Improvements in and relating to compositions, methods and uses, for: alleviating conditions caused by, or related to, chronic inflammation; and / or reducing chronic inflammation

Abstract

Nutraceutical compositions, uses and methods for: treating chronic or joint inflammation related conditions, wherein said compositions comprise marine collagen, green-lipped mussel (Perna canaliculus) extract and fucoidan for: alleviating the symptoms. 25 Our Ref: 319097AU ABSTRACT2023202817 05 May 2023 25

Description

Technical Field The present invention relates to nutraceutical compositions, methods and uses, for alleviating conditions caused by, or related to, chronic inflammation; and / or reducing chronic inflammation. Background Art There is a need in the art for cost effective nutraceutical oral compositions, and / or alternative methods, for: - helping to alleviate one or more symptoms of joint inflammation; - reducing joint inflammation; - reducing a person’s production of TNF-a and / or IL-6; and / or - reducing osteoarthritis or alleviating one or more of symptoms of osteoarthritis. Generally speaking, inflammation is the process by which the immune system reacts to infection from outside invaders, such as bacteria and viruses, in order to protect the host. However, in autoimmune diseases such as arthritis, the immune system elicits inflammation when there are no outside invaders to fight off, as the immune system wrongly concludes regular tissues are infected, thereby causing tissue damage via chronic inflammation. Inflammation can be either acute, which is short-lived and goes away within hours or days, or chronic, which is long-lasting for months or years, even after the first trigger is gone. Chronic inflammation associated conditions include cancer, heart disease, diabetes, asthma, Alzheimer’s disease, inflammatory bowel disease (IBD), osteoarthritis (OA), etc. Specifically, IBD is a disorder that involves chronic inflammation of the digestive tract. There are two types of IBD: ulcerative colitis involving inflammation and ulcers along the superficial lining of the colon and rectum; and Crohn's disease involving inflammation of the deeper lining of the digestive tract. 2023202817 05 May 2023 In addition, the symptoms of chronic inflammation can include pain (abdominal, chest, joint or general), fatigue (such as in systemic lupus), fever (such as in tuberculosis), joint stiffness (such as in rheumatoid arthritis), mouth sores (such as in HIV infection), skin rash (such as in psoriasis), etc. It also an objective to provide an alternative to current treatments to one or more of the above listed conditions which has less side effects or contraindications, particularly if used over prolonged periods. For example, typical OA pain relief includes codeine or hydrocodone which can cause side effects such as nausea, constipation, dizziness, and addiction. It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice. Marine collagen Collagen consists of amino acids bound together to form a triple helix of elongated fibril known as a collagen helix. It is mostly found in connective tissue such as cartilage, bones, tendons, ligaments, and skin, and plays key structural roles by supporting the formation, tensile strength, and flexibility of joints (Geahchan et al). There are several types of collagens. In particular, type II collagen (C 2) is a fibrillar protein made up of three long chains of amino acids that form a tightly packed network of fibrils and fibres. It is the main component of cartilage that gives cartilage its tensile strength and elasticity, thereby enabling it to support the joints. Marine collagen is derived from marine sources such as fish tissues. Specifically, collagen extracted from fish skin is rich in C 2. Its oral ingestion is believed to reduce autoimmunity to the body's own C 2, resulting in less inflammation in instances of osteoarthritis (OA) and rheumatism, together with benefits to joint health. Studies have shown that marine collagen and its derivatives have anti-inflammatory effects that are beneficial in both osteoporosis and OA prevention and treatment. Specifically, Bourdo et al. demonstrated the beneficial effects of marine collagen hydrolysates in counteracting each step of the OA pathogenesis. In addition, marine collage may also target other bone-related diseases as it is capable of increasing bone mineral density, mineral deposition, and importantly, osteoblast maturation and proliferation (Geahchan et al). Green-lipped mussel Green-lipped mussel (GLM), also known as Perna canaliculus, is endemic to New Zealand. When taken orally in whole powder or oil / lipid extract formats, GLM has been found to reduce pain and ameliorate other debilitating symptoms, associated with inflammatory 2023202817 05 May 2023 diseases such as OA, without causing the adverse side effects of non-steroidal antiinflammatories (NSAIDs). The underlying mechanisms explaining the effects of GLM, are in the anti-inflammatory activity of bioactive lipids in GLM, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). These lipids mediate the anti-inflammatory response by inhibiting both the cyclo-oxygenase (COX) and lipo-oxygenase (LOX) cascades of arachidonic acid (AA) metabolism, thereby resulting in a decrease in the synthesis of pro-inflammatory prostaglandins and leukotrienes. Furthermore, GLM is reported to contain novel bioactive lipids such as pro-resolving lipid mediators which reduce inflammation through counter-regulating pro-inflammatory cytokines, clearing apoptotic neutrophils, and inducing wound healing and tissue regeneration: thereby also ameliorating the effects and / or symptoms of inflammation including OA. Several clinical studies concluded that GLM, at a dose of around 1000mg per day, provided moderate and clinically meaningful treatment effects on OA pain (Abshirini et al. 2021). Fucoidan Fucoidan is an acidic polysaccharide mainly composed of fucose, galactose and sulfate with smaller amounts of mannoses, uronic acid, glucose, rhamnose, arabinose, and xylose, and is commonly extracted from brown seaweeds like Ascopbyllum nodosum, Cladosiphon okamuranus, Fucus vesiculosus, Laminaria japonica, Saccharina japonica, Sargassum thunbergia, Undaria pinnatifida etc. (Fitton 2011). Fucoidan is found in the cell walls of the seaweed plant and serves to protect it from external stresses. As such, fucoidan has been shown to provide similar protective benefits for both human and animal health. Fucoidan extracts have been utilised in a wide range of therapeutic health care preparations. Specifically, fucoidan has been shown to be effective in preventing the progression of monosodium iodoacetate (MIA)-induced OA in rats, by alleviating the symptoms and inflammatory cytokine activation (Lee et al. 2015). Similarly, fucoidan has been found to improve meniscal / ligamentous injury and obesity-induced OA in rats (Sudirman et al. 2018). In addition, fucoidan extracts from brown marine algae have been shown to have a range of anti-inflammatory effects in an open label combined phase I and II pilot scale study. Specifically, when taken orally over twelve weeks, the symptoms of OA were found to decrease in a dose-dependent manner, demonstrating the efficacy of fucoidans in treating OA (Myers et al. 2010). 2023202817 05 May 2023 It is apparent in the art that marine collagen, GLM extract and fucoidan have different mechanisms of action. Marine collagen, in particular C 2, supports and strengthens bone tissues and reduces inflammation related to bone tissues. GLM mainly exerts its action via its lipid components, which work on the COX pathway and affect pro-inflammation cytokines to reduce inflammation of the joint environment. Fucoidan works on the inhibition of inflammatory cytokine activation to reduce joint inflammation. Therefore, marine collagen, GLM and fucoidan each works on a different pathway to alleviate OA effects and / or symptoms. The present invention is related to such a nutraceutical combination, comprising marine collagen, GLM extract and fucoidan. KR102287247B1 discloses a composition for animal joint health, comprising methylsulfonylmethane, GLM extract and chondroitin sulfate as the main components, with fucoidan being an optional component. By way of contrast, the present invention comprises marine collagen, GLM extract and fucoidan in a specific w / w ratio. Nutraceutical compositions, methods and uses, of the present invention provide surprising anti-inflammatory effects and possible symptom reduction as shown in the in vitro experiments. It is an object of the present invention to provide methods and a composition for assisting with the prevention, treatment, or alleviation of the conditions mentioned herein. It is an object of the present invention to provide an alternative way to maintain joint health. All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although one or more prior art publications may be referred to herein, any such references do not constitute an admission that any of these documents form part of the common general knowledge in the art, in Australia, New Zealand or in any other country. Throughout this specification, the word “comprise”, or variations thereof such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. 2023202817 05 May 2023 Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only. Definitions The term ‘collagen’ as used herein refers to a protein comprising amino acids bound together to form a triple helix of elongated fibril known as a collagen helix, and is the main structural protein in the extracellular matrix, mostly found in connective tissue such as cartilage, bones, tendons, ligaments, and skin. The term ‘green-lipped mussel’ as used herein refers to Perna canaliculus, which is also known as the New Zealand mussel, the greenshell mussel, kuku, and kutai, and is a bivalve mollusc in the Mytilidae family. The term ‘fucoidan’ as used herein refers to an acidic polysaccharide mainly composed of fucose, galactose and sulfate with smaller amounts of mannoses, uronic acid, glucose, rhamnose, arabinose, and xylose, and is commonly extracted / isolated from brown seaweeds like Ascopbyllum nodosum, Cladosiphon okamuranus, Fucus vesiculosus, Laminaria japonica, Saccharina japonica, Sargassum thunbergia, Undaria pinnatifida etc. The term ‘osteoarthritis’ or ‘OA’ as used herein refers to a type of degenerative joint disease resulting from breakdown of joint cartilage and underlying bone, with the most common symptoms being joint pain and stiffness. The term ‘lipid’ as used herein refers to a micro biomolecule that is soluble in nonpolar solvents, which are typically hydrocarbons used to dissolve other naturally occurring hydrocarbon lipid molecules that do not, or do not easily dissolve in water. For example, fatty acids are common components of complex lipids. The term ‘extract’ as used herein refers to the resulting oil / lipid product of an extraction process, and this may be in the form of a powder or liquid. The term ‘effective amount’ as used herein refers to an amount of an active component of the present invention which when the oral composition is taken in a dosage quantity will be effective in helping to alleviate the symptoms of chronic inflammation in a mammal. The term ‘effective amount’ when used herein may also in some embodiments refer to a therapeutically effective amount of an active component of the present invention which when the oral composition is taken in a dosage quantity can alleviate or mitigate the symptoms of chronic inflammation. 2023202817 05 May 2023 In some embodiments, the effective amount will be the same as the dosage quantity. In other embodiments, the effective amount may be half, quarter, or a third, of the dosage quantity. In other embodiments, the effective amount may be greater than the dosage quantity in the range of two to ten times of the dosage quantity. The term ‘dosage form’, also known as ‘unit dose’, as used herein refers to nutraceutical products in the form in which they are marketed for use, with a specific mixture of active ingredients and inactive components (excipients), in a particular configuration (such as a capsule shell, for example), and apportioned into a particular dose. For example, dosage forms for oral administration may include pills, such as tablets or capsules, syrups, powders, pastes, or injectable suspensions. This list should not be seen as limiting. The term ‘dosage quantity’ as used herein refers to the relative amount of the active components of the oral composition of the present invention which when taken at a particular time or series of times can induce a positive biological or medical response in a human or other mammal. In preferred embodiments the term ‘dosage quantity’ as used herein refers to the following observed effects: - can help alleviate or mitigate, the symptoms; and / or - can alleviate or mitigate, the symptoms. It should be appreciated that the dosage quantity may be adjusted as required depending on the subject to be treated. For example only, a dosage quantity may be a single tablet of the present invention; or dosage quantity may be a part thereof - e.g. half a tablet; or a dosage quantity may be multiple tablets e.g. 2 tablets. As mentioned, a dosage quantity is envisaged to be taken more than once a day to maintain the availability of the active components within a person or animal. It should be appreciated that the relative amount of the active components may be adjusted as required depending on the subject to be treated. The examples given herein should be seen as exemplary and base lines for re-adjusting the oral composition and / or dosage amount as required as would be taken into account by a person skilled in the art. The terms ‘treatment’, ‘treat’ and ‘treating’ as used herein refer to a subject taking the nutraceutical composition of the present invention after the onset of a clinical chronic manifestation of a disease state or condition, so as to eliminate or reduce the symptoms, or clinical manifestation of the disease state or condition. 2023202817 05 May 2023 Summary of Invention The present invention is concerned with reducing chronic inflammation. More specifically, in one aspect the present invention is concerned with reducing the production of Tumour Necrosis Factor alpha (TNF-a) and / or lnterleukin-6 (IL-6), inflammatory cytokines produced during inflammation. While it is known that marine collagen, GLM extract and fucoidan can each positively affect the cartilage structures and reduce pain in OA patients, studies have shown that their individual effects on OA are via different modes of action. The inventor was therefore interested to explore whether or not a combination of just these three ingredients could have additional positive effects on people suffering from chronic inflammatory conditions including OA. According to a first aspect of the present invention, there is provided a nutraceutical composition comprising: • marine collagen; •      green-lipped mussel (Perna canaliculus) extract; and •       fucoidan. Preferably, marine collagen, green-lipped mussel (Perna canaliculus) extract and fucoidan are respectively present in a 1:1:1 or 1:1:3 w / w ratio in the nutraceutical composition. Preferably, the use of the nutraceutical composition is for reducing chronic or joint inflammation. Preferably, the use of the nutraceutical composition is for reducing the symptoms of chronic or joint inflammation, such as pain, fatigue, fever, joint stiffness, mouth sores, and skin rash. Preferably, the use of the nutraceutical composition is for alleviating the symptoms and / or effects of osteoarthritis. Preferably, the use of the nutraceutical composition is for reducing the production of TNF-a and / or IL-6. According to a second aspect of the present invention, there is provided a dosage form of a nutraceutical composition comprising: • at least substantially 50mg of marine collagen; and • at least substantially 450mg of green-lipped mussel (Perna canaliculus) extract; and • at least substantially 50mg of fucoidan. 2023202817 05 May 2023 Preferably, the dosage form of the nutraceutical composition is in the form of a capsule or a tablet. According to a third aspect of the present invention, there is provided a method of reducing one or more symptoms associated with the following conditions: • chronic inflammation; • osteoarthritis; and • joint inflammation; by the steps of taking two capsules or tables of the nutraceutical composition once a day. Preferably, the nutraceutical composition is in a suitable dosage form. Preferably, the nutraceutical composition is to be taken daily in a dosage quantity comprising substantially 100mg marine collage, 900mg green-lipped mussel (Perna canaliculus) extract and optionally 100mg fucoidan. Preferably, the nutraceutical composition reduces inflammation and / or reduces the production of TNF-a and / or IL-6. According to a fourth aspect of the present invention, there is provided a method of improving joint health comprising orally administering a dosage quantity of: • marine collagen; • green-lipped mussel (Perna canaliculus) extract; and • fucoidan. According to a fifth aspect of the present invention, there is provided a method treating, alleviating or preventing: osteoarthritis or other chronic inflammatory conditions; and / or to treating, preventing, or reducing inflammation or an inflammatory response, and / or to reducing the production of TNF-a and / or IL-6; comprising the step of administering: a dosage form of a nutraceutical composition as claimed in claim 7, or a dosage quantity of a nutraceutical composition as claimed in claim 1. 2023202817 05 May 2023 The present invention comprising marine collage, GLM extract and fucoidan at proper dosages allows each of the active ingredients to work on their respective pathways, thereby exerting beneficial effects in alleviating OA effects and / or symptoms. Brief Description of Drawings Figure 1 shows the viability of mouse macrophage cell line RAW 264.7 in the presence of C 2 and the combination of C 2 and GLM extract; Figure 2 shows the percentage in TNF-a inhibition in mouse macrophage cell line RAW 264.7 stimulated by LPS in the presence of C 2, GLM extract, and the combination of C 2 and GLM extract; Figure 3 shows the TNF-a production in mouse macrophage cell line RAW 264.7 treated with LPS, GLM extract, C 2, fucoidan, and the combination comprising GLM extract, C 2 and fucoidan at 1:1:1 or 1:1:3 ratios respectively; Figure 4 shows the inhibition of TNF-a production in mouse macrophage cell line RAW 264.7 pre-stimulated with LPS, then treated with the positive control DXA and samples GLM extract, C 2, fucoidan, the combination comprising GLM extract, C 2 and fucoidan at 1:1:1 or 1:1:3 ratios respectively; Figure 5 shows the IL-6 production in mouse macrophage cell line RAW 264.7 treated with LPS, GLM extract, C 2, fucoidan, and the combination comprising GLM extract, C 2 and fucoidan at 1:1:1 or 1:1:3 ratios respectively; Figure 6 shows the inhibition of IL-6 production in mouse macrophage cell line RAW 264.7 pre-stimulated with LPS, then treated with the positive control DXA and samples GLM extract, C 2, fucoidan, the combination comprising GLM extract, C 2 and fucoidan at 1:1:1 or 1:1:3 ratios respectively; Figure 7 shows the TNF-a production in human THP-1 monocytes treated with LPS, GLM extract, C 2, fucoidan, and the combination comprising GLM extract, C 2 and fucoidan at 1:1:1 or 1:1:3 ratios respectively; Figure 8 shows the inhibition of TNF-a production in human THP-1 monocytes prestimulated with LPS, then treated with the positive control DXA and samples GLM extract, C 2, fucoidan, the combination comprising GLM extract, C 2 and fucoidan at 1:1:1 or 1:1:3 ratios respectively; Figure 9 shows the TNF-a production in human THP-1 macrophages treated with 2023202817 05 May 2023 LPS, GLM extract, C 2, fucoidan, and the combination comprising GLM extract, C 2 and fucoidan at 1:1:1 or 1:1:3 ratios respectively; and Figure 10 shows the inhibition of TNF-a production in human THP-1 macrophages prestimulated with LPS, then treated with the positive control DXA and samples GLM extract, C 2, fucoidan, the combination comprising GLM extract, C 2 and fucoidan at 1:1:1 or 1:1:3 ratios respectively. Best Modes for Carrying out the Invention EXAMPLE 1 - Viability of mouse cell line A murine macrophage cell line (RAW 264.7) was obtained from the American Type Cell Culture (ATCC). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics (100 unit / mL penicillin, 100 mg / mL streptomycin) at 37 °C with 5% CO2. Cells were passaged every 48-72 hours. Mouse macrophage cell line RAW264.7 was incubated with 50 pg / ml C 2 alone (Collagen) or 50 pg / ml C 2 in combination with 50 pg / ml GLM extract (Mussel Extract + Collagen) to determine if cell viability was affected. MTT assay was used to determine cell viability. Cells were seeded at densities of 5000 cells / well, for 6 to 24 h, in the incubator in 96-well plates. 100 pL of fresh complete culture medium containing various concentrations of treatment compounds was added to corresponding wells. After incubation, the medium was carefully removed and replaced with 100 pL of fresh complete culture medium. An aliquot of 10 pL of MTT stock solution was added to each well and the plates were placed in the 37°C incubator. The supernatant was gently removed from the wells. An aliquot of 150 pL of DMSO was added to each well and mixed thoroughly using an orbit plate shaker. After incubating at 37°C for 20 to 30 min, the plate was shaken briefly and absorbance was measured by a plate reader (FLUOstar Omega, Alphatech Systems Ltd., Auckland, New Zealand), at 540 nm. As shown in Figure 1, cell viability of mouse macrophage cell line RAW264.7 was not affected by either C 2 alone or the combination of C 2 with GLM extract. EXAMPLE 2 - Inhibition of TNF-a production in mouse cell line by the combination comprising GLM extract and C 2 The anti-inflammatory effects of the combination comprising C 2 and GLM extract were tested in cultured mouse macrophage cell line RAW264.7 cultured in Dulbecco’s Modified Eagle Medium (DMEM) media. 0.01 pg / ml lipopolysaccharide (LPS), also known as endotoxin, was used to induce inflammatory-like responses in cultured RAW264.7 cells. 2023202817 05 May 2023 Upon stimulation, RAW264.7 cells overproduced around 5,000 ng / ml tumour necrosis factor-a (TNF-a), indicating inflammation. The combination comprising C 2 and GLM extract was added at concentrations of 5, 12.5, 25 or 50 pg / ml to the LPS-stimulated RAW264.7 cells by dissolving the combination in the cell culture media. C 2 (from fish skin in this case from shark skin) and GLM extract were prepared from Oceanfit™ GLM powder in Professor Jun Lu’s Laboratory. GLM was dissolved in 2% ethanol (proven to be non-cytotoxic) and C 2 was dissolved in water directly. The two solvents were then mixed to create concentrations of the combination at 5, 12.5, 25 or 50 pg / ml. Concentrations of the combination at 5, 12.5, 25 or 50 pg / ml substantially correspond to the estimated range of body fluid concentrations of persons weighing between 50kg to 100kg who have taken the combination comprising 100mg marine collagen and 900mg GLM extract. After 24 hours incubation, TNF-a concentration in the cell culture media was measured using a commercially available TNF-a enzyme-linked immunosorbent assay (ELISA) kit according to manufacturer’s instruction. All concentrations of the combination comprising C2 and GLM extract displayed approximately 20% inhibition of TNF-a production as shown in Figure 2, suggesting that the combination comprising C 2 and GLM extract is able to alleviate inflammatory conditions by reducing the production of TNF-a. In particular, the most effective concentrations of the combination are 25 and 50 pg / ml. In particular, at 25 pg / ml, there is a clear synergistic inhibition can be observed. In addition, 1 pg / ml dexamethasone, a glucocorticoid, was used as a positive control to show inhibition of TNF-a production in LPS-stimulated RAW264.7 cells. The experiments were conducted in triplicates. EXAMPLE 3 - Inhibition of TNF-a production in mouse cell line by the combination comprising GLM extract, C 2 and fucoidan The anti-inflammatory effects of the combination comprising GLM extract, C 2, and fucoidan were tested in cultured mouse macrophage cell line RAW264.7 cultured in DMEM media. 0.01 pg / ml LPS was used to induce inflammatory-like responses in cultured RAW264.7 cells. After 24 hours incubation, TNF-a concentration in the cell culture media was measured using a commercially available TNF-a ELISA kit according to manufacturer’s instruction. The experiments were conducted in triplicates. Specifically, as controls, RAW264.7 cells were first treated with GLM extract, C 2, fucoidan, the combination comprising GLM extract, C 2, and fucoidan at 1:1:1 and 1:1:3 ratios respectively, at either 30 or 60 pg / ml. As shown in Figure 3, RAW264.7 cells treated with 2023202817 05 May 2023 0.01 pg / ml LPS produced around 1,000 ng / ml TNF-a indicating inflammation, whereas cells treated with the samples produced some but not significant TNF-a in comparison to LPS treatment. As shown in Figure 4, following the pre-stimulation using 0.01 pg / ml LPS, RAW264.7 cells were treated with 1 pg / ml DXA as a positive control to show inhibition of TNF-a production in LPS-stimulated RAW264.7 cells. In parallel, RAW264.7 cells were also treated with GLM extract, C 2, fucoidan, and the combination comprising GLM extract, C 2, and fucoidan at 1:1:1 and 1:1:3 ratios respectively, at either 30 or 60 pg / ml by dissolving the combination in the cell culture media. Fucoidan, C 2 (from fish skin in this case shark skin) and GLM extract were prepared from Oceanfit™ GLM powder in Professor Jun Lu’s Biomedical Laboratory at Auckland University of Technology. GLM was dissolved in 2% ethanol (proven to be non-cytotoxic) and C 2 was dissolved in water directly. The three solvents were then mixed to create concentrations of the combination at 30 or 60 pg / ml. Concentrations of the combination at 30 or 60 pg / ml substantially correspond to the estimated range of body fluid concentrations of persons weighing between 50kg to 100kg who have taken the combination comprising 100mg marine collagen, 900mg GLM extract, and 100mg fucoidan. Surprisingly, the combination of GLM extract, C 2 and fucoidan at the ratio of 1:1:1 displayed greater inhibition of TNF-a production than that of the positive control DXA, suggesting the combination comprising C 2 and GLM extract, C 2 and fucoidan at 1:1:1 ratio is able to alleviate inflammatory conditions by significantly reducing the production of TNF-a. In particular, the most effective concentration of the combination is 30 pg / ml. However, the combination displays a clear synergistic inhibition in comparison to the GLM extract, C 2 or fucoidan alone, whether at the ratio of 1:1:1 or 1:1:3, or at the concentration of 30 or 60 pg / ml. Table 1. TNF-a production in unstimulated RAW264.7 cells Treatment of RAW264.7 cells TNF-a production (ng / ml) Untreated 14.30 0.01 pg / ml LPS 991.9 30 pg / ml GLM 0 60 pg / ml GLM 0 2023202817 05 May 2023 30 pg / ml C 2 14.0 60 pg / ml C 2 45.7 30 pg / ml fucoidan 598.3 60 pg / ml fucoidan 606.2 30 pg / ml G:C:F=1:1:1 103.1 60 pg / ml G:C:F=1:1:1 180.6 30 pg / ml G:C:F=1:1:3 249.7 60 pg / ml G:C:F=1:1:3 246.5 Table 2. TNF-a production in LPS-pre-stimulated RAW264.7 cells Treatment of LPS-pre-stimulated RAW264.7 cells TNF-a production (ng / ml) 1 pg / ml DXA 407.3 30 pg / ml GLM 890.8 60 pg / ml GLM 946.4 30 pg / ml C 2 955.1 60 pg / ml C 2 904.8 30 pg / ml fucoidan 1290.0 60 pg / ml fucoidan 1329.4 30 pg / ml G:C:F=1:1:1 155.0 60 pg / ml G:C:F=1:1:1 183.1 30 pg / ml G:C:F=1:1:3 578.7 60 pg / ml G:C:F=1:1:3 522.3 EXAMPLE 4 - Inhibition of IL-6 production in mouse cell line by the combination 2023202817 05 May 2023 comprising GLM extract, C 2 and fucoidan The anti-inflammatory effects of the combination comprising GLM extract, C 2, and fucoidan were tested in cultured mouse macrophage cell line RAW264.7 cultured in DMEM media. 0.01 pg / ml LPS was used to induce inflammatory-like responses in cultured RAW264.7 cells. After 24 hours incubation, lnterleukin-6 (IL-6) concentration in the cell culture media was measured using a commercially available IL-6 ELISA kit according to manufacturer’s instruction. The experiments were conducted in triplicates. Specifically, as controls, RAW264.7 cells were first treated with GLM extract, C 2, fucoidan, the combination comprising GLM extract, C 2, and fucoidan at 1:1:1 and 1:1:3 ratios respectively, at either 30 or 60 pg / ml. As shown in Figure 5, RAW264.7 cells treated with 0.01 pg / ml LPS produced around 1,200 ng / ml IL-6 indicating inflammation, whereas cells treated with the samples produced some but not significant TNF-a in comparison to LPS treatment. As shown in Figure 6, following the pre-stimulation using 0.01 pg / ml LPS, RAW264.7 cells were treated with 1 pg / ml DXA as a positive control to show inhibition of TNF-a production in LPS-stimulated RAW264.7 cells. In parallel, RAW264.7 cells were also treated with GLM extract, C 2, fucoidan, and the combination comprising GLM extract, C 2, and fucoidan at 1:1:1 and 1:1:3 ratios respectively, at either 30 or 60 pg / ml by dissolving the combination in the cell culture media. Fucoidan, C 2 (from fish skin in this case from shark skin) and GLM extract were prepared from Oceanfit™ GLM powder in Professor Jun Lu’s Laboratory. GLM was dissolved in 2% ethanol (proven to be non-cytotoxic) and C 2 and fucoidan were dissolved in water directly. The three solvents were then mixed to create concentrations of the combination at 30 or 60 pg / ml. Concentrations of the combination at 30 or 60 pg / ml substantially correspond to the estimated range of body fluid concentrations of persons weighing between 50kg to 100kg who have taken the combination comprising 100 mg marine collagen, 900 mg GLM extract, and 100 mg fucoidan. Surprisingly, yet consistent with the results of Example 3, the combination of GLM extract, C 2 and fucoidan at the ratio of 1:1:1 displayed greater inhibition of IL-6 production than that of the positive control DXA, suggesting the combination comprising C 2 and GLM extract, C 2 and fucoidan at 1:1:1 ratio is able to alleviate inflammatory conditions by significantly reducing the production of IL-6. The combination displays a clear synergistic inhibition in comparison to the GLM extract, C 2 or fucoidan alone, whether at the ratio of 1:1:1 or 1:1:3, or at the concentration of 30 or 60 pg / ml. 2023202817 05 May 2023 Table 3. IL-6 production in unstimulated RAW264.7 cells Treatment of RAW264.7 cells IL-6 production (ng / ml) Untreated 14.265 0.01 pg / ml LPS 1237.7 30 pg / ml GLM 8.0 60 pg / ml GLM 24.1 30 pg / ml C 2 10.4 60 pg / ml C 2 11.9 30 pg / ml fucoidan 507.1 60 pg / ml fucoidan 836.8 30 pg / ml G:C:F=1:1:1 171.3 60 pg / ml G:C:F=1:1:1 122.1 30 pg / ml G:C:F=1:1:3 426.0 60 pg / ml G:C:F=1:1:3 278.2 Table 4. IL-6 production in LPS-pre-stimulated RAW264.7 cells Treatment of LPS-pre-stimulated RAW264.7 cells IL-6 production (ng / ml) 1 pg / ml DXA 262.8 30 pg / ml GLM 786.7 60 pg / ml GLM 820.0 30 pg / ml C 2 819.4 60 pg / ml C 2 852.9 30 pg / ml fucoidan 1241.8 2023202817 05 May 2023 60 pg / ml fucoidan 1295.1 30 pg / ml G:C:F=1:1:1 237.9 60 pg / ml G:C:F=1:1:1 234.5 30 pg / ml G:C:F=1:1:3 977.3 60 pg / ml G:C:F=1:1:3 755.6 EXAMPLE 5 - Inhibition of TNF-a production in THP-1 monocytes by the combination comprising GLM extract, C 2 and fucoidan The anti-inflammatory effects of the combination comprising GLM extract, C 2, and fucoidan were tested in cultured human monocyte cell line THP-1 cultured in DMEM media. 0.01 pg / ml LPS was used to induce inflammatory-like responses in cultured THP-1 monocytes. After 24 hours incubation, TNF-a concentration in the cell culture media was measured using a commercially available TNF-a ELISA kit according to manufacturer’s instruction. The experiments were conducted in triplicates. Specifically, as controls, THP-1 monocytes were first treated with GLM extract, C 2, fucoidan, the combination comprising GLM extract, C 2, and fucoidan at 1:1:1 and 1:1:3 ratios respectively, at either 30 or 60 pg / ml. As shown in Figure 7, THP-1 monocytes treated with 0.01 pg / ml LPS produced over 250 ng / ml TNF-a indicating inflammation, whereas cells treated with the samples produced some but not significant TNF-a in comparison to LPS treatment. As shown in Figure 8, following the pre-stimulation using 0.01 pg / ml LPS, THP-1 monocytes were treated with 1 pg / ml DXA as a positive control to show inhibition of TNF-a production in LPS-stimulated THP-1 monocytes. In parallel, THP-1 monocytes were also treated with GLM extract, C 2, fucoidan, and the combination comprising GLM extract, C 2, and fucoidan at 1:1:1 and 1:1:3 ratios respectively, at either 60 or 100 pg / ml by dissolving the combination in the cell culture media. Fucoidan, C 2 (from fish skin in this case shark skin) and GLM extract were prepared from Oceanfit™ GLM powder in Professor Jun Lu’s Laboratory. GLM was dissolved in 2% ethanol (proven to be non-cytotoxic) and C 2 and fucoidan were dissolved in water directly. The three solvents were then mixed to create concentrations of the combination at 60 or 100 pg / ml. Concentrations of the combination at 60 or 100 pg / ml substantially correspond to the estimated range of body fluid concentrations of persons weighing between 50kg to 100kg 2023202817 05 May 2023 who have taken the combination comprising 100 mg marine collagen, 900 mg GLM extract and 100 mg fucoidan. Surprisingly, the combination of GLM extract, C 2 and fucoidan at the ratio of 1:1:1 displayed greater inhibition of TNF-a production than that of the positive control DXA, suggesting the combination comprising C 2 and GLM extract, C 2 and fucoidan at 1:1:1 ratio is able to alleviate inflammatory conditions by significantly reducing the production of TNF-a. The combination displays a clear synergistic inhibition in comparison to the GLM extract, C 2 or fucoidan alone, whether at the ratio of 1:1:1 or 1:1:3, or at the concentration of 60 or 100 pg / ml. Table 5. TNF-a production in unstimulated THP-1 monocytes T reatment of THP-1 monocytes TNF-a production (ng / ml) Untreated 1.0 0.01 pg / ml LPS 278.2 60 pg / ml GLM 10.7 100 pg / ml GLM 9.0 60 pg / ml C 2 2.0 100 pg / ml C 2 2.0 60 pg / ml fucoidan 92.7 100 pg / ml fucoidan 73.4 60 pg / ml G:C:F=1:1:1 29.3 100 pg / ml G:C:F=1:1:1 36.3 60 pg / ml G:C:F=1:1:3 80.5 100 pg / ml G:C:F=1:1:3 95.9 Table 6. TNF-a production in LPS-pre-stimulated THP-1 monocytes 2023202817 05 May 2023 Treatment of LPS-pre-stimulated THP-1 monocytes TNF-a production (ng / ml) 1 pg / ml DXA 95.1 60 pg / ml GLM 247.7 100 pg / ml GLM 193.2 60 pg / ml C 2 250.6 100 pg / ml C 2 282.1 60 pg / ml fucoidan 301.1 100 pg / ml fucoidan 329.0 60 pg / ml G:C:F=1:1:1 35.7 100 pg / ml G:C:F=1:1:1 35.2 60 pg / ml G:C:F=1:1:3 152.9 100 pg / ml G:C:F=1:1:3 165.8 EXAMPLE 5 - Inhibition of TNF-a production in THP-1 macrophages by the combination comprising GLM extract, C 2 and fucoidan The anti-inflammatory effects of the combination comprising GLM extract, C 2, and fucoidan were tested in cultured human macrophage cell line THP-1 cultured in DMEM media. THP-1 macrophages were transformed from THP1 monocytes using the standard protocol provided by the manufacturer. 0.01 pg / ml LPS was used to induce inflammatory-like responses in cultured THP-1 macrophages. After 24 hours incubation, TNF-a concentration in the cell culture media was measured using a commercially available TNF-a ELISA kit according to manufacturer’s instruction. The experiments were conducted in triplicates. Specifically, as controls, THP-1 macrophages were first treated with GLM extract, C 2, fucoidan, the combination comprising GLM extract, C 2, and fucoidan at 1:1:1 and 1:1:3 ratios respectively, at either 30 or 60 pg / ml. As shown in Figure 9, THP-1 macrophages treated with 0.01 pg / ml LPS produced over 150 ng / ml TNF-a indicating inflammation, whereas cells treated with the samples produced some but not significant TNF-a in comparison to LPS treatment. 2023202817 05 May 2023 As shown in Figure 10, following the pre-stimulation using 0.01 pg / ml LPS, THP-1 macrophages were treated with 1 pg / ml DXA as a positive control to show inhibition of TNF-a production in LPS-stimulated THP-1 macrophages. In parallel, THP-1 macrophages were also treated with GLM extract, C 2, fucoidan, and the combination comprising GLM extract, C 2, and fucoidan at 1:1:1 and 1:1:3 ratios respectively, at either 60 or 100 pg / ml by dissolving the combination in the cell culture media. Fucoidan, C 2 (from fish skin in this case shark skin) and GLM extract were prepared from Oceanfit™ GLM powder in Professor Jun Lu’s Laboratory. GLM was dissolved in 2% ethanol (proven to be non-cytotoxic) and C 2 and fucoidan were dissolved in water directly. The three solvents were then mixed to create concentrations of the combination at 60 or 100 pg / ml. Concentrations of the combination at 60 or 100 pg / ml substantially correspond to the estimated range of body fluid concentrations of persons weighing between 50kg to 100kg who have taken the combination comprising 100 mg marine collagen, 900 mg GLM extract, and 100 mg fucoidan. Surprisingly, the combination of GLM extract, C 2 and fucoidan at the ratio of 1:1:1 displayed greater inhibition of TNF-a production than that of the positive control DXA, suggesting the combination comprising C 2 and GLM extract, C 2 and fucoidan at 1:1:1 ratio is able to alleviate inflammatory conditions by significantly reducing the production of TNF-a. The combination displays a clear synergistic inhibition in comparison to the GLM extract, C 2 or fucoidan alone, whether at the ratio of 1:1:1 or 1:1:3, or at the concentration of 60 or 100 pg / ml. Table 7. TNF-a production in unstimulated THP-1 macrophages T reatment of THP-1 macrophages TNF-a production (ng / ml) Untreated 0.56 0.01 pg / ml LPS 158.9 60 pg / ml GLM 0.1 100 pg / ml GLM 0.6 60 pg / ml C 2 0.3 100 pg / ml C 2 0.3 60 pg / ml fucoidan 194.1 2023202817 05 May 2023 100 pg / ml fucoidan 200.3 60 pg / ml G:C:F=1:1:1 54.1 100 pg / ml G:C:F=1:1:1 47.9 60 pg / ml G:C:F=1:1:3 126.0 100 pg / ml G:C:F=1:1:3 122.1 Table 8. TNF-a production in LPS-pre-stimulated THP-1 macrophages Treatment of LPS-pre-stimulated THP-1 macrophages TNF-a production (ng / ml) 1 pg / ml DXA 43.8 60 pg / ml GLM 161.1 100 pg / ml GLM 138.4 60 pg / ml C 2 171.5 100 pg / ml C 2 158.5 60 pg / ml fucoidan 228.0 100 pg / ml fucoidan 217.4 60 pg / ml G:C:F=1:1:1 35.1 100 pg / ml G:C:F=1:1:1 41.2 60 pg / ml G:C:F=1:1:3 121.5 100 pg / ml G:C:F=1:1:3 126.6 The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features. 2023202817 05 May 2023 Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the appended claims. 2023202817 05 May 2023 REFERENCES: Abshirini et al. Green-lipped (greenshell™) mussel (Perna canaliculus) extract supplementation in treatment of osteoarthritis: a systematic review. Inflammopharmacology (2021) Atashrazm et al. Fucoidan and Cancer: A Multifunctional Molecule with Anti-Tumor Potential. Mar Drugs (2015) Bourdon et al. Marine Collagen Hydrolysates Downregulate the Synthesis of Pro-Catabolic and Pro-Inflammatory Markers of Osteoarthritis and Favor Collagen Production and Metabolic Activity in Equine Articular Chondrocyte Organoids IntJ Mol Sci. (2021) Fitton. Therapies from Fucoidan; Multifunctional Marine Polymers. Mar Drugs (2011) Geahchan et al. Marine Collagen: A Promising Biomaterial for Wound Healing, Skin AntiAging, and Bone Regeneration. Mar Drugs (2022) Lee et al. Fucoidan Prevents the Progression of Osteoarthritis in Rats. J Med Food (2015) Myers et al. A combined phase I and II open label study on the effects of a seaweed extract nutrient complex on osteoarthritis. Biologies (2010) Sudirman et al. Effect of Fucoidan on Anterior Cruciate Ligament Transection and Medial Meniscectomy Induced Osteoarthritis in High-Fat Diet-Induced Obese Rats. Nutrients (2018)

Claims

2023202817 05 May 2023WHAT WE CLAIM IS:

1. A nutraceutical composition comprising:• marine collagen; and• green-lipped mussel (Perna canaliculus) extract.• fucoidan.

2. A nutraceutical composition as claimed in claim 2 wherein marine collagen, greenlipped mussel (Perna canaliculus) extract and fucoidan are respectively present in a 1:1:1 to 1:1:3 w / w ratio.

3. The use of a nutraceutical composition as claimed in any one of the preceding claims for reducing chronic or joint inflammation.

4. The use of a nutraceutical composition as claimed in any one of the preceding claims for reducing the symptoms of chronic or joint inflammation, such as pain, fatigue, fever, joint stiffness, mouth sores, and skin rash.

5. The use of a nutraceutical composition as claimed in claim 1 for alleviating the symptoms and / or effects of osteoarthritis.

6. The use of a nutraceutical composition as claimed in claim 1 for reducing the production of TNF-a and / or IL-6.

7. A dosage form of a nutraceutical composition comprising:• at least substantially 50mg of marine collagen;• at least substantially 450mg green-lipped mussel (Perna canaliculus) extract; and• at least substantially 50mg of fucoidan.

8. A dosage form as claimed in claim 7 wherein the dosage form is in the form of a capsule or a tablet.

9. A method of reducing one or more symptoms associated with the following conditions:- chronic inflammation;- osteoarthritis;2023202817 05 May 2023- joint inflammation;by the step of taking two capsules or tablets as claimed in claim 9 once a day.

10. A nutraceutical composition as claimed in any one of the preceding claims, wherein the composition is for oral consumption.

11. A nutraceutical composition as claimed in any one of the preceding claims, wherein the composition is in a suitable dosage form.

12. A nutraceutical composition as claimed in any one of the preceding claims, wherein the composition is to be taken daily in a dosage quantity comprising substantially 100mg marine collagen, 900mg green-lipped mussel (Perna canaliculus) extract and optionally 100mg fucoidan.

13. A nutraceutical composition as claimed in any one of the preceding claims, wherein the composition reduces inflammation and / or reduces the production of TNF-a and / or IL-6.

14. A method of improving joint health comprising orally administering a dosage quantity of:• marine collagen;• green-lipped mussel (Perna canaliculus) extract; and• fucoidan;15. A method of treating, alleviating or preventing: osteoarthritis or other chronic inflammatory conditions; and / or to treating, preventing, or reducing inflammation or an inflammatory response, and / or to reducing the production of TNF-a and / or IL-6; comprising the step of administering: a dosage form of a nutraceutical composition as claimed in claim 7, or a dosage quantity of a nutraceutical composition as claimed in claim 1.

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