Identification Of Pericarpium Citri Reticulatae 'Chachi'?Pericarpium Citri Reticulatae And Peri Carpium Citri Reticulatae Viride Based On DNA Barcode And Metabolomics Analysis

ObjectiveCitrus is a kind of traditional Chinese medicine with many varieties which easy to confuse clinical medicines,difficult to distinguish between genuine and authentic medicines,and difficult to guarantee the quality of genuine medicines,which seriously affects the effect of traditional Chinese medicine and is not conducive to improving the quality of medicines.In this experiment,Pericarpium Citri Reticulatae chachiensis(“Guangchenpi”in Chinese,GCP),Pericarpium Ci tri Reticulatae(“Chenpi”in Chinese,CP)and Pericarpium Citri Reticulatae viride("Qingpi " in Chinese,QP)were taken as the research objects.The“bi-molecular”identification technology based on molecular biology identification of DNA barcode technology and Chinese medicine secondary metabolite detection technology is used to distinguish between qualitative and quantitative analysis of DNA genetic information.At the molecular level,we study the types,differences and quality differences of traditional Chinese medicines,in order to establish an integrated method for quickly distinguishing between Pericarpium Citri Reticulatae chachiensis,Pericarpium Citri Reticulatae and Pericarpium Citri Reticulatae viride.Methods1.Analysis of genetic information differences between GCP,CP and QP by DNA barcoding:A total of 81 samples of the provenance of GCP,CP and QP were used as experimental materials.The total DNA of the sample was extracted by the plant DNA extraction kit method and the kit improvement method.By comparing the five commonly used DNA barcode candidate fragments:ITS2,ITS,psbA-trnH,rbcL and matK sequences,for PCR amplification efficiency,interspecific variation within the candidate sequence,sequence feature structure,BLAST similarity search algorithm,phylogenetic tree,etc.for the identification of GCP and CP,QP.2.Untargeted Metabolomics analysis by using UPLC-QTOF-MS/MS to study the chemical composition analysis between GCP and CP.The secondary metabolite detection technology of traditional Chinese medicine was used to carry out the quality identification of the GCP and CP.The ultra-high performance liquid chromatography and multi-stage mass spectrometry techniques were used to obtain the secondary metabolite information of GCP and CP.Analytical method was established using UPLC-QTOF-MS/MS technology:The chromatographic separation was performed on a Waters ACQUITYTM UPLCTM HSS T3 column(100×2.1 mm,1.7 ?m)at 30?.The mobile phase consisted of solvent A(water containing 0.1%formic acid)and solvent B(Acetonitrile),and the elution program was optimized as follows:0-3.5 min,5-15%B;3.5-6 min,15-25%B;6-15 min,25-40%B;15-18 min,40-55%B;18-20 min,55-85%B;20-22 min,85-95%B.The flow rate was set to 0.2 mL·min-1,with an injection volume set to 2 ?L.The MS analysis was performed on a TripleTOFTM 5600+ mass spectrometer equipped with a DuoSprayTM Electron Spray Ionization(ESI)source in both positive and negative modes.the ESI condition was applied with the following parameters:ion spray voltage,4500 V in positive mode(-4500 V in negative mode);ion source temperature,500 0 C;curtain gas,25 psi;nebulizer gas(GS1)50 psi;heater gas(GS2),50 psi;and declustering potential(DP),80 V(-80 V in negative mode).The mass range was set between m/z 120-1200 in both positive and negative modes.The MS scanning program consisted of a TOF MS survey scan and MS/MS scans of eight most intense ions using the information-dependent acquisition(IDA)mode,that was set for structural analysis of potential biomarkers.The IDA collision energy(CE)was set at 35 eV in positive mode(-35 eV in negative mode),and the collision energy spread(CES)was(±)10 eV.By collecting and arranging the mass spectrometry cleavage of flavonoids from the GCP,CP and QP,and combining with the general mass spectrometry rules,the chemical components of GCP,CP and QP were analyzed on-line to obtain secondary metabolites information,multivariate statistical methods,principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA)were used to analyze the LC-MS data of samples to find the markers that can effectively distinguish the GCP from the CP.3.Chemical discrimination of CP and QP by using UPLC-Q-Exactive Orbi trap MS/MS based untargeted metabolomics approaches.The analytical method was established:The chromatographic separation was carried out on a Waters ACQUITYTTM UPLCTM HSS T3 column(100 X 2.1 mm,1.7 ?m);The mobile phase consisted of phase A is 0.1%formic acid and phase B is acetonitrile.The gradient program started as follows:0-3 min,10-15%B;3-15 min,15-40%B;15-20 min,40-60%B:20-24 min,60-85%B;24-27 min,85-90%B;27-30 min,90%B;The flow rate was 0.2 mL·min-1 and the column temperature was set at 30?;the injection volume is 2?L.The analytes was performed by mass spectrometry with Turbo Ionspray ionization(ESI);the distribution adopts two modes of positive and negative ions,The mass range was set between m/z 120-1200 in both positive and negative modes.The main ion source parameter of mass spectrum were described as follows:sheath gas 40;auxiliary gas 15;Sweep gas 0;Spray voltage positive ion 3.5 kV,negative ion 2.5kV;Capillary temp 350?;Aux gas heater temp 350?;lens(S-Lens RF level):50.Combining with the general mass spectrometry rules,the chemical components of CP and QP were analyzed on-line to obtain secondary metabolites information,multivariate statistical methods,principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA)were used to analyze the LC-MS data of samples to find the markers that can effectively distinguish the GCP from the CP from the QP.Results1.This experiment successfully extracted and amplified sequencing samples of five kinds of stripe candidate sequences.Through screening and analysis of five sequence bands,ITS2 sequence had the best identification effect.The difference of Neighbor-Joining tree and the secondary structure characteristics based on ITS2 sequence showed that there were some differences between Xinhui Guangchenpi(Citrus reticulata 'Chachi')and other three sources of Chenpi.The success rate of ITS sequence sequencing was low,and NJ phylogenetic tree constructed based on ITS sequence can not distinguish between Guangchenpi,Chenpi and Qingpi.The chloroplast genome sequence(psbA-trnH,rbcL,matK)was conservative,while the sequence information of Guangchenpi and other three Chenpi was similar.The samples of Chenpi and Qingpi belong to the same source and different harvesting periods.The DNA barcode sequences of different developmental stages of the same species are same.Therefore,the five DNA sequence fragments of Chenpi and Qingpi from the same source are identical.DNA barcode is not suitable for identifying and distinguishing them.2.Untargeted Metabolomics analysis based on UPLC-QTOF-MS/MS analysis technique by Using principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA)of multivariate statistical methods,OPLS-DA model can effectively distinguish the samples of Guangchenpi and Chenpi.Through the S-plot of OPLS-DA model and the variable weight VIP values were screened and analyzed and the compounds with statistical differences were determined by Mann-Whitney U test.Then,according to the accurate molecular mass combined with the secondary mass spectrometry fragments and related literature research reports,the sample compound components were identified.Finally,34 kinds of compounds(VIP>1.5,P<0.05)based on polymethoxylated flavonoids were screened from GCP and CP samples in the positive and negative ion mode as the marker components in the samples of GCP and CP.And 26 different components were screened in the positive ion mode,17 chemical components were detected in the negative ion mode,and 9 components were detected in both positive and negative ions.3.UPLC-Q-Exactive Orbitrap MS/MS analysis technology was used to identify 79 chemical components in CP and QP both in positive and negative ion mode,including 74 flavonoids,3 kinds of limonoids,2 kinds of alkaloids.Then,the OPLS-DA model was established by untargeted metabolomics analysis to discriminate between CP and QP.A total of 27 kinds of compounds(VIP>1.5,P<0.05)mainly composed of polymethoxyflavonoids and limonoids were screened as the marker components,including 19 compounds were screened in positive ion mode and 15 compounds in negative ion mode,and 6 components were detected in both positive and negative ions.ConclusionIn this experiment,the"bimolecular" identification technology based on molecular biology identification DNA barcode technology and metabonomics analysis technology was used to study the samples from Xinhui Guangchenpi(Citrus reticulata'Chachi,GCP)and three other varieties.Five candidate DNA barcode sequence fragments were used to identify GCP and three other varieties('Dahongpao','Unshiu','Tangerina').The identification effect of Citrus reticulata varieties is not good,and it is difficult to distinguish them accurately by DNA barcode.The sequence of ITS and ITS2 of nuclear gene was quite different among the four cultivars.The sequence of chloroplast gene fragment(psbA-trnH,rbcL,matK)was relatively conservative.Combined with the success rate of sequence amplification and sequencing and the difference of sequence structure,ITS2 sequence is the best one for single sequence identification.Its sequence secondary structure and NJ tree can distinguish GCP(Citrus reticulata 'Chachi')from two kinds of CP(Citrus reticulata'Unshiu',Citrus reticulata'Tangerina'),but it is not enough to accurately identify GCP and CP which from Citrus reticulata'Dahongpao'.ITS2 sequence combined with its secondary structure characteristics can be used as the identification of GCP as a reference,but it is not enough to achieve the purpose of accurate identification of GCP and three other sources.The UPLC-QTOF-MS/MS sample analysis method based on metabonomics was stable and could be used for qualitative analysis of experimental samples accurately.The OPLS-DA model was constructed to screen and identify the differentiated components.34 components were identified GCP and three other sources.And a method for identifying GCP and three other sources was established.Because CP and QP belong to different harvesting periods of homologous medicinal materials,the DNA sequences of the same species at different development stages or different growth stages are identical,the identification of CP and QP by DNA barcode failed.The UPLC-Q-Exactive Orbitrap MS/MS method can be used for qualitative analysis of the chemical constituents of CP and QP samples.Based on metabonomics analysis,27 differential compounds were screened by OPLS-DA model of multivariate statistics,and an analytical method based on chemical constituent difference analysis was established to identify CP and QP.Through exploratory research on identification methods based on DNA barcode and metabonomics technology,this experiment can provide experience and reference for combination identification technology based on DNA barcode and metabonomics technology for identification of traditional Chinese medicines.