ESSENTIAL OILS AND PLANT EXTRACTS

RIRDC Completed Projects in 2007 - 2008 and Research in Progress as at June 2008

ESSENTIAL OILS AND PLANT EXTRACTS

October 2008 RIRDC Publication No 08/066

© 2008 Rural Industries Research and Development Corporation. All rights reserved. ISBN 1 74151 655 2 ISSN 1440-6845 RIRDC Completed Projects in 2007 – 2008 and Research in Progress as at June 2008 - Essential Oils and Plant Extracts Publication No 08/066 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. 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. RIRDC Essential Oils and Plant Extracts Research Manager Dr Roslyn Prinsley Rural Industries Research and Development Corporation Level 2 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: (02) 6271 4120 Fax: (02 6271 4199 Email: [email protected] 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] Website: http://www.rirdc.gov.au

Published in October 2008 by Union Offset

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Foreword RIRDC produces Research in Progress summaries of continuing projects and those completed during 2007-2008. Our intention is to:

• give stakeholders early access to the results of ongoing and completed work to inform their decisions, and

• to inform researchers of results to shape research directions

The complete report on all programs is on our website at http:www.rirdc.gov.au This report is an addition to RIRDC’s diverse range of over 1800 research publications, which are available for viewing, downloading or purchasing online thorough our website: http:// www.rirdc.gov.au/fullreports/index.html• purchases at http:/www.rirdc.gov.au/eshop

Peter O’Brien Managing Director Rural Industries Research and Development Corporation

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http://www.rirdc.gov.au/fullreports/index.html

http://www.rirdc.gov.au/eshop

Contents

1.4 ESSENTIAL OILS COMPLETED PROJECTS PROJECT No

PROJECT TITLE

RESEARCHER

PHONE

ORGANISATION

PAGE No

Improved Post Harvest Treatments PRJ-000467 Quality assurance protocols to

minimise pesticide residues in essential oil

Robert Menary (03) 6226 2723 University of Tasmania 1

Improved Production Systems PRJ-000468 Increased yields from peppermint

crops through improved micronutrient nutrition

Robert Menary (03) 6226 2723 University of Tasmania 2

PRJ-000598 Commercial production of milkweed a plant with anti-cancer properties

Larry Cooper (07) 3824 9541 Qld Department of Primary Industries and Fisheries

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PRJ-000019 Spatial analysis of plant-host relationships in tropical sandalwood

Liz Barbour (08) 9475 8888 Forest Products Commission

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Support new ideas for products PRJ-000795 Fabrication of Electronic Materials

from the Australian Essential OilsMohan Jacob (07) 4781 4379 James Cook University 5

PRJ-000703 Analysis of Dodonea viscosa as a potential remedy for anti-inflammatory and

Andrew Pengelly (02) 4349 4490 University of Newcastle

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1.4 ESSENTIAL OILS RESEARCH IN PROGRESS PROJECT No

PROJECT TITLE

RESEARCHER

PHONE

ORGANISATION

PAGE No

Improve industry and research capacity PRJ-000020 Production of TALGA lavender

industry newsletterMichael Basil (02) 4843 3334 Australian Lavender

Growers’ Association Inc

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Improved Post Harvest Treatments PRJ-000021 Develop Australian Standards For

Oil Of Australian Lavandin Cultivars

Ian Southwell (02) 6624 2453 Phytoquest 11

Improved Production Systems PRJ-000773 Growing the essential oils industry Susan Hinton 0417 038 274 Essential Oils of

Tasmania 12

Support new ideas for products PRJ-000017 Aroma and Flavour Products from

Plant WasteRobert Menary (03) 6226 2723 University of Tasmania 13

PRJ-000018 Management of postharvest diseases using Australian essential oils

Elena Lazar (02) 4348 1935 NSW Department of Primary Industries

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PRJ-000464 Clinical trial of kunzea oil for onychomycoses treatment-commercial potential

Glenn Jacobson (03) 6226 2202 University of Tasmania 15

PRJ-000462 Production of Newsletter of the Essential Oil Producers Association of Australia

Ian Southwell (02) 6624 2453 Phytoquest 16

PRJ-000841 Growing Xi Shu and extracting an anti-cancer drug (Camptothecin)

Craig Davis (07) 3406 8611 Qld Department of Primary Industries and Fisheries

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PRJ-000763 Using frontier technologies for the quality assurance of medicinal herbs

Eddie Pang (03) 9925 2283 Royal Melbourne Institute of Technology

18

PRJ-000778 Development and economic assessment of Artemisia for production of anti-malarial extract

Rowland Laurence (03) 6425 0508 Botanical Resources Australia Pty Ltd

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Completed Projects - EOI-Improved Post Harvest Treatments Project Title

Quality assurance protocols to minimise pesticide residues in essential oil

RIRDC Project No.:

PRJ-000467

Start Date: 5/31/2004 Finish Date: 8/30/2007 Researcher: Robert Menary Organisation: University of Tasmania Phone: (03) 6226 2723 Fax: (03) 6226 7609 Email: [email protected] Objectives

* Develop a paper based on a chemical management plant for an approved

supplier program * Optimise analytical procedures for analysis of pesticides in essential oils * Monitor pesticide behaviour in cropping cycle - Undertake hazard analysis

for pesticides used in crop production Background

The Australian essential oils industry has an obligation to provide quality products that meet the requirements in relation to pesticide content that are specified by the customer. Additionally, strict regulations in regard to allowable pesticide levels are now in place in many of the European Union and North American countries where Australian essential oils and extracts are routinely sold. Consequently the necessity for a comprehensive Quality Assurance Program for the essential oils industry that reduces the risk of pesticide levels exceeding either the customer requirement or regulatory guidelines was apparent.

Research

The research in this project related to development of a HACCP based hazard reduction program for the minimisation of pesticide use and the prevention of products with unacceptable pesticide levels reaching the market place. Firstly a collaborative meeting between staff from Essential Oils of Tasmania (EOT), researchers at the University of Tasmania, Farmers and Consultants (Icon Global Link) resulted in identification of all steps in the production cycle. This was used to develop a HACCP based hazard reduction plan and areas where research was required for implementation of the plan were identified. This research involved method development in regard to methods for pesticide analysis and protocols for the development of equipment handling and calibration.

Outcomes

A HACCP based Standards Enforcement Plan and a related Approved Supplier Program were developed for use by the essential oils industry and farmers. Method development for the analysis of pesticides using a targeted HPLC MS/ MS approach was undertaken and Limits of Detection (LOD) and Limits of Quantification (LOQ) were subsequently determined in specific plant matrices as part of the analysis of field trial samples.

Implications

The implementation of the recommendations contained within this report will minimise the risk of pesticide contamination in extracts and oils. However ongoing research is necessary for the development of analytical methods for pesticides that are newly proposed as having application in the essential oils industry or where customers require lower detection limits than are presently available for a particular pesticide.

Publications

Minimising pesticide residues in essential oils, RIRDC publication No. 08/121

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Completed Projects - EOI-Improved Production Systems Project Title

Increased yields from peppermint crops through improved micronutrient nutrition

RIRDC Project No.:

PRJ-000468


Determine current micronutrient status of commercial fields and undertake diagnosis and recommendation for remedial action.

Background

Peppermint oil yields in Southern Australia are highly variable and this is impacting on the long term viability of the industry. The average yield of 50 kg/ha, despite attempts to improve management practice, has not been sufficient to sustain profitability for farmers. Improved nutrition is likely to deliver the yield required to meet criteria for an increased production. Particularly as the improved nutrition will enable a second cut to be made due to increased growth. The steam distillation facilities are also required for fennel, parsley, chamomile and clary sage but the volume of herb to sustain viability of the steam distillation facilities primarily comes from peppermint. Without peppermint, the steam distillation units would not be viable.

Research

Plant tissue and soil analysis for macro and micro nutrients from all commercial production areas have shown that deficiencies of S, B, Cu and Mo commonly occur. A nutrition trial was conducted in the greenhouse on soil collected from one of the commercial areas. This trial confirmed that deficiencies of S, Cu, B and Mo existed and established calibration data for detecting nutrient deficiencies. A fertiliser trial in the field confirmed the oil yield and plant responses to S, B, Cu and Mo.

Outcomes

Based on the evidence of nutrient deficiencies, an ongoing fertiliser program has been devised for growers. Diagnostic tools to develop scientifically based fertiliser recommendations for correction of deficiencies have also been devised and are now available to field advisory staff.

Implications

The project is likely to improve gross margins to a level which will encourage new plantings. Furthermore, balanced nutrition will allow sufficient growth for a double cutting program which has been hampered in the past by poor plant growth.

Publications

A CD entitled, “Aspects of Peppermint Nutrition” has been produced which describes visible symptoms as an aid to detection of deficiencies in the field. This was distributed to all peppermint growers in Tasmania at the completion of project UT-37A. Publications will follow when the research outcomes have been fully applied to commercial practice.

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Commercial production of milkweed a plant with anti-cancer properties

RIRDC Project No.:

PRJ-000598

Start Date: 7/1/2005 Finish Date: 2/29/2008 Researcher: Larry Cooper Organisation: The State of Queensland Acting through the Department of Primary Industries

and Fisheries Phone: (07) 3824 9541 Fax: (07) 3286 3094 Email: [email protected] Objectives

Milkweed (Euphorbia peplus) has bioactives known as PEP compounds and these are suitable for use in treating various cancers, particularly skin cancer. This research will build on the system for commercially growing of E. peplus, which was developed as a result of previous projects funded by RIRDC. Cost effective 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. Research to date was focused on production of material for clinical trials. This research will result in the development of intensive production techniques for commercial scale production and extraction. The growing sector which will benefit from this research would be small holdings and holdings with some covered or protected areas such as shade or plastic tunnel. The growing E. peplus would aso be attractive to organic growers.

Background

Milkweed (Euphorbia peplus) has bioactives known as PEP compounds and these are suitable for use in treating various cancers, particularly skin cancer. Research to date was focused on production of material for clinical trials. There is a need for cost effective production techniques to enable the even and reliable production of PEP compounds all year round for a larger market. This research will result in the development of intensive production techniques for commercial scale production and extraction.

Research

Research concentrated on enhancing the methods of production for commercial growers of E. peplus. The investigation were carried out in field and protected environment studies at DPI&Fs Redlands Research Station, Cleveland. Optimal row width and planting density - Replicated field trials investigated the effect of planting density, plant type (seedling or seed) and plastic mulch on overall plant weight harvested per unit area and PEP compound content. Fertiliser regimes - Fertiliser trials in small, replicated pot experiments investigated macronutrients such as nitrogen, potassium and phosphorus looking at plant weight, health and PEP content using treatments designed after discussion with DPI&F plant physiologists, soil scientists and existing growers. Time of harvest - There appears to be no dramatic response to either temperature or solar radiation in the pattern of production of PEP005 and PEP008.

Outcomes

From a production point of view the best systems are to use plastic mulch (efficient water use and weed control), trickle irrigation (efficient water use), plug seedlings followed by direct seeding (easier and more consistent establishment) and a fertiliser regime which includes high rates of nitrogen and low rate of phosphorous and potassium (maximise plant size and PEP compound production). Seed supply system, grower meetings and extension activities were also carried out.

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Spatial analysis of plant-host relationships in tropical sandalwood

RIRDC Project No.:

PRJ-000019

Start Date: 7/1/2007 8:00:00 AM Finish Date: 5/30/2008 5:00:00 PM Researcher: Liz Barbour Organisation: Forest Products Commission Phone: (08) 9475 8888 Fax: (08) 9475 8899 Email: [email protected] Objectives

To determine the critical parameters of a host plant that stimulate tropical sandalwood wood production.

Background

The production of sandalwood oil is the fastest growing essential oil and plant extract industry in Western Australia. The Forest Products Commission and former entities of this Western Australian State agency has been researching tree crops in the Ord River Irrigation Area since 1987. The majority of the trials are located in Cununurra clays. This project is an analysis of one of these trials.

Research

In 1999 a trial was established which included 6 long-term hosts , Khaya senegalensis and Cedrela odorata from the Meliaceae and Cathormium umbellatum, Dalbergia latifolia , Pongamia umbellatum and Pterocarpus indicus from the Leguminosae. The pot host was Alternantha and the short-term host was Acacia trachycarpa. The trial was planted in a Randomised Complete Block design with 5 replicates. The ratio of sandalwood to short-term host was 1:1 and 1:2 for the long term hosts. After 4 years growth, due to over-crowding, the long term hosts were thinned to a ratio of 1:1 sandalwood to long term hosts. Trial assessment was undertaken at 2, 3 and 9 years of age.

Outcomes

Good initial establishment of the pot host, Alternantha and the short-term host Acacia trachycarpa were successful in maintaining high survival and growth for the first years of the trial. The trial changed dramatically with two events between year 3 and 9. The first event was the artificial reduction of the secondary hosts to half their original planned stocking and the second, the natural death of the Acacia trachycarpa. The final measurement showed a clear difference in Tropical sandalwood growth with those supported by Leguminosae species being superior to the Meliaceae hosts. There were also differences in performance within the Leguminosae with Dalbergia latifolia and Pongamia pinnata showing the greatest potential for sustained sandalwood growth. In 9 years of growth, tree wood volume per hectare is estimated to be 1.5 m3 per hectare with a survival of between 357 – 390 sph after an initial establishment of 462 sph. The reason for their superiority was masked by the changing of sandalwood to host ratios.

Implications

This trial has demonstrated that quality Tropical sandalwood can be grown in the Cununurra clays with a pot host of Alternantha, a primary host of Acacia trachycarpa and a secondary host of a Leguminosaea, preferably Dalbergia latifolia or Pongamia pinnata.

Publications

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Completed Projects - EOI-Support new ideas for products Project Title

Fabrication of Electronic Materials from the Australian Essential Oils

RIRDC Project No.:

PRJ-000795

Start Date: 7/1/2006 Finish Date: 4/30/2008 Researcher: Mohan Jacob Organisation: James Cook University Phone: 07-47814379 Fax: 07-47815177 Email: [email protected] Objectives

The main objective of this project is to find a non-medical application for essential oils and hence encourage Australian farmers to produce more essential oils. The project aims to: 1. Fabricate high quality Plasma Polymerised thin films from Australian

essential oils (limonene, á-pinene, Tea Tree, Lavender and Eucalyptus oils) 2. Study the electrical and optical properties of the polymer thin films and find

suitable applications and iii)Test the polymer thin films as a protective/anticorrosive layer in devices.

Background

The total polymer industry around the world will be worth billions of dollars and employ millions of people. Most of the commercially available polymers are made out of chemicals and are not biodegradable, and are hazardous to the environment. Synthetic polymers are hazardous to the environment and could contain carcinogenic materials. Bio-based polymers will in the future lead towards a healthy and environmentally friendly life. This innovative research will have immediate impact on the economic and social background of Australian farmers by transforming the essential oil industry via development of novel applications for high value added products. During the last few years research funding agencies realised the relevance of organic semiconductors to replace the conventional semiconducting material – Silicon. This particular project is investigating the fabrication of polymer thin films, which will be of use to the Electronic Industry. This work was to fabricate polymer material from agricultural products for the implementation in electronic products. The project outcomes will benefit the Electronic Industry and Agricultural Industries.

Research

Several essential oils, such as Tea Tree Oil, Sandalwood Oil, Eucalyptus Oil, Alpha-Pinene, d-Limonene, Lavender Oil (a separate PhD project) and five different major components of Tea Tree Oil, were tested. With the exception of Sandalwood oil, all other oils investigated were successfully polymerised. The thin film thickness, surface profile and surface roughness, optical, electrical and chemical properties of the fabricated polymer thin film were studied. Several samples were deposited under varying deposition conditions such as input RF power, pressure, time and dopants. The investigations showed the properties of the fabricated polymer thin films could be tailored to suit different optical and electrical applications. Polymer thin films fabricated from the Tea Tree Oil were also investigated to understand the biocompatibility of the material.

All the fabricated thin films were transparent to the optical wavelengths and the film thickness varied from 200nm to 2000nm. The film thickness could be varied by changing the deposition conditions to cater the requirement for a given application. Over all, the refractive index varied from 1.53 to 1.7 in the wavelength range 200 nm to 1000nm. The refractive index is above that of the glass surface. The material could be used in many lens applications. The surface profile shows that the fabricated polymer thin films are very smooth (roughness around 1 nm or smaller), uniform and defect free. In terms of

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surface properties and smoothness, the materials fabricated from Tea Tree Oil and its components were found to be superior to that of alpha-pinene and d-Limonene. The hardness of the material varied from material to material ranging from 0.11 to 0.65 for the samples fabricated under the normal deposition conditions, with Terpenene-4-ol exhibiting the highest hardness. The hardness of the material can be varied by changing the input RF power. Materials fabricated at low RF power were very soft whilst the ones manufactured at high RF power were hard. Therefore, materials of different hardness could be fabricated from the essential oil to meet the demands of a particular application. The chemical properties of the monomer and polymer were studied. The polymer thin films show very consistent and stable material properties. There was no inconsistency observed in terms of chemical, optical or electrical properties between the samples fabricated based on monomers from different sources. The electrical properties were studied especially by looking at the energy gap. All the materials investigated exhibited energy gap in the range 2.6 to 3.1eV.

The bio-compatibility study of the material was carried out. The results of this study are very interesting and the material clearly showed the biocompatibility. In few mice sinus formation was observed even though it was not critical.

Outcomes

1) Several essential oils were tested to understand the feasibility of polymerising the material and to develop strategies to fabricate polymer thin films.

2) Thin films of a wide range of thickness were fabricated, which ranges from 200nm to 2000nm.

3) The Optical properties of the essential oil based polymer thin films were studied; refractive indexes of the developed materials are above that of glass.

4) The Chemical properties were studied to understand the stability of the material especially in terms of the variation of the contents from different sources. The properties were not affected by the slight variations in the base material properties.

5) The band gap of the material shows that the material could be classified in the semiconducting range.

6) The biocompatibility study shows that the material could be used in bio-medical applications.

7) The properties of the material were not altered with time and hence could be used in many protective layer coating applications.

8) As a result of this work, we have established new collaborative work with RMIT, Swinburne and ANSTO.

Implications

This study proved that it is possible to polymerise essential oils and the fabricated materials could be the potential candidates for many electronics and bio-medical applications. A more detailed study is essential to understand the polymerisation process and the feasibility of implementation in many practical applications especially biomedical applications. Already we have established the basic facility and infrastructure to pursue the advanced and systematic research in this area. If we get additional funds to support a research assistant and a PhD student, we could advance further in this area.

Publications

Two papers are published in Journals in 2007. New findings and the fabricated material properties will be published without affecting the commercial prospects of the project and RIRD will be acknowledged. Easton, Jacob and Krupka,“Non-destructive complex permittivity measurement of low permittivity thin film materials”, Measurement Science and Technology, vol. 18 pp. 2869–2877 (2007).

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M.V. Jacob, C.D. Easton, G.S. Woods, C.C. Berndt, Fabrication of a novel organic polymer thin film, Thin Solid Films, doi:10.1016/j.tsf.2007.07.151

C. D. Easton and M. V. Jacob, “Evaluation of the different methods used for determining the energy gap and optical band gap of amorphous polymer thin films”, IUMRS-ICAM: 10th International Conference on Advanced Materials October 8-13, 2007.

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Completed Projects - EOI-Support new ideas for products Project Title

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

RIRDC Project No.:

PRJ-000703

Start Date: 7/1/2003 Finish Date: 11/30/2007 Researcher: Andrew Pengelly Organisation: The University of Newcastle Phone: 02 4349 4490 Fax: 02 4348 4145 Email: [email protected] Objectives

(a) To develop analytical techniques to analyse for saponins, flavonoids, coumarins and diterpenes found in Dodonaea viscosa var Angustifolia (b) To determine levels of each constituent group in various plant sections and determine the effects of some variables such as growing location (c) To test extracts with a known biochemical matrix for antiinflammatory and antibacterial properties.

Background

Dodonaea viscosa is an evergreen woody perennial herb found in many parts of the world, most notably in and around the rangeland regions of New South Wales. Ethnopharmacology reporting in Australia and elsewhere indicates a variety of therapeutic uses, in particular as a topical application with anti-inflammatory and analgesic properties. Recent phytochemical studies have confirmed a positive correlation between several groups of active constituents and the traditional usage. Numerous constituents such as triterpenoid saponins and flavonoids have been identified elsewhere, however little work has been conducted on Australian specimens. There is currently a large consumer demand for natural products that relieve pain and inflammation associated with chronic conditions, and this species has the potential to produce a natural extract possessing these characteristics.

Research

An analytical method based on High Performance Liquid Chromatography was designed and validated using the common flavonoid quercetin as a calibration standard, in order to quantify flavonoid levels in a range of D. viscosa samples. Comparisons were made between four distinct geographical regions in New South Wales, and between specimens transplanted from their natural location (Burnbrae) to the Ourimbah campus site with other Burnbrae specimens. Comparisons were also made between plant part extracted and extraction solvent. Samples were tested for anti-inflammatory activity using Cox-2 and PGE2 assays, for anti-oxidant activity using spectrophotometer and microplate assays, and wound healing activity using a human fibroblast proliferation assay.

Outcomes

Specimens from Burnbrae, the most accessible of the regional sites, contained slightly superior levels of flavonoids compared to the other sites, hence it was selected as the main harvest site as well as providing genetic stock for cultivation purposes. There was little variation in overall flavonoid levels during the months tested for, however flowers and capsules had very low levels. Leaves extracted with ethanol provided sufficient flavonoid levels to account for purported therapeutic actions. While there was no evidence of influence on Cox-2 or PGE2 activity, D. viscosa extracts provided significant anti-oxidant activity, particularly when prepared by the hot water method. Initial results also point to an ability to stimulate human dermal fibroblasts and hence enhance wound healing.

Implications

On the basis of these results we have confirmed that Australian D.viscosa has sufficient levels of active constituents to account for its reputation as a

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traditional medicine. It is readily harvested and extracts prepared using simple techniques have demonstrated antioxidant and wound healing activity in vitro. The constituent levels and biological activity appear to be retained when wild plant stock is domesticated, making it a potential candidate for cultivation, were it to become the basis of a commercialised topical application.

Publications

Pengelly, A. Lucock, M. D. and Roach, P.D. A Method For Assessing The Variation In Flavonoid Levels Of Dodonaea viscosa Subspecies Angustifolia, A Traditional Wound Healing Medicine (Poster and abstract). Phytochemical Society of Europe 50th Annual Conference, Cambridge UK. April 2007. Pengelly, A. 2007. Wound healing properties of traditional medicines. NHAA 6th International Conference on Phytotherapeutics, Canberra. Proceedings in press.

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Research in Progress - EOI-Improve industry and research capacity Project Title

Production of TALGA lavender industry newsletter

RIRDC Project No.:

PRJ-000020

Start Date: 7/1/2007 Finish Date: 10/30/2009 Researcher: Michael Basil Organisation: Australian Lavender Growers Association Inc Phone: 02 4843 3334 Fax: Email: [email protected] Objectives

To provide four per annum Lavender newsletters with more appropriate technical content that what is available at present

Current Progress

Since the start of the project, four newsletters have been printed and mailed to industry recipients. These were the Winter 2007 (424), Spring 2007 (424), Summer 2008 (429), and Autumn 2008 (440) editions. The Winter 2008 Journal is currently being finalised for submission.

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Research in Progress - EOI-Improved Post Harvest Treatments Project Title

Develop Australian Standards For Oil Of Australian Lavandin Cultivars

RIRDC Project No.:

PRJ-000021

Start Date: 7/1/2007 Finish Date: 8/31/2009 Researcher: Ian Southwell Organisation: Phytoquest Phone: (02) 6624 2453 Fax: Email: [email protected] Objectives

1. Collect and analyse lavandin oils from Australian lavandin oil producers and collate the data for the purpose of developing new Australian Standards to suit the range of lavandin oil cultivars which are currently grown and for which no Standard(s) exist 2. Use the collected data to determine if the present Australian Standard AS 5028-2002 for Lavandin GROSSO oil satisfactorily accommodates the typical GROSSO oils derived from Australian grown plants , and to recommend revisions if it does not.

Current Progress

Lavandin oil producers have been notified of the details of the project in through the association (TALGA), through essential oil and private networks (eg EOPAA, SAA) and by media release. The industry has generally been slow to respond. As only 6 samples had been submitted by 11/2007, the first agreed milestone, it was agreed to await further samples. A further seven (total 13) had been submitted by May 2008 so these samples were submitted to the first of the three analytical laboratories for analysis, re-bottling and distribution to the other three participating laboratories.

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Research in Progress - EOI-Improved Production Systems Project Title

Growing the essential oils industry

RIRDC Project No.:

PRJ-000773

Start Date: 9/1/2006 Finish Date: 5/30/2009 Researcher: Susan Hinton Organisation: Essential Oils of Tasmania Phone: 0417 038274 Fax: 03 6343 3470 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 germplasm with the highest potential available.

Current Progress

Crop Manuals Grower feedback on the first drafts indicated more in depth information was required. This information is being collated and incorporated into the second drafts. Crop Database The crop management database is currently being refined after implementation. We have external computer consultants developing a web based system for EOT staff and for growers to have real time access to the crop database. This will allow EOT staff to make in field agronomic recommendations and growers to access these recommendations immediately. This system will become the primary communication tool between EOT and the growers, e.g. providing timely information on any crop alerts, updates, reports and any news or events. Germplasm Development A designated area has been created for the germplasm work. In this area we have selections of peppermint and lavender. Samples have been harvested and distilled from the peppermint and lavender selections. Analysis of the oil samples will be conducted in the next month and the results will determine which selections will be further investigated. At this site we have sown a number of parsley and fennel plants from which selections will be made.

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Research in Progress - EOI-Support new ideas for products Project Title

Aroma and Flavour Products from Plant Waste

RIRDC Project No.:

PRJ-000017


A series of experiments and trials will be conducted in order to obtain an increased yield of extract by processing waste boronia flowers following the primary extraction. This process and the associated methods developed as part of the project will be a model for future development in other industries. A preliminary investigations will be conducted here with regard to the potential for application of these technologies to peppermint and fennel.

Current Progress

Analysis of nine commercial trials and two pilot scale trials were completed based either on the use of a fungal isolate or on incubation of the marc without added fungus. The success, in terms of extract yield, obtained with the commercial trials emphasized the need to comprehensively analyse the data from these trials and to investigate the use of AR2000 for the production of an extract that more fully utilized the glycosidic conjugates. This work has delayed the work on isolation of the conjugates. These changes were highlighted in the 2007 Annual Progress Report. The extracts obtained through the commercial processes were examined on the basis of analysis of volatiles, analysis of glycosides and organoleptic assessment. A GC / FID method was developed based on GC / MS identification for the analysis of volatiles. Laboratory based investigations of the use of pectinase treatment investigated variations in the amount of added enzyme, pH, temperature, and removal of polyphenols prior to incubation. Investigations were undertaken with regard to fennel and peppermint through an examination of the commercial harvest and distillation processes and laboratory analysis of samples. Glycosides of flavour and aroma volatiles were detected in peppermint. Incubations of both peppermint and fennel marc were undertaken at a pilot scale, in order to assess the capacity for an increased oil yield from the waste material. Parsley and dill were omitted from the project when it was reduced from a 3 year to a 2 year project.

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Management of postharvest diseases using Australian essential oils

RIRDC Project No.:

PRJ-000018

Start Date: 1/1/2008 Finish Date: 1/1/2009 Researcher: Elena Lazar Organisation: New South Wales Department of Primary Industries for and on behalf of the

State of NSW Phone: (02) 4348 1935 Fax: Email: [email protected] Objectives

The objectives of the project are: 1. To compare the antifungal properties of lemon myrtle oil against citral and aniseed myrtle oil against trans-anethole on selected horticultural postharvest pathogens in vitro. 2. To evaluate the optimal application methods for the selected oils/components in vivo against inoculated and noninoculated products. 3. To establish the efficacy of the oils/components in vivo - treatments necessary to achieve at least 50% reduction in postharvest disease. 4. To trial whether treatment with the selected oils/components under these conditions taints the taste of the product. 5. To submit for publication results of this work in peer-reviewed plant pathology/postharvest journals.

Current Progress

Post Harvest Pathogens selected: One of the key contributors to postharvest losses of fruit and vegetables is fungal infection. To evaluate the antifungal activity of the three essential oils (Lemon Myrtle, Aniseed Myrtle and Tea Tree) and the two requested standards (citral and trans-anethole) four main postharvest pathogens- Monilinia fructicola, Botrytis cinerea, Fusarium oxysporum, and Geotrichum candidum -had been selected for initial in vitro trials.These pathogens are considered of significance to Australian horticultural industry in terms of losses produced during transport and storage of fresh produce. In vitro methodology established: As little research has been conducted into the use of Lemon Myrtle and Aniseed Myrtle essential oils against the selected pathogens using fumigation, pilot trials were conducted in February/March to refine the methodology to be used. The process in which culture plates would be fumigated has been determined and it is believed improves on methods documented in other published research. The concentration ranges of each essential oil and standards to be tested have been established for each pathogen using the pilot trials. Each pathogen showed a different level of sensitivity to each essential oil/standard with preliminary results suggesting that the essential oils/standards provide excellent control of some pathogens on mycelium growth at very low concentrations, whereas for other pathogens higher concentrations that would be economically viable, are needed to reduce significant mycelium growth. In vitro trials should be concluded by the end of June, after which the oils determined to be most effective will be used as a fumigant in in vivo experiments. Taste panel trials will also be conducted towards the end of the year. We are waiting for more results before communicating the outcomes to the public.

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Clinical trial of kunzea oil for onychomycoses treatment-commercial potential

RIRDC Project No.:

PRJ-000464

Start Date: 9/1/2006 Finish Date: 12/31/2008 Researcher: 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

We have screened 129 patients with onychomycosis through RHH podiatry department and 100 had positive mycology, of which 96 started the trial. The gender breakdown of the subjects enrolled in the trial shows 69% male and 31% female. Of the 129 patients screened 51% of the subjects had Distal and lateral onychomycosis. Of the recruited subjects 46% had tinea pedis and 34% had nail trauma. Of the 96 patients started in the trial, 57 patients had their third assessment [20±5 weeks] completed at the time of sub-analysis. Both treatment groups showed significant improvement (p < 0.001) in both clinical parameters [clinical scores, measured area(s) of infection]. At this point no statistical difference was observed between the treatment groups. Infected nail area decreased from 1.84±0.86 cm2 to 1.48±0.76 cm2 in (Loceryl, n=52) and 2.18±1.09 cm2 to 1.38±0.72 cm2 in (kunzea oil, n=44) groups, mean improvement was 16% and 33% respectively. So far 4 subjects have dropped out from the trial, 2 subjects from the kunzea group and 2 from the loceryl group due to non-medical reasons. Cases of adverse reactions reported so far, 2 in the kunzea oil group and 1 in the loceryl group.

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Production of Newsletter of the Essential Oil Producers Association of Australia

RIRDC Project No.:

PRJ-000462

Start Date: 1/2/2006 Finish Date: 5/30/2009 Researcher: Ian Southwell Organisation: Phytoquest Phone: (02) 6624 2453 Fax: Email: [email protected] Objectives

To produce three issues of the newsletter over a period of 2 years.

Current Progress

Newsletter No. 16 was submitted to RIRDC in November 2007 and printed and distributed in late March early April 2008 (16 pages). Data for Newsletter No. 17 has been collected and is at present being collated for submission to RIRDC in early July 2008. 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 and especially the current EU REACH legislation where pre-registration of oils and products imported to Europe in quantities > 1 tonne is required by 30 November 2007

* 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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Growing Xi Shu and extracting an anti-cancer drug (Camptothecin)

RIRDC Project No.:

PRJ-000841

Start Date: 6/4/2007 Finish Date: 5/1/2010 Researcher: Craig Davis Organisation: The State of Queensland Acting through the Department of Primary Industries

and Fisheries Phone: (07) 3406 8611 Fax: (07) 3406 8677 Email: [email protected] Objectives

To propoagate and grow a plot of Chinese happy trees (Xi shu or Campotheca acuminata) To devise methods for the regular harvesting of the young growth To develop a process for the extraction of campothecin from the plant material

Current Progress

In August 2007, ten seeds (previously imported by the Brisbane City Council Botanic Gardens on behalf of the QDPI) were placed into tissue culture. Nine of the seeds were healthy, and two of them resulted in nice naked embryos. In the end, one embryo initiated but it grew abnormally. It seems there was a viability problem with the seeds. Since then, a further 2000 seeds have been imported from the USA. 1000 of these seeds were planted in 5 batches of 200 (separated over a period of 4 weeks). It appears that the seed was old as all seeds have rotted. A further 2000 seeds were imported from France, and all of these have recently been planted. These seeds look more fresh, and so we are more hopeful but far from optimistic. About 40 seeds have germinated thus far, and it appears that more seeds are in the process of germinating. Attempts to import seeds from China have been unsuccessful. Discussions with the Sydney and Adelaide Botanic Gardens have also been protracted due to IP co-ownership, and non-transfer of the plant material issues. Cuttings were collected from the most advanced seedlings in late May 2008 to determine whether a vegetative propagation system could be used to bulk-up Camptotheca acuminata. This approach appears to have great promise, because most of the mother plants have already formed new shoots and some of the cuttings have already formed roots. If cuttings production proves successful, we will meet our second-year milestones very easily. If it proves unsuccessful, we will need to consider discontinuing the project. We will have a better understanding of the situation by 30 June.

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Using frontier technologies for the quality assurance of medicinal herbs

RIRDC Project No.:

PRJ-000763

Start Date: 07/01/06 Finish Date: 05/30/09 Researcher: Eddie Pang Organisation: Royal Melbourne Institute of Technology Phone: (03) 9925 2283 Fax: Email: Objectives

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, 2. Develop a database of DNA fingerprints for economically important species and chemotypes of medicinal herbs.

Current Progress

The prototype microarray was thoroughly tested with a number of species from the six main clades of flowering plants. From the analysis of data, we were able to select a set of polymorphic features which were predictive of each of these clades. Two new microarrays were subsequently produced to expand the functionality of the original array to include a number of Western herbs, including Passiflora incarnata, Salvia spp, Echinaceae and Artemisia annua. These herbs were sourced from The Southern Cross University and from Botanical Resources Australia. The first of these arrays was designed for Family- and species-level identification of the largest angiosperm group, the Asterids, while the second array was designed for chemotyping medicinal Salvia species (both Western and Chinese), specifically for Salvia miltiorrhiza (Danshen). Each array is comprised of 300 features. Validation of these arrays, especially of the chemotyping array showed that the subtraction process was successful in producing a large number of polymorphic features. DNA fingerprints are currently being generated for each Salvia species (10 species) and chemotypes (10 chemotypes). This study should be completed by the end of the year. Concurrently, a microarray is currently being prepared for fingerprinting another large clade of the angiosperms, the Rosids. It is anticipated that the array will be completed, and ready for validation studies in September.

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

RIRDC Project No.:

PRJ-000778

Start Date: 1/01/06 Finish Date: 12/31/09 Researcher: Rowland Laurence Organisation: Botanical Resources Australia Pty Ltd Phone: (03) 6425 0508 Fax: Email: [email protected] Objectives

This project, funded from January 2006, attempts to develop in Australia a commercially viable production method for the herb, Artemisia annua, the source of the peroxide anti-malarial, artemisinin and its substituted compounds. Thus the work attempts to maximise the yield of artemisinin present in the plant and its extract and to provide an economic analysis and recommendations regarding the crop’s potential for commercialisation to the project’s stakeholders, the RIRDC and Botanical Resources Australia (BRA).

Current Progress

A review report (3000 words) submitted to RIRDC in April, 2008 showed that good progress is being made in the project, which is on schedule to achieve its objectives in the proposed timeframe. Extrapolation from small plot work suggests that commercially viable artemisinin yields may be achievable. However, the production of such yields from mechanised operations is yet to be confirmed and pilot commercial sowings are underway to this end. Future milestones will be met as planned if this work is successful through 2008-09. Atemisinin yields from mechanically harvested areas will be assessed. Sample extracts will be provided to buyers for price indicators and the objective of economic evaluation of the crop for commercial production will be achieved.

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ESSENTIAL OILS AND PLANT EXTRACTS

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