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ITS sequence analysis used for molecular identification of the Bupleurum species from northwestern China.

Abstract

Radix Bupleuri (Chaihu), a famous Traditional Chinese Medicine, is derived from the dried roots of Bupleurum chinense DC. and B. scorzonerifolium Willd. But nowadays, other nine species and varieties from genus Bupleurum are also used as Radix Bupleuri in northwestern China. The authentic identification of dried roots of B. chinense and B. scorzonerifolium, however, is difficult to just base on the appearance and morphology. A molecular genetic method was developed to help discriminate the original species of Radix Bupleuri. The ITS sequences (~600 bp) were amplified by the PCR technique from genomic DNA isolated from all the collected samples. According to analyzing the information given by ITS sequences, the conclusion can be made that ITS sequence could serve as markers for authentication of Radix Bupleuri.

[c] 2007 Elsevier GmbH. All rights reserved.

Keywords: ITS sequence; Radix Bupleuri; Chaihu; Traditional Chinese medicine; Identification

Introduction

Radix Bupleuri, a well-known Traditional Chinese Medicine, has been utilized to treat common cold with fever, influenza, hepatitis, malaria and menoxenia for more than 2000 years. Its original herbs belong to Bupleurum L., which is one of the largest genera of the family Apiaceae (Umbelliferae). The genus comprises annual and perennial species, from small herbs a few centimeters tall to shrubs up to 3 m high (Neves and Watson, 2004). In China, 44 species, 17 varieties and seven forma have been reported, and nearly half of them are native species (Shan and She, 1979; Pan et al., 2002). The Pharmacopeia of the People's Republic of China (Shao, 2005) states that Bupleurum chinense DC. and B. scorzonerifolium Willd. were officially regarded as the standard medical plants. But due to the slightly different morphologic appearance of Bupleurum plants and their dried roots, there were 36 Bupleurum species and varieties under the same name of Radix Bupleuri in different districts and markets, including B. longiradiatum Turcz. with toxic ingredients and B. hamiltonii Balak with very little active constituents (Wang and Zhang, 1994; Pan, 1996; Pan et al., 2002). In northwestern China, there are approximately 11 species utilized nowadays. Inevitably, this situation will compromise the values of Radix Bupleuri and even imperil the safety of the consumers.

To identify crude drugs precisely is a crucial base for chemical and pharmacological research of Traditional Chinese Medicine and for their clinical applications. Traditional means of Radix Bupleuri (Chaihu) authentication rely on the inspection of morphological markers such as shape, color, texture and odor (Wang and Zhang, 1994). Its accuracy depends heavily on the examiner's experience. Additionally, medicinal plants are normally processed and dried, some morphological and anatomical characteristics, as well as some chemical constituents, are changed. The determination of botanical origins of crude drugs as well as their quality control through anatomical and chemotaxonomical studies are, therefore, very difficult (Zhao et al., 2003). Therefore, more objective and definitive methods are necessary. Herein, the ITS region of nuclear ribosomal DNA was employed to assist identification for its valuable source of phylogenetic information, particularly at the infragenetic and infrafamilial level (Baldwin, 1992, 1993; Baldwin et al., 1995). The nrDNA ITS region has also been used successfully in various studies as genetic markers for identification (Xu et al., 2001; Liu et al., 2001; Zeng et al., 2003; Ding and Fang, 2005; Wang and Wang, 2005). It is the first time to identify all the Bupleurum species used in northwestern China by ITS sequence analysis.

Materials and methods

Acquisition of plant materials

Fresh plants were kept in silica gel upon collection; the voucher specimens were deposited in the School of Pharmacy, Fudan University, Shanghai, China. Bupleurum taxa, totally 20 populations representing 11 species, covering all species currently used in northwestern China, were examined for sequence variations in the ITS sequence region of nuclear rDNA (Table 1). Samples included fresh plants, crude drugs and herbaria. Details of origin material, GenBank accession numbers and locations of herbarium vouchers are provided in Table 1.

Extraction of genomic DNA

The materials were frozen with liquid nitrogen and milled into a fine powder. Total cellular DNA was extracted from the ground powder by using a modified cetyltrimethylammonium bromide method from Doyle and Doyle (1987). The concentration of genomic DNA was measured by Eppendorf Biophotometer (Eppendorf Biotech Int'l Trade Company Ltd.).

PCR amplification

DNA amplifications were performed in 25 [micro]l reaction mix containing approximately 20ng genomic DNA, 2.5 [micro]l 10 x PCR buffer ([Mg.sup.2+] Plus), 1 [micro]l dNTP (2.5 mmol/L each), 1 U TaKaRa Taq (TaKaRa Biotechnology Co. Ltd.), 1 [micro]mol of primers 'ITS 5P' (Moller and Cronk, 1997; the same as 'modified ITS 5' in Downie and Katz-Downie, 1996) and 'ITS 4' (White et al., 1990), then we added dd[H.sub.2]O to reach 25 [micro]l. The PCR profile consisted of an initial 5 min at 93 [degrees]C, 2 min at 55 [degrees]C then followed by 30s at 93 [degrees]C, 45s at 55 [degrees]C, 45s at 70[degrees]C, repeated for 35 cycles and with an extension of 5 min at 70 [degrees]C in MJ-PTC100[TM] (Eastwin Life Sciences, Inc.).

Successful PCR amplification produced a single DNA band of approximately 700 bp long on a 1.2% agarose gel and was visualized by ethidium bromide staining under UV. PCR products were subsequently purified using the Gel Extraction Mini Kit (Watson Biotechnologies, Inc.).

Sequence analysis

The PCR products were subcloned into the pMD18-T vector (TaKaRa Biotechnology Co. Ltd.). Competent XL10-gold cells were transformed with the ligation products, and the colonies identified by color selection were picked and grown in 4 ml of Luria-Berteni liquid medium overnight. The correct DNA inserts were verified by bacteria colonies PCR; and the plasmid DNAs from verified colonies were isolated with the Wizard Minipreps DNA Extraction system (Promega). ABI Prism[R], BigDye[TM] Terminator and Cycle Sequencing Ready Reaction Kit were used for sequence reaction with the M13 specific primer; the sequence was detected by the ABI PRISM 3700 DNA Analyzer (Applied Biosystems, Foster City, CA). All three colonies for each specimen were sequenced in forward and reverse reactions for sequence confirmation. Ambiguous positions were verified against the original electropherograms.

The complete sequence of the ITS region for each specimen was then stored as a separated text file, and later deposited at GenBank (Table 1). Sequence boundaries of ITS1, 5.8S and ITS2 were determined following the submitted sequence data from GenBank (U88138, U88139, U88140, U88141). Carum carvi as an outgroup species was also from GenBank (AF077878).

Clustal X, version 1.8 (Thompson et al., 1997), was used for multiple alignment of complete sequences. Pairwise distances (sequence divergence) between taxa and base frequencies (G + C content) were determined using MEGA 3.1 (Kumar et al., 2004).

Phylogenetic analysis

Phylogenetic analyses were performed using PAUP*, versions 4.0b10 (Swofford, 2003). The maximally parsimonious (MP) trees were sought using the heuristic search option with the starting tree obtained by 100 random-addition replicates and tree bisection reconnection branch swapping. Characters were equally weighted and bootstrap probability of support for each branch was calculated by 1000 resamplings.

Maximum likelihood (ML) analysis was performed by quartet puzzling using TREE-PUZZLE 5.0 (Schmidt et al., 2002). The HKY model with gamma distribution approximated by four categories of substitution rate and other parameters with default options were used in the ML analysis.

Distance tree was obtained by neighbor-joining analyses, using two of the distance measures available in MEGA 3.1: Jukes and Cantor (1969) and Kimura 2-parameter (K2P) (Kimura, 1980) models. For K2P, substitution rates were assumed to follow gamma distribution. Neighbor-joining bootstrap values were calculated from 1000 replicates.

Results and discussion

Sequence analysis

All the 21 sequences were included in the final analysis. Some sequences obtained from the same population were identical (B. krylovianum) or showed little variation (B. aureum), but some samples for a single species such as B. chinense and B. marginatum var. stenophyllum varied obviously. The sequences of B. chinense collected from five different districts (three of Shangxi and two of Gansu) produced 27 mutations (four indels included), while B. marginatum var. stenophyllum from two different districts produced 20 mutations (two indels included). The result suggested that these mutations were possibly related to the species' broad distribution, especially to B. chinense.

Alignment of all sequences resulted in a matrix of 614 positions, including ITS1, 5.8S and ITS2. Characteristics of these sequences, including length, G + C content, number of indels and variable bases, are summarized in Table 2. ITS1 ranged from 213 bp (S4) to 219 bp (DY), and ITS2 from 226 bp (Y7 and 02) to 228 bp. The total length of ITS1 + ITS2 ranged from 441 bp to 447 bp. ITS1, with a maximum of 50.4% divergence across all taxa (20.1% in Bupleurum), is slightly shorter and more variable in length than ITS2, which has a maximum of 54.2% divergence across all taxa (14.2% in Bupleurum). The length and levels of variability of ITS1 and ITS2 accorded with those reported in other studies of Apiaceae (Downie et al., 1998; Katz-Downie et al., 1999). The 5.8S is 163 bp long in all taxa. As expected, the 5.8S gene showed little variation, with 5.1% divergence across all taxa (only 5% in Bupleurum). The overall of ITS divergence was approximately 14% within Bupleurum.

Traditionally, the main morphological differences for identifying these 11 local-used Chaihu are the characters observed from their original plants: the width of involucel bracts, the shape of leaves, the number of umbellules and the vitta number at the vallecula and commissure of fruits; and the characters from the major morphological features of their dried roots: the shape and texture of roots, straight or detached, the color of root and cut surface, with/without fiber-like petiole residues, rich in xylem fiber or not. But generally, the morphological criteria are often subtle and ambiguous. In B. chinense and B. yinchowense, it is hard to distinguish them according to their morphological resemblances. And, withal, all these phenotype characteristics inevitably change according to the environment and different processes, such as drying, heating or long-term storage. Under some circumstances, the herbs have to be ground into powder form before medication. This could make morphological identification impossible. As had been analyzed above, more steady genetic information can be inferred from ITS sequence, like B. chinense (S1) and B. yinchowense (G6) again, 15 mutations (including two intels) within their ITS sequences make the discrimination much easier when combining with the morphological observation. By comparing all the ITS sequences studied in this paper, it turned out that almost every species had their own specific variable bases; therefore, ITS sequence could serve confidently as informative traits of diagnosing these local-used Chaihu. Furthermore, the availability of their genetic sequences could also lead to the development of gene chip application in their identification.

Phylogenetic analysis

Parsimony analysis of 614 characters, including ITS1 and ITS2 and the 5.8S region, resulted in 14 retained MP trees, each 292 steps long, with a consistency index (CI) of 0.89 and a retention index (RI) of 0.83. The strict consensus of these trees, with accompanying bootstrap, is shown in Fig. 1.

Regardless of methods (parsimony, likelihood or neighbor joining), all phylogenetic analyses of ITS confirmed that Bupleurum is monophyletic. In the cluster Bupleurum, all the phylogenetic trees (Figs. 1 and 2) resulted in two major clades (A and B), with more than 99% bootstrap support. This result was congruent with their disjunct distribution in China. B. marginatum var. stenophyllum (clade B) is a species mainly produced in southwestern China, whereas others mostly distribute in the northwest.

Clade A was mainly divided into three minor clades (C, D and E), each of which with more than 78% bootstrap value. Contrary to Clade C, the phylogenetic relationship of the species in clade D did not correspond to the taxonomically delimited groups in previous classifications, according to what the phylogenetic trees (MP, ML, NJ trees) inferred. Clade D comprised two species (C1 and C41-C42) collected from Xinkiang, China; however, B. krylovianum (including C2, C3-1, C3-2, C3-3), which was obtained from Xinkiang as well, was not included in the same clade (clade D). Traditionally, the characteristics of involucel bracts and cauline leaves were the major identified characteristics of these three species. Unlike B. tianschanicum (C1) and B. krylovianum (C2), B. aureum (C4) had large involucel bracts, which were ovate or circular, and large cauline leaves, amplexicaul at base, cordate or with large ligules. It is for the first time that species from the Sinkiang area have been studied in genetic analysis, although there are many Bupleurum species being investigated (Neves and Watson, 2004). Therefore, their phylogenetic relationship within the whole genus Bupleurum does merit further study.

[FIGURE 1 OMITTED]

Clade E was constitutive of two species (B. chinense and B. yinchowense). They have very close morphological properties and ellipsoidal shape pollens (Shu and She, 2001; Pan, 2006). As analyzed in the sequences divergency, B. chinense varied obviously even in the same population.

The ML tree was obtained by TREE-PUZZLE 5.0 (Schmidt et al., 2002). The best results (not shown) were largely consistent with those from MP and NJ methods with respect to the major clades distinguished.

The NJ tree (Fig. 2) topology was not affected by different models of sequence evolution (Jukes-Cantor or K2P); however, slight differences were produced with higher gamma distribution or if substitution rates were set as equal. The NJ tree produced when assuming equal rates of substitution is similar to the MP tree (Fig. 1).

[FIGURE 2 OMITTED]

In all the MP, ML and NJ analyses, Bupleurum appears as a strongly supported monophyletic group. The division of the genus into two major clades (Figs. 1 and 2) is consistent among all trees. The main difference among the MP, ML and NJ trees is the position of clade E. This clade appears as the first branch in the MP tree (Fig. 1), but as sister clade to clade D in the NJ tree and in the ML tree, i.e., the relationship within clades was not so certain, hence it needs more samples and more analyses to demonstrate it much clearly.

The source status of Radix Bupleuri

Although B. chinense DC. and B. scorzonerifolium Willd. as the source of Radix Bupleuri (Chaihu) have been recommended in the Chinese Pharmacopoeia (Shao, 2005) for a long time, the roots of several species from Bupleurum genus frequently occurred in the prescriptions of Oriental Traditional Medicine. The other reason to result in such situation is the shrinking storage of the original plants of Radix Buplueri, such as B. scorzonerifolium Willd. By analyzing their genetic properties and phylogenetic relationships, we suggested that the species that have close genetic properties and phylogenetic relationships with official species could serve as new sources of the original plants of Radix Buplueri, after chemical analysis on the main effective ingredients--saikosaponins and volatile oils, etc. (Li et al., 1995; Yang et al., 2005; Pan et al., 2002). Based on the data obtained from phylogenetic analysis and phytochemical analysis on saikosaponin a, c, d, B. yinchowense could probably serve as the alternative source of Radix Bupleuri. In terms of the three species collected in Sinkiang, only little chemical research has been done to support their use as Radix Bupleuri; therefore, further study is needed to find more supporting data.

Acknowledgments

We are grateful to Dao-guang Zhang, Zhi-bin Xu and Feng-lin Liu for the specimens collecting. We thank Dr. Ze Zhang for advice on phylogenetic analysis. We also thank an anonymous reviewer and Prof. Dr. Wagner for helpful comments on the manuscript.

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Zhi-Ye Yang (a), Zhi Chao (c), Ke-Ke Huo (b), Hui Xie (a), Zhi-Peng Tian (b), Sheng-Li Pan (a,*)

(a) School of Pharmacy, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, People's Republic of China

(b) School of Life Science, Fudan University, Shanghai 200433, People's Republic of China

(c) Southern Medical University, Guangzhou 510515, People's Republic of China

Received 20 November 2006; accepted 28 February 2007

*Corresponding author. Tel: +8621 5423 7454.

E-mail address: slpan@shmu.edu.cn (S.-L. Pan).
Table 1. Accessions of Bupleurum species sampled for the ITS studies

Species Code name Specimen number

B. chinense (cultivated) S1 0308016(SHMU)
B. chinense S3 0308020(SHMU)
B. chinense S4 0300001(SHMU)
B. chinense (cultivated) G2 040721001(SHMU)
B. chinense G3 040724001(SHMU)
B. scorzonerifolium O1 0307001(SHMU)
B. smithii G4 040727001(SHMU)
B. commelynoideum var. G5 040728001(SHMU)
 flaviflorum
B. yinchouwense G6 040723001(SHMU)
B. sibiricum M1 0519(SHMU)
B. marginatum var. Y7 0506(SHMU)
 stenophyllum
B. marginatum var. O2 0308055(SHMU)
 stenophyllum
B. tianschanicum C1 20060812(SHMU)
B. krylovianum C2 20060813(SHMU)
B. krylovianum C3-1 20060816(SHMU)
B. krylovianum C3-2 20060817(SHMU)
B. krylovianum C3-3 20060818(SHMU)
B. aureum C41 20060814(SHMU)
B. aureum C42 20060815(SHMU)
B. longiradiatum DY --
Carum carvi GL --

Species Geographical origin GenBank Acc.

B. chinense (cultivated) Center Primary School of Dachi DQ285452
 Xiang, Zhenba county, Shangxi,
 China
B. chinense Yellow Lawn of Dachi Xiang, DQ285449
 Zhenba county, Shangxi, China
B. chinense Taibai mountain, Shangxi, China DQ285450
B. chinense (cultivated) Xiaolongshan forest area, DQ285448
 Tianshui city, Ganshu, China
B. chinense Maiji xiang, Beidao district, DQ285451
 Tianshui city, Ganshu, China
B. scorzonerifolium Kun ming, Yunnan, China DQ285465
B. smithii Yangzhai village, Mapo Xiang, DQ285455
 Yuzhong county, Ganshu, China
B. commelynoideum var. Baibao village, Mapo Xiang, DQ285456
 flaviflorum Yuzhong county, Ganshu, China
B. yinchouwense Baihua forestry center, DQ285454
 Xiaolongshan forest area,
 Ganshu, China
B. sibiricum Daqing Mountain, Inner DQ285457
 Mongolia, China
B. marginatum var. West mountain, Kunming city, DQ285468
 stenophyllum Yunnan, China
B. marginatum var. Yunnan, China DQ285466
 stenophyllum
B. tianschanicum Si Xiang, Tekes County, EF101155
 Sinkiang, China
B. krylovianum Hemu Xiang, Burqin county, EF101156
 Sinkiang, China
B. krylovianum Hemu Xiang, Burqin county, EF114436
 Sinkiang, China
B. krylovianum Hemu Xiang, Burqin county, EF114437
 Sinkiang, China
B. krylovianum Hemu Xiang, Burqin county, EF114438
 Sinkiang, China
B. aureum Hemu Xiang, Burqin county, EF101157
 Sinkiang, China
B. aureum Hemu Xiang, Burqin county, EF101158
 Sinkiang, China
B. longiradiatum South Korea AY551291
Carum carvi Russia AF077878

Note: The first 12 sequences were obtained in the authors' laboratory
before, and the last one acted as an outgroup species of this study.

Table 2. Sequence characteristics of the internal transcribed spacers
ITS1 and ITS2, and of the 5.8S subunit of nuclear rDNA of Bupleurum and
outgroup species Carum carvi

Sequence characteristics ITS1 5.8S ITS2

Length range in all taxa (bp) 213-219 163 223-228
Length range in Bupleurum (bp) 213-219 163 226-228
Mean length in Bupleurum (bp) 215.7 163 227.7
Length range of outgroup species 215 163 223
 (bp)
Aligned length (bp) 223 163 228
G + C content range (mean) in 27.5-30.2 30.9-32.8 25.4-26.8
 Bupleurum (%) (28.8) (32.6) (25.9)
G + C content range (mean) in all 27.5-30.2 30.9-32.8 23.1-26.8
 taxa (%) (28.8) (32.6) (25.8)
Sequence divergence in Bupleurum (%) 0-20.1 0-5 0-14.2
Sequence divergence in all taxa (%) 0-50.4 0-5.1 0-54.2
Size of intels in Bupleurum (bp) 1-2 0 1-2
Size of intels in all taxa (bp) 1-2 0 1-2
Number (and %) of constant sites 114 (51.1) 150 (92.0) 127 (55.7)
Number (and %) of variable sites 106 (47.5) 13 (7.9) 100 (43.9)
Number (and %) of parsimony 46 (20.6) 3 (1.8) 38 (16.7)
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Title Annotation:internal transcribed spacers
Author:Yang, Zhi-Ye; Chao, Zhi; Huo, Ke-Ke; Xie, Hui; Tian, Zhi-Peng; Pan, Sheng-Li
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:9CHIN
Date:Jun 1, 2007
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