SYNERGISTIC COMPOSITIONS

Abstract

The invention concerns novel compositions, comprising at least 2 different sources of ω-3 fatty acids, including wherein hoki roe at 5% (w/w) in powder or oil form and green-lipped mussel in powder or oil form. The compositions are particularly useful as/in supplements and/or animal or human food stuffs. The invention may further relate to the maintenance or treatment of human and veterinary conditions, such as use in the improvement of inflammation or joint related disorders that maybe associated with inflammation.

Claims

1. A composition comprising a combination of two powder or two oil components including: a green-lipped mussel (GLM) powder or oil; and at least 5% (w/w) of a Hoki Roe (HR) powder or oil.

2. A composition comprising: a blend of at least two different powder or oil components each comprising ω-3 fatty acids, wherein a first of the at least two different powder or oil components comprises 5% (w/w) or more of HR powder or oil and wherein the blend of the at least two different powder or oil components together provides at least 3.3% total ω-3 fatty acids in the composition.

3. The composition of claim 2, wherein a second component, of the at least two different powder or oil components, is a GLM oil or powder.

4. The composition of any of claims 1 to 3, wherein the HR powder or oil is present in a range from 5% to 95% (w/w) of the total composition.

5. The composition of any previous claim, wherein the GLM powder or oil comprises a total fat content from 7% to 13% (w/w).

6. The composition of any previous claim, wherein the GLM powder or oil comprises a total ω-3 fatty acid content in a range of 2.0% to 5.0% (w/w).

7. The composition of any previous claim, wherein the HR powder or oil comprises a total fat content of 14% to 40% (w/w).

8. The composition of any previous claim, wherein the HR powder or oil comprises a total ω-3 fatty acid content from 2% to 20% (w/w).

9. A food stuff, nutraceutical or supplement comprising the composition of any preceding claim.

10. The food stuff or supplement of claim 9, further comprising one or more excipients selected from Glucosamine and/or Hyaluronic Acid.

11. The composition, food stuff or supplement according to any previous claim, for use in the treatment or maintenance of health, preferably joint health, of a human or animal.

12. The composition according to claim 11, wherein the treatment or maintenance involves support of anti-inflammatory pathways in the human or animal.

13. A method of treating a subject, the method comprising administering to the subject the composition of claim 1, wherein the subject is a human or an animal.

14. The method of claim 13, wherein the method treats inflammation or maintains low levels of inflammation in the subject, wherein the subject is an animal.

15. The method of claim 13, wherein the method maintains health of the subject.

16. The method of claim 13, wherein the method maintains joint health of the subject.

17. A food stuff, nutraceutical, or supplement comprising the composition of claim 2.

18. The food stuff, nutraceutical, or supplement of claim 17, further comprising at least one excipient chosen from glucosamine or hyaluronic acid.

19. A method of treating a subject, the method comprising administering to the subject the composition of claim 2, wherein the subject is a human or an animal.

20. The method of claim 19, wherein the method treats inflammation or maintains low levels of inflammation in the subject, wherein the subject is an animal.

21. The method of claim 19, wherein the method maintains joint health of the subject.

Description

BRIEF DESCRIPTION

[0025] FIG. 1 is a graph illustrating a dose response curve for gold standard specification of GLM;

[0026] FIG. 2 is a graph illustrating the varying GLM quality sample work;

[0027] FIG. 3 is a graph illustrating the ω-3/COX2 ratio data;

[0028] FIG. 4 is a graph illustrating marine sample screening work comparing GLM, Paua, Blue mussels and HR;

[0029] FIG. 5 is a graph illustrating the effects at specific % combinations showing the predicted effect on inflammatory processes and the actual effect;

[0030] FIG. 6 is a graph illustrating the concentration-dependent inhibition or modulation of COX2 of the 3 different sampled powders;

[0031] FIG. 7 is a graph illustrating anti-inflammatory activity exhibited vs % powder blend and compared with the H powder and X powder expected trend line;

[0032] FIG. 8 is a graph illustrating the anti-inflammatory activity of x+H blends as compared to the levels of total ω-3 fatty acids as compared to H powder and X powder expected trend line; and

[0033] FIG. 9 is a graph illustrating the combination of these two components in oil form (at various blend percentages) is more effective at modulating COX2 activity than would be expected from the trend line.

DETAILED DESCRIPTION

[0034] GLM is used to provide EFAs into the body. They support the anti-inflammatory process by being preferentially acted upon in the anti-inflammatory system, resulting in anti-inflammatory mediators.

[0035] In the absence of these ω-3 EFAs, other EFAs, notably arachidonic acid, will be used as substrates, resulting in pro-inflammatory mediators.

[0036] A dose response is seen in effect depending upon the quantity of GLM used, i.e. as the concentration is increased, the activity of an inflammatory process (COX2 activity) drops away—see FIG. 1.

[0037] The inventors have identified that when GLM is utilised at the same concentration but crucially has a lower EFA specification, a decreased effect on an inflammatory process is seen—see FIG. 2.

[0038] Further the inventors have identified a correlation between ω-3 EFAs and activity of an inflammatory process i.e. with increasing ω-3 EFAs, activity of an inflammatory process decreases-see FIG. 3.

[0039] It has further been identified that certain marine species are more effective at decreasing levels of an inflammatory process, despite having similar levels of total EFA, suggesting the bioactivity is connected to particular EFAs, as compared to all tested—see FIG. 4.

[0040] HR has been identified as one source that appears to have higher profiles of certain EFAs that is similar to GLM in some EFAs. The potential to utilise HR as a replacement/top up for reduced EFA spec GLM and reduced concentration of full spec GLM was therefore further investigated.

[0041] Based on the relative effects of GLM and HR on an inflammatory process, a number of combinations were tested of reduced concentration GLM/HR added or reduced EFA GLM/HR added. The expected additive effect was calculated based on the inflammatory performance of the original GLM & HR samples, with the aim to maintain the effect expected from a full concentration, full spec GLM—see FIG. 5.

[0042] In fact, what the inventors identified is a greater effect on the inflammatory processes in the combination than would be expected from sum of the individual component parts. The invention demonstrates a clear synergistic effect that can be used to maintain a better level of support for the anti-inflammatory system.

[0043] EFAs are thought to be important as a component in any composition which is intended to help support natural anti-inflammatory processes related to human or animal joints, as described here before.

[0044] In particular ω-3 EFAs appear to act as dual competitors of arachidonic acid oxygenation by both the cyclooxygenase (COX) and lipoxygenase (LOX) pathway and thus are useful in said applications.

[0045] EFA and ω-3 fatty acids particularly can be found in the green-lipped mussel (GLM) or green shell mussel (GSM; Perna canaliculus). As described previously, alternatives or additives were sought for investigation due to the variation in the specification of GLM powders and the fact that alone, many existing powder sources of GLM did not necessarily provide a specification with a sufficient content of ω-3 fatty acids.

[0046] A variety of marine sources were reviewed as potential sources of desirable ω-3 fatty acid components. Generally marine-derived high ω-3 fatty acid sources are oils from shellfish or fish organs.

[0047] A range of these types of oils were screened as potential substitutes for part of a composition comprising GLM in order to improve the total ω-3 fatty acid content. While being good sources of ω-3 fatty acids per se it was found that it was difficult to blend the oils with the GLM powder to provide a homogenous powder suitable for formulation into applicable products.

[0048] However, one such source of ω-3 fatty acids was the dried roe powder from Macruronus novaezelandiae, or the white fish hoki. Thus Hoki Roe (HR) powder was tested to determine whether its addition to the GLM powder would improve the content of the EFA, particularly the ω-3 fatty acids.

[0049] The suitability of HR powder was determined by measuring the levels of the ω-3 fatty acids in the GLM powder before addition of HR powder, then after addition, to examine if the ω-3 levels were elevated to the required specification by the combination. The required specification was provided by a GLM powder known to give a commercially desired therapeutic outcome, with a comparatively high ω-3 fatty acid specification.

[0050] To ensure that the novel blend of powders also exhibited desired anti-inflammatory activity (despite the inclusion of lower ω-3 content GLM) in vitro enzyme activity was tested and compared to a standard desirable specification product, as used by the applicants.

[0051] A commonly used in vitro process of measuring of anti-inflammatory activity was used, namely by determining the level of in vitro competition of the COX2 enzyme, which is known to be a pathway involved significantly in the inflammation process, discussed herein before.

[0052] Methods

[0053] New Zealand green lipped mussel (GLM) powder and New Zealand HR powder were obtained from commercial sources and lipid extracts were obtained from the individual powders, or blends of the powders, using the Bligh and Dyer method. This method is described in the following published reference concerning the same (Bligh and Dyer, A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology 1959, 37: 911-917). The fatty acid components of the lipid extracts were measured using AOAC method 963.22.

[0054] Results

[0055] A first set of data was generated by measuring and comparing the amount of ω-3 fatty acids in a standard reference source of GLM product (X) known by the applicant to have an acceptable specification and thus the required amount of ω-3 fatty acid suitable and desirable for use. A further source of GLM powder (x) known to be of a lower specification was also measured.

[0056] The GLM x powder, alone, presented insufficiently, measuring at significantly less ω-3 fatty acid than the reference powder GLM (X) with low COX2 activity or modulation and was thus below the required specification required for the commercially desirable applications mentioned herein before.

[0057] A new combination was tested to determine how ω-3 fatty acid levels were impacted when the below spec product x was supplemented with an amount of a different powder known to be rich in ω-3.

[0058] HR powder (H) had previously been selected from a variety of sources, on the basis that it had good potential for elevating the total ω-3 fatty acid content and is able to be easily blended into the GLM powder.

[0059] In order to establish if a lower specification GLM powder could be raised to the required specification by addition of HR powder it was theoretically determined that at least 5% (w/w), preferably 8% (w/w) of a specific HR powder (BN:HR114) added to a lower specification GLM powder (x) would raise the total ω-3 content score (the measure of % fatty acid in the total fat of the product) to a level that meets the required specification.

[0060] It was determined by measurement that by including 8% (w/w) HR powder (H) into a blend with the below specification GLM powder (x), the ω-3 fatty acid content was increased to 3.5% (w/w), very close to the level shown for the standard powder X (BN32132) as in the Table 1:

TABLE-US-00001 TABLE 1 GLM powder HR powder Total ω-3 Batch component component score Composition number (g) (g) (g/100 g) GLM powder - 100 0 3.1 below specification (x) HR powder BN:HR114 0 100 5.1 (H) GLM powder - 95 5 3.3 below specification plus HR (x + H) GLM powder - BN32127 92 8 3.5 below specification plus HR (x + H) GLM powder - BN32132 100 0 3.6 commercially acceptable specification (X)

[0061] Biological Activity and Graphic Analysis

[0062] Different quality specifications of GLM (that varied in the levels of ω-3 fatty acids) were combined with HR powder in different ratios. We then measured total ω-3 fatty acids in the combination product and the COX inhibitory or modulating scores of those powders compared to the source GLM (x) and Hoki powders (H).

[0063] Next, anti-inflammatory activity of the above examples was measured to determine the likely potential for biological impact of the combination as compared to the expected activity based on the new combinations of sourced ω-3 fatty acids.

[0064] The COX2 inhibition or modulation was determined by incubating lipid extracts of the powder blend combinations with commercially available mammalian COX2 enzyme and the enzyme's activity was measured as the co-oxidation of N,N,N,N′-tetramethyl-p-phenylenediamine (TMPD) by prostaglandin G2 (PGG2) to produce oxidised TMPD, which is blue in colour and readily detectable at 611 nm.

[0065] The COX2 inhibitory or modulatory activity of the powder blend combinations was determined from the lipid extract concentration-dependent inhibition or modulation of the COX2 enzyme, or by the anti-inflammatory activity (AI) score of the powder sample, calculated as the inverse of the concentration of lipid extract required to inhibit or modulate the COX2 enzyme by 50% (IC.sub.50) multiplied by the total weight of lipid extracted from the powder blend combinations.

[0066] As can be seen from the Figures, it was determined that concentration-dependent inhibition or modulation of COX2 by the below specification GLM powder (x) blended with 8% (w/w) HR powder (H) to produce x+H (BN32127) powder was significantly greater than a GLM powder X (BN 32132) alone, which had displayed a similar ω-3 fatty acid level (as was shown in Table 1) and thus would have been assumed to be similar.

[0067] Rather surprisingly, as shown in FIG. 6 specifically, the concentration-dependent inhibition or modulation of COX2 by x+H (BN 32127) was even greater than a 100% HR powder H (BN:HR114), which exhibited an ω-3 level that was significantly greater than x+H (as shown in Table 1).

[0068] As can be seen in FIG. 7, it was further established that anti-inflammatory (AI) activity exhibited by x+H at various percentage of % H blend (diamond data points) was greater than would be expected from the (AI) activity one would expect when plotting a linear trend line showing activity of the GLM powder X (far left square data points) and HR powder H alone (far right square data points). The linear trend line between the two indicates what might be expected from a simple additive effect of the two powders suggesting the two have an enhanced effect that in combination goes far beyond the addition of the individual effect.

[0069] This trend was further observed in FIG. 8, since the anti-inflammatory (AI) activity of x+H at various % powder blends (diamond data points) was also greater than would be expected from: the levels of total Omega-3 fatty acids in the H and X alone (as was shown in FIG. 3).

[0070] The large square data points display the linear trend line relationship indicating what would be expected from a simple additive effect achieved by combining the two powders. However, as can be seen, the blended powder exceeded the expected AI activity quite significantly, indicating the novel combination provides for an enhanced and thus synergistic effect, rather than a mere accumulative or additive effect.

[0071] The inventors have additionally tested oil-based combinations of these components and established that anti-inflammatory activity exhibited at various percentage of H blend (circle data points) was also greater than would be expected from the additive activity of the oil components. This confirmed that oil based component combination was also effective in producing an enhanced bioactivity as compared to the individual components.

[0072] Table 2 shows the results of the oil-based component testing below:

TABLE-US-00002 Level of inflammation HR oil GLM oil (COX2 IC.sub.50 μg/mL) 0.0% 100.0% 28.50 100.0% 0.0% 51.50 5.0% 95.0% 22.80 10.0% 90.0% 25.90 25.0% 75.0% 23.50 50.0% 50.0% 41.60 75.0% 25.0% 29.70 90.0% 10.0% 32.40 95.0% 5.0% 40.80

[0073] FIG. 9 illustrates the significance of these results (shown in Table 2) in graphical form by plotting the relative COX2 activity one might expect from the 100% GLM oil (far left square) and 100% HR oil (far right square) when either is used alone. The linear trend line between the two plotted data square points indicates what might be expected from a simple additive effect of the two oils.

[0074] The plotted results from the actual activity observed in various combinations as also found in Table 2 above (plotted as circle data points) suggest the oil component combination also has an enhanced synergistic effect that goes beyond the additive effect of the individual oil components.