,this paper optimized the DNA extraction process of YYQFW by magnetic bead method.A shotgun metabarcoding method with high throughput sequencing combined with four barcodes(ITS2,psb A-trn H,mat K and rbc L)was established,which provided a new method for species identification of YYQFW.Objective:This paper selected YYQFW as the research object,optimized the DNA extraction process of traditional patent medicine based on magnetic bead method,combined with traditional identification and shotgun metabarcoding method for species identification of YYQFW,aiming to establish a more comprehensive and accurate identification system for traditional patent medicine.Methods:1.The traditional identification methods stipulated in the 2020 edition of Chinese Pharmacopoeia,namely microscopic detection,thin layer chromatography(TLC)and high performance liquid chromatography(HPLC),were used to identify commercially available samples of YYQFW.The internal tissue structure and cell morphology of YYQFW were observed under biological microscope.paeonol,paeoniflorin and Scrophulariae Radix were used as chemical controls in TLC.Paeonol was used as developing agent with cyclohexan-ethyl acetate(13:2),and Paeonol and Radix Scrophularia were used as developing agent with ethyl acetate-butyl ketone-formic acid-water(5:3:1:1).Reference substance and test solution in HPLC were prepared according to Chinese Pharmacopoeia,and the reference solution was injected for five consecutive times to check whether it met the requirements of system suitability.The control solution and the test solution were each injected 10 μL into the liquid chromatograph for determination,and the average content of paeonol was calculated twice,respectively.2.Eight medicinal materials of YYQFW were collected by referring to the 2020 edition of Chinese Pharmacopoeia,and another Panacis Quinquefolii Radix(Xiyangshen)was collected as a positive control.Firstly,morphological and character identification were performed on all medicinal materials,and then the reference sequences of ITS2,psb A-trn H,mat K and rbc L fragments of medicinal materials were obtained by DNA barcoding technology and compared by BLAST.The reference sequence and the downloaded sequence in Genbank were used to construct phylogenetic tree to further ensure the accuracy of the medicinal materials.3.Optimizing the DNA extraction process of YYQFW based on magnetic bead method through the conditions of pre-treatment stage,cleavage stage and precipitation stage.The washing effects of DD water and nuclear separation solution were investigated in the pre-treatment stage.In the cracking stage,the cracking effect of(CTAB,SDS and CTAB-SDS)and the influence of water bath conditions on DNA concentration were investigated respectively.Precipitation phase of methanol,ethanol,isopropyl alcohol,methanol-sodium acetate,ethanol-sodium acetate,isopropyl alcohol-sodium acetate and the precipitation effect of 10,20 and 30 μL magnetic beads on DNA purity were investigated.The DNA extraction process optimized based on magnetic beads was compared with the plant kit,so as to determine the best DNA extraction scheme of YYQFW.4.HSZY166 and HSZY178 mock samples of YYQFW were prepared according to the 2020 version of pharmacopoeia.Panacis Quinquefolii Radix was added into HSZY178 with the minimum dosage equal to that in YYQFW.Four commercially pharmaceutical samples were collected and numbered as A17,HSZY007,HSZY141 and HSZY142,respectively.Based on the optimized DNA extraction process of magnetic bead method,two mock samples and four commercially pharmaceutical samples were extracted and shotgun sequencing was conducted.After data quality control,enrichment,assembly,annotation,mapping and other steps,the selected and reliable OTUs were compared by BLAST.The representative OTUs were selected to make phylogenetic tree,and the species composition of traditional patent medicine was analyzed by annotation and visualization.In order to verify the accuracy of shotgun metabarcoding method,the assembly sequences obtained from mock samples were compared with the reference sequences.The sensitivity of the technology was verified by testing Panacis Quinquefolii Radix added to HSZY178.Shotgun metabarcoding method was used to test prescription ingredients in commercially pharmaceutical to verify the applicability of this technique.Thus,a species identification method of traditional patent medicine YYQFW based on shotgun metabarcoding technique was established.Results:1.Microscopic examination showed that the samples of commercially pharmaceutical showed black-brown cells(Rehmanniae Radix),calcium oxalate aciculate crystals gathered in bundles(Ophiopogonis Radix),the parenchyma cells around the fiber bundles contained calcium oxalate square crystals(Glycyrrhizae Radix et Rhizoma),brown stone cells showed rectangular shape(Scrophulariae Radix)and flat spherical gland scales(Menthae Haplocalycis Herba).In accordance with the microscopic characteristics of YYQFW in the 2020 edition of Chinese Pharmacopoeia.TLC results showed that the spots of paeonol,Scrophulariae Radix and paeoniflorin could be detected in four commercially pharmaceutical samples.HPLC results showed that the paeonol content of A17,HSZY007,HSZY141 and HSZY142 in each pill of YYQFW were 8.2 mg,9.1 mg,6.7 mg and 6.2mg,respectively,which were all higher than the 5.8 mg prescribed in pharmacopoeia and in line with the formulation standard.2.All medicinal materials and positive control materials were confirmed to be authentic by morphological and character identification.In addition,reference sequences of all medicinal materials could be obtained successfully by combining ITS2,psb A-trn H,mat K and rbc L fragments of DNA barcodes.The results of BLAST comparison and phylogenetic tree analysis further ensured the accuracy of the primitive of medicinal materials.3.Finally,the optimized DNA extraction process of YYQFW based on magnetic bead method was determined as follows: DD water was the best washing method in the pre-treatment stage,CTAB cracking solution was used in water bath at 65 ℃ for two hours in the cracking stage,isopropanol-sodium acetate was used in the precipitation stage to precipitate DNA better than other precipitation methods,and the effect was better when the amount of magnetic beads was controlled at 10-20 μL.Compared with the plant kit,it was found that the DNA concentration and purity of YYQFW obtained by magnetic bead method were significantly higher than those obtained by plant kit.High quality DNA can satisfy the subsequent shotgun sequencing,which is helpful to improve the accuracy of the quality evaluation technology of traditional patent medicine.4.The DNA of commercially pharmaceutical and mock samples of YYQFW was extracted by magnetic bead optimization method.Illumina sequencing platform was used for shotgun sequencing.127,20,8 and 18 OTUs of ITS2,psb A-trn H,mat K and rbc L could be obtained after assembly.In the mock sample HSZY178,all prescription component sequences were obtained from the ITS2 fragment.A total of 31 OTUs fungi were obtained based on ITS2 fragment,belonging to 12 genera,mainly Aspergillus,Fusarium and Cladosporium.In addition,OTUs for non-prescription ingredient such as Acalypha australis(Tiexiancai)and Setaria viridis(Gouweicao)and so on.Therefore,shotgun metabarcoding can be used as a new molecular detection method to supplement the traditional identification methods,so as to establish a more comprehensive species identification system.Conclusions:In traditional identification methods,microscopic identification can only detect the microscopic characteristics of five kinds of medicinal materials,and there may be other species with the same characteristics of microscopic structure,which cannot ensure the real existence of prescription ingredients.TLC and HPLC could only be used for qualitative and quantitative studies on paeonol,paeoniflorin and Scrophulariae Radix in YYQFW,but could not reach the identification of all medicinal materials.However,shotgun metabarcoding can not only identify all medicinal materials,but also detect non-prescription components such as impurity species and fungi in traditional patent medicines.Therefore,combining shotgun metabarcoding with traditional identification methods,a more comprehensive identification system of traditional patent medicine can be established.