Molecular identification of Achyranthis Bidentatae Radix by using DNA barcoding

Abstract

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.

Inspecting the True Identity of Herbal Materials from Cynanchum Using ITS2 Barcode

“…To solve this problem, researchers increased the amount of root- and rhizome-based herbs in their experiments to improve the success rate of extraction. For instance, Hou et al ( 2016 ) increased the amount of materials to 40 mg and successfully obtained the DNA of Achyranthis Bidentatae Radix. Han et al ( 2017 ) used 50 mg of herbal materials in DNA extraction.…”

“…Second, the extracts were concentrated into one spin column after freezing at −20°C for 20 min to concentrate the DNA. Third, 10% PVP-40 was used to remove polyphenols as the same as Hou's study for the sake of eliminating polysaccharides and ensuring DNA purity (Hou et al, 2016 ). Finally, heating DNA at 56°C overnight was performed to increase the cleavage of materials and the dissolution rate of DNA.…”