JP2018510910A - Water-soluble mussel extract - Google Patents

Abstract

The present invention relates to a water-soluble mussel extract produced by enzymatic hydrolysis of mussel material. The extract contains a high proportion of low molecular weight peptides and exhibits anti-inflammatory properties. [Selection figure] None

Description

This application claims priority based on New Zealand provisional application No. 706298, filed Mar. 24, 2015, which is hereby incorporated by reference.
1. The present invention relates to water-soluble mussel extracts, compositions containing them and their use in nutritional supplements and pharmaceutical applications.

2. BACKGROUND OF THE INVENTION Shellfish, especially mussels, are known to be rich in compounds that maintain health. Shellfish extract is an established dietary supplement category in the global market.

  Some extracts of shellfish have an anti-inflammatory effect and other drug effects, which are often claimed to arise from the lipid fraction. Inflammation is associated with a wide variety of conditions including arthritis, atherosclerosis and cancer. Current medical care 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 dietary supplement preparations for shellfish have been described. The actual chemical composition of such a preparation is
(A) raw materials including shellfish species, their age, and the environmental conditions in which they were grown, and (b) the process used to obtain the preparation—for example, selectively excluding and retaining certain types of compounds The step of breaking or changing depends on a number of factors including that will affect the chemical composition of the final preparation.

So far, shellfish supplement preparations have focused on whole animal extracts or lipid fractions of their materials. Supercritical CO ₂ extraction of lyophilized material is widely used to produce mussel lipid extracts such as those sold under the trade name Lyprinol ™, especially green-shelled mussel lipid extracts Has been.

  However, mussel whole body powders are not suitable for inclusion in many foods because they are insoluble and generally have an unpleasant “fish-like” flavor. Mussel lipid extracts are also insoluble, and the “fish-like” flavor / odor can be exacerbated by oxidation, which is likely to occur during many standard food processing processes. They generally must be administered as lipid-filled capsules to hide unpleasant odors and flavors.

  Accordingly, it would be advantageous to provide a medicinal water-soluble mussel extract that does not suffer from the disadvantages associated with the corresponding mussel whole body powder and / or lipid extract.

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

3. SUMMARY OF THE INVENTION In one aspect, the present invention provides a water soluble mussel extract.
In one embodiment, the water soluble mussel extract comprises at least about 45% peptide, preferably a low molecular weight peptide, based on solids.

  In one embodiment, the water-soluble mussel extract comprises about 45% to about 65% peptide by weight, based on solids, preferably about 50% to about 60% peptide based on solids.

In one embodiment, more than 90%, preferably more than 95% by weight of the peptide is less than 5 kDa.
In one embodiment, more than 75%, preferably more than 80% by weight of the peptide is less than 2 kDa.

In one embodiment, more than 35%, preferably more than 40% by weight of the peptide is less than 1 kDa.
In one embodiment, the water soluble mussel extract comprises no more than about 5% lipid by weight based on solids.

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 comprising:
(A) hydrolyzing an aqueous suspension of mussel material using one or more proteolytic enzymes;
(B) denaturing the enzyme in the hydrolysis mixture;
(C) removing insoluble material from the hydrolysis mixture to provide a water soluble mussel extract.

In one embodiment, the hydrolysis mixture is adjusted to pH 4 after enzyme denaturation.
In one embodiment, the water soluble mussel extract is purified and / or concentrated.
In one embodiment, the mussels are greenshell mussel (or green-lipped mussel) (GSM)-Perna canaliculus, blue mussel-Mytilus edulis, And ribbed mussel (maori name kopakopa)-selected from the group including but not limited to Aulacomya atra maoriana. Preferably, the mussel is GSM.

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

In another aspect, the present invention provides a dietary supplement composition comprising the water soluble mussel extract of the present invention and one or more ingestible excipients.
In one embodiment, the dietary supplement composition comprises maltodextrin and / or an antioxidant.

In one embodiment, the dietary supplement composition comprises a food composition, a food additive composition, a health supplement or a medical food.
In another aspect, the present invention provides a pharmaceutical composition comprising the water-soluble mussel extract of the present 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 of reducing inflammation in a subject in need, comprising administering to the subject a therapeutically effective amount of a water-soluble mussel extract of the invention.

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

In one embodiment, the condition associated with inflammation is a chronic condition.
In one embodiment, the condition associated with inflammation is inflammatory bowel disease, including asthma, encephalitis, Crohn's disease and ulcerative colitis, chronic obstructive pulmonary disease (COPD), allergic disease, fibrosis, juvenile arthritis, Psoriatic arthritis, arthritis including rheumatoid arthritis and osteoarthritis, psoriasis, polymyalgia, tendonitis, bursitis, laryngitis, gingivitis, gastritis, otitis, celiac disease, diverticulitis, atheromatous arteries Selected from the group comprising sclerosis, heart disease, obesity, diabetes, cancer and Alzheimer's disease.

In one embodiment, the condition associated with inflammation increases the level of inflammatory TNFα and / or IL-1β.
In one embodiment, the water-soluble mussel extract of the present invention is administered orally.

  Embodiments of the present invention will now be described with reference to the drawings.

4). Brief Description of Drawings
2 is a graph showing positive (dexamethasone) and negative (dimethyl sulfoxide, DMSO) assay controls for inhibition of IL-1β in TLR4 (LPS) stimulated monocyte THP-1 cells. FIG. 2 is a graph showing the effect of a water-soluble extract from GSM residue powder on the inhibition of IL-1β in TLR4 (LPS) stimulated monocyte THP-1 cells. 2 is a graph showing the effect of two aqueous extracts from GSM whole body powder on the inhibition of IL-1β in TLR4 (LPS) stimulated monocyte THP-1 cells. Two enzyme combinations were tested for proteolysis. It is a graph which shows the effect of the water-soluble extract from a fresh mussel whole body with respect to inhibition of IL-1 (beta) in TLR4 (LPS) stimulation monocyte THP-1 cell. FIG. 2 is a graph showing the effect of a fresh GSM whole body water-soluble extract (with a defatting step) on IL-1β inhibition in TLR4 (LPS) stimulated monocyte THP-1 cells. FIG. 6 is a graph showing the effect of fresh Copacopa whole body derived water-soluble extract on the inhibition of IL-1β in TLR4 (LPS) stimulated monocyte THP-1 cells. 2 is a graph showing positive (dexamethasone) and negative (DMSO) assay controls for inhibition of TNFα in TLR4 (LPS) stimulated monocyte THP-1 cells. FIG. 2 is a graph showing the effect of a water-soluble extract from GSM residue powder on inhibition of TNFα in TLR4 (LPS) stimulated monocyte THP-1 cells. 2 is a graph showing the effect of two aqueous extracts from GSM whole body powder on the inhibition of TNFα in TLR4 (LPS) stimulated monocyte THP-1 cells. It is a graph which shows the effect of the water-soluble extract from a fresh mussel whole body with respect to the inhibition of TNF (alpha) in TLR4 (LPS) stimulation monocyte THP-1 cell. FIG. 6 is a graph showing the effect of a fresh GSM whole body water-soluble extract (with a defatting step) on TNFα inhibition in TLR4 (LPS) stimulated monocyte THP-1 cells. FIG. 6 is a graph showing the effect of a fresh Copacopa whole body-derived water-soluble extract on the inhibition of TNFα in TLR4 (LPS) stimulated monocyte THP-1 cells. FIG. 6 is a graph showing the effect of a water-soluble extract (pilot scale) from GSM residue powder on the inhibition of IL-1β in TLR4 (LPS) stimulated monocyte THP-1 cells. FIG. 6 is a graph showing the effect of a water-soluble extract (pilot scale) from GSM residue powder on the inhibition of TNFα in TLR4 (LPS) stimulated monocyte THP-1 cells. 2 is a graph showing the effect of unhydrolyzed GSM residue powder on the inhibition of IL-1β in TLR4 (LPS) stimulated monocyte THP-1 cells. 2 is a graph showing the effect of unhydrolyzed GSM whole body powder on inhibition of IL-1β in TLR4 (LPS) stimulated monocyte THP-1 cells. 2 is a graph showing the effect of non-hydrolyzed GSM residue powder on inhibition of TNFα in TLR4 (LPS) stimulated monocyte THP-1 cells. 2 is a graph showing the effect of non-hydrolyzed GSM whole body powder on inhibition of TNFα in TLR4 (LPS) stimulated monocyte THP-1 cells. It is a positive product ion-based chromatogram generated by LC-MS analysis of the water-soluble extract PAR58. FIG. 2 is a positive product ion-based chromatogram generated by LC-MS analysis of unhydrolyzed GSM residue powder. FIG. 2 is a positive product ion-based chromatogram generated by LC-MS analysis of unhydrolyzed GSM whole body powder. It is a negative product ion base chromatogram generated by LC-MS analysis of the water-soluble extract PAR58. FIG. 2 is a negative product ion-based chromatogram generated by LC-MS analysis of unhydrolyzed GSM residue powder. FIG. 5 is a negative product ion-based chromatogram generated by LC-MS analysis of unhydrolyzed GSM whole body powder.

5. Detailed Description of the Invention 5.1 Definitions As used herein, “a” or “an” means at least one, unless expressly stated otherwise.

As used herein, the term “about” refers to a value within 10% above and below the value modified by that term.
The term “comprising”, as used herein and in the claims, means “consisting at least in part”. When interpreting the description in this specification and the claims that include the term “comprising”, there may also be other features than the features that follow this term in each description. Related terms such as “including” and “including” should be interpreted similarly.

  As used herein, the term “therapeutically effective amount” is used to reduce or ameliorate the severity, duration, or one or more of its symptoms in the context of administering a therapy to a subject. To prevent the progression of the condition, to cause regression of the condition, to prevent recurrence, development or onset of the condition, or to enhance or improve the prophylactic or therapeutic effect of another therapy Mean enough.

  As used herein, the terms “manage”, “managing” and “management” do not result in a cure of the condition in the context of administration of the therapy to the subject, but the subject obtains from the therapy Refers to beneficial effects. For example, state management includes preventing state deterioration.

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

  As used herein, the terms “treat”, “treatment” and “treating” refer to the progression of a condition, severe, resulting from the administration of a therapy in the context of the administration of the therapy to a subject. Refers to reduction or improvement of sex and / or duration, or improvement of one or more of its symptoms.

As used herein, the term “water-soluble” means that the material has a solubility of at least 45% (w / v) when applied to the material.
Where reference is made herein to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing features of the present invention. Unless specifically stated otherwise, references to such external documents shall be construed as permitting such documents or such information sources to be prior art or form part of the common general knowledge in the art in any authority. Should not be done.

In the description herein, reference may be made to subject matter that is not within the scope of the claims of this application. The subject matter should be readily identifiable by those skilled in the art and may assist in practicing the invention as defined in the claims of this application.
5.2 Method for Producing the Water-Soluble Mussel Extract of the Invention The water-soluble mussel extract of the present invention is readily prepared using conventional processing techniques, as described below.

The present invention is a method for producing a water-soluble mussel extract comprising:
(A) hydrolyzing an aqueous suspension of mussel material using one or more proteolytic enzymes;
(B) denaturing the enzyme in the hydrolysis mixture;
(C) removing insoluble material from the hydrolysis mixture to provide a water soluble mussel extract.

  The term “mussel” as used herein refers to an edible bivalve from 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 mussels are selected from the group including but not limited to GSM, mussel and copakopa. Preferably, the mussel is GSM.
In one embodiment, the mussel material is a “fresh” mussel “whole body”. This can be obtained by collecting the material that remains after removal of the mussel shells, legs and whiskers. In another embodiment, the mussel material is a dry mussel powder. This is generally obtained by drying a fresh mussel whole body, usually by freeze-drying, and grinding into a powder.

  In another embodiment, the mussel material is a mussel supercritical residue. The term “residue” generally refers to organic material remaining after extraction of biological material such as plant or animal material. As used herein, the term “supercritical residue” means an organic material that remains after supercritical fluid extraction.

As used herein, the term “mussel supercritical residue” means an organic material that remains after supercritical fluid extraction of mussel meat or mussel powder.
In one embodiment, the mussel material is a mussel supercritical residue, preferably a GSM supercritical residue.

In the first step of the method, an aqueous suspension of mussel material is hydrolyzed using one or more proteolytic enzymes.
Hydrolysis is performed by incubating an aqueous suspension of mussel material with the proteolytic enzyme (s) under conditions optimized for the enzyme or enzyme system used. Usually, the pH should be adjusted to about 5.5 to about 8. Optionally, an antioxidant, such as Oxyless U, can be added to the aqueous suspension.

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

  During hydrolysis, proteins present in the mussel material are hydrolyzed to low molecular weight peptides. Hydrolysis must be performed enzymatically to deliver peptides of the appropriate molecular weight range. For example, chemical hydrolysis with acid is non-selective. Without being bound by theory, it is believed that the anti-inflammatory properties of the water-soluble mussel extract of the present invention are present in certain 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 enzyme can be of any type, such as endo, exoprotease / peptidase, aminopeptidase, serine protease, metalloprotease, cysteine protease, and the like. In one embodiment, each proteolytic enzyme has a slightly acidic to near neutral pH optimum. In one embodiment, the proteolytic enzyme is a non-animal enzyme.

  Suitable proteolytic enzymes include papain, Alcalase (subtilisin), Enzidase FP (endo / exopeptidase from selected strains of Aspergillus oryzae var.) And Enzidase Neutral (Bacillus amyloliquefaciens) Metallo-neutral endopeptidase derived from controlled fermentation of non-genetically modified strains).

In one embodiment, the proteolytic enzyme is selected from the group comprising papain, Enzidase FP, Enzidase Neutral, Alcalase and the like.
In one embodiment, the proteolytic enzyme comprises papain and Enzidase FP.

In another embodiment, the proteolytic enzyme comprises 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 hydrolysis depends on the activity of the enzyme (s) used and the mussel material.

The hydrolysis step is performed with agitation to optimize the contact between the enzyme (s) and the mussel material.
After hydrolysis is complete, the enzyme is 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 30 to about 40 minutes. In another embodiment, the hydrolysis mixture is heated at about 80 ° C. to about 95 ° C. for about 20 to about 40 minutes.

  In one embodiment, an aqueous suspension of hydrolyzed mussel material can optionally be treated to remove lipids before denaturing the enzyme. Lipid removal may be achieved using any method known in the art, for example using physical techniques such as centrifugation or chemical extraction.

  In chemical extraction, water is removed from the aqueous suspension of hydrolyzed mussel material, which is then contacted with a solvent in which the lipid fraction is soluble. Subsequently, the defatted product (mussel residue) is resuspended in water to denature the hydrolase.

  In another embodiment, an aqueous suspension of hydrolyzed mussel material is lyophilized to form a powder that is rinsed with an organic solvent to remove lipids. 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 hydrolyzed mussel material is dried and extracted with a supercritical solvent such as CO ₂ or CO ₂ / ethanol to remove the lipid fraction. Subsequently, the defatted product (mussel residue) is resuspended in water to denature the hydrolase.

  If the mussel material contains a significant amount of lipid, an optional degreasing step may be used. If the mussel material has a low lipid content (eg fresh mussel whole body or powder), or if mussel residues are used, the defatting step can be omitted.

  After denaturation of the hydrolase, the pH of the mixture may be adjusted to about 3.5 to about 4.2, preferably about 4, to prevent microbial spoilage. The pH can be adjusted using agents such as phosphoric acid, citric acid or hydrochloric acid.

The insoluble material present in the hydrolysis mixture is then removed, resulting in the water-soluble mussel extract of the present invention.
Insoluble material can be removed by any method known in the art. In one embodiment, insoluble material is removed by centrifuging the hydrolysis mixture and collecting the supernatant.

Generally, the pH is adjusted prior to removal of the insoluble material, but if the latter contains shell fragments, this insoluble material should first be removed to prevent solubilization of the shell by acid.
The water-soluble mussel extract of the present invention may be treated to remove impurities contributing to undesirable color and / or odor, for example, by contact with activated carbon. Charcoal can be contained in a container to pass the product through it. Alternatively, carbon, residual 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 yield a powdered extract. Drying can be performed using any technique known in the art, including lyophilization.

Agents such as maltodextrins and antioxidants may be added to the water-soluble mussel extract of the present invention.
Examples 1-8 describe a method for producing the water-soluble mussel extract of the present invention.
5.3 Water-soluble mussel extract of the present invention The above-described method of the present invention produces a water-soluble mussel extract enriched in peptides, particularly low molecular weight peptides. As used herein, the term “low molecular weight peptide” means a peptide that weighs 5 kDa or less.

  The extract has an acceptable flavor without bitterness. The extract can be frozen without a cold precipitation reaction. The powder solubility characteristics of mussel extract do not change during dry storage.

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

  The water-soluble mussel extract of the present invention can be seen from FIGS. 19-24, which show the results of LC-MS analysis performed on the water-soluble GSM mussel extract of the present invention, as well as GSM residue and whole body powder starting materials. Contains more compounds than the non-hydrolyzed starting material.

In one embodiment, the water soluble mussel extract comprises at least about 45% peptide, preferably a low molecular weight peptide, based on solids.
In another embodiment, the water soluble mussel extract comprises at least about 45 wt% to about 65 wt% peptide, preferably about 50 wt% to about 60 wt% peptide, based on solids.

In one embodiment, more than 90%, preferably more than 95% by weight of the peptide is less than 5 kDa.
In one embodiment, more than 75%, preferably more than 80% by weight of the peptide is less than 2 kDa.

In one embodiment, more than 35%, preferably more than 40% by weight of the peptide is less than 1 kDa.
Some carbohydrate materials, lipids and other materials and minerals may also be present.

  The peptide content of the extract is calculated based on the “solid” present so that it remains unaffected by its concentration. The “solid” present in the extract constitutes the material that remains when any solvent present, including water, is removed. “Solids” present include peptides, carbohydrates and lipids and any other non-solvent material.

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

In one embodiment, the water-soluble mussel extract of the present invention is a dry powder.
The weight percent of peptides present in the extract is calculated excluding solid excipients added to the extract such as maltodextrin and antioxidants.

  In one embodiment, the present invention provides a water-soluble mussel extract comprising at least about 45% peptide by weight based on solids, wherein more than 90% by weight of the peptide is less than 5 kDa. provide.

In another embodiment, the invention provides a water-soluble mussel extract comprising about 50% to about 60% peptide by weight, based on solids, wherein more than 95% by weight of the peptide is less than 5 kDa Provide sex mussel extract.
5.4 Use of Water-Soluble Peptide Extracts of the Invention Inflammation is a process involving a complex biological cascade of molecules and cellular signals that alter the physiology of an organism. While acute inflammation protects and heals the body after physical injury or infection, chronic inflammation results in changes that play an important 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 family, TNFα and prostaglandins. These mediators induce up-regulation of other pro-inflammatory cytokines. IL-1, IL-6 and TNFα are major inflammatory biomarkers found at higher levels in inflammatory cells than in quiescent 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 radicals, superoxide anions, singlet oxygen and hydrogen peroxide are generated. These species kill invading microorganisms and parasites.

  Dietary supplements are through several mechanisms including blocking the expression of pro-inflammatory cytokines such as IL-1 and TNFα, inhibiting ROS-generating enzyme activity, or increasing ROS scavenging capacity. The inflammatory process can be inhibited or reduced.

  As presented in Example 9, the water-soluble mussel extract of the present invention has been found to be active against known markers of inflammation and is therefore believed to reduce or prevent inflammation. As shown in Tables 10 and 11, the activity shown is several times higher than the anti-inflammatory activity of non-hydrolyzed starting mussel material such as whole body powder or residue.

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

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

The present invention also provides a water soluble mussel extract of the present invention for use in reducing inflammation in a patient in need thereof.
The present invention also provides a water soluble mussel extract of the present invention for use in preventing, treating or managing a condition associated with inflammation in a patient 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 the inflammatory markers TNFα and / or IL-1β.

  Examples of conditions associated with inflammation that can be prevented, treated or managed according to the present invention include inflammatory bowel diseases, including asthma, encephalitis, Crohn's disease and ulcerative colitis, chronic obstructive pulmonary disease (COPD), allergies Sexual disorders, fibrosis, juvenile arthritis, psoriatic arthritis, arthritis including 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 present invention also provides a method for reducing the level of the inflammatory markers TNFα and / or IL-1β in a subject in need, comprising administering to the subject a therapeutically effective amount of the water-soluble mussel extract of the present invention. A method comprising:

  A subject in need of prevention, treatment or management of a condition associated with inflammation is a subject diagnosed with, at risk of, or recovered from such a condition. A subject may be susceptible to a condition and / or may be at risk of a condition due to genetic and / or environmental factors.

In one embodiment, the subject is a mammal, preferably a human. In another embodiment, the subject is a pet or a horse.
Administration of the water soluble mussel extract of the present invention may be via a pharmaceutical composition or a dietary supplement composition.

In one aspect, the present invention provides a dietary supplement composition comprising the water-soluble mussel extract of the present invention and one or more ingestible excipients.
In one embodiment, the dietary supplement composition of the present invention is a food composition, a food additive composition, a health supplement or a medical food composition.

  The term “ingestible” as used herein generally means that it is suitable for consumption by animals and humans, or has been approved by government regulatory agencies in that regard.

  The term “food” as used herein means any material that has been processed, semi-processed, or unprocessed and is intended for consumption by animals, including humans. Including but not limited to beverages and chewing gum. The food composition of the present invention comprises the water-soluble mussel extract of the present invention in combination with food.

  The term “food additive” as used herein refers to any substance that is not normally consumed as a food product by itself but is added to a food product for technical purposes. Examples are natural and artificial sweeteners, colorants, curing and pickling agents, flavoring agents, emulsifiers, lipid substitutes, stabilizers, swelling agents, lubricants, humectants, preservatives, stabilizers and thickeners. . The food additive composition of the present invention comprises the water soluble mussel extract of the present invention in combination with one or more food additives.

  The term “health supplement”, as used herein, is intended to supplement a diet that includes one or more of the following ingredients: vitamins, minerals, herbs, metabolites, extracts, and the like. Refers to the product. Health supplements are not normally intended to be used as a single item of meal and can be taken independently of any food. The dietary supplement of the present invention comprises the water soluble mussel extract of the present invention in combination with one or more dietary supplements.

  The term “medical food” as used herein refers to food that is ingested or formulated for enteral administration under the supervision of a physician. Medical food is intended for dietary management in situations with characteristic nutritional requirements. Examples of medical foods include, but are not limited to, single designated nutritional foods, oral rehydration fluids, and products intended for use in dietary management of metabolic disorders. The medical food composition of the present invention comprises the water-soluble mussel extract of the present invention in combination with a medical food.

  In one embodiment, the dietary supplement composition of the present invention is a food composition. The water-soluble extract of the present invention is particularly suitable for use as a food composition because it has a pleasant odor and flavor and can therefore be added directly to food. Unlike other mussel products, it does not need to be encapsulated. Example 11 describes the analysis of volatile organic compounds present in two water-soluble extracts of the present invention. The results shown in Table 13 and Table 14 are consistent with a pleasant flavor composition.

  In one embodiment, the food composition is a bakery product including but not limited to bread, pizza dough, cakes, muffins, donuts, biscuits, tortillas, wraps, naan, noodles and pasta.

  In another embodiment, the food composition is a liquid food comprising milk, fruit juice, smoothie, yogurt, soup and soft drink. The water-soluble mussel extract of the present invention can also be added to dry mixes such as instant soups, beverages, puddings and cake mixes.

In one aspect, the present invention provides a pharmaceutical composition comprising the water-soluble mussel extract of the present 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, particularly humans.

  The term “excipient” as used herein refers to a vehicle, carrier, diluent, adjuvant, stabilizer, or the vehicle with which the water-soluble mussel extract of the present invention is stored, transported or administered. Contains a filler. Suitable excipients are well known to those skilled in pharmacy and include starch (and derivatives thereof such as maltodextrin), glucose, sucrose, flour, silica gel, glycerol, sodium chloride, water, ascorbic acid and ethanol. However, it is not limited to these. Whether a particular excipient is suitable for incorporation into the pharmaceutical composition of the invention will depend on the mode of administration of the dosage form.

The pharmaceutical compositions of the present 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 usually vary depending on the usage. Examples of dosage forms include tablets, capsules, pills, pellets, capsules containing liquids, troches, lozenges, dispersions, suppositories, nasal sprays or inhalants, gels, suspensions Examples include, but are not limited to, suspensions, emulsions, solutions and elixirs.

  In one embodiment, the present invention provides a water-soluble mussel extract of the present invention in an oral dosage form. Because of their ease of administration, oral dosage forms are preferred both as a dietary supplement composition and as a pharmaceutical composition. A typical oral dosage form is prepared by combining the dry water soluble mussel extract of the present invention with at least one excipient suitable for use in a solid oral dosage form. Subsequently, the product is 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 disintegrants.
Examples of binders include corn starch, potato starch or other starches, gelatin, gums such as Arabic and Guar, sodium alginate, powdered tragacanth, cellulose and carboxymethylcellulose, pregelatinized cellulose, hydroxypropylcellulose and microcrystalline Examples thereof include, but are not limited to, cellulose. Examples of fillers include, but are not limited to, talc, calcium carbonate granules or powder, microcrystalline cellulose, powdered cellulose, dextrate, kaolin, mannitol, sorbitol and starch. The disintegrant ensures that the tablet disintegrates when exposed to an aqueous environment. Examples include, but are not limited to, agar, calcium carbonate, alginic acid, croscarmellose sodium, microcrystalline cellulose, pregelatinized starch, clay and gum. Lubricants include, but are not limited to, calcium stearate, light 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 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 provided as a dry product for reconstitution with water or another suitable vehicle prior to use. .

  The amount of the water-soluble mussel extract of the present invention that is effective in the prevention, treatment or management of conditions associated with inflammation depends on the nature and severity of the disease or condition and the route to which the extract is to be administered, as well as the subject. Varies depending on factors specific to the subject such as age, weight, sex and medical history.

  In general, dosage forms used in the acute treatment of conditions associated with inflammation contain larger amounts of water-soluble mussel extract than are used in the treatment of chronic conditions. Similarly, parenteral dosage forms may contain less water-soluble mussel extract than oral dosage forms. The formulation of a particular dosage form is readily understood by those skilled in pharmacy. We have described Remington's Pharmaceutical Sciences, Allen et al., 22nd edition. Reference is made to ISBN 978-0-85711-062.

  One skilled in the art can predict effective doses from dose response curves derived from in vitro or animal model studies. In general, an effective amount of the water-soluble mussel extract of the present invention is from about 100 mg to about 3000 mg per day as a single daily dose or as a divided dose throughout the day.

  Various aspects of the invention will now be described in a non-limiting manner with reference to the following examples.

6). Example 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 are from Immers Filtration Minerals Inc. (San Jose, California, USA). Enzidase papain 6000L, Enzidase FP and Enzidase Neutral were from Zymus (Auckland, NZ). Alcalase was made by Novozymes (Bausbea, Denmark).

All other chemicals and reagents were standard laboratory supplies.
Size exclusion HPLC setting column: Yarra ™ 3 μm SEC-2000 (Phenomenex).
Sample preparation:
1. Dissolve the lyophilized extract sample at a concentration of 10 mg / mL in 100 mL of sodium phosphate buffer pH 6.8.

2. The solution is diluted in a ratio of 4 parts sample and 1 part 10% SDS in phosphate buffer to give a final concentration of 2% SDS.
3. Heat at 50 ° C. for 5 minutes.

4. Centrifuge at 13000 rpm for 5 minutes.
5. Fill the vial with the supernatant and perform HPLC.
Operating conditions for peptide SE-HPLC:

6.2 Extraction of mussels to produce the water-soluble extract of the present invention Various mussel sources were processed according to the examples provided below. The process and sample code are summarized in Table 1 below.

Example 1: Extraction of green mussel supercritical residue (GSM residue extract) PAR15
The residue (160 g) resulting from supercritical CO ₂ extraction of lyophilized green mussels was suspended in 600 ml of water. Oxyless U antioxidant (0.25 g) was added. Papain (5 ml) was added and the mixture was heated to 55 ° C. in a shaking water bath for 1 hour. Next, Enzidase FP (0.5 g) was added and hydrolysis continued for an additional hour at 55 ° C. in a shaking water bath.

  The hydrolysis mixture was heated at 90 ° C. for an additional 30 minutes, cooled to 55 ° C., and then centrifuged at 10,000 rpm for 30 minutes, after which the supernatant (565 ml) was collected. The pH of the supernatant was adjusted to pH 4 using phosphoric acid. Activated carbon was added (2 g). The supernatant was filtered through a Seitz 900 filter board (20 g pre-coated (Celite HyFlo) +20 g body feed (Celite 545)) using diatomaceous earth. The filtrate was collected (510 ml) and mixed with maltodextrin (27.2 g) and Oxyless U antioxidant (0.25 g) until dissolved. The resulting solution was lyophilized to give 126.2 g of a solid product (GSM residue extract).

The GSM residue extract was completely water soluble with a pleasant flavor. 60% of the solid present in the starting supercritical CO ₂ residue was recovered.
Using the same enzyme system (PAR13, 19 and 30), once the Enzidase FP was replaced with Alcalase (PAR12), the procedure was repeated several times.

  The molecular weight profile of the product as analyzed by SE-HPLC is shown in Table 2 below.

Example 2: Fresh green mussel whole body extraction (fresh GSM whole body extract) PAR37
Homogenized mussel-derived homogenized meat (500 g) was suspended in 400 ml of water. Oxyless U antioxidant (0.25 g) was added. Alcalase (2.5 ml) and Enzidase Neutral (2.5 ml) were added and the mixture was heated to 60 ° C. in a shaking water bath for 3 hours. Next, Enzidase FP (0.125 g) was added and hydrolysis continued for an additional hour at 60 ° C. in a shaking water bath.

  The hydrolysis mixture was heated at 95 ° C. for an additional 40 minutes, cooled to 55 ° C., and subsequently centrifuged at 10,000 rpm for 30 minutes, after which the supernatant (795 ml) was collected. The pH of the supernatant was adjusted to pH 4 using phosphoric acid. Activated carbon was added (2 g). The supernatant was filtered through a Seitz 900 filter board (precoat 20 g + body feed 20 g) using diatomaceous earth. The filtrate was collected (750 ml) and mixed with maltodextrin (27.2 g) and Oxyless U antioxidant (0.25 g) until dissolved. The resulting solution was lyophilized to give a solid product (fresh GSM whole body extract).

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

Example 3: Extraction of GSM whole body powder (GSM whole body powder extract) PAR36
Lyophilized powdered green mussel (160 g) was suspended in 600 ml of water. Oxyless U antioxidant (0.25 g) was added to adjust the pH to 6.0-8.0. Alcalase (2.5 ml) and Enzidase Neutral (2.5 ml) were added and the mixture was heated to 60 ° C. in a shaking water bath for 3 hours. Next, Enzidase FP (0.125 g) was added and hydrolysis continued for an additional hour at 60 ° C. in a shaking water bath.

  The hydrolysis mixture was heated at 95 ° C. for an additional 40 minutes, cooled to 55 ° C., and subsequently centrifuged at 10,000 rpm for 30 minutes, after which the supernatant (590 ml) was collected. The pH of the supernatant was adjusted to pH 4 using phosphoric acid. Activated carbon was added (2 g). The supernatant was filtered through a Seitz 900 filter board (precoat 20 g + body feed 20 g) using diatomaceous earth. The filtrate was collected (550 ml) and mixed with maltodextrin (27.2 g) and Oxyless U antioxidant (0.25 g) until dissolved. The resulting solution was lyophilized to obtain a solid product (GSM whole body powder extract).

The GSM whole body powder extract was completely water soluble. 66% of the solids present in the starting mussel material were recovered.
The process was repeated with Alcalase and Enzidase Neutral replaced with papain (PAR36a).

  The molecular weight profile of the product as analyzed by SE-HPLC is shown in Table 4 below.

Example 4: Extraction of fresh mussel whole body (fresh mussel whole body extract) PAR40
Homogenized mussel-derived homogenized meat (516 g) was suspended in 300 ml of water. Oxyless U antioxidant (0.25 g) was added. The pH was adjusted to 6.0-8.0 followed by the addition of Alcalase (2.5 ml) and Enzidase Neutral (2.5 ml) and the mixture was heated to 60 ° C. in a shaking water bath for 3 hours. . Next, Enzidase FP (0.125 g) was added and hydrolysis continued for an additional hour at 60 ° C. in a shaking water bath.

  The hydrolysis mixture was heated at 95 ° C. for an additional 30 minutes, cooled to 55 ° C., and subsequently centrifuged at 10,000 rpm for 30 minutes, after which the supernatant (746 ml) was collected. The pH of the supernatant was adjusted to pH 4 using phosphoric acid. Activated carbon was added (2 g). The supernatant was filtered through a Seitz 900 filter board (precoat 20 g + body feed 20 g) using diatomaceous earth. The filtrate was collected (700 ml) and mixed with maltodextrin (27.2 g) and Oxyless U antioxidant (0.25 g) until dissolved. The resulting solution was lyophilized to obtain a solid product (fresh mussel whole body extract).

  The fresh mussel whole body extract was completely water soluble. 83% of the solids present in the shellfish material were recovered. The molecular weight profile of the product as analyzed by SE-HPLC is shown in Table 5 below.

Example 5: Fresh GSM whole body extraction including a degreasing step (fresh GSM whole body defatted extract) PAR41
Homogenized meat from GSM (507 g) was suspended in 400 ml of water. Oxyless U antioxidant (0.25 g) was added. The pH was adjusted to 6.5-8.0 followed by the addition of Alcalase (2.5 ml) and Enzidase Neutral (2.5 ml) and the mixture was heated to 60 ° C. in a shaking water bath for 3 hours. . Next, Enzidase FP (0.125 g) was added and hydrolysis continued for an additional hour at 60 ° C. in a shaking water bath.

  Subsequently, the hydrolyzed material (890 ml) was lyophilized to recover 100 g of powder. The powder was rinsed with cold acetone (3 × 250 ml) to remove lipids. The powder was recovered by filtering through paper during rinsing. The final defatted powder was dried under nitrogen.

  The defatted powder was resuspended in 400 ml of water, heated at 95 ° C. for 40 minutes and subsequently cooled to 55 ° C. Next, the hydrolyzed aqueous suspension was centrifuged at 10,000 rpm for 30 minutes, and then the supernatant (415 ml) was recovered. The pH of the supernatant was adjusted to pH 4 using phosphoric acid. Activated carbon was added (2 g). The supernatant was filtered through a Seitz 900 filter board (precoat 20 g + body feed 20 g) using diatomaceous earth. The filtrate was collected (375 ml) and mixed with maltodextrin (27.2 g) and Oxyless U antioxidant (0.25 g) until dissolved. The resulting solution was lyophilized to give a solid product (fresh GSM whole body defatted extract).

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

Example 6: Factory-scale extraction of GSM supercritical residue (factory GSM residue extract) PAR43
Oxyless U antioxidant (80 g) was added to cold (19 ° C.) water (500 L). Papain (200 g) was subsequently stirred in the residue (50.0 kg) resulting from supercritical CO ₂ extraction of lyophilized green mussels. The mixture was heated to 55 ° C. and digested for 2 hours. Next, Enzidase FP (160 g) was added and hydrolysis continued at 55 ° C. for an additional 1.5 hours.

  The hydrolysis mixture was heated at 80-88 ° C. for an additional 20 minutes, cooled to 60 ° C., and then centrifuged through a liquid-sludge separator (twice), after which the supernatant (450 L) was recovered. . The pH of the supernatant was adjusted to 4 using phosphoric acid. Activated carbon was added (200 g). The supernatant was filtered through a filter press using diatomaceous earth (precoat 6.0 kg + body feed 6.0 kg). The filtrate was collected (500 L) and mixed with maltodextrin (7.0 kg) and Oxyless U antioxidant (80 g) until dissolved. The resulting solution was lyophilized to obtain 24.0 kg of a solid (GSM residue extract) excluding additives.

The GSM residue extract was completely water soluble and was accompanied by a pleasant fresh sea flavor. 50% of the solid present in the starting supercritical CO ₂ residue was recovered.
The molecular weight profile of the product as analyzed by SE-HPLC is shown in Table 7 below.

Example 7: Fresh Copacopa whole body extraction (fresh Copacopa whole body extract) PAR45
Homogenized Copacopa meat (501 g) was suspended in 400 ml of water. Oxyless U antioxidant (0.25 g) was added. The pH (6) was not adjusted. Papain (5 ml) was added and the mixture was heated to 55 ° C. for 2 hours in a shaking water bath. Next, Enzidase FP (0.5 g) was added and hydrolysis continued for an additional 1.5 hours at 55 ° C. in a shaking water bath.

  The hydrolysis mixture was heated at 95 ° C. for an additional 30 minutes, cooled to 55 ° C., and then centrifuged at 10,000 rpm for 30 minutes, after which the supernatant (720 ml) was collected. The pH of the supernatant was adjusted to 4 using phosphoric acid. Activated carbon was added (2 g). The supernatant was filtered through a Seitz 900 filter board (20 g pre-coated (Celite HyFlo) +20 g body feed (Celite 545)) using diatomaceous earth. The filtrate was collected (575 ml) and mixed with maltodextrin and Oxyless U antioxidant. The resulting solution was lyophilized to obtain a solid product (fresh Copacopa whole body extract).

The fresh Copacopa whole body extract was completely water soluble. 55% of the solid present in the Copacopa raw material was recovered.
The molecular weight profile of the product as analyzed by SE-HPLC is shown in Table 8 below.

Example 8: Extraction of GSM residue powder (pilot scale) PAR58
Oxyless U (24 g) was added to 150 L of cold water. Papain (500 mL) (Zymus Enzidase papain 6000 L) was added with continuous stirring. GSM supercritical residue (15.0 kg) was added and the suspension was heated at 50 ° C. for 15 hours. Enzidase FP (50 g) was added and hydrolysis continued for an additional 2 hours at 55 ° C.

The suspension was then heated at 80 ° C. for 20 minutes and then cooled.
The suspension was centrifuged through a liquid-sludge separator and 60 g of activated carbon was added during the process. The supernatant was collected again and its pH was changed to 4 using phosphoric acid. The supernatant was centrifuged through a liquid sludge separator. The final supernatant was collected (95 L). Maltodextrin (2.4 kg) and Oxyless U (24 g) were added and mixed until dissolved.

Subsequently, the water-soluble extract of the present invention was lyophilized to obtain a water-soluble solid product. 69% of the solid present in the mussel residue was recovered. The flavor was pleasant (delicious).
The procedure was repeated using the same enzyme system (papain 500 mL), digested at 50 ° C. for 21 hours, followed by an additional 2 hours of hydrolysis at 55 ° C. with 45 g of Enzidase FP (PAR63).

83% of the solids present in the supercritical GSM residue were recovered. The product was water soluble and had a pleasant delicious flavor.
The molecular weight profile of the product as analyzed by SE-HPLC is shown in Table 9 below.

  The approximate composition of PAR58 was compared to the approximate composition of the GSM residue starting material. The sample was dried at 103 ° C. for 16 hours (gravimetric measurement). AOAC 945.15, 19th edition. The ash content was determined by ignition in a muffle furnace at 600 ° C. for 6 hours (weight measurement). AOAC 942.05, 19th edition. Total nitrogen was determined using Dumas combustion. AOAC 1992.15, 19th edition. Total protein was calculated based on total nitrogen x 6.25. AOAC 1992.15, 19th edition. Total protein values are also given based on a factor of 6.75 (which is more suitable for highly hydrolyzed proteins where significant water has been added to the peptide bond). Lipids are described in Bligh, E .; G. , & Dyer, W. J. et al. (1959), “A rapid method of total lipid extraction and purification”, Can. J. et al. Biochem. and Physiol. 37 (8), pages 911 to 917.

  Table 10 below shows the results.

The value is g / 100 g of lyophilized material without any additives.
6.3 Anti-inflammatory properties of water-soluble extracts of the invention Example 9: Inhibition of TLR4 (LPS) -stimulated IL-1β and TNFα secretion in monocyte THP-1 cells by water-soluble extracts of the invention IL-1β and TNFα is an important mediator of the inflammatory response. They initiate a cascade of events that regulate the body's natural defenses against invading microorganisms and 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.

  Thus, chronic inflammation is suppressed by limiting IL-1β and TNFα expression and biological activity in chronic inflammatory conditions. In addition, various immune cells are known to express IL-1β and TNFα during chronic inflammation. However, the triggers for their expression are very diverse and they are the result of abnormal immune cell signaling to invading microorganisms.

  As part of the natural defense system, humans have developed complex cell surface markers including a group known as Toll-like receptors (TLRs) that recognize foreign bodies and trigger appropriate cellular defense processes.

Bacterial ligands that activate TLR4 in monocyte immune cells are used to up-regulate IL-1β and TNFα expression in suppressing the expression of these pro-inflammatory cytokines in chronic inflammatory conditions The efficiency of mussel extract was explored.
Method: Monocyte cell line THP-1 (2 × 10 ⁶ cells / mL) grown in RPMI (Roswell Park Memorial Laboratory) medium containing 10% FBS (fetal calf serum) Under culture conditions, TLR4-lipopolysaccharide [LPS] (0.5-50 ng / mL) was incubated for 6 hours. Supernatants were collected and measured for IL-1β and TNFα production using a commercially available ELISA. The effect of mussel extract on IL-1β and TNFα secretion was examined using an optimal dose of TLR4 (approximately 70% of maximum bioactivity). The bioassay was validated using dexamethasone (0.001-10 μM or 0-3.925 μg / mL), a known immunosuppressant.

  The effectiveness of the mussel extract in reducing IL-1β and TNFα expression in a chronic inflammatory scenario was evaluated. THP-1 cells were initially stimulated with an optimal dose of TLR4 for 30 minutes to induce upregulation of IL-1β and TNFα expression. Next, mussel extract (usually 1-100 μg / mL) was added and incubated with the cells for an additional 6 hours. Subsequently, the culture medium was collected (by centrifuging the cells) and measured for IL-1β and TNFα secretion by ELISA. Data were calculated as cytokine pg / mL and presented as percent inhibition of TLR4-stimulated IL-1β or TNFα secretion.

The results are shown in FIGS. All tested water-soluble extracts of the present invention showed anti-inflammatory activity (FIGS. 1-14).
FIGS. 15-18 are the results of a control experiment in which non-hydrolyzed mussel material was tested in the above assay.

Figures 15 and 16 show the effect of GSM residue powder and GSM whole body powder on the inhibition of IL-1β in TLR4 (LPS) stimulated monocyte THP-1 cells.
Figures 17 and 18 show the effect of the same material on the inhibition of TNFα in TLR4 (LPS) stimulated monocyte THP-1 cells.

  Table 10 below shows the effect of various GSM products on the inhibition of IL-1β in TLR4 (LPS) stimulated monocyte THP-1 cells when applied at 100 μg / mL in the assay. The lyophilized product was resuspended in either phosphate buffered saline (PBS) or dimethyl sulfoxide (DMSO) prior to addition to the assay.

  Each column represents the result compared to the effect of the soluble extract assigned the value 1 (highest inhibition).

  Table 11 below shows the effect of various GSM products on the inhibition of TNFα in TLR4 (LPS) stimulated monocyte THP-1 cells when applied at 100 μg / mL in the assay. The lyophilized product was resuspended in either phosphate buffered saline (PBS) or dimethyl sulfoxide (DMSO) prior to addition to the assay.

  Each column represents the result compared to the effect of the soluble extract assigned the value 1 (highest inhibition).

6.4 Analysis of water-soluble extract of the present invention Example 10: LC-MS analysis method of GSM residue, whole body powder and water-soluble extract of the present invention Each sample was assigned and 0.5% formic acid using vortex Mixing in aqueous solution dissolved the polar constituents, but some samples remained solids that were not dissolved as shown in Table 12 below.

  The LC-MS system consists of the Thermo Electron Corporation (San Jose, CA, USA) Finnigan Surveyor MS pump, the Thermo Accela Open autosampler (PAL HTC-xt with DLW) and the Finnigan Turbo PD with detectors It consisted of a 130 column heater (Phenomenex, Torrance, CA, USA).

  An aqueous normal phase chromatography (Cogent ™ Diamond-Hydride ™, 4 μm, 100 μm, 150 × 2.1 mm, MicroSolv) with 2 μL aliquots of each prepared extract maintained at 30 ° C. at a flow rate of 200 μl / min. (Trademark) Technologies (USA) separated using a mobile phase composed of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). A gradient was applied: 0-2 minutes, 5% A; 30-39.5 minutes, 90% A; 40-45 minutes, 5% A.

  The data are based on API-MS (LTQ, 2D linear ion trap, Thermo-) using electrospray ionization (ESI) in negative and positive modes with respect to the precursor mass, with a product ion range for MS of 100-2000 amu. (Finigan, San Jose, CA, USA).

The results are shown in FIGS. The LS-MS chromatogram of the water-soluble extract of the present invention (FIGS. 19 and 22) is more than that seen in the LC-MS chromatogram of the starting mussel material (FIGS. 20, 21, 23 and 24). A more complex composition with a peak of.
Example 11: Solid-phase microextraction of water-soluble GSM extracts-GC-MS analysis Volatile organic compounds in mussel extracts PAR58 and PAR63 were modified by modified Tukeyy, Day and Miller methods (Tuckey, NPL, L.L. , Day, JR, and Miller, MR (2013), New Zealand Greenshell ™ mussel (Perna canalique) in refrigerated storage using solid phase microextraction gas chromatography-mass spectrometry. ), Food Chemistry, 136 (1), 218-223) and analyzed by SPME-GC-MS. A sample of lyophilized 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 analyzed in duplicate. After incubating the vial for 4 minutes at 40 ° C. with agitation, the volatiles were removed for 20 minutes using SPME fibers (Supelco, Bellfont, PA, USA) coated with 85 μm carboxene poly (dimethylsiloxane). Adsorbed. A blank fiber sample was run prior to and between each analytical operation to ensure a clean baseline. The adsorbed volatile material was analyzed by GC-MS. The GC-MS system consists of a GC-2010 gas chromatograph coupled with a GCMS-QP2010 mass spectrometer (Shimadzu, Kyoto, Japan) and equipped with an AOC-5000 automatic injector (PAL, CTC Analytics, Zwingen, Switzerland). It was. The conditions for GC-MS analysis were as follows: injection temperature 250 ° C .; splitless mode; 2 minute desorption in the injector; Rtx-5Sil MS capillary column—30 m × 0.25 mm ID × 0.25 μm film thickness ( Restek, Bellfont, PA, USA); furnace temperature program: start 50 ° C., hold for 1 minute, increase to 160 ° C. at 10 ° C./minute, increase to 250 ° C. at 40 ° C./minute, hold at 250 ° C. for 1 minute; He Carrier gas, flow rate 1.90 mL / min; MS detector temperature, 260 ° C. Peaks were identified by comparison with a mass spectral database (Wiley 7.0 library, Wiley, New York, NY, USA). The results are shown below in Table 13 and Table 14.

7). INDUSTRIAL APPLICABILITY The water-soluble mussel extract of the present invention and the composition containing them can be added to foods or health supplements in order to enhance health effects. Their water solubility and pleasant taste allow them to be incorporated directly into a wide range of ingestible products. The product is likely to exist in the form of tablets, capsules, beverage health “shots”, premix soup tonics, and as a component of functional foods such as delicious health food bars.

  They can also be used as pharmaceuticals to help prevent, treat or manage conditions associated with inflammation.

Claims (18)

  Water-soluble mussel extract.   The water-soluble mussel extract of claim 1, comprising at least about 45% peptide by weight, based on solids.   The water-soluble mussel extract of claim 1, comprising from about 45 wt% to about 65 wt% peptide, based on solids.   The water-soluble mussel extract according to any one of claims 1 to 3, wherein more than 90% by weight of the peptide is less than 5 kDa.   The water-soluble mussel extract according to any one of claims 1 to 4, comprising no more than about 5% by weight lipid based on solids.   The water-soluble mussel extract according to any one of claims 1 to 5, wherein the mussel is selected from the group comprising green mussels (GSM), mussels and ribbed mussels. A method for producing a water-soluble mussel extract,
(A) hydrolyzing an aqueous suspension of mussel material using one or more proteolytic enzymes;
(B) denaturing the enzyme in the hydrolysis mixture;
(C) removing insoluble material from the hydrolysis mixture to yield a water soluble mussel extract.   8. The method of claim 7, wherein the proteolytic enzyme has a slightly acidic to near neutral pH optimum.   8. The method of claim 7, wherein the proteolytic enzyme is selected from the group comprising papain, Alcalase, Enzidase FP and Enzidase Neutral.   10. A method according to any one of claims 7 to 9, wherein the mussel material is selected from the group comprising fresh whole mussel, mussel powder and mussel supercritical residue.   11. A method according to any one of claims 7 to 10, wherein the mussel is selected from the group comprising green mussels, mussels and ribbed mussels.   A water-soluble mussel extract produced by the method according to any one of claims 7 to 11.   A pharmaceutical composition comprising the water-soluble mussel extract according to any one of claims 1 to 6 and one or more pharmaceutically acceptable excipients.   A dietary supplement composition comprising the water-soluble mussel extract according to any one of claims 1 to 6 and one or more ingestible excipients.   15. A dietary supplement composition according to claim 14, which is a food composition, food additive composition, health supplement or medical food.   A method of reducing inflammation in a subject in need comprising administering to the subject a therapeutically effective amount of a water-soluble mussel extract according to any one of claims 1-6.   A method for preventing, treating or managing a condition associated with inflammation in a subject in need comprising administering a therapeutically effective amount of the water-soluble mussel extract according to any one of claims 1 to 6 to the subject. A method comprising:   The conditions associated with the inflammation include inflammatory bowel diseases including asthma, encephalitis, Crohn's disease and ulcerative colitis, chronic obstructive pulmonary disease (COPD), allergic diseases, fibrosis, juvenile arthritis, psoriatic arthritis, Arthritis including rheumatoid arthritis and osteoarthritis, psoriasis, polymyalgia, tendonitis, bursitis, laryngitis, gingivitis, gastritis, otitis, celiac disease, diverticulitis, atherosclerosis, heart 18. A method according to claim 17 selected from the group comprising disease, obesity, diabetes, cancer and Alzheimer's disease.

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