WO2017124149A1 - Blacklip abalone (haliotis rubra) extract - Google Patents

Blacklip abalone (haliotis rubra) extract Download PDF

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Publication number
WO2017124149A1
WO2017124149A1 PCT/AU2017/050041 AU2017050041W WO2017124149A1 WO 2017124149 A1 WO2017124149 A1 WO 2017124149A1 AU 2017050041 W AU2017050041 W AU 2017050041W WO 2017124149 A1 WO2017124149 A1 WO 2017124149A1
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WIPO (PCT)
Prior art keywords
extract
fraction
abalone
subject
condition
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PCT/AU2017/050041
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English (en)
French (fr)
Inventor
Simone OSBORNE
Original Assignee
Commonwealth Scientific And Industrial Research Organisation
Fisheries Research And Development Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from AU2016900175A external-priority patent/AU2016900175A0/en
Application filed by Commonwealth Scientific And Industrial Research Organisation, Fisheries Research And Development Corporation filed Critical Commonwealth Scientific And Industrial Research Organisation
Priority to CN201780012466.5A priority Critical patent/CN109069547A/zh
Priority to AU2017208715A priority patent/AU2017208715B2/en
Priority to NZ744397A priority patent/NZ744397B2/en
Priority to KR1020187023498A priority patent/KR20180103129A/ko
Publication of WO2017124149A1 publication Critical patent/WO2017124149A1/en
Priority to ZA2018/04847A priority patent/ZA201804847B/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/618—Molluscs, e.g. fresh-water molluscs, oysters, clams, squids, octopus, cuttlefish, snails or slugs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • A23L17/40—Shell-fish
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • A23L17/50—Molluscs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/20—Natural extracts
    • A23V2250/204—Animal extracts
    • A23V2250/2042—Marine animal, fish extracts

Definitions

  • the present invention relates to novel extracts and extract fractions of blacklip abalone (Haliotis rubra) processing waste. Processes for the preparation of the extract and fractions thereof and use of the extract or fraction as an ingredient, medicament, dietary supplement or nutraceutical are also described.
  • blacklip abalone Haliotis rubra
  • the present invention is predicated, at least in part, on the discovery that commercially viable biologically active components can be recovered from the non-shell processing waste from blacklip abalone (Haliotis rubra) meat production.
  • blacklip abalone Haliotis rubra
  • non-shell processing waste such as offal, meat trimmings, waste water streams and cooking juices generated from the processing of blacklip abalone are a source of anti-inflammatory and anti -thrombotic agents.
  • extracts obtained from blacklip abalone processing waste produced significant and consistent anti -thrombotic and antiinflammatory activity.
  • the present invention advantageously provides an extract of blacklip abalone (Haliotis rubra), or fraction thereof, comprising one or more biologically active components, which extract is derived from blacklip abalone processing waste.
  • the processing waste is raw (or fresh) processing waste.
  • the one or more biologically active components are selected from anti -thrombotic agents and anti-inflammatory agents.
  • the extract is fractionated to produce a fraction enriched in protein components. In some embodiments the extract is fractionated to produce a fraction enriched in anionic polysaccharide components.
  • the invention provides a use of an extract, or fraction thereof, according to the invention in the preparation or manufacture of a medicament, dietary supplement, nutraceutical, functional food or functional food ingredient.
  • the present invention provides a medicament, dietary supplement, nutraceutical, functional food or functional food ingredient comprising an extract of the invention, or fraction thereof.
  • the present invention provides a pharmaceutical composition comprising an extract according to the invention, or fraction thereof, and one or more pharmaceutically acceptable carriers or excipients.
  • the present invention provides an anti-inflammatory agent comprising an extract or fraction enriched in protein components according to the invention.
  • the present invention provides a method of treating an IL-6 mediated condition in a subject, the method comprising administering to the subject an effective amount of an extract or fraction enriched in protein components according to the invention.
  • the present invention provides a method of treating an inflammatory condition in a subject, the method comprising administering to the subject an effective amount of an extract or fraction enriched in protein components according to the invention.
  • the present invention provides a method of treating an autoimmune condition in a subject, the method comprising administering to the subject an effective amount of an extract or fraction enriched in protein components according to the invention.
  • the present invention provides a use of an extract or fraction enriched in protein components according to the invention in the manufacture of a medicament for treating an IL-6-mediated condition, an inflammatory condition or an autoimmune condition, for example rheumatoid arthritis.
  • the present invention further provides an anti -thrombotic or anti -coagulant agent comprising an effective amount of an extract or fraction enriched in anionic polysaccharide components according to the invention.
  • the present invention provides a method of inhibiting thrombosis, or of inhibiting thrombin activity in a subject, the method comprising administering to the subject an effective amount of an extract or fraction enriched in anionic polysaccharide components according to the invention.
  • the present invention provides the use of an extract or fraction enriched in anionic polysaccharide components according to the invention in the manufacture of a medicament for inhibiting thrombosis or thrombin activity.
  • the present invention provides a process for preparing an extract or fraction thereof according to the invention, the method comprising digesting blacklip abalone processing waste using an enzymatic or chemical digestion process and isolating the extract from the digested waste.
  • the inventors have discovered that it may be advantageous to enrich, or increase the concentration of certain component or components, such as anionic polysaccharide components or protein components.
  • a process for preparing a fraction according to the invention may also comprise fractionating the extract, preferably to increase the concentration of anionic polysaccharide components or protein components.
  • Figure 1 shows graphically the in vitro anti-inflammatory activity in response to various abalone processing waste extracts.
  • Anti-inflammatory activity indicated by a decrease in nitric oxide production by LPS stimulated mouse macrophages (RAW 264.7 cells).
  • Data indicated by "*” display significant statistical differences (p ⁇ 0.05) to the assay positive control (quercetin) using a one-way ANOVA with Dunnett's multiple comparisons test.
  • Figure 2 shows graphically the in vitro thrombin inhibition activity in response to various abalone processing waste extracts.
  • Thrombin inhibition activity indicated by a decrease in thrombin activity using the chromogenic substrate ChromozymTH.
  • Data indicated by "*” display significant statistical differences (p ⁇ 0.05) to the assay positive control (0.0625 mg/mL heparin) using a one-way ANOVA with Dunnett's multiple comparisons test.
  • Figures 3A and 3B show graphically anion exchange separation of various abalone processing waste extracts.
  • Abalone extracts 1-1 ( Figure 3A) and 2-1 ( Figure 3B) were separated into 30 fractions using anion exchange chromatography and a linear NaCl gradient.
  • Total protein black
  • sulphated polysaccharides (grey) were estimated in the fractions.
  • Figure 4 shows graphically in vitro anti-inflammatory activity in response to anion exchange chromatography fractions obtained from various abalone processing waste extracts that were pooled.
  • Anti-inflammatory activity indicated by a decrease in nitric oxide production by LPS stimulated mouse macrophages (RAW 264.7 cells).
  • Data indicated by "*" display significant statistical differences (p ⁇ 0.05) to the assay positive control 10 ⁇ quercetin using a one-way ANOVA with Dunnett's multiple comparisons test.
  • Data with ⁇ display significant statistical differences (p ⁇ 0.05) to the assay positive control 1 ⁇ quercetin using a one-way ANOVA with Dunnett's multiple comparisons test.
  • Figure 5 shows graphically in vitro heparin cofactor II-mediated thrombin inhibition in response to pooled anion exchange chromatography fractions obtained from various abalone processing waste extracts.
  • Thrombin inhibition indicated by a decrease in thrombin activity using the chromogenic substrate ChromozymTH.
  • Data indicated by "*” display significant statistical differences (p ⁇ 0.05) to the assay positive control (0.0625 mg/mL heparin) using a one-way ANOVA with Dunnett's multiple comparisons test.
  • Data indicated by " A " display significant statistical differences (p ⁇ 0.05) to the assay positive control (0.001 mg/mL heparin) using a one-way ANOVA with Dunnett's multiple comparisons test.
  • Figures 6A and 6B show graphically the clinical score in mice following collagen II induced arthritis (CIA). Mice were orally administered control feed; prophylactic/therapeutic abalone dose 1; prophylactic/therapeutic abalone dose 2 and therapeutic prednisolone. Prophylactic treatments are summarised in Figure 6A. Therapeutic treatments are summarised Figure 6B.
  • Figure 7 shows graphically the individual clinical scores at Day 20 for each treatment group, including mean clinical scores (with data range).
  • Figure 8 shows graphically the LSMEANS of clinical scores from onset of symptoms (Day 6) until Day 20 for CIA treatment groups. Statistical significance, as determined using the GLM Procedure of SAS to estimate separately the least squares means (LSMEANS) for CIA treatment (group) effect, is shown in comparison to control feed: **** (p ⁇ 0.0001); *** (p ⁇ 0.001) and * (p ⁇ 0.05).
  • Figure 9 shows graphically the LSMEANS of Circulating IL-6 (cytokine) levels in blood serum collect on day 21. Statistical significance, as determined using the GLM Procedure of SAS to estimate separately the least squares means (LSMEANS) for treatment (group) effect, is shown in comparison to control feed + CIA: **** (p ⁇ 0.0001); ** (p ⁇ 0.01).
  • Figure 10 shows graphically the serum collagen-II antibody levels in blood serum collected on day 21. Antibody levels expressed as a ratio to no CIA control: control diet; abalone dose 1; abalone dose 2 and prednisolone.
  • FIG 11 shows graphically the LSMEANS total synovitis scores at Day 21 for CIA and non-CIA treatment groups. Statistical significance, as determined using the GLM Procedure of SAS to estimate separately the least squares means (LSMEANS) for treatment (group) effect, is shown in comparison to control feed + CIA: **** (p ⁇ 0.0001); ** (p ⁇ 0.01). Detailed Description of the Preferred Embodiments
  • the term "about” refers to a quantity, level, concentration, value, size, or amount that varies by as much as 10% or even as much as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, to a reference quantity, level, concentration, value, size, or amount.
  • agent is used herein to denote 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 portion thereof, e.g., a peptide), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.
  • a biological macromolecule such as a nucleic acid, an antibody, a polysaccharide or oligosaccharide, a protein or portion thereof, e.g., a peptide
  • an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.
  • the activity of such agents may render it suitable as a "therapeutic agent” which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.
  • autoimmune disease refers to a disease or condition arising from an inappropriate immune response against the body's own components, such as tissues and other components.
  • IL-6 levels are elevated in autoimmune disease.
  • Non- limiting exemplary autoimmune diseases that may be treated with the extracts described herein include rheumatoid arthritis, inflammatory bowel diseases (especially ulcerative colitis and Crohn's disease), systemic lupus erythromatosus, systemic sclerosis, polymyositis, vasculitis syndrome including giant cell arteritis, takayasu aeteritis, cryoglobulinemia, myeloperoxidase-antineutrophilcytoplasmic, eczema, psoriasis, myasthenia gravis, antibody- associated crescentic glomerulonephritis, rheumatoid vasculitis, relapsing polychondritis, acquired hemophilia A,
  • blacklip abalone processing waste refers to waste produced during the processing of blacklip abalone to produce blacklip abalone meat (muscle, foot) for consumption.
  • Processing waste includes parts of the soft tissue of the abalone discarded during processing of the animal to produce abalone meat, including meat trimmings and internal organs as well as waste liquids generated during washing processes, cooking juices and undersized whole abalone rejected during grading.
  • the processing waste may be in the form of, for example, a solid, powder, paste or liquid; it may be raw, treated or cooked; and may be fresh, or preserved for example by freezing, bagging, canning or drying.
  • biologically active components or “bioactive molecules” and similar when used herein refers to components of the extract that have a physiological effect when administered to an animal or investigated in vitro.
  • cancer means a disease or condition involving unregulated and abnormal cell growth.
  • Non-limiting cancers that may be treated with the extracts described herein include multiple myeloma, leukemia, pancreatic cancer, breast cancer, colorectal cancer, cachexia, melanoma, cervical cancer, ovarian cancer, lymphoma, gastrointestinal, lung cancer, prostate cancer, renal cell carcinoma, metastatic kidney 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 exemplary cancer-related conditions include non-small cell lung cancer-related fatigue and cancer related anorexia.
  • a protein component of a composition can be enriched by chromatographic techniques including fractionation such that the protein component of the composition is increased about 1.5-fold, about 2-fold, about 3-fold, about 5-fold, about 10- fold, about 15-fold, about 30-fold, about 50-fold, or about 100-fold.
  • an "IL-6 mediated disease or condition” refers to a disease or condition in which at least some of the symptoms and/or progression of the disease or condition is caused by IL-6-mediated signaling.
  • Non-limiting exemplary IL-6 mediated diseases or conditions include inflammatory diseases, malignant diseases (including cancer and cancer-related conditions), infections, and autoimmune diseases.
  • IL-6 mediated diseases include, but are not limited to, Castleman's disease, ankylosing spondylitis, coronary heart disease, cardiovascular disease in rheumatoid arthritis, pulmonary arterial hypertension, chronic obstructive pulmonary disease (COPD), atopic dermatitis, psoriasis, eczema, sciatica, type II diabetes, obesity, giant cell arteritis, inflammatory bowel diseases (especially ulcerative colitis and Crohn's disease), acute graft- versus-host disease (GVHD), non-ST elevation myocardial infarction, anti -neutrophil cytoplasmic antibody (ANCA) associated vasculitis, neuromyelitis optica, chronic glomerulonephritis, Takayasu arteritis, graft versus host disease and transplant rejection (including in renal transplantation).
  • ANCA anti -neutrophil cytoplasmic antibody
  • infection refers to a disease or condition caused by a pathogen, such as a bacteria, virus, fungus, etc.
  • pathogen such as a bacteria, virus, fungus, etc.
  • Non-limiting exemplary infections that may be treated with the extracts described herein include human immunodeficiency virus (HIV), human T- lymphotropic virus (HTLV), cerebral malaria, urinary tract infections, and meningococcal infections.
  • HIV human immunodeficiency virus
  • HTLV human T- lymphotropic virus
  • cerebral malaria cerebral malaria
  • urinary tract infections and meningococcal infections.
  • inflammatory disease refers to a disease or condition involving an inflammatory response.
  • the inflammatory response may be acute and/or chronic.
  • chronic inflammation involves an increase in the level of IL-6.
  • Non-limiting inflammatory diseases that may be treated with the extracts described herein include rheumatoid arthritis, juvenile idiopathic arthritis, systemic-onset juvenile idiopathic arthritis, osteoarthritis, sepsis, asthma, interstitial lung disease, inflammatory bowel disease, systemic sclerosis, intraocular inflammation, Graves disease, endometriosis, systemic sclerosis, adult- onset still disease, amyloid A amyloidosis, polymyalgia rheumatic, remitting seronegative symmetrical synovitis with pitting edema, Behcet's disease, uveitis, graft-versus-host diseases, and TNFR-associated periodic syndrome.
  • protein components when used here,
  • anionic polysaccharide refers to negatively charged polysaccharides such as sulphated polysaccharides, and includes glycosaminoglycan polysaccharides (GAG) and glycosaminoglycan-like polysaccharides (GAG-like).
  • Nutraceutical when used herein refers to an edible product derived from food sources. Nutraceutical s are considered to offer health benefits, and can, for example, form the basis of functional food, food ingredients, dietary supplements, drinks, and animal feeds.
  • anti -thrombotic agent is an agent that reduces the formation of blood clots and can be used for the prevention and treatment of, for example, arterial and venous thrombosis.
  • anti -thrombotic agents include anti-coagulant agents and antiplatelet agents.
  • the anti-thrombotic effect may, for example, be mediated through heparin co-factor II or anti thrombin (AT).
  • anticoagulant agent is an agent which acts to reduce blood clotting.
  • Anticoagulant agents find application in the prevention and initial treatment of venous thromboembolism, including deep vein thrombosis (DVT) and pulmonary embolism (PE).
  • Anti -coagulant agents can also be used in prophylaxis of ischaemic complications, in treatment of unstable angina, and treatment of non-Q-wave myocardial infarction.
  • Anticoagulant agents can be used in prophylaxis or treatment of DVT following, for example, joint replacement surgery or abdominal surgery.
  • Anti-coagulant agents can also be used to prevent or treat DVT which may occur due to restricted mobility as a result of injury or illness; or which may be a complication of a malignant disease such as a cancer.
  • Anti-coagulant agents may also be used as a prophylactic in situations where there is a patient history of DVT or pulmonary embolism.
  • Anti-coagulant agents can also be used to inhibit blood clotting in a patient undergoing surgical intervention, for example intravascular catheterisation procedures or cardiac surgery.
  • Other therapeutic uses of anti -coagulants include treatment of atrial fibrillation, congestive heart failure, myocardial infarction and genetic or acquired hypercoagulability.
  • antiplatelet agent or "antiaggregant agent” is an agent which acts to reduce platelet aggregation in the blood, and thus inhibits thrombus formation.
  • Antiplatelet agents are effective in the arterial circulation, where anti -coagulants may not be significantly effective. Platelet aggregation can lead to, for example, heart attack, angina or stroke.
  • Antiplatelet agents can be used in prophylaxis or treatment of thrombotic cerebrovascular disease, such as stroke, mini-stroke, ischaemic stroke or haemorrhagic stroke; or cardiovascular disease such as hypertensive heart disease, rheumatic heart disease, cardiomyopathy, atrial fibrillation, congenital heart disease, endocarditis, aeortic aneurysms, peripheral artery disease, venous thrombosis or coronary artery disease, e.g. angina or myocardial infarction.
  • thrombotic cerebrovascular disease such as stroke, mini-stroke, ischaemic stroke or haemorrhagic stroke
  • cardiovascular disease such as hypertensive heart disease, rheumatic heart disease, cardiomyopathy, atrial fibrillation, congenital heart disease, endocarditis, aeortic aneurysms, peripheral artery disease, venous thrombosis or coronary artery disease, e.g. angina or my
  • thrombosis refers to intravascular coagulation of the blood in the circulatory system, being the heart, arteries, veins and capillaries.
  • thrombin refers to an enzyme which acts as a catalyst for several transformations in the blood coagulation cascade. It also promotes platelet activation and aggregation.
  • Suitable animals that fall within the scope of the invention include, but are not restricted to, humans, primates, livestock animals (e.g., sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs) and captive wild animals (e.g., foxes, deer, dingoes, birds, reptiles).
  • livestock animals e.g., sheep, cows, horses, donkeys, pigs
  • laboratory test animals e.g., rabbits, mice, rats, guinea pigs, hamsters
  • companion animals e.g., cats, dogs
  • captive wild animals e.g., foxes, deer, dingoes, birds, reptiles.
  • malignant disease includes cancer and cancer-related conditions.
  • treatment refers to administering an agent to obtain a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of effecting a partial or complete cure for a disease and/or symptoms of the disease.
  • the effect may be therapeutic in terms of a partial or complete cure for a disease or condition (e.g., a disease or condition mediated by IL-6) and/or adverse effect attributable to the disease or condition.
  • a condition or disease in a mammal particularly in a human, and include: (a) preventing the disease or condition or a symptom of a disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it (e.g., including diseases or conditions that may be associated with or caused by a primary disease or condition; (b) inhibiting the disease or condition, i.e., arresting its development; (c) relieving the disease or condition, i.e., causing regression of the disease or condition; (d) relieving a symptom of the disease or condition and/or (e) reducing the frequency of a symptom of the disease or condition.
  • pharmaceutically acceptable carrier excipient or diluent
  • a solid or liquid filler diluent or encapsulating substance that can be safely used in systemic administration.
  • extracts obtained from blacklip abalone processing waste, or fractions thereof comprise biologically active components that have anti-thrombotic or anti-inflammatory activity.
  • Extracts are prepared from waste streams obtained from the processing of blacklip abalone meat for consumption.
  • the extracts are prepared from the processing waste using a process involving enzyme or chemical digestion, heat inactivation and clarification steps.
  • the extract is derived from offal.
  • the processing waste is raw.
  • the processing waste is preserved.
  • the processing waste is in the form of a liquid.
  • the processing waste is in the form of a solid, for example a paste or a powder.
  • the processing waste may be canned, chilled or frozen.
  • the blacklip abalone is wild harvest abalone.
  • the one or more biologically active components are selected from anionic polysaccharide components and protein components.
  • the protein components comprise collagen.
  • the anionic polysaccharide components comprise one or more sulphated polysaccharide components.
  • the anionic polysaccharide components comprise one or more glycosaminoglycan-like polysaccharides.
  • the present invention provides a process for preparing an extract according to the invention, the method comprising digesting blacklip abalone processing waste using an enzymatic or chemical digestion process and isolating the extract from the digested waste.
  • the process comprises enzymatic digestion.
  • Exemplary enzymes for enzymatic digestion include bromelain and/or papain.
  • the processing waste samples are digested for about 16-18 hours.
  • the digest volume contains about 10% w/v processing waste.
  • the digest volume contains about 2% w/v of one or more enzymes, for example about 1% w/v bromelain and about 1% w/v papain.
  • the digests are heat inactivated for about 10 minutes at about 95°C before cooling. In some embodiments the digests are fractionated to separate different fractions of the digest. In some embodiments the fractionation produces a fraction enriched in protein components. In some embodiments the fractionation produces a fraction enriched in anionic polysaccharide components.
  • the fractionation may be achieved using any suitable technique. Suitable techniques are well known to the skilled person, and illustrative examples include centrifugation, extraction, continuous liquid-liquid extraction, pervaporation, filtration including 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.
  • the process comprises the steps of: digesting blacklip abalone processing waste in the presence of a digesting enzyme to produce an aqueous fraction and a precipitate; and separating the aqueous fraction from the precipitate to thereby produce the extract.
  • a process of the invention further comprises filtering the aqueous fraction to produce a filtrate.
  • the filtrate is desalted.
  • the filtrate is desalted using a 3 kDa filter to produce the extract as a 3 kDa retentate.
  • Blacklip abalone extracts can be separated into fractions using, for example, ion exchange chromatography and size exclusion chromatography.
  • the process further comprises separating the extract into one or more fractions enriched in protein components or anionic polysaccharide components, preferably using ion exchange chromatography and/or size exclusion chromatography.
  • the composition of an extract or extract fraction of the invention with respect to amount of protein components and anionic polysaccharide components is readily determined by methods known in the art, such as those described in the Examples herein.
  • the present invention provides a blacklip abalone extract, or extract fraction, enriched in protein components.
  • the protein components comprise protein collagen.
  • the present invention provides a blacklip abalone extract, or extract fraction, enriched in anionic polysaccharide components.
  • the anionic polysaccharide components comprise one or more sulphated polysaccharides.
  • the anionic polysaccharide components comprise one or more glycosaminoglycan-like polysaccharide components.
  • the present invention provides a blacklip abalone extract, or extract fraction, obtained or obtainable by a process of the invention.
  • the extracts and extract fractions of the present invention comprise one or more biologically active components.
  • the one or more biologically active components are selected from anti -thrombotic agents and anti-inflammatory agents.
  • the anti -thrombotic agents comprise one or more anionic polysaccharide components.
  • the anti-thrombotic agents comprise one or more glycosaminoglycan-like polysaccharide components.
  • the one or more biologically active components are selected from anti-inflammatory agents.
  • the anti-inflammatory agents comprise one or more protein components.
  • extracts and fractions obtained from blacklip abalone processing waste produce significant and consistent antithrombotic and anti-inflammatory activity in vitro. Furthermore, these extracts and fractions have also been found to significantly reduce the symptoms of severe arthritis in vivo in a mouse model of rheumatoid arthritis.
  • mice fed powdered abalone extracts prior to and during the onset of rheumatoid arthritis experienced less severe symptoms than mice fed a basal diet.
  • blacklip abalone processing waste extract according to the present invention produced positive and significant results in a mouse model of rheumatoid arthritis.
  • prophylactic administration of abalone extract significantly decreased toe, paw and carpus swelling and deformity ( Figures 7 and 8).
  • prophylactic and therapeutic administration of abalone extract significantly reduced total synovitis observed in joints and paws ( Figure 1 1).
  • the extracts and fractions of the present invention are therefore considered to be useful in the treatment or prevention of IL-6 mediated diseases or conditions as described for example above.
  • the fraction is a fraction enriched in protein components.
  • the IL-6 mediated disease or condition is an autoimmune disease or condition such as but not limited to rheumatoid arthritis, inflammatory bowel diseases (especially ulcerative colitis and Crohn's disease), eczema, psoriasis and myasthenia gravis, or transplant rejection (including in renal transplantation).
  • the extracts and fractions of the invention are useful in treating or preventing rheumatoid arthritis.
  • the present invention provides a method of treating an IL-6 mediated condition in a subject, the method comprising administering to the subject an effective amount of the extract according to the invention, preferably a fraction enriched in protein components.
  • the present invention further provides a method of treating an inflammatory condition in a subject, the method comprising administering to the subject an effective amount of an extract according to the invention, preferably a fraction enriched in protein components.
  • the inflammatory condition is an autoimmune condition.
  • the present invention provides a use of an extract of the invention, preferably a fraction enriched in protein components, in the manufacture of a medicament for treating an IL-6-mediated condition, an inflammatory condition, or an autoimmune condition.
  • the condition is rheumatoid arthritis.
  • the present invention also provides for use of an extract or fraction of the invention as a medicament.
  • the medicament is formulated for oral administration.
  • an extract of the invention in the manufacture of a dietary supplement, nutraceutical, functional food or functional food ingredient.
  • extracts and fractions from blacklip abalone processing waste produced significant and consistent anti -thrombotic activity in vitro.
  • the extracts or fractions of the present invention are considered to have antithrombotic or anti-coagulant properties, and are therefore considered to be useful in treatment or prevention of thrombosis, or of inhibiting thrombin activity.
  • the present invention further provides an anti -thrombotic or anticoagulant agent comprising an effective amount of an extract of the invention, preferably a fraction enriched in anionic polysaccharide components.
  • an antithrombotic effect is mediated through heparin cofactor II or antithrombin.
  • the present invention provides a method of inhibiting thrombosis, or of inhibiting thrombin activity in a subject, the method comprising administering to the subject an effective amount of an extract according to the invention, preferably a fraction enriched in anionic polysaccharide components.
  • the present invention provides the use of an extract according to the invention, preferably a fraction enriched in anionic polysaccharide components, in the manufacture of a medicament for inhibiting thrombosis or thrombin activity.
  • the subjects, individuals or patients to be treated are mammalian subjects including but not limited to humans, primates, livestock animals such as sheep, cattle, pigs, horses, donkeys and goats; laboratory test animals such as mice, rats, rabbits and guinea pigs; companion animals such as cats and dogs or captive wild animals such as those kept in zoos.
  • the subject is a human.
  • an "effective amount” means an amount necessary at least partly to attain the desired response, or to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition being treated.
  • the amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • An effective amount in relation to a human patient for example, may lie in the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage.
  • the dosage is preferably in the range of ⁇ g to 1 g per kg of body weight per dosage, such as is in the range of lmg to lg per kg of body weight per dosage. In one embodiment, the dosage is in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage is in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage is in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per kg of body weight per dosage. In yet another embodiment, the dosage is in the range of 1 ⁇ to 1 mg per kg of body weight per dosage. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals, or the dose may be proportionally reduced according to the situation.
  • treatment does not necessarily imply that a subject is treated until total recovery. “Treatment” may also reduce the severity of an existing condition.
  • the term “prophylaxis” does not necessarily mean that the subject will not eventually contract a disease condition.
  • the term “prophylaxis” may be considered to include delaying the onset of a particular condition. Accordingly, treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition.
  • an extract of the invention may be administered together with another therapy. Administration may be in a single composition or in separate compositions simultaneously or sequentially such that each compound or therapy is active within the same time period in the body.
  • An extract of the invention may be administered with one or more further nutraceutical products such as, for example, Omega-3 oils, glucosamine or chondroitin sulphate.
  • An extract, particularly an extract fraction enriched in protein components may also be administered with an 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,
  • the extracts and fractions of the invention are considered to be beneficial to health and/or well being, accordingly there is also provided a method for enhancing or maintaining the well being of a subject, the method comprising administering to the subject an effective amount of an extract or fraction of the invention.
  • the extract or fraction of the invention may be taken alone as a neat extract. However, in some circumstances it is preferable to formulate the extract for consumption.
  • the extract may be formulated as a dietary supplement, nutraceutical, functional food or functional food ingredient.
  • the present invention also provides a medicament, dietary supplement, nutraceutical, functional food or functional food ingredient comprising an extract or fraction according to the invention.
  • the extract or fraction of the invention may be formulated as a food or drink product. Animal food is also contemplated as part of the invention.
  • the dietary supplement, nutraceutical, functional food or functional food ingredient further comprises one or more acceptable excipients, fillers or carriers.
  • the extract or fraction of the invention may be used in combination with one or more further active ingredients selected from, for example, nutraceuticals, functional foods or functional food ingredients. The content ratio of active ingredients may be appropriately determined depending on the purpose of the use.
  • the dietary supplement, nutraceutical, functional food or functional food ingredient may be appropriately used according to conventional methods.
  • an extract of the present invention when producing a solid or liquid food, an extract of the present invention may be added at up to 50% w/w, and more preferably up to 30%, 20% or 10% w/w. Generally, when producing a drink, an extract of the present invention may be added at up to 20% w/w, and more preferably up to 15%, 10% or 5%) w/v.
  • Suitable excipients, fillers or carriers are known in the art and include starches, sugars, edible oils, water, glycerine, gums, methyl cellulose and the like.
  • Dietary supplement, nutraceutical, functional food or functional food ingredient compositions of the invention may also comprise additional components such as flavouring agents, colourings, sweetening agents, thickeners, pH modifiers, stabilizers, or preservatives.
  • Dietary supplement, nutraceutical, functional food or functional food ingredient compositions of the present invention may further comprise additional active ingredients such as nutrients, vitamins, or electrolytes.
  • Nutraceutical, functional food or food ingredient compositions may be formulated, for example, as powders, granules, liquids, pastes, oils or gels.
  • Dietary supplements may be formulated, for example, as pills, tablets, capsules, softgels, gelcaps, pastes or powders. Dietary supplements may also be formulated as liquids, such as solutions or suspensions.
  • the required daily dose of an extract or fraction according to the present invention can be determined according to the specific needs of the individual.
  • a typical daily intake is considered to be from 100 mg to 500 mg/kg body weight/day.
  • the daily intake of a fraction is less than 100 mg/kg body weight/day.
  • the extract or fraction of the invention may be incorporated into conventional foodstuffs. Accordingly, the present invention further provides a foodstuff comprising an extract of the invention.
  • compositions comprising an extract or fraction according to the invention, and one or more pharmaceutically acceptable carriers or excipients.
  • the composition is for use in treating or preventing an inflammatory disease or condition.
  • the composition is for use in treating an autoimmune disease or condition such as rheumatoid arthritis.
  • the fraction is enriched in protein components.
  • the fraction is enriched in anionic polysaccharide components.
  • an extract or fraction of the invention may be administered alone, in some embodiments the active extract or fraction is provided in the form of a pharmaceutical composition.
  • a pharmaceutical composition comprising an extract or fraction of the invention and at least one pharmaceutically acceptable carrier, excipient or diluent.
  • compositions of the present invention or the compositions used in the methods of the present invention may be formulated and administered using methods known in the art. Techniques for formulation and administration may be found in Remington: The Science and Practice of Pharmacy, Loyd V. Allen, Jr (Ed), The Pharmaceutical Press, 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 thereof.
  • compositions or formulations include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation.
  • the compounds of the invention may thus be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, all for oral use, in the form of suppositories for rectal coadministration; or in the form of sterile injectable solutions for parenteral (including subcutaneous) use.
  • Such pharmaceutical compositions and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.
  • Formulations containing 10 milligrams of active ingredient or, more broadly, 0.1 to 200 milligrams, per tablet, are accordingly 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 routine to those skilled in the art that the following dosage forms may comprise, as the active component, an extract of the invention.
  • pharmaceutically acceptable carriers can be either solid or liquid.
  • Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
  • a solid carrier can be one or more substances which may also act as diluents, flavouring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
  • the extract or fraction is formulated for oral administration.
  • the extract or fraction is formulated as a powder, tablet, pill, capsule, or dispersible granules.
  • the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.
  • the powders and tablets preferably contain from 5-70% percent of the active compound.
  • Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
  • the term "preparation” is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it.
  • Liquid form preparations for oral administration include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions.
  • Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilizing and thickening agents, as desired.
  • Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well known suspending agents.
  • solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration.
  • liquid forms include solutions, suspensions, and emulsions.
  • These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
  • the active ingredients may be provided in the form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP).
  • a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP).
  • formulations adapted to give sustained release of the active ingredient may be employed.
  • the pharmaceutical preparations are preferably in unit dosage forms.
  • the preparation is subdivided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as blister pack tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
  • Sample 1 liquid; processed and bagged abalone offal; wild harvest abalone
  • Sample 2 Solid (paste); processed and canned abalone offal; wild harvest abalone
  • Sample 3 Solid (powder); processed abalone offal; wild harvest abalone
  • Sample 4 Solid; raw (fresh) abalone offal; wild harvest abalone
  • Extracts were prepared from the processing waste using a method comprising digestion, centrifugation, filtration and desalting. All processing waste samples were digested overnight (16 - 18 hours) at 50°C using two food grade enzymes, bromelain and papain (commercially available from, for example, Enzyme Solutions Pty Ltd, Vic 3136). All solid waste samples were cut into small pieces (1 - 2 cm 2 ). Digests volumes ranged from 50 - 1000 mL (in water) and contained 10 - 20 % w/v processing waste (5 - 200 g) and 0.5 - 1% w/v addition of each enzyme (0.25 - 10.0 g) (Osborne SA et al, Glycobiology.
  • digests were heat inactivated at 95°C for 10 - 20 minutes and then cooled on ice before being centrifuged at 5,940 - 50,000 g for 10 - 30 minutes to remove any fat (top) layer and undigested sediment (pellet).
  • the aqueous layer (supernatant) was retained and sequentially filtered using 2 ⁇ and 1 ⁇ (Whatman glass microfiber) pre-filters followed by a final filtration with a 0.45 ⁇ (Millipore mixed cellulose ester) filter.
  • Filtered samples were then desalted using a 3 kDa (Amicon Ultra- 15, Ultracel low binding regenerated cellulose membrane) centrifugal filter device producing retentate (>3 kDa and containing low/no salt) and filtrate ( ⁇ 3 kDa and containing salt) extract samples.
  • the retentate samples were selected for further analysis due to the possible interference of salt in quantitative and bioactivity assays.
  • Total protein content was estimated in the extracts using the Pierce BCA (bicinchoninic acid) Protein Assay Kit (Thermo Fisher Scientific, VIC, 3179, Australia) according to manufacturer's instructions. Briefly, 25 ⁇ of standards (bovine serum albumin) and extracts were diluted in water and added in triplicate to a 96 well plate (Nunc®, polystyrene). 200 ⁇ of BCA working reagent was prepared (by mixing Reagent A with Reagent B using 50: 1 ratio) and added to each well. The plate was mixed and incubated at 37°C for 30 minutes before 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 extracts.
  • Total collagen content was estimated in the extracts using the QuickZyme Biosciences Total Collagen Assay Kit (Bio-Scientific, Kirrawee, NSW, Australia) according to manufacturer's instructions. Briefly, 125 ⁇ standards (rat tail collagen) and extracts were added to 125 ⁇ 12 M HC1 and hydrolysed for 20 hours at 95°C in screw capped tubes. Following the hydrolysis, 35 ⁇ hydrolysed samples and standards were transferred to the provided 96 well plate, mixed with 75 ⁇ assay buffer and incubated at room temperature (-21 °C) for 20 minutes. 75 ⁇ detection reagent was then added to each well and the resulting solution mixed and incubated at 60°C for 30 minutes. The plate absorbance was read at 570 nm. A linear standard curve was constructed and used to interpolate the total collagen concentration in the extracts. All assays were performed in either duplicate or triplicate.
  • DMMB 1,9-dimethylmethylene blue assay
  • the DMMB dye is a metachromatic dye that detects sulphated glycosaminoglycans (Osborne SA et al, Glycobiology. 2008 Mar; 18(3):225-34) and many other types of sulphated and anionic polysaccharides (Cinelli LP et al, . Comp Biochem Physiol B Biochem Mol Biol. 2009 Sep; 154(l): 108-12; Keler T et al, Anal Biochem. 1986 Jul; 156(1): 189-93. 19-20).
  • the Blyscan Sulphated Glycosaminoglycan Assay is also based on the DMMB dye. Sulphated polysaccharide concentration was estimated in the extracts according to manufacturer's instructions. Briefly, 25 ⁇ standard (chondroitin sulphate from bovine cartilage) and samples were added in triplicate to a V bottom polypropylene plate (PerkinElmer, Melbourne 3150, Australia). 250 ⁇ Blyscan dye reagent was added to each well. The plate was then placed on an orbital shaker for 30 minutes at room temperature before being centrifuged at 2090 g for 10 minutes. The supernatant (liquid) was removed and replaced with 250 ⁇ Blyscan dye dissociation reagent.
  • the plate was mixed on an orbital shaker for 30 minutes before 200 ⁇ of each standard and sample was transferred to a polystyrene 96 well plate (Nunc®) for absorbance readings at 656 nm.
  • a linear standard curve was constructed and used to interpolate the sulphated polysaccharide concentration in the extracts.
  • Bioactivity in terms of anti-inflammatory activity, was indicated using an in vitro cell based assay.
  • Bacterial lipopolysaccharide (LPS) was used to induce an inflammatory cellular state in the murine macrophage cell line, RAW 264.7 (Sosroseno W et al, Oral Microbiol Immunol. 2002 Apr; 17(2):72-8).
  • Inflammation was indicated by the cellular production of nitric oxide (NO) as measured using the Griess assay.
  • NO nitric oxide
  • Anti-inflammatory activity was measured in response to the quercetin positive control (Sigma Aldrich Corporation) and extracts as previously described (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), with some minor modifications.
  • the RAW 264.7 cells (American Type Culture Collection, www.ATCC.com) were routinely cultured in RPMI-1640 media (Thermo Fisher Scientific, Vic 3179, Australia) supplemented with 100 U/mL penicillin, 100 ⁇ g/mL streptomycin (Life Technologies, Thermo Fisher Scientific, Vic 3179, Australia) and 10% v/v fetal bovine serum ⁇ in vitro) and grown at 37°C and 5% C0 2 in humidified air.
  • the antiinflammatory assay was performed over two days. On day one the cells were seeded into 96 well plates at a density of 6xl0 4 cells/well.
  • thrombin inhibition was measured using an in vitro kinetic assay with the chromogenic substrate, Chromozym TH (CH-TH) (Dupouy D et al, Thromb Haemost. 1988 Oct 31;60(2):236-9).
  • CH-TH Chromozym TH
  • Thrombin is a serine protease that is involved in blood clotting by converting soluble fibrinogen into insoluble strands of fibrin.
  • CH-TH is cleaved by thrombin producing a yellow coloured compound (called p-nitroaniline).
  • Thrombin can be inhibited by heparin cofactor II (HCII) and antithrombin (AT) through a variety of sulphated polysaccharides.
  • HCII heparin cofactor II
  • AT antithrombin
  • a decrease in the production of p-nitroaniline indicates antithrombotic activity, that may also lead to anti -clotting activities
  • HCII Heparin cofactor II
  • Antithrombin (AT)-mediated thrombin inhibition was measured in the extracts and fractions using a 384 well-format, robot-assisted assay. In this kinetic assay, thrombin cleaves Chromozym TH, producing two molecules: a residual peptide and 4-nitraniline that can be measured by absorbance change at 405 nm.
  • the assay standard heparin (final concentration range: 0.016 - 1.04 ⁇ g/mL) or molecules present within the samples bind to ATIII (0.2 ⁇ g/mL) forming a ternary complex with thrombin (2.03 ng/mL) preventing it from cleaving chromozym TH and forming 4-nitraniline.
  • a heparin standard was diluted and with final concentrations 0.016 - 1.04 ⁇ g/mL.
  • Prothrombin time was measured by adding 100 iL of citrated plasma to a glass clotting tube and incubating at 37°C on the heating block of a Hyland-Clotek clotting machine (Hyland, USA) for 5 minutes. The machine measures time in seconds until clot formation. 50 iL of different concentrations of abalone extracts were added to the tube, using saline as a blank. The total volume was adjusted to 150 ⁇ _, with plasma before adding 100 ⁇ L ⁇ of Thromborel S® to initiate clotting (Siemens, 545477, USA).
  • Activated partial thromboplastin time was determined by adding 100 ⁇ ⁇ of citrated plasma, 100 ⁇ ⁇ Triniclot (TriniCLOT aPTT, HS, Tcoag, Ireland Limited) and different concentrations of abalone extracts to a clotting tube. The final volume was adjusted to 250 ⁇ ⁇ by adding saline. The clotting tube was incubated at 37°C in the heating block of a Hyland-Clotek clotting machine for 5 minutes before 50 ⁇ ⁇ 50 mM Ca+2 was added to initiate clotting.
  • anion exchange chromatography was used to separate the biologically active molecules on the basis of charge.
  • Bioactivity in terms of in vivo anti-inflammatory activity, was investigated using an established and validated animal model of rheumatoid arthritis, namely a collagen type II- induced arthritis (CIA) model in mice (van Duivenvoorde LM et al, Arthritis. Rheum. 2004 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).
  • CIA collagen type II- induced arthritis
  • prednisolone was also included in the trial as a positive treatment control.
  • Score 4 deformity with ankylosis of the carpus/tarsus.
  • Anti-inflammatory activity measured in response to the abalone processing waste extracts, a commercial abalone extract and an assay positive control (quercetin), is presented in Table 2. Unless specified, cell viability remained above 80% (or below 20% cell death or toxicity) following application of the abalone extracts or other samples. Given that a 70% decrease in nitric oxide production was the maximum effect of the positive control, results comparable to this decrease were considered positive. All abalone extracts and the commercial abalone extract produced positive results decreasing the production of nitric oxide in the RAW 264.7 cells by 40 - 80%. In particular, sample 2-2 displayed the greatest anti-inflammatory activity by significantly decreasing nitric oxide production more than the assay positive (quercetin) control (Figure 1).
  • Heparin cofactor Il-mediated thrombin inhibition measured in response to the abalone processing waste extracts and an assay positive control (heparin), is presented in Table 3. In these assays, the abalone extracts were assayed without dilution to observe maximum anti-thrombotic potential.
  • Antithrombin and heparin cofactor Il-mediated thrombin inhibition measured in response to the abalone processing waste extracts and an assay positive control (heparin), is presented in Table 4. In these assays, the abalone extracts were assayed with several dilutions to observe the full anti-thrombotic potential. Table 1: Estimated total protein, collagen and sulphated polysaccharide contents in abalone processing waste samples
  • Table 4 In vitro antithrombin (AT) and heparin cofactor II (HCII)-mediated thrombin inhibition from extracts prepared from abalone processing waste
  • Anti-inflammatory activity measured in response to the abalone anion exchange chromatography pooled fractions and an assay positive control (quercetin), is presented in Table 5. Cell viability remained above 80% (or below 20% cell death or toxicity) following application of the pooled fractions and positive control.
  • prophylactic treatment with the abalone extract, dose 1 and 2 decreased mean clinical scores by -50% (p ⁇ 0.0001) and 20% (p ⁇ 0.05).
  • Therapeutic treatment with prednisolone significantly decreased the clinical score by - 70% (p ⁇ 0.0001).

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