رمزگذاری میله ای دی ان آی Buplerum در طب چینی DNA barcoding Chinese medicinal Bupleurum

Chaihu (Bupleuri radix), the dried roots of Bupleurum chinense DC. and B. scorzonerifolium Willd., is a famous Traditional Chinese medicinal herb with a history of medical use for more than 2000 years. In Traditional Chinese Medicine (TCM), Chaihu is thought to regulate the exterior and interior metabolisms, disperse evil heat from superficies, sooth the liver, and promote yang and qi (representing “life energy” or “life force” in TCM theory). Deficiency in yang and qi leads to insufficiency in the body’s functionality resulting in a lowered metabolism, and malfunctioning of the entire body or certain organs. Symptoms like low spirit, tired and weak, reduced appetite, chronic diarrhea, prolapses (such as proctoptosis, gastroptosis, hysteroptosis), are all manifestations of deficiency and sinking of yang and qi. Chaihu is an important component in prescriptions to treat such symptoms through promoting yang and qi. According to Chinese Pharmacopoeia, Chaihu is effective in treating common cold with fever, influenza, hepatitis, malaria, menoxenia, hysteroptosis and proctoptosis. (National Pharmacopoeia Committee, 2010). Bupleurum L. (Apiaceae) comprises approximately 150 species worldwide. In China, about 44 species, 17 varieties and 7 forma have been identified, distributed widely across the country in 27 provinces (municipalities or autonomous regions)(She andWatson 2005; Pan 2006). Although only the 2 species mentioned above are officially approved for clinical use in Chinese Pharmacopoeia, as many as 25 species, 6 varieties, and 1 form of Bupleurum are found in use for medicinal purpose, including B. longiradiatum Turcz. with toxic ingredients and B. hamiltonii Balak with almost undetectable saikosaponin contents. In some areas, even 5 or 6 different species of local Bupleurum are used under the same name of Chaihu. This confusion (intentional or not) results in rather variable qualities of the raw materials for Chaihu in Chinese market. Accurate identification of the crude drugs is crucial for safe clinical applications of Chaihu in TCM practices and for its chemical and pharmacological researches. However, the conventional anatomical and chemotaxonomical studies provide limited value in determining the botanical origins and controlling the quality of Chaihu (Pan 1996), and a more reliable method for that purpose is urgently needed. Efforts had been made to discuss the feasibility and applicability of internal transcribed spacer (ITS) sequences for determining the identity of Bupleurum medicinal plants (Yang et al. 2007; Xie et al. 2009). We found that the homologous alignment of ITS sequences was lower than 75% between members of the genus Bupleurum and associated out-groups, while the within-group alignment was greater than 87%; samples ofthe same herb showed an ITS similarity of at least 99%. We thus proposed that ITS sequences be used as reliable molecular markers for the identification of Chaihu; but we also found 6 major groups of different species with ITS sequence homology greater than 99%, i.e., these species could not be distinguished by ITS sequence (Xie et al. 2009). DNA barcoding is a method of species identification which involves sequencing a standard and specific DNA region. It was first proposed by Hebert et al. (2003a) and has been demonstrated as a powerful tool for species identification. The cytochrome c oxidase subunit 1 mitochondrial region (COI) is emerging as the standard barcode region for distinguish a wide range of animals (Hebert et al. 2003b, 2004; Ward et al. 2005; Smith et al. 2007), but its evolution rate is too slow for application in plants. In 2005, Kress proposed the combined use of psbA-trnH and ITS of nuclear ribosomal DNA for identifying plants; and Chase et al. (2005) and Newmaster et al. (2006) recommended rbcL for that purpose. The combination of rbcL and matK was proposed as a core barcode to identify land plants by CBOL Plant Working Group (2009). But as the amplifi- cation and identification efficiencies of the proposed regions vary with different plant groups, there has been an ongoing effort to screen for universal barcoding markers for plants. In a large sample volume study, Chen et al. (2010) found that nuclear internal transcribed spacer 2 (ITS2) regions had not only a high amplification efficiency, but also a high identification efficiency up to 90%, and they proposed that ITS2 be used as a potential barcode for plant identification. In this study, we tested 4 candidate loci, namely the chloroplast genes rbcL, matK, psbA-trnH, and ITS2 regions for their validity as DNA barcodes to identify species in Chinese medicinal Bupleurum. We aimed to establish a DNA barcode system of Chinese Bupleurum archiving the overall information of the samples of crude drug Chaihu to facilitate the identification of Chaihu, and to provide better insights into the genetic relationship and phylogeny of Bupleurum species.