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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

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

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.

Links

Last updated: January 2008
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http://www.rirdc.gov.au/reports/NAP/08-0109um.html