Using Frontier Technologies for the Quality Assurance of Medicinal Herbs RIRDC Publication No. 11/093 RIRDCInnovation for rural Australia Using Frontier Technologies for the Quality Assurance of Medicinal Herbs by Associate-Professor Eddie Pang November 2011 RIRDC Publication No. 11/093 RIRDC Project No. PRJ-000763 © 2011 Rural Industries Research and Development Corporation. All rights reserved. ISBN 978-1-74254-273-7 ISSN 1440-6845 Using Frontier Technologies for the Quality Assurance of Medicinal Herbs Publication No. 11/093 Project No. PRJ-000763 The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable regions. You must not rely on any information contained in this publication without taking specialist advice relevant to your particular circumstances. While reasonable care has been taken in preparing this publication to ensure that information is true and correct, the Commonwealth of Australia gives no assurance as to the accuracy of any information in this publication. The Commonwealth of Australia, the Rural Industries Research and Development Corporation (RIRDC), the authors or contributors expressly disclaim, to the maximum extent permitted by law, all responsibility and liability to any person, arising directly or indirectly from any act or omission, or for any consequences of any such act or omission, made in reliance on the contents of this publication, whether or not caused by any negligence on the part of the Commonwealth of Australia, RIRDC, the authors or contributors. The Commonwealth of Australia does not necessarily endorse the views in this publication. This publication is copyright. Apart from any use as permitted under the Copyright Act 1968, all other rights are reserved. However, wide dissemination is encouraged. Requests and inquiries concerning reproduction and rights should be addressed to the RIRDC Publications Manager on phone 02 6271 4165. Researcher Contact Details A/Prof Eddie Pang Royal Melbourne Institute of Technology School of Applied Sciences PO BOX 71 BUNDOORA VIC 3083 Phone: +61 3 99257137 Fax: +61 3 99257110 Email: [email protected] In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 2, 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6271 4100 Fax: 02 6271 4199 Email: [email protected]. Web: http://www.rirdc.gov.au Electronically published by RIRDC in November 2011 Print-on-demand by Union Offset Printing, Canberra at www.rirdc.gov.au or phone 1300 634 313 ii Foreword Ensuring the safety and efficacy of herbal medicines has increased in relevance following negative media coverage, particularly in relation to the adulteration and substitution of herbal products. Calls for the stricter regulation of the herbal industry have subsequently increased. However, to police the industry, regulators require the necessary tools for the rapid identification of herbal species and products. As many commercial herbal products are available in either powdered or shredded form, authentication by morphological and histological methods is extremely difficult, if not impractical. Chemical analysis is restricted in many circumstances as growth environment significantly affects the profile and amount of chemical compounds in herbal medicine plants. Recently, the identification of herbal products via DNA-based fingerprinting has been developed to complement the existing methods of microscopic examination and chemical analyses. A fingerprinting microarray was developed in this three-year project to identify important medicinal species from around the world, with an emphasis on herbal species employed in Western and Chinese medicine. In this project, distinct fingerprints were generated for more than 40 herbal species from the flowering plant clades, Asterids and Rosids. Additionally, a sub-array was constructed to fingerprint different chemotypes of Salvia miltiorrhiza (Danshen). A significant advantage of the developed microarrays was their ability to fingerprint species of medicinal and non-medicinal plants which were not used in microarray construction—potentially widening their application to general plant fingerprinting. Further, employing a modular sub-array design, the fingerprinting microarray may be further expanded to include other important species from the Monocots, Core Eudicots and Magnoliids. The existing microarrays are fully functional and with the eventual incorporation of the remaining sub- arrays will be a valuable tool for the medicinal plant industry for quality assurance and identification purposes. Once this is in place, the technology will be published in the scientific literature and a fee- for-service facility would be offered to the medicinal herb industry, i.e. for growers, manufacturers, pharmacological companies, or the Therapeutic Goods Administration—any body interested in validating the identity of medicinal herbs. It is also envisioned that this array will be useful for researchers and herb breeders interested in the genetic diversity of their breeding lines. This project was funded from RIRDC Core Funds which are provided by the Australian Government. Additional funding and in-kind support was also provided by MediHerb, Botanical Resources Australia, Southern Cross University and RMIT University. This report, an addition to RIRDC’s diverse range of over 2000 research publications, forms part of our Essential Oils and Plant Extracts R&D program which aims to provide the knowledge and skills base for industry to provide high, consistent and known qualities in their essential oils and plant extracts products that respond to market opportunities and enhance profitability. Most of RIRDC’s publications are available for viewing, free downloading or purchasing online at www.rirdc.gov.au. Purchases can also be made by phoning 1300 634 313. Craig Burns Managing Director Rural Industries Research and Development Corporation iii About the Author Associate-Professor Eddie Pang obtained a Bachelor of Agricultural Science with Honours at the University of Melbourne, and subsequently a PhD in Plant Breeding from the same institution in 1992. After working at Agriculture Victoria (Department of Primary Industries) for several years as a pea geneticist, he returned to academia, firstly at the University of Melbourne, and subsequently at RMIT University, where he has lectured and conducted research into molecular plant breeding since 1998. Acknowledgments My research team and I would like to gratefully acknowledge the funding received from the RIRDC, without which the research detailed in this report would not have been possible. We would like to acknowledge the financial assistance granted to us by MediHerb Pty Ltd, and for the invaluable advice and guidance of Dr Reg Lehman of MediHerb (later part of the Integria Healthcare Group). We would also like to gratefully acknowledge the advice, and the provision of plant materials by Dr Hans Wohlmuth of Southern Cross University and Mr Tim Groom of Botanical Resources Australia. Lastly, we would like to thank the University for the provision of a VRII grant as seed funding for this project. Abbreviations BSA bovine serum albumin DNA deoxyribonucleic acid EDTA ethylenediaminetetraacetic acid gDNA genomic DNA PCA principal component analysis PCR polymerase chain reaction PMT photomultiplier tube SDA subtracted diversity array SDS sodium dodecylsulphate SSC standard saline citrate SSH suppression subtraction hybridisation USDA United States Department of Agriculture iv Contents Foreword ............................................................................................................................................... iii About the Author .................................................................................................................................. iv Acknowledgments ................................................................................................................................. iv Abbreviations ........................................................................................................................................ iv Executive Summary ............................................................................................................................. vii Introduction ........................................................................................................................................... 1 Objectives ............................................................................................................................................... 2 Methodology ........................................................................................................................................... 3 Refining the fingerprinting microarray ...................................................................................... 3 Construction of the Asterid sub-array ........................................................................................ 5 Construction of the Rosid sub-array ........................................................................................... 6 Construction of the Salvia miltiorrhiza chemotyping sub-array ................................................ 6 Construction of the Echinacea fingerprinting sub-array ...........................................................