A New Essential Oil – Agonis fragrans Chemotype Selection and Evaluation
A report for the Rural Industries Research and Development Corporation by Christopher J. Robinson August 2006 RIRDC Publication No 06/090 RIRDC Project No GSD -1A
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© 2006 Rural Industries Research and Development Corporation. All rights reserved. ISBN 1 74151 353 7 ISSN 1440-6845 Chemotype Selection and Evaluation of Agonis fragrans oil Publication No. 06/090 Project No GSD -1A The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable industries. The information should not be relied upon for the purpose of a particular matter. Specialist and/or appropriate legal advice should be obtained before any action or decision is taken on the basis of any material in this document. The Commonwealth of Australia, Rural Industries Research and Development Corporation, the authors or contributors do not assume liability of any kind whatsoever resulting from any person's use or reliance upon the content of this document. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186. Researcher Contact Details Chris Robinson c/o 444 Albany Highway ALBANY WA 6330 Phone: 08 98 928486 Fax: 08 98 412707 Email: [email protected]
In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 2, Pharmacy Guild House 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6272 4819 Fax: 02 6272 5877 Email: [email protected]. Web : http://www.rirdc.gov.au Published in August 2006 Printed on environmentally friendly paper by Canprint
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Foreword Although Western Australia has one of the most diverse floras in the world (ca. 12,000 taxa), with enormous potential for native plant based industries, to date there has been little research into the development of the essential oils of these species. Over the last decade there has been growing interest in the commercial production and uses of the essential oil of Agonis fragrans, which is a species native to the south coast of WA. This project aimed to investigate chemotype variation of Agonis fragrans across its natural range to ensure that any further commercial development of its essential oil was based upon a suitable chemotype. The project also aimed to confirm antimicrobial status of the oil, identify uses and clarify regulatory processes to increase its commercial application. This research is important to ensure vital background data is available to the emerging industry based upon Agonis fragrans oil. The species itself was only described in 2001 and no scientific data on its chemistry and uses had previously been completed. The report is intended to benefit essential oil producers and users. It is essentially a collation of sub reports on various aspects of research into Agonis fragrans oil. Readers requiring specific information on the research methodologies used are referred to the appendices which contain the complete sub-reports. This project was funded by RIRDC’s core funds which are provided by the government of Australia. Funding support was also provided by the Great Southern Development Commission, Albany. This report is an addition to RIRDC’s diverse range of over 1500 research publications. It forms part of our Essential Oil and Plant Extracts R&D sub-program which aims to support the growth of a profitable and sustainable essential oils and natural plant extracts industry in Australia. Most of our publications are available for viewing, downloading or purchasing online through our website: • downloads at www.rirdc.gov.au/fullreports/index.html • purchases at www.rirdc.gov.au/eshop Peter O’Brien Managing Director Rural Industries Research and Development Corporation
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Acknowledgments The support and funding provided by RIRDC has been critical in facilitating the research contained in this report which will strengthen and enhance the commercial reality of Agonis fragrans oil. RIRDC Research Manger Tony Byrne was always available, responsive and flexible in his dealings with the project partners. Peter Grayling of CALM provided the initial GC analysis of 55 leaf samples which facilitated more targeted sampling for GCMS analysis. The Buxton family of Redmond allowed harvest of their Agonis fragrans plantation for oil production. The Day family of The Paperbark Company provided great enthusiasm and the necessary commercial reason to continue the research into this new oil. Great Southern Development Commission contributed to funding and provided an administrative base through which the partners could develop this project.
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Contents Foreword ............................................................................................................................................... iii Acknowledgments................................................................................................................................. iv Contents.................................................................................................................................................. v Executive Summary ............................................................................................................................. vi Introduction ........................................................................................................................................... 1 Project Objectives.................................................................................................................................. 1 Production and Analysis of Oil Samples from Plantations and Natural Populations..................... 2 Comparative Antimicrobial Tests........................................................................................................ 2 Identification of Best or Superior Chemotypes .................................................................................. 2 Identification of Potential Uses ............................................................................................................ 3 Assessment of Immunostimulatory Ability for Aquaculture............................................................. 4 Identification of and Progression Toward Recognition by Regulatory Authority .......................... 4 Determination of Traditional Use ........................................................................................................ 4 Determination of Anti Inflammatory Ability...................................................................................... 5 Discussion............................................................................................................................................... 6 Implications and Recommendations.................................................................................................... 8 Appendices ............................................................................................................................................. 9
Appendix 1: A Report on the Field Investigation of Market Opportunities for Agonis Oil. .............. 9 Appendix 2: Gas Chromatography of 55 Agonis fragrans leaf samples in ethanol .......................... 13 Appendix 3: GCMS analysis and “Comments on the chemistry of the oils”.................................... 19 Appendix 4: Composition of an Anti-microbial Essential Oil from Agonis fragrans....................... 24 Appendix 5: “Antimicrobial activity of Agonis fragrans oil”........................................................... 32 Appendix 6: “Regulatory Overview Agonis Oil” ............................................................................. 44 Appendix 7: Effects of Agonis fragrans oil on mononuclear cell immune responses ...................... 60
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Executive Summary What this report is about This report provides information on research to facilitate realisation of the commercial potential of a new essential oil, Agonis fragrans. Who the report is targeted at The report is particularly targeted at potential investors in the production of agonis oil and potential users of the product. It will also be useful as a first step in investigation of the regulatory requirements for the oil. Background The oil contained in the foliage of Agonis fragrans, an erect shrub native to SW Western Australia, was first recognised as having commercial potential as an essential oil in 1996. Since then, a market investigation confirmed commercial interest, but identified a lack of supply as a limitation to further development. In 2001, The Paperbark Company established a plantation of Agonis fragrans and is now producing, promoting and selling increasing quantities of the oil. Aims This project aimed to investigate chemotype variation of the species across its natural range to ensure that any further commercial development was based upon a suitable chemotype. The project also aimed to confirm antimicrobial status of the oil, identify uses and clarify regulatory processes to increase its commercial application. Methods used The methodology involved production and analysis of oil samples from plantations and across range of natural populations and identification of best or superior chemotypes; comparative antimicrobial tests; assessment of immunostimulatory ability for aquaculture; and testing anti-inflammatory ability. Results Initial GC analysis of leaf tissue from 55 plants (five plants from each of eleven wild populations) indicated some variation in constituents. The major variation was two plants in one population which contained no 1,8 cineole, which was present as a major constituent in all other plants. Preliminary GC analysis was used to select six plants for leaf tissue GCMS analysis. This investigation showed the variation in constituents was not unusual (apart from the no cineole sample). At least two varieties were considered suitable for further investigation and commercial development. In-vitro studies conducted by UWA Microbiology confirmed that Agonis fragrans oil has significant anti-microbial activity, similar to that of other recognised anti-microbial essential oils, including tea tree oil (from Melaleuca alternifolia) and that it may have clinical potential as an antimicrobial agent. These studies indicated that there was no significant variation in results between the oils tested, which however did not include the no-cineole sample which was unable to be tested. It does confirm that the chemotype currently being produced and marketed is at least equal to any other. In-vitro studies conducted by the School of Paediatrics and Child Health, University of Western Australia, found that Agonis oil inhibits secretion of the IFNγ, involved in inflammatory response to tissue injury or infection, which could support the notion that this oil potentially has anti-inflammatory properties. However more studies are required, in particular to determine the effects on other cell types such as neutrophils and purified macrophages, important in inflammatory immune responses as well as safety issues. Implications During the course of this project, significant commercial developments between the industry partner, The Paperbark Company and international interests have emerged. Extensive clinical trials to support a commercial launch of Agonis fragrans oil are now being conducted on behalf of a core commercial
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development group, and may confirm a range of reported clinical uses such as remedial therapies for joint, muscle and arthritis pain and respiratory infections. Research (by the core commercial development group) is also being undertaken into the likely emotionally balancing activities of the oil, possibly due to the inherent balance of the major constituent groups. The regulatory overview undertaken as part of the project has given a clear path to the regulatory process required to establish Agonis fragrans oil as a new chemical entity. The Paperbark Company is already using this report as a guide through appropriate processes.
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Introduction Although Western Australia has one of the most diverse floras in the world (ca. 12,000 taxa), with enormous potential for native plant based industries, to date there has been little development of the essential oils of these species. This project focused on the commercialisation of the oil of a south coast native myrtaceous species, Agonis fragrans. In 1996, a RIRDC funded project (Management of Bush Stands for Cut flower and foliage), recognised this species contained a fragrant essential oil with significant antimicrobial properties and that the plant itself responded with vigorous growth to various management strategies in the field. A subsequent a report funded by the Great Southern Development Commission (A Report on the Field Investigation of Market Opportunities for Agonis Oil. Executive Summary presented in Appendix 1) confirmed market potential for the oil, but identified a lack of supply to respond to demand for testing and product development. Plantations to increase supply have since been established by The Paperbark Company at Harvey and Water Corporation (on treated waste water) at Albany. Commercial development and marketing of Agonis fragrans oil and associated products is now taking place particularly through The Paperbark Company. Australian clinical aromatherapist, Mark Webb, who regularly visits WA, had made preliminarily identification of Agonis fragrans oil as having multiple uses in his field to warrant clinical trials. The research carried out by this project was to ensure that if commercialisation of Agonis fragrans oil occurs, it will be in a logical and progressive manner. It may also provide a blueprint for testing and developing commercial potential of the essential oil of other WA native species. This realisation of potential will bring commercial opportunity and associated employment and will foster Western Australia’s fledgling essential oil industry. The publication of results, especially those of antimicrobial and clinical trials will further expose this oil to a wider audience and greater commercial prospects.
Project Objectives The fundamental aim of this project was to facilitate realisation of the commercial potential of a new essential oil, Agonis fragrans. The research proposed was 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 regulatory options to identify appropriate actions to enhance future marketability and expansion of uses. Regulatory processes and requirements have in the past been notoriously complex and require specific effort to resolve appropriate direction for product development. Separate objectives were:
Production and analysis of oil samples from plantations and across range of natural populations Comparative antimicrobial tests Identification of best or superior chemotypes Identification of potential uses Assessment of immunostimulatory ability for aquaculture Identification of regulatory processes and options Determination of anti-inflammatory ability Determination of any evidence of traditional use
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Production and Analysis of Oil Samples from Plantations and Natural Populations An extensive field survey was conducted in October 2003. This involved collecting three gram leaf samples in a fixed volume of ethanol from five individual plants from each of 11 populations from across the geographic range (as determined by Department of Conservation and Land Management [CALM] database: Florabase). Representative samples were collected from each population and herbarium voucher specimens were prepared and have been submitted to WA Herbarium, via CALM Albany Regional Herbarium. Each plant sampled was labeled in the field with a solid aluminum tag. The leaf in ethanol samples were dispatched to Peter Grayling of CALM Science in WA for GC analysis. Grayling provided 55 chromatographs (see Appendix 2), each with accompanying table of constituents and percentage composition. Twenty eight components were recorded, including seventeen unknowns. These results were forwarded to Prof. Tom Riley (UWA Microbiology) and John Day (The Paperbark Company) for consideration. A number of significant variations were revealed, especially two plants from Beardmore Road population west of Walpole, which had no cineole (usually over 20%). Other plants were also identified which had high levels of major components and others with high levels of unknowns. Following consideration of the GC chromatographs six plants of interest for further study were selected and sampled for more expensive GCMS analysis. Material from the selected plants was collected in the field and leaf samples sent to Dr. Ian Southwell, Principal Research Scientist, Essential Oils, Wollongbar Agricultural Institute for GCMS analysis. Results of the GCMS investigation and “Comments on the chemistry of the oils” by Ian Southwell and Robert Lowe are presented in Appendix 3. To facilitate antimicrobial and anti-inflammatory research by UWA, oil samples from three selected wild populations were prepared. This involved revisiting field sites to collect bulk leaf material. This was transported to Albany where a 25 litre steam still (manufactured by Essential Harvests, Beechworth, Victoria) was used to distil the oil samples. Another three samples of plantation grown oil (of three separate provenances) were added by John Day (The Paperbark Company) and the six samples were then delivered to UWA. Duplicate samples were also forwarded to Ian Southwell for GCMS analysis.
Comparative Antimicrobial Tests Six oil samples (from three wild populations and three plantations) were sent to the School of Biomedical and Chemical Sciences within the University of Western Australia for determination and comparison of antimicrobial activity. The no cineole sample from Beardmore Road which was produced in a very small quantity (due to low number of plants adjacent to original sample) was of insufficient volume to be tested. Methods and results are presented in Appendix 5. The combined results of this work and the GCMS analysis are being prepared for publication in Journal of Essential Oil Research - a preliminary draft of “Variation within the Leaf Essential Oil and Antimicrobial Activity of Agonis fragrans” is presented in Appendix 4.
Identification of Best or Superior Chemotypes The work conducted by Grayling (Appendix 2) and Southwell (Appendix 3) indicate that the chemical variation within Agonis fragrans is not unusual. Southwell states that “Although it is difficult to distinguish separate chemical varieties……variation is evident”. However, of the samples selected for antimicrobial testing (based on variability), Carson et al (Appendix 5) state that “there was no significant variation in the MIC (minimum inhibitory concentration) or MBC (minimum bacterial concentration) results between the oils”.
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The work of Carson et al confirms that Agonis fragrans oil is antimicrobial and does have clinical potential. The industry partner, The Paperbark Company supplied the three bulk oils and is therefore now able to assess its oil relative to other potential chemotypes.
Identification of Potential Uses The work conducted by Dr. Ian Southwell, Principal Research Scientist, Essential Oils, Wollongbar Agricultural Institute includes (Appendix 3) comments on the potential uses of the oil: As a fragrance, the common type oil has a powerful cineole dominated aroma whereas the myrtenol type has a weaker, less distinctive note. The latter 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. John Day of the Paperbark Company has been producing and marketing Agonis fragrans oil since 2003. Anecdotal reports by his customers indicate that that it is favoured in aromatherapy and has specific application in relieving pain caused by arthritis. Other clinical practitioners, within Australia and abroad, have found its application has been successful in treating a range of medical conditions from sore throat to non-specific, chronic fatigue syndrome. The Paperbark Company provided the following comments: The oil extracted from the leaves of Agonis fragrans would appear to have significant potential in the therapeutic market and this is where most attention to date has been applied. This sector of the market only accounts for about 6% of the essential oils used worldwide. However, there is increasing interest being shown by the fragrance sector. Anti-microbial and anti-inflammatory testing has been undertaken, with results from the former being very encouraging. The pleasant scent of this oil will likely support the use of this oil in this area. Early work has identified the major constituents of 1-8 cineole, alpha pinene and linalool as being indicative of possible application in remedial therapies for joint, muscle and arthritis pain (Mark Webb pers com). The constituents are also indicative of application to remedial therapies for respiratory infections (Mark Webb pers com). In addition to the anti-microbial aspects of the oil, there is considerable research being undertaken into the likely emotionally balancing activities of certain chemotype/constituent varieties of the oil. One of the primary aspects which has been identified as contributing to this seemingly significant property is the “balance” of a particular chemotype of the genus. The chemical constituents are loosely grouped as oxides (1-8cineole), monoterpenes and monoterpenols, all in near perfect balance. This aspect of the oil is very exciting and is initially where a lot of the early attention is being directed. There is a growing interest in the use of the oil in fragrance applications. Several small local users are currently using the oil, and a significant export of this oil for this application has recently been made. Overseas comment has also been received about the potential in the flavouring industry.
The Paperbark Company is now in sensitive negotiations with other parties (international) who are conducting further clinical trails as part of building a base for international market launch. Results of these clinical trials are not currently available and may not be released in the near future. There is concern that to release clinical results now may encourage commercial interest by other parties which may result in uncontrolled planting of differing chemotypes and erroneous and fanciful claims made relative to this new oil. At this stage, this could significantly complicate negotiations
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and the orderly development of this new product. Adverse initial publicity will damage public awareness and future marketability. The report by Found and Associates (Appendix 6) stated “it is quite likely that Agonis Oil could be important as a general purpose antiseptic, acne treatment, disinfectant or skin wash. Activity against staphylococcus organisms may prove useful against methicillin resistant staph aureus (MRSA) which has become a life threatening problem for hospital workers.”
Assessment of Immunostimulatory Ability for Aquaculture During project development, Curtin University (Professor Louis Evans, Aquatic Science Research Unit) agreed to participate, with $1000 allocated to assist an honours student to conduct aquaculture immunostimulatory studies. Following observations of marron (Cherax tenuimanus, a commercially cultivated edible crustacean, native to WA) chewing leaves and bark from Agonis branches fallen into aquaculture ponds, and knowledge that marron naturally occur in waters stained brown by leaf compounds from native stream side vegetation (including Agonis species), it was postulated that Agonis oil may contribute to marron health. Maintenance of marron health in commercial culture is a major production issue. However, the student who was to conduct this work resigned in early 2004 and no suitable replacement was offered, and the work did not take place.
Identification of and Progression Toward Recognition by Regulatory Authority An industrial chemist, Found and Associates, was engaged to document regulatory options and requirements to enhance future marketability and expansion of uses for Agonis fragrans oil. The Regulatory Overview is presented in Appendix 6. At this stage there has been no application to the Theraputic Goods Administration) TGA for registration of Botanical Name for Agonis fragrans on the Australian Approved Names (AAN) register. John Found’s report included a copy of the relevant application form for the registration of the proposed botanical name. This seems to be straight forward, being purely regulatory, and doesn’t require any toxicity data. It does require supporting material as part of the application, this being typically the extract from Nuystia 13(3):567-570 (2001), Agonis fragrans (Myrtaceae), a new species from Western Australia. The only complication with this application may be the recently proposed change of genus name from Agonis to Taxandria, and the earlier references to the plant by the Western Australian Herbarium as Agonis sp Coarse Tea Tree and Agonis sp Coarse Agonis. Found and Associates’ report gives a reasonable picture of the requirements for getting appropriate regulatory approvals for this oil to be used in therapeutic and cosmetic applications, although the process still remains somewhat mysterious to the uninitiated. In parallel with the above AAN application, an application needs to be made to the Cosmetic Toiletry and Fragrance Association (CTFA) so that an International Nomenclature of Cosmetic Ingredients (INCI) name will be allocated. Again this seems fairly straight forward. The major area of complication and expense in introducing this oil as a therapeutic good will be in testing its safety and efficacy. Found and Associates’ report identifies in excess of $34,000 to achieve this and it would not surprise to find many additional expenses.
Determination of Traditional Use Melaleuca alternifolia (tea tree) oil has recognised traditional use by aboriginal people on the east coast for treatment of skin infections, burns, rashes and similar problems which enhances its marketability. During the development of this project, the local Noongar community was consulted
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about researching possible local indigenous use of Agonis fragrans plant material (and hence its oil) for medicinal or other purposes. Although no such use has been documented, there is in fact little or no documentation of all aspects of traditional life in south coast WA, which was settled early in the colonisation of WA and indigenous culture was rapidly and enormously changed. The Indigenous Landcare Facilitor, based in the Agriculture Department, was involved in developing this project and had suggested a means to investigate traditional use by arranging dialogue with Noongar elders who are reputed to retain some knowledge of traditional life. Access to elders is covered by certain etiquettes and apparently should only be through a third person such as the Indigenous Landcare Facilitator. This person was approached repeatedly on many separate occasions to facilitate access to the elders. Although many promises were made to arrange discussions they were never acted upon, and traditional use of Agonis oil remains unexplored.
Determination of Anti Inflammatory Ability Anecdotal information from clinical therapists has indicated that Agonis fragrans oil is an effective anti inflammatory agent. The process to investigate and prove its efficacy and safety for human application is both extensive and expensive. Preliminary in-vitro studies were carried out through the School of Paediatrics and Child Health, University of Western Australia. This study (see Appendix 7) tested the potential anti-inflammatory effects of oil from Agonis fragrans, by examining effects on cytokine responses of peripheral blood mononuclear cells in vitro. Inflammation occurs as a result of host immune responses to injury, infection or foreign antigen. This response is characterised by the production of inflammatory cytokines by immune cells including monocytes, macrophages and lymphocytes which are collectively referred to as mononuclear cells. These cells recirculate continuously between the blood, tissue and the lymphatic system and contribute to local inflammation. The study found that Agonis oil inhibits secretion of the IFNγ, involved in inflammatory response to tissue injury or infection, which could support the notion that Agonis fragrans oil potentially has anti-inflammatory properties, however more studies are required, in particular effects on other cell types such as neutrophils and purified macrophages, important in inflammatory immune responses as well as safety issues.
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Discussion Although the work of Southwell indicated that there is variance of Agonis fragrans oil between and within populations, he considered that the variation is not unusual. The major variation found was a subpopulation (within one of the eleven populations sampled) that contained no cineole. The work conducted by University of WA Microbiology, 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. Most importantly for the outcomes of this project “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. The anti inflammatory study suggests that Agonis oil inhibits several aspects of immune response. In particular it found that Agonis fragans oil inhibits cytokine IFNγ production by mononuclear cells. IFNγ, along with other pro-inflammatory cytokines (IL6 and IL12), is thought to be involved in the pathogenesis of inflammation, auto-immune disease and atopic dermatitis. The finding that Agonis oil inhibits secretion of this cytokine could support the notion of anti-inflammatory properties. Systemic mononuclear cells do traffic to the periphery so it is plausible that Agonis oil used on the skin may have anti-inflammatory effects, however more studies are required to test this. Clinical trials which were to be funded by this project have not taken place, due primarily to other commercial developments. However, the Paperbark Company is aware from communications from clinical practitioners that clinical use indicates application in remedial therapies for joint, muscle and arthritis pain and respiratory infections. Additionally, research is being conducted by a commercial partner of The Paperbark Company into the likely emotionally balancing activities the oil, possibly due to the inherent balance of oxides, monoterpenes and monoterpenols. During the course of this project, continued production and promotion of Agonis fragrans oil by The Paperbark Company has resulted in significant interest in commercialisation and marketing of this oil by international parties. A core commercial development group has been established. This group proposes to cover all aspects of the production and distribution of this new product, namely growing, distilling, identification and testing, high level clinical trials and research, distribution and marketing. The core group will introduce small quantities to the market in the first year, followed by larger, closely controlled quantities in following years. Some of this work will be done locally, but much will be done overseas. It is intended however that Australia will be high on the list of priorities for full scale distribution once the oil has completed its initial acceptance trials. These significant developments have resulted in commercial sensitivities which have resulted in the delay of publication of results of clinical trials. It is expected that clinical trials, as indicated by anecdotal reports from reputable clinicians, will confirm that Agonis fragrans oil has considerable clinical application. However, as yet, little is known of the safety profile of these oils and much work would be required before Agonis fragrans oil could be evaluated in vivo, the definitive way in which to determine its clinical potential. As indicated above, much of this work will be done by the core commercial development group in association with The Paperbark Company. Currently, there is a shortage of the oil relative to levels of existing and projected demand. In order to address this supply issue, The Paperbark Company is expanding plantation to near its limit in 2005. This is still a relatively small area. Discussions have commenced with neighbour farmer groups and others, in confidence, for expanded development and plantings in 2006. Interestingly, Southwell indicated that 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
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species of pine beetle and melon fly and as a flavouring agent. None of these suggested uses has been tested. The regulatory overview provided has given a clear path to the regulatory process required to establish Agonis fragrans oil as a new chemical entity. The Paperbark Company is already using this report as a guide through appropriate processes.
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Implications and Recommendations The research presented here has confirmed that Agonis fragrans oil has significant anti-microbial activity, similar to that of other recognised anti-microbial essential oils, including tea tree oil (from Melaleuca alternifolia) and that it may have clinical potential as an antimicrobial agent. There was no significant variation in results between the oils tested, which however did not include the no-cineole sample which was unable to be tested. It does confirm that the chemotype currently being produced and marketed is at least equal to any other. Further clinical trials are required to prove the oil for human application, however these are currently being conducted. The anti inflammatory study carried out through School of Biomedical & Chemical Sciences, University of Western Australia found that Agonis oil inhibits secretion of the IFNγ, involved in inflammatory response to tissue injury or infection, which could support the potential of anti-inflammatory properties of Agonis fragrans oil, however more studies in inflammatory immune responses (such as effects on other cell types such as neutrophils and purified macrophages) and safety issues are required. 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.
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Appendices Appendix 1: A Report on the Field Investigation of Market Opportunities for Agonis Oil. Robinson Rural Consulting 1999
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A Report on the Field Investigation of Market Opportunities for
Agonis Oil. Submitted to: The Albany Agonis Steering Committee
Great Southern Development Commission 110 Serpentine Road ALBANY WA 6330
Submitted by: Robinson Rural Consulting
96 Aberdeen Street ALBANY WA 6330
Date : 14 September 1999
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Executive Summary
• A market field investigation was carried out to determine the potential market opportunities for Agonis oil.
• The study included 33 interviews – 19 in person interviews and 14 telephone interviews – with
potential customers, researchers and other relevant industry personnel based in Melbourne, Sydney, Perth and South West of WA.
• “Relatively firm” orders for approximately 130 - 150 kilograms of Agonis oil, at a subsidised
introductory price of about $45/kg, have been received as a result of the marketing field investigation. The real price of the oil is likely to be significantly more than the subsidised price of about $45/kg. A number of firms have also dispatched samples of the oil to their laboratories around the world for further assessment. In essence the 130-150 kg relatively firm orders are to allow further market testing to take place.
• As a result of the interviews, one major international company, Warner Lambert Ltd. agreed to
analyse the samples for possible product development as a: - Preservative with skin products - Liquid disinfectant - Antiseptic cream
Another major flavour and fragrance company, International Flavours and Fragrances Ltd, is assessing the oil at their international laboratories for its perfumery potential.
• Agonis oil: has a pleasant and distinctive fragrance, displays some antimicrobial activity and is
new and “a bit signature” in the market place; has a different chemical composition to Melaleuca alternifolia (tea tree) oil and could be marketed as a sister product to it; is a local product unique to the south west of Western Australia; and has some traditional Noongar Aboriginal uses.
• The potential market for Agonis oil could be segmented into the following broad product
groups: - Antimicrobial health care - Fragrance, Aromatherapy, Aerosols - Degreasers and Detergents - Veterinary Care - Cosmetics/Soaps - Wholesale Oil - Local Cottage Industry/Giftware/Tourism
Preliminary screening of the product segments suggested that the Antimicrobial Health Care, Fragrance/Aromatherapy/Aerosols, and the Local Cottage Industry/Giftware/Tourism will probably be the best market segments to pursue.
• A market opportunity could exist for Agonis oil if it was possible to build its own combined fragrance, antimicrobial and solvent attributes, as well as its image as a natural product that has traditional Aboriginal use and as a plant that grows in a pristine corner of WA. Further market testing of samples of the oil is warranted. However, the extent of the market demand for the oil itself will not be known until the results of tests being done by the firms surveyed are known, and further test marketing of the oil around the world is completed.
• There was a mixed response to the oil from the sample of companies surveyed. Some, for
example Warner Lambert and IFF, etc were keen to progress investigations. Others expressed scepticism as to whether there would be a sufficiently strong market demand for Agonis oil to warrant developing the industry. Unless a major company decides to run with Agonis oil, the
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industry is likely to only develop on a small scale and only if someone has the will and capital to do it.
• Agonis oil is a completely different product to oil from Melaleuca alternifolia and whilst
Agonis oil can out compete it on the basis of fragrance, Agonis oil should be established as a separate product and should not attempt to compete directly with it. There has been about $300 million spent on developing Melaleuca alternifolia industry to date and, even so, it is facing considerable problems of over production and falling prices. A price of less than $30/kg is expected for Melaleuca alternifolia next year.
• It appears as though it should be possible to develop simple fragrances and lotions without
having to gain Therapeutic Goods Act (TGA) Listing provided no therapeutic claims are made. However, with careful labelling it may be possible to say a product “may” have some antibacterial advantages. Eventually TGA listing could be beneficial. Depending on the product selected, it may be possible to avoid TGA listing in the initial instance.
• The name of the Agonis oil should reflect the benefits it provides to its customers and the
selection of an appropriate Noongar Aboriginal name for fragrance, etc may be appropriate. Further research is required to find a suitable name.
• The ideal situation would be to develop the industry in tandem with an international
manufacturer and marketer such as Warner Lambert or IFF. Companies such as these are likely to have; the requisite capital; expertise in formulating, manufacturing, marketing; and the distribution networks. The fallback position would be to develop the industry on a local cottage giftware/tourism basis. In both instances the Agonis oil industry would provide a significant number of new long term jobs to the region, provided the enterprises were shown to have reasonable prospects of profitability.
• Whilst the results of the assessment of the Agonis oil by several multinational companies
surveyed are still to be received, this market field investigation does suggest that there are grounds for developing a small supply base of agonis oil to meet the further market testing and product development. It will be difficult to market the oil without some guaranteed supply. Some risk capital will be required and RIRDC or other New Industry Development Seed Funding should be sought for this purpose.
• A feasibility study, using the market intelligence collected in this field market investigation,
and the results flowing from it, now needs to be completed to determine the potential viability of the industry and the basis upon which any further development of the industry needs to take place. The supply side of the equation needs to be determined.
• The assistance of the Albany Agonis Steering Committee; those interviewed; the Department
of Employment, Workplace Relations and Small Business: the Great Southern Development Commission; and the New Industries Program of Agriculture Western Australia, with this Field Market Investigation is gratefully acknowledged.
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Appendix 2: Gas Chromatography of 55 Agonis fragrans leaf samples in ethanol Peter Grayling. 2004
WHICHER A6840 A6841 A6842 A6843 A6844 Averagea thujene 0.4 0 0 0 0.3 0.14a pinene 18.4 21.4 16.7 16.8 20 18.66mycene 1.5 1.3 1.6 1.5 1.6 1.5B pinene 1.1 1.1 1.1 1 1.5 1.16limonene 1.9 1.8 2.3 2.3 1.8 2.02B phellp cymene 2.1 2.5 2 2 1.8 2.081,8 cineol 27.8 30.4 34.8 33.4 30.8 31.44unknwn 1 1.2 1 1.3 1.5 1.4 1.28linalool 9.5 11.2 6.8 8.3 7.8 8.72terp 4 ol 3.7 3.7 4.2 3.8 3.8 3.84a terpinol 6.5 7.9 8.2 8.4 7.6 7.72unknwn 2 3.4 5 6.4 6.2 4 5unknwn 3 1.1 0.6 1 0.5 1.4 0.92unknwn 4 2.3 2.3 1.4 2.9 2.1 2.2unknwn 5 0.9 0.4 0.9 0.2 0.8 0.64unknwn 6 2.6 2.4 0.7 1.5 0.9 1.62unknwn 7 0.2 0 0.3 0.3 0.3 0.22unknwn 8unknwn 9 7 3.2 4.7 3.8 3.4 4.42unkwn 10 5.6 2.2 2.7 1.9 5.7 3.62unkwn 11unknwn12 1.1 0 0.2 0.2 1 0.5unknwn13 0.8 0.2 0.2 0.1 0.325unknwn14unkwn14a 0.7 1.3 2.5 3.3 1.7 1.9unknwn15unknwn16 0 0.5 0 0 0 0.1
VLAM A6845 A6846 A6847 A6848 A6849 Averagea thujene 0 0 1.6 0 4.8 1.28a pinene 20 25.1 34.7 20.6 22.9 24.66mycene 1 2.1 2.8 1 1.1 1.6B pinene 1.3 1.6 0.8 1.4 2.2 1.46limonene 2 2.1 1.8 2.7 1.8 2.08B phell #DIV/0!p cymene 1.8 1.7 1.3 2.3 2.3 1.881,8 cineol 26.6 26 12.4 24.8 16.7 21.3unknwn 1 1 1.4 1.1 1.5 0.8 1.16linalool 7.7 11.1 3.5 12.1 2.6 7.4terp 4 ol 2.4 6 1.9 2.1 4.8 3.44a terpinol 5.9 8.6 3.3 5.9 3.7 5.48unknwn 2 6.8 0.8 0.5 10 0.3 3.68unknwn 3 0.4 1.3 0.2 0.3 0.7 0.58unknwn 4 0.8 1.4 0.3 1.5 0.3 0.86unknwn 5 0.5 0 0.6 0.4 0.3 0.36unknwn 6 0.7 0 0 0.4 0 0.22unknwn 7 0 0 0 0 0 0unknwn 8 #DIV/0!unknwn 9 0.4 1.2 5.9 0.7 1.6 1.96unkwn 10 1.9 0.9 4 1.8 3.8 2.48unkwn 11 0.2 0.2 0.3 0.9 0.2 0.36unknwn12 1.8 0.4 3.5 1.3 3.1 2.02unknwn13 4.3 0.7 5.9 1.8 5.1 3.56unknwn14 6.1 1.1 6.7 1.7 7.4 4.6unkwn14a 1.4 1.9 0 3.8 0.2 1.46unknwn15 0.8 0.2 0.9 0.4 1.1 0.68unknwn16 3.7 1.2 6 0.5 7.9 3.86
14
STEWART A6850 A6851 A6852 A6853 A6854 Averagea thujene 0 0.4 0.2 0.7 2.2 0.7a pinene 25.6 21 19.3 12.3 28.3 21.3mycene 2 2.7 3.1 2.7 2.7 2.64B pinene 1.4 0.9 1.2 1.6 1.7 1.36limonene 2.7 2.3 2.4 1.9 2.3 2.32B phellp cymene 2.1 1.9 1.8 3.5 2.5 2.361,8 cineol 26.9 28.6 28.3 29.4 26.7 27.98unknwn 1 1.5 2.5 2.1 4.7 2.2 2.6linalool 12.1 4.1 13.9 9.1 4.7 8.78terp 4 ol 2.3 3.1 3.5 2.6 3.5 3a terpinol 6.3 6.9 7.3 6.7 6.2 6.68unknwn 2 7.1 4.2 5.9 5.2 4.6 5.4unknwn 3 0.8 0.7 1.7 1.4 1.1 1.14unknwn 4 0.9 0.7 1.4 0.2 1.1 0.86unknwn 5 0.3 0 0.7 0.6 0 0.32unknwn 6 1 1.6 0 0.9 1.3 0.96unknwn 7 0 0.5 0 0 0.1 0.12unknwn 8 0 0.3 0 0.3 0.1 0.14unknwn 9 0.4 1.3 1.2 1.3 3 1.44unkwn 10 1.8 4.2 2.8 3.4 0 2.44unkwn 11 0.2 0 0 0 0.1 0.06unknwn12 0.4 1.3 0.5 1.2 0.5 0.78unknwn13 0.5 2.5 0.3 2.1 1.4 1.36unknwn14 0.7 3.1 0 2.8 1.2 1.56unkwn14a 2.5 2.3 1.7 2.9 0.7 2.02unknwn15 0.4 0.3 0.3 0.3 0.4 0.34unknwn16 2.5 0.3 2.2 1.6 1.65
South Coast Rd A6855 A6856 A6857 A6858 A6859 Averagea thujene 0.4 0.5 0.4 0 0.2 0.3a pinene 16.7 18.1 12.3 18 16.7 16.36mycene 2 2.6 2.9 3.9 2.7 2.82B pinene 1.5 1.1 0.9 1.3 1.4 1.24limonene 2.7 1.9 2.4 2.2 1.9 2.22B phell #DIV/0!p cymene 2 1.5 1.7 0.6 1 1.361,8 cineol 32.3 24 29.5 30.5 31.4 29.54unknwn 1 1.6 2 2.5 1.2 1.7 1.8linalool 7.1 12.1 13.5 7.8 9.2 9.94terp 4 ol 3.7 2.4 3 2.3 4.1 3.1a terpinol 7.2 6.1 7 7.7 8 7.2unknwn 2 6 5.4 5.7 7.3 5.9 6.06unknwn 3 0.7 0.4 1.8 1.8 2 1.34unknwn 4 2 2.6 2.1 1.8 1.9 2.08unknwn 5 2.1 0 1 1 0.9 1unknwn 6 1.2 2.2 1.6 1.2 0.8 1.4unknwn 7 0.2 0.2 0 0 0.1 0.1unknwn 8 0.2 0.5 0 0 0 0.14unknwn 9 1 2.2 4.3 3.3 2.3 2.62unkwn 10 3.1 4.3 0.4 0 1.5 1.86unkwn 11 0.1 0 0.1 0 0 0.04unknwn12 1 1.3 0.4 0.4 0.8 0.78unknwn13 0.8 1.9 0.9 1.4 0.8 1.16unknwn14 0.9 2.1 0.9 1.8 1 1.34unkwn14a 3.4 3 4.5 3.6 2.1 3.32unknwn15 0 0.2 0 0 0.3 0.1unknwn16 0.2 1.5 0.1 0.9 1.2 0.78
15
Beardmore A6860 A6861 A6864 Average A6862 A6863 Averagea thujene 1.1 0.9 1 1 0 0 0a pinene 18 21.6 16.8 18.8 53.5 46.1 49.8mycene 2.6 2.1 4.4 3.03333333 0 0 0B pinene 1.4 1.4 1.3 1.36666667 0.5 3.6 2.05limonene 1.5 1.4 1.4 1.43333333 2.4 2.2 2.3B phell #DIV/0! 0 0 0p cymene 0.5 0.3 0.6 0.46666667 0 1 0.51,8 cineol 19.6 16.5 19.2 18.4333333 0 0 0unknwn 1 0.8 0.9 1 0.9 0 0 0linalool 2.4 1.7 1.5 1.86666667 12.6 14.9 13.75terp 4 ol 3.2 2.9 3.5 3.2 0.5 0 0.25a terpinol 5.2 4.5 5.2 4.96666667 2.1 2.8 2.45unknwn 2 6 4.9 5.4 5.43333333 12.3 12.6 12.45unknwn 3 0.4 0 0.2 0.2 1.1 1.1 1.1unknwn 4 2.1 1.9 2 2 0.4 0.6 0.5unknwn 5 0.5 0.4 0.4 0.43333333 3.9 1.8 2.85unknwn 6 0.1 1 0.3 0.46666667 0.4 0.7 0.55unknwn 7 0 0.3 0 0.1 0 0.2 0.1unknwn 8 #DIV/0! 0 0 0unknwn 9 2 2.3 2.1 2.13333333 0 0.6 0.3unkwn 10 2.2 2.1 2.4 2.23333333 1 0.6 0.8unkwn 11 0.1 0.1 0.1 0 0.4 0.2unknwn12 3.3 3.6 3.5 3.46666667 0 2 1unknwn13 6.3 6.4 6.1 6.26666667 3 3.2 3.1unknwn14 9 9.1 8.3 8.8 3.9 1.5 2.7unkwn14a 0.7 0.2 0.45 1 2.6 1.8unknwn15 1.5 2 1.6 1.7 0 0.7 0.35unknwn16 10.4 11 11.5 10.9666667 1.2 1.1 1.15
Walpole A6865 A6866 A6867 A6868 A6869 Averagea thujene 0.3 0 0.1 0.4 0.3 0.22a pinene 14.4 14.6 16.5 14.5 16.6 15.32mycene 1.4 1.4 1.7 1.3 2.5 1.66B pinene 9.9 9.9 10.7 9.8 10.7 10.2limonene 1.5 2 2.3 1.4 1.3 1.7B phell #DIV/0!p cymene 1.2 1.5 1.1 1.7 1 1.31,8 cineol 19.6 19.7 17.8 19.9 17.2 18.84unknwn 1 1 1.3 1.2 0.5 1.4 1.08linalool 7.8 9 7.8 7.6 7.8 8terp 4 ol 2.8 2.9 2.4 3.1 2.4 2.72a terpinol 5.3 5.5 5.2 5.3 4.6 5.18unknwn 2 4.3 4.7 5 4.2 4.5 4.54unknwn 3 1 1 0.7 0.7 0.5 0.78unknwn 4 2.5 2.5 2.7 1.8 2.4 2.38unknwn 5 1 0.9 0.5 1.1 0.6 0.82unknwn 6 0.3 0.5 1 1.1 1.6 0.9unknwn 7 2.1 2.2 2.1 2.1 2 2.1unknwn 8 1.8 1.7 1.8 1.5 1.5 1.66unknwn 9 2.3 2 1.9 1.7 2.2 2.02unkwn 10 3.7 3.2 3.2 3.1 4.5 3.54unkwn 11 #DIV/0!unknwn12 2.2 2.4 2.2 2.2 1.7 2.14unknwn13 3.9 3.3 3.4 4.6 3.8 3.8unknwn14 4.3 3.4 3.9 4.9 3.7 4.04unkwn14a 1.2 0.9 1.2 0.4 0.9 0.92unknwn15 0.6 0.4 0.4 1.2 0.6 0.64unknwn16 3.5 2.9 3.5 4 3.5 3.48
16
Conspicuous Cliffs A6870 A6871 A6872 A6873 A6874 Averagea thujene 0.5 1.2 2.1 2.2 2.2 1.64a pinene 13.9 14.5 21.6 22.2 18.3 18.1mycene 1.1 0.4 0.5 0.6 1.3 0.78B pinene 9 1.3 1.2 1.5 1.6 2.92limonene 1.5 2.4 1.5 1.5 2 1.78B phell #DIV/0!p cymene 2.6 2.6 1.9 1.9 1.7 2.141,8 cineol 20.2 25.6 15.9 15.5 22.4 19.92unknwn 1 1 0.2 0.9 1 0.8 0.78linalool 6.3 1.6 11.2 10.2 2 6.26terp 4 ol 2.6 4.1 2.8 2.9 4 3.28a terpinol 5.5 6.3 4.3 4.3 5.8 5.24unknwn 2 4.6 4.9 2.4 2.5 5.8 4.04unknwn 3 0.4 0.4 0.3 0.3 0.4 0.36unknwn 4 0.6 0.4 2.6 2.4 0.6 1.32unknwn 5 0.3 0.6 0.6 0.3 0.6 0.48unknwn 6 0.9 0.6 0.5 1.8 0.4 0.84unknwn 7 0 4.4 4.1 3.8 4.7 3.4unknwn 8 0 3.7 3.1 2.8 3.9 2.7unknwn 9 6.1 1.3 4.1 4 1.3 3.36unkwn 10 0.8 1.4 0.3 0.3 1.6 0.88unkwn 11 0 0 0.2 0.2 0.6 0.2unknwn12 2.9 1.9 2.4 1.8 1.5 2.1unknwn13 5.4 10.8 5 11 8.05unknwn14 6.9 11.3 0 5.4 5.9unkwn14a 0.4 0.2 0.1 0.1 0.2unknwn15 1.1 1.5 0.9 1 1 1.1unknwn16 5.5 7 3.7 4.6 4.5 5.06
Kernutts Old A6875 A6876 A6877 A6878 A6879 Averagea thujene 0.6 2.5 0.6 0.6 0.6 0.98a pinene 23.4 21.1 23 15.1 16.1 19.74mycene 2.1 2.6 1.9 1.7 2.2 2.1B pinene 2.1 2 1.8 1.8 2 1.94limonene 2.3 2 2.6 2.3 2.3 2.3B phell #DIV/0!p cymene 2.2 1.8 2.8 3 2.7 2.51,8 cineol 28.3 23.9 28.6 30.7 30.9 28.48unknwn 1 1.7 1.9 1.3 1.6 1.7 1.64linalool 10.8 9.6 10.3 9.1 10.6 10.08terp 4 ol 3.9 4 3.9 4.9 4.6 4.26a terpinol 7.3 6.5 7.3 7.8 7.6 7.3unknwn 2 5 4.3 5.6 3.2 3.3 4.28unknwn 3 0.9 0.5 0.6 0.6 0.8 0.68unknwn 4 1.8 1.8 1.7 0.4 0.7 1.28unknwn 5 0 0.3 0.3 1.3 1.4 0.66unknwn 6 0 0.9 0.5 0 0.4 0.36unknwn 7 0 0.5 0 0 0 0.1unknwn 8 #DIV/0!unknwn 9 2.2 1.9 2 1.7 1.4 1.84unkwn 10 3.1 2.2 2.7 1.4 1.3 2.14unkwn 11 #DIV/0!unknwn12 0.7 1.4 0.7 1.9 1.6 1.26unknwn13 0 1.9 0.2 2.2 1.4 1.14unknwn14 0 2.2 0 3.1 2.1 1.48unkwn14a 1 0.5 0.8 0.5 1.3 0.82unknwn15 0 0.6 0.1 0.9 0.5 0.42unknwn16 0.5 2.9 0.8 4.3 2.6 2.22
17
Kernutts Young A6880 A6881 A6882 A6883 A6884 Averagea thujene 0.8 0.9 0 0.4 0 0.42a pinene 21.4 25.8 26.2 19.6 23.5 23.3mycene 5.1 6.1 4.5 3.8 5.3 4.96B pinene 0.6 1.4 1.5 1 1.4 1.18limonene 2.1 1.8 1.9 1.8 1.4 1.8B phell #DIV/0!p cymene 2.1 0.5 0.8 1.2 0.7 1.061,8 cineol 27.8 23.9 25.6 26.4 27.6 26.26unknwn 1 3.1 3.3 3.5 2.8 2.6 3.06linalool 6.8 9.1 8.6 10.5 8.1 8.62terp 4 ol 2.5 1.7 1.9 2.2 1.9 2.04a terpinol 6.8 5.4 5.4 5.6 6.7 5.98unknwn 2 4.2 3.8 2.8 4.1 4.3 3.84unknwn 3 0.7 0.4 1 1.9 0.7 0.94unknwn 4 0.5 2.3 2 1.6 2.2 1.72unknwn 5 1.4 0.7 0.7 0.6 0.85unknwn 6 1 4.7 3.7 3.5 4.3 3.44unknwn 7 0.5 0.3 0 0 0 0.16unknwn 8 #DIV/0!unknwn 9 1.4 1.5 1.7 2.2 1.5 1.66unkwn 10 1.6 2.3 3.1 4 3.6 2.92unkwn 11 #DIV/0!unknwn12 0.7 0 0.2 0.8 0 0.34unknwn13 2.6 0 0.2 0.9 0 0.74unknwn14 2.2 0.2 0.8 1.1 1.075unkwn14a 1.3 3.2 3.4 3.5 4 3.08unknwn15 0.3 0.5 0.6 0 0 0.28unknwn16 2.6 0.2 0 0.3 0 0.62
Randall Rd A6885 A6886 A6887 A6888 A6889 Averagea thujene 0 0.6 0 0.6 0.7 0.38a pinene 18.8 18.8 20 20 17.5 19.02mycene 1.9 1.5 2.2 2.1 3.9 2.32B pinene 1.9 1.7 1.6 1.8 1.4 1.68limonene 2 2.3 2.2 2.1 2.7 2.26B phell #DIV/0!p cymene 1.2 2.8 1.6 2 2 1.921,8 cineol 28.1 30.8 27.8 29.8 27.3 28.76unknwn 1 1.3 0.9 0.7 1.6 1.5 1.2linalool 10.6 9.3 8.9 7.6 12 9.68terp 4 ol 3.4 3.6 3.4 3.4 3.2 3.4a terpinol 6.9 7.1 6.4 6.9 6 6.66unknwn 2 4.3 4.2 4.9 4.4 4.6 4.48unknwn 3 1 0.8 1.1 0.8 1.2 0.98unknwn 4 1 0.9 1.9 1.5 2 1.46unknwn 5 1 0.7 0.7 1.8 0.6 0.96unknwn 6 0.3 0 0 0.4 1.4 0.42unknwn 7 0 0 0.5 0 0 0.1unknwn 8 0.2 0.4 0.3 0.5 0.2 0.32unknwn 9 3 2.3 2.2 2.2 2.5 2.44unkwn 10 2.1 1.7 1 1.5 0.8 1.42unkwn 11 0.2 0 0.3 0 0.5 0.2unknwn12 1.7 0.9 4.1 1.1 1.9 1.94unknwn13 1.8 1.9 2 1.6 1.3 1.72unknwn14 2.8 3 2.7 2.3 1.7 2.5unkwn14a 0.5 0.2 0.2 0.6 1.9 0.68unknwn15 0.6 0.7 0.2 0.4 0.3 0.44unknwn16 3.4 2.7 3 2.8 1.2 2.62
18
Marbellup North A6890 A6891 A6892 A6893 A6894 Averagea thujene 0.7 0.4 0.5 0.7 0.4 0.54a pinene 22.8 24 23.9 26.1 25 24.36mycene 2.7 1 1.1 2.8 2.2 1.96B pinene 0.8 1.3 0.4 1.3 0.6 0.88limonene 1.7 2 1.9 1.9 1.8 1.86B phell #DIV/0!p cymene 2.5 2.4 2.6 0.9 1.5 1.981,8 cineol 23.4 22.3 23 21.6 22.1 22.48unknwn 1 1.9 1.8 1.6 2.4 2.1 1.96linalool 14.6 13 14.5 11.2 12.5 13.16terp 4 ol 3 2.9 2.8 2.5 2.4 2.72a terpinol 5.3 5.8 5.6 5.2 5.5 5.48unknwn 2 3.3 3 3.9 3.6 3.3 3.42unknwn 3 1 0.7 0.8 0.5 0.6 0.72unknwn 4 2.1 2.2 2.7 2.4 2.3 2.34unknwn 5 0.7 0.5 0.4 0.5 0.4 0.5unknwn 6 0.4 0.6 0.2 1.3 0.7 0.64unknwn 7 0 0 0 0.1 0.3 0.08unknwn 8 0.3 0 0.1 0 0.5 0.18unknwn 9 1.8 2 1.5 2 2.4 1.94unkwn 10 2.8 3 2.7 3.2 3.2 2.98unkwn 11 0.3 0 0.1 0 0.2 0.12unknwn12 2 2.2 2.7 1.8 2.4 2.22unknwn13 1.7 2.3 1.8 2 2.1 1.98unknwn14 1.8 3 2.3 2.5 2.6 2.44unkwn14a 0.9 0.7 0.8 2 1.2 1.12unknwn15 0.2 0.5 0 0 0.1 0.16unknwn16 1.3 2.5 2.1 1.6 1.5 1.8
19
Appendix 3: GCMS analysis and “Comments on the chemistry of the oils” Ian Southwell and Robert Lowe. 2004
20
Agonis fragrans Project.
Comments on the chemistry of the oils. Ian Southwell and Robert Lowe. Six single plant extracts and six mixed plant, single provenance oils were analysed by GC and GCMS to determine constituents as shown in the Table. The extracts reflected the quality of the distilled oils with the only major differences being caused by non steam-volatiles (eg epi-cryptomeridiol, cryptomeridiol and neophytodiene) present in the extracts that did not distill to be present in the essential oils. Quantitatively, the concentrations of cyclodecadienes (eg bicyclogermacrene, hedycaryol) were significantly higher in the extracts than in the oils. The single plant extracts showed one plant (6863) devoid of 1,8-cineole which, along with α-pinene was the major constituent of all other plants. In this plant the 1,8-cineole seemed to have been replaced by higher concentrations of α-pinene, linalool and myrtenol. Another plant (6881) was devoid of the sesquiterpenoid eudesmols (α-, β- and γ- ) and their putative precursor cryptomeridiols. In addition one plant (6873) contained significant traces (<4%) of the santalenes (α- and β- ) and α-trans-bergamotene all of which are present in the most volatile fractions of W.A. sandalwood oil. Another plant (6864) contained lower concentrations of linalool than the other five. As the distilled oils were from bulk collections, it was not possible to directly correlate the single plant extracts with the distilled oils. Some trends were evident however. The plant devoid of cineole was well represented in oil sample Bulk Y (cineole 0.6%). This oil reflected the high myrtenol and linalool content of the extracted plant but an enhanced α-pinene level was not evident. The low linalool plant was well represented in oil sample Bulk X (linalool 3.3%) and the low eudesmol plant by Bulk oils A-C. The presence of the sandalwood oil constituents was not seen in any oil suggesting that their presence may have been due to contamination. As potentially commercial oils, the most common are those with high cineole, α-pinene, linalool and low myrtenol (ABC and Z). Even these four can be divided further on eudesmol and myrtenol content. The low linalool Bulk X is closely related to this common type. The low cineole – high myrtenol type (Bulk Y) is clearly a separate chemical variety. As a fragrance, the common type oil has a powerful cineole dominated aroma whereas the myrtenol type has a weaker, less distinctive note. The latter 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 (http://www.pherobase.com/database/compounds-detail-myrtenol.html) and as a flavouring agent (http://www.inchem.org/documents/jecfa/jeceval/jec_1446.htm). In conclusion, the chemical variation within Agonis fragrans is not unusual and provides plant breeders and commercial developers with at least two varieties for further investigation and commercial development. Experimental Area percent concentrations of the volatile components were determined on a (i) Hewlett Packard 6890 series gas chromatograph with an Alltech AT35 60m x 0.25mm, 0.25µm film thickness, mid polarity FSOT column and a (ii) Shimadzu 14B series gas chromatograph fitted with an HP5-MS 29.5m x 0.25mm, 0.25µm film thickness, FSOT column. Carrier gas was hydrogen (55cm/s), injection port temperature 200°C with a 1:50 split, flame ionization detector set at 300°C and temperature programming from 60°C (3 min.) to 240°C at 9°C/min. Component identifications were performed on a Hewlett Packard 6890 series GC/MS fitted with an HP5-MS 29.5m x 0.25mm, 0.25µm film thickness, FSOT column with helium (36 cm/s) as carrier gas, injection port (split 1:50) at 250°C,
21
mass selective detector (HP 5973) at 250°C (source) and 150°C (quad) with transfer line 280°C and ion source filament voltage of 70 eV. Retention indices were measured with respect to n-alkane standards on the HP5-MS column. Component identifications were made on the basis of mass spectral fragmentation, retention time comparison with authentic constituents and mass spectral and retention matching with commercial (NIST, Wiley and Adams) and in-house libraries.
22
AGONIS fragrans - extracts and oils
WAI No. RP04-291 292 293 294 295 296Cust.I.D. R.I. 6893 6894 6870 6873 6881 6883
Type (HP5 GC/MS) extract extract extract extract extract extractα-thujene 927 0.5 0.4 1.6 0.3 0.4α-pinene 933 38.5 15.1 11.6 18.0 17.7 16.6sabinene 973 3.7 0.5 0.8 3.4 1.6β-pinene 976 0.4 1.8 8.3 0.9 2.9 2.6myrcene 990 2.0 1.4 0.9 2.9 2.7α-phellandrene 1001 0.1 0.1 0.1 0.1α-terpinene 1015 0.2 0.3 0.3 0.7 0.5limonene 1028 2.0 1.4 1.0 1.1 1.9 1.8p-cymene 1024 0.2 1.5 0.3 1.11,8-cineole 1031 18.9 17.5 12.4 25.3 25.7trans-β-ocimene 1049 0.6γ-terpinene 1060 0.5 0.8 1.5 1.2 1.7 1.6trans-sab. hydrate 1068 0.3 0.1terpinolene 1088 0.1 0.2 0.2linalool 1099 18.3 1.7 7.7 10.5 13.1 11.2δ-terpineol 1169 0.1 0.1terpinen-4-ol 1179 1.8 2.0 2.1 2.4 3.1α-terpineol 1192 1.1 4.6 4.3 3.0 6.2 6.3myrtenol 1198 12.1 4.3 4.5 1.8 3.9 3.6citronellol 1228 0.4nerol 1229 1.0 0.5 0.7 0.3trans-myrtanol 1264 1.0 0.2 0.6 0.7 0.9geraniol 1254 2.0 1.0 2.7 1.9 1.5α-copaene 1382 0.4methyl geranate 1323 0.8 0.3 0.2 0.6dihydro cinnamic acid 1339 0.1methyl cinnamate 1388 0.6α-santalene 1426 3.0α-trans-bergamotene 1441 3.9caryophyllene 1430 0.9 2.1 6.8 4.6 2.1 2.9β-santalene 1467 0.9aromadendrene 1450cadina-3,5-diene 1460 0.2humulene 1465 2.2 2.5 0.4 1.2 3.0 1.2bicyclogermacrene 1507 4.5 2.0 2.5 6.7 3.0 0.7δ-cadinene 1532 0.7 0.1cis-calamanene 1533 0.4 0.3 0.2 1.1 0.1 0.1cadina-1,4-diene 1542 0.4 0.3 0.3 0.4 0.1epi-globulol 1582spathulenol 1590 0.2 0.2 0.1globulol 1598 0.3 0.5 0.2caryophyllene oxide 1598 0.7 0.5viridiflorol 1607 0.3rosifoliol 1615humulene oxide 1625elemol 1617 1.4 0.3 0.1hedycaryol 1629 2.5eudesma-5-en-11-ol 1637 0.2γ-eudesmol 1645 1.2 3.1 1.9 1.0 0.9iso-spathulenol 1653 0.2β-eudesmol 1667 1.4 5.5 4.2 2.2 2.1α-eudesmol 1670 2.0 5.1 4.5 2.4 1.8epi-cryptomeridiol 1790 0.5 0.4 0.4cryptomeridiol 1831 0.5 6.6 2.8 2.5 2.1neophytadiene 1837 0.2 0.7 0.4 1.2 0.2TOTAL IDENTIFIED (%) 89.1 92.9 92.5 89.7 96.4 95.7
Stat.phase,Detector: Black - BP5, FID Red(italics) - AT35, FID
23
AGONIS fragrans - extracts and oils
WAI No. RP04-319 320 321 322 323 324Cust.I.D. Bulk A Bulk B Bulk C Bulk X Bulk Y Bulk Z
Type oil oil oil oil oil oilα-thujene 0.3 0.5 1.5 0.4α-pinene 28.0 25.1 24.2 20.9 21.5 13.7sabinene 0.6 1.5 0.4 2.8 0.2 0.8β-pinene 1.9 1.8 1.6 2.1 0.4 1.4myrcene 2.1 1.8 1.6 2.6 0.3 2.7α-phellandrene 0.2 0.1 0.2 0.2 0.2α-terpinene 0.8 0.7 0.7 0.7 0.7limonene 1.9 3.4 2.9 1.2 1.1 1.8p-cymene 2.5 2.4 2.6 2.8 2.9 2.51,8-cineole 28.3 29.8 34.2 31.7 0.6 34.1trans-β-ocimene 0.1 0.1γ-terpinene 2.1 2.4 2.0 2.0 0.4 1.9trans-sab. hydrateterpinolene 0.5 0.4 0.5 0.4 0.3 0.4linalool 11.9 10.4 9.2 3.3 25.3 14.7δ-terpineol 0.4 0.4 0.4 0.4 0.8 0.4terpinen-4-ol 2.9 3.8 3.5 3.9 0.5 3.2α-terpineol 5.9 5.2 5.9 5.9 2.4 5.6myrtenol 3.4 1.7 4.1 5.5 20.0 4.5citronellolnerol 0.9 1.0 0.8 0.9 1.6 1.6trans-myrtanolgeraniol 1.5 1.7 0.9 1.8 1.9 1.1α-copaenemethyl geranatedihydro cinnamic acidmethyl cinnamateα-santaleneα-trans-bergamotenecaryophyllene 0.5 0.2 0.4 0.5 0.5 0.7β-santalenearomadendrene 0.1cadina-3,5-dienehumulene 0.7 0.3 0.4 0.7 1.3 0.5bicyclogermacrene 0.9 0.9 0.8 0.6 2.9 0.5δ-cadinenecis-calamanene 0.7cadina-1,4-diene 0.1epi-globulol 0.3spathulenol 0.1 0.3 0.1globulol 0.1 1.0 0.3caryophyllene oxideviridiflorol 0.1 1.4 0.2rosifoliol 0.4humulene oxide 0.1 0.6elemolhedycaryoleudesma-5-en-11-ol 0.5γ-eudesmol 0.9 1.0 0.3iso-spathulenol 0.2 0.1β-eudesmol 0.1 1.5 1.4 0.5α-eudesmol 0.1 1.1 1.3 0.5epi-cryptomeridiolcryptomeridiolneophytadieneTOTAL IDENTIFIED (%) 98.3 95.1 98.0 96.3 94.1 95.5
24
Appendix 4: Composition of an Anti-microbial Essential Oil from Agonis fragrans Robert F. Lowe, Michael F. Russell, Ian A. Southwell, Christopher J. Robinson and John Day
25
Composition of an Anti-microbial Essential Oil
from Agonis fragrans
Robert F. Lowe, Michael F. Russell, Ian A. Southwell
Wollongbar Agricultural Institute, Wollongbar, NSW, 2477, Australia
Christopher J. Robinson
Greening Australia, Albany, WA, 6330, Australia
John Day
The Paperbark Company, Claremont, WA, 6010, Australia
Abstract
The leaf essential oils of Agonis fragrans, isolated by steam distillation
and solvent extraction, were analysed by GC and GC/MS. The major
components identified in five of the six samples investigated were 1,8-cineole
(28 - 34% and 12 - 26% respectively) and α-pinene (14 – 28 % and 12 - 18%
respectively). The remaining sample was almost devoid of 1, 8-cineole (1% in
oil and 0% in extract) with higher concentrations of α-pinene (22 and 39%),
linalool (25 and 18%) and (+)-(1S, 5R)-myrtenol (20 and 12% respectively).
The compositional variation within the species indicated that a breeding
project could identify and use the best composition for commercial
development.
26
Key Word Index
Agonis fragrans, Myrtaceae, Course Agonis, Course Tea Tree, essential
oil composition, ethanolic extract, 1,8-cineole, (+)-(1S, 5R)-myrtenol,
linalool, α-pinene, antimicrobial activity.
Introduction
The genus Agonis (Myrtaceae) (1), closely related to Melaleuca and
Leptospermum, is limited in natural occurrence to the south west of Western
Australia. These woody shrubs or small trees are cultivated for their
ornamental shrub and cutflower value. Agonis fragrans, previously
recognised as Agonis sp. Coarse Tea Tree and as Agonis sp. Coarse Agonis, is
a newly described (2), fine-leaved, lignotuberous shrub reaching 2.4m in
height on acid peaty sand in seasonally waterlogged margins of broad valleys.
This investigation reports the essential oil composition of a further potentially
commercial essential oil from the Australian flora.
Experimental
Plant Material: Agonis fragrans voucher specimens from each
population were lodged with the Perth Herbarium in Western Australia. Six
single plant extracts and six mixed plant, single provenance distilled oils were
compared. Samples 6863 and 6864 (Voucher CJR 1332) were from
Beardmore Road west of Walpole (34°49'S, 116°32'E), 6870 and 6873
(Voucher CJR 1334) from Conspicuous Cliffs Road east of Walpole (35°01'S,
116°52'E), 6881 (Voucher CJR 1335) from Kernutt’s Road north-east of
Denmark (34°55'S, 117°24'E) and sample 6888 (Voucher CJR 1336) from
Randall Road east of Denmark (34°58'S, 117°24'E). Oil samples are from
bulk collections of either plantation trees or wild plants. Samples A and C
were derived from different provenance plantation-grown bulk harvests
located north-west of Harvey (33°02'S, 115°45'E), sample B was from
plantation grown harvest from “Buxton’s Farm”, Redmond West Road in
Redmond (34° 55'S, 117° 30'E) and from seed originating from Redmond.
Sample X was from wild trees in Beardmore Road (34°49'S, 116°32'E), Y,
from an isolated provenance of trees at the same location and Z from plants
from Randall Road east of Denmark (34°58'S, 117°24'E).
27
Oil Isolation: Oil samples were obtained by hydrodistillation of bulk
leaf with terminal stems in a Clevenger-type distillation apparatus. Micro-
ethanolic extractions of the leaf were performed as previously described (3 –
5).
Oil Composition: Optical rotations were determined on an Optical
Activity AA-5 series automatic polarimeter in ethanol. IR spectra were
determined as liquid films on a Perkin Elmer 16PC FTIR. Volatile component
identifications were performed on a Hewlett Packard 6890 series GC/MS
fitted with an HP5-MS 29.5m x 0.25mm, 0.25µm film thickness, FSOT
column with helium (36 cm/s) as carrier gas, injection port (split 1:50) at
250°C, mass selective detector (HP 5973) at 250°C (source) and 150°C (quad)
with transfer line 280°C and ion source filament voltage of 70 eV. Retention
indices were measured with respect to n-alkane standards on the HP5-MS
column. Component identifications were made on the basis of mass spectral
fragmentation, retention time comparison with authentic constituents and
mass spectral and retention matching with commercial (NIST (6), Wiley (7),
Adams (8)) and in-house libraries. Area percent concentrations of the
components were determined on one of two gas chromatographs. For column
compatibility with the GC/MS, the first choice was a Shimadzu GC-14B
chromatograph fitted with a HP5-MS 30m x 0.25mm, 0.25µm film thickness,
non-polar FSOT column. Carrier gas was hydrogen (55cm/s), injection port
temperature 200°C with a 1:50 split, flame ionization detector set at 300°C
and temperature programming from 50°C (5 min.) to 250°C at 10°C/min.
Where quantitation could be improved, components were determined on a
Hewlett Packard 6890 series GC with an Altech AT35 60m x 0.25mm,
0.25µm film thickness, mid polarity FSOT column. Carrier gas, injection port,
detector and temperature program conditions were as per the Shimadzu GC-
14B. The identity of (+)-1S,5R-myrtenol was confirmed by separation from
the oil by elution from an alumina column using 10% diethyl ether in hexane
followed by comparison of GC/MS, GCRI, FTIR and [α]D data with those of
authentic (+)-1S,5R-myrtenol.
Results and Discussion
Agonis fragrans is a source of an aromatic and medicinal commercial
essential oil with no previously recorded investigations of essential oil
chemistry. In this study leaf-extract and oil components were identified by
GC/MS by comparison with mass spectra and retention indices of authentic
28
constituents (Table I). Major oil components in the 1,8-cineole variant were
found to be α-pinene (14-28%), 1, 8-cineole (28-34%) and linalool (3-15%).
Extract composition was similar to that of the distilled oils with the only
major differences being caused by non steam-volatile constituents such as
neophytodiene or steam labile constituents such as epi-cryptomeridiol and
cryptomeridiol. In addition, the concentrations of steam labile
cyclodecadienes (eg bicyclogermacrene, hedycaryol) were significantly
higher in the extracts than in the oils.
The single-plant extracts showed one plant (6863) devoid of 1, 8-
cineole which was typical of an isolated provenance which yielded an oil with
only 0.6% 1,8-cineole. The decreased 1,8-cineole concentrations were
balanced by increased concentrations of α-pinene, linalool and (+)-(1S, 5R)-
myrtenol. The identity of the latter alcohol was confirmed following isolation
by elution from an alumina column with 10% diethyl ether in hexane. GC
retention index (8), mass spectral (8,9), infrared spectral (9) and optical
rotation (+ 44.6° [86% pure]; lit.(10) + 49.7°) data were identical to data from
authentic material. This provenance provided the best source (20.0% in the
oil, 12 % in the extract) of (+)-(1S, 5R)-myrtenol. This bicyclic
monoterpenoid alcohol is used as a flavour and fragrance material and also
elicits a response from many insects (11) (eg bark beetle aggregation
pheromones (12), vine weevil (Otiorhynchus sulcatus) EAG response (13)).
Although common in essential oils, myrtenol does not seem to occur, with the
exception of Lippia multiflora (27%) (14), in concentrations greater than
20%.
Other minor variations were evident amongst our sampling. Some
provenances (A-C) had little or no α-, β- and γ-eudesmol or their putative
precursor cryptomeridiols (6881). Another plant (6864, X) contained lower
concentrations of linalool than all other five provenances.
Although it is difficult to distinguish separate chemical varieties
amongst the individual and bulk specimens assessed in this investigation,
variation is evident. As a potentially commercial fragrance oil, the most
abundant populations are those with high 1,8-cineole, α-pinene, linalool and
low myrtenol concentrations (6870, 6873, 6881, 6888; A, B, C, Z). The less
abundant low linalool and low cineole/high myrtenol variants also have the
potential for commercial development. The latter type seems to have more
potential in non-fragrance applications and may find use as a source of
myrtenol or as an insect attractant with potential use in lure-and-kill traps for
plantation pest control and as a flavouring agent (15). The anti-microbial and
29
anti-inflammatory investigations will be the subject of a separate
communication (16).
Acknowledgements
We are grateful to Christine Carson, Jan Dunstan, Jasmine Roper, Heidi Lehman, Tom Riley, Kate Hammer and Susan Prescott, University of Western Australia, for the release of anti-microbial and anti-inflammatory findings, Rural Industries Research and Development Corporation for financially supporting this project and to Peter Grayling, Department of Conservation and Land Management, Western Australia for valuable preliminary GC analysis of 55 plants from 11 populations.
References
1. W. R. Elliot and D. L. Jones, Encyclopaedia of Australian Plants
Suitable for Cultivation. Vol 2. pp 242 – 244, Lothian Publishing Company,
Melbourne (1982).
2. J. R. Wheeler, N. G. Marchant and C. J. Robinson, Agonis fragrans
(Myrtaceae), a new species from Western Australia. Nuytsia 13(3), 567-570.
(2001).
3. I. A. Southwell, C. D. A. Maddox and M. P. Zalucki, Metabolism of 1,
8-cineole in tea tree (Melaleuca alternifolia and Melaleuca linariifolia) by
pyrgo beetle (Paropsistema tigris). J. Chem. Ecol., 21, 439 – 453. (1995).
4. I. A. Southwell, Tea Tree Constituents, Ch. 2 in Tea Tree, The Genus
Melaleuca Edits., I. A. Southwell and R. F. Lowe, Vol 9 in series Medicinal
and Aromatic Plants – Industrial Profiles Edit.,. R. Hardman, pp 29 – 62,
Harwood Academic Publishers, Amsterdam (1999).
5. G. R. Baker, R. F. Lowe and I. A. Southwell. Comparison of oil
recovered from tea tree leaf by ethanol extraction and steam distillation. J.
Agric. Food Chem., 48 4041 – 4043 (2000).
6. National Institute of Standards and Technology, NIST/EPA/NIH 98
Mass Spectral Library, Gaithersburg, MD, (1998).
7. F. W. McLafferty, Wiley Register of Mass Spectral Data, 6th Edn. J.
Wiley & Sons, New York, (1994).
8. R. P. Adams, Identification of Essential Oil Components by Gas
Chromatography / Quadrupole Mass Spectroscopy. Allured Publishing
Corporation, Carol Stream, (2001).
30
9. A. A. Swigar and R. M. Silverstein, Monoterpenes. Infrared, Mass, 1H
NMR and 13C NMR Spectra, and Kovats Indices. p 72, Aldrich, Milwaukee
(1981).
10. G. Rauchschwalbe and M. Schlosser, Selektive Synthesen mit
Organometallen IV: Gezielte Hydroxylierung von Allylstellungren. Helv.
Chim. Acta, 58, 1094-1099 (1975).
11. A. M. El-Sayed, The Pherobase: Database of Insect Pheromones and
Semiochemicals.<http://www.pherobase.com/database/compounds-detail-
myrtenol.html> viewed 17 Nov. 2004. (2004).
12. J. H. Borden, A. M. Pierce, H. D. Pierce Jr., L. J. Chong, A. J. Stock
and A. C. Oehlschlager, Semiochemicals Produced by Western Balsam Bark
Beetle, Dryocoetes confuses Swaine (Coleoptera: Scolytidae). J. Chem. Ecol.
13, 823-836 (1987).
13. R. W. H. M. van Tol and J. H. Visser, Olfactory Antennal Responses of
the Vine Beetle Otiorhynchus salcatus to plant volatiles. Entomologia
Experimentalis et Applicata, 102, 49-64 (2002).
14. C. Menut, G. Lamaty, D. K. Sohounlhoue, J. Dangou, and J. M.
Bessiere, Aromatic Plants of Tropical West Africa. III. Chemical Composition
of Leaf Essential Oil of Lippia multiflora Moldenke from Benin. J. Essent. Oil
Res., 7, 331-333 (1995).
15. G. Mosciano, M. Fasano, J. Michalski and S. Saduri, Organoleptic
Characteristics of Flavor Materials, Perf. & Flav., 16 (4), 45-47 (1991).
16. T. V. Riley, Personal communication (2005).
31
Table I : Percentage composition of the leaf essential oils of high (n=5) and low (n=1) 1,8-cineole variants of Agonis fragrans
Distilled oils Extracted oils
Component R.I. High cineole Low
cineole High cineole Low cineole
HP5 - TIC Mean Range Sample Y Mean Range
Sample 6863
α-thujene 927 0.5 0.0-1.5 0.6 0.3-1.6 α-pinene 933 22.4 13.7-28.0 21.5 15.8 11.6-18.0 38.5 sabinene 973 1.2 0.4-2.8 0.2 2.0 0.5-3.7 β-pinene 976 1.8 1.4-2.1 0.4 3.3 0.9-8.3 0.4 myrcene 990 2.2 1.6-2.7 0.3 2.0 0.9-2.9 α-phellandrene 1001 0.2 0.1-0.2 0.1 0.0-0.1 α-terpinene 1015 0.7 0.7-0.8 0.4 0.2-0.7 p-cymene 1024 2.6 2.4-2.8 2.9 0.6 0.0-1.5 limonene 1028 2.2 1.2-3.4 1.1 1.4 1.0-1.9 2.0 1,8-cineole 1031 31.6 28.3-34.2 0.6 20.0 12.4-25.7 trans-β-ocimene 1049 0.0 0.0-0.1 0.6 γ-terpinene 1060 2.1 1.9-2.4 0.4 1.4 0.8-1.7 0.5 δ-terpineol 1169 0.4 0.4-0.4 0.8 0.0 0.0-0.1 terpinen-4-ol 1179 3.5 2.9-3.9 0.5 2.3 1.8-3.1 terpinolene 1088 0.4 0.3-0.5 0.3 0.1 0.0-0.2 α-terpineol 1192 5.7 5.2-5.9 2.4 4.9 3.0-6.3 1.1 myrtenol 1198 3.8 1.7-5.5 20.0 3.6 1.8-4.5 12.1 linalool 1099 9.9 3.3-14.7 25.3 8.8 1.7-13.1 18.3 citronellol 1228 0.1 0.0-0.4 nerol 1229 1.0 0.8-1.6 1.6 0.5 0.0-1.0 geraniol 1254 1.4 0.9-1.8 1.9 1.8 1.0-2.7 trans-myrtanol 1264 0.5 0.0-0.9 1.0 methyl geranate 1323 0.4 0.0-0.8 α-copaene 1382 0.4 methyl cinnamate 1388 0.1 0.0-0.6 caryophyllene 1430 0.5 0.2-0.7 0.5 3.7 2.1-6.8 0.9 humulene 1465 0.5 0.3-0.7 1.3 1.7 0.4-3.0 2.2 bicyclogermacrene 1507 0.7 0.5-0.9 2.9 3.0 0.7-6.7 4.5 δ-cadinene 1532 0.0 0.0-0.1 0.7 cis-calamanene 1533 0.7 0.4 0.1-1.1 0.4 cadina-1,4-diene 1542 0.1 0.2 0.0-0.4 0.4 epi-globulol 1582 0.3 spathulenol 1590 0.0 0.0-0.1 0.3 0.1 0.0-0.2 globulol 1598 0.1 0.0-0.3 1.0 0.2 0.0-0.5 caryophyllene oxide 1598 0.2 0.0-0.7 viridiflorol 1607 0.1 0.0-0.2 1.4 0.1 0.0-0.3 rosifoliol 1615 0.4 elemol 1617 0.4 0.0-1.4 humulene oxide 1625 0.0 0.0-0.1 0.6 hedycaryol 1629 0.5 0.0-2.5 guaia-5-en-11-ol 1637 0.5 0.0 0.0-0.2 γ-eudesmol 1645 0.6 0.3-0.9 1.0 1.4 0.0-3.1 1.2 iso-spathulenol 1653 0.0 0.0-0.1 0.2 0.0 0.0-0.2 β-eudesmol 1667 0.4 0.0-1.5 1.4 2.8 0.0-5.5 1.4 α-eudesmol 1670 0.3 0.0-1.1 1.3 2.8 0.0-5.1 2.0 epi-cryptomeridiol 1790 0.3 0.0-0.5 cryptomeridiol 1831 2.8 0.0-6.6 0.5 neophytadiene 1837 0.5 0.2-1.2 Total Recovered 96.8 94.1 91.8 89.1
32
Appendix 5: “Antimicrobial activity of Agonis fragrans oil”
Christine F. Carson et al. 2004
33
Antimicrobial activity of Agonis fragrans oil
Christine F. Carson1, Kate A. Hammer1 & Thomas V. Riley1,2
1Microbiology (M502)
School of Biomedical & Chemical Sciences
The University of Western Australia
35 Stirling Highway
Crawley WA 6009
2Division of Microbiology & Infectious Diseases
The Western Australian Centre for Pathology & Medical Research
Nedlands WA 6009
RIRDC project number GSD-1A
34
Abbreviations
ATCC American Type Culture Collection
CFU colony forming units
MIC minimum inhibitory concentration
MIC50 minimum concentration that inhibits 50% of the isolates tested
MIC90 minimum concentration that inhibits 90% of the isolates tested
MBC minimum bactericidal concentration
MBC50 minimum concentration that kills 50% of the isolates tested
MBC90 minimum concentration that kills 90% of the isolates tested
MHB Mueller-Hinton broth
MHB-T Mueller-Hinton broth supplemented with 0.002% Tween 80
µl microlitre
ml millilitre
NCTC National Collection of Type Cultures
PBS-T phosphate buffered saline supplemented with 0.001% Tween 80
spp. species
vol/vol volume to volume
w/vol weight to volume
35
Antimicrobial activity of Agonis fragrans oil
Oils Five samples of Agonis fragrans oil were investigated for their antimicrobial activity. The oils
were selected on the basis of their composition and represented the range of oils whose
composition had previously been characterised. The oils selected were oils A, B, C, X and Z.
Bacteria
The antimicrobial activity of the oils was assessed against a range of clinical and reference
isolates of bacteria. Clinical isolates were obtained from the Division of Microbiology and
Infectious Diseases, The Western Australian Centre for Pathology & Medical Research
(PathCentre), Nedlands, Western Australia, Australia. Reference isolates were obtained from
the culture collections of Microbiology, School of Biomedical & Chemical Sciences, The
University of Western Australia, Crawley, Western Australia, Australia and PathCentre. The
reference isolates tested were: Acinetobacter baumannii NCTC 7844 ATCC 15308,
Escherichia coli NCTC 10418 ATCC 10536, Pseudomonas aeruginosa NCTC 10662 ATCC
25668, Serratia marcescens NCTC 1377 ATCC 274, Staphylococcus aureus NCTC 6571
ATCC 9144 and Staphylococcus epidermidis NCTC 7944.
Isolates were stored at -80°C in brain heart infusion broth (Oxoid Ltd., Basingstoke, United
Kingdom) supplemented with 2% glycerol. When required, isolates were retrieved by plating
out a loopful onto blood agar and incubating aerobically at 37°C for 24-36 hours.
Broth microdilution method for determining MICs and MBCs Serial two-fold dilutions of the Agonis fragrans oils were prepared in plastic, flat-bottomed,
96-well microtitre trays (Nunc, Roskilde, Denmark). Stock solutions of each Agonis fragrans
oil at twice the desired final concentration, 8% vol/vol, were prepared in Mueller-Hinton broth
(MHB) (Oxoid) supplemented with 0.002% Tween 80 (MHB-T). The highest final
concentration tested was 4% (vol/vol). Stock solutions were serially diluted across the
microtitre trays in MHB-T. The last column did not receive any Agonis fragrans oil and
contained MHB-T only. Once the serial dilutions were complete, the tray was inoculated with
test organisms.
Bacterial suspensions were prepared by inoculating one to two colonies from an overnight
culture on blood agar into 5-10 ml of MHB and incubating at 37°C for 18-24 hours. These
suspensions were diluted in saline and adjusted using a nephelometer to approximately 85%
transmission or 0.5 McFarland turbidity standards which corresponded to approximately 108
36
CFU/ml. Inocula for the microdilution assay were prepared by diluting this suspension one
hundred-fold in MHB so that upon addition to the microdilution tray the final concentration of
organisms was approximately 5 × 105 CFU/ml. Inocula concentrations were confirmed by
Miles-Misra counts; quadruplicate samples of 10 µl of 10-3 dilutions of each inoculum were
plated onto blood agar, incubated at 37°C for 18-24 hours and the resulting number of
colonies counted.
Once inoculated, trays were incubated at 37°C for 18-24 hours after which the MICs were
determined. MICs were determined visually with the aid of a reading mirror as the lowest
concentration of oil that resulted in an optically clear well indicating that growth of the test
organism had been prevented. Once the MICs had been read, 10 µl samples were taken
from each well, plated onto Mueller Hinton agar and incubated overnight. The minimum
bactericidal concentration (MBC) was determined from these sub-cultures as the lowest
concentration of oil which killed 99.9% of the inoculum.
The MIC of oil that inhibited 50% (MIC50) and 90% (MIC90) of the isolates tested was
determined for genera or species where 10 or more isolates were tested. Similarly, the
MBC50 and MBC90 were also determined.
Time-kill method
The antimicrobial activity of oils A and X was assessed using a time-kill method. For tests
with S. aureus, a 2% (vol/vol) concentration of each Agonis fragrans oil was prepared in
phosphate buffered saline supplemented with 0.001% Tween 80 (PBS-T). PBS-T was added
to control tubes. Suspensions of S. aureus NCTC 6571 were prepared by inoculating one to
two colonies from an overnight culture on blood agar into 10 ml of Mueller-Hinton broth
(MHB) (Oxoid) and incubating at 37°C for 16-20 hours. The concentration of organisms in
this culture was determined by serially 10-fold diluting a 0.1 ml sample in 0.85% (w/vol)
saline and plating duplicate samples onto blood agar. The plates were incubated at 37°C for
24-48 hours after which the number of colonies was counted and the original concentration
of organisms calculated. This concentration was used to calculate the starting concentration
for each test and control suspension.
At time zero, a volume of 0.1 ml of the bacterial culture was added to the Agonis fragrans oil
and control tubes so that the final volume in each was 10 ml. The tubes were mixed
vigorously for approximately 20 seconds after which a 0.1 ml sample was taken at 30
seconds and serially diluted ten-fold in 0.85% (w/vol) saline. Duplicate samples of 100 µl
were spread plated onto blood agar and incubated at 37°C for 24-48 hours after which the
number of colonies was counted and the number of surviving organisms determined.
37
Samples were taken at 30, 60 and 120 minutes, diluted and plated in the same manner.
There are no validated inactivating agents for essential oils. Dilution was used to arrest
treatment and reduce carryover. The minimum dilution used was 1 in 10. Experiments were
conducted twice and the mean concentration of organisms ± standard deviation for each time
point calculated.
This method was also used to evaluate the cidal activity of oils A and X against Escherichia
coli NCTC 10418. The concentrations of Agonis fragrans oil tested were 1%, 0.25% and
0.125% (vol/vol). Experiments were conducted once.
Results
The MICs and MBCs observed for the five selected Agonis fragrans oils ranged from 0.12-
4% with the exception of those for P. aeruginosa which exceeded 4%, the maximum
concentration tested in this work (Table 1). There was no significant variation in the MIC or
MBC results between the oils. The MIC90 for S. aureus was 0.5-2% depending on which oil
was tested, oil X having the lowest of 0.5%.
Results from the time-kill tests against S. aureus (Figure 1) show that there was no
significant difference in the cidal activity of oils A and X against this organism. Both oils
resulted in an approximately 4 log10 reduction in organism numbers over the two hour assay.
This is despite the modest difference in the MIC90s for these oils which are 2% and 0.5%,
respectively.
Against E. coli, both oils exhibited considerable cidal activity with 1% Agonis fragrans oil A or
X resulting in no recoverable organisms by 30 seconds treatment (data not shown). The
lower treatment concentrations of 0.25% and 0.125% also resulted in dramatic reductions in
viability with 0.25% of either oil effecting a 1 log10 kill within 30 seconds and resulting in no
organisms being recovered beyond the 30 second sample (Figure 3).
38
Table 1. Antimicrobial susceptibility data for selected Agonis fragrans oils
Susceptibility data
Organism (n) MIC MBC
Oil Range MIC50 MIC90 Range MBC50 MBC90
Acinetobacter spp. (6) A 0.12 nd nd 0.12 nd nd
Klebsiella spp. (11) 0.5 - 1 0.5 1 0.5 - 1 0.5 1
P. aeruginosa (11) >4 >4 >4 >4 >4 >4
S. marcescens (6) 0.25 - 0.5 nd nd 0.25 - 1 nd nd
S. aureus (10) 1 - 2 1 2 2 – 4 2 4
S. epidermidis (10) 1 - 2 2 2 2 - >4 4 >4
Acinetobacter spp. (6) B 0.12 nd nd 0.12 nd nd
Klebsiella spp. (11) 0.5 - 2 0.5 2 0.5 - 2 1 2
P. aeruginosa (11) >4 >4 >4 >4 >4 >4
S. marcescens (6) 0.25 - 0.5 nd nd 0.25 - 0.5 nd nd
S. aureus (10) 1 1 1 1 - 4 2 4
S. epidermidis (10) 1 - 2 2 2 2 - >4 4 >4
Acinetobacter spp. (6) C 0.12 nd nd 0.12 - 0.25 nd nd
Klebsiella spp. (11) 0.25 - 2 0.5 1 0.5 - 2 0.5 2
P. aeruginosa (11) >4 >4 >4 >4 >4 >4
S. marcescens (6) 0.25 - 1 nd nd 0.25 - 1 nd nd
S. aureus (10) 0.5 - 1 1 1 1 - 4 2 4
S. epidermidis (10) 1 - 2 2 2 2 - >4 4 >4
Table 1. (cont.) Antimicrobial susceptibility data for selected Agonis fragrans oils
39
Susceptibility data
Organism (n) MIC MBC
Oil Range MIC50 MIC90 Range MBC50 MBC90
Acinetobacter spp. (6) X 0.12 - 0.25 nd nd 0.12 - 0.25 nd nd
Klebsiella spp. (11) 0.5 - 2 0.5 1 0.5 - 2 1 2
P. aeruginosa (11) 4 - >4 >4 >4 >4 >4 >4
S. marcescens (6) 0.5 - 1 nd nd 0.5 - 1 nd nd
S. aureus (10) 0.5 - 1 0.5 0.5 0.5 - 2 2 2
S. epidermidis (10) 0.5 0.5 0.5 1 - 2 2 2
Acinetobacter spp. (6) Z 0.06 - 0.12 nd nd 0.12 - 0.12 nd nd
Klebsiella spp. (11) 0.25 - 1 0.5 1 0.25 – 2 0.5 1
P. aeruginosa (11) 0.25 - >4 >4 >4 4 - >4 >4 >4
S. marcescens (6) 0.25 - 0.5 nd nd 0.25 - 0.5 nd nd
S. aureus (10) 0.5 - 1 1 1 1 – 2 2 2
S. epidermidis (10) 0.5 - 1 1 1 1 – 4 2 4
40
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
1.00E+09
0 15 30 45 60 75 90 105 120
Time (min)
CFU
/ml
Agonis oil A 2%Agonis oil X 2%Control
Figure 1. Time-kill curves of S. aureus NCTC 6571 in control suspensions (▲) and after treatment with Agonis fragrans oil A (♦) or Agonis fragrans oil X ( ) at 2% (vol/vol), the MIC for this organism. The organisms were suspended in PBS-T and incubated at 37°C. Each symbol indicates the mean ± standard deviation for two replicates. The lower detection threshold was 103 CFU/ml. The MICs of Agonis fragrans oil A and X against this organism are 2% and 0.5%, respectively.
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
1.00E+11
0 30 60 90 120
Time (min)
CFU
/ml
TTO 0.5%TTO 0.25% (MIC)TTO 0.125%Control
Figure 2. Time-kill curves of S. aureus NCTC 6571 in control suspensions (▲) and after treatment with 0.5% tea tree oil (♦), 0.25% tea tree oil (MIC) ( ) or 0.125% tea tree oil (●) (all vol/vol). The organisms were suspended in PBS-T at room temperature (22°C). Each symbol indicates the mean ± standard deviation for at least three replicates. The lower detection threshold was 103 CFU/ml. Adapted from Carson et al., 2002 Antimicrobial Agents & Chemotherapy 46:1914-1920.
109
108
107
106
105
104
103
102
101
1011
1010
109
108
107
106
105
104
103
102
101
41
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
0 15 30 45 60 75 90 105 120
Time (min)
CFU
/ml
Agonis oil A 0.25%Agonis oil X 0.25%Agonis oil A 0.125%Agonis oil X 0.125%Control
Figure 3. Time-kill curves of E. coli NCTC 10418 in control suspensions (▲) and after treatment with Agonis fragrans oil A at 0.25% (♦) or 0.125% (◊) or Agonis fragrans oil X at 0.25% ( ) or 0.125% ( ) (all vol/vol). The organisms were suspended in PBS-T and incubated at 37°C. The lower detection threshold was 103 CFU/ml.
108
107
106
105
104
103
102
101
42
Discussion The MICs and MBCs observed for the five Agonis fragrans oils in this work are similar to
those reported for other essential oils considered to have antimicrobial activity including
Melaleuca alternifolia (tea tree) oil, Cymbopogon citratus (lemongrass) oil and Origanum
vulgare (oregano) oil (Hammer, Carson & Riley, 1996; Sivropoulou et al., 1996; Hammer,
Carson & Riley, 1999). As with many other essential oils and conventional antimicrobial
agents, Pseudomonas aeruginosa was the least susceptible organism tested with 4%, the
maximum test concentration, failing to inhibit or kill most isolates.
Despite suggestions from the MIC broth dilution data that oils A and X may differ in their
degree of antimicrobial activity against S. aureus, the time-kill experiments indicated that
their cidal activity against this organism did not differ. Both oils at a concentration of 2%
reduced the number of viable organisms by approximately 4 log10 at a similar rate over two
hours. Since 2% was the MIC of Agonis fragrans oil A for S. aureus and 0.5% was the MIC of
Agonis fragrans oil X, the indifference between the two time-kill profiles suggests that once a
threshold of maximum killing is reached, increasing the concentration of oil may not increase
the level of activity. More work would be required to clarify this observation. Interestingly,
compared to treatment with the MIC of tea tree oil (see Figure 2), treatment with the MIC of
Agonis fragrans oil A reduced the viability of S. aureus to a greater extent, although the
experimental conditions were not identical. For E. coli, data from the time-kill experiments
indicated that both Agonis fragrans oils A and X at 0.25% were able to reduce the number of
viable bacteria by a factor of 10 after just 30 seconds of treatment. That no organisms could
be recovered from the 30 minute sample shows that at least an additional 3 log10 kill had
occurred by 30 minutes. While 0.125% Agonis fragrans oil had less effect at 30 seconds than
0.25% oil, by 30 minutes the effect was similar with no organisms recoverable.
Collectively, these data suggest that Agonis fragrans oil may have clinical potential as an
antimicrobial agent. However, 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. Much work would be
required before Agonis fragrans oil could be evaluated in vivo, the definitive way in which to
determine its clinical potential.
43
References Hammer, K. A., Carson, C. F. & Riley, T. V. (1996) Susceptibility of transient and commensal
skin flora to the essential oil of Melaleuca alternifolia (tea tree oil). American Journal of
Infection Control 24:186-189.
Hammer, K. A., Carson, C. F. & Riley, T. V. (1999) Antimicrobial activity of essential oils and
other plant extracts. Journal of Applied Microbiology 86:985-990.
Sivropoulou, A., Papanikolaou, E., Nikolaou, C., Kokkini, S., Lanaras, T. & Arsenakis, M.
(1996) Antimicrobial and cytotoxic activities of Origanum essential oils. Journal of
Agricultural & Food Chemistry 44:1202-1205.
44
Appendix 6: “Regulatory Overview Agonis Oil” Found & Associates 2005
45
GREAT SOUTHERN REGIONAL DEVELOPMENT CORP Regulatory Overview
AGONIS OIL
John Found and Associates
46
TABLE OF CONTENTS
P A R T 1 A
Summary ..................................................................................................................................................
P A R T 1 B
Sample Specification ...............................................................................................................................
P A R T 1 C
Therapeutic Indications ..........................................................................................................................
P A R T 1 D
Regulatory Process ..................................................................................................................................
P A R T 1 E
Process Costs............................................................................................................................................
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1a. Summary The following is a summary of the regulatory requirements for new chemical entities
All chemical substances are regulated by various authorities and each country has its own regulatory system. New chemical entities are treated with a great deal of caution and their use must substantiated by a considerable amount of scientific data. New substances are subjected to higher levels of scrutiny than ever before in an attempt to minimise harmful effects to humans, animals or the environment. The preliminary steps to obtaining official approval are largely concerned with the adequate scientific identification of the substance. The Agonis oil will require detailed analysis using gas chromatography/mass spectrometry to identify its components. Once the chemical make up of the product is established other quality data will be required to sufficiently characterise the product. After the chemical and physical properties are documented consideration should be given to the establishment of relative toxicity data. These tests usually include such parameters as acute oral toxicity (LD50) , acute dermal toxicity and skin irritancy. Regulatory Authorities Applications would be required to be made to the following; National Industrial Chemicals Notification and Assessment Scheme (NICNAS) Therapeutic Goods Administration (TGA) Cosmetic Toiletry and Fragrance Association (CTFA) Chemical Abstracts Society (CAS) Once the chemical and physical data is complete applications may be made for adoption onto the various registers. Since some approvals require a pre-existing registration status it is important that the project is structured so that critical information is available at the appropriate time. Overseas Regulatory Authorities Notification schemes for new chemical substances, manufactured or imported within EU industries, were first introduced during the nineteen-seventies by individual Member States. Notification allowed a priority assessment of potential occupational/consumer risks and environmental impact. A harmonised pan-European notification system was introduced for new substances as part of the 6th Amendment to Directive 67/548/EEC, concerned with the classification, packaging, and labelling of dangerous substances. The 6th Amendment (Directive 79/831/EEC) was adopted in September 1979 and came into force in all Member States on 18th September 1981. A 7th Amendment to Directive 67/548/EEC (Directive 92/32/EEC) was adopted in April 1992 with effect from November 1993 and introduced a risk assessment for new notified substances. Over 6000 notifications in total, representing more than 3700 substances, have been submitted since 1981. Inherent of legislation are principles for notification, including criteria for exemption. A fundamental definition makes distinction between new and existing chemicals which are delimited by the enforcement date of Directive 79/831/EEC. Existing chemicals are listed in European Inventory of Existing commercial Chemical Substances (EINECS) published in Official Journal of the European Communities on 15th June 1990. EINECS contains 100 196 entries, comprising substances introduced between 1st January 1971 and 18th September 1981. New chemicals, introduced subsequently, form a cumulative index, European LIst of Notified Chemical Substances (ELINCS) periodically updated as an Official Journal. Exemption categories include consumer products pertaining to pharmaceutics, cosmetics and foodstuffs. The Directive is not applicable to pesticides, radioactive materials, wastes, and substances used in scientific research. Beside details on the notifier/manufacturer and the identity of the chemical (IUPAC name, CAS
48
number etc.), a technical dossier for new substance notification should provide information on the substance as such (e.g. production process, proposed uses) and results from analysis of physical and chemical properties, and test reports from toxicological and ecotoxicological assays. Proposals for Classification and Labelling should be submitted, including recommended precautions relating to safety. Risk Assessment should be drafted. Requisite dossier detail increases according to substance quantity, viz: 10kg, 100kg, 1000kg, per year per manufacturer (7th Amendment Annexes VIIC, VIIB, VIIA ("base-set"), respectively) with further toxicological and ecotoxicological testing required at amounts exceeding 100 and 1000 tonnes per year per manufacturer (Annex VIII). Industry has to submit the notification dossier to the national Competent Authority (Notification-Units). Dossiers are evaluated by Competent Authority and forwarded to ECB Work Area New Substances in standard format using software incorporating Data Entry Screens (DES) and Structured Notification Interchange Format (SNIF). The programs enable harmonised data assembly and diskette exchange of summary information on new notified substances. Aspects of dossier information are confidential, particularly chemical spectra and structures, which are not transmitted or archived in electronic form. Dossier handling is managed through New Chemicals Database (NCD) maintained in a security area at ECB with authorised access only. The Work Area maintains quality assurance of SNIF/NCD content and co-ordinates diskette ddistribution among EU Competent Authorities and Norway. In general, there is the principle of mutual acceptance between the Competent Authorities of the Member States. However, everybody participating in the system may comment in a written procedure. Issues related to implementation of Directives are discussed in meetings organised and chaired by the ECB (Technical and Scientific Meetings, Working Group Meetings) or DG Environment (Competent Authority Meetings). The decisions taken at these meetings are collected in a "Manual of Decisions", which provides guidance for industry and other bodies involved in the notification of new substances. Essentially, Agonis oil will be exempted from European registration because
a) its use will be largely pharmaceutical and cosmetic b) production volumes will be less than the threshold for the type of product.
49
1b1. Specification The following is a sample of a typical raw material specification that would be required by TGA as part of its review process
Definition XYZ oil is obtained by steam distillation and rectification from the fresh leaves or the fresh terminal branchlets of various species of XYZ rich in 1,8-cineole. The species mainly used are XYZ (1) Labill., XYZ (2) F. von Mueller and XYZ (3) R.T. Baker. Characters A colourless or pale-yellow liquid with an aromatic and camphoraceous odour and a pungent and camphoraceous taste. Identification First identification Second identification A. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R. Test solution Dissolve 0.1 g of the substance to be examined in toluene R and dilute to 10 ml with the same solvent. Reference solution Dissolve 50 ml of cineole R in toluene R and dilute to 5 ml with the same solvent. Apply to the plate as bands 10 ml of each solution. Develop over a path of 15 cm using a mixture of 10 volumes of ethyl acetate R and 90 volumes of toluene R. Allow the plate to dry in air and spray with anisaldehyde solution R and examine in daylight while heating at 100°C to 105°C for 5 min to 10 min. The chromatogram obtained with the reference solution shows in the middle a zone due to cineole. The chromatogram obtained with the test solution shows a main zone similar in position and colour to the zone due to cineole in the chromatogram obtained with the reference solution. It also shows an intense violet zone (hydrocarbons) near the solvent front. Other weaker zones may be present. B. Examine the chromatograms obtained in the test for chromatographic profile. The chromatogram obtained with the test solution shows five peaks similar in retention time to the five peaks in the chromatogram obtained with the reference solution. Tests Relative density (2.2.5) 0.906 to 0.925. Refractive index (2.2.6) 1.458 to 1.470. Optical rotation (2.2.7)
50
The angle of optical rotation is 0° to +10°. Solubility in alcohol (2.8.10) It is soluble in 5 volumes of alcohol (70 per cent V/V) R. Aldehydes Place 10 ml in a glass-stoppered tube 25 mm in diameter and 150 mm long and add 5 ml of toluene R and 4 ml of alcoholic hydroxylamine solution R. Shake vigorously and titrate immediately with 0.5M potassium hydroxide in alcohol (60 per cent V/V) until the red colour changes to yellow. Continue the titration with shaking; the end-point is reached when the pure yellow colour of the indicator is permanent in the lower layer after shaking vigorously for 2 min and allowing separation to take place. The reaction is complete in about 15 min. Repeat the titration using a further 10 ml of the substance to be examined and, as a reference solution for the end-point, the titrated liquid from the first determination to which has been added 0.5 ml of 0.5M potassium hydroxide in alcohol (60 per cent V/V). Not more than 2.0 ml of 0.5M potassium hydroxide in alcohol (60 per cent V/V) is required in the second titration. Chromatographic profile Examine by gas chromatography (2.2.28). Test solution The substance to be examined. Reference solution Dissolve 80 ml of a-pinene R, 10 ml of b-pinene R, 10 ml of a-phellandrene R, 10 ml of limonene R, 0.8 ml of cineole R and 10 mg of camphor R in 10 ml of acetone R. The chromatographic procedure may be carried out using: a fused-silica column 60 m long and about 0.25 mm in internal diameter coated with macrogol 20 000 R as the bonded phase, helium for chromatography R as the carrier gas at a flow rate of 1.5 ml/min, a flame-ionisation detector, a split ratio of 1:100, maintaining the temperature of the column at 60°C for 5 min, then raising the temperature at a rate of 5°C per minute to 200°C and maintaining at 200°C for 5 min; maintaining the temperature of the injection port and that of the detector at 220°C. Inject about 0.5 ml of the reference solution. When the chromatogram is recorded in the prescribed conditions the components elute in the order indicated in the composition of the reference solution. Record the retention times of these substances. The assay is not valid unless: the number of theoretical plates calculated for the peak due to limonene at 110°C is at least 30 000, the resolution between the peaks corresponding to limonene and cineole is at least 1.5.
51
Inject about 0.5 ml of the test solution. Using the retention times determined from the chromatogram obtained with the reference solution, locate the components of the reference solution on the chromatogram obtained with the test solution. Determine the percentage content of each of these components by the normalisation procedure. The percentages are within the following ranges: a-pinene: 2 to 8 per cent, b-pinene: less than 0.5 per cent, a-phellandrene: less than 1.5 per cent, limonene: 4 to 12 per cent, 1,8-cineole: not less than 70 per cent, camphor: less than 0.1 per cent. Storage Store in a well-filled, airtight container , protected from heat. The following chromatogram is published for information. Ph Eur
52
1c. Therapeutic Indications The therapeutic indications for a particular essential oil must be based upon the characteristics of its component parts. Agonis Oil will probably have similar characteristics to the eucalypt and melaleuca species. These oils have been found to have good antiseptic and counter inflammatory as well as anti microbial properties. In order to substantiate anti microbial claims the product should be subjected to a challenge test. The test trials varying concentrations of the oil against live, standardised, bacteria of different species. This type of trial may be conducted by the University of WA and also the Princess Margaret Hospital for Children. Anti-inflammatory trials can be performed by the University of Queensland and are usually conducted on live mice or rats. It is quite likely that Agonis Oil could be important as a general purpose antiseptic, acne treatment, disinfectant or skin wash. Activity against staphylococcus organisms may prove useful against methicillin resistant staph aureus (MRSA) which has become a life threatening problem for hospital workers. The following is a brief history of therapeutic use for Eucalyptus Oil, the indications for Agonis oil will be very similar. Eucalyptus Oil Eucalyptus oil is a colourless or pale yellow liquid with a characteristic aromatic camphoraceous odour and a pungent camphoraceous cooling taste. It is obtained by rectifying the oil distilled from the fresh leaves or terminal branches of various species of Eucalyptus (E. globulus, E. fructicetorum, and E. smithii are used). It contains not less than 70% w/w of eucalyptol (note - cineole is the AAN) and is soluble 1 in 5 with 70% alcohol.
History and Patterns of Previous Use
Eucalyptus oil is used as an antiseptic, antispasmodic stimulant agent in bronchitis, asthma, and minor respiratory complaints. A 1% ointment has been used in rhinitis and a 25% liniment as a rubefacient. The vapours are often inhaled in asthma, pharyngitis, and related conditions. In aromatherapy the oil is used externally to soothe coughing, promote scar formation in burns and injuries, and as an antirheumatic. Internally it is recommended by aromatherapists for asthma, diabetes, measles, and rheumatism. For external use the Commission E recommended concentrations are: 5-20 percent in oil and semisolid preparations; 5-10 percent in aqueous-alcoholic preparations (Blumenthal et al. 1998).
Adverse Reactions
There is a total of 7 adverse reactions reported on the Adverse Drug Reactions System for products containing eucalyptus oil - note that most products contain multiple ingredients. In four of these reports, prescription medicines were taken concurrently. Most adverse reactions cannot be attributed to eucalyptus oil with any certainty because of the multi-ingredient nature of the preparations and concurrent prescription drug use.
Eucalyptus oil and cineole are generally non-irritating, non-sensitising, and non-phototoxic both in animal tests and in patch tests with human volunteers.
Biological Activity
Eucalyptus oil is categorised as an antipruritic (topical). The German Commission E Monograph lists the actions of eucalyptus oil as: secretomotory, expectorant, mildly antispasmodic, and mild local hyperemic. Recent studies have demonstrated eucalyptus oil and cineole to be generally non-irritating, non-sensitising, and non-phototoxic to the skin.
Toxicology
53
Studies in rats showed that cineole, the main constituent of eucalyptus oil, probably cannot cross the blood-milk barrier in amounts sufficient to affect hepatic microsomal enzymes in the offspring. Eucalyptus oil was not teratogenic when administered subcutaneously to pregnant mice. An in vitro assay indicated that eucalyptus tincture was not mutagenic.
Evaluation
There are more than 500 species of Eucalyptus belonging to the family Myrtaceae. The major species of commercial interest are E. globulus Labill., E. fruticetorum F. von Muell. (E. polybractea), and E. smithii R.T. Baker which are used for the distillation of essential oil (eucalyptus oil) (Corrigan 1992).
Eucalyptus oil is a colourless or pale yellow liquid with a characteristic aromatic camphoraceous odour and a pungent, camphoraceous, cooling taste. It is obtained by rectifying the oil distilled from the fresh leaves or terminal branches of various species of Eucalyptus (E. globulus, E. fructicetorum, and E. smithii are used). It contains not less than 70% w/w of eucalyptol (note - cineole is the AAN) and is soluble 1 in 5 with 70% alcohol (Ph Eur 2002).
The main constituents of interest for Eucalyptus are the essential oil and the tannins. The essential oil may be distilled from the fresh or dried leaves. Eucalyptus oils for medicinal purposes are usually distilled from E. globulus giving 1.5-3.5% of oil, of which 70-85% is cineole (Corrigan 1992). Other constituents present are mostly monoterpene hydrocarbons (a-pinene, d-limonene, p-cymene, /3-pinene, a-phellandrene, camphene, y-terpinene, etc., with the first three in major amounts), with lesser amounts of sesquiterpenes (e.g., aromadendrene, allo-aromadendrene, globulol, epiglobulol, ledol, and viridiflorol), aldehydes (e.g., myrtenal), ketones (e.g., carvone and pinocarvone), and others (Leung and Foster 1996). History and Patterns of Previous Human Use Leaves and oil are reported to be used as antiseptic and febrifuge, and as expectorant and stimulant in respiratory ailments; also used for wounds, burns, ulcers, and cancers. In Chinese medicine, leaves and oil are used for similar purposes. In addition, aqueous extracts and decoctions of the leaves are used to treat aching joints, bacterial dysentery, ringworms, pulmonary tuberculosis, and others; successful clinical studies on some of these uses have been reported. The oil is used as a fragrance ingredient in topical balms and massage oils (Leung and Foster 1996). The world market for eucalyptus oil is 2000-3000 tonnes a year (Abbott 1989). Eucalyptus oil and its major component cineole are used as flavour ingredients in many food products, including alcoholic and nonalcoholic beverages, frozen dairy desserts, candy, baked goods, gelatins and puddings, meat and meat products, and others. Average maximum use levels reported are generally low, with the highest being about 0.002% (19.5 ppm) for cineole in candy (Leung and Foster 1996). Both eucalyptus oil and cineole are extensively used as expectorants and/or flavouring agents in cold and cough medicines (e.g., cough drops and syrups), vapouriser fluids, antiseptic liniments, ointments, toothpastes, and mouthwashes. They are also widely used as fragrance components in soaps, detergents, creams, lotions, and perfumes, with maximum use levels of 1.0 and 1.6% in perfumes reported for eucalyptus oil and cineole, respectively. Eucalyptus oil and cineole are used in dentistry as components of certain root canal sealers and also used as solvents for root canal fillings (Leung and Foster 1996). The oil is used as an antiseptic, antispasmodic stimulant agent in bronchitis, asthma, and minor respiratory complaints. A 1% ointment has been used in rhinitis and a 25% liniment as a rubefacient. The vapours are often inhaled in asthma, pharyngitis, and related conditions. In aromatherapy the oil is used externally to soothe coughing, promote scar formation in burns and injuries, and as an antirheumatic. Internally it is recommended by aromatherapists for asthma, diabetes, measles, and rheumatism (Corrigan 1992; Blumenthal et al. 1998). Eucalyptus oil and eucalyptol (cineole) were granted GRAS (generally recognised as safe) status in the
54
USA in 1965 and both are approved for food use by the US Food and Drug Administration. The Council of Europe permits the use of the oil as a spice or seasoning. Eucalyptol is included in the Council list of admissible artificial flavouring substances at a level of 15 ppm. In France, an infusion of F.. globulus leaf is accepted for product registration purposes as being 'traditionally used during benign acute bronchial disorders' and as such no toxicological study is required. However, when the powdered leaf is supplied, a reduced toxicological study is required (Corrigan 1992). For external use the Commission E recommended concentrations are: 5-20 percent in oil and semisolid preparations; 5-10 percent in aqueous-alcoholic preparations (Blumenthal et al. 1998). Allen et al. (2000) indicate a recommended topical dosage of eucalyptus oil for adults and for children aged two years and older of 0.5-3% concentration applied to the affected area not more than three or four times a day. Biological Activity The oil does have slight antibacterial action as measured by the Rideal-Walker test (E. globulus 3.0, E. australiana 5.8, and E. cinearifolia 4.8). The oil from E. viminalis reportedly has activity against influenza viruses A2 and A in chickens (Corrigan 1992). Kovar et al. (1987) have reported blood levels of the major constituent of eucalyptus oil, cineole after inhalation and oral administration of the essential oil of rosemary (Rosmarinus officinalis). After oral dosing the blood level reached a peak after 5 min. The elimination of the cineole from the blood after inhalation was biphasic with a short half-life of 6 min during the first 10 min and a half-life of about 45 min during a second phase, reflecting possible different elimination rates from a central and from a peripheral compartment. In relation to the use of eucalyptus oil in aromatherapy, Meyer and Meyer (1959) found that the oil was rapidly absorbed across the intact shaved skin of mice. Eucalyptus oil and cineole reportedly have antiseptic (antibacterial) and expectorant properties; strongly antibacterial against several strains of Streptococcus. Eucalyptus oil has been reported to promote the formation of tumours (papillomas) by 9,10-dimethyl-l,2-benzanthracene (Meyer and Meyer 1959). More recent studies have demonstrated eucalyptus oil and cineole to be generally nonirritating, non-sensitising, and non-phototoxic to the skin (Leung and Foster 1996). Eucalyptus oil is categorised as an antipruritic (topical) in Merck (1996). The German Commission E Monograph lists the actions of eucalyptus oil as: secretomotory, expectorant, mildly antispasmodic, and mild local hyperemic (Blumenthal et al. 1998). The antiviral effect of Australian tea tree and eucalyptus oil against herpes simplex virus was examined by Schnitzler et al. (2001). Cytotoxicity of tea tree oil and eucalyptus oil was evaluated in a standard neutral red dye uptake assay. Toxicity of tea tree oil and eucalyptus oil was moderate for RC-37 cells and approached 50% (TCSO) at concentrations of 0.006% and 0.03%, respectively. Antiviral activity of tea tree oil and eucalyptus oil against herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) was tested in vitro on RC-37 cells using a plaque reduction assay. The 50% inhibitory concentration (ICSO) of tea tree oil for herpes simplex virus plaque formation was 0.0009% and 0.0008% and the ICSO of eucalyptus oil was determined at 0.009% and 0.008% for HSV-1 and HSV-2, respectively. Australian tea tree oil exhibited high levels of virucidal activity against HSV-1 and HSV-2 in viral suspension tests. At noncytotoxic concentrations of tea tree oil plaque formation was reduced by 98.2% and 93.0% for HSV-1 and HSV-2, respectively. Noncytotoxic concentrations of eucalyptus oil reduced virus titers by 57.9% for HSV-1 and 75.4% for HSV-2. Virus titers were reduced significantly with tea tree oil, whereas eucalyptus oil exhibited distinct but less antiviral activity. In order to determine the mode of antiviral action of both essential oils, either cells were pretreated before viral infection or viruses were incubated with tea tree oil or
55
eucalyptus oil before infection, during adsorption or after penetration into the host cells. Plaque formation was clearly reduced, when herpes simplex virus was pretreated with the essential oils prior to adsorption. These results indicate that tea tree oil and eucalyptus oil affect the virus before or during adsorption, but not after penetration into the host cell. Thus tea tree oil and eucalyptus oil are capable to exert a direct antiviral effect on HSV. Schnitzler et al. (2001) comment that although the active antiherpes components of Australian tea tree and eucalyptus oil are not yet known, their possible application as antiviral agents in recurrent herpes infection is promising. Pathogenesis and symptoms of inflammatory processes are accompanied and/or initiated by the production of reactive oxygen species (Grassman et al. 2000). The effects of essential oils on these processes have been studied with the aid of biochemical model reactions simulating these pathological events. It can be shown that eucalyptus oil ameliorates inflammatory processes by interacting with aggressive oxygen radicals of the OH-type and interfere with leukocyte activation. These activities partially allow attenuation of oxidative attack and damage introduced by infections or environmental impacts. The effects of peppermint oil and eucalyptus oil preparations on neurophysiological, psychological and experimental algesimetric parameters were investigated in 32 healthy subjects in a double-blind, placebo-controlled, randomised cross-over design (Gobel et al. 1994). Four different test preparations were applied to large areas of the forehead and temples using a small sponge and their effect was evaluated by comparing baseline and treatment measure. The combination of peppermint oil, eucalyptus oil and ethanol increased cognitive performance and had a muscle-relaxing and mentally relaxing effect, but had little influence on pain sensitivity. Toxicology Acute oral toxicity values for eucalyptus oil indicate that it has fairly low oral toxicity (Table 3). However, the lowest published lethal dose (LDLo) and lowest published toxic doses (TDLo) for man and child indicate that eucalyptus oil can be quite toxic.
Table 3. Results of acute toxicity assays for eucalyptus oil (from RTECS 2002). Species Test - Exposure Route Dose/Duration Toxic effects Mouse LD50 - Oral 2738 mg/kg Only LD50 reported Rabbit LD50 . 2480 mg/kg Only LD50 reported Rat LD50 - Oral 2480 mg/kg Only LD50 reported Mouse LD50 - Intraperitoneal 562 mg/kg Only LD50 reported Rabbit Draize - Topical 500 mg/24H Moderately severe Man LDLo - Oral 375 mg/kg Somnolence, nausea, Child TDLo - Oral 218 mg/kg Ciliary spasm,
somnolence,
When taken internally, eucalyptus oil is toxic, and ingestion of as little as 3.5 mL has been reported as fatal (Leung and Foster 1996). Fatalities have occurred with 4 to 480 milliliters (Gurr and Scroggie 1965; MacPherson 1925). Death has occurred after ingestions ranging from 4 to 24 milliliters of the essential oil; however, recoveries have also been reported after ingesting the same amounts (Duke et al. 2002). In two series of children (109 and 14) with eucalyptus oil ingestion, ingestion of 2 to 3 milliliters of 100% eucalyptus oil was associated with minor CNS depression. Ingestion of 5 milliliters or more was associated with significant CNS depression or coma (Tibballs 1995; Spoerke et al. 1989).
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The symptoms of poisoning with eucalyptus oil include gastrointestinal symptoms such as epigastric burning, nausea and vomiting, and CNS depression, including coma. Cyanosis, ataxia, miosis, pulmonary damage, delirium, and convulsions may occur. Deaths have been reported (Martindale
Other Toxicology
A tincture of the leaves was not mutagenic in the Salmonella/microsome assay using S. typhimurium strains TA 100 and TA98 (Schimmer 1994). Eucalyptus oil was not teratogenic when administered subcutaneously to pregnant mice (135 mg/kg bwt) daily on days 6-15 of gestation (Pages 1990). Studies in rats showed that cineole probably cannot cross the bloodmilk barrier in amounts sufficient to affect hepatic microsomal enzymes in the offspring, but it is able to penetrate the placental tissue and to reach a concentration in foetal blood adequate for stimulating hepatic enzyme activity after a dose of 500 mg/kg given subcutaneously (Jori and Briatico 1973).
Adverse reactions
Opdyke (1975) concludes that eucalyptus oil and cineole are generally non-irritating, nonsensitising, and non-phototoxic both in animal tests and in patch tests with human volunteers. However, Mitchell and Rook (1979) include several reports of urticaria (from handling foliage), dermatitis, and skin irritation relating to a number of Eucalyptus species. Rudzki et al. (1976) report that eucalyptus oil can cause contact allergy.
Eucalyptus oil is mildly irritating to skin (MacPherson 1925; Adams 1969). Redness, irritation, and a burning sensation was reported in a 4-year-old placed in a bath containing an unknown concentration of eucalyptus oil. The irritation resolved within one hour (Spoerke et al. 1989).
The symptoms of oral poisoning with eucalyptus oil include gastrointestinal symptoms such as epigastric burning, nausea and vomiting, and CNS depression, including coma. Cyanosis, ataxia, miosis, pulmonary damage, delirium, and convulsions may occur. Deaths have been reported. Oily solutions of eucalyptus oil are unsuitable for use in nasal sprays as the vehicle inhibits ciliary movements and may cause lipoid pneumonia (Martindale 2003; Leung and Foster 1996).
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REFERENCES
Abbott PS (1989) Commercial eucalyptus oil production. Proceedings of the Eucalyptus Oil Production Seminar held at Gnowangerup 14 February 1989. Department of Agriculture, Western Australia, www.agric.wa.gov.au.
Adams RM (1969) Treatment dermatitis. I. Secondary contact dermatitis. Postgrad Med 45: 95-8.
Blumenthal M, Busse WR, Goldberg A, Gruenwald J, Hall T, Riggins CW, Rister RS (eds) (1998) The Complete German Commission E Monographs. Therapeutic Guide to Herbal Medicines. American Botanical Council, Austin, TX. Corrigan D (1992) Eucalyptus species. In PAGM De Smet, K Keller, R Hansel, RF Chandler (eds) Adverse Effects of Herbal Drugs 1. Springer-Verlag, Berlin. Duke JA, Bogenschutz-Godwin MJ, duCellier J (eds) (2002) Eucalyptus globulus. Handbook of Medicinal Herbs, 2nd edition. CRC Press, Boca Raton, FL. Gobel H, Schmidt G, Soyka D (1994) Effect of peppermint and eucalyptus oil preparations on neurophysiological and experimental algesimetric headache parameters. Cephalalgia 14(3): 228-34. Grassmann J, Hippeli S, Dornisch K, Rohnert U, Beuscher N, Elstner EF (2000) Antioxidant properties of essential oils. Possible explanations for their anti-inflammatory effects. Arzneimittel-Forschung 50(2): 135-9. Gurr RW, Scroggie JG (1965) Eucalyptus oil poisoning treated by dialysis and mannitol infusion. AustAnn Med 4: 238. Jori A, Briatico G (1973) Effects of eucalyptol on microsomal enzyme activity of foetal and newborn rats. Biochem Pharmacol 22: 543-4.
Leung AY, Foster S (1996) Encyclopedia of Common Natural Ingredients Used in Food, Drugs,and Cosmetics, 2nd edition. John Wiley & Sons, Inc., New York. MacPherson J (1925) The toxicology of eucalyptus oil. Med JAust 2: 108-10. Martindale (2003) Micromedex Healthcare Series Vol. 115. Mitchell J, Rook A (1979) Botanical Dermatology. Greengrass, Vancouver. Opdyke DLJ (1975) Eucalyptus oil. Food Cosmet Toxicol 13: 107-8. Rudzki E, Grzywa Z, Bruo W (1976) Sensitivity to 35 essential oils. Contact Dermatitis 2: 196. Rudzki E, Kleniewska D (1970) The epidemiology of contact dermatitis in Poland. Br J Dermatol 83: 543-5. Schimmer O (1994) An evaluation of 55 commercial plant extracts in the Ames mutagenicity test. Pharmazie 49: 448-51. Schnitzler P, Schon K, Reichling J (2001) Antiviral activity of Australian tea tree oil and eucalyptus oil against herpes simplex virus in cell culture. Pharmazie 56(4): 343-7. Spoerke DG, Vandenberg S, Smolinske S (1989) Eucalyptus oil: 14 cases of exposure. Yet Hum Toxicol 31: 166-8. Tibballs J (1995) Clinical effects and management of eucalyptus oil ingestion in infants and young children. Med JAust 163: 177-80.
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1 d Regulatory Process This product contains actives that are the subject of compendial standards. Expert reports were not procured for this application
• Regulatory compliance lies entirely in the adequate and accurate identification of the substance and the establishment of its safety and efficacy.
• An application must be made to the CTFA for inclusion in their register. The adoption as a new substance will ensure that an INCI (International Nomenclature for Cosmetic Ingredients) name will be allocated.
• Simultaneously, acute and chronic oral and dermal toxicity testing should be undertaken. Scantox Ltd can conduct these trials in a timely manner, they are based in Scandinavia.
• The oil should then be entered on the CAS (Chemical Abstracts Society) registry whereby a CAS number will be allocated.
• The manufacturing company will also be required to register with NICNAS as a manufacturer of chemical substances. Once registered an application may be made to have Agonis Oil adopted onto the Australian Inventory of Chemical Substances (AICS).
• After the product is identified an application may be made for the allocation of a botanical name with TGA. This process is purely name allocation and does not require toxicity data.
• Following the name allocation an application may be made for a new chemical substance to TGA. This application requires a considerable amount of safety and efficacy data and is the key element in the acceptance of the product for therapeutic use.
59
1 e Process Costs Regulatory Authority Application Fees Local Preparation
Cost
CTFA $71.00 $240.00
CAS $285.00 $240.00
NICNAS $11,700.00 $480.00
Scantox $12,600.00 $480.00
TGA Botanical Name Nil $480.00
TGA New Substance $5,040.00 $4,900.00
Total $29,696.00 $6,820.00
Note: The regulatory costs are based upon prices quoted in August 2004. Some authorities have an annual indexing policy, while other prices are based upon international currency exchange rates.
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Appendix 7: Effects of Agonis fragrans oil on mononuclear cell immune responses Jan Dunstan et al. School of Paediatrics and Child Health, University of Western Australia
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Effects of Agonis fragrans oil on mononuclear cell immune responses Jan Dunstan, Jasmine Hale, Heidi Lehman and Susan Prescott School of Paediatrics and Child Health, University of Western Australia
Introduction Inflammation occurs as a result of host immune responses to injury, infection or foreign antigen. This response is characterised by the production inflammatory cytokines by immune cells including monocytes, macrophages and lymphocytes which are collectively referred to as mononuclear cells. These cells recirculate continuously between the blood, tissue and the lymphatic system and contribute to local inflammation. In this study we tested the potential anti-inflammatory effects of oil from Agonis fragrans, by examining effects on cytokine responses of peripheral blood mononuclear cells in vitro.
Hypothesis Agonis oil, emulsified by sonification and added to cell culture of stimulated peripheral blood mononuclear cells (PBMC) will inhibit lymphocyte proliferation and cytokine response. Specific aims
1. To determine the highest concentration at which Agonis oil is non-cytotoxic in 48 hour cell cultures of PBMC.
2. To compare T cell proliferation and cytokine response of mitogen stimulated MC from healthy adults incubated with non-cytotoxic levels of Agonis oil with those cultured without oil added.
3. To compare T cell proliferation and cytokine response of allergen stimulated MC from allergic (skin prick test positive) adults incubated with non-cytotoxic levels of Agonis oil with those cultured without oil added.
Methods Preparation of Agonis oil Five preparations of oil labelled Agonis oil A, B, C, X and Y were provided by Tom Riley and Kate Hammer for testing. Tea tree oil was emulsified by sonication in Hanks balanced salt solution (HBSS) containing 10% fetal calf serum as previously described (1) and stored at –20C in 1ml aliquots. Mononuclear cell isolation Mononuclear cells (MC) were isolated from peripheral blood collected from healthy volunteers using Lymphoprep (Nycomed Pharma, Norway) gradient centrifugation (2). MC were cryopreserved until the time of assay and then thawed, washed in RPMI and resuspended in serum free tissue culture medium (AIM V). It has been shown previously that cryopreservation and thawing does not distort MC cellular immune responses (3), (4, 5). Lymphoproliferation For lymphoproliferation, MC (109/L) were cultured for 6 days in serum free medium (2, 3) in microwells either alone or with allergens [house dust mite extract (HDM) 30 µg/ml] (CSL, Australia) or phytohaemaglutinen (PHA) mitogen 1 µg/ml (HA16, Murex, Biotech Ltd, UK). 3H-thymidine incorporation 16-18 hours prior to harvest was used to determine DNA synthesis. The median of three wells was used to determine the difference in disintergrations per minute (dpm) between the stimulated cultures and the background counts. A difference of >1000 dpm was considered a positive response.
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Cytokine analysis For cytokine analysis 1 x 109 CBMC/L were cultured in AIM V (Gibco, Life Technology, UK) serum free medium (3) for 48 hours with or without PHA or HDM (as above). IL-10, IL-13 and IFNγ were quantified by an in-house ELISA using a time resolved fluorometry (TRF) detection system (DELPHIA, PerkinElmer, Life Sciences, MA, USA) (Dunstan 2003). For detection of IL-10, rat IgG1 anti-human IL-10 monoclonal antibody (clone JES3-9D7; Pharmingen) was used for capture and biotinylated rat IgG2a anti-IL10 monoclonal antibody (Clone JES3-12G8; Pharmingen) for detection. For detection of IL-13, rat IgG1 anti-human IL-13 monoclonal antibody (clone JES10-5A2; Pharmingen) was used for capture and biotinylated mouse IgG1,Κ anti-IL13 monoclonal antibody (Clone B69-2; Pharmingen) for detection. For detection of IFNγ, mouse IgG1,Κ anti-human IFNγ monoclonal antibody (clone NIB42; Pharmingen) was used for capture and biotinylated mouse IgG1,Κ anti-IFNγ monoclonal antibody (Clone 4S.B3; Pharmingen) for detection. The biotinylated antibody was detected using Europium-labelled streptavidin and fluorescence was quantified using a fluorometer (WALLAC VICTOR2, PerkinElmer, Life Sciences, MA, USA). Standard curves, generated using serial dilutions of recombinant human IL-10, IL-13 or IFNγ (Pharmingen), were linear between 3 and 30,000 pg/ml. Cytokine data was expressed as the difference between the stimulated culture and the control.
Assessing the viability of MC cultured in Agonis oil The viability of MC incubated for 48 hours with Agonis oil was determined by trypan blue exclusion (0.4%) and by flow cytometry of MC after removal of cell culture supernatant for cytokine analysis. Lymphocytes were labelled with anti-CD3 conjugated fluoroscein isothiocyanate (FITC) (Pharmingen, Becton Dickinson [BD], CA, USA). Dead cells were labelled with propidium iodide (PI). The percentage of dead cells was examined using FACSCalibar (Becton Dickenson) and CellQuest software.
Statistical analysis All statistical analyses were performed using SPSS software (Version 11 for Macintosh). The cytokine and proliferation data were generally not normally distributed and were unable to be normalized with logarithmic transformation. These data are displayed as median and interquartile range and a non-parametric test for paired data (Mann-Whitney signed ranks test) was used to determine effects of Agonis oil on cytokine and T cell proliferation levels compared to the control. Data were also expressed as a percentage of control levels. P value < 0.05 was considered statistically significant for all analyses. Results Viability of mononuclear cells cultured in the presence of Agonis oil Agonis oil A was used to perform a dose response to determine the concentration of oil emulsified by sonication in HBSS plus 10% fetal calf serum which was cytotoxic when incubated with stimulated mononuclear cells. Agonis oil was cytotoxic to 48 hour cell cultures at a concentration 0.004% but not at 0.002% or less (Figure 1). These findings were confirmed by trypan blue exclusion (data not shown). Agonis oils B, C, X and Y were also not cytotoxic at 0.002%.
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Figure 1: Effect of Agonis oil on the viability of peripheral blood mononuclear cells. The graph shows the percentage of dead mononuclear cells in unstimulated (open bars) and PHA stimulated (closed bars) cultures incubated with different concentrations of Agonis oil, determined by flow cytometric analysis of cells staining with propidium iodide.
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Agonis oil at (or below) a concentration of 0.002% in MC cultures was not cytotoxic as detected by CD3+ T cell responses to mitogen (PHA) or allergen (HDM) after 48 hours (Table 1). Table 1: Live CD3+ T cells from cell cultures incubated for 48 hours with 0.002% Agonis oil expressed as the percentage of the total T cells gated Stimulant No Oil Oil A Oil B Oil C Oil X Oil Z PHA 83.33 80.68 85.09 81.38 83.63 78.88 HDM 90.51 86.59 85.49 90.89 87.07 89.32 Effect of Agonis oil on mitogen stimulated lymphoproliferation and cytokine production All descriptive data and graphs showing the effects of 0.002%, 0.001% and 0.0005% concentrations of the five Agonis oils on lymphoproliferation and cytokine response provided are contained in the two attached excel spreadsheets. Agonis oil at concentration 0.002% or lower did not appear to have effect on HDM or PHA stimulated lymphoproliferation although the sample size (n=5) was small to perform statistics reliably. The greatest inhibitory effect of the Agonis oil was on the production of Th 1 cytokine IFNγ. All of the Agonis oils tested reduced the production of Th1 cytokine IFNγ by PHA stimulated MC at 0.002% concentration (Oil A and B, P=0.006; Oil C, P=0.003, Oil X, P=0.005 and Oil Z P=0.05). Agonis oil X was also inhibitory at 0.001% (P=0.016) but not at 0.0005%.
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Figure 2: Effect of Agonis oils (A, B, C, X, Z) on IFNγ response induced by PHA stimulation. Results represent medians of 12-14 subjects as a percent of the control. *P<0.05.
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Production of T cell regulatory cytokine IL-10 in MC cultures was also reduced in the presence of 0.002% of Agonis oils A (P=0.012), B (P=0.071), C (P=0.023) and X (P=0.001) for cultures stimulated with PHA (Figure 3). Figure 3: Effect of Agonis oils (A, B, C, X, Z) on IL-10 response induced by PHA stimulation. Results represent medians of 12-14 subjects as a percent of the control. *P<0.05.
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The greatest effect of the Agonis oil on Th2 cytokine IL-13 was at 0.0005% concentration (Oil B (P=0.009), X (P=0.037) and Z (P=0.015) for cultures stimulated with PHA (Figure 4). Inhibitory effects were also seen at 0.002% of Agonis oils B (P=0.010), X (P=0.034) and Z (P=0.075) stimulated with PHA.
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Figure 4: Effect of Agonis oils (A, B, C, X, Z) on IL-13 response induced by PHA stimulation. Results represent medians of 12-14 subjects as a percent of the control. *P<0.05.
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Effect of Agonis oil on mitogen stimulated lymphoproliferation and cytokine production
The effects of Agonis oil on allergen (HDM) stimulated PBMCs from atopic adults are difficult to determine because the sample size (n=5-6) is too small to reliably use non-parametric paired data statistic. The graphs on the excel spreadsheet indicate potential inhibitory effects at 0.002% concentration on IFNγ and IL-10 response but not IL-13, although we would need to test more samples to do this. Summary of findings:
1. Agonis oil was non-cytotoxic to peripheral blood mononuclear cells when added at concentrations of 0.002% v/v or less to RPMI (10% fetal calf serum) tissue culture medium. It was cytotoxic at (or above) concentrations of 0.004% v/v.
2. Incubation with non-cytotoxic levels of Agonis oil (0.002%) significantly inhibited mitogen-induced Th1 cytokine IFNγ and T cell regulatory cytokine IL-10 responses by MC from healthy adults
3. Mitogen-induced Th2 cytokine IL-13 responses by were also significantly inhibited when low concentrations (0.0005%) of Agonis oil were added to cultures.
4. There was no effect of Agonis oil on mitogen-stimulated lymphoproliferation. 5. There were possible effects of Agonis oil on allergen (house dust mite) stimulated cytokine
and lymphoproliferative responses in allergic adults, however more subjects are needed to confirm this.
Discussion The findings of this study suggest that Agonis oil inhibits several aspects of immune response. In particular we found that Agonis fragans oil inhibits cytokine IFNγ production by mononuclear cells. IFNγ, along with other pro-inflammatory cytokines (IL6 and IL12), is thought to be involved in the pathogenesis of inflammation, auto-immune disease and atopic dermatitis (6). The finding that Agonis oil inhibits secretion of this cytokine could support the notion of anti-inflammatory properties. Systemic mononuclear cells do traffic to the periphery so it is plausible that Agonis oil used on the skin may have anti-inflammatory effects however more studies are required to test this. The significance of the finding that Agonis oil also inhibited cytokine IL10 is not known. IL10 is generally associated with regulation of T cell responses and is important in the development of immune tolerance. Cytokine IL-13 is associated with allergic type responses (7) and inhibition of this
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cytokine in allergic individuals may indicate another mechanism by which Agonis oil modulates the immune response. Lymphocytes proliferate at a faster rate when stimulated with either mitogens or allergens and we can be measured this experimentally by the increase in tritiated thymidine incorporation into the DNA following stimulation. In this study Agonis oil at a concentration of 0.002% inhibited cytokine secretion but had no effect on the rate of proliferation. This lack of effect of Agonis oil on lymphocyte proliferation supports our conclusion that effects on cytokine response are not due to cell death, since cell death would have resulted in decreased lymphocyte proliferation. Conclusion: The finding that Agonis oil inhibits secretion of the IFNγ, involved in inflammatory response to tissue injury or infection, could support the potential of anti-inflammatory properties Agonis fragrans oil, however more studies are required. In particular effects on other cell types such as neutrophils and purified macrophages, important in inflammatory immune responses as well as safety issues. References 1. Brand C, Ferrante A, Prager RH, Riley TV, Carson CF, Finlay-Jones JJ, et al. The water-soluble components of the essential oil of Melaleuca alternifolia (tea tree oil) suppress the production of superoxide by human monocytes, but not neutrophils, activated in vitro. Inflamm Res 2001;50(4):213-9. 2. Prescott SL, Macaubas C, Holt BJ, Smallacombe TB, Loh R, Sly PD, et al. Transplacental priming of the human immune system to environmental allergens: universal skewing of initial T cell responses toward the Th2 cytokine profile. J Immunol 1998;160(10):4730-7. 3. Upham JW, Holt BJ, Baron-Hay MJ, Yabuhara A, Hales BJ, Thomas WR, et al. Inhalant allergen-specific T-cell reactivity is detectable in close to 100% of atopic and normal individuals: covert responses are unmasked by serum-free medium. Clin Exp Allergy 1995;25(7):634-42. 4. MacAubas C, Sly PD, Burton P, Tiller K, Yabuhara A, Holt BJ, et al. Regulation of T-helper cell responses to inhalant allergen during early childhood. Clin Exp Allergy 1999;29(9):1223-1231. 5. Ausiello CM, Lande R, Urbani F, la Sala A, Stefanelli P, Salmaso S, et al. Cell-mediated immune responses in four-year-old children after primary immunization with acellular pertussis vaccines. Infect Immun 1999;67(8):4064-71. 6. Elenkov IJ, Chrousos GP. Stress hormones, proinflammatory and antiinflammatory cytokines, and autoimmunity. Ann N Y Acad Sci 2002;966:290-303. 7. Wills-Karp M. Interleukin-13 in asthma pathogenesis. Immunol Rev 2004;202:175-90.
