A comparative study on the triterpene saponins of 47 samples of Ginseng drugs derived from 12 Panax taxa was conducted using a reverse-phase high-performance liquid chromatography (HPLC)method. Eleven ginsenosides, which represent 4 types of typical sapogenins, were chosen as standards for quantitative determination in order to characterize the chemical constituent pattern of each Ginseng drug and investigate the relationship between genetic varieties and chemical constituent pattern. The results showed that the ginsenoside compositions in Ginseng drugs of different origins were of considerable variability. Total saponin contents varied by 10-fold from the highest drug to the lowest one. Chikusetsu-ninjin derived from P. japonicus (Japan) was found to have the highest content (192.80 - 296.18 mg/g) and Ginseng from P. ginseng to be the lowest (5.78 - 15.63 mg/g). Two main groups (I and II) suggested by phytochemical data were clearly observed; group I mainly containing dammarane saponins consisted of P. ginseng, P. quinquefolius, P. notoginseng, P. vietnamensis and P. vietnamensis var. fuscidiscus; and group II containing a large amount of oleanolic acid saponins was com-posed of P.japonicus (apan), P. zingiberensis, P.japonicus (China),P. japonicus var. angustifolius, P. japonicus var. major, P. japonicus var. bipinnatifidus and P. stipuleanatus. The ratios of the subtotal of dammarane saponins to that of oleanolic acid saponins (D/0) were found to be > 1.9 and < 0.25 for groups I and II, respectively. The drug samples derived from the same botanical origin revealed similar constituent patterns, in other words, each Panaxtaxon showed its own characteristic chromatographic profile,which appeared in the specific shape of an 11-direction radar graph constructed on the basis of the result of quantitative analysis. Similarities of chemical constitution were seen among the closely phylogenetically-related taxa, including P. ginseng and P.quinquefolius, P. vietnamensis and P. vietnamensis var.fuscidiscus,P. japonicus (China) and its varieties were demonstrated, except P. japonicus (Japan) and P. zingiberensis.

Fig. 4. The Phylogenetic Trees Using the UPGMA Method among Six Curcuma SpeciesA, the tree reconstructed based on trnK gene sequences; B, the tree reconstructed based on combined 18S rRNA-trnK gene sequence data. The trees were outgroup-rooted using the sequence data of Hedychium spicatum. Branch lengths were calculated by Kimura's two-parameter method and mapped along each branch. CN, The Chinese population; JP, The Japanese population; pl, purple-cloud type; gl, pubescent type.

Panax vietnamensis was discovered recently in Vietnam. Its bamboo-like rhizomes, called Vietnamese Ginseng, have attracted considerable attention because of their specific pharmacological activities. In order to define the taxonomic position of this new species and include it in the molecular authentication of Ginseng drugs, the 18S ribosomal RNA gene and matK gene sequences of P. vietnamensis were determined and compared with those of its related taxa, P. japonicus var. major and P. pseudo-ginseng subsp. himalaicus, besides previously reported P. ginseng, P. japonicus and P. quinquefolius. The 18S rRNA gene sequences were found to be 1809 bps in length. The sequence of P. vietnamensis was identical to that of P. quinquefolius, and presented one base substitution from those of both P. japonicus var. major and P. pseudo-ginseng subsp. himalaicus. The matK gene sequences of 6 taxa were found to be 1509 bps in length. The sequence of P. vietnamensis differed from those of P. japonicus var. major, P. pseudo-ginseng subsp. himalaicus, P. ginseng, P. japonicus and P. quinquefolius at 4, 5, 9, 9 and 10 nucleotide positions, respectively. The phylogenetic tree reconstructed by the combined 18S rRNA-matK gene analysis using the maximum parsimony method showed that P. vietnamensis was sympatric with other Panax species and had a close relationship with P. japonicus var. major and P. pseudo-ginseng subsp. himalaicus.

Herbal drugs derived from rhizomes of Curcuma plants including Curcuma longa, C. zedoaria, etc. have attracted attention because of their antiinflammatory, 1) antihepatotoxic, 2) and neuroprotective activity.3) These drugs were originally used in traditional Asian medicines, and in Chinese medicine they are mainly used for treating various syndromes due to obstruction of blood circulation and retention of blood stasis such as arthralgia, psychataxia, and dysmenorrhea. 4) In China and Japan, more than 10 Curcuma species are distributed or cultivated, and the underground parts of six species have been used medicinally. In the Chinese herbal literature, 5) each Curcuma drug is prescribed for a specific ailment, although at present their clinical application is complicated because of the morphological similarity of the drugs as well as the plants. To develop the ultimate identification of Curcuma plants, we previously carried out molecular analysis of six Curcuma species based on 18S ribosomal RNA gene and trnK gene sequences. 6) As a result, two types of 18S rRNA gene sequences and five types of trnK gene sequences were demonstrated, and the identical sequence was observed between C. phaeocaulis and the Chinese population of C. zedoaria, and the Japanese population of C. zedoaria and C. kwangsiensis with a purple-colored band in the leaf blades (purple-cloud type), respectively. We report here the sequence analysis of Curcuma drugs available in Chinese and Japanese markets in both gene regions to identify their botanical origins. Moreover, to develop a more convenient method for identification of Curcuma plants and drugs, amplification-refractory mutation system (ARMS) analysis of both gene regions was investigated using species-specific primers designed by nucleotide differences among Curcuma species. Since five new species have been recently recorded in Chinese botanical papers 7,8) based on minute morphological differences from existing species, their relationship with other species was investigated at the molecular taxonomic to adjust the genus Curcuma in China. MATERIALS AND METHODS MaterialsThe following 11 Curcuma species comprising 20 specimens were examined: C. longa, C. phaeocaulis, C. zedoaria (Chinese and Japanese populations), C. kwangsiensis (purple-cloud and pubescent types), C. wenyujin, C. aromatica, C. sichuanensis, C. chuanezhu, C. chuanyujin, C. chuanhuangjiang, and C. yunnanensis (Table 1). Twenty-five Curcuma drugs were purchased from Chinese markets near the cultivation areas, the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China), Tochimoto Tenkaido Co., Ltd. (Osaka, Japan), and Uchida Wakanyaku Co., Ltd. (Tokyo, Japan) ( Table 2). The authors identified the plant specimens with the help of Professor D. Fang and Dr. X. H. Xiao. All materials were stored in the Museum of Materia Medica, Institute of Natural Medicine, Toyama Medical and Pharmaceutical University (TMPW), Japan.Extraction and Purification of Total DNA and PCR Amplification Total DNA was ex...

Ephedrae herba has been used in traditional Chinese and Japanese (Kampo) medicine from ancient times, with the primary resource being in China. In the present study, a field survey as well as molecular and chemical assessments were conducted on Ephedra plants in Mongolia to clarify whether they could be an alternative resource of the Ephedrae herba used in Japanese Kampo medicine. Ephedra sinica, E. equisetina, E. przewalskii, E. regeliana, E. monosperma and an unknown taxon (ESP) collected in Mongolia were divided into 9 genotypes on the basis of nucleotide sequences of 18S ribosomal RNA (rRNA) gene and trnK gene. E. sinica, E. equisetina, and E. monosperma presented completely identical sequences to the corresponding species from China. The sequences of trnK gene and 18S rRNA gene provide a useful index for identification and taxonomic classification of Mongolian Ephedra plants. Quantitative analysis of 5 ephedrine alkaloids revealed that almost all Mongolian Ephedra plants contained high amounts of total ephedrine alkaloids (TAs, 1.86-4.90%) and a high percentage of pseudoephedrine in TAs differed obviously from the Chinese. E. sinica and E. equisetina found in eastern and central Mongolia, showing total contents of ephedrine and pseudoephedrine higher than 1.43%, were potential new resources of Japanese Pharmacopoeia grade Ephedrae herba.Key words Ephedra; Mongolia; molecular identification; ephedrine alkaloid; HPLC analysis Biol. Pharm. Bull. 32(7) 1235-1243 (2009) © 2009 Pharmaceutical Society of Japan * To whom correspondence should be addressed. e-mail: katsukok@inm.u-toyama.ac.jp present study, we determined the nucleotide sequences of these two regions and compared them with 8 clearly identified Chinese Ephedra plants for objective identification and to aid in the taxonomic resolution of Ephedra plants in Mongolia. In addition, clarification of the chemical equality of Mongolian Ephedra plants and the available Ephedrae herba is needed, because the constitutions of ephedrine alkaloids differ among species and there are also some species without these alkaloids.2) Subsequently, quantitative analysis on five major ephedrine alkaloids was conducted by HPLC-photodiode array detector (PDA) method for quality evaluation. lia showed curved or twisted stems and some had allopheric features as shown in Fig. 2. Here, we tentatively treated these samples as Ephedra sp. (ESP) and included all of them in the following molecular and chemical analyses. It is noteworthy that due to fewer morphological characteristics being available in "Flora of Mongolia" 11) and the "Key to the vascular plants of Mongolia," 12) floristic literature with valuable morphological information on Ephedra plants of neighboring regions were used as reference in morphological identification, such as "Flora Xinjiangensis," MATERIALS AND METHODS Materials19) "Flora of China" and "Flora of Siberia." 20) All the vouchers and samples from Mongolia are stored in the Museum of Materia Medica, Institute of Natural Medicine, University of Toyama...