Authentication of Pinellia ternata and its Adulterants Based on PCR with Specific Primers

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

Tubers of Pinellia ternata are one of the well known traditional Chinese medicines. According to the Chinese Pharmacopoeia, the remedy is commonly used as an antitussive and expectorant. The shapes of young tubers from species of P. ternata are similar to those of P. pedatisecta and Arisaema heterophyllum, but different in medicinal properties. In order to provide molecular evidence for genuine origin identification of P. ternata species, the mannose-binding lectin sequences of P. ternata and its adulterants P. pedatisecta and A. heterophyllum were cloned using genomic walker technology. Based on the sequence analyses, we designed a pair of species-specific primers to authenticate P. ternata. For PCR-selective restriction (PCR-SR), we identified two distinctive sites which can be recognized by the restriction endonucleases BamHI and NcoI in the open reading frame sequences of P. ternata, P. pedatisecta and A. heterophyllum. Our results indicate that the methods of PCR and PCR-SR are effective, accurate and applicable for identification of the bulbs of P. ternata.

Pinellia ternata, P. pedatisecta and Arisaema heterophyllum are toxic traditional Chinese medicines (TCM) that all come from the Araceae family [1]. The tubers of P. ternata, P. pedatisecta and A. heterophyllum were designated Banxia, Huzhang and Tiannanxing in the China Pharmacopoeia (2000) [2]. P. ternata is one of the most important TCM to cure cough, and it is an expectorant, antitussive and anti-vomiting agent [3]. P. pedatisecta and A. heterophyllum, often misused as P. ternata in China due to the morphological similarity, are different in medicinal properties. Obviously, the accurate identification of the tuber of P. ternata is a prerequisite for the quality control of TCM [4], [5].

A great deal of research has been done to evaluate the quality of P. ternata by means of morphology, histology, chemistry and biochemistry, but these methods do not allow us to ensure the dependability of quality control [6], [7]. As a result of recent developments in nucleotide sequencing technology, the polymerase chain reaction (PCR)-based DNA direct sequencing has become one of the most frequently utilized molecular approaches with regard to phylogenetic relationships and species identification of organisms [8]. In this approach, small amounts of DNA are amplified by PCR and the reaction products are analyzed by electrophoresis and sequencing. In recent years, the ribulose 1,5-bisphosphate carboxylase large subunit (rbcL) sequence from chloroplast genome and ribosomal RNA (rRNA) gene coding region or repeating unit sequence from nuclear genome has been used to authenticate P. ternata [9], [10], [11], [12], [13], [14]. However, the rbcL sequence and rRNA gene coding region or repeating unit sequence are highly conserved across the plant kingdom. The PCR amplification of these sequences in many plant species, such as Panax ginseng and P. ternata, produces products of the same sizes [13]. Therefore, the reliability and application of these methods to distinguish P. ternata from its adulterants are limited.

In this study, we report a validation method using a special encoding gene sequence as molecular marker for distinguishing P. ternata from its adulterants. We cloned the mannose-binding lectin gene from P. ternata (pta), A. heterophyllum (aha) and P. pedatisecta (ppa), respectively. Comparative sequence alignment revealed that the open reading frame (ORF) region sequences of mannose-binding lectins from the three species were highly homologous, but their 5′ and 3′ flanking region sequences were not homologous.

Based on the analysis of the three species’ mannose-binding lectin sequences, four gene-specific primers, P1 and P2, P3 and P4 (marked with arrows in Fig. [1], Table [1]), were synthesized for the amplification of the specific sequence of pta, and for the amplification of the ORF region sequences of pta, ppa and aha, respectively. A single fragment of about 750 bp in length was amplified by PCR from the three species using primers P3 and P4. However, a single fragment of about 1000 bp in length was amplified only by PCR from P. ternata using primers P1 and P2.

From the analysis of restriction maps in the mannose-binding lectins from the three species, only one restriction site of endonuclease BamHI (G*GATCC) was present in the mannose-binding lectin of P. ternata. Two restriction sites of endonucleases BamHI and NcoI (C*CATGG) were present in the mannose-binding lectin from P. pedatisecta, which were not present in the mannose-binding lectin of A. heterophyllum. The PCR products amplified with primers P3 and P4 were digested with BamHI and NcoI, which resulted in two fragments sized 475 bp and 275 bp, three fragments sized 364 bp, 275 bp and 111 bp, and only one fragment in digested PCR products of P. ternata, P. pedatisecta and A. heterophyllum, respectively.

Obviously, an effective method to distinguish P. ternata from its adulterants is important to the healthy development of the herbal industry. The accurate identification of these medicinal materials is a prerequisite for the quality control of TCM.

Materials and Methods

The tubers of A. heterophyllum (he, No. A2004002) were collected in Chongqing, China, while those of P. ternata (te, No. P2004008) and P. pedatisecta (pe, No. P2004016) were collected in Nanchong, China. The voucher specimens were deposited in the herbarium of the Shanghai Botanical Garden, 1111 Longwu Road, Shanghai 200 231, P. R. China. The collected tubers were grown in pots in the greenhouse under standard conditions. Leaves were collected from two-month-old germinated seedlings and stored at -70 °C until use.

Genomic DNA was extracted from the three species using a modified protocol of Lin et al. (2005) [15]. GenomeWalker DNA libraries were constructed using the Universal GenomeWalker^TM Kit (Clontech; Palo Alto, CA, USA).

DNA fragments were amplified by PCR using specified primers GSPF and GSPR (Table [1]). The primers were designed and synthesized according to the cDNA sequences of pta (Pinellia ternata agglutinin) (GenBank Acc. No. AY191305) using the software of primer-premier 5.0 (Premier Biosoft International; Palo Alto, CA, USA).

The amplification program of upstream sequences of pta, ppa and aha genomic DNA consisted of two PCR amplifications per library. The primary PCR used the outer adaptor primer (AP) and an inner, gene-specific primer (5GSP1). The primary PCR mixture was diluted and used as the template for nested PCR with the nested adaptor primer (NAP) and a nested gene-specific primer (5GSP2).

The amplification program of downstream sequences of pta, ppa and aha genomic DNA consisted of two PCR amplifications per library. The primary PCR used the outer AP primer and an inner, gene-specific primer (3GSP1). The nested PCR used the NAP primer and a nested gene-specific primer (3GSP2).

The sequences of 3′ and 5′ genomic DNA end products of pta, ppa and aha were aligned with Vector NTI^TM Suite 8.0 (InforMax Inc.; Bethesda, MD, USA) to obtain the predicted DNA fragment. Subsequently, three pairs of PCR primers, PTAF1 and PTAR1, PPAF1 and PPAR1, AHAF1 and AHAR1 (Table [1]), were designed according to the aligned sequences and synthesized for the amplification of the complete DNA sequences of pta, ppa and aha. The PCR reaction was performed in a PTC-100^TM programmable PCR machine (MJ Research Inc.; Waltham, MA, USA) for 30 cycles (94 °C for 30 sec, 52 °C for 1 min, 72 °C for 3 min) followed by extension for 7 min at 72 °C.

Based on the sequencing results, a pair of PCR primers, P1 and P2, were designed and synthesized for the amplification of a specific sequence of pta, while a pair of PCR primers, P3 and P4, were designed and synthesized for the amplification of the ORFs of pta, ppa and aha. The PCR reaction was performed in a PTC-100^TM programmable PCR machine (MJ Research) for 30 cycles (94 °C for 30 sec, 55 °C for 1 min, 72 °C for 2 min) followed by extension for 7 min at 72 °C. All the primer sequences used are listed in Table [1].

All the PCR products were purified using a Gel Extraction Mini Kit (Watson Biotechnologies Inc.; Shanghai, P. R. China), ligated into pMD18-T vectors (Takara; Dalian, P. R. China), transformed into Escherichia coli strain DH5α and then sequenced (Shanghai Sangon Biological Engineering Technology and Service Co. Ltd., P. R. China).

PCR products were purified and completely digested in separate reactions with two restriction enzymes (BamHI and NcoI) (New England Biolabs Inc.; Ipswich, MA, USA). The reaction volume (20 μL) contained 5 μL PCR products, 1 μL endonucleases (BamHI or NcoI), 1 μL BSA (1 %), 5 μL endonuclease buffer and 8 μL deionized water. After incubation at 37 °C overnight, an aliquot (10 μL) of each digested sample was run on a 1.0 % agarose/EB gel to determine whether the PCR products were completely digested.

Acknowledgements

This work was funded by China National ”863” High-Tech Program, China Ministry of Education, and Shanghai Science and Technology Committee.

References

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  Search in Google Scholar
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  Search in Google Scholar
• 12 Liu Y P, Cao H, Komatsu K, But P PH. Quality control for Chinese herbal drugs using DNA probe technology.  Acta Pharm Sin. 2001;  36 475-80
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  Thieme ConnectPubMedSearch in Google Scholar
• 15 Lin J, Liu J, Sun X F, Zhou X W, Fei J, Tang K X. An efficient method for rapid amplification of Arisaema heterophyllum agglutinin gene using a genomic walking technique.  Prep Biochem Biotechnol. 2005;  2 155-67
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Prof. Kexuan Tang

State Key Laboratory of Genetic Engineering

School of Life Sciences

Morgan-Tan International Center for Life Sciences

Fudan-SJTU-Nottingham Plant Biotechnology R&D Center

Fudan University

220 Handan Road

Shanghai 200433

People’s Republic of China

Phone: +86-21-6564-2772

Fax: +86-21-6564-3552

Email: kxtang@fudan.edu.cn; kxtang1@163.com

References

• 1 Li H. Flora of China. Beijing; Science Press 1979: p 1-242
  Search in Google Scholar
• 2 Chinese Pharmacopoeia C ommission. Pharmacopoeia of the People’s Republic of China. Beijing; Chemical Industry Press 2000: p 89
  Search in Google Scholar
• 3 Marki T, Takahashi K, Shibata S. An anti-emetic principle of Pinellia ternata .  Planta Med. 1987;  53 410-4
  PubMedSearch in Google Scholar
• 4 Hu S L. A survey on the medicinal history of Pinellia ternata Breit.  J Chin Mater Med. 1989;  14 646-8
  PubMedSearch in Google Scholar
• 5 Kurata K, Tai T, Yang Y, Kinoshita K, Koyama K, Takahashi K. et al . Quantitative analysis of anti-emetic principle in the tubers of Pinellia ternata by enzyme immunoassay.  Planta Med. 1998;  64 645-8
  PubMedSearch in Google Scholar
• 6 Wu Y. The identification of Pinellia ternata and it adulterant, Arisaema heterophyllum .  Lishizhen Med Mater Med Res. 2003;  14 33-4
  PubMedSearch in Google Scholar
• 7 Zhao X Q, Lui Q M. The identification of Pinellia ternata and it multiple adulterant.  Shandong J Trad Chin Med. 1997;  16 131
  PubMedSearch in Google Scholar
• 8 Wang C Z, Li P, Ding J Y, Jin G Q, Yuan C S. Identification of Fritillaria pallidiflora using diagnostic PCR and PCR-RFLP based on nuclear ribosomal DNA internal transcribed spacer sequences.  Planta Med. 2005;  71 384-6
  Thieme ConnectPubMedSearch in Google Scholar
• 9 Kondo K, Terabayaahi S, Higuchi M, Komatsu Y, Okada M. Discrimination between Banxia and Tiannanxing based on rbcL sequence.  Nat Med. 1998;  52 253-8
  PubMedSearch in Google Scholar
• 10 Liu Y P, Cao H, Wang X T. Application of gene technology in quality control of Chinese drugs (I): identification of Pinellia ternata species from Yuncheng, Shandong using DNA sequencing.  Chin J Pharm Anal. 2001;  21 423-7
  PubMedSearch in Google Scholar
• 11 Cao H, Liu Y P, Bi P X, Shao P Z. DNA analysis and molecular identification of Pinellia ternata . In: Chinese Pharmaceutical Association and Editorial Board of Chinese Pharmaceutical Journal, editor Forum on modern pharmaceutical analysis. Beijing; Xinhua Press 2001: p 348-52
  Search in Google Scholar
• 12 Liu Y P, Cao H, Komatsu K, But P PH. Quality control for Chinese herbal drugs using DNA probe technology.  Acta Pharm Sin. 2001;  36 475-80
  PubMedSearch in Google Scholar
• 13 Chung H S, Um J Y, Kim M S, Hong S H, Kim S M, Kim H K. et al . Determination of the site of origin of Pinellia ternata roots based on RAPD analysis and PCR-RFLP.  Hereditas. 2002;  136 126-9
  CrossrefPubMedSearch in Google Scholar
• 14 Carles M, Cheung M K, Moganti S, Dong T T, Tsim K W. et al . A DNA microarray for the authentication of toxic traditional Chinese medicinal plants.  Planta Med. 2005;  71 580-4
  Thieme ConnectPubMedSearch in Google Scholar
• 15 Lin J, Liu J, Sun X F, Zhou X W, Fei J, Tang K X. An efficient method for rapid amplification of Arisaema heterophyllum agglutinin gene using a genomic walking technique.  Prep Biochem Biotechnol. 2005;  2 155-67
  PubMedSearch in Google Scholar

Prof. Kexuan Tang

State Key Laboratory of Genetic Engineering

School of Life Sciences

Morgan-Tan International Center for Life Sciences

Fudan-SJTU-Nottingham Plant Biotechnology R&D Center

Fudan University

220 Handan Road

Shanghai 200433

People’s Republic of China

Phone: +86-21-6564-2772

Fax: +86-21-6564-3552

Email: kxtang@fudan.edu.cn; kxtang1@163.com

• Supplementary Material