Molecular Identification Of DNA Barcode In Chinese Pharmacopoeia

In the Pharmacopioea of the People’s Republic of China (Chinese Pharmacopoeia for short)2010edition, herbs and decoction pieces identification methords mainly contains morphological identification, microscopic identification, and physicochemical identification. DNA barcoding has been developing for more than ten years. Nowadays, this technique has been accepted by the public and applied to different fields for its application value. Based on the study on comprehensive samples, the DNA barcoding systems have been established to identify Chinese material medica (CMM), i.e. ITS2as a core barcode and psbA-trnH as a complementary locus for identification of Planta Medica, and COI as a core barcode and ITS2as a complementary locus for identification of Animal Medica. Currently, the Guiding Principle of DNA Barcoding Molecular Identification of Chinese Material Medica has been adopted by the Pharmacopoeia of the People’s Republic of China (2010, enlarged text). This study identified the CMM recorded in the Chinese Pharmacopoeia according to the guiding principle for the first time. In this study, Notopterygii Rhizoma et Radix, Lycii Fructus and Rhodiolae Crenulatae Radix et Rhizoma were involved to discuss the standard operation procedure and actual application value of this approach. Based on the results of experimental research, the main results are as follows:(1)65samples including Notopterygii Rhizoma et Radix and its adulterants were used to get the genomic DNA and the ITS/ITS2sequences according to the standard protocol of DNA barcoding molecular identification method. Results indicated that the lengths of ITS regions were603-604bp, while the lengths of ITS2regions were228bp. The haplotypes of ITS/ITS2regions of Notopterygii Rhizoma et Radix were the same as those of the original plant leaves. The intra-specific genetic distances were smaller than inter-specific ones in ITS/ITS2regions of Notopterygium incisum and N. franchetii. The NJ trees showed that N. incisum, N. franchetii and its adulterants can be easily differentiated according to their monophyly. Therefore, ITS/ITS2regions as DNA barcodes can stably and accurately distinguish Notopterygii Rhizoma et Radix from its adulterants.(2) We used ITS2sequence as a DNA barcode for distinguishing L. barbarum, L. chinense, and L. ruthencium. The genomic DNA from77samples collected from the field and market was extracted. Ideal ITS2barcodes were obtained by PCR amplification and sequencing. The results suggested that the length of the L. barbarum, L. chinense and L. ruthenicum ITS2sequences was230,212-229, and229bp before alignment, respectively. Based on the ITS2barcode, the neighbor-joining (NJ) tree, BLAST1, and nearest distance methods were able to differentiate the three above-mentioned species. Therefore, we present the ITS2barcode as a powerful tool for the authentication of Lycii Fructus.(3) Commonly known as Hongjingtian, Rhodiola crenulata is called "Plateau Ginseng" by the Orient. It is the only species for Rhodiolae Crenulatae Radix et Rhizoma accepted by the Chinese Pharmacopoeia.82samples from10Rhodiola species were used to construct the DNA barcode database of Rhodiola crenulata and its closely related species. We used this database to identify100commercial samples purchased in drug stores and hospitals from31provinces in China. About90%of the samples could get ideal ITS2barcodes by PCR amplification and sequencing. The result showed that40%test samples’best hit was R. crenulata. Another38.9%test samples’best hit was R. serrata. The rest samples matched other Rhodiola species. This indicated that the decoction pieces of Rhodiolae Crenulatae Radix et Rhizoma on the market were complex sources. Therefore, we present the ITS2barcode as a powerful tool for the market supervision of "Plateau Ginseng".As a conclusion, DNA barcoding molecular identification of traditional Chinese material medica can be used to authenticate Notopterygii Rhizoma et Radix, Lycii Fructus, Rhodiolae Crenulatae Radix et Rhizoma and their adulterants, respectively. This study provides an important evidence for other Chinese material medica in the Pharmacopioea of the People’s Republic of China. DNA barcoding is the powerful tool for the TCM market supervision and the ensurance of the clinical safety in utilization of traditional Chinese medicines.