Biol. Pharm. Bull. 29(6) 1207—1211 (2006)

used in Chinese and Japanese traditional (Kampo) medicines. Although about 35 species belonging to the genus Ephedra are distributed in Eurasia, North America, and South America, only a few species contain pharmacologically active ephedrine alkaloids and are used for medicinal purposes. The Japanese Pharmacopoeia defines Ephedra Herb as dried aerial parts of Ephedra sinica, E. intermedia and E. equisetina. Morphological and anatomical characteristics have long been investigated to identify and/or discriminate these species since Konoshima described the morphology of E. equisetina collected in China. However, because of their relatively simple organization it is sometimes difficult to identify original species of Ephedra Herb, especially when they do not bear flowers or fruits. Chemical identification of Ephedra plants mainly based on their alkaloid compositions or HPLC fingerprints has also been attempted, but such chemical traits are inevitably affected by environmental and intra-specific variations. For correct identification of biological materials, DNA profiling (DNA-based polymorphic assay) has several advantages over morphological and chemical analyses because genotypes rather than phenotypes are directly assayed, and so that the results are not affected by environmental factors. Long et al. compared nucleotide sequences of intergenic transcribed spacer (ITS) 1 and ITS2 of nuclear ribosomal DNA (rDNA) and of the chloroplast trnL–trnF region including trnL intron, 3 -exon of trnL, and trnL/trnF spacer from eight Ephedra species. They found that the ITS sequence of E. sinica was identical with that of E. intermedia, whereas there were several polymorphic sites between these species and E. przewalskii. However, most of these polymorphic sites were later found to be subjected to intra-specific variation within either E. intermedia or E. przewalskii. As for the chloroplast genome there were no polymorphic nucleotide sites in the trnL intron among E. sinica, E. intermedia and E. przewalskii. The only difference in these species was that two nucleotide deletions were present in the trnL/trnF spacer of E. sinica. Thus, neither ITS of rDNA nor the trnL–trnF region is suitable as a DNA marker to identify medicinal Ephedra species by itself. The chlB gene encodes a subunit B of light-independent photochlorophyllide reductase that catalyzes reduction of protochlorophyllide to chlorophyllide in chlorophyll biosynthesis, and is located in the chloroplast genome of gymnosperms, algae and photosynthetic bacteria but not in angiosperms. The chlB has a higher evolutionary rate than the rbcL (large subunit of ribulose bisphosphate carboxylase/ oxygenase) gene which has been widely used as a molecular marker in plant phylogenetic analyses, and might be suitable as a DNA marker for identification and/or discrimination of Ephedra species and the crude drugs derived therefrom. In the present investigation we analyzed the nucleotide sequences of the chloroplast chlB gene amplified from the herbarium and crude drug specimens of Ephedra plants and established a novel protocol for DNA authentication of Ephedra Herb based on their chlB and rDNA ITS 1 sequences. Furthermore, we showed that the protocol could be successfully applied to identify the original species of Ephedra Herb obtained in the Chinese market.