AU2022202246A1 - Water-soluble mussel extract - Google Patents

Abstract

The invention relates to a water-soluble mussel extract produced by enzymatic hydrolysis of mussel material. The extract comprises a high proportion of low molecular weight 5 peptides and demonstrates anti-inflammatory properties.

Description

WATER-SOLUBLE MUSSEL EXTRACT

This application is a divisional of Australian patent application 2016236863, which is the national phase entry of PCT International application PCT/NZ2016/050047 (published as WO 2016/153363), and claims priority from New Zealand provisional application 706298, filed 24 March 2015, each of which are incorporated herein by reference.

1. FIELDOFTHE INVENTION The present invention relates to water-soluble mussel extracts, compositions comprising the same, and their uses in nutraceutical and medicinal applications.

2. BACKGROUND OF THE INVENTION Shellfish, in particular, mussels, are known to be rich in compounds that support health. Shellfish extracts are an established nutraceutical category in the global marketplace.

Some extracts of shellfish have anti-inflammatory and other medicinal effects, often claimed to originate from the lipid fraction. Inflammation is associated with a wide variety of conditions, including arthritis, atherosclerosis and cancer. Current medical treatment of inflammation relies heavily on non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, many of which have undesirable side-effects. Therefore, nutraceutical shellfish preparations with fewer side-effects are highly desirable.

Many nutraceutical preparations of shellfish have been described. The actual chemical composition of such preparations depends on many factors including:

(a) the source material, including the species of shellfish, its age, and the environmental conditions in which it was grown; and

(b) the process used to obtain the preparation - for example, steps that selectively eliminate, retain, destroy or change certain classes of compounds will affect the chemical make-up of the final preparation.

To date, nutraceutical preparations of shellfish have focussed on whole animal extracts or the lipid fraction of the material. Supercritical CO2 extraction of freeze-dried material has been used extensively to produce mussel lipid extracts, particularly green-shelled mussel lipid extracts, such as those sold under the brand name Lyprinol TM .

However, whole mussel powders are insoluble and generally have an unpleasant "fishy" taste which makes them unsuitable for inclusion in many food products. Mussel lipid extracts are also insoluble and the "fishy" taste/odour can worsen with oxidation, which is likely to occur during many standard food processing steps. They must generally be administered as lipid-filled capsules, to disguise the unpleasant smell and taste.

Therefore, it would be advantageous to provide a water-soluble mussel extract with medicinal benefits, which is not subject to the disadvantages associated with comparable whole mussel powder and/or lipid extracts.

It is an object of the present invention to provide such an extract, or to at least provide the public with a useful choice.

3. SUMMARY OF THE INVENTION In one aspect, the invention provides a water-soluble mussel extract.

In one embodiment, the water-soluble mussel extract comprises at least about 45 wt% peptides on a solids basis, preferably, low molecular weight peptides.

In one embodiment, the water-soluble mussel extract comprises about 45 wt% to about 65 wt % peptides on a solids basis, preferably about 50 wt% to about 60 wt% peptides.

In one embodiment, greater than 90 wt%, preferably greater than 95 wt%, of the peptides are less than 5 KDa.

In one embodiment, greater than 75 wt%, preferably greater than 80 wt%, of the peptides are less than 2 KDa.

In one embodiment, greater than 35 wt%, preferably greater than 40 wt%, of the peptides are less than 1 KDa.

In one embodiment, the water-soluble mussel extract comprises no more than about 5 wt% lipid on a solid basis.

In one embodiment, the water-soluble mussel extract has anti-inflammatory activity.

In another aspect, the invention provides a method for producing a water-soluble mussel extract, the method comprising the steps of:

(a) hydrolysing an aqueous suspension of mussel material using one or more proteolytic enzymes;

(b) denaturing the enzymes in the hydrolysis mixture, and

(c) removing the insoluble material from the hydrolysis mixture to provide a water soluble mussel extract.

In one embodiment, the hydrolysis mixture is adjusted to pH 4 following denaturation of the enzymes.

In one embodiment, the water-soluble mussel extract is purified and/or concentrated.

In one embodiment, the mussel is selected from the group including but not limited to greenshell (or green-lipped) mussel (GSM) - Perna canaliculus, blue mussel - Mytilus edulis, and ribbed mussel (Maori name kopakopa) - Aulacomya atra maoriana. Preferably, the mussel is GSM.

In one embodiment, the mussel material is whole fresh mussel. In another embodiment, the mussel material is mussel powder produced by drying whole fresh mussel. In another embodiment, the mussel material is mussel supercritical marc, produced by supercritical fluid extraction of mussel powder or other mussel material.

In another aspect, the invention provides a nutraceutical composition comprising the water-soluble mussel extract of the invention and one or more consumable excipients.

In one embodiment, the nutraceutical composition comprises maltodextrin and/or anti oxidants.

In one embodiment, the nutraceutical composition comprises a food composition, food additive composition, dietary supplement or medical food.

In another aspect, the invention provides a pharmaceutical composition comprising the water-soluble mussel extract of the invention and one or more pharmaceutically acceptable excipients.

In one embodiment, the pharmaceutical composition comprises an oral dosage form, preferably a powder.

In another aspect, the invention provides a method for reducing inflammation in a subject in need thereof, the method comprising administering to the subject, a therapeutically effective amount of a water-soluble mussel extract of the invention.

In another aspect, the invention provides a method for preventing, treating or managing a condition associated with inflammation, in a subject in need thereof, the method comprising administering to the subject, a therapeutically effective amount of a water soluble mussel extract of the invention.

In one embodiment, the condition associated with inflammation is a chronic condition.

In one embodiment the condition associated with inflammation is selected from the group comprising asthma, encephalitis, inflammatory bowel disease including Crohn's disease and Ulcerative Colitis, chronic obstructive pulmonary disease (COPD), allergic disorders, fibrosis, arthritis including juvenile arthritis, psoriatic arthritis, rheumatoid arthritis and osteoarthritis, psoriasis, polymyalgia, tendonitis, bursitis, laryngitis, gingivitis, gastritis, otitis, celiac disease, diverticulitis, atherosclerosis, heart disease, obesity, diabetes, cancer and Alzheimer's disease.

In one embodiment, the condition associated with inflammation increases the levels of inflammatory TNFa and/or IL-1p.

In one embodiment, the water-soluble mussel extract of the invention is administered orally.

4. BRIEF DESCRIPTION OF THE FIGURES Embodiments of the invention will now be described with reference to the drawing in which:

Figure 1 is a graph showing the positive (dexamethasone) and negative (dimethyl sulfoxide, DMSO) assay controls for the inhibition of IL-10 in TLR4 (LPS) stimulated, monocytic THP-1 cells.

Figure 2 is a graph showing the effect of water-soluble extract from GSM marc powder on the inhibition of IL-10 in TLR4 (LPS) stimulated, monocytic THP-1 cells.

Figure 3 is a graph showing the effect of two water-soluble extracts from whole GSM powder on the inhibition of IL-10 in TLR4 (LPS) stimulated, monocytic THP-1 cells. Two enzyme combinations were tested for protein hydrolysis.

Figure 4 is a graph showing the effect of water-soluble extract from fresh whole blue mussels on the inhibition of IL-10 in TLR4 (LPS) stimulated, monocytic THP-1 cells.

Figure 5 is a graph showing the effect of water-soluble extract from fresh whole GSM (with defatting step) on the inhibition of IL-10 in TLR4 (LPS) stimulated, monocytic THP-1 cells.

Figure 6 is a graph showing the effect of water-soluble extract from fresh whole kopakopa on the inhibition of IL-10 in TLR4 (LPS) stimulated, monocytic THP-1 cells.

Figure 7 is a graph showing the positive (dexamethasone) and negative (DMSO) assay controls for the inhibition of TNFa in TLR4 (LPS) stimulated, monocytic THP-1 cells.

Figure 8 is a graph showing the effect of water-soluble extract from GSM marc powder on the inhibition of TNFa in TLR4 (LPS) stimulated, monocytic THP-1 cells.

Figure 9 is a graph showing the effect of two water-soluble extracts from whole GSM powder on the inhibition of TNFa in TLR4 (LPS) stimulated, monocytic THP-1 cells.

Figure 10 is a graph showing the effect of water-soluble extract from fresh whole blue mussels on the inhibition of TNFa in TLR4 (LPS) stimulated, monocytic THP-1 cells.

Figure 11 is a graph showing the effect of water-soluble extract from fresh whole GSM (with defatting step) on the inhibition of TNFa in TLR4 (LPS) stimulated, monocytic THP-1 cells.

Figure 12 is a graph showing the effect of water-soluble extract from fresh whole kopakopa on the inhibition of TNFa in TLR4 (LPS) stimulated, monocytic THP-1 cells.

Figure 13 is a graph showing the effect of water-soluble extract from GSM marc powder (pilot scale) on the inhibition of IL-10 in TLR4 (LPS) stimulated, monocytic THP-1 cells.

Figure 14 is a graph showing the effect of water-soluble extract from GSM marc powder (pilot scale) on the inhibition of TNFa in TLR4 (LPS) stimulated, monocytic THP-1 cells.

Figure 15 is a graph showing the effect of unhydrolysed GSM marc powder on the inhibition of IL-10 in TLR4 (LPS) stimulated, monocytic THP-1 cells.

Figure 16 is a graph showing the effect of unhydrolysed GSM whole powder on the inhibition of IL-10 in TLR4 (LPS) stimulated, monocytic THP-1 cells.

Figure 17 is a graph showing the effect of unhydrolysed GSM marc powder on the inhibition of TNFa in TLR4 (LPS) stimulated, monocytic THP-1 cells.

Figure 18 is a graph showing the effect of unhydrolysed GSM whole powder on the inhibition of TNFa in TLR4 (LPS) stimulated, monocytic THP-1 cells.

Figure 19 is a positive product ion base chromatogram produced by LC-MS analysis of water-soluble extract PAR 58.

Figure 20 is a positive product ion base chromatogram produced by LC-MS analysis of unhydrolysed GSM marc powder.

Figure 21 is a positive product ion base chromatogram produced by LC-MS analysis of unhydrolysed GSM whole powder.

Figure 22 is a negative product ion base chromatogram produced by LC-MS analysis of water-soluble extract PAR 58.

Figure 23 is a negative product ion base chromatogram produced by LC-MS analysis of unhydrolysed GSM marc powder.

Figure 24 is a negative product ion base chromatogram produced by LC-MS analysis of unhydrolysed GSM whole powder.

5. DETAILED DESCRIPTIONOFTHE INVENTION

5.1 Definitions As used herein "a" or "an" means at least one, unless clearly indicated otherwise.

As used herein, the term "about" refers to a value that is no more than 10% above or below the value being modified by the term.

The term 'comprising' as used in this specification and claims means 'consisting at least in part of'. When interpreting statements in this specification and claims which include the term 'comprising', other features besides the features prefaced by this term in each statement can also be present. Related terms such as 'comprise' and 'comprised' are to be interpreted in similar manner.

As used herein, the term "therapeutically effective amount", in the context of the administration of therapy to a subject, means the amount which is sufficient to reduce or ameliorate the severity, duration of a condition or one or more symptoms thereof, prevent advancement of the condition, cause regression of the condition, prevent the recurrence, development or onset of the condition or enhance or improve the prophylactic or therapeutic effect of another therapy.

As used herein, the terms "manage", "managing" and "management" in the context of the administration of therapy to a subject refer to the beneficial effects that a subject derives from the therapy, while not resulting in a cure of the condition. For example, management of the condition includes preventing a worsening of the condition.

As used herein, the terms "prevent", "preventing" and "prevention" in the context of the administration of therapy to a subject refer to the prevention or inhibition of the recurrence, onset or development of the condition resulting from administration of the therapy.

As used herein, the terms "treat", "treatment" and "treating" in the context of the administration of therapy to a subject refer to the reduction or amelioration of the progression, severity and/or duration of the condition, or the amelioration of one or more symptoms thereof, resulting from administration of the therapy.

As used herein, the term "water-soluble" when applied to a material, means that the material has at least 45% (w/v) solubility.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

In the description in this specification reference may be made to subject matter that is not within the scope of the claims of the current application. That subject matter should be readily identifiable by a person skilled in the art and may assist in putting into practice the invention as defined in the claims of this application.

5.2 The method of producing the water-soluble mussel extract of the invention

The water-soluble mussel extract of the invention is readily prepared using conventional processing techniques, as described below.

The invention provides a method for producing a water-soluble mussel extract, the method comprising the steps of:

(a) hydrolysing an aqueous suspension of mussel material using one or more proteolytic enzymes;

(b) denaturing the enzymes in the hydrolysis mixture, and

(c) removing the insoluble material from the hydrolysis mixture to provide a water soluble mussel extract.

The term "mussel" as used herein, refers to the edible bivalves of the marine family Mytilidae. In one embodiment, the mussel used in the method of the invention is a New Zealand mussel.

In one embodiment, the mussel is selected from the group including but not limited to GSM, blue mussel and kopakopa. Preferably, the mussel is GSM.

In one embodiment, the mussel material is "whole fresh" mussel. This can be obtained by collecting the material left following removal of the mussel shell, foot and beard. In another embodiment, the mussel material is dried mussel powder. This is generally obtained by drying whole fresh mussel, usually by freeze-drying, and grinding into a powder.

In another embodiment, the mussel material is mussel supercritical marc. The term "marc" generally refers to the organic material remaining after extraction of a biological material such as plant or animal material. As used herein, the term "supercritical marc" means the organic material remaining following supercritical fluid extraction.

As used herein, the term "mussel supercritical marc" means the organic material remaining following supercritical fluid extraction of mussel flesh or mussel powder.

In one embodiment, the mussel material is mussel supercritical marc, preferably GSM supercritical marc.

In the first step of the method, an aqueous suspension of mussel material is hydrolysed with one or more proteolytic enzymes.

Hydrolysis is carried out by incubating an aqueous suspension of mussel material with the proteolytic enzyme(s) under conditions optimised for the enzyme or enzyme system used. Typically, the pH should be adjusted to somewhere between about 5.5 to about 8. Optionally, anti-oxidants can be added to the aqueous suspension, for example, Oxyless U.

The aqueous suspension will typically be heated to about 30 to about 65 0 C for 1 to 24 hours, depending on the enzyme system used. Generally, the mussel material is suspended in cold water, and the mixture heated to the required temperature. However, the mussel material can alternatively be suspended in water already heated.

During hydrolysis, the proteins present in the mussel material will be hydrolysed to low molecular weight peptides. Hydrolysis must be carried out enzymatically, to deliver peptides of the appropriate molecular weight range. Chemical hydrolysis, for example, with acid, is non-selective. While not being bound by theory, it is believed that the anti inflammatory properties of the water-soluble mussel extract of the invention reside in specific low molecular weight peptides present. Processing steps that destroy these peptides or prevent their formation, will alter the anti-inflammatory activity of the extract.

The proteolytic enzymes can be of any type, for example, endo-, exo proteases/peptidases, aminopeptidases, serine proteases, metalloproteases, cysteine proteases and the like. In one embodiment, the proteolytic enzymes each have a pH optimum of slightly acidic to about neutral. In one embodiment, the proteolytic enzymes are non-animal enzymes.

Suitable proteolytic enzymes include papain, Alcalase (subtilisin), Enzidase FP (an endo/exo peptidase derived from a selected strain of Aspergillus oryzae var.) and Enzidase Neutral (a metallo neutral endopeptidase derived from a controlled fermentation of a non-genetically modified strain of Bacillus amyoliquefaciens).

In one embodiment, the proteolytic enzymes are selected from the group comprising papain, Enzidase FP, Enzidase Neutral, Alcalase and the like.

In one embodiment, the proteolytic enzymes comprise papain and Enzidase FP.

In another embodiment, the proteolytic enzymes comprise Alcalase, Enzidase Neutral and Enzidase FP.

The temperature of the aqueous suspension of mussel material during hydrolysis should not be so hot as to degrade the enzyme or inhibit its activity. The duration of the hydrolysis will depend on the activity of the enzyme(s) used and the mussel material.

The hydrolysis step is performed under stirring, to optimise contact between the enzyme(s) and the mussel material.

Once hydrolysis is complete the enzymes are denatured. In one embodiment, the hydrolysis mixture is heated under conditions sufficient to denature the proteolytic enzyme(s). In one embodiment, the hydrolysis mixture is heated at about 800 C to about 95 0 C for about 30 to about 40 min. In another embodiment, the hydrolysis mixture is heated at about 80 0 C to about 95 0 C for about 20 to about 40 min.

In one embodiment, the aqueous suspension of hydrolysed mussel material can optionally be treated to remove lipids before the enzymes are denatured. Lipid removal can be effected using any method known in the art, for example, using physical techniques such as centrifugation or chemical extraction.

In a chemical extraction, the water is removed from the aqueous suspension of hydrolysed mussel material, which is then contacted with a solvent in which the lipid fraction is soluble. The defatted product (mussel marc) is then re-suspended in water and the hydrolysis enzymes denatured.

In another embodiment, the aqueous suspension of hydrolysed mussel material is freeze dried to form a powder, which is rinsed with an organic solvent to remove the lipid. Examples of solvents that can be used include but are not limited to, acetone, ethanol, isopropyl alcohol, hexane, ethyl acetate and dimethylformamide.

In another embodiment, the aqueous suspension of hydrolysed mussel material is dried and extracted with a supercritical solvent such as CO 2 or C02/ethanol to remove the lipid fraction. The defatted product (mussel marc) is then re-suspended in water and the hydrolysis enzymes denatured.

The optional defatting step may be used where the mussel material is reasonably high in lipids. Where the mussel material has a low lipid content, for example, whole fresh or powdered blue mussels or when mussel marc is used, the defatting step can be omitted.

Following denaturation of the hydrolysis enzymes, the pH of the mixture may be adjusted to about 3.5 to about 4.2, preferably about 4, to prevent microbial spoilage. Agents such as phosphorinic acid, citric acid or hydrochloric acid can be used to adjust the pH.

The insoluble material present in the hydrolysis mixture is then removed to provide the water-soluble mussel extract of the invention.

The insoluble material can be removed by any method known in the art. In one embodiment, the insoluble material is removed by centrifuging the hydrolysis mixture and recovering the supernatant.

Generally the pH is adjusted before the removal of the insoluble material but if the latter contains shell fragments, this insoluble material should be removed first to prevent solubilisation of the shell by acid.

The water-soluble mussel extract of the invention may be treated to remove impurities contributing to undesirable colour and/or odour, for example, by contact with activated charcoal. The charcoal can be contained in a vessel and the product passed through it. Alternatively, the carbon, remaining fines and lipid residues can be removed using a filter press or equivalent, coated with diatomaceous earth.

The water-soluble mussel extract may also be concentrated by removing some or all of the water present.

In one embodiment, the water-soluble mussel extract is dried to provide a powdered extract. Drying can be performed using any technique known in the art including freeze drying.

Agents such as maltodextrin and anti-oxidants may be added to the water-soluble mussel extract of the invention.

Examples 1 - 8 describe methods of producing the water-soluble mussel extracts of the invention.

5.3 The water-soluble mussel extract of the invention The method of the invention described above produces a water-soluble mussel extract which is rich in peptides, particularly low molecular weight peptides. As used herein, the term "low molecular weight peptides" means peptides that are 5 kDa or less in weight.

The extract has an acceptable taste with no bitterness. The extract can be frozen with no cryo-precipitation. The powder solubility characteristics of the mussel extract do not change on dry storage.

The water-soluble mussel extract of the invention mainly comprises peptides, in particular, low molecular weight peptides. As can be seen in the molecular weight profiles of the extracts produced in Examples 1-8, over 90% of peptides present in the extracts are smaller than 5kDa.

A water-soluble extract of the invention comprises many more compounds than the unhydrolysed starting material, as can be seen in Figures 19-24 which show the results of LC-MS analysis carried out on water-soluble GSM mussel extracts of the invention, and GSM marc and whole powder starting materials.

In one embodiment, the water-soluble mussel extract comprises at least about 45 wt% peptides on a solids basis, preferably, low molecular weight peptides.

In another embodiment, the water-soluble mussel extract comprises at least about 45 wt% to about 65 wt % peptides on a solids basis, preferably about 50 wt% to about 60 wt% peptides.

In one embodiment, greater than 90 wt%, preferably greater than 95 wt%, of the peptides are less than 5 KDa.

In one embodiment, greater than 75 wt%, preferably greater than 80 wt%, of the peptides are less than 2 KDa.

In one embodiment, greater than 35 wt%, preferably greater than 40 wt%, of the peptides are less than 1 KDa.

Some carbohydrate material, lipids and other material and minerals may also be present.

The peptide content of the extract is calculated on the basis of the "solids" present, so as to remain unaffected by its concentration. The "solids" present in the extract constitute the material remaining when any solvents present, including water are removed. The "solids" present include peptides, carbohydrates and lipids as well as any other non solvent material.

The solubility of the extract of the invention is at least 45% (w/v). The solubility can be determined by saturating water with the extract (1:1 ratio), centrifuging, then drying the resulting supernatant to establish the dissolved weight.

In one embodiment, the water-soluble mussel extract of the invention is a dried powder.

The wt % of peptides present in the extract is calculated excluding solid excipients added to the extract such as maltodextrin and anti-oxidants.

In one embodiment, the invention provides a water-soluble mussel extract comprising at least about 45 wt% peptides, on a solids basis, wherein greater than 90 wt% of the peptides are less than 5 KDa.

In another embodiment, the invention provides a water-soluble mussel extract comprising about 50 wt% to about 60 wt % peptides on a solids basis, wherein greater than 95 wt%, of the peptides are less than 5 KDa.

5.4 Uses of the water-soluble peptide extract of the invention Inflammation is a process involving a complex biological cascade of molecular and cellular signals that alter the physiology of the organism. While acute inflammation protects and heals the body following physical injury or infection, chronic inflammation results in changes that play a key role in many degenerative diseases. Chronic inflammation is primarily mediated by monocytes and long-lived macrophages. Macrophages release chemical mediators including IL-1, IL-6 families, TNFa and prostaglandins. These mediators trigger up-regulation of other pro-inflammatory cytokines. IL-1, IL-6 and TNFa are major inflammatory biomarkers that are found in higher levels in inflamed cells than in resting cells.

Phagocytosis of bacteria or foreign particles is associated with an increase in oxygen uptake by neutrophils, called a respiratory burst. During this period, reactive oxygen species (ROS) such as hydroxyl radical, superoxide anion, singlet oxygen and hydrogen peroxide are produced. These species kill the invading microorganism or parasite.

Nutraceuticals can inhibit or reduce the inflammation process via several mechanisms including blocking expression of pro-inflammatory cytokines such as IL-1 and TNFa, inhibiting ROS generating enzyme activity or increasing ROS scavenging ability.

As set out in Example 9, the water-soluble mussel extracts of the invention have been found active against known markers of inflammation and hence, are believed to reduce or prevent inflammation. The activity shown is several times greater than the anti inflammatory activity of the unhydrolysed starting mussel material, such as whole power or marc, as shown in Tables 10 and 11.

In one aspect the invention provides a method for reducing inflammation in a subject in need thereof, the method comprising administering to the subject, a therapeutically effective amount of a water-soluble mussel extract of the invention.

In another aspect, the invention provides a method for preventing, treating or managing a condition associated with inflammation, in a subject in need thereof, the method comprising administering to the subject, a therapeutically effective amount of a water soluble mussel extract of the invention.

In another aspect, the invention provides a use of a water-soluble mussel extract of the invention in the manufacture of a medicament for reducing inflammation in a subject in need thereof.

In another aspect, the invention provides a use of a water-soluble mussel extract of the invention, in the manufacture of a medicament for preventing, treating or managing a condition associated with inflammation, in a subject in need thereof.

The invention also provides a water-soluble mussel extract of the invention for use in reducing inflammation in a subject in need thereof.

The invention also provides a water-soluble mussel extract of the invention for use in preventing, treating or managing a condition associated with inflammation, in a subject in need thereof.

In one embodiment, the condition associated with inflammation is a chronic condition.

In another embodiment, the condition associated with inflammation increases the level of inflammatory markers TNFa and/or IL-1p.

Examples of conditions associated with inflammation that can be prevented, treated or managed in accordance with the invention include but are not limited to asthma, encephalitis, inflammatory bowel disease including Crohn's disease and Ulcerative Colitis, chronic obstructive pulmonary disease (COPD), allergic disorders, fibrosis, arthritis including juvenile arthritis, psoriatic arthritis, rheumatoid arthritis and osteoarthritis, psoriasis, polymyalgia, tendonitis, bursitis, laryngitis, gingivitis, gastritis, otitis, celiac disease, diverticulitis, atherosclerosis, heart disease, obesity, diabetes, cancer and Alzheimer's disease.

The invention also provides a method for decreasing the level of inflammatory markers TNFa and/or IL-1p, in a subject in need thereof, the method comprising administering to the subject, a therapeutically effective amount of a water-soluble mussel extract of the invention.

A subject in need of prevention, treatment or management of a condition associated with inflammation is a subject diagnosed with such a condition, at risk of such a condition, or that has recovered from such a condition. A subject may be predisposed and/or at risk of the condition because of genetic and/or environmental factors.

In one embodiment the subject is a mammal, preferably a human. In another embodiment, the subject is a companion animal or horse.

Administration of the water-soluble mussel extract of the invention may be via a pharmaceutical composition or nutraceutical composition.

In one aspect, the invention provides a nutraceutical composition comprising the water soluble mussel extract of the invention and one or more consumable excipients.

In one embodiment, the nutraceutical composition of the invention is a food composition, food additive compositions, dietary supplement, or medical food composition.

The term "consumable" as used herein, means generally suitable for, or approved by a Government regulatory agency, for consumption by animals and humans.

The term "food" as used herein, means any substance, whether processed, semi processed or raw, which is intended for consumption by animals including humans and includes, but is not limited to, drink and chewing gum. A food composition of the invention comprises the water-soluble mussel extract of the invention in combination with food.

The term "food additive" as used herein, refers to any substance not normally consumed as a food by itself but added to food for a technical purpose. Examples are natural and artificial sweeteners, colouring agents, curing and pickling agents, flavours, emulsifiers, fat replacers, firming agents, leavening agents, lubricants, humectants, preservatives, stabilisers and thickeners. A food additive composition of the invention comprises the water-soluble mussel extract of the invention in combination with one or more food additives.

The term "dietary supplement" as used herein, refers to a product intended to supplement the diet that includes one or more of the following ingredients: vitamins, minerals, herbs, metabolites, extracts and the like. A dietary supplement is not typically intended to be used as the sole item of a meal, but can be consumed independently of any food. A dietary supplement of the invention comprises the water-soluble mussel extract of the invention in combination with one or more dietary supplements.

The term "medical food" as used herein refers to a food which is formulated to be consumed or administered enterally under the supervision of a medical practitioner. Medical foods are intended for the dietary management of conditions which have distinctive nutritional requirements. Examples of medical foods include, but are not limited to, sole source nutrition products, oral rehydration solutions and products intended for use in dietary management of metabolic disorders. A medical food composition of the invention comprises the water-soluble mussel extract of the invention in combination with a medical food.

In one embodiment, the nutraceutical composition of the invention is a food composition. The water-soluble extract of the invention is particularly suited for use as a food composition because it smells and tastes pleasant, and so can be added directly to food. Unlike other mussel products, it does not need to be encapsulated. Example 11 describes analysis of the volatile organic compounds present in two water-soluble extracts of the invention. The results, shown in Tables 13 and 14 are consistent with a pleasant tasting composition.

In one embodiment, the food composition is a bakery product including but not limited to breads, pizza bases, cakes, muffins, doughnuts, biscuits, tortilla, wraps, naans, noodles and pasta.

In another embodiment, the food composition is a liquid food including milk, fruit juice, smoothies, yogurt, soups and soft drinks. The water-soluble mussel extract of the invention can also be added to dry mixes such as instant soups, drinks, pudding and cake mixes.

In one aspect, the invention prodvides a pharmaceutical composition comprising the water-soluble mussel extract of the invention and one or more pharmaceutically acceptable excipients.

The term "pharmaceutically acceptable" as used herein, means approved by a Government regulatory agency for use in animals, in particular, humans.

The term "excipient" as used herein, includes vehicles, carriers, diluents, adjuvants, stabilizers or fillers with which the water-soluble mussel extract of the invention is stored, transported or administered. Suitable excipients are well known to those skilled in the art of pharmacy and include but are not limited to, starch (and its derivatives such as maltodextrin), glucose, sucrose, flour, silica gel, glycerol, sodium chloride, water, ascorbic acid and ethanol. Whether a particular excipient is suitable for incorporation into the pharmaceutical composition of the invention will depend on the way the dosage form is to be administered.

The pharmaceutical composition of the invention can be administered by any suitable route including but not limited to, parenteral, oral, intranasal, topical, transdermal, transmucosal and rectal.

The composition and shape of the dosage form will typically vary depending on usage. Examples of dosage forms include, but are not limited to, tablets, capsules, pills, pellets, capsules containing liquids, troches, lozenges, dispersions, suppositories, nasal sprays or inhalers, gels, suspensions, emulsions, solutions and elixirs.

In one embodiment, the invention provides an oral dosage form of the water-soluble mussel extract of the invention. Because of their ease of administration, whether as a nutraceutical or pharmaceutical composition, an oral dosage form is preferred. Typical oral dosage forms are prepared by combining the dried water-soluble mussel extract of the invention with at least one excipient suitable for use in solid oral dosage forms. The product is then shaped into the desired dosage form.

Such excipients include but are not limited to diluents, granulating agents, wetting agents, suspending agents, fillers, lubricants, binders and disintegrating agents.

Examples of binders include but are not limited to corn starch, potato starch or other starches, gelatin, gums such as acacia and guar, sodium alginate, powdered tragacanth, cellulose and its derivatives including carboxymethyl cellulose, pre-gelatinized cellulose, hydroxypropyl cellulose and microcrystalline cellulose. Examples of fillers include, but are not limited to talc, calcium carbonate granules or powder, microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, sorbitol and starch. Disintegrants ensure that a tablet disintegrates when exposed to an aqueous environment. Examples include, but are not limited to agar-agar, calcium carbonate, alginic acid, croscarmellose sodium, microcrystalline cellulose, pre-gelatinized starch, clays and gums. Lubricants include but are not limited to calcium stearate, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, sodium lauryl sulfate, hydrogenated vegetable oil and agar. Wetting agents include but are not limited to lecithin and polyoxyethylene stearate. Suspending agents include but are not limited to sodium carboxymethylcellulose, methylcellulose and sodium alginate.

Tablets can be prepared by compression or molding. Oral use dosage forms of the invention also include chewing tablets, hard gelatin capsules or soft gelatin capsules. Liquid preparations for oral administration include but are not limited to solutions, syrups or suspensions and may be presented as a dry product for reconstitution with water or another suitable vehicle before use.

The amount of the water-soluble mussel extract of the invention that will be effective in the prevention, treatment or management of a condition associated with inflammation will vary with the nature and severity of the disease or condition and the route by which the extract is to be administered, as well as factors specific to the subject such as their age, weight, sex and past medical history.

Generally, the dosage form used in the acute treatment of a condition associated with inflammation will comprise a larger amount of the water-soluble mussel extract of the invention than would be used in the treatment of the chronic condition. Similarly, a parenteral dosage form may contain less water-soluble mussel extract than would an oral dosage form. Formulation of the specific dosage form would be readily understood by a person skilled in the art of pharmacy. We refer to Remington's Pharmaceutical Sciences, Allen, et al., 2 2nd Ed. ISBN 978-0-85711-062-6.

A person skilled in the art can predict an effective amount from dose-response curves derived from in vitro or animal model tests. Generally, an effective amount of the water soluble mussel extract of the invention will be about 100 mg to about 3000 mg per day, either as a single daily dose or as divided doses throughout the day.

Various aspects of the invention will now be illustrated in non-limiting ways by reference to the following examples.

6. EXAMPLES

6.1 Materials and methods Oxyless U was sourced from Naturex (Avignon, France). Maltodextrin (Maltrin 100 or 150) was from New Zealand Starch (Auckland, NZ). Celite HyFlo and Celite 545 were from Imerys Filtration Minerals Inc. (San Jose, California, USA). Enzidase papain 6000L, Enzidase FP and Enzidase Neutral were from Zymus (Auckland, NZ). Alcalase was from Novozymes (Bagsvaerd, Denmark). All other chemicals and reagents were standard laboratory supplies.

Size exclusion HPLC setup Column: Yarral" 3pm SEC-2000 (Phenomenex). Sample preparation: 1. Dissolve freeze dried extract samples at the concentration of 10 mg/mL in 100 mM sodium phosphate buffer pH 6.8. 2. Dilute the solution in the ratio of 4 parts sample in phosphate buffer and one part 10% SDS to give a final concentration of 2% SDS. 3. Heat at 50°C for 5 minutes. 4. Centrifuge at 13 000 rpm for 5 minutes. 5. Load the supernatant into vials to run on the HPLC.

Running conditions for the SE-HPLC of peptides:

Parameter Setting Running buffer 100 mM sodium phosphate buffer, pH 6.8, with 0.5% SDS

Flow rate 1 mL/min Temperature 20 0 C Injection Volume Recommend 10 pL but up to 100 pL if necessary.

Loading Recommend loading approx 100 pg Run Time 20 minutes Detection 205 nm Maximum pressure 1000 psi

6.2 Extraction of mussels to produce water-soluble extracts of the invention Various mussel sources were processed in accordance with the Examples provided below. The processes and sample codes are summarised in Table 1 below.

Table 1: Summary of sample codes and corresponding process

Lipid removed Sample code Example Sourcematerial (PAR)

Yes, before enzyme 1 GSM marc 15, 13, 19, 30, 12 hydrolysis

2 GSM whole fresh No 37

3 GSM whole powder No 36, 36a

4 Blue mussel whole fresh No 40

5 GSM whole fresh Yes, after enzyme 41 hydrolysis

Yes, before enzyme 6 GSM marc (factory scale) Yds 43 hydrolysis

7 Kopakopa whole fresh No 45

Yes, before enzyme 8 GSM marc (pilot scale) 58, 63 hydrolysis

Example 1: Extraction of green-shell mussel supercritical marc (GSM marc extract) PAR 15

Marc resulting from supercritical CO 2 extraction of freeze-dried green-shell mussel (160 g) was suspended in 600 ml water. Oxyless U antioxidant (0.25 g) was added. Papain (5 ml) was added and the mixture heated to 55 0 C in a shaking water bath for 1 hr. Enzidase FP (0.5 g) was then added and hydrolysis continued for a further 1 hr at 55 0 C in a shaking water bath.

The hydrolysis mixture was heated for a further 30 min at 900 C, cooled to 550 C, then centrifuged at 10000 rpm for 30 minutes before recovery of the supernatant (565 ml). The pH of the supernatant was adjusted to pH 4 with phosphoric acid. Activated carbon was added (2g). The supernatant was filtered through Seitz 900 filter board using diatomaceous earth (20 g pre-coat (Celite HyFlo) + 20 g body feed (Celite 545)). The filtrate was recovered (510 ml) and mixed with maltodextrin (27.2 g) and Oxyless U antioxidant (0.25 g) until dissolved. The resulting solution was freeze-dried to produce 126.2 g solid product (GSM marc extract)

The GSM marc extract was fully water-soluble with a pleasant taste. 60% of the solids present in the starting supercritical C02 marc were recovered.

The procedure was repeated several times using the same enzyme system (PAR 13, 19, and 30) and once replacing Enzidase FP with Alcalase (PAR 12).

The molecular weight profile of the products, as analysed by SE-HPLC, is shown in Table 2 below:

Table 2: Molecular weight profile of peptides of GSM marc extract

Size range: Relative peak area (%) Expt >5kDa 2 -5kDa 1 - 2kDa <1kDa <5kDa

PAR 15 0.9 18.0 45.2 35.9 99.1

PAR 13 1.0 19.0 45.0 35.1 99.1

PAR 19 0.7 18.0 45.2 36.1 99.3

PAR 30 0.8 16.2 44.6 38.5 99.3

PAR 12 0.6 8.3 50.0 41.1 99.4

Example 2: Extraction of whole fresh green-shell mussel (GSM whole fresh extract) PAR 37

Homogenised flesh from green-shell mussel (500 g) was suspended in 400 ml water. Oxyless U antioxidant (0.25 g) was added. Alcalase (2.5 ml) and Enzidase Neutral (2.5 ml) were added and the mixture heated to 60 0 C in a shaking water bath for 3 hrs. Enzidase FP (0.125 g) was then added and hydrolysis continued for a further 1 hr at 60 0 C in a shaking water bath.

The hydrolysis mixture was heated for a further 40 min at 95 0 C, cooled to 550 C then centrifuged at 10000 rpm for 30 minutes before recovery of the supernatant (795 ml). The pH of the supernatant was adjusted to pH 4 with phosphoric acid. Activated carbon was added (2g). The supernatant was filtered through Seitz 900 filter board using diatomaceous earth (20 g pre-coat + 20 g body feed). The filtrate was recovered

(750 ml) and mixed with maltodextrin (27.2 g) and Oxyless U antioxidant (0.25 g) until dissolved. The resulting solution was freeze-dried to produce the solid product (GSM whole fresh extract).

The GSM whole fresh extract was fully water-soluble. 56% of the solids present in the shellfish raw material were recovered. The molecular weight profile of the product, as analysed by SE-HPLC, is shown in Table 3 below:

Table 3: Molecular weight profile of peptides of GSM whole fresh extract

Size range: Relative peak area (%) >5kDa 2 -5kDa 1 - 2kDa <1kDa <5ka

1.4 14.1 40.0 44.5 98.6

Example 3: Extraction of GSM whole powder (GSM whole powder extract) PAR 36

Freeze-dried powdered green-shell mussel (160 g) was suspended in 600 ml water. Oxyless U antioxidant (0.25 g) was added and the pH adjusted from 6.0 to 8.0. Alcalase (2.5 ml) and Enzidase Neutral (2.5 ml) were added and the mixture heated to 60 0 C in a shaking water bath for 3 hrs. Enzidase FP (0.125 g) was then added and hydrolysis continued for a further 1 hr at 600 C in a shaking water bath.

The hydrolysis mixture was heated for a further 40 min at 95 0 C, cooled to 550 C, then centrifuged at 10000 rpm for 30 minutes before recovery of the supernatant (590 ml). The pH of the supernatant was adjusted to pH 4 with phosphoric acid. Activated carbon was added (2g). The supernatant was filtered through Seitz 900 filter board using diatomaceous earth (20 g pre-coat + 20 g body feed). The filtrate was recovered (550 ml) and mixed with maltodextrin (27.2 g) and Oxyless U antioxidant (0.25 g) until dissolved. The resulting solution was freeze-dried to produce the solid product (GSM whole powder extract).

The GSM whole powder extract was fully water-soluble. 66% of the solids present in the starting mussel material were recovered.

The process was repeated replacing Alcalase and Enzidase Neutral with Papain (PAR 36a)

The molecular weight profile of the product, as analysed by SE-HPLC, is shown in Table 4 below:

Table 4: Molecular weight profile of peptides of GSM whole powder extract

Size range: Relative peak area (%) Expt >5kDa 2 -5kDa 1 - 2kDa <1kDa Tota

PAR 36 2.1 13.9 40.2 43.8 97.9

PAR 36a 2.6 15.1 41.9 40.3 97.3

Example 4: Extraction of whole fresh blue mussel (blue mussel whole fresh extract) PAR 40

Homogenised flesh from blue mussel (516 g) was suspended in 300 ml water. Oxyless U antioxidant (0.25 g) was added. The pH was adjusted from 6.0 to 8.0 then Alcalase (2.5 ml) and Enzidase Neutral (2.5 ml) added and the mixture heated to 60 0 C in a shaking water bath for 3 hrs. Enzidase FP (0.125 g) was then added and hydrolysis continued for a further 1 hr at 600 C in a shaking water bath.

The hydrolysis mixture was heated for a further 30 min at 95 0 C, cooled to 55 °C, then centrifuged at 10000 rpm for 30 minutes before recovery of the supernatant (746 ml). The pH of the supernatant was adjusted to pH 4 with phosphoric acid. Activated carbon was added (2g). The supernatant was filtered through Seitz 900 filter board using diatomaceous earth (20 g pre-coat + 20 g body feed). The filtrate was recovered (700 ml) and mixed with maltodextrin (27.2 g) and Oxyless U antioxidant (0.25 g until dissolved. The resulting solution was freeze-dried to produce the solid product (blue mussel whole fresh extract).

The blue mussel whole fresh extract was fully water-soluble. 83% of the solids present in the shellfish raw material were recovered. The molecular weight profile of the product, as analysed by SE-HPLC, is shown in Table 5 below:

Table 5: Molecular weight profile of peptides of blue mussel whole fresh extract

Size range: Relative peak area (%) >5kDa 2 -5kDa 1- 2kDa <1kDa Tota

0.3 12.3 42.3 45.1 99.7

Example 5: Extraction of whole fresh GSM including a defatting step (GSM defatted whole fresh extract) PAR 41

Homogenised flesh from GSM(507 g) was suspended in 400 ml water. Oxyless U antioxidant (0.25 g) was added. The pH was adjusted from 6.5 to 8.0 then Alcalase (2.5 ml) and Enzidase Neutral (2.5 ml) added and the mixture heated to 60 0 C in a shaking water bath for 3 hrs. Enzidase FP (0.125 g) was then added and hydrolysis continued for a further 1 hr at 600 C in a shaking water bath.

The hydrolysed material (890 ml) was then freeze-dried and 100g of powder recovered. The powder was rinsed with cold acetone (3 x 250 ml) to remove the lipids. The powder was recovered between rinses by filtering through paper. The final defatted powder was dried under nitrogen.

The defatted powder was re-suspended in 400 ml water, heated at 95C for 40 min and then cooled to 55 0 C. The hydrolysed aqueous suspension was then centrifuged at 10000 rpm for 30 minutes before recovery of the supernatant (415 ml). The pH of the supernatant was adjusted to pH 4 with phosphoric acid. Activated carbon was added (2g). The supernatant was filtered through Seitz 900 filter board using diatomaceous earth (20 g pre-coat + 20 g body feed). The filtrate was recovered (375 ml) and mixed with maltodextrin (27.2 g) and Oxyless U antioxidant (0.25 g) until dissolved. The resulting solution was freeze-dried to produce the solid product (defatted GSM whole fresh extract).

The defatted GSM whole fresh extract was fully water-soluble. 55% of the solids present in the shellfish raw material were recovered. The molecular weight profile of the product, as analysed by SE-HPLC, is shown in Table 6 below:

Table 6: Molecular weight profile of peptides of defatted GSM whole fresh extract

Size range: Relative peak area(%) >5kDa 2 -5kDa 1- 2kDa <1kDa Tota

1.1 14.7 41.5 42.7 98.9

Example 6: Factory scale extraction of GSM supercritical marc (factory GSM marc extract) PAR 43

Oxyless U antioxidant (80 g) was added to cold (19 0 C) water (500 L). Papain (200 g) was stirred in, followed by marc (50.0 kg) resulting from supercritical C02 extraction of freeze-dried greenshell mussel. The mixture was heated to 55 0 C and digested for 2 hr. Enzidase FP (160 g) was then added and hydrolysis continued for a further 1.5 hr at 55°C.

The hydrolysis mixture was heated for a further 20 min at 80-88 0 C, cooled to 60 °C, then centrifuged through a liquid-sludge separator (2X) before recovery of the supernatant (450 L). The pH of the supernatant was adjusted to 4 with phosphoric acid. Activated carbon was added (200g). The supernatant was filtered through a filter press using diatomaceous earth (6.0 kg pre-coat + 6.0 kg body feed). The filtrate was recovered (500 L) and mixed with maltodextrin (7.0 kg), and Oxyless U antioxidant (80 g) until dissolved. The resulting solution was freeze-dried to produce 24.0 kg solids, excluding additives (GSM marc extract).

The GSM marc extract was fully water-soluble with a pleasant fresh sea taste. 50% of the solids present in the starting supercritical C02 marc were recovered.

The molecular weight profile of the product, as analysed by SE-HPLC, is shown in Table 7 below:

Table 7: Molecular weight profile of peptides of factory GSM marc extract

Size range: Relative peak area (%) >5kDa 2 -5kDa 1 - 2kDa <1kDa <5ka

3.4 28.4 35.5 36.7 96.6

Example 7: Extraction of whole fresh kopakopa (kopakopa whole fresh extract) PAR 45

Homogenised kopakopa flesh (501 g) was suspended in 400 ml water. Oxyless U antioxidant (0.25 g) was added. The pH (6) was not adjusted. Papain (5 ml) was added and the mixture heated to 55 0 C in a shaking water bath for 2 hrs. Enzidase FP (0.5 g) was then added and hydrolysis continued for a further 1.5 hr at 550 C in a shaking water bath.

The hydrolysis mixture was heated for a further 30 min at 95 0 C, cooled to 55 °C, then centrifuged at 10000 rpm for 30 minutes before recovery of the supernatant (720 ml). The pH of the supernatant was adjusted to 4 with phosphoric acid. Activated carbon was added (2g). The supernatant was filtered through Seitz 900 filter board using diatomaceous earth (20 g pre-coat (Celite HyFlo) + 20 g body feed (Celite 545)). The filtrate was recovered (575 ml) and mixed with maltodextrin and Oxyless U antioxidant. The resulting solution was freeze-dried to produce the solid product (kopakopa whole fresh extract).

The kopakopa whole fresh extract was fully water-soluble. 55% of the solids present in the kopakopa raw material were recovered.

The molecular weight profile of the product, as analysed with SE-HPLC, is shown in Table 8 below:

Table 8: Molecular weight profile of peptides of kopakopa whole fresh extract

Size range: Relative peak area (%)

Expt >5kDa 2 - 5kDa 1- 2kDa <1kDa Tota

PAR 45 0.5 7.9 45.1 46.6 99.6

Example 8: Extraction of GSM marc powder (pilot scale) PAR 58

Oxyless U (24g) was added to 150L cold water. Papin (500ml) (Zymus Enzidase papain 6000L) was added with continuous stirring. GSM supercritical marc (15.0 kg) was added and the suspension heated at 500 C for 15 hours. Enzidase FP (50g) was added and hydrolysis continued for a further 2 hours at 550 C.

The suspension was then heated at 800 C for 20 minutes then allowed to cool.

The suspension was centrifuged through a liquid-sludge separator, with 60g activated carbon added during the process. The supernatant was again recovered and its pH changed to 4 with phosphoric acid. The supernatant was centrifuged through a liquid sludge separator. The final supernatant was recovered (95L). Maltodextrin (2.4kg) and Oxyless U (24g) were added and mixed until dissolved.

The water-soluble extract of the invention was then freeze-dried to produce a water soluble solid product. 69% of the solids present in the mussel marc were recovered. The taste was pleasant (savoury).

The procedure was repeated using the same enzyme system (500ml papain, digested for 21 hours at 50°C followed by 45g Enzidase FP hydrolysed for a further 2 hours at 55°C (PAR 63).

83% of the solids present in the supercritical GSM marc were recovered. The product was water-soluble and had a pleasant, savoury taste.

The molecular weight profiles of the products as analysed with SE-HPLC are shown in Table 9.

Table 9: Molecular weight profile of peptides of pilot scale GSM marc extracts

Expt >5kDa 2 - 5kDa 1- 2kDa <1kDa Toka

PAR 58 7.01 30.73 38.54 23.74 93.01

PAR 63 2.44 25.31 41.61 30.65 97.57

The proximate composition of PAR 58 was compared with that of the GSM marc starting material. The samples were dried for 16 hours at 1030 C, gravimetry. AOAC 945.15, 19th Edition. Ash was determined by ignition in muffle furnace 6000 C, 6 hours, gravimetry. AOAC 942.05, 19th Edition. Total Nitrogen was determined using Dumas combustion. AOAC 992.15, 19th Edition. Total Protein was calculated based on the total Nitrogen x 6.25. AOAC 992.15, 19th Edition. Values for Total Protein are also given based on a factor of 6.75 (which is more suitable for highly hydrolysed proteins where significant water has been added across the peptide bonds). Lipid was determined using the method of Bligh, E. G., & Dyer, W. J. (1959) "A rapid method of total lipid extraction and purification", Can. J. Biochem. and Physiol., 37(8), 911-917.

Table 10 below shows the results.

Table 10: Proximate composition for PAR 58 and GSM marc powder

PAR 58 GSM marc powder Moisture 10.6 4.3 Ash 19.6 28.0 Total Nitrogen 6.6 7.3 Total Protein 41.3 45.6 (N x 6.25) Total Protein 44.6 49.3 (N x 6.75) Lipid 1.1 6.1

The values are g/100g of freeze-dried material without any additives.

6.3 Anti-inflammatory properties of water-soluble extracts of the invention

Example 9: Inhibition of TLR4 (LPS) stimulated IL-113 and TNFa secretion in monocytic THP-1 cells by water-soluble extracts of the invention

IL-1p and TNFa are key mediators of the inflammatory response. They initiate a cascade of events that regulate the body's natural defences against invading microbes or trauma. Under normal conditions these pro-inflammatory cytokines are localized and short-lived. However, under chronic inflammatory conditions their expression is prolonged, and in certain situations, spread throughout the body. This perpetuates the pro-inflammatory response and underlies the abnormal recruitment and activation of immune cells.

Therefore limiting IL-1p and TNFa expression and biological activity in chronic inflammatory conditions will suppress the chronic inflammation. Moreover, a variety of immune cells are shown to express IL-1p and TNFa during chronic inflammation. However, the triggers for their expression are very diverse and they are the consequence of abnormal immune cell signalling to invading microbes.

As part of the natural defence system, humans have developed sophisticated cell surface markers, including a group known as Toll-like receptors (TLRs) that recognize foreign material and trigger the appropriate cell defence process.

Bacterial ligands that activate TLR4 in monocytic immune cells were utilised to up regulate the expression of IL-1p and TNFa to explore the efficacy of mussel extracts in suppressing the expression of these pro-inflammatory cytokines in chronic inflammatory conditions.

Methodology: Monocytic cell-line-THP-1 (2x10 6 cell/mL) grown in RPMI (Roswell Park Memorial Institute) media containing 10% FBS (fetal bovine serum) were incubated under mammalian cell culture conditions with TLR4-lipopolysaccharide [LPS] (0.5 50 ng/mL) for 6 hours. The supernatant was collected and measured for IL-1p and TNFa production using commercial ELISAs. Optimal doses of TLR4 (~70% of maximal bioactivity) were used to examine the effect of mussel extracts on IL-1p and TNFa secretion. A known immune suppressor - dexamethasone (0.001-10 uM or 0-3.925 pg/mL) was used to validate this bioassay.

The effectiveness of mussel extracts in attenuating the expression of IL-1p and TNFa in a chronic inflammatory scenario was assessed. THP-1 cells were initially stimulated with optimal doses of TLR4 for 30 mins to evoke the up-regulation of IL-1p and TNFa expression. Mussel extract (typically 1-100 pg/mL) was then added and incubated with the cells for a further 6 hrs. The culture media was then collected (by spinning down the cells) and measured for IL-1P and TNFa secretion by ELISA. Data was calculated as cytokine pg/mL and presented as percentage inhibition of TLR4-stimulated IL-1p or TNFa secretion.

The results are shown in Figures 1-18. All of the water-soluble extracts of the invention tested, demonstrated anti-inflammatory activity (Figures 1-14).

Figures 15-18 are the results of control experiments where unhydrolysed mussel materials were tested in the assays described above.

Figures 15 and 16 show the effect of GSM marc powder and GSM whole powder in the inhibition of IL-1 P in TLR4 (LPS) stimulated, monocyclic THP-1 cells.

Figures 17 and 18 show the effect of the same materials on the inhibition of TNFa in TLR4 (LPS) stimulated, monocyclic THP-1 cells.

Table 10 below shows the effect of various GSM products on the inhibition of IL-1p in TLR4 (LPS) stimulated, monocytic THP-1 cells, when applied at 100 pg/mL in the assay. The freeze-dried products were re-suspended in either phosphate-buffered saline (PBS) or dimethyl sulfoxide (DMSO) prior to addition to the assay.

For each column, the results are expressed relative to the effect of soluble extracts (highest inhibition), assigned a value of 1.

Table 10: Relative inhibition

Product GSM PBS DMSO Whole powder 0.4 - 0.5 0.4 - 0.5 Marc powder 0.2 - 0.3 0.5 - 0.6 Water-soluble extract of the invention

Table 11 below shows the effect of various GSM products on the inhibition of TNFU in TLR4 (LPS) stimulated, monocytic THP-1 cells, when applied at 100 pg/mL in the assay. The freeze-dried products were re-suspended in either phosphate-buffered saline (PBS) or dimethyl sulfoxide (DMSO) prior to addition to the assay.

For each column, the results are expressed relative to the effect of soluble extracts (highest inhibition), assigned a value of 1.

Table 11: Relative inhibition

Product GSM PBS DMSO Whole powder 0.2 - 0.3 0.4 - 0.5 Marc powder 0.1 - 0.2 0.1 - 0.2 Water-soluble extract of 1 1 the invention

6.4 Analysis of water-soluble extracts of the invention

Example 10: LC-MS analysis of GSM marc, whole powder and water-soluble extracts of the invention

Methods

Samples were proportioned and mixed with the aid of vortex in 0.5% formic acid aq. to dissolve polar components, however some samples maintained some undissolved solids as shown in Table 12 below.

Table 12: Concentration and solubility of samples

Sample mg.ml- 1 Insolubles Par 44 21.0 Low Par 58 10.9 Low Par36 Low GSM marc 10.4 High GSM whole 13.8 High

The LC-MS system consisted of a Thermo Electron Corporation (San Jose, CA, USA) Finnigan Surveyor MS pump, Thermo Accela Open Auto sampler (PAL HTC-xt with DLW), Finnigan Surveyor PDA plus detector and a ThermaSphere TS-130 column heater (Phenomenex, Torrance, CA, USA).

A 2 pL aliquot of each prepared extract was separated with a mobile phase consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) by aqueous normal phase chromatography (Cogent T " Diamond-Hydride T , 4 pm, 100A, 150 x 2.1 mm, M

MicroSolv T Technologies Corporation, USA) maintained at 30°C with a flow rate of 200pl/min. A gradient was applied: 0-2 min, 5% A; 30-39.5 min, 90% A; 40-45 min, 5% A.

Data was acquired by API-MS (LTQ, 2D linear ion-trap, Thermo-Finnigan, San Jose, CA, USA) with electrospray ionisation (ESI) in the negative and positive mode for precursor masses in the range m/z 100-2000 amu with product ions to MS.

The results are shown in Figures 19-24. The LC-MS chromatograms for the water-soluble extracts of the invention (Figures 19 and 22) indicate a more complex composition, with many more peaks than are seen in the LC-MS chromatograms of the starting mussel material (Figures 20, 21, 23 and 24).

Example 11: Solid-phase micro-extraction- GC-MS analysis of water-soluble GSM extracts

Volatile organic compounds in the mussel extracts PAR 58 and PAR 63 were analysed by SPME-GC-MS, using the method of Tuckey, Day, and Miller (Tuckey, N. P. L., Day, J. R., TM & Miller, M. R. (2013). Determination of volatile compounds in New Zealand Greenshel mussels (Perna canaliculus) during chilled storage using solid phase microextraction gas chromatography-mass spectrometry. Food Chemistry, 136(1), 218-223 ) with modifications. A sample of freeze-dried extract (1.20 g) was placed in a 20 mL glass vial fitted with a self-sealing septum. Two vials were prepared for each extract and each vial was analysed in duplicate. The vial was incubated at 40 °C for 4 min with agitation, prior to adsorption of volatiles for 20 min using a 85 pm carboxen poly(dimethyl siloxane)-coated SPME fibre (Supelco, Bellefonte, PA, USA). A blank fibre sample was run prior to and between each analytical run to ensure a clean baseline. The adsorbed volatiles were analysed by GC-MS. The GC-MS system consisted of a GC-2010 gas chromatograph coupled with a GCMS-QP2010 mass-spectroscopy unit (Shimadzu, Kyoto, Japan) and equipped with AOC-5000 automatic injector (PAL, CTC Analytics, Zwingen, Switzerland). The conditions for GC-MS analysis were as follows: injection temperature 250 °C; splitless mode; 2 min desorption in the injector; Rtx-5Sil MS capillary column - 30 m x 0.25 mm ID x 0.25 pm film thickness (Restek, Bellefonte, PA, USA); oven temperature program: 50 °C start, hold for 1 min, increase at 10 °C/min to 160 °C, increase at 40 °C/min to 250 °C, hold at 250 °C for 1 min; He carrier gas, flow rate 1.90 mL/min; MS detector temperature, 260 °C. The peaks were identified by comparison with the mass spectra database (Wiley 7.0 library, Wiley, New York, NY, USA). The results are shown below in Tables 13 and 14.

Table 13: Relative amounts of volatile organic compounds in PAR 58

Volatile organic compound Odour description Area

% Dimethyl sulfide Cabbage, sulfurous 1.4 Acetic acid Vinegar, sour 15.7 3-Methylbutanal Chocolate, malt 1.6 Acetoin Buttery, creamy 11.5 Propanoic acid Pungent, soy, body odour 33.4 N-methylformamide Faint fishy, ammonia-like 3.8 Butanoic acid Sharp, cheesy, sweaty 1.7 Dimethyl sulfoxide Faint garlic, oyster 5.2 2-Furanmethanol Faint burnt 6.7 Methional Cooked potato 1.2 Hexanoic acid Pungent, musty, cheesy 1.9 2-Methyl-2-propyl-1,3-propanediol Odourless 4.8

Table 14: Relative amounts of volatile organic compounds in PAR 63

Volatile organic compound Odour description Area

% Trimethylamine Fishy 2.0 Acetic acid Vinegar, sour 16.7 Acetoin Buttery, creamy 26.7 2-Propenoic acid Acrid, tart 2.3 N-methylformamide Faint fishy, ammonia-like 3.8 Butane-2,3-diol Odourless (faint sweet) 4.6 3-Methylbutanoic acid Cheesy, sweaty 2.9 2-Furanmethanol Faint burnt 1.6 Hexanoic acid Pungent, musty, cheesy 3.7 4-Methyl-2-hexanol Sweaty 3.9 Heptanoic acid Rancid 1.0 Nonanal Fatty, citrus, green 3.8 Butylamine Fishy, ammonia-like 2.8 Octanoic acid Cheesy, sweaty 2.0 Nonanoic acid Green, fatty 5.6 Toluene, 2,4-diisocyanate Sharp, pungent 2.6 3-Hydroxy-2,2,4-trimethylpentyl ester of Faint acidic, cheesy, sour isobutanoic acid 1.0

7. INDUSTRIAL APPLICABILITY The water-soluble mussel extracts of the invention and compositions comprising them can be added to food or dietary supplements to increase their health benefits. Their water-solubility and pleasant taste allows them to be directly incorporated into a wide range of consumable products. Products are likely to be in the form of: tablets, capsules, beverage health 'shots', premix soup tonics, and as components of functional foods, e.g. savoury health food bars.

They can also be used as pharmaceutical agents to help prevent, treat or manage conditions that are associated with inflammation.

Claims (18)

CLAIMS:

1. A water-soluble mussel extract.

2. A water-soluble mussel extract of claim 1 comprising at least about 45 wt% peptides on a solid basis.

3. A water-soluble mussel extract of claim 1 comprising about 45 wt% to about 65 wt% peptides on solids basis.

4. A water-soluble mussel extract of any one of claims 1-3 wherein greater than 90 wt% of peptides are less than 5 KDa.

5. A water-soluble mussel extract of any one of claims 1-4 which comprises no more than about 5 wt% lipids on a solids basis.

6. A water-soluble mussel extract of any one of claims 1-5 wherein the mussel is selected from the group comprising greenshell mussel (GSM), blue mussel and ribbed mussel.

7. A method for producing a water-soluble mussel extract, the method comprising the steps of:

(a) hydrolysing an aqueous suspension of mussel material using one or more proteolytic enzymes; (b) denaturing the enzymes in the hydrolysis mixture, and (c) removing the insoluble material from the hydrolysis mixture to provide a water-soluble mussel extract.

8. The method of claim 7 wherein the proteolytic enzymes have pH optimum of slightly acidic to about neutral.

9. The method of claim 7 wherein the proteolytic enzymes are selected from the group comprising papain, Alcalase, Enzidase FP and Enzidase Neutral.

10. A method of any one of claims 7-9 wherein the mussel material is selected from the group comprising whole fresh mussel, mussel powder and mussel supercritical marc.

11. A method of any one of claims 7-10 wherein the mussel is selected from the group comprising greenshell mussel, blue mussel and ribbed mussel.

12. A water-soluble mussel extract produced by a method of any one of claims 7-11.

13. A pharmaceutical composition comprising a water-soluble mussel extract of any one of claims 1-6 and one or more pharmaceutically acceptable excipients.

14. A nutraceutical composition comprising a water-soluble mussel extract of any one of claims 1-6 and one or more consumable excipients.

15. A nutraceutical composition of claim 14, which is a food composition, food additive composition, dietary supplement or medical food.

16. A method of reducing inflammation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a water soluble mussel extract of any one of claims 1-6.

17. A method for preventing treating or managing a condition associated with inflammation, in a subject in need thereof, the method comprising administering to the subject, a therapeutically effective amount of a water-soluble mussel extract of any one of claims 1-6.

18. A method of claim 17, wherein the condition associated with inflammation is selected from the group comprising asthma, encephalitis, inflammatory bowel disease including Crohn's disease and Ulcerative Colitis, chronic obstructive pulmonary disease (COPD), allergic disorders, fibrosis, arthritis including juvenile arthritis, psoriatic arthritis, rheumatoid arthritis and osteoarthritis, psoriasis, polymyalgia, tendonitis, bursitis, laryngitis, gingivitis, gastritis, otitis, celiac disease, diverticulitis, atherosclerosis, heart disease, obesity, diabetes, cancer and Alzheimer's disease.