DNA Barcoding Raw Medicinal Materials Of Chinese Patent Drugs

DNA barcoding is currently used for rapid and accurate identification of traditional Chinese medicine. It is not restricted by morphological characteristics and physiological conditions and allows species authentication without specialist taxonomic knowledge. Therefore it achieves a renaissance in traditional Chinese medicine identification from morphology to DNA. Currently, the Guiding Principle of DNA Barcoding Molecular Identification of Chinese Material Medica has been adopted by the Chinese Pharmacopoeia to help guarantee the security and veracity of Chinese medicine raw material. In this study, DNA barcoding was employed to identify the raw material medicine of Chinese patent drugs of China Resources of Sanjiu Medical & Pharmaceutical Co., Ltd., such as Ilex asprella, Murrayae Folium et Cacumen and ’Xueteng’. The standard operation procedure and application value of DNA Barcoding System were discussed. Based on the experimental data, the DNA barcoding system for identifying raw medicinal materials of Chinese patent drugs of Sanjiu Medical & Pharmaceutical Co., Ltd was established. The main results include:Firstly, ITS2 sequence was used to identify 93 samples of Ilex asprella and its closely related species. The BLAST method, genetic distances and neighbor-joining (NJ) phylogenetic tree were used to assess the sequence identification efficiency. The PCR amplification success rate of the ITS2 region was 100%. The intra-specific genetic distances of I, asprella were ranged from 0 to 0.030 and the minimum inter-specific of I, asprella and its closely related species was 0.043. Furthermore, the samples of I. asprella formed into a single branch with high bootstrap value in NJ tree. The identification efficiency of ITS2 barcode using BLAST was 100%. Therefore, the ITS2 region can accurately and stably distinguish I. asprella from its closely related species, which provides an important contribution to its safe and effective use.Secondly, ITS2 sequence was applied to identify a total of 98 samples of eight Murraya species. The nearest genetic distances, UPGMA phylogenetic tree, BLAST method and SNPs analysis were used to evaluate the identification ability of ITS2 sequence. The results indicated that K2P genetic distance analysis can not effectively distinguish M. exotica, M. paniculata and M. alata. The UPGMA tree showed that different species can be differentiated according to their monophyly and the identification efficiency of ITS2 barcode using BLAST was high. Besides, three stable SNPs in ITS2 were existed between M. exotica and other Murraya species, which can be used to rapidly identify M. exotica. So, ITS2 sequence can be used to distinguish Murrayae Folium et Cacumen from its closely related species.Thirdly, Spatholobi Caulis, Sargentodoxae Caulis and Kadsurae Caulis were recored in Chinese Pharmacopoeia and they all were named as ’Xueteng’. In this research, ITS2 and psbA-trnH were used to identify 110 samples of 8 species of "Xueteng". The sequence similarity was computed using DNAMAN, and the NJ tree was built to evaluate the identification efficiency. The results indicated that the intra-specific sequence similarities of ITS2 and psbA-trnH regions of Spatholobus suberectus, Sargentodoxa cuneata and Kadsura interior were all higher than those of the corresponding inter-specific ones. Furthermore, Spatholobi Caulis, Sargentodoxae Caulis, Kadsurae Caulis formed into a single branch with high bootstrap value in NJ tree respectively and could be distinguished from other species efficiently. Therefore, DNA barcoding molecular identification method is able to accurately identify Spatholobi Caulis, Sargentodoxae Caulis, Kadsurae Caulis and its adulterants and can be used as an effective complement to the traditional identification methods. The two-dimensional DNA barcoding images of the ITS2 sequences will be used to monitor the circulation regulation of Xueteng.Finally, the DNA barcoding system for identifying raw medicinal materials of Sanjiu Medical & Pharmaceutical Co., Ltd was established based on the experimental data. The specially designed database can be applied to identify the commonly used raw medicinal materials of Sanjiu Medical & Pharmaceutical Co., Ltd. It helps to guarantee accurate species identification, and accelerates the progress of standardization in identifying the raw medicinal materials of the company.