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Lepidium Meyenii Walp.) and Its Adulterants by a DNA-Barcoding Approach Based on the ITS Sequence

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Lepidium Meyenii Walp.) and Its Adulterants by a DNA-Barcoding Approach Based on the ITS Sequence Chinese Journal of Natural Chinese Journal of Natural Medicines 2015, 13(9): 06530659 Medicines doi: 10.3724/SP.J.1009.2015.00653 ·Research articles· Identification of maca (Lepidium meyenii Walp.) and its adulterants by a DNA-barcoding approach based on the ITS sequence CHEN Jin-Jin1, 2, ZHAO Qing-Sheng1, LIU Yi-Lan1, 2, ZHA Sheng-Hua1, 2, 3, ZHAO Bing1* 1 National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China; 2 University of Chinese Academy of Sciences, Beijing 100049, China; 3 Beijing Tong Ren Tang Health Pharmaceutical Co., Ltd., Beijing 100062, China Available online 20 Sep., 2015 [ABSTRACT] Maca (Lepidium meyenii) is an herbaceous plant that grows in high plateaus and has been used as both food and folk medicine for centuries because of its benefits to human health. In the present study, ITS (internal transcribed spacer) sequences of forty-three maca samples, collected from different regions or vendors, were amplified and analyzed. The ITS sequences of nineteen potential adulterants of maca were also collected and analyzed. The results indicated that the ITS sequence of maca was consistent in all samples and unique when compared with its adulterants. Therefore, this DNA-barcoding approach based on the ITS sequence can be used for the molecular identification of maca and its adulterants. [KEY WORDS] Lepidium meyenii; Maca; ITS; DNA-barcoding; Molecular identification [CLC Number] R282.5 [Document code] A [Article ID] 2095-6975(2015)09-0653-07 even corn have been found to be used to adulterate into maca Introduction products [13], which would have a adverse effect on the Maca (Lepidium meyenii Walp.), a Peruvian plant of the healthy development of the maca industry. Therefore, a rapid, Brassicaceae family, has been used as both food and specific, and accurate method is needed to identify maca and traditional medicine in the Andes for a long time [1-2]. its adulterants in commercial samples. [13-15] Biochemical and pharmacological studies have shown that There are a number of reports mentioning the maca has various health-related properties, including beneficial identification of maca by phenotypic or phytochemical effects on fertility [3], sexual behavior [4], energy [5], memory [6], characteristics. However, phenotypic characteristics might be osteoporosis [7], prostate [8], and skin [9]. Therefore, maca has totally destroyed during post-harvest processing, and chemical been introduced to Asia, Europe and North America during components are highly affected by the environment, [16-17] the last decade, with an increasing consumption worldwide post-harvest processing, and storage . Therefore, it is [10-11]. Since 2002, maca has been transplanted successfully in necessary to develop a more accurate, reliable method for maca Yunnan, Shanxi, and Xinjiang provinces of China, and has identification. formed large-scale plantings [12]. With the rapid development DNA barcoding is a new molecular diagnostic technique of the commercialization of maca, turnip, radish, potato, and for identifying species. ITS (internal transcribed spacer) sequences had been proven to be a very useful DNA barcode for identifying species in seed plants [18-19]. However, until now, there has been no report on the identification of maca [Received on] 05-Jun-2014 based on its ITS sequences. As considerable intra-specific [Research funding] This work was supported by the High-Tech Research Program–863 Program in China (No. 2012AA021702-4). variations in ITS sequences have been reported in many plant [20-21] [*Corresponding author] Tel/Fax: 86-10-62574372, E-mail: bzhao@ species , there also are different species sharing the same [22] ipe.ac.cn ITS . In the present study, the ITS of forty-three maca These authors have no conflict of interest to declare. samples from different locations were amplified and – 653 – CHEN Jin-Jin, et al. / Chin J Nat Med, 2015, 13(9): 653659 sequenced, and the intra-species variations in ITS sequences originating from different locations in China(Table 1). All the were analyzed.. The inter-species variation of ITS sequences specimens were authenticated by Prof. WANG Yun-Chun between maca and its adulterants were also assessed. The from Institute of Process Engineering, Chinese Academy of results from the present study provided an accurate method Sciences, Beijing, China. Meanwhile, macamides, the marker for molecular identification of maca. compounds of maca, were detected using HPLC (Shimadzu LC-20AT, Kyoto, Japan) to authenticate these specimens Materials and Methods further [23]. The sample of ‘yuangen’ (local name of turnip) Materials was obtained from Qinghai Province, China. In addition, the Forty-three maca samples were examined in the present ITS sequences of fifteen other Lepidium species and four study, including four powders imported from Peru, eight fresh common adulterants were collected from GenBank roots, sixteen dried slices or fruits, and fifteen essential tablets (http://www.ncbi.nlm.nih.gov/genbank/) as shown in Table 2. Table 1 Maca samples used in the present study Sample Sample Type Source Type Source No. No. 1 Fresh root Shangri-La County, Yunnan Province 25 Essential tablet Tiantian Biotech Co., Ltd. Taiyuan Biological Technology Development Co.,, 2 Fresh root Yongshan County, Yunnan Province 26 Essential tablet Ltd. 3 Fresh root Dongchuan City, Yunnan Province 27 Essential tablet Lijiang Green Enhancer Biological Plant Co., Ltd. 4 Fresh root Dahai Village, Huize County, Yunnan Province 28 Essential tablet Yunnan Tiangen Maca Industry Co., Ltd. Yunping Village, Dongchuan City, Yunnan Lijiang Baisuifang Biotechnology Development 5 Fresh root 29 Essential tablet Province (3 600 m) Co., Ltd. 6 Fresh root Dongchuan City, Yunnan Province 30 Essential tablet Yunnan Guzhiji Nourishment Co., Ltd. Yunping Village, Dongchuan City, Yunnan 7 Fresh root 31 Essential tablet Lijiang Longjian Biotechnology Co., Ltd. Province (3 400 m) Hongqi Village, Shangri-La County, Yunnan Shantou Shuangjun Biological Engineering Co., 8 Fresh root 32 Essential tablet Province Ltd. Hongqi Village, Shangri-La County, Yunnan Yunnan Tianyuan Huama Bio-technology Co., Ltd. 9 Dried fruit 33 Essential tablet Province (pure maca) Yunnan Tianyuan Huama Bio-technology Co., Ltd. 10 Dried fruit Pamirs, Xinjiang Uygur Autonomous Region 34 Essential tablet (Ginseng extract added) Yunnan Tianyuan Huama Bio-technology Co., Ltd. 11 Dried fruit Nakhi Garden, Taobao online shop 35 Essential tablet (Almond extract added) Lijiang Evergreen Maca Biotechnology R&D Co., 12 Dried fruit Shanxi Guhua Ecological Technology Co., Ltd. 36 Essential tablet Ltd. Yunnan Maca Manor Agricultural Science and 13 Dried fruit 37 Essential tablet Yunnan MaiKa Biotechnology Co., Ltd. Technology Co., Ltd. 14 Dried slice Lijiang Longjian Biotechnology Co., Ltd. 38 Essential tablet Yunnan MaiKa Biotechnology Co., Ltd. 15 Dried slice Yunnan Guzhiji Nourishment Co., Ltd. 39 Essential tablet La Molina, Taobao online shop 16 Dried slice Yunnan Tiangen Maca Industry Co., Ltd. 40 Powder Life-flo health 17 Dried slice Tiantian Biotech Co., Ltd. 41 Powder Amax International Co., Ltd. Lijiang Baisuifang Biotechnology 18 Dried slice 42 Powder 2 Dragon Trading Co., Ltd. Development Co., Ltd. Lijiang Green Enhancer Biological Plant Co., 19 Dried slice 43 Powder Kindly provided by Prof. LI Yu-Lai Ltd. 20 Dried slice Taiyuan Biol. Technol. Development Co., Ltd. Lijiang Evergreen Maca Biotechnol. R&D Co., 21 Dried slice Ltd. 22 Dried slice Yunnan Tianyuan Huama Biotechnol. Co., Ltd. 23 Dried slice Kunming Hongyi Biotechnology Co., Ltd. 24 Dried slice Yunnan MaiKa Biotechnology Co., Ltd. DNA extraction, amplification, and sequencing concentration on 0.8% agarose gels stained with Goldview The materials were ground into powders for total DNA (SBS Genetech Co., Ltd., Beijing, China). Then the DNA extraction. Total DNA was extracted from maca samples (0.2 samples were diluted to approximately 20 ng·μL−1 as working g) with the GenStar Plant DNA Kit according to the solution. manufacturer’s instructions (GenStar Biosolutions Co., Ltd., The PCR amplification was performed in a volume of Beijing, China). The DNA concentrations were estimated by 25 μL containing 2.5 μL 10 × buffer (with Mg2+), 0.5 mmol·L−1 comparing band intensity with the DNA marker of known dNTP, 1.5 U Taq DNA polymerase, 10 pmol·L−1 each primer, and – 654 – CHEN Jin-Jin, et al. / Chin J Nat Med, 2015, 13(9): 653659 Table 2 Overview of the studied species and information of their ITS sequences GenBank a Taxonomy Length (bp) Variations GC content No. Name accession ID b c (%) No. of ITS ITS ITS1 ITS2 5.8S Sites Rate 1 L. meyenii 153348 JX908826 621 267 190 164 - - 55.7 2 L. africanum 153324 AY254529 621 267 190 164 25 4.0 56.2 3 L. apetalum 153459 JF976770 621 267 190 164 16 2.6 55.6 4 L. bonariense 153333 HM134831 620 267 189 164 7 1.1 55.6 5 L. campestre 65351 AF055197 620 267 189 164 46 7.4 55.3 6 L. draba 153317 EF367913 620 267 189 164 45 7.2 55.0 7 L. lacerum 743983 FN821676 621 267 190 164 24 3.9 55.4 8 L. montanum 153349 EF367869 622 267 191 164 16 2.6 55.0 9 L. papilliferum 454201 EF368005 622 267 191 164 16 2.6 55.0 10 L. perfoliatum 153358 EF368007 619 266 189 164 51 8.2 55.1 11 L. phlebopetalum 153359 AY254528 617 267 186 164 103 16.6 51.9 12 L. ruderale 153365 JF976776 621 267 190 164 16 2.6 55.6 13 L. sisymbrioides 106949 DQ997561 622 268 190 164 13 2.1 55.5 14 L. solandri 413682 DQ997557 622 268 190 164 13 2.1 55.5 15 L. squamatum 228869 AY254533 623 273 186 164 110 17.7 49.6 16 L. virginicum 59292 HM134830 621 267 190 164 5 0.8 55.1 17 Brassica rapa 51350 GQ268060 608 257 187 164 126 20.3 51.8 18 Raphanus sativus 41679 AY746462 620 267 189 164 120 19.3 55.5 19 Zea mays 4579 AF019817 597 213 220 164 332 53.5 67.7 20 Solanum tuberosum 4113 KF022369 642 235 254 153 240 38.6 63.2 a Variations between maca and the other species; b No.
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