Chondroitin sulfate biopolyamine complex, method for preparing the same, and use thereof

Abstract

The present invention relates to a chondroitin sulfate biopolyamine complex, a method for preparing the same, and use thereof. The present invention provides a chondroitin sulfate biopolyamine complex which is a complex of chondroitin sulfate and a biopolyamine, wherein the chondroitin sulfate and the biopolyamine are non-covalently bonded, and the biopolyamine includes one, two or a combination of three or more of spermine, spermidine, putrescine and cadaverine. The chondroitin sulfate biopolyamine complex provided by the present invention has significantly superior anti-inflammatory activity than ordinary sodium chondroitin sulfate, and can be used for the prevention and treatment of inflammatory diseases, particularly arthritis and joint injuries, and the repair of bone tissue, and has effects such as reduction of blood lipids, antioxidation, delay of aging, and extension of lifespan.

Description

Technical Field

[0001] Priority and Related Applications This application claims priority based on Chinese Patent Application No. 202210551251.3, titled "Chondroitin Sulfate Biopolyamine, Its Preparation Method and Use", filed on May 18, 2022, and incorporates all of its contents, including the appendix, herein by reference.

[0002] The present invention belongs to the field of natural medicines. Specifically, it relates to the extraction of chondroitin sulfate, and further to a chondroitin sulfate biopolyamine complex, its preparation method and use.

Background Art

[0003] Chondroitin sulfate (CS) is one of the main components of the proteoglycan family, a typical substance of animal mucopolysaccharides, widely present in various animal tissues, especially rich in cartilage and connective tissues. Chondroitin sulfate is used not only as a raw material for pharmaceuticals, but also as a raw material for health foods and cosmetics. It is extracted from animal cartilage tissue, with rich sources and various structures. Chondroitin sulfate can be used for the treatment of arthritis, neuralgia, neuropathic migraine, etc., and also has an auxiliary therapeutic effect on chronic nephritis, chronic hepatitis, keratitis and corneal ulcer, etc. Some studies on the prevention and treatment of diseases such as coronary heart disease, angina pectoris, and myocardial infarction have also been reported. Among them, chondroitin sulfate has a certain effect on reducing pain and symptoms caused by arthritis and suppressing the progression of lesions.

[0004] However, although numerous clinical studies on chondroitin sulfate have been conducted worldwide, its clinical effect is not very significant, the dosage is high, and furthermore, some clinical studies have suggested little effect. Therefore, it is rarely used as a prescription drug for the treatment of bone tissue-related diseases (Citation 3). The important reason for this controversy is that there are differences in the purity, type, and subject group of chondroitin, and moreover, the physiological activity of chondroitin sulfate itself is very limited, making it insufficient to show a stable and reliable significant difference. In addition, chondroitin sulfate, as a bio-derived polysaccharide, has extremely high safety, and no obvious toxic side effects have been discovered at present. On the other hand, most other anti-inflammatory drugs generally have significant side effects and are not suitable for long-term use. Therefore, it is of great significance to greatly enhance the physiological activity of chondroitin sulfate and develop new chondroitin sulfate substances with high efficacy and high safety.

[0005] Researchers have made a lot of efforts to enhance the activity of chondroitin. Previous reports have shown that low-molecular-weight chondroitin sulfate can reduce viscosity, enhance tissue permeability, and improve physiological activity. Recently, it has been reported that low-molecular-weight polysaccharides such as low-molecular-weight chondroitin sulfate are also being studied. Among them, Citation 1 discloses a method for preparing low-molecular-weight chondroitin products by a polysaccharide decomposition method using a metal solid-phase catalyst, a method for preparing low-molecular-weight polysaccharides, and the catalyst used therefor. Citation 2 discloses the use of low-molecular-weight chondroitin sulfate in the production of topical preparations for the treatment of acne. However, overall, there are limitations in improving the anti-inflammatory activity by low-molecular-weight chondroitin sulfate, and so far, low-molecular-weight chondroitin has rarely been commercialized.

[0006] Regarding the preparation method of chondroitin sulfate, Citation 3 mentions that "in the products obtained by the current preparation methods of chondroitin sulfate or mixtures containing it, mainly due to the large proportion of bio-derived raw materials, diverse manufacturing processes, large differences in molecular structure, and different molecular weights, there are problems such as unstable quality and efficacy, and even side effects."

[0007] Therefore, currently, researchers have conducted many attempts to enhance the activity of chondroitin sulfate. However, further research is still awaited regarding stable and highly active chondroitin sulfate or a complex containing the same, as well as the preparation method.

[0008] For example, in some reports of Reference 4 and Reference 5, combinations of chondroitin sulfate and polyamines are also used. However, in Reference 4, the combination of chondroitin sulfate and polyamines is only used as a matrix, and the physiological activity of the complex itself (medicine) has not been examined. In addition, since the proportion of polyamine in the composition is high, micron-sized particles are formed and the stability is low. In contrast, in the present application, a water-soluble complex with excellent stability is formed. Further, Reference 5 discloses a supramolecular complex of a polyanionic polymer and spermidine, but the influence of the molecular weight distribution of chondroitin, the type of polyamine, the protein content, etc. on the activity of the complex has not been examined.

Prior Art Documents

Patent Documents

[0009] Reference 1: CN111495428A Reference 2: CN111110695A Reference 3: Discrepancies in Composition and Biological Effects of Different Formulations of Chondroitin Sulfate. Molecules 2015, 20, 4277 - 4289 Reference 4: Poly-ion Complex of Chondroitin Sulfate and Spermine and Its Effect on Oral Chondroitin Sulfate Bioavailability. Chem. Pharm. Bull. 2016, 64, 390―398 Reference 5: CN105797159A

Summary of the Invention

Problems to be Solved by the Invention

[0010] As described above, in order to improve the pharmacological activity of chondroitin sulfate, researchers have made various attempts. According to previous reports, low-molecular-weight chondroitin sulfate can reduce viscosity, enhance tissue permeability, and increase physiological activity, but there are limitations in improving anti-inflammatory activity as a whole. Moreover, to date, low-molecular-weight chondroitin has hardly been commercialized. In addition, there is no method for extracting or preparing a stable and highly active chondroitin sulfate or a complex containing the same.

[0011] According to the research of the present inventors, the activity of chondroitin extracted varies greatly depending on differences in extraction conditions, processes, etc. It has been found that some chondroitin shows significantly superior anti-inflammatory activity compared to other chondroitin. Through further research on its structure, as a result of confirming the structural composition of such chondroitin, the present invention has been proven to be a chondroitin sulfate biopolyamine complex provided by the present invention, which has anti-inflammatory activity far superior to that of ordinary chondroitin.

[0012] Therefore, in order to solve the above problems, the present invention provides a chondroitin sulfate biopolyamine complex having ultra-high activity that can significantly improve the anti-inflammatory (especially arthritis) activity of chondroitin sulfate and is expected to fill the blank area in the clinical treatment of osteoarthritis, and a method for preparing the same. It has also been found to have effects such as reducing blood lipids, antioxidant, delaying aging, and extending lifespan. At present, there are no reports on the super-strong anti-inflammatory activity of the chondroitin sulfate biopolyamine complex, and effects such as repair of joint damage, reduction of blood lipids, antioxidant, delaying aging, and extending lifespan.

Means for Solving the Problems

[0013] In a first aspect of the present invention, there is provided a chondroitin sulfate - biopolyamine complex which is a complex of chondroitin sulfate and a biopolyamine, wherein the chondroitin sulfate and the biopolyamine are non - covalently bonded, and the biopolyamine includes one, two, or a combination of three or more of spermine, spermidine, putrescine, and cadaverine. In some embodiments, the chondroitin sulfate is chondroitin sulfate in acid form or chondroitin sulfate in salt form. In some embodiments, the proportion of chondroitin sulfate having a weight - average molecular weight of 50,000 or more in terms of GPC integration ratio is 0%. In some embodiments, the proportion of chondroitin sulfate having a weight - average molecular weight of 25,000 - 50,000 in terms of GPC integration ratio is 40% or less. In some preferred embodiments, the proportion of chondroitin sulfate having a weight - average molecular weight of 25,000 - 50,000 in terms of GPC integration ratio is 35% or less. In some more preferred embodiments, the proportion of chondroitin sulfate having a weight - average molecular weight of 25,000 - 50,000 in terms of GPC integration ratio is 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less, or 0%. In some embodiments, for the chondroitin sulfate, in terms of the GPC integration ratio, the upper limit of the proportion of chondroitin sulfate having a weight average molecular weight of 400 to 25,000 is 80% or more, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or 100%, and the lower limit of the proportion of chondroitin sulfate having a weight average molecular weight of 400 to 25,000 is 40% or more, preferably 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% or more. In some embodiments, for the chondroitin sulfate, in terms of the GPC integration ratio, the proportion of chondroitin sulfate having a weight average molecular weight of 400 or less is 15% or less, preferably 3% or less, more preferably 1% or less, and most preferably 0%. In some embodiments, for the chondroitin sulfate, the upper limit of the weight average molecular weight is 25,000 or less, preferably 24,000, 23,000, 22,000, 21,000, 20,000, 19,000, 18,000, 17,000, 16,000, 15,000, 14,000, 13,000, 12,000, 11,000, 10,000, 9,000, or 8,000 or less, and the lower limit of the weight average molecular weight is 400, 500, 600, 700, or 800 or more. The upper limit of the proportion of chondroitin sulfate is 80% or more, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or 100%, and the lower limit is 40% or more, preferably 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% or more. In some embodiments, in terms of the GPC integration ratio, the proportion of chondroitin sulfate having a weight average molecular weight of 400 to 10,000 is 40% to 100%, preferably 50% to 100%, 60% to 100%, 70% to 100%, or 80% to 100%. In some more preferred embodiments, in terms of the GPC integration ratio, the molecular weight distribution of the chondroitin sulfate is as follows: the proportion of chondroitin sulfate having a weight average molecular weight exceeding 50,000 is 0%; the proportion of chondroitin sulfate having a weight average molecular weight of 25,000 to 50,000 is 0 to 40%; the proportion of chondroitin sulfate having a weight average molecular weight of 400 to 10,000, preferably the proportion of chondroitin sulfate having a weight average molecular weight of 400 to 8,000, is 40% to 100%; and the proportion of chondroitin sulfate having a weight average molecular weight of 400 or less is 15% or less. In some embodiments, the weight average molecular weight of the chondroitin sulfate - biopolyamine complex is 400 to 50,000. In some preferred embodiments, the weight average molecular weight of the chondroitin sulfate - biopolyamine complex is 400 to 25,000. In all embodiments, the sum of the proportion of chondroitin sulfate having a weight average molecular weight of 25,000 to 50,000, the proportion of chondroitin sulfate having a weight average molecular weight of 400 to 25,000, and the proportion of chondroitin sulfate having a weight average molecular weight of 400 or less is 100%. In some embodiments, with respect to the total weight (g) of the chondroitin sulfate biopolyamine complex, the biopolyamine contained in the chondroitin sulfate biopolyamine complex is 200 μmol / g or less, preferably 199 μmol / g, 198 μmol / g, 197 μmol / g, 196 μmol / g, 195 μmol / g, 194 μmol / g, 193 μmol / g, 192 μmol / g, 191 μmol / g, 190 μmol / g, 189 μmol / g, 188 μmol / g, 187 μmol / g, 186 μmol / g, 185 μmol / g, 184 μmol / g, 183 μmol / g, 182 μmol / g, 181 μmol / g or 180 μmol / g or less, and the contained biopolyamine is 0.5 μmol / g or more, preferably 0.6 μmol / g, 0.7 μmol / g, 0.8 μmol / g, 0.9 μmol / g, 1 μmol / g or more. In some embodiments, the mass percentage of protein in the chondroitin sulfate biopolyamine complex is less than 8%, preferably less than 5%, more preferably less than 3%, most preferably less than 1%, and even more preferably 0%.

[0014] A second aspect of the present invention is a method for preparing the chondroitin sulfate biopolyamine complex according to the first aspect of the present invention, including the step of mixing chondroitin sulfate and a biopolyamine, and containing the biopolyamine at 0.5 to 200 μmol / g with respect to the total weight (g) of the chondroitin sulfate biopolyamine complex, and providing the preparation method.

[0015] A third aspect of the present invention is a method for preparing the chondroitin sulfate biopolyamine complex according to the first aspect of the present invention, including the step of mixing an extract containing chondroitin sulfate and a polyamine separated and extracted from a raw material with ethanol, wherein the pH of the extract is 4 to 6, and the volume ratio of the extract to ethanol is 1:1 to 3, and providing the preparation method of the chondroitin sulfate biopolyamine complex. In some embodiments, the step of separating and extracting chondroitin sulfate and polyamine from a raw material is An enzymatic decomposition or acid decomposition step of decomposing a raw material by enzymatic decomposition or acid decomposition to obtain an enzymatic decomposition solution or an acid decomposition solution, and a separation and extraction step of simultaneously or stepwise extracting chondroitin sulfate and polyamine from the enzymatic decomposition solution or the acid decomposition solution. In some embodiments, when extracting chondroitin sulfate and polyamine stepwise, the polyamine in the enzymatic decomposition solution or the acid decomposition solution is separated by chromatography or an extraction method, and the residue after separation of the polyamine is treated by one or more of an enzymatic decomposition method, a protein precipitation method, chromatography, and an alcohol precipitation method to separate chondroitin sulfate from the residue. Optionally, after separating chondroitin sulfate, a step of reducing the molecular weight of chondroitin sulfate may further be included. In some embodiments, when extracting chondroitin sulfate and polyamine simultaneously, the protein in the enzymatic decomposition solution or the acid decomposition solution is precipitated by a protein precipitation method to separate chondroitin sulfate and polyamine. In some embodiments, the raw material includes animal tissue, plant tissue, and a microbial culture fermentation broth.

[0016] A fourth aspect of the present invention provides a chondroitin sulfate biopolyamine complex prepared by the preparation method described in the second or third aspect of the present invention.

[0017] A fifth aspect of the present invention provides the use of a chondroitin sulfate biopolyamine complex in any one of the following (a) to (g). (a) Use in the preparation of an anti-inflammatory drug, (b) Use in the preparation of a pharmaceutical for the treatment and / or prevention of an inflammatory disease, (c) Use in the preparation of a pharmaceutical for reducing blood lipids, (d) Use in the preparation of a pharmaceutical for the treatment and / or prevention of hyperlipidemia, (e) Use in the preparation of a pharmaceutical for the treatment and / or repair of joint injuries, (f) Use in the preparation of an antioxidant, (g) Use in the preparation of a medicament for delaying aging and / or extending lifespan.

[0018] Here, the chondroitin sulfate biopolyamine complex is the chondroitin sulfate biopolyamine complex described in the first aspect or the fourth aspect of the present invention and / or the chondroitin sulfate biopolyamine complex prepared by the preparation method described in the second aspect or the third aspect of the present invention. In some embodiments, the inflammatory disease includes inflammation caused by an inflammatory inducer and / or inflammation caused by inflammatory cells, interleukins and / or tumor necrosis factors. Preferably, the inflammatory disease is one or more selected from allergy, eczema, myocardial infarction, cerebral infarction, Alzheimer's disease, dermatitis or arthritis. In some embodiments, the hyperlipidemia includes primary hyperlipidemia and / or secondary hyperlipidemia. In some embodiments, the hyperlipidemia includes hypertriglyceridemia and / or hypercholesterolemia. In some embodiments, the hyperlipidemia includes hyperlipidemia-related diseases. Optionally, the hyperlipidemia-related disease may include a cardiovascular disease. Optionally, the cardiovascular disease includes one or more of arteriosclerosis, coronary artery disease, angina pectoris, carotid artery disease, stroke, cerebral arteriosclerosis, myocardial infarction, cerebral infarction, restenosis after balloon angioplasty, hypertension, intermittent claudication, dyslipidemia, postprandial hyperlipidemia and xanthoma. In some embodiments, the joint injury includes joint injury caused by inflammation, aging, exercise, or injury.

[0019] The sixth aspect of the present invention provides the use of the chondroitin sulfate biopolyamine complex described in the first aspect or the fourth aspect of the present invention and / or the chondroitin sulfate biopolyamine complex prepared by the preparation method described in the second aspect or the third aspect of the present invention in the preparation of a health food or a cosmetic.

[0020] The seventh aspect of the present invention provides a pharmaceutical composition, a health food, or a cosmetic containing the chondroitin sulfate biopolyamine complex described in the first aspect or the fourth aspect of the present invention and / or the chondroitin sulfate biopolyamine complex prepared by the preparation method described in the second aspect or the third aspect of the present invention.

Advantages of the Invention

[0021] The chondroitin sulfate biopolyamine complex provided by the present invention has significantly superior anti-inflammatory activity compared to ordinary sodium chondroitin sulfate, and can be used for the prevention and treatment of inflammatory diseases, particularly arthritis and joint injuries, and the repair of bone tissue, and has effects such as reducing blood lipids, antioxidation, delaying aging, and extending lifespan.

Brief Description of the Drawings

[0022]

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Mode for Carrying Out the Invention

[0023] Hereinafter, the content of the present invention will be described in detail. The technical features described below will be described based on typical embodiments and specific examples of the present invention, but the present invention is not limited to these embodiments and specific examples. The description is as follows.

[0024] In this specification, the numerical range represented by "numerical value A to numerical value B" means a range including the limit values A and B.

[0025] As used herein, "substantially" or "substantively" means that the standard deviation from the theoretical model or theoretical data is within 5%, preferably within 3%, more preferably within 1%.

[0026] As used herein, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0027] As used herein, "any" or "optionally" means that the event or situation described thereafter may or may not occur, and the description includes both the case where the event occurs and the case where it does not occur.

[0028] As used herein, references to "some specific / preferred embodiments", "another specific / preferred embodiment", "embodiment", etc. mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in relation to the embodiment are included in at least one embodiment described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that the elements described can be combined in any suitable form in various embodiments.

[0029] In the present invention, the terms "comprising" and "having" and their variants are intended to cover non-exclusive inclusion. For example, a process, method, device, product, or apparatus comprising a series of steps is not limited to the exemplified steps or modules, and may further include steps not exemplified, or may further include other steps specific to these processes, methods, products, or apparatuses.

[0030] In the present invention, "a plurality" means two or more. "And / or" represents the relationship between related things, meaning that three relationships may exist. For example, A and / or B may represent three cases: only A exists, both A and B exist, and only B exists. The symbol " / " usually represents that the related things are in an "or" relationship.

[0031] In the present invention, the term "weight average molecular weight (Mw)" refers to the relative molecular weight, the statistical average molecular weight based on mass, and the average molecular weight per unit weight. For example, it can be measured by methods such as light scattering method and gel chromatography. In some embodiments, the weight average molecular weight is measured by high performance gel permeation chromatography, and the weight average molecular weight of chondroitin sulfate·chondroitin sulfate polyamine is measured using dextran molecular weight standard (National Institutes for Food and Drug Control) as a standard substance.

[0032] In the present invention, the term "chondroitin sulfate" (hereinafter also referred to as "CS") means various sulfated glycosaminoglycans having various molecular weights and present in various animal tissues. The sugar backbone of CS is composed of disaccharide units [4)-β-D-GlcA-(1→3)-β-D-GalNAc-(1→] repeatedly linked by β-glycosidic bonds (1→4). In the present invention, chondroitin sulfate includes an acid form and a salt form of the polysaccharide.

[0033] CS is usually a mixture, and CS extracted from terrestrial animals is mainly composed of chondroitin sulfate A (CSA) and chondroitin sulfate C (CSC). In addition, CS extracted from marine animals further includes types such as chondroitin sulfate D (CSD) and chondroitin sulfate E (CSE). In the present invention, CSA generally refers to CS characterized by CSA-type disaccharide units [4)-β-D-GlcA-(1→3)-β-D-GalNAc4SO 3 --(1→] (the sulfate group is at the O-4 position of galactose). In the present invention, CSC generally refers to CSC-type disaccharide units [4)-β-D-GlcA-(1→3)-β-D-GalNAc6SO 3--(1→] refers to CS (the sulfate group is at the O-6 position of galactose). CSD generally refers to the disaccharide unit of the CSD type [4)-β-D-GlcA2SO 3 -(1→3)-β-D-GalNAc6SO 3 --(1→] refers to CS (the sulfate groups are at the O-2 of glucuronic acid and the O-6 position of galactose). CSE generally refers to the disaccharide unit of the CSE type [4)-β-D-GlcA-(1→3)-β-D-GalNAc4,6SO 3 --(1→] refers to CS (the sulfate groups are at the O-4 and O-6 positions of galactose).

[0034] In the present invention, the "GPC integration ratio" refers to the GPC integration ratio corresponding to each molecular weight range, which is obtained by examining each molecular weight and the corresponding integration ratio in the chromatogram of the test sample using the slicing function of GPC software.

[0035] In the present invention, the term "biogenic amines (BA)" is a general term for amino-containing low-molecular-weight organic compounds having physiological activity. It can be regarded as a substance in which 1 to 3 hydrogen atoms in an ammonia molecule are substituted by an alkyl group or an aryl group, and it is a low-molecular-weight organic alkali of aliphatic, aliphatic or heterocyclic types that are commonly present in animals, plants and foods. Biogenic amines can be classified into aliphatic amines such as putrescine, cadaverin, spermine, spermidine, etc., aromatic amines such as tyramine, phenylethylamine, etc., and heterocyclic amines such as histamine, tryptamine, etc. according to their structures. In addition, biogenic amines can also be classified into monoamines containing histamine, tyramine, tryptamine, phenylethylamine, etc., and polyamines (also referred to as biological polyamines) containing cadaverin, putrescine, spermine and spermidine according to their composition components.

[0036] In the present invention, the terms "prevention" or "treatment" refer to reducing the risk of contracting or progression of a disease or medical condition (i.e., in a patient who has a tendency to develop a disease or is exposed to a disease but does not yet show symptoms of the disease, not developing at least one of the clinical symptoms of the disease). For example, treatment may include (i) prevention of a disease, disorder and / or medical condition in a patient who is susceptible to but not yet diagnosed with such disease, disorder and / or medical condition; (ii) suppression of the disease, disorder and / or medical condition, i.e., suppressing its progression; or (iii) alleviation of the disease, disorder and / or medical condition, i.e., alleviating the disease, disorder and / or medical condition.

[0037] In the present invention, the term "effective amount" means an amount of a compound sufficient to achieve such treatment or prevention when administered to a subject for the treatment or prevention of a disease. The "effective amount" can be determined by variations in the compound, the disease and its severity, as well as the age and weight of the subject to be treated. The "therapeutically effective amount" means an amount effective for a therapeutic treatment. The "preventively effective amount" means an amount effective for a preventive treatment.

[0038] In the present invention, the term "administration" means physically introducing a drug to a subject using any of a variety of methods and delivery systems known to those skilled in the art. Examples of administration routes include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes such as injection or infusion.

[0039] In the present invention, the terms "subject", "individual", and "patient" are well known in the art and refer to any subject in need of treatment, particularly a mammalian subject, and can be used interchangeably herein. Specific examples include, but are not limited to, humans and other primates (including non-human primates such as chimpanzees, other apes, and monkey species). The terms individual, subject, and patient do not themselves refer to a specific age, gender, race, etc.

[0040] As described herein, the term "inflammatory disease" refers to a general term for diseases in which inflammation is the main destructive factor. Examples of inflammatory diseases include, but are not limited to, edema, dermatitis, acne, oral ulcers (e.g., inflammatory oral ulcers), allergies, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastric ulcers, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, systemic lupus erythematosus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis of shoulder, tendinitis, tenosynovitis, myositis, hepatitis, cystitis, nephritis, Sjögren's syndrome, and multiple sclerosis. In the present invention, "inflammatory disease" may also include diseases in which inflammation is involved (e.g., diseases belonging to inflammatory complications).

[0041] In the present invention, the term "chronic inflammatory disease" refers to various pathological conditions and disorders characterized by the presence of chronic inflammation. Examples of chronic inflammatory diseases include, but are not limited to, dermatomyositis, Graves' disease, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus (SLE), sarcoidosis, Sjögren's syndrome, amyloidosis, Hashimoto thyroiditis, vasculitis, rheumatoid arthritis, reactive arthritis, polymyositis, scleroderma, Addison's disease, vitiligo, pernicious anemia, glomerulonephritis, severe celiac disease, type 1 diabetes, psoriasis, pulmonary fibrosis, and eczema. In the present invention, "chronic inflammatory disease" may include diseases in which chronic inflammation is involved (e.g., including diseases belonging to chronic inflammatory complications). Such "chronic inflammatory diseases" may include myocardial infarction, cerebral infarction, Alzheimer's disease, and the like.

[0042] In the present invention, the term "arthritis" is a general term for diseases accompanied by inflammatory changes occurring in the joint region due to bacterial infection, trauma, etc. Arthritis is roughly classified into acute arthritis and chronic arthritis. Acute arthritis can be further classified as follows. (1) Serous arthritis: Usually caused by trauma, but there are also cases with unknown causes, and generally occurs in only one joint. (2) Serofibrinous arthritis: Occurs simultaneously with acute rheumatoid arthritis, and turbid exudate accumulates in the joint cavity. This can cause movement disorders even after the inflammation subsides due to the formation of a pseudomembrane. (3) Suppurative arthritis: Occurs in open wounds of joints, or in infectious diseases such as gonorrhea, typhoid fever, scarlet fever, and sepsis where polyarthritis occurs. Infants aged 1 to 2 months can develop into dislocation due to untreatable bone damage. Adults often develop into osteomyelitis when the suppurative part ruptures and pus enters the joint, which is called secondary suppurative arthritis. Chronic arthritis can be further classified as follows. (1) Special type of inflammation: Usually refers to tuberculous arthritis, syphilitic arthritis, or gouty arthritis caused by metabolic disorders of uric acid that occur frequently in middle-aged men. (2) Polyarthritis: Most commonly seen in rheumatoid arthritis. This can transition from acute serous arthritis, occur as polyarthritis during the course of pneumonia, syphilis, and gonorrhea, or be a type of sepsis. In addition, Still's disease also belongs to this category. (3) Degenerative arthritis: Usually occurs due to a degenerative aging process or trauma. (4) Hemophilic arthritis: Occurs in hemophilia patients due to bleeding within the joint. Degenerative arthritis is also called osteoarthritis and is a local arthritis caused by degenerative changes in articular cartilage, mainly occurring in middle-aged and elderly people.

[0043] In the present invention, the term "rheumatoid arthritis" is a chronic systemic inflammatory disease of unknown cause that can infect many organs. In this process, an inflammatory reaction first occurs in the synovium around the joint, and then gradually spreads to the adjacent cartilage and bone, causing joint destruction and deformation. Extra-articular clinical symptoms include anemia, Sjogren's syndrome, subcutaneous nodules, pulmonary fibrosis, vasculitis, skin ulcers, etc.

[0044] In the present invention, the term "hyperlipidemia" refers to a disease characterized by an abnormal increase in serum lipids. The lipid fraction in circulating blood includes, for example, total cholesterol, specific lipoproteins, and triglycerides. Serum lipoproteins are carriers for lipids in circulation and are classified by their density into chylomicrons, very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and high density lipoprotein (HDL). The term "hyperlipidemia" includes primary and secondary hyperlipidemia. Primary hyperlipidemia generally occurs due to genetic defects, while secondary hyperlipidemia generally occurs due to other factors such as various disease states, drugs, and dietary factors. For example, secondary hyperlipidemia can be caused by diabetes. Alternatively, hyperlipidemia can result from a combination of both primary and secondary hyperlipidemia causes. Hyperlipidemia can include hypertriglyceridemia, hypercholesterolemia, or a combination thereof. "Hypertriglyceridemia" refers to a condition where the serum total triglyceride level is elevated above the desired level. "Hypercholesterolemia" refers to a condition where the serum cholesterol level is elevated above the desired level. In some embodiments, hypercholesterolemia is a condition where serum total cholesterol, HDL cholesterol (HDL-C), or LDL cholesterol (LDL-C) exceeds the desired level. Hyperlipidemia further increases the risk of cardiovascular disease and atherosclerosis and may induce the development of cardiovascular disease and atherosclerosis. The term "cardiovascular disease" includes vascular diseases of the circulatory system resulting from abnormally high lipid concentrations in the blood vessels. The term "atherosclerosis" refers to a disease of the arteries in which fat accumulates on the inner walls of the arteries, ultimately blocking blood flow.

[0045] The term "pharmaceutically acceptable" (or "pharmacologically acceptable") refers to molecular entities and compositions that do not produce adverse side effects, allergic reactions or other harmful reactions when administered to animals or humans. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coating agents, antibacterial agents, isotonic agents and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants, etc. that can be used as vehicles for pharmaceutically acceptable substances.

[0046] The present invention provides a chondroitin sulfate biopolyamine complex and a method for preparing the chondroitin sulfate biopolyamine complex from biological tissues. The chondroitin sulfate biopolyamine complex provided by the present invention and the chondroitin sulfate biopolyamine complex obtained by the method of the present invention (simply referred to as CSX in the present invention) have a weight average molecular weight of 400 to 50,000. The chondroitin sulfate biopolyamine complex has significantly better anti-inflammatory activity than ordinary sodium chondroitin sulfate. The present invention further relates to the use of the chondroitin sulfate biopolyamine complex obtained by the method of the present invention, which is applicable to the prevention, treatment of chronic inflammation, reduction of blood lipids, particularly arthritis and joint damage, and repair of bone tissue.

[0047] Hereinafter, the chondroitin sulfate biopolyamine complex provided by the present invention, its preparation method and use will be described in detail.

[0048] <Chondroitin sulfate biopolyamine complex> In some embodiments of the present invention, there is provided a chondroitin sulfate - biopolyamine complex which is a complex of chondroitin sulfate and a biopolyamine, and in which the chondroitin sulfate and the biopolyamine are bound. In some specific embodiments, the chondroitin sulfate and the biopolyamine are bound via a non - covalent bond (non - covalently bound). This complex functions not as a matrix for other drugs or active ingredients, but as a drug itself. The chondroitin sulfate - biopolyamine complex provided by the present invention is a natural medicine, but can also be synthesized by chemical methods.

[0049] In some embodiments, the chondroitin sulfate in the chondroitin sulfate - biopolyamine complex contains one or a combination of two or more of chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate D, and chondroitin sulfate E. n represents a natural number from 1 to 100 or 90, or 80, or 70, or 60, or 50, or 40, or 30, or 20, or 16.

Chemical formula

[0050] In some specific embodiments, due to the differences in chondroitin sulfate (for example, CSA, CSC, CSD, and CSE), the chondroitin sulfate - biopolyamine complex may include any one having a structure represented by Formula I (CSA), Formula II (CSC), Formula III (CSD), and Formula IV (CSE) below, or an arbitrarily combined one of them at an arbitrary ratio.

Chemical formula

[0051] Since chondroitin sulfate usually exists as a mixture, it should be understood that the chondroitin sulfate biopolyamine complex provided by the present invention, particularly the chondroitin sulfate biopolyamine complex prepared by extraction from natural raw materials such as bone tissue, is usually a mixture of various chondroitin sulfates bound to various biopolyamines.

[0052] In some embodiments of the present invention, the biopolyamine includes one, two, or a combination of three or more of spermine, spermidine, putrescine, and cadaverine. In some specific embodiments of the present invention, the biopolyamine includes one of spermine, spermidine, putrescine, and cadaverine.

[0053] Examples of the chondroitin sulfate biopolyamine complex containing one biopolyamine include a complex of chondroitin sulfate and spermine, a complex of chondroitin sulfate and spermidine, a complex of chondroitin sulfate and putrescine, and a complex of chondroitin sulfate and cadaverine. In other specific embodiments of the present invention, the biopolyamine includes at least two of spermine, spermidine, putrescine, and cadaverine.

[0054] In more specific embodiments of the present invention, the biopolyamine is spermine and spermidine; spermine and putrescine; spermine and cadaverine; spermidine and putrescine; spermidine and cadaverine; putrescine and cadaverine; spermine, spermidine, and putrescine; spermine, spermidine, and cadaverine; spermine, putrescine, and cadaverine; Spermidine, putrescine and cadaverine; or Spermine, spermidine, putrescine and cadaverine, comprising a combination selected from.

[0055] As demonstrated in the examples and test examples described later of the present invention, chondroitin sulfate biopolyamine complexes containing two or more kinds of biopolyamines generally have superior activities in all aspects than chondroitin sulfate biopolyamine complexes containing only one kind of biopolyamine. In addition, chondroitin sulfate biopolyamine complexes containing one kind of biopolyamine also have significantly superior activities than conventional chondroitin sulfate. Such activities include, but are not limited to, anti-inflammatory (especially arthritis) activity, reduction of blood lipids, antioxidant, delay of aging, extension of lifespan, etc.

[0056] In some embodiments of the present invention, the chondroitin sulfate biopolyamine complex has a weight average molecular weight of 400 to 50,000. Preferably, the chondroitin sulfate biopolyamine complex has a weight average molecular weight of 400 to 25,000.

[0057] In some embodiments of the present invention, the chondroitin sulfate has a ratio of chondroitin sulfate with a weight average molecular weight of 50,000 or more of 0% in terms of GPC integration ratio

[0058] In some embodiments of the present invention, the chondroitin sulfate has a ratio of chondroitin sulfate with a weight average molecular weight of 25,000 to 50,000 of 40% or less in terms of GPC integration ratio.

[0059] In some preferred embodiments of the present invention, the chondroitin sulfate has a ratio of chondroitin sulfate with a weight average molecular weight of 25,000 to 50,000 of 35% or less in terms of GPC integration ratio.

[0060] In a more preferred embodiment of the present invention, in terms of the GPC integration ratio, the proportion of chondroitin sulfate having a weight average molecular weight of 25,000 to 50,000 is 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less, or 0%.

[0061] In some embodiments of the present invention, in terms of the GPC integration ratio, the upper limit of the proportion of chondroitin sulfate having a weight average molecular weight of 400 to 25,000 is 80% or more. Preferably, in terms of the GPC integration ratio, the upper limit of the proportion of chondroitin sulfate having a weight average molecular weight of 400 to 25,000 is 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or 100%.

[0062] In some embodiments of the present invention, in terms of the GPC integration ratio, the lower limit of the proportion of chondroitin sulfate having a weight average molecular weight of 400 to 25,000 is 40% or more. Preferably, in terms of the GPC integration ratio, the lower limit of the proportion of chondroitin sulfate having a weight average molecular weight of 400 to 25,000 is 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% or more.

[0063] In some embodiments of the present invention, in terms of the GPC integration ratio, the proportion of chondroitin sulfate having a weight average molecular weight of 400 or less is 15% or less, preferably 3% or less, more preferably 1% or less, and most preferably 0%.

[0064] The upper limit of the weight-average molecular weight is 25,000 or less, preferably 24,000, 23,000, 22,000, 21,000, 20,000, 19,000, 18,000, 17,000, 16,000, 15,000, 14,000, 13,000, 12,000, 11,000, 10,000, 9,000, or 8,000 or less, and the lower limit of the weight-average molecular weight is 400, 500, 600, 700, or 800 or more. The upper limit of the proportion of chondroitin sulfate is 80% or more, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or 100%, and the lower limit is 40% or more, preferably 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% or more.

[0065] The total proportion of chondroitin sulfate with a weight-average molecular weight of 25,000 to 50,000, the proportion of chondroitin sulfate with a weight-average molecular weight of 400 to 25,000, and the proportion of chondroitin sulfate with a weight-average molecular weight of 400 or less is 100%.

[0066] In some preferred embodiments of the present invention, for the chondroitin sulfate, in terms of the GPC integration ratio, the proportion of chondroitin sulfate with a weight-average molecular weight of 400 to 10,000 is 40% to 100%, preferably 50% to 100%, 60% to 100%, 70% to 100%.

[0067] In some more preferred embodiments of the present invention, in terms of the GPC integration ratio, the molecular weight distribution of the chondroitin sulfate is as follows: the proportion of chondroitin sulfate with a weight average molecular weight exceeding 50,000 is 0%, the proportion of chondroitin sulfate with a weight average molecular weight of 25,000 to 50,000 is 0 to 40%, the proportion of chondroitin sulfate with a weight average molecular weight of 400 to 10,000, preferably the proportion of chondroitin sulfate with a weight average molecular weight of 400 to 8,000 is 40% to 100%, and the proportion of chondroitin sulfate with a weight average molecular weight of 400 or less is 15% or less.

[0068] As described hereinafter and in the examples, the chondroitin sulfate biopolyamine complex provided by the present invention can be prepared from natural raw materials, and the chondroitin sulfate and biopolyamine present in the natural raw materials can be coprecipitated to form a complex bound by non-covalent bonds between the two. Therefore, the mixing ratio of chondroitin sulfate and biopolyamine present in the complex is the same as or close to the ratio of the two in the natural raw materials used, and it is understood that any mixing ratio below the saturation state of the biopolyamine bound to chondroitin sulfate has activity.

[0069] In some embodiments, based on the total weight (g) of the chondroitin sulfate biopolyamine complex, the biopolyamine contained in the chondroitin sulfate biopolyamine complex is 200 μmol / g or less; preferably, 199 μmol / g, 198 μmol / g, 197 μmol / g, 196 μmol / g, 195 μmol / g, 194 μmol / g, 193 μmol / g, 192 μmol / g, 191 μmol / g, 190 μmol / g, 189 μmol / g, 188 μmol / g, 187 μmol / g, 186 μmol / g, 185 μmol / g, 184 μmol / g, 183 μmol / g, 182 μmol / g, 181 μmol / g, or 180 μmol / g or less.

[0070] In some preferred embodiments, with respect to the total weight (g) of the chondroitin sulfate biopolyamine complex, the biopolyamine contained in the chondroitin sulfate biopolyamine complex is 0.5 μmol / g or more; preferably, 0.6 μmol / g, 0.7 μmol / g, 0.8 μmol / g, 0.9 μmol / g, 1 μmol / g or more.

[0071] In some more preferred embodiments, with respect to the total weight (g) of the chondroitin sulfate biopolyamine complex, the chondroitin sulfate biopolyamine complex contains the biopolyamine in an amount of 0.5 to 200 μmol / g, preferably 0.6 to 200 μmol / g, 0.7 to 200 μmol / g, 0.8 to 200 μmol / g, 0.9 to 200 μmol / g, more preferably 1 to 200 μmol / g, 1 to 195 μmol / g, 1 to 190 μmol / g, 1 to 185 μmol / g, 1 to 180 μmol / g. In some preferred embodiments, the biopolyamine is a combination of one, two or more of spermine, spermidine, putrescine and cadaverine in any ratio.

[0072] In some embodiments, the chondroitin sulfate is chondroitin sulfate in acid form (chondroitin sulfate acid) or chondroitin sulfate in salt form. In some preferred embodiments, the chondroitin sulfate is chondroitin sulfate in acid form. Chondroitin sulfate in acid form binds more tightly to the biopolyamine and can bind to more polyamine molecules, enabling the chondroitin sulfate biopolyamine complex to have more excellent physiological activity.

[0073] As used herein, "low molecular weight CS", "low molecular weight chondroitin", or "low molecular weight chondroitin sulfate" refers to chondroitin sulfate having a weight average molecular weight of 400 to 8,000, and is obtained by decomposing chondroitin sulfate by methods such as enzymatic decomposition, acid decomposition, oxidative free radical decomposition, and radiation decomposition. The chondroitin sulfate biopolyamine complex containing low molecular weight CS is referred to as low molecular weight CSX or low molecular weight chondroitin sulfate biopolyamine complex. As used herein, "ordinary chondroitin" or "ordinary CS" refers to ordinary chondroitin obtained commercially.

[0074] <Method for preparing chondroitin sulfate biopolyamine complex and chondroitin sulfate biopolyamine obtained by the preparation method> The present invention has found that chondroitin sulfate polyamine has significantly improved activity in many aspects compared to chondroitin sulfate and can be extracted and prepared from natural raw materials. However, in the conventional extraction method of chondroitin sulfate, in order to ensure the properties such as the purity and color of chondroitin, usually, processes such as ion resin exchange purification, decolorization with hydrogen peroxide, and alcohol precipitation and dissolution more than twice are adopted. Although these processes improve the chondroitin purity, they almost completely remove polyamines. The complexation of chondroitin sulfate and biopolyamine and its effects are not mentioned at all. The conventional method usually requires a high ionic strength, which greatly affects non-covalent bonds, and even if a small amount of polyamine is present, the biopolyamine complex of the present invention cannot be formed.

[0075] In view of the above problems, in some aspects of the present invention, there is provided a method for preparing a chondroitin sulfate biopolyamine complex, which includes a step of mixing an extract containing chondroitin sulfate and polyamine separated and extracted from a raw material with ethanol, wherein the pH of the extract is 4 to 6, and the volume ratio of the extract to ethanol is 1:1 to 3.

[0076] In the method for preparing a chondroitin sulfate biopolyamine complex provided by the present invention, under certain conditions, an extract containing chondroitin sulfate and polyamine separated and extracted from a raw material is mixed with ethanol, thereby obtaining a chondroitin sulfate biopolyamine complex that has significantly better anti-inflammatory activity than ordinary chondroitin sulfate and has effects such as repair of joint damage, reduction of blood lipids, antioxidation, delay of aging, and extension of lifespan.

[0077] In some specific embodiments, the pH of the extract may be 4, 4.5, 5, 5.5, or 6. In some preferred embodiments, the pH of the extract is 5. Different from the neutral pH used in the extraction and precipitation of conventional chondroitin sulfate, the extract under the above pH conditions is advantageous for the formation of the chondroitin sulfate biopolyamine complex. For example, it can increase the content of biopolyamine that can coprecipitate with chondroitin sulfate to form a complex, thereby promoting the formation of the chondroitin sulfate biopolyamine complex.

[0078] In some specific embodiments, the volume ratio of the extract to ethanol is 1:1 to 2.5, such as 1:1 or 1:2.5. In some preferred embodiments, the volume of ethanol accounts for 60 to 70% of the volume of the mixture of the extract and ethanol. At this volume ratio, it is more suitable for the formation of the chondroitin sulfate biopolyamine complex. Different from the method of pursuing high-purity chondroitin sulfate at this volume ratio, although some chondroitin sulfate is lost, the content of polyamine can be increased, protein impurities can be reduced, and the formation of the chondroitin sulfate biopolyamine complex can be promoted. In contrast, under conventional conditions, a chondroitin sulfate biopolyamine complex is usually not obtained. In some specific embodiments, the ethanol is anhydrous ethanol.

[0079] (Origin of raw material) In the present invention, the origin of the raw material is not particularly limited, and any raw material containing chondroitin sulfate and / or polyamine may be used, such as animal tissues, plant tissues, and microbial culture fermentation broths.

[0080] In some embodiments, the raw material may be animal connective tissues including cartilage, bone, tendon, fascia, and blood vessel walls. The animal may be a terrestrial animal such as cattle, pigs, chickens, or a marine animal such as fish. Those skilled in the art can understand that the above raw materials contain chondroitin sulfate and polyamine components. In some embodiments, the raw material may further include raw materials containing polyamine components such as soybeans and wheat germ.

[0081] The types of chondroitin sulfate contained in the chondroitin sulfate biopolyamine complex obtained from the origin of the raw material also vary. Usually, CS extracted from terrestrial (animal) origin is mainly a mixture of CSA and CSC, CS extracted from marine (animal) origin is mainly a mixture of CSA, CSC, and CSD, and a part also contains CSE. And depending on the raw materials of different animal species, the proportions of various types of CS contained also vary. Therefore, in the chondroitin sulfate biopolyamine complex obtained by the preparation method of the chondroitin sulfate biopolyamine complex provided by the present invention, the chondroitin sulfate may be any one or an arbitrarily combined mixture of CSA, CSC, CSD, and CSE in any proportion.

[0082] In some preferred embodiments, chicken bones can be selected as the raw material. Chicken bones generally contain a large amount of biopolyamines, are cheaper, have stable supply quality, and are easy to obtain in large quantities.

[0083] (Pretreatment) Before implementing the method of the present invention, it is usually necessary to perform a pretreatment step to make the raw materials suitable for implementing the method of the present invention (for example, enzymatic hydrolysis, acid hydrolysis, separation and extraction, etc.). There are no particular restrictions on the pretreatment method, and those skilled in the art can select an appropriate pretreatment method according to different raw materials. For example, for raw materials such as bones and cartilages, the pretreatment method may include boiling, removal of impurities, washing, drying, grinding and pulverization, etc. In the case of soybeans, the pretreatment method may include grinding and pulverization.

[0084] (Separation and extraction of chondroitin sulfate and polyamine in raw materials) Before mixing with ethanol and precipitating to form a chondroitin sulfate biopolyamine complex, by separating and extracting chondroitin sulfate and polyamine in the raw materials, the ratio of chondroitin sulfate and polyamine in the raw materials can be increased, a higher purity chondroitin sulfate biopolyamine complex can be obtained, the presence of impurities such as inorganic salts and proteins can be reduced, and the influence of impurities on the subsequent complexation and precipitation processes can be decreased.

[0085] In some embodiments of the present invention, when separating and extracting chondroitin sulfate and polyamine in the raw materials, first, the raw materials are enzymatically hydrolyzed or acid hydrolyzed, and the raw materials are decomposed by enzymatic hydrolysis or acid hydrolysis to obtain an enzymatic hydrolysis solution or an acid hydrolysis solution so that components such as chondroitin sulfate and / or polyamine in the raw materials are sufficiently released.

[0086] Insufficient enzymatic hydrolysis or acid hydrolysis affects the release of polyamine. Therefore, in some embodiments, the raw materials are sufficiently enzymatically hydrolyzed or acid hydrolyzed. Those skilled in the art can select an appropriate amount of enzymatic hydrolysis or acid hydrolysis reagent to sufficiently enzymatically hydrolyze or acid hydrolyze the raw materials.

[0087] In some embodiments, the enzymes selectable for enzymatic degradation include one or more of papain, alkaline protease, neutral protease, acidic protease, pepsin, trypsin, chymotrypsin, bromelain, and ficin. In some preferred embodiments, 4,000 U or more, preferably 10,000 U or more, 16,000 U or more, 20,000 U or more, 30,000 U or more, 40,000 U or more, 50,000 U or more of the enzyme is added per gram of the pretreated sample. The lower the enzyme activity, the less able the protein can be completely hydrolyzed and the less able the polyamine molecules can be released from the raw material. And in the process of precipitating the protein with trichloroacetic acid, the polyamine binds to the large protein and is more likely to precipitate, further reducing the extraction amount of the polyamine and affecting the activity of the product. In some embodiments, the temperature of enzymatic degradation is 50°C to 70°C, preferably 55°C to 65°C, such as 55°C, 60°C, 65°C. In some embodiments, the time of enzymatic degradation is 1 to 24 hours, preferably 3 to 24 hours, 3 to 16 hours, such as 3 hours, 6 hours, 16 hours. In some embodiments, the enzymatic degradation may be carried out once. In another embodiment, the enzymatic degradation may be carried out 2 times or 3 times or more.

[0088] In some embodiments, for acid degradation, organic acids and / or inorganic acids can be used. In some embodiments, the organic acid may include one or more of trifluoroacetic acid, trichloroacetic acid, formic acid, and acetic acid. In some embodiments, the inorganic acid may include one or more of hydrochloric acid, sulfuric acid, and nitric acid. In some embodiments, in order to improve the acid degradation efficiency, fully decompose the raw material, and release components such as chondroitin sulfate and / or polyamine, methods such as ultrasonic extraction, addition, and grinding can be combined to assist in acid degradation.

[0089] In some embodiments of the present invention, chondroitin sulfate and polyamine are separated and extracted from an enzymatically digested solution or an acid digested solution obtained by enzymatic digestion or acid digestion. In some embodiments, chondroitin sulfate and polyamine can be separated and extracted step by step. By extracting step by step, polyamine can be extracted to the maximum extent, the weight ratio of polyamine in the chondroitin sulfate - biopolyamine complex can be increased, and further impurities (such as proteins and inorganic salts) can be reduced. Also, further operations can be performed on chondroitin sulfate alone, such as further purification, removal of chondroitin sulfate in salt form, or reduction of the molecular weight of chondroitin sulfate. In another embodiment, chondroitin sulfate and polyamine can be separated and extracted simultaneously.

[0090] In some specific embodiments, when chondroitin sulfate and polyamine are separated and extracted simultaneously, usually the raw material is treated by enzymatic digestion. In some embodiments, an acid is added to the enzymatically digested solution to precipitate the proteins in the enzymatically digested solution, and the proteins in the enzymatically digested solution can be removed. In some specific embodiments, after removing the proteins in the enzymatically digested solution, the concentration of chondroitin sulfate in the obtained preliminary extract may be in the range of about 6 - 7% (w / v). In this concentration range, it is more advantageous for obtaining the chondroitin sulfate - biopolyamine complex in subsequent steps. In this concentration range, the content of biopolyamine contained in the preliminary extract of chondroitin sulfate is higher, which is more advantageous for complexation with chondroitin sulfate.

[0091] After concentrating and / or adjusting the pH of the solution from which proteins have been removed, ethanol is added for complexation and precipitation to obtain a chondroitin sulfate - biopolyamine complex. In some specific embodiments, as the acid used for precipitating proteins, trichloroacetic acid, perchloric acid, nitric acid, etc. can be selected.

[0092] When chondroitin sulfate and polyamines are separated and extracted step by step, in some embodiments, the polyamines in the sample are separated by chromatography or extraction method, and then the residue is subjected to one or more of enzymatic degradation, protein precipitation, chromatography, and alcohol precipitation to purify the chondroitin sulfate in the residue. The purified chondroitin sulfate is mixed with the separated polyamines, ethanol is added for complexation and precipitation to obtain a chondroitin sulfate-biogenic polyamine complex.

[0093] In some specific embodiments, the chromatography is ion exchange chromatography. In some specific embodiments, the extraction method is an organic solvent extraction method or a supercritical extraction method. In a more specific embodiment, the organic solvent used in the organic solvent extraction method includes one or more of n-butanol, dichloromethane, chloroform, and diethyl ether. In another more specific embodiment, the supercritical extraction method is a supercritical carbon dioxide oxidation extraction method.

[0094] In some specific embodiments, when chondroitin sulfate and polyamines are separated and extracted step by step, it further includes a step of reducing the molecular weight of chondroitin sulfate. In some embodiments, for example, for chondroitin sulfate purified by one or more of enzymatic degradation, protein precipitation, chromatography, and alcohol precipitation, the method described in CN111495428A (such as Example 2) is used for molecular weight reduction to obtain low molecular weight chondroitin sulfate. In some embodiments, the low molecular weight chondroitin sulfate is mixed with the separated polyamines, ethanol is added for complexation and precipitation to obtain a chondroitin sulfate-biogenic polyamine complex.

[0095] In some specific embodiments, in the extract containing the chondroitin sulfate-biogenic polyamine complex obtained by the above method for preparing the chondroitin sulfate-biogenic polyamine complex, after separation from chondroitin sulfate, the detected total content (mass ratio) of polyamines is 0.01% - 5%.

[0096] (Other processes) For other processes of the method for preparing the chondroitin sulfate biopolyamine complex, there are no particular restrictions, but they can be adjusted or selected according to the actual apparatus or as necessary. In some embodiments, by including a purification step of the product, a higher purity chondroitin sulfate biopolyamine complex can be obtained. However, it is understood that the chondroitin sulfate biopolyamine complex obtained by the above preparation method can exert its function.

[0097] In another embodiment of the present invention, the chondroitin sulfate biopolyamine complex provided by the present invention can be prepared by mixing chondroitin sulfate and a biopolyamine, and the biopolyamine is contained in the chondroitin sulfate biopolyamine complex in an amount of 0.5 to 200 μmol / g based on the total weight of the chondroitin sulfate biopolyamine complex.

[0098] In some specific embodiments, the chondroitin sulfate and the biopolyamine may be commercially available products. In another specific embodiment, the chondroitin sulfate and the biopolyamine can be extracted from the above-mentioned raw materials respectively.

[0099] In a more specific embodiment, the chondroitin sulfate and the biopolyamine can be mixed in a solvent and dried for preparation. In some exemplary embodiments, the solvent is water. In some exemplary embodiments, the drying is freeze-drying.

[0100] Some aspects of the present invention further provide a chondroitin sulfate biopolyamine complex prepared by the above preparation method.

[0101] <Pharmaceutical compositions, health foods, and cosmetics> Some aspects of the present invention further provide a pharmaceutical composition comprising the chondroitin sulfate biopolyamine complex or a chondroitin sulfate biopolyamine complex obtained by the method for preparing the chondroitin sulfate biopolyamine complex, and a pharmaceutically acceptable carrier.

[0102] Some other aspects of the present invention further provide a health food comprising the chondroitin sulfate biopolyamine complex or a chondroitin sulfate biopolyamine complex obtained by the method for preparing the chondroitin sulfate biopolyamine complex.

[0103] Some other aspects of the present invention further provide a cosmetic comprising the chondroitin sulfate biopolyamine complex or a chondroitin sulfate biopolyamine complex obtained by the method for preparing the chondroitin sulfate biopolyamine complex.

[0104] <Anti-inflammatory Use and Method of Chondroitin Sulfate Biopolyamine Complex> Some aspects of the present invention relate to the use of a chondroitin sulfate biopolyamine complex in the preparation of an anti-inflammatory agent, wherein the chondroitin sulfate biopolyamine complex is the chondroitin sulfate biopolyamine complex or a chondroitin sulfate biopolyamine complex obtained by the method for preparing the chondroitin sulfate biopolyamine complex.

[0105] In some embodiments, it relates to the use of a chondroitin sulfate biopolyamine complex in the preparation of a medicament for the treatment and / or prevention of an inflammatory disease, wherein the chondroitin sulfate biopolyamine complex is the chondroitin sulfate biopolyamine complex or a chondroitin sulfate biopolyamine complex obtained by the method for preparing the chondroitin sulfate biopolyamine complex.

[0106] In some embodiments of the present invention, the inflammatory disease is an inflammatory disease caused by various inflammation-inducing factors and / or an inflammatory disease caused by inflammatory cells, inflammatory factors such as Interleukin (IL) and / or Tumor Necrosis Factor (TNF). In some specific embodiments, the inflammatory factor includes one or two of interleukin 6 (IL-6) and interleukin 1β (IL-1β).

[0107] In some embodiments of the present invention, the inflammatory disease is a chronic inflammatory disease.

[0108] In some embodiments of the present invention, the inflammatory disease is one or more selected from allergy, eczema, myocardial infarction, cerebral infarction, Alzheimer's disease, dermatitis or arthritis.

[0109] In some embodiments of the present invention, the inflammatory disease is arthritis. In some more specific embodiments of the present invention, the arthritis is chronic arthritis, such as rheumatoid arthritis. More specifically, the arthritis is rheumatoid arthritis.

[0110] Some aspects of the present invention are methods for treating and / or preventing an inflammatory disease, comprising the step of administering to a subject a therapeutically effective amount or a prophylactically effective amount of a chondroitin sulfate biopolyamine complex, wherein the chondroitin sulfate biopolyamine complex is the above chondroitin sulfate biopolyamine complex or a chondroitin sulfate biopolyamine complex obtained by the above method for preparing a chondroitin sulfate biopolyamine complex.

[0111] <Use and Method for Reducing Blood Lipids of Chondroitin Sulfate Biopolyamine Complex> Some other aspects of the present invention further found that the above chondroitin sulfate biopolyamine complex or a chondroitin sulfate biopolyamine complex obtained by the above method for preparing a chondroitin sulfate biopolyamine complex has the effect of reducing blood lipids.

[0112] Accordingly, in some embodiments, there is provided the use of a chondroitin sulfate biopolyamine complex in the preparation of a medicament for reducing blood lipids, and in another embodiment, there is provided the use of a chondroitin sulfate biopolyamine complex in the preparation of a medicament for the treatment and / or prevention of hyperlipidemia, wherein the chondroitin sulfate biopolyamine complex is the chondroitin sulfate biopolyamine complex or a chondroitin sulfate biopolyamine complex obtained by the method for preparing the chondroitin sulfate biopolyamine complex.

[0113] In some specific embodiments, the hyperlipidemia may be primary hyperlipidemia and / or secondary hyperlipidemia. In some specific embodiments, the hyperlipidemia may be hypertriglyceridemia and / or hypercholesterolemia.

[0114] In some specific embodiments, the medicament for the treatment and / or prevention of hyperlipidemia can also be applied to conditions associated with (e.g., inducing or exacerbating) hyperlipidemia, such as, but not limited to, cardiovascular diseases such as arteriosclerosis, coronary artery disease, angina pectoris, carotid artery disease, stroke, cerebral arteriosclerosis, myocardial infarction, cerebral infarction, restenosis after balloon angioplasty, hypertension, intermittent claudication, dyslipidemia, postprandial hyperlipidemia, and xanthomatosis.

[0115] Some aspects of the present invention are methods for treating and / or preventing hyperlipidemia, comprising the step of administering to a subject a therapeutically effective amount or a prophylactically effective amount of a chondroitin sulfate biopolyamine complex, wherein the chondroitin sulfate biopolyamine complex is the chondroitin sulfate biopolyamine complex or a chondroitin sulfate biopolyamine complex obtained by the method for preparing the chondroitin sulfate biopolyamine complex.

[0116] Another aspect of the present invention is the use of a chondroitin sulfate biopolyamine complex in the preparation of a health food for assisting in blood lipid regulation (lowering total cholesterol and triglycerides) and lowering blood lipids, which contributes to maintaining healthy levels of blood lipids (cholesterol / triglycerides), wherein the chondroitin sulfate biopolyamine complex is the chondroitin sulfate biopolyamine complex or a chondroitin sulfate biopolyamine complex obtained by the method for preparing the chondroitin sulfate biopolyamine complex as described above.

[0117] <Other uses of the chondroitin sulfate biopolyamine complex> The present invention has further found that the chondroitin sulfate biopolyamine complex or a chondroitin sulfate biopolyamine complex obtained by the method for preparing the chondroitin sulfate biopolyamine complex as described above has effects such as treating and repairing joint damage, antioxidation, delaying aging, and extending lifespan.

[0118] Therefore, in some embodiments, it provides the use of a chondroitin sulfate biopolyamine complex in the preparation of a pharmaceutical for treating and / or repairing joint damage, wherein the chondroitin sulfate biopolyamine complex is the chondroitin sulfate biopolyamine complex or a chondroitin sulfate biopolyamine complex obtained by the method for preparing the chondroitin sulfate biopolyamine complex as described above.

[0119] In some specific embodiments, the joint damage may be joint damage caused by inflammation, aging, exercise, injury, etc.

[0120] Another aspect of the present invention is the use of a chondroitin sulfate biopolyamine complex in the preparation of a health food, wherein the chondroitin sulfate biopolyamine complex is the chondroitin sulfate biopolyamine complex or a chondroitin sulfate biopolyamine complex obtained by the method for preparing the chondroitin sulfate biopolyamine complex. In some specific embodiments, there is provided the use of a chondroitin sulfate biopolyamine complex in the preparation of a health food that contributes to the improvement of bone density.

[0121] In some embodiments, there is provided the use of a chondroitin sulfate biopolyamine complex in the preparation of a pharmaceutical for antioxidant, anti-aging and / or life extension purposes, wherein the chondroitin sulfate biopolyamine complex is the chondroitin sulfate biopolyamine complex or a chondroitin sulfate biopolyamine complex obtained by the method for preparing the chondroitin sulfate biopolyamine complex.

[0122] In some specific embodiments, the pharmaceutical achieves an antioxidant effect by removing oxygen free radicals.

[0123] Another aspect of the present invention is the use of a chondroitin sulfate biopolyamine complex in the preparation of a health food that contributes to antioxidant, wherein the health food helps to maintain the balance between the oxidation process and the antioxidant process in the human body, and the chondroitin sulfate biopolyamine complex is the chondroitin sulfate biopolyamine complex or a chondroitin sulfate biopolyamine complex obtained by the method for preparing the chondroitin sulfate biopolyamine complex.

[0124] Another aspect of the present invention is the use of a chondroitin sulfate biopolyamine complex in the preparation of cosmetics, wherein the chondroitin sulfate biopolyamine complex is the chondroitin sulfate biopolyamine complex or a chondroitin sulfate biopolyamine complex obtained by the method for preparing the chondroitin sulfate biopolyamine complex described above. In some specific embodiments, it provides the use of a chondroitin sulfate biopolyamine complex in the preparation of cosmetics that contribute to antioxidant effects. Examples and Test Examples

Examples

[0125] Hereinafter, the present invention will be further described with reference to examples and test examples, but the present invention is not limited thereto. Specific materials used in the embodiments of the present invention and their sources are shown below. However, these are merely examples and do not limit the present invention. It should be understood that materials identical or similar to the following types, model numbers, qualities, properties or functions of reagents and equipment can be used to implement the present invention. The experimental methods in the following examples and test examples are conventional methods unless otherwise specified. Materials, reagents, etc. used in the following examples and test examples can all be obtained commercially unless otherwise specified.

[0126] In the following examples, the method for measuring the molecular weight of chondroitin sulfate / chondroitin sulfate biopolyamine complex is as follows. The molecular weight and its distribution of chondroitin sulfate / chondroitin sulfate biopolyamine complex were measured using high-performance gel permeation chromatography (GPC). Dextran molecular weight standard sample was used as the standard sample. The mobile phase was 0.2 M aqueous sodium sulfate solution. The chromatography conditions are as follows. Column: TSKgel G3000PWXL 7.8*300mm Flow rate: 0.5 mL / min Detector: Differential refractive index detector Column temperature: 35°C Injection volume: 30 μL.

[0127] In the following examples, the measurement of polyamine is carried out in accordance with GB5009.208-2016.

[0128] The basic principle of the following examples is that there is a strong non-covalent bond between biological polyamine and chondroitin sulfate. Therefore, by adjusting the conditions such as pH and concentration of biological polyamine and chondroitin sulfate in the solution, and precipitating them by the method of ethanol precipitation, the purpose of extraction and purification can be achieved. However, since there are many other molecules (amino acids, peptides, proteins, fats, inorganic salts, etc.) in biological tissues, different extraction processes are used according to different materials and needs to exclude the interference of other substances and further improve the purity and polyamine content.

[0129] Example: Extraction of chondroitin sulfate biological polyamine complex Example 1. Expression and verification of chondroitin sulfate biological polyamine complex In this example, the chondroitin sulfate biological polyamine complex was integrated, separated and precipitated from animal bone tissue. Before confirming that the activity is derived from chondroitin sulfate polyamine substance, various analyses and evaluations were carried out in this example.

[0130] 1. Extraction In this example, chicken bones were used as raw materials. (1) Pretreatment of raw materials The fresh raw bones were boiled to remove impurities such as blood, grease, and remaining meat residues, and then the bones were washed with clean water, dried, and ground into powder.

[0131] (2) Separation and extraction of chondroitin sulfate and polyamine in raw materials 2.1 Enzymatic hydrolysis Weighed 5 g of pretreated chicken bones each, and added 100 mL of water with pH 5.7 - 6.0 and 2% enzyme respectively based on the mass of the raw chicken bones as follows. 0.1 g of papain (200 U / mg, Bisons Biotech Co., Ltd.) (NO.A - 2); 0.1 g of papain (2,000 U / mg, Adamas reagent) (NO.A-3); 0.1 g of papain (800 U / mg, Bisons Biotech) (NO.A-4); or, 0.1 g of trypsin (250 U / mg, Aladdin Reagent Company) and 0.1 g of pepsin (3,000 U / mg, Aladdin Reagent Company) (NO.A-6). Stir evenly, raise the temperature of the system to 65 °C, keep it warm for 3 hours for reaction, and enzymatically decompose the pretreated chicken bones to obtain an enzymatic decomposition solution. 2.2 Separation and extraction of chondroitin sulfate and polyamine After the enzymatic decomposition reaction was completed, the enzymatic decomposition solutions obtained in Step 2.1 were each filtered with filter paper. To the filtrates obtained after filtration, 5.0 g of trichloroacetic acid (Aladdin Reagent Company, China) was added respectively, kept in an ice bath for 1 hour, mixed well, centrifuged at 8,000 rpm / min for 5 minutes, and the supernatant was taken out to remove excess protein.

[0132] (3) Complexation and precipitation of chondroitin sulfate biopolyamine complex The supernatants obtained in Step 2.2 were each adjusted to a pH of about 5 with solid sodium hydroxide and concentrated under reduced pressure to about 10 mL of yellow liquid (without solid precipitation) respectively. 25 mL of absolute ethanol was added to each of the yellow liquids for alcohol precipitation, shaken, centrifuged to obtain extracts (chondroitin sulfate biopolyamine complexes) respectively. After that, the centrifuged alcohol precipitation solids were washed once again with absolute ethanol, centrifuged, and then vacuum dried at 40 °C overnight to obtain 600 mg of white solid as the extract (which was later verified to be the chondroitin sulfate biopolyamine complex).

[0133] 2. Analysis of the extract Analysis and verification were carried out on the extracts extracted after the above different enzymatic treatments. First, the inhibition level of IL-6, i.e., the anti-inflammatory activity, was measured. Specifically, mouse monocytes RAW264.7 were used. The cells were digested and seeded at 10,000 cells per well in a 96-well plate. After cell adhesion, dexamethasone (DXM) and CSX were diluted in DMEM medium containing 10% FBS with 0.1 μg / ml of LPS. The final concentration of dexamethasone was 0.1 mg / ml, and the final concentration of CSX was 0.1 mg / ml. The cells were cultured for 18 - 24 hours, the supernatant was aspirated, and the IL-6 level in the supernatant was detected by enzyme-linked immunosorbent assay (Elisa).

[0134] Figure 14 is a diagram showing the IL-6 inhibition levels after the action of each extract and conventional chondroitin. NO.A-2, A-3, A-4, A-6 are the extracts extracted after the respective enzyme treatments, NO.A-1 is the chondroitin provided by the company (Wuxing Biology, Hunan), and NO.A-5 is the chondroitin purchased from a reagent company (Aladdin, Shanghai). From Figure 14, it can be seen that the extract NO.A-3 has a significantly better inhibitory effect on IL-6 than other extracts or chondroitin, followed by NO.A-6, but there is no significant difference among the chondroitins of NO.A-1, A-4, and A-5. When the chondroitin extract (NO.A-3) with excellent anti-inflammatory activity was selected for 1H nuclear magnetic resonance spectrum test (solvent: heavy water, 600 MHz, Agilent, USA), it was found that in addition to the hydrogen atom signal of the chondroitin molecule, obvious new signals appeared at chemical shifts of 1.7, 1.9, and 3.0 ppm (Figure 15). Figure 16 is a partial enlarged view of the characteristic peak of the 1H nuclear magnetic resonance spectrum of each extract up to (1.7 ppm). In Figure 16, A, B, C, and D are the extracts using the above different extraction methods, corresponding to the NO.A-6, A-4, A-3, and A-2 extracts respectively. The relative integral area is C > A > B > D, and the order of the inhibitory activities of the corresponding extracts against inflammatory factors is A-3 > A-6 > A-4 > A-2, and the integral area of the characteristic peak corresponds to the magnitude of the activity.

[0135] To further explore the origin of the activity, the components in the extract were separated, and chondroitin and substances with chemical shifts of 1.7, 1.9, and 3.0 ppm were separated. The separation method is as follows. 100 g of A-3 extract and 200 g of cetylpyridinium chloride (CPC) (InNoChem LOT: KYGA540) were added to 2 L of purified water. Chondroitin and CPC underwent electrostatic interaction to produce a white precipitate. After stirring at room temperature for 1 hour and then centrifuging (7000 rpm, 10 °C, 15 minutes), it was found that it precipitated as a complex of CPC and chondroitin, while the supernatant contained the substance components with chemical shifts of 1.7, 1.9, and 3.0 ppm. The above chondroitin and CPC were co-precipitated, added to a 10% aqueous NaCl solution, and heated and stirred at 60 °C to dissociate the precipitate. Then, three times the solution volume of absolute ethanol was added, and it precipitated as chondroitin sulfate. After repeating the ethanol precipitation operation twice, the precipitate after filtration was dried, and 81 g of a white solid with a purity of 95.5% was obtained. It was found by nuclear magnetic resonance test that the characteristic peaks of the above chemical shifts disappeared. Regarding the supernatant containing substance components with chemical shifts of 1.7, 1.9, and 3.0 ppm, it was washed three times with dichloromethane (200 mL / time) to remove excess CPC, the aqueous phase was recovered, approximately 280 mL of absolute ethanol was added to produce a precipitate (substances such as salts, proteins, and excess chondroitin), which was removed by centrifugation, and the supernatant was freeze-dried for 48 hours to obtain a white solid. After dissolving the white solid, it was further separated using a gel chromatography column under the following conditions. The separation apparatus was an AKTA pure protein purification system (USA), and the column was a GE Superdex G30increase 10 / 300GL (USA). The mobile phase was water, and the flow rate was 0.5 ml / min. The detection wavelengths were 210 nm and 260 nm. Fractions were collected respectively (the chromatogram is shown in Figure 17, and the numbers in the figure are the collected fraction numbers). When the activity measurement against the inflammatory factor IL-6 was performed again by the above method, it was found that Fraction No. 3 was much higher than other components. Fraction A-3 was further concentrated, and nuclear magnetic resonance and mass spectrometry were performed. It was found that they were three types of biogenic polyamines, spermine, spermidine, and putrescine, and it was confirmed that the molar ratio of these three types of amines was close to 1:1:1.

[0136] To further verify the existence forms of CS and polyamines in the extract, the binding of CS and polyamines was confirmed by the following experiment.

[0137] 1. The No. 3 extract was dialyzed with a cut-off molecular weight of the dialysis bag of 10 kD for 5 days, and the water was changed every 8 hours. After dialysis, the retention solution was freeze-dried. When the polyamine content was measured, no decrease in the polyamine content was observed.

[0138] 2. Reaction of fluorescein isothiocyanate (FITC) and chondroitin polyamine Experimental method: 50 mg of the No.A-3 extract was dissolved in 10 mL of purified water, adjusted to pH 9 - 10 with 6 mol / L sodium hydroxide solution, then 1 mL of an aqueous solution containing 10 mg of FITC was added, wrapped with tin foil, and reacted overnight at room temperature in the dark. After dialysis for 5 days using a 1000D dialysis membrane, three volumes of absolute ethanol were added to obtain a yellow precipitate. After filtration, it was washed once with absolute ethanol to obtain a yellow solid, which was dried under vacuum overnight. The same operation was carried out using normal chondroitin as a control. Normal chondroitin is white, while active chondroitin is yellow, showing an obvious color difference. This indicates that a condensation reaction occurs between the FITC molecule and the amino group of the polyamine molecule. Although some amino groups have a weakened bond with CS due to the formation of amides, unreacted amino groups with a strong binding force to CS still exist. Since the polyamine molecule is still bound to the polymer chain of CS, an obvious color remains after dialysis. On the other hand, since normal chondroitin cannot react with the FITC molecule, the free FITC molecules are removed. Normal chondroitin is bovine-derived chondroitin provided by Hunan Wuxing Biotechnology Company, with a weight-average molecular weight of 21k, a proportion of over 50,000 being 0%, a proportion of 25,000 - 50,000 being 31%, a proportion of 400 - 25,000 being 69%, and a proportion of chondroitin sulfate with a molecular weight of less than 400 being 0%.

[0139] 3. ITC reaction To verify the presence and magnitude of the affinity between chondroitin molecules and polyamine molecules, the binding constant between chondroitin molecules and polyamine molecules was measured by Isothermal Titration Calorimetry (ITC) (MicroCal TM iTC200). The chondroitin molecule was titrated with the polyamine molecule as a ligand, and the parameters were set so that the binding molar ratio was 1:1. The chondroitin molecule was dissolved in water at a concentration of 20 μM, and the polyamine molecule was also dissolved in water at a concentration of 200 μM. Titration was performed 17 times with 2 μL per drop. As shown in Fig. 19 of the experimental results, the binding constant KD = 1 / KA = 5 μM, the stoichiometric ratio N = 1.74, and the enthalpy ΔH = 2.29 Kcal / mol between chondroitin molecules and polyamine molecules. It was found that chondroitin has a strong binding force with polyamine molecules.

[0140] As a result of the above tests, the main active components in extracts NO.A-2, A-3, A-4, and A-6 are chondroitin sulfate biopolyamine complexes containing spermine, spermidine, and putrescine. When separated and measured, the results of the polyamine content (molar mass ratio), chondroitin sulfate mass ratio, protein mass ratio, and weight-average molecular weight with respect to the total weight of the extract are shown in Table 1 below. The measurement method of the mass ratio of chondroitin sulfate refers to sodium chondroitin sulfate in Chinese Pharmacopoeia 2020, and the standard sample is chondroitin sulfate (National Institutes for Food and Drug Control). The measurement method of the protein content was measured by GPC using the Lowry molecular weight. However, the large molecular weights of A-2 and A-4 may be due to the fact that the covalent bonds between proteins and multiple polysaccharide molecules have not been completely broken. The presence of proteins may lead to a decrease in the anti-inflammatory activity of the complex and even a pro-inflammatory effect. For example, NO.A-2 has a certain pro-inflammatory effect, which is considered to be due to the too high content of impurities such as proteins. On the other hand, NO.A-6 does not have a good anti-inflammatory effect, which may also be due to the high protein content.

[0141]

Table 1

[0142] To further verify the origin of the activity, it was formulated according to the proportion of polyamines in extract NO.A-3. In addition, three types of polyamines and chondroitin were each formulated in the same molar amount, and the IL-6 inhibitory ability was measured by the method described above. The formulation method was to dissolve chondroitin sulfate (Sigma, USA) in water, and then add spermine tetrahydrochloride (Acros, USA), spermidine trihydrochloride (Acros, USA), and putrescine dihydrochloride (Acros, USA) respectively or simultaneously, stir uniformly, and then lyophilize to obtain a white solid. Here, the weight-average molecular weight of chondroitin sulfate is 20,100, the proportion of molecular weight 400 - 25,000 is 71%, the proportion of 25,000 - 50,000 is 29%, and the proportion of 400 or less is 0%.

[0143] Figure 18 is a diagram showing the measurement of the anti-inflammatory activity of various substances against IL-6. NO.B-1 is a combination of biogenic polyamines (spermine + spermidine + putrescine, the mixing ratio of the three polyamines is the same as that of extract NO.B-4, and the total molar amount of the three polyamines is the same as the total molar amount of the polyamines contained in the amount of extract NO.B-4 used), NO.B-2 is a mixture of CS and polyamines (CS + spermine + spermidine + putrescine, the mixing ratio of the three polyamines is the same as that in the extract of NO.B-4, the total molar amount of the three polyamines is the same as the total molar amount of the polyamines in the extract of NO.B-4, and the amount of CS used is the same as the mass obtained by subtracting the polyamines from the extract of NO.B-4), NO.B-3 is CS + spermine (the molar amount of spermine is the same as the total molar amount of the polyamines contained in the extract of NO.B-4, and the amount of CS used is the same as the mass obtained by subtracting the polyamines from the extract of NO.B-4), NO.B-4 is the extract of the above NO.A-3, NO.B-5 is CS + spermidine (the molar amount of spermidine is the same as the total molar amount of the polyamines contained in the extract of NO.B-4, and the amount of CS used is the same as the mass obtained by subtracting the polyamines from the extract of NO.B-4), NO.B-6 is CS + putrescine (the molar amount of putrescine is the same as the total molar amount of the polyamines contained in the amount of extract NO.B-4 used, and the amount of CS used is the same as the mass obtained by subtracting the polyamines from the extract of NO.B-4), and NO.B-7 is CS (the amount used is the same as the mass of the extract of NO.B-4). From the above results, it was found that when the polyamine content is the same, the composite has the same activity as the extract, is higher than the composition of the same amount of the above biogenic polyamines (not mixed with CS), and is much higher than CS alone. The ratio of the total molar amount of polyamines in the blending combination was the same as that of A-3.

[0144] From the above results, it was found that when the polyamine content is the same, the composite has the same activity as the extract, and the inhibition rate against inflammatory factors is about 3.5 times that of the same amount of the above biogenic polyamine composition (not mixed with CS), and is much higher than CS alone.

[0145] In the conventional method for extracting chondroitin sulfate, it is common to remove other components in the raw material in order to obtain higher-purity chondroitin sulfate. Different from the conventional technology, in the present invention, after the raw material has been sufficiently acted on by an enzyme, chondroitin sulfate is precipitated under certain conditions using absolute ethanol, and unexpectedly, a complex of chondroitin sulfate and polyamine having unexpectedly significantly improved activity compared to chondroitin sulfate is obtained. Therefore, in the subsequent examples, the amount of polyamine obtained from the raw material is increased by a stepwise extraction method and complexed with the chondroitin sulfate obtained from the raw material to obtain chondroitin sulfate polyamine from natural raw materials.

[0146] Example 2. Papain enzymatic hydrolysis - resin purification extraction + absolute ethanol complexation and precipitation In this example, porcine cartilage was used as the raw material to prepare a chondroitin sulfate biopolyamine complex. (1) Pretreatment of raw material The pretreatment method of the raw material was the same as that in Example 1.

[0147] (2) Separation and extraction of chondroitin sulfate and polyamine in the raw material 2.1 Enzymatic hydrolysis 100 g of pretreated porcine cartilage was weighed, 500 mL of water and 2% papain (2,000 U / mg) based on the mass of the raw material porcine cartilage were added, the pH was adjusted to 6 - 7, the temperature was raised to 55 °C, and stirring was carried out for 16 hours. The enzymatic hydrolysis system was cooled to room temperature to obtain an enzymatic hydrolyzate. 2.2 Chromatography of biopolyamine The enzymatic hydrolyzate obtained in 2.1 was placed on diatomaceous earth and vacuum suction filtration was carried out to filter and remove insoluble impurities such as bone residues to obtain a pale yellow transparent solution. Weighed 100 mL of weakly acidic cation exchange resin (product number D152 was used in this example), put it into an empty column tube, and washed and filled it with purified water. Passed 500 mL of the pale yellow transparent solution (filtrate) obtained above through the column and collected the permeate. Then, washed the resin column with 200 mL of 4% (m / v) dilute hydrochloric acid and collected the eluate as eluate A. After adsorption and elution with the cation exchange column as described above, the polyamine substance can be concentrated and purified and concentrated in eluate A. 2.3 Separation and extraction of chondroitin sulfate The permeate obtained in Step 2.2 was distilled under reduced pressure, concentrated to 100 mL, 5 g of trichloroacetic acid was added to precipitate proteins, the precipitate was removed by centrifugation, and the supernatant was left. Added three volumes of absolute ethanol to the permeate in which proteins were precipitated with trichloroacetic acid, precipitated chondroitin sulfate by alcohol precipitation, shaken, and centrifuged. The lower layer solid obtained by centrifugation was washed once with absolute ethanol, centrifuged, and the obtained solid was vacuum dried at 45 °C overnight to obtain a crude product of sodium chondroitin sulfate. To further purify chondroitin sulfate, the obtained crude product of sodium chondroitin sulfate was prepared into an aqueous solution with a mass-volume ratio of 20%, 50 mL of strongly basic anion exchange resin (D280 was used in this example) was added, stirred and adsorbed, and the adsorption time was 20 - 30 minutes. Then, a 9% (m / v) sodium chloride solution 1 - 1.5 times the resin volume was added for elution to obtain eluate B. Eluate B was again subjected to alcohol precipitation of chondroitin sulfate with three volumes of absolute ethanol, shaken, and centrifuged. The lower layer solid obtained by centrifugation was washed once with absolute ethanol, centrifuged, and the obtained solid was vacuum dried at 45 °C overnight to obtain sodium chondroitin sulfate. The purity was measured to be 90.1%, and the measurement method was the same as in Example 1.

[0148] (3) Low molecular weight of chondroitin sulfate (solid-phase catalytic decomposition) The dried solid (sodium chondroitin sulfate) obtained in Step 2.3 was dissolved in 100 mL of water to obtain a solution, which was then processed according to Chinese Patent Application No. CN111495428A (Title of the Invention: Method for Preparing Low-Molecular-Weight Polysaccharide and Catalyst Used Therefor). Specifically, 0.1 g of a resin catalyst and 2.1 mL of 30% hydrogen peroxide were added to the solution, and after reacting at 50 °C for 6 hours, a low-molecular-weight chondroitin sulfate solution was obtained.

[0149] (4) Complexation and precipitation of chondroitin sulfate biopolyamine complex To the low-molecular-weight chondroitin sulfate solution obtained in Step (3), the eluate A obtained in Step 2.2 was added, the pH was adjusted to about 5, 2.5 volumes of absolute ethanol were added for alcohol precipitation, shaken, centrifuged, the lower-layer solid obtained by centrifugation was washed once with absolute ethanol, and the solid obtained by centrifugation was vacuum-dried at 45 °C overnight to obtain 6.0 g of a milky-white solid, which was a chondroitin sulfate biopolyamine complex.

[0150] As a result of measurement, this substance was a chondroitin sulfate biopolyamine complex containing spermine and spermidine. As a result of detection after separation, the total content (molar mass ratio) of polyamine in the extract was 23.26 μmol / g. Among them, spermine was 4.31 μmol / g, spermidine was 7.07 μmol / g, putrescine was 5.59 μmol / g, and cadaverine was 6.29 μmol / g. The weight-average molecular weight of low-molecular chondroitin was 4.3 k, the proportion exceeding 50,000 was 0%, the proportion of 25,000 - 50,000 was 0%, and the proportion of 400 - 25,000 was 100%. Specifically, the content of molecular weight 8,000 - 25,000 was 14%, the content of 5,000 - 8,000 was 30%, the content of 400 - 5,000 was 56%, and the content of less than 400 was 0%.

[0151] Example 3. Acid extraction - organic solvent extraction method + absolute ethanol complexation and precipitation In this example, chicken tissue (including chicken bones, chicken heads, and chicken viscera) was used as the raw material to prepare a chondroitin sulfate biopolyamine complex.

[0152] (1) Pretreatment of raw materials Meat was removed from fresh chicken tissue (including chicken bones, chicken heads, and chicken internal organs).

[0153] (2) Separation and extraction of chondroitin sulfate and polyamine in raw materials 2.1 Acid decomposition 100 g of pretreated chicken tissue (including chicken bones, chicken heads, and chicken internal organs) was weighed, and ultrasonic extraction was performed twice with 1 M dilute hydrochloric acid for a total of 2 hours (300 mL for 1 hour and 200 mL for 1 hour). After extraction, it was filtered using filter paper. The tissue filtration residue was washed twice with 50 mL of purified water to prepare the tissue residue. 2.2 Extraction of biogenic polyamine The total liquid from Step 2.1 was combined to obtain a total of 600 mL of slightly turbid pale yellow liquid, which was concentrated to 50 mL by rotary evaporation, centrifuged, and the supernatant was taken out. The pH was adjusted to about 12 with sodium hydroxide, centrifuged, and the supernatant was extracted twice with 10 mL of n-butanol to extract the biogenic polyamine mixture. The organic phases were combined, adjusted to acidity with dilute hydrochloric acid, and concentrated to dryness until a grayish-brown solid was obtained to obtain a crude extract of biogenic polyamine. 2.3 Separation and extraction of chondroitin sulfate 2.3.1 Enzymatic decomposition To the tissue filtration residue from Step 2.1, 100 mL of water with a pH of 5.7 - 6.0 and 2% papain (2,000 U / mg) based on the mass of the tissue filtration residue were added, stirred evenly, the system was heated to 65°C, and after reacting for 3 hours while maintaining the temperature, 1 g of papain was added and the reaction was continued for 3 hours to obtain an enzymatic decomposition solution. 2.3.2 Separation and extraction After filtering the enzymatic hydrolysate of Project 2.3.1, 20.0 g of trichloroacetic acid was added to the filtrate obtained after filtration, stored in an ice bath for 1 hour, mixed well, centrifuged at 8,000 rpm / min for 5 minutes, and the supernatant was taken out to remove proteins. The pH was adjusted to about 5 using solid sodium hydroxide, three times the amount of absolute ethanol was added for alcohol precipitation, shaken, centrifuged, the filtrate was temporarily stored, the alcohol precipitation solid obtained by centrifugation was washed once again with absolute ethanol, centrifuged, and the solid obtained by centrifugation was vacuum dried at 40 °C overnight to obtain a white solid of chondroitin sulfate.

[0154] (3) Low molecular weight of chondroitin sulfate (solid-phase catalytic decomposition) The dried white solid (chondroitin sulfate) obtained in Project 2.3.2 was dissolved in 100 mL of water to obtain a solution, and low molecular weight treatment was carried out according to Chinese Patent Application No. CN111495428A (Method for preparing low molecular weight polysaccharide and catalyst used therefor). Specifically, 0.05 g of resin catalyst and 1.1 mL of 30% hydrogen peroxide were added to the solution, and after reacting at 50 °C for 3 hours, a low molecular weight chondroitin sulfate solution was obtained.

[0155] (4) Complexation and precipitation of chondroitin sulfate biopolyamine complex Using the low molecular weight chondroitin sulfate solution obtained in Step (3), the crude extract of biopolyamine (grayish-brown solid) in Project 2.2 was dissolved, the pH was adjusted to about 5, 2.5 times the volume of absolute ethanol was added for alcohol precipitation, shaken, centrifuged, the lower layer solid obtained by centrifugation was washed once with absolute ethanol, and the solid obtained by centrifugation was vacuum dried at 45 °C overnight to obtain 6.0 g of a milky white solid of chondroitin sulfate biopolyamine complex.

[0156] As a result of measurement, this substance is a chondroitin sulfate biopolyamine complex containing spermine, spermidine, putrescine and cadaverine. As a result of detection after separation, the total content (molar mass ratio) of polyamines in the extract was 56.91 μmol / g. The spermine was 28.16 μmol / g, the spermidine was 9.82 μmol / g, the putrescine was 8.07 μmol / g, and the cadaverine was 10.86 μmol / g. The weight-average molecular weight of low-molecular chondroitin was 6.2k. The proportion exceeding 50,000 was 0%, the proportion of 25,000 - 50,000 was 0%, the proportion of 400 - 25,000 was 100%. Specifically, the proportion of 8,000 - 25,000 was 24%, the proportion of 5,000 - 8,000 was 26%, the proportion of 400 - 5,000 was 43%, and the proportion of less than 400 was 0%.

[0157] Example 4. Acid extraction - organic solvent extraction method + complexation and precipitation with absolute ethanol In this example, fish bones and soybeans were used as raw materials to prepare a chondroitin sulfate biopolyamine complex. Soybeans are rich in spermine and spermidine. The purpose of adding soybean raw materials as raw materials is to improve the polyamine content in the product.

[0158] (1) Pretreatment of raw materials The raw material fish bones and soybeans were pulverized by a stirrer.

[0159] (2) Separation and extraction of chondroitin sulfate and polyamines in raw materials 2.1 Acid decomposition 100 g of pretreated fish bones and 10 g of pretreated soybeans were weighed, and ultrasonic extraction was performed twice with 1M dilute hydrochloric acid for a total of 2 hours (300 mL for 1 hour, 200 mL for 1 hour). After extraction, filtration was performed using filter paper. The tissue filtration residue was washed twice with 50 mL of purified water to prepare a tissue residue. 2.2 Extraction of biopolyamines Combine all the liquid in Project 2.1 to obtain a total of 600 mL of slightly turbid pale yellow liquid. Concentrate it to 50 mL by rotary evaporation, centrifuge it, take out the supernatant, adjust the pH to about 12 with sodium hydroxide, centrifuge it, and extract the supernatant twice with 10 mL of n-butanol to extract the biological polyamine mixture. Combine the organic phases and concentrate them to dryness until a grayish-brown solid is obtained to obtain a crude extract of biological polyamine. 2.3 Separation and extraction of chondroitin sulfate 2.3.1 Enzymatic hydrolysis Add 100 mL of water with a pH of 5.7 - 6.0 and 2% papain (2,000 U / mg) based on the mass of the tissue filtration residue in the tissue filtration residue of Project 2.1, stir evenly, heat the system to 65 °C, keep it warm for 3 hours to carry out the reaction, then add 1 g of papain and continue the reaction for 3 hours to obtain an enzymatic hydrolysis solution. 2.3.2 Separation and extraction Filter the enzymatic hydrolysis solution obtained in Step 2.3.1 with filter paper. Add 20.0 g of trichloroacetic acid to the filtrate obtained after filtration, store it in an ice bath for 1 hour, mix well, centrifuge it at 8,000 rpm / min for 5 minutes, take out the supernatant to remove proteins. Weigh 100 mL of strongly acidic cation exchange resin (product number 001*7 was used in the example), add it to an empty column tube, wash it with purified water and fill it. Pass the supernatant after centrifugation through the column and collect the permeate. The permeate was chondroitin sulfate.

[0160] (3) Complexation and precipitation of chondroitin sulfate biological polyamine complex Dissolve the grayish-brown solid in Step 2.2 in the permeate obtained in Step 2.3.2 and adjust the pH to 5.0. Add 2.5 times of absolute ethanol for alcohol precipitation, shake it, centrifuge it, wash the alcohol precipitation solid obtained by centrifugation once again with absolute ethanol, centrifuge it, and vacuum dry the solid obtained by centrifugation at 40 °C overnight to obtain a white solid which is the chondroitin sulfate biological polyamine complex.

[0161] As a result of measurement, this substance is a chondroitin sulfate biopolyamine complex containing spermine, spermidine, putrescine and cadaverine. As a result of detection after separation, the total content (molar mass ratio) of polyamines in the extract was 129.7 μmol / g, with spermine being 5.2 μmol / g, spermidine being 70.3 μmol / g, putrescine being 49.7 μmol / g, and cadaverine being 4.6 μmol / g. The weight-average molecular weight of chondroitin was 22k, with the proportion over 50,000 being 0%, the proportion between 25,000 and 50,000 being 37%, the proportion between 400 and 25,000 being 63%, the proportion between 8,000 and 25,000 being 53%, the proportion between 5,000 and 8,000 being 8%, the proportion between 400 and 5,000 being 2%, and the proportion below 400 being 0%.

[0162] Test Example Test Example 1: Evaluation of the therapeutic effect of chondroitin sulfate on chronic inflammation by adjuvant arthritis mouse test To analyze whether CSX can suppress the inflammatory response in mice, a mouse adjuvant arthritis model was established (0.01 mL of complete Freund's adjuvant (CFA, Sigma) was injected daily into the right hind toe of each mouse (Vital River, BALB / c), and after constructing the arthritis model for 1 week, administration was carried out.). Subsequently, forced oral administration was performed (dosage: 3 mg / mouse / day, note: Group 1A is CS, Group 1B is low-molecular-weight CS, and low-molecular-weight CS is CS obtained by the decomposition method of Example 2 (weight-average molecular weight 4.3k, molecular weight distribution is the same as that of Example 2), and Group 1C is CSX, the product of Example 3.). After 30 days, blood was collected from the orbital cavity, serum was prepared, and IL-6 and IL-1β inflammatory factors were measured using a Mouse IL-6 ELISA kit (product number: VAL604) and a Mouse IL-1 ELISA kit (R&D Systems, product number: MLB00C).

[0163] As shown in FIGS. 1A and 1B, Group 1C significantly decreased the levels of IL-6 and IL-1β in mouse serum. The serum IL-6 and IL-1β levels in the normal group (non-model group) were extremely low, while the serum IL-6 and IL-1β levels in the control group (model group) were significantly increased. The serum IL-6 level in Group 1C was already equivalent to that of the normal group (non-model group), indicating a strong anti-inflammatory effect.

[0164] Furthermore, the toe width of mice was measured to analyze the swelling level of the toes. Referring to FIG. 2A, in Groups 1B and 1C, the toe swelling of mice significantly decreased as the administration time increased. Among them, in Group 1C, the toe swelling was significantly alleviated at the 22nd day of administration, and it was found that individual mice were close to the normal group. Thus, it was found that Group 1C has the effect of significantly reducing the toe swelling caused by the adjuvant. When the toes of mice were dissected and analyzed, referring to FIG. 2B, it was found that significant red swelling and lesions due to the inflammatory reaction appeared in the legs of the control group (model group). In Group 1C, no significant inflammatory reaction lesions were observed, and there was no red swelling phenomenon.

[0165] As a result, it was found that using CSX at 3 mg / day / mouse significantly improved mouse arthritis. Next was low-molecular-weight CS, and the conventional CS was the worst, with inflammation still being high even after 30 days.

[0166] Test Example 2: Therapeutic effect on arthritis (rheumatoid arthritis) In the rat rheumatoid arthritis model, CSX has a significant therapeutic effect on rheumatoid arthritis. Mainly, CSX can significantly improve the swelling of organs such as the toes, liver, and spleen in the inflammatory model rats, reduce the inflammatory phenotypes of lymphocytes and neutrophils in the blood, and has a therapeutic effect similar to methotrexate (MTX). It can basically treat joint and foot bone injuries caused by chronic inflammation, has a better therapeutic effect than MTX, and has no harmful side effects like MTX.

[0167] In this test example, the effect of CSX in rheumatoid arthritis rats (Vital River, SD) was analyzed. Preparation of rat rheumatoid arthritis model: A rat rheumatoid arthritis model was prepared using bovine type II collagen (CII, Solarbio). CII was mixed with an equal volume of complete Freund's adjuvant (CFA, Chondrex) to prepare an emulsion. The final concentration of CII in the emulsion was 1.0 mg / mL. Six rats were randomly selected as the blank control group (i.e., normal group / blank group), and the remaining rats were used to prepare the rheumatoid arthritis model. Rats other than those in the blank control group were injected with a total of 0.5 mL of the emulsion at multiple sites on the back and the base of the tail. Ten days later, the same dose of the collagen and adjuvant emulsion mixture was injected at the same site for the first time to enhance immunity. Grouping and administration: Normal group (blank group), model group, positive control group (MTX, methotrexate solution 0.2 mg / kg, administered orally every other day), drug group (CSX obtained in Example 2, dosage 200 mg / kg, administered orally daily).

[0168] The swelling of the rat toe was observed 1 week, 2 weeks, 3 weeks, and 4 weeks after administration. The thickness of the toes of the right hind limb of the rats in each group was measured and recorded. Specific method: After restricting the activity of the rat, the right hind limb of the rat was gently extended, and after marking the toe measurement site of the rat with a marker pen, the toe thickness was measured with calipers.

[0169] Referring to FIGS. 3A and 3B, in the drug group (CSX), it can be seen that the degree of swelling of the rat toes significantly decreased without side effects after 7 days of administration, and after 14 days of administration, the swelling of the toes further decreased. The toes of the rats in the model group were always in a significantly swollen state. Although the administration was continued until 28 days, the swelling of the toes remained at a relatively stable level. The fact that there was no further decrease may be related to the hyperplasia of connective tissue caused by swelling modeling. In the positive control group (MTX), although it showed the expected effect of suppressing the swelling of the rat toes, side effects such as diarrhea and weight loss were observed. From the above results, it was shown that CSX has a significant effect of suppressing the swelling of the toes caused by rheumatoid arthritis in rats. And there was no significant toxicity in rats.

[0170] Referring to FIGS. 4A to 4E, after collecting the venous blood of rats after 14 days of administration and performing a routine blood test, it was found that the absolute value of lymphocytes in the positive control group (MTX) was significantly decreased compared with the model group, and the percentage of lymphocytes in the drug group (CSX) was significantly decreased compared with the model group. However, there was no statistical difference in the absolute value of lymphocytes between the drug group (CSX) and the model group. In addition, the absolute value of neutrophils in the positive control group (MTX) was significantly decreased compared with the model group, and the drug group (CSX) also showed a significant decrease. There was no statistical difference in the absolute value of white blood cells between the model group, the drug group (CSX), and the positive control group (MTX).

[0171] Referring to FIGS. 5A to 5C, after dissecting the rats after 28 days of administration, the liver, spleen, and thymus were collected, and after accurately weighing them, the organ index was measured based on the ratio of the body weight of the rats to the weight of each organ. The results showed that CSX can significantly reduce the liver enlargement of rats. The spleen index reflects that CSX can significantly reduce the spleen enlargement of rats, suggesting a reduction in the occurrence of chronic inflammation. There was no statistically significant difference in the thymus index.

[0172] According to the above results, it was found that CSX can significantly improve the degree of swelling in the toes, liver, spleen, etc. of the inflammation model rats, reduce the inflammatory phenotypes of lymphocytes and neutrophils in the blood, and has a therapeutic effect similar to MTX.

[0173] Referring to Figures 6 and 7, the CT images of the ankle joint site of the rats showed that the interface of the joint site of the rats in the blank group (normal group) was clear, the joint connection sites were regular, and the bone surface was smooth. For the rats in the model group, the joint connection interface was unclear, the joints were irregular, the bone surface was not smooth, and bone hyperplasia, swelling, etc. were observed. Although there was also a certain degree of damage in the joint site of the rats in the drug group (CSX), the joint interface was clearer than that in the model group, the joints were more regular, and the bone surface was smoother. The bone surface of the joints of the rats in the positive control group (MTX) was relatively smoother than that in the control group, and the joint connection surface was closer than that in the model group. However, the degree of joint damage was more severe than that of the rats in the drug group (CSX).

[0174] Referring to FIGS. 8A to 8F, the CT results showed that the bone mineral density (BMD) of the model group was significantly lower than that of the blank group (normal group). After CSX administration, the decrease in bone density induced by inflammation could be effectively alleviated. The specific surface area of the ankle joint in the model group was significantly increased compared with the blank group, but after CSX and MTX administration, the bone surface (BS) of the rat ankle joint was significantly decreased. Among them, the decrease in the rats of the drug group (CSX) was more significant. The rats of the drug group (CSX) showed a significant decrease in the bone volume (BV) of the ankle joint and a significant decrease in the ratio of bone surface area to bone volume (BS / BV). Trabecular Separation / Spacing (Tb.Sp) refers to the average width of the medullary cavity between trabeculae. An increase in Tb.Sp may cause osteoporosis. In the rats of the model group, Tb.Sp was significantly increased, but after CSX administration, the Tb.Sp of the rats decreased, suggesting that CSX has the effect of reducing osteoporosis caused by inflammation. The trabecular thickness (Tb.Th) of the drug group (CSX) increased compared with the model group, which also reflects that CSX can suppress osteoporosis induced by inflammation. From the above CT results, it was found that CSX has the function of significantly suppressing osteoporosis caused by inflammation, increasing bone density, and improving the smoothness of the bone surface, and has a better effect than MTX.

[0175] Also, it can be seen from the experiment that the positive control group (MTX) showed diarrhea symptoms at the dosage of this experiment, while the drug group (CSX) showed no obvious abnormalities. In the acute toxicity test in mice, no toxic reaction was observed in the drug group (CSX) even with a single administration of up to 2000 mg / kg, suggesting that CSX may be safer.

[0176] The above CT results indicate that CSX can basically treat joint and foot bone injuries caused by chronic inflammation and has a stronger effect than MTX.

[0177] Test Example 3: Activity of Reducing Blood Lipids In this test example, to evaluate the effect of CSX on reducing blood lipids, after feeding C57 / b6 mice a high-fat diet (60% fat calorie diet (Research diets)) for 3 weeks, they were force-fed orally for 43 days (Note: Group 2A is normal CS, Group 2B is low-molecular-weight CS, and low-molecular-weight CS is CS obtained by the decomposition method in Example 2 (weight-average molecular weight 4.3k, molecular weight distribution is the same as in Example 2), and Group 2C is the product CSX of the NO.A-3 extract in Example 1. The dosage for all was 6 mg / mouse, force-fed orally daily), and the levels of total cholesterol (T-CHO), triglyceride (TG), and high / low-density lipoprotein cholesterol (H / LDL-C) in the mice were evaluated. The blood lipid-free model group was the group fed a normal diet, the blood lipid model group was the group fed a high-fat diet, the PBS group was the only force-fed group, and the normal diet group was the group fed a normal diet after 3 weeks of modeling. Normal chondroitin is bovine-derived chondroitin provided by Hunan Wuxing Biotechnology Co., Ltd., with a weight-average molecular weight of 21k, a proportion of over 50,000 molecular weight of 0%, a proportion of 25,000 - 50,000 of 31%, a proportion of 400 - 25,000 of 69%, and a proportion of chondroitin sulfate with a molecular weight of 400 or less of 0%.

[0178] Referring to FIGS. 9A - 9D, compared with the blood lipid model group, in Groups 2A, 2B, and 2C, the total cholesterol decreased significantly, and among them, the decrease effect in Group 2C was the most significant. In Groups 2A and 2B, the total triglyceride did not show a significant decrease compared to the blood lipid model group, but in Group 2C, it decreased significantly. In Groups 2A, 2B, and 2C, the low-density lipoprotein cholesterol decreased significantly more than the blood lipid model group, and the decrease in Group 2C was the most significant. Compared with the blood lipid model group, the high-density lipoprotein cholesterol decreased significantly in Groups 2A and 2B, but not significantly in Group 2C. From the animal test in this test example, it was found that CSX has the effect of significantly reducing serum total cholesterol, serum total triglyceride, and low-density lipoprotein.

[0179] Test Example 4: Anti-skin inflammation activity In this test example, to evaluate the anti-inflammatory effect of CSX on skin inflammation, a mouse ear swelling model was used, and the right ear of the mouse was sensitized three times. The sensitizer was phorbol ester (Psaitong reagent, China), with a concentration of 0.125 mg / mL. The sensitization method was to apply 20 μL (10 μL on each side) to the right ear of the mouse once, and each sensitization was performed at 48-hour intervals. Group 4A was an aqueous solution of CSX (the product of Example 3), Group 4B was an aqueous solution formulated with low-molecular-weight CS and spermidine (the molar mass ratio of spermidine was the same as the molar ratio of the polyamine in Group 4A, and the molar amount of spermidine was the same as the total molar amount of the polyamine contained in the extract of Example 3. The amount of CS used was the same as the mass obtained by subtracting the polyamine from the extract. The low-molecular-weight CS was the CS obtained by the decomposition method of Example 2 (weight-average molecular weight 4.3k, molecular weight distribution was the same as that of Example 2), and Group 4C was the negative control group (ultrapurified water). Group 4D was an aqueous solution formulated with normal CS and spermidine (the molar mass ratio of spermidine was the same as the molar ratio of the polyamine in Group 4A, that is, the molar amount of spermidine was the same as the total molar amount of the polyamine contained in the extract of Example 3, and the amount of CS used was the same as the mass obtained by subtracting the polyamine from the extract.). The normal CS was chondroitin provided by Hunan Wuxing Biotechnology Company, with a weight-average molecular weight of 48k, of which the content with a molecular weight exceeding 50,000 was 34%, the content between 25,000 and 50,000 was 55%, and the content between 400 and 25,000 was 11%. The solution concentrations of 4A, 4B, and 4D were all 2% in mass-to-volume ratio, and they were administered continuously once a day for 6 days. The administration method was surface coating, and the administration amount was 20 μL / mouse. The evaluation indicators were mainly the degree of ear swelling and body weight of the mouse. The evaluation method for the degree of ear swelling of the mouse was to decapitate the mouse by cervical dislocation, cut both ears, take symmetrical ears with a 6-mm diameter punch, measure the weight with an electronic balance, and take the weight difference between the left and right ears as the degree of swelling.

[0180] The results are shown in Table 2 and Table 3 below. It can be seen that both the product CSX of Example 3 and the complex of low-molecular-weight CS and spermidine had significant anti-inflammatory effects, and although the effect of the product of Example 3 was more excellent, both were significantly superior to the negative control group. In addition, CSX did not affect the body weight of mice, and no obvious side effects were observed. It was found that the complex of low-molecular-weight CS and spermidine was superior to the combined use of normal CS and spermidine.

[0181]

Table 2

[0182]

Table 3

[0183] Test Example 5: Antioxidant Activity To evaluate the antioxidant effect of CSX, in this test example, human liver HEPG2 cells were used as a model, and the effect of CSX on intracellular reactive oxygen species free radicals was measured by a fluorescence probe method. CSX-1 used in this test example is the product of the NO.A-3 extract of Example 1, and CSX-2 is a mixture of low-molecular-weight CS and polyamines (spermine, spermidine, and putrescine) in the same ratio as CSX-1 (the mixing ratio of the three types of polyamines is the same as that of the NO.A-3 extract of Example 1, and the total molar amount of polyamines is the same as the total molar amount of polyamines contained in the NO.A-3 extract of Example 1. The amount of CS used is the same as the mass obtained by subtracting polyamines from the extract). The low-molecular-weight CS is CS (weight-average molecular weight 4.3k, molecular weight distribution is the same as that of Example 2) by the decomposition method in Example 2.

[0184] The principle of this experiment is the detection of reactive oxygen species using the fluorescent probe DCFH-DA. DCFH-DA itself does not have fluorescence and can freely pass through the cell membrane. Once it enters the cell, it can be hydrolyzed by intracellular esterase to generate DCFH. On the other hand, since DCFH cannot permeate the cell membrane, the probe can be easily introduced into the cell. Reactive oxygen species in the cell can oxidize non-fluorescent DCFH to fluorescent DCF. By detecting the fluorescence of DCF, the level of reactive oxygen species in the cell can be known.

[0185] Cells in the logarithmic growth phase were collected, and the cell state was observed under a microscope. They were washed twice with sterile PBS, the supernatant was discarded, 3 mL of PBS was added, 0.5 mL of 2.5% trypsin was added, digested at 37 °C for 5 minutes, 3 mL of serum-containing medium was added to stop the reaction, aspirated into a 15 mL centrifuge tube, centrifuged at 1,000 rpm, the supernatant was discarded, an appropriate amount of medium was added to count the cells, 2,000 - 10,000 cells were dispensed into each well of a 96-well plate, cultured at 37 °C for 6 hours until the cells adhered to the wall, drugs of each concentration were added to give stimulation, after 24 hours, the plate was taken out, DCFH-DA was diluted 1:1000 with serum-free culture medium so that the final concentration of the probe was 10 μM. The cell culture medium was removed, and diluted DCFH-DA in an appropriate volume was added. Incubated in a cell culture tank at 37 °C for 20 minutes. To thoroughly remove the DCFH-DA that had not entered the cells, the cells were washed 3 times with serum-free cell culture medium. Using an excitation wavelength of 488 nm and an emission wavelength of 525 nm, the fluorescence intensity before and after stimulation was measured using a flow cytometer. From the results of the cytological experiment, it was shown that CSX (especially low-molecular-weight CSX) has significant antioxidant ability at a final concentration of 0.25 mg / mL. Compared with the control group (untreated cells), it can be seen that in the CSX-1 administration group, the number of reactive oxygen species (ROS) positive cells decreased from 36.5% to 25.7%, and in CSX-2, it further decreased to 1.17%. Please refer to Figure 10.

[0186] Test Example 6: Measurement of the Activity of Alzheimer's Disease-Related Inflammation In research on neurodegenerative diseases such as Alzheimer's disease (AD), the inflammatory process plays a fundamental role in the onset process of AD, and the inflammatory reaction mediated by microglia in the central nervous tissue has been shown to be one of the major factors in neurodegenerative diseases, especially Alzheimer's disease. Inflammatory components related to AD include brain cells such as microglia and astrocytes. IL-6 released from microglia is considered to be one of the major factors mediating AD production.

[0187] To detect the inhibitory effect of CSX on microglial inflammation, human microglia HMC3 was treated with CSX (prepared in Example 2). As a result, it was shown that CSX could significantly inhibit the expression level of IL-6 in microglia induced by LPS. Please refer to Figure 11.

[0188] Test Example 7: Inhibitory effect of CSX on inflammatory response at the cellular level In mouse monocytes RAW264.7, induction by lipopolysaccharide (LPS) increases the expression levels of many intracellular inflammatory factors such as IL-6, TNF-α, and IL-1β. In this test example, it was analyzed whether CSX could inhibit the production of cellular inflammatory factors. The CSX used in this test example is the product of Example 3.

[0189] LPS (Solarbio) at a final concentration of 0.1 μg / mL was added to DMEM 10% FBS (Life techNology) medium. CSX was prepared in 10% FBS DMEM containing LPS and adjusted to a final concentration of 0.6 mg / mL, and 100 μl was added per well. This was repeated 3 times for each gradient. The amount of dexamethasone (DXM) used was the stock solution at 2 mg / mL. It was diluted at a ratio of 1000 times in 10% FBS DMEM containing LPS, and 100 μl was added per well. This was repeated 3 times for each gradient. A cell LPS-unstimulated group and a cell LPS-stimulated group were set up. Each was repeated 3 times. After culturing for 24 hours, the supernatant was carefully aspirated and directly analyzed by enzyme-linked immunosorbent assay (ELISA).

[0190] 12A to 12C, it was shown that CSX can significantly inhibit the expression of IL-6, IL-1β and TNF-α in mouse monocytes / macrophages induced by LPS. The inhibitory effect on IL-6 is superior to that of dexamethasone. It was demonstrated that CSX has an excellent inhibitory effect on the expression level of inflammatory factors at the cellular level.

[0191] Test Example 8: Aging delay / life extension activity To evaluate the lifespan extension activity of CSX, guppies were used as the study subjects, and the survival time of the offspring was measured. The experimental fish was one artificially cultured pregnant guppy (product name "Hongyun Dangtou") purchased from Beijing Shilihe Flower and Bird Fish Market. After one week of culture, the experimental fish produced more than 50 fry, and after stable breeding for three days, the fry were divided into two groups for culture, each group containing 25 fry. The CSX used in this test example is the product of Example 3.

[0192] Rearing conditions: Each tank contained approximately 10L of water that had been sun-dried for a week, the temperature was kept constant at 26°C, and a water circulation filtration system and oxygen supply device were installed. Water was replenished to 10L every two days, and approximately 1 / 3 (approximately 3-4L) of the water was replaced weekly. Barrel drinking water was used as water. An appropriate amount of food for fry was given once a day. CSX was added to tank No. 1: After replacing the water every week, 30mg / L of CSX was added. Normal rearing in tank No. 2: Water was replaced every week and no CSX was added. The number of dead fry was observed and counted every day, and the average survival time of the fry in each tank was calculated from the sum of (days of survival * number of fish) / total number of fish.

[0193] As a result, the average survival time in tank No. 1 was 31.88 days, and the average survival time in tank No. 2 was 27.48 days, indicating that 30 mg / L CSX can extend the survival time of artificially reared guppy fry by 16.01%. The results are shown in Figure 13.

[0194] To further evaluate the aging delay / life extension activity of CSX, Drosophila melanogaster (bred in this laboratory) was used as the research object, and its survival time was measured. The experimental Drosophila melanogaster was the w1118 wild-type Drosophila melanogaster bred in the laboratory, and the basic feed formulation was a liquid feed containing 2.2% sucrose, 8% malt extract, 1.8% yeast, and 1.2% butyl paraben. Three different formulations of feed were given to female and male Drosophila melanogaster respectively. Formulation 1 was the basic feed formulation, formulation 2 was the basic feed formulation added with 0.1 g / L of CSX (the product of Example 2), and formulation 3 was the basic feed formulation added with a combination of spermidine hydrochloride and spermidine hydrochloride (10 times the molar amount of the polyamine in formulation 2). Six groups including 3 groups of females and 3 groups of males were set up for each formulation, with 20 flies in each group, all of which were virgin females and males that had not mated within 4 hours after eclosion. The number of dead Drosophila melanogaster was observed and counted daily, 400 μl of the corresponding feed was replenished daily for each group, and the average survival time of Drosophila melanogaster in each group was calculated from the total of (survival days * number of flies) / total number of flies.

[0195] Referring to FIGS. 21A and 21B, the average survival time of Drosophila melanogaster bred with formulation 1 was 46.2 - 49 days, the average survival time of Drosophila melanogaster bred with formulation 2 was 54.5 - 59 days, and the average survival time of Drosophila melanogaster bred with formulation 3 was 50.6 - 52.5 days. It was found that CSX could extend the lifespan of Drosophila melanogaster by 18.0% - 20.4% compared with the normal feed. In addition, although the total molar amount of polyamine in CSX of formulation 2 was only one-tenth of the free polyamine in formulation 3, the lifespan extension effect of the CSX group was 7.7 - 12.38% higher than that of the general polyamine.

[0196] Test Example 9: Anti-inflammatory Activity Test In this test example, the anti-inflammatory activity against IL-6 by various substances was measured, and the measurement method was the same as that in Example 1. All the low-molecular-weight CS used in this test example was CS obtained by the decomposition method of Example 2 (weight-average molecular weight 4.3k, molecular weight distribution the same as that in Example 2). Here, No.C-1 is low-molecular-weight CS, No.C-2 is spermine, No.C-3 is spermidine, No.C-4 is spermidine + spermine, C-5 is spermidine + spermine + putrescine, No.C-6 is low-molecular-weight CS + spermine, No.C-7 is low-molecular-weight CS + spermidine, No.C-8 is low-molecular-weight CS + spermidine + spermine, No.C-9 is low-molecular-weight CS + spermidine + spermine + putrescine. The total molar amounts of the polyamines of No.C3 to C12 above are the same, and when two or more polyamines are used, the molar ratios of each polyamine are the same. From the above results, it was found that the activity of the complex of CS and polyamine is significantly superior to that of low-molecular-weight CS alone or polyamine alone. The effect of a combination of multiple types of amines is superior to that of amine alone. For details, please refer to Figure 22.

[0197] Test Example 10: Comparison of Low-Molecular-Weight CS with Bio-Polyamine and Non-Bio-Polyamine Activity In this test example, the anti-inflammatory activity against IL-6 by the combination of low molecular weight CS and various biogenic polyamines was measured, and the measurement method was the same as in Example 1. All of the low molecular weight CS was CS obtained by the decomposition method of Example 2 (weight average molecular weight 4.3k, molecular weight distribution the same as in Example 2). All of the low molecular weight CS used in this test example was CS obtained by the decomposition method of Example 2 (weight average molecular weight 4.3k, molecular weight distribution the same as in Example 2). As shown in Fig. 23, No.D-1 is low molecular weight CS + spermine, No.D-2 is low molecular weight CS + spermidine, No.D-3 is low molecular weight CS + putrescine, No.D-4 is low molecular weight CS + cadaverine, D-5 is low molecular weight CS + histamine, D-6 is low molecular weight CS + tryptamine, D-7 is low molecular weight CS + 1,7-diaminoheptane, D-8 is low molecular weight CS + pentaethylenehexamine. The total molar concentration mass ratio of polyamines in each group is the same at 2 μmol / g, and when two or more polyamines are used, the molar ratio of each polyamine is the same. From the above results, the combinations of low molecular weight CS and various polyamines have different activities. Among them, spermine and spermidine have equivalent activities, are superior to putrescine, and are even more superior to cadaverine. In contrast, histamine and tryptamine, as well as 1,7-diaminoheptane and pentaethylenehexamine which are non-biogenic polyamines, have an inflammation-inducing effect, and among them, histamine has the strongest effect.

[0198] Test Example 11: Comparison of the activities of chondroitin sulfate biogenic polyamine complexes with different biogenic polyamine contents In this test example, low-molecular-weight CS and spermine were blended at different ratios, and the stability of their combination was measured. All of the low-molecular-weight CS was the CS obtained by the decomposition method of Example 2 (weight-average molecular weight 4.3k, molecular weight distribution the same as that of Example 2). The experimental method is as follows. An aqueous solution of low-molecular-weight CS at 40 mg / ml was prepared, and an aqueous solution of spermine hydrochloride was prepared at the concentrations shown in Table 4 below. Two solutions of the same volume were uniformly mixed to obtain the blended solutions of No. E-1 to E-8. The transmittance (wavelength 600 nm) of the solution was measured using an ultraviolet spectrophotometer. Among them, the transmittance of No. E-7 and E-8 decreased significantly, which is considered to be due to the formation of a colloidal solution from the nanometer to the micron level. The stability of the blended solution was measured by two methods. 1. It was allowed to stand for 7 days to observe its stability. 2. To simulate the stability in blood, 20 μl of the blended solution was added to 1 ml of rabbit plasma solution (Beijing Land Bridge Biotechnology), and the change in the solution was observed. The results are shown in Table 4 below. From the above results, it can be seen that when the content of spermine is too high (No. E-7, E-8), it is likely to react with chondroitin to form a precipitate, and the system becomes unstable. Also, when the content of spermine is too high (No. E-6, E-7, E-8), it is likely to electrostatically bind to serum proteins, resulting in the formation of a large amount of precipitate, and it was found that its cytotoxicity is significant in cell tests. From this, it can be seen that a combination with too high a content of spermine has poor stability, high toxicity, and no practical value. No. E-6, E-7, and E-8 were synthesized according to the method of Reference 4.

[0199]

Table 4

[0200] Test Example 12: Proving that the effects of other polysaccharides and other weight-average molecular weight CS do not extend to the combination of the present application In this test example, the anti-inflammatory activity against IL-6 by the combination of CS with each molecular weight distribution and other anionic polysaccharides and polyamines was measured in the same manner as in Example 1. The polyamine in the combination is a combination of putrescine, spermine, and spermidine, and the total molar concentration mass ratio is 2 μmol / g for all. When two or more polyamines are included, the molar ratio of each polyamine is the same. Here, NO.F-1 is CS + polyamine, and CS obtained by the decomposition method of Example 2 (weight average molecular weight is 4.3k, and the molecular weight distribution is the same as in Example 2) was used. No.F-2 is CS + polyamine, and the CS used is a product provided by Hunan Wuxing Biotechnology Co., Ltd., with a weight average molecular weight of 48k, a ratio of over 50,000 being 34%, a ratio of 25,000 - 50,000 being 55%, and a ratio of 400 - 25,000 being 11%. No.F-3 is CS + polyamine, and the CS used is a product provided by Hunan Wuxing Biotechnology Co., Ltd., with a weight average molecular weight of 35k, a ratio of 50,000 or more being 12%, a ratio of 25,000 - 50,000 being 68%, and a ratio of 400 - 25,000 being 20%. No.F-4 is CS + polyamine, and the CS used is a product of Hunan Wuxing Biotechnology Co., Ltd. manufactured by the enzymatic decomposition method, with a weight average molecular weight of 879, a ratio of 400 - 20,00 being 99%, a ratio of 400 - 1,000 being 54%, a ratio of 1,000 - 2,000 being 45%, a ratio of less than 400 being 1%, and a ratio of over 2,000 being 0%. NO.F-5 is CS + polyamine, and the CS used is a product provided by Hunan Wuxing Biotechnology Co., Ltd., with a weight average molecular weight of 27k, a ratio of over 50,000 being 0%, a ratio of 25,000 - 50,000 being 53%, and a ratio of 400 - 25,000 being 47%. NO.F-6 is CS + polyamine, and the CS used is a product provided by Hunan Wuxing Biotechnology Co., Ltd. manufactured by the enzymatic decomposition method, with a weight average molecular weight of 598, a ratio of less than 400 being 11%, a ratio of over 2,000 being 0%, a ratio of 400 - 2,000 being 89%, a ratio of 400 - 1,000 being 85%, and a ratio of 1,000 - 2,000 being 4%.No.F-7 is CS complete hydrolysate + polyamine. The weight-average molecular weight of the CS complete hydrolysate is 415, the proportion less than 400 is 56%, and the proportion more than 400 is 44%. The CS complete hydrolysate is obtained by hydrolyzing chondroitin sulfate with 6M hydrochloric acid at 100 °C for 4 hours. NO.F-8 is hyaluronic acid + polyamine, and NO.F-9 is trehalose + polyamine. The measurement results are shown in Figure 24. From the above results, it was found that the activities of the composites of low-molecular-weight CS of NO.F-1, F-4, F-5, and F-6 and polyamine are significantly superior to those of CS with other molecular weight distributions or combinations of other anionic polysaccharides and polyamine.

[0201] Test Example 13: Prove that the molar ratios of various polyamines do not have a significant impact on activity In this test example, the anti-inflammatory activity against IL-6 by the combination of CS and polyamine with each molar ratio was measured in the same manner as in Example 1. All CS were obtained by the decomposition method of Example 2 (weight-average molecular weight 4.3k, molecular weight distribution the same as in Example 2). The polyamine in the combination is a combination of putrescine, spermine, and spermidine, and the total molar concentration mass ratio is 2 μmol / g for all. The molar ratio of polyamine in NO.G-1 is spermine:spermidine:putrescine = 1:1:1, the molar ratio of polyamine in NO.G-2 is spermine:spermidine:putrescine = 5:1:1, the molar ratio of polyamine in NO.G-3 is spermine:spermidine:putrescine = 1:5:1, and the molar ratio of polyamine in NO.G-4 is spermine:spermidine:putrescine = 1:1:5. The measurement results are shown in Figure 25. From the above results, it was found that in the case of CS having the same molecular weight and molecular weight distribution, when the total molar amount of polyamine is the same, the difference in the molar ratio of each polyamine does not have a significant impact on activity.

Claims

1. A chondroitin sulfate - biopolyamine complex, which is a complex of chondroitin sulfate and a biopolyamine, wherein the chondroitin sulfate and the biopolyamine are non - covalently bonded, and the biopolyamine comprises one, two or a combination of three or more of spermine, spermidine, putrescine and cadaverine.

2. The chondroitin sulfate - biopolyamine complex according to claim 1, wherein the chondroitin sulfate is chondroitin sulfate in acid form or chondroitin sulfate in salt form.

3. The chondroitin sulfate has, in terms of GPC integration ratio, a proportion of chondroitin sulfate with a weight - average molecular weight of 50,000 or more of 0%, and the chondroitin sulfate has, in terms of GPC integration ratio, a proportion of chondroitin sulfate with a weight - average molecular weight of 25,000 - 50,000 of 40% or less, preferably, a proportion of chondroitin sulfate with a weight - average molecular weight of 25,000 - 50,000 of 35% or less, more preferably, a proportion of chondroitin sulfate with a weight - average molecular weight of 25,000 - 50,000 of 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less, or 0%, and the chondroitin sulfate has, in terms of GPC integration ratio, an upper limit of the proportion of chondroitin sulfate with a weight - average molecular weight of 400 - 25,000 of 80% or more, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or 100%, and a lower limit of the proportion of chondroitin sulfate with a weight - average molecular weight of 400 - 25,000 of 40% or more, preferably 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60% or more, and the chondroitin sulfate has, in terms of GPC integration ratio, a proportion of chondroitin sulfate with a weight - average molecular weight of 400 or less of 15% or less, preferably 3% or less, more preferably 1% or less, and most preferably 0%. and / or The upper limit of the weight-average molecular weight is 25,000 or less, preferably 24,000, 23,000, 22,000, 21,000, 20,000, 19,000, 18,000, 17,000, 16,000, 15,000, 14,000, 13,000, 12,000, 11,000, 10,000, 9,000, or 8,000 or less, and the lower limit of the weight-average molecular weight is 400, 500, 600, 700, or 800 or more. The upper limit of the proportion of chondroitin sulfate is 80% or more, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or 100%, and the lower limit is 40% or more, preferably 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% or more. The sum of the proportion of chondroitin sulfate having a weight-average molecular weight of 25,000 to 50,000, the proportion of chondroitin sulfate having a weight-average molecular weight of 400 to 25,000, and the proportion of chondroitin sulfate having a weight-average molecular weight of 400 or less is 100%. The chondroitin sulfate biopolyamine complex according to claim 1 or 2.

4. In the chondroitin sulfate, in terms of the GPC integration ratio, the proportion of chondroitin sulfate having a weight-average molecular weight of 400 to 10,000 is 40% to 100%, preferably 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%. Preferably, in terms of the GPC integration ratio, the molecular weight distribution of the chondroitin sulfate is as follows: The proportion of chondroitin sulfate having a weight-average molecular weight exceeding 50,000 is 0%, the proportion of chondroitin sulfate having a weight-average molecular weight of 25,000 to 50,000 is 0 to 40%, the proportion of chondroitin sulfate having a weight-average molecular weight of 400 to 10,000, preferably the proportion of chondroitin sulfate having a weight-average molecular weight of 400 to 8,000 is 40% to 100%, and the proportion of chondroitin sulfate having a weight-average molecular weight of 400 or less is 15% or less. The chondroitin sulfate biopolyamine complex according to any one of claims 1 to 3.

5. The weight average molecular weight of the chondroitin sulfate biopolyamine complex is 400 to 50,000, Preferably, the weight average molecular weight of the chondroitin sulfate biopolyamine complex is 400 to 25,000, and the chondroitin sulfate biopolyamine complex according to any one of claims 1 to 4 is characterized in that.

6. Based on the total weight (g) of the chondroitin sulfate biopolyamine complex, the biopolyamine contained in the chondroitin sulfate biopolyamine complex is 200 μmol / g or less, preferably 199 μmol / g, 198 μmol / g, 197 μmol / g, 196 μmol / g, 195 μmol / g, 194 μmol / g, 193 μmol / g, 192 μmol / g, 191 μmol / g, 190 μmol / g, 189 μmol / g, 188 μmol / g, 187 μmol / g, 186 μmol / g, 185 μmol / g, 184 μmol / g, 183 μmol / g, 182 μmol / g, 181 μmol / g or 180 μmol / g or less, and the content of the biopolyamine is 0.5 μmol / g or more, preferably 0.6 μmol / g, 0.7 μmol / g, 0.8 μmol / g, 0.9 μmol / g, 1 μmol / g or more, and the chondroitin sulfate biopolyamine complex according to any one of claims 1 to 5 is characterized in that.

7. The mass percentage of protein in the chondroitin sulfate biopolyamine complex is less than 8%, preferably less than 5%, more preferably less than 3%, most preferably less than 1%, and still more preferably 0%, and the chondroitin sulfate biopolyamine complex according to any one of claims 1 to 6 is characterized in that.

8. A method for preparing the chondroitin sulfate biopolyamine complex according to any one of claims 1 to 7, comprising a step of mixing chondroitin sulfate and a biopolyamine, and containing 0.5 to 200 μmol / g of the biopolyamine in the chondroitin sulfate biopolyamine complex based on the total weight (g) of the chondroitin sulfate biopolyamine complex, and the preparation method is characterized in that.

9. A method for preparing the chondroitin sulfate biopolyamine complex according to any one of claims 1 to 7, A preparation method comprising a step of mixing an extract containing chondroitin sulfate and polyamine separated and extracted from a raw material with ethanol, wherein the pH of the extract is 4 to 6, and the volume ratio of the extract to ethanol is 1:1 to 3.

10. The step of separating and extracting chondroitin sulfate and polyamine from a raw material includes an enzymatic decomposition or acid decomposition step of decomposing the raw material by enzymatic decomposition or acid decomposition to obtain an enzymatic decomposition solution or an acid decomposition solution, and a step of separating and extracting chondroitin sulfate and polyamine from the enzymatic decomposition solution or the acid decomposition solution simultaneously or stepwise, and the preparation method according to claim 9, characterized in that it includes the above steps.

11. When extracting chondroitin sulfate and polyamine stepwise, the polyamine in the enzymatic decomposition solution or the acid decomposition solution is separated by chromatography or an extraction method, and the residue after separating the polyamine is treated by one or more of an enzymatic decomposition method, a protein precipitation method, chromatography, and an alcohol precipitation method to separate chondroitin sulfate from the residue. Optionally, after separating chondroitin sulfate, the preparation method according to claim 10 further includes a step of reducing the molecular weight of chondroitin sulfate.

12. When extracting chondroitin sulfate and polyamine simultaneously, the protein in the enzymatic decomposition solution or the acid decomposition solution is precipitated by a protein precipitation method to separate chondroitin sulfate and polyamine, and the preparation method according to claim 10, characterized in that it includes the above steps.

13. The preparation method according to any one of claims 9 to 12, characterized in that the raw material includes animal tissues, plant tissues, and microbial culture fermentation broths.

14. A chondroitin sulfate biopolyamine complex prepared by the preparation method according to any one of claims 8 to 12.

15. Use of the chondroitin sulfate biopolyamine complex in any one of the following (a) to (g), (a) Use in the preparation of anti-inflammatory pharmaceuticals, (b) Use in the preparation of pharmaceuticals for the treatment and / or prevention of inflammatory diseases, (c) Use in the preparation of pharmaceuticals for reducing blood lipids, (d) Use in the preparation of pharmaceuticals for the treatment and / or prevention of hyperlipidemia, (e) Use in the preparation of pharmaceuticals for the treatment and / or repair of joint injuries, (f) Use in the preparation of antioxidant pharmaceuticals, Use in the preparation of a medicament for delaying aging and / or extending lifespan, The use wherein the chondroitin sulfate biopolyamine complex is the chondroitin sulfate biopolyamine complex according to any one of claims 1 to 7, 14 and / or the chondroitin sulfate biopolyamine complex prepared by the preparation method according to any one of claims 8 to 13.

16. The inflammatory disease includes an inflammatory disease caused by an inflammation-inducing factor and / or an inflammatory disease caused by inflammatory cells, interleukins and / or tumor necrosis factors. Preferably, the inflammatory disease is one or more selected from allergy, eczema, myocardial infarction, cerebral infarction, Alzheimer's disease, dermatitis or arthritis, or The hyperlipidemia includes primary hyperlipidemia and / or secondary hyperlipidemia, or The hyperlipidemia includes hypertriglyceridemia and / or hypercholesterolemia, or The hyperlipidemia includes hyperlipidemia-related diseases. Optionally, the hyperlipidemia-related diseases include cardiovascular diseases. Optionally, the cardiovascular diseases include one or more of arteriosclerosis, coronary artery disease, angina pectoris, carotid artery disease, stroke, cerebral arteriosclerosis, myocardial infarction, cerebral infarction, restenosis after balloon angioplasty, hypertension, intermittent claudication, dyslipidemia, postprandial hyperlipidemia and xanthoma, or The use according to claim 15, wherein the joint injury includes joint injury caused by inflammation, aging, exercise or injury.

17. Use in the preparation of a health food or cosmetic of the chondroitin sulfate biopolyamine complex according to any one of claims 1 to 7, 14 and / or the chondroitin sulfate biopolyamine complex prepared by the preparation method according to any one of claims 8 to 13.

18. A pharmaceutical composition, health food or cosmetic comprising the chondroitin sulfate biopolyamine complex according to any one of claims 1 to 7, 14 and / or the chondroitin sulfate biopolyamine complex prepared by the preparation method according to any one of claims 8 to 13.

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