The Use of Polymerase Chain Reaction (PCR) for the Identification of Ephedra DNA in Dietary Supplements

Abstract

As part of a continuing research effort to develop chemical and genetic authentication profiles of botanicals, an investigation was performed with the goal to detect, identify and verify Ephedra sinica Stapf DNA in dietary supplements such as plant mixtures and tablets/capsules. We amplified and sequenced the chloroplast psbA-trnH spacer from 21 Ephedra spp. and from two of their closest relatives, Gnetum gnemon L. and Welwitschia mirabilis Hook. Based on sequence comparisons, we identified regions unique to all of the Ephedra spp. samples analyzed. We concluded that the psbA-trnH spacer sequence could be used as a molecular marker. Based on this spacer sequence, we designed Ephedra spp.-specific primers that can help to identify Ephedra spp. DNA in plant mixtures containing as little as 1/1,000 part of Ephedra spp. tissue. We used a DNA extraction method that allows for quick DNA isolation from plant mixtures for PCR analysis.

Ephedra - PCR - molecular marker - chloroplast DNA - psbA-trnH - dietary supplements

References

• 1 Price R P. Systematics of the Gnetales: a review of morphological and molecular evidence.  Int J Plant Sci. 1996;  157 (Suppl) 40-9
• 2 Blumenthal M, Goldberg A, Brinckmann J. In: Expanded Commission E monographs. Blumenthal M, Goldberg A, Brinckmann J, editors Newton; Integrative Medicine Communications 2000: pp 110-7
• 3 Haller C A, Benowitz N L. Adverse cardiovascular and central nervous system events associated with dietary supplements containing Ephedra alkaloids.  N Engl J Med. 2000;  343 1833-8
• 4 Techen N, Crockett S L, Khan I A, Scheffler B E. Authentication of medicinal plants using molecular biology techniques to compliment conventional methods.  Curr Med Chem. 2004;  11 1391-401
• 5 Schaneberg B T, Crockett S L, Bedir E, Khan I A. The role of chemical fingerprinting: application to Ephedra .  Phytochemistry. 2002;  62 911-8
• 6 Luis L, Ramírez A H, Ramírez R, Vélez I D, Mendoza-León A. Nuclear DNA sequence specific to Leishmania (Viannia) subgenus: a molecular marker for species identification.  Parasitology. 2000;  122 405-14
• 7 Kapteyn J, Simon J E. The use of RAPDs for assessment of identity, diversity, and quality of Echinacea . In: Trends in new erops and new uses, Janick J, Whipkey A, editors Alexandria; ASHS Press 2002: pp 509-13
• 8 Parani M, Rajesh K, Lakshmi M, Parducci L, Szmidt , AE Parida A. Species identification in seven small millet species using polymerase chain reaction - restriction fragment length polymorphism of trnS-psbC gene region.  Genome. 2001;  44 495-9
• 9 Yang D Y, Fushimi H, Cai S Q, Komatsu K. Molecular analysis of Rheum species used as Rhei Rhizoma based on the chloroplast matK gene sequence and its application for identification.  Biol Pharm Bull. 2004;  27 375-83
• 10 Wang X D, Wang Z P, Zou Y P. An improved procedure for the isolation of nuclear DNA from leaves of wild grapevine dried with silica gel.  Plant Mol Biol Rep. 1996;  14 369-73
• 11 Sang T, Crawford D J, Stuessy T F. Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae).  Am J Bot. 1997;  84 1120-36
• 12 Nicholas K B, Nicholas HB J r., Deerfield DW I I. GeneDoc: analysis and visualization of genetic variation.  Embnet News. 1997;  4 1-4
• 13 Higgins D, Thompson J, Gibson T, Thompson J D, Higgins D G, Gibson T J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.  Nucleic Acids Res. 1994;  22 4673-80
• 14 Altschul S F, Gish W, Myers E W, Lipman D J. Basic local alignment search tool.  J Mol Biol. 1990;  215 403-10
• 15 Dilworth E, Frey J E. A rapid method for high throughput DNA extraction from plant material for PCR amplification.  Plant Mol Biol Rep. 2000;  18 61-4
• 16 Loomis W D. Overcoming problems of phenolics and quinones in the isolation of plant enzymes and organelles.  Methods Enzymol. 1974;  31 528-44
• 17 Rott M E, Lawrence T S, Wall E M, Green M J. Detection and quantification of roundup ready soy in foods by conventional and real-time polymerase chain reaction.  J Agric Food Chem. 2004;  52 5223-32
• 18 Tengel C, Schuessler P, Setzke E, Balles J, Sprenger-Haussels M. PCR-based detection of genetically modified soybean and maize in raw and highly processed foodstuffs.  Biotechniques. 2001;  31 426-9
• 19 Techen N, Crockett S, Khan I A, Scheffler B A. Authentication of medicinal plants using molecular biology techniques to compliment conventional methods.  Curr Med Chem. 2004;  11 1391-401
• 20 Pusch C M, Giddings I, Scholz M. Repair of degraded duplex DNA from prehistoric samples using Escherichia coli DNA polymerases I and T4 DNA ligase.  Nucleic Acids Res. 1998;  26 857-9
• 21 Abourashed E A, El-Alfy A T, Khan I A, Walker L. Ephedra in perspective-a current review.  Phytother Res. 2003;  17 703-12