Purification method

Abstract

The invention relates to a method for purifying type II collagen from cartilage tissue. The invention also relates to a collagen type II composition, and the use of the collagen type II composition as a dietary supplement or treatment for cartilage-related conditions. Methods of treating or preventing cartilage-related conditions are also provided.

Description

Technical Field

[0001] The present invention relates to a method for purifying type II collagen from cartilage tissue. The present invention also relates to a type II collagen composition, and the use of the type II collagen composition as a dietary supplement or treatment for cartilage-related disorders. Background Art

[0002] Type II collagen has shown promising beneficial effects on human health, such as improving joint function and reducing joint inflammation (Harris et al., J. Diet Suppl. 2021, 1:1-16). It has also shown promise as a treatment for cartilage-related disorders such as osteoarthritis and rheumatoid arthritis (Scarpellini et al., J Orthop Traumatol. 2008; 9(2):81-7, and Crowley et al., Int J Med Sci. 2009; 9; 6(6):312-21). The beneficial effects of type II collagen are thought to be due to the binding of tolerogenic epitopes present in native type II collagen to Peyer's patches in the intestine. Through these interactions, type II collagen is thought to elicit an immune response that reduces joint inflammation and discomfort (Harris et al., J. DietSuppl. 2021, 1:1-16).

[0003] Examples of type II collagen products that are commercially available and reported to contain native type II collagen include Collavant (formerly known as Bioberica, S.A.U.; Barcelona, Spain) and (Lonza ConsumerHealth Inc, USA). Both of these products are reported to be derived from chicken breastbone cartilage.

[0004] Common methods for obtaining type II collagen from cartilage tissue include washing, drying, and grinding the cartilage tissue, followed by treatment with guanidine hydrochloride and / or enzymes (such as trypsin, pepsin, and / or elastase) to remove proteoglycans and glycosaminoglycans, and dissolving the type II collagen. Cartilage tissue is typically obtained from livestock or poultry. Treatment of cartilage tissue with guanidine hydrochloride and / or enzymes can result in the denaturation and / or hydrolysis of collagen in the cartilage. Therefore, type II collagen products prepared by such methods typically contain low levels of native type II collagen.

[0005] Therefore, there is still a need for methods suitable for obtaining type II collagen, particularly methods for obtaining native type II collagen suitable for use in drugs and dietary supplements. Another object of the present invention is to provide an improved or alternative method or composition, and / or at least to provide useful alternatives for the public. Summary of the Invention

[0006] The present invention provides a method for purifying type II collagen from cartilage tissue, the method comprising:

[0007] i) providing a cartilage tissue sample;

[0008] ii) contacting the sample with a salt solution under conditions that permit at least one component of the cartilage tissue to dissolve in the salt solution, wherein the at least one component of the cartilage tissue is selected from proteoglycans, glycosaminoglycans, and calcium salts (such as calcium phosphate); and

[0009] iii) removing the salt solution and the at least one component of the cartilage tissue from the sample;

[0010] wherein step iii) removes at least about 25% of the glycosaminoglycans from the sample; and wherein, after step iii), the sample comprises type II collagen in which at least about 50% (w / w) is undenatured type II collagen.

[0011] The sample provided after step iii) of the method of the present invention is referred to herein as a type II collagen composition. The inventors have found that the type II collagen composition obtained from the method of the present invention contains undenatured type II collagen in an amount and purity that makes it particularly suitable for use as a drug or dietary supplement.

[0012] The inventors have also found that the method of the present invention is particularly effective in purifying undenatured type II collagen from fish cartilage, particularly from fish parts that are typically discarded during the commercial processing of fish for consumption. This feature of the present invention enables the production of low-cost undenatured type II collagen products with a limited environmental impact.

[0013] For the avoidance of doubt, the term "undenatured type II collagen" as used herein refers to type II collagen that retains its native triple-helix structure. This is in contrast to denatured type II collagen, which has lost its native triple-helix structure. An example of denatured type II collagen is type II collagen that has been hydrolyzed into peptide fragments (commonly referred to as collagen hydrolysates). Undenatured type II collagen typically has a molecular weight of about 300 kDa, while the molecular weight of collagen hydrolysates is in the range of about 2 kDa to about 9 kDa. Typically, undenatured type II collagen is insoluble in aqueous solutions, while denatured type II collagen is soluble in aqueous solutions. Unless otherwise specified, the term "type II collagen" as used herein should be understood to refer to undenatured type II collagen, denatured type II collagen, or a mixture thereof.

[0014] The present invention also provides a type II collagen composition obtained by the method of the present invention. The type II collagen composition of the present invention can be used as a drug, for example, a drug for treating or preventing cartilage-related diseases. The type II collagen composition of the present invention can also be used as a dietary supplement, for example, a dietary supplement for promoting joint health.

[0015] The present invention also provides a method for treating cartilage-related diseases, and a type II collagen composition for preparing a drug for treating cartilage-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shows the amount of glycosaminoglycan (GAG) extracted from the cartilage tissue of hoki nasal cartilage or chicken breast bone under various conditions. The conditions shown on the x-axis including "CS" are used with chicken breast bone cartilage. The conditions shown on the x-axis that do not include "CS" or include "HN" are used with hoki nasal cartilage. In Figure 1 where: AcAc / AcOH = acetic acid; TBP = tributyl phosphate; chaps = (3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate); and GuHCl = guanidine hydrochloride.

[0017] Figure 2 Shows the amounts of GAG and hydroxyproline extracted from the cartilage tissue of hoki nasal cartilage or chicken breast bone under various conditions. In Figure 2 where: HN = hoki nasal cartilage; CS = chicken breast bone cartilage; AcOH = acetic acid; and GuHCl = guanidine hydrochloride.

[0018] Figure 3 Shows the amount of GAG extracted from the cartilage tissue of dogfish (DF), hoki nasal cartilage (HN), and chicken breast bone (CS) with a sodium sulfate solution.

[0019] Figure 4 Shows the total hydroxyproline in the cartilage tissue samples obtained from dogfish (DF), hoki nasal cartilage (HN), and chicken breast bone (CS). The treated cartilage tissue samples (DF, HN, and CS treated) were treated with a sodium sulfate solution before analysis.

[0020] Figure 5 Shows the percentage of collagen in the cartilage tissue sample, which is undenatured collagen. The cartilage tissue analyzed in this experiment was obtained from dogfish (DF), hoki nasal cartilage (HN), and chicken breast bone (CS). The treated cartilage tissue samples (DF, HN, and CS treated) were treated with a sodium sulfate solution before analysis.

[0021] Figure 6Shows the extraction of GAG from (A) chicken breast bone and (B) hoki nasal cartilage tissue using 0.5 M sulfate and chloride salt solutions at pH 4, pH 7, and pH 9. * Indicates precipitation of the material after addition of the salt solution to the treated cartilage tissue sample.

[0022] Figure 7 Shows the extraction of GAG from (A) chicken breast bone and (B) hoki nasal cartilage tissue using 1.0 M sulfate and chloride salt solutions at pH 4, pH 7, and pH 9. * Indicates precipitation of the material after addition of the salt solution to the treated cartilage tissue sample.

[0023] Figure 8 Shows the percentage of undenatured collagen based on the percentage of hydroxyproline in the pellets formed after treating pellet A samples with trypsin. The proportion of hydroxyproline in the pellets is an indirect measure of the content of undenatured collagen in the cartilage tissue samples after treatment with the extraction reagent. Pellet A samples were generated by treating hoki nasal cartilage tissue samples with 0.5 M sodium sulfate solution at a pH of about 4, 7, or 9, or 1 M magnesium sulfate solution at a pH of about 4, 7, or 9. Figure 6 and 7 The amounts of GAG extracted from the cartilage tissue samples by each solution as shown in Figure 8 overlap and are shown as "x".

[0024] Figure 9 Shows scanning electron micrographs of collagen produced using 1 M MgSO4 or pepsin treatment. (A) MgSO4 treatment, magnified 534 times. (B) Pepsin treatment, magnified 534 times. (C) MgSO4 treatment, magnified 133,000 times. (D) Pepsin treatment, magnified 133,000 times. DETAILED DESCRIPTION

[0025] As described herein, the inventors have developed a method for purifying type II collagen, particularly undenatured type II collagen, from cartilage tissue. The method of the present invention is carried out in vitro. As described above, the method comprises the following steps:

[0026] i) providing a cartilage tissue sample;

[0027] ii) contacting the sample with a salt solution under conditions that allow at least one component of the cartilage tissue to dissolve in the salt solution, wherein the at least one component is selected from proteoglycans, glycosaminoglycans, and calcium salts (such as calcium phosphate); and

[0028] iii) removing the salt solution and the at least one dissolved component from the sample;

[0029] wherein step iii) removes at least about 25% of the glycosaminoglycan from the sample; and wherein, after step iii), the sample comprises type II collagen in which at least about 50% (w / w) is undenatured type II collagen.

[0030] Also provided herein is a method for purifying type II collagen from cartilage tissue, the method comprising:

[0031] i) providing a sample of cartilage tissue from a teleost fish of the family Merlucciidae (e.g., blue hake, also known as Macruronus novaezelandiae or hoki), a chondrichthyan fish (e.g., dogfish, blue shark, and mako shark), or a bird (e.g., poultry such as chicken, turkey, duck, goose, guinea fowl, and pigeon);

[0032] ii) contacting the sample with a chloride salt solution or a sulfate solution under conditions that allow at least one component of the cartilage tissue to dissolve in the salt solution, wherein the at least one component of the cartilage tissue is selected from proteoglycans, glycosaminoglycans, and calcium salts (e.g., calcium phosphate); and

[0033] iii) removing the salt solution and the at least one component of the cartilage tissue from the sample.

[0034] For the avoidance of doubt, as used herein the term "comprising" means "consisting at least in part of". When interpreting statements in this specification that include this term, the features starting with this term in each statement or claim need to be present, and other features may also be present. The related terms "comprises" and "comprised" should be interpreted similarly. As used herein, the term "and / or" means "and" or "or", or both.

[0035] Furthermore, for the avoidance of doubt, the term "purifying type II collagen from cartilage tissue" as used herein can be understood as a process of removing proteoglycans, glycosaminoglycans, and / or calcium salts (e.g., calcium phosphate) from a cartilage tissue sample. In step iii) of the method of the present invention, proteoglycans, glycosaminoglycans, and / or calcium salts (e.g., calcium phosphate) are removed from the cartilage tissue sample.

[0036] Generally, the method of the present invention removes at least about 0.1%, 0.2%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.8%, 99.9% or 100% of proteoglycans, glycosaminoglycans and / or calcium salts (such as calcium phosphate) from a cartilage tissue sample.For example, the method of the present invention can remove about 0.1% to about 50%, about 0.2% to about 50%, about 0.5% to about 50%, about 1% to about 50%, about 5% to about 50%, about 10% to about 50%, about 15% to about 50%, about 20% to about 50%, about 25% to about 50%, about 30% to about 50%, about 35% to about 50%, about 40% to about 50%, about 45% to about 50%, about 0.1% to about 60%, about 0.2% to about 60%, about 0.5% to about 60%, about 1% to about 60%, about 5% to about 60%, about 10% to about 60%, about 15% to about 60%, about 20% to about 60%, about 25% to about 60%, about 30% to about 60%, about 35% to about 60%, about 40% to about 60%, about 45% to about 60%, about 0.1% to about 70%, about 0.2% to about 70%, about 0.5% to about 70%, about 1% to about 70%, about 5% to about 70%, about 10% to about 70%, about 15% to about 70%, about 20% to about 70%, about 25% to about 70%, about 30% to about 70%, about 35% to about 70%, about 40% to about 70%, about 45% to about 70%, about 0.1% to about 80%, about 0.2% to about 80%, about 0.5% to about 80%, about 1% to about 80%, about 5% to about 80%, about 10% to about 80%, about 15% to about 80%, about 20% to about 80%, about 25% to about 80%, about 30% to about 80%, about 35% to about 80%, about 40% to about 80%, about 45% to about 80%, about 0.1% to about 90%, about 0.2% to about 90%, about 0.5% to about 90%, about 1% to about 90%, about 5% to about 90%, about 10% to about 90%, about 15% to about 90%, about 20% to about 90%, about 25% to about 90%, about 30% to about 90%, about 35% to about 90%, about 40% to about 90%, about 45% to about 90%, about 0.1% to about 99%, about 0.2% to about 99%, about 0.5% to about 99%, about 1% to about 99%, about 5% to about 99%, about 10% to about 99%, about 15% to about 99%, about 20% to about 99%, about 25% to about 99%, about 30% to about 99%, about 35% to about 99%, about 40% to about 99%, and about 45% to about 99%, about 0.1% to about 100%, about 0.2% to about 100%, about 0.5% to about 100%, about 1% to about 100%, about 5% to about 100%, about 10% to about 100%, about 15% to about 100%, about 20% to about 100%, about 25% to about 100%, about 30% to about 100%, about 35% to about 100%, about 40% to about 100%, and about 45% to about 100% of proteoglycans, glycosaminoglycans, and / or calcium salts (such as calcium phosphate) from a cartilage tissue sample.

[0037] Typically, the calcium salt removed from cartilage tissue is calcium phosphate.

[0038] Preferably, the method of the present invention removes from about 10% to about 100% of proteoglycans, glycosaminoglycans, and / or calcium salts (such as calcium phosphate) from a cartilage tissue sample. For example, a cartilage tissue sample containing about 100 mg of proteoglycans before performing the method of the present invention may contain from about 0 mg to about 90 mg of proteoglycans after step iii) of the method. Or, for example, a cartilage tissue sample containing about 100 mg of glycosaminoglycans before performing the method of the present invention may contain from about 0 mg to about 90 mg of glycosaminoglycans after step iii) of the method. Or, for example, a cartilage tissue sample containing about 100 mg of calcium salt (such as calcium phosphate) before performing the method of the present invention may contain from about 0 mg to about 90 mg of calcium salt (such as calcium phosphate) after step iii) of the method.

[0039] Methods for determining the content of proteoglycans, glycosaminoglycans, and / or calcium salts (such as calcium phosphate) in a cartilage tissue sample are known in the art. For example, assays based on dimethylmethylene blue (DMMB), mass spectrometry, sodium dodecyl sulfate-polyacrylamide (SDS) gel electrophoresis, spectrophotometry, and / or colorimetric protein assays (such as Coomassie blue-based assays) can be used to detect and quantify proteoglycans and / or glycosaminoglycans. Assays based on ammonium molybdate and / or arsenazo III (CAS No. 1668-00-4), and / or inductively coupled plasma mass spectrometry (ICP-MS) can be used to detect and quantify calcium salts, such as calcium phosphate. Other techniques, such as ELISA, liquid chromatography-mass spectrometry, and liquid chromatography-mass spectrometry (LC-MS) can also be used. Commercial assay kits for detecting and quantifying proteoglycans, glycosaminoglycans, and / or calcium salts (such as calcium phosphate) are also widely available. Other suitable methods are also described in the examples disclosed herein.

[0040] The present inventors have found that by removing proteoglycans, glycosaminoglycans and / or calcium salts (such as calcium phosphate) from a cartilage tissue sample, the percentage (w / w) of type II collagen in the sample increases. Typically, after step iii) of the method of the present invention, the percentage (w / w) of type II collagen in the cartilage tissue sample is at least about 0.1%, 0.2%, 0.5%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200% greater than the percentage (w / w) of type II collagen in the cartilage tissue sample before step iii) of the method. Preferably, after step iii) of the method of the present invention, the percentage (w / w) of type II collagen in the cartilage tissue sample is greater than the percentage (w / w) of type II collagen in the cartilage tissue sample before step iii) of the method by at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, more preferably greater than the percentage (w / w) of type II collagen in the cartilage tissue sample before step iii) of the method by at least about 40%, 50%, 60%, 70%, 80%, 90% or 100%. For example, after step iii) of the method of the present invention, the percentage (w / w) of type II collagen in the cartilage tissue sample can be greater than the percentage (w / w) of type II collagen in the cartilage tissue sample before step iii) of the method by about 40% to about 100%, about 45% to about 100%, about 45% to about 90%, about 45% to about 85%, or about 45% to about 80%.

[0041] Typically, after step iii) of the method of the present invention, the percentage (w / w) of type II collagen in the cartilage tissue sample is greater than the percentage (w / w) of type II collagen in the cartilage tissue sample before step iii) of the method by about 40% to about 100%. For example, a cartilage tissue sample that includes about 10% (w / w) type II collagen before performing the method of the present invention can include about 14% (w / w) to about 20% (w / w) type II collagen after step iii) of the method of the present invention. Or, for example, a cartilage tissue sample that includes about 10 mg of type II collagen per 100 mg of cartilage tissue sample before performing the method of the present invention can contain about 14 mg to about 20 mg of type II collagen per 100 mg of sample after step iii) of the method.

[0042] For the avoidance of doubt, unless otherwise specified, the term "type II collagen" as used herein shall be understood to refer to native type II collagen, denatured type II collagen, or a mixture thereof. Thus, the method of the present invention can increase the percentage (w / w) of native type II collagen, denatured type II collagen, or a mixture thereof in a cartilage tissue sample by the percentage disclosed above. Preferably, the method of the present invention increases the percentage (w / w) of native type II collagen in a cartilage tissue sample by the percentage disclosed above. For example, after step iii) of the method of the present invention, the percentage (w / w) of native type II collagen in the cartilage tissue sample can be at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the percentage (w / w) of native type II collagen in the cartilage tissue sample before step iii) of the method.

[0043] For the avoidance of doubt, the term "w / w" as used herein refers to the weight of a particular substance in a mixture by weight. For example, a sample containing 10% (w / w) native type II collagen should be understood to contain 10 mg of native type II collagen per 100 mg of the sample. Unless otherwise specified, any reference herein to the mass of type II cartilage, native type II collagen, or a cartilage tissue sample shall be understood to refer to the dry mass of type II cartilage, native type II collagen, or a cartilage tissue sample.

[0044] Methods for determining the content of type II collagen in a sample (i.e., the sum of the content of native and denatured type II collagen) and the content of native type II collagen are known in the art. For example, Harris et al. (J. Diet Suppl. 2021, 1:1-16, which is incorporated herein by reference) described determining the content of type II collagen in a sample by hydrolyzing the proteins in the sample with hydrochloric acid and measuring the hydroxyproline content. Harris et al. also described using an enzyme-linked immunosorbent assay (ELISA)-based method to determine the content of native type II collagen in a sample, which uses monoclonal antibodies specific for native type II collagen. Alternative methods for determining the content of native type II collagen in a sample include trypsin digestion sensitivity assays, such as those described by Veres et al. and Bank et al. (Matrix Biol, 2014, 33:54-59 and Matrix Biol, 1997, 16(5):233-243, which are incorporated herein by reference). The content of type II collagen and native type II collagen in a sample can also be determined by measuring the average molecular weight of the collagen in the composition, for example, by using methods known in the art, such as liquid chromatography, mass spectrometry, and liquid chromatography-mass spectrometry (LC-MS). Commercial assay kits suitable for determining the content of type II collagen and native type II collagen in a sample are widely available. Other suitable methods are also described in the examples disclosed herein.

[0045] Cartilage tissue

[0046] Cartilage tissue has a matrix structure containing collagen, chondrocytes, proteoglycans, and glycosaminoglycans (GAGs), such as chondroitin sulfate, heparan sulfate, heparin, dermatan sulfate, keratan sulfate, and hyaluronic acid. Generally, cartilage tissue contains about 60-80% water, 10-20% collagen, 20-30% proteoglycans and glycosaminoglycans, and 1-5% chondrocytes. Cartilage tissue of cartilaginous fish (i.e., Chondrichthyes) such as sharks typically also includes calcium salts (e.g., calcium phosphate). Cartilaginous fish cartilage can contain about 15-25% calcium salts.

[0047] Type II collagen is typically present in hyaline cartilage tissue, where it serves as a matrix molecule providing structural integrity. Hyaline cartilage tissue is found on joint surfaces and as a structural component of the rib neck and bronchi. In cartilaginous fish, cartilage can constitute a significant portion of the skeleton. Preferably, the cartilage tissue sample used in the method of the present invention is a hyaline cartilage tissue sample.

[0048] Typically, the cartilaginous tissue sample used in the method according to the present invention is obtained from a fish, a bird, a mammal, a mollusk or an echinoderm. For example, the cartilaginous tissue sample can be a bony fish (i.e., Osteichthyes), such as cod, haddock, pollock, hake, tuna, salmon (e.g., Atlantic salmon (Salmo salar)), sturgeon or trout, or a cartilaginous fish, such as a shark, a ray, a skate or a sawfish. Alternatively, for example, the cartilaginous tissue sample can be from a bird, such as a poultry (e.g., chicken, turkey, duck, goose, guinea fowl and pigeon). Alternatively, for example, the cartilaginous tissue sample can be from a mammal, such as a domestic animal (e.g., domestic cattle, domestic pig, horse, donkey, zebu, Bali cattle, yak, buffalo, gayal, sheep, reindeer, camel, llama, alpaca, rabbit and guinea pig). Alternatively, for example, the cartilaginous tissue sample can be from a mollusk, such as a squid, a cuttlefish or an octopus. Alternatively, for example, the cartilaginous tissue sample can be from an echinoderm, such as a sea cucumber, a starfish or a sea urchin. Preferably, the cartilaginous tissue sample is from a fish, a bird or a mammal.

[0049] When the cartilaginous tissue sample is from a bony fish, the fish can be a fish classified in the order Gadiformes, such as cod, haddock, pollock or hake (e.g., blue hake, also known as Macruronus novaezelandiae or Hoki, or European hake (Merluccius merluccius), also known as European cod). Alternatively, for example, the bony fish can be a fish classified in the order Salmoniformes, such as trout or salmon (e.g., Atlantic salmon). Preferably, the bony fish is blue hake.

[0050] When the cartilaginous tissue sample is from a cartilaginous fish, the fish can be a shark. For example, the cartilaginous fish can be a spiny dogfish (Squalus acanthias), a blue shark (Prionace glauca) or a mako shark (e.g., shortfin mako (Isurus oxyrinchus) and longfin mako).

[0051] When the cartilaginous tissue sample is from a bird, the bird can be a poultry. For example, the bird can be a chicken or a turkey. Preferably, the bird is a chicken.

[0052] When the cartilaginous tissue sample is from a mammal, the mammal can be a domestic animal. For example, the mammal can be domestic cattle.

[0053] The inventors have found that, according to the present invention, teleost fish and birds are particularly suitable sources of cartilage tissue for use. Thus, in certain preferred embodiments, the cartilage tissue sample is from a teleost fish or a bird, such as blue whiting or chicken.

[0054] Cartilage tissue samples can be obtained from animals that are discarded as by-catch during the harvesting or collection of animals or plants, or cartilage tissue samples can be obtained from animal body parts that are typically discarded when the animal is processed for consumption. The discarded by-catch and animal parts provide a low-cost source of cartilage suitable for use in the present invention. Examples of animal parts that can be used as a source of cartilage tissue include fish heads, spines, fins, and breastbones (e.g., chicken or turkey breastbones) or parts thereof. It has been found that nasal cartilage from teleost fish, cartilage from bird breastbones, and cartilage from shark spines are particularly suitable for use according to the present invention.

[0055] In one exemplary embodiment, the cartilage tissue sample is from cod, haddock, pollock, salmon (e.g., Atlantic salmon (Salmo salar)), European hake (also known as Merluccius merluccius), or blue whiting (also known as Macruronus novaezelandiae).

[0056] In another exemplary embodiment, the cartilage tissue sample is nasal cartilage tissue from blue whiting. In another exemplary embodiment, the cartilage tissue sample is from the spine or a part thereof of a shark (e.g., dogfish, blue shark, or mako shark). In another exemplary embodiment, the cartilage tissue sample is from a chicken breastbone.

[0057] Salt solution

[0058] The salt solution used in the method of the present invention can have a pH of from about 3 to about 10, such as from about 3 to about 9. It has been found that a salt solution with a pH of from about 3 to about 10 can effectively dissolve proteoglycans, glycosaminoglycans, and calcium salts (such as calcium phosphate) in a cartilage tissue sample while maintaining or increasing the percentage (w / w) of undenatured type II collagen in the sample. Typically, the pH of the salt solution is from about 3 to about 10, from about 3.5 to about 10, 4 to about 10, from about 4.5 to 10, from about 5 to about 10, from about 5.5 to about 10, from about 6 to about 10, from about 6.5 to about 10, from about 7 to about 10, from about 7.5 to about 10, from about 8 to about 10, from about 8.5 to about 10, from about 9 to about 10, from about 9.5 to about 10, from about 3 to about 9.5, from about 3.5 to about 9.5, 4 to about 9.5, from about 4.5 to 9.5, from about 5 to about 9.5, from about 5.5 to about 9.5, from about 6 to about 9.5, from about 6.5 to about 9.5, from about 7 to about 9.5, from about 7.5 to about 9.5, from about 8 to about 9.5, from about 8.5 to about 9.5, from about 9 to about 9.5, from about 3 to about 9, from about 3.5 to about 9, 4 to about 9, from about 4.5 to 9, from about 5 to about 9, from about 5.5 to about 9, from about 6 to about 9, from about 6.5 to about 9, from about 7 to about 9, from about 7.5 to about 9, from about 8 to about 9, from about 3 to about 8.5, from about 3.5 to about 8.5, 4 to about 8.5, from about 4.5 to 8.5, from about 5 to about 8.5, from about 5.5 to about 8.5, from about 6 to about 8.5, from about 6.5 to about 8.5, from about 7 to about 8.5, from about 7.5 to about 8.5, from about 8 to about 8.5, 3 to about 8, from about 3.5 to about 8, about 4 to about 8, from about 4.5 to 8, from about 5 to about 8, from about 5.5 to about 8, from about 6 to about 8, from about 6.5 to about 8, from about 7 to about 8, from about 7.5 to about 8, from about 3 to about 7.5, from about 3.5 to about 7.5, about 4 to about 7.5, from about 4.5 to 7.5, from about 5 to about 7.5, from about 5.5 to about 7.5, from about 6 to about 7.5, from about 6.5 to about 7.5, from about 7 to about 7.5, from about 3 to about 7, from about 3.5 to about 7, about 4 to about 7, from about 4.5 to 7, from about 5 to about 7, from about 5.5 to about 7, from about 6 to about 7, from about 6.5 to about 7, from about 3 to about 6.5, from about 3.5 to about 6.5, about 4 to about 6.5, from about 4.5 to 6.5, from about 5 to about 6.5, from about 5.5 to about 6.5, from about 6 to about 6.5, from about 3 to about 6, from about 3.5 to about 6, about 4 to about 6, from about 4.5 to 6, from about 5 to about 6, from about 5.5 to about 6, from about 3 to about 5.5, from about 3.5 to about 5.5, about 4 to about 5.5, from about 4.5 to 5.5, about 4 to about 5, from about 4.5 to about 5, from about 3 to about 4.5, or from about 3.5 to about 4.5.

[0059] In certain embodiments, the salt solution has a pH of from about 3.5 to about 7, or from about 4 to about 7 (e.g., a pH of about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.7, about 6.8, about 6.9, or about 7). For example, the salt solution can have a pH of from about 3.5 to about 6, from about 3.5 to about 5, from about 3.5 to about 4.5, from about 4 to about 7, from about 4 to about 6, or from about 4 to about 5. In certain preferred embodiments, the salt solution has a pH of from about 3.5 to about 4.5 (e.g., a pH of about 4).

[0060] In some embodiments, the salt solution (e.g., a sodium sulfate salt solution) has a pH of from about 3 to about 5 or from about 8 to about 10, preferably from about 3.5 to about 4.5 or from about 8.5 to about 9.5, more preferably about 4 or about 9. In some embodiments, the salt solution (e.g., a magnesium sulfate salt solution) has a pH of from about 4 to about 6 or from about 8 to about 10, preferably from about 4.5 to about 5.5 or from about 9 to about 9.75, more preferably about 5 or about 9.

[0061] The salt solution can be an inorganic salt solution or an organic salt solution. For example, the salt solution can be a salt solution of a chloride, sulfate, or acetate. Suitable salts include, for example, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, ammonium chloride, sodium sulfate, magnesium sulfate, ammonium sulfate, and sodium acetate. Preferably, the salt solution is a chloride salt solution or a sulfate solution. For example, the salt solution can be a sodium chloride, potassium chloride, magnesium chloride, calcium chloride, ammonium chloride, sodium sulfate, magnesium sulfate, ammonium sulfate salt solution. The inventors have found that sodium sulfate solution and sodium chloride solution are particularly effective in solubilizing proteoglycans, glycosaminoglycans, and / or calcium salts (e.g., calcium phosphate) in cartilage tissue. Thus, in certain embodiments, the salt solution is a sodium sulfate solution or a sodium chloride solution. Preferably, the salt solution is a sodium sulfate solution.

[0062] In some embodiments, the salt is a metal salt (e.g., a sodium salt or a magnesium salt, such as sodium chloride, sodium sulfate, magnesium chloride, or magnesium sulfate) or an ammonium salt (e.g., ammonium chloride or ammonium sulfate). In some embodiments, the salt is a metal salt (e.g., a sodium salt or a magnesium salt, such as sodium chloride, sodium sulfate, magnesium chloride, or magnesium sulfate).

[0063] In some embodiments, the salt is a chaotropic salt. In some embodiments, the salt solution does not contain a chaotropic salt. A chaotropic salt is a salt that disrupts hydrogen bonds within and / or between proteins and thus can denature proteins in solution. A commonly used chaotropic salt is guanidine chloride (also known as guanidine hydrochloride). A non-chaotropic salt is a salt that does not have chaotropic properties. A kosmotropic salt (also known as a chaotropic salt) stabilizes hydrogen bonds, resulting in increased protein stability.

[0064] Typically, the salt concentration of the salt solution is from about 0.1 M to about 2 M. For example, from about 0.2 M to about 2 M, from about 0.3 M to about 2 M, from about 0.4 M to about 2 M, from about 0.4 M to about 1.75 M, 0.5 M to about 1.5 M, from about 0.75 M to about 1.5 M, 0.75 M to about 1.25 M, from about 0.8 M to about 1.2 M, or from about 0.9 M to about 1.1 M.

[0065] It has been found that when the salt is present at a concentration of from about 0.4 M to about 2 M, chloride salt solutions and sulfate salt solutions are particularly effective for dissolving glycosaminoglycans and calcium salts (such as calcium phosphate) in cartilage tissue. Thus, in certain embodiments, the salt solution is a chloride salt solution or a sulfate salt solution having a chloride or sulfate concentration of from about 0.4 M to about 2 M. For example, the chloride or sulfate can be present at a concentration of from about 0.4 M to about 1.75 M, 0.5 M to about 1.5 M, from about 0.75 M to about 1.5 M, 0.75 M to about 1.25 M, from about 0.8 M to about 1.2 M, or from about 0.9 M to about 1.1 M. Alternatively, for example, the chloride or sulfate can be present at a concentration of about 0.4 M, about 0.5 M, about 0.75 M, about 1 M, about 1.5 M, or about 2 M. It has been found that a salt solution having a chloride or sulfate concentration of about 1 M is particularly effective. Thus, in certain preferred embodiments, the chloride or sulfate is present at a concentration of about 1 M.

[0066] In some particularly contemplated embodiments, sodium sulfate can be present at a concentration of at least about 0.2 M, such as at least about 0.3 M, at least about 0.4 M, at least about 0.5 M, at least about 0.6 M, at least about 0.7 M, at least about 0.8 M, at least about 0.9 M, at least about 1 M, at least about 1.1 M, or at least about 1.2 M, and useful ranges can be selected from any of these values (e.g., about 0.2 M to about 1.2 M, about 0.2 M to about 1.1 M, about 0.2 M to about 1 M, about 0.2 M to about 0.9 M, about 0.2 M to about 0.8 M, about 0.2 M to about 0.7 M, about 0.2 M to about 0.6 M, about 0.3 M to about 1.2 M, about 0.3 M to about 1.1 M, about 0.3 M to about 1 M, about 0.3 M to about 0.9 M, about 0.3 M to about 0.8 M, about 0.3 M to about 0.7 M, about 0.3 M to about 0.6 M, about 0.4 M to about 1.2 M, about 0.4 M to about 1.1 M, about 0.4 M to about 1 M, about 0.4 M to about 0.9 M, about 0.4 M to about 0.8 M, about 0.4 M to about 0.7 M, about 0.4 M to about 0.6 M), preferably about 0.4 M.

[0067] In some particularly contemplated embodiments, magnesium sulfate can be present at a concentration of at least about 0.2 M, such as at least about 0.3 M, at least about 0.4 M, at least about 0.5 M, at least about 0.6 M, at least about 0.7 M, at least about 0.8 M, at least about 0.9 M, at least about 1 M, at least about 1.1 M, at least about 1.2 M, at least about 1.3 M, or at least about 1.4 M, and useful ranges can be selected from any of these values (e.g., about 0.2 M to about 1.4 M, about 0.2 M to about 1.3 M, about 0.2 M to about 1.2 M, about 0.2 M to about 1.1 M, about 0.2 M to about 1 M, about 0.3 M to about 1.4 M, about 0.3 M to about 1.3 M, about 0.3 M to about 1.2 M, about 0.3 M to about 1.1 M, about 0.3 M to about 1 M, about 0.4 M to about 1.4 M, about 0.4 M to about 1.3 M, about 0.4 M to about 1.2 M, about 0.4 M to about 1.1 M, about 0.4 M to about 1 M, about 0.5 M to about 1.4 M, about 0.5 M to about 1.3 M, about 0.5 M to about 1.2 M, about 0.5 M to about 1.1 M, about 0.5 M to about 1 M, about 0.6 M to about 1.4 M, about 0.6 M to about 1.3 M, about 0.6 M to about 1.2 M, about 0.6 M to about 1.1 M, about 0.6 M to about 1 M, about 0.7 M to about 1.4 M, about 0.7 M to about 1.3 M, about 0.7 M to about 1.2 M, about 0.7 M to about 1.1 M, about 0.7 M to about 1 M, about 0.8 M to about 1.4 M, about 0.8 M to about 1.3 M, about 0.8 M to about 1.2 M, about 0.8 M to about 1.1 M, about 0.8 M to about 1 M), preferably about 0.8 M.

[0068] In some particularly contemplated embodiments, sodium sulfate is present at a concentration of about 0.2 M to about 0.6 M (preferably about 0.4 M) and a pH of about 8.5 to about 9.5 (preferably about 9). In some particularly contemplated embodiments, magnesium sulfate is present at a concentration of about 0.6 M to about 1.0 M (preferably about 0.8 M) and a pH of about 4.5 to about 5.5 or about 8.5 to about 9.75 (preferably about 5 or about 9).

[0069] Preferably, the salt solution is an aqueous salt solution. That is, the solvent of the salt solution comprises water. The solvent of the salt solution may comprise water and a water-miscible organic solvent, such as ethanol, acetone, acetonitrile or methanol. Typically, the salt solution does not contain or substantially does not contain an organic solvent, such as a water-miscible organic solvent.

[0070] Details of the method steps

[0071] The method of the present invention may include the step of removing foreign substances such as muscle fibers and ligaments from the cartilage tissue sample. The step of removing foreign substances may include washing the cartilage tissue with an acid, such as acetic acid, citric acid, lactic acid or hydrochloric acid. Additionally or alternatively, the step of removing foreign substances may include washing the cartilage tissue with a base, such as a hydroxide solution. Additionally, or alternatively, the step of removing foreign substances may include washing the cartilage tissue with water, for example, the cartilage tissue may be rinsed or soaked with water, or the cartilage tissue may be subjected to a high-pressure water spray or jet. Additionally, or alternatively, foreign substances may be removed by sonication of the cartilage tissue sample.

[0072] Generally, the step of removing foreign substances from the cartilage tissue sample is carried out at room temperature or at a temperature below room temperature, such as at 4 °C to 10 °C.

[0073] The step of removing foreign substances from the cartilage tissue sample may be repeated 1, 2, 3 or more times. Each step may use the same method to remove foreign substances from the cartilage tissue sample, or use different methods to remove foreign substances from the cartilage tissue sample. For example, the cartilage tissue sample may be washed with an acid twice, or washed with an acid once and then washed with a base once.

[0074] The method of the present invention may further include the step of reducing the cartilage tissue sample into particles. For example, the cartilage tissue may be cut into small pieces or crushed into particles. Typically, the cartilage tissue is dehydrated before being reduced into particles, such as by lyophilization. The cartilage tissue may be reduced to particles having a diameter of about 1 mm to about 10 mm, such as about 1 mm to about 2 mm. Preferably, the step of reducing the cartilage tissue into particles is carried out before the step of contacting the cartilage tissue with the salt solution (i.e., step ii) of the method.

[0075] The method of the present invention may further include a step of sterilizing the cartilage tissue. A suitable sterilization method is a method that removes bacterial and viral contaminants from the cartilage tissue without destroying the native structure of type II collagen in the cartilage tissue sample. Examples of suitable sterilization methods include washing the cartilage tissue with an oxidizing agent such as hydrogen peroxide or sodium hypochlorite, or exposing the cartilage tissue to radiation, such as gamma radiation sterilization, ultraviolet (UV) radiation sterilization, or electron beam radiation sterilization.

[0076] As previously mentioned, the cartilage tissue of cartilaginous fish (i.e., Chondrichthyes) such as sharks typically also includes calcium salts (e.g., calcium phosphate). Cartilaginous fish cartilage may contain about 15-25% calcium salts. In some embodiments, for example when using the cartilage tissue of cartilaginous fish, the method includes a demineralization step, i.e., a step of removing or reducing the amount of calcium salts present in the cartilage tissue sample. Preferably, the demineralization step is carried out before step ii).

[0077] In some embodiments, before step ii), the cartilage tissue sample is subjected to demineralization treatment. In some embodiments, the demineralization treatment includes contacting the cartilage tissue sample with a solution comprising EDTA, hydrochloric acid, phosphoric acid, acetic acid, lactic acid, or any combination of any two or more thereof. In some specifically contemplated embodiments, the solution comprises EDTA, acetic acid, and / or lactic acid; preferably acetic acid and / or lactic acid. In some embodiments, EDTA, hydrochloric acid, phosphoric acid, acetic acid, and / or lactic acid are present at a concentration of at least about 0.1M, at least about 0.2M, at least about 0.3M, at least about 0.4M, at least about 0.5M, at least about 0.6M, at least about 0.7M, at least about 0.8M, at least about 0.9M, at least about 1M, at least about 1M, at least about 1.5M, at least about 2M, at least about 2.5M, at least about 3M, at least about 3.5M, at least about 4M, at least about 4.5M, or at least about 5M, and useful ranges can be selected from any of these values (about 0.1M to about 5M, about 0.1M to about 4M, about 0.1M to about 3M, about 0.1M to about 2M, about 0.1M to about 1M, about 0.2M to about 5M, about 0.2M to about 4M, about 0.2M to about 3M, about 0.2M to about 2M, about 0.2M to about 1M, about 0.3M to about 5M, about 0.3M to about 4M, about 0.3M to about 3M, about 0.3M to about 2M, about 0.3M to about 1M, about 0.4M to about 5M, about 0.4M to about 4M, about 0.4M to about 3M, about 0.4M to about 2M, about 0.4M to about 1M). Preferably, the solution includes about 0.3M to about 1.0M, more preferably about 0.5M of acetic acid and / or lactic acid.

[0078] A sterilization step may be employed before the step of reducing the cartilage tissue sample to particles. Additionally or alternatively, the cartilage tissue sample may be sterilized after being reduced to particles. For example, the tissue may be sterilized after being reduced to particles but before contacting with the salt solution in step ii) of the method, or after being reduced to particles and after contacting with the salt solution in step ii) of the method. The method of the present invention may include one or more disinfection steps. For example, the method may include a disinfection step using an oxidizing agent and a disinfection step using radiation.

[0079] For the avoidance of doubt, the term "under conditions that permit at least one component of the cartilage tissue to dissolve in the salt solution" in step ii) of the method of the present invention should be understood as conditions that promote the dissolution of proteoglycans, glycosaminoglycans, and / or calcium salts (such as calcium phosphate) in the salt solution. Such conditions include, for example, the temperature of the salt solution and the cartilage tissue sample, the contact time between the salt solution and the cartilage tissue, the volume of the salt solution, and the mass of the cartilage tissue sample. For example, in certain exemplary embodiments, the cartilage tissue sample is exposed to the salt solution at about 10°C, about 15°C, or about 20°C for about 6 hours, about 12 hours, about 18 hours, about 24 hours, or about 48 hours. For example, the cartilage tissue sample may be exposed to the salt solution at about 20°C for about 24 hours. Those of ordinary skill in the art can readily determine the conditions suitable for dissolving proteoglycans, glycosaminoglycans, and / or calcium salts (such as calcium phosphate) in the cartilage tissue sample.

[0080] Typically, step iii) of the method includes centrifuging, filtering, and / or gravity separating the cartilage tissue and salt solution mixture. This step separates the salt solution and any dissolved components of the cartilage tissue from the insoluble components of the cartilage tissue sample (such as undenatured type II collagen). The insoluble components of the sample may be further processed (such as air drying, freeze drying, and / or formulated into a pharmaceutical or dietary composition) to provide a type II collagen composition suitable for administration to animals such as humans.

[0081] Each of steps ii) and iii) may be performed one or more times. For example, step ii) may be performed using the salt solution disclosed herein and then repeated with a different salt solution disclosed herein. In certain embodiments, step ii) may be performed with a salt solution having a pH of about 4 to about 7 and then repeated with a salt solution having a pH of about 7 to about 9. Salt solutions suitable for step ii) and for repeating step ii) are disclosed herein. Step iii) may be performed after step ii) has been repeated, or directly after each performance of step ii).

[0082] Type II collagen composition

[0083] The sample provided after step iii) of the method of the present invention is referred to herein as a type II collagen composition. The type II collagen composition obtained by the method of the present invention comprises undenatured type II collagen. Undenatured type II collagen has been found to have a variety of health benefits, such as improving joint function and reducing joint inflammation. It also shows promise as a treatment for cartilage-related conditions such as osteoarthritis and rheumatoid arthritis.

[0084] The method of the present invention has been found to be particularly effective in providing a type II collagen composition comprising a high percentage of undenatured type II collagen (e.g., at least 5% (w / w) undenatured type II collagen). Thus, the type II collagen composition of the present invention can be administered at a lower dose compared to type II collagen compositions containing a smaller proportion of undenatured type II collagen.

[0085] The type II collagen composition of the present invention generally comprises from about 1% to about 50% (w / w) of undenatured type II collagen. For example, the type II collagen may comprise about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 75% or 100% (w / w) of undenatured type II collagen. For example, the type II collagen may comprise from about 0.1% to about 50%, from about 0.2% to about 50%, from about 0.5% to about 50%, from about 1% to about 50%, from about 5% to about 50%, from about 10% to about 50%, from about 15% to about 50%, from about 20% to about 50%, from about 25% to about 50%, from about 30% to about 50%, from about 35% to about 50%, from about 40% to about 50%, from about 45% to about 50%, from about 0.1% to about 60%, from about 0.2% to about 60%, from about 0.5% to about 60%, from about 1% to about 60%, from about 5% to about 60%, from about 10% to about 60%, from about 15% to about 60%, from about 20% to about 60%, from about 25% to about 60%, from about 30% to about 60%, from about 35% to about 60%, from about 40% to about 60%, from about 45% to about 60%, from about 0.1% to about 70%, from about 0.2% to about 70%, from about 0.5% to about 70%, from about 1% to about 70%, from about 5% to about 70%, from about 10% to about 70%, from about 15% to about 70%, from about 20% to about 70%, from about 25% to about 70%, from about 30% to about 70%, from about 35% to about 70%, from about 40% to about 70%, from about 45% to about 70%, from about 0.1% to about 80%, from about 0.2% to about 80%, from about 0.5% to about 80%, from about 1% to about 80%, from about 5% to about 80%, from about 10% to about 80%, from about 15% to about 80%, from about 20% to about 80%, from about 25% to about 80%, from about 30% to about 80%, from about 35% to about 80%, from about 40% to about 80%, from about 45% to about 80%, from about 0.1% to about 90%, from about 0.2% to about 90%, from about 0.5% to about 90%, from about 1% to about 90%, from about 5% to about 90%, from about 10% to about 90%, from about 15% to about 90%, from about 20% to about 90%, from about 25% to about 90%, from about 30% to about 90%, from about 35% to about 90%, from about 40% to about 90%, from about 45% to about 90%, from about 0.1% to about 99%, from about 0.2% to about 99%, from about 0.5% to about 99%, from about 1% to about 99%, from about 5% to about 99%, from about 10% to about 99%, from about 15% to about 99%, from about 20% to about 99%, from about 25% to about 99%, from about 30% to about 99%, from about 35% to about 99%, from about 40% to about 99%, from about 45% to about 99%, and from about 50% to about 100% (w / w) of undenatured type II collagen.Preferably, the type II collagen composition comprises at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15% or at least 20% (w / w) of undenatured type II collagen. More preferably, at least 5% (w / w) of undenatured type II collagen.

[0086] In some embodiments, the type II collagen may comprise from about 50% to about 100% (w / w) of undenatured type II collagen, such as from about 50% to about 95%, from about 50% to about 90%, from about 55% to about 100%, from about 55% to about 95%, from about 55% to about 90%, from about 60% to about 100%, from about 60% to about 95%, from about 60% to about 90%, from about 65% to about 100%, from about 65% to about 95%, from about 65% to about 90%, from about 70% to about 100%, from about 70% to about 95%, from about 70% to about 90%, from about 75% to about 100%, from about 75% to about 95%, from about 75% to about 90%, from about 80% to about 100%, from about 80% to about 95%, or from about 80% to about 90% (w / w) of undenatured type II collagen.

[0087] The content of undenatured type II collagen in the compositions of the present invention can be determined using methods known in the art, for example, by Harris et al. (J. Diet Suppl. 2021, 1: 1-16) or by trypsin digestion sensitivity assays, such as those described by Veres et al. and Bank et al. (Matrix Biol, 2014, 33: 54-59 and Matrix Biol, 1997, 16(5): 233-243). Alternatively, for example, the content of undenatured type II collagen can be determined by measuring the average molecular weight of the collagen in the composition as described herein.

[0088] For the avoidance of doubt, the term "w / w" as used herein refers to the weight of a particular substance in a mixture by weight. For example, a type II collagen composition containing 10% (w / w) of undenatured type II collagen should be understood to contain 10 grams of undenatured type II collagen per 100 grams of type II collagen composition. Unless otherwise stated, any reference herein to the mass of type II cartilage, undenatured type II collagen or a cartilage tissue sample should be understood to refer to the dry mass of the type II cartilage or cartilage tissue sample.

[0089] As described above, native type II collagen is generally insoluble in aqueous solutions, while denatured and / or hydrolyzed type II collagen is soluble in aqueous solutions. The solubility of the type II collagen composition is determined as described in Example 6. In some embodiments, the solubility of the type II collagen composition of the present invention is less than about 80%, such as less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or about 0%, and useful ranges can be selected from any of these values (e.g., about 0% to about 70%, about 0% to about 50%, about 0% to about 30%, about 0% to about 10%, about 0% to about 5%, about 0% to about 4%, about 0% to about 3%, about 1% to about 70%, about 1% to about 50%, about 1% to about 30%, about 1% to about 10%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 2% to about 70%, about 2% to about 50%, about 2% to about 30%, about 2% to about 10%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 70%, about 3% to about 50%, about 3% to about 30%, about 3% to about 10%, or about 3% to about 5%).

[0090] In some embodiments, the method does not include treatment with a protease. In some embodiments, the purified type II collagen has not been treated with a protease.

[0091] In certain applications, it may be desirable to produce soluble type II collagen that retains its triple helix structure. The type II collagen composition can be treated with a proteolytic enzyme such as pepsin to cleave the N- and C-terminal telopeptides, releasing soluble triple helix type II collagen, referred to as "telopeptide-free collagen".

[0092] The present applicant has found that in order to effectively produce telopeptide-free collagen, GAG must first be removed. Thus, the method of the present invention can enhance the extraction of telopeptide-free collagen from cartilage tissue. In addition, when using chaotropic agents such as guanidine hydrochloride or urea to remove GAG, it may be necessary to remove the chaotropic agent before treatment with the proteolytic enzyme, or they may interfere with proteolysis. Conversely, without wishing to be bound by theory, it is believed that the method of the present invention can allow subsequent proteolytic treatment without the need (or with reduced need) to remove extraction reagents.

[0093] In some embodiments, the method of purifying type II collagen from cartilage tissue further comprises: iv) treating the sample with a proteolytic enzyme (preferably pepsin) to produce telopeptide - free collagen. In some embodiments, the method produces at least about 30 μg of telopeptide - free collagen / mg of cartilage, such as at least about 35 μg, at least about 40 μg, at least about 45 μg, at least about 50 μg, at least about 55 μg, at least about 60 μg, at least about 65 μg, at least about 70 μg, at least about 75 μg, at least about 80 μg, at least about 85 μg, at least about 90 μg, at least about 95 μg, or at least about 100 μg of telopeptide - free collagen / mg of cartilage, and the useful range can be selected from any of these values (e.g., about 30 to about 100, about 30 to about 90, about 30 to about 80, about 35 to about 100, about 35 to about 90, about 35 to about 80, about 40 to about 100, about 40 to about 90, about 40 to about 80, about 45 to about 100, about 45 to about 90, about 45 to about 80, about 50 to about 100, about 50 to about 90, about 50 to about 80, about 55 to about 100, about 55 to about 90, about 55 to about 80, about 60 to about 100, about 60 to about 90, about 60 to about 80, about 65 to about 100, about 65 to about 90, about 65 to about 80, about 70 to about 100, about 70 to about 90, about 70 to about 80, about 75 to about 100, about 75 to about 90, or about 75 to about 80).

[0094] Also provided is the telopeptide - free collagen obtained by this method.

[0095] The type II collagen composition obtained from the method of the present invention may contain proteoglycans, glycosaminoglycans, and / or calcium salts (such as calcium phosphate). Preferably, the content of proteoglycans, glycosaminoglycans, and / or calcium salts (such as calcium phosphate) in the type II collagen composition is lower than the content of proteoglycans, glycosaminoglycans, and / or calcium salts (such as calcium phosphate) in the cartilage tissue sample before performing the method of the present invention.

[0096] In an exemplary embodiment, the type II collagen composition of the present invention contains less than 50% (w / w) of proteoglycans and / or glycosaminoglycans. For example, less than 45%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 2.5%, or less than 1% (w / w) of proteoglycans and / or glycosaminoglycans. In certain embodiments, the type II collagen composition of the present invention contains less than about 45% (w / w) of proteoglycans and / or glycosaminoglycans.

[0097] In certain embodiments, the type II collagen composition of the present invention contains less than 50% (w / w) of a calcium salt (e.g., calcium phosphate). For example, less than 45%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 2.5%, or less than 1% (w / w) of a calcium salt (e.g., calcium phosphate).

[0098] Generally, the type II collagen composition of the present invention comprises less than 20%, less than 15%, less than 10%, less than 5%, less than 2.5%, or less than 1% (w / w) of water. Preferably, less than 10%, less than 5%, less than 2.5%, or less than 1% (w / w) of water. For example, the type II collagen composition of the present invention can be substantially free of water.

[0099] Although the type II collagen composition of the present invention can be administered alone, it is preferably formulated into a composition suitable for human and / or animal consumption.

[0100] The type II collagen composition of the present invention can be incorporated into various formulations for therapeutic administration. More specifically, the type II collagen composition of the present invention can be formulated into a pharmaceutical composition by combining the type II collagen composition with a suitable pharmaceutically acceptable carrier or diluent. Thus, in certain embodiments, the type II collagen composition of the present invention comprises one or more carriers (including excipients and diluents), such as one or more pharmaceutically acceptable carriers (including pharmaceutically acceptable excipients and diluents).

[0101] Various pharmaceutically acceptable carriers and formulations are described in standard pharmaceutical treatises, such as Remington's Pharmaceutical Sciences by E.W. Martin. See also Wang, Y.J. and Hanson, M.A., Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42:2S, 1988.

[0102] For oral administration, the type II collagen composition can be administered alone or in combination with one or more carriers or diluents to prepare capsules, tablets, edible films, lozenges, and powders. The type II collagen composition can also be in the form of boluses, troches, or pastes. The composition can optionally be in a form that provides slow or controlled release of type II collagen once administered to a subject.

[0103] Pharmaceutical compositions suitable for therapeutic administration include those suitable for oral, parenteral (including subcutaneous, intradermal, intraosseous infusion, intramuscular, intravascular (bolus or infusion) and intramedullary), intraperitoneal, transmucosal, transdermal, rectal and topical (including dermal, buccal, sublingual and intraocular) administration, although the most suitable route may depend on the characteristics of the subject to whom the composition is administered, such as species, age, weight, sex, medical condition, severity of the medical condition, and other relevant medical and physical factors.

[0104] Compositions for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending and thickening agents. The compositions may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the immediate addition of a sterile liquid carrier, for example, saline or water for injection, prior to use. Temporary injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the aforedescribed types. Exemplary compositions for parenteral administration include injectable solutions or suspensions which may contain, for example, a suitable non-toxic, parenterally acceptable diluent or solvent such as mannitol, 1,3-butanediol, water, Ringer's solution, isotonic sodium chloride solution, or other suitable dispersing or wetting agents and suspending agents, including synthetic mono- or di-glycerides of fatty acids, and fatty acids such as oleic acid or Cremophor. Compositions for nasal, aerosol or inhalation administration include solutions in saline which may contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters which enhance bioavailability, and / or other solubilizing or dispersing agents such as those known in the art.

[0105] Compositions for rectal administration may be presented in the form of suppositories with carriers such as cocoa butter, synthetic glycerides or polyethylene glycols. Such carriers are generally solid at ordinary temperature but liquefy and / or dissolve in the rectal cavity to release the drug.

[0106] Compositions for topical administration in the mouth, for example, buccal or sublingual administration, include lozenges comprising the active ingredient in a flavored base such as sucrose and acacia or tragacanth, and lozenges comprising the active ingredient in a base such as gelatin and glycerin or sucrose and acacia. Exemplary compositions for topical administration include topical carriers such as Plastibase (a compound ointment base composed of liquid paraffin and polyethylene) (mineral oil gelled with polyethylene).

[0107] The type II collagen composition of the present invention can also be formulated as a food additive, food ingredient, health functional food, dietary supplement, medical food, nutritional product or food supplement. For example, it can exist in the form of a beverage, food bar, syrup, spread, sauce, paste, jelly, pudding, soup, capsule, tablet, edible film, lozenge or powder.

[0108] In an exemplary embodiment, the type II collagen composition comprises one or more excipients selected from microcrystalline cellulose, stearic acid and silica. In certain exemplary embodiments, the type II collagen composition can be in the form of a tablet having a core and a coating, wherein the core comprises type II collagen, microcrystalline cellulose, stearic acid and silica, and the coating comprises hypromellose.

[0109] It should be understood that in addition to the above ingredients, the compositions of the present invention may also include other conventional reagents in the art that are contemplated for the type of composition being discussed. In certain embodiments, the composition may comprise one or more additional therapeutic agents or dietary supplements. Examples of other therapeutic agents and dietary supplements that may be present in the compositions of the present invention include, but are not limited to, antihistamines, anti-inflammatory drugs, disease-modifying antirheumatic drugs (DMARDs), corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDs), glucosamine, chondroitin and S-adenosyl-methionine.

[0110] Treatment

[0111] The present invention provides a type II collagen composition for use as a medicament and / or dietary supplement. In certain embodiments, the type II collagen composition of the present invention can be administered to a subject suffering from a cartilage-related disorder such as osteoarthritis and rheumatoid arthritis. Alternatively, or additionally, the type II collagen composition can be administered to a subject known or suspected to be at risk of developing a cartilage-related disorder.

[0112] As used herein, "cartilage-related disorder" refers to a disorder related to the function of cartilage tissue in a human or animal body. Examples of cartilage-related disorders include joint stiffness, joint discomfort, joint inflammation, reduced joint mobility, exercise-induced joint pain and arthritis (such as osteoarthritis and rheumatoid arthritis). Further examples include bursitis, gout, tendinitis, joint sprains and joint strains.

[0113] In certain embodiments, the type II collagen composition can be consumed by a subject as a dietary supplement to promote joint health, for example by reducing or preventing joint stiffness, joint discomfort and / or improving joint mobility.

[0114] The present invention also provides a method for treating or preventing cartilage-related disorders, which method comprises the step of administering to a subject in need a dose of the type II collagen composition of the present invention. The present invention also provides a type II collagen composition for use in the preparation of a medicament, such as a medicament for treating or preventing cartilage-related disorders.

[0115] For the avoidance of doubt, the term "subject" as used herein refers to a living vertebrate. In certain embodiments, the subject is a mammal, for example, the subject can be a human. In certain embodiments, the subject is a human.

[0116] The therapeutic dose of the type II collagen composition prepared according to the present invention is a dose sufficient to treat or prevent cartilage-related disorders. By way of example, the composition can be administered at a dose of from 0.1 mg or less to 5000 mg or more per day. Exemplary human doses can be from 1 mg to 200 mg per day. The dose can be determined based on the total type II collagen content of the composition or the total undenatured type II collagen content of the composition. Preferably, the dose is determined based on the total undenatured type II collagen content of the composition. It should be understood that the type II collagen can be administered as a single daily dose or can be administered as a number of discrete separate doses, which may be appropriate. A ordinarily skilled physician can readily determine and administer the therapeutic dose or an appropriate fraction thereof required to achieve the desired therapeutic effect.

[0117] Numbered paragraphs

[0118] The present disclosure will now be further described by the following numbered paragraphs.

[0119] 1. A method for purifying type II collagen from cartilage tissue, the method comprising:

[0120] i) providing a sample of cartilage tissue;

[0121] ii) contacting the sample with a salt solution having a pH of from about 3 to about 9 under conditions that permit at least one component of the cartilage tissue to dissolve in the salt solution, wherein the at least one component of the cartilage tissue is selected from proteoglycans, glycosaminoglycans, and calcium salts; and

[0122] iii) removing the salt solution and the at least one component of the cartilage tissue from the sample.

[0123] 2. The method according to paragraph 1, wherein the salt solution has a pH of from about 3.5 to about 4.5, such as a pH of about 4.

[0124] 3. The method according to paragraph 1 or paragraph 2, wherein the salt solution is a sulfate solution or a chloride salt solution.

[0125] 4. A method according to any one of paragraphs 1 to 3, wherein the salt solution has a sulfate or chloride concentration of from about 0.4 M to about 2 M.

[0126] 5. A method according to any one of paragraphs 1 to 4, wherein the salt solution is an aqueous salt solution.

[0127] 6. A method according to any one of paragraphs 1 to 5, wherein the cartilage tissue sample is obtained from a fish, bird, mammal, mollusk or echinoderm.

[0128] 7. A method according to any one of paragraphs 1 to 6, wherein the cartilage tissue sample is from a fish.

[0129] 8. A method according to paragraph 7, wherein the fish is a teleost fish, for example the fish is cod, haddock, pollock, salmon (e.g., Atlantic salmon (Salmo salar)), European hake (also known as Merluccius merluccius) or blue hake (also known as Macruronus novaezelandiae or hoke).

[0130] 9. A method according to paragraph 7, wherein the fish is a cartilaginous fish, for example a shark, ray, skate or sawfish.

[0131] 10. A method according to paragraph 7 or 8, wherein the component dissolved in the salt solution is a glycosaminoglycan.

[0132] 11. A method according to paragraph 9, wherein the component dissolved by the salt solution is a calcium salt, such as calcium phosphate.

[0133] 12. A method according to any one of paragraphs 1 to 11, wherein step iii) removes from about 10% to 100% of the proteoglycans, glycosaminoglycans and / or calcium salts from the sample.

[0134] 13. A method according to any one of paragraphs 1 to 12, wherein the percentage (w / w) of type II collagen in the sample is about 40% to about 100% higher than the percentage (w / w) of type II collagen in the sample before step iii), and preferably the percentage (w / w) of non-denatured type II collagen in the sample is about 40% to about 100% higher than the percentage (w / w) of non-denatured type II collagen in the sample before step iii).

[0135] 14. A type II collagen composition obtained by a method according to any one of paragraphs 1 to 13.

[0136] 15. The composition according to paragraph 14, wherein the composition comprises a pharmaceutically acceptable excipient.

[0137] 16. The composition according to paragraph 13 or 14, which is used as a medicine.

[0138] 17. The composition according to paragraph 13 or 14, which is used for treating or preventing cartilage-related disorders.

[0139] 18. Use of the composition according to paragraph 13 or 14 as a dietary supplement.

[0140] 19. A method for treating or preventing cartilage-related disorders, the method comprising the step of administering a dose of the composition according to paragraph 13 or 14 to a subject in need thereof.

[0141] 20. Use of the composition according to paragraph 13 or 14 for the preparation of a medicine.

[0142] 21. The use according to paragraph 20, wherein the medicine is used for treating cartilage-related disorders.

[0143] Equivalent

[0144] The present invention has been described herein in a broad and general manner. Those of ordinary skill in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are exemplary, and the actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications using the teachings of the present invention. Those skilled in the art will recognize, or be able to determine using only routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Accordingly, it should be understood that the foregoing embodiments are presented by way of example only, and within the scope of the appended claims and their equivalents, the present invention may be practiced in a manner different from that specifically described and claimed. The present invention relates to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the scope of the present invention. In addition, each narrower genus and subgenus grouping falling within the general disclosure also forms part of the present invention. This includes the general description of the present invention, with the proviso or negative limitation removing any subject matter from that genus, whether or not the excised material is specifically recited herein.

[0145] Incorporated by reference

[0146] The contents of articles, patents, and patent applications mentioned or cited herein, as well as all other literature and electronically available information, are hereby incorporated by reference in their entirety to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference. The applicant reserves the right to physically incorporate any and all materials and information from any such article, patent, patent application, or other physical and electronic documents into this application.

[0147] The following examples illustrate the present invention.

[0148] Example

[0149] Example 1: Development of Purification Method

[0150] Method for treating cartilage tissue:

[0151] Chicken breastbone cartilage and hoki nasal cartilage were purchased from a butcher and a fishing company, respectively. The chicken breastbone cartilage and hoki nasal cartilage were manually removed and washed overnight in 0.1 M acetic acid at 10 °C while mixing to remove the external ligaments / skin. The cartilage was recovered using a sieve and rinsed with water.

[0152] The cartilage samples were freeze-dried and ground into 1 mm particles using a blender, and then ground using a grinder.

[0153] 50 mg of the freeze-dried and ground cartilage tissue sample was mixed with 1 mL of an extraction reagent (4 M guanidine hydrochloride solution; 1%, 0.25%, or 0.1% trypsin solution; 2% tributyl phosphate solution; 0.1 - 0.4 M sodium sulfate solution pH 3.9 - 10; 0.1 - 0.2 M sodium acetate solution pH 3.9 - 10; 2% triton-X solution; 2% CHAP solution; or 0.1 M acetic acid solution). The mixture was mixed by inversion at 20 °C or 40 °C for 24 hours. Then the sample was centrifuged (17,000 xg, 5 minutes), and the supernatant was transferred to another tube. The granular material from this method step is hereinafter referred to as pellet A.

[0154] GAG was precipitated from the supernatant by adding 500 μL of acetone. The precipitate was granulated using centrifugation, and the supernatant was removed. The granular material from this method step is hereinafter referred to as pellet B. Pellet B was resuspended in phosphate-buffered saline buffer (pH 7.4) before analysis.

[0155] 1a) Glycosaminoglycan extraction:

[0156] The amount of GAG in pellet B was measured using the dimethylmethylene blue assay (DMMB), which is based on the method described by Coulson-Thomas and Gesteira (Bio-protocol, 4(18):e1236, which is incorporated herein by reference). The resuspended pellet B samples were compared to a standard curve of chondroitin 4 sulfate. The amount of GAG extracted from the cartilage tissue samples was expressed in Figure 1 and 2 as μg GAG / mL of extraction reagent, normalized by the mass of the freeze-dried and ground cartilage tissue used in the extraction.

[0157] Results:

[0158] The extraction reagents and conditions used (i.e., 20 °C or 40 °C) were effective in extracting GAG from hoki nasal cartilage or chicken breast bone cartilage (see Figure 1 ). The amount of GAG extracted with sodium sulfate solution or sodium acetate solution was found to be similar to that extracted with trypsin or guanidine hydrochloride solution.

[0159] 1b) Collagen hydrolysis in cartilage samples:

[0160] Hydroxyproline is an amino acid found almost exclusively in collagen. Hydroxyproline extracted from cartilage tissue samples after treatment with extraction reagent is a marker of collagen hydrolysis, which can indicate a reduction in undenatured collagen in the sample. High levels of GAG extraction and low levels of hydroxyproline extraction indicate effective removal of GAG by the extraction reagent without disrupting the undenatured collagen content of the sample.

[0161] The hydroxyproline level was measured using the HPLC method described by Henderson et al. (Rapid, Accurate, Sensitive and Reproducible HPLC Analysis of Amino Acids. Agilent Technologies, Application Note, Publication No: 5980-1193). The samples were hydrolyzed in 6M HCl and derivatized with fluorenylmethoxycarbonyl chloride (FMOC-Cl), and then run on a 2.6 μm EVO C18 column. The fluorescence intensity of the FMOC group was measured at ex. / em. 266 / 305 nm. The area of the peak corresponding to FMOC-hydroxyproline was measured relative to a known amount of FMOC-hydroxyproline.

[0162] Results:

[0163] It was found that the sodium sulfate extraction reagent extracts high levels of GAG and low levels of hydroxyproline (see Figure 2 ), indicating that the extraction conditions do not damage the collagen contained in the sample. This is in contrast to the extraction conditions using trypsin solution, which results in high levels of GAG extraction and hydroxyproline, indicating that trypsin hydrolyzes the collagen in the sample into hydroxyproline.

[0164] Example 2: Evaluation of sodium sulfate extraction reagent

[0165] Hoki nose, dogfish backbone, and chicken breastbone cartilage samples were processed using the cartilage tissue treatment method described in Example 1. At 20 °C, 50 mg of freeze-dried and ground cartilage tissue sample was mixed with 1 mL of extraction reagent (0.4 M sodium sulfate solution, pH 4) for 20 hours and mixed by inverting.

[0166] After treatment with the extraction reagent, the samples were processed according to the cartilage tissue treatment method described in Example 1 to provide pellets A and B for analysis.

[0167] 2a) Extraction of glycosaminoglycans in cartilage samples:

[0168] The amount of GAG in pellet B obtained from hoki nose, dogfish backbone, and chicken breastbone cartilage tissue samples was analyzed using the method described in Example 1a. The amount of GAG extracted from each cartilage tissue sample is expressed as μg GAG / mL extraction reagent (left y-axis) in Figure 3 , or μg GAG / mL extraction reagent normalized by the mass of the freeze-dried and ground cartilage tissue used in the extraction (right y-axis).

[0169] Results:

[0170] It was found that the extraction reagent extracts approximately 30 mg and 50 mg of GAG per gram of freeze-dried and ground chicken breastbone and hoki nose cartilage tissue, respectively. No significant amount of GAG was extracted from the freeze-dried and ground dogfish cartilage tissue (see Figure 3 ).

[0171] 2b) Collagen content in cartilage samples:

[0172] The trypsin digestion sensitivity assay was used to measure the amount of undenatured collagen in the granular cartilage tissue samples (i.e., pellet A samples) after the extraction method. Undenatured collagen is more resistant to trypsin hydrolysis than denatured collagen. After trypsin treatment, samples with high levels of undenatured collagen are expected to have low levels of free hydroxyproline in the sample.

[0173] This assay method is based on the methods described by Veres et al. and Bank et al. (Matrix Biol 33:54-59 and Matrix Biol 16(5):233-243, which are incorporated herein by reference). Briefly, the pellet A samples generated by the extraction method (i.e., treating cartilage tissue with 0.4 M sodium sulfate solution (pH 3.9) at 20 °C for 18 h and mixing by inversion) were lyophilized to ensure that the exact weight of each sample could be measured. As a control, lyophilized and ground cartilage tissue samples that did not undergo the extraction method were used. (Lonza) was used as a further control. The samples were reported to contain 40 mg of cartilage and 1.2 mg of undenatured type II collagen.

[0174] The pellet A samples and the samples were mixed with 14,7000 units of porcine trypsin (type IX-S) / mg in 100 mM phosphate buffer (pH 7.4) and digested at 20 °C for 24 h. The reaction mixture was centrifuged, and the supernatant was immediately removed and frozen until analysis. The pellets and the supernatant were hydrolyzed in 6 N HCl at 110 °C for 24 h, and then the hydroxyproline content was measured. The amount of hydroxyproline in the pellets after trypsin treatment is an indirect measurement of the undenatured collagen content in the pellets and thus an indirect measurement of the undenatured collagen content in the cartilage tissue samples after the extraction method. The sum of the amounts of hydroxyproline in the pellets and the supernatant is an indirect measurement of the total collagen content in the cartilage tissue samples after the extraction method.

[0175] Results:

[0176] It was found that compared with the control samples that did not undergo the extraction method (i.e., "untreated" samples), the cartilage tissue samples that underwent the extraction method (i.e., "treated" samples) contained more collagen per mg of dry sample, indicating that the extraction method removed non-collagen components (such as GAG and calcium phosphate) from the cartilage (see Figure 4 ). In particular, compared with the untreated dogfish cartilage tissue samples (untreated DF), the treated dogfish samples (treated DF) had a 1.7-fold higher collagen content; the treated hoki nose samples (treated HN) had a 1.45-fold higher collagen content compared with the untreated hoki nose samples (untreated HN); compared with the samples, the processed chicken breast bone samples (treated CS) had a 2.2-fold higher collagen content (see Figure 4 ). Applying conversion factors based on the hydroxyproline content of pure type II collagen (9.5 and 10.22 for marine and chicken type II collagen) to convert hydroxyproline (μg / mg dry sample) to collagen percentage, the collagen percentage in each cartilage sample was estimated to be approximately 35% to approximately 100%, where in the untreated dogfish cartilage tissue samples and The lowest in [description missing], and the highest in the processed chicken breast bone cartilage tissue samples (Cumming et al., 2019, Marine Drugs 17(4):223, Naffa et al., 2019. Journal American Leather Chemists Association 114:29 - 37, and Don et al., 2013, Modern Food Science and Technology 29:2538 - 2541+2419).

[0177] Figure 5 It is shown that, compared with the control samples without the extraction method (i.e., "untreated" samples), all the samples with the extraction method (i.e., "treated" samples) maintained a higher proportion of undenatured collagen relative to the total collagen content.

[0178] Example 3: Effects of salt and pH on the content of undenatured collagen and GAG extraction in cartilage tissue

[0179] 3a) Cartilage tissue treatment method:

[0180] Remove Hoki (blue cod, also known as blue hake) nasal cartilage and chicken breast bone from the frozen carcass by hand. Then wash the extracted cartilage tissue samples with 0.5M acetic acid at 10 °C overnight to remove ligaments and foreign tissues. Then freeze and lyophilize the cartilage tissue samples. Then grind the lyophilized material into particles of 1 mm to 2 mm.

[0181] Add 1 mL of extraction reagent to 50 mg of the freeze - dried and ground cartilage tissue samples. The extraction reagents used are solutions of sodium chloride, ammonium chloride, or magnesium chloride; or solutions of sodium sulfate, ammonium sulfate, or magnesium sulfate. Test each salt solution at a 0.5M or 1M salt concentration at pH 4, 7, or 9. Invert and mix the mixture at 20 °C for 24 h. Then centrifuge the mixture (17,000 xg, 5 min). Remove the supernatant and analyze the glycosaminoglycan content as described in 3b). Analyze the undenatured collagen content of the granular material as described in 3c).

[0182] 3b) Glycosaminoglycan extraction in cartilage samples:

[0183] To precipitate the glycosaminoglycans in the supernatant removed from the treated cartilage tissue samples, 500 μL of acetone was added to each supernatant sample. The precipitate was pelleted using centrifugation. The pellet was resuspended in phosphate buffered saline buffer (pH 7.4), and the glycosaminoglycan content was measured using the dimethylmethylene (DMMB) blue assay method as described by Coulson-Thomas et al. (Bio-protocol, 2014, Vol. 4, No. 18). The readings were compared with a standard curve of chondroitin 4-sulfate.

[0184] 3c) Collagen content in cartilage samples:

[0185] The amount of undenatured collagen in the granulated cartilage tissue samples treated with 1 M magnesium sulfate solution having a pH of about 4, 7 or 9 or with 0.5 M sodium sulfate solution was indirectly measured using a trypsin digestion sensitivity assay. The assay method was the same as that described in Example 2b).

[0186] Results:

[0187] It was found that the sulfates (sodium sulfate, ammonium sulfate and magnesium sulfate) and chloride salts (sodium chloride, ammonium chloride and magnesium chloride) tested extracted GAG from hoki nasal cartilage and chicken breast bone cartilage. Sulfates were generally found to be more effective than chloride salts in extracting GAG (see Figure 6 and Figure 7 ).

[0188] It was found that hoki nasal cartilage tissue samples treated with an extraction reagent having a pH of about 4 or 7 contained higher levels of undenatured collagen than samples treated with an extraction reagent having a pH of about 9. It was found that a salt solution having a pH of about 4 extracted GAG from cartilage tissue samples while maintaining a good level of undenatured collagen in the cartilage tissue samples (see Figure 8 ).

[0189] Example 4: Effect of salt concentration and pH on GAG extraction

[0190] Using the cartilage tissue treatment method described in Example 1, hoki nasal cartilage was treated with Na2SO4 or MgSO4 at various concentrations and pHs as extraction reagents. The amount of GAG extracted was determined as described in Example 1.

[0191] Results

[0192] As shown in Tables 1 and 2, GAG was effectively extracted from cartilage tissue over a wide range of salt concentrations and pHs.

[0193] Table 1. Effect of salt concentration on GAG extraction.

[0194]

[0195] Table 2. Effect of pH on GAG extraction.

[0196]

[0197] * Since the solubility of MgSO4 decreases at high pH, 0.8 M MgSO4 was measured at pH 9.75 instead of pH 10.

[0198] Example 5: Cartilage source.

[0199] 5a) GAG content from cartilage sources

[0200] The total amount of GAG / mg of freeze-dried cartilage was determined for hoki nose, dogfish (mineralized cartilage), chicken breast bone, lamb trachea, calf trachea, and bovine articular cartilage. Briefly, a known amount of tissue was treated with a proteolytic enzyme (endoprotease, ) for 24 hours to completely dissolve all proteinaceous material. GAG was precipitated from the solution by adding 5 volumes of acetone per 1 volume of sample. The precipitate was recovered by centrifugation and resuspended in phosphate buffer, and the amount of GAG was determined using the dimethylmethylene blue (DMMB) assay.

[0201] The efficacy of GAG extraction using various cartilage sources was tested. As described in Example 1, cartilage from chicken breast bone, lamb trachea, calf trachea, and bovine joints was prepared using 0.4 M Na2SO4 at pH 4 or 9, or 0.8 M MgSO4 at pH 5 or 9 as the extraction reagent. The amount of GAG extracted per mg of cartilage was quantified as described in Example 1. The percentage of undenatured collagen was determined based on the percentage of hydroxyproline in the insoluble fraction after trypsin treatment as described in Example 2b.

[0202] Results

[0203] The GAG content of various cartilage sources is shown in Table 3.

[0204] Table 3. GAG content of various cartilage sources.

[0205]

[0206] Table 4 shows the efficacy of Na2SO4 and MgSO4 in extracting GAG from various cartilage sources.

[0207] Table 4. GAG extraction in other cartilage sources.

[0208]

[0209] The content of undenatured collagen (as a percentage of total collagen) in samples of chicken breast bone and bovine articular cartilage extracted with 0.4 M Na2SO4 at pH 4 and 0.8 M MgSO4 at pH 9 is shown in Table 5.

[0210] Table 5. Percentage of undenatured collagen.

[0211]

[0212] 5b) Shark cartilage

[0213] The cartilage tissue of chondrichthyes (i.e., Chondrichthyes, such as sharks) contains calcium salts (such as calcium phosphate). Chondrichthyes cartilage may contain about 15 - 25% calcium salts. The effects of demineralization treatment on GAG removal and shark cartilage denaturation were tested.

[0214] Shark cartilage was pretreated with various demineralization treatments, and then processed using the cartilage tissue treatment method described in Example 1, with 1M MgSO4 pH4 as the extraction reagent. The amount of extracted GAG was determined as described in Example 1, and the percentage of undenatured collagen was determined as described in Example 2.

[0215] Results

[0216] The effects of various demineralization pretreatments on the extraction of GAG from shark cartilage using 1M MgSO4 pH4 are shown in Table 6. Undenatured collagen is shown as a percentage of total collagen.

[0217] Table 6. Effects of demineralization treatment on GAG extraction from shark cartilage.

[0218]

[0219] Example 6: Comparative extraction methods.

[0220] Using the cartilage tissue treatment method described in Example 1, freeze - dried hoki cartilage samples were processed with 1M MgSO4 at pH4 or pH9 as the extraction reagent.

[0221] The control samples were processed by the following method:

[0222] Ground freeze - dried hoki cartilage was treated with 50 mM Tris pH8, 4M guanidine hydrochloride, 0.1% PMSF, 2 mM EDTA for 5 days to remove proteoglycans. The insoluble material was granulated by centrifugation, washed twice with 0.1M acetic acid, and treated with 0.1% pepsin in 0.2M acetic acid for 24 hours. The soluble fraction was dialyzed against 0.1M acetic acid, and collagen was precipitated using sodium chloride.

[0223] The percentage of soluble protein in the collagen samples was determined by suspending in 0.5M acetic acid and incubating at 20 °C for 1 hour with mixing. Then it was centrifuged at 3260×g for 15 minutes, and the protein concentration of the supernatant was measured using the biuret assay. The collagen samples were also observed by scanning electron microscopy.

[0224] result

[0225] The percentage of soluble protein in untreated Hoki cartilage, collagen prepared using 1 M MgSO4 as described in Example 1, and comparative pepsin-treated collagen is shown in Table 7. Native (untreated) cartilage and collagen prepared using MgSO4 were highly insoluble, whereas collagen prepared using pepsin digestion was highly soluble.

[0226] Table 7. Percentage of soluble protein.

[0227]

[0228] Scanning electron micrographs of MgSO4 and pepsin treated collagen are shown in Figure 9 Middle. At low magnification (534×), MgSO4-treated collagen appears as discrete particles, while pepsin-treated collagen appears as irregular spots. At high magnification (133,000×), MgSO4-treated collagen appears fibrillar, while pepsin-treated collagen lacks any visible fibrillar structure.

[0229] Example 7: Salt treatment increases the production of pepsin-soluble type II collagen (atelopeptide collagen).

[0230] To prepare soluble type II collagen that retains its triple helical structure, pepsin is used to cleave the N- and C-terminal telopeptides, releasing the triple helical type II collagen. This is called atelocollagen. In order to effectively extract collagen, the glycosaminoglycans must first be removed.

[0231] Various extraction reagents were tested for their ability to remove GAGs and produce triple helical type II collagen upon subsequent treatment with pepsin. Hoki nasal cartilage was prepared as described in Example 1 and glycosaminoglycans were extracted over 2 days at 8°C using the following extraction reagents:

[0232] ·water

[0233] 4M urea

[0234] 4M guanidine hydrochloride, 50mM tris, pH 8.0

[0235] 0.4M Na2SO4, 10mM sodium acetate, pH 4

[0236] 0.8M MgSO4, 10mM sodium acetate, pH 4

[0237] Glycosaminoglycan extraction was quantified as described in Example 1. Collagen was treated by limited pepsin digestion and the amount of soluble protein (minus pepsin itself) was determined.

[0238] Result

[0239] The efficacy of the extraction reagent on the amounts of GAG extraction and pepsin-soluble protein (telopeptide-free collagen) is shown in Table 8. Treatment with Na2SO4 or MgSO4 was as effective as guanidine hydrochloride in extracting GAG and more than twice as effective as urea. Pretreatment with Na2SO4 or MgSO4 enhanced the extraction of soluble type II collagen with pepsin.

[0240] Table 8. GAG extraction and pepsin-soluble protein concentration in hoki cartilage.

[0241]

Claims

1. A method for purifying type II collagen from cartilage tissue, the method comprising: i) providing a cartilage tissue sample; ii) contacting the sample with a salt solution under conditions that permit at least one component of the cartilage tissue to dissolve in the salt solution, wherein the at least one component of the cartilage tissue is selected from proteoglycans, glycosaminoglycans, and calcium salts; and iii) removing the salt solution and the at least one component of the cartilage tissue from the sample; wherein step iii) removes at least about 25% of the glycosaminoglycans from the sample; and wherein, after step iii), the sample comprises type II collagen in which at least about 50% (w / w) is undenatured type II collagen.

2. The method according to claim 1, wherein step iii) removes at least about 35%, preferably at least about 45% of the glycosaminoglycans from the sample.

3. The method according to claim 1 or 2, wherein after step iii), the sample comprises type II collagen, at least about 70% (w / w) of which is undenatured type II collagen, preferably at least about 80% (w / w).

4. The method according to any one of claims 1 to 3, wherein the sample after step iii) has a solubility of less than about 80%, preferably less than about 30%, more preferably less than about 5%.

5. The method according to any one of claims 1 to 4, wherein the salt solution has a pH of from about 3 to about 10, preferably from about 3.5 to about 4.5, such as a pH of about 4.

6. The method according to any one of claims 1 to 5, wherein the salt solution is a sulfate solution or a chloride salt solution.

7. The method according to any one of claims 1 to 6, wherein the salt is sodium sulfate or magnesium sulfate.

8. The method according to any one of claims 1 to 7, wherein the salt solution has a sulfate or chloride concentration of from about 0.4M to about 2M.

9. The method according to any one of claims 1 to 8, wherein the salt solution is an aqueous salt solution.

10. The method according to any one of claims 1 to 9, wherein the cartilage tissue sample is obtained from a fish, bird, mammal, mollusk, or echinoderm.

11. The method according to any one of claims 1 to 10, wherein the cartilage tissue sample is from a fish.

12. The method according to claim 11, wherein the fish is a teleost fish, such as the fish is cod, haddock, pollock, salmon (e.g., Atlantic salmon (Salmo salar)), European hake (also known as Merluccius merluccius (European hake)) or blue hake (also known as Macruronus novaezelandiae or hoke).

13. The method according to claim 11, wherein the fish is a cartilaginous fish, such as a shark, ray, skate, or sawfish.

14. The method according to claim 13, wherein the cartilage tissue sample is demineralized before step ii), and the demineralization treatment comprises contacting the cartilage tissue sample with a solution comprising EDTA, hydrochloric acid, phosphoric acid, acetic acid, lactic acid, or any combination of any two or more thereof.

15. The method according to any one of claims 1 to 10, wherein the cartilage tissue sample is obtained from a chicken, a domestic cow, or a sheep.

16. The method according to claim 11 or 12, wherein the component dissolved by the salt solution is glycosaminoglycan.

17. The method according to claim 13 or 14, wherein the component dissolved by the salt solution is a calcium salt, such as calcium phosphate.

18. The method according to any one of claims 1 to 17, wherein step iii) removes from about 10% to 100% of the proteoglycan and / or calcium salt from the sample.

19. The method according to any one of claims 1 to 18, wherein the percentage (w / w) of type II collagen in the sample is about 10% to about 100% higher than the percentage (w / w) of type II collagen in the sample before step iii), and preferably the percentage (w / w) of undenatured type II collagen in the sample is about 20% to about 100% higher than the percentage (w / w) of undenatured type II collagen in the sample before step iii).

20. A type II collagen composition obtained by the method according to any one of claims 1 to 19.

21. The composition according to claim 20, the composition comprising a pharmaceutically acceptable excipient.

22. The composition according to claim 20 or claim 21, which is used as a drug.

23. The composition according to claim 20 or claim 21, which is used for treating or preventing cartilage-related disorders.

24. Use of the composition according to claim 20 or claim 21 as a dietary supplement.

25. A method for treating or preventing cartilage-related disorders, the method comprising the step of administering a dose of the composition according to claim 20 or claim 21 to a subject in need thereof.

26. Use of the composition according to claim 20 or claim 21 in the preparation of a drug for treating cartilage-related disorders treatable by administering type II collagen.

27. The composition for the use according to claim 23, the method according to claim 25, or the use according to claim 26, wherein the cartilage-related disorder is arthritis, such as osteoarthritis and / or rheumatoid arthritis.

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