Identification of the crude drug atractylodes rhizome (Byaku-jutsu) and atractylodes lancea rhizome (So-jutsu) using chloroplast TrnK sequence as a molecular marker.
Novel methods for molecular authentication of Atractylodes-derived crude drugs (Jutsu) were established based on PCR-restriction fragment length polymorphism (RFLP) and direct sequencing of chloroplast trnK. Two regions inside the chloroplast trnK were selected as molecular markers for identification and discrimination of Atractylodes Rhizome (Byaku-jutsu) and Atractylodes Lancea Rhizome (So-jutsu). The Region 1 fragment (260 bp) amplified from So-jutsu and Wa-byaku-jutsu (Atractylodes Rhizome derived from A. japonica) gave 2 bands of 180 bp and 80 bp on agarose gel electrophoresis after digestion with a restriction endonuclease HinfI, whereas the fragment amplified from Kara-byaku-jutsu (Atractylodes Rhizome derived from A. ovata) remained undigested, which allowed unambiguous identification of Kara-byaku-jutsu. By direct sequencing of Region 2 (436 bp) and comparison of the nucleotide sequence data sets we could not only discriminate Byaku-jutsu and So-jutsu but also identify the original plant species of each crude drug specimen. A simple and reliable protocol for rapid preparation of DNA suitable for PCR from as little as 1 mg of Atractylodes-derived crude drugs was also described.
Mizukami
H
H
Faculty of Pharmaceutical Sciences, Nagoya City University, Japan. hajimem@phar.nagoya-cu-ac.jp
Okabe
Y
Y
Kohda
H
H
Hiraoka
N
N
D019659:Asteraceae / Q000737:chemistry / Q000235:genetics*; D002736:Chloroplasts / Q000235:genetics; D018744:DNA, Plant / Q000032:analysis; D004365:Drugs, Chinese Herbal / Q000032:analysis*; D017343:Genes, Plant; D005819:Genetic Markers; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D017421:Sequence Analysis
D018744:DNA, Plant; D004365:Drugs, Chinese Herbal; D005819:Genetic Markers