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...

Ephedrae herba, called "Mao" in Japanese and "Mahuang" in Chinese, has been used in Kampo and traditional Chinese medicine from ancient times for inducing perspiration, dispersing cold, relieving cough, etc., and is included in a number of popular Kampo formulas. In Japanese Pharmacopoeia (JP), Ephedra herb is prescribed as the aerial parts of Ephedra sinica STAPF, E. intermedia SCHRENK et C. A. MEYER and E. equisetina BUNGE. In Japan, supplies of Ephedra plants (resource of "Mao") depend primarily on import from China. However, the Chinese government has curtailed the export of Ephedra plants since 1999 to prevent desertification. In order to search for alternative resources in neighboring regions, we carried out field investigations on Ephedra plants in Mongolia during 2002Mongolia during -2004, and subsequently conducted molecular and chemical evaluations. The results 1) revealed that E. sinica and E. equisetina in eastern and central Mongolia had potential as new resources of Ephedrae herba of JP grade, and other species could be resources of ephedrine alkaloids.However, during those investigations, we found that many Ephedra populations in the southern area had curved or twisted stems and were therefore very difficult to identify from external features. We treated them tentatively as Ephedra sp., and successive molecular analysis suggested hybrid origins of these plants. In order to obtain more detailed information and evidence, we conducted another field survey in the southwestern area of Mongolia in 2006 (Fig. 1), and molecular and chemical evaluation was done as before on the newly collected samples. In the molecular analysis, besides the formerly investigated chloroplast trnK gene and nuclear 18S ribosomal RNA (rRNA) gene sequence, partial nuclear internal transcribed spacer (ITS) sequences were also determined and compared, because this region was demonstrated to have a higher rate of nucleotide variation, 2) especially at the 3Ј-terminal of the ITS1 region in the case of Ephedra plants.The ITS sequence has also been proved as potentially effective in detecting the hybrid origin of plants or species, as well as in identifying reticulate evolution by showing additive peaks on a sequencing electropherogram, i.e., at the variable sites, different nucleotides from both parental types presented simultaneously. In our previous paper, 1) we reported that additive peaks at the nucleotide position 1709th of 18S rRNA gene sequences were observed in most samples of E. sinica and Epheda sp., with variable relative-intensity between the two peaks. However the additive peaks, sometimes with a weak signal of the 2nd peak, were difficult to distinguish from high noise sites. Rauscher et al. have tested the sensitivity of direct sequencing for detecting the presence of more than one type of sequence by using an experimental mixture of different sequence samples.3) Therefore, we investigated the detection efficiency in our experimental system referring to their method in order to facilitate the judgment of additive pea...