program 1.4 essential oils & plant extracts...peppermint in favour of crops with higher gross...

RIRDC Completed Projects in 2006-2007 and Research in Progress as at June 2007

Program 1.4

Essential Oils & Plant Extracts

August 2007

RIRDC 07/004

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© 2007 Rural Industries Research and Development Corporation. All rights reserved. ISBN 1 74151 404 5

ISSN 1440-6845

"RIRDC Completed Projects in 2006-2007 Research in Progress as at June 2007 - 1.4 Essential Oils and Plant Extracts" Publication No: 07/004 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 industries. The information should not be relied upon for the purpose of a particular matter. Specialist and/or appropriate legal advice should be obtained before any action or decision is taken on the basis of any material in this document. The Commonwealth of Australia, Rural Industries Research and Development Corporation, the authors or contributors do not assume liability of any kind whatsoever resulting from any person's use or reliance upon the content of this document.

This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6271 4165.

RIRDC Essential Oils and Plant Extracts Dr Roslyn Prinsley RIRDC Level 2, Pharmacy Guild House 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6271 4120 Fax: 02 6271 4199 Email: [email protected] RIRDC Publications Rural Industries Research and Development Corporation Level 2, Pharmacy Guild House 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6271 4165 Fax: 02 6271 4199 Email: [email protected] Website: http://www.rirdc.gov.au Published in August 2007 Printed on environmentally friendly paper by Canprint

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Foreword This Research in Progress, June 2007, contains short summaries of continuing projects as well as those that were completed during 2006-2007 for RIRDC’s Essential Oils and Plant Extracts Sub-program. This program aims to support the growth of a profitable and sustainable essential oils and natural plant extracts industry in Australia. The complete report on all the programs is only available in electronic format on our website at http://www.rirdc.gov.au This report is an addition to our extensive catalogue of over 1600 research reports, videos and CD-Roms of projects supported by RIRDC. Please contact us for the latest publications catalogue of view it on our website: Downloads at www.rirdc.gov.au Purchases at www.rirdc.gov.au/eshop Peter O’Brien Managing Director Rural Industries Research and Development Corporation

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Contents

1.4 ESSENTIAL OILS

COMPLETED PROJECTS

PROJECT No PROJECT TITLE RESEARCHER PHONE ORGANISATION

PAGE No

Development and Improvement of Products

DAQ-328A A literature review and assessment of Morinda citrifolia (Noni) Ms. Helen Macpherson (07) 3406 8589

Department of Primary Industries and Fisheries (Qld)

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Improved Production Systems

UT-38A Rejuvenation of peppermint crops through establishment of quality nursery stock

Prof. Robert Menary (03) 6226 2723 University of Tasmania 3

UT-40A Improved oil quality and yield from Tasmanian-grown French lavender Prof. Robert Menary (03) 6226 2723 University of Tasmania 4

Industry Development

AGL-5A Sandalwood industry overview and research needs analysis Mr Michael Clarke (02) 9817 5888 AgEconPlus Pty Ltd 5

1.4 ESSENTIAL OILS

RESEARCH IN PROGRESS PROJECT

No PROJECT TITLE RESEARCHER PHONE ORGANISATION PAGE

No Development and Improvement of Products

BRA-4A Development and economic assessment of Artemisia for production of anti-malarial extract

Dr. Rowland Laurence (03) 6428 2900 University of Tasmania 7

ISO-1A Production of newsletter of the Essential Oil Producers Association of Australia Dr Ian Southwell (02) 6624 2453 Ian Southwell 8

RMI-18A Using frontier technologies for the quality assurance of medicinal herbs Dr Eddie Pang (03) 9925 7137 RMIT University 9

UJC-13A Fabrication of Electronic Materials from the Australian Essential Oils Dr Mohan Jacob (07) 4781 4379 James Cook University 11

UNC-21A Analysis of Dodonea viscosa as a potential remedy for anti-inflammatory and antibacterial applications

Mr Andrew Pengally (02) 4353 3193 The University of Newcastle 12

UT-51A Clinical trial of kunzea oil for onychomycoses treatment-commercial potential

Dr Glenn Jacobson (03) 6226 2202 The University of Tasmania 13

Improved Production Systems

ANU-74A Diagnostic tools for quality enhancement in Australian essential oil industries Dr William Foley (02) 6125 2866 Australian National University 14

DAQ-329A Commercial production of milkweed, a plant with anticancer properties Mr. Larry Cooper (07) 3824 9541 Department of Primary Industries and Fisheries (Qld)

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EOT-1A Growing the essential oils industry Ms Susan Hinton 0417 038 274 Essential Oils of Tasmania 16

UT-49A Increased yields from peppermint crops through improved micronutrient nutrition Prof. Robert Menary (03) 6226 2723 University of Tasmania 17

Regulatory Approvals

UT-45A Quality assurance protocols to minimise pesticide residues in essential oils Prof. Robert Menary (03) 6226 2723 University of Tasmania 18

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COMPLETED PROJECTS − Development and Improvement of Products

Project Title

Morinda citrifolia (Noni) A Literature review & assessment

RIRDC Project No.:

DAQ-328A

Researcher: Ms Helen Macpherson Organisation: Dept of Primary Industries and Fisheries

19 Hercules St Hamilton Qld 4007

Phone: (07) 3406 8589 Fax: (07) 3406 8677 Email: [email protected]

Objectives To investigate the potential for a new value adding industry by conducting an

extensive literature review of the plant Morinda citrifolia (noni) and its juice extract.

Background Noni grows widely throughout the Pacific and is native to Australia. There are many claims as to the efficacy of noni juice from its use by traditional healers but there appears to be little in the way of scientifically reproducible studies. A group of growers have commenced planting noni trees and have formed an association (Australian Noni Producers Inc.) to initiate the growth of this opportunity to produce plant extracts from both the native and exotic varieties of this species. The association has identified that information is required to progress the development of this industry.

Research By searching, compiling and reviewing the extensive existing literature search on the plant Morinda citrifolia (noni) and its juice extract, the researchers were able to assess the broad range of supposed therapeutic effects, agronomic best practices, the fermentation process and the market opportunities and potential returns to Australian producers.

Outcomes Noni has an extremely wide range of environmental tolerances so production should be possible in a wide range of tropical environments in Australia, but yields will be greatest on highly fertile soils with abundant available water. Considerable variation exists in noni plant and fruit characteristics but there is no recognised germplasm collection in existence. This work has demonstrated that the basic mechanisms and associated microflora of the reported Noni juice fermentation process is not well understood. Further research is required to identify the mechanisims and microflora involved in the reported fermentation process to develop commercial process parameters, processing aids (starter cultures) and industry standards. Various compounds have been identified in noni fruit in relation to possible health benefits. In vitro research and limited animal experiments have shown that noni has antimicrobial, anti-cancer, antioxidant, anti-inflammatory, analgesic and cardiovascular activity. Further research is required to fully characterise the composition of noni fruit, noni juice and noni powder. The limited research available on bioactivity studies needs to be confirmed and further developed.

Implications A wide range of potential production systems for noni should be investigated, including those with low inputs located away from north Queensland’s wet tropical coast as they may require less capital investment and thus represent

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lower risk. There is a good opportunity for exploration in Australia and overseas to locate plants with desired characteristics including fruit size, plant habit, disease resistance, concentration of medicinally active components etc. As with modern fermentation techniques, optimum processing parameters combined with a quality starter culture (with known characteristics under controlled conditions) would allow the processor to potentially produce a reliable consistent safe product every time. Subsequently, a product with consistent quality has more potential to retain repeat business in a competitive market providing more opportunity for success of the business. Noni product composition needs to be better understood, particularly as the cultivars and growing areas become more diverse. Significant research needs to be undertaken to confirm the bioactivity of the various products (animal studies, clinical trials, etc).

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COMPLETED PROJECTS − Improved Production Systems Project Title

Rejuvenation of peppermint crops through establishment of quality nursery stock

RIRDC Project No.: UT-38A Researcher: Professor R.C. Menary and Dr L M Falzari Organisation: University of Tasmania Phone: (03) 6226 2723 Fax: (03) 6226 7609 Email: [email protected]

Objectives 1. To identify a selection of peppermint clones that produce a high yield of oil,

conforming to the ISO standard for oil of peppermint.

2. To test three potential propagation methods for suitability in the production of commercial nursery areas from which propagules can be lifted for the establishment of commercial crops.

Background Peppermint is a key species in the Australian essential oil industry. Productivity of peppermint crops is important both to the primary producers growing the crop and to the wholesalers of the oil. If yields continually fall below a certain economic threshold growers will receive insufficient income and will cease to produce peppermint in favour of crops with higher gross margins. Traders unable to source sufficient locally produced oil and will shift to imported oils. Recently, there has been a decline in peppermint productivity. Two potential contributors to peppermint are plant genotype and nursery propagation method.

Research To examine the effect of genotype on oil production a glasshouse screening trial was conducted on 200 peppermint plants. A field trial tested the effect of propagation method on the yield and quality of oil.

Outcomes There were two key findings. Increased productivity in mint crops can be achieved by rejuvenating the planting material through using tip cuttings as propagation material. There are significant genetic differences between peppermint plants within the Tasmanian peppermint population. These differences are potentially contributing to a decline in oil production within the crops. At the completion of this project, several potential peppermint genotypes have been selected and are available for testing under commercial growing conditions.

Implications It is recommended that the industry use the tip cuttings to establish nursery sites, which can be expended into future commercial crops. Each new crop should be expanded from a newly established nursery so as to take advantage of the rejuvenating effect of tip cutting propagation. These nurseries should be established from the most productive genotypes. Urgent testing of the selected genotypes is necessary, to determine their performance under standard commercial production. It is strongly recommended that the industry immediately commence testing the selections to avoid further propagation of unproductive clones in this perennial crop.


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COMPLETED PROJECTS − Improved Production Systems Project Title

Improved oil quality and yield from Tasmanian-grown French lavender

RIRDC Project No.: UT- 40A Researcher: Professor Robert Menary Organisation: University of Tasmania Phone: (03) 6226 2723 Fax: (03) 6226 7609 Email: [email protected]

Objectives 1. Identify a series of Lavender clones that conform to the ISO standards,

including low camphor, and establish these in a nursery that is available to the industry. 2. Monitor changes in oil yield and chemical levels during the development of the flowers 3. Gain some insight into the possibility of increased outputs through more intensive production.

Background Lavender has been grown at Bridestowe in Northern Tasmania for the production of French lavender oil since about 1922. Recently it was noted that the levels of camphor in the distilled oil had been rising close to a level that would make the oil unsuitable for sale on the international market . In order to produce lavender oil for the international markets, strategies were needed to produce oil that complied with all of the chemical requirements of the international standards organisations (ISO) in sufficient marketable quantities.

Research The research in this project had four main thrusts. Firstly, clones were chosen that were low in camphor. These clones were then propagated using tissue culture and potting to create a nursery of low camphor clones. Thirdly, any changes in the oil content of flowers were measured during two growing seasons. Fourthly, lavender was grown under conditions of increased nitrogen and water application and the oil content measured.

Outcomes Low camphor clones were selected for all four commercial cultivars (MS, JP, PC and RB). The chemical characteristics of the oil produced from these clones were then assessed and a possible strategy for producing high quality oil was recommended. Measurement of the oil content during flower development showed a high level of complexity. Oil components changed over the growing season with some components increasing whilst others decreased. Some levels of difference in the development of the oil were observed between the four commercial cultivars. Trials using increase nitrogen and water application to the plants demonstrated that the oil had a different composition to that obtained under dry land conditions.

Implications It is recommended that further trials are conducted with the selected clones in order to establish commercial plantings of the clones in a ratio that will lead to a blended oil that meets the international standards. Further work with time of harvest and intensive agricultural practices may also lead to improved oil quality. The optimum harvest data compiled in this report can be used as the basis for experiments leading to a harvest time practice similar to that practiced for other Tasmanian steam distilled oils.


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COMPLETED PROJECTS − Industry Development Project Title

Sandalwood industry overview and research needs analysis

RIRDC Project No.:

AGL-5A

Researcher: Mr Michael Clarke Organisation: AgEconPlus Pty Ltd Phone: (02) 9817 5888 Fax: (02) 9816 4840 Email: [email protected]

Objectives Produce an overview/scoping study of the Australian sandalwood industry and

identify research needs and gaps.

Background There is currently widespread interest in the sandalwood industry, including broad based interest from the public following extensive marketing of sandalwood Managed Investment Schemes (MIS). Provision of an independent scoping report on the sandalwood industry was seen by RIRDC to be both appropriate and opportune.

Research The study was completed using desk-based research techniques including:

• Finalisation of the project scope and objectives with RIRDC;

• Review of the relevant Australian and available international literature;

• Consultation with Western Australian (WA) Forest Products Commission (FPC), sandalwood researchers, Sandalwood Research News, manufacturers and marketers of essential oils, private consultants and others working in the area;

• Situation assessment documentation for both the Indian sandalwood (S. album) and WA sandalwood (S. spicatum) industries;

• Research needs analysis including preparation of a gaps analysis and assessment of the potential RIRDC role in research funding;

• Draft report documentation and circulation to RIRDC and key industry stakeholders;

• Incorporation of comments received on the draft report; and

• Submission of a final report to RIRDC. The research was completed between June and September 2006.


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Outcomes The research needs analysis reveals that:

1. There is an organised and active research community investigating sandalwood in Australia. The research community includes agencies of the Western Australian government, the private sector and universities.

2. Research outputs are channelled through a number of publications including the Sandalwood Research Newsletter and the Avon Sandalwooder.

3. To date, Australian research has focussed on Indian sandalwood (S. album) and projects have had a strong applied research focus.

4. Industry consultation revealed concern that much of the research being completed in Australia is proprietary and not available to potential investors.

5. Strong support was recorded for additional investment in sandalwood R&D and a greater role for RIRDC was requested.

6. Key research breakthroughs for both Indian and Western Australian sandalwood were identified as faster maturing and higher yielding varieties and production systems. For Western Australian sandalwood, product and market development, especially in relation to the S. spicatum nut, was identified as a priority.

7. Industry constraints were a corollary of research breakthroughs with the length of time between plantation establishment and harvest remaining the major barrier to profitable production. The importance of plantation design for S. spicatum was also highlighted.

8. Least adequately researched areas for both species were markets and plant genetics.

9. Future Indian sandalwood research should target processing, plant physiology/genetics, markets, communication and industry development and training.

10. Future Western Australian research should target market research, processing, plant physiology/genetics and sustainability.

Implications The Australian sandalwood industry is set to dominate world supply of

sandalwood oil. Substantial gaps in the industry’s knowledge in relation to processing, plant physiology, markets, sustainability and industry development remain. There is a role for RIRDC in the provision of public good research that fills the breach between industry need and non-proprietary research interests. The findings from this report should inform future priorities in the Essential Oils and Plant Extracts sub-program.

Publications AGL-5A (RIRDC Publication 06/131)

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RESEARCH IN PROGRESS − Development and Improvement of Products Project Title

Development and economic assessment of Artemisia for production of anti-malarial extract

RIRDC Project No.:

BRA-4A

Finish Date: 31/12/09 Researcher: Dr. Rowland Laurence Organisation: University of Tasmania

(Honorary Research Associate) Phone: (03) 6428 2900 Fax: (03) 6425 4468 Email: [email protected]; [email protected]

Objectives To develop commercially viable techniques for the production of marketable

extracts of artemisia, through the investigation of ways to maximise the yield of artemisinin, the anti-malarial compound, present in Artemisia annua L.

Current Progress This project, funded from January 2006, attempts to determine a production method for Artemisia annua, which will be economically viable in Australia. Key areas for investigation are seed handling and sowing techniques, weed control and harvesting systems. Trials were carried out in the 2006-07 season in attempts to improve the low levels of field emergence, which were previously achieved. Treatments, including soil additives and mulches, together with seed pre-germination, aimed to maintain the soil surface moisture levels required by this small and fragile seed. While some treatments improved emergence, none were as effective as sufficiently frequent irrigation. The previous season’s initial assessment of the herbicide tolerance of A. annua transplants has been extended to small, directly sown plots, with some limited success. Small plots of A. annua transplants were used to investigate the changes in plant artemisinin levels with sowing date and harvest time. Transplants were also used to make an initial assessment of the effectiveness of a commercial harvester to strip the artemisia leaves from stems, with encouraging results. Investigations in 2007-08 will continue to seek the reliable establishment of A. annua seed sown directly into larger plots and to improve therein the methods of chemical weed control.

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Production of newsletter of the Essential Oil Producers Association of Australia

RIRDC Project No.:

ISO-1A

Start Date: 02/01/06 Finish Date: 30/06/07 Researcher: Dr Ian Southwell Organisation: 22 Canterbury Chase

GOONELLABAH NSW 2480 Phone: (02) 6624 2453 Email: [email protected]

Objectives To publish a newsletter for the essential oil industry that reports on matters of

interest to growers, producers, distillers, formulators, traders, marketers and researchers of essential oils and related natural products.

Current Progress

Newsletters Nos 13, 14 and 15 were published in February 2006 (12 pages), September 2006 (12 pages) and May 2007 (20 pages). The newsletter continued to update members with current information and bring them together with a means whereby they can share key facets of the industry. Such information dissemination is stimulating the growth and development of the natural products industry. Thus the newsletters became valuable vehicles for the adoption of R&D findings as commercial reality. Each issue included details about:

• Recent conferences dealing with essential oils (both research oriented

as well as industry oriented); • Company news and general trade information; • Regulatory matters such as NICAS developments, SCCP opinions

especially for for Tea tree, new European Union rules for the labelling of allergens etc;

• Standards Australia matters: new standards (Australian as well as international);

• Information on recently published articles on essential oils of interest to Australian Producers;

• RIRDC news items. • Details of conferences on essential oils and related matters scheduled

for the next 12 months.


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Using frontier technologies for the quality assurance of medicinal herbs

RIRDC Project No.:

RMI-18A

Start Date: 01/07/06 Finish Date: 30/06/09 Researcher: Associate Professor Eddie Pang Organisation: RMIT University Phone: (03) 9925 7137 Fax: (03) 9925 7110 Email: [email protected]

Objectives The rising demand for medicinal herbs increases the pressure for quality

assurance, one of the major problems in the medicinal herb industry. Many medicinal herbal products are in powered or shredded form and it is almost unfeasible to ensure authentication by morphological and histological methods. Botanical identification by chemical methods is limited by variables such as storage conditions, sample freshness, and the varying techniques used for post harvest processing. A number of DNA-based genetic marker systems have been developed and are considered more reliable. However, the reliance of those methods on gel electrophoresis makes them less advantageous over highly novel array based fingerprinting methods. DNA array methodology presents a better alternative that meets the high throughput needs of the medicinal herbs industry. The objectives of this project are: 1. Refine the prototype DNA fingerprinting microarray developed for

Chinese medicinal herbs, expanding its functionality to include other herbs important to the Australian herbal industry, e.g. Ayurvedic, Western and Pan-American herbs.

• A prototype DNA microarray has been successfully developed with funds from an RMIT University Research Investment grant for fingerprinting locally grown Chinese medicinal herbs. We propose to expand and refine this prototype array such that it may be used as a single platform for identifying species, varieties and chemotypes of medicinal herbs used in Australia.

2. Develop a database of DNA fingerprints for economically important

species and chemotypes of medicinal herbs. • Using the enhanced microarray, we plan to fingerprint over 20 species

of medicinal herbs and their chemotypes. Generated molecular fingerprints and other relevant information will be stored in a comprehensive database.


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Current Progress

The initial phase of the project involved extracting DNA from Western, and additional species of Chinese medicinal herbs. We have completed extracting total DNA from fresh leaves of significant Chinese medicinal herbs (122 species). We initially encountered a few difficulties in extracting substantial amounts of good quality DNA from certain plant species due to their phenolic/polysaccharide content. However, these difficulties were overcome when we employed an optimised CTAB extraction technique. Southern Cross University provided us with additional Chinese, Ayurvedic, Western and Pan-American herbs. Out of 80 different samples, we have extracted DNA from 50 samples and will be able to process remaining samples within a fortnight. This will conclude the initial phase of the project and the second phase will be the construction of a subtracted DNA library. A PhD student was recruited via the APA Scholarship Scheme to work on producing a component of the microarray which is capable of discriminating between different chemotypes of red sage (Salvia miltiorrhiza) and medicinal passionfruit (Passiflora sp.). We have also published some of our initial findings (based on prototype array) in a leading scientific journal and are in the process of preparing second manuscript for publication.

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RESEARCH IN PROGRESS − Development and Improvement of Products

Project Title

Fabrication of electronic materials from the Australian essential oils

RIRDC Project No.:

UJC-13A

Researcher: Dr Mohan Jacob Organisation: James Cook University Phone: (07) 4781 4379 Fax: (07) 4781 5177 Email: [email protected] Objectives 1. Fabricate high quality Plasma Polymerised thin films from essential oils

available in Australia. 2. Procure an Ellipsometer and study the optical properties of the fabricated thin films to find suitable applications. 3. Fabrication of thin film and study the semi-conducting properties.

Background More than 60 billion pounds of polymer products is used per year only in USA, which includes the polymers like Polyethylene. The total polymer industry around the world is worth billions of dollars and employs several million people. Most of the commercially available polymers are made out of chemicals and not biodegradable, and are hazardous to the environment. The aim of this research is to fabricate polymers from natural resources and hence adding value to our resources.

Research A plasma polymerisation unit has been fabricated and installed to develop polymer thin films from essential oils. The deposition parameters were changed to optimise the thin film properties. Different essentials oils were tested to find the feasibility of polymerisation. a-Pinene and d-Limonene and tea tree oil was used as monomers for fabrication of the thin films. The Atomic Force Microscope was used to study the surface properties and found that very smooth films can be obtained from tea tree oil. We have also tried different substrate materials. An ellipsometer was ordered to study the optical properties. We have installed an electrical characterisation set up for the measurement of electrical properties.

Outcomes Even though we could polymerise the monomers, the quality of the thin film was different. Some of the cheapest essential oils like a-pinene and d-Limonene could produce polymer thin films under ideal conditions but the roughness of the surface was very high. The optical properties of the thin films derived from tea tree oil was studied and found that the refractive index of the thin film is close to that of glass. A setup is made ready for electrical characterisation.

Implications The properties of the thin polymer films show that they are potential candidates for electrical and biomedical applications. More tests are under progress to study the behaviour of the films under ideal and varying physical and environmental conditions. Initial studies show a biodegradable nature of the fabricated thin film.

Publications We are waiting for more results before communicating the results in the public domain.

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Analysis of Dodonaea viscosa as a potential remedy for anti-inflammatory applications

RIRDC Project No.:

UNC-21A

Start Date: 01/07/03 Finish Date: 30/06/07 Researcher: Mr Andrew Pengelly Organisation: School of Environmental & Life Sciences

University of Newcastle Phone: (02) 4353 3193 Fax: (02) 4349 4490 Email: [email protected]

Objectives 1. To develop analytical techniques to analyse for saponins, flavonoids,

coumarins and diterpenes found in Dodonaea viscosa var Angustifolia. 2. To determine levels of each constituent group in various plant sections and determine the effects of some variables such as growing location 3. To test extracts with a known biochemical matrix for anti-inflammatory properties.

Current Progress

Phytochemistry Previously, a quantitative method based on quercetin equivalents for assessing total flavonoids in D. viscosa was described. The procedure continues to be applied to test for seasonal variation in flavonoid levels in two growing locations. Variation between male and female plants is also being assessed. To date, the male samples have consistently contained higher flavonoid levels. In addition, I have just obtained a set of flavonoid standards previously identified in D. viscosa. The standards are from a world authority in the field. These are being used to further characterise and quantify flavonoids in Australian D. viscosa. The interim results were presented at the Phytochemistry Society of Europe’s annual conference at Cambridge University, in the form of a poster. In a separate undertaking further phytochemical analysis for diterpenes and other constituents is underway. Bioassays Previous difficulties encountered in culturing fibroblasts have been overcome, and tissue culture experiments are now in progress. The objective of this procedure is to assess fibroblast proliferation activity for D. viscosa, as a means of substantiating the observed wound healing properties, in conjunction with an already demonstrated peroxide inhibition activity. In addition, a soy lipoxygenase assay will soon be established to assess inhibition of inflammatory mediators.


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Clinical trial of kunzea oil for onychomycoses treatment- Commercial potential

RIRDC Project No.:

UT-51A

Start Date: 01/09/06 Finish Date: 31/12/08 Researcher: Dr Glenn Jacobson Organisation: University of Tasmania Phone: (03) 6226 2202 Fax: (03) 6226 2870 Email: [email protected]

Objectives This study will compare the effectiveness and tolerability of kunzea oil with

amorolfine nail lacquer, in the treatment of onychomycosis. Our hypothesis is that due its potent antifungal activity and high penetration of the nail bed, kunzea oil is a better treatment for onychomycosis than amorolfine. The proposed trial of the oil will result in the clinical validation of the oil for the treatment onychomycosis. Clinical proof of efficacy can then be used in the marketing of the oil as a therapeutic agent. Industry and commercial partners will facilitate commercialisation of this product should clinical efficacy be demonstrated.

Current Progress

Progress has been made to significantly recoup the time delays noted in the previous annual report where contractual delays meant finance accounts were not opened until 28th November 2006 and the holiday period during January 2007 slowed progress. From the GANNT chart (B13): All tasks requiring completion have been met (shown below) and the project is currently treating and recruiting patients. However, recruitment has been slower than initially expected through clinic advertising alone. Recruitment is to be accelerated through extensive local media advertising of the trial in June now that the recruitment/pathology confirmation/medication supply logistics have been streamlined. Targeted recruitment at beauty salons (performing nail pedicures) is to be undertaken. All consumables have been purchased (including nail files, lacquer bottles with brushes, labels printed, amorolfine lacquer) and treatment outcome measure paraphernalia including cameras and memory cards, Opsite flexi fix film. Patient recruitment has commenced (12 patients recruited week ending 25/05/07; pathology results received for 5 patients, 2 of these with confirmed fungi as per inclusion criteria; waiting on pathology for 7 patients). All trial documentation (case report forms, quality of life questionnaires etc) have been finalised and are in use.


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RESEARCH IN PROGRESS - Improved Production Systems Project Title

Diagnostic tools for quality enhancement in Australian essential oil industries

RIRDC Project No.:

ANU-74A

Start Date: 01/06/06 Finish Date: 30/05/07 Researcher: Dr William Foley Organisation: Australian National University Phone: (02) 6125 2866 Fax: (02) 6125 5573 Email: [email protected]

Objectives The project aims to exploit the recent discovery of genes that control the

production of terpenes in Melaleuca. The discovery of these genes provides a means of identifying the genetic differences responsible for the chemotypic variation among species and genotypes of Melaleuca plantation cultivars. expensive and slow. This project will correlate variations in the terpene profiles of Melaleuca chemotypes with variations in the sequences of terpene synthase genes. With this information it will be possible to identify diagnostic genetic variation that can ultimately be converted into diagnostic assays for use by breeders.

Current Progress We have completed the collection of Melaleuca alternifolia leaf samples, covering its natural distribution. The leaf oils have been analysed and the presence of individuals of all known chemotypes has been confirmed. RNA has been extracted from individuals representing all chemotypes, and the construction of cDNA libraries has commenced. DNA has been extracted from all samples collected, and has been tested for purity and quantity. We currently have consistent results in isolating four distinct terpene synthases from genomic DNA, covering up to 60% of the open reading frame with sufficient specificity to allow for direct sequencing. Furthermore, we have been able to apply recently obtained sequence information from Eucalyptus as well as publicly available Melaleuca sequence to isolating the mevalonate kinase and isopentenyl diphosphate isomerase genes responsible for upstream steps in the terpene biosynthesis pathway. Presently, population wide sequencing of close to 100 individuals is underway to ascertain variation in the sequences available, while further effort is being put into completing the sequence information of all genes to obtain 100% coverage. We are thus close to being able to correlate variations in gene sequences with variations in oil profiles to provide a diagnostic test of high value oils in Melaleuca.


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Commercial production of milkweed, a plant with anticancer properties

RIRDC Project No.:

DAQ-329A

Start Date: 01/11/05 Finish Date: 28/02/08 Researcher: Mr Larry Cooper Organisation: QLD Department of Primary Industries and Fisheries Phone: (07) 3824 9541 Fax: (07) 3286 3094 Email: [email protected]

Objectives Research will concentrate on enhancing the methods of commercial production

of E. peplus. Within the proposed project, cost effective, large-scale production techniques will be enhanced to enable producers to meet market needs and enable the even and reliable production of PEP compounds all year round for a larger market. Efficient production systems for both intensive cropping in artificial structures and less intensive broad-acre field production will be developed. To date, some of the production has been poor quality and erratic. This project will also seek to identify safe ‘organic’ or ‘low synthetic input’ methods, while enhancing the overall yield of the PEP compounds.

Current Progress Trials initiated in December 2006 to examine the effects of nutrients on PEP005 content were abandoned in Feb 2007 due to the researcher having health problems. A new trial was initiated in May 2007 and treatments are currently being applied. All plants receive trace elements and low rates on NPK and treatments of higher rates of NPK are added over time. Determination of PEP compound concentrations will occur after all harvesting is completed. Seed storage testing and supply system to growers is working well, and a additional seedling supplier has been engaged. The seed supply is now stored at DPI&F Science Centre, Indooroopilly. A second meeting of researchers, growers and Peplin was held on 2nd December 2006 at Stanthorpe and was attended by 9 growers, 3 researchers and 2 staff from Peplin. Discussions and presentations focussed on plugs/direct seed, irrigation, spacing of plants and harvesting issues. Results were the design of the nutrition trial and plans for a further trial to examine ‘Time of day’ for harvesting on PEP005 content. Growers networked and shared ideas freely.


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RESEARCH IN PROGRESS - Improved Production Systems Project Title Growing the Essential Oils Industry RIRDC Project No.:

EOT-1A

Start Date: 01/09/06 Finish Date: 30/05/09 Researcher: Ms Susan Hinton Organisation: Essential Oils of Tasmania Pty Ltd

PO Box 162 Kingston TAS 7051

Phone: 0417 038 274 Fax: (03) 6229 2957 Email: [email protected]

Objectives This project aims to support the expansion and international competitiveness of

the essential oils industry in Australia by establishing an industry development team to ensure full implementation of past research knowledge on a range of crops including peppermint, fennel, parsley, boronia and lavender. The emphasis will be on developing and fine tuning best practice procedures for commercial production of essential oil crops through detailed high quality crop manuals incorporating the latest information supported by readily accessible crop advisory systems. The project will also allow ongoing field trial work, in some cases to refine data from previous research, as it is progressively incorporated into commercial management systems. A minor aspect of this will be assessment and securing of genetic resources to provide producers with germ plasm with the highest potential available.

Current Progress (200 words maximum)

Existing information, including cultural notes, the Mint Manager(RIRDC 2000 – publication no. 00/20) and research reports have been reviewed and draft pro-forma manuals for peppermint, fennel, parsley, lavender and boronia have been developed.

Requirements for visual display material have been determined. Some photos clearly depicting disease symptoms have been taken during the 2006/07 season and more will be taken during the 2007-08 season, for example, the various stages of puccinia infection on peppermint and boronia or cercosporidium infection on fennel.

The review has also identified situations where production information is incomplete or research results need verification before committing to commercial programs, and the appropriate field trials have started.

Sampling programs have been started for the development nutrient management protocols to allow fertiliser recommendations to be fine-tuned. Soil moisture monitoring equipment is being used to develop profiles of crop water use and irrigation scheduling programs for each crop.

The assessment and securing of genetic resources has commenced. The aim is to provide producers with germplasm with the highest potential available. Areas have been planted to peppermint and lavender, with a number of selections of each, to be evaluated. Seed has been collected from fennel and will be planted and evaluated. A number of parsley varieties have been sourced, for evaluation.


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Improved yields from Tasmanian peppermint crops through improved micronutrient nutrition

RIRDC Project No.:

UT - 49A

Start Date: 01/07/05 Finish Date: 30/02/07 Researcher: Professor Robert Menary Organisation: University of Tasmania Phone: (03) 6226 2723 Fax: (03) 6226 7609 Email: [email protected]

Objectives Determine current micronutrient status of commercial fields and undertake

diagnosis and recommendation for remedial action.

Current Progress A field trial to investigate responses of peppermint to S, Mo, Cu and B has been harvested in two successive seasons. In the first season, oil yield in herb was significantly increased in the presence of B. There was no overall increase in oil yield per ha. In the 2nd, oil yield per ha was significantly increased where S and Mo was applied as fertiliser. Nutrient calibration trials provided data to establish critical levels in tissues for B, S, Cu and Mo. Using this data the presence of deficiencies of these elements was established during the last season. Oil compositional changes have been followed for two seasons. There have been no significant changes in oil composition that could be directly related to fertiliser treatment. An important outcome of the work has been the detection and correction of deficiencies in commercial fields of peppermint based on experimental data. This has led to increased yields and improved economic viability of the industry


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RESEARCH IN PROGRESS – Regulatory Approvals Project Title

Quality assurance protocols to minimise pesticide residues in essential oils

RIRDC Project No.:

UT - 45A

Start Date: 01/06/04 Finish Date: 30/06/07 Researcher: Professor Robert Menary Organisation: University of Tasmania Phone: 03 6226 2723 Fax: 03 6226 7609 Email: [email protected]

Objectives 1. Develop a paper based chemical management plant for an approved supplier

program. 2. Optimise analytical procedures for analysis of pesticides in essential oils. 3. Monitor pesticide behaviour in cropping cycle. 4. Undertake hazard analysis for pesticides used in crop production.

Current Progress) Researchers at the University of Tasmania, together with Essential Oils of Tasmania as the industry partner, and Tasmanian farmers have developed a HACCP based approach to minimising pesticide residues in essential oils and plant extracts. The process identified a range of critical control points and control points within the production chain. The pesticide related critical control points are grower selection, pesticide analysis, and traceability of products throughout the system. Research was conducted in relation to the control points in order to set critical limits and make recommendations for improvements. An important area of focus was pesticide analysis. In consultation with the industry, analytical systems, primarily using HPLC MS / MS methods were further developed in comparison to research contained in previous RIRDC reports. A series of field trials were also conducted to assist with the determination of critical limits in relation to withholding periods following pesticide application. A further research focus was an exploration of calibration and cleaning procedures. The developed procedures are contained within a document titled the Approved Supplier Program. The aim of the document is to provide farmers with strategies for minimising pesticide usage in order for the product to comply with the maximum residue limits defined by buyers and, international standards and regulations.

program 1.4 essential oils & plant extracts...peppermint in favour of crops with higher gross...

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