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The Development of Deoxyribonucleic Acid (DNA) Based Methods for the Identification and Authentication of Medicinal Plant Material

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The Development of Deoxyribonucleic Acid (DNA) Based Methods for the Identification and Authentication of Medicinal Plant Material DE MONTFORT UNIVERSITY, LEICESTER The development of Deoxyribonucleic Acid (DNA) based methods for the identification and authentication of medicinal plant material. Caroline Howard Ph.D. June 2010 The development of Deoxyribonucleic Acid (DNA) based methods for the identification and authentication of medicinal plant material. Caroline Howard A thesis submitted in partial fulfilment of the requirement for the degree of Doctor of Philosophy to De Montfort University Faculty of Health and Life Sciences June 2010 De Montfort University i Caroline Howard Abstract Herbal medicines are growing in popularity in the Western world and are becoming more stringently regulated under new EU legislation. Within the arena of herbal medicines, St. John’s Wort (SJW), Hypericum perforatum, is a top ten best seller with clinical evidence to support its use as an anti-depressant. A fundamental requirement of the new legislation is to prove the identity of the plant material in question. This is currently achieved via morphological and chemical methods, neither of which are ideal. A wide range of DNA based methods have been applied to this arena, standardisation is required to realise the potential of DNA based techniques. The DNA barcoding initiative aims to produce sequence data for all plant species, capable of species identification. The proposal is to use these data to design fast and effective DNA based methods of identification. For assay design, the putative barcode region nrITS was selected as a platform. Three assays were designed; A PCR assay designed to hyper variable sequences within a barcode region. This assay is capable of distinguishing SJW from other closely related species. A quantitative qPCR assay designed to measure total DNA and specific SJW DNA within a mixed sample. A multiplex PCR incorporating fluorescently labelled primers, allowing amplicon detection by capillary electrophoresis. This assay identifies four separate Hypericum species, including SJW, with a mixed sample in one reaction. The suitability of the nrITS and three other barcode regions is assessed based on sequence data generated for 32 vouchered samples of different Hypericum species, and a Lithuanian sample set of 22 and 16 H. perforatum and H. maculatum samples respectively. The matK is currently unusable, the rbcL highly conserved, trnH-psbA problematically variable and the nrITS proved to be ideal for assay design. De Montfort University ii Caroline Howard Acknowledgements I would like to thank my entire supervisory team for initiating this project, seeking funding and supporting every aspect of my research. In particular, Dr Adrian Slater for conceiving the project, and for guiding and advising with infinite patience, this was tested. Dr Paul Bremner for his optimistic and global approach, and Dr Mark Fowler for his insightful critique and advice. Thanks are due to the many contributors of samples to this research. Mark Carine at the Natural History Museum London for providing vouchered DNA samples and general guidance. Asta Judzentiene of the Institute of Chemistry Vilnius, Lithuania, for providing Hypericum samples. Santiago Madriñán, Univ. de los Andes, for permission to use DNA from vouchered specimens which was supplied by the Kew DNA Databank. I would also like to thank our collaborators from Leicester University, Dr Eleanor Graham and Eleni Socratous for their work and expertise. Thanks are also due to my colleagues in the Cell Signalling Laboratory of De Montfort University where I have conducted this research. I would also like to thank our Research Assistant, Sarah Smith, who worked on this project both with and without payment. Her hard work was and is very much appreciated. Finally I would like to thank my husband Alex, who has been patient and supportive throughout my research, and all of my friends and family. De Montfort University iii Caroline Howard Publications and Presentations Original Papers Molecular Identification of Hypericum perforatum L. by PCR amplification of the ITS and 5.8S rDNA Region. Caroline Howard, Paul D. Bremner, Mark R. Fowler, Belinda Isodo, Nigel W. Scott and Adrian Slater. Planta Medica 2009: 75; 8, 864-869. Primer Design for DNA Based Identification of Medicinal Plants. C. Howard, P.D. Bremner, M.R. Fowler, N.W. Scott and A. Slater. Acta Horticulturae: In Press. Presentations PlantID – A System for the Identification of Medicinal Plant Material by DNA Profiling. Caroline Howard, Eleni Socratous, Sarah Smith, Eleanor Graham, Mark R Fowler, Nigel W Scott, Paul D Bremner and Adrian Slater. RRJ Arroo (ed.) (2010) Trends in Natural Products Research: Abstracts of the Phytochemical Society of Europe. Phytochemical Society of Europe, Leicester. ISBN 978-0-9565472-0-0. Meeting held at De Montfort University Leicester, April 2010 . Development of DNA sequence-based tests to discriminate Hypericum species. Caroline Howard, Mark Fowler, Paul Bremner, Nigel Scott and Adrian Slater. Invited talk given at the 2nd Annual Hypericum Meeting at the IPK in Gatersleben Germany, November 2009. Molecular Identification of Hypericum perforatum L. by PCR amplification of the rDNA ITS region. Caroline Howard, Paul Bremner, Mark Fowler and Adrian Slater. The African Journal of Traditional Complementary and Alternative Medicine 2009; World Conference on Medicinal and Aromatic Plants, Cape Town South Africa, November 2008. Molecular Identification of St John’s Wort by PCR amplification of the ITS1 Region: Implications for medicinal plant identification. Caroline Howard, Paul D. Bremner, Mark R. Fowler and Adrian Slater. Journal of Pharmacy and Pharmacology 2008; 60: A10. British Pharmaceutical Conference, September 2008. DNA based identification of Hypericum perforatum /Medicinal Plants. Caroline Howard. Oral presentation to a meeting of King’s College London, Royal Botanic Gardens Kew and The London School of Pharmacy, February 2008. Posters Molecular identification and quantitation of Hypericum perforatum in mixed samples. Caroline Howard, Paul D Bremner, Mark R Fowler, Nigel W Scott and Adrian Slater. Planta Medica De Montfort University iv Caroline Howard 2009: 75; 9, 1000. 55th International Congress and Annual Meeting of the Society for Medicinal Plant Research and Natural Product Research, Geneva 2009. De Montfort University v Caroline Howard Contents Abstract .......................................................................................................................................... ii Acknowledgements ....................................................................................................................... iii Publications and Presentations ..................................................................................................... iv Figures ............................................................................................................................................ x Tables ........................................................................................................................................... xv 1 Introduction .......................................................................................................................... 1 1.1 Medicinal Plant Usage and Regulation ......................................................................... 1 1.1.1 Medicinal Plant Usage ........................................................................................... 1 1.1.2 Medicinal Plant Legislation ................................................................................... 2 1.2 Current Medicinal Plant Identification Methods .......................................................... 4 1.2.1 Morphology ........................................................................................................... 5 1.2.2 Chemical Methods ................................................................................................ 5 1.3 DNA-Based Identification of Medicinal Plant Material ................................................. 7 1.3.1 Randomly Amplified Polymorphic DNA (RAPD) .................................................... 7 1.3.2 Sequence Characterised Amplified Region (SCAR) ............................................... 9 1.3.3 Restriction Fragment Length Polymorphism (RFLP) ........................................... 10 1.3.4 Amplified Fragment Length Polymorphism (AFLP) ............................................. 11 1.3.5 Amplification-Refractory Mutation System (ARMS) ........................................... 13 1.3.6 Simple Sequence Repeats (SSRs) ........................................................................ 13 1.3.7 Direct Sequencing ............................................................................................... 14 1.3.8 Advantages of DNA based Techniques ............................................................... 14 1.3.9 DNA Barcoding .................................................................................................... 15 1.4 Hypericum perforatum L., St John’s Wort ................................................................... 20 1.4.1 Efficacy ................................................................................................................ 20 1.4.2 Active Compounds .............................................................................................. 21 1.4.3 Drug Interactions ................................................................................................ 23 1.4.4 Morphological and Chemical Identification .......................................................
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