NZ510407A - Mussel extract derivative with anti-inflammatory activity - Google Patents

Abstract

Extracts from the New Zealand Green-Lipped Mussel (Perna canaliculus), including, an anti-inflammatory extract of carbohydrate(s) and lipid(s) and an anti-inflammatory extract being an extract of proteins, lipids and carbohydrates which at least to some extent has been de-proteinated. Methods of preparing such extracts are also disclosed.

Description

51 OA 07 NEW ZEALAND PATENTS ACT, 1953 No: 510407/517625 Date: 08 March 2001/06 March 2002 Intellectual Property Office of N.z.

MAY 2002 RECEIVED COMPLETE SPECIFICATION "Mussel Extract Derivative" We, HEALTHERIES OF NEW ZEALAND LIMITED, a company duly incorporated under the laws of New Zealand of 505 Mt Wellington Highway, Auckland, New Zealand, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: The present invention relates to an anti-inflammatory composition, methods of preparing such an anti-inflammatory composition and to related methods, uses and means.

The New Zealand Green-Lipped Mussel {Perna canaliculus) has been subject to close scrutiny over an extensive number of years in respect of its therapeutic activities.

Various extracts of such mussels have been disclosed in respect of which a therapeutic activity has been alleged.

New Zealand Patent Specification 211928 (NZ 211928) discloses a method of stabilising an extract from such mussels reliant upon the addition of an organic aliphatic acid or an alkyl metal or alkaline earth metal salt.

NZ 270754 describes a mixture of finely ground freeze dried extract from such mussels suspended in a fish oil in a maimer yielding a substantially uniform particle distribution.

NZ 314867 discloses a synergistic anti-inflammatory composition including a substance containing carbohydrates extracted from the New Zealand Green-Lipped Mussel and one or more glycosaminoglycans.

The full content of the foregoing patent specifications is hereby here included by way of reference.

A mussel extract product sold in New Zealand under the brand SEATONE™ by McFarlane Laboratories as a health food to be taken at a recommended regime of five 230 mg capsules per day has been available for some time. Such a product includes the BIOLANE™ ingredient being an extract from live Green-Lipped Mussels. Such a product by virtue of its anti-inflammatory efficacy is found to enhance mobility.

The BIOLANE™ mussel extract ingredient of the SEATONE™ product, unlike mussel powder made from frozen mussel flesh, has an anti-inflammatory activity.

The SEATONE™ product (and this includes its BIOLANE™ mussel extract ingredient) is not recommended to people allergic to shellfish. We believe allergies will be reduced by such de-proteination.

We have determined that the BIOLANE™ mussel extract (ignoring ash and other small inclusions eg, vitamins) is approximately 50% w/w proteins with the remaining 50% w/w being made up of about 25% w/w carbohydrates and about 10% w/w lipids.

These existing products we believe are prepared by process we believe involves: a) passing of fresh mussel through a bath of an organic acid, b) crushing and separation of the shell, c) liquid extract separated from solids by centrifuging, d) liquid extract freeze dried, and e) milled to make a powder.

NZ 314867 (McFarlane) discloses an extraction procedure for the substance containing carbohydrates from the New Zealand Green-Lipped Mussel as follows: a) the flesh is extracted from live unfrozen mussels and homogenised approximately 10 volumes of approximately 45% aqueous phenol followed by stirring for about 45 minutes at about 20°C. b) that homogenised mixture is then subjected to centrifuging for approximately 10 minutes at substantially 250G. c) the upper aqueous layer (containing the carbohydrate-containing product) is then aspirated. d) the carbohydrate-containing product is then precipitated from that aqueous media with about 3 volumes of approximately 95% ethanol. e) the carbohydrate-containing product is then isolated by centrifuging for about 15 minutes at approximately 2300G. f) the carbohydrate-containing product from that centrifuging is then resuspended in the minimum amount of distilled water. g) the carbohydrate-containing product is then reprecipitated. h) there then follows dialysing of a suspension of the carbohydrate-containing product against distilled water for approximately 24 hours, i)the purified carbohydrate containing product is then subject to freeze drying, and j)there then follows a milling procedure to produce the solid anti-inflammatory substance in the form of a powder.

Such a process we believe (as with SEATONE™ product BIOLANE™ mussel extract ingredient) has a protein fraction as well as lipids and carbohydrates.

We believe the efficacy of such mussel extract on a dosage taken basis can be enhanced by removal of any significant non inflammatory component. Such a component we believe to be much of the protein content of such a product. We attribute most of the anti-inflammatory effect to the lipid and/or carbohydrate content of the extract.

We also believe that the anti-inflammatory efficacy of any efficacious component (whether lipid and/or carbohydrate) within such a product need not be diminished by subjecting the protein inflammatory extract of reduced protein content from the New Zealand Green-Lipped Mussel.

Accordingly in a first aspect the present invention consists in a protein reduced or protein devoid anti-inflammatory extract of carbohydrate(s) and lipid(s) from the New Zealand Green-Lipped Mussel (Perna canaliculus).

In another aspect the present invention consists in an anti-inflammatory extract from the New Zealand Green-Lipped Mussel (Perna canaliculus), being an extract of proteins, lipids and carbohydrates, which extract has at least to some extent been de-proteinated.

In still a further aspect the present invention consists in an extract from the New Zealand Green-Lipped Mussel which is at least substantially of carbohydrate(s) and lipid(s) components extracted,without any substantial degree of denaturing of the carbohydrate(s) and/or lipid(s), from the flesh of the mussel and which carbohydrates and/or lipids are each present in the extract in excess of any protein content.

In yet a further aspect the present invention consists in an anti-inflammatory extract of carbohydrate(s) and lipid(s) from the New Zealand Green-Lipped Mussel (.Perna canaliculus) where the carbohydrate(s) and/or lipid(s) have been through a deproteination procedure.

In still a further aspect the present invention consists in a method of improving the efficacy on a weight for weight basis of any dosage form of an anti-inflammatory extract from the New Zealand Green-Lipped Mussel (Perna canaliculus) which comprises de-proteinating at least in part an extract of (i) anti-flammatory lipid(s) and anti-inflammatory carbohydrate(s) and (ii) proteins.

In still a further aspect the present invention consists in a downstream process for a New Zealand Green-Lipped Mussel extract ingredient or product form thereof, wherein said ingredient or product form thereof either; (a) has a protein fraction; and/or (b) has a significant non-anti inflammatory component; and/or (c) is made essentially according to the following method; (i) the flesh is extracted from live unfrozen mussels and homogenised approximately 10 volumes of approximately 45% aqueous phenol followed by stirring for about 45 minutes at about 20°C; (ii) that homogenised mixture is then subjected to centrifuging for approximately 10 minutes at substantially 250G; Intel factual Property Office of NZ (iii) the upper aqueous layer (containing the carbohydrate-containing product) is then aspirated; (iv) the carbohydrate-containing product is then precipitated from that aqueous media with about 3 volumes of approximately 95% ethanol; (v) the carbohydrate-containing product is then isolated by centrifuging for about 15 minutes at approximately 2300G; (vi) the carbohydrate-containing product from that centrifuging is then resuspended in the minimum amount of distilled water; (vii) the carbohydrate-containing product is then reprecipitated; (viii) there then follows dialysing of a suspension of the carbohydrate-containing product against distilled water for approximately 24 hours; (ix) the purified carbohydrate containing product is then subject to freeze drying; and (x) there then follows a milling procedure to produce the solid anti-inflammatory substance in the form of a powder; and wherein said process is a de-proteination procedure that does not denature to any substantial extent anti-inflammatory carbohydrate(s) and/or lipid(s) thereof.

In still another aspect the present invention consists in a method of providing an extract of the New Zealand Green-Lipped Mussel (Perna canaliculus) which has a greater w/w anti-inflammatory effect and/or a reduced allergenic content than a New Zealand Green Lipped Mussel extract ingredient or product form there of which; (a) has a protein fraction; and/or (b) has a significant non-anti inflammatory component; and/or (c) is made essentially according to the following method; (i) the flesh is extracted from live unfrozen mussels and homogenised approximately 10 volumes of approximately 45% aqueous phenol followed by stirring for about 45 minutes at about 20°C; (ii) that homogenised mixture is then subj ected to centrifuging for approximately 10 minutes at substantially 250G; (iii) the upper aqueous layer (containing the carbohydrate-containing product) is then aspirated; (iv) the carbohydrate-containing product is then precipitated from that aqueous media with about 3 volumes of approximately 95% ethanol; (v) the carbohydrate-containing product is then isolated by centrifuging for about 15 Inteiiactual Properly Office of UZ - 6 NOV 2003 minutes at approximately 2300G; (vi) the carbohydrate-containing product from that centrifuging is then resuspended in the minimum amount of distilled water; (vii) ' the carbohydrate-containing product is then reprecipitated; (viii) there then follows dialysing of a suspension of the carbohydrate-containing product against distilled water for approximately 24 hours; (ix) the purified carbohydrate containing product is then subject to freeze drying; and (x) there then follows a milling procedure to produce the solid anti-inflammatory substance in the form of a powder; wherein said method involves deriving said extract or product form thereof or an extract which nonetheless provides an anti-inflammatory mixture of carbohydrate(s) and/ lipid(s) and extracted protein(s), and subjecting such extract to a de-proteination procedure that does not substantially denature or otherwise reduce the anti-inflammatory efficacy or the anti-inflammatory component or components of the carbohydrate(s) and lipid(s).

In still a further aspect the present invention consists in an anti-inflammatory composition comprising the lipid and carbohydrate fractions of the New Zealand green lipped mussel in combination.

Preferably the composition is substantially free of protein from the green lipped mussel or is a blend of carbohydrates and lipids from the green lipped mussel each substantially free of protein. As used herein "substantially free" means at least less than 10% w/w of the overall composition.

In still a further aspect the present invention consists in a method of preparing a composition as aforesaid which comprises isolating from the New Zealand green lipped mussel a carbohydrate fraction substantially free of protein and a lipid fraction substantially free of protein and thereafter blending the two to provide the composition.

In another aspect the present invention consists in an anti-inflammatory extract prepared by any procedure as aforesaid.

Preferably the de-proteination procedure is substantially as hereinafter described with reference to any one of Figures 1 to 4 of the accompanying drawings.

In still a further aspect the present invention consists in a method of de-proteination of an extract of the New Zealand Green-Lipped Mussel, said process comprising taking a said extract having a protein content as well as a carbohydrate and lipid content, fnisjb-.tital Prooerty O nies of NZ ~B NOV 2003 reacting in aqueous conditions the protein fraction with urea or one or more salts or compounds and/or mixed salts or compounds of the elements boron, manganese and/or potassium, and thereafter isolating from the or a precipitate of the reaction mixture (or part thereof) the at least substantially de-proteinated mussel extract.

In one embodiment urea (eg; at a 4 molar concentration) or other suitable amide is used.

In another, more preferred embodiment, a mineral or mineral salts is (are) used. Such salts can be of any suitable cation (eg; sodium, potassium, manganese, boron, etc.) Suitable anions for such preferably water soluble salts at ambient temperatures abound. Examples include sodium chloride (eg; 1M, 4M, etc. solutions), potassium salt solutions, (eg; 1 M potassium acetate, etc.) manganese salt solution (eg; 0.5M, etc. solutions) with anions including any one or more of, for example, halides (eg; CI"), the nitrate; the sulphate and the carbonate. Other options are suitable boron compounds in solution or boron salt solutions (eg; 0.5M, etc, solutions) including solutions of boric acid or reaction products thereof, and mixtures of any of the aforementioned solutions (eg; with 0.5M manganese cations, 0.5M potassium cations, and 0.5M boron ions).

In a further embodiment at least one manganese salt and/or at least one boron salt or compound and/or at least one potassium salt is (are) used.

In yet a further embodiment at least the mineral salts used result from an aqueous solution of boric acid, potassium acetate, and manganese sulphate.

In yet another aspect the mineral salts used result from an aqueous solution of boric acid, potassium acetate, and manganese sulphate in the cation ratio of about 4:17:1.

The present invention also consists in a method as aforesaid when performed at least substantially as herein described with reference to any one or more of the examples or Figures 1 to 4 of the accompanying drawings.

In still a further aspect the present invention consists in an extract prepared by a process as previously defined.

In still a further aspect the present invention consists in, as a food stuff, food supplement and/or an orally administrable composition, an extract as defined in the proceeding paragraph.

A preferred form of the present invention will now be described with reference to the accompanying drawings in which Figure 1 is a preferred flow diagram of the procedure commencing with the BIOLANE™ Property Office of NZ - 6 KOV 2033 freeze-dried Green-Lipped Mussel Extract used in the SEATONE™ product, Figures 2A and 2B are variants of a preferred overall procedure from still live fresh mussels, and Figure 3 is a more preferred flow diagram to that of Figure 1, and Figure 4 is a more preferred overall procedure from still live fresh mussels.

In one form of the present invention the procedure to provide the material to be subject to de-proteination is preferably performed by a procedure at least substantially to completion as disclosed herein in respect of the BIOLANE™ Mussel Extract used in the SEATONE™ product or in the aforementioned NZ 314867 (McFarlane).

Preferably the deproteination procedure is as disclosed in Figure 1. This rises to a yield of de-proteinated mussel extract of greater than 20%. We believe in use the extractions will provide (with respect to the starting point of the BIOLANE™ Mussel Extract powder ingredient of the SEATONE™ product) a yield of from 23% to 30% w/w in the form of a largely de-proteinated extract.

We have determined that the anti-inflammatory activity of such a derivative product is effective as an anti-inflammatory agent (increases of about 64% w/w over the BIOLANE™ mussel extract ingredient of the SEATONE™ product have been shown) owing to no significant degree of denaturing of the anti-inflammatory components and the removal of the unwanted inclusions.

In practice, it is desirable to avoid the freeze-dry at the end of the BIOLANE™ mussel extract process or the McFarlane process. This greatly improves the economies from the initial live mussel flesh through to the de-proteinated product. See Figures 2A and Figures 2B.

Figure 2A shows a less preferred procedure of acidification (eg; with a weak organic acid such as tartaric) prior to centrifuging. The procedure of Figure 2B is to be preferred as it ensures better product stability.

Figure 3 shows a more preferred form to that of Figure 1 where, instead of the addition of urea for the purpose of the reaction in aqueous conditions with the protein, fraction mineral salts are added. This change in the procedure is preferred for the deproteinisation since both manganese cations and boron cations are anti-inflammatory and their use instead of urea obviates the need for dialysis or equivalent process to remove residual urea in the production process.

Indeed, in the procedure from the live fresh mussels, Figure 4 shows an alternative to the procedures shown in Figure 2B where, if desired (and we believe it is more preferable), the Intellectual Property Office of NZ - 6 NOV 2053 - - ; - ■ "* ^ ;acidification can follow the centrifuging and be immediately prior to the addition of the mineral salts (instead of urea or an amide) for the purpose of protein solubilisation. ;It can be seen that by use of the cations of manganese and the cations of boron, which need not be removed owing to their having an advantageous presence rather than a deleterious presence (i.e. they are in fact anti-inflammatory in their own right), there is no need in a procedure of Figure 3 or Figure 4 for a dialysis for the purpose of removal of unwanted species which, in the process of Figures 1 and 2, was unreacted urea. ;With the processes of the present invention residual levels of the protein solubilising agent(s) are below that (those) for tolerability or recommended daily allowances. ;Procedures of the present invention satisfy the objects earlier stated. ;Examples: ;The invention is further described with reference to the following examples. It will be appreciated that the invention is not to be construed as limited to the examples. ;Example 1 - Extraction with Urea Solution: ;Urea solution (2 molar concentration, 172 ml) was added to mussel slurry (458 g) and stirred at room temperature for 7 hours. The mixture was allowed to stand overnight at 4°C. The mixture was centrifuged at 5000 rpm at 30 min. The supernatent was discarded and the solid pellet was collected and dialysed as a concentrated slurry against distilled. The retentate of this dialysis was freeze dried. The final product was a fine light brown powder (yield 11.3 g, 2.5% wt/wt of slurry). ;Analysis of the powder (Folch extraction) showed the lipid content to be 19.5% wt/wt. In comparison, the liquid content of freeze dried mussel powder (no urea extraction, centrifugation or dialysis) was only 8.7% wt/wt. ;The anti-flammatory activity of the water soluble portion of the powder was determined by a carrageenan foot-pad swelling model and produced a 53% reduction in swelling. In comparison, freeze dried mussel powder (no urea extraction, centrifugation or dialysis) produced only 20% inhibition in swelling. ;Example 2 - Extraction with Sodium Chloride Solution: ;Sodium chloride solution (4 molar concentration, 167 ml) was added to mussel slurry (446 g) and stirred at room temperature for 7 hours. The mixture was allowed to stand overnight at ;•wr■ ■fTT»r«iiiMtfja— ■■■ immmiW ;i INTELLECTUAL PROPERTY ;p opqnp ;- 5 KAH 2004 ;1 ;J RECEIVED ;- 10- ;Sodium chloride solution (4 molar concentration, 167 ml) was added to mussel slurry (446 g) and stirred at room temperature for 7 hours. The mixture was allowed to stand overnight at 4°C. The mixture was centrifuged at 5000 rpm for 30 min. The supernatent was discarded and the solid pellet was collected and dialysed as a concentrated slurry against distilled. The retentate of this dialysis was freeze dried. The final product was a fine light brown powder (yield 14.5 g, 3.3% wt/wt of slurry). Again, the reduced yield when compared to the centrifuged/dialysed control demonstrates that material was removed using the extraction step. ;Analysis of the powder (Folch extraction) showed the lipid content to be 20.4% wt/wt. The anti-flammatory activity of the water soluble portion of the powder was determined by a carrageenan foot-pad swelling model and produced a 56% reduction in swelling. ;Example 3 - Extraction with Boron/Potassium/Manganese Salt Solution: ;A salt solution of containing 1 molar concentration of cations (boron, potassium and manganese) was prepared in the following proportions: boric acid (37 ml, 1 M), potassium acetate (154 ml, 1 M, sufficient to adjust the pH up to 7.0) and manganese sulphate (9 ml, 1 M). This salt solution (179 ml) was added to mussel slurry (478 g) and stirred at room temperature for 7 hours. The mixture was allowed to stand overnight at 4°C. The mixture was centrifuged at 5000 rpm for 30 min. The supernatent was discarded and the solid pellet was collected and freeze dried. The final product was a fine light brown powder (yield 24.1 g, 5% wt/wt of slurry). ;Bioassays of the Residual Product Following De-Proteination: ;The residual products, following separation from the solubilised protein fraction of the mussel slurry and freeze drying, were subjected to a bio assay which determines the degree of inhibition of oedema in a rat paw which has been injected with carrageenan. This is a standard method as employed by the pharmaceutical industry for the determination of anti-flammatory activity in test materials. ;Each of the trial derivatives (derivative refers to the residual products mentioned above) was subjected to the bioassay and the degree of inhibition of oedema determined. In addition, the initial slurry product was freeze dried and assayed as a control. The degree of inhibition of oedema is a direct measure of the anti-inflammatory activity of the test substance. ;The procedure for the bioassay involves 12 rats, 6 for control and 6 for the test substance. ;Each of the rats has the volume of each of its two rear feet measured by the volume of water ;InteMsctual Property ;Office of f42 ;- 6 ^OV 2003 ;the injection of carrageenan into each rear paw. The control animals just have the carrageenan injected into each rear paw. After three hours the maximum swelling will have occurred and at this time the volume of each of the rear paws of all the animals is measured again. The difference in volume between the test and the control animals determines the mean level of inhibition caused by the test substance over the twelve paws involved. ;The results of the bioassay of the test derivatives was conducted separately on an aqueous extract and ethanolic extract to allow a soluble injectable test substance to be used. The net inhibition is assessed as the sum of the two individual inhibitions. ;The results indicated that there was little difference between the use of 8 molar urea and 4 molar urea as the solubilising agent, 63.6% and 62.9% inhibition respectively. A better result was achieved with the use of 4 molar sodium chloride, 70.1 % inhibition. A 1 molar sodium chloride solubilising agent produced a 18.9% inhibition. ;The level of anti-inflammatory activity achieved with these de-proteinated derivatives suggests a dual benefit due to a concentration factor plus the removal of an inhibitory factor from the original slurry rather than just a concentration effect. ;iLLECTlj'u" PROPERTY OFRCF Or- M.Z ;n,nr\ i. ;r M * "" 5 '

Claims (22)

CLAIMS:

1. An anti-inflammatory extract of carbohydrate(s) and lipid(s) from the New Zealand Green-Lipped Mussel (Perna canaliculus).

2. An anti-inflammatory extract from the New Zealand Green-Lipped Mussel (Perna canaliculus), being an extract of proteins, lipids and carbohydrates, which extract has at least to some extent been de-proteinated.

3. An extract from the New Zealand Green-Lipped Mussel which is at least substantially of carbohydrate(s) and lipid(s) components extracted, without any substantial degree of denaturing of the carbohydrate(s) and/or lipid(s), from the flesh of the mussel and which carbohydrates and/or lipids are each present in the extract in excess of any protein content.

4. An anti-inflammatory extract of carbohydrate(s) and lipid(s) from the New Zealand Green-Lipped Mussel (Perna canaliculus) where the carbohydrate(s) and/or lipid(s) have been through a deproteination procedure.

5. A method of improving the efficacy on a weight for weight basis of any dosage form of an anti-inflammatory extract from the New Zealand Green-Lipped Mussel (Perna canaliculus) which comprises de-proteinating at least in part an extract of (i) anti-flammatory lipid(s) and anti-inflammatory carbohydrate(s) and (ii) proteins.

6. A downstream process for processing a New Zealand Green-Lipped Mussel extract ingredient or product form thereof, wherein said ingredient or product form thereof either: (a) has a protein fraction; and/or (b) has a significant non-anti inflammatory component; and/or (c) is made essentially according to the following method; (i) the flesh is extracted from live unfrozen mussels and homogenised approximately 10 volumes of approximately 45% aqueous phenol followed by stirring for about 45 minutes at about 20°C; (ii) that homogenised mixture is then subj ected to centrifuging for approximately 10 minutes at substantially 250G; (iii) the upper aqueous layer (containing the carbohydrate-containing product) is then aspirated; (iv) the carbohydrate-containing product is then precipitated from that aqueous media with about 3 volumes of approximately 95% ethanol; (v) the carbohydrate-containing product is then isolated by centrifuging for about 15 minutes at approximately 2300G; INTELLECTUAL PROPERTY OFFICE OF N.Z. 16 DEC 2003 RECEIVED (vi) the carbohydrate-containing product from that centrifuging is then resuspended in the minimum amount of distilled water; (vii) the carbohydrate-containing product is then reprecipitated; (viii) there then follows dialysing of a suspension of the carbohydrate-containing product against distilled water for approximately 24 hours; (ix) the purified carbohydrate containing product is then subject to freeze drying; and (x) there then follows a milling procedure to produce the solid anti-inflammatory substance in the form of a powder; and wherein said process is a de-proteination procedure that does not denature to any substantial extent anti-inflammatory carbohydrate(s) and/or lipid(s) thereof.

7. A method of providing an extract of the New Zealand Green-Lipped Mussel (Perna canaliculus), which has a greater w/w anti-inflammatory effect and/or a reduced allergenic content than a New Zealand Green Lipped Mussel extract ingredient or product form there of which (a) has a protein fraction; and/or (b) has a significant non-anti inflammatory component; and/or (c) is made essentially according to the following method; (i) the flesh is extracted from live unfrozen mussels and homogenised approximately 10 volumes of approximately 45% aqueous phenol followed by stirring for about 45 minutes at about 20°C; (ii) that homogenised mixture is then subj ected to centrifuging for approximately 10 minutes at substantially 250G; (iii) the upper aqueous layer (containing the carbohydrate-containing product) is then aspirated; (iv) the carbohydrate-containing product is then precipitated from that aqueous media with about 3 volumes of approximately 95% ethanol; (v) the carbohydrate-containing product is then isolated by centrifuging for about 15 minutes at approximately 2300G; (vi) the carbohydrate-containing product from that centrifuging is then resuspended in the minimum amount of distilled water; (vii) the carbohydrate-containing product is then reprecipitated; (viii) there then follows dialysing of a suspension of the carbohydrate-containing product against distilled water for approximately 24 hows; Property OftioQ of NZ - e f'ov m (ix) the purified carbohydrate containing product is then subject to freeze drying; and (x) there then follows a milling procedure to produce the solid anti-inflammatory substance in the form of a powder; wherein said method involves deriving said extract or product form thereof or an extract which nonetheless provides an anti-inflammatory mixture of carbohydrate(s) and lipid(s) and extracted protein(s), and subjecting such extract to a de-proteination procedure that does not substantially denature or otherwise reduce the anti-inflammatory efficacy or the anti-inflammatory component or components of the carbohydrate(s) and lipid(s).

8. An anti-inflammatory composition comprising the lipid and carbohydrate fractions of the New Zealand green lipped mussel in combination.

9. A composition of claim 8 which is substantially free of protein from the green lipped mussel or is a blend of carbohydrates and lipids from the green lipped mussel each substantially free of protein.

10. A method of preparing a composition of claim 8 or 9 which comprises or includes isolating from the New Zealand green lipped mussel a carbohydrate fraction substantially free of protein and a lipid fraction substantially free of protein and thereafter blending the two to provide the composition.

11. An anti-inflammatory extract prepared by any procedure as of any one of claims 5 to 7.

12. An extract of claim 11 wherein the procedure is a de-proteination procedure is substantially as hereinafter described with reference to any one of Figures 1 to 4 of the accompanying drawings.

13. A method of de-proteination of an extract of the New Zealand Green-Lipped Mussel, said process comprising taking a said extract having a protein content as well as a carbohydrate and lipid content, reacting in aqueous conditions the protein fraction with urea or one or more salts or compounds and/or mixed salts or compounds of the elements boron, manganese and/or potassium, and thereafter isolating from the or a precipitate of the reaction mixture or part thereof the at least substantially de-proteinated mussel extract.

14. A method of claim 13 wherein urea or other suitable amide is used.

15. A method of claim 13 wherein a mineral or mineral salts is (are) used. INTELLECTUAL PROPERTY OFFICE OF N.Z. 16 DEC 2003 RECEIVED

16. A method of claim 15 wherein at least one manganese salt and/or at least one boron salt is used.

17. A method of claim 15 wherein at least one manganese salt and/or at least one boron salt or compound and/or at least one potassium salt is (are) used.

18. A method of claim 17 wherein the mineral salts used result from an aqueous solution of boric acid, potassium acetate, and manganese sulphate.

19. A method of claim 18 wherein the mineral salts used result from an aqueous solution of boric acid, potassium acetate, and manganese sulphate, in the cation ratio of about 4:17:1.

20. A method of any one of claims 13 to 15 when performed at least substantially as hereinbefore described with reference to any one or more of the examples or Figures 1 to 4 of the accompanying drawings.

21. An extract prepared by a process of any one of claims 13 to 17.

22. As a foodstuff, food supplement and/or an orally administerably composition, an extract of claim 18. °AY OF jy PER AGENTS intellectual Property Office of NZ - 6 2053 h:\libraiy\patents\djj\specs\427992ca