Jul 20, 2018
Version 1
DNA Barcoding of Aristolochia Plants V.1
- Piroonrat Dechbumroong1,
- Surattana Aumnouypol1,
- Jessada Denduangboripant2,
- Suchada Sukrong1
- 1DNA Barcoding of Thai Medicinal Plants Research Unit, Department of Pharmacognosy and Pharmaceutical Botany, Chulalongkorn University Drug and Health Products Innovation Promotion Center, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand;
- 2Department of Biology, Faculty of Sciences, Chulalongkorn University, Bangkok, Thailand
Protocol Citation: Piroonrat Dechbumroong, Surattana Aumnouypol, Jessada Denduangboripant, Suchada Sukrong 2018. DNA Barcoding of Aristolochia Plants. protocols.io https://dx.doi.org/10.17504/protocols.io.rt3d6qn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 20, 2018
Last Modified: July 20, 2018
Protocol Integer ID: 13915
Keywords: Aristolochia, DNA barcode,
Abstract
The anecdotal evidence is outstanding on the uses of Aristolochia plants as traditional medicines and dietary supplements in many regions of the world. However, herbal materials derived from Aristolochia species have been identified as potent human carcinogens since the first case of severe renal disease after ingesting these herbal preparations. Any products containing Aristolochia species have thus been banned on many continents, including Europe, America and Asia. Therefore, the development of a method to identify these herbs is critically needed for customer safety. The present study evaluated DNA barcoding of the rbcL, matK, ITS2 and trnH-psbA regions among eleven Aristolochia species collected in Thailand. Polymorphic sites were observed in all four DNA loci. Among those eleven Aristolochia species, three species (A. pierrei, A. tagala and A. pothieri) are used as herbal materials in Thai folk medicine, namely, in Thai “Krai-Krue”. “Krai-Krue” herbs are interchangeably used as an admixture in Thai traditional remedies without specific knowledge of their identities. A species-specific multiplex PCR based on nucleotide polymorphisms in the ITS2 region was developed as an identification tool to differentiate these three Aristolochia species and to supplement the HPTLC pattern in clarifying the origins of herbal materials. The combination of multiplex PCR and HPTLC profiling achieves accurate herbal identification with the goal of protecting consumers from the health risks associated with product substitution and contamination.
Plant materials
Plant materials
Thirty-eight specimens of eleven Aristolochia taxa were collected from various regions of Thailand. All specimens were identified by Associate Professor Thatree Phadungcharoen at the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Thailand. Herbarium specimens were prepared and kept at the Museum of Natural Medicines, Faculty of Pharmaceutical Science, Chulalongkorn University.
Genomic DNA extraction
Genomic DNA extraction
Total genomic DNA was extracted from 80 to 100 mg of fresh leaves of the plants or from 15 to 25 mg of dried commercially available crude drug samples. DNA was extracted by a DNeasy Plant Mini Kit (Qiagen, Germany) according to the manufacturer's protocol. DNA quality and quantity were determined by agarose gel electrophoresis; the gels were stained with ethidium bromide and visualized under UV light.
PCR amplification and electrophoresis
PCR amplification and electrophoresis
The rbcL, ITS2, and trnH-psbA of Aristolochia were amplified by amplification primers. Nucleotide sequences of the trnK-matK regions of A. pierrei (accession number DQ296649), A. grandiflora (accession number DQ532052), and A. gigantea (accession numbers JX485569 and DQ882187) were retrieved from GenBank for primer design. New primers, matK-Aris-F1, matK-Aris-F2 and matK-Aris-R1, were used to amplify the complete matK sequence of the genus Aristolochia.
| DNA locus | Sequencing primer | Sequence (5'→3') | |
| rbcL | rbcL_aF* | ATG TCA CCA CAA ACA GAG ACT AAA GC | |
| rbcL-Aris-327R | TTC AAA AAG GTC TAA AGG GTA AGC | ||
| rbcL_636F | GCG TTG GAG AGA TCG TTT CT | ||
| rbcL_R23* | TTT TAG TAA AAG ATT GGG CCG | ||
| matK | matK-Aris-F1* | ATC CCC TAT TCC TTC AGT TCA A | |
| matK-Aris-F2* | CCT TGT TTT GAC TGT ATC GCA C | ||
| matK-Aris-F458 | ATA CCC CAC CCC ATC CAT CTG | ||
| matK-Aris-F967 | CAC TTG TGG TCT CAA CCG GG | ||
| matK-Aris-R1* | GCA CAC GGC TTT CCC TAT G | ||
| trnH-psbA | psbAF* | GTT ATG CAT GAA CGT AAT GCT C | |
| trnHR* | CGC GCA TGG TGG ATT CAC AAA TC | ||
| ITS2 | ITS1* | TCC GTA GGT GAA CCT GCG G | |
| ITS3* | GCA TCG ATG AAG AAC GCA GC | ||
| ITS4* | TCC TCC GCT TAT TGA TAT GC | ||
| ITS-Aris-390F | AAT TGC AGA ATC CCG CGA AC |
The PCR amplification was performed in 50 μL of reaction mixture consisting of 5X PCR buffer, 25 mM MgCl2, 2.5 mM each dNTP, 10 mM each primer, 5U GoTaq® Flexi DNA polymerase (Promega, USA), and 10–100 ng of total DNA as a template. PCR amplifications were carried out in a C1000™ Thermal Cycler (Bio-Rad, USA) using cycling conditions of 96°C for 3 min; followed by 30 cycles of 96°C for 1 min, 55°C for 1 min and 72°C for 2 min (for rbcL and matK) and 45 sec (for ITS2 and trnH-psbA); and a final extension at 72°C for 10 min. The amplified products were detected by 1.2% agarose gel electrophoresis in 1X TAE buffer and were visualized by ethidium bromide staining.
Sequence analysis
Sequence analysis
The sequencing process was performed by capillary sequencing (AIT Biotech, Singapore) with sequencing primers for each region. The sequences were aligned, edited and analysed using BioEdit Sequence Alignment Editor Version 7.2.5. The obtained sequences were assembled to create consensus sequences using the DNASTAR® (Version 8.0.2) program (USA). The sequences were then submitted to the DDBJ/EMBL/GenBank nucleotide sequence databases, and their accession numbers.