Application of the Multiplex PCR Method for Discrimination of Artemisia iwayomogi from Other Artemisia Herbs (original) (raw)

2008, Biological and Pharmaceutical Bulletin

Artemisia plants, particularly A. iwayomogi, A. capillaris, A. princeps, and A. argyi, are important medicinal materials that are utilized in traditional Asian medicines. 2) A. iwayomogi and A. capillaris, referred to in Korea as 'Haninjin' and 'Injinho', respectively, 2) have been utilized traditionally for the treatment of diuresis and as anti-inflammatory agents, as is also the case in China and Japan. 2,3) Meanwhile, both A. princeps and A. argyi are referred to as 'Aeyup,' and are employed in the treatment of colic pain, vomiting, and irregular uterine bleeding. 2,4) In traditional Korean medicine markets, a portion of certain specific Artemisia herbs tend to be distributed interchangeably with other Artemisia herbs. 2) For instance, some 'Haninjin' is distributed under the name 'Aeyup.' However, 'Aeyup' may occasionally be substituted with 'Haninjin' or 'Injinho' in medicinal prescriptions. To address this problem, an efficient method for the identification of Artemisia herbs is clearly necessary. Currently, the discrimination of one medicinal herb from another is a process which involves subjective methods, frequently predicated on the morphological features of the plant and/or the observations of a trained individual. However, these methods are not always sufficient for the identification of plants that morphologically resemble each other, particularly when the plants are in a dried and/or sliced state, the common forms of plants when purveyed in an herbal market. Therefore, objective methods, such as those predicated on differences in DNA sequences between organisms, 5) should be developed. In a previous work, 6) we showed that a sequence-characterized amplified region (SCAR) marker 7) based on the nucleotide sequence of a specific random amplified polymorphic DNA (RAPD) product amplified uniquely from 'Aeyup' (both A. princeps and A. argyi) proved to be a reliable DNA marker for the discrimination of 'Aeyup' from other Artemisia herbs. As some Artemisia herbs can be well-distinguished from each other by RAPD, a SCAR marker of 'Haninjin' (A. iwayomogi) developed by the same strategy used for the SCAR markers of 'Aeyup' might facilitate the discrimination of 'Haninjin' from other Artemisia herbs, particularly 'Injinho' and 'Aeyup.' In this work, we developed SCAR markers of Artemisia herbs, including 'Haninjin.' We then applied these SCAR markers to the development of a method that could be applied not only to identify tested Artemisia herbs as 'Haninjin' (A. iwayomogi), but also to determine which Artemisia herbs are being tested concurrently with A. iwayomogi. Using this method, we were able to differentiate 'Haninjin' from other Artemisia herbs with more certainty as compared with a technique predicated on the amplification of a single DNA marker, such as a SCAR marker. Furthermore, we were able to apply this method to a search for Artemisia herbs that are substituted for 'Haninjin' and/or mixed with 'Haninjin'. In service of these research purposes, we developed a multiplex PCR method 8) using a primer set to amplify a SCAR marker of 'Haninjin', along with two other primer sets to amplify the 'Injinho' and 'Aeyup' SCAR markers, in order to amplify each SCAR marker of the Artemisia herbs in a single