June 2006 Biol. Pharm. Bull. 29(6) 1207—1211 (2006) 1207 Sequence Analysis of Chloroplast chlB Gene of Medicinal Ephedra Species and Its Application to Authentication of Ephedra Herb a a b b b Yahong GUO, Ayako TSURUGA, Shigeharu YAMAGUCHI, Koji OBA, Kasumi IWAI, c,1) ,a Setsuko SEKITA and Hajime MIZUKAMI* a Graduate School of Pharmaceutical Sciences, Nagoya City University; 3–1 Tanabe-dori, Mizuho-ku, Nagoya 467–8603, Japan: b Research and Development Department, Asgen Pharmaceutical Co., Ltd.; 2–28–8 Izumi, Higashi-ku, Nagoya 461–8531, Japan: and c Tsukuba Medicinal Plant Research Station, National Institute of Health Sciences; 1 Hachimandai, Tsukuba, Ibaraki 305–0843, Japan. Received December 26, 2005; accepted February 15, 2006 Chloroplast chlB gene encoding subunit B of light-independent protochlorophyllide reductase was amplified from herbarium and crude drug specimens of Ephedra sinica, E. intermedia, E. equisetina, and E. przewalskii. Se- quence comparison of the chlB gene indicated that all the E. sinica specimens have the same sequence type (Type S) distinctive from other species, while there are two sequence types (Type E1 and Type E2) in E. equisetina. E. intermedia and E. prezewalskii revealed an identical sequence type (Type IP). E. sinica was also identified by di- gesting the chlB fragment with Bcl I. A novel method for DNA authentication of Ephedra Herb based on the se- quences of the chloroplast chlB gene and internal transcribed spacer of nuclear rRNA genes was developed and successfully applied for identification of the crude drugs obtained in the Chinese market. Key words chloroplast chlB; DNA authentication; Ephedra Herb; polymerase chain reaction-restriction fragment length poly- morphism Ephedra Herb is an important crude drug which has been nucleotide deletions were present in the trnL/trnF spacer of used in Chinese and Japanese traditional (Kampo) medi- E. sinica.5) Thus, neither ITS of rDNA nor the trnL–trnF re- cines. Although about 35 species belonging to the genus gion is suitable as a DNA marker to identify medicinal Ephedra are distributed in Eurasia, North America, and Ephedra species by itself. South America, only a few species contain pharmacologi- The chlB gene encodes a subunit B of light-independent cally active ephedrine alkaloids and are used for medicinal photochlorophyllide reductase that catalyzes reduction of purposes. The Japanese Pharmacopoeia defines Ephedra protochlorophyllide to chlorophyllide in chlorophyll biosyn- Herb as dried aerial parts of Ephedra sinica, E. intermedia thesis, and is located in the chloroplast genome of gym- and E. equisetina.2) Morphological and anatomical character- nosperms, algae and photosynthetic bacteria but not in an- istics have long been investigated to identify and/or discrimi- giosperms.7) The chlB has a higher evolutionary rate than the nate these species since Konoshima described the morphol- rbcL (large subunit of ribulose bisphosphate carboxylase/ ogy of E. equisetina collected in China.3) However, because oxygenase) gene which has been widely used as a molecular of their relatively simple organization it is sometimes diffi- marker in plant phylogenetic analyses,8) and might be suit- cult to identify original species of Ephedra Herb, especially able as a DNA marker for identification and/or discrimina- when they do not bear flowers or fruits. Chemical identifica- tion of Ephedra species and the crude drugs derived there- tion of Ephedra plants mainly based on their alkaloid compo- from. sitions or HPLC fingerprints has also been attempted,4) but In the present investigation we analyzed the nucleotide se- such chemical traits are inevitably affected by environmental quences of the chloroplast chlB gene amplified from the and intra-specific variations. herbarium and crude drug specimens of Ephedra plants and For correct identification of biological materials, DNA established a novel protocol for DNA authentication of profiling (DNA-based polymorphic assay) has several advan- Ephedra Herb based on their chlB and rDNA ITS 1 se- tages over morphological and chemical analyses because quences. Furthermore, we showed that the protocol could be genotypes rather than phenotypes are directly assayed, and so successfully applied to identify the original species of that the results are not affected by environmental factors. Ephedra Herb obtained in the Chinese market. Long et al. compared nucleotide sequences of intergenic transcribed spacer (ITS) 1 and ITS2 of nuclear ribosomal MATERIALS AND METHODS DNA (rDNA) and of the chloroplast trnL–trnF region includ- ing trnL intron, 3Ј-exon of trnL, and trnL/trnF spacer from Herbarium and Crude Drug Specimens Ten herbar- eight Ephedra species.5) They found that the ITS sequence of ium specimens and 22 crude drug specimens were selected E. sinica was identical with that of E. intermedia, whereas from the collection of Central Research Laboratories, there were several polymorphic sites between these species Tsumura & Co. (Table 1). Their morphological features were and E. przewalskii. However, most of these polymorphic sites re-evaluated based on the anatomical traits such as presence were later found to be subjected to intra-specific variation or absence of pith and of fiber bundles in the cortex and within either E. intermedia or E. przewalskii.6) As for the abundance of cuticles in the epidermis to confirm their chloroplast genome there were no polymorphic nucleotide species. The pharmacognostical evaluation of the samples sites in the trnL intron among E. sinica, E. intermedia and E. was performed by Drs. H. Yamaji, K. Kondo and S. Tera- przewalskii. The only difference in these species was that two bayashi of Central Research Laboratories, Tsumura & Co. ∗ To whom correspondence should be addressed. e-mail: [email protected] © 2006 Pharmaceutical Society of Japan 1208 Vol. 29, No. 6 Table1. Herbarium and Crude Drug Specimens Used in the Present Investigation Sample Plant Voucher Place of Date of Herbarium specimen No. name ID collection collection or crude druga) 1 Ephedra sinica THS 41925 Ibaraki, Japan (cultivation) 1999.6 HS 2 THS 42471 Shanxi 1999.8 HS 3 THS 43342 Liaoning 2000.8 HS 4 THS 42443 Liaoning 1984.9 HS 5 THS 42440 Liaoning 1985.6 HS 6 THS 42532 Liaoning 1999.9 HS 7 TMS 10069 Jilin 1984.12 CD 8 TMS 11094 Inner Mongolia 1987.12 CD 9 TMS 11624 Jilin 1989.6 CD 10 TMS 13963 Liaoning 1994.1 CD 11 TMS 14139 Inner Mongolia 1995.6 CD 12 TMS 14697 Inner Mongolia 1996.4 CD 13 TMS 16929 Shanxi 1999.8 CD 14 TMS 8739 Inner Mongolia 1983.6 CD 15 E. intermedia THS 32595 Gansu 1988.9 HS 16 TMS 14695 Qinghai 1996.4 CD 17 TMS 16404 Gansu 1998.4 CD 18 TMS 8585 Gansu 1982.12 CD 19 TMS 8586 Gansu 1982.12 CD 20 TMS 8587 Gansu 1982.12 CD 21 TMS 8686 Gansu 1983.6 CD 22 TMS 8687 Heilongjiang 1983.6 CD 23 TMS 17483 Heilongjiang 2000.1 CD 24 E. equisetina THS 40637 Xinjiang 1997.8 HS 25 TMS 10135 Russia 1985.1 CD 26 TMS 16416 Russia 1983.1 CD 27 TMS 6874 Shaanxi 1982.2 CD 28 TMS 8378 Russia 1982.1 CD 29 E. przewalskii THS 32593 Gansu 1988.9 HS 30 THS 40638 Xinjiang 1997.8 HS 31 TMS 6469 Xinjiang 1989.6 CD 32 TMS 8314 Gansu 1984.12 CD a) HSϭherbarium specimen; CDϭcrude drug. and voucher samples of these specimens were deposited in Central Research Laboratories, Tsumura & Co. For DNA au- thentication, 21 crude drug samples were obtained in the Chinese market during the period between 1993 and 2003. The voucher samples of the crude drugs were stored in the Research and Development Department, Asgen Pharmaceuti- cal Co., Ltd. Preparation of DNA Total DNA was prepared from the powdered samples (about 40 mg) using a DNeasy Plant Mini Kit (Qiagen). DNA content in the preparation was estimated by a DyNA Quant 200 Fluorometer (Amersham) using calf thymus DNA (Sigma) as a standard. Amplification of chlB Gene Polymerase chain reaction Fig. 1. Sequence Strategy of the Chloroplast chlB Genes from Ephedra (PCR) primers (1F and 4R) were designed based on the chlB Species gene sequence of E. altissima retrieved from the DDBJ/ Primers 1F to 4F and 1R to 4R indicate the annealing position of the forward and re- EMBL/Genebank Nucleotide Sequence Database (accession verse, respectively, primers for PCR amplification of the chlB gene and its fragments. no. U21315) so that the whole open reading frame (ORF) of the chlB gene can be amplified. In addition, the 1.5 kb chlB GTTTTCCCAGTCACGACACTGTTGTTTTTGGTGA- ORF was also divided into four partially overlapped frag- TGC-3Ј; 1Rϭ5Ј-ATTTAGGTGACACTATAGAATACCC- ments (Fragment 1 to 4) of about 0.5 kb and each fragment GATGATATTTACAGAAGG-3Ј;2Rϭ5Ј-ATTTAGGTGA- was individually amplified using internal primers (2F, 3F, 4F, CACTATAGAATACTGAAAACCAAACAGCTTGGG-3Ј; 1R, 2R, and 3R) designed based on the chlB sequence deter- 3Rϭ5Ј-ATTTAGGTGACACTATAGAATACTCGTACCC- mined. The nucleotide sequences of the PCR primers were as CATAAAAGGACG-3Ј; 4Rϭ5Ј-ATTTAGGTGACACTATA- follows: 1Fϭ5Ј-GTTTTCCCAGTCACGACATGAAATT- GAATACCAATTGTAATCTTAACTACACCC-3Ј. The un- AGCTTATTGGAT-3Ј; 2Fϭ5Ј-GTTTTCCCAGTCACGAC- derlined sequence in the forward and reverse primers shows ATTCAAGCAGCAGATAGGAC-3Ј; 3Fϭ5Ј-GTTTTCCC- the M13 universal forward primer and SP6 primer sequence, AGTCACGACTCGGTGAATCCTTTTGCTTC-3Ј; 4Fϭ5Ј- respectively, for direct sequencing of the PCR products. The June 2006 1209 approximate positions where the primers anneal to the tem- and the filtrate was subjected to HPLC column (Develosil plate DNA are shown in Fig. 1. ODS-5, 4.6ϫ150 mm, Nomura Chemicals) with isocratic The PCR mixture contained 10 mM Tris–HCl (pH 8.8), elution by 27 mM sodium lauryl sulfate/acetonitrile/phos- 25 mM potassium chloride, 5 mM ammonium sulfate, 2 mM phoric acid (640 : 360 : 1). Flow rate was 1.3 ml/min and the magnesium sulfate, 0.2 mM each dNTP, 0.4mM of each elution was monitored at 210 nm.