Identification of Dendrobium Species Used for Herbal Medicines Based on Ribosomal DNA Internal Transcribed Spacer Sequence

Abstract

Dendrobium is one of the largest genera in the Orchidaceae, of which about 1200 species are distributed widely in the Asia-Pacific region. 1,2) Owing to the enormous number of species and great diversification of morphological characters, many taxonomic and phylogenetic problems of this genus remain unresolved.Dried stems of specific Dendrobium species, so-called Dendrobii Herba, have been used in traditional Chinese medicine for benefiting the stomach, promoting the production of body fluid, nourishing "yin," and antipyresis.3,4) Stems of some Dendrobium species contain compounds that exhibit antioxidant activity and antitumor activity.5-7) Accordingly, the demand for Dendrobii Herba has increased rapidly, and the wild resources of Dendrobium have decreased severely due to over-collection. Seventy-four Dendrobium species are distributed in China. However, in practice, Dendrobium species that are not described in either of these references are used clinically, and other orchid genera such as Pholidota have also been used as adulterants.9-11) Such a lax situation in commercial distribution could result in the risk of inconsistent therapeutic effect and even be a threat against consumer safety. Therefore, discrimination of Dendrobii Herba is important for quality control. However, it is difficult to identify Dendrobium species from dried stems by traditional methods based on morphological, anatomical and/or chemical analyses [12][13][14] because these characteristics are not always useful markers at the species level and often vary depending on growing conditions.To date, several molecular analytical methods have been applied to identify herbal medicines.15) For example, DNA sequencing, DNA microarrays, random amplified polymorphic DNA (RAPD), and DNA fingerprinting have been used for identification of Dendrobium species. [16][17][18][19][20][21][22][23][24][25][26][27] Unfortunately, these methods have only been applied to a limited number of Dendrobium species, and molecular markers that can be applied more universally to Dendrobium have not been developed. To recognize herbal medicines accurately at the species level, a comprehensive DNA sequence database that covers Dendrobium species potentially used for medicine is desired, and to create such a database, the following conditions are required: (1) precise identification of the plant species, (2) comprehensive data banking of reference sequences for the relevant plant group at the species level, and (3) employment of sequence markers that can discriminate interspecific differences.We constructed a sequence database of the internal transcribed spacer (ITS)1-5.8S-ITS2 region for 196 Dendrobium species, all of which were identified accurately based on the morphological characters of vegetative and reproductive organs. The database covers most of the Dendrobium species from China and adjacent regions. The sequence readout of ITS1 and ITS2 regions of nuclear ribosomal DNA appeared to contain sufficient variation to discriminate Dendrobium at the interspecific level...

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