RESUMO
Hyoscyami Semen, the mature dried seed of Hyoscyamus niger L., has long been used as a traditional Chinese medicine to treat human diseases. Hyoscyami Semen is found in local markets in China. In markets, sellers and buyers commonly inadvertently mix the seeds of H. niger with the seeds of related species such as Hygrophila salicifolia (Vahl) Nees, Astragalus complanatus R. Br., Cuscuta australis R. Br., Cuscuta chinensis Lam., and Impatiens balsamina L. because of their similar morphologies or similar names. Thus, developing a reliable method for discriminating H. niger seeds from its adulterants is necessary to reduce confusion and ensure the safe use of Hyoscyami Semen. The present study was designed to evaluate the efficiency of high-resolution melting analysis combined with DNA barcoding (Bar-HRM) with internal transcribed spacer 2 to discriminate H. niger. Our results show that Bar-HRM successfully identified the adulterants and detected the proportion of H. niger DNA extract within an admixture. In particular, HRM detected H. niger DNA extract in A. complanatus DNA extract at concentrations as low as 1%. In conclusion, the Bar-HRM method developed in the present study for authenticating H. niger is rapid and cost-effective. It can be used in the future to guarantee the purity of Hyoscyami Semen for the clinical use.
Assuntos
China , Código de Barras de DNA Taxonômico , Métodos , DNA Intergênico , Química , Genética , DNA de Plantas , Química , Genética , Análise Discriminante , Contaminação de Medicamentos , Medicamentos de Ervas Chinesas , Química , Hyoscyamus , Genética , Sementes , Genética , Temperatura de TransiçãoRESUMO
The misuse of toxic drugsisseriousone of the threats of public health. In this study, toxic Hyoscyami Semen and its adulterants were identified by DNA barcoding. The genomic DNA was extracted from 61 samples including Hyoscyami Semen and its adulterants by reagent kit method. Their ITS2 sequences were amplified, and purified PCR products were sequenced. Sequence assembly and consensus sequence generation were performed using CodonCode Aligner v 4.25. The genetic distances, variable sites and the neighbor-joning (NJ) phylogenetic tree were computed by MEGA 6.0 in accordance with the Kimura 2-parameter(K2P) model. The results showed that the intra-specific genetic distances of Hyoscyamusniger were 0.005 which were smaller than inter -specific ones (0.360) of H. niger and their adulterants. The NJ tree showed that H. niger was clustered into one monophyletic branch, and clearly separated with other species. Therefore, ITS2 sequence was able to identify Hyoscyami Semen and its adulterants to ensure the safty of medicines.