Essential Oils & Plant Extracts

Transcription

1 RIRDC Completed Projects in and Research in Progress as at June 2005 Sub-Program 1.4 Essential Oils & Plant Extracts August 2005 RIRDC Publication No 05/059

2 2005 Rural Industries Research and Development Corporation. All rights reserved. ISBN ISSN "RIRDC Completed Projects in Research in Progress as at June Essential Oils and Plant Extracts" Publication No: 05/059 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 RIRDC Essential Oils and Plant Extracts Mr Tony Byrne RIRDC Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: Fax: tony.byrne@rirdc.gov.au RIRDC Publications Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: Fax: cecile.ferguson@rirdc.gov.au Website: Published in August 2005 Printed on environmentally friendly paper by Canprint ii

3 Foreword This Research in Progress, June 2005, contains short summaries of continuing projects as well as those that were completed during 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 This report is an addition to our extensive catalogue of over 1500 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 Purchases at Peter O Brien Managing Director Rural Industries Research and Development Corporation iii

4 Contents PROJECT No 1.4 ESSENTIAL OILS RESEARCH IN PROGRESS PROJECT TITLE RESEARCHER PHONE ORGANISATION PAGE No UCS-31A UNC-21A UT-39A Development and Improvement of Products Improvement of lavender varieties by manipulation of chromosome number Analysis of Dodonea viscosa as a potential remedy for anti-inflammatory and antibacterial applications Development of an internationally competitive saffron industry Dr. Nigel Urwin (02) Charles Sturt University 1 Dr. Douglas Stuart (02) The University of Newcastle 3 Prof. Robert Menary (03) University of Tasmania 5 UT-38A UT-40A Improved Production Systems Rejuvenation of peppermint crops through establishment of quality nursery stock Improved oil quality and yield from Tasmanian-grown French lavender Prof. Robert Menary Prof. Robert Menary (03) University of Tasmania 7 (03) University of Tasmania 8 UT-45AA Regulatory Approvals Quality assurance protocols to minimise pesticide residues in essential oils Prof. Robert Menary (03) University of Tasmania 9 PROJECT No 1.4 ESSENTIAL OILS COMPLETED PROJECTS PROJECT TITLE RESEARCHER PHONE ORGANISATION PAGE No ABN-2A Understanding of Markets Assessment of market potential for selected oil and plant extract products Mr Gee Yap (02) Australian Business Ltd 10 DAQ-304A GSD-1A UCS-30A UT-35A UT-34A Development and Improvement of Products Growing milkweed, a plant with prospective anti-cancer properties Chemotype selection and evaluation of Agonis fragrans oil Bioactivity of lavandula essential oils, hydrosols and plant extracts Development of a celery oil and extract industry in Tasmania Preliminary evaluation of some medicinal herbs Dr. Craig Davis (07) Mr. Chris Robinson (08) Dr. Heather Cavanagh Prof. Robert Menary Dr. Rowland Laurence Dept of Primary industries (Qld) Great Southern Development Commision (02) Charles Sturt University 16 (03) University of Tasmania 18 (03) University of Tasmania UT-37A Improved Production Systems Increased yields from Tasmanian peppermint crops through improved micronutrient nutrition Prof. Robert Menary (03) University of Tasmania 22 PSE-3A Industry Development Production of newsletter of the essential oil producers association of Australia Dr Erich Lassak (02) Phytochemical Services 23 iv

5 RIRDC RESEARCH IN PROGRESS Development and Improvement of Products Project Title Improvement of lavender varieties by manipulation of chromosome number RIRDC Project No.: UCS-31A Start Date: 01-Aug-02 Finish Date: 01-Aug-05 Dr Nigel Urwin Charles Sturt University School of Agriculture Locked Bag 588 WAGGA WAGGA NSW 2678 Phone: (02) Fax: (02) Objectives This project aims to breed improved cultivars of Lavandula angustifolia (lavender) and Lavandula x intermedia (lavadin) both of which are grown commercially for their essential oil, dried and cut flowers. Plant tissue culture techniques and the use of mitotic spindle inhibitors will be used to induce polyploidy. The quality of oils produced by all lavender cultivars resulting from and used as starting material for the project will be tested by a perfumer. The specific outcomes of the proposed research are as follows: Tetraploid and/or octoploid L. angustifolia and L. x intermedia cultivars, with improved oil yield and vigor. Double haploid L. angustifolia and fertile L. x intermedia cultivars, which can be propagated by seed. A new species of lavender (fertile L. x intermedia) for future breeding. Current Progress Progress towards objectives 1. Generation of polyploid L. angustifolia and L x intermedia cultivars. L. angustifolia cultivars Bee, Swampy and L x intermedia cultivars Grosso and Seal have all been treated with colchicine to induce polyploidy both in vivo (cuttings) and in vitro (shoot culture). Surviving plants of Bee and Swampy have been observed and seed collected from these. Several Bee plants having larger flowers indicating polyploidy have been identified and these are being evaluated by flow cytometry to determine nuclear DNA content. L x intermedia Grosso and Seal plants surviving colchicine treatment produced seed late 2004 and early It therefore appears induction of polyploidy in these previously sterile hybrids restores fertility. The ploidy level of these seedlings has yet to be confirmed by flow cytometry but metaphase cells of root tip squashes have been shown to have approximately 100 chromosomes, indicating they are tetraploid. These are essentially a new species and can be incorporated into future lavender breeding programs. 2. Generation of polyploid L. angustifolia from seed. Tetraploid L. angustifolia cultivars which were generated from colchicine treatment of seed are now growing under field conditions as are some of their progeny. We are observing genetic variability in these and have distilled oil from one tetraploid cultivar and several controls. This oil is being evaluated by gas chromatography and by a perfumer. More seed 1

6 types have been similarly treated to produce a variety of tetraploids with different genetic backgrounds. Partly because of the success of this project, a research infrastructure block grant from Charles Sturt University was obtained and a Beckman Coulter Quanta 488 flow cytometer has been purchased. This is being used to measure ploidy levels and absolute nuclear DNA content (Cvalue) in various lavender cultivars generated by this project. 3. Generation of double haploid L. angustifolia. This objective is the only one which has not been achieved to date despite repeated attempts to culture haploid plants from microspores and anthers on a variety of media and plant hormone combinations. 2

7 RIRDC RESEARCH IN PROGRESS Development and Improvement of Products Project Title Analysis of Dodonea viscosa as a potential remedy for anti-inflammatory and antibacterial applications RIRDC Project No.: UNC-21A Start Date: 01-Jul-03 Finish Date: 31-Jul-07 Mr Andrew Pengelly School of Applied Sciences The University of Newcastle Brush Road OURIMBAH NSW 2258 Phone: (02) Fax: (02) andrew.pengelly@newcastle.edu.au Objectives To develop analytical techniques to analyse for saponins, flavonoids, coumarins and diterpenes found in Dodonaea viscose var Angustifolia. To determine levels of each constituent group in various plant sections and determine the effects of some variables such as growing location To test extracts with a known biochemical matrix for antiinflammatory and antibacterial properties. Current Progress As reported in the annual progress report to RIRDC for 2004, I arranged for a series of bioassays to be conducted for possible anti-inflammatory, antioxidant and cytotoxic activity at the Natural Products Pharmacology Unit, Southern Cross University, in accordance with milestone 6 of the original proposal. The results of these bioassays were obtained, and none of the samples demonstrated anti-inflammatory capacity based on COX-II expression or PGE 2 inhibition. Some antioxidant capacity was demonstrated, predominantly in the acetone extract, suggesting the activity is associated with lipophilic constituents. At higher concentrations, methanolic and acetone extracts also demonstrated cytotoxicity. The three batch samples provided for these studies derived from distinct geographical zones within New South Wales. In general the variation in biological activity between batches was minimal the main instance of variation involving batch C2 which had lower cytotoxic and antioxidant activity but slightly higher anti-inflammatory activity. The batch BB12 (from the original harvest site) showed the most activity overall and probably can be selected for a representative sample in future tests. For anti-inflammatory studies C2 should also be considered. Given the positive anti-oxidant results for the acetone extract, it was decided to conduct further tests using a simple bench top antioxidant model developed on the Ourimbah campus for assessing the antioxidant capacity of green tea. Soxlet extraction of Dodonaea leaves was performed using three solvents dichloromethane, hexane and petroleum ether. The results of this assay are currently being assessed. Given the surprisingly negative results for the anti-inflammatory assays, I am looking to set up a simple assay that can be conducted in our lab in 3

8 order to test these results. Meanwhile I am also pursuing other potential mechanisms that may underlie the traditional and anecdotal reports of wound healing and pain relieving properties for this species. This will involve setting up assays that test for proliferation of fibroblasts, collagen contraction and / or antimicrobial effects. Earlier I had embarked on two field trips to collect specimens of Dodonaea viscosa from different geographical regions within New South Wales and southern Queensland. The resultant herbarium specimens that were prepared have been submitted in person to Dr. West at the National Herbarium, and have each been provided with accession numbers. The identity of the New South Wales specimens is confirmed as D. viscosa subsp. Angustifolia, however the specimens from Queensland were identified as D. viscosa subsp. Spatulata. 4

9 RIRDC RESEARCH IN PROGRESS Development and Improvement of Products Project Title Development of an internationally competitive saffron industry RIRDC Project No.: UT-39A Start Date: 08-Jul-03 Finish Date: 07-Jul-06 Prof Robert Menary University of Tasmania School of Agricultural Science GPO Box HOBART TAS 7001 Phone: (03) Fax: (03) Objectives To develop management strategies and chemical analysis protocols to maximize saffron quality and ensure the Australian product meets ISO standard. To identify potential perfume products from waste flowers after stigma removal and establish viable extraction protocols for these products. The 2 objectives above will be achieved through a detailed knowledge of the chemistry of carotenoid degradation in Crocus. This will occur by applying both current knowledge of this chemistry in the stigmas (leading to saffron biosynthesis) and by the new knowledge gained from the study of this chemistry in the petals. To investigate the content of colchicine and its glucoside in waste bulbs and then establish viable extraction protocols for it's commercial use. Work closely with management and marketing arm of Tas-Saff. Pty.Ltd. to apply new technology to the current product and facilitate introduction of new products to the market. Current Progress Further analysis of the results obtained from the drying experiments conducted during the 2004 saffron harvest was undertaken during the remainder of 2004 and this gave some broad parameters for potential improvements in drying technique. Following on from this, a series of experiments to more precisely determine optimum drying conditions were designed for the 2005 harvest and these experiments have just been completed. Analysis of these results is as yet incomplete, although the initial indications are that sufficient information has been gained to allow a detailed protocol for the optimum drying of saffron to be drawn up. It is intended that this protocol will be tested on a commercial scale during the (April) 2006 harvest. Included in this work was the use of a prototype, saffron specific dryer designed to allow more precise temperature control, rapid heating and cooling and variable airflow control than both conventional ovens and the equipment currently being used by the local industry. The prototype proved successful, although it s use in these experiments has shown of a number of improvements that can be made to the design. Construction of a new model will occur before the 2006 harvest. Investigation of potential new products has been focussed on solvent 5

10 extraction of the waste flower parts as distillations have given too low a yield of product. Feedback on the potential use of trial extract product is currently being sought from flavour and fragrance experts overseas. The chemical characterisation of this trial product is continuing. 6

11 RIRDC RESEARCH IN PROGRESS Improved Production Systems Project Title Rejuvenation of peppermint crops through establishment of quality nursery stock RIRDC Project No.: UT-38A Start Date: 18-May-03 Finish Date: 31-Jul-06 Prof Robert Menary University of Tasmania School of Agricultural Science GPO Box HOBART TAS 7001 Phone: (03) Fax: (03) Objectives The objective of the project is to establish a source of productive, clean peppermint material and the best method of propagation to achieve high yields. The project will deliver both an established nursery of clean, high yielding peppermint stock and a report to the industry on the best method for future production of such a nursery. Current Progress The genetic variability of the 200 mother plants collected in the first season was tested in a glasshouse trial. Plant oil content and oil composition were assessed by microextraction of leaf pairs. The trial showed clonal differences in both plant growth rate and plant oil content. The data were used to develop an index of oil production that could be used to rank the clones for quality, however the index needs to be validated as a selection tool before it can be used with confidence to establish the elite clone bank. Eighteen clones, covering the range of indices calculated from the glasshouse trial, were chosen. Four harvests of these plants were made from plants growing in pots in a shadehouse and one harvest from the same clones growing in the field. Steam-distilled oils produced by distilling whole plants of these selections will be used to calculate similar indices, to those calculated for the oleoresins. The correlation between the indices from the oleoresins and steam-distilled oils will determine the value of the indices from the oleoresins as a selection tool. A field trial was established to test three parent materials as alternative propagation methods for the nursery areas. These parent materials were stolon material from field grown parents, tip cuttings from glasshouse grown stock and micropropagated plants initiated from meristems of glasshouse grown stock. In the first growing season, the trial was assessed for plant growth rate, plant oil content and oil yield per unit area. Oil content was similar for plants produced from tip cuttings and stolon material but the tip cuttings outperformed the stolon material in both plant growth rate and oil production per unit area. The oil content of the micropropagated plants was much lower but because they exhibited a much stronger growth rate, they outperformed both the tip cuttings and stolon material in terms of oil yield per unit area. However, because this trial is designed to assess the material as sources of propagation for nurseries, the most important variable to be measured is the number of propagules per unit area. This measurement will be made in the autumn. 7

12 RIRDC RESEARCH IN PROGRESS Improved Production Systems Project Title Improved oil quality and yield from Tasmanian-grown French lavender RIRDC Project No.: UT-40A Start Date: 01-Nov-03 Finish Date: 30-Nov-06 Prof Robert Menary University of Tasmania School of Agricultural Science GPO Box HOBART TAS 7001 Phone: (03) Fax: (03) Objectives The production of French lavender oil conforming to the ISO standard is possible only if suitable clones are harvested at the appropriate time. The primary objective of this proposal is to screen the available lavender germplasm for those clones producing oils of a desirable composition. These will be used to establish a nursery of elite mother stock from which material will be available for future industry expansion. Optimum harvest date for each clone will be determined by monitoring changes in oil composition through the season. The industry partner will conduct a field trial to determine the feasibility of a more intensive production system with increased nitrogen and irrigation inputs. The university will undertake the monitoring of oil yield and quality from this trial. Current Progress In the first flowering season, the available germplasm was screened and 90 clones, believed to produce oil conforming to the selection criteria, were chosen. This year, a tissue culture clone bank of these clones was established. The success rate of the initiation process was 92% ie 83 clones are currently held in storage. An improved multiplication medium has been developed for the eventual propagation of these clones. The initial screening by headspace gas chromatography provided a rapid, minimal labour technique for assessing a large number of individual clones. These screening data were examined and clones covering the range of detected camphor levels selected. During this season, samples of flowers from these individuals were taken to validate the initial screening. Oleoresins from these samples will be compared with the headspace composition. For a few individuals, enough flowers were collected for steam distillation, as a further validation. The assessment of optimum harvest time in the first season showed that oil yield peaks at approximately the time of completion of the commercial harvest, followed later by a second flush of oil production. Oil composition data are variable but the trend appears to be for camphor levels to fall somewhat throughout the season. The data from the current season will be collated with those from the first season in due course. A trial examining the effect of a more intensive production system, with higher inputs of nitrogen and irrigation has been established. 8

13 RIRDC 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-Jun-04 Finish Date: 30-Aug-07 Prof Robert Menary University of Tasmania Phone: (03) Fax: (03) 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. Current Progress Elements of all four objectives are underway and have advanced significantly in recent months. A paper based chemical management plan is in its final stages; this has consisted of several drafts that have now been consolidated to a paper-based version that has been bound and is ready for presentation to industry partners. A HACCP based approved supplier program specifically for pesticide residues in essential oil crops is near completion. Additional target compounds identified as a priority by industry i.e. Lontrel (clopyralid), Omite (propargit), Goal (oxyflurofen), Stomp (pendimethalin), Treflan (trifluralin) and Linuron have been successfully introduced and are complementary to existing HPLC-MS-MS pesticide screening methods. Sample collection for stage one field trials have been completed for the analysis of Treflan (trifluralin) in peppermint; Lontral (clopyralid), Linuron and Gesagard (prometryn) in parsley; dimethoate in fennel and simazine in blackcurrant. Laboratory analysis is complete for peppermint and underway for the remaining crops. Spray boom calibration procedures have been identified as an important control point in a quality based pesticide program. Work is underway with selected growers and the University Farm to optimise a calibration procedure for standard boom spray delivery systems. 9

14 Completed Projects Understanding of Markets Project Title: Assessment of market potential for selected oil and plant extract products RIRDC Project No.: ABN-2A Gee Yap Australian Business Ltd Level 13, 140 Arthur Street NORTH SYDNEY, NSW, 2060 Phone: (02) Fax: (02) Objectives Background Research Outcomes Assess the market potential and viability for profitable production in Australia for the following herbs. Valerian (Valeriana officinalis L.) Arnica (Arnica montana L.) Skullcap (Scutellaria lateriflora L.) Echinacea (E. angustifolia, E. pallida, E. purpurea) Goldenseal (Hydrastis canadensis L.) This project was formulated to support RIRDC s R&D Plan for Essential Oils and Plant Extracts 2002/2006 by providing: A clearer focus on which new crops that Australia s R&D efforts should be applied. A road map for growers and processors in terms of viable market opportunities. Potential to establish a new Australian essential oil/plant extracts industry based on researched market opportunities. The research encompassed the following. Developing a profile of global market opportunities for Echinacea, Valerian, Goldenseal, Arnica and Skullcap. Identifying of niche market segments in which Australia might effectively compete. Providing insights into cost structures through the supply chain from farm gate to end-users, and through this, an assessment of the viability to cost effectively produce in Australia. At the consumer level, the sale of medicinal herb products (including essential oils and plant extracts) in Australia and round the world continues to enjoy strong growth as consumers are increasingly turning to natural remedies for the treatment of minor ailments and preventive healthcare. Processors and manufacturers have reacted well to increased consumer demand with a proliferation of new products and wider distribution channels. For example, in Australia the vitamins and dietary/health supplements category alone grew a staggering 132% between 1998 and 2003 to reach a new high of A$511.5 million whilst the overall OTC healthcare reached a record A$1.8 billion. Despite increasing demand for natural remedies, most local growers continue to struggle to achieve required returns from the supply of highly demanded raw materials. The global trade for medicinal herbs (including essential oils and extracts) is well organised and local growers find it difficult to compete against more established growers from North 10

15 America and Europe. The small Australian market for dry plant materials and extracts further exacerbates the problem as it prevents growers from producing medicinal herbs in any significant volume. It remains unclear if Australia has a true competitive advantage in the production of medicinal herbs and extracts. Growers from North America and Europe can compete on three vital fronts; quality, price and supplying consistency thus giving little opportunity for local growers to establish themselves. The ability for global suppliers to supply medicinal herbs at prices below Australian production cost makes it extremely difficult for local growers to be competitive. Results of the research showed that E. angustifolia, E. purpurea, Skullcap and Goldenseal will generate the best possible returns in view of current strong prices and a proven ability for these herbs to successfully grow in Australia. However, the research also identified that local growers have attempted to capitalise on these opportunities in the past but without much success largely due overseas competition and inability for the domestic market to absorb any significant volume. The Australian medicinal herb cultivation industry is at a cross-road. If the industry is to grow, growers must help improve market confidence and acceptability of their crops among processors. Better communication and interaction is needed between growers and processors to allow for an integrated approach for the supply of medicinal herbs in Australia. In addition, growers collectively also need to address a range of industry development challenges including: Better co-ordination and co-operation across industry stakeholders including growers, government, processor and manufacturers; Better access and retention of true botanicals, cultivars knowledge and technical expertise; Improving transparency and flow of information between industry stakeholders Improving quality and consistency of delivery Developing critical mass/volume to achieve economies of scale Better access to market information Developing quality assurance systems to guarantee herb authenticity, integrity and safety; and Developing a commercial model/template that industry can adopt It is through addressing these challenges that will allow the processors the confidence and preference to source locally grown herbs over imports. Overcoming the above challenges will not be easy and will require significant investment which can be hard to justify given the size of the industry. Implications This research will create discussion among industry stakeholders if Echinacea, Arnica, Valerian, Skullcap, Goldenseal, and on a wider context, medicinal herbs are worthwhile of cultivation in Australia. More importantly, the research will help growers understand some of the complexities associated with commercialising new medicinal herbs and the resources needed in order to effectively service the Australian and overseas markets. Publications Gee Yap, Assessment of market potential for selected oil and plant extract products. RIRDC Publication number pending. 11

16 Completed Projects Development and Improvement of Products Project Title: Growing milkweed, a plant with prospective anti-cancer properties RIRDC Project No.: DAQ-304A Dr Craig Davis Department of Primary Industries (Qld) Centre for Food Technology 19 Hercules Street HAMILTON QLD 4007 Phone: (07) Fax: (07) craig.davis@dpi.qld.gov.au Objectives Background Research This project explored the potential for commercial propagation, growth and harvesting of milkweed. This production on a commercial scale will tap under-utilised land and labour resources by providing agronomic systems leading to the extraction, purification and production of high quality active pharmaceutical ingredients which will be used to provide effective control of the most expensive cancers to treat (common skin cancers) both in the Australian market and in other International markets. Peplin Biotech has discovered a novel class of natural compounds, purified from Euphorbia, which show great potential in the laboratory as a potent treatment for a wide range of human cancers including breast and prostate cancer, leukaemia, melanoma and other skin cancers, as well as lung, colorectal and cervical cancer. Methods have been developed by Peplin Biotech for the extraction and purification of compounds from E.peplus in the laboratory, and these are currently being scaled-up for commercial production. It is anticipated that the overall cost of producing the active ingredient in pure form will result in a commercially viable treatment, provided the cost of agricultural production can be kept low. The limiting factor in capturing commercial value from these discoveries is the availability of plant feedstock for extraction of the active compounds. Developing large-scale agricultural production can solve this problem. Currently, the plant is sown by hand and tended in plots of sq m. Harvesting of seed and plant is also carried out by hand. Sufficient information has now been obtained to show that there is no inherent difficulty in large-scale production in a variety of locations and environments in Australia. This project helped to develop methods suitable for broad-acre, mechanised production. The experimental system for the intensive cropping of Euphorbia peplus has been used to assess the effect of time of harvest on the quality and quantity of active ingredient from Euphorbia peplus. Research at the QDPI Redland Research Station developed a profile of the levels of desired compounds during the whole year. This research was mimicked in Toowoomba to assess the effect of different climate conditions on the growth of the plant and the production of the chemicals of interest. An analytical testing method was established to routinely test for PEP5 in plant material collected from the various trials. The assay system was optimised to provide completely reliable and reproducible results. In addition, the chemical profile and bioactivity of other Euphorbia species was surveyed and harvesting methods for E. peplus were compared. 12

17 E. peplus remains the species of choice for the industry partner (Peplin Biotech) because of its ease of cultivation, history of safe community use and demonstrated efficacy in a clinical trial of the crude sap and in animal models using the purified compounds. Outcomes An analytical testing method to routinely test for PEP5 in plant material collected from the various trials was established. An experimental system for the intensive cropping of E.peplus was developed The effect of time of harvest (Summer/Winter) on the quality and quantity of active ingredient from E.peplus was determined. The effect of different climate conditions on the growth of the plant and the production of the chemicals of interest was assessed. 13

18 Completed Projects Development and Improvement of Products Project Title: Chemotype selection and evaluation of Agonis fragans oil RIRDC Project No.: GSD-1A Mr Chris Robinson Great Southern Development Commision 444 Albany Highway ALBANY WA 6330 Phone: (08) Fax: (08) Objectives The fundamental aim is to facilitate realisation of the commercial potential of a new essential oil, Agonis fragrans. The research proposed here is to ensure that at this early stage of commercialisation that the best chemotype(s) is identified now to ensure maximum development potential. Inherent in best chemotype identification is demonstration of chemical composition, antimicrobial ability, assessment of therapeutic and other uses linked with examination of possible traditional uses and product stability. The project also seeks to begin exploration of the TGA options to identify appropriate actions to enhance future marketability and expansion of uses. TGA processes and requirements have in the past been notoriously complex and require specific effort to resolve appropriate direction for product development. Additional antimicrobial testing and preliminary work on antiinflammatory activity. Outcomes Chemical composition of Agonis fragrans oil has been thoroughly investigated. Although there is variance in composition of Agonis fragrans oil between and within populations, the variation is not unusual. The major variation found was a subpopulation (within one of the eleven populations sampled) that contained no cineole. University of WA Microbiology Dept. confirmed that Agonis fragrans oil does have significant antimicrobial activity, and that the activity is similar to other essential oils considered to have antimicrobial activity including Melaleuca alternifolia (tea tree) oil, Cymbopogon citratus (lemongrass) oil and Origanum vulgare (oregano) oil. There was no significant variation in the MIC or MBC results between the oils, which indicates that all chemotypes selected for testing were equally useful and anti-microbial in their activity. Due to low volumes of oil available of the no cineole variant, this was not tested with the other five oils for antimicrobial activity. Preliminary clinical use of Agonis fragrans oil reported to this project indicates possible application in remedial therapies for joint, muscle and arthritis pain and for respiratory infections. Research is also being undertaken into the likely emotionally balancing activities of the oil, contributed to by the near perfect balance of chemical constituents which are loosely grouped as oxides (1-8cineole), monoterpenes and monoterpenols. There is also reported interest in the use of the oil in fragrance applications, both locally and overseas. Overseas comment 14

19 has been received about the potential in the flavouring industry. The myrtenol (no cineole) type has more potential in non-fragrance applications and may find applications relating to its use in insect communication with some species of pine beetle and melon fly and as a flavouring agent. The regulatory overview provided has given a clear path to the regulatory process required to establish Agonis fragrans oil as a new chemical entity. Implications The preliminary results of this project have served to underpin commercial negotiations between the industry partner (The Paperbark Co) and an international commercial development group. The confirmation of antimicrobial activity of Agonis fragrans oil sets the foundation for further commercialisation of this oil. Clinical trials currently being conducted by international interests in collaboration with the industry partner are expected to further enhance the market position of this oil. The UWA antimicrobial work indicated that although the data suggest that Agonis fragrans oil may have clinical potential as an antimicrobial agent, little is known of the safety profile of these oils and there are insufficient in vitro data for in vivo work to proceed at this stage. They state that much work would be required before Agonis fragrans oil could be evaluated in vivo, the definitive way in which to determine its clinical potential. The chemotype currently being grown in plantation, compared favourably with other chemotypes, and as there is currently a shortage of oil supply relative to existing and projected levels of demand, planting of this chemotype can proceed with confidence. The Paperbark Company is expanding plantation to near its limit in 2005 and plantings on other properties are being negotiated for Further work may be indicated to determine if the myrtenol (no cineole) type has commercial potential in non-fragrance applications such as insect communication and as a flavouring agent. Publications Variation within the Leaf Essential Oil and Antimicrobial Activity of Agonis fragrans. Robert F. Lowe et al DRAFT to Journal of Essential Oil Research 15

20 Completed Projects Development and Improvement of Products Project Title: Bioactivity of Lavandula essential oils, hydrosols and plant extracts RIRDC Project No.: UCS-30A Dr Heather Cavanagh & Dr Jenny Wilkinson Charles Sturt University School of Biomedical Sciences Locked Bag 588 WAGGA WAGGA NSW 2678 Phone: (02) & (02) Fax: (02) Objectives This project addresses the key issues of Lavandula plant extract bioactivity, efficacy and safety and will provide novel marketing and export opportunities for these products and support their development as viable alternative primary crops. This project aims to identify products which have potential medical/veterinary/agricultural applications and identify new commercial applications for these plant products. Outcomes The antifungal, antibacterial and antiparasitic activity of a range of Lavandula essential oils, via direct contact and/or volatile exposure, was assessed in addition to preliminarily investigations into the antimicrobial activity of related hydrosols. Both the oils and their volatile components displayed antifungal activity, with some difference in activity between oils against specific fungi observed. For example Lavandula angustifolia and Lavandula x intermedia oils demonstrated the greatest effect against A. nidulans and T. mentagrophytes while L. stoechas was particularly effective against the two agricultural fungi, L. maculans and S. sclerotiorum. No significant difference was observed between the antifungal activity of L. angustifolia essential oils derived from European and Australian grown plants and no evidence of antifungal activity was observed with any of the hydrosols. These results suggest that essential oils from various lavender varieties may be useful in the treatment of fungal infections. Similarly, all of the bacteria tested in this project, except Pseudomonas aeruginosa, were susceptible to one or more of the Lavandula oils, indicating that the inclusion of lavender oils in cosmetics, pharmaceuticals and cleaning products may be beneficial. The only oil to show minimal activity against P. aeruginosa was a European grown L. angustifolia. All ten oils tested demonstrated some level of activity against C. albicans. These results support the anecdotal use of lavender essential oil as an antimicrobial product, however as some differences between oils for different Lavandula species were also noted, it may be possible to target the use of particular lavender oils for particular organisms. This study has also demonstrated that low ( 1%) concentrations of L. angustifolia and L. x intermedia oil can completely eliminate the parasites Trichomonas vaginalis, Giardia duodenalis and the fish parasite Hexamita inflata in culture. Both oils tested in this study exhibited strong, 16

21 and rapid anti-parasitic activity at 1% and 0.5% and the mode of action appeared to be via lysis of the cells. At 1% and 0.5% there were few differences between the activity of the oils and these are unlikely to be clinically or biologically significant. In contrast significant differences were observed at oil concentration of 0.1% with L. angustifolia demonstrating better activity against G. duodenalis and H. inflata. Implications The findings indicate that a variety of Lavandula essential oils may have potential use as antimicrobials against bacteria, human and agricultural fungi. Use of the volatile components rather than direct contact may provide a pathway to greater use and overcome many of the allergic/toxicity problems associated with direct contact. Whether lavender essential oils can be used as a viable treatment for infected water sources or as a treatment of parasitic infection remains unknown. Questions that remain to be addressed include whether the residual lavender oil presents a risk of cell toxicity due to ingestion or skin contact, how lavender might be best added to water sources and whether the active constituents(s) can be identified and isolated to produce a standardised pharmaceutical. Further research is required in this area to assess the viability of Lavandula oils (or their derivatives) for use in the prevention and/or treatment of parasitic diseases in a range of settings including agriculture, veterinary and aquaculture industries. Publications Submitted for peer-reviewed publication 1. Moon, T, Cavanagh, HMA, Wilkinson, JM. Which is better for disc diffusion assays of essential oils IsoSensitest or nutrient agar? Journal of Essential Oil Research (Submitted Dec 2004) 2. Moon, T, Cavanagh, HMA, Wilkinson, JM. Antifungal activity of Australian grown lavender essential oils. Phytotherapy Research (Submitted Jan 2005) In preparation for peer-reviewed publication 3. Moon, T, Wilkinson, JM, Cavanagh, HMA. Investigation of the activity of lavender essential oil against Giardia duodenalis, Trichomonas vaginalis and Hexamita inflata 4. Moon, T, Cavanagh HMA, Wilkinson, JM. Essential oils from Australian grown Lavandula spp.: A comparison of antibacterial activity Non-refereed Publications 5. Moon T, Cavanagh HMA, Wilkinson JM (2004) Lavender as an antibacterial essential oil - are all lavenders equal? The Lavender Bag, 24, Moon T, Chan YF, Wilkinson JM and Cavanagh HMA (2004) Antifungal activity of Lavandula essential oil and oil volatiles. Australian Infection Control National Conference, abstracts p46. [Oral and poster presentation, winner of best oral presentation] 7. Moon T, Cavanagh HMA, Wilkinson JM (2004) Lavender as an antibacterial essential oil - are all lavenders equal? Australian Infection Control Journal [poster presentation] 17

22 Completed Projects Development and Improvement of Products Project Title: Development of a celery oil and extract industry in Tasmania RIRDC Project No.: UT-35A Prof Robert Menary University of Tasmania School of Agricultural Science GPO Box HOBART TAS 7001 Phone: (03) Fax: (03) r.menary@utas.edu.au Objectives The inclusion of celery (Apium graveolens L.) oil and extract products in the essential oils industry within Tasmania will increase the diversity of crops available to growers and provide a buffer against price fluctuations in local and international markets. Any refinement of current extraction processes, as a result of the development of the new products has the potential to benefit the entire industry. Background Celery essential oil and extract products are traded with increasing demand in international markets. Celery seed oil is the best known of these. It has a characteristic warm, spicy, pervasive aroma that is produced primarily by the components β-selinene and several phthalides. Previous marketing success with parsley herb oil, established a procedure for developing similar products. Samples of celery herb oil were developed for market evaluation in Europe and USA during the 1980 s. The response to these was very positive. Additionally, celery seed oil is currently imported into Australia, for encapsulation and sold as a therapeutic good. The current research project aimed to select celery varieties to be grown for the production of high quality flavour and fragrance oil and for the pharmaceutical industry. Both domestic and international markets are targeted. Research Outcomes The research began with assessments of potential varieties. Concurrent with that research was the necessary development of suitable methods for obtaining and assessing the oils and extracts. Steam distilled oils of both herb and seed were examined, in addition to extracts from both seeds and roots. Recommendations based on current commercial methods were developed to allow celery to slot into the current suite of essential oil and extract crops produced by local growers. The variety best suited to local production for both herb essential oil for flavour and fragrance and the pharmaceutical industry was Salad Green. The crop should be sown early in the autumn and pre-harvest sampling for oil quality should commence at full bloom. Harvest should be scheduled by organoleptic assessment of the oils. The duration of commercial distillation should be extended for as long as economically possible, because the valuable components are distilled 18

23 late in the process. Consideration should be given to solvent extraction of the marc, especially if the crop is distilled after seed maturity has been reached. Extraction of the roots will further increase the value of the crop. Implications Publications This new crop should fit well into the current industry. Yields attained through good agronomic management, should be sufficient when coupled with a conservative farm-gate price for the crop to reach similar gross margins to pyrethrum or poppies. The following publications are being prepared from the data generated by this research and will be submitted to refereed journals: Comparison of two capillary GC columns for FID quantification of the major components of celery essential oils and extracts Evaluation of seed and herb oils from six varieties of celery (Apium graveolens L.) Yield and composition of celery essential oil with increasing duration of distillation Changes in composition of celery herb oil with harvest date Comparison of solvents and preparation method for the extraction of celery roots Comparison of solvents and preparation method for the extraction of celery seeds 19

24 Completed Projects Development and Improvement of Products Project Title: Preliminary evaluation of some medicinal herbs RIRDC Project No.: UT-34A Dr. Rowland Laurence Tasmanian Institute of Agricultural Research, P O Box 3523 BURNIE Tasmania 7320 Phone: (03) Fax: (03) rowland.laurence@utas.edu.au Objectives This project attempted to provide preliminary data to the stakeholders on the growth and marketable yield of four species of medicinal herbs grown in Tasmania, in order to allow decisions by the industry stakeholder on further investments in one or more of these crops. Some additional market information was sought as part of the work. This focused on the European situation, complementing previous market research in the USA. Background Research Outcomes The potential to expand the medicinal herbs and extracts industries in Australia has been recognized for some years and the industry stakeholder in this project, Botanical Resources Australia, sees such development as complementary to its current involvement in the contract growing, processing and export of refined pyrethrum product. The strengths on which the latter have been built are likely to be easily applied to other extractive crops. The project, begun in 2001, considers four species: astragalus, bilberry, feverfew and stevia. The project investigated the suitability of these species for profitable, commercial production in south-eastern Australia by providing preliminary evidence of their ability to grow in a local, temperate climate and indications of commercial plant and extract yield potential. Activities involved acquisition of genotypes, plant propagation, analyses of seasonal growth and of extracts where available. The project sought also to supply additional market information on market trends in Europe. Astragalus grew well in the temperate Tasmanian climate. Plants overwintered well after a first growing season but less well after the second and the reasons for this require further study. Levels of astragalosides, one set of active chemical compounds found in roots and used in traditional Chinese medicine, were all high compared with published data, which result suggests that the crop is worthy of further development. After a difficult establishment period, field plantings of bilberry flowered and produced a few fruit in their third year of growth, which is consistent with its growth in native habitats. Growth and yield responses of bilberry to seasonal temperature and shading regimes need to be better determined and, given the development work required on the harvest mechanisation, the plant is a longer term commercial prospect. Feverfew grew and over-wintered very well in the temperate climate. Levels of the principal active constituent, parthenolide, varied considerably with season and plant management and some high levels were achieved. While the market for feverfew at the time of writing this report is well supplied, these encouraging results have convinced the industry stakeholder, BRA, to maintain feverfew plantings beyond the life 20

25 of the current project. Implications Publications A current detraction to the development of stevia is the banning of the importation of stevia products by Australia and some other countries. Therefore, its further development is unlikely in the short term. Astragalus and feverfew are worthy of further development in response to sufficient positive market signals and bilberry will require considerable development over the long term. The work has been publicised in regional media and at field days. 21

26 Completed Projects Improved Production Systems Project Title: Increased yields from Tasmanian peppermint crops through improved micronutrient nutrition RIRDC Project No.: UT-37A Prof Robert Menary University of Tasmania School of Agricultural Science GPO Box HOBART TAS 7001 Phone: (03) Fax: (03) Objectives Background Research Outcomes Implications Publications To undertake a preliminary survey o commercial fields to assess nutrient status of soils and peppermint plants Use established critical levels in other and related crops to predict the likelihood of deficiencies Produce diagnostic material for growers and recommend fertiliser practice to reduce the severity of the problem. Peppermint oil yields in southern Australia are highly variable and this is impacting on the long term viability of the industry. The average yield is 50 kg/ha, despite attempts to improve management practice and this is not sufficient to sustain the current farm gate price. Improved nutrition is likely to deliver the yield required to meet criteria for profitability. 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 comes from peppermint. Without peppermint, the steam distillation units would not be viable. Plant tissue and soil were analysed for macro and micro nutrients from all commercial production areas. Levels were compared with those reported in the literature for peppermint and related crops. Deficiencies of S, B, Cu and Mo were identified as nutrients which were likely to be in the deficiency range. A nutrition trial was conducted in the glasshouse on soil collected from one of the commercial areas. This trial confirmed that deficiencies of S, Cu, B and Mo were present. Based on preliminary evidence of nutrient deficiencies, a pilot scale fertiliser program was devised for growers to use in the season. Growers were also given a CD of deficiency symptoms. A field day was held to explain the methodology used and the approach to new fertiliser practice. 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 will follow when statistical treatment of results become possible through a continuation of the grant. 22