Composition for promoting synthesis of chondroitin sulfate

Abstract

The purpose of the present invention is to provide a composition for promoting chondroitin sulfate synthesis and a method for promoting chondroitin sulfate synthesis. The present invention relates to a composition for promoting chondroitin sulfate synthesis, the composition containing quercetin or a glycoside thereof as an active ingredient.

Description

[Technical field]

[0001] The present invention relates to a composition for promoting chondroitin sulfate synthesis. The present invention also relates to a method for promoting the synthesis of chondroitin sulfate. [Background technology]

[0002] Chondroitin sulfate is a type of acidic mucopolysaccharide, and exists in animals as proteoglycan covalently bound to a core protein, usually called a core protein. Chondroitin sulfate is present in many animal tissues, such as cartilage, connective tissues such as skin, and brain tissue, and is particularly abundant in the extracellular matrix of cartilage.

[0003] Cartilage is a connective tissue consisting of chondrocytes and the matrix surrounding them, and forms joints, skeletons, etc. in animals such as humans. For example, articular cartilage thinly covers the surface of bones in joints and plays a role in smoothing joint movement. When articular cartilage is reduced due to aging, etc., problems such as joint pain occur. Patent Document 1 describes a composition containing oleuropein and / or hydroxytyrosol and further containing quercetin for use in preventing or treating cartilage destruction. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2016-520603 Summary of the Invention [Problem to be solved by the invention]

[0005] The composition described in Patent Document 1 is intended to prevent or treat (suppress or reduce) cartilage destruction. However, once cartilage is reduced due to aging or the like, it is not possible to increase the reduced cartilage by simply preventing cartilage destruction. Patent Document 1 does not consider any substance that is effective in promoting cartilage synthesis. For example, a component that can promote the synthesis of chondroitin sulfate, a component of the cartilage matrix, contributes to promoting cartilage synthesis and is useful for increasing cartilage.

[0006] An object of the present invention is to provide a composition for promoting chondroitin sulfate synthesis, which promotes the synthesis of chondroitin sulfate. Another object of the present invention is to provide a method for promoting the synthesis of chondroitin sulfate. [Means for solving the problem]

[0007] As a result of intensive research aimed at solving the above-mentioned problems, the inventors have discovered that quercetin or its glycoside has the effect of promoting the expression of glycosyltransferase involved in the synthesis of chondroitin sulfate, and is useful for increasing the amount of chondroitin sulfate and maintaining cartilage function, etc.

[0008] That is, the present invention relates to the following compositions for promoting chondroitin sulfate synthesis, methods for promoting the synthesis of chondroitin sulfate, and the like, although the present invention is not limited thereto. [1] A composition for promoting chondroitin sulfate synthesis, comprising quercetin or its glycoside as an active ingredient. [2] The composition for promoting chondroitin sulfate synthesis described in [1] above, which promotes expression of a glycosyltransferase involved in the synthesis of chondroitin sulfate. [3] A composition for promoting chondroitin sulfate synthesis according to [2] above, wherein the glycosyltransferase involved in the synthesis of chondroitin sulfate is one or more selected from the group consisting of chondroitin sulfate N-acetylgalactosaminyltransferase 1, chondroitin sulfate N-acetylgalactosaminyltransferase 2, xylosetransferase 2, β-1,3-galactosyltransferase 1, chondroitin sulfate synthase 1 and chondroitin polymerization factor 2. [4] A composition for promoting chondroitin sulfate synthesis according to [2] or [3] above, wherein the glycosyltransferase involved in the synthesis of chondroitin sulfate is chondroitin sulfate N-acetylgalactosaminyltransferase 1 and / or chondroitin sulfate N-acetylgalactosaminyltransferase 2. [5] The composition for promoting chondroitin sulfate synthesis according to any one of the above [1] to [4], which is a food or drink, a cosmetic, or a quasi-drug. [6] A method for promoting the synthesis of chondroitin sulfate by administering quercetin or its glycoside. [7] The method according to [6] above, wherein the synthesis of chondroitin sulfate is promoted by promoting expression of a glycosyltransferase involved in the synthesis of chondroitin sulfate. [8] Use of quercetin or a glycoside thereof for promoting the synthesis of chondroitin sulfate. [9] The use according to the above-mentioned [8], which promotes the synthesis of chondroitin sulfate by promoting the expression of a glycosyltransferase involved in the synthesis of chondroitin sulfate. Effect of the Invention

[0009] According to the present invention, there is provided a composition for promoting chondroitin sulfate synthesis, which promotes the synthesis of chondroitin sulfate. According to the present invention, there is provided a method for promoting the synthesis of chondroitin sulfate. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a graph showing the amount of chondroitin sulfate in HCH cells to which 5 μM quercetin was added. [Diagram 2] Figure 2 is a graph showing the expression levels of glycosyltransferase genes and Ext1 gene involved in the synthesis of chondroitin sulfate in the knee joint cartilage region of mice administered quercetin glycoside (QG) (p: Csgalnact1 gene, q: Csgalnact2 gene, r: Chpf gene, s: Xylt2 gene, t: Ext1 gene). [Diagram 3]FIG. 3 is a graph showing the amount of chondroitin sulfate in the femoral joint of mice administered QG. [Figure 4] Fig. 4 is a graph showing the area of ​​cartilage in the knee joint of mice administered QG. The relative area shown in Fig. 4 is the relative value of the area of ​​cartilage in the knee joint of the control group, where the area of ​​the cartilage in the knee joint of the control group is set to 1. [Diagram 5] Figure 5A is a graph showing the expression level of the Csgalnact1 gene in cells to which quercetin has been added. Figure 5B is a graph showing the expression level of the Csgalnact2 gene in cells to which quercetin has been added. Figure 5C is a graph showing the expression level of the Xylt2 gene in cells to which quercetin has been added. Figure 5D is a graph showing the expression level of the B3galt1 gene in cells to which quercetin has been added. [Figure 6] Figure 6A is a graph showing the expression level of the Chsy1 gene in cells to which quercetin has been added. Figure 6B is a graph showing the expression level of the Chpf2 gene in cells to which quercetin has been added. Figure 6C is a graph showing the expression level of the Ext1 gene in cells to which quercetin has been added. Figure 6D is a graph showing the expression level of the Ext2 gene in cells to which quercetin has been added. [Figure 7] FIG. 7 is a graph showing the amount of chondroitin sulfate in OSCV2 cells to which 5 μM quercetin was added. [Figure 8] FIG. 8 shows the names of enzymes involved in the synthesis pathways of chondroitin sulfate (CS) and heparan sulfate (HS). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The composition for promoting chondroitin sulfate synthesis of the present invention contains quercetin or a glycoside thereof as an active ingredient. Hereinafter, the composition for promoting chondroitin sulfate synthesis of the present invention will also be simply referred to as the composition of the present invention.

[0012] In this specification, "quercetin" refers to quercetin, a compound belonging to flavonol, a type of polyphenol. In the present invention, "quercetin glycoside" refers to the above-mentioned glycoside of quercetin, and specifically, is a general term for a series of compounds in which one or more sugars are glycosidically bonded to the hydroxyl group at the 3-position of quercetin. Quercetin glycoside is a compound represented by the following general formula. In the following general formula, (X)n represents a sugar chain. X represents a sugar (monosaccharide), and n is an integer of 1 or more. Quercetin glycoside may be one type of compound, or two or more types of compounds.

[0013] [ka]

[0014] The sugar constituting the sugar chain represented by X that is glycosidically bonded to quercetin is, for example, glucose, rhamnose, galactose, glucuronic acid, etc., and preferably glucose or rhamnose. In addition, n is not particularly limited as long as it is 1 or more, but is preferably 1 to 16, more preferably 1 to 8. When n is 2 or more, the X moiety may be composed of one type of sugar or may be composed of multiple types of sugar. In other words, when n is 2 or more, (X)n may be a sugar chain composed of one type of sugar or may be a sugar chain composed of multiple types of sugar. The quercetin glycoside in the present invention also includes existing quercetin glycosides that have been treated with enzymes or the like to be glycosyltransferred. The quercetin glycoside in the present invention specifically includes rutin, enzyme-treated rutin, quercitrin, isoquercitrin, etc. In the present invention, it is particularly preferable to use an enzyme-treated product of rutin as the quercetin glycoside. Preferred examples of the enzyme-treated product of rutin include isoquercitrin obtained by enzymatically treating rutin to remove the rhamnose sugar chain portion, isoquercitrin obtained by treating isoquercitrin with a glycosyltransferase to which a sugar chain consisting of 1 to 7 glucose units is bound, and a mixture thereof as the main component. In one embodiment, the quercetin glycoside is preferably a compound in which 1 to 8 glucose units are glycosidically bound to the hydroxyl group at the 3-position of quercetin (e.g., isoquercitrin, a glycoside in which 1 to 7 glucose units are bound to isoquercitrin), etc. When ingested, quercetin glycoside is broken down into aglycone (quercetin) in the intestinal tract and then absorbed into the body.

[0015] In the present invention, there is no particular limitation on the origin or production method for obtaining quercetin or its glycoside. For example, plants that are known to contain a large amount of quercetin or its glycoside include buckwheat, sophora japonica, capers, apples, tea, onions, grapes, broccoli, mulukhiyah, raspberries, cowberries, cranberries, opuntia, leafy vegetables, and citrus fruits, and quercetin or its glycoside can be obtained from these plants. Quercetin or its glycosides may be chemically synthesized. In the present invention, plant-derived raw materials such as plant extracts containing quercetin or its glycosides may be contained in the composition as long as the effects of the present invention are achieved. Purified or isolated quercetin or its glycosides may be used.

[0016] Quercetin or its glycoside is a compound that is contained in natural products and foods and beverages and has been consumed. Therefore, from the viewpoint of safety, it is considered that there are few problems even if quercetin or its glycoside is taken every day. According to the present invention, a composition for promoting chondroitin sulfate synthesis containing a highly safe substance as an active ingredient can be provided.

[0017] Chondroitin sulfate is a carbohydrate chain that has a repeating disaccharide unit in which D-glucuronic acid is linked to N-acetyl-D-galactosamine, and sulfate is linked to the repeating disaccharide unit. The carbohydrate portion of chondroitin sulfate is synthesized by glycosyltransferase. Figure 8 shows the names of the enzymes involved in the synthesis pathways of chondroitin sulfate (CS) and heparan sulfate (HS) (Reference: Seikagaku 87(6): 744-748 (2015)). In cartilage and other tissues, the addition of chondroitin sulfate (CS) chains to the core protein of proteoglycans begins with the sequential transfer of xylose (Xyl), two molecules of galactose (Gal), and D-glucuronic acid (GlcA) to the serine residue (Ser) in the chondroitin sulfate linkage sequence in the core protein, synthesizing the Xyl-Gal-Gal-GlcA sugar chain. The transfer of Xyl to the serine residue is carried out by xylosetransferase (XylT), the transfer of Gal to Xyl by β-1,4-galactosyltransferase (B4GalT), the transfer of the second Gal to the first Gal by β-1,3-galactosyltransferase (B3GalT), and the transfer of GlcA to Gal by β-1,3-glucuronyltransferase (B3GaT). Next, N-acetyl-D-galactosamine (GalNAc) is transferred to the GlcA at the non-reducing end (the side that is elongating). This GalNAc transfer is carried out by chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGalNacT1) or chondroitin sulfate N-acetylgalactosaminyltransferase 2 (CSGalNacT2). GlcA and GalNAc are then alternately transferred to the GalNAc at the non-reducing end, and the glycan is elongated. This glycan elongation process is carried out by CSGalNacT1, CSGalNacT2, chondroitin polymerizing factor 2 (Chpf2), and chondroitin sulfate synthases 1, 2, and 3 (ChSy1, ChSy2, and ChSy3). CSGalNacT1 and CSGalNacT2 have the transfer activity of transferring GalNAc to GlcA. Chpf2 has the transfer activity of transferring GlcA to GalNAc. ChSy1, ChSy2, and ChSy3 show the transfer activity of transferring GalNAc to GlcA and the transfer activity of transferring GlcA to GalNAc. Chondroitin sulfate synthesis can be promoted by promoting the expression of glycosyltransferases involved in the synthesis of chondroitin sulfate.

[0018] As shown in Figure 8, in the addition of heparan sulfate (HS) chains, GalNAc is transferred to the non-reducing end of the Xyl-Gal-Gal-GlcA glycan attached to the serine residue of the core protein by exostocin 1 (Ext1), exostocin 2 (Ext2), Ext-like1 (Extl1), Ext-like2 (Extl2), and Ext-like3 (Extl3). Then, GlcA and GalNAc are alternately transferred to the GalNAc at the non-reducing end, and the glycan is elongated.

[0019] As shown in the examples below, when cells were cultured in the presence of quercetin, the expression levels of genes (glycosyltransferase genes) encoding glycosyltransferases involved in the synthesis of chondroitin sulfate, such as the gene encoding CSGalNacT1 (Csgalnact1) and the gene encoding CSGalNacT2 (Csgalnact2), increased compared to when quercetin was not present. In addition, when cells such as chondrocytes were cultured in the presence of quercetin, a significant increase in the amount of chondroitin sulfate was observed compared to the control. In addition, when animals were given quercetin glycoside, the expression levels of genes encoding glycosyltransferases involved in the synthesis of chondroitin sulfate, such as Csgalnact1 and Csgalnact2, increased significantly in the knee joint, and the amount of chondroitin sulfate in the joint increased significantly. Furthermore, the area of ​​articular cartilage increased in animals that had received quercetin glycoside.

[0020] Therefore, quercetin or its glycoside has the effect of promoting the synthesis of chondroitin sulfate and the effect of promoting the expression of glycosyltransferase involved in the synthesis of chondroitin sulfate. Quercetin or its glycoside can be used as an active ingredient for promoting the expression of glycosyltransferase involved in the synthesis of chondroitin sulfate in order to promote the synthesis of chondroitin sulfate. In the present invention, the promotion of the synthesis of chondroitin sulfate and the promotion of the expression of glycosyltransferase involved in the synthesis of chondroitin sulfate are preferably the promotion of the synthesis of chondroitin sulfate in the body of a subject and the promotion of the expression of the glycosyltransferase in the body of a subject.

[0021] Examples of glycosyltransferases involved in the synthesis of chondroitin sulfate include chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGalNacT1), chondroitin sulfate N-acetylgalactosaminyltransferase 2 (CSGalNacT2), xylosetransferase (XylT), β-1,3-galactosyltransferase (B3GalT), β-1,4-galactosyltransferase (B4GalT), chondroitin sulfate synthases 1 to 3 (ChSy1 to 3), chondroitin polymerizing factor 2 (Chpf2), etc. Five types of glycosyltransferases involved in the synthesis of heparan sulfate chains (Ext1, Ext2, EXTL1 (Extl1), EXTL2 (Extl2), EXTL3 (Extl3)) are not included in the glycosyltransferases involved in the synthesis of chondroitin sulfate in the present invention.

[0022] The composition for promoting chondroitin sulfate synthesis of the present invention can promote the expression of glycosyltransferases involved in the synthesis of chondroitin sulfate. In one embodiment, the composition of the present invention can be used to promote the synthesis of chondroitin sulfate by promoting the expression of glycosyltransferases involved in the synthesis of chondroitin sulfate. The glycosyltransferases involved in the synthesis of chondroitin sulfate are preferably one or more selected from the group consisting of chondroitin sulfate N-acetylgalactosaminyltransferase 1, chondroitin sulfate N-acetylgalactosaminyltransferase 2, xylosetransferase 2, β-1,3-galactosyltransferase 1 (BGalT1), chondroitin sulfate synthase 1, and chondroitin polymerization factor 2. Preferably, the composition of the present invention can promote the expression of one or more of these glycosyltransferases. In a preferred embodiment, the composition of the present invention can promote the expression of chondroitin sulfate N-acetylgalactosaminyltransferase 1 and / or chondroitin sulfate N-acetylgalactosaminyltransferase 2, and can be used for such a purpose. CSGalNacT1 and CSGalNacT2 are considered to be important enzymes that are the starting point of chondroitin sulfate synthesis. More preferably, the composition of the present invention can promote the expression of chondroitin sulfate N-acetylgalactosaminyltransferase 1, chondroitin sulfate N-acetylgalactosaminyltransferase 2, xylosetransferase 2, and chondroitin polymerization factor 2. The promotion of expression of the glycosyltransferase includes the promotion of expression of a gene encoding the glycosyltransferase and the promotion of expression of the enzyme at the protein level, and may be either or both of these. The promotion of expression of the gene includes the promotion of expression of mRNA, preferably the promotion of transcription to mRNA. The promotion of expression of the glycosyltransferase at the protein level includes the promotion of translation.

[0023] In one embodiment, the composition of the present invention can be preferably used for promoting the expression of glycosyltransferase involved in the synthesis of chondroitin sulfate in chondrocytes, promoting the synthesis of chondroitin sulfate in chondrocytes, etc. By promoting the synthesis of chondroitin sulfate in cartilage, the synthesis of cartilage can be promoted. In one embodiment, the composition of the present invention can be used for promoting the synthesis of cartilage. By promoting the synthesis of cartilage, it is possible to increase cartilage. Promoting cartilage synthesis is useful, for example, in preventing the decrease of cartilage.

[0024] It has been reported that promoting the synthesis of chondroitin sulfate in the brain promotes the generation of new neurons in the hippocampus, and promoting the synthesis of chondroitin sulfate in osteoblasts can promote bone formation. Quercetin or its glycoside has an effect of promoting the expression of glycosyltransferase involved in the synthesis of chondroitin sulfate in nerve cells or osteoblasts, and can promote the synthesis of chondroitin sulfate. Quercetin or its glycoside promotes the synthesis of chondroitin sulfate in nerve cells (preferably hippocampal nerve cells), and is useful for protecting nerves. By protecting nerves, cognitive function improving effects and the like can be expected. Quercetin or its glycoside promotes the synthesis of chondroitin sulfate in osteoblasts, and is useful for promoting bone formation. In one embodiment, the composition of the present invention can be used for protecting nerves or promoting bone formation.

[0025] The compositions of the present invention can be applied in either therapeutic (medical) or non-therapeutic (non-medical) applications, the latter being a concept that does not include medical procedures, i.e., surgery, treatment or diagnosis of humans. The composition of the present invention can be in the form of a food or drink, a cosmetic, a drug, a quasi-drug, a feed, etc. The composition of the present invention may be a food or drink, a cosmetic, a drug, a quasi-drug, a feed, etc. that is used for promoting chondroitin sulfate synthesis by itself, or may be a material or preparation that is used by being blended with these. The composition for promoting chondroitin sulfate synthesis of the present invention can be provided in the form of an agent, for example, but is not limited to this form. The agent can be provided as a composition as it is, or as a composition containing the agent. In one aspect, the composition for promoting chondroitin sulfate synthesis of the present invention can also be called a chondroitin sulfate synthesis promoter. The composition of the present invention may be either an oral composition or a parenteral composition, but is preferably an oral composition. By orally ingesting or administering the composition of the present invention to a subject, or by parenterally administering the composition, the synthesis of chondroitin sulfate in the subject can be promoted. Oral compositions include food and drink, oral medicines, quasi-drugs, and feed, and are preferably food and drink or oral medicines, and more preferably food and drink. Parenteral compositions include cosmetics, parenteral medicines, and parenteral quasi-drugs.

[0026] The composition of the present invention may contain any additives and any components in addition to quercetin or its glycoside, so long as the effects of the present invention are not impaired. These additives and components may be selected depending on the form of the composition, and those that can be generally used for foods and beverages, cosmetics, medicines, quasi-drugs, feed, etc. may be used. When the composition of the present invention is used as a food and beverage, medicine, quasi-drug, feed, etc., the method of production is not particularly limited, and it may be produced by a general method.

[0027] For example, when the composition of the present invention is made into a food or drink, various foods and drinks can be prepared by blending quercetin or its glycoside with ingredients that can be used in foods and drinks (e.g., food ingredients, food additives used as necessary, etc.). The foods and drinks are not particularly limited, and examples thereof include general foods and drinks, health foods, health drinks, functional foods, foods for specified health uses, dietary supplements, foods and drinks for sick people, etc. The above-mentioned health foods, functional foods, foods for specified health uses, dietary supplements, etc. can be used in various formulation forms, such as fine granules, tablets, granules, powders, capsules, chewable agents, dry syrups, syrups, liquids, beverages, and liquid diets.

[0028] When the composition of the present invention is used as a cosmetic, quercetin or its glycoside may be blended with a carrier, additive, etc. that are acceptable for cosmetic use. The product form of the cosmetic is not particularly limited.

[0029] When the composition of the present invention is used as a drug or quasi-drug, for example, quercetin or its glycoside can be mixed with a pharmacologically acceptable carrier, additives added as necessary, and the like to produce a drug or quasi-drug in various dosage forms. Such carriers, additives, and the like may be any pharmacologically acceptable carriers that can be used in drugs or quasi-drugs, and examples of such carriers, additives, and the like include one or more of excipients, binders, disintegrants, lubricants, antioxidants, colorants, and the like. Examples of the administration (ingestion) form of a drug or quasi-drug include oral or parenteral (transdermal, transmucosal, enteral, injection, etc.) administration forms. When the composition of the present invention is used as a drug or quasi-drug, it is preferable to use an oral drug or quasi-drug. Examples of dosage forms for oral administration include liquids, tablets, powders, fine granules, granules, sugar-coated tablets, capsules, suspensions, emulsions, chewables, and the like. Examples of dosage forms for parenteral administration include injections, infusions, and skin topicals (patches, creams, ointments, etc.). The pharmaceutical agent may be a non-human animal drug.

[0030] When the composition of the present invention is used as a feed, quercetin or its glycoside may be added to the feed. The feed also includes feed additives. Examples of the feed include livestock feed for cows, pigs, chickens, sheep, horses, etc.; small animal feed for rabbits, rats, mice, etc.; and pet food for dogs, cats, small birds, etc.

[0031] The content of quercetin or its glycoside in the composition of the present invention is not particularly limited and can be set depending on the form, etc. The content of quercetin or its glycoside in the composition of the present invention is, for example, preferably 0.01% by weight or more, more preferably 0.1% by weight or more, and preferably 80% by weight or less, more preferably 50% by weight or less, in terms of quercetin. In one embodiment, the content of quercetin or its glycoside in the composition of the present invention is preferably 0.01 to 80% by weight, more preferably 0.1 to 50% by weight, in terms of quercetin. The content of quercetin or its glycosides can be measured according to a known method, for example, HPLC method.

[0032] The composition of the present invention is generally ingested or administered to a subject. The route of administration of the composition of the present invention is not particularly limited, and the composition can be ingested or administered by an appropriate method according to its form. In one embodiment, the composition of the present invention is preferably taken orally (administered orally). The dosage (which can also be called the intake amount) of the composition of the present invention is not particularly limited, and may be an amount that can obtain the effect of promoting the synthesis of chondroitin sulfate and the effect of promoting the expression of glycosyltransferase involved in the synthesis of chondroitin sulfate, and may be appropriately set according to the dosage form, administration method, etc.

[0033] In one embodiment, when the composition of the present invention is ingested or administered to a human (adult), the dosage of quercetin or its glycoside is preferably 0.3 mg or more, more preferably 1.0 mg or more, even more preferably 10 mg or more, and preferably 4000 mg or less, more preferably 2000 mg or less, and even more preferably 1000 mg or less per 60 kg body weight per day, as a quercetin equivalent value, for a human (adult), and is preferably 0.3 to 4000 mg, more preferably 1.0 to 2000 mg, even more preferably 1.0 to 1000 mg, and particularly preferably 10 to 1000 mg per 60 kg body weight per day. It is preferable to ingest or administer the above amount at least once a day, for example, once to several times a day (for example, 2 to 3 times) in divided doses. In one embodiment, it is preferable to orally ingest or administer the above amount of quercetin or its glycoside to a human. In one embodiment of the present invention, the composition of the present invention can be used to ingest or administer the above amount of quercetin or its glycosides per 60 kg body weight per day to a human.

[0034] The composition of the present invention is preferably one that is continuously ingested or administered. By continuously ingesting or administering quercetin or its glycoside, it is expected that the effect of promoting chondroitin sulfate synthesis will be enhanced. In one embodiment, the composition of the present invention is preferably continuously ingested or administered for 1 week or more, more preferably 4 weeks or more, and even more preferably 8 weeks or more.

[0035] The composition of the present invention can be used for preventing or improving a condition or disease that can be prevented or improved by promoting the synthesis of chondroitin sulfate. Such conditions or diseases include conditions or diseases caused by a decrease in the amount of chondroitin sulfate, such as osteoarthritis of the knee, osteoarthritis of the hip, spinal canal stenosis, osteoporosis, etc. In one embodiment, the composition of the present invention can be used for preventing or improving cognitive decline, and for preventing or improving dementia. In this specification, the prevention of a condition or disease includes preventing onset, delaying onset, reducing the incidence rate, reducing the risk of onset, etc. The improvement of a condition or disease includes recovering a subject from a condition or disease, reducing the symptoms of a condition or disease, improving the symptoms of a condition or disease, delaying or preventing the progression of a condition or disease, etc.

[0036] The subject to which the composition of the present invention is ingested or administered (also referred to as an administration subject) is not particularly limited, and is preferably a human or a non-human mammal, more preferably a human. In one embodiment, subjects of administration include subjects who need or desire promotion of chondroitin sulfate synthesis, and subjects who need or desire prevention or improvement of conditions or diseases caused by a decrease in the amount of chondroitin sulfate, etc. The decrease in the amount of chondroitin sulfate may be a decrease in the amount of chondroitin sulfate due to aging, or a decrease in the amount of chondroitin sulfate due to aging in middle-aged and elderly people. In one embodiment, the subject of administration in the present invention includes middle-aged and elderly people. Middle-aged and elderly people include elderly people. Among middle-aged and elderly people, elderly people are preferred as the subject. In the present invention, middle-aged and elderly people may be, for example, people aged 40 years or older. Elderly people may be, for example, people aged 60 years or older or 65 years or older. In one embodiment, the composition of the present invention is suitably used as a composition for promoting chondroitin sulfate synthesis for middle-aged and elderly people. The composition of the present invention can also be used in healthy individuals for the purpose of preventing conditions or diseases that can be expected to be prevented or improved by promoting chondroitin sulfate synthesis.

[0037] The composition of the present invention may be labeled with a function exhibited by promoting chondroitin sulfate synthesis. The composition for promoting chondroitin sulfate synthesis of the present invention may be labeled with one or more functions, such as "maintaining cartilage mass," "suppressing cartilage wear," "suppressing cartilage degeneration," "reducing knee joint problems," "maintaining knee joint function," "improving knee joint troubles during movement," "relieving discomfort in the knee joint," "improving movement involving bending and straightening the knee," "maintaining knee joint function that deteriorates with age," and "protecting the cartilage of the knee joint, thereby facilitating bending and straightening the knee of those who feel a decrease in mobility, such as walking, standing, and sitting, and helping to improve walking ability." The above-mentioned function may be labeled with a description that the above-mentioned function is obtained by promoting the synthesis of chondroitin sulfate. In one embodiment, the composition of the present invention is preferably a food or drink to which the above-mentioned label is attached. The above-mentioned label may be a label indicating that the composition is used to obtain the above-mentioned function. The label may be attached to the composition itself, or to a container or packaging of the composition.

[0038] The present invention also encompasses the following methods and uses. A method for promoting the synthesis of chondroitin sulfate by administering quercetin or its glycoside. Use of quercetin or a glycoside thereof for promoting the synthesis of chondroitin sulfate. The above method and use may be a therapeutic method or use, or a non-therapeutic method or use. The administration (ingestion) of quercetin or its glycoside can increase the expression level of glycosyltransferase involved in the synthesis of chondroitin sulfate. The administration of quercetin or its glycoside can promote the synthesis of chondroitin sulfate. The above method may be a method for promoting the synthesis of chondroitin sulfate by promoting the expression of glycosyltransferase involved in the synthesis of chondroitin sulfate. The above use may be the use of quercetin or its glycoside to promote the synthesis of chondroitin sulfate by promoting the expression of glycosyltransferase involved in the synthesis of chondroitin sulfate. The promotion of the synthesis of chondroitin sulfate may be, for example, the promotion of the synthesis of chondroitin sulfate in cartilage. Quercetin or its glycoside can be used for the prevention or improvement of a condition or disease that can be expected to be prevented or improved by the promotion of the synthesis of chondroitin sulfate.

[0039] In the above-mentioned method and use, it is preferable to administer (ingest) quercetin or its glycoside to a subject at least once a day, for example, once to several times a day (for example, 2 to 3 times a day). The above-mentioned use is preferably for humans or non-human mammals, more preferably for humans. The quercetin or its glycoside is as described above.

[0040] In the above-mentioned method and use, quercetin or its glycoside may be used in an amount (which may be called an effective amount) that provides a chondroitin sulfate synthesis promoting effect. The preferred dosage, administration target, administration method, etc. of quercetin or its glycoside are the same as those of the above-mentioned composition for promoting chondroitin sulfate synthesis of the present invention. Quercetin or its glycoside may be administered as it is, or may be administered as a composition containing it. For example, the above-mentioned composition of the present invention may be used. Quercetin or its glycoside is preferably administered orally (ingested).

[0041] Quercetin or its glycoside can be used for the production of foods and beverages, cosmetics, pharmaceuticals, quasi-drugs, feeds, etc., which are used to promote the synthesis of chondroitin sulfate. In one aspect, the present invention also includes the use of quercetin or its glycoside for the production of a composition for promoting chondroitin sulfate synthesis. The composition for promoting chondroitin sulfate synthesis and its preferred aspects are the same as those of the composition of the present invention described above. EXAMPLES

[0042] The present invention will be described in more detail below with reference to examples. A series of animal experiments was carried out in compliance with the Animal Welfare and Management Law and other related laws and regulations, and was based on a plan approved by the head of the institution after review by the in-house animal experiment committee. However, the present invention is not limited to these examples.

[0043] <Example 1: Effect of quercetin on the promotion of chondroitin sulfate synthesis (in vitro)> (1) Cell culture HCH cells (chondrocytes) were cultured in Dulbecco's Modified Eagle Medium (Sigma-Aldrich) containing 10% fetal bovine serum until they became 100% confluent.

[0044] (2) Quantitation of chondroitin sulfate in cells HCH cells were cultured after adding 5 μM quercetin to the culture medium. Quercetin was added dissolved in dimethyl sulfoxide (DMSO). Four days after addition, the amount of chondroitin sulfate in the cultured cells was quantified using a Cell ELISA system with an anti-chondroitin sulfate antibody (CS-56). Control cells were cultured in the same way except for the addition of DMSO, and the amount of chondroitin sulfate was quantified. The difference in the mean values ​​of each group was examined using Student's t test, with a significance level of 5% or less (*: p<0.05 vs control).

[0045] The results are shown in Figure 1 (*: p<0.05 vs. control). Figure 1 is a graph showing the amount of chondroitin sulfate in HCH cells to which 5 μM quercetin was added. In Figure 1, the relative CS level on the vertical axis is the relative value of the amount of chondroitin sulfate (CS) in cells to which quercetin was added, when the amount of CS in cells to which DMSO was added as a control was set to 1 (all values ​​are the mean ± standard deviation of N=4). In Figure 1, "Quercetin" refers to cells cultured in a medium containing quercetin. A significant increase in the amount of chondroitin sulfate produced in HCH cells was confirmed by the addition of 5 μM quercetin.

[0046] <Method for quantifying gene expression> In the following examples, gene expression levels in cells or tissues were measured by isolating RNA from the cells or tissues and using the following method. cDNA synthesis was performed from the isolated RNA using High-Capacity cDNA Reverse Transcriptional Kits (Thermo Fisher Scientific). Quantitative PCR was performed using TaqMan Fast Universal PCR Mastermix (Thermo Fisher Scientific) in a Quantstudio Real Time PCR System (Thermofisher) to quantify the expression level of a gene (glycosyltransferase gene) encoding a glycosyltransferase involved in the synthesis of chondroitin sulfate or heparan sulfate. The difference in the mean values ​​of each group was examined using Dunnett's t test, and a value of 5% or less was considered significant (*: p<0.05 vs. quercetin 0 μM). The types of genes quantified in Examples 2 and 3, the proteins encoded by the genes, and the PCR primers and probes used for the measurement are shown in Table 1. The position of each enzyme subjected to gene analysis in the glycosaminoglycan synthesis pathway is shown in FIG. 8.

[0047] [Table 1]

[0048] <Example 2: Effect of quercetin glycoside on promoting chondroitin sulfate synthesis (in vivo)> (1) Administration of quercetin glycoside to mice 36-week-old C57BL / 6J wild-type (WT) mice and Csgalnact1 gene knockout (T1-KO) mice were divided into a control group that was given distilled water or a group that was given distilled water containing 4.5 g / L quercetin glycoside (QG) (QG administration group) based on weight. The WT mice (WT-QG) and T1-KO mice (KO-QG) in the QG administration group were given distilled water containing the above quercetin glycoside for 18 weeks. The daily intake of quercetin glycoside per body weight was 117 mg / kg in terms of quercetin. The control group WT mice (WT-control) and T1-KO mice (KO-control) were given distilled water for 18 weeks. All mice were kept alone in cages, and a device capable of measuring spontaneous running on a saucer-type wheel (Muromachi Kikai Co., Ltd.) was placed in the cage to induce a certain amount of spontaneous exercise. Knee joint tissue was collected 18 weeks after the start of drinking water administration. Using the collected knee joint tissue, gene expression levels, chondroitin sulfate levels, and articular cartilage levels were measured. In Example 2, enzyme-treated isoquercitrin obtained by treating isoquercitrin with glycosyltransferase and in which a sugar chain consisting of 1 to 7 glucose units is bonded to isoquercitrin was used as the quercetin glycoside.

[0049] (2) Quantification of gene expression levels using mouse tissues RNA was isolated from the collected knee joints using RNeazy Mini Kit (QIAGEN). Using the isolated RNA, cDNA synthesis was performed using the method described above for quantifying gene expression levels, and the expression levels of glycosyltransferase genes involved in the synthesis of chondroitin sulfate or heparan sulfate were quantified by quantitative RT-PCR (N=12). The genes quantified were Csgalnact1, Csgalnact2, Chpf2, Xylt2, and Ext1, as shown in Table 1. The difference in the mean values ​​between the groups was examined using Student's t test, and a value of 5% or less was considered significant (*: p<0.05 vs. control).

[0050] The results are shown in Fig. 2 (*: p < 0.05 vs control). Fig. 2 is a graph showing the expression levels of glycosyltransferase genes (Csgalnact1, Csgalnact2, Chpf2, and Xylt2) and Ext1 involved in chondroitin sulfate synthesis in the knee joint tissues of mice administered with quercetin glycoside (QG) (mean ± standard deviation, N = 12). In Fig. 2, p represents Csgalnact1, q represents Csgalnact2, r represents Chpf2, s represents Xylt2, and t represents Ext1. In Fig. 2, the relative mRNA level on the vertical axis is the relative value of the mRNA amount of each group's gene when the mRNA amount of the gene in the control group (WT-control) of WT mice is set to 1.

[0051] In WT mice, a significant increase in the gene expression levels of Csgalnact1, Csgalnact2, Chpf2, and Xylt2 was confirmed in the QG-administered group (WT-QG) compared with the control group (WT-control). On the other hand, no change in the gene expression level of Ext1 was confirmed. In T1-KO mice, a significant increase in the gene expression levels of Csgalnact2, Chpf2, and Xylt2 was confirmed in the QG-administered group (KO-QG) compared with the control group (KO-control). On the other hand, no change in the gene expression level of Ext1 was confirmed.

[0052] (3) Quantification of chondroitin sulfate amount using mouse tissues The protein component was removed from the collected knee joint tissues by trichloroacetic acid precipitation, and the glycosaminoglycan fraction was concentrated by ethanol precipitation. After desalting, the glycosaminoglycan was decomposed with chondroitinase and fluorescently labeled with 2-aminobenzamide. The chondroitin sulfate of the disaccharide unit was quantified by high-performance liquid chromatography (Shimadzu Corporation) under the following conditions (N = 6).

[0053] <HPLC conditions> Column: DOCOSIL SP100 5.0μm 4.6×100mm (Senshu Science Co., Ltd.) Guard column: Myghtysil RP-18GP 5.0 μm, 4.6 × 10 mm (Kanto Chemical Co., Ltd.) Eluate: A. Distilled water B. 0.2M NaCl C.10mM Tetrabutyl ammonium hydrogen sulfate D. 50% acetonitrile Gradient conditions: 0-10min: A(70%), B(1%), C+D(29%)(C(12%), D(17%)) 10-11min: A(67%), B(4%), C+D(29%)(C(12%), D(17%)) 11-20min: A(61%), B(10%), C+D(29%)(C(12%), D(17%)) 20-26min: A(53%), B(18%), C+D(29%)(C(12%), D(17%)) 26-28min: A(35%), B(36%), C+D(29%)(C(12%), D(17%)) 28-37min: A(18%), B(53%), C+D(29%)(C(12%), D(17%)) 37-57min: A(70%), B(1%), C+D(29%)(C(12%), D(17%)) Flow rate (eluent): 1.1mL / min Reaction solution: A.0.5% 2-cyanoacetate solution B. 0.25M NaOH Flow rate (reaction solution): 0.7mL / min Injection volume: 50μL Fluorescence monitor: EX 346nm, EM 410nm, AT 64, Gain x100 Integrator: End Time 40min, Speed ​​2min / min, AT 512

[0054] Differences in the mean values ​​between groups were tested using Student's t test, with a significance level of 5% or less (*: p<0.05 vs. control).

[0055] The results are shown in Figure 3 (*: p<0.05 vs. control). Figure 3 is a graph showing the amount of chondroitin sulfate in the knee joints of mice administered quercetin glycoside (QG) (mean ± standard deviation of N=6). In Figure 3, the relative CS level on the vertical axis is the relative value of the chondroitin sulfate (CS) amount in each group, when the CS amount in the control group of WT mice is set to 1. In WT and T1-KO mice, a significant increase in the amount of chondroitin sulfate was confirmed in the QG-administered groups (WT-QG and KO-QG) compared to the control groups (WT-control and KO-control). The intake of quercetin glycoside increased the amount of chondroitin sulfate in the knee joints.

[0056] (4) Quantification of articular cartilage volume using mouse tissue The collected knee joint tissue was fixed in formalin and prepared as serial paraffin sections. The central area, which is the widest part of the joint, was surfaced, and 20 sections were selected from each tissue at 100 micrometer intervals. They were then stained with HE and measured so that the area variation range was the same. The area of ​​the articular cartilage was quantified using image analysis software Image J (N=18). The difference in the mean values ​​of each group was examined using Student's t test, and a value of 5% or less was considered significant (*: p<0.05 vs. control).

[0057] The results are shown in Figure 4 (*: p<0.05 vs. control). Figure 4 is a graph showing the area of ​​cartilage in the knee joints of mice administered quercetin glycoside. The relative areas shown in Figure 4 are the relative values ​​of the cartilage area of ​​each group, when the area of ​​the cartilage in the control group of WT mice is set to 1. In WT mice, a significant increase in the articular cartilage area was confirmed in the QG-treated group (WT-QG) compared to the control group (WT-control). On the other hand, in T1-KO mice, although there was a tendency for the area to increase in the QG-treated group (KO-QG) compared to the control group (KO-control), no significant change was observed.

[0058] <Example 3: Effect of quercetin on the promotion of chondroitin sulfate synthesis 2 (in vitro)> (1) Cultivation of various cells C6 cells (glial cells), SKOV3 cells (ovarian adenoma cells), C1300 cells (neuroblasts), KINGS1 cells (glial cells), NM-CG1 cells (glial cells), MC3T3-E1 cells (osteoblasts), and OSCV2 cells (osteoclasts) were cultured in Dulbecco's Modified Eagle Medium (Sigma-Aldrich) containing 10% fetal bovine serum until they became 100% confluent.

[0059] (2) Quantification of gene expression levels using cells DMSO or 0.5 μM, 1 μM, or 5 μM quercetin was added to various cells (C6 cells, SKOV3 cells, C1300 cells, KINGS1 cells, NM-CG1 cells, and MC3T3-E1 cells). RNA was isolated from the cells 2 hours after addition using RNeazy Mini Kit (QIAGEN). Using the isolated RNA, cDNA synthesis and quantitative PCR were performed by the method described in the above-mentioned method for quantifying gene expression levels, and the expression levels of glycosyltransferase genes involved in the synthesis of chondroitin sulfate or heparan sulfate were quantified. The difference in the mean values ​​of each group was examined using Dunnett's t test, and a value of 5% or less was considered significant (*: p<0.05 vs. quercetin 0 μM). The genes quantified were Csgalnact1, Csgalnact2, Xylt2, B3galt1, Chsy1, Chpf2, Ext1, and Ext2 listed in Table 1.

[0060] The results are shown in Figures 5A, 5B, 5C, 5D, 6A, 6B, 6C, and 6D (*: P<0.05, vs. 0 μM quercetin). Figure 5A is a graph showing the expression level of the Csgalnact1 gene in cells to which quercetin was added. Figure 5B is a graph showing the expression level of the Csgalnact2 gene in cells to which quercetin was added. Figure 5C is a graph showing the expression level of the Xylt2 gene in cells to which quercetin was added. Figure 5D is a graph showing the expression level of the B3galt1 gene in cells to which quercetin was added. Figure 6A is a graph showing the expression level of the Chsy1 gene in cells to which quercetin was added. Figure 6B is a graph showing the expression level of the Chpf2 gene in cells to which quercetin was added. Figure 6C is a graph showing the expression level of the Ext1 gene in cells to which quercetin was added. Figure 6D is a graph showing the expression level of the Ext2 gene in cells to which quercetin was added. In Figures 5A to 5D and 6A to 6D, a indicates C6 cells, b indicates SKOV3 cells, c indicates C1300 cells, d indicates KINGS1 cells, e indicates NM-CG1 cells, and f indicates MC3T3-E1SK cells. In Figures 5A to 5D and 6A to 6D, the expression levels of genes are shown as relative mRNA levels. The relative mRNA levels are the relative values ​​(mean ± standard deviation of N=8) of the mRNA levels in cells to which quercetin was added (quercetin concentration 0.5 μM, 1 μM, or 5 μM) when the mRNA level of each gene in cells to which DMSO was added as a control (quercetin concentration 0 μM) was set to 1.

[0061] Addition of 5 μM quercetin significantly increased the gene expression levels of Csgalnact1 and Csgalnact2 in all cells (Figures 5A and 5B). Significant increases in gene expression levels were confirmed by addition of 5 μM quercetin in the following cells: Xylt2 in C6 cells, C1300 cells, KINGS1 cells, NM-CG1 cells, and MC3T3-E1 cells (Figure 5C), B3galt1 in SKOV3 cells, KINGS1 cells, and MC3T3-E1 cells (Figure 5D), Chsy1 in SKOV3 cells, C1300 cells, KINGS1 cells, NM-CG1 cells, and MC3T3-E1 cells (Figure 6A), and Chpf2 in SKOV3 cells, C1300 cells, KINGS1 cells, and MC3T3-E1 cells (Figure 6B). On the other hand, no significant changes in the gene expression levels of Ext1 and Ext2, which are involved in the synthesis of heparan sulfate, were observed in any of the cells (FIGS. 6C and 6D).

[0062] (3) Quantitation of chondroitin sulfate in cells 5μM quercetin was added to the medium of OSCV2 cells and they were cultured. Four days after addition, the amount of chondroitin sulfate in the cultured cells was quantified using a Cell ELISA system with an anti-chondroitin sulfate antibody (CS-56). Control cells were cultured in the same way except for the addition of DMSO, and the amount of chondroitin sulfate was quantified. The difference in the mean values ​​of each group was examined using Student's t test, and a value of 5% or less was considered significant (*: p<0.05 vs. control).

[0063] The results are shown in Figure 7 (*: p<0.05 vs. control). Figure 7 is a graph showing the amount of chondroitin sulfate in OSCV2 cells to which 5 μM quercetin was added. In Figure 7, the relative CS level on the vertical axis is the relative value of the amount of chondroitin sulfate (CS) in cells to which quercetin was added, when the amount of CS in cells to which DMSO was added as a control was set to 1 (all values ​​are the mean ± standard deviation of N=4). In Figure 7, "Quercetin" refers to cells cultured in a medium containing quercetin. A significant increase in the amount of chondroitin sulfate was confirmed by the addition of 5 μM quercetin.

[0064] From the above results, it was found that quercetin or its glycoside has the effect of promoting the expression of glycosyltransferase involved in the synthesis of chondroitin sulfate, and the effect of promoting the synthesis of chondroitin sulfate. Quercetin or its glycoside has the effect of increasing the amount of articular cartilage.

Claims

1. A composition for promoting chondroitin sulfate synthesis, comprising quercetin or its glycoside as an active ingredient.

2. The composition for promoting chondroitin sulfate synthesis according to claim 1, which promotes the expression of a glycosyltransferase involved in the synthesis of chondroitin sulfate.

3. The composition for promoting chondroitin sulfate synthesis according to claim 2, wherein the glycosyltransferase involved in the synthesis of chondroitin sulfate is one or more selected from the group consisting of chondroitin sulfate N-acetylgalactosaminyltransferase 1, chondroitin sulfate N-acetylgalactosaminyltransferase 2, xylosetransferase 2, β-1,3-galactosyltransferase 1, chondroitin sulfate synthase 1 and chondroitin polymerization factor 2.

4. 4. The composition for promoting chondroitin sulfate synthesis according to claim 2 or 3, wherein the glycosyltransferase involved in the synthesis of chondroitin sulfate is chondroitin sulfate N-acetylgalactosaminyltransferase 1 and / or chondroitin sulfate N-acetylgalactosaminyltransferase 2.

5. The composition for promoting chondroitin sulfate synthesis according to any one of claims 1 to 4, which is a food or drink, a cosmetic, or a quasi-drug.

Images