Chondroitin sulfate biopolyamine complex, method of preparation thereof, and uses

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 pharmaceuticals. 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 and a representative substance of animal mucopolysaccharides. It is widely present in various animal tissues, especially rich in cartilage and connective tissues. Chondroitin sulfate is not only used 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, migraine, etc., and also has an auxiliary therapeutic effect on chronic nephritis, chronic hepatitis, keratitis and corneal ulcer. Some studies on the prevention and treatment of diseases such as coronary heart disease, angina pectoris, 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, despite numerous clinical studies worldwide, chondroitin sulfate has rarely been used as a prescription drug for the treatment of bone tissue-related diseases due to its limited clinical efficacy, high dosages, and, in some studies, little to no effect (Reference 3). A key reason for this controversy is the inconsistency in chondroitin purity, type, and subject groups, coupled with the very limited physiological activity of chondroitin sulfate itself, making it insufficient to demonstrate stable and reliable statistical significance. Furthermore, chondroitin sulfate, as a bio-derived polysaccharide, possesses extremely high safety, and no clear toxic side effects have been identified to date. In contrast, most other anti-inflammatory drugs generally have significant side effects and are unsuitable for long-term use. Therefore, developing novel chondroitin sulfate compounds with significantly increased physiological activity, high efficacy, and high safety is of paramount importance.

[0005] Researchers have made many efforts to enhance the activity of chondroitin. Previous reports have shown that low molecular weight chondroitin sulfate reduces viscosity, increases tissue permeability, and improves physiological activity. More recently, it has been reported that low molecular weight polysaccharides of low molecular weight chondroitin sulfate are also being investigated. In particular, Reference 1 discloses a method for preparing low molecular weight polysaccharides and the catalyst used therein, for obtaining low molecular weight chondroitin products by polysaccharide decomposition using a metal solid-phase catalyst. Reference 2 discloses the use of low molecular weight chondroitin sulfate in the manufacture of topical formulations for acne treatment. However, overall, there are limitations to the improvement of anti-inflammatory activity by low molecular weight chondroitin sulfate, and so far, low molecular weight chondroitin has hardly been commercialized.

[0006] Regarding the preparation method of chondroitin sulfate, reference 3 states that "the products obtained by current methods for preparing chondroitin sulfate or mixtures containing it are mainly derived from biological raw materials, and the manufacturing processes are diverse, resulting in significant differences in molecular structure and molecular weight, which leads to problems such as unstable quality and efficacy, and even side effects."

[0007] Therefore, while researchers have made many attempts to enhance the activity of chondroitin sulfate, further research is still needed to develop stable and highly active chondroitin sulfate or complexes containing it, as well as methods for preparing them.

[0008] For example, while some reports in References 4 and 5 use combinations of chondroitin sulfate and polyamine, in Reference 4, the combination of chondroitin sulfate and polyamine is only used as a matrix, and the physiological activity of the complex itself (pharmaceutical) is not investigated. Furthermore, because the proportion of polyamine in the composition is high, micron particles are formed, resulting in low stability. In contrast, the present invention forms a water-soluble complex with excellent stability. Also, 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, and the protein content on the activity of the complex is not investigated. [Prior art documents] [Patent Documents]

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

[0010] As mentioned above, researchers have undertaken a wide range of attempts to improve the pharmacological activity of chondroitin sulfate. Previous reports indicate that low molecular weight chondroitin sulfate can reduce viscosity, increase tissue permeability, and enhance physiological activity, but there are limitations to improving overall anti-inflammatory activity, and to date, low molecular weight chondroitin has hardly been commercialized. Furthermore, there are no methods for extracting or preparing stable and highly active chondroitin sulfate or complexes containing it.

[0011] Our research has shown that the activity of extracted chondroitin varies greatly depending on the extraction conditions and processes, and that some chondroitins exhibit significantly superior anti-inflammatory activity compared to others. Further research into their structure has confirmed the structural composition of such chondroitins, and it has been proven that the chondroitin sulfate biopolyamine complex provided by this invention has anti-inflammatory activity far exceeding that of ordinary chondroitin.

[0012] Therefore, in order to solve the above problems, the present invention provides a highly active chondroitin sulfate bio-polyamine complex and a method for preparing the same, which can significantly improve the anti-inflammatory (especially arthritis) activity of chondroitin sulfate and is expected to fill a gap in the clinical treatment of osteoarthritis. Furthermore, it has been found to have effects such as lowering blood lipids, antioxidant effects, aging delay, and life extension. Currently, there are no reports on the extremely potent anti-inflammatory activity of chondroitin sulfate bio-polyamine complexes, nor on their effects such as joint damage repair, blood lipid reduction, antioxidant effects, aging delay, and life extension. [Means for solving the problem]

[0013] In a first aspect of the present invention, a chondroitin sulfate biopolyamine complex is provided, 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 three or more combinations 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 with a weight-average molecular weight of 50,000 or more, as measured by the GPC integral ratio, is 0%. In some embodiments, the chondroitin sulfate is such that the proportion of chondroitin sulfate with a weight-average molecular weight of 25,000 to 50,000 is 40% or less in terms of GPC integral ratio. In some preferred embodiments, the chondroitin sulfate is such that the proportion of chondroitin sulfate with a weight-average molecular weight of 25,000 to 50,000 is 35% or less in terms of GPC integral ratio. In some more preferred embodiments, the chondroitin sulfate is such that, in terms of GPC integral ratio, the proportion of chondroitin sulfate with 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%. In some embodiments, the chondroitin sulfate is such that, in terms of GPC integral ratio, the upper limit of the proportion of chondroitin sulfate with 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%. The lower limit of the percentage of chondroitin sulfate that is 99% or more, or 100%, and has 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, the chondroitin sulfate is such that the proportion of chondroitin sulfate with a weight-average molecular weight of 400 or less in the GPC integral ratio is 15% or less, preferably 3% or less, more preferably 1% or less, and most preferably 0%. In some embodiments, 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 percentage 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 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, the chondroitin sulfate is such that, in terms of GPC integral 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%, or 80% to 100%. In some more preferred embodiments, the molecular weight distribution of the chondroitin sulfate in terms of GPC integral ratio is as follows: 0% of chondroitin sulfate has a weight-average molecular weight greater than 50,000; 0-40% has a weight-average molecular weight of 25,000-50,000; 40-100% has a weight-average molecular weight of 400-10,000, preferably 400-8,000; and 15% or less has a weight-average molecular weight of 400 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 proportions of chondroitin sulfate with a weight-average molecular weight of 25,000 to 50,000, the proportions of chondroitin sulfate with a weight-average molecular weight of 400 to 25,000, and the proportions of chondroitin sulfate with a weight-average molecular weight of 400 or less is 100%. In some embodiments, the amount of biopolyamine contained in the chondroitin sulfate biopolyamine complex is 200 μmol / g or less relative to the total weight (g) of the chondroitin sulfate biopolyamine complex, 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 The concentration is 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 amount of bio-polyamine contained is 0.5 μmol / g or more, preferably 0.6 μmol / g, 0.7 μmol / g, 0.8 μmol / g, 0.9 μmol / g, or 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 still more preferably 0%.

[0014] A second aspect of the present invention provides a method for preparing a chondroitin sulfate biopolyamine complex according to the first aspect of the present invention, comprising the step of mixing chondroitin sulfate and a biopolyamine, wherein the biopolyamine is contained in a quantity of 0.5 to 200 μmol / g based on the total weight (g) of the chondroitin sulfate biopolyamine complex.

[0015] A third aspect of the present invention provides a method for preparing a chondroitin sulfate biopolyamine complex according to the first aspect of the present invention, comprising the step of mixing an extract containing chondroitin sulfate and polyamine separated and extracted from raw materials 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. In some embodiments, the step of separating and extracting chondroitin sulfate and polyamine from the 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, 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 extraction method, and the residue after polyamine separation is treated by one or more of enzymatic decomposition method, protein precipitation method, chromatography and 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 the protein precipitation method to separate chondroitin sulfate and polyamine. In some embodiments, the raw material includes animal tissue, plant tissue and 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 the chondroitin sulfate biopolyamine complex in any one of the following (a) to (g). (a) Use in the preparation of anti-inflammatory drugs, (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 antioxidants, (g) Use in the preparation of pharmaceuticals for the purpose of slowing aging and / or extending life.

[0018] Here, the chondroitin sulfate biopolyamine complex is the chondroitin sulfate biopolyamine complex described in the first or fourth aspect of the present invention and / or the chondroitin sulfate biopolyamine complex prepared by the preparation method described in the second or third aspect of the present invention. In some embodiments, the inflammatory disease includes inflammation caused by inflammatory factors and / or inflammation caused by inflammatory cells, interleukins and / or tumor necrosis factors, and preferably the inflammatory disease is one or more selected from allergies, 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, which may optionally include cardiovascular diseases, and which may optionally include one or more of the following: 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 xanthomatous disease. In some embodiments, the joint injury includes joint injury due to inflammation, aging, exercise, or injury.

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

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

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

[0022] [Figure 1A] This figure shows the measurement results of IL-6 in the serum of mice in each group in Test Example 1. [Figure 1B] This figure shows the detection results of IL-1β in the serum of mice in each group in Test Example 1. [Figure 2A] This figure shows the curves of change in the degree of toe swelling in mice in each group in Test Example 1. [Figure 2B] These are photographs and anatomical images of the toes of mice in each group in Test Example 1. The arrows indicate the apparent site of inflammation. [Figure 3A] This figure shows the toe thickness data of rats in each group in Test Example 2. [Figure 3B] These are photographs of the toes of rats in each group in Experiment Example 2. The arrows indicate the apparent site of inflammation. [Figure 4A-4E] The figures show the results of routine blood analysis of rats in each group in Test Example 2. Figure 4A shows the absolute value of lymphocytes, Figure 4B shows the percentage of lymphocytes, Figure 4C shows the absolute value of neutrophils, Figure 4D shows the percentage of neutrophils, and Figure 4E shows the absolute value of white blood cells. [Figure 5A-5C]The figures show the results of organ index measurements for each group of rats in Test Example 2, with Figure 5A being the liver index, Figure 5B being the spleen index, and Figure 5C being the thymus index. [Figure 6] These are CT images of the ankle joints of rats in each group in Test Example 2. [Figure 7] These are magnified CT images of the ankle joints of rats in each group in Test Example 2. The arrows indicate the joints of some of the joints in the model group. [Figures 8A-8F] The figures show the CT results of rats in each group in Test Example 2. Figure 8A shows bone mineral density, Figure 8B shows bone surface area, Figure 8C shows bone volume, Figure 8D shows the ratio of bone surface area to bone volume, Figure 8E shows the intertrabecular space, and Figure 8F shows the trabecular width. [Figures 9A-9D] The figures show the blood lipid detection results for each group of rats in Test Example 3. Figure 9A shows the level of total serum cholesterol in mice, Figure 9B shows the level of total serum triglycerides in mice, Figure 9C shows the level of low-density lipoprotein in mice, and Figure 9D shows the level of high-density lipoprotein in mice. [Figure 10] This figure shows the number of reactive oxygen species-positive cells in Test Example 5, with the control group on the left and the CSX-administered group on the right. [Figure 11] This figure shows the results of detecting the anti-inflammatory activity of CSX against Alzheimer's disease-related inflammation in Test Example 6. [Figure 12A-12C] This figure shows the inflammatory response suppression effect of CSX at the cellular level in Test Example 7, with Figure 12A representing IL-6, Figure 12B representing IL-1β, and Figure 12C representing TNF-α. [Figure 13] This is a schematic diagram showing the results of CSX's aging-delaying and life-extending activity in Test Example 8. [Figure 14] This figure shows the IL-6 inhibition by each extract in Example 1 and conventional chondroitin. [Figure 15] This figure shows the H nuclear magnetic resonance spectrum of the extract in Example 1. [Figure 16]These are magnified partial views of the hydrogen spectra of chondroitin in each extract from Example 1 (chemical shift 3.0 ppm). [Figure 17] This is a separation chromatogram of the supernatant containing polyamine components from the extract in Example 1. [Figure 18] This figure shows the inhibition of IL-6 by various substances in Example 1. [Figure 19] This figure shows the binding force between chondroitin molecules and polyamine molecules in Example 1. [Figure 20] This is a schematic flowchart of the method for preparing the chondroitin sulfate biopolyamine complex according to the present invention. [Figures 21A-21B] This is a schematic diagram showing the results of CSX's activity in delaying aging and extending lifespan in Drosophila melanogaster in Test Example 8. [Figure 22] This figure shows the inhibition of IL-6 by various substances in Test Example 9. [Figure 23] This figure shows the inhibition of IL-6 by various substances in Test Example 10. [Figure 24] This figure shows the inhibition of IL-6 by various substances in Test Example 12. [Figure 25] This figure shows the inhibition of IL-6 by various substances in Test Example 13. [Modes for carrying out the invention]

[0023] The present invention will now be described in detail. The technical features described below will be explained based on representative embodiments and specific examples of the present invention, but the present invention is not limited to these embodiments and specific examples. Further details are provided below.

[0024] In this specification, a numerical range expressed as "Numerical A to Numerical B" means the range that includes the limit values ​​A and B.

[0025] In this specification, "substantially" or "effectively" means that the standard deviation from the theoretical model or theoretical data is within 5%, preferably within 3%, and more preferably within 1%.

[0026] In this specification, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0027] In this specification, “optional” or “optionally” means that the event or situation described thereafter may or may not occur, and that such description includes both cases in which the event occurs and cases in which it does not occur.

[0028] In this specification, references to “several specific / preferred embodiments,” “another specific / preferred embodiment,” and “embodiments” mean that certain elements (e.g., features, structures, properties, and / or characteristics) described in relation to an 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 preferred form in various embodiments.

[0029] In the present invention, the terms “including” and “having” and their variations 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 other steps specific to these processes, methods, products, or apparatus.

[0030] In this invention, "multiple" means two or more. "And / or" indicates a relationship between related things, and it means that there may be three such relationships. For example, A and / or B may represent three cases: A only exists, both A and B exist, and B only exists. The symbol " / " usually indicates that related things are in an "or" relationship.

[0031] In this invention, the term "weight-average molecular weight (Mw)" refers to relative molecular weight, statistically average molecular weight on a mass basis, or average molecular weight per unit weight. For example, it can be measured by methods such as light scattering and gel chromatography. In some embodiments, the weight-average molecular weight is measured by high-speed gel permeation chromatography, and the weight-average molecular weight of chondroitin sulfate and chondroitin sulfate polyamine is measured using a dextran molecular weight standard (China Food and Drug Administration) as a standard substance.

[0032] In this invention, the term "chondroitin sulfate" (hereinafter also referred to as "CS") refers to a variety of sulfated glycosaminoglycans with diverse molecular weights that are 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→) that are repeatedly linked by β-glycosidic bonds (1→4). In this invention, chondroitin sulfate includes the acid and salt forms of polysaccharides.

[0033] Chondroitin sulfate (CS) is usually a mixture, and CS extracted from terrestrial animals mainly consists of chondroitin sulfate A (CSA) and chondroitin sulfate C (CSC). CS extracted from marine animals further includes types such as chondroitin sulfate D (CSD) and chondroitin sulfate E (CSE). In this invention, CSA generally refers to CS characterized by the CSA-type disaccharide unit [4)-β-D-GlcA-(1→3)-β-D-GalNAc4SO3--(1→) (the sulfate group is located at the O-4 position of galactose). In this invention, CSC generally refers to CS characterized by the CSC-type disaccharide unit [4)-β-D-GlcA-(1→3)-β-D-GalNAc6SO3--(1→) (the sulfate group is located at the O-6 position of galactose). D generally refers to CS characterized by the CSD type disaccharide unit [4)-β-D-GlcA2SO3-(1→3)-β-D-GalNAc6SO3--(1→) (sulfate groups are located at the O-2 position of glucuronic acid and the O-6 position of galactose). CSE generally refers to CS characterized by the CSE type disaccharide unit [4)-β-D-GlcA-(1→3)-β-D-GalNAc4,6SO3--(1→) (sulfate groups are located at the O-4 and 6 positions of galactose).

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

[0035] In this invention, the term "biogenic amines (BA)" is a general term for low molecular weight organic compounds containing amino acids that have physiological activity. They can be considered as substances in which 1 to 3 hydrogen atoms in an ammonia molecule are substituted with alkyl or aryl groups, and are aliphatic, aliphatic, or heterocyclic low molecular weight organic alkalis that are commonly found in plants, animals, and food. Biogenic amines are classified structurally into aliphatic amines such as putrescine, cadaverin, spermine, and spermidine; aromatic amines such as tyramine and phenylethylamine; and heterocyclic amines such as histamine and tryptamine. Furthermore, biogenic amines can also be classified according to their composition into monoamines, which include histamine, tyramine, tryptamine, and phenylethylamine, and polyamines (also called biopolyamines), which include cadaverine, putrescine, spermine, and spermidine.

[0036] In the present invention, the terms “prevention” or “treatment” mean reducing the risk of developing or progressing a disease or condition (i.e., preventing the development of at least one clinical symptom of a disease in a patient who is prone to developing a disease or who may be exposed to a disease but has not yet shown symptoms of the disease). For example, treatment may include (i) prevention of a disease, disorder and / or condition in a patient who is prone to a disease, disorder and / or condition but has not yet been diagnosed with the disease, disorder and / or condition; (ii) suppression of the disease, disorder and / or condition, i.e., suppression of its progression; or (iii) reduction of the disease, disorder and / or condition, i.e., mitigation of the disease, disorder and / or condition.

[0037] In this invention, the term "effective dose" means the amount of a compound sufficient to achieve treatment or prevention of a disease when administered to a subject. The "effective dose" may be determined by the compound, the disease and its severity, and changes such as the age and weight of the subject being treated. "Therapeutic effective dose" means the amount effective for a therapeutic treatment. "Preventive effective dose" means the amount effective for preventive treatment.

[0038] In the present invention, the term "administration" means the physical introduction of a drug into a target using any of the various 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 this invention, the terms “subject,” “individual,” and “patient” are commonly known in the art and refer to any subject, particularly mammalian subjects, that require treatment, and can be used interchangeably herein. Specific examples include, but are not limited to, humans and other primates (including, for example, non-human primates such as chimpanzees, other apes, and monkey species). The terms “individual,” “subject,” and “patient” do not, in themselves, refer to any specific age, sex, race, etc.

[0040] As described herein, the term “inflammatory disease” is a general term for diseases in which inflammation is the primary destructive factor. Examples of inflammatory diseases include, but are not limited to, edema, dermatitis, acne, oral ulcers (e.g., inflammatory oral ulcers), allergies, atopic dermatitis, 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 the 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 dermatomyositis, Graves' disease, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus (SLE), tuberculosis, Sjögren's syndrome, amyloidosis, Hashimoto's thyroiditis, vasculitis, rheumatoid arthritis, reactive arthritis, polymyositis, scleroderma, Addison's disease, vitiligo, pernicious anemia, glomerulonephritis, severe abdominal diseases, type 1 diabetes mellitus, psoriasis, pulmonary fibrosis, and eczema. In the present invention, "chronic inflammatory disease" may include diseases involving chronic inflammation (for example, diseases belonging to chronic inflammatory complications). Such "chronic inflammatory diseases" may include myocardial infarction, cerebral infarction, and Alzheimer's disease.

[0042] In this invention, the term "arthritis" refers to a general term for diseases involving inflammatory changes in the joint area caused by bacterial infection, trauma, etc. Arthritis is broadly classified into acute arthritis and chronic arthritis. Acute arthritis can be further classified as follows: (1) Serous arthritis: Usually caused by trauma, but some cases have an unknown cause and generally occur in only one joint. (2) Serous fibrinous arthritis: Occurs simultaneously with acute rheumatoid arthritis, and cloudy exudate accumulates in the joint cavity. This can cause movement disorders even after the inflammation subsides due to the formation of a pseudomembrane. (3) Septic arthritis: Polyarthritis occurs in open joint wounds or infectious diseases such as gonorrhea, typhoid fever, scarlet fever, and sepsis. In infants 1-2 months old, it can develop into dislocation due to incurable bone damage. In adults, the suppurative area often ruptures, pus enters the joint, and develops into osteomyelitis of the periosteum; this is called secondary septic arthritis. Chronic arthritis can be further classified as follows. (1) Special types of inflammation: Usually refers to tuberculous arthritis or syphilitic arthritis, or gouty arthritis caused by uric acid metabolic disorders, which are more common in middle-aged men. (2) Polyarthritis: Chronic rheumatoid arthritis is the most common. This can develop from acute serous arthritis, occur as polyarthritis in the course of pneumonia, syphilis and gonorrhea, or be a type of sepsis. Still's disease also falls into this category. (3) Osteoarthritis: Usually occurs as a result of degenerative aging or trauma. (4) Hemophilic arthritis: Occurs in hemophilia patients due to bleeding within the joints. Degenerative arthritis, also called osteoarthritis, is localized arthritis caused by degenerative changes in articular cartilage and mainly occurs in middle-aged and elderly people.

[0043] In this invention, the term "rheumatoid arthritis" refers to a chronic systemic inflammatory disease of unknown cause that can infect many organs. In this process, an inflammatory response initially occurs in the synovial membrane surrounding the joints, and then gradually spreads to adjacent cartilage and bone, causing joint destruction and deformation. Extra-articular clinical symptoms include anemia, Sjögren's syndrome, subcutaneous nodules, pulmonary fibrosis, vasculitis, and skin ulcers.

[0044] In this 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, certain 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 is generally caused by a genetic defect, while secondary hyperlipidemia is generally caused by other factors such as various disease conditions, 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 causes. Hyperlipidemia may include hypertriglyceridemia, hypercholesterolemia, or a combination thereof. "Hypertriglyceridemia" refers to a condition in which serum total triglyceride levels are elevated above a desired level. "Hypercholesterolemia" refers to a condition in which serum cholesterol levels are elevated above a desired level. In some embodiments, hypercholesterolemia is a condition in which serum total cholesterol, HDL cholesterol (HDL-C), or LDL cholesterol (LDL-C) is above a desired level. Hyperlipidemia may further increase 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 levels of lipids in the blood vessels. The term "atherosclerosis" refers to arterial diseases in which fat accumulates in the inner walls of the arteries, eventually blocking blood flow.

[0045] The term “pharmaceutically acceptable” (or “pharmacologically acceptable”) means molecular entities and compositions that, when administered to animals or humans, do not produce adverse side effects, allergic reactions, or other harmful reactions. As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, dispersions, coatings, antimicrobial agents, isotonic and absorption retarders, buffers, excipients, binders, lubricants, gels, surfactants, etc., that can be used as a vehicle for a pharmacochemically acceptable substance.

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

[0047] The following describes in detail the chondroitin sulfate biopolyamine complex provided by the present invention, its preparation method, and its use.

[0048] <Chondroitin sulfate biopolyamine complex> In some aspects of the present invention, a chondroitin sulfate biopolyamine complex is provided, which is a complex of chondroitin sulfate and a biopolyamine, wherein the chondroitin sulfate and the biopolyamine are bound together. In some specific embodiments, the chondroitin sulfate and the biopolyamine are bound via a non-covalent bond. This complex functions as a drug itself, rather than as a matrix for other drugs or active ingredients. The chondroitin sulfate biopolyamine complex provided by the present invention is a natural pharmaceutical product, but can also be synthesized by chemical methods.

[0049] In some embodiments, the chondroitin sulfate in the chondroitin sulfate biopolyamine complex includes one or more combinations 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. [ka]

[0050] In some specific embodiments, depending on the type of chondroitin sulfate (e.g., CSA, CSC, CSD, and CSE), the chondroitin sulfate biopolyamine complex may have any of the structures represented by formulas I(CSA), II(CSC), III(CSD), and IV(CSE) below, or any combination thereof in any proportion. [ka] In equations I through IV, "polyamine" means polyamine / biological polyamine. n represents a natural number from 1 to 100, or 90, 80, 70, 60, 50, 40, 30, 20, or 16.

[0051] Since chondroitin sulfate usually exists as a mixture, it should be understood that the chondroitin sulfate biopolyamine complex provided in this 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 comprises one, two, or three or more combinations of spermine, spermidine, putrescine, and cadaverine. In some specific embodiments of the present invention, the biopolyamine comprises one of spermine, spermidine, putrescine, and cadaverine.

[0053] As a chondroitin sulfate biopolyamine complex containing one type of biopolyamine, A complex of chondroitin sulfate and spermine. A complex of chondroitin sulfate and spermidine. A complex of chondroitin sulfate and putrescine, and One example is a complex of chondroitin sulfate and cadaverine. In another specific embodiment of the present invention, the biopolyamine comprises at least two of spermine, spermidine, putrescine, and cadaverine.

[0054] In a more specific embodiment 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, Includes combinations selected from.

[0055] As demonstrated in the examples and test cases described later in this invention, chondroitin sulfate biopolyamine complexes containing two or more biopolyamines generally exhibit superior activity in all aspects compared to chondroitin sulfate biopolyamine complexes containing only one biopolyamine. Furthermore, chondroitin sulfate biopolyamine complexes containing one biopolyamine also exhibit significantly superior activity compared to conventional chondroitin sulfate. Such activities include, but are not limited to, anti-inflammatory (especially arthritis) activity, blood lipid reduction, antioxidant activity, aging delay, and life extension.

[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 is such that the proportion of chondroitin sulfate having a weight-average molecular weight of 50,000 or more in the GPC integral ratio is 0%.

[0058] In some embodiments of the present invention, the chondroitin sulfate is such that the proportion of chondroitin sulfate with a weight-average molecular weight of 25,000 to 50,000 is 40% or less in terms of GPC integral ratio.

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

[0060] In a more preferred embodiment of the present invention, the chondroitin sulfate is such that, in terms of GPC integral ratio, the proportion of chondroitin sulfate with 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, the chondroitin sulfate is such that the upper limit of the proportion of chondroitin sulfate having a weight-average molecular weight of 400 to 25,000 in the GPC integral ratio is 80% or more. Preferably, the chondroitin sulfate is such that the upper limit of the proportion of chondroitin sulfate having a weight-average molecular weight of 400 to 25,000 in the GPC integral ratio 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, the chondroitin sulfate has a lower limit of 40% or more in terms of the GPC integral ratio, where the proportion of chondroitin sulfate with a weight-average molecular weight of 400 to 25,000 is 40% or more. Preferably, the chondroitin sulfate has a lower limit of 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% or more in terms of the GPC integral ratio, where the proportion of chondroitin sulfate with 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, the chondroitin sulfate is such that the proportion of chondroitin sulfate with a weight-average molecular weight of 400 or less in the GPC integral ratio is 15% or less, preferably 3% or less, more preferably 1% or less, and most preferably 0%.

[0064] The upper limit of the proportion of chondroitin sulfate is such that 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 lower limit 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 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 sum of the proportions of chondroitin sulfate with a weight-average molecular weight of 25,000 to 50,000, the proportions of chondroitin sulfate with a weight-average molecular weight of 400 to 25,000, and the proportions of chondroitin sulfate with a weight-average molecular weight of 400 or less is 100%.

[0066] In some preferred embodiments of the present invention, the chondroitin sulfate is such that, in terms of GPC integral 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%, or 80% to 100%.

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

[0068] As described later and in the examples, the chondroitin sulfate biopolyamine complex provided by the present invention can be prepared from natural raw materials, and a complex can be formed by coprecipitation of chondroitin sulfate and biopolyamine present in the natural raw materials, with the two being bound by non-covalent bonds. Therefore, it is understood that any mixing ratio of chondroitin sulfate and biopolyamine present in the complex that is the same as or close to the ratio of the two in the natural raw materials used, and that is below the saturation state of the biopolyamine bound to chondroitin sulfate, will be active.

[0069] In some embodiments, the amount of bio-polyamine contained in the chondroitin sulfate bio-polyamine complex is 200 μmol / g or less relative to the total weight (g) of the chondroitin sulfate bio-polyamine complex; 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, the amount of the biopolyamine contained in the chondroitin sulfate biopolyamine complex is 0.5 μmol / g or more relative to the total weight (g) of the chondroitin sulfate biopolyamine complex; preferably, 0.6 μmol / g, 0.7 μmol / g, 0.8 μmol / g, 0.9 μmol / g, or 1 μmol / g or more.

[0071] In some more preferred embodiments, the chondroitin sulfate biopolyamine complex contains 0.5 to 200 μmol / g of the biopolyamine relative to the total weight (g) of the chondroitin sulfate biopolyamine complex, 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, or 1 to 180 μmol / g. In some preferred embodiments, the biopolyamine is a combination of one, two, or three or more of spermine, spermidine, putrescine, and cadaverine in any proportion.

[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 biopolyamines and can bind to more polyamine molecules, causing the chondroitin sulfate biopolyamine complex to have better physiological activity.

[0073] In this specification, "low molecular weight CS," "low molecular weight chondroitin," or "low molecular weight chondroitin" 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, or radiolysis. A 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. In this specification, "ordinary chondroitin" or "ordinary CS" refers to ordinary chondroitin available commercially.

[0074] <Method for preparing chondroitin sulfate biopolyamine complex and chondroitin sulfate biopolyamine obtained by the preparation method> This invention has found that chondroitin sulfate polyamine has significantly improved activity compared to chondroitin sulfate in many respects, and that chondroitin sulfate polyamine can be prepared by extraction from natural raw materials. However, conventional extraction methods for chondroitin sulfate typically employ processes such as ion resin exchange purification, decolorization with hydrogen peroxygen, and two or more alcohol precipitation dissolutions in order to ensure the purity and color characteristics of chondroitin. While these processes improve the purity of chondroitin, they also remove almost all of the polyamine. The complexation of chondroitin sulfate with bio-polyamines and its effects are not mentioned at all. Conventional methods usually require high ionic strength, which greatly affects non-covalent bonding, and even if a small amount of polyamine is present, the bio-polyamine complex of this invention cannot be formed.

[0075] In view of the above problems, in some aspects of the present invention, a method for preparing the chondroitin sulfate biopolyamine complex is provided, comprising the step of mixing an extract containing chondroitin sulfate and polyamine separated and extracted from raw materials 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 the chondroitin sulfate bio-polyamine complex provided by the present invention, under certain conditions, an extract containing chondroitin sulfate and polyamine separated and extracted from raw materials is mixed with ethanol to form a chondroitin sulfate bio-polyamine complex, which is a complex of chondroitin sulfate and polyamine that has significantly superior anti-inflammatory activity compared to ordinary chondroitin sulfate, as well as effects such as joint damage repair, blood lipid reduction, antioxidant, aging delay, and life extension.

[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. Unlike the neutral pH used for conventional extraction and precipitation of chondroitin sulfate, extracts with the above pH conditions are favorable for the formation of chondroitin sulfate biopolyamine complexes, and can increase the content of biopolyamines that can co-precipitate with chondroitin sulfate to form complexes, thereby promoting the formation of chondroitin sulfate biopolyamine complexes.

[0078] In some specific embodiments, the volume ratio of the extract to ethanol is 1:1 to 2.5, for example, 1:1 or 1:2.5. In some preferred embodiments, the volume of ethanol accounts for 60 to 70% of the volume of the extract-ethanol mixture, and this volume ratio is more suitable for the formation of chondroitin sulfate biopolyamine complexes. Unlike methods that pursue high purity chondroitin sulfate, this volume ratio results in some loss of chondroitin sulfate, but it can increase the polyamine content, reduce protein impurities, and further promote the formation of chondroitin sulfate biopolyamine complexes. In contrast, under conventional conditions, chondroitin sulfate biopolyamine complexes are usually not obtained. In some specific embodiments, the ethanol is anhydrous ethanol.

[0079] (Origin of raw materials) In the present invention, the origin of the raw materials is not particularly limited, and any raw materials containing chondroitin sulfate and / or polyamines may be used, for example, animal tissue, plant tissue, and microbial culture fermentation liquid.

[0080] In some embodiments, the raw material may be animal connective tissue, including cartilage, bone, tendons, fascia, and blood vessel walls. The animal may be a terrestrial animal such as a cow, pig, or chicken, or a marine animal such as a fish. Those skilled in the art will understand that the raw material contains 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 vary depending on the origin of the raw materials. Typically, CS extracted from terrestrial (animal) sources is mainly a mixture of CSA and CSC, while CS extracted from marine (animal) sources is mainly a mixture of CSA, CSC, and CSD, and also contains some CSE. Furthermore, the proportion of the diverse types of CS contained differs depending on the type of animal raw material. Therefore, in the chondroitin sulfate biopolyamine complex obtained by the method for preparing the chondroitin sulfate biopolyamine complex provided by the present invention, the chondroitin sulfate may be any combination of CSA, CSC, CSD, and CSE in any proportion.

[0082] In some preferred embodiments, chicken bones can be selected as the raw material, as they generally contain a large amount of bio-polyamines, are cheaper, have a stable supply quality, and are readily available in large quantities.

[0083] (Pre-processing) Before carrying out the method of the present invention, it is usually necessary to perform a pretreatment step to make the raw materials suitable for carrying out the method of the present invention (e.g., enzymatic hydrolysis, acid hydrolysis, separation and extraction). The pretreatment method is not particularly limited, and those skilled in the art can select an appropriate pretreatment method depending on the different raw materials. For example, for raw materials such as bone and cartilage, the pretreatment method may include boiling, removal of impurities, washing, drying, and grinding. In the case of soybeans, the pretreatment method may include grinding.

[0084] (Separation and extraction of chondroitin sulfate and polyamines in the raw materials) By separating and extracting chondroitin sulfate and polyamine from the raw materials before mixing and precipitation with ethanol to form a chondroitin sulfate biopolyamine complex, the proportion of chondroitin sulfate and polyamine in the raw materials can be increased, resulting in a higher purity chondroitin sulfate biopolyamine complex. This also reduces the presence of impurities such as inorganic salts and proteins, and minimizes the influence of impurities on subsequent complexation and precipitation processes.

[0085] In some embodiments of the present invention, when separating and extracting chondroitin sulfate and polyamine from the raw material, the raw material is first decomposed by enzymatic or acidic hydrolysis to obtain an enzymatic or acidic hydrolyzed solution, so as to sufficiently release components such as chondroitin sulfate and / or polyamine from the raw material.

[0086] Since insufficient enzymatic or acid hydrolysis affects the release of polyamines, in some embodiments, the raw materials are thoroughly enzymatically or acidically hydrolyzed. Those skilled in the art can select an appropriate amount of enzymatic or acid hydrolysis reagent to thoroughly enzymatically or acidically hydrolyze the raw materials.

[0087] In some embodiments, the selectable enzymes for enzymatic degradation include one or more of papain, alkaline proteases, neutral proteases, acidic proteases, pepsin, trypsin, chymotrypsin, bromelain, and fikain. In some preferred embodiments, 4,000 U or more of the enzyme, 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, or 50,000 U or more, is added per gram of pre-treated sample. The lower the enzyme activity, the less likely the protein is to be completely hydrolyzed, and the less likely polyamine molecules are to be released from the raw material. Furthermore, in the process of precipitation of the protein with trichloroacetic acid, polyamines tend to bind to larger proteins and precipitate, further reducing the amount of polyamine extracted and affecting the activity of the product. In some embodiments, the temperature for enzymatic degradation is 50°C to 70°C, preferably 55°C to 65°C, for example, 55°C, 60°C, or 65°C. In some embodiments, the enzymatic digestion time is 1 to 24 hours, preferably 3 to 24 hours, 3 to 16 hours, for example, 3 hours, 6 hours, or 16 hours. In some embodiments, enzymatic digestion may be performed once. In other embodiments, enzymatic digestion may be performed two or three or more times.

[0088] In some embodiments, organic acids and / or inorganic acids can be used for acid decomposition. 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, acid decomposition can be assisted by a combination of methods such as ultrasonic extraction, addition, and grinding in order to improve the efficiency of acid decomposition, thoroughly decompose the raw material, and release components such as chondroitin sulfate and / or polyamines.

[0089] In some embodiments of the present invention, chondroitin sulfate and polyamines are separated and extracted from the enzymatic or acidic hydrolyzed saturates obtained by enzymatic or acidic hydrolysis. In some embodiments, chondroitin sulfate and polyamines can be separated and extracted in steps. Stepwise extraction maximizes the extraction of polyamines, increases the weight ratio of polyamines in the chondroitin sulfate biopolyamine complex, and further reduces impurities (such as proteins and inorganic salts). Furthermore, chondroitin sulfate can be subjected to further operations, such as further purification, removal of chondroitin sulfate in salt form, or depolymerization of chondroitin sulfate. In another embodiment, chondroitin sulfate and polyamines can be separated and extracted simultaneously.

[0090] In some specific embodiments, when chondroitin sulfate and polyamines are simultaneously separated and extracted, the raw materials are usually treated by enzymatic hydrolysis. In some embodiments, an acid can be added to the enzymatic hydrolysis solution to precipitate the proteins in the solution and remove them. In some specific embodiments, after removing the proteins from the enzymatic hydrolysis solution, the concentration of chondroitin sulfate in the resulting preliminary extract may be in the range of approximately 6-7% (w / v). This concentration range is advantageous for obtaining the chondroitin sulfate biopolyamine complex in subsequent steps. In this concentration range, the biopolyamine content in the preliminary extract of chondroitin sulfate is higher, which is advantageous for complexation with chondroitin sulfate.

[0091] After the solution from which the protein has been removed is concentrated and / or its pH is adjusted, ethanol is added to complexize and precipitate it to obtain a chondroitin sulfate biopolyamine complex. In some specific embodiments, the acid used to precipitate the protein can be selected from trichloroacetic acid, perchloric acid, nitric acid, etc.

[0092] In stepwise separation and extraction of chondroitin sulfate and polyamine, in some embodiments, the polyamine in the sample can be separated by chromatography or extraction, and then the residue can be purified to obtain chondroitin sulfate by one or more of the following methods: enzymatic decomposition, protein precipitation, chromatography, or alcohol precipitation. The purified chondroitin sulfate and the separated polyamine are mixed, and ethanol is added to complexize and precipitate them to obtain a chondroitin sulfate biopolyamine complex.

[0093] In some specific embodiments, the chromatography is ion exchange chromatography. In some specific embodiments, the extraction method is organic solvent extraction or supercritical fluid extraction. 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 fluid extraction method is supercritical carbon dioxide extraction.

[0094] In some specific embodiments, when chondroitin sulfate and polyamine are separated and extracted stepwise, the process further includes a step of reducing the molecular weight of the chondroitin sulfate. In some embodiments, chondroitin sulfate purified by one or more methods, such as enzymatic decomposition, protein precipitation, chromatography, and alcohol precipitation, can be reduced in molecular weight using the method described in CN111495428A (e.g., Example 2) to obtain low molecular weight chondroitin sulfate. In some embodiments, the low molecular weight chondroitin sulfate and the separated polyamine are mixed, and ethanol is added to complexize and precipitate to obtain a chondroitin sulfate biopolyamine complex.

[0095] In some specific embodiments, the chondroitin sulfate biopolyamine complex-containing extract obtained by the above method for preparing the chondroitin sulfate biopolyamine complex showed a total polyamine content (mass ratio) of 0.01% to 5% after separation from chondroitin sulfate and subsequent detection.

[0096] (Other processes) Other steps in the method for preparing the chondroitin sulfate biopolyamine complex are not particularly limited, but can be adjusted or selected as needed or based on the actual equipment. In some embodiments, a higher purity chondroitin sulfate biopolyamine complex can be obtained by including a product purification step. However, it is understood that the chondroitin sulfate biopolyamine complex obtained by the above preparation method can exert its effects.

[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, wherein the chondroitin sulfate biopolyamine complex contains 0.5 to 200 μmol / g of the biopolyamine based on the total weight of the chondroitin sulfate biopolyamine complex.

[0098] In some specific embodiments, the chondroitin sulfate and the bio-polyamine may be commercially available products. In another specific embodiment, the chondroitin sulfate and the bio-polyamine can each be extracted from the raw materials described above.

[0099] In a more specific embodiment, chondroitin sulfate and a biopolyamine can be mixed in a solvent and dried to prepare the product. 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 chondroitin sulfate biopolyamine complexes 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 described above or a chondroitin sulfate biopolyamine complex obtained by a method for preparing the chondroitin sulfate biopolyamine complex described above, and a pharmaceutically acceptable carrier.

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

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

[0104] <Anti-inflammatory uses and methods of chondroitin sulfate biopolyamine complexes> Some aspects of the present invention provide the use of a chondroitin sulfate biopolyamine complex in the preparation of an anti-inflammatory drug, wherein the chondroitin sulfate biopolyamine complex is the chondroitin sulfate biopolyamine complex described above or a chondroitin sulfate biopolyamine complex obtained by the method for preparing the chondroitin sulfate biopolyamine complex described above.

[0105] In some embodiments, the use of a chondroitin sulfate biopolyamine complex in the preparation of a pharmaceutical for the treatment and / or prevention of inflammatory diseases is provided, wherein the chondroitin sulfate biopolyamine complex is the chondroitin sulfate biopolyamine complex described above or a chondroitin sulfate biopolyamine complex obtained by a method for preparing the chondroitin sulfate biopolyamine complex described above.

[0106] In some embodiments of the present invention, the inflammatory disease is an inflammatory disease caused by various pro-inflammatory factors, and / or an inflammatory disease caused by inflammatory cells, interleukins (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 allergies, 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 polyarthritis. More specifically, the arthritis is rheumatoid arthritis.

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

[0111] <Use and method of chondroitin sulfate biopolyamine complex for lowering blood lipids> In some other aspects of the present invention, it has been found that the chondroitin sulfate biopolyamine complex described above, or the chondroitin sulfate biopolyamine complex obtained by the method for preparing the chondroitin sulfate biopolyamine complex described above, has the effect of lowering blood lipids.

[0112] Accordingly, in some embodiments, the use of a chondroitin sulfate biopolyamine complex in the preparation of a pharmaceutical for lowering blood lipids is provided, and in other embodiments, the use of a chondroitin sulfate biopolyamine complex in the preparation of a pharmaceutical for the treatment and / or prevention of hyperlipidemia is provided, wherein the chondroitin sulfate biopolyamine complex is the chondroitin sulfate biopolyamine complex described above or a chondroitin sulfate biopolyamine complex obtained by a method for preparing the chondroitin sulfate biopolyamine complex described above.

[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, pharmaceuticals for the treatment and / or prevention of hyperlipidemia may also be applied to conditions associated with (e.g., inducing or exacerbating) hyperlipidemia, including, 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 xanthomatous disease.

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

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

[0117] <Other uses of chondroitin sulfate biopolyamine complex> The present invention further discovers that the chondroitin sulfate biopolyamine complex described above, or the chondroitin sulfate biopolyamine complex obtained by the method for preparing the chondroitin sulfate biopolyamine complex described above, has effects such as therapeutic and repairing joint damage, antioxidant activity, aging retardation, and life extension.

[0118] Accordingly, in some embodiments, the use of chondroitin sulfate biopolyamine complexes in the preparation of pharmaceuticals for the treatment and / or repair of joint injuries is provided, wherein the chondroitin sulfate biopolyamine complex is the chondroitin sulfate biopolyamine complex described above or a chondroitin sulfate biopolyamine complex obtained by a method for preparing the chondroitin sulfate biopolyamine complex described above.

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

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

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

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

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

[0124] Another aspect of the present invention provides 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 described above or a chondroitin sulfate biopolyamine complex obtained by the method for preparing the chondroitin sulfate biopolyamine complex described above. Some specific embodiments provide the use of a chondroitin sulfate biopolyamine complex in the preparation of cosmetics that contribute to antioxidant activity. Examples and Test Examples [Examples]

[0125] The present invention will be further described below with reference to examples and test examples, but the present invention is not limited thereto. Specific materials used in embodiments of the present invention and their origins 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 types, models, qualities, properties, or functions of the reagents and equipment described below may be used to carry out the present invention. The experimental methods in the following examples and test examples are conventional methods unless otherwise specified. Unless otherwise specified, the materials and reagents used in the following examples and test examples are all commercially available.

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

[0127] In the following examples, polyamines are measured in accordance with GB5009.208-2016.

[0128] The basic principle of the following examples is that, due to the strong non-covalent bonding between biological polyamines and chondroitin sulfate, the pH and concentration of the biological polyamines and chondroitin sulfate in the solution are adjusted, and precipitation is performed by ethanol precipitation to achieve the purpose of extraction and purification. However, since biological tissues contain many other molecules (amino acids, peptides, proteins, fats, inorganic salts, etc.), different extraction processes are used depending on the different materials and needs in order to eliminate interference from other substances and further improve purity and polyamine content.

[0129] Example: Extraction of chondroitin sulfate biopolyamine complex Example 1. Expression and verification of chondroitin sulfate biopolyamine complex. In this embodiment, a chondroitin sulfate polyamine complex was integrated, separated from animal bone tissue, and precipitated. Before confirming that the activity originated from the chondroitin sulfate polyamine substance, various analyses and evaluations were performed in this embodiment.

[0130] 1. Extraction In this example, chicken bones were used as the raw material. (1) Pretreatment of raw materials Fresh raw bones were boiled to remove impurities such as blood, fat, and remaining meat scraps. After that, the bones were washed with clean water, dried, and then ground and polished.

[0131] (2) Separation and extraction of chondroitin sulfate and polyamines from raw materials 2.1 Enzymatic Decomposition The pre-treated chicken bones were weighed in 5g portions, and 100mL of water with a pH of 5.7-6.0 and 2% enzyme relative to the mass of the raw chicken bones were added to each portion as follows. 0.1g papain (200U / mg, Hissho Biological Science and Technology) (NO.A-2); 0.1g papain (2,000 U / mg, Adamas reagent) (NO.A-3); 0.1g papain (800U / mg, Bishou Biotechnology) (NO.A-4); or, 0.1g trypsin (250U / mg, Aladdin Reagent Company) and 0.1g pepsin (3,000U / mg, Aladdin Reagent Company) (NO.A-6). The mixture was uniformly stirred, the system temperature was raised to 65°C, and the reaction was maintained at a constant temperature for 3 hours. The pre-treated chicken bones were then enzymatically hydrolyzed to obtain an enzymatic hydrolyzed solution. 2.2 Separation and Extraction of Chondroitin Sulfate and Polyamines After the enzymatic hydrolysis reaction was complete, the enzymatic hydrolysates obtained in step 2.1 were filtered through filter paper. 5.0 g of trichloroacetic acid (Aladdin Reagent Company, China) was added to each of the filtrates obtained after filtration, and the mixture was kept in an ice bath for 1 hour. After thorough mixing, the mixture was centrifuged at 8000 rpm / min for 5 minutes, and the supernatant was removed 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 approximately 5 with solid sodium hydroxide, and then concentrated under reduced pressure to approximately 10 mL of yellow liquid (no solid precipitate). 25 mL of anhydrous ethanol was added to each of the yellow liquids to induce alcohol precipitation. The mixture was shaken and centrifuged to obtain extracts (chondroitin sulfate biopolyamine complex). The centrifuged alcohol precipitates were then washed once again with anhydrous ethanol, centrifuged, and vacuum-dried overnight at 40°C to obtain 600 mg of the white solid extract (which was later verified to be chondroitin sulfate biopolyamine complex).

[0133] 2. Analysis of the extract The extracts obtained after the different enzymatic treatments described above were analyzed and validated. First, the inhibitory level of IL-6, i.e., anti-inflammatory activity, was measured. Specifically, mouse monocytes RAW264.7 were used. The cells were digested and inoculated into a 96-well plate at a rate of 10,000 cells per well. After cell adhesion, dexamethasone (DXM) and CSX were diluted in 10% FBS-containing DMEM medium containing 0.1 μg / ml of LPS. The final concentrations of dexamethasone and CSX were 0.1 mg / ml. After 18-24 hours of incubation, the supernatant was aspirated, and the IL-6 level in the supernatant was detected by enzyme-linked immunosorbent assay (Elisa).

[0134] Figure 14 shows the IL-6 inhibition levels after action with each extract and conventional chondroitin. NO.A-2, A-3, A-4, and A-6 are extracts obtained after the enzymatic treatment described above. NO.A-1 is chondroitin provided by a company (Wuxing Bio, Hunan), and NO.A-5 is chondroitin purchased from a reagent company (Aladdin, Shanghai). From Figure 14, it can be seen that extract NO.A-3 had a significantly superior inhibitory effect on IL-6 compared to the other extracts or chondroitin, followed by NO.A-6, but there was no significant difference in chondroitin NO.A-1, A-4, and A-5. Chondroitin extract (NO.A-3) exhibiting excellent anti-inflammatory activity was selected and subjected to 1H nuclear magnetic resonance spectroscopy (solvent: heavy water, 600 MHz, Agilent, USA). In addition to the hydrogen atom signal of the chondroitin molecule, clear new signals appeared at chemical shifts of 1.7, 1.9, and 3.0 ppm (Figure 15). Figure 16 shows a magnified section of the 1H nuclear magnetic resonance spectrum from the characteristic peak (1.7 ppm) for each extract. In Figure 16, A, B, C, and D are extracts obtained using the different extraction methods described above, corresponding to extracts NO.A-6, A-4, A-3, and A-2, respectively. The relative integrated area is C > A > B > D, and the order of inhibitory activity against inflammatory factors for the corresponding extracts is A-3 > A-6 > A-4 > A-2, with the integrated area of ​​the characteristic peak corresponding to the magnitude of activity.

[0135] To further investigate the origin of the activity, components in the extract were separated, and chondroitin and substances with chemical shifts of 1.7, 1.9, and 3.0 ppm were isolated. The separation method was as follows: 100 g of A-3 extract and 200 g of cetylpyridinium chloride (Hexadecylpyridinium Chloride, CPC) (InNoChem LOT: KYGA540) were added to 2 L of purified water. Electrostatic reaction between chondroitin and CPC produced a white precipitate. After stirring at room temperature for 1 hour, centrifugation (7000 rpm, 10°C, 15 minutes) was performed, and a complex of CPC and chondroitin precipitated. The supernatant contained chemical shifts of 1.7, 1.9, and 3.0 ppm. The chondroitin and CPC were co-precipitated and added to a 10% NaCl aqueous solution. The precipitate was then heated and stirred at 60°C to dissociate. Three times the volume of solution, anhydrous ethanol was added, and chondroitin sulfate precipitated. After repeating the ethanol precipitation procedure twice, the precipitate after filtration was dried to obtain 81 g of a white solid with a purity of 95.5%. Nuclear magnetic resonance (NEM) testing revealed the disappearance of the characteristic peaks of the above chemical shifts. The supernatant containing chemical shifts of 1.7, 1.9, and 3.0 ppm was washed three times with dichloromethane (200 mL / wash) to remove excess CPC, the aqueous phase was collected, approximately 280 mL of anhydrous 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. Each fraction was collected (chromatograms are shown in Figure 17, with the numbers in the figure indicating the fraction numbers), and the activity against the inflammatory factor IL-6 was measured again using the method described above. It was found that fraction No. 3 showed a much higher activity than the other components. Fraction A-3 was further concentrated, and nuclear magnetic resonance and mass spectrometry were performed, revealing that it consisted of three types of biopolyamines: spermine, spermidine, and putrescine. It was confirmed that the molar ratio of these three amines was close to 1:1:1.

[0136] To further investigate the forms of CS and polyamine in the extract, the following experiment confirmed the binding of CS and polyamine.

[0137] 1. Extract No. 3 was dialyzed, with the cutoff molecular weight of the dialysis bag set to 10 kD. Dialysis was performed for 5 days, with water changed every 8 hours. After dialysis, the retained solution was freeze-dried. When the polyamine content was measured, no decrease in polyamine content was observed.

[0138] 2. Reaction of fluorescein isothiocyanate (FITC) with chondroitin polyamine Experimental method: 50 mg of No. A-3 extract was dissolved in 10 mL of purified water, and the pH was adjusted to 9-10 with a 6 mol / L sodium hydroxide solution. Then, 1 mL of aqueous solution containing 10 mg of FITC was added, and the mixture was wrapped in tin foil and allowed to react overnight at room temperature, away from light. After dialyzing for 5 days using a 1000D dialysis membrane, three times the volume of anhydrous ethanol was added to obtain a yellow precipitate. After filtration, the solution was washed once with anhydrous ethanol to obtain a yellow solid, which was vacuum-dried overnight. The same procedure was performed using normal chondroitin as a control. Normal chondroitin was white, while activated chondroitin was yellow, showing a clear difference in color. This indicates that a condensation reaction is occurring between the FITC molecule and the amino groups of the polyamine molecule. Some amino groups have weakened bonds with CS due to amide formation, but unreacted amino groups with strong bonding to CS still exist. The polyamine molecule remains attached to the high-molecular-weight chain of CS, so a clear color persists even after dialyzing. On the other hand, ordinary chondroitin cannot react with FITC molecules, so the freed FITC molecules are removed. The ordinary chondroitin is bovine chondroitin provided by Hunan Wuxing Biotechnology Co., Ltd., with a weight-average molecular weight of 21k, 0% of which has a molecular weight over 50,000, 31% between 25,000 and 50,000, 69% between 400 and 25,000, and 0% of which is chondroitin sulfate with a molecular weight of 400 or less.

[0139] 3. ITC reaction To verify the presence and magnitude of affinity between chondroitin molecules and polyamine molecules, isothermal titration calorimetry (ITC) (Microcalcium) was performed. TM The binding constants of chondroitin molecules and polyamine molecules were measured using iTC200. Chondroitin molecules were titrated using polyamine molecules as ligands, and the parameters were set to achieve a 1:1 molar ratio. Chondroitin molecules were dissolved in water at a concentration of 20 μM, and polyamine molecules were similarly dissolved in water at a concentration of 200 μM. The titration was performed a total of 17 times with 2 μL per drop. As shown in Figure 19, the experimental results showed that the binding constant between chondroitin molecules and polyamine molecules was KD = 1 / KA = 5 μM, the stoichiometric ratio N = 1.74, and the enthalpy ΔH = 2.29 kcal / mol. This indicates that chondroitin has a strong binding affinity to polyamine molecules.

[0140] The results of the above tests showed that the main active ingredients in extracts NO.A-2, A-3, A-4, and A-6 were chondroitin sulfate biopolyamine complexes containing spermine, spermidine, and putrescine. After separation and measurement, the results for polyamine content (molar mass ratio), chondroitin sulfate mass ratio, protein content ratio, and weight-average molecular weight relative to the total weight of the extract are shown in Table 1 below. The method for measuring the chondroitin sulfate mass ratio was based on the Chinese Pharmacopoeia 2020, with chondroitin sulfate sodium as the standard sample (China Food and Drug Administration). Protein content was measured by GPC using Lowry molecular weight. The higher molecular weights of A-2 and A-4 may be due to the incomplete breakdown of covalent bonds between the protein and multiple polysaccharide molecules. The presence of protein may lead to a decrease in the anti-inflammatory activity of the complex, and even to a pro-inflammatory effect. For example, NO.A-2 exhibits a certain pro-inflammatory effect, which is thought to be due to an excessively high content of impurities such as protein. On the other hand, NO.A-6 does not exhibit good anti-inflammatory effects, which may be due to its high protein content.

[0141] [Table 1]

[0142] To further investigate the origin of the activity, the formulation was adjusted according to the proportion of polyamines in extract NO.A-3. Additionally, the IL-6 inhibitory activity was measured by the aforementioned method when three types of polyamines and chondroitin were each formulated in equal molar amounts. The formulation method involved dissolving chondroitin sulfate (Sigma, USA) in water, then adding spermine tetrahydrochloride (Acros, USA), spermidine trihydrochloride (Acros, USA), and putrescine dihydrochloride (Acros, USA) individually or simultaneously. After homogeneous stirring, the mixture was freeze-dried to obtain a white solid. Here, the weight-average molecular weight of chondroitin sulfate was 20,100, with 71% of the molecules having a molecular weight of 400-25,000, 29% having a molecular weight of 25,000-50,000, and 0% having a molecular weight of 400 or less.

[0143] Figure 18 shows the measurement of the anti-inflammatory activity of various substances against IL-6. NO.B-1 is a combination of biopolyamines (spermine + spermidine + putrescine, the ratio of the three polyamines is the same as in extract NO.B-4, and the total molar amount of the three polyamines is the same as the total molar amount of polyamines contained in the amount of extract used for NO.B-4), NO.B-2 is a combination of CS and polyamines (CS + spermine + spermidine + putrescine, the ratio of the three polyamines is the same as in the extract for NO.B-4, the total molar amount of the three polyamines is the same as the total molar amount of polyamines in the extract for NO.B-4, and the amount of CS used is the same as the amount of extract for NO.B-4 minus the polyamines), NO.B-3 is CS + spermine (the molar amount of spermine is NO. The total molar amount of polyamines in the B-4 extract is the same as the amount of CS used (the amount of CS used is the same as the amount of polyamines subtracted from the NO.B-4 extract), NO.B-4 is the NO.A-3 extract mentioned above, NO.B-5 is CS + spermidine (the molar amount of spermidine is the same as the total molar amount of polyamines in the NO.B-4 extract, the amount of CS used is the same as the amount of polyamines subtracted from the NO.B-4 extract), NO.B-6 is CS + putrescine (the molar amount of putrescine is the same as the total molar amount of polyamines in the amount of NO.B-4 extract used, the amount of CS used is the same as the amount of polyamines subtracted from the NO.B-4 extract), and NO.B-7 is CS (the amount used is the same as the mass of NO.B-4 extract). From these results, it was found that when the polyamine content is the same, the complex has activity equivalent to the extract, is higher than the equivalent amount of the above bio-polyamine composition (without CS), and is far higher than CS alone. The proportion of the total molar amount of polyamines in the compound combination was the same as A-3.

[0144] From the above results, it was found that when the polyamine content is the same, the complex has activity equivalent to that of the extract, and the inhibition rate against inflammatory factors is approximately 3.5 times higher than that of the above bio-polyamine composition (without CS) in equivalent amounts, which is far higher than CS alone.

[0145] Conventional methods for extracting chondroitin sulfate typically involve removing other components from the raw material to obtain higher purity chondroitin sulfate. Unlike conventional techniques, the present invention has discovered that by precipitating the chondroitin sulfate under certain conditions using anhydrous ethanol after the raw material has undergone sufficient enzymatic action, a complex of chondroitin sulfate and polyamine with surprisingly significantly improved activity compared to chondroitin sulfate alone can be obtained. Therefore, in subsequent examples, the amount of polyamine obtained from the raw material is increased by a stepwise extraction method, and this is combined 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 + anhydrous ethanol complexing and precipitation. In this example, porcine cartilage was used as a raw material to prepare a chondroitin sulfate biopolyamine complex. (1) Pretreatment of raw materials The pretreatment method for the raw materials was the same as in Example 1.

[0147] (2) Separation and extraction of chondroitin sulfate and polyamines from raw materials 2.1 Enzymatic Decomposition 100g of pre-treated pork cartilage was weighed, 500mL of water and 2% papain (2,000U / mg) relative to the mass of the raw pork cartilage were added, the pH was adjusted to 6-7, the temperature was raised to 55°C, and the mixture was stirred for 16 hours. The enzymatic hydrolysis system was then cooled to room temperature to obtain the enzymatic hydrolysate. 2.2 Chromatography of Biological Polyamines The enzymatically decomposed product obtained in 2.1 was placed on diatomaceous earth and filtered by vacuum suction to remove insoluble impurities such as bone residue, yielding a pale yellow, transparent solution. 100 mL of weakly acidic cation exchange resin (product code D152 was used in this example) was measured out, placed in an empty column tube, washed with purified water, and packed. 500 mL of the pale yellow transparent solution (filtrate) obtained above was passed through the column, and the permeate was collected. Subsequently, the resin column was washed with 200 mL of 4% (m / v) dilute hydrochloric acid, and the eluate was collected as eluate A. After adsorption and elution using the cation exchange column as described above, the polyamine substance can be concentrated and purified and concentrated into eluate A. 2.3 Separation and Extraction of Chondroitin Sulfate The permeate obtained in step 2.2 was distilled under reduced pressure and concentrated to 100 mL. 5 g of trichloroacetic acid was added to precipitate the protein, and the precipitate was removed by centrifugation, leaving the supernatant. To obtain a permeate obtained by precipitating proteins with trichloroacetic acid, three times the volume of anhydrous ethanol was added, and chondroitin sulfate was precipitated by alcohol precipitation. The mixture was shaken and centrifuged. The lower solid obtained by centrifugation was washed once with anhydrous ethanol, and after centrifugation, the resulting solid was vacuum-dried overnight at 45°C to obtain a crude product of sodium chondroitin sulfate. To further purify chondroitin sulfate, the crude chondroitin sulfate sodium obtained was prepared as a 20% aqueous solution by mass-volume ratio. 50 mL of strongly basic anion exchange resin (D280 was used in this example) was added, and the mixture was stirred to adsorb the solution for 20-30 minutes. Then, 1-1.5 times the volume of the resin was added to a 9% (m / v) sodium chloride solution to eluate the solution and obtain eluate B. Elution B was again precipitated with 3 times the volume of anhydrous ethanol to precipitate the chondroitin sulfate, which was then shaken and centrifuged. The lower solid obtained by centrifugation was washed once with anhydrous ethanol, and after centrifugation, the resulting solid was vacuum-dried overnight at 45°C to obtain chondroitin sulfate sodium. The purity was measured to be 90.1%, and the measurement method was the same as in Example 1.

[0148] (3) Decomposition of chondroitin sulfate into smaller molecules (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 Invention: Method for Preparing Low Molecular Weight Polysaccharides and Catalyst Used Therein). In short, 0.1 g of resin catalyst and 2.1 mL of 30% hydrogen peroxide were added to the solution, and the mixture was reacted at 50°C for 6 hours to obtain a low molecular weight chondroitin sulfate solution.

[0149] (4) Complexation and precipitation of chondroitin sulfate biopolyamine complex To the low molecular weight chondroitin sulfate solution obtained in step (3), eluate A obtained in step 2.2 was added, the pH was adjusted to approximately 5, and 2.5 times the volume of anhydrous ethanol was added to precipitate the mixture with alcohol. The mixture was shaken, centrifuged, and the lower solid obtained by centrifugation was washed once with anhydrous ethanol. The solid obtained by centrifugation was vacuum-dried overnight at 45°C to obtain 6.0 g of a milky white solid that is a chondroitin sulfate biopolyamine complex.

[0150] Measurements revealed that this substance is a chondroitin sulfate biopolyamine complex containing spermine and spermidine. After separation, detection revealed that the total polyamine content (molar mass ratio) in the extract was 23.26 μmol / g. Of this, 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 the low molecular weight chondroitin was 4.3k, with 0% of the content exceeding 50,000, 0% between 25,000 and 50,000, and 100% between 400 and 25,000. Specifically, 14% of the content was between 8,000 and 25,000, 30% between 5,000 and 8,000, 56% between 400 and 5,000, and 0% below 400.

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

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

[0153] (2) Separation and extraction of chondroitin sulfate and polyamines from raw materials 2.1 Acid decomposition 100g of pre-treated chicken tissue (including chicken bones, heads, and internal organs) was weighed and subjected to two ultrasonic extractions using 1M dilute hydrochloric acid for a total of 2 hours (300mL for 1 hour, 200mL for 1 hour). After extraction, the tissue was filtered using filter paper. The tissue filtration residue was washed twice with 50mL of purified water to prepare the tissue residue. 2.2 Extraction of biological polyamines The entire solution from step 2.1 was combined to obtain a total of 600 mL of pale yellow, slightly cloudy liquid. This liquid was concentrated to 50 mL by rotary evaporation, centrifuged, and the supernatant was removed. The pH was adjusted to approximately 12 with sodium hydroxide, and the solution was centrifuged again. The supernatant was extracted twice with 10 mL of n-butanol to extract the biopolyamine mixture. The organic phases were combined, the dilute hydrochloric acid was adjusted to acidity, and the mixture was concentrated to dryness until a grayish-brown solid was obtained, yielding a crude extract of biopolyamines. 2.3 Separation and Extraction of Chondroitin Sulfate 2.3.1 Enzymatic Decomposition To the tissue filtration residue from step 2.1, add 100 mL of water with a pH of 5.7-6.0 and 2% papain (2,000 U / mg) relative to the mass of the tissue filtration residue. Stir uniformly, raise the temperature of the system to 65°C, and maintain the temperature for 3 hours to allow the reaction to proceed. Then, add 1 g of papain and continue the reaction for another 3 hours to obtain the enzymatic hydrolysate. 2.3.2 Separation and extraction After filtering the enzymatic hydrolysate from step 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, and centrifuged at 8,000 rpm / min for 5 minutes. The supernatant was removed to remove the protein. The pH was adjusted to approximately 5 using sodium hydroxide solid, and three times the amount of anhydrous ethanol was added to precipitate the alcohol. The mixture was shaken, centrifuged, and the filtrate was temporarily stored. The alcohol precipitate obtained by centrifugation was washed once again with anhydrous ethanol, centrifuged, and the solid obtained by centrifugation was vacuum-dried overnight at 40°C to obtain a white solid which is chondroitin sulfate.

[0154] (3) Decomposition of chondroitin sulfate into smaller molecules (solid-phase catalytic decomposition) The dried white solid (chondroitin sulfate) obtained in step 2.3.2 was dissolved in 100 mL of water to obtain a solution, and a low molecular weight treatment was performed according to Chinese patent application No. CN111495428A (Method for preparing low molecular weight polysaccharides and catalyst used therein). In short, 0.05 g of resin catalyst and 1.1 mL of 30% hydrogen peroxide were added to the solution, and the mixture was reacted at 50°C for 3 hours to obtain a low molecular weight chondroitin sulfate solution.

[0155] (4) Complexation and precipitation of chondroitin sulfate biopolyamine complex The crude extract of the bio-polyamine from step 2.2 (a grayish-brown solid) was dissolved using the low molecular weight chondroitin sulfate solution obtained in step (3), the pH was adjusted to approximately 5, and 2.5 times the volume of anhydrous ethanol was added to precipitate the mixture with alcohol. The mixture was shaken and centrifuged. The lower solid obtained by centrifugation was washed once with anhydrous ethanol, and the solid obtained by centrifugation was vacuum-dried overnight at 45°C to obtain 6.0 g of a milky white solid that is a chondroitin sulfate bio-polyamine complex.

[0156] Measurements revealed that the substance is a chondroitin sulfate biopolyamine complex containing spermine, spermidine, putrescine, and cadaverine. After separation, detection revealed that the total polyamine content (molar mass ratio) in the extract was 56.91 μmol / g. The spermine content was 28.16 μmol / g, spermidine was 9.82 μmol / g, putrescine was 8.07 μmol / g, and cadaverine was 10.86 μmol / g. The weight-average molecular weight of low-molecular-weight chondroitin was 6.2k, with 0% of samples having a molecular weight over 50,000, 0% between 25,000 and 50,000, and 100% between 400 and 25,000. Specifically, 24% were between 8,000 and 25,000, 26% between 5,000 and 8,000, 43% between 400 and 5,000, and 0% were below 400.

[0157] Example 4. Acid extraction-organic solvent extraction method + anhydrous ethanol complex and precipitation. In this example, a chondroitin sulfate biopolyamine complex was prepared using fish bones and soybeans as raw materials. Soybeans are rich in spermine and spermidine, and the purpose of adding soybeans as a raw material is to improve the polyamine content in the product.

[0158] (1) Pretreatment of raw materials The raw materials, fish bones and soybeans, were crushed using a mixer.

[0159] (2) Separation and extraction of chondroitin sulfate and polyamines from raw materials 2.1 Acid decomposition 100g of pre-treated fish bones and 10g of pre-treated soybeans were weighed and subjected to ultrasonic extraction twice using 1M dilute hydrochloric acid for a total of 2 hours (300mL for 1 hour, 200mL for 1 hour). After extraction, the samples were filtered using filter paper. The tissue filtration residue was washed twice with 50mL of purified water to prepare the tissue residue. 2.2 Extraction of biological polyamines The entire solution from step 2.1 was combined to obtain a total of 600 mL of pale yellow, slightly cloudy liquid. This liquid was concentrated to 50 mL by rotary evaporation, centrifuged, and the supernatant was removed. The pH was adjusted to approximately 12 with sodium hydroxide, and the solution was centrifuged again. The supernatant was extracted twice with 10 mL of n-butanol to extract the biopolyamine mixture. The organic phases were combined and concentrated to dryness until a grayish-brown solid was obtained to obtain a crude extract of biopolyamines. 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) relative to the mass of the tissue filtration residue were added, and the mixture was uniformly stirred. The system was then heated to 65°C and maintained at this temperature for 3 hours to allow the reaction to proceed. After that, 1 g of papain was added and the reaction was continued for 3 hours to obtain an enzymatic hydrolysate. 2.3.2 Separation and extraction The enzyme hydrolysate obtained in step 2.3.1 was filtered through filter paper. 20.0 g of trichloroacetic acid was added to the filtrate, stored in an ice bath for 1 hour, mixed well, and centrifuged at 8,000 rpm / min for 5 minutes. The supernatant was removed to remove the protein. 100 mL of strongly acidic cation exchange resin (product code 001*7 was used in this example) was weighed, added to an empty column tube, washed with purified water, and packed. The supernatant after centrifugation was passed through the column, and the permeate was collected. The permeate was chondroitin sulfate.

[0160] (3) Complexation and precipitation of chondroitin sulfate biopolyamine complex The grayish-brown solid from step 2.2 was dissolved in the permeate obtained in step 2.3.2, and the pH was adjusted to 5.0. Anhydrous ethanol was added 2.5 times to induce alcohol precipitation, the mixture was shaken, and the mixture was centrifuged. The alcohol precipitate solid obtained by centrifugation was washed once again with anhydrous ethanol, centrifuged, and the solid obtained by centrifugation was vacuum-dried overnight at 40°C to obtain a white solid which is a chondroitin sulfate biopolyamine complex.

[0161] Measurements revealed that this substance is a chondroitin sulfate biopolyamine complex containing spermine, spermidine, putrescine, and cadaverine. After separation, detection revealed that the total polyamine content (molar mass ratio) in the extract was 129.7 μmol / g, with spermine at 5.2 μmol / g, spermidine at 70.3 μmol / g, putrescine at 49.7 μmol / g, and cadaverine at 4.6 μmol / g. The weight-average molecular weight of chondroitin was 22k, with 0% of molecules exceeding 50,000, 37% between 25,000 and 50,000, 63% between 400 and 25,000, 53% between 8,000 and 25,000, 8% between 5,000 and 8,000, 2% between 400 and 5,000, and 0% below 400.

[0162] Test example Test Example 1: Evaluation of the therapeutic effects of each chondroitin sulfate on chronic inflammation using an adjuvant arthritis mouse test. To analyze whether CSX can suppress the inflammatory response in mice, a mouse adjuvant arthritis model was created (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) to establish an arthritis model for one week before administration). Subsequently, forced oral administration was performed (dosage: 3 mg / mouse / day; note: Group 1A was CS, Group 1B was low molecular weight CS, the low molecular weight CS was obtained by the degradation method of Example 2 (weight-average molecular weight 4.3 k, molecular weight distribution similar to Example 2), and Group 1C was CSX, the product of Example 3). After 30 days, blood was collected from the orbit, serum was prepared, and IL-6 and IL-1β inflammatory factors were measured using the Mouse IL-6 ELISA kit (product number: VAL604) and the Mouse IL-1 ELISA kit (R&D Systems, product number: MLB00C).

[0163] As shown in Figures 1A and 1B, Group 1C significantly reduced the levels of IL-6 and IL-1β in mouse serum. Serum IL-6 and IL-1β levels in the normal group (no model group) were extremely low, while serum IL-6 and IL-1β levels in the control group (model group) were significantly elevated. Serum IL-6 levels in Group 1C were already comparable to those of the normal group (no model group). This demonstrates a potent anti-inflammatory effect.

[0164] Furthermore, the width of the mice's toes was measured, and the level of toe swelling was analyzed. Referring to Figure 2A, in groups 1B and 1C, toe swelling in mice decreased significantly with increasing administration time. In group 1C, toe swelling was considerably reduced by day 22 of administration, and individual mice were found to be close to the normal group. Thus, group 1C was found to have a significant effect in reducing toe swelling caused by the adjuvant. Anatomical analysis of the mouse toes revealed, as shown in Figure 2B, that the control group (model group) exhibited significant red swelling and lesions due to an inflammatory response in the legs. Group 1C did not show significant inflammatory lesions or red swelling.

[0165] The results showed that using 3 mg / day / mouse CSX significantly improved mouse arthritis, followed by low molecular weight CSX, while conventional CSX was the worst, with inflammation still high even after 30 days.

[0166] Test Example 2: Therapeutic effect on arthritis (rheumatoid arthritis) In a rat model of rheumatoid arthritis, CSX has shown remarkable therapeutic effects against the disease. Specifically, CSX significantly improves swelling of organs such as the toes, liver, and spleen in inflammatory model rats, reduces inflammatory phenotypes in lymphocytes and neutrophils in the blood, and exhibits therapeutic effects similar to methotrexate (MTX). It can essentially treat joint and foot bone damage caused by chronic inflammation, demonstrating superior therapeutic efficacy compared to MTX, without the harmful side effects associated with MTX.

[0167] This study analyzed the effects of CSX in rheumatoid arthritis rats (Vital River, SD). Preparation of the rat rheumatoid arthritis model: A rat rheumatoid arthritis model was prepared using bovine type II collagen (CII, Solarbio). An emulsion was prepared by mixing CII with an equal volume of complete Freund's adjuvant (CFA, Chondrex), resulting in a final CII concentration of 1.0 mg / mL. Six rats were randomly selected as a blank control group (i.e., normal group / blank group), and the remaining rats were used to prepare the rheumatoid arthritis model. All rats except those in the blank control group received a total of 0.5 mL of the emulsion injected into multiple locations on their backs and at the base of their tails. Ten days later, the same dose of the collagen and adjuvant emulsion mixture was injected into the same sites 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, forced oral administration every other day), drug group (CSX obtained in Example 2, dose 200 mg / kg, forced oral administration daily).

[0168] Swelling of the rats' toes was observed one, two, three, and four weeks after administration, and the thickness of the toes on the right hind limb of each group of rats was measured and recorded. Specific method: After restricting the rats' activity, the right hind limb of each rat was gently extended, the measurement point on the rat's toe was marked with a marker pen, and then the thickness of the toe was measured with calipers.

[0169] Referring to Figures 3A and 3B, in the drug group (CSX), the degree of toe swelling in rats decreased significantly after 7 days of administration without side effects, and further decreased after 14 days of administration. The toes of rats in the model group were always significantly swollen. Administration was continued for 28 days, but the toe swelling remained at a relatively stable level. The lack of further reduction may be related to connective tissue hyperplasia due to swelling modeling. In the positive control group (MTX), the expected effect of suppressing toe swelling in rats was observed, but side effects such as diarrhea and weight loss were seen. From these results, it was shown that CSX has a significant effect in suppressing toe swelling caused by rheumatoid arthritis in rats. Furthermore, there was no significant toxicity in the rats.

[0170] Referring to Figures 4A to 4E, venous blood was collected from rats after 14 days of administration, and routine blood tests were performed. The absolute number of lymphocytes was significantly lower in the positive control group (MTX) compared to the model group, and the percentage of lymphocytes was significantly lower in the drug group (CSX) compared to the model group. However, the absolute number of lymphocytes in the drug group (CSX) did not differ statistically from that of the model group. Furthermore, the absolute number of neutrophils was significantly lower in the positive control group (MTX) compared to the model group, and also significantly lower in the drug group (CSX). There were no statistically significant differences in the absolute number of white blood cells between the model group, the drug group (CSX), and the positive control group (MTX).

[0171] Referring to Figures 5A to 5C, rats were dissected after 28 days of administration, and the liver, spleen, and thymus were collected and accurately weighed. Organ indices were then measured based on the ratio of rat body weight to the weight of each organ. The results showed that CSX significantly reduced hepatomegaly in rats. The spleen index reflected CSX's significant reduction in splenomegaly in rats, suggesting a reduction in the development of chronic inflammation. There was no statistically significant difference in the thymus index.

[0172] Based on these results, CSX significantly improved the degree of swelling in the toes, liver, and spleen of inflammatory model rats, and reduced the inflammatory phenotype of lymphocytes and neutrophils in the blood, demonstrating a therapeutic effect similar to that of MTX.

[0173] Referring to Figures 6 and 7, CT images of rat ankle joints showed that the blank group (normal group) rats had clearly defined joint interfaces, regular joint surfaces, and smooth bone surfaces. The model group rats had unclear joint interfaces, irregular joints, rough bone surfaces, and exhibited bone hyperplasia and swelling. The drug group (CSX) rats also showed some degree of joint damage, but their joint interfaces were clearer than the model group, their joints were more regular, and their bone surfaces were smoother. The positive control group (MTX) rats had relatively smoother joint bone surfaces than the control group, and their joint surfaces were even tighter than the model group. However, the degree of joint damage was more severe than in the drug group (CSX) rats.

[0174] Referring to Figures 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). CSX administration can effectively mitigate inflammation-induced bone mineral density loss. While the specific surface area of ​​the ankle joint in the model group was clearly higher than in the blank group, the bone surface area (BS) of the ankle joint in rats clearly decreased after administration of CSX and MTX. The decrease was more pronounced in the drug group (CSX). The drug group (CSX) rats showed a significant decrease in bone volume (BV) and a significant decrease in the bone surface area-to-bone volume ratio (BS / BV). Trabecular separation / spacing (Tb.Sp) refers to the mean width of the medullary cavity between trabeculae. An increase in Tb.Sp may lead to osteoporosis. In the rat model group, Tb.Sp significantly increased, but after CSX administration, Tb.Sp decreased in the rats, suggesting that CSX has an effect of reducing inflammation-induced osteoporosis. Trabecular thickness (Tb.Th) increased in the drug group (CSX) compared to the model group, reflecting that CSX can suppress inflammation-induced osteoporosis. From these CT results, it was found that CSX significantly suppresses inflammation-induced osteoporosis, increases bone density, and improves bone surface smoothness, demonstrating superior efficacy compared to MTX.

[0175] Furthermore, the experiment revealed that the positive control group (MTX) developed diarrhea at the dosage used in this experiment, while the drug group (CSX) showed no obvious abnormalities. In acute toxicity tests in mice, the drug group (CSX) showed no toxic reactions even with a single dose of up to 2000 mg / kg, suggesting that CSX may be safer.

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

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

[0178] Referring to Figures 9A to 9D, compared with the blood lipid model group, total cholesterol was significantly reduced in groups 2A, 2B, and 2C, with the reduction being most pronounced in group 2C. While total triglycerides did not show a significant decrease in groups 2A and 2B compared to the blood lipid model group, they were significantly reduced in group 2C. Low-density lipoprotein cholesterol was significantly reduced in groups 2A, 2B, and 2C compared to the blood lipid model group, with the reduction being most pronounced in group 2C. Compared to the blood lipid model group, high-density lipoprotein cholesterol was significantly reduced in groups 2A and 2B, but not significantly reduced in group 2C. From this animal study, it was found that CSX has the effect of significantly reducing serum total cholesterol, serum total triglycerides, and low-density lipoprotein cholesterol.

[0179] Test Example 4: Anti-inflammatory activity In this study, to evaluate the anti-inflammatory effect of CSX on skin inflammation, a mouse ear swelling model was used, and the right ear of mice was sensitized three times. The sensitizer was phorbol ester (Psaitong reagent, China), at a concentration of 0.125 mg / mL. The sensitization method involved applying 20 μL (10 μL per surface) to the right ear of the mouse each time, with each sensitization performed at 48-hour intervals. Group 4A was an aqueous solution of CSX (product of Example 3), Group 4B was an aqueous solution of low molecular weight CS and spermidine (the molar mass ratio of spermidine was the same as the molar ratio of polyamine in Group 4A, and the molar amount of spermidine was the same as the total molar amount of polyamine contained in the extract of Example 3. The amount of CS used was the same as the mass obtained by subtracting polyamine from the extract, and 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), and Group 4C was the negative control group (ultra-purified water). Group 4D was an aqueous solution of normal CS and spermidine (the molar mass ratio of spermidine was the same as the molar ratio of polyamine in Group 4A, i.e., the molar amount of spermidine was the same as the total molar amount of polyamine contained in the extract of Example 3). The amount of CS used was equal to the mass of the extract minus the polyamines. The standard CS is chondroitin provided by Hunan Wuxing Biotechnology Co., Ltd., with a weight-average molecular weight of 48k, of which 34% is of molecular weight over 50,000, 55% is of 25,000-50,000, and 11% is of 400-25,000. The solution concentrations of 4A, 4B, and 4D were all 2% by mass-volume ratio and administered once a day for a total of 6 consecutive days. The administration method was surface application, and the dose for each was 20 μL / mouse. The evaluation indicators were mainly the degree of ear swelling and body weight of the mice. To evaluate the degree of ear swelling of the mice, after the mice were killed by cervical vertebral dislocation, both ears were cut off, symmetrical ears were taken using a 6 mm diameter hole punch, and their weight was measured using an electronic balance. The difference in weight between the left and right ears was defined as the degree of swelling.

[0180] The results are shown in Tables 2 and 3 below. Both the product CSX from Example 3 and the complex of low molecular weight CS and spermidine exhibited significant anti-inflammatory effects. The product from Example 3 was more effective, and both were significantly superior to the negative control group. Furthermore, CSX did not affect the body weight of the mice, and no obvious side effects were observed. The complex of low molecular weight CS and spermidine was found to be superior to the combined use of regular CS and spermidine.

[0181] [Table 2]

[0182] [Table 3]

[0183] Test Example 5: Antioxidant Activity To evaluate the antioxidant effect of CSX, this study used human liver HEPG2 cells as a model, and measured the effect of CSX on intracellular reactive oxygen species free radicals using a fluorescent probe method. CSX-1 used in this study was the product of the NO.A-3 extract from Example 1, and CSX-2 was a mixture of low molecular weight CS and polyamines (spermine, spermidine, and putrescine) in the same proportions as CSX-1 (the proportions of the three polyamines were the same as in the NO.A-3 extract from Example 1, and the total molar amount of polyamines was the same as the total molar amount of polyamines contained in the NO.A-3 extract from Example 1. The amount of CS used was the same as the mass of the extract minus the polyamines). The low molecular weight CS was obtained by the decomposition method in Example 2 (weight-average molecular weight 4.3k, molecular weight distribution the same as 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 fluoresce and can freely pass through the cell membrane. Once inside the cell, it can be hydrolyzed by intracellular esterases to produce DCFH. On the other hand, DCFH cannot pass through the cell membrane, so the probe can be easily introduced into the cell. Intracellular reactive oxygen species can oxidize non-fluorescent DCFH to fluorescent DCF. By detecting the fluorescence of DCF, the level of reactive oxygen species inside the cell can be determined.

[0185] Cells in the logarithmic growth phase were collected and their cellular 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, and the mixture was digested at 37°C for 5 minutes. 3 mL of serum-containing medium was added to stop the reaction, the mixture was aspirated into a 15 mL centrifuge tube, centrifuged at 1,000 rpm, the supernatant was discarded, an appropriate amount of medium was added, and the cells were counted. 2,000 to 10,000 cells were dispensed into each well of a 96-well plate, incubated at 37°C for 6 hours to allow the cells to adhere to the cell wall, stimulated by adding drugs of varying concentrations, and after 24 hours, the plate was removed. DCFH-DA was diluted 1:1000 with serum-free culture medium to achieve a final probe concentration of 10 μM. The cell culture medium was removed, and DCFH-DA diluted to an appropriate volume was added. The cells were incubated in a cell culture vessel at 37°C for 20 minutes. To ensure sufficient removal of DCFH-DA not present in the cells, the cells were washed three 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. Cytological experiments showed that CSX (especially low molecular weight CSX) possesses significant antioxidant capacity at a final concentration of 0.25 mg / mL. Compared to the control group (untreated cells), the number of reactive oxygen species (ROS) positive cells decreased from 36.5% to 25.7% in the CSX-1 treatment group, and further decreased to 1.17% in the CSX-2 group. See Figure 10.

[0186] Test Example 6: Measurement of anti-Alzheimer's disease-related inflammation activity Research on neurodegenerative diseases such as Alzheimer's disease (AD) has shown that inflammatory processes play a fundamental role in the development of AD, and that microglia-mediated inflammatory responses in the central nervous system are one of the major factors in neurodegenerative diseases, particularly Alzheimer's disease. AD-related inflammatory components include brain cells such as microglia and astrocytes. IL-6, released from microglia, is considered one of the major factors mediating AD production.

[0187] To detect the inhibitory effect of CSX on microglial inflammation, human microglia HMC3 were treated with CSX (prepared in Example 2). The results showed that CSX significantly inhibited the expression level of IL-6 in LPS-induced microglia. See Figure 11.

[0188] Test Example 7: CSX-mediated inhibitory effect on inflammatory response at the cellular level. Mouse monocytes RAW264.7 increase the expression levels of many intracellular inflammatory factors, such as IL-6, TNF-α, and IL-1β, upon induction with lipopolysaccharide (LPS). In this study, we analyzed whether CSX could inhibit the production of inflammatory factors in cells. The CSX used in this study 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 LPS-containing 10% FBS DMEM to a final concentration of 0.6 mg / mL, and 100 μl was added per well. This was repeated three times for each gradient. Dexamethasone (DXM) was used at a concentration of 2 mg / mL of the mother liquor. It was diluted 1000-fold in LPS-containing 10% FBS DMEM, and 100 μl was added per well. This was repeated three times for each gradient. Cells were divided into a non-LPS-stimulated group and a cell-LPS-stimulated group. Each group was repeated three times. After 24 hours of incubation, the supernatant was carefully aspirated and analyzed by direct enzyme-linked immunosorbent assay (ELISA).

[0190] Referring to Figures 12A to 12C, it was shown that CSX can significantly inhibit the expression of IL-6, IL-1β, and TNF-α in LPS-induced mouse monocyte macrophages. The inhibitory effect on IL-6 was superior to that of dexamethasone. CSX was demonstrated to be superior in suppressing the expression levels of inflammatory factors at the cellular level.

[0191] Test Example 8: Aging Delay / Life Extension Activity To evaluate the life-extending activity of CSX, guppies were used as the study subject, and the survival time of their offspring was measured. The experimental fish was one artificially farmed pregnant guppy (product name "Hongyun Dangtou") purchased from the Beijing Shilihe Bird, Fish and Insect Market. After one week of farming, this experimental fish produced more than 50 fry. After stable rearing for 3 days, the fry were divided into two groups, with 25 fry in each group. 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 changed weekly. Barrel drinking water was used as the water source. An appropriate amount of fry food was given once a day. CSX was added to tank 1: After changing the water weekly, 30mg / L of CSX was added. Tank 2 was raised under normal conditions: the water was changed weekly, but no CSX was added. The number of fry deaths was observed and tallied daily, and the average survival time of fry in each tank was calculated from the sum of (survival days * number of fry) / total number of fry.

[0193] As a result, the average survival time in tank 1 was 31.88 days, and the average survival time in tank 2 was 27.48 days, demonstrating that 30 mg / L of 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-delaying / life-extending activity of CSX, we studied Drosophila melanogaster (bred in our laboratory) and measured their survival time. The experimental Drosophila melanogaster were w1118 wild-type Drosophila melanogaster bred in the laboratory, and the basal diet formulation was a liquid feed containing 2.2% sucrose, 8% malt extract, 1.8% yeast, and 1.2% butylparaben. Female and male Drosophila were each given three different diet formulations. Formulation 1 was the basal diet formulation, Formulation 2 was the basal diet formulation with 0.1 g / L of CSX (product of Example 2) added, and Formulation 3 was the basal diet formulation with a combination of spermidine hydrochloride and spermine hydrochloride (10 times the molar amount of polyamine in Formulation 2) added. Each formulation included six groups, consisting of three groups of females and three groups of males, with 20 individuals in each group. All were female or male Drosophila melanogaster that had not mated within four hours of emergence. The number of dead Drosophila melanogaster flies was observed and tallied daily. 400 μl of feed corresponding to each group was supplied daily, and the average survival time for each group was calculated by dividing the sum of (survival days * number of individuals) by the total number of individuals.

[0195] Referring to Figures 21A and 21B, the average survival time of Drosophila melanogaster fed with Formula 1 was 46.2–49 days, that of Formula 2 was 54.5–59 days, and that of Formula 3 was 50.6–52.5 days. Compared to normal feed, CSX was found to extend the lifespan of Drosophila melanogaster by 18.0%–20.4%. Although the total molar amount of polyamines in CSX of Formula 2 was only one-tenth of that in Formula 3 (free polyamines), the life-extending effect of the CSX group was 7.7–12.38% higher than that of general polyamines.

[0196] Test example 9: Anti-inflammatory activity measurement test In this test example, the anti-inflammatory activity of each type of substance against IL-6 was measured, and the measurement method was the same as in Example 1. All low molecular weight CS used in this test example is CS obtained by the decomposition method of Example 2 (weight-average molecular weight 4.3k, molecular weight distribution is the same as 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, and No. C-9 is low molecular weight CS + spermidine + spermine + putrescine. The total molar amount of polyamines in Nos. C3 to C12 is the same, and when two or more polyamines are used, the molar ratio of each polyamine is the same. From the above results, it was found that the activity of the CS and polyamine complex is significantly superior to that of low molecular weight CS alone or polyamine alone. The combined effect of multiple amines is superior to that of amines used alone. See Figure 22 for details.

[0197] Test Example 10: Comparison of low molecular weight polyamines with bio-polyamine and non-bio-polyamine activity In this test example, the anti-inflammatory activity against IL-6 was measured by combining low molecular weight CS with various biopolyamines, and the measurement method was the same as in Example 1. All low molecular weight CS were obtained by the degradation method of Example 2 (weight-average molecular weight 4.3k, molecular weight distribution is the same as in Example 2). All low molecular weight CS used in this test example were obtained by the degradation method of Example 2 (weight-average molecular weight 4.3k, molecular weight distribution is the same as in Example 2). The results are shown in Figure 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, and D-8 is low molecular weight CS + pentaethylenehexamine. The total molar concentration mass ratio of polyamines in each group was the same at 2 μmol / g, and when two or more polyamines were used, the molar ratio of each polyamine was the same. From the above results, the combination of low molecular weight CS and each type of polyamine showed different activities, with spermine and spermidine exhibiting comparable activity, superior to putrescine, and even superior to cadaverine. In contrast, histamine and tryptamine, as well as the non-biogenic polyamines 1,7-diaminoheptane and pentaethylenehexamine, had pro-inflammatory effects, with histamine exhibiting the strongest effect among them.

[0198] Test Example 11: Comparison of the activity of chondroitin sulfate biopolyamine complexes with different biopolyamine content. In this test example, low molecular weight CS and spermine were mixed in different proportions, and the stability of the combinations was measured. All low molecular weight CS was obtained by the decomposition method of Example 2 (weight-average molecular weight 4.3k, molecular weight distribution is the same as in Example 2). The experimental method is as follows: A 40 mg / ml aqueous solution of low molecular weight CS was prepared, and an aqueous solution of spermine hydrochloride was prepared at the concentrations shown in Table 4 below. The two solutions of the same volume were uniformly mixed to obtain the mixed solutions No. E-1 to E-8. The transmittance of the solutions (wavelength 600 nm) was measured using an ultraviolet spectrophotometer. Of these, the transmittance of No. E-7 and E-8 decreased significantly, which is thought to be due to the formation of a colloidal solution at the nanometer to micron level. The stability of the mixed solutions was measured by two methods: 1. The solutions were left to stand for 7 days and their stability was observed. 2. To simulate stability in blood, 20 μl of the mixed solution was added to 1 ml of rabbit plasma solution (Beijing Luqiao Biological), and the changes in the solution were observed. The results are shown in Table 4 below. From the above results, it can be seen that when the spermine content is too high (No. E-7, E-8), it reacts with chondroitin and precipitates easily, making the system unstable. Furthermore, when the spermine content is too high (No. E-6, E-7, E-8), it readily binds electrostatically to serum proteins, producing a large amount of precipitate, and cell tests revealed significant cytotoxicity. From this, it can be seen that combinations with excessively high spermine content are unstable, highly toxic, and have no practical value. No. E-6, E-7, and E-8 were synthesized according to the method in reference 4.

[0199] [Table 4]

[0200] Test Example 12: Demonstrates that the effects of other polysaccharides and other weight-average molecular weight CS are inferior to those of the present combination. In this test example, the anti-inflammatory activity against IL-6 was measured using the same method as in Example 1 for each molecular weight distribution of CS and combinations of other anionic polysaccharides and polyamines. The polyamines in the combinations were putrescine, spermine, and spermidine, with a total molar concentration mass ratio of 2 μmol / g for all of them. When two or more polyamines were included, the molar ratio of each polyamine was 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 molecular weight distribution is the same as in Example 2) was used. No.F-2 is CS + polyamine, and the CS used was a product provided by Hunan Wuxing Biotechnology Co., Ltd., with a weight-average molecular weight of 48k, a proportion of over 50,000 molecular weight of 34%, a proportion of 25,000-50,000, and a proportion of 400-25,000 of 11%. No. F-3 is CS + polyamine, and the CS used is a product provided by Hunan Wuxing Biotechnology Co., Ltd. It has a weight-average molecular weight of 35k, with 12% of the molecules having a molecular weight of 50,000 or more, 68% between 25,000 and 50,000, and 20% between 400 and 25,000. No. F-4 is CS + polyamine, and the CS used is a product of Hunan Wuxing Biotechnology Co., Ltd., manufactured by enzymatic hydrolysis, with a weight-average molecular weight of 879, with 99% between 400 and 2000, 54% between 400 and 1,000, 45% between 1,000 and 2,000, 1% less than 400, and 0% greater than 2,000. NO.F-5 is a CS+ polyamine, and the CS used is a product provided by Hunan Wuxing Biotechnology Co., Ltd. It has a weight-average molecular weight of 27k, with 0% of the molecules having a molecular weight greater than 50,000, 53% between 25,000 and 50,000, and 47% between 400 and 25,000. NO.F-6 is a CS+ polyamine, and the CS used is a product provided by Hunan Wuxing Biotechnology Co., Ltd., manufactured by enzymatic hydrolysis, with a weight-average molecular weight of 598, with 11% having a molecular weight less than 400, 0% having a molecular weight greater than 2,000, 89% between 400 and 2,000, 85% between 400 and 1,000, and 4% between 1,000 and 2,000.No. F-7 is a complete hydrolysate of CS + polyamine, with a weight-average molecular weight of 415 for the complete hydrolysate of CS, where 56% is less than 400 and 44% is greater than 400. The complete hydrolysate of CS was 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 these results, it was found that the activity of the low molecular weight CS and polyamine complexes No. F-1, F-4, F-5, and F-6 is significantly superior to that of CS with other molecular weight distributions or combinations of other anionic polysaccharides and polyamines.

[0201] Test Example 13: Proof that the molar ratio of various polyamines does not significantly affect the activity. In this test example, the anti-inflammatory activity against IL-6 was measured using the same method as in Example 1 for combinations of CS and polyamines in molar proportions. 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 polyamines in the combinations were putrescine, spermine, and spermidine, with a total molar concentration mass ratio of 2 μmol / g for all. The molar ratio of polyamines in NO.G-1 was spermine:spermidine:putrescine = 1:1:1, in NO.G-2 it was spermine:spermidine:putrescine = 5:1:1, in NO.G-3 it was spermine:spermidine:putrescine = 1:5:1, and in NO.G-4 it was spermine:spermidine:putrescine = 1:1:5. The measurement results are shown in Figure 25. From the results above, it was found that, in the case of CS having the same molecular weight and molecular weight distribution, if the total molar amount of polyamines is the same, differences in the molar ratio of each polyamine do not significantly affect the activity.

Claims

1. A chondroitin sulfate biopolyamine complex 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 three or more combinations of spermine, spermidine, putrescine, and cadaverine. The chondroitin sulfate in question has a GPC integral ratio in which the proportion of chondroitin sulfate with a weight-average molecular weight of 50,000 or more is 0%, and The chondroitin sulfate in question has a GPC integral ratio in which the proportion of chondroitin sulfate with a weight-average molecular weight of 25,000 to 50,000 is 40% or less. The chondroitin sulfate in question has a GPC integral ratio such that the upper limit of the proportion of chondroitin sulfate with a weight-average molecular weight of 400 to 25,000 is 100%. Furthermore, the lower limit of the proportion of chondroitin sulfate having a weight-average molecular weight of 400 to 25,000 is 40%, and The chondroitin sulfate in question has a GPC integral ratio such that the proportion of chondroitin sulfate with a weight-average molecular weight of 400 or less is 15% or less. The sum of the proportions of chondroitin sulfate with a weight-average molecular weight of 25,000 to 50,000, the proportions of chondroitin sulfate with a weight-average molecular weight of 400 to 25,000, and the proportions of chondroitin sulfate with a weight-average molecular weight of 400 or less is 100%. The amount of the biopolyamine contained in the chondroitin sulfate biopolyamine complex is 200 μmol / g or less relative to the total weight (g) of the chondroitin sulfate biopolyamine complex. Furthermore, the content of the biopolyamine contained in the chondroitin sulfate biopolyamine complex is 0.5 μmol / g or more. Chondroitin sulfate biopolyamine complex.

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 in question has a GPC integral ratio in which 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%, or 80% to 100%. Preferably, the molecular weight distribution of the chondroitin sulfate in terms of GPC integral ratio is as follows: The chondroitin sulfate biopolyamine complex according to claim 1 or claim 2, wherein 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 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.

4. The weight-average molecular weight of the chondroitin sulfate biopolyamine complex is 400 to 50,000. Preferably, the chondroitin sulfate biopolyamine complex according to claim 1 or claim 2, characterized in that the weight-average molecular weight of the chondroitin sulfate biopolyamine complex is 400 to 25,000.

5. The chondroitin sulfate biopolyamine complex according to claim 1 or claim 2, wherein 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%.

6. A method for preparing a chondroitin sulfate biopolyamine complex according to claim 1, A preparation method comprising the step of mixing chondroitin sulfate and a biopolyamine, characterized in that the chondroitin sulfate biopolyamine complex contains 0.5 to 200 μmol / g of the biopolyamine based on the total weight (g) of the chondroitin sulfate biopolyamine complex.

7. A method for preparing a chondroitin sulfate biopolyamine complex according to claim 1, A preparation method comprising the step of mixing an extract containing chondroitin sulfate and polyamine separated and extracted from raw materials with ethanol, characterized in that the pH of the extract is 4 to 6 and the volume ratio of the extract to ethanol is 1:1 to 3.

8. The process of separating and extracting chondroitin sulfate and polyamine from the raw materials is as follows: An enzymatic or acidic hydrolysis step is performed to decompose the raw material by enzymatic or acidic hydrolysis to obtain an enzymatic hydrolyzed solution or an acidic hydrolyzed solution, The preparation method according to claim 7, characterized by comprising a chondroitin sulfate and polyamine separation and extraction step of simultaneously or stepwise extracting chondroitin sulfate and polyamine from the enzymatic hydrolysate or acid hydrolysate.

9. When extracting chondroitin sulfate and polyamines in steps, the polyamines in the enzymatic or acidic hydrolyzed solution are separated by chromatography or extraction, and the residue after polyamine separation is treated with one or more of the following methods: enzymatic hydrolysis, protein precipitation, chromatography, and alcohol precipitation, thereby separating chondroitin sulfate from the residue. The preparation method according to claim 8, further comprising the step of optionally separating chondroitin sulfate and then reducing the molecular weight of the chondroitin sulfate.

10. The preparation method according to claim 8, characterized in that, when chondroitin sulfate and polyamine are extracted simultaneously, the proteins in the enzymatic or acidic hydrolyzed solution are precipitated by a protein precipitation method to separate the chondroitin sulfate and polyamine.

11. The preparation method according to any one of claims 7 to 10, characterized in that the raw materials include animal tissue and plant tissue and microbial culture fermentation liquid.

12. The use of any of the following (a) to (g) of the chondroitin sulfate biopolyamine complex, (a) Use in the preparation of anti-inflammatory drugs, (b) Use in the preparation of pharmaceuticals for the treatment and / or prevention of inflammatory diseases, (c) Use in the preparation of pharmaceuticals for lowering 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 drugs, (g) Use in the preparation of pharmaceuticals for the purpose of slowing aging and / or extending lifespan, The chondroitin sulfate biopolyamine complex is the chondroitin sulfate biopolyamine complex described in claim 1 or claim 2 and / or the chondroitin sulfate biopolyamine complex prepared by the preparation method described in any one of claims 6 to 10.

13. The inflammatory disease includes inflammatory diseases caused by inflammatory factors, and / or inflammatory diseases caused by inflammatory cells, interleukins, and / or tumor necrosis factors, and preferably, the inflammatory disease is one or more selected from allergies, eczema, myocardial infarction, cerebral infarction, Alzheimer's disease, dermatitis, or arthritis, or The aforementioned hyperlipidemia includes primary hyperlipidemia and / or secondary hyperlipidemia, or The aforementioned hyperlipidemia includes hypertriglyceridemia and / or hypercholesterolemia, The aforementioned hyperlipidemia includes hyperlipidemia-related diseases, and optionally, the hyperlipidemia-related diseases include cardiovascular diseases, and optionally, the aforementioned cardiovascular diseases include one or more of the following: 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 xanthomatous disease, or The use according to claim 12, wherein the joint injury includes joint injury due to inflammation, aging, exercise, or injury.

14. Use in the preparation of health foods or cosmetics of the chondroitin sulfate biopolyamine complex according to claim 1 or claim 2 and / or the chondroitin sulfate biopolyamine complex prepared by the preparation method according to any one of claims 6 to 10.

15. A pharmaceutical composition, health food, or cosmetic comprising the chondroitin sulfate biopolyamine complex described in claim 1 or claim 2.

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