Anti-inflammatory Efficacy of Emu Oil

Transcription

1 Anti-inflammatory Efficacy of Emu Oil Refinement of an in vitro assay by Dr Christine A Lunam January 2008 RIRDC Publication No 08/010 RIRDC Project No UF-13A

2 2008 Rural Industries Research and Development Corporation. All rights reserved. ISBN ISSN Anti-inflammatory efficacy of emu oil: refinement of an in vitro assay Publication No. 08/010 Project No. UF-13A 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 Researcher Contact Details Dr Christine Lunam Department of Anatomy & Histology Flinders University PO Box 2100 ADELAIDE SA 5001 Phone: Fax: chris.lunam@flinders.edu.au 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: Fax: rirdc@rirdc.gov.au. Web: Published in January 2008 Printed by Canprint ii

3 Foreword The aim of this short term study is to refine an in vitro test to provide a quantitative measure of the potential anti-inflammatory efficacy of emu oil. Subsequent work will then investigate factors that affect the anti-inflammatory activity of emu oil using the in vitro assay as a measure of its potential anti-inflammatory efficacy. To maximise the market potential for emu oil, the Emu Industry needs to overcome two problems. The first is to identify those factors that result in the different levels of its anti-inflammatory activity, thereby allowing the consistent production of oil with known anti-inflammatory efficacy. The second problem is to develop a sensitive in vitro assay to provide a quantitative measure of the potential antiinflammatory activity of each batch of oil. This report describes the effect of emu oil on the in vitro production of inflammatory mediators from stimulated human lymphocytes. This report also provides a methodology to form a stable emulsion of emu oil that is non-toxic to human lymphocytes. This project was funded from RIRDC Core Funds which are provided by the Australian Government for the program area of New Animal Products. This report, an addition to RIRDC s diverse range of over 1700 research publications, forms part of our New Animal Products R&D program, which aims to accelerate the development of viable new animal industries. Most of our publications are available for viewing, downloading or purchasing online through our website: downloads at purchases at Peter O Brien Managing Director Rural Industries Research and Development Corporation iii

4 Acknowledgments Dr Peter McInnes for his support and encouragement to undertake this study. Mr Geoff Lean, (Industry Partner) for his enthusiasm, the fruitful discussions of the work and hands-on assistance in preparation of the emulsified oil samples at Flinders University. Dr Peter Macardle, Director of the Department of Immunology, Allergy and Arthritis at Flinders Medical Centre for his advice on development of the in vitro assay and interpretation of the data, for overseeing the assays, and for supplying the culture media and many other reagents used in the preparation of the cultures. Ms Rachel Hall for preparation of the cultures and conducting the cytokine assays. Thank you also Rachel for supplying the information on the methodology and data generated from the assays. Mr Chris Gregory, (Industry Partner) managing director of Emu Tracks Australasia Pty Ltd for supplying the emu oils used in this study. Dr John Plummer, Chief Medical Scientist in the Department of Anaesthesia & Pain Management at Flinders Medical Centre for his advice on the preparation of the oil samples. Dr Michael Story (Industry Partner) for discussions regarding the initial preparation of the emulsified oil samples and for supplying the cyclodextrins used in the initial oil preparations. New Animal Products Research & Development Program, RIRDC, for funding the work. iv

5 Contents Foreword... iii Acknowledgments... iv Contents... v Executive Summary... vi 1. Introduction Background and overall aims Specific aims Emu oil industry perspective Methods Emulsification of the emu oil Lymphocyte source Lymphocyte viability Selection of oil samples Measurement of cytokine release Results Effect of emulsified oil on lymphocyte viability Mitogen stimulation of cytokine release Effect of emu oil on cytokine release Discussion References... 8 v

6 Executive Summary What the report is about & who it is targeted at To maximise the market potential for emu oil, the Australian Emu Industry needs to overcome two problems. The first is to identify those factors that result in the different levels of its antiinflammatory activity, thereby allowing the consistent production of oil with known anti-inflammatory efficacy. The second problem is to develop a sensitive in vitro assay to measure the anti-inflammatory activity of each batch of oil to grade its potential anti-inflammatory efficacy. The inflammatory reaction involves the release of a range of inflammatory mediators (cytokines) from lymphocytes. It is hypothesised, that as an anti-inflammatory agent, emu oil will suppress the production of these cytokines from human lymphocytes. It is further hypothesised that the amount of suppression of the cytokine mediators of the inflammatory response from activated human T lymphocytes will directly correlate with the anti-inflammatory activity of individual oil samples. This report discusses the work undertaken to investigate these two hypotheses. Aims & methodology The aim of this project was to develop an in vitro assay to provide a quantitative measure of the effect of emu oil on the production of cytokines from human lymphocytes. To develop this assay the following specific aims were addressed. Establish a concentration of emulsified emu oil in fetal calf serum that is non-toxic to the cultured human lymphocytes Examine the effect of emu oil on the in vitro production of inflammatory mediators from stimulated human lymphocytes. Determine whether different batches of emu oil differentially suppress the in vitro production of inflammatory mediators from stimulated human lymphocytes. Implications & recommendations Emu oil at 1% v/v was found to form a stable emulsion when sonicated in 10% fetal calf serum. Furthermore this emulsion (at 1%) proved to be non-toxic to human lymphocytes. Emu oil, when applied to stimulated human lymphocytes altered the production of cytokine inflammatory mediators. Different batches of emu oil had different effects on the production of individual cytokines. This study demonstrated it is possible to form a stable emulsion of emu oil that is non-toxic to human lymphocytes. Emu oil was found to alter the production of pro-inflammatory cytokines from activated human lymphocytes in vitro. Furthermore individual oils had different effects on the production of the cytokines. This finding of suppression of pro-inflammatory cytokines from activated human lymphocytes supports animal studies which demonstrated that emu oil has anti-inflammatory activity when applied topically. In addition, that individual oils can either suppress or stimulate specific cytokines to different extents supports the possibility that different emu oils may have different antiinflammatory efficacy. Therefore, the in vitro assay developed in this short-term study shows promise as a measure of the potential anti-inflammatory efficacy of individual oils. Refinement of the in vitro assay is however necessary to be able to definitively quantify the effects of emu oil on the production of the cytokines. These refinements are given under 'Further work'. Finally, to be able to use this in vitro assay as a measure of the potential anti-inflammatory efficacy of emu oil it is necessary to correlate the effect of each oil on cytokine production from activated human lymphocytes to its ability to suppress inflammation in vivo. vi

7 Further work Further refinement of the in vitro assay is neccessary to establish a quantitative measure of the effect of individual emu oil samples on cytokine release from activated human lymphocytes. These refinements are tabulated below. Increase the number of emu oils tested and conduct the in vitro assay in triplicate for each oil to provide adequate samples for statistical analysis of the data. Determine the toxicity of the mitogen (DHA) on lymphocyte viability so that the effect of the each emulsified oil sample on cytokine production can be definitively quantitated. Reduce the variation in the known concentration of emu oil in the emulsion by using pipette tips especially designed for viscous fluids. Assess the potential toxicity of emu oil between 1 and 2% (v) on lymphocyte viability. Determine whether emu oil has a dose-related effect on production of the different cytokines. This is necessary to be able to determine whether the effect on cytokine production is a doserelated effect of the emulsified oil or is a result of inherent unique properties of the individual oils. To validate this in vitro assay as a measure of the potential anti-inflammatory efficacy of emu oil, it is necessary to correlate the effect of each oil on cytokine production from activated human lymphocytes to its ability to suppress inflammation in vivo. vii

8 1. Introduction 1.1 Background and overall aims To date scientific evidence for the anti-inflammatory activity of emu oil has been largely confined to animal models of inflammation. Topical application of emu oil has been reported to reduce inflammation in mice (López et al. 1999; Yoganathan et al. 2003) and rats (Snowden & Whitehouse 1997). Furthermore, oils sourced from emus from different habitats and rendered by different processes vary in their anti-inflammatory efficacy when applied to the skin of rats (Snowden & Whitehouse 1997; Whitehouse et al. 1998). The overall aim of this short term study is to refine an in vitro test to provide a quantitative measure of the potential anti-inflammatory efficacy of different batches of emu oil. Subsequent work will then investigate factors that may influence the anti-inflammatory activity of emu oil using the in vitro assay as a measure of its potential anti-inflammatory efficacy. Factors to be investigated in future work will include the age and sex of the birds at the time of slaughter as well as different rendering processes of the oil. The inflammatory reaction involves the release of a range of inflammatory mediators from lymphocytes. These include the cytokines interleukin (IL)-8, IL-1β, IL-6, IL-10, IL-12p70 and tumour necrosis factor-α (TNF-α). It is hypothesised, that as an anti-inflammatory agent, emu oil will suppress the production of these cytokines from activated human T lymphocytes. We further hypothesise that the amount of suppression of the cytokine mediators of the inflammatory response from activated human T lymphocytes will directly correlate with the anti-inflammatory activity of individual oil samples. To develop an in vitro assay, it is essential that the oil is emulsified in a medium that is not toxic to the human T lymphocytes. In preliminary studies in our laboratory at Flinders University we have been able to emulsify the oil samples by sonication in culture medium containing 10% fetal calf serum at 37ºC. The current work examines the effect of emu oil on the viability of human T lymphocytes and cytokine production with mitogen activation. 1.2 Specific aims Establish a concentration of emulsified emu oil in fetal calf serum that is non-toxic to the cultured human lymphocytes. Examine the effect of emu oil on the in vitro production of inflammatory mediators from stimulated human lymphocytes. Determine whether different batches of emu oil differentially suppress the in vitro production of inflammatory mediators from stimulated human lymphocytes. 1.3 Emu oil industry perspective The Australian Emu Industry needs to develop its products to maintain its sustainability. Currently income is received from the skins, meat and oil. Due to the high costs of both transport to the abattoir and subsequent processing of the emus it is essential to maximise profits from the sale of each of the skins, meat and oil. 1

9 To maximise the market potential for emu oil, the Industry needs to overcome two problems. The first is to identify those factors that result in the different levels of its anti-inflammatory activity, thereby allowing the consistent production of oil with known anti-inflammatory efficacy. The second problem is to develop a sensitive in vitro assay to measure the anti-inflammatory activity of each batch of oil to grade its potential anti-inflammatory efficacy. Market research to date has highlighted the commercial potential of emu oil in pharmaceutical (high grade) and cosmetic (lower grade) applications. Assuming 5% market penetration by emu oil indicates a demand for over 750,000 litres of oil per annum. At $70/litre this would result in sales of Australian product in excess of $50 million per annum. Initial indications are that oil with high antiinflammatory efficacy would command a price of at least $200 per litre on a world market. This compares to the current market price of $50-70 per litre for emu oil with relatively low antiinflammatory levels. Enhancing the anti-inflammatory levels of emu oil raises its potential to compete with non-specific anti-inflammatory drugs (NSAIDs) that can have severe side effects. Indications are that the market NSAIDs in the treatment of arthritis in major developed countries is in excess of US$10 billion per annum. It is reasonable to expect that a successful natural alternative with no untoward side effects could target a market share of 2% approximating US$200 million. The information on market research was kindly provided by the Industry partners. 2

10 2. Methods 2.1 Emulsification of the emu oil Oil samples (1ml) were sonicated for 20 seconds in RPMI-1460 medium (SAFC Biosciences TM ) containing 10% fetal bovine serum (Gibco ), 13mM NaHCO 3, 2mM glutamine, 300 µg/ml streptomycin and 8 µg/ml penicillin (complete RPMI medium). The emulsified oil samples were maintained at 37 C. Emulsions were prepared in duplicate at concentrations of 1%, 0.1%, 0.01% and 0.001% v/v. As the oil was extremely viscous care was used to pipette the initial pure oil into the fetal calf serum. Initial studies in our laboratory examined the effects of different concentrations of β-cyclodextrin, α- cyclodextrin or λ-cyclodextrin (0.1, 1% and 10%) on emulsification of the emu oil. As the oil was found to form a stable emulsion with sonication in the RPMI-1460 medium in the absence of any of the cyclodextrins, they were excluded from future preparations. 2.2 Lymphocyte source Two different sources of lymphocytes were used in this study. These were the Jurkat lymphocyte line and human peripheral blood lymphocytes. The Jurkat cell line was chosen as it is maintained in the Department of Immunology, Allergy & Arthritis at Flinders Medical Centre and thus provided an unlimited source of cells. Jurkat is a human T lymphocyte cell line that has been transformed to survive in culture. Human peripheral blood lymphocytes were separated from whole blood by density gradient separation using Ficoll Paque TM Plus (GE Healthcare). Blood was taken from healthy volunteers by venipuncture (FMC ethics approval FcREC 067/78.25). 2.3 Lymphocyte viability For in vitro testing of the different concentrations of the oil on lymphocyte viability, 0.5ml of each oil sample was incubated overnight at 37ºC in 5% CO 2, with 10 5 cells/ml in 0.5ml of the complete RPMI medium. All samples were prepared in duplicate. At the end of the incubation period the lymphocytes were centrifuged at 500g for 5 minutes to pellet the cells. The cells were then resuspended and incubated for 15 minutes at room temperature in 100µml of 10% fetal calf serum to which was added 7.5µM propidium iodide (ICN Biochemicals). Viability of the lymphocytes was analysed by propidium iodide exclusion on a BD FACSCanto flow cytometer. In the initial experiments, designed to determine the effect of the oil on lymphocyte viability, the different concentrations of oil were prepared from aliquots of the same sample. 2.4 Selection of oil samples In the second set of experiments, the incubation medium contained 1% v/v emulsified oil, with the exception of one incubation medium that contained 2% v/v of one of the oil samples. The emulsified samples were prepared from four different batches of emu oil sourced from different emus and prepared by varying rendering processes. These oils were chosen in an attempt to maximise the chance of the potential different anti-inflammatory activity of the samples. Due to restraints of both time to conduct the project and the number of samples that could be analysed with the cytokine assay kit available, the number of samples that could be examined was limited. Consequently it was only possible to emulsify one oil sample in duplicate. In addition, one of the oils (coded #5) was selected at random from the four batches and prepared at 1% (5a) and 2% v/v (5b). This was to determine whether the higher concentration of the oil had a greater effect on inhibiting the production of any of the cytokines compared to 1% v/v of emulsified oil. All the emulsified oil 3

11 samples were coded prior to analysis and incubations were performed in duplicate. The four different batches of oil were coded #1, #4, #5, #6 and #12, where #s 1and 12 were taken from the same batch of oil. 2.5 Measurement of cytokine release Prior to incubation, lymphocytes were stimulated with the mitogens phytohemagglutinin 0.1ng/ml PHA; Sigma) or 10ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma). These were added to the medium to stimulate cytokine release into the supernatant from the lymphocytes. The effect of the mitogens on cytokine release was examined in separate experiments on lymphocytes obtained from both the Jurkat cell line and those separated from whole blood. Cultured lymphocytes were incubated with either both or only one of the mitogens. After culturing the cells overnight, the supernatant was retrieved by centrifugation and assayed for IL- 8, IL-1β, IL-6, IL-10, TNF-α and IL-12p70 using a BD Cytometric Bead Array (CBA) Human Inflammation Kit (BD Biosciences, USA). The amount of each cytokine in the culture supernatant was measured by the intensity of fluorescence using by a BD FACSCanto flow cytometer 4

12 3 Results 3.1 Effect of emulsified oil on lymphocyte viability The Jurkat cells (human T lymphocyte cell line) and those separated from whole blood, remained viable at all concentrations (1%, 0.1%, 0.01% and 0.001%v/v) of the oil after incubation. As 1% oil v/v proved to be non-toxic to the lymphocytes this concentration was used in all future experiments. 3.2 Mitogen stimulation of cytokine release The Jurkat cells showed only a minimal increase in the level of IL-8 production in response to either the phorbol ester (PMA) or PHA compared to the unstimulated cells. No increase in the levels of any other cytokines was observed with stimulation with either mitogen. This low level of cytokine release from the Jurkat cells rendered them inappropriate for testing the effects of emu oil on cytokine release. Unstimulated peripheral blood lymphocytes naturally secreted IL-8 and IL-6 at high levels whereas IL-1β and TNF were released at intermediate levels. The unstimulated cells showed no release of either IL-10 or IL-12p70. Stimulation with PHA resulted in a marked increase in the levels of all the cytokines IL-1β, IL-6, IL-10, IL-8 and TNF-α, except IL-12p70 which was not released with PHA stimulation (Figure 3.1). A small decrease in cell viability was observed with PHA as there was an increase in the percentage of necrotic cells compared to the untreated cells. Stimulation with PMA +/- PHA resulted in a reduction in cytokine release compared to stimulation with PHA alone. This effect may be due to higher toxicity of the PMA compared to PHA resulting in cell death. Consequently stimulation of the peripheral blood lymphocytes by PHA was used in all future experiments. Untreated cells PHA stimulated cells Figure 3.1 Scatter plots showing the effects of PHA stimulation on cytokine production from human peripheral blood lymphocytes. The axes show mean fluorescence intensity, where B-585/42 is R- phycoerythrin and B-670LP is fluorescein isothiocyanate. An increase in cytokine production is seen by a right shift of the fluorescent markers with PHA stimulation. 5

13 3.3 Effect of emu oil on cytokine release Cells stimulated with PHA and additionally cultured with the 1% oil, showed an increase in cell viability compared to PHA treatment alone. All four batches of the 1% v/v oil had variable effects on cytokine levels (Table 3.1). All the oils suppressed PHA-stimulation of IL-10 production. With the exception of suppression of IL-10 levels, two of the oils, coded #1(#12) and #6 had no effect on the production of IL-8, IL-1β or IL-6, although production of TNF-α increased by approximately 30%. A similar response on cytokine production was observed with the duplicate oil of the same batch as #1 (coded #12). In contrast, oils numbered 4 and 5 (shown in Figure 3.2), sampled from different batches, decreased the production of IL-1β, IL-6, and TNF-α. Levels of each of these three cytokines in the culture supernatant decreased by greater than 50% with oil coded #5. Increasing the concentration of the oil from 1% to 2% v/v almost totally suppressed the levels of all cytokines. A greater percentage of the cells appeared to be necrotic compared to the cells similarly incubated in 1% oil although this effect was definitively quantitated. As IL-12p70 production remained low with PHA stimulation, the effect of the oil on this cytokine could not be determined. Table 3.1 Mean fluorescent intensities of cytokines in the culture supernatant following treatment with emu oil Treatment IL-8 IL-1β IL-6 IL-10 TNF-α IL-12p70 1% Oil (no cells/ no PHA) Untreated cells PHA-treated cells % Oil +cells (no PHA) % Oil #1 + PHA % Oil #4 + PHA % Oil #5a + PHA % Oil #5b + PHA % Oil #6 + PHA % Oil #12 + PHA PHA stimulated cells PHA stimulated cells + oil Figure 3.2 Scatter plots showing effects of 1% oil v/v on cytokine production with PHA-stimulation of peripheral lymphocytes. A reduction in cytokine levels is seen by a left shift of the fluorescent markers following incubation with the emu oil. 6

14 4. Discussion Two of the four oils suppressed the production of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. The suppression of these pro-inflammatory cytokines is consistent with a study using a mouse model of inflammation, which reported a decrease in both IL-1α and TNF-α following topical application of emu oil (Yoganathan et al. 2003). Yoganathan and colleagues also reported that the decrease in these pro-inflammatory cytokines was associated with a decrease in inflammation. In addition, different preparations of emu oil were found to vary in their anti-inflammatory activity against adjuvant-induced arthritis in rats (Snowden & Whitehouse 1997; Whitehouse et al. 1998). A plausible hypothesis, taking into account these reports in the literature together with the data from the present study, is that the variable ability of the different oils to suppress cytokine production from the stimulated lymphocytes may be related to differences in the anti-inflammatory activity between the oils. However, whilst the remaining two oils had no apparent affect on the levels of the proinflammatory cytokines IL-8, IL-1β, IL-6, the production of TNF-α increased by approximately 30%. This increased production of TNF-α complicates any extrapolation of the cytokine levels in the assay to the potential anti-inflammatory activity of the oil. In our pilot study only four different oils were tested and a single oil was emulsified in duplicate. Approximately a 15% variability in cytokine production was observed between the two preparations. Although care was taken to pipette the viscous oil into the fetal calf serum, it is possible that some variation in the levels of cytokines detected in the supernatant was due to slightly different amounts of oil in the initial emulsion. Another factor that may have affected the levels of the cytokine levels in the culture supernatant is the viability of the mitogen-stimulated lymphocytes. PHA did reduce lymphocyte viability, although cells stimulated with PHA and additionally cultured with the 1% oil, showed an increase in cell viability compared to PHA treatment alone. It is therefore possible that at a concentration of 1% the oil may have exerted a protective effect against PHA-induced injury. Treatment of the PHA-stimulated lymphocytes with 2% emu oil (v/v) almost totally suppressed the levels of all cytokines. One explanation for this effect is that a constituent(s) of emu oil is suppressing cytokine production. In support of this hypothesis, this particular oil, selected at random prior to incubation, markedly reduced production of IL-1β, IL-6, and TNF-α at 1% (v/v). The possibility cannot be discounted however, that the higher concentration of the oil may have been toxic to the cells. In support of this, although not definitively quantitated, a greater percentage of the cells appeared to be necrotic compared to the cells similarly incubated in 1% oil. Further work is required to assess the potential toxicity of emu oil between 1 and 2% (v/v) on lymphocyte viability and to determine whether emu oil has a dose-related effect on production of the different cytokines. In summary, emu oil at 1% v/v was found to form a stable emulsion when sonicated in 10% fetal calf serum. Furthermore this emulsion (at 1%) proved to be non-toxic to human lymphocytes. Emu oil, when applied to PHA-stimulated human lymphocytes altered the production of cytokine inflammatory mediators. Different batches of emu oil had variable effects of cytokine production. Further work is required to develop this assay which promises to provide a rapid and quantitative measure of the effect of emu oil on cytokine production. 7

15 5. References López, A, Sims ED, Ablett, RF, Skinner, RE, Léger, LW, Lariviere CM, Jamieson, LA, Marténez- Burnes, J & Zawadzka, GG, 1999, Effect of emu oil on auricular inflammation induced with croton oil in mice, American Journal of Veterinary Research, vol. 60(12), pp Snowden, JM & Whitehouse, MW, 1997, Anti-inflammatory activity of emu oil in rats, Inflammopharmacology, vol. 5, pp Whitehouse, MW, Turner AG, Davis CKC Roberts, MS, Emu oils(s): A source of non-toxic transdermal anti-inflammatory agents in aboriginal medicine, Inflammopharmacology, vol. 6, pp 1-8. Yoganathan, S, Nicolosi, R, Wilson, T, Handelman, G, Scollin, P, Tao, R, Binford, P & Orthoefer, F, 2003, Anatogonism of croton oil inflammation by topical emu oil in CD-1 mice, Lipids, vol. 38, pp