Research On The Difference Of Four Kinds Of Atractylodes And Atractylodes

Purpose:The ITS2 sequence was used to identify A.lancea.,A.Chine sis.,A.japonica.,A.coreana.and A.macrocephalae.,and to determine the genetic relationship between them.The crude polysaccharide content and fine polysaccharide content of the four species of Atractylodis rhizoma and Atractylodis macrocephalae rhizoma were determined by water extraction and alcohol precipitation and HPLC.The monosaccharide composition of polysaccharides and the monosaccharide composition of medicinal materials were studied.The chemical composition of four species of Atractylodis rhizoma and Atractylodis macrocephalae rhizoma was studied by TLC chromatography.GC-MS was used to analyze the similarities and differences of the volatile oil components of the four species of Atractylodis rhizoma and Atractylodis macrocephalae rhizoma.Using ACQUITY UPLCTM-Q-TOF system analyzes the chemical similarities and differences of the four species of Atractylodis rhizoma and Atractylodis macrocephalae rhizoma.Through the above studies,the similarities between the four species of Atractylodis rhizoma and Atractylodis macrocephalae rhizoma are explored from the perspective of genetic relationship and chemical composition,providing a reference for clinical use.Material and method:Materials:A.lancea.was picked from Hubei Yingshan,A.Chinesis.and A.japonica.were picked from Liaoning Kuandian.A.coreana.was picked from Liaoning Qingyuan.A.macrocephalae.was picked from Zhejiang Xinchang.The above plants are cultivated by local farmers.Approved by Professor Li Feng of the Department of Traditional Chinese Medicine Appraisal,Liaoning University of Chinese Medicine,the roots and rhizomes of A.lancea.,A.Chinesis.,A.japonica.,A.coreana.and A.macrocephalae.are identified respectively.Methods1.Extract genomic DNA from 40 samples of A.lancea.,A.Chinesis.,A.japonica.,A coreana.and A.macrocephalae.from different wild cultivars,and 7 wild germplasm samples from A.coreana..Amplify the ITS2 sequence by PCR and perform bidirectional sequencing.All sequences obtained The alignment analysis(clustal W)was performed with the software MEGA 5.0,and the 5.8S and 28S sequences at both ends were removed to obtain the complete ITS2 sequence and a systematic clustering tree(NJ tree)was constructed2.HPLC-ELSD method was used to detect the types of free monosaccharides and the contents of each monosaccharide in the four herbs of Atractylodis rhizoma and Atractylodis macrocephalae rhizoma.Four kinds of Atractylodis rhizoma and Atractylodis macrocephalae rhizoma medicinal materials were processed by water extraction and alcohol precipitation method to obtain crude polysaccharides,and the phenol-sulfuric acid colorimetric method was used,Glc was used as a standard control,and four types were measured using a UV-spectrophotometer external standard one-point method.The content of crude polysaccharides in four herbs of Atractylodis rhizoma and Atractylodis macrocephalae rhizomawas availuble.The crude polysaccharides obtained by water extraction and alcohol precipitation were subjected to protein precipitation treatment by Sevage method to obtain refined polysaccharides,and then hydrolyzed by TFA,and the types and contents of monosaccharides were detected by HPLC-ELSD method3.Four kinds of Atractylodis rhizoma and Atractylodis macrocephalae rhizoma medicinal powder were added with appropriate amount of methanol,and then ultrasonic extraction was performed.The filtrate and methanol solution of atractyloxin and atractone were subjected to GF254 thin-layer plate spot control,and the room temperature was controlled at 25?.Toluene-ethyl acetate(7:2)as developing agent,and the fluorescence was observed at 254 nm.Spray with 10%sulfuric acid ethanol solution,heat at 105? until the spots become clear,and observe the fluorescence under natural light and 365nm,respectively4.The steam oil distillation method was used to extract the four volatile oils of Atractylodis rhizoma and Atractylodis macrocephalae rhizoma.GC-MS was used to analyze the volatile oil components of the four Atractylodis rhizoma and Atractylodis macrocephalae rhizoma.The obtained data was analyzed,and the relative percentage content of each component was calculated using the peak area normalization method5.The ACQUITYUPLC TM-Q-TOF system was used to make qualitative analysis of the components in four species of Atractylodis rhizoma and Atractylodis macrocephalae rhizoma,combined with principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA)to identify the four types of Atractylodis rhizoma and Atractylodis macrocephalae rhizoma in chemical composition.Results:1.The ITS2 sequences of four types of Atractylodis rhizoma and Atractylodis macrocephalae rhizoma are 232 bp in length.According to the results of the secondary structure of NJ tree and ITS2,with the exception of the A.Chinesis.and A.coreana.,the other several medicinal materials can be clearly distinguished,showing a good single lineage.According to the results of the NJ tree,the cultivated lines and wild germplasm of A.coreana.can also be well gathered together2.The free monosaccharides detected in A.lancea.and A.japonica.were Fru and Glc,mainly Fru,with the content of 10.83%and 19.18%.The free monosaccharides in the herbs of A.Chinesis.,A.coreana.andA.macrocephalae.were only detected at 1.58%,1.43%and 8.48%.There was no significant difference in polysaccharide content among the four species of Atractylodis rhizoma and Atractylodis macrocephalae rhizoma.There are differences in the composition of the refined polysaccharides of the four species of Atractylodis rhizoma and Atractylodis macrocephalae rhizoma,and the molar ratios of the individual sugars are:Rha:Fuc:Fru:Glc?1:0.83:3.76:0.83(A.lancea.);Rha:rock Fuc:Fru:Glc?1:0.77:2.46:0.46(A.Chinesis.);Rha:Fuc:Fru:Glc?1:1.05:5.88:0.97(A.japonica.);Rha:Fuc:Fru:Glc=1:0.67:2.76:0.73(A.coreana.);Rha:Fuc:Fru:Glc=1:0.7:3.24:0.49(A.macrocephalae.).3.In visible light,the TLC spots of A.lancea.and A.Chinesis.are similar.The TLC spots of A.japonica.,A.coreana.and A.macrocephalae.are different.With atractyloxin as a reference,A.lancea.,A.Chinesis.and A.coreana.appeared the spot at same space in atractyloxin spot.With atractone as the reference,A.japonica.and A.macrocephalae.appeared the spot at same space in atractone spot.Under 365nm fluorescence,the spots in A.lancea.and A.Chinesis.were consistent basically,while the spots in A.japonica.and A.macrocephalae.were similar.Under 254nm fluorescence,the spots in A.lancea.and A.Chinesis.were similar.4.A total of 74 compounds were identified from the volatile oils of four species of Atractylodis rhizoma and Atractylodis macrocephalae rhizoma,and naphthophenone compounds were identified for the first time from A.Chinesis.and A.coreana.The results of cluster analysis showed that the chemical components of A.lancea.,A.Chinesis.,A.japonica.and A.macrocephalae.were similar,while A.coreana.was clustered into a single group.5.A total of 30 compounds were identified from the four species of Atractylodis rhizoma and Atractylodis macrocephalae rhizoma,including 13 glycoside compounds,3 polyene compounds,12 terpenes compounds,1 benzoquinone compound and 1 phenolic acid compound.It was found that 5 components existed only in A.lancea.,which were ?-xiangdanone and 2-[(2E)-3,5-dimethyl-2,4-hexadienyl]-6-methyl-2.,5-Cyclohexadiene-1,4-benzoquinone,Spartanol,Garlicene,and Velleral.There are two components that are only present in the A.Chinesis.,which are protocatechualdehyde,(1,5E,11E)-ten Tridecatriene-7,9-diyne-3,4-diacetate;1 component is only present in A.japonica.,which is limonoid;2 components are only present in A.macrocephalae.,which are(4S)-p-menth-1-ene-7,8-diol 8-O-?-D-glucopyranoside and atractyloside G.The PCA results show that the four species ofAtractylodis rhizoma and Atractylodis macrocephalae rhizoma have large similarities in main components.The OPLS-DA results show that A.japonica.and A.macrocephalae.can be grouped into one class separately.A.lancea.,A.Chinesis.and A.coreana.still cannot be separated,and the three have certain similaritiesConclusion:By studying the genetic relationship and chemical composition of theFour kinds of Atractylodis rhizoma and Atractylodis macrocephalae rhizoma,we found that A coreana.and A.Chinesis.have a high degree of similarity,especially in terms of chemical composition.A.japonica.is similar to A.lancea.in sugar components,and similar to A.macrocephalae.in thin layer chromatography.The results of chemical composition analysis of temperament and liquid quality are classified separately,so further research is needed.