May 2004 Biol. Pharm. Bull. 27(5) 661—669 (2004) 661 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 a,b a b ,a Dong-Ye YANG, Hirotoshi FUSHIMI, Shao-Qing CAI, and Katsuko KOMATSU* a Research Center for Ethnomedicines, Institute of Natural Medicine, Toyama Medical and Pharmaceutical University; 2630 Sugitani, Toyama 930–0194, Japan: and b Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University; Beijing 100083, China. Received December 24, 2003; accepted January 16, 2004 Previously, we have determined marker nucleotides on the chloroplast matK gene to identify Rheum palma- tum, R. tanguticum and R. officinale used as Rhei Rhizoma officially. In the present study, we further developed a convenient and efficient identification method on the basis of marker nucleotides with Amplification Refractory Mutation System analysis. On the basis of the nucleotide substitutions at positions 367 and 937 among the three -species on the matK gene, at each position two kinds of reverse primers with complementary 3؅-terminal nu cleotides were designed. Upon PCR amplification using three sets of primers and template DNA from each species, one or two fragments (202 bp or/and 770 bp) were detected. As the resultant three fragment profiles were species-specific, the procedure enabled us to classify the botanic origins of 22 drug samples of Rhei Rhizoma. Key words Rheum; matK gene; amplification refractory mutation system (ARMS) analysis; polymerase chain reaction–restric- tion fragment length polymorphism (PCR-RFLP) analysis; Rhei Rhizoma; identification Rhei Rhizoma (“Dahuang” in Chinese) has been used for revealed that the key nucleotides for identifying section the treatment of constipation and various syndromes caused Palmata, including official species, existed at positions 587, by the obstruction of blood circulation, such as dysmenor- 707, and 838, and the marker nucleotides for identifying rhea, etc. in traditional Chinese medicine.1,2) Its pharmaco- three official species such as R. palmatum, R. tanguticum and logical effects have been known to be variable according to R. officinale were at 367 and 937, while those for distinguish- its botanic origin and the composition of its chemical con- ing the three intraspecies groups of R. palmatum were at 619, stituents.3,4) In the Japanese Pharmacopoeia,5) Rhei Rhizoma 769, 883, and 1061. In the present paper, based on the above is prescribed as Rheum palmatum, R. tanguticum, R. offici- marker nucleotides, polymerase chain reaction–restriction nale, and R. coreanum of section Palmata, family Polygo- fragment length polymorphism (PCR-RFLP)14,15) and ampli- naceae,6) while the first three species are prescribed as fication refractory mutation system (ARMS)14—16) analyses Dahuang in the Chinese Pharmacopoeia.1) However, due to were designed in order to develop a convenient and efficient the morphologic similarity of the aerial parts and frequent identification method for Rhei Rhizoma. On the matK gene occurrence of intermediate forms, the taxonomy of this sequence, we found restriction enzyme sites detecting three genus and the correct identification of Rheum species and marker nucleotides, one at position 587 which is useful for Rhei Rhizoma are very difficult. Recently, DNA-based poly- identifying section Palmata, and another two at positions 769 morphic assay was found to provide valuable information and 1061 for distinguishing the three groups of R. palmatum necessary to resolve such taxonomic problems.7—9) The (Fig. 1). For these two purposes, PCR-RFLP analysis was chloroplast matK gene sequence has been widely employed carried out. On the other hand, for the purpose of identifica- as a powerful tool in examining inter- and intragenus phy- tion of R. palmatum, R. tanguticum and R. officinale, ARMS logeny due to its high substitution rate.10—12) In our previous analysis using three sets of designed primers17,18) was per- paper,13) molecular analysis of the matK gene sequences of formed. After PCR-RFLP and ARMS methods were estab- 56 plant specimens of nine Rheum species, especially of 47 lished using plant specimens of eight Rheum species, their specimens of the above three species used as the official application for the identification of Rhei Rhizoma was inves- Dahuang in China (Table 1), was performed to clarify their tigated. phylogenetic relationship and to determine the identification markers of the three species. Although the matK gene se- MATERIALS AND METHODS quence of genus Curcuma of family Zingiberaceae was quite conservative intragenus,8) and that of genus Panax of family Materials Forty-seven plant specimens of R. palmatum, Araliaceae was stable intraspecies,9) those of the three Rheum R. tanguticum and R. officinale, and eight specimens of R. species were variable not only interspecies but also in- rhaponticum, R. franzenbachii, R. undulatum, R. przewalskyi traspecies (Table 2). However, the phylogenetic tree con- and R. kialense were used in this study (Table 1). Twenty-two structed using the unweighted pair group method with arith- drug samples of Rhei Rhizoma (Dahuang) were purchased metic mean (UPGMA method) showed that the specimens of from Chinese markets near the fields of Rheum species, and R. tanguticum and those of R. officinale belonged to their from Uchida Wakanyaku Co., Ltd. (Tokyo, Japan) and Tochi- own subclades, respectively, while those of R. palmatum be- moto Tenkaido Co., Ltd. (Osaka, Japan) (Table 3). Both plant longed to the three subclades related to their production specimens and drug samples are deposited in the Museum of areas. The result of sequence comparison of the matK gene Materia Medica, Institute of Natural Medicine, Toyama Med- ∗ To whom correspondence should be addressed. e-mail: [email protected] © 2004 Pharmaceutical Society of Japan 662 Vol. 27, No. 5 Table 1. Plant Specimens Used in This Study Date of GenBank Species Locality of voucher Voucher no. Statement Code no. Cladea) collection accession no. Section Palmata A. LOS. Rheum palmatum L. Qinghai Prov., Banma (班瑪) Co. 2000.7.20 K. Komatsu et al. QS127-2, cult. Pq1, 2 RPI AB115669 China QS129 Dongqinghan (東傾溝) 2000.7.21 K. Komatsu et al. QS171-5 cult. Pq3 RPI AB115670 Sichuan Prov., Heping (和坪), 2000.8.7 K. Komatsu et al. QS351 cult. Ps4 RPII AB115672 China Shimian (石棉) Co. Heping, Shimian Co. 2000.8.7 K. Komatsu et al. QS352-6 cult. Ps5 RPI AB115671 Caoke (草科), Shimian Co. 2000.8.8 K. Komatsu et al. QS354-1 cult. Ps6 RPIII AB115676 Caoke, Shimian Co. 2000.8.8 K. Komatsu et al. QS354-2 cult. Ps7 RPIII AB115677 Zheduo (折多山), 2000.8.8 K. Komatsu et al. wild Ps8, 9, 10 RPIII AB115678 Kangding (康定) Co. QS365-2, -4, -7 Geka (葛 ), Daofu (道孚) Co. 2000.8.9 K. Komatsu et al. QS405 cult. Ps11 RPI AB115670 Geka, Daofu Co. 2000.8.12 K. Komatsu et al. QS479 cult. Ps12 RPIII AB115679 Tagong (塔公), Kangding Co. 2000.8.12 K. Komatsu et al. QS480 cult. Ps13 RPIII AB115680 Xinduqiao (新都橋), 2000.8.12 K. Komatsu et al. QS481-1 cult. Ps14 RPIII AB115680 Kangding Co. Xinlong (新龍) Co. 2001.4.28 H. Cao QS0738 wild Ps15 RPI AB115671 Yulong (玉隆), Dege (徳格) Co. 2000.8.11 K. Komatsu et al. QS416-1, -4 wild Ps16, 17 RPI AB115670 Shiqu (石渠) Co. 2000.8.14 Y. Sahashi QS2 cult. Ps18 RPIII AB115680 Yunnan Prov., Heqing (鶴慶), Dali (大理) Co. 1999.7.30 K. Komatsu et al. Y229, Y231, cult. Py19, 20, 21, 22 RPIII AB115681 China Y232, Y233 Heqing, Dali Co. 1999.7.30 K. Komatsu et al. Y230 cult. Py23 RPII AB115673 Heqing, Dali Co. 1999.7.30 X. Wang Y1 cult. Py24 RPII AB115673 Gansu Prov., Li (礼) Co. 2001.7.30 D.Y. Yang G01 cult. Pg25 RPII AB115673 China Gezigou (格子溝), Li (礼) Co. 2001.7.30 D.Y. Yang G02-1 cult. Pg26 RPII AB115673 Shuanshui (銓水), Li Co. 2001.7.31 D.Y. Yang G04-1 cult. Pg27 RPIII AB115677 Shuanshui, Li Co. 2001.7.31 D.Y. Yang G04-6 cult. Pg28 RPIII AB115677 Nanyang (南陽), 2001.7.31 D.Y. Yang G05-1 cult. Pg29 RPII AB115673 Tanchang (宕昌) Co. Nanyang, Tanchang Co. 2001.7.31 D.Y. Yang G05-2 cult. Pg30 RPIII AB115677 Chengjiao (城郊), Min (岷) Co. 2001.8.2 D.Y. Yang G06, G07 cult. Pg31, 32 RPII AB115673 Chengjiao, Min Co. 2001.8.2 D.Y. Yang G08-1 cult. Pg33 RPIII AB115677 Hanban (愍班), 2001.8.4 D.Y. Yang G09 cult. Pg34 RPII AB115674 Zhouqu (舟曲) Co. Baidian (白点山), Zhouqu Co. 2001.8.4 D.Y. Yang G10-1, -2, -6 wild Pg35, 36, 37 RPII AB115675 R. tanguticum Qinghai Prov., Qunjia (群加), 2000.7.17 K. Komatsu et al. QS1 cult. T1 RT AB115682 MAXIM. ex BALF. China Huangzhong (湟中) Co. Mashidang (麻什当), 2000.7.24 K. Komatsu et al. QS201-1 wild T2 RT AB115682 Huangnan (黄南) Co. Reshui (熱水), 2000.8.2 K. Komatsu et al. QS307-1, -2 wild T3, 4 RT AB115683 Dulan (都蘭) Co. R. officinale BAILLON Sichuan Prov., Fengtong (蜂桶), 2000.8.19 K. Komatsu et al. QS500-3, cult. O1, 2, 3 ROC AB115684 China Wanyuan (万源) Co. QS501-1, -3 Piwo (皮窩), Wanyuan Co. 2000.8.20 K. Komatsu et al. QS510 cult. O4 ROC AB115685 Piwo, Wanyuan Co. 2000.8.20 K. Komatsu et al. QS514-2, -3 wild O5, 6 ROC AB115686 Section Rhapontica A. LOS. R. rhaponticum L. Qinghai Prov., Dulan (都蘭) Co. 2000.8.1 K. Komatsu et al. QS306 cult. R RR AB115688 China R. franzenbachii Hebei Prov., Anguo (安国) 2002.9.16 K. Komatsu 1 cult. F RR AB115689 MU¨ NT.