WO2021124912A1 - コンドロイチン硫酸合成促進用組成物 - Google Patents
コンドロイチン硫酸合成促進用組成物 Download PDFInfo
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- WO2021124912A1 WO2021124912A1 PCT/JP2020/045091 JP2020045091W WO2021124912A1 WO 2021124912 A1 WO2021124912 A1 WO 2021124912A1 JP 2020045091 W JP2020045091 W JP 2020045091W WO 2021124912 A1 WO2021124912 A1 WO 2021124912A1
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/60—Sugars; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
Definitions
- 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 and the like.
- Chondroitin sulfate is a type of acidic mucopolysaccharide and exists in animals as a proteoglycan covalently bound to a core protein usually called a core protein. Chondroitin sulfate is present in many tissues such as animal cartilage, connective tissues such as skin, and brain tissues, and is particularly abundant in the extracellular matrix of cartilage.
- Cartilage is a connective tissue composed of chondrocytes and a substrate surrounding them, and forms joints, skeletons, etc. in animals such as humans.
- articular cartilage thinly covers the surface of joint bones and plays a role in smoothing the movement of joints.
- Patent Document 1 describes a composition containing oleuropein and / or hydroxytyrosol and further containing quercetin for use in preventing or treating cartilage destruction.
- Patent Document 1 The composition described in Patent Document 1 is for preventing or treating (suppressing or reducing) cartilage destruction.
- the reduced cartilage cannot be increased by preventing the cartilage destruction.
- a substance effective for promoting cartilage synthesis has not been investigated.
- a component capable of promoting the synthesis of chondroitin sulfate which is a component of a cartilage substrate, contributes to the promotion of cartilage synthesis and is useful for increasing cartilage.
- quercetin or its glycoside has an action of promoting the expression of a glycosyltransferase involved in the synthesis of chondroitin sulfate, an increase in the amount of chondroitin sulfate, and It was found to be useful for maintaining cartilage function.
- the present invention is not limited to this, but the present invention relates to the following composition for promoting chondroitin sulfate synthesis, a method for promoting the synthesis of chondroitin sulfate, and the like.
- a composition for promoting chondroitin sulfate synthesis containing quercetin or a glycoside thereof as an active ingredient.
- Glycosyltransferases involved in the synthesis of chondroitin sulfate are chondroitin sulfate N-acetylgalactosaminyl transferase 1, chondroitin sulfate N-acetylgalactosaminyl transferase 2, xylose transferase 2, ⁇ -1,3-galactosyl transferase 1.
- the glycosyltransferase involved in the synthesis of chondroitin sulfate is chondroitin sulfate N-acetylgalactosaminyl transferase 1 and / or chondroitin sulfate N-acetylgalactosaminyl transferase 2 according to the above [2] or [3].
- Composition for promoting chondroitin sulfate synthesis [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, which administers quercetin or a glycoside thereof.
- the method according to [6] above which promotes the synthesis of chondroitin sulfate by promoting the expression of glycosyltransferase involved in the synthesis of chondroitin sulfate.
- composition for promoting the synthesis of chondroitin sulfate which promotes the synthesis of chondroitin sulfate.
- method for promoting the synthesis of chondroitin sulfate there is provided.
- FIG. 1 is a graph showing the amount of chondroitin sulfate in HCH cells supplemented with 5 ⁇ M quercetin.
- FIG. 2 is a graph showing the expression levels of the glycosyltransferase gene and the Ext1 gene involved in the synthesis of chondroitin sulfate in the knee joint cartilage region of mice administered with quercetin glycoside (QG) (p: Csgalnact1 gene, q: Csgalnact2 gene, r: Chpf gene, s: Xylt2 gene, t: Ext1 gene).
- FIG. 3 is a graph showing the amount of chondroitin sulfate in the femoral joint of mice to which QG was administered.
- FIG. 1 is a graph showing the amount of chondroitin sulfate in HCH cells supplemented with 5 ⁇ M quercetin.
- FIG. 2 is a graph showing the expression levels of the glycosyltransferase
- FIG. 4 is a graph showing the area of the cartilage portion of the knee joint portion of the mouse to which QG was administered.
- the relative area shown in FIG. 4 is a relative value of the area of the QG administration group when the area of the cartilage portion of the knee joint portion of the control group is 1.
- FIG. 5A is a graph showing the expression level of the Csgalnact1 gene in cells supplemented with quercetin.
- FIG. 5B is a graph showing the expression level of the Csgalnact2 gene in cells supplemented with quercetin.
- FIG. 5C is a graph showing the expression level of the Xylt2 gene in cells supplemented with quercetin.
- FIG. 5D is a graph showing the expression level of the B3galt1 gene in cells supplemented with quercetin.
- FIG. 6A is a graph showing the expression level of the Chsy1 gene in cells supplemented with quercetin.
- FIG. 6B is a graph showing the expression level of the Chpf2 gene in cells supplemented with quercetin.
- FIG. 6C is a graph showing the expression level of the Ext1 gene in cells supplemented with quercetin.
- FIG. 6D is a graph showing the expression level of the Ext2 gene in cells supplemented with quercetin.
- FIG. 7 is a graph showing the amount of chondroitin sulfate in OSCV2 cells supplemented with 5 ⁇ M quercetin.
- FIG. 8 is a diagram showing the names of enzymes associated with the synthetic pathways of chondroitin sulfate (CS) and heparan sulfate (HS).
- composition for promoting chondroitin sulfate synthesis of the present invention contains quercetin or a glycoside thereof as an active ingredient.
- the composition for promoting chondroitin sulfate synthesis of the present invention is hereinafter simply referred to as the composition of the present invention.
- quercetin means quercetin, which is a compound belonging to flavonols, which is a type of polyphenol.
- quercetin glycoside means the above-mentioned quercetin glycoside, and specifically, is a general term for a series of compounds in which one or more sugars are glycosidic bonded to the hydroxyl group at the 3-position of quercetin.
- the quercetin glycoside is a compound represented by the following general formula.
- (X) n in the following general formula represents a sugar chain.
- X represents a sugar (monosaccharide), and n is an integer of 1 or more.
- the quercetin glycoside may be one kind of compound or two or more kinds of compounds.
- the sugar constituting the sugar chain represented by X that is glycosidic-bonded to quercetin is, for example, glucose, rhamnose, galactose, glucuronic acid, etc., and is preferably glucose or rhamnose.
- n is not particularly limited as long as it is 1 or more, but is preferably 1 to 16, and more preferably 1 to 8.
- the X portion may consist of one type of sugar or may consist of a plurality of types of sugars.
- (X) n may be a sugar chain composed of one kind of sugar or a sugar chain composed of a plurality of kinds of sugars.
- the quercetin glycoside in the present invention also includes a quercetin glycoside obtained by treating an existing quercetin glycoside with an enzyme or the like to transfer the glycoside.
- the quercetin glycoside referred to in the present invention includes rutin, enzyme-treated rutin, quercitrin, isoquercitrin and the like.
- isoquercitrin obtained by enzymatically treating rutin to remove the rhamnose sugar chain portion and isoquercitrin obtained by treating isoquercitrin with a glycosyltransferase from 1 to 7 glucoses.
- examples thereof include those to which the sugar chains are bound and those having a mixture thereof as a main component.
- a quercetin glycoside a compound in which 1 to 8 glucoses are glycosidic bonded to the hydroxyl group at the 3-position of quercetin (for example, isoquercitrin, a glycoside in which 1 to 7 glucoses are bound to isoquercitrin). Body) and the like are preferable.
- the ingested quercetin glycoside is absorbed into the body after the sugar is cleaved in the intestinal tract to become aglycone (quercetin).
- quercetin or a glycoside thereof there is no particular limitation on the origin and production method for obtaining quercetin or a glycoside thereof.
- plants rich in quercetin or its glycosides buckwheat, enju, caper, apple, tea, onion, grape, broccoli, moroheiya, raspberry, cowberry, cranberry, optian, leafy vegetables, citrus and the like are known.
- Quercetin or its glycosides can be obtained from these plants.
- a chemically synthesized product can also be used.
- a plant-derived raw material such as a plant extract containing quercetin or a glycoside thereof may be contained in the composition as long as the effects of the present invention are exhibited. Purified or isolated quercetin or glycosides thereof may be used.
- Quercetin or its glycosides are compounds contained in natural products and foods and drinks and have eating experience. Therefore, from the viewpoint of safety, it is considered that quercetin or its glycoside has few problems even if it is ingested every day, for example. According to the present invention, it is possible to provide a composition for promoting chondroitin sulfate synthesis containing a highly safe substance as an active ingredient.
- Chondroitin sulfate has a structure in which sulfuric acid is bound to a sugar chain having a repeating structure of disaccharide units in which D-glucuronic acid is bound to N-acetyl-D-galactosamine.
- the sugar chain portion of chondroitin sulfate is synthesized by glycosyltransferase.
- FIG. 8 shows the names of enzymes associated with the synthetic pathways of chondroitin sulfate (CS) and heparan sulfate (HS) (Reference: Biochemistry 87 (6): 744-748 (2015)).
- chondroitin sulfate (CS) chain to the core protein of proteoglycan first involves xylose (Xyl) and two molecules of galactose (Gal) at the serine residue (Ser) in the chondroitin sulfate binding sequence in the core protein. ), D-glucuronic acid (GlcA) is sequentially transferred to synthesize a sugar chain of Xyl-Gal-Gal-GlcA.
- This transfer of GalNAc is carried out by chondroitin sulfate N-acetylgalactosaminyl transferase 1 (CSGalNacT1) or chondroitin sulfate N-acetylgalactosaminyl transferase 2 (CSGalNacT2).
- GlcA and GalNAc are sequentially and alternately transferred to GalNAc at the non-reducing end, and the sugar chain is elongated.
- This sugar chain elongation process is carried out by CSGalNacT1, CSGalNacT2, chondroitin polymerization factor 2 (Chpf2), chondroitin sulfate synthases 1, 2 and 3 (ChSy1, ChSy2 and ChSy3).
- CSGalNacT1 and CSGalNacT2 have a transposable activity to transfer GalNAc to GlcA.
- Chpf2 has a glycosyl transfer activity that transfers GlcA to GalNAc.
- ChSy1, ChSy2 and ChSy3 show a glycosyl transfer activity that transfers GalNAc to GlcA and a glycosyl transfer activity that transfers GlcA to GalNAc.
- extoscin 1 (Ext1) is added to the non-reducing end of the sugar chain of Xyl-Gal-Gal-GlcA added to the serine residue of the core protein.
- Exostocin 2 (Ext2)
- Ext-like1 (Extl1)
- Ext-like2 Extl2
- Ext-like3 Extl3
- chondroitin sulfates such as the gene encoding CSGalNacT1 (Csgalnact1) and the gene encoding CSGalNacT2 (Csgalnact2) are compared with the case where kelcetin is not present.
- the expression level of the gene encoding the glycosyltransferase involved in the synthesis increased.
- chondrocytes were cultured in the presence of quercetin, a significant increase in the amount of chondroitin sulfate was observed as compared with the control.
- quercetin or its glycoside has an action of promoting the synthesis of chondroitin sulfate and an action 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.
- promotion of the synthesis of chondroitin sulfate and promotion of the expression of glycosyltransferase involved in the synthesis of chondroitin sulfate are preferably promotion of the synthesis of chondroitin sulfate in the body of the subject and promotion of the expression of the glycosyltransferase in the body of the subject. ..
- chondroitin sulfate N-acetylgalactosaminyl transferase 1 CSGalNacT1
- chondroitin sulfate N-acetylgalactosaminyltransferase 2 CSGalNacT2
- XylT xylose transferase
- B3GalT 3-galactosyltransferase
- ⁇ -1,4-galactosyltransferase B4GalT
- chondroitin sulfate synthase 1-3 ChSy1-3
- Chopf2 chondroitin polymerization factor 2
- glycosyltransferases involved in the synthesis of heparan sulfate chains are the glycosyltransferases involved in the synthesis of chondroitin sulfate in the present invention. Is not included.
- composition for promoting chondroitin sulfate synthesis of the present invention can promote the expression of glycosyltransferase involved in the synthesis of chondroitin sulfate.
- the compositions 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.
- glycosyltransferases involved in the synthesis of chondroitin sulfate are preferably chondroitin sulfate N-acetylgalactosaminyl transferase 1, chondroitin sulfate N-acetylgalactosaminyl transferase 2, xylose transferase 2, ⁇ -1,3-galactosyltransferase 1.
- chondroitin sulfate N-acetylgalactosaminyl transferase 1
- xylose transferase 2 ⁇ -1,3-galactosyltransferase 1.
- BGalT1 one or more selected from the group consisting of chondroitin sulfate synthase 1 and chondroitin transferase 2.
- the compositions of the present invention can promote the expression of one or more of these glycosyltransferases.
- compositions of the present invention can promote the expression of chondroitin sulfate N-acetylgalactosaminyltransferase 1 and / or chondroitin sulfate N-acetylgalactosaminyltransferase 2 for such purposes.
- CSGalNacT1 and CSGalNacT2 are considered as important enzymes that serve as the starting point for chondroitin sulfate synthesis.
- the composition of the present invention can promote the expression of chondroitin sulfate N-acetylgalactosaminyl transferase 1, chondroitin sulfate N-acetylgalactosaminyl transferase 2, xylose transferase 2, and chondroitin polymerization factor 2.
- the promotion of the expression of the glycosyltransferase includes the promotion of the expression of the gene encoding the glycosyltransferase and the promotion of the expression of the enzyme at the protein level, and may be one or both of these. Examples of the promotion of the expression of the above gene include promotion of the expression of mRNA, and preferably promotion of transcription into mRNA. Promotion of expression of glycosyltransferases at the protein level includes promotion in translation.
- the composition of the present invention is preferably used for promoting the expression of a glycosyltransferase involved in the synthesis of chondroitin sulfate in chondrocytes, for promoting the synthesis of chondroitin sulfate in chondrocytes, and the like. it can.
- a glycosyltransferase involved in the synthesis of chondroitin sulfate in cartilage the synthesis of cartilage can be promoted.
- the compositions of the invention can be used to promote cartilage synthesis. By promoting cartilage synthesis, it becomes possible to increase cartilage. Promoting cartilage synthesis is useful, for example, in preventing cartilage loss.
- Quercetin or its glycoside has an action of promoting the expression of glycosyltransferase involved in the synthesis of chondroitin sulfate in nerve cells and osteoblasts, and can promote the synthesis of chondroitin sulfate. Quercetin or its glycosides are useful for promoting the synthesis of chondroitin sulfate in nerve cells (preferably hippocampal nerve cells) and protecting nerves.
- compositions of the invention can be used to protect nerves or promote bone formation.
- composition of the present invention can be applied to either therapeutic use (medical use) or non-therapeutic use (non-medical use).
- Non-therapeutic is a concept that does not include medical practice, ie human surgery, treatment or diagnosis.
- the composition of the present invention can be in the form of foods and drinks, cosmetics, pharmaceuticals, quasi-drugs, feeds and the like.
- the composition of the present invention may itself be a food or drink, a cosmetic, a pharmaceutical product, a quasi-drug, a feed, etc. used for promoting chondroitin sulfate synthesis, or a material used in combination with these. It may be a preparation or the like.
- the composition for promoting chondroitin sulfate synthesis of the present invention can be provided in the form of an agent as an example, but is not limited to this form.
- the agent can be provided as it is as a composition or as a composition containing the agent.
- the composition for promoting chondroitin sulfate synthesis of the present invention can also be referred to as a chondroitin sulfate synthesis accelerator.
- the composition of the present invention may be either an oral composition or a parenteral composition, but is preferably an oral composition.
- the synthesis of chondroitin sulfate can be promoted in a subject by orally ingesting or administering the composition of the present invention or by parenterally administering the composition of the present invention.
- oral composition examples include foods and drinks, oral medicines, quasi-drugs, and feeds, preferably foods and drinks or oral medicines, and more preferably foods and drinks.
- parenteral compositions include cosmetics, parenteral medicines, and parenteral quasi-drugs.
- composition of the present invention may contain any additive and any component in addition to quercetin or a glycoside thereof, as long as the effects of the present invention are not impaired.
- additives and ingredients can be selected according to the form of the composition and the like, and generally, those that can be used for foods and drinks, cosmetics, pharmaceuticals, quasi-drugs, feeds and the like can be used.
- the composition of the present invention is a food or drink, a pharmaceutical product, a quasi-drug, a feed or the like, the production method thereof is not particularly limited and can be produced by a general method.
- composition of the present invention when used as a food or drink, quercetin or a glycoside thereof is mixed with ingredients that can be used in the food or drink (for example, food materials, food additives used as necessary, etc.).
- Various foods and drinks can be used.
- Foods and drinks are not particularly limited, and examples thereof include general foods and drinks, health foods, health drinks, foods with functional claims, foods for specified health use, dietary supplements, foods and drinks for the sick, and the like.
- the above-mentioned health foods, foods with functional claims, foods for specified health use, health supplements, etc. include, for example, fine granules, tablets, granules, powders, capsules, chewables, dry syrups, syrups, liquids, beverages, fluids. It can be used as various formulations such as food.
- composition of the present invention when used as a cosmetic, quercetin or a glycoside thereof can be mixed with a carrier, an additive or the like that is acceptable for the cosmetic.
- the product form of the cosmetic is not particularly limited.
- composition of the present invention is a pharmaceutical product or a quasi-drug
- a pharmacologically acceptable carrier for example, quercetin or a glycoside thereof
- a pharmacologically acceptable carrier for example, quercetin or a glycoside thereof
- Such carriers, additives and the like may be pharmacologically acceptable as long as they can be used in pharmaceutical products or quasi-drugs, for example, excipients, binders, disintegrants, lubricants, etc.
- antioxidants, colorants and the like can be mentioned.
- Examples of the administration (ingestion) form of the drug or quasi-drug include oral or parenteral (transdermal, transmucosal, enteral, injection, etc.) administration forms.
- composition of the present invention is a drug or a quasi-drug
- it is preferably an oral drug or a quasi-drug.
- Dosage forms for oral administration include liquids, tablets, powders, fine granules, granules, sugar-coated tablets, capsules, suspensions, emulsions, chewables and the like.
- Dosage forms for parenteral administration include injections, infusions, external preparations for skin (patches, creams, ointments, etc.) and the like.
- the drug may be a non-human veterinary drug.
- quercetin or a glycoside thereof may be added to the feed.
- the feed also contains feed additives.
- examples of the feed include livestock feed used for cattle, pigs, chickens, sheep, horses and the like; small animal feed used for rabbits, rats, mice and the like; pet food used for dogs, cats, small birds and the like.
- the content of quercetin or its glycoside contained in the composition of the present invention is not particularly limited and can be set according to its form and the like.
- the content of quercetin or a glycoside thereof in the composition of the present invention is preferably 0.01% by weight or more, more preferably 0.1% by weight or more in the composition in terms of quercetin, for example. , 80% by weight or less, more preferably 50% by weight or less.
- the content of quercetin or a glycoside thereof is preferably 0.01 to 80% by weight, more preferably 0.1 to 50% by weight in the composition of the present invention in terms of quercetin.
- the content of quercetin or its glycoside can be measured according to a known method, and for example, an HPLC method or the like can be used.
- compositions of the present invention are usually 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.
- the composition of the present invention is preferably taken orally (orally administered).
- the dose (which can also be referred to as ingestion) of the composition of the present invention is not particularly limited, and may be an amount such that 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 can be obtained. It may be appropriately set according to the administration form, administration method and the like.
- the dose of quercetin or its glycoside is preferably 0.3 mg or more per 60 kg of body weight per day in terms of quercetin. , More preferably 1.0 mg or more, still more preferably 10 mg or more, still preferably 4000 mg or less, more preferably 2000 mg or less, still more preferably 1000 mg or less.
- the dose of quercetin or a glycoside thereof, as a quercetin equivalent value is preferably 0.3 to 4000 mg, more preferably 1.0 to 2000 mg per 60 kg of body weight per day for humans (adults). , More preferably 1.0 to 1000 mg, and particularly preferably 10 to 1000 mg.
- compositions of the invention can be used to ingest or administer the above amounts of quercetin or glycosides thereof per day to a human body weight of 60 kg.
- composition of the present invention is preferably continuously ingested or administered. Continuous ingestion or administration of quercetin or its glycosides is expected to enhance the effect of promoting chondroitin sulfate synthesis. In one embodiment, the composition of the present invention is preferably continuously ingested or administered for 1 week or longer, more preferably 4 weeks or longer, and even more preferably 8 weeks or longer.
- composition of the present invention can be used for the prevention or amelioration of a condition or disease that can be expected to be prevented or ameliorated by promoting the synthesis of chondroitin sulfate.
- a condition or disease include a condition or disease caused by a decrease in the amount of chondroitin sulfate, and examples thereof include knee osteoarthritis, coxarthrosis, spinal stenosis, and osteoporosis.
- the compositions of the present invention can be used for the prevention or amelioration of cognitive decline and for the prevention or amelioration of dementia.
- prevention of a condition or disease includes preventing the onset, delaying the onset, reducing the incidence, reducing the risk of developing the disease, and the like. Improvement of a condition or disease is to recover the subject from the condition or disease, reduce the symptoms of the condition or disease, improve the symptoms of the condition or disease, delay the progression of the condition or disease, or prevent it. Etc. are included.
- administration targets include subjects that require or desire to promote chondroitin sulfate synthesis, and subjects that require or desire to prevent or ameliorate a condition or disease caused by a decrease in the amount of chondroitin sulfate.
- the decrease in the amount of chondroitin sulfate may be a decrease in the amount of chondroitin sulfate due to aging, or may be a decrease in the amount of chondroitin sulfate due to aging in middle-aged and elderly people.
- the administration target in the present invention includes middle-aged and elderly people.
- Middle-aged and elderly people include the elderly.
- the elderly are preferable as the target.
- the middle-aged person may be, for example, a person aged 40 years or older.
- the elderly person may be, for example, a person aged 60 years or older or a person aged 65 years or older.
- 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 for healthy subjects, for example, for the purpose of preventing or preventing a condition or disease that can be expected to be prevented or improved by promoting chondroitin sulfate synthesis.
- the composition of the present invention may be labeled with a function exerted by promoting chondroitin sulfate synthesis.
- the composition for promoting chondroitin sulfate synthesis of the present invention includes, for example, "maintenance of cartilage amount”, “suppression of cartilage wear”, “suppression of cartilage degeneration”, “reduction of defects in knee joint” and “maintenance of knee joint function”.
- the display of the above function may describe that the above function is a function obtained by promoting the synthesis of chondroitin sulfate.
- the composition of the present invention is preferably a food or drink with the above indication. Further, the above display may be a display to the effect that it is used to obtain the above function.
- the label may be affixed to the composition itself or to the container or packaging of the composition.
- the present invention also includes the following methods and uses.
- the methods and uses may be therapeutic or non-therapeutic methods or uses.
- Administration (ingestion) of quercetin or its glycosides can increase the expression level of glycosyltransferases involved in the synthesis of chondroitin sulfate.
- Administration of quercetin or its glycosides 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 a glycoside thereof for promoting the synthesis of chondroitin sulfate by promoting the expression of glycosyltransferase involved in the synthesis of chondroitin sulfate.
- the promotion of chondroitin sulfate synthesis may be, for example, promotion of chondroitin sulfate synthesis in cartilage.
- Quercetin or its glycoside can be used for the prevention or amelioration of a condition or disease that can be expected to be prevented or ameliorated by promoting the above-mentioned chondroitin sulfate synthesis.
- quercetin or a glycoside thereof it is preferable to administer (ingest) quercetin or a glycoside thereof to a subject at least once a day, for example, once to several times a day (for example, 2 to 3 times).
- the above use is preferably in humans or non-human mammals, more preferably in humans. Quercetin or its glycosides are as described above.
- quercetin or a glycoside thereof in an amount (which can be said to be an effective amount) that can promote chondroitin sulfate synthesis may be used.
- the preferable dose, administration target, administration method and the like of quercetin or a glycoside thereof 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 the same.
- the composition of the present invention described above may be used.
- Quercetin or its glycosides are preferably orally administered (ingested).
- Quercetin or its glycosides can be used for the production of foods and drinks, cosmetics, pharmaceuticals, quasi-drugs, feeds and the like used to promote the synthesis of chondroitin sulfate.
- the present invention also includes the use of quercetin or a glycoside thereof for producing a composition for promoting chondroitin sulfate synthesis.
- the composition for promoting chondroitin sulfate synthesis and its preferred embodiments are the same as the above-mentioned composition of the present invention.
- Example 1 Chondroitin sulfate synthesis promoting effect 1 (in vitro) by quercetin> (1)
- Cell culture HCH cells chondrocytes
- Dulvecco's Modified Eagle Medium Sigma-Aldrich
- FIG. 1 is a graph showing the amount of chondroitin sulfate in HCH cells supplemented with 5 ⁇ M quercetin.
- the relative CS level on the vertical axis is a relative value of the amount of CS in the cell to which quercetin was added, when the amount of chondroitin sulfate (CS) in the cell to which DMSO was added as a control was 1.
- quercetin is a cell cultured in a quercetin-added medium. The addition of 5 ⁇ M quercetin confirmed a significant increase in chondroitin sulfate production in HCH cells.
- RNA was isolated from cells or tissues and using the following method. From the isolated RNA, cDNA synthesis was performed using High-Capacity cDNA Reverse Transcriptional Kits (Thermo Fisher Scientific). Quantitative PCR using TaqMan Fast Universal PCR Mastermix (Thermo Fisher Scientific) at Quant Studio Real Time PCR System (Thermofisher), and chondroitin sulphate (Thermo Fisher Scientific). The expression level of the transposase gene) was quantified.
- Table 1 shows 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 measurements.
- FIG. 8 shows the position of each enzyme subjected to gene analysis in the glycosaminoglycan synthesis pathway.
- Example 2 Chondroitin sulfate synthesis promoting effect (in vivo) by quercetin glycoside> (1) Administration of quercetin glycosides to mice 36-week-old C57BL / 6J wild-type (WT) mice and Csgalnact1 gene knockout (T1-KO) mice are fed with distilled water (control group) or 4 Distilled water containing .5 g / L quercetin glycoside (QG) was divided into a drinking water group (QG administration group) so as to have an equal weight.
- WT wild-type mice
- the WT mice (WT-QG) and T1-KO mice (KO-QG) in the QG-administered group were ingested distilled water containing the above-mentioned quercetin glycoside for 18 weeks.
- the daily intake of quercetin glycosides per body weight was 117 mg / kg in terms of quercetin.
- the WT mice (WT-control) and T1-KO mice (KO-control) in the control group were fed with distilled water for 18 weeks. All the cages were bred independently, and a saucer type device (Muromachi Machinery Co., Ltd.) that can measure spontaneous rotation was placed in the cages to promote a certain amount of spontaneous movement.
- Example 2 As the quercetin glycoside, an enzyme-treated isoquercitrin obtained by treating isoquercitrin with a glycosyltransferase and in which a sugar chain consisting of 1 to 7 glucoses was bound to isoquercitrin was used. ..
- 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 osteoarthritis tissue of mice administered with quercetin glycoside (QG).
- QG quercetin glycoside
- p indicates Csgalnact1
- q indicates Csgalnact2
- r indicates Chpf2
- s indicates Xylt2
- t indicates Ext1.
- the relative mRNA level on the vertical axis is a relative value of the mRNA amount of the gene in each group when the mRNA amount of the gene in the control group (WT-control) of the WT mouse is 1.
- QG quercetin glycoside
- the relative CS level on the vertical axis is a relative value of the CS amount of each group when the amount of chondroitin sulfate (CS) in the control group of WT mice is 1.
- FIG. 4 is a graph showing the area of the cartilage portion of the knee joint portion of the mouse to which the quercetin glycoside was administered.
- the relative area shown in FIG. 4 is a relative value of the area of the cartilage portion of each group when the area of the cartilage portion of the control group of the WT mouse is 1.
- WT-QG QG-administered group
- WT-control control group
- T1-KO mice although there was an increasing tendency in the QG-administered group (KO-QG) as compared with the control group (KO-control), no significant change was observed.
- Example 3 Chondroitin sulfate synthesis promoting effect 2 (in vitro) by quercetin> (1) Culture of various cells C6 cells (glial cells), SKOV3 cells (ovarian adenoma cells), C1300 cells (neuroblasts), KINGS1 cells (glia cells), NM-CG1 cells (glia cells), MC3T3 -E1 cells (osteoblasts) and OSCV2 cells (osteolithic cells) were cultured in Dulvecco's Modified Eagle Medium (Sigma-Aldrich) containing 10% bovine fetal serum until they were 100% confluent.
- Dulvecco's Modified Eagle Medium Sigma-Aldrich
- the difference in the mean value of each group was tested using Dunnett's t-test test, and 5% or less was considered significant (*: p ⁇ 0.05 vs quercetin 0 ⁇ M).
- the quantified genes are Csgalnact1, Csgalnact2, Xylt2, B3galt1, Chsy1, Chpf2, Ext1 and Ext2 shown in Table 1.
- FIGS. 5A, 5B, 5C, 5D, 6A, 6B, 6C and 6D The results are shown in FIGS. 5A, 5B, 5C, 5D, 6A, 6B, 6C and 6D (*: P ⁇ 0.05, vs quercetin 0 ⁇ M).
- FIG. 5A is a graph showing the expression level of the Csgalnact1 gene in cells supplemented with quercetin.
- FIG. 5B is a graph showing the expression level of the Csgalnact2 gene in cells supplemented with quercetin.
- FIG. 5C is a graph showing the expression level of the Xylt2 gene in cells supplemented with quercetin.
- FIG. 5D is a graph showing the expression level of the B3galt1 gene in cells supplemented with quercetin.
- FIG. 5A is a graph showing the expression level of the Csgalnact1 gene in cells supplemented with quercetin.
- FIG. 5B is
- FIGS. 6A is a graph showing the expression level of the Chsy1 gene in cells supplemented with quercetin.
- FIG. 6B is a graph showing the expression level of the Chpf2 gene in cells supplemented with quercetin.
- FIG. 6C is a graph showing the expression level of the Ext1 gene in cells supplemented with quercetin.
- FIG. 6D is a graph showing the expression level of the Ext2 gene in cells supplemented with quercetin.
- a is a C6 cell
- b is a SKOV3 cell
- c is a C1300 cell
- d is a KINGS1 cell
- e is an NM-CG1 cell
- f is an MC3T3-E1SK cell.
- gene expression levels are shown at relative mRNA levels.
- Chpf2 was found in SKOV3 cells, C1300 cells, KINGS1 cells and MC3T3-E1 cells (Fig. 6B) in gene expression levels due to the addition of 5 ⁇ M kercetin. A significant increase was confirmed. On the other hand, in Ext1 and Ext2 involved in the synthesis of heparan sulfate, no significant change in the gene expression level was observed in any of the cells (FIGS. 6C and 6D).
- FIG. 7 is a graph showing the amount of chondroitin sulfate in OSCV2 cells supplemented with 5 ⁇ M quercetin.
- the relative CS level on the vertical axis is a relative value of the amount of CS in the cell to which quercetin was added, when the amount of chondroitin sulfate (CS) in the cell to which DMSO was added as a control was 1.
- quercetin is a cell cultured in a quercetin-added medium. The addition of 5 ⁇ M quercetin confirmed a significant increase in the amount of chondroitin sulfate.
- quercetin or its glycoside has an action of promoting the expression of glycosyltransferase involved in the synthesis of chondroitin sulfate and an action of promoting the synthesis of chondroitin sulfate.
- Quercetin or its glycosides had the effect of increasing the amount of articular cartilage.
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Abstract
Description
〔1〕ケルセチン又はその配糖体を有効成分として含有するコンドロイチン硫酸合成促進用組成物。
〔2〕コンドロイチン硫酸の合成に関与する糖転移酵素の発現を促進する上記〔1〕に記載のコンドロイチン硫酸合成促進用組成物。
〔3〕コンドロイチン硫酸の合成に関与する糖転移酵素が、コンドロイチン硫酸N-アセチルガラクトサミニルトランスフェラーゼ1、コンドロイチン硫酸N-アセチルガラクトサミニルトランスフェラーゼ2、キシローストランスフェラーゼ2、β-1,3-ガラクトシルトランスフェラーゼ1、コンドロイチン硫酸合成酵素1及びコンドロイチン重合因子2からなる群より選択される1以上である上記〔2〕に記載のコンドロイチン硫酸合成促進用組成物。
〔4〕コンドロイチン硫酸の合成に関与する糖転移酵素が、コンドロイチン硫酸N-アセチルガラクトサミニルトランスフェラーゼ1及び/又はコンドロイチン硫酸N-アセチルガラクトサミニルトランスフェラーゼ2である上記〔2〕又は〔3〕に記載のコンドロイチン硫酸合成促進用組成物。
〔5〕飲食品、化粧料又は医薬部外品である上記〔1〕~〔4〕のいずれかに記載のコンドロイチン硫酸合成促進用組成物。
〔6〕ケルセチン又はその配糖体を投与する、コンドロイチン硫酸の合成を促進する方法。
〔7〕コンドロイチン硫酸の合成に関与する糖転移酵素の発現を促進することによりコンドロイチン硫酸の合成を促進する上記〔6〕に記載の方法。
〔8〕コンドロイチン硫酸の合成を促進するための、ケルセチン又はその配糖体の使用。
〔9〕コンドロイチン硫酸の合成に関与する糖転移酵素の発現を促進することによりコンドロイチン硫酸の合成を促進する上記〔8〕に記載の使用。
摂取されたケルセチン配糖体は、腸管内で糖が切断されてアグリコン(ケルセチン)になってから体内へ吸収される。
ケルセチン又はその配糖体は、化学合成品を使用することもできる。本発明においては、本発明の効果を奏することになる限り、ケルセチン又はその配糖体を含む植物の抽出物等の植物由来原料等を組成物に含有させてもよい。精製又は単離されたケルセチン又はその配糖体を使用してもよい。
図8に、コンドロイチン硫酸(CS)及びヘパラン硫酸(HS)の合成経路と関連する酵素の名称を示す(参考文献:生化学 87(6): 744-748 (2015))。
軟骨等においては、プロテオグリカンのコアタンパク質へのコンドロイチン硫酸(CS)鎖の付加は、まずコアタンパク質内のコンドロイチン硫酸結合配列内のセリン残基(Ser)にキシロース(Xyl)、2分子のガラクトース(Gal)、D-グルクロン酸(GlcA)が順番に転移してXyl-Gal-Gal-GlcAの糖鎖が合成される。セリン残基へのXylの転移は、キシローストランスフェラーゼ(XylT)によって、XylへのGalの転移は、β-1,4-ガラクトシルトランスフェラーゼ(B4GalT)によって、1つ目のGalへの2つ目のGalの転移は、β-1,3-ガラクトシルトランスフェラーゼ(B3GalT)によって、GalへのGlcAの転移はβ-1,3-グルクロニルトランスフェラーゼ(B3GaT)によって行われる。次に、非還元末端(伸長していく側)のGlcAにN-アセチル-D-ガラクトサミン(GalNAc)が転移する。このGalNAcの転移は、コンドロイチン硫酸N-アセチルガラクトサミニルトランスフェラーゼ1(CSGalNacT1)又はコンドロイチン硫酸N-アセチルガラクトサミニルトランスフェラーゼ2(CSGalNacT2)によって行われる。その後、非還元末端のGalNAcにGlcA、GalNAcが順次交互に転移していき、糖鎖が伸長する。この糖鎖伸長過程は、CSGalNacT1、CSGalNacT2、コンドロイチン重合因子(Chondroitin polymerizing factor)2(Chpf2)、コンドロイチン硫酸合成酵素1、2及び3(ChSy1、ChSy2及びChSy3)によって進行する。CSGalNacT1及びCSGalNacT2は、GlcAにGalNAcを転移する転移活性を有する。Chpf2は、GalNAcにGlcAを転移する糖転移活性を有する。ChSy1、ChSy2及びChSy3は、GlcAにGalNAcを転移する糖転移活性と、GalNAcにGlcAを転移する糖転移活性を示す。コンドロイチン硫酸の合成に関与する糖転移酵素の発現を促進すると、コンドロイチン硫酸の合成を促進することができる。
上記糖転移酵素の発現促進には、糖転移酵素をコードする遺伝子の発現促進及び当該酵素のタンパク質レベルでの発現促進が含まれ、これらのいずれか又は両方であってよい。上記遺伝子の発現促進として、mRNAの発現促進が挙げられ、好ましくはmRNAへの転写促進である。糖転移酵素のタンパク質レベルでの発現促進には、翻訳における促進が含まれる。
ケルセチン又はその配糖体は、神経細胞や骨芽細胞においてコンドロイチン硫酸の合成に関与する糖転移酵素の発現を促進する作用を有し、コンドロイチン硫酸の合成を促進することができる。ケルセチン又はその配糖体は、神経細胞(好ましくは海馬の神経細胞)においてコンドロイチン硫酸の合成を促進し、神経を保護するために有用である。神経を保護することにより、認知機能改善効果等が期待できる。ケルセチン又はその配糖体は、骨芽細胞においてコンドロイチン硫酸の合成を促進し、骨形成を促進するために有用である。一態様において、本発明の組成物は、神経を保護するため、又は、骨形成を促進するために使用することができる。
本発明の組成物は、飲食品、化粧料、医薬品、医薬部外品、飼料等の形態とすることができる。本発明の組成物は、それ自体がコンドロイチン硫酸合成促進のために用いられる飲食品、化粧料、医薬品、医薬部外品、飼料等であってもよく、これらに配合して使用される素材又は製剤等であってもよい。
本発明のコンドロイチン硫酸合成促進用組成物は、一例として、剤の形態で提供することができるが、本形態に限定されるものではない。当該剤をそのまま組成物として、又は、当該剤を含む組成物として提供することもできる。一態様において、本発明のコンドロイチン硫酸合成促進用組成物は、コンドロイチン硫酸合成促進剤ということもできる。
本発明の組成物は、経口用組成物、非経口用組成物のいずれであってもよいが、好ましくは経口用組成物である。本発明の組成物を対象に経口で摂取させる又は投与することにより、又は、非経口投与することにより、対象においてコンドロイチン硫酸の合成を促進することができる。経口用組成物としては、飲食品、経口用の医薬品、医薬部外品、飼料が挙げられ、好ましくは飲食品又は経口用医薬品であり、より好ましくは飲食品である。非経口用組成物として、化粧料、非経口用医薬品、非経口用医薬部外品が挙げられる。
ケルセチン又はその配糖体の含有量は、公知の方法に従って測定することができ、例えば、HPLC法などを用いることができる。
一態様において、投与対象として、コンドロイチン硫酸合成促進を必要とする又は希望する対象及びコンドロイチン硫酸量の減少に起因する状態又は疾患の予防又は改善を必要とする又は希望する対象等が挙げられる。コンドロイチン硫酸量の減少は、加齢によるコンドロイチン硫酸量の減少であってよく、中高年者における加齢によるコンドロイチン硫酸量の減少であってよい。
一態様において、本発明における投与対象として、中高年者が挙げられる。中高年者は、高齢者を含む。中高年者の中でも、対象として高齢者が好ましい。本発明において、中高年者は、例えば、40歳以上のヒトであってよい。高齢者は、例えば、60歳以上又は65歳以上のヒトであってよい。一態様において、本発明の組成物は、中高年者用のコンドロイチン硫酸合成促進用組成物として好適に使用される。
本発明の組成物は、例えば、コンドロイチン硫酸合成促進により予防又は改善が期待できる状態又は疾患の予防等を目的として、健常者に対して使用することもできる。
一態様において、本発明の組成物は、上記の表示が付された飲食品であることが好ましい。また上記の表示は、上記の機能を得るために用いる旨の表示であってもよい。当該表示は、組成物自体に付されてもよいし、組成物の容器又は包装に付されていてもよい。
ケルセチン又はその配糖体を投与する、コンドロイチン硫酸の合成を促進する方法。
コンドロイチン硫酸の合成を促進するための、ケルセチン又はその配糖体の使用。
上記方法及び使用は、治療的な方法又は使用であってもよく、非治療的な方法又は使用であってもよい。ケルセチン又はその配糖体を投与する(摂取させる)と、コンドロイチン硫酸の合成に関与する糖転移酵素の発現量を増加させることができる。ケルセチン又はその配糖体を投与すると、コンドロイチン硫酸の合成を促進することができる。上記方法は、コンドロイチン硫酸の合成に関与する糖転移酵素の発現を促進することによりコンドロイチン硫酸の合成を促進する方法であってよい。上記使用は、コンドロイチン硫酸の合成に関与する糖転移酵素の発現を促進することによりコンドロイチン硫酸の合成を促進するための、ケルセチン又はその配糖体の使用であってよい。コンドロイチン硫酸の合成促進は、例えば、軟骨におけるコンドロイチン硫酸の合成促進であってよい。ケルセチン又はその配糖体は、上記のコンドロイチン硫酸合成促進により予防又は改善が期待できる状態又は疾患の予防又は改善のために使用することができる。
(1)細胞の培養
HCH細胞(軟骨細胞)を10%ウシ胎児血清含有Dulbecco’s Modified Eagle Medium(Sigma-Aldrich社)で100%コンフルエントになるまで培養した。
HCH細胞の培地に5μMのケルセチンを添加し、培養した。ケルセチンは、Dimethyl sulfoxide(DMSO)に溶解して添加した。添加4日後の培養細胞のコンドロイチン硫酸量を抗コンドロイチン硫酸抗体(CS-56)によるCell ELISAシステムで定量した。コントロールの細胞は、DMSOを添加する以外は同じ方法で培養を行い、コンドロイチン硫酸を定量した。各群の平均値の差はStudent’s t test検定を用いて検定し、5%以下を有意とした(*:p<0.05 vs コントロール)。
以下の実施例において、細胞又は組織における遺伝子発現量の測定は、細胞又は組織からRNAを単離し、以下の方法で行った。
単離したRNAから、High-Capacity cDNA Reverse Transcriptional Kits(Thermo Fisher Scientific社)を用いてcDNA合成を行った。Quantstudio Real Time PCR System(Thermofisher)にて、TaqMan Fast Universal PCR Mastermix(Thermo Fisher Scientific社)を使用して定量的PCRを行い、コンドロイチン硫酸又はヘパラン硫酸の合成に関わる糖転移酵素をコードする遺伝子(糖転移酵素遺伝子)の発現量を定量した。各群の平均値の差はDunnett’s t test検定を用いて検定し、5%以下を有意とした(*:p<0.05 vs ケルセチン0μM)。実施例2及び3で定量した遺伝子の種類、該遺伝子がコードするタンパク質、並びに、測定のために使用したPCRプライマー及びプローブを表1に示した。また遺伝子解析を実施した各酵素のグリコサミノグリカン合成経路における位置づけを図8に示す。
(1)マウスへのケルセチン配糖体の投与
36週齢のC57BL/6Jの野生型(WT)マウス及びCsgalnact1遺伝子ノックアウト(T1-KO)マウスを、蒸留水を飲水させる群(コントロール群)又は4.5g/Lケルセチン配糖体(QG)を含有する蒸留水を飲水させる群(QG投与群)に体重が均等になるように分けた。QG投与群のWTマウス(WT-QG)及びT1-KOマウス(KO-QG)には、上記のケルセチン配糖体を含有する蒸留水を18週間摂取させた。1日当たり体重当たりのケルセチン配糖体の摂取量は、ケルセチン換算で117mg/kgであった。コントロール群のWTマウス(WT-コントロール)及びT1-KOマウス(KO-コントロール)には、蒸留水を18週間摂取させた。ケージはすべて単独飼育として、ケージ内には運動負荷をかけるためソーサー型輪転自発走行を計測できる機器(室町機械(株))を置いて一定量の自発運動を促した。飲水投与開始18週間後に、膝関節部組織を採取した。採取した膝関節部組織を用いて、遺伝子発現量、コンドロイチン硫酸量及び関節軟骨量を測定した。実施例2において、ケルセチン配糖体には、イソクエルシトリンを糖転移酵素で処理して得られる、イソクエルシトリンにグルコース1~7個からなる糖鎖が結合した酵素処理イソクエルシトリンを使用した。
採取した膝関節部から、RNeazy Mini Kit(QIAGEN社)を用いてRNAを単離した。単離したRNAを用いて、上記の遺伝子発現量の定量方法に記載の方法でcDNA合成を行い、定量的RT-PCRにて、コンドロイチン硫酸又はヘパラン硫酸の合成に関与する糖転移酵素遺伝子の発現量を定量した(N=12)。定量した遺伝子は、表1に記載のCsgalnact1、Csgalnact2、Chpf2、Xylt2及びExt1である。各群の平均値の差はStudent’s t test検定を用いて検定し、5%以下を有意とした(*:p<0.05 vs コントロール)。
採取した膝関節部組織からトリクロロ酢酸沈殿によりタンパク質成分を除去し、エタノール沈殿によりグリコサミノグリカン画分を濃縮した。脱塩後、コンドロイチナーゼによりグリコサミノグリカンを分解し、2-アミノベンズアミドで蛍光標識した2糖単位のコンドロイチン硫酸を、下記の条件で、高速液体クロマトグラフィー((株)島津製作所)で定量した(N=6)。
カラム:DOCOSIL SP100 5.0μm 4.6×100mm((株)センシュー科学)
ガードカラム:Myghtysil RP-18GP 5.0μm,4.6×10mm(関東化学(株))
溶出液:
A.蒸留水
B.0.2M NaCl
C.10mM Tetrabutyl ammonium hydrogen sulfate
D.50% Acetonitril
グラジエント条件:
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%))
流速(溶出液):1.1mL/min
反応液:
A.0.5% 2-cyanoacetate solution
B.0.25M NaOH
流速(反応液):0.7mL/min
注入量:50μL
蛍光モニター:EX 346nm、EM 410nm、AT 64、Gain ×100
インテグレーター:End Time 40min、Speed 2min/min、AT 512
採取した膝関節部組織をホルマリン固定し、連続パラフィン切片として試料作製を行った。関節部において最も領域の広い中心部を面出しするとともに、そこから100マイクロメートル毎の切片を1組織当たり20枚選択し、HE染色によってそれぞれの面積変動幅が同一となるように計測した。関節軟骨部の面積を画像解析ソフトimage Jによって定量した(N=18)。各群の平均値の差はStudent’s t test検定を用いて検定し、5%以下を有意とした(*:p<0.05 vs コントロール)。
WTマウスでは、コントロール群(WT-コントロール)と比べて、QG投与群(WT-QG)において関節軟骨部面積の有意な増加が確認された。一方でT1-KOマウスでは、コントロール群(KO-コントロール)に比べてQG投与群(KO-QG)で増加傾向にあるものの、有意な変動は認められなかった。
(1)各種細胞の培養
C6細胞(グリア系細胞)、SKOV3細胞(卵巣腺腫細胞)、C1300細胞(神経芽細胞)、KINGS1細胞(グリア系細胞)、NM-CG1細胞(グリア系細胞)、MC3T3-E1細胞(骨芽細胞)、OSCV2細胞(破骨細胞)を10%ウシ胎児血清含有Dulbecco’s Modified Eagle Medium(Sigma-Aldrich社)で100%コンフルエントになるまで培養した。
各種細胞(C6細胞、SKOV3細胞、C1300細胞、KINGS1細胞、NM-CG1細胞、MC3T3-E1細胞)にDMSO又は0.5μM、1μM若しくは5μMのケルセチンを添加した。添加2時間後の細胞からRNeazy Mini Kit(QIAGEN社)を用いてRNAを単離した。単離したRNAを用いて、上記の遺伝子発現量の定量方法に記載の方法で、cDNA合成及び定量的PCRを行い、コンドロイチン硫酸又はヘパラン硫酸の合成に関わる糖転移酵素遺伝子の発現量を定量した。各群の平均値の差はDunnett’s t test検定を用いて検定し、5%以下を有意とした(*:p<0.05 vs ケルセチン0μM)。定量した遺伝子は、表1に記載のCsgalnact1、Csgalnact2、Xylt2、B3galt1、Chsy1、Chpf2、Ext1及びExt2である。
図5A~図5D及び図6A~図6D中、aはC6細胞、bはSKOV3細胞、cはC1300細胞、dはKINGS1細胞、eはNM-CG1細胞、fはMC3T3-E1SK細胞をそれぞれ示す。図5A~図5D及び図6A~図6Dにおいて、遺伝子の発現量は、相対mRNAレベルで示した。相対mRNAレベルは、コントロールとしてDMSOを添加した細胞(ケルセチン濃度0μM)における各遺伝子のmRNA量を1としたときの、ケルセチンを添加した細胞(ケルセチン濃度0.5μM、1μM又は5μM)におけるmRNA量の相対値(N=8の平均±標準偏差)である。
OSCV2細胞の培地に5μMのケルセチンを添加し、培養した。添加4日後の培養細胞のコンドロイチン硫酸量を抗コンドロイチン硫酸抗体(CS-56)によるCell ELISAシステムで定量した。コントロールの細胞は、DMSOを添加する以外は同じ方法で培養を行い、コンドロイチン硫酸を定量した。各群の平均値の差はStudent’s t test検定を用いて検定し、5%以下を有意とした(*:p<0.05 vs コントロール)。
Claims (9)
- ケルセチン又はその配糖体を有効成分として含有するコンドロイチン硫酸合成促進用組成物。
- コンドロイチン硫酸の合成に関与する糖転移酵素の発現を促進する請求項1に記載のコンドロイチン硫酸合成促進用組成物。
- コンドロイチン硫酸の合成に関与する糖転移酵素が、コンドロイチン硫酸N-アセチルガラクトサミニルトランスフェラーゼ1、コンドロイチン硫酸N-アセチルガラクトサミニルトランスフェラーゼ2、キシローストランスフェラーゼ2、β-1,3-ガラクトシルトランスフェラーゼ1、コンドロイチン硫酸合成酵素1及びコンドロイチン重合因子2からなる群より選択される1以上である請求項2に記載のコンドロイチン硫酸合成促進用組成物。
- コンドロイチン硫酸の合成に関与する糖転移酵素が、コンドロイチン硫酸N-アセチルガラクトサミニルトランスフェラーゼ1及び/又はコンドロイチン硫酸N-アセチルガラクトサミニルトランスフェラーゼ2である請求項2又は3に記載のコンドロイチン硫酸合成促進用組成物。
- 飲食品、化粧料又は医薬部外品である請求項1~4のいずれか一項に記載のコンドロイチン硫酸合成促進用組成物。
- ケルセチン又はその配糖体を投与する、コンドロイチン硫酸の合成を促進する方法。
- コンドロイチン硫酸の合成に関与する糖転移酵素の発現を促進することによりコンドロイチン硫酸の合成を促進する請求項6に記載の方法。
- コンドロイチン硫酸の合成を促進するための、ケルセチン又はその配糖体の使用。
- コンドロイチン硫酸の合成に関与する糖転移酵素の発現を促進することによりコンドロイチン硫酸の合成を促進する請求項8に記載の使用。
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