WO2023074894A1

WO2023074894A1 - Cartilage regeneration promoter

Info

Publication number: WO2023074894A1
Application number: PCT/JP2022/040711
Authority: WO
Inventors: 善之水品; 公輔田中; 淳也戸口田; 啓之吉富; 永輝金
Original assignee: 小林製薬株式会社; 国立大学法人京都大学
Priority date: 2021-11-01
Filing date: 2022-10-31
Publication date: 2023-05-04
Also published as: TW202333660A

Abstract

The purpose of the present invention is to provide a novel component having a cartilage regeneration-promoting effect. This cartilage regeneration promoter containing hydroxycitric acid and/or a salt thereof is capable of promoting cartilage regeneration.

Description

cartilage regeneration promoter

The present invention relates to a cartilage regeneration promoting agent.

Joint pain due to cartilage disorders such as osteoarthritis caused by degenerative degeneration of cartilage and cartilage damage due to trauma is a major factor that limits daily activities, and in the aging society, it is an increasingly important issue to deal with. . There is no fundamental conservative treatment for cartilage disorders, and oral administration of non-steroidal anti-inflammatory drugs (NSAIDs) and intra-articular injection of steroids are used as symptomatic treatments for pain. Intraarticular injection of hyaluronic acid, one of the glycosaminoglycans (GAGs) that are important components of cartilage, is also widely used, and although it has been shown to have a certain effect on pain, its effect on cartilage regeneration is limited. uncertain.

Glucosamine is one of the amino sugars that make up GAG. Glucosamine is contained in the cell walls of crustaceans such as shrimps and crabs, insects such as beetles, and fungi, and is one of monosaccharides widely present in nature as a constituent unit of chitin. In addition, its important role in vivo has been studied, such as its existence as a constituent sugar of mucopolysaccharides in vivo. Glucosamine is not only a component of cartilage and connective tissue, but is also reported to have an anti-inflammatory effect via neutrophils, and is reported as an effective component for arthritis (Patent Document 1).

Although it has not been clarified to what extent orally ingested glucosamine can participate in the metabolism of articular cartilage, glucosamine is actively added to foods, quasi-drugs, or pharmaceuticals for the purpose of treating or preventing cartilage disorders. Attempts are being made to take advantage of For example, a composition for improving arthralgia containing collagen, methylsulfonylmethane, glucosamine, and chondroitin, wherein the composition contains collagen in an intake of 2000 mg or more per day, relieves symptoms of arthralgia such as arthritis. It has been proposed as a composition for improving arthralgia, an agent for improving arthralgia, or a food, which can alleviate, treat, and prevent joint pain (Patent Document 2).

On the other hand, it is known that the hydroxycitric acid contained in the garcinia pericarp suppresses the storage of excess carbohydrates as body fat and promotes fat consumption. In addition, it has been reported that fat metabolism can be promoted more effectively by combining a Garcinia extract with caffeine (Patent Document 3).

Japanese Patent Publication No. 2004-529860 JP 2009-051833 A JP 2001-258506 A

The effect of glucosamine in the treatment or prevention of cartilage disorders has attracted attention, and various glucosamine-containing compositions have been studied, but the level of the effect is still insufficient. Therefore, the present inventors believed that a component having an effect of promoting cartilage regeneration would be expected to be more effective in treating or preventing cartilage disorders.

An object of the present invention is to provide a cartilage regeneration-promoting agent having an excellent cartilage regeneration-promoting action.

When the present inventors searched for new ingredients that have cartilage regeneration-promoting effects through cartilage differentiation induction experiments, they discovered that hydroxycitric acid and/or its salts have excellent cartilage regeneration-promoting effects. The present invention has been completed through further studies based on such findings.

That is, the present invention provides inventions in the following aspects.
Section 1. A cartilage regeneration promoter containing hydroxycitric acid and/or a salt thereof.
Section 2. Item 2. The cartilage regeneration promoter according to Item 1, wherein the hydroxycitric acid is (-) hydroxycitric acid and/or (+) allo-hydroxycitric acid.
Item 3. Item 3. The cartilage regeneration promoter according to

Item

1 or 2, wherein the hydroxycitric acid is a non-lactone body and/or a lactone body.
Section 4. Item 4. The cartilage regeneration promoting agent according to any one of Items 1 to 3, which is used for promoting cartilage synthesis.
Item 5. Item 5. The agent for promoting cartilage regeneration according to any one of Items 1 to 4, which contains a plant extract containing hydroxycitric acid and/or a salt thereof.
Item 6. Item 6. The cartilage regeneration promoter according to Item 5, wherein the plant extract is Garcinia extract.
Item 7. Item 6. The cartilage regeneration promoter according to Item 5, wherein the plant extract is a hibiscus extract.
Item 8. Item 8. The cartilage regeneration-promoting agent according to any one of Items 1 to 7, which is used for the treatment or prevention of cartilage disorders.
Item 9. Item 9. The cartilage regeneration promoter according to any one of Items 1 to 8, wherein the dose to humans is 0.3 g to 1.5 g/day/60 kg in terms of hydroxycitric acid.
Item 10. A chondrocyte differentiation promoting agent that promotes differentiation of undifferentiated cells into chondrocytes, containing hydroxycitric acid and/or a salt thereof.
Item 11. Item 11. The chondrocyte differentiation promoting agent according to Item 10, wherein the hydroxycitric acid is (-) hydroxycitric acid and/or (+) allo-hydroxycitric acid.
Item 12. Item 12. The chondrocyte differentiation promoting agent according to Item 10 or 11, wherein the hydroxycitric acid is a non-lactone body and/or a lactone body.
Item 13. Item 13. The chondrocyte differentiation promoting agent according to any one of Items 10 to 12, which contains a plant extract containing hydroxycitric acid and/or a salt thereof.
Item 14. Item 14. The chondrocyte differentiation promoting agent according to Item 13, wherein the plant extract is Garcinia extract.
Item 15. Item 14. The chondrocyte differentiation promoting agent according to Item 13, wherein the plant extract is a hibiscus extract.
Item 16. Item 16. The chondrocyte differentiation promoting agent according to any one of Items 10 to 15, which is exposed to the cells at a concentration of 0.08 to 0.4 mM in terms of hydroxycitric acid.
Item 17. A cartilage degradation inhibitor containing hydroxycitric acid and/or a salt thereof.
Item 18. A protective agent for maintaining cartilage thickness, containing hydroxycitric acid and/or a salt thereof.
Item 19. An agent for relieving joint pain, discomfort and/or discomfort, containing hydroxycitric acid and/or a salt thereof.
Item 20. Use of hydroxycitric acid and/or a salt thereof for producing a cartilage regeneration promoting agent.
Item 21. Hydroxycitric acid and/or its salts are chondrocyte differentiation promoters that promote differentiation from undifferentiated cells to chondrocytes, cartilage degradation inhibitors, protective agents for maintaining cartilage thickness, or joint pain. , for the manufacture of an agent for relieving discomfort and/or discomfort.
Item 22. Use of a composition containing hydroxycitric acid and/or a salt thereof for promoting cartilage regeneration.
Item 23. Said use promotes chondrocyte differentiation to promote differentiation from undifferentiated cells to chondrocytes, inhibits cartilage degradation, protects to maintain cartilage thickness, or relieves joint pain, discomfort, and/or discomfort 23. Use according to paragraph 22, which is a palliative use.
Item 24. A cartilage regeneration method, comprising administering an effective amount of hydroxycitric acid and/or a salt thereof to a subject in need of cartilage regeneration.

According to the present invention, a cartilage regeneration-promoting agent having excellent cartilage regeneration-promoting action is provided.

Fig. 2 shows the results of cartilage matrix production brought about by induction of differentiation into chondrocytes by hydroxycitric acids in two-dimensional culture of human iPS cell-derived mesenchymal stem cells. Fig. 2 shows the results of chondrocyte proliferation brought about by induction of differentiation into chondrocytes by hydroxycitric acids in two-dimensional culture of human iPS cell-derived mesenchymal stem cells. Fig. 2 shows the results of cartilage matrix production and chondrocyte proliferation brought about by induction of chondrocyte differentiation with various concentrations of hydroxycitric acid in two-dimensional culture of human iPS cell-derived mesenchymal stem cells. Fig. 2 shows the results of chondrocyte differentiation induction by hydroxycitric acids in three-dimensional culture of human bone marrow-derived mesenchymal stem cells. Fig. 2 shows the results of cartilage matrix production brought about by induction of differentiation into chondrocytes by a garcinia extract in two-dimensional culture of human iPS cell-derived mesenchymal stem cells. Fig. 2 shows the results of chondrocyte proliferation brought about by induction of differentiation into chondrocytes by a garcinia extract in two-dimensional culture of human iPS cell-derived mesenchymal stem cells. Fig. 2 shows the results of chondrocyte differentiation induction by Garcinia extract in three-dimensional culture of human bone marrow-derived mesenchymal stem cells. Fig. 2 shows the results of chondrocyte differentiation induction by hydroxycitric acids, garcinia extract and hibiscus extract in three-dimensional culture of human bone marrow-derived mesenchymal stem cells. Fig. 2 shows the results of cartilage matrix production brought about by induction of differentiation into chondrocytes by hydroxycitric acids and garcinia extract and hibiscus extract in three-dimensional culture of human bone marrow-derived mesenchymal stem cells. Fig. 3 shows the results of chondrocyte proliferation induced by induction of differentiation into chondrocytes by hydroxycitric acids and garcinia extract and hibiscus extract in three-dimensional culture of human bone marrow-derived mesenchymal stem cells. Fig. 2 shows changes in body weight of cartilage-damaged rats to which Garcinia extract was administered. Fig. 2 shows photographs of the cartilage surface of the trochlea of the knee femur on the day of rat cartilage injury surgery and after administration of garcinia extract (16 days after surgery). Fig. 2 shows a stained image (a) of a cross-sectional specimen crossing a drilled hole in knee cartilage of a cartilage-damaged rat to which a garcinia extract was administered, and a measurement result (b) of the depth of the drilled hole. Fig. 3 shows the measurement results of cartilage synthesis marker (CPII) and cartilage degradation marker (C2C) levels in the blood of cartilage-damaged rats to which Garcinia extract was administered. Fig. 2 shows a stained image of a cross-sectional sample of cartilage of knee femur prepared from a rat knee osteoarthritis-induced model to which a garcinia extract was administered. Fig. 2 shows a stained image of a cross-sectional sample of knee femoral cartilage prepared from a mouse knee joint cartilage degeneration model to which a garcinia extract was administered. Fig. 2 shows the cartilage degeneration score of a mouse knee joint cartilage degeneration model to which a garcinia extract was administered. Fig. 3 shows measurement results of cartilage synthesis marker (CPII) amount and cartilage degradation marker (C2C) amount in blood of mouse knee joint cartilage degeneration model to which garcinia extract was administered. 1 shows the relationship between the cartilage degeneration score and blood cartilage synthesis marker (CPII) amount in a mouse knee joint cartilage degeneration model to which a garcinia extract was administered, and the human-equivalent dose of hydroxycitric acid.

1. Cartilage regeneration-promoting agent The cartilage regeneration-promoting agent of the present invention contains hydroxycitric acid, derivatives thereof, and/or salts thereof (hereinafter also referred to as "hydroxycitric acids"). Hydroxycitric acids are known as ingredients that promote fat metabolism. In the agent for promoting cartilage regeneration of the present invention, hydroxycitric acids act as active ingredients for promoting cartilage regeneration.

Hydroxycitric Acids Hydroxycitric acid is an α-hydroxy tribasic acid (1,2-dihydroxypropane-1,2,3-tricarboxylic acid) with two asymmetric centers and can be formed into two pairs of diastereoisomers or four forms two different isomers. Specifically, hydroxycitric acid includes (-) hydroxycitric acid, (+) hydroxycitric acid, (-) allo-hydroxycitric acid, and (+) allo-hydroxycitric acid. These isomers may be used singly or in combination of two or more. Among these isomers, (-) hydroxycitric acid and (+) allo-hydroxycitric acid are preferred from the viewpoint of obtaining a more favorable effect of promoting cartilage regeneration.

　Hydroxycitric acid and its salts are specifically represented by the following general formulas (I) and (II).

In the present invention, among hydroxycitric acid and salts thereof, the compound represented by the above formula (I) is also described as "non-lactone form" or "free form", and the compound represented by formula (II) (that is, A dehydrated condensed cyclized product of a free form) is also referred to as a "lactone form".

When the compound represented by the above formulas (I) and (II) is hydroxycitric acid, all of M ¹ , M ² and M ³ in the above formulas (I) and (II) are hydrogen atoms.

When the compounds represented by formulas (I) and (II) are salts of hydroxycitric acid, M ¹ , M ² and M ³ in formula (I) and M ¹ and M ² in formula (II) are simultaneously It never becomes a hydrogen atom. Also, the salt of hydroxycitric acid is not particularly limited as long as it is pharmaceutically or cosmetically acceptable.

Specifically, when the compounds represented by the formulas (I) and (II) are salts of hydroxycitric acid, M ¹ , M ² and M ³ in the formula (I) and M in the formula (II) ¹ and ^M2 each independently represent an alkali metal or alkaline earth metal, or an organic base. Examples of alkali metals include potassium and sodium. Alkaline earth metals include calcium. Organic bases include monoethanolamine, diethanolamine, triethanolamine, aminomethylpropanol, and aminomethylpropanediol groups. These salts may be used singly or in combination of two or more.

In the agent for promoting cartilage regeneration of the present invention, either one of a free form and a lactone form may be used as hydroxycitric acid and its salt, or both may be used in combination. and lactone bodies, one may be selected and used, or two or more may be used in combination. Among these free forms and lactone forms of hydroxycitric acid and salts thereof, lactone forms are preferred from the viewpoint of obtaining a more excellent effect of promoting cartilage regeneration.

　Hydroxycitric acid derivatives are derived from hydroxycitric acid as a base, with introduction of functional groups, oxidation, reduction, and atom replacement. More specific examples of hydroxycitric acid derivatives include hydroxycitric acid-2-octanoate, hydroxycitric acid-2-caprate, hydroxycitric acid-2-laurate, hydroxycitric acid-2-myristate, hydroxycitric acid- 2-palmitate, hydroxycitrate-2-stearate, hydroxycitrate-2-behenoate, hydroxycitrate-2-isopalmitate, hydroxycitrate-2-isostearate, hydroxycitrate-2-hexyldecanoate, hydroxycitric acid-2-linolate, hydroxycitric acid monomethyl ester-2-myristate, hydroxycitric acid monomethyl ester-2-palmitate, hydroxycitric acid monomethyl ester-2-stearate and the like.

Salts of hydroxycitric acid derivatives include alkali metal or alkaline earth metal salts or organic base salts of the above hydroxycitric acid derivatives.

In addition, in the cartilage regeneration promoting agent of the present invention, as hydroxycitric acids, one of the four types of hydroxycitric acid, hydroxycitric acid salts, hydroxycitric acid derivatives, and hydroxycitric acid derivative salts may be used. Alternatively, two or more may be used in combination, and from among the compounds included in each of the above hydroxycitric acid, hydroxycitric acid salts, hydroxycitric acid derivatives, and hydroxycitric acid derivative salts, one may be selected and used, or two or more compounds may be used in combination. Among these hydroxycitric acids, hydroxycitric acid salts are preferable, and alkali metal salts and alkaline earth metal salts of hydroxycitric acid are more preferable, from the viewpoint of obtaining a more excellent effect of promoting cartilage regeneration. Alkaline earth metal salts of hydroxycitric acid are more preferred, and calcium salts of hydroxycitric acid are particularly preferred.

In the agent for promoting cartilage regeneration of the present invention, hydroxycitric acids may be obtained from natural products or chemically synthesized. Natural products include Garcinia species belonging to the family Hypericum genus Fukugi (specific examples include Garcinia cambogia, Garcinia indica, Garcinia atroviridis, Garcinia mangostana ), Garcinia subelliptica, etc.), and plants such as Hibiscus (Hibiscus L.) of the family Malvaceae, preferably the genus Garcinia, more preferably Garcinia cambogia (Garcinia cambogia). cambogia). Plants may be those produced by cultivation or those collected from nature. The part of the plant to be used is not limited as long as it contains hydroxycitric acids, but the pericarp is preferred. Methods for obtaining hydroxycitric acids from plants are conventionally known. The cartilage regeneration-promoting agent of the present invention may contain isolated and purified hydroxycitric acids from natural products, or may contain crudely purified hydroxycitric acids from natural products.

Examples of crudely purified products of hydroxycitric acids include processed products of plants, preferably processed products of pericarp. Specific examples of processed plant products include dried plant products, pulverized plant products (including fresh and dried products), and plant extracts. As the processed plant product, one derived from a single plant may be used, or two or more different plant-derived products may be used in combination. Among these processed products of plants, plant extracts are preferred. Specific examples of plant extracts include non-concentrated extracts (not concentrated), soft extracts (liquid concentrates) and powdered extracts (dry extracts).

Among the crudely purified products of the above-mentioned plant processed products, from the viewpoint of obtaining a more excellent cartilage regeneration promoting effect, preferred are plant extracts, and more preferred are extracts obtained from the genus Garcinia (Garcinia extract) (especially Preferred are extracts obtained from Garcinia cambogia) and hibiscus extracts obtained from Malvaceae. The plant extract may be a squeezed juice, a solvent extract, or a fraction containing hydroxycitric acids from the solvent extract. Although the method for obtaining the plant extract is not particularly limited, it can be obtained, for example, as follows. The plant extract is prepared, for example, in the raw state of the pericarp of a plant such as Garcinia species or the petals of hibiscus, or in the state of a dried product such as the pericarp of a plant such as Garcinia species or the petals of hibiscus. It can be prepared according to a conventional extraction method such as solvent extraction, supercritical extraction, etc., after being cut or pulverized in size or further as required. Examples of extraction solvents include water (including hot water and hot water), organic solvents (lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, n-propanol, isopropanol, n-butanol; propylene glycol, 1,3-butylene, polyhydric alcohols such as glycol; ketones such as acetone; esters such as diethyl ether, dioxane, acetonitrile, ethyl acetate; xylene, benzene, chloroform, etc.), mixtures thereof, preferably water, lower alcohol and mixtures thereof, more preferably heated water such as warm water or hot water, and still more preferably hot water. These solvents may be used singly or in combination of two or more.

Furthermore, as a method of obtaining a plant extract containing hydroxycitrate using a raw material plant containing hydroxycitric acid, a plant extract containing alkali metal salt and/or alkaline earth metal salt (for example, calcium hydroxycitrate) is used. When obtaining, calcium carbonate, calcium lactate, calcium salts such as eggshell calcium) are obtained), and after obtaining a plant extract containing hydroxycitric acid, the plant extract is added to alkali metals and / Or a method of converting hydroxycitric acid in the extract to a salt by treating with a basic compound of an alkaline earth metal.

When the obtained plant extract (squeezed juice, solvent extract, fraction containing hydroxycitric acids of solvent extract, etc.) is included in the cartilage regeneration promoting agent, the plant extract is used as a non-concentrated extract that is not concentrated as it is. It may be in the form of a concentrated liquid soft extract, or it may be an extract powder obtained by further drying a non-concentrated extract or soft extract. Drying processes include spray-drying and freeze-drying.

The amount of hydroxycitric acids in 100% by weight of the dry weight of the crudely purified hydroxycitric acids (preferably processed plant products, more preferably plant extracts) is, for example, 10% by weight or more, preferably 30% by weight or more, more preferably 50% by weight or more, and still more preferably 55% by weight or more. Although the upper limit of the amount of hydroxycitric acids in 100% by weight in terms of dry weight is not particularly limited, examples thereof include 80% by weight or less or 70% by weight or less.

The method for obtaining an isolated and purified product of hydroxycitric acids is not particularly limited, but includes a method of further purifying the fraction containing hydroxycitric acids from the above extract. The purification treatment can be carried out according to a conventional method, as long as it is a method of isolating hydroxycitric acids to further increase the degree of purification. Examples thereof include separation treatment such as chromatography, recrystallization treatment, and the like.

The amount of hydroxycitric acid compounded in the agent for promoting cartilage regeneration of the present invention is not particularly limited, and is appropriately set according to the formulation form and application of the agent for promoting cartilage regeneration. is 60 to 90% by weight.

The hydroxycitric acids contained in the agent for promoting cartilage regeneration of the present invention have the effect of promoting cartilage synthesis, which has not been observed with glucosamine. In addition, the hydroxycitric acids contained in the cartilage regeneration-promoting agent of the present invention are excellent in suppressing the degradation of cartilage. Therefore, the agent for promoting cartilage regeneration of the present invention can regenerate damaged (defective or degenerated) cartilage by both promoting cartilage synthesis and inhibiting cartilage differentiation.

Other Ingredients The cartilage regeneration promoting agent of the present invention may or may not contain other ingredients in addition to hydroxycitric acids, as long as the effects of the present invention are not impaired, depending on the form of application. good too. Such other components include, for example, physiologically active substances and additives.

The physiologically active substance preferably includes components effective for improving cartilage and joints. Specific examples include collagen, type II collagen, non-denaturing active type 2 collagen, collagen peptide, methylsulfonylmethane (MSM ), glucosamine, glucosamine hydrochloride, N-acetylglucosamine, N-acetylglucosamine sulfate, chondroitin, chondroitin sulfate, galactosamine, N-acetylgalactosamine, glucuronic acid, uronic acid, proteoglycan, non-denatured proteoglycan, heparin, heparan sulfate, iduron acid, keratan sulfate, dermatan sulfate, S-adenosylmethionine, creatine, theanine, piperine, maslinic acid, 5-aminolevulinic acid phosphate, cat's claw, black ginger, boswellia serrata, artichoke, amino acids, vitamin A, Vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D2, vitamin D3, vitamin E, vitamin K, folic acid, hyaluronic acid, bonito-derived elastin peptide, imidazole dipeptide, quercetin glycoside, krill oil-derived EPA・DHA, Moringa seed-derived glucomoringin, Japanese knotweed, devil's claw, chicken leg-derived hyaluronic acid production promoter (HAS-II), soy isoflavone, β-cryptoxanthin, bonepep, concentrated whey active protein (CBP), 3-hydroxy -3-methylbutyrate (HMB), calcium bis-3-hydroxy-3-methylbutyrate monohydrate (HMB calcium), calcium maltobionate, calcium, magnesium, zinc, iron, selenium, potassium, estrogen, calcitonin, Aspirin, steroidal anti-inflammatory agents, non-steroidal anti-inflammatory agents and the like. These physiologically active substances may be used singly or in combination of two or more.

Additives include pharmaceutically or food acceptable excipients, disintegrants, diluents, lubricants, flavoring agents, coloring agents, sweetening agents, corrigents, suspending agents, wetting agents, and emulsifying agents. , dispersants, adjuvants, preservatives, buffers, binders, stabilizers, extenders, thickeners, pH adjusters, surfactants, coating agents, nutritional components, and the like. These additives may be used singly or in combination of two or more.

Form of Formulation The form and properties of the cartilage regeneration-promoting agent of the present invention are not particularly limited as long as it contains the hydroxycitric acids described above.

The dosage form of the agent for promoting cartilage regeneration of the present invention includes both oral and parenteral dosage forms. Therefore, the agent for promoting cartilage regeneration of the present invention is prepared as an oral preparation, an injection, a drip, a nasal drop, a percutaneous absorbable preparation (external preparation), and the like. Since the cartilage regeneration-promoting agent of the present invention is used for the purpose of cartilage regeneration, it is preferably an oral preparation that can be easily administered (taken) on a daily and/or continuous basis.

Further, the properties of the cartilage regeneration promoting agent of the present invention may be liquid or solid. Examples of liquids include liquids, beverages, emulsions, suspensions, spirits, syrups, elixirs, soft extracts, etc.), and examples of solids include tablets, pills, Powders, fine granules, granules, tablets, capsules (including hard capsules and soft capsules), lozenges, chewables, dry extracts and the like. When the agent for promoting cartilage regeneration of the present invention is in a solid form, it may be in a sustained or sustained release dosage form, or may be mixed with water or the like at the time of administration (ingestion).

The agent for promoting cartilage regeneration of the present invention is used as general food and drink, food with health claims (including food for specified health use, food with nutrient function claims, food with functional claims, supplements, etc.), food for the sick, pharmaceuticals, and quasi-drugs. It can also be used as an additive to foods, pharmaceuticals, quasi-drugs, and the like. In particular, from the viewpoint of casual ingestion on a daily and/or continuous basis, it can be used as a supplement containing hydroxycitric acids, or a supplement containing a plant extract containing hydroxycitric acids, preferably a supplement containing garcinia extract. preferable.

Manufacturing Method The method for manufacturing the cartilage regeneration-promoting agent of the present invention uses the above-mentioned hydroxycitric acids and other ingredients that are blended as necessary, according to various forms and properties, and the purpose of use. Normal formulation procedures may be followed.

Use The agent for promoting cartilage regeneration of the present invention promotes cartilage regeneration by administration (ingestion). Regeneration of cartilage refers to the process of synthesizing cartilage, i.e., differentiating undifferentiated cells into chondrocytes, proliferating them, and synthesizing the cartilage matrix, thereby replacing defective or degenerated cartilage tissue with normal cartilage tissue (hyraline tissue). Cartilage) to reconstruct. The promotion of cartilage regeneration refers to promoting the regeneration of cartilage, suppressing further degradation of cartilage tissue, and promoting reconstruction of cartilage tissue.

Therefore, the cartilage regeneration-promoting agent of the present invention suppresses and/or regenerates the wear and tear of cartilage, enhances the ability to form cartilage, and cartilage components (specifically, cartilage matrix) for subjects requiring cartilage regeneration. ) and/or can be used for the purpose of maintaining the state of healthy cartilage.

More specific examples of the above object of the cartilage regeneration promoting agent of the present invention are when walking (particularly when walking long distances in a certain period of time), when going up and down stairs, and when putting on and taking off socks. knee flexion and extension (i.e. smoothness of knee motion), flexibility, mobility, and and/or to help (i.e., support) in relieving annoyance.

Targets requiring cartilage regeneration are preferably mammals, more specifically, humans; pets such as dogs and cats; and domesticated animals such as horses and cows.

Hydroxycitric acids, which are the active ingredients of the agent for promoting cartilage regeneration of the present invention, have not only the effect of promoting cartilage synthesis but also the effect of suppressing cartilage degradation. One aspect of the preferred application of is application for the purpose of treatment of cartilage damage to a subject having cartilage damage (an example of a subject requiring cartilage regeneration ability). Treatment of cartilage disorders includes not only cure but also reduction of cartilage disorders. Cartilage disorders are not particularly limited as long as they are pathological conditions caused by loss or degeneration of cartilage, and include osteoarthritis, traumatic cartilage injury, and arthritis due to various causes. The target cartilage site is not particularly limited, but includes various joint sites in the body, such as knee joints, hip joints, elbow joints, shoulder joints, wrist joints, ankle joints, and temporomandibular joints.

In addition, hydroxycitric acids, which are active ingredients of the agent for promoting cartilage regeneration of the present invention, have excellent cartilage synthesis-promoting action and cartilage degradation-inhibiting action. One aspect of preferred application of the regeneration promoter includes application for the purpose of preventing cartilage damage to subjects at risk of cartilage damage (examples of subjects requiring cartilage regeneration ability). Subjects at risk of cartilage damage include elderly people at risk of osteoarthritis, which is often seen in humans over the age of 50, especially over the age of 65, and sports enthusiasts at risk of cartilage damage, which is seen in sports injuries. Home or professional athletes, other people who overuse their joints on a daily basis, or those who experience the natural wear and tear of cartilage with age, preferably with the natural wear and tear of cartilage with age. There are people who are When the cartilage regeneration-promoting agent of the present invention is applied to a person whose cartilage is naturally worn down with age, the cartilage regeneration-promoting agent of the present invention exerts an effect of suppressing degradation of cartilage tissue and promoting reconstruction. Preferably, it can be used for the purpose of maintaining the thickness of the knee cartilage that has become thinner with age, and alleviating knee pain, discomfort, and/or discomfort in daily life.

Hydroxycitric acids, which are the active ingredients of the agent for promoting cartilage regeneration of the present invention, also have the effect of differentiating undifferentiated cells into chondrocytes. In view of such effects of the present invention, one aspect of the preferred application of the agent for promoting cartilage regeneration of the present invention is a subject having severe cartilage damage in which a defect in the cartilage itself (that is, cartilage deficiency) is observed (cartilage For the purpose of treatment to regenerate the cartilage itself for a subject that requires regenerative power. Treatment of severe cartilage disorders includes not only cure but also reduction of cartilage disorders. Specific examples of severe cartilage disorders include traumatic cartilage defects caused by sports or traffic accidents.

Dose The dose of the agent for promoting cartilage regeneration of the present invention is, for example, 0.1 g/day/60 kg or more, preferably 0.2 g/day/60 kg or more, more preferably 0.2 g/day/60 kg or more, in terms of hydroxycitric acid equivalent, as a dose to humans. 0.3 g/day/60 kg or more, more preferably 0.4 g/day/60 kg or more, and still more preferably 0.5 g/day/60 kg or more. Although the upper limit of the dose is not particularly limited, for example, 18 g/day/60 kg or less, preferably 10 g/day/60 kg or less, more preferably 2 g/day/60 kg or less, further preferably 1.5 g/day/60 kg or less, More preferably 1.2 g/day/60 kg or less, still more preferably 0.9 g/day/60 kg or less, and particularly preferably 0.7 g/day/60 kg or less. In the case of a cartilage regeneration-promoting agent containing a garcinia extract, the dosage for humans is 0.16 g/day/60 kg or more, preferably 0.3 g/day/60 kg or more, preferably 0.3 g/day/60 kg or more. It is preferably 0.5 g/day/60 kg or more, more preferably 0.6 g/day/60 kg or more, and still more preferably 0.8 g/day/60 kg or more. The upper limit of the dose is not particularly limited, but is, for example, 30 g/day/60 kg or less, preferably 17 g/day/60 kg or less, more preferably 3.3 g/day/60 kg or less, still more preferably 2.5 g/day/60 kg. Below, more preferably 2 g/day/60 kg or less, still more preferably 1.5 g/day/60 kg or less, and particularly preferably 1.2 g/day/60 kg or less.

The method of administration (ingestion) of the agent for promoting cartilage regeneration of the present invention is not particularly limited. ~3 doses can be given orally.

2. chondrocyte differentiation promoting agent, cartilage degradation inhibitor, protective agent for maintaining cartilage thickness, and joint pain, discomfort, and/or discomfort alleviating agent As described above, hydroxycitric acid, its derivatives, and salts thereof have an action of promoting the differentiation of undifferentiated cells into chondrocytes, an action of promoting cartilage synthesis, and an action of inhibiting cartilage degradation, so they are chondrocyte differentiation promoters, cartilage degradation inhibitors, and maintain cartilage thickness. It is also useful as an active ingredient in a protective agent for joint pain, discomfort, and/or discomfort. Therefore, the present invention provides chondrocyte differentiation promoting agents, cartilage degradation inhibitors, protective agents for maintaining cartilage thickness, and joints containing hydroxycitric acid, derivatives thereof, and/or salts thereof. Also provided are pain, discomfort, and/or discomfort relievers.

Hydroxycitric acid as an active ingredient in chondrocyte differentiation promoters, cartilage degradation inhibitors, protective agents for maintaining cartilage thickness, and joint pain, discomfort, and/or discomfort relief agents, Details of derivatives and/or salts thereof, details of other components whether or not they are contained, formulation forms, manufacturing methods, uses, and dosages are the same as those described in "1. Cartilage regeneration promoter" above.

Furthermore, chondrocyte differentiation promoting agents can also be used in vitro for the purpose of promoting chondrocyte differentiation by direct exposure to undifferentiated cells. In this case, the concentration of the chondrocyte differentiation promoting agent when exposed to undifferentiated cells is not particularly limited. 0.3 mM, more preferably 0.1-0.32 mM, still more preferably 0.15-0.25 mM, even more preferably 0.18-0.22 mM.

The present invention will be more specifically described below by showing Examples, but the present invention is not limited to these.

Test Example 1: Chondrocyte differentiation-inducing activity, cartilage matrix production activity, and chondrocyte proliferation activity of hydroxycitric acids (in vitro)
1. Primary screening method/cultured cells PLoS ONE, (US), 2014, 9(12), e112291. Mesenchymal stem cells were induced via neural crest cells from the cells, and an equal amount of cell cryoprotectant CP-1 (manufactured by Kyokuto Pharmaceutical Co., Ltd.) was added and cryopreserved.

• Two-dimensional culture of chondrocyte differentiation and cartilage matrix production Human iPS cell-derived mesenchymal stem cells that had been thawed and proliferated were seeded at 1 x 10 ⁵ cells/well in a 96-well plate (manufactured by Corning). After 24 hours, the culture supernatant was removed and replaced with a chondrocyte differentiation induction medium according to Document A. In a chondrocyte differentiation induction medium, (-) hydroxycitric acid (HCA, free form) (manufactured by ChromaDex, Inc.); K) (manufactured by Sigma-Aldrich) or (-) hydroxycitrate-3 calcium (HCA-Ca) (manufactured by Sigma-Aldrich); - lactone or garcinia acid) (Sigma-Aldrich) was added to a final concentration of 60 μg/mL. To the positive control group, 10 ng/mL of BMP-7 (manufactured by R&D Systems), an osteogenic factor, was added. Neither the test substance nor the osteogenic factor was added to the control group. After culturing for about 10 days, the cells were fixed with 4% paraformaldehyde/phosphate buffer (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). After fixation, staining was performed for 1 hour with an alcian blue solution (manufactured by Muto Kagaku), and the dye was eluted using a 6M guanidine hydrochloride solution (manufactured by Tokyo Kasei Kogyo). The eluate was transferred to a 96-well plate, and the OD value at 620 nm of each well was measured using a microplate reader to measure the amount of matrix produced by the differentiation-induced chondrocytes. Specifically, when the average OD value in the control group was set to 1, the average OD value in each group was derived as the cartilage matrix production ratio.

Fig. 1A shows the results of cartilage matrix production brought about by induction of differentiation into chondrocytes in two-dimensional culture of human iPS cell-derived mesenchymal stem cells. As shown in FIG. 1A, 200 μM (−) hydroxycitric acid (HCA-free form) improved the cartilage matrix production ratio (1.25 times or more as compared to the control) compared to the control without the material. This also indicates that prior to cartilage matrix regeneration, 200 μM (−) hydroxycitric acid (HCA, free body) induced differentiation of human iPS cell-derived mesenchymal stem cells into chondrocytes that produce cartilage matrix. ing. In addition, the potassium salt and calcium salt of 200 μM (-) hydroxycitric acid improved the cartilage matrix production ratio by 1.05 and 1.08 times, respectively, as compared with the free form. This also indicates that the hydroxycitric acid salt has higher chondrocyte differentiation-inducing activity and cartilage matrix-producing activity than the free body (HCA). Similarly, the lactone body (HCA-lactone) also showed a higher cartilage matrix production ratio than the free body, indicating higher chondrocyte differentiation-inducing activity and cartilage matrix production activity.

Also when hydroxycitric acid-2-palmitate was used in place of hydroxycitric acid and its salts, chondrocyte differentiation-inducing activity and cartilage matrix production activity were observed.

• Chondrocyte Differentiation and Number of Chondrocytes in Two-Dimensional Culture Human iPS cell-derived mesenchymal stem cells that had been thawed and proliferated were seeded in 96-well plates (manufactured by Corning) at 1×10 ⁵ cells/well. After 24 hours, the culture supernatant was removed and replaced with a chondrocyte differentiation induction medium according to Document A. In a chondrocyte differentiation induction medium, (-) hydroxycitric acid (HCA, free form) (manufactured by ChromaDex, Inc.); K) (manufactured by Sigma-Aldrich) or (-) hydroxycitrate-3 calcium (HCA-Ca) (manufactured by Sigma-Aldrich); - lactone or garcinia acid) (Sigma-Aldrich) was added to a final concentration of 60 μg/mL. 10 ng/mL of BMP-7 (manufactured by R&D Systems), which is an osteogenic factor, was added to the positive control group. Neither the test substance nor the osteogenic factor was added to the control group. After culturing for about 10 days, chondrocyte differentiation induction medium prepared in 10% Cell Counting Kit-8 (manufactured by Dojin Kagaku Kenkyusho) was added and cultured for 1 hour. After that, the OD value at 450 nm of each well of this 96-well plate was measured with a microplate reader to measure the number of proliferation-induced chondrocytes. Specifically, when the average OD value in the control group was set to 1, the average OD value in each group was derived as the chondrocyte proliferation ratio.

Fig. 1B shows the results of chondrocyte proliferation brought about by induction of differentiation into chondrocytes in two-dimensional culture of human iPS cell-derived mesenchymal stem cells. In FIG. 1B, the same tendency as in FIG. 1A was observed. Specifically, 200 μM (-) hydroxycitric acid (HCA-free form) improved the chondrocyte proliferation rate compared to no material (control). This also indicates that 200 μM (−) hydroxycitric acid (HCA, free body) induced the differentiation of human iPS cell-derived mesenchymal stem cells into chondrocytes prior to chondrocyte proliferation. In addition, the potassium salt and calcium salt of 200 μM (-) hydroxycitric acid were found to have an effect of improving the chondrocyte proliferation ratio superior to that of the free form, and among them, the calcium salt showed an even more excellent effect of improving the chondrocyte proliferation ratio. was accepted. This also indicates that the hydroxycitric acid salt has higher chondrocyte differentiation-inducing activity and chondrocyte proliferation activity than the free form (HCA). Similarly, the lactone body (HCA-lactone) also showed higher chondrocyte proliferation activity than the free body, indicating that the chondrocyte proliferation activity was higher.

Chondrocyte proliferation activity was also observed when hydroxycitric acid-2-palmitate was used in place of hydroxycitric acid and its salts.

Concentration dependence of chondrocyte differentiation-inducing activity of hydroxycitric acid, etc. The production of cartilage matrix and the number of chondrocytes by two-dimensional culture of chondrocyte differentiation were performed in the same manner as described above. The results are shown in Figure 1C.

As shown in FIG. 1C, excellent cartilage matrix production ratio and chondrocyte proliferation ratio were observed at 0.1 to 0.4 mM, preferably 0.1 to 0.3 mM, particularly preferably 0.2 mM. . That is, the concentration of hydroxycitric acid at which chondrocyte differentiation-inducing activity, cartilage matrix-producing activity, and chondrocyte-proliferating activity is expressed is 0.1 to 0.4 mM, preferably 0.1 to 0.3 mM, and particularly preferably. was shown to be 0.2 mM (53 μg/ml).

2. Secondary screening method (three-dimensional culture)
・Cultured cells Tissue Engineering, (US), 2010, 16(1), p81-91. According to DOI:10.1089=ten.tec.2008.0693, mesenchymal stem cells were isolated from human normal bone marrow in a special medium, Cells were grown. Proliferated cells were collected, added with an equal amount of cell cryoprotectant CP-1 (Kyokuto Pharmaceutical Co., Ltd.), and cryopreserved.

• Induction of chondrocyte differentiation by three-dimensional culture It is known that dedifferentiation occurs when chondrocytes are cultured two-dimensionally. As a culture system for chondrocytes that is closer to nature, a technique called three-dimensional culture has been adopted. Human bone marrow-derived mesenchymal stem cells that had been thawed and proliferated were dispensed into 15 mL tubes (manufactured by TPP) at 2.5×10 ⁵ cells and centrifuged at 1000 rpm for 3 minutes at room temperature. The supernatant was removed and replaced with 0.5 mL of chondrocyte differentiation induction medium according to Reference A. At the same time, (-) hydroxycitric acid (HCA-free form) and its salts (HCA-K and HCA-Ca) were added as test substances at a concentration of 100 µM (0.1 mM). After culturing for 21 days, the cells were fixed with 4% paraformaldehyde/phosphate buffer for 12 hours, dehydrated with 70% ethanol, and embedded in paraffin to prepare specimens. The prepared specimen was subjected to proteoglycan staining with safranin O, and then the morphology of differentiated chondrocytes was observed using an optical microscope (10x magnification). Furthermore, using the image analysis software ImageJ, the relative area (red area ratio) was also calculated with respect to the area that was colored red by proteoglycan staining with safranin O, with the area in the control being set to 1.

Fig. 2 shows the morphology and red area ratio of differentiated chondrocytes obtained by three-dimensional culture of human bone marrow-derived mesenchymal stem cells. As shown in FIG. 2, 100 μM (-) hydroxycitric acid (HCA free form) and its salts (HCA K and HCA Ca), and 100 μM HCA- which is the lactone form of (-) hydroxycitric acid With lactone, proteoglycan staining with safranin O was observed, and differentiated chondrocytes were observed, as compared with no material (control).

From the above results, it was shown that hydroxycitric acid and its salts have excellent chondrocyte differentiation-inducing activity. In addition, hydroxycitric acid derivatives were also found to have chondrocyte differentiation-inducing activity.

Test Example 2: Chondrocyte differentiation-inducing activity, cartilage matrix production activity, and chondrocyte proliferation activity of Garcinia extract (in vitro)
(Preparation of Garcinia extract)
The pericarp of Garcinia cambogia was dried and extracted with hot water by adding 10 times the amount of water and eggshell calcium. After filtering the resulting extract, it was concentrated under reduced pressure using a rotary evaporator, and the obtained concentrate was further spray-dried. The dried extract was pulverized to obtain powdered Garcinia extract. This garcinia extract (garcinia cambogia extract) was used in all test examples below. In this Garcinia extract, the hydroxycitric acid ((-) hydroxycitric acid) contained in the pericarp of Garcinia cambogia is contained in the form of a calcium salt due to the use of eggshell calcium at the time of extraction. . The content of active ingredients in this garcinia extract is 60% by weight in terms of hydroxycitric acid.

(in vitro test)
1. Primary screening method The test substance was changed to garcinia extract (final concentration of 60 μg / mL in terms of dry weight of extract. In the following, the amount of garcinia extract represents the amount of garcinia extract in terms of dry weight.) In the same manner as in Example 1, the effect of chondrocyte differentiation-induced cartilage matrix production and chondrocyte proliferation was tested.

• Chondrocyte Differentiation and Cartilage Matrix Production in Two-Dimensional Culture FIG. 3A shows the results of cartilage matrix production brought about by induction of differentiation into chondrocytes in two-dimensional culture of human iPS cell-derived mesenchymal stem cells. The positive controls, 10 ng/mL BMP7 and 60 μg/mL Garcinia extract, improved the cartilage matrix production ratio (specifically, 1.25-fold or more than the control) compared to the no material (control). This also indicates that prior to cartilage matrix production, 10 ng/mL BMP7 and 60 μg/mL Garcinia extract induced differentiation of human iPS cell-derived mesenchymal stem cells into chondrocytes that produce cartilage matrix.

In FIG. 3A, calcium hydroxycitrate (HCA-Ca) was used as a test substance at the same concentration (specifically, 60 μg/mL), and cartilage matrix production accompanying chondrocyte differentiation induction was measured in the same manner as described above. The results obtained are also shown. Garcinia extract and hydroxycitric acid at the same concentration were found to have a lower cartilage matrix production ratio than HCA/Ca, although Garcinia extract had a lower amount of hydroxycitric acid (p < 0 .01) was improving. In other words, it was shown that Garcinia extract enhances the activity of inducing differentiation into chondrocytes and the activity of producing cartilage matrix more than HCA·Ca.

• Chondrocyte Differentiation and Number of Chondrocytes in Two-Dimensional Culture FIG. 3B shows the results of chondrocyte proliferation brought about by induction of differentiation into chondrocytes by Garcinia extract in two-dimensional culture of human iPS cell-derived mesenchymal stem cells. In FIG. 3B, the same trend as in FIG. 3A was observed. Specifically, the positive control 10 ng / mL BMP7 and 60 μg / mL Garcinia extract improved the chondrocyte proliferation ratio compared to no material (control). It has been shown.

FIG. 3B shows the results of measuring the number of chondrocytes in the same manner as described above using garcinia extract or calcium hydroxycitrate (HCA/Ca) as test substances at the same concentration (specifically 60 μg/mL). are also shown. Garcinia extract and hydroxycitric acid at the same concentration were found to have a lower chondrocyte proliferation ratio than HCA/Ca, although Garcinia extract had a smaller amount of hydroxycitric acid (p < 0 .05) was improving. In other words, it was shown that Garcinia extract enhances chondrocyte differentiation-inducing activity and chondrocyte proliferation activity more than HCA·Ca.

2. Secondary screening method (three-dimensional culture)
In addition, chondrocyte differentiation was induced by three-dimensional culture of human bone marrow-derived mesenchymal stem cells in the same manner as in Test Example 1, except that Garcinia extract was added as a test substance at a concentration of 30 µg/mL. Observation of cell morphology and derivation of red area ratio were performed.

Fig. 3C shows the morphology and red area ratio of chondrocytes obtained by three-dimensional culture of human bone marrow-derived mesenchymal stem cells using garcinia extract or calcium hydroxycitrate (HCA/Ca). Proteoglycan staining by Safranin O was observed with 30 μg/mL Garcinia extract, as with 100 ng/mL BMP7 of the positive control, and differentiated chondrocytes were observed. Furthermore, with the Garcinia extract, darker proteoglycan staining and better chondrocyte differentiation were observed compared to 30 μg/mL HCA·Ca.

Test Example 3: Chondrocyte differentiation-inducing activity, cartilage matrix production activity, and chondrocyte proliferation activity (in vitro) by various hydroxycitric acids and plant extracts

(Preparation of hibiscus extract)
Petals of Hibiscus (Hibiscus sabdariffa L. var ruber (Malvaceae)) were dried and extracted by adding 10 times the amount of aqueous ethanol. After filtering the resulting extract, it was concentrated under reduced pressure using a rotary evaporator, and the obtained concentrate was further spray-dried. The dried extract was pulverized to obtain a powdered hibiscus extract. The content of (+) HCA/free form (ie, (+) allo-hydroxycitric acid free form) in the hibiscus extract is less than 60% by weight.

(Garcinia extract)
The Garcinia extract prepared in Test Example 2 was used.

(Preparation of various hydroxycitric acids)
(-) HCA free form and (-) HCA lactone were purified from Garcinia extract by HPLC. (+) HCA free form, ie (+) allo-hydroxycitric acid free form and (+) HCA lactone, ie (+) allo-hydroxycitric acid lactone form, are obtained from hibiscus extract, JP-A-2006-306863. Purified by column chromatography according to the method described in .

Three-dimensional culture of human bone marrow-derived mesenchymal stem cells in the presence of 0.05 mM (about 10 μg/mL of various HCAs, 10 μg/mL of garcinia extract, or 10 μg/mL of hibiscus extract) in the same manner as in Test Example 2 did

(Result 1-chondrocyte differentiation-inducing activity)
The differentiated chondrocyte cluster (pellet) was stained in the same manner as in Test Example 1, and the red area ratio was derived. The results are shown in FIG. 4A.

As shown in Fig. 4A, chondrocyte differentiation was induced in all HCAs of (+) form, (-) form, free form and lactone form, as well as garcinia extract and hibiscus extract.

(Result 2-Cartilage Matrix Production Activity)
After digesting one cartilage cell mass (pellet) with 100 μL of a digestive enzyme papain solution (manufactured by Sigma-Aldrich, papain was solubilized with 100 mM phosphate buffer pH 7.4, 5 mM L-cystein hydrochloride monohydrate, and 10 mM EDTA), The amount of GAG (glycosaminoglycan) was measured using a GAG quantification kit (manufactured by Blyscan). A higher amount of GAG is an indicator of a higher amount of cartilage matrix production. The results are shown in Figure 4B.

As shown in FIG. 4B, all of the (+) forms, (−) forms, free forms and lactone forms of HCAs, as well as garcinia extract and hibiscus extract, showed an increase in the amount of GAG compared to the control. , was found to promote cartilage matrix production.

(Result 3—Chondrocyte proliferation activity)
After digesting the chondrocyte mass (pellet) with the digestive enzyme papain, the amount of dsDNA (double-stranded DNA) was measured using Pico Green ds DNA Assay Kit (manufactured by Invitrogen). More dsDNA is an indicator of more chondrocytes proliferating. The results are shown in Figure 4C.

As shown in Fig. 4C, it was confirmed that chondrocyte proliferation was promoted in the garcinia extract and hibiscus extract, since the amount of dsDNA increased compared to the control.

Test Example 4: Rat knee joint cartilage regeneration test with garcinia extract (in vivo)
A garcinia extract was administered to articular cartilage of the femoral trochlea of rats after drilling (cartilage damage), and cartilage regeneration was examined.

(test target)
Ten 8-week-old female SD rats (Japan SLC) were prepared, and after acclimatization for one week, a 1.0 mm diameter drill was drilled on the cartilage surface of the femoral trochlea of the left knee joint using a microdrill. Surgery was performed to create one hole and one drilled hole of 1.5 mm diameter.

(Test schedule and test method)
Water was administered to 5 rats in the control group, and 2000 mg/kg/day of the garcinia extract used in Test Example 1 was administered to 5 rats in the test group (this dose is converted to the human equivalent dose of garcinia extract). (60 kg per human using a divisor of 6.2 for rats, which is 19.4 g/day) once daily orally. Rats were weighed on days 0 (the day of surgery), 4 and 16 days. 16 days after the start of administration, all the rats were dissected, and after photographing the surface of the left knee articular cartilage, which was the surgical site, the femur was excised. After fixation with 4% paraformaldehyde/phosphate buffer, dehydration with 70% ethanol and embedding in paraffin, specimens were prepared. The prepared specimen was subjected to HE staining, Alcian blue staining and Safranin O staining. In addition, blood was collected from rats at the time of dissection, serum was prepared, and then quantified using a commercially available cartilage degradation marker C2C and a cartilage synthesis marker CPII kit (IBEX).

(Result 1 - body weight)
FIG. 5 shows changes in body weight of rats. As shown in FIG. 5, weight gain was observed in rats administered water and rats administered garcinia extract for 16 days. Garcinia extract is known to have a fat-burning effect and a diet (weight loss) effect, but under the conditions of this test example, no significant difference was observed in body weight change between water-administered rats and Garcinia extract-administered rats. .

(Result 2-operated part)
FIG. 6 shows photographs of the operated part of the cartilage surface of the femoral trochlea on the day of the operation (Day 0) and 16 days after the operation (Day 16). In FIG. 6, locations indicated by arrows indicate locations where drill holes are drilled. In the photograph of the surgical site on the day of surgery, the upper hole is a drilled hole with a diameter of 1.5 mm, and the lower hole is a drilled hole with a diameter of 1.0 mm. 16 days after the operation, the openings of both holes were clearly confirmed in the water-administered rats, but the openings of the two holes were restored to a state close to the surface of the surrounding cartilage in the rats administered the garcinia extract. It was confirmed.

(Result 3-Tissue sample analysis)
Sections were made across both of the two drilled holes in the trochlear cartilage. A stained image of the cross-sectional specimen is shown in FIG. 7(a). In FIG. 7(a), locations indicated by arrows indicate locations where drilled holes are made. As shown in FIG. 7, the defect was filled in both the water-administered rat and the garcinia extract-administered rat, but in the garcinia extract-administered rat, the surface layer was covered with cartilage tissue, and the formation of hyaline cartilage was confirmed.

More specifically, in HE staining, the cell nucleus is visible as a small dark purple dot, and in the garcinia extract-administered rat, a dot indicating the cell nucleus was observed on the surface layer of the filled drill hole. It was confirmed that it existed. In addition, the tissue formed in the surface layer of the rats administered the garcinia extract was stained blue with alcian blue and red with safranin O, so it was determined to be cartilage tissue composed of differentiated and proliferated chondrocytes. However, in water-administered rats, no staining indicating cartilage tissue was observed at the drilled portion.

Fig. 7(b) shows the results of measuring the remaining depth of the drilled hole. As shown in FIG. 7(b), the average value of 5 water-administered rats was 105 μm, whereas the average value of 5 Garcinia extract-administered rats was 32 μm (p<0.01). significant difference).

From the above results, it was confirmed that the administration of Garcinia extract promotes the regeneration of cartilage tissue and treats cartilage damage. Although the mechanism of cartilage regeneration promotion obtained by administration of garcinia extract is not clear, mesenchymal stem cells (MSC) in the bone marrow accumulate at the defect site from the drilled hole, and they are induced to differentiate into chondrocytes by the garcinia extract, resulting in cartilage. It is conceivable that this may indicate tissue regeneration.

(Result 4-blood cartilage metabolism marker measurement)
FIG. 8 shows the measurement results of the blood cartilage synthesis marker (CPII) level and the cartilage degradation marker (C2C) level. As shown in FIG. 8, it was confirmed that Garcinia extract administration significantly increased cartilage synthesis marker levels and significantly decreased cartilage degradation markers compared to water administration (p<0.05 significant difference). In other words, the garcinia extract was found to have both cartilage synthesizing action and cartilage degradation inhibiting action.

Test Example 5: Onset prevention test for rat osteoarthritis-induced model with garcinia extract (in vivo)
Cartilage regeneration was investigated by administering garcinia extract or glucosamine to a rat model in which articular cartilage degeneration due to joint instability was induced by amputation of the medial collateral ligament of the knee joint.

(test target)
14 8-week-old female SD rats (Japan SLC) were prepared, and after acclimatization for one week, the inside of the left knee joint was incised, Nagoya Gakuin University Review, (Japan), 2018, 6, p1-9 According to ., an operation (OA operation) was performed to cut the medial collateral ligament.

(Test schedule and test method)
Two rats were administered water without administration (control); six rats were administered 258 mg/kg/day of the garcinia extract used in Test Example 1 (this dose was the same as that of the garcinia extract Converted to a human equivalent dose (using a divisor of 6.2 for rats and 60 kg per human), it is 2.5 g / day), once a day from the day after OA surgery, daily oral administration and 6 other rats were orally administered glucosamine at 258 mg/kg/day once daily. Twenty-eight days after the start of administration, the rats were dissected, the left femur was extracted, and tissue sample analysis was performed.

(Results-Tissue sample analysis)
Cross-sectional specimens of left femoral knee articular cartilage were prepared. A safranin O-stained image of the cross-sectional specimen is shown in FIG. As shown in FIG. 9, in both rats to which water was administered, articular cartilage, which should be stained red with safranin O, disappeared. On the other hand, in the rats to which the garcinia extract was administered, articular cartilage stained red with safranin O was observed in 4 rats, confirming that cartilage degeneration was suppressed. Also, only two of the rats administered glucosamine had articular cartilage preserved.

From the above results, it was shown that garcinia extract is more effective than glucosamine in suppressing cartilage degeneration due to joint instability. This result indicates that the cartilage degradation inhibitory action of the garcinia extract, together with the cartilage synthesis-promoting action, is considered to have maintained articular cartilage.

Test Example 6: Cartilage regeneration test (in vivo) for accelerated mouse knee cartilage degeneration model with Garcinia extract
Cartilage regeneration was examined when the same doses of hydroxycitric acid (HCA) and Garcinia extract were administered to a mouse model of accelerated knee cartilage degeneration.

(test target)
Osteoarthritis Cartilage, 2012, 20(8), 887-895. DOI:10.1016/j.joca.2012.04.012. , 20 m/min, 20 min/day, mice were forced to walk for 2 weeks to promote degeneration of knee articular cartilage. In this way, we constructed an animal experimental model without surgery, which is similar to cartilage degeneration associated with aging in humans, that is, knee cartilage wear and tear.

(Test schedule and test method)
Hydroxycitric acid or Garcinia extract at a dose of 512 mg/kg/day (this dose is converted to a human equivalent dose of hydroxycitric acid or Garcinia extract (using a divisor of 12.3 for mice, giving 60 kg per human) ), both at 2.5 g/day) were administered orally for 8 weeks. After 8 weeks of administration, the animals were dissected and blood was collected, and safranin O-stained specimens were prepared from the articular cartilage tissue of the right femur. The results of tissue sample analysis are shown in FIG.

In addition, observing the knee joint cross-section specimen in FIG. 10, (1) staining intensity of safranin O (red), (2) degree of erosion formation, (3) degree of crack formation (roughening), (4) surface layer (5) degree of irregularity of cell arrangement (column), (6) loss of chondrocytes, and (7) exposure of subchondral bone were scored on a scale of 0 to 4. A normal condition without degeneration of cartilage is scored 0, and a diseased condition in which cartilage is completely lost is scored 28. The results of scoring are shown in FIG.

Furthermore, in the same manner as in Test Examples 3 and 4 above, the cartilage synthesis marker (CPII) and cartilage degradation marker (C2C) levels in the blood of mice were measured. The results are shown in FIG.

(result)
As is clear from FIG. 10, formation of hyaline cartilage and regeneration of articular cartilage tissue were observed in both cases of administration of hydroxycitric acid and administration of garcinia extract.

In addition, as is clear from FIG. 11, the cartilage degeneration score was smaller in the administration of hydroxycitric acid and the garcinia extract than in the administration of water, indicating cartilage tissue regeneration. In particular, the score of cartilage degeneration with garcinia extract administration was significantly smaller than that with hydroxycitric acid administration (p<0.05), indicating even better cartilage tissue regeneration.

Furthermore, as is clear from FIG. 12, both the administration of hydroxycitric acid and the administration of garcinia extract confirmed an increase in the amount of cartilage synthesis markers and a decrease in cartilage degradation markers. In particular, it was confirmed that administration of the garcinia extract resulted in a further increase in cartilage synthesis marker level and a further decrease in cartilage degradation marker level compared to administration of hydroxycitric acid. In other words, at the same dose, the garcinia extract was found to have stronger cartilage synthesizing action and cartilage degradation inhibitory action than the hydroxycitric acid, although the garcinia extract was less than the hydroxycitric acid.

Stem Cells International, 2017, Article ID 9312329, DOI: 10.1155/2017/9312329 has demonstrated the presence of mesenchymal stem cells (MSCs) in the synovium and synovial fluid of human knee and hip joints. In a model of accelerated knee joint cartilage degeneration by forced walking in mice without surgery, it is possible that the administration of garcinia extract differentiated MSCs in the synovial membrane and synovial fluid into chondrocytes, resulting in cartilage tissue regeneration. be done. From the above, it is considered that the ingestion of Garcinia extract promotes cartilage tissue regeneration even in the case of natural age-related cartilage abrasion in humans.

Test Example 7: Hydroxycitric Acid Dose Dependence of Cartilage Regeneration in Mouse Knee Cartilage Degeneration Accelerated Model by Garcinia Extract (in vivo)
Test examples, except that the dose of garcinia extract (hydroxycitric acid content is 60% by weight) was 0 mg/kg/day, 103 mg/kg/day, 206 mg/kg/day, or 512 mg/kg/day A cartilage regeneration test was performed on a mouse knee cartilage degeneration accelerated model in the same manner as in 6, and a cartilage degeneration score was derived and a cartilage synthesis marker (CPII) in blood was measured.

The doses of 0 mg/kg/day, 103 mg/kg/day, 206 mg/kg/day, and 512 mg/kg/day of the garcinia extract administered to the mice in this test example were converted to 0 mg/day in terms of hydroxycitric acid. /kg/day, 62 mg/kg/day, 124 mg/kg/day, and 307 mg/kg/day. These doses are 0 g/day, 0.5 g/day, 1 g/day and 2.5 g/day and converted to human equivalent doses of hydroxycitric acid (60 kg per human using a divisor of 12.3 for mice) of 0 g/day, 0.3 g/day, and 0 g/day, respectively. .6 g/day and 1.5 g/day. FIG. 13 shows the relationship between the cartilage degeneration score and blood cartilage synthesis marker (CPII) concentration and the human-equivalent dose of hydroxycitric acid.

As is clear from FIG. 13, the relationship between the dose of hydroxycitric acid and the degree of cartilage synthesis effect and cartilage tissue regeneration effect shows that the concentration of hydroxycitric acid exposed to the cells shown in FIG. A similar trend was observed in relation to Specifically, from the results of FIG. 13, the preferred human equivalent dose of hydroxycitric acid of Garcinia extract for cartilage synthesis and cartilage regeneration is 0.3 g to 1.5 g/day, especially 0.3 g to 1.5 g/day. The highest cartilage synthesizing effect and cartilage tissue regeneration effect were observed at 6 g/day.

Claims

A cartilage regeneration promoter containing hydroxycitric acid and/or its salt.
The cartilage regeneration promoter according to claim 1, wherein the hydroxycitric acid is (-) hydroxycitric acid and/or (+) allo-hydroxycitric acid.
The cartilage regeneration promoter according to claim 1, wherein the hydroxycitric acid is a non-lactone body and/or a lactone body.
The cartilage regeneration promoter according to claim 1, which is used for promoting cartilage synthesis.
The cartilage regeneration promoter according to claim 1, which contains a plant extract containing the hydroxycitric acid and/or its salt.
The cartilage regeneration promoter according to claim 5, wherein the plant extract is Garcinia extract.
The cartilage regeneration promoter according to claim 5, wherein the plant extract is a hibiscus extract.
The cartilage regeneration promoter according to claim 1, which is used for the treatment or prevention of cartilage disorders.
The cartilage regeneration promoter according to claim 1, wherein the dose to humans is 0.3 g to 1.5 g/day/60 kg in terms of hydroxycitric acid.
A chondrocyte differentiation promoting agent that promotes differentiation of undifferentiated cells into chondrocytes, containing hydroxycitric acid and/or salts thereof.
The chondrocyte differentiation promoting agent according to claim 10, wherein the hydroxycitric acid is (-) hydroxycitric acid and/or (+) allo-hydroxycitric acid.
The chondrocyte differentiation promoting agent according to claim 10, wherein the hydroxycitric acid is a non-lactone body and/or a lactone body.
The chondrocyte differentiation promoting agent according to claim 10, which contains a plant extract containing the hydroxycitric acid and/or its salt.
The chondrocyte differentiation promoting agent according to claim 13, wherein the plant extract is Garcinia extract.
The chondrocyte differentiation promoting agent according to claim 13, wherein the plant extract is a hibiscus extract.
The chondrocyte differentiation promoting agent according to claim 10, which is exposed to cells at a concentration of 0.08 to 0.4 mM in terms of hydroxycitric acid.
A cartilage degradation inhibitor containing hydroxycitric acid and/or its salt.
A protective agent for maintaining cartilage thickness, containing hydroxycitric acid and/or its salt.
An agent for relieving joint pain, discomfort, and/or discomfort containing hydroxycitric acid and/or its salts.