Study On Identification Method Of Chinese Herbal Medicine Based On DSS Molecular Marker

Objective:1.The aim of the study is to develop DSS markers that can accurately identify 20 Chinese herbal medicines,and to provide a new molecular identification method for the accurate identification of Chinese herbal medicines and their adulterants.2.The purpose of this study is to establish a PCR-RFLP method for the identification of Atractylodis Macrocephalae Rhizoma,so as to provide a new method for the molecular authentication of Atractylodis Macrocephalae Rhizoma and A.lancea,A.chinensis,A.japonica and A.coreana.3.To establish the PCR-RFLP method for the molecular identification of Periplocae Cortex,in order to provide the reference for distinguishing Periplocae Cortex and its adulterants,Acanthopanacis Cortex and Lycii Cortex.4.To establish a specific PCR method for accurate and simple identification of Hibisci Syriaci Cortex is the purpose,so as to provide a reference for solving the confusion of the name of uncommon medicinal herbs and the difficult identification among related species.Methods:1.In this study,12 Chinese herbal medicines in Anhui Province Processing Procedures of Prepared Slices of Chinese Crude Drugs and 8 toxic medicinal herbs in the Chinese Pharmacopoeia(2020 edition)were selected as the research objects.DNA of 20 Chinese herbal medicines and their adulterants were extracted.Candidate DSS markers of 20 Chinese herbal medicines were screened based on the(CGIR).Specific primers were designed according to the position of DSS markers in the genome,and PCR amplification and sequencing were performed.The sequenced sequences were compared with the corresponding candidate DSS markers,and DSS markers of 20 Chinese herbal medicines were verified.2.Based on the DSS markers of Atractylodes macrocephala obtained by our research group in the previous stage,the DSS of Atractylodes macrocephala was analyzed to screen the different enzyme restriction sites of Atractylodes macrocephala and A.lancea,A.chinensis,A.japonica and A.coreana.PCR amplification primers were designed according to the two ends of the different enzyme restriction sites and PCRRFLP analysis was performed.The identification conditions and digestion time of PCR-RFLP reaction were optimized.3.Based on the DSS markers of Periplocae Cortex verified,the DSS of Periplocae Cortex was analyzed to find the different enzyme restriction sites of Acanthopanar gracilistμlus,Acanthopanax gracilistylus,Lycium chinense and L.barbarum.PCR amplification primers were designed according to the two ends of the different enzyme restriction sites and PCR-RFLP analysis was performed.And the identification conditions and digestion conditions of PCR-RFLP reaction were optimized.4.Based on the DSS markers of Hibiscus syriacus verified in Chapter 1,and SNP sites between Hibiscus syriacus and four related adulterants were screened,and specific identification primers of Hibisci Syriaci Cortex were designed.The PCR method was established to identify Hibisci Syriaci Cortex.The PCR reaction conditions were optimized,and the tolerance was explored.The applicability of different sources of Hibisci Syriaci Cortex and its related adulterants was investigated.Results: 1.One DSS marker that could be used for identification of Chinese herbal medicines and their adulterants,such as Moutan Cortex,Ilicis Asprellae Radix,Hibisci Syriaci Cortex,Notoginseng Flos,Sophorae Tonkinensis et Rhizoma,Periplocae Cortex,Anemones Raddeanae Rhizoma,Carotae Fructus,Menispermi Rhizoma.One DSS marker that could be used for identification of Chinese herbal medicines and related species,such as Penthori Herba,Zanthoxyli Semen.Two DSS markers that could be used for identification of Chinese herbal medicine and related species,such as Macleayae Cordatae Herba.One DSS marker that could be used for identification of Chinese herbal medicines and counterfeits,such as Sinopodophyllum hexandrum.Two DSS markers that could be used for identification of Chinese herbal medicines and adulterants were obtained for Talini Paniculati Radix,Moschatae Calyx,Campsis Grandiflorae Radix.Two DSS marker that could be used for identification of Chinese herbal medicines and counterfeits,such as Dichroae Radix,Tribuli Fructus.Three DSS markers that could be used for identification of Chinese herbal medicines and adulterants were obtained for Forsythiae Semen.2.A PCR-RFLP identification method for Atractylodis Macrocephalae Rhizoma was established using DSS marker of A.macrocephala.When the annealing temperature was 62℃ and the cycle number was 30,PCR amplification was performed on A.macrocephala,A.lancea,A.chinensis,A.japonica and A.coreana.with the discriminant primes of A.macrocephala and the target bands of 245 bp were generated.The PCR products were digested by Stu I restriction endonuclease,and only the samples of A.macrocephala could be digested into two fragments.3.A PCR-RFLP identification method was established for Periplocae Cortex using DSS marker of Periplocae Cortex.When the annealing temperature was 59 ℃ and the number of cycles was 40,PCR amplification was performed on the original plants of Periplocae Cortex,Acanthopanacis Cortex and Lycii Cortex with the identification primer of Periplocae Cortex,and target bands of about 430 bp were generated.When all PCR products were digested by Cla I restriction endonuclease,only the samples of Periplocae Cortex could be digested into two fragments.4.A specific PCR identification method was established for Hibisci Syriaci Cortex.When the annealing temperature was 61℃ and the number of cycles was 33,identification primers of Hibisci Syriaci Cortex were used for PCR amplification of Hibisci Syriaci Cortex and its four related adulterants.The gel electrophoresis results showed that all Hibisci Syriaci Cortex samples had about 270 bp target bands,while all related adulterants had no corresponding band in the corresponding position of the electrophoretic map.Conclusion: 1.DSS markers of 20 Chinese herbal medicines based on CGIR can be used for authenticity identification.2.The PCR-RFLP method established based on DSS markers can be used for the identification of Atractylodes macrocephala and A.lancea,A.chinensis,A.japonica and A.coreana.3.The PCR-RFLP method based on DSS markers can be used for the identification of Periplocae Cortex and its adulterants,Acanthopanacis Cortex and Lycii Cortex.4.AS-PCR method established based on DSS markers can quickly and accurately identify Hibisci Syriaci Cortex and related adulterants.