Molecular Authentication of Geo-Authentic Scrophularia Ningpoensis*
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Chen et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2011 12(5):393-398 393 Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology) ISSN 1673-1581 (Print); ISSN 1862-1783 (Online) www.zju.edu.cn/jzus; www.springerlink.com E-mail: [email protected] Molecular authentication of geo-authentic Scrophularia ningpoensis* Chuan CHEN1,2, Li-na DUAN1,2, Xiao-long ZHOU3, Bing-long CHEN3, Cheng-xin FU†‡1,2 (1Key Laboratory of Conservation Biology for Endangered Wildlife of Ministry of Education, College of Life Sciences, Zhejiang University, Hangzhou 310058, China) (2Laboratory of Systematic and Evolutionary Botany and Biodiversity, Institute of Plant Sciences and Conservation Center for Gene Resources of Endangered Wildlife, Zhejiang University, Hangzhou 310058, China) (3Pan’an Institute of Traditional Chinese Medicine, Pan’an 322300, China) †E-mail: [email protected] Received May 16, 2010; Revision accepted Nov. 15, 2010; Crosschecked Apr. 12, 2011 Abstract: Scrophularia ningpoensis has long been used in the Chinese Materia Medica for inflammation. Like other herbal medicines, S. ningpoensis collected from different localities may considerably differ in their therapeutic efficacy, and the one grown in Zhejiang Province is recognized as geo-authentic. However, it is difficult to confirm the geo- graphical authenticity by similar morphological characteristics. In the present study, inter-simple sequence repeat (ISSR) markers were conducted to detect S. ningpoensis from different origins. A 1 259-bp fragment amplified by primer UBC874 was found only in geo-authentic ones. By cloning and sequencing that specific band, sequence characterized amplified region (SCAR) markers were designed to distinguish geo-authentic S. ningpoensis from others. This is a rapid and easy method that can be used to identify the geographical authenticity of S. ningpoensis. Key words: Inter-simple sequence repeat (ISSR), Sequence characterized amplified region (SCAR), Scrophularia ningpoensis, Chinese Materia Medica, Traditional Chinese medicine doi:10.1631/jzus.B1000179 Document code: A CLC number: Q949.95 1 Introduction buncles (Reid, 1996), and constipation (Yen, 1992). Recent research revealed that this medicinal species, Scrophularia ningpoensis Hemsley, known as which has high antiangiogenic activity, also can be “Zhexuanshen”, used in the Chinese Materia Medica used as an anticancer agent (Sagar et al., 2006). (CMM), belonging to the family Scrophulariaceae, The major bioactive components of S. ning- has a long history of widespread use in China (Ka- poensis have been reported to be harpagoside, an- jimoto et al., 1989; Fernández et al., 1996; Miyase goroside C, acteoside, and cinnamic acid (Liu et al., and Mimatsu, 1999; Giner et al., 2000). S. ningpoensis 1995; Miyazawa et al., 1998; de Santos Galíndez et named by Forbes and Hemsley (1890) based on the al., 2002; Díaz et al., 2004). However, determined specimens collected in Tiantong County, Ningbo City by bioactive components, the quality and efficacy of of Zhejiang Province, is endemic to China and now is CMM depend significantly on its geographical ori- widely cultivated in China as well. It is used to treat gin (Woo et al., 1999). The chemical differences of inflammation, laryngitis, tonsillitis, abscesses of car- Radix Scrophulariae among various production re- gions were demonstrated to different extents. That grown in Zhejiang Province has better medicinal ‡ Corresponding author * Project supported by the National Basic Research Program (973) of effect and is recognized as geo-authentic (Wang and China (No. 2007CB411600), the National Natural Science Foundation Wang, 2007). Several methods based on high- of China (No. 31070205), and the Key Agricultural Program of Pan’an County of Zhejiang Province, China (No. 2005ZB01) performance liquid chromatography (HPLC) or © Zhejiang University and Springer-Verlag Berlin Heidelberg 2011 combined with liquid chromatography-electrospray 394 Chen et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2011 12(5):393-398 ionisation-mass spectrometry (LC-ESI-MS) were cific fragment only in populations originated from developed to quality and quantify the bioactive Zhejiang Province. Then based on that specific frag- compounds in S. ningpoensis (Liu et al., 2007; Zhu ment, we designed a pair of diagnostic primers to et al., 2008). Our previous studies on HPLC finger- identify S. ningpoensis of Zhejiang Province. prints of S. ningpoensis have revealed that three of the four major bioactive compounds, harpagoside, an- goroside C, and cinnamic acid, were largely variable 2 Materials and methods among samples collected from different regions 2.1 Plant materials and DNA extraction (Yang et al., 2010). The materials from Zhejiang Province produced the highest contents of the bioactive A total of 189 samples of S. ningpoensis origi- compounds, which have the most anti-inflammatory nated from seven different Provinces were used in this effect (Yang et al., 2010). Medicinal parts (roots) of S. study, wherein 85 individuals from three geographi- ningpoensis originating from different geographical cal origins were surveyed for ISSR and all samples areas share similar morphological characters. There- were tested by designed SCAR primers (Table 1). The fore, it is very difficult to distinguish S. ningpoensis voucher specimens were deposited in the Herbarium of Zhejiang from others by using morphological of the Zhejiang University (HZU). DNA was isolated methods. from silica-gel dried leaf by a modified hexadecyl The quality control of CMM is important for safe trimethyl ammonium bromide (CTAB) method and effective use (Chung et al., 2006). Medicinal (Doyle, 1991). plants collected from different localities are consid- erably different in their therapeutic efficacy (Woo Table 1 Sampling localities and codes of S. ningpoensis Locality Sample et al., 1999). Recent developments in molecular bi- Originated location ISSR ology techniques make DNA markers be useful for code size Yaochuan, Pan’an County, YC 15 √ the identification and standardization of CMM (Yang Zhejiang et al., 2001). Our group has established species- Renchuan, Pan’an County, RC 15 √ specific polymerase chain reaction-restriction frag- Zhejiang Guangmingcun, Pan’an County, ment length polymorphism (PCR-RFLP) methods for GM 15 √ Zhejiang identifications of Actinidia macrosperma and Sino- Shanghu, Pan’an County, PA 10 √ podophyllum hexandrum (Gong et al., 2006; Zhao et Zhejiang al., 2007), and sequence characterized amplified re- Xianju County, Zhejiang XJ 10 √ gion (SCAR) markers for Sinocalycanthus chinensis Hubei HB 10 √ (Ye et al., 2006). Shanxi SX 10 √ As to the identification of geo-authentic CMM, Jinfo Mountain, Chongqing JF 19 chemical fingerprints are the most used method, but the Jingang Mountain, Jiangxi JX 15 whole genome patterns are proven to be useful, accu- Pingjiang County, Hunan HN 15 rate, and convenient as well. For instance, different Jiuhua Mountain, Anhui AH 15 arbitrarily primed (AP)-PCR fingerprints are used to Tianmu Mountain, Zhejiang TM 15 distinguish samples of Astragalus membranaceus Dapan Mountain, Zhejiang DP 10 originated from different localities (Yip and Kwan, Matou County, Jiangxi MT 15 2006). For Codonopsis pilosula, AP-PCR and random amplification of polymorphic DNA (RAPD) finger- 2.2 ISSR-PCR amplification prints revealed different patterns according to different geographic origins (Zhang et al., 1999). Similarly, Out of 100 ISSR markers (UBC primer set No. 9, Vitex rotundifolia samples from 14 different regions Biotechnology Laboratory, University of British were divided by inter-simple sequence repeat (ISSR) Columbia, Vancouver, Canada; http://www.ubc.ca/), markers (Hu et al., 2007). In this study, we used ISSR twelve primers (Table 2) that produced the strongest, method to detect the whole genome of S. ningpoensis clearest, and most reproducible bands were selected from different geographical origins and found a spe- for further study. A 25 μl PCR amplification run Chen et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2011 12(5):393-398 395 contained 25 ng of genomic DNA, 2.5 μl 10× buffer, (Version 4.0.5 Gene Codes Corporation, Ann Arbor, 2 mmol/L MgCl2, 0.2 mmol/L dNTPs, 0.4 μmol/L of MI, USA). primers, and 2.0 U Taq DNA polymerase (Shanghai 2.4 Primer design and SCAR-PCR Sangon Biotechnology Co. Ltd., Shanghai, China). ISSR-PCR amplifications were performed in a Ge- Based on the sequencing results, a pair of prim- neAmp® PCR System 9700 thermal cycler (Applied ers (Table 3) was designed using the software of Biosystems, Foster City, USA) with programme: 94 °C primer-primer 5.0 (Premier Biosoft International; for 4 min; 45 cycles of 94 °C for 30 s, 49.4–65.0 °C Palo Alto, CA, USA) and synthesized by Shanghai for 45 s, and 72 °C for 1.5 min; 72 °C for 10 min (the Sangon Biotechnology Co., Ltd. The diagnostic PCR specific annealing temperature for every ISSR primer by primers CC874u and CC874d was carried out by is in Table 2). For every PCR run, a negative control programme: 94 °C for 5 min; 35 cycles of 94 °C for without template DNA was also included. And every 30 s; 59 °C for 45 s; 72 °C for 1.5 min; 72 °C for PCR amplification was repeated at least twice. PCR 10 min. The reaction mixture is the same as ISSR- products were electrophoresed on 1.5% (v/v) agarose PCR, containing 0.2 μmol/L of the upper primer and gels along with DNA Marker DL2000 (TaKaRa Bio- 0.2 μmol/L of the lower primer. PCR products were technology Co. Ltd., Dalian, China), then