This application claims priority from New Zealand provisional application 706301, filed 24 March 2015, which is incorporated herein by reference.

1. FIELD OF THE INVENTION

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

2. BACKGROUND OF THE INVENTION

Shellfish 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 type of shellfish, its age, and the environmental conditions under which it was grown; and

(b) the process used to obtain the composition - for example, steps that selectively eliminate or retain certain types of compounds and steps that actively destroy or change some types of compound will affect the chemical make-up of the final composition .

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-lipped mussel lipid extracts. Nutraceutical compositions prepared in this manner are sold under the brand name Lyprinol™.

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 a lipid-filled capsule, to disguise the unpleasant smell and taste.

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

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. SU MMARY OF TH E INVENTION

In one aspect the invention provides a water-soluble paua extract.

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

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

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

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

(a) hydrolysing an aqueous suspension of paua 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 paua extract.

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

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

In one embodiment, the paua is selected from the group including but not limited to Haliotis iris (Blackfoot paua), Haliotis australis (Silver paua/Yellow foot paua) and Haliotis virgenea.

In one embodiment, the paua material is guts and/or gonad left after removal of the edible muscle tissue.

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

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

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 paua 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 paua 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 administerin to the subject, a therapeutically effective amount of a water- soluble paua extract of the invention.

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

In one embodiment the condition associated with inflammation comprises 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- Ιβ.

In one embodiment, the water-soluble paua 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 figures in which :

Figure 1 is a graph showing the positive (dexamethasone) and negative (dimethyl sulfoxide, DMSO) assay controls for the inhibition of IL- Ιβ in TL 4 (LPS) stimulated, monocytic THP- 1 cells.

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

Figure 3 is a graph showing the effect of water-soluble extract from whole paua powder on the inhibition of IL- Ι β in TLR4 (LPS) stimulated, monocytic THP- 1 cells.

Figure 4 is a graph showing the effect of water-soluble extract from paua marc powder (pilot scale) on the inhibition of IL- Ιβ in TLR4 (LPS) stimulated, monocytic THP-1 cells.

Figure 5 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 6 is a graph showing the effect of water-soluble extract from paua marc powder on the inhibition of TNFa in TLR4 (LPS) stimulated, monocytic THP- 1 cells.

Figure 7 is a graph showing the effect of water-soluble extract from whole paua powder on 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 paua marc powder (pilot scale) on the inhibition of TNFa in TLR4 (LPS) stimulated, monocytic THP- 1 cells.

Figure 9 is a graph showing the effect of unhydrolysed paua marc powder on the inhibition of IL-Ιβ in TLR4 (LPS) stimulated, monocytic THP-1 cells.

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

Figure 11 is a graph showing the effect of unhydrolysed paua marc powder on the inhibition of TNFa in TLR4 (LPS) stimulated, monocytic THP-1 cells.

Figure 12 is a graph showing the effect of unhydrolysed paua whole powder on the inhibition of TNFa in TLR4 (LPS) stimulated, monocytic THP-1 cells.

Figure 13 is a positive product ion base peak chromatograph produced by LC-MS analysi of a water-soluble extract of the invention (PAR 44).

Figure 14 is a positive product ion base peak chromatograph produced by LC-MS analysi of a water-soluble extract of the invention (PAR 36).

Figure 15 is a positive product ion base peak chromatograph produced by LC-MS analysi of unhydrolysed paua marc powder.

Figure 16 is a positive product ion base peak chromatograph produced by LC-MS analysi of unhydrolysed paua whole powder.

Figure 17 is a negative product ion base peak chromatograph produced by LC-MS analysis of a water-soluble extract of the invention (PAR 44) .

Figure 18 is a negative product ion base peak chromatograph produced by LC-MS analysis of a water-soluble extract of the invention (PAR 36) . Figure 19 is a negative product ion base peak chromatograph produced by LC-MS analysis of unhydrolysed paua marc powder.

Figure 20 is a negative product ion base peak chromatograph produced by LC-MS analysis of unhydrolysed paua whole powder.

5. DETAILED DESCRIPTION OF THE 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 prog ression, 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 paua extract of the invention

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

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

(a) hydrolysing an aqueous suspension of paua 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 paua extract.

The term "paua" as used herein, refers to the edible marine gastropod molluscs of the family Haliotidae (there is only genus Haliotis), known in the United States and Australia as abalone, and in the United Kingdom as ormer shells. In one embodiment, the paua material is dried paua powder (whole powder) . This is generally obtained by drying the guts and gonad of fresh paua, usually by freeze-drying, and grinding into a powder but can include paua flesh .

In another embodiment, the paua material is paua supercritical marc (marc powder) . 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 "paua supercritical marc" means the organic material remaining following supercritical fluid extraction of paua material, preferably guts and gonads.

In one embodiment, the paua material is paua supercritical marc, preferably Haliotis iris.

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

Hydrolysis is carried out by incubating an aqueous suspension of paua 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° C for 1 to 24 hours, depending on the enzyme system used . Generally, the paua material is suspended in cold water, and the mixture heated to the required temperature. However, the paua material can alternatively be suspended in water already heated .

During hydrolysis, the proteins present in the paua 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 antiinflammatory properties of the water-soluble paua 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, metal loproteases, 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 amyloliquefaciens) .

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

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

The temperature of the aqueous suspension of paua 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 paua material.

The hydrolysis step is performed under stirring, to optimise contact between the enzyme(s) and the paua 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 80° C to about 95° C for about 20 to about 40 min.

In one embodiment, the aqueous suspension of hydrolysed paua 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 paua material, which is then contacted with a solvent in which the lipid fraction is soluble. The defatted product is then re-suspended in water and the hydrolysis enzymes denatured . In another embodiment, the aqueous suspension of hydrolysed paua 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 paua material is dried and extracted with a supercritical solvent such as CO2 or CC ethanol to remove the lipid fraction. The defatted product is then re-suspended in water and the hydrolysis enzymes denatured .

The optional defatting step may be used where the paua material is reasonably high in lipids. Where the paua material has a low lipid content, 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 paua 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 paua 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 paua extract may also be concentrated by removing some or all of the water present. In one embodiment, the water-soluble paua 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 paua extract of the invention .

Examples 1 - 3 describe methods of producing the water-soluble paua extracts of the invention.

5.3 The water-soluble paua extract of the invention

The method of the invention described above produces a water-soluble paua 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 paua extract do not change on d ry storage.

The water-soluble paua extract of the invention mainly comprises peptides, in particular, low molecular weight peptides. As can be seen from the molecular weight profiles of the extracts produced in Examples 1-3, over 90% of the peptides present in the extracts are smaller than 5KDa.

A water-soluble paua extract of the invention comprises many more compounds than the unhydrolysed paua material, as can be seen in Figures 13 - 20, which show the results of LC-MS analysis carried out on extracts of the invention, as well as paua marc and paua whole powder starting materials.

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

In another embodiment, the water-soluble paua 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 paua 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 paua 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 paua 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 4, the water-soluble paua 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 antiinflammatory activity of the unhydrolysed starting paua material, such as whole powder or marc, as shown in Tables 5 and 6.

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 paua 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 paua extract of the invention.

In another aspect, the invention provides a use of a water-soluble paua 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 paua 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 paua extract of the invention for use in reducing inflammation in a subject in need thereof. The invention also provides a water-soluble paua 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- Ιβ.

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- Ιβ, in a subject in need thereof, the method comprising administering to the subject, a therapeutically effective amount of a water-soluble paua 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 paua 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 paua 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 paua 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 paua 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 paua 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 paua 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 suited for use in food applications because it smells and tastes acceptable, and so can be added directly to food . Unlike other paua products, it does not need to be encapsulated. 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 d rinks. The water-soluble paua 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 provides a pharmaceutical composition comprising the water-soluble paua 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 paua 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 paua 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 paua 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 paua 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 paua 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 paua 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., 22 ^nd 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 paua 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 : Yarra™ 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:

6.2 Extraction of paua to produce water-soluble extracts of the invention

Various paua (H. iris) 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 processes

Sample code

Example Source material

(PAR)

1 Paua marc 39

2 Paua whole powder 36

3 Paua marc (pilot scale) 44

Example 1: Extraction of paua supercritical marc (PAR 39)

Marc resulting from supercritical CO2 extraction of freeze-dried paua ( 160 g) was suspended in 600 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°C in a shaking water bath for 3 hr. Enzidase FP (0.125 g) was then added and hyd rolysis continued for a further 1 hr at 60°C in a shaking water bath.

The hydrolysis mixture was heated for a further 30 min at 95°C, then centrifuged at 10000 rpm for 30 minutes before recovery of the supernatant (500 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 (450 ml) and mixed with maltodextrin and Oxyless U antioxidant. The resulting solution was freeze-d ried to produce the solid product (paua marc extract) The paua marc extract was fully water soluble. 42% of the solids present in the paua marc were recovered. The molecular weight profile of the product is shown in Table 2 below:

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

Example 2: Extraction of paua whole powder (PAR 36)

Freeze-dried powdered paua gut material (160 g) was suspended in 600 ml water. Oxyless U antioxidant (0.25 g) was added and the pH adjusted from 5.5 to 8.0. Alcalase (2.5 ml) and Enzidase Neutral (2.5 ml) were added and the mixture heated to 60°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°C in a shaking water bath.

The hydrolysis mixture was heated for a further 40 min at 95°C, then centrifuged at 10000 rpm for 30 minutes before recovery of the supernatant (505 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 (435 ml) and mixed with maltodextrin and Oxyless U antioxidant. The resulting solution was freeze-d ried to produce the solid product (paua whole powder extract) .

The GSM whole powder extract was fully water soluble. 41% of the solids present in the paua material were recovered. The molecular weight profile of the product is shown in Table 3 below:

Table 3: Molecular weight profile of peptides of paua whole powder extract

Size range: Relative peak area (%)

Total

>5kDa 2 -5kDa l - 2kDa <lkDa

<5kDa

2.2 16.5 38.7 42.6 97.8 Example 3: Extraction of paua supercritical marc (pilot scale) PAR 44

Marc resulting from supercritical CO2 extraction of paua (10.0 kg) was suspended in 150 L water. Oxyless U antioxidant ( 16 g) was added and the pH adjusted from 5.6 to 8.0. Alcalase (148 ml) and Enzidase Neutral (160 ml) were added and the mixture heated to 59°C in a shaking water bath for 3 hrs. Enzidase FP (32 g) was then added and hydrolysis continued for a further 1 hr at 60°C in a shaking water bath.

The hydrolysis mixture was heated for a further 20 min at 80°C, then centrifuged through a liquid-sludge separator (2x) before recovery of the supernatant ( 135 L). The pH of the supernatant was adjusted to pH 4 with phosphoric acid. Activated carbon was added (60g) . The supernatant was filtered through a Seitz 900 filter board using diatomaceous earth (940g pre-coat + 1070 g body feed) . The filtrate was recovered (144 L) and mixed with maltodextrin ( 1.6 kg) and Oxyless U antioxidant ( 16 g) until dissolved . The resulting solution was freeze-dried to produce 6.3 kg of solid product (paua marc extract).

The paua marc extract was fully water soluble. 66% of the solids present in the paua material were recovered . The molecular weight profile of the product is shown in Table 4 below:

Table 4: Molecular weight profile of peptides of paua marc extract

The proximate composition of PAR 44 was obtained. The sample was dried for 16 hours at 103°C, gravimetry. AOAC 945.15, 19th Edition. Ash was determined by ignition in muffle furnace 600°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. The results are shown in Table 5 below.

Table 5: Proximate composition for PAR 44

PAR 44

Moisture 4.9

Ash 13.6

Total Nitrogen 7.8

Total Protein

48.8

(N x 6.25)

Total Protein

52.7

(N x 6.75)

Lipid 4.8

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

6.3 Anti-inflammatory activity of water-soluble paua extracts of the invention

Example 4: Inhibition of TLR4 (LPS) stimulated IL- Ιβ and TNF secretion in monocytic THP-1 cells

IL-Ιβ 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-Ι β 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- Ιβ 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-Ιβ and TNFa to explore the efficacy of paua extracts in suppressing the expression of these pro-inflammatory cytokines in chronic inflammatory conditions.

Methodology: Monocytic cell-line-THP-1 (2xl0 ⁵ 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-Ιβ and TNFa production using commercial ELISAs. Optimal doses of TLR4 (~70% of maximal bioactivity) were used to examine the effect of paua extracts on IL-Ιβ 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 paua extracts in attenuating the expression of IL-Ιβ 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-Ιβ and TNFa expression. Paua 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-Ιβ and TNFa secretion by ELISA. Data was calculated as cytokine pg/mL and presented as percentage inhibition of TLR4-stimulated IL-Ιβ or TNFa secretion.

The results are shown in Figures 1-8. All of the water-soluble paua extracts tested demonstrated anti-inflammatory activity.

Figures 9-12 show the effect of unhydrolysed paua materials in the assays described above.

Table 5 below shows the effect of various paua products on the inhibition of IL-Ιβ 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 5: Relative inhibition

Table 6 below shows the effect of various paua products on the inhibition of TNFa in TLR4 (LPS) stimulated, monocytic THP- 1 cells, when applied at 100 μς/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 6: Relative inhibition

Example 5: LC-MS analysis of paua 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 7 below.

Table 7: Concentration and solubility of samples

Sample mg.ml ¹ Insolubles

Par 44 21.0 Low

Par 36 11.1 Low

paua whole 11.4 High

paua marc 10.8 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™ Diamond-Hydride™, 4 μηη, ΙΟθΑ, 150 x 2.1 mm, MicroSolv™ Technologies Corporation, USA) maintained at 30°C with a flow rate of 200μΙ/ηηίη. 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 13 - 20. The LC-MS chromatograms for the water- soluble extracts of the invention (Figures 13, 14, 17 and 18) indicate a more complex composition, with many more peaks than are seen in the LC-MS chromatograms of the starting paua material (Figures 15, 16, 19 and 20).

7. INDUSTRIAL APPLICABILITY

The water-soluble paua 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 acceptable 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.