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

Abstract

Rhei Rhizoma ("Dahuang" in Chinese) has been used for the treatment of constipation and various syndromes caused by the obstruction of blood circulation, such as dysmenorrhea, etc. in traditional Chinese medicine.1,2) Its pharmacological effects have been known to be variable according to its botanic origin and the composition of its chemical constituents. 3,4) In the Japanese Pharmacopoeia, 5) Rhei Rhizoma is prescribed as Rheum palmatum, R. tanguticum, R. officinale, and R. coreanum of section Palmata, family Polygonaceae,6) while the first three species are prescribed as Dahuang in the Chinese Pharmacopoeia.1) However, due to the morphologic similarity of the aerial parts and frequent occurrence of intermediate forms, the taxonomy of this genus and the correct identification of Rheum species and Rhei Rhizoma are very difficult. Recently, DNA-based polymorphic assay was found to provide valuable information necessary to resolve such taxonomic problems.7-9) 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). However, the phylogenetic tree constructed using the unweighted pair group method with arithmetic mean (UPGMA method) showed that the specimens of R. tanguticum and those of R. officinale belonged to their own subclades, respectively, while those of R. palmatum belonged to the three subclades related to their production areas. The result of sequence comparison of the matK gene revealed that the key nucleotides for identifying section Palmata, including official species, existed at positions 587, 707, and 838, and the marker nucleotides for identifying three official species such as R. palmatum, R. tanguticum and R. officinale were at 367 and 937, while those for distinguishing the three intraspecies groups of R. palmatum were at 619, 769, 883, and 1061. In the present paper, based on the above marker nucleotides, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) 14,15) and amplification refractory mutation system (ARMS) [14][15][16] analyses were designed in order to develop a convenient and efficient identification method for Rhei Rhizoma. On the matK gene sequence, we found restriction enzyme sites detecting three marker nucleotides, one at position 587 which is useful for identifying section Palmata, and another two at positions 769 and 1061 ...

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

“…21) At the same time, the key nucleotide markers for identifying 3 official species of rhubarb such as R. palmatum, R. tanguticum and R. officinale, and those for distinguishing the 3 intraspecies groups of R. palmatum were determined, by which most rhubarb samples were identified correctly. 22) In our future paper, the chemical constituent pattern of rhubarb samples from different sources and the relationship between that and 946…”

“…Standard Compounds Anthraquinones (1-5), anthraquinone glucosides (6-10), dianthrones (11,12), phenylbutanones (13,14), stilbenes (15, 16), flavan-3-ols (17, 18), procyanidins (19,20), galloylglucoses (21)(22)(23)(24), acylglucoses (25)(26)(27) and gallic acid (28) were isolated from the rhubarb sample (TMPW no. 19929), and are listed in Fig.…”

“…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).…”

Molecular Analysis of the Genus Mitragyna Existing in Thailand Based on rDNA ITS Sequences and Its Application to Identify a Narcotic Species: Mitragyna speciosa

“…4). Since in many samples, DNA was highly degraded into small pieces probably due to the oxidative and hydrolytic process during preservation period, 22,23) longer PCR products were difficult to be obtained. Therefore, a new primer set (ITS-1F located in 18S rDNA region and In-18S-25S-3ЈR located in 5.8S rDNA region) was designed, which produced a short fragment, to allow identification of crude drug samples.…”

Identification of Dendrobium Species by Dot Blot Hybridization Assay

“…[3][4][5][6] High polymorphisms of rDNA can be easily found between inter-species and even intraspecies. The DNA sequences of several traditional Chinese medicines (TCMs) have been reported; identifying markers were generated via restriction fragment length polymorphism (RFLP) [7][8][9] or polymerase chain reaction (PCR) methods [10][11][12] based on the polymorphic sequences. A system of designing PCR primers or probes is necessary to differentiate the TCMs in a quality control schedule.…”