Arnebia decumbens (Vent.) Coss. et Kralik, A. euchroma (Royle) Johnst and A. guttata Bunge, three commonly used traditional Chinese medicinal plants have been widely used for the clinical treatment of inflammatory diseases caused by fungal, bacterial, oxidation, and other related pathogens. However, precise identification at the similar species level is usually challenging due to the influence of the source of medicinal materials, traditional ethnic medicine and medicinal habits. Here we developed a comprehensive and efficient identification system for three source spices of Arnebiae Radix based on DNA barcoding and HPLC fingerprinting. A total of 599 samples from thirty-five wild populations were collected and identified by using DNA barcodes of ITS2 regions, and the chemotypes of seven naphthoquinoneswere revealed by HPLC quantitative analysis including principal component analysis and hierarchical clustering analysis. Our results showed that the ITS2 sequences can distinguish three source spices of Arnebiae Radix from adulterants. However, it was difficult to identify them by HPLC-specific chromatograms combined with chemometric analysis. These results indicated that DNA barcoding was a more powerful method than HPLC fingerprinting for the identification of related species that were genetically similar. DNA barcoding analysis could be a promising and reliable tool to accurately confirm the identities of medicinal materials, especially for those whose sources are multiple and difficult to be identified by conventional chromatography.
ABSTRACT. Achyranthis Bidentatae Radix has a long history in China as a commonly used herb that can be used to treat various diseases, including those related to the liver, muscles, bones, and kidneys. Recently, an increase in the number of adulterants has been reported, which affects the clinical safety of Achyranthis Bidentatae Radix. To identify adulterants of Achyranthis Bidentatae Radix, we collected samples from major regions and conducted an in-depth genetic comparison of the herb and its commonly used adulterants. We amplified and sequenced three genomic regions, internal transcribed spacer (ITS), psbA-trnH, and internal transcribed spacer 2 (ITS2), to confirm whether ITS2 is a suitable identifier for Achyranthis Bidentatae Radix. Results showed that the ITS2 sequence length of Achyranthis Bidentatae Radix was 199 bp, with no variation between samples. The inter-specific genetic distance of ITS2 between Achyranthis Bidentatae Radix and its adulterants was 0.390. Neighbor-joining trees showed that Achyranthis Bidentatae Radix and its adulterants are easily differentiated by monophyly. In conclusion, ITS2 regions accurately and effectively distinguished between Achyranthis Bidentatae Radix and its adulterants.
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