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RIRDC Completed Projects in 2008 - 2009 and Research in Progress as at June 2009 RIRDC Publication No. 09/101 TEA TREE OIL RIRDC Innovation for rural Australia
RIRDC Completed Projects in 2008 - 2009
and Research in Progress as at June 2009
RIRDC Publication No. 09/101
TEA TREE OIL
RIRDCInnovation for rural Australia
TEA TREE OIL
RIRDC Completed Projects in 2008- 2009 and Research in Progress as at June 2009
September 2009 RIRDC Publication No 09/101
© 2009 Rural Industries Research and Development Corporation. All rights reserved. ISBN 1 74151 900 4 ISSN 1440-6845 RIRDC R&D Projects Completed in 2008-09 and Research in Progress as at June 2009 - Tea Tree Oil Publication No 09/101 The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable regions. You must not rely on any information contained in this publication without taking specialist advice relevant to your particular circumstances. While reasonable care has been taken in preparing this publication to ensure that information is true and correct, the Commonwealth of Australia gives no assurance as to the accuracy of any information in this publication. The Commonwealth of Australia, the Rural Industries Research and Development Corporation (RIRDC), the authors or contributors expressly disclaim, to the maximum extent permitted by law, all responsibility and liability to any person, arising directly or indirectly from any act or omission, or for any consequences of any such act or omission, made in reliance on the contents of this publication, whether or not caused by any negligence on the part of the Commonwealth of Australia, RIRDC, the authors or contributors. The Commonwealth of Australia does not necessarily endorse the views in this publication. This publication is copyright. Apart from any use as permitted under the Copyright Act 1968, all other rights are reserved. However, wide dissemination is encouraged. Requests and inquiries concerning reproduction and rights should be addressed to the RIRDC Publications Manager on phone 02 6271 4165. RIRDC Tea Tree Oil Research Manager Roslyn Prinsley Rural Industries Research and Development Corporation Level 2, 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: (02) 6271 4120 Fax: (02) 6271 4199 Email: [email protected] In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 2, 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: (02) 6271 4100 Fax: (02) 6271 4199 Email: [email protected] Website: http://www.rirdc.gov.au Electronically published by RIRDC in September 2009 Print-on-demand by Union Offset Printing, Canberra at www.rirdc.gov.au or phone 1300 634 313
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Foreword RIRDC produces Research in Progress summaries of continuing projects and those completed during 2008-2009. Our intention is to:
• give stakeholders early access to the results of ongoing and completed work to inform their decisions, and
• inform researchers of results to shape research directions.
The complete report on all programs is on our website at http://www.rirdc.gov.au This program aims to support the continued development of an environmentally sustainable and profitable Australian tea tree oil industry that has established international leadership in marketing, value-adding, product reliability and production. There are four program objectives. These are: • Enhancing production systems to maintain the competitiveness of Australian growers • Identifying regulatory regimes and market barriers, and enhancing the ability of industry to meet
safety standards • Demonstrating proof of concept/efficacy for innovative applications of tea tree oil • Fostering communication that increases understanding and thereby encourages greater use of tea
tree oil. This report is an addition to RIRDC’s diverse range of over 1900 research publications, which are available for viewing, downloading or purchasing online through our website: www.rirdc.gov.au. Purchases can also be made by phoning 1300 634 313.
Peter O’Brien Managing Director Rural Industries Research and Development Corporation
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Contents
1.1 TEA TREE OIL – COMPLETED PROJECTS PROJECT No
PROJECT TITLE
RESEARCHER
PHONE
ORGANISATION
PAGE No
Demonstrating proof of concept/efficacy for innovative applications of tea tree oil PRJ-000005 Effects of tea tree oil on
microbial adhesionKate Hammer (08) 9346 1986 University of
Western Australia 1
1.2 TEA TREE OIL – RESEARCH IN PROGRESS PROJECT No
PROJECT TITLE
RESEARCHER
PHONE
ORGANISATION
PAGE No
Demonstrating proof of concept/efficacy for innovative applications of tea tree oil PRJ-000500 Improved tea tree varieties for
a competitive marketTrevor Olesen (02) 6626 1237 NSW Department
of Primary Industries
2
PRJ-002334
Tea tree oil for control of sheep ectoparasites
Peter James (07) 3362 9409 QLD DEEDI 3
PRJ-002395 Anticancer Activity of Melaleuca alternifolia (tea tree) oil
Thomas Riley (08) 9346 3690 University of Western Australia
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PRJ-002403
Microbial adaptation and tolerance to tea tree oil
Christine Carson (08) 9346 3288 University of Western Australia
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PRJ-000002 Use of tea tree oil against buffalo flies in cattle
Lex Turner (07) 5464 8749 Qld Department of Primary Industries and Fisheries
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PRJ-000459 Tea tree oil to prevent staphylococcal infections in dialysis patients
Thomas Riley (08) 9346 3690 University of Western Australia
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Enhancing production systems to maintain the competitiveness of Australian growers PRJ-002803
Genetic tools for improving Melaleuca alternifolia oils
Professor William J. Foley
(02) 6125 2866 Australian National University
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Completed Projects - TTO- Demonstrating proof of concept/efficacy for innovative applications of tea tree oil Project Title
Effects of tea tree oil on microbial adhesion
RIRDC Project No.:
PRJ-000005
Start Date: 1/07/2007 Finish Date: 29/05/2009 Researcher: Kate Hammer Organisation: University of Western Australia Phone: (08) 9346 1986 Fax: (08) 9346 2912 Email: [email protected] Objectives
Establishing efficacy and new modes of antimicrobial action are key to the ongoing development and profitability of the Australian tea tree oil industry. Pre-clinical or proof-of-concept work such as the research proposed for this study is critical for encouraging more clinical work to be undertaken.
Background
Adhesion is a critical step in the infection process. The ability of organism to adhere to human cells or inert surfaces such as indwelling devices is a crucial event that has the potential to lead to infection and disease. There is good scientific evidence to support the use of tea tree oil for the treatment of oral candidiasis and vaginal thrush, conditions both caused by the yeast Candida albicans and both reliant to some extent on the successful colonisation of the epithelium to initiate the disease process. Therefore, the hypothesis being investigated in this study is that tea tree oil reduces the adhesion of Candida yeasts to both inert and biological surfaces.
Research
* Adhesion to inert surfaces was investigated by incubating organisms with varying concentrations of tea tree oil in a polystyrene 96-well microtitre tray for a period of time, removing non-adherent cells and quantifying the adherent cells by staining. * Adhesion to biological surfaces was investigated by incubating Candida cells with buccal epithelial cells, HeLa cells or A549 cells and quantifying adhered Candida by microscopy of flow cytometry. * Cell surface hydrophobicity was examined by the Microbial Adhesion to Hydrocarbons (MATH) method and flow cytometry.
Outcomes
* Tea tree oil reduced the adhesion of Candida yeasts to both polystyrene and to human cells. Furthermore, reduced adhesion was not a product of reduced viability. * Tea tree oil also reduced the adhesion of Staphylococcus aureus and Escherichia coli to polystyrene. However, viability was also reduced, meaning that specific anti-adhesive action was unlikely to be occurring. * Reduced adhesion to polystyrene or human cells may be a result of decreased cell surface hydrophobicity in combination with the alteration of cell-surface proteins resulting from treatment with tea tree oil.
Implications
This study provides new information about the ways in which tea tree oil acts against microorganisms. This may benefit industry by increasing consumer acceptability and demand for tea tree oil. This study may bolster the tea tree oil industry which will in turn benefit communities that are affected by the tea tree oil industry. Individuals that already use, or are interested in using tea tree oil products will benefit from this additional knowledge about the mode of antimicrobial action of tea tree oil.
Publications
"Melaleuca alternifolia (tea tree) oil reduces cell surface hydrophobicity and adhesion in Candida albicans" Manuscript in preparation.
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Research in Progress - TTO- Demonstrating proof of concept/efficacy for innovative applications of tea tree oil Project Title
Improved tea tree varieties for a competitive market
RIRDC Project No.:
PRJ-000500
Start Date: 1/07/2006 Finish Date: 1/07/2009 Researcher: Trevor Olesen Organisation: New South Wales Department of Primary Industries for and on behalf of the
State of NSW Phone: (02) 6626 2422 Fax: Email: [email protected] Objectives
This research has as a key objective, to: release improved seed and clones to maximise profit and market access for Australian tea tree oil producers. In addition to increasing oil yield through selection and breeding, the project aims to investigate genetic aspects of low-allergenic oil, and insect resistance.
Current Progress
Seed from the tea tree breeding project has been made available to the industry since 1997. Seed sales total 9.3kg (enough to plant over 1300 ha). Released seed is from the best provenances, together with improved seed from both seedling and clonal orchards. Over 1.1kg of seed from the clonal orchard have been sold since 2004 when yield gains of over 70% (averaged over 4 harvests) were confirmed for this seedlot. Demand for improved seed dramatically increased during 2008. To supply the demand, 1.3kg of seed from the second-generation partial seedling seed orchard were sold. All sales for 2009 will be sourced from the second-generation seedling seed orchard at Wollongbar. Sales are expected to exceed 1.3kg this year. A large (>34,000 trees) yield trial was established in 2008 to compare the performance of all seed releases to that of industry standards. Trees from the clonal and second-generation seed orchards were again assessed for oil characteristics. Inferior trees will be removed prior to this years flowering to further improve the genetic quality of orchard seed. Seedlots for a third-generation orchard are currently being collected. A cooperative (with ANU PhD student Baskorowati) study on the reproductive biology of M. alternifolia was undertaken. One key finding of direct relevance to the project was that tea tree orchards should be sited where winter temperatures are low, with minima below 5oC. Earlier and more intense flowering under these conditions will shorten generation times and increase seed yields. A second cooperative (with SCU MSc student Prastyono) study comparing the financial performance of clonal vs improved seedling plantations at different stocking rates was undertaken. In brief, a plantation of elite clones at a stocking of 33,333 plants/ha was predicted to give the greatest profit at any of the oil prices tested, followed by plantations using improved seedlings at a stocking rate of 33,333 plants/ha.
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Research in Progress - TTO- Demonstrating proof of concept/efficacy for innovative applications of tea tree oil
Project Title
Tea tree oil for control of sheep ectoparasites
RIRDC Project No.:
PRJ-002334
Start Date: 1/10/2008 Finish Date: 25/07/2011 Researcher: Peter James Organisation: The State Of Queensland Acting Through The Department of Employment,
Economic Development and Innovation Phone: (07) 3362 9409 Fax: (07) 3362 9429 Email: [email protected] Objectives
Increasing the market for tea tree oil by: • Demonstrating the effectiveness of tea tree oil (TTO) formulations in controlling sheep lice (Bovicola ovis) at concentrations that make development of a commercial formulation economically viable • Demonstrating the effectiveness of a TTO based formulation in treating flystrikes and protecting wounds against new strikes • Providing data towards the assessment of the commercial feasibility of development of TTO-based sheep ectoparasiticides and that can support registration of TTO products suitable for use in conventional and organic production systems.
Current Progress
Lice and flystrike formulations with acceptable stability and wool wetting properties, suitable for application to sheep have been developed. Laboratory studies have confirmed the formulations effect against sheep lice and sheep blowflies and have determined suitable concentrations and strategies of application for use in live animal studies. Live animal studies have commenced.
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Research in Progress - TTO- Demonstrating proof of concept/efficacy for innovative applications of tea tree oil
Project Title
Anticancer Activity of Melaleuca alternifolia (tea tree) oil
RIRDC Project No.:
PRJ-002395
Start Date: 2/10/2007 Finish Date: 11/05/2010 Researcher: Thomas Riley Organisation: University of Western Australia Phone: (08) 9346 3690 Fax: (08) 9346 2912 Email: [email protected] Objectives
The major objectives of this study are to: 1. Optimise the in vivo administration of topical TTO treatment in subcutaneous tumour bearing mice to establish its maximum efficacy, and 2. Identify and investigate the in vivo mechanism of action of TTO on tumours.
Current Progress
We are making significant progress in our aims to examine the antitumour mechanism(s) of action of topical tea tree oil. Microscopic analysis of skin sections after haematoxylin and eosin staining found that 10%TTO in dimethylsulphoxide (DMSO) topical treatment in mice resulted in epidermal skin irritation and dermal inflammation characterised by an abundance of neutrophils. Dermal inflammation increased with 2-3 topical 10%TTO/DMSO treatments, with diverse immune cells present including macrophages, mast cells and lymphocytes but not eosinophils. This is currently being assessed by quantitative analysis of the cells. This irritant effect is likely to be the same as that occasionally seen in humans when TTO at a concentration of 10% and above is applied to skin. No permanent damage is done and if this is the only side-effect of the anticancer activity of TTO then it would appear to be a small price to pay. Importantly, preliminary examination of livers from 10% TTO/DMSO treated mice revealed no signs of liver toxicity, indicating that there is not likely to be any systemic toxicity. We also examined the topical effect of a mixture of TTO’s five major components (as a 10% solution in DMSO): 40% terpinen-4-ol, 20% γ-terpinene, 10% α-terpinene 5% 1,8-cineole and 5% p-cymene in our tumour bearing mouse model. This combination induced a significant period of tumour growth inhibition and a day of significant tumour regression, with similar skin irritation as with 10% TTO/DMSO. Overall this was seen as a positive result and provided further evidence of the anticancer effect of TTO. Immune analysis experiments revealed 10% TTO/DMSO failed to regress or inhibit the growth of AE17 tumours in immunodeficient SCID mice. This was not surprising as SCID mice are specifically bred lacking an adaptive T and B cell immune response. These findings suggest the immune response is important in facilitating TTO’s antitumour effect and we are currently further investigating the mechanisms by FACS and cytokine analysis.
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Research in Progress - TTO- Demonstrating proof of concept/efficacy for innovative applications of tea tree oil
Project Title
Microbial adaptation and tolerance to tea tree oil
RIRDC Project No.:
PRJ-002403
Start Date: 6/08/2008 Finish Date: 31/08/2009 Researcher: Christine Carson Organisation: University of Western Australia Phone: (08) 9346 3288 Fax: (08) 9346 2912 Email: [email protected] Objectives
The main objectives of the proposed work are to: 1. Investigate whether the adaptive changes offer cross-protection for other compounds or agents including conventional antibiotics and confirm or refute previous findings, 2. Investigate whether exposure to tea tree oil elicits stress responses in microorganisms including whether or not exposure induces efflux mechanisms, and 3. Compare any adaptive responses seen to those shown previously for other agents such as organic solvents and determine whether any of the responses or combination of responses is unique to tea tree oil.
Current Progress
Growth curves for 30 staphylococcal isolates have been completed. Briefly, standardized suspensions of each isolate were grown in broth in the presence of a range of tea tree oil concentrations and the growth monitored spectrophotometrically at 15 minute intervals for 18 h. Each isolate has been tested on at least two separate occasions. Two additional attempts have been made to repeat the work by McMahon et al. (2007) that suggested application tea tree oil at sub-lethal concentrations may contribute to the development of antibiotic resistance in human pathogens. Most test isolates did not survive the three days of serial sub-culture in 0.25% tea tree oil. In one attempt, none of the 30 isolates was viable 24 hours after transfer into the third bottle containing 0.25% tea tree oil. In the second attempt, 4 of the 10 methicillin-susceptible Staphylococcus aureus (MSSA), 1 of the 10 methicillin resistant S. aureus (MRSA) and 4 of the 10 coagulase negative staphylococci (CNS) were viable after 24 hours in the third bottle of tea tree oil. Five isolates each of MSSA, MRSA and CNS have been habituated to 0.075% tea tree oil and their antibiotic susceptibility profile determined. Compared to control organisms, no significant changes occurred. Ongoing work is attempting to determine whether habituation to 0.075% tea tree oil has “stressed” these isolates or activated well-known resistance mechanisms. Results from one method using a fluorescent dye suggest that efflux mechanisms have not been induced. Additional replicates of this experiment are required as is corroboration by growth in the presence of efflux substrates and/or efflux inhibitors. Reagents for the preliminary experiments required for the real-time reverse transcription PCR methods are being prepared. In summary, the work completed so far suggests that exposure to sub-inhibitory concentrations of tea tree oil has no effect on susceptibility or resistance to conventional antibiotics. Reference: McMahon et al. Habituation to sub-lethal concentrations of tea tree oil (Melaleuca alternifolia) is associated with reduced susceptibility to antibiotics in human pathogens. J Antimicrob Chemother 2007; 59: 125-127.
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Research in Progress - TTO- Demonstrating proof of concept/efficacy for innovative applications of tea tree oil
Project Title
Use of tea tree oil against buffalo flies in cattle
RIRDC Project No.:
PRJ-000002
Start Date: 19/09/2006 Finish Date: 16/09/2009 Researcher: Lex Turner Organisation: The State Of Queensland Acting Through The Department of Employment,
Economic Development and Innovation Phone: (07) 5464 8749 Fax: (07) 5464 8778 Email: [email protected] Objectives
The aim of this project is to scientifically investigate the efficacy of tea tree oil against buffalo flies and the effects of buffalo flies on cattle.
Current Progress
The cattle trial was completed in the expected time frame with no unexpected issues after enough buffalo flies were detected to conduct the work. The trial involved the agreed number of animals and paddocks. The cattle treated with the tea tree oil have been euthanased under APVMA guidelines and samples from these cattle have been submitted for residue testing. These residue testing results are not yet available but will be included in the final report. Time and weather data has recently been supplied to the biometrician to determine any weather and time of day effects and I am awaiting final biometrical analysis. A general comparison of eye lesions due to the fly worry has been completed. This will be included in the final report. We are awaiting tissue sample residue analysis. These details will also be included in the final report. The trial has been completed and the final report is in progress.
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Research in Progress - TTO- Demonstrating proof of concept/efficacy for innovative applications of tea tree oil Project Title
Tea tree oil to prevent staphylococcal infections in dialysis patients
RIRDC Project No.:
PRJ-000459
Start Date: 4/7/2006 Finish Date: 10/11/2008 Researcher: Thomas Riley Organisation: University of Western Australia Phone: (08) 9346 3690 Fax: Email: Objectives
The project aims are: (1) To demonstrate that tea tree oil products are efficacious in the prevention of staphylococcal infections associated with dialysis catheters, (2) To demonstrate that tea tree oil products are a suitable alternative to existing products used to prevent staphylococcal infections associated with dialysis catheters and (3) To provide clinical data on the efficacy and safety of tea tree oil products.
Current Progress
There have been a number of issues that have delayed the commencement of the trial. 1. The trial was not approved by Human Research Ethics Committee (HREC) and the Research Governance Unit (RGU) at Sir Charles Gairdner Hospital (SCGH) until 28 February 2008. 2. The Clinical Trial Notification (CTN) was not signed by Professor McEachern on behalf of The University of Western Australia until 10 September 2008. 3. In early September 2008 Dr Christine Carson who was the trial co-ordinator decreased her working hours from 2.5 days per week to just 1 day per week and it was not possible to carry out further work on the trial until another co-ordinator could be found. 4. On 9 March 2009 Dr Kerry Carson was appointed trial co-ordinator and submitted the CTN for to the Therapeutic Goods Administration (TGA). 5. On 27 March the CTN form was returned by the TGA as it required that it be re-submitted on a new version which had been introduced in June 2008. 6. The new form has been submitted to the SCGH HREC for signature. The form must then be signed by the site director at SCGH, Professor McEachern on behalf on UWA and by WA Home Dialysis Therapies who will collect data from the peritoneal dialysis patients. 7. The trial cannot commence until the TGA have given their authorisation.
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Research in Progress - TTO-Enhancing production systems to maintain the competitiveness of Australian growers
Project Title
Genetic tools for improving Melaleuca alternifolia oils
RIRDC Project No.:
PRJ-002803
Start Date: 1/03/08 Finish Date: 26/02/10 Researcher: Professor William J. Foley Organisation: Australian National University Phone: (02) 6125 2866 Fax: (02) 6125 5573 Email: [email protected] Objectives
This project aims to improve the breeding of Melaleuca alternifolia to achieve better essential oil profiles and better yields of oil. To do so, it will develop new knowledge on the genes that influence the yield and profile of terpenes. Specifically it aims to (i) validate a series of diagnostic tools based on gene variants associated with specific oil profiles (ii) develop diagnostic tests based on gene variants of four candidate genes (IPPI, DXS, DXR, GPPS) associated with oil yields (iii) provide the opportunity for industry to protect IP associated with genes responsible for their major products.
Current Progress
In M. alternifolia, three major compounds determine the established chemotypes: terpinen-4-ol, 1,8-cineole and terpinolene. In the chemotypes that are dominated by only one of these three compounds, different monoterpene synthases appear in the foliar RNA pool. In chemotype 5, the major monoterpene synthase is responsible for the biosynthesis of cineole, α-pinene, limonene and α-terpineol. In chemotype 2, the major monoterpene synthase synthesises terpinolene, while in chemotype 1, we have a sabinene hydrate synthase. This corresponds to measurements of in-vivo non enzymatic rearrangements in chemotype 1 tea tree leaf, whereby the sabinene hydrate of young leaf gradually rearranges into terpinen-4-ol, α- and γ-terpinene. Primers were designed to differentiate between the three different monoterpene synthases. The gene encoding for sabinene hydrate synthase can only be found in the genome of individuals showing significant amounts of terpinen-4-ol in their leaf oil. The probes designed for cineole and terpinolene synthases showed the presence of these genes in the genomes of all individuals, indicating that the concentrations of cineole and terpinolene is likely to involve the regulatory pathway. Sequence comparisons between the three genes shows a high similarity between cineole and sabinene hydrate synthases. As few as 17 non-synonymous mutations separate the two genes. This represents groundbreaking insight into the formation of the most important compound for the tea tree industry. While cineole, and thereby cineole synthase is abundant in Myrtaceae, the gene responsible for the unique chemistry of tea tree oil is a recent allelic variant of this gene. These findings have allowed us to design diagnostic probes that can distinguish between the different chemotypes reliably.
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RIRDC Publication No. INSERT PUB NO. HERE
TEA TREE OILThis program aims to support the continued development of an environmentally sustainable and profitable Australian tea tree oil industry that has established international leadership in marketing, value-adding, product reliability and production.
There are four program objectives. These are:
• Enhancing production systems to maintain the competitiveness of Australian growers
• Identifying regulatory regimes and market barriers, and enhancing the ability of industry to meet safety standards
• Demonstrating proof of concept/efficacy for innovative applications of tea tree oil
• Fostering communication that increases understanding and thereby encourages greater use of tea tree oil.
The Rural Industries Research and Development Corporation (RIRDC) manages and funds priority research and translates results into practical outcomes for industry.
Our business is about developing a more profitable, dynamic and sustainable rural sector. Most of the information we produce can be downloaded for free or purchased from our website: www.rirdc.gov.au, or by phoning 1300 634 313 (local call charge applies).
RIRDCInnovation for rural Australia
Contact RIRDC:Level 2
15 National CircuitBarton ACT 2600
PO Box 4776Kingston ACT 2604
Ph: 02 6271 4100Fax: 02 6271 4199
Email: [email protected]: www.rirdc.gov.au
Most RIRDC books can be freely downloaded or purchased from www.rirdc.gov.au or by phoning 1300 634 313 (local call charge applies).
www.rirdc.gov.au
RIRDC Publication No. 09/101
ForewordEffects of tea tree oil on microbial adhesionImproved tea tree varieties for a competitive marketTea tree oil for control of sheep ectoparasitesAnticancer Activity of Melaleuca alternifolia (tea tree) oilMicrobial adaptation and tolerance to tea tree oilUse of tea tree oil against buffalo flies in cattleTea tree oil to prevent staphylococcal infections in dialysis patientsGenetic tools for improving Melaleuca alternifolia oils
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