Molecular Analysis of Rheum Species Used as Rhei Rhizoma Based on the Chloroplast matK Gene Sequence and Its Application for Identification

Abstract

Rhei Rhizoma, called Dahuang in Chinese, is one of the important herbal drugs widely used as a purgative and antiinflammatory agent. In traditional Chinese formulations and over-the-counter drugs, Dahuang is also used for the treatment of oketsu (various syndromes caused by the obstruction of blood circulation such as dysmenorrhea, etc.). 1,2) In the Chinese Pharmacopoeia, 1) the official Dahuang is prescribed for the dried rhizome and root of Rheum palmatum L., R. tanguticum MAXIM. ex BALF., and R. officinale BAILLON of the family Polygonaceae. On the other hand, the Japanese Pharmacopoeia 3) prescribes not only the above three species but also R. coreanum NAKAI, and their interspecific hybrids as the botanical origins for Rhei Rhizoma. Since Rheum plants are self-incompatible in nature 4) and have been hybridized at random since ancient times, morphologically intermediate forms (in the shape of the leaf blade, color of flowers, etc.) are frequently present. This situation made the taxonomic identification of Rheum plants very difficult. Accordingly, Dahuang derived from Rheum species has not been identified correctly and its quality has varied depending on differences in markets and production areas. Some Dahuang from Gansu Province, China, contained less sennoside A than that indicated in the Japanese Pharmacopoeia (our unpublished data). Moreover, the underground parts of R. franzenbachii MUNT., R. rhaponticum L., R. undulatum L., and R. spiciforme ROYLE, which have been used as folk medicines under the names Shan-dahuang, Tu-dahuang, Wadaio, and Chu-tsa in China, Japan, and Tibet, respectively, were sometimes confused with official Dahuang. Identification of Dahuang was primarily based on the histologic characteristics of Rheum rhizomes, 5,6) although these characteristics are influenced by plant growth and environmental conditions. Recently, DNAbased polymorphic assay were found to provide valuable information necessary to resolve taxonomic problems.7,8) Yang et al. described the nucleotide sequences of the chloroplast trnL/trnF gene region of 13 Rheum species to discriminate R. palmatum, R. tanguticum, and R. officinale from adulterated species. 9) However, their phylogenetic relationship and the identification markers for each species have not been clarified. The matK gene sequence has been widely employed as a powerful tool in examining inter-and intragenus phylogeny due to its high substitution rate.10,11) As a part of our study on the quality evaluation of Dahuang, DNA analysis of the matK gene region was performed on R. palmatum, R. tanguticum, R. officinale, and six other Rheum species to clarify their phylogenetic relationship, and further to develop a correct identification method for plants and drugs. Moreover, the 18S ribosomal RNA gene sequences of nine species were elucidated. MATERIALS AND METHODSMaterials Fifty-six plant specimens investigated were identified carefully according to the definition of Kao 12,13) as nine Rheum species belonging to four sections. The nine species are: R. palmatu...

Development of a High Performance Liquid Chromatographic Method for Systematic Quantitative Analysis of Chemical Constituents in Rhubarb

“…The botanical origins of each sample were correctly identified by the molecular biological methods previously reported. 21,22) The rhubarb sample (Qinghai Prov., TMPW no. 19929) used for the isolation of standard compounds was purchased from Uchida Wakanyaku Co., Ltd. (Japan).…”

Assessment of genetic diversity among four orchids based on ddRAD sequencing data for conservation purposes

“…Different molecular techniques were employed to measure the genetic diversity among plant species including some gene specific sequences such as matK, rbcL, ITS regions (Hilu and Liang 1997;Ma and Yin 2009;Cao et al 2001;Koch et al 2001;Zhu et al 2003;Yang et al 2004;Zhang et al 2013;Chen et al 2014;Xing 2014). Recently the high-throughput SNP genotyping techniques based on NGS method are used to analyze the genetic diversity (Kumar et al 2012;Shirasawa et al 2016).…”

Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Amplification Refractory Mutation System (ARMS) Analyses of Medicinally Used Rheum Species and Their Application for Identification of Rhei Rhizoma

“…Previously, 13) we investigated the phylogenetic relationship of the Rheum species, especially of R. palmatum, R. tanguticum and R. officinale of section Palmata, used as Rhei Rhizoma officially on the basis of their matK gene sequences. The phylogenetic tree suggested that R. tanguticum and R. officinale were monophyletic, but R. palmatum was mainly composed of 3 groups (I, II, III) which were related to the production area.…”

“…On the other hand, one drug sample named "Ya-huang" (D21), which is available in the Japanese market, was found to be derived from group I of R. palmatum, not as expected from group III. 13) Although the Ya-huang samples imported from China have been known to be produced in Jiulong and Shimian Counties, the central part of Sichuan Province, 5,20) their production areas have been moved recently to the northwestern part. In fact, the partial matK gene sequence was found to be identical to that of wild plants from Xinlong County, in the northwestern part of Sichuan Province (Table 2: Ps15).…”

“…[7][8][9] The chloroplast matK gene sequence has been widely employed as a powerful tool in examining inter-and intragenus phylogeny due to its high substitution rate. [10][11][12] In our previous paper, 13) molecular analysis of the matK gene sequences of 56 plant specimens of nine Rheum species, especially of 47 specimens of the above three species used as the official Dahuang in China (Table 1), was performed to clarify their phylogenetic relationship and to determine the identification markers of the three species. Although the matK gene sequence of genus Curcuma of family Zingiberaceae was quite conservative intragenus, 8) and that of genus Panax of family Araliaceae was stable intraspecies, 9) those of the three Rheum species were variable not only interspecies but also intraspecies (Table 2).…”