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 dr ugs (Jutsu) were established based on PCR-restriction fragment length polym orphism (RFLP) and direct sequencing of chloroplast trnK. Two regions insid e the chloroplast trnK were selected as molecular markers for identificatio n and discrimination of Atractylodes Rhizome (Byaku-jutsu) and Atractylodes Lancea Rhizome (So-jutsu), The Region 1 fragment (260 bp) amplified from S o-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 diges tion 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 descri bed.