Molecular Analysis and Chemical Evaluation of Ephedra Plants in Mongolia

Abstract

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...

Genetic Diversity of Ephedra Plants in Mongolia Inferred from Internal Transcribed Spacer Sequence of Nuclear Ribosomal DNA

“…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.…”

“…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 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.…”

“…the same as in our previous report. 1) Samples collected in 2006 were identified by Dr. C. Sanchir of the Institute of Biology, Mongolian Academy of Science, and by the authors themselves as Ephedra sinica STAPF, E. equisetina BUNGE, E. monosperma GMELIN ex C. A. MEYER, E. glauca REGEL, E. regeliana FLORIN, E. przewalskii STAPF, and E. lomatolepis SCHRENK. Samples M1454 and M1455 were the cultivated samples collected in Guulin, Govi-Altay prov., the former sprouted from seeds collected in eastern Mongolia, and the latter sprouted from seeds collected in Inner Mongolia, China.…”

Formulations, characterization, in vitro and ex vivo release of Ephedra extract from topical preparations using dialysis cellulose membrane and natural rabbit skin

“…Ephedrine activates the central nervous system, increases blood pressure, dilates the bronchial tubes, and increase the pulse rate. Pseudoephedrine is used for the relief of nasal congestion in its synthetic form (17)(18)(19). The herb Ephedra, or ma-huang as the aerial part of the herb is identified in conventional Chinese medicine, is the oldest medicinal herbs mentioned in literature.…”