KR20180103129A - Black lips abalone (HALIOTIS RUBRA) extract - Google Patents

Abstract

The present invention relates to a method for the treatment of black lips abalone ( Haliotis The present invention relates to new extracts and extractive fractions of processed wastes of rubra . In addition, the method for producing the extract and its fractions, the ingredients of the extract and the fractions thereof, medicines, dietary supplements, and uses as functional foods are described.

Description

Black lips abalone (HALIOTIS RUBRA) extract

The present invention relates to a method for the treatment of black lips abalone ( Haliotis The present invention relates to new extracts and extractive fractions of processed wastes of rubra . In addition, the method for producing the extract and its fractions, the ingredients of the extract and fractions thereof, medicines, dietary supplements, and uses thereof as a neutraceutical are described.

Seafood processing wastes are produced only by the occurrence of thousands of tons of shells, shells and organs abandoned in Australia each year. In particular, the manufacture of abalone for sale as food produces an unbalanced amount of processing waste, since about one third of the animals, i.e., only the foot muscles, are commercially available as abalone meat. The remaining two thirds include shells, meat trimmings and intestines (bowel, organs) and are usually discarded.

Also, the processing of the abalone requires a cleaning step to produce the waste in the form of a wastewater stream that is usually discarded. In addition, the manufacture of some abalone meat for sale creates additional waste associated with preservation in the form of liquid (or cooked juice) open, usually in the form of abandoned dishes and packaging (canned or packaged). In addition, the processing waste may include small rollovers that are rejected during the classification.

The transport of abandoned rolled waste is expensive, the waste treatment is problematic and unpleasant. In addition, there are additional problems of potential environmental impacts associated with the treatment of large amounts of seafood waste.

It is necessary to find viable alternative uses for abalone processing wastes in an effort to add value for abalone resources, reduce waste treatment, and minimize environmental impacts. There is also a need to find practical alternatives to waste processing streams to add value to the abalone meat processing industry and to improve economic benefits.

The present invention is based, at least in part, on the discovery that commercially viable biologically active ingredients from non-shell processing wastes can be recovered in the production of Haliotis rubra meat. Surprisingly, it has been found that non-husked processing wastes such as viscera, meat trimmings, wastewater streams and cooking juices produced in the processing of black lips abalone are sources of anti-inflammatory and anti-thrombogenic agents. In particular, the extracts obtained from the black lip abalone waste produced significant and consistent anti-thrombotic and anti-inflammatory activity.

Based on these findings, the present inventors have developed extracts, extract fractions, compositions and methods for use in the treatment of various diseases including inflammatory and thrombotic diseases described below.

Thus, in a first aspect, the present invention provides a method of treating a black lip abalone ( Haliotis) , comprising one or more biologically active ingredients, rubra or fractions thereof. In one aspect, the process wastes are raw (or fresh) process wastes.

In some embodiments, the at least one biologically active ingredient is selected from an antithrombotic agent and an anti-inflammatory agent.

In some embodiments, the extract is fractionated to produce a fraction in which the protein component is concentrated. In some embodiments, the extract is fractionated to produce an enriched fraction of the anionic polysaccharide component.

In another aspect, the invention provides the use of the extract or its fractions according to the invention in the preparation or preparation of pharmaceuticals, dietary supplements or nutraceuticals, functional foods or functional food ingredients.

In another aspect, the invention provides a medicament, dietary supplement or nutraceutical, functional food or functional food ingredient comprising the extract of the present invention or a fraction thereof.

In yet another aspect, the invention provides a pharmaceutical composition comprising an extract or fraction thereof according to the invention, and at least one pharmaceutically acceptable carrier or excipient.

In another aspect, the present invention provides an anti-inflammatory agent comprising an extract or a fraction enriched in a protein component according to the present invention.

In another aspect, the invention provides a method of treating an IL-6 mediated disease in a patient, the method comprising administering to the patient an effective amount of an extract or fraction enriched in a protein component according to the present invention.

In another aspect, the invention provides a method of treating an inflammatory disease in a patient, the method comprising administering to the patient an effective amount of an extract or fraction enriched in a protein component according to the present invention.

In another aspect, the invention provides a method of treating an autoimmune disease in a patient, the method comprising administering to the patient an effective amount of an extract or fraction enriched in a protein component according to the present invention.

In another aspect, the invention provides the use of an extract or fraction of a protein component according to the invention in the manufacture of a medicament for the treatment of an IL-6-mediated disease, inflammatory disease or autoimmune disease, such as rheumatoid arthritis .

The present invention further provides an antithrombotic agent or an anti-coagulant comprising an effective amount of an extract or a fraction enriched in an anionic polysaccharide component according to the present invention.

In another aspect, the invention provides a method of inhibiting thrombosis, or thrombin activity, in a patient comprising administering to the patient an effective amount of an extract or fraction enriched in an anionic polysaccharide component according to the present invention.

In another aspect, the present invention provides the use of an extract or fraction enriched in an anionic polysaccharide component according to the present invention in the manufacture of a medicament for inhibiting thrombosis or thrombin activity.

In another aspect, the present invention provides a method of producing an extract or a fraction thereof according to the present invention, which process comprises digesting the processing waste of a black lips abalone using an enzymatic or chemical digestion process, And separating the extract from the waste. The inventors have found that it may be advantageous to increase or concentrate the concentration of certain components or components such as anionic polysaccharide components or protein components. Thus, the process for preparing fractions according to the present invention may comprise fractionating the extract and preferably increasing the concentration of the anionic polysaccharide component or protein component.

Figure 1 schematically illustrates in vitro inflammatory activity against various abalone waste extracts. Anti-inflammatory activity is indicated by a reduction in nitric oxide production by LPS-stimulated mouse macrophages (RAW 264.7 cells). Data indicated by "*" were statistically significant (p <0.05) for the analytical positive control (quercetin) using Dunnett's multiple comparison test and one-way ANOVA see.
Figure 2 schematically illustrates the in vitro thrombin inhibitory activity against various abalone waste extracts. The thrombin inhibitory activity is indicated by a reduction in thrombin activity using the chromogenic substrate ChromozymTH. Data indicated by "*" show a statistically significant difference (p <0.05) for analytical positive control (0.0625 mg / mL heparin) using Dunnett's multiple comparison test and one-way ANOVA.
Figures 3a and 3b schematically illustrate the anion exchange separation of various abalone waste extracts. Abalone extract 1-1 (Figure 3a) and 2-1 (Figure 3b) were separated into 30 fractions using anion exchange chromatography and linear NaCl gradient. Total protein (black) and sulfated polysaccharide (gray) are estimated from the fraction.
Figure 4 schematically illustrates the in vitro antiinflammatory activity against anion exchange chromatography fractions obtained from various rolled aborted waste extracts pooled. Anti-inflammatory activity is indicated by a reduction in nitric oxide production by LPS-stimulated mouse macrophages (RAW 264.7 cells). Data shown as "*" show statistically significant differences (p <0.05) for 10 μM quercetin in the assay positive control using multiple comparison tests and one-way ANOVA in Dunnett. The data for ^ showed significant statistical significance (p <0.05) for 1 μM quercetin in the positive control group using Dunnett's multiple comparison test and one-way ANOVA.
Figure 5 schematically illustrates in vitro heparin cofactor II-mediated thrombin inhibition for polled anion exchange chromatography fractions obtained from various abalone waste extracts. Thrombin inhibition is indicated by a reduction in thrombin activity using the chromogenic substrate ChromozymTH. Data indicated by "*" show a statistically significant difference (p <0.05) for analytical positive control (0.0625 mg / mL heparin) using Dunnett's multiple comparison test and one-way ANOVA. Data indicated by "^" showed significant statistical significance (p <0.05) for the analytical positive control (0.001 mg / mL heparin) using Dunnett's multiple comparison test and one-way ANOVA.
Figures 6a and 6b diagrammatically show the clinical score of the mice after collagen II induced arthritis (CIA). Mice were fed with control; Prophylactic / therapeutic rollover dose 1; Prophylactic / therapeutic rollover dose 2 and therapeutic prednisolone are administered orally. Prophylactic treatment is summarized in Figure 6a. Therapeutic treatment is summarized in Figure 6b.
Figure 7 schematically illustrates each clinical score at 20 days for each treatment group, including the average clinical score (in the data range).
Figure 8 schematically illustrates the LSMEANS of the clinical scores from the onset of symptoms (6 days) to 20 days for the CIA treatment group. The statistical significance measured using SAS's GLM procedure to estimate LSMEANS (least squares means) of the CIA treatment (group) effect is shown in comparison with the control group's diet: **** (p <0.0001); *** (p <0.001) and * (p <0.05).
Figure 9 schematically illustrates the IL-6 (cytokine) level LSMEANS circulating in blood serum collections at day 21. The statistical significance measured using the SAS's GLM procedure to estimate LSMEANS of the treatment (group) effect is shown in comparison with the control group's diet: **** (p <0.0001); ** (p &lt; 0.01).
Figure 10 schematically shows serum collagen-II antibody levels in blood serum collected on day 21; The antibody level is the ratio of control to non-CIA, control diet; Abalone dose 1; Rollover dose 2 and prednisolone.
Figure 11 schematically illustrates the LSMEANS total synovitis score at 21 days for CIA and non-CIA treatment groups. The statistical significance measured using the SAS GLM procedure to estimate the LSMEANS of the treatment (group) effect is shown in comparison with the control diet + CIA: **** (p <0.0001); ** (p &lt; 0.01).

Justice

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

The articles "a" and "an" are used herein to denote one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

As used herein, "and / or" refers to any and all possible combinations of one or more of the listed items, as well as a lack of combination when interpreted as an alternative (or).

The term "about ", as used herein, is intended to encompass all or part of any of the above referenced documents, including but not limited to, 10% or 9%, 8%, 7%, 6%, 5%, 4% Level, concentration, value, size, or amount that varies by 2%, 1%, 2%, and 1%.

The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a polysaccharide or oligosaccharide, a protein or a portion thereof such as a peptide), or a bacteria, plant, fungus, ) Is used to mean an extract formed from a biological material such as a cell or tissue. The activity of such agents may make them suitable as "therapeutic agents" which are biological, physiological, or pharmaceutically active substances (or substances) that act partly or wholly in the patient.

As used herein, an " autoimmune disease "refers to a disease or disorder that causes an immune response inappropriate to the body's own components, such as tissue and other components. In some embodiments, IL-6 levels elevate autoimmune disease. Non-limiting examples of autoimmune diseases that can be treated with the extracts described herein include rheumatoid arthritis, inflammatory bowel disease (especially ulcerative colitis and Crohn's disease), systemic lupus erythematosus, scleroderma, giant cell arteritis, Takayasu aeteritis, cryoglobulinemia, neutrophil-antineutrophilcytoplasmic, eczema, psoriasis, myasthenia gravis, antibody-related crescentic glomerulonephritis, rheumatic vasculitis, recurrent polychondritis, A Hemophilia, and hematocrit, including autoimmune hemolytic anemia.

Throughout this specification, unless the context requires otherwise, the word "comprises, " " comprises, " or" comprising, " Group, but does not exclude any other step or group of elements or steps or elements.

The term "blacklip abalone processing waste" as used herein refers to wastes produced during the processing of black lips abalone to produce black lips abalone (muscle, foot) for consumption. Processing wastes include some of the soft tissues of abalone that are discarded during animal processing to produce abalone, including wastewater generated during meat trimming and containment and rinsing, juices for cooking and small abalone rejected in grading do. The processing waste may be in the form of, for example, solid, powder, paste or liquid; It can be processed or cooked as it is raw; Fresh, or preserved, for example, by freezing, packaging, canning or drying.

The term "biologically active components" or "bioactive molecules ", and the like, as used herein, refers to components of an extract that have physiological effects when irradiated or administered to an animal.

As used herein, "cancer" refers to a disease or disorder, including irregular and abnormal cell growth. Non-limiting cancers that may be treated with the extracts described herein include, but are not limited to, multiple myeloma, leukemia, pancreatic cancer, breast cancer, colon cancer, cachexia, malignant melanoma, cervical cancer, ovarian cancer, lymphoma, , Lung cancer, prostate cancer, kidney cancer, metastatic renal cancer, solid tumors, non-small cell lung carcinoma, non-Hodgkin's lymphoma, bladder cancer, oral cancer, myeloproliferative neoplasm, B-cell lymphoproliferative disease, and plasma cell leukemia. Non-limiting examples of cancer-related diseases include non-small cell lung cancer-related fatigue and cancer-related anorexia.

As used herein, the terms "enrich," "enriched," "enriching," and the like refer to the process of increasing the concentration of components (eg, protein components) of a composition. For example, the protein component of the composition may be about 1.5 times, about 2 times, about 3 times, about 5 times, about 10 times, about 15 times, about 30 times, about 50 times, or about 100 times Lt; RTI ID = 0.0 &gt; fractions. &Lt; / RTI &gt;

An "IL-6 mediated disease or condition ", as used herein, refers to a disease or disorder caused by an IL-6-mediated signal, at least a portion of the symptoms and / or progression of the disease or disorder . Non-limiting examples of IL-6 mediated diseases or disorders include inflammatory diseases, malignant diseases (including cancer and cancer related diseases), infections, and autoimmune diseases. Also, non-limiting examples of IL-6 mediated diseases include, but are not limited to, Castleman's disease, ankylosing spondylitis, coronary heart disease, cardiovascular disease of rheumatoid arthritis, pulmonary hypertension pulmonary arterial hypertension, COPD, atopic dermatitis, psoriasis, eczema, sciatica, type II diabetes, obesity, giant cell arteritis, inflammatory bowel disease (especially ulcerative colitis and Crohn's disease) (GVHD), non-ST elevation myocardial infarction, antinuclear antibodies (ANCA) -related vasculitis, neuromyelitis optica, chronic glomerulonephritis , Takayasu's arteritis, graft versus host disease, and transplant rejection (including kidney transplant).

As used herein, "infection" refers to a disease or disease caused by a pathogen, such as bacteria, viruses, fungi, and the like. Non-limiting examples of infections that can be treated with the extracts described herein include human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), cerebral malaria, urinary tract infection, and meningococcal infection.

As used herein, "inflammatory disease" refers to a disease or condition involving an inflammatory response. The inflammatory response may be acute and / or chronic. In some embodiments, chronic inflammation involves an increase in the level of IL-6. Non-limiting inflammatory diseases that can be treated with the extracts described herein include rheumatoid arthritis, juvenile idiopathic arthritis, systemic-onset juvenile idiopathic arthritis, osteoarthritis, sepsis, Inflammatory bowel disease, inflammatory bowel disease, scleroderma, intraocular inflammation, Graves disease, endometriosis, scleroderma, adult-onset still disease, amyloidosis, A amyloidosis, polymyalgia rheumatic, remitting seronegative symmetrical synovitis with pitting edema, Behcet's disease, uveitis, graft versus host disease, and TNFR-associated periodic syndrome syndrome.

The term "protein components" as used herein includes polypeptides and oligopeptides. In one aspect, the protein component comprises collagen.

The term "anionic polysaccharide " as used herein refers to a negatively charged polysaccharide such as a sulfated polysaccharide and includes glycosaminoglycan polysaccharide (GAG) and glycosaminoglycan-like polysaccharide (GAG-like).

The term "nutraceutical " as used herein refers to an edible product derived from a food source. Nuclear medicine is considered to provide a health benefit and may be formed based on, for example, functional foods, food ingredients, dietary supplements, drinks, and animal feed.

As used herein, the term "anti-thrombotic agent" is an agent that reduces the formation of thrombus and can be used, for example, in the prevention and treatment of thrombosis of arteries and veins. Examples of antithrombotic agents include anticoagulants and antiplatelet agents. The antithrombotic effect can be regulated, for example, via heparin cofactor II or antithrombin (AT).

As used herein, the term "anticoagulant agent" is an agent that acts to reduce thrombosis. Anti-coagulants are used in the prevention and early treatment of venous thromboembolism, including deep vein thrombosis (DVT) and pulmonary embolism (PE). In addition, anti-coagulants can be used to prevent ischaemic complications, to treat unstable angina, and to treat non-Q-wave myocardial infarction. Anti-coagulants can be used for the prevention or treatment of DVT, for example, after joint replacement surgery or abdominal surgery. It may also occur due to injury or mobility limited as a result of illness; Or for the prevention or treatment of DVT, which may be a complication of malignant diseases such as cancer. In addition, anti-coagulants can be used prophylactically in situations where a patient history of DVT or pulmonary embolism is present. The anti-coagulant may also be used to inhibit thrombosis in a patient undergoing surgery, such as an intravascular catheterization procedure or cardiac surgery. Other therapeutic uses of anti-coagulants include treatment of atrial fibrillation, congestive heart failure, myocardial infarction and genetic or acquired coagulopathy.

The term " antiplatelet agent "or" antiaggregant agent ", as used herein, is an agent that acts to reduce platelet aggregation in blood and inhibits the formation of thrombus. Antiplatelet agents are effective in arterial circulation, where anti-coagulants may not be as effective. Platelet aggregation can cause, for example, heart attack, angina pectoris or stroke. Antiplatelet agents include thrombotic cerebrovascular disease such as stroke, mini-stroke, ischemic stroke or hemorrhagic stroke; Or at least one of the following: hypertensive heart disease, rheumatic heart disease, cardiomyopathy, atrial fibrillation, congenital heart disease, endocarditis, aortic aneurysms, peripheral artery disease, Can be used for the prevention or treatment of venous thrombosis or coronary artery disease such as angina pectoris or myocardial infarction.

The term "thrombosis " as used herein refers to intravascular coagulation of blood in the circulatory organs of the heart, arteries, veins and capillaries. The term "thrombin" refers to an enzyme that acts as a catalyst for some modifications in the blood coagulation cascade. It also promotes platelet activation and coagulation.

The terms " individual ", "patient ", and" subject "are used interchangeably herein to refer to each of human or other animal origins, Lt; / RTI &gt; However, it should be understood that this term does not mean that the symptoms are present. Suitable animals within the scope of the invention include, but are not limited to, humans, primates, livestock (e.g., sheep, cow, horse, donkey, pig), laboratory test animals (such as rabbits, mice, rats, guinea pigs, ), Pets (e.g., cats, dogs), and captive wild animals (e.g., foxes, deer, dingoes, birds, reptiles).

As used herein, "malignant disease" includes cancer and cancer-related diseases.

As used herein, the terms " treatment ", "treating ", and the like refer to administering an agent to achieve a desired pharmacological and / or physiological effect. This effect may be prophylactic in terms of preventing the disease or its symptoms in whole or in part, or may be therapeutic in terms of partially or completely treating the symptoms of the disease and / or disease. This effect may be therapeutic in terms of partial or complete treatment of the disease or disorder (e. G., A disease or disorder mediated by IL-6) and / or an adverse effect attributable to the disease or disorder. The term also covers any treatment of a mammal, particularly a human disease or disorder, and includes (a) a disease or condition, such as a disease or condition associated with or caused by a primary disease or disease Preventing a patient suffering from such a disease or disease from being diagnosed as having a disease or disease or a disease or condition; (b) inhibiting the disease or disease, ie preventing its development; (c) relieving the disease or disorder, i. e., causing regression of the disease or disorder; (d) alleviating the symptoms of the disease or disorder, and / or (e) reducing the frequency of symptoms of the disease or disorder.

"Pharmaceutically acceptable carrier, excipient or diluent" means a solid, liquid filler, diluent or encapsulating material that can be safely used for systemic administration.

Extract of black lips abalone

The present inventors have found that extracts obtained from black lips abalone waste, or fractions thereof, contain biologically active ingredients having anti-thrombotic or anti-inflammatory activity.

The extract is prepared from a waste stream obtained from the processing of black lipped meat for consumption. The extracts are prepared from the processing wastes using enzymes or processing including chemical digestion, thermal deactivation and purification steps.

Thus, in a first aspect, there is provided an extract of black lips rollover, or fractions thereof, comprising at least one biologically active ingredient, derived from black lips rolled wastes. In some embodiments, the extract is derived from viscera. In some embodiments, the processing waste is raw. In some embodiments, the processing waste is preserved. In some embodiments, the processing waste is in liquid form. In some embodiments, the processing waste is in the form of a solid, e.g., a paste or a powder. In some embodiments, the processed wastes can be canned, chilled or frozen. In some embodiments, the black lip overturn is an abalone harvested in the wild.

In some embodiments, the at least one biologically active component is selected from an anionic polysaccharide component and a protein component. In some embodiments, the protein component comprises collagen. In some embodiments, the anionic polysaccharide component comprises at least one sulfated polysaccharide component. In some embodiments, the anionic polysaccharide component comprises at least one glycosaminoglycan-like polysaccharide.

In another aspect, the present invention provides a method of making an extract according to the present invention, which method comprises digesting the processing waste of a black lips abalone using an enzymatic or chemical digestion process and separating the extract from the digested waste . In some embodiments, the method comprises enzymatic digestion. Exemplary enzymes for enzymatic digestion include bromelain and / or papain. In some embodiments, the processed waste sample is digested for about 16-18 hours. In some embodiments, the digest volume contains about 10% w / v processing waste. In some embodiments, the package volume contains about 2% w / v of one or more enzymes, such as about 1% w / v bromelain and about 1% w / v papain. In some embodiments, the digests are heat inactivated at about 95 캜 for about 10 minutes prior to cooling. In some embodiments, the package is fractionated and separated into other fractions of the fraction. In some embodiments, the fraction produces a fraction in which the protein component is concentrated. In some embodiments, the fraction produces a fraction in which the anionic polysaccharide component is concentrated.

Fractions can be achieved using any suitable technique. Suitable techniques are well known to those skilled in the art and include, for example, centrifugation, extraction, continuous liquid-liquid extraction, dialysis evaporation, membrane filtration, extractive filtration and ultrafiltration membrane separation, reverse osmosis, electrodialysis, distillation, crystallization , Ion exchange chromatography, size exclusion chromatography (gel filtration, gel permeation), and absorption chromatography.

In some embodiments, the method of manufacturing comprises digesting black lips rolled waste in the presence of digestive enzymes to produce aqueous fractions and precipitates; And separating the aqueous fraction from the precipitate to produce an extract. In some embodiments, the method of the present invention further comprises the step of filtering the aqueous fraction to produce a filtrate. In some embodiments, the filtrate is desalted. In some embodiments, the filtrate is desalted using a 3 kDa filter to produce an extract as a 3 kDa retentate.

The black lip rollover extract can be separated into fractions using, for example, ion exchange chromatography and size exclusion chromatography. In some embodiments, the method further comprises separating the extract, preferably using ion exchange chromatography and / or size exclusion chromatography, into one or more fractions in which the protein component or anionic polysaccharide component is concentrated. The components of the extract or extract fraction of the present invention relative to the amount of protein component and anionic polysaccharide component are readily determined by methods known in the art as described in the Examples herein.

Thus, in another aspect, the present invention provides a black lipped-skin extract, or extract fraction, in which the protein component is concentrated. In some embodiments, the protein component comprises protein collagen.

In another aspect, the present invention provides an extract of black lips, or an extract fraction, in which the anionic polysaccharide component is concentrated. In some embodiments, the anionic polysaccharide component comprises at least one sulfated polysaccharide. In some embodiments, the anionic polysaccharide component comprises at least one glycosaminoglycan-like polysaccharide component.

In another aspect, the present invention provides a black lip rollover extract, or extract fraction, which can be obtained or obtained by the method of the present invention.

The method of the present invention

The extracts and extract fractions of the present invention comprise at least one biologically active ingredient. In some embodiments, the at least one biologically active ingredient is selected from an antithrombotic agent and an anti-inflammatory agent. In some embodiments, the antithrombotic agent comprises at least one anionic polysaccharide component. In some embodiments, the antithrombotic agent comprises one or more glycosaminoglycan-like polysaccharide components. In some embodiments, the one or more biologically active ingredients are selected from anti-inflammatory agents. In some embodiments, the anti-inflammatory agent comprises one or more protein components.

In accordance with the present invention, it has been found that the extracts and fractions obtained from black lips rollover wastes produce significant and constant anti-thrombotic and anti-inflammatory activity in vitro. It has also been found that these extracts and fractions significantly reduce the symptoms of severe arthritis in vivo in a mouse model of rheumatoid arthritis.

In particular, mice had less severe symptoms than rats fed the primary feed, before the onset of rheumatoid arthritis and during the onset of arthritis.

In addition, the present inventors have found that, based on clinical, histological and immunological basis, the black lip rolled waste extract according to the present invention is positive in mouse models of rheumatoid arthritis and produces remarkable results. Based on clinical results, prophylactic administration of abalone extract significantly reduces toe, foot and wrist swelling and anomalies from the onset of arthritis symptoms to the end of clinical trials (FIGS. 7 and 8). Based on the histological basis, prophylactic and therapeutic administration of the abalone extract significantly reduces the total scleritis observed in the joints and feet (Figure 11). Finally, based on the immunological basis, prophylactic administration of Abalone extract and therapeutic administration of Abalone extract significantly reduced circulating levels of IL-6 (FIG. 9), which plays an important role in chronic inflammation and rheumatoid arthritis (Gabay C, Interleukin-6 and chronic inflammation, Arthritis Res Ther., 2006; 8 Suppl 2: S3; Hennigan S et al , Interleukin-6 inhibitors in the treatment of rheumatoid arthritis. 4): 767-75) and showed a remarkable likelihood of black lips abalone extract in the treatment of inflammatory diseases such as rheumatoid arthritis.

Thus, the extracts and fractions of the present invention are considered useful, for example, in the treatment or prevention of IL-6 mediated diseases or disorders as described above. In some embodiments, the fraction is a fraction in which the protein component is concentrated. In some embodiments, the IL-6 mediated disease or condition includes, but is not limited to, rheumatoid arthritis, inflammatory bowel disease (especially ulcerative colitis and Crohn's disease), eczema, psoriasis and myasthenia gravis, Lt; / RTI &gt; or autoimmune diseases or diseases.

In some embodiments, the extracts and fractions of the present invention are useful for the prevention or treatment of rheumatoid arthritis.

Accordingly, the present invention provides a method of treating an IL-6 mediated disease in a patient, comprising administering to the patient an effective amount of an extract, preferably a fraction enriched in the protein component, according to the present invention.

The present invention provides a method of treating an inflammatory disease in a patient comprising administering to the patient an effective amount of an extract of the present invention, preferably a fraction enriched for the protein component. In one embodiment, the inflammatory disease is an autoimmune disease.

Also provided is a method of treating an autoimmune disease in a patient comprising administering to the patient an effective amount of an extract of the invention, preferably a fraction enriched for the protein component.

In another aspect, the present invention provides the use of the extract of the present invention, preferably a fraction enriched in protein components, in the manufacture of a medicament for treating an IL-6 mediated disease, an inflammatory disease, or an autoimmune disease.

In some embodiments, the disease is rheumatoid arthritis.

Further investigation of antiinflammatory activity in vivo using a mouse model of rheumatoid arthritis was used to evaluate prophylactic and therapeutic doses of low and high dose abalone extracts. Oral administration significantly reduced the symptoms of arthritis, including decreased swelling and malformations, while maintaining animal weight.

Thus, the present invention provides the use of the extract or fraction of the present invention as a medicament. In some embodiments, the medicament is formulated for oral administration. Also provided is the use of the extract of the present invention in the manufacture of dietary supplements, nutraceuticals, functional foods or functional food ingredients.

In addition, the inventors have found that extracts and fractions from black lips rolled wastes produce significant and constant antithrombotic activity in vitro. The extract or fraction of the present invention is considered to have antithrombotic or anticoagulant properties and is considered useful for the treatment or prevention of thrombosis or for the inhibition of thrombin activity.

Accordingly, the present invention further provides an antithrombotic or anticoagulant comprising an effective amount of an extract of the present invention, preferably a fraction enriched in an anionic polysaccharide component. In some embodiments, the antithrombotic effect is modulated via heparin cofactor II or antithrombin.

In another aspect, the present invention provides a method of inhibiting thrombosis or inhibiting thrombin activity in a patient comprising administering to the patient an effective amount of an extract of the present invention, preferably a fraction enriched in an anionic polysaccharide component, .

In another aspect, the invention provides the use of an extract according to the invention, preferably a fraction enriched in an anionic polysaccharide component, in the manufacture of a medicament for inhibiting thrombosis or thrombin activity.

Subjects, individuals or patients to be treated include, but are not limited to, livestock such as humans, primates, sheep, cows, pigs, horses, donkeys, goats; Laboratory test animals such as mice, rats, rabbits and guinea pigs; Cats and dogs, or zoo animals in zoos. In certain embodiments, the patient is a human.

"Effective amount" means an amount that is at least partially required to obtain a favorable response to a particular disease to be treated, delay the onset, inhibit the progress, or stop all. The amount will depend on the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated, the degree of protection desired, formulation of the composition, evaluation of the medical situation, and other relevant factors. This amount is expected to be in a relatively broad range that can be determined through routine experimentation. An effective amount for a human patient can range, for example, from about 0.1 ng / kg body weight to 1 g / kg body weight / dose. The dosage is preferably in the range of 1 mg to 1 g / kg of body weight / dose, such as in the range of 1 μg to 1 g / kg body weight / dose. In one embodiment, the dosage is in the range of 1 mg to 500 mg / kg body weight / dose. In another embodiment, the dosage is in the range of 1 mg to 250 mg / kg body weight / dose. In another embodiment, the dosage is in the range of 1 mg to 100 mg / kg body weight / dose, for example up to 50 mg / kg body weight / dose. In another embodiment, the dosage is in the range of 1 [mu] g to 1 mg / kg body weight / dose. The dosage regimen may be adjusted to provide an optimal therapeutic response. For example, some dispensed doses may be administered daily, weekly, monthly or at other suitable time intervals, or the dose may be proportionally reduced depending on the situation.

The terms "treatment" and "prevention" are to be considered in their broadest sense. The term "treatment" does not necessarily mean that the patient is treated until fully recovered. In addition, "treatment" can reduce the severity of an existing disease. The term "prevention" does not necessarily mean that the patient will eventually be prevented from suffering from the disease. The term "prevention" can be considered to include delaying the onset of a particular disease. Thus, treatment and prevention include preventing or reducing the symptoms of a particular disease or the risk of developing a particular disease.

In some embodiments, the extract of the present invention may be administered with other therapies. The compounds or therapies may be administered simultaneously or sequentially to a single composition or to individual compositions so that each compound or therapy is active within the same time in the body.

The extract of the present invention may be administered with one or more additional nutraceutical products such as, for example, omega-3 oil, glucosamine or chondroitin sulfate. In addition, the extracts, especially the extract fractions, in which the protein components are concentrated can be used as anti-inflammatory agents including steroids such as cortisone, hydrocortisone, prednisolone, methylprednisolone, prednisone, budesonide, mometasone, triamcinolone and aclometasone , And non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, diflunisal, salsalate, ibuprofen, napoxen, fenoprofen, ketoprofen, flubiprofen, oxaprozin, loxoprofen, indomethacin, sulindac, etodolac, ketorolac, Diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, methane, and the like. Metenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, celecoxib, rofecoxib, valdecoxib, parecoxib, etore May be administered together with etoricoxib and ferocoxib.

Extracts and fractions of the present invention are considered to be beneficial to health and / or well-being, thus providing a method for enhancing or maintaining the well-being of a patient, comprising administering to the patient an effective amount of an extract or fraction of the invention, .

The composition of the present invention

The extract or fraction of the present invention can be taken alone as a clean extract. However, in some conditions, it is desirable to formulate an extract for consumption. For example, the extract may be formulated as a dietary supplement, a nutraceutical, a functional food, or a functional food ingredient.

In another aspect, the present invention provides pharmaceuticals, dietary supplements, neutrons, functional foods or functional food ingredients comprising the extract or fraction according to the invention.

The extract or fraction of the present invention can be formulated as a food or beverage product. Animal feed is also contemplated as part of the present invention.

In some embodiments, the dietary supplement, nutraceutical, functional food or functional food ingredient further comprises one or more acceptable excipients, fillers or carriers. In some embodiments, the extract or fraction of the present invention may be used in combination with one or more other active ingredients selected from, for example, a nutraceutical, a functional food or a functional food ingredient. The content ratio of the active ingredient can be appropriately determined depending on the purpose of use. Dietary supplements, nutraceuticals, functional foods or functional food ingredients can be suitably used according to conventional methods. Generally, when preparing solid or liquid food, the extract of the present invention can be added up to 50% w / w, more preferably up to 30%, 20% or 10% w / w. In general, when the beverage is prepared, the extract of the present invention can be added up to 20% w / w, more preferably up to 15%, 10% or 5% w / v.

Suitable excipients, fillers or carriers are well known in the art and include starches, sugars, cooking oil, water, glycerin, gum, methylcellulose and the like.

In addition, the dietary supplement, nutraceutical, functional food or functional food ingredient composition of the present invention may contain additional ingredients such as flavorings, colorants, sweeteners, thickeners, pH adjusters, stabilizers or preservatives.

The dietary supplement, nutraceutical, functional food or functional food ingredient composition of the present invention may further comprise additional active ingredients such as nutrients, vitamins, or electrolytes.

Nutraceutical, functional food or food ingredient compositions can be formulated, for example, as powders, granules, liquids, pastes, oils or gels.

Dietary supplements can be formulated, for example, as peels, tablets, capsules, soft gels, gelcaps, pastes or powders. In addition, dietary supplements can be formulated into liquids, such as solutions or suspensions.

The required daily dose of the extract or fraction according to the invention may be determined according to the particular needs of the individual. A typical daily intake is considered as 100 mg to 500 mg / kg body weight / day. In one aspect, the daily intake of the fraction is less than 100 mg / kg body weight / day.

In some embodiments, the extract or fraction of the present invention can be introduced as a conventional food. Accordingly, the present invention further provides a food comprising the extract of the present invention.

In another aspect of the present invention, there is provided a pharmaceutical composition comprising an extract or fraction according to the invention, and at least one pharmaceutically acceptable carrier or excipient. In some embodiments, the composition is used to treat or prevent an inflammatory disease or disorder. In some embodiments, the composition is used to treat an autoimmune disease or disease, such as rheumatoid arthritis. In some embodiments, the fraction is enriched in protein components. In some embodiments, the fraction is concentrated in the anionic polysaccharide component.

For use in therapy, the extract or fraction of the present invention may be administered alone, but in some embodiments the active extract or fraction may be provided in the form of a pharmaceutical composition.

Accordingly, in another aspect of the present invention, there is provided a pharmaceutical composition comprising an extract or fraction of the present invention and at least one pharmaceutically acceptable carrier, excipient or diluent.

The pharmaceutical composition of the present invention or the composition used in the method of the present invention can be formulated and administered using methods known in the art. Techniques for formulation and administration Remington: The Science and Practice of Pharmacy , Loyd V. Allen, Jr (Ed), The Pharmaceutical Press, is found in ^{London, 22 nd Edition, September 2012} .

The carrier (s) must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient.

The pharmaceutical composition or formulation may be in a form suitable for oral, rectal, nasal, topical (including ball and sublingual), vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration, or in a form suitable for administration by inhalation or aeration . Thus, in addition to conventional additives, carriers, excipients or diluents, the compounds of the present invention may be presented in the form of pharmaceutical compositions and unit dosages thereof, and in this form may be formulated as solid, such as tablets or filled capsules, Capsules filled with suspensions, emulsions, elixirs, or liquids, in the form of suppositories for rectal administration; Or parenterally (including by subcutaneous) in the form of a sterile injectable solution. Such pharmaceutical compositions and their unit doses may contain conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may be formulated to contain any of the active ingredients May include a suitable effective amount. Thus, formulations containing 10 milligrams of active ingredient per tablet, or more broadly 0.1 to 200 milligrams, are suitable representative unit dosage forms. The compounds of the present invention can be administered in a wide variety of oral and parenteral dosage forms. It will be ordinary to those skilled in the art that the following dosage forms may comprise the extract of the present invention as an active ingredient.

For preparing pharmaceutical compositions from the extracts or fractions of the present invention, the pharmaceutically acceptable carrier may be either solid or liquid. Solid formulations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. The solid carrier can be one or more substances that can act as diluents, flavors, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or encapsulating materials.

In a preferred aspect, the extract or fraction is formulated for oral administration. Suitably the extract or fraction is formulated as a powder, tablet, pill, capsule, or dispersible granule.

In tablets, the active ingredient is compacted in the desired shape and size, and mixed with a carrier having the necessary binding capacity in the appropriate proportions.

The powders and tablets preferably contain 5-70% of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoa butter, and the like. The term "preparation" is intended to include the formulation of the active compound with the encapsulating material as a carrier, in which the active ingredient, with or without a carrier, is surrounded by the carrier and thus provides a capsule associated with the carrier.

Liquid form preparations for oral administration include solutions, suspensions, and emulsions such as water or water-propylene glycol solutions. Aqueous solutions suitable for oral use can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavors, stabilizers and thickening agents as necessary. Aqueous suspensions suitable for oral use can be prepared by dispersing the finely divided active ingredient in water with a viscous material such as natural or synthetic gum, resin, methylcellulose, sodium carboxymethylcellulose, or other well known suspending agents have.

Also included are solid formulations which are rapidly converted to liquid form preparations prior to use for oral administration. Such liquid forms include solutions, suspensions, and emulsions. Such formulations may additionally contain active ingredients, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents and the like.

Alternatively, the active ingredient may be provided in the form of a powder mixture of the compound in a suitable powder base such as a dry powder, such as lactose, starch, hydroxypropylmethylcellulose and starch derivatives such as polyvinylpyrrolidone (PVP).

If desired, formulations adapted to provide sustained release of the active ingredient may be applied.

The pharmaceutical preparations are preferably unit dosage forms. In this form, the agent is subdivided into unit doses containing appropriate quantities of the active ingredient. The unit dosage form can be a packaged preparation, and the packaging contains separate amounts of the formulation, such as blister pack tablets, capsules, and powders in glass bottles or ampoules. In addition, the unit dosage form may be a capsule, tablet, cachet, or lozenge itself, or it may be any suitable number of these in packaging form.

It will be apparent to those skilled in the art that various modifications and variations will become apparent. All such variations and modifications as would be apparent to one skilled in the art are intended to be within the spirit and scope of the present invention as broadly described.

Certain preferred embodiments will be described by way of the following non-limiting examples in order that the present invention may be readily understood and may exhibit practical effects.

Example

The following black lips rolled waste samples are used:

Sample 1: liquid; Built and packed abalone; Abalone harvested in the wild

Sample 2: solid (paste); Built and canned abalone; Abalone harvested in the wild

Sample 3: solid (powder); Processed abalone; Abalone harvested in the wild

Sample 4: Solid; Raw abalone (raw); Abalone harvested in the wild

All samples except powder samples are transported fresh at 4 ° C or transferred onto dry ice before storage at -20 ° C until needed. Powder samples are stored at room temperature (~ 21 ° C).

Extract formulation

The extract is prepared from the processing waste using methods including digestion, centrifugation, filtration and desalting. The entire processed waste sample is digested overnight at 50 ° C. (16-18 hours) using two food grade enzymes, bromelin and papain (eg, available from Enzyme Solutions Pty Ltd, Vic 3136). The entire solid waste sample is cut into small pieces (1 - 2 cm ² ). The digestible volume is in the range of 50-1000 mL (in water), and the additional enzyme (0.25 - 10.0 g) of each of the 10-20% w / v processed waste (5 - 200 g) and 0.5 - 1% (Osborne SA et al , Glycobiology. 2008 Mar; 18 (3): 225-34). After overnight incubation, digestion is heat-inactivated at 95 ° C for 10-20 minutes and then cooled on ice and then centrifuged at 5,940-50,000 g for 10-30 minutes to remove any fat (upper) The precipitate (pellet) was removed. The aqueous layer (the supernatant) was maintained and dried in order using 2 μm and 1 μm (Whatman glass microfiber) pre-filters and 0.45 μm (Millipore mixed cellulose ester) Filtered. The filtered sample was then desalted using a 3 kDa (Amicon Ultra-15, Ultracel low binding regenerated cellulose membrane) centrifugal filter device and the residue (> 3 kDa and low salt Containing / non-salt) and filtrate (<3 kDa and salt containing) extract samples. Residual samples were screened for further analysis due to possible interference of the salts in quantitative and bioavailability assays.

As used herein, the term "extract" refers to a 3 kDa residue sample unless otherwise stated.

Total protein estimate

Total protein content is estimated from extracts using the Pierce BCA (bicinchoninic acid) Protein Assay Kit (Thermo Fisher Scientific, VIC, 3179, Australia) according to the manufacturer's instructions. Briefly, 25 μl of standard (bovine serum albumin) and extracts were diluted with water and added to 96-well plates (Nunc®, polystyrene) three times. 200 [mu] l of BCA working reagent was prepared (by mixing Reagent A and Reagent B in a 50: 1 ratio) and added to each well. Plates were mixed and incubated for 30 min at 37 [deg.] C, then the plate absorbance was read at 562 nm. A linear standard curve was constructed and used to interpolate the total protein concentration in the diluted extract.

Total Collagen Estimation

Total collagen content is estimated from extracts using the QuickZyme Biosciences Total Collagen Assay Kit (Bio-Scientific, Kirrawee, NSW, Australia) according to the manufacturer's instructions. Briefly, 125 μl of standard (rat tail collagen) and extract were added to 125 μl 12 M HCl and hydrolysed in a screw-capped tube at 95 ° C for 20 hours. After hydrolysis, 35 μl of hydrolyzed samples and standards were transferred to the provided 96-well plate, mixed with 75 μl assay buffer and incubated for 20 minutes at room temperature (~ 21 ° C). Then, 75 μl of detection reagent was added to each well, and the resulting solution was mixed and incubated at 60 ° C. for 30 minutes. Plate absorbance was read at 570 nm. A linear standard curve was constructed and used to interpolate the total collagen concentration in the extract. All analyzes were performed twice or three times.

Sulfation  Polysaccharide estimation

Sulfated polysaccharide content was estimated using two different assays.

First, the content of sulfated polysaccharides was estimated using basic 1,9-dimethyl methylene blue analysis (DMMB), and the Blyscan Sulfated Glycosaminoglycan Assay (Labtek Pty Ltd, QLD 4500, Australia ) Was used to determine the dilutions needed to more accurately measure the sulfated polysaccharide content.

DMMB dyes have been found to be useful in the synthesis of sulfated glycosaminoglycans (Osborne SA et al , Glycobiology. 2008 Mar; 18 (3): 225-34) and various other forms of sulfuric acid and anionic polysaccharides (Cinelli LP et al ., Comp Biochem Physiol B . Biochem Mol Biol 2009 Sep; 154 (1): 108-12; Keler T et al, Anal Biochem 1986 Jul; 156 (1):.. 189-93 is otitis paints (metachromatic dye) for detecting the 19-20) .

In addition, the bliccane sulfosuccinic acid glycosaminoglycan analysis is based on DMMB dyes. Concentrations of sulfated polysaccharides are estimated from extracts according to the manufacturer's instructions. Briefly, 25 μl of standard (chondroitin sulfate from bovine cartilage) and sample were added 3 times to a V bottom bottom polypropylene plate (PerkinElmer, Melbourne 3150, Australia). 250 [mu] l of blisscreen dye reagent was added to each well. The plates were then placed on an orbital shaker for 30 minutes at room temperature and centrifuged at 2090 g for 10 minutes. The supernatant (liquid) was removed and replaced with 250 μl of Blisscane dye dissociation reagent. After mixing the plates on the orbital shaker for 30 minutes, 200 μl of each standard and sample were transferred to a polystyrene 96-well plate (Nunc®) for absorbance reading at 656 nm. A linear standard curve was constructed and used to interpolate the sulfated polysaccharide concentration in the extract.

In vitro anti-inflammatory activity

In terms of anti-inflammatory activity, viability was demonstrated using in vitro cell-based assays. Inflammatory cell status was induced in the murine macrophage cell line, RAW 264.7 (Sosroseno W et al , Oral Microbiol Immunol. 2002 Apr; 17 (2): 72-8) using bacterial lipopolysaccharide (LPS). Inflammation was expressed as cell formation of nitric oxide (NO) as measured using a Greek reagent. Decreased production of NO by positive control or by overturned waste extracts exhibits anti-inflammatory activity.

The anti-inflammatory activity was compared with some of the previously described extracts with some minor modifications (Cho JY et al, Eur J Pharmacol 2000 Jun 23; 398 (3): 399-407; Kim AR et al, Arch Pharm Res. 2005 Mar; 28 (3): 297-304) and a quercetin positive control (Sigma Aldrich Corporation). RAW 264.7 cells (American Type Culture Collection, www.ATCC.com) were incubated with 100 U / mL penicillin, 100 μg / mL streptomycin (Life Technologies, Thermo Fisher Scientific, Vic 3179, Australia) and 10% v / v fetal serum Cultured in RPMI-1640 medium (Thermo Fisher Scientific, Vic 3179, Australia) supplemented (in vitro) and grown at 37 ° C and 5% CO ₂ in humid air. The anti-inflammatory assay was performed over two days. For one day, the cells were seeded on a 6 × 10 ⁴ 96-well plates at a cell / well density. 300 ng / mL LPS in culture was added to cells with positive control (six point dose response of quercetin using a 0-100 μM concentration range) or extract. Cells were cultured for 48 hours, and on day 2, NO secreted by RAW 264.7 cells was measured in culture by grease reaction (50 μL of cell culture was added to 50 μL of grease reagent (Sigma Aldrich Corporation)) . Standard curves were also made with sodium nitrate (0 - 50 μM) and used to interpolate NO production in cell culture media. All analyzes were performed 3 times (n = 3). In addition, the cell viability was measured using a MTS CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (Promega Corporation, VIC 3122, Australia) in response to LPS, quercetin and extracts according to the manufacturer's instructions Respectively.

In vitro thrombin inhibition

In terms of thrombin inhibition, viability was measured using an in vitro kinetic assay using a chromogenic substrate, Chromozym TH (CH-TH) (Dupouy D et al , Thromb Haemost. 1988 Oct 31; 60 (2): 236-9). Thrombin is a serine protease associated with blood clotting by converting soluble fibrinogen into an insoluble fibrin strand. CH-TH was decomposed by thrombin to produce a yellowish compound (called p-nitroaniline). Thrombin can be inhibited by heparin cofactor II (HCII) and antithrombin (AT) through various sulfated polysaccharides. Reduced production of p-nitroaniline exhibits antithrombotic activity which may lead to anticoagulant activity.

The heparin cofactor II (HCII) -mediated thrombin inhibition was measured in 96 well plates (Osborne SA et al , Glycobiology. 2008 Mar; 18 (3): 225-34) Dupouy D et al, Thromb Haemost 1988 Oct 31; 60 (2): 236-9)). Briefly, HCII, assay buffer (0.02 M Tris-HCl pH 7.4 / 0.15 M NaCl / 1 mg / mL PEG), and extracts or commercial specifications (pig heparin from Sigma Aldrich Corporation) were mixed and incubated for 2 minutes at room temperature Followed by the addition of 150 nM thrombin. This solution was mixed gently for 1 minute and then 100 [mu] M CH-TH was added. The assay was incubated for 40 min at 37 ° C and absorbance was measured at 405 nm (SpectraMax PLUS 384 microplate reader, Molecular Devices) at 2 min intervals. HCII-mediated thrombin inhibition by extract or heparin standards was expressed as a percentage reduction in thrombin activity after 40 minutes. Each sample was analyzed twice (n = 3) and expressed as the mean of the standard error.

Antithrombin (AT) -mediated thrombin inhibition was measured in extracts and fractions using a 384 well-format, robotic-assisted assay. In this kinetic analysis, thrombin degraded Chromozym TH to generate two molecules: the residual peptide, which can be measured by absorbance change at 405 nm, and 4-nitroaniline. Analytical standard Heparin (final concentration range: 0.016-1.04 μg / mL) or the molecule is present in a sample that binds to ATIII (0.2 μg / mL) and forms a three component complex with thrombin (2.03 ng / To decompose and to form 4-nitroaniline. The heparin standard is diluted and has a final concentration of 0.016 - 1.04 μg / mL. ATIII-mediated thrombin inhibition by extract or heparin standards was expressed as a percentage reduction in thrombin activity after 40 minutes. Each sample was analyzed 3 times (n = 3) and expressed as the mean of standard error.

In vitro plasma and blood coagulation assay

Prothrombin time (PT) analysis

100 [mu] L citrated plasma was added to a glass clotting tube and incubated for 5 min at 37 [deg.] C on a heating block of Hyland-Clotek noodles (Hyland, USA) . The instrument measured elapsed time until solidification formation. Abalone extracts at various concentrations of 50 [mu] L were added to the tubes using saline as a blank. After adjusting the total volume to 150 μL with plasma, 100 μL of Thromborel S® was added to initiate coagulation (Siemens, 545477, USA).

Active portion Thromboplastin  time( aPTT ) analysis

The activated partial thromboplastin time (aPTT) was measured by adding 100 μL citrate plasma, 100 μL Triniclot (TriniCLOT aPTT, HS, Tcoag, Ireland Limited) and various concentrations of rolled extract to the coagulation tube. The final volume was adjusted to 250 μL by addition of saline. The clotted tubes were incubated for 5 min on a heating block of Hyland-Clotek noodles at 37 ° C, and then 50 μL of 50 mM Ca + 2 was added until the initial coagulation.

Ion exchange chromatography

Biologically active molecules were separated based on their charge using anion exchange chromatography to separate biologically active molecules and to produce fractions in which collagen or anionic polysaccharides are concentrated.

Anion separation was performed on the rolled waste extract using packed columns of Q Sepharose Fast Flow or Q Sepharose Big Beads (GE Healthcare, Little Chalfont, UK). The extract was applied to a column of Buffer A (water) and eluted from Buffer B (2M NaCl) using a linear gradient from 0-100% Buffer B. After washing with buffer A (i.e., unbound molecules), sample application and flow through were collected with gradient fractions.

Rheumatism  Mouse model of arthritis

In terms of in vivo anti-inflammatory activity, viability is investigated using established and valid animal models of rheumatoid arthritis, namely collagen type II-induced arthritis (CIA) model of mice (van Duivenvoorde LM et al , Arthritis. Oct; 50 (10): 3354-64 ; Leung BP et al, J. Immunol 2004 Jul 1; 173 (1):... 145-50; van Holten J et al, Arthritis Res Ther 2004; 6 (3) : R239-49). Two overtube extract doses, 100 mg and 500 mg / kg body weight / day (doses 1 and 2, respectively) were introduced into the pellet diet and before and during 21 days of collagen II induced arthritis (CIA) , Or after the induction and onset of CIA symptoms (therapeutic dose). In addition, all doses of Abalone extract were administered to mice without CIA and any side effects of the Abalone extract were monitored.

Specifically, 108 male DBA-1 mice (10-12 weeks old) were weighed and divided into 9 groups (n = 12 mice / group). The test was performed in two batches of 54 mice (n = 6 mice / group). Animals were anesthetized with isoflurane prior to injection of chicken collagen type II in the Freunds complete adjuvant (Sigma Aldrich Company) at two sites about 1 cm apart from the tail base. And the introduction of the CIA. Animals were then monitored daily for 3 days and weighed weekly before booster immunization was given on the 21st day after the first injection (at 2 sites about 2 cm away from the tail base). After re-vaccination, the animals were monitored and weighed, and the arthritis symptoms were scored every 3 days for a total of 3 weeks. In addition, anti-inflammatory drug, prednisolone, was included in the study as a positive treatment control.

The group received the following treatment:

1. Control food; No CIA

2. Dose 1; No CIA

3. Dose 2; No CIA

4. Control food + CIA

5. Dose 1 (prophylactic) + CIA

6. Dose 2 (prophylactic) + CIA

7. Dose 1 (therapeutic) + CIA

8. Dose 2 (therapeutic) + CIA

9. Prednisolone (therapeutic + CIA)

Test Timeline

-29 days Preventive feeding begins

-21 days First inoculation

0 days Re-vaccination

6 days Start of symptoms and start of therapeutic feeding

21st closing

Clinical scores were determined using a standard arthritis scoring system as follows:

Score 0: No arthritis

Score 1: Only one or two toes affected

Score 2: swelling of three or more toes and / or feet (metacarpus / metatarsus)

Score 3: Swelling of wrist (carpus) / ankle (tarsus)

Score 4: Malformations with joint stiffness of wrist / ankle bone.

At the end of the day, blood samples were collected by cardiac puncture under anesthesia before euthanasia. Blood samples were allowed to clot for 1 hour at room temperature, rotated, and serum collected. Anti-collagen II antibodies and cytokines were determined by ELISA. Also, at the end of the day, one knee joint and hind paw were taken from each animal, fixed in 10% neutral buffered formalin, calcined in 10% formic acid in 5% formalin and paraffin embedded. The thalamic region of the knee was stained with toluidine blue and fast green and haematoxylin and eosin. Histology sections were scored using a standard histopathology grading system.

Statistical analysis

Unless otherwise noted, the overall statistical analysis is performed using one-way ANVOA and then post hoc comparison using Tukey's or Dunnett's multiple comparison test. A P-value less than 0.05 is considered significant. This calculation is performed using Windows Graphic PadPrince 6 software (GraphPad, San Diego California USA, www.graphpad.com ).

Estimation of total protein, collagen and sulfated polysaccharide content

Estimates of the total protein, collagen and sulfated polysaccharide content of the overturned wastes extracts produced are shown in Table 1. A total of four different types of overturned wastes were investigated.

In vitro anti-inflammatory activity

The anti-inflammatory activity measured on the abalone processing waste extract, the commercial abalone extract and the assay positive control (quercetin) is shown in Table 2. Unless mentioned, cell viability is maintained at 80% or more (or 20% or less cell death or toxicity) after application of abalone extract or other samples. Assuming a 70% reduction in nitric oxide production was the greatest effect of the positive control group, comparable results to this reduction were considered positive. Total abalone extract and commercial abalone extract produced positive results that reduced nitric oxide production by 40-80% in RAW 264.7 cells. In particular, Sample 2-2 showed the greatest anti-inflammatory activity by significantly reducing nitric oxide production than the assay positive (quercetin) control (FIG. 1).

In vitro thrombin inhibition

Heparin cofactor II-mediated thrombin inhibition as measured against abalone wastes extract and assay positive control (heparin) is shown in Table 3. In these analyzes, the abalone extracts were analyzed without dilution and the maximum antithrombotic potential was observed.

Total abalone extracts except for sample 3-1 showed thrombin inhibition as compared to the analytical positive control (heparin) (FIG. 2).

The anti-thrombin and heparin cofactor II-mediated thrombin inhibition measured for abalone wastes extract and analytical positive control (heparin) is shown in Table 4. In these analyzes, the Abalone extract was diluted several times and analyzed, and the possibility of total antithrombotic activity was observed.

Table 1: (1) Sample 3-1 (powder) and commercial offal extract (powder) were administered to the analysis based on dry weight (mg powder dissolved in water); (2) Significant cytotoxicity was observed following application of sample 4-1; (3) Quercetin was administered to the assay based on micromolar ([mu] M).

Ion exchange chromatography

On the assumption that the sulfated polysaccharide contents in the abalone extract cause negative charge on the abalone extract, anion exchange chromatography was selected and the abalone extract was fractionated into other molecular pools. Pierce BCA Total Protein and DMMB assays were used to determine the total protein content and the estimated sulfated polysaccharide in the pooled fractions. Anion separation of Abalone extract 1-1 and 2-1 is shown in Fig. The elution profile indicates that most of the protein is weakly bound to the column to elute at low concentrations of NaCl while the sulfated carbohydrate is strongly bound to the column and requires a higher concentration of NaCl to elute from the column. To collect similarly charged groups of molecules, the passage liquid was collected and the fractions pooled as described below. Prior to further analysis, the entire anion sample was desalted using a 3 kDa centrifugal filter device (Amicon Ultra-15, Ultracel low binding regenerated cellulose membrane) and NaCl added by linear salting. Also, after the sample was loaded, the cleaning step was performed using only water before the NaCl gradient started. In addition, cleaned samples were collected, analyzed, and found to be free of proteins or sulfated polysaccharides.

1. Through amount

2. Pool 1: Fraction 1 - 4

3. Pool 2: Fraction 5 - 18

4. Pool 3: Fraction 19 - 25

5. Pool 4: Fraction 26-30

In vitro anti-inflammatory activity

The anti-inflammatory activity measured for abalone anion exchange chromatography pooled fractions and the assay positive control (quercetin) is shown in Table 5. &lt; tb &gt; &lt; TABLE &gt; After application of the pooled fractions and positive control, cell viability was maintained at 80% or more (or 20% or less cell death or toxicity).

Most of the antiinflammatory activity was maintained in anion exchange permeate samples (flow) with reduced nitric oxide production of 47-64% (Figure 4). The anti-inflammatory activity for 1-1, 2-1 and 2-2 pass-through samples was higher than that of the highest positive control (10 μM quercetin). The 1-2 pass sample was significantly lower than the highest assay positive control but was much more active than the lowest positive assay (quercetin 1 μM). The pooled fractions had a 23-38% reduction in nitric oxide production. The anti-inflammatory response to pooled fractions was equal or significantly lower than that of the lowest assay positive control, confirming the maintenance of anti-inflammatory activity in anion exchange-through samples.

In vitro thrombin inhibition

Heparin cofactor II-mediated thrombin inhibition, as measured against abalone anion exchange chromatography flow and pooled fractions, and assay positive control (heparin), is shown in Table 6. In this assay, abalone extracts were analyzed without dilution to determine the maximum antithrombotic potential.

Most of the thrombin inhibition was maintained in pool 2-4 of the anion exchange sample. In particular, pool 3 decreased thrombin activity by 50-93% (FIG. 5). Thrombin inhibition for the 1-1, 1-2, and 2-2 pool 3 samples is comparable to the peak positive assay (0.0625 mg / mL heparin). 2-1 full 3 samples were significantly lower than the highest analytical positive control but were much more active than the lowest positive analytical control (0.001 mg / mL heparin). Only Pool 1 and flow samples showed a 1-9% reduction in thrombin activity, markedly lower than the lowest assay positive control, and the maintenance of thrombin inhibition by pool 2-4 of the anion exchange sample.

In vivo anti-inflammatory activity

In vivo antiinflammatory activity was analyzed using a collagen-induced mouse model of rheumatoid arthritis (CIA). Large-scale abalone extract, Sample 3-1, was included in the basic diet in two doses (100 and 500 mg / kg body weight / day) and was fed to the mice before and after the introduction of arthritis. In addition, the positive drug control, prednisolone, was given to the mice after induction of arthritis in a dose of 1 mg / kg body weight / day. The treatment effects of abortion and prednisolone on arthritis severity were assessed through clinical outcomes, incidence, final weight and level of circulating collagen II antibody. Using the standard arthritic scoring system, clinical trials showed a range of severity of symptoms throughout testing (Figure 6).

On the end of the study (20 days), no side effects were observed in mice that did not progress to arthritis (Fig. 7), indicating that oral ingestion of abalone does not produce a pro-inflammatory symptom. In addition, comparison of treatment groups with CIA negative controls was performed by comparing the least squares of clinical scores from the start (6 days) to 20 days using the SAS GLM procedure during disease progression (http://support.sas .com / en / support-home.html; https://support.sas.com/documentation/cdl/en/statug/63033/HTML/default/viewer.htm#glm_toc.htm) (FIG. 8). This comparison revealed that prophylactic treatment with abalone extract, doses 1 and 2, reduced the mean clinical score by ~50% (p < 0.0001) and 20% (p < 0.05). Therapeutic treatment with abalone extract dose 1 reduced the mean clinical score by ~ 30% (p <0.001), but therapeutic treatment with abalone extract dose 2 resulted in an average clinical score of 5% (p < 0.4374). Therapeutic treatment with prednisolone significantly reduced the clinical score by ~70% (p <0.0001).

Animal weights were collected throughout the trial and maintained steadily in the CIA group regardless of treatment type (data not shown). Also, circulating levels of cytokines (IL2, IL-6, IL-10, IFN-y and TNF-a) and collagen type II antibodies were measured in serum samples collected on day 21. Although IL-2, IL-10, IFN-y and TNF-a remained low in the overall treatment group (data not shown), IL-6 was significantly higher after arthritis induction (Fig. After administration of the abalone extract and prednisolone, the circulating levels of IL-6 were significantly reduced in the whole treatment group, except after therapeutic dosing of 1 abalone extract dose. Circulating collagen type II antibody levels remained high in the whole CIA group (Figure 10). Histologic evaluation also showed a decrease in total scleritis after a prophylactic rollover dose of 2 (FIG. 11).

Anticoagulant activity

In vitro anticoagulant activity was measured using plasma clotting assays prothrombin time (PT) and activated partial thromboplastin time (aPTT). Samples 1-2 and 2-2 showed delayed coagulation times in both analyzes by the dose-dependent method (shown in Table 7), indicating that the abalone extract acts as an anticoagulant molecule in blood and plasma.

In addition, the in vitro anticoagulant activity was measured for fractions prepared from the abalone extract. Anion exchange fractions were prepared from Sample 2-2 and pooled as shown in Table 8. The pools containing the highest amounts of sulfated carbohydrates and small amounts of protein were delayed in most of the coagulation times in the aPTT and PT assays (as shown in Table 9), indicating that the anticoagulant activity is in agreement with the sulfated polysaccharide content .

The disclosures of all patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety.

The citation of any reference herein is not to be construed as an admission that such reference is available as the "prior art" of the present application.

Throughout this specification, it is the object of the present invention to describe a preferred embodiment of the invention without restricting the invention to any one aspect or set of specific features. Accordingly, those skilled in the art will appreciate that various modifications and changes may be made in the specific embodiments illustrated without departing from the scope of the invention, in light of the present disclosure. All such modifications and variations are intended to be included within the scope of the appended claims.

Claims (28)

A black lip abalone ( Haliotis) , comprising at least one biologically active ingredient, derived from the processing waste of black lips abalone rubra , or fractions thereof.
The method according to claim 1,
Wherein the extract or fraction is derived from raw processing waste as raw material.
3. The method according to claim 1 or 2,
Wherein the at least one biologically active ingredient is selected from antithrombotics and anti-inflammatory agents.
3. The method according to claim 1 or 2,
Wherein the at least one biologically active ingredient is selected from a protein and an anionic polysaccharide.
5. The method according to any one of claims 1 to 4,
Wherein the extract or fraction is a concentrated protein component.
5. The method according to any one of claims 1 to 4,
Wherein the extract or fraction is an enriched anionic polysaccharide component.
Use of the extract or fraction according to any one of claims 1 to 6 as a medicament, dietary supplement or nutraceutical, functional food or functional food ingredient.
A pharmaceutical, dietary supplement or nutraceutical, functional food or functional food ingredient comprising the extract or fraction according to any one of claims 1 to 6.
7. A pharmaceutical composition comprising an extract or a fraction according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier or excipient.
An anti-inflammatory agent comprising the extract or fraction of any one of claims 1 to 5.
A method of treating an IL-6 mediated disease in a patient,
Wherein the method comprises administering to the patient an effective amount of an extract or a fraction according to any one of claims 1 to 5.
A method for treating an inflammatory disease in a patient,
Wherein the method comprises administering to the patient an effective amount of an extract or a fraction according to any one of claims 1 to 5.
13. The method of claim 12,
Wherein said inflammatory disease is an autoimmune disease.
A method of treating an autoimmune disease in a patient,
Wherein the method comprises administering to the patient an effective amount of an extract or a fraction according to any one of claims 1 to 5.
Use of an extract or a fraction according to any one of claims 1 to 5 in the manufacture of a medicament for the treatment of IL-6-mediated diseases.
Use of an extract or a fraction according to any one of claims 1 to 5 in the manufacture of a medicament for the treatment of inflammatory diseases.
Use of an extract according to any one of claims 1 to 5 in the manufacture of a medicament for the treatment of autoimmune diseases.
18. The method according to any one of claims 11 to 17,
Wherein said disease is rheumatoid arthritis.
An antithrombotic or anticoagulant comprising the extract or fraction of any one of claims 1 to 4 or 6.
20. The method of claim 19,
The antithrombotic effect is regulated via heparin cofactor II or antithrombin.
As a method for suppressing thrombosis in a patient,
Said method comprising administering to the patient an effective amount of an extract or a fraction according to any one of claims 1 to 4 or 6.
As a method for inhibiting thrombin activity in a patient,
Said method comprising administering to the patient an effective amount of an extract or a fraction according to any one of claims 1 to 4 or 6.
Use of an extract or a fraction according to any one of claims 1 to 4 and 6 in the manufacture of a medicament for inhibiting thrombosis or thrombin activity.
7. A method for producing an extract or a fraction according to any one of claims 1 to 6,
Said method comprising the steps of digesting the processing waste of a black lips abalone using an enzymatic or chemical digestion process and separating the extract from the digested waste.
24. The method of claim 23,
Digesting the processing waste of black lips abalone in the presence of digestive enzymes to produce aqueous fractions and precipitates; And
Separating the aqueous fraction from the precipitate to produce an extract;
/ RTI &gt;
25. The method of claim 24,
Filtering the aqueous fraction to produce a filtrate.
26. The method of claim 25,
Further comprising desalting the filtrate.
27. The method according to any one of claims 23 to 26,
Further comprising separating the extract into one or more fractions in which the protein component or the anionic polysaccharide component is concentrated.

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