JP2013245168A - Aggrecan degradation inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an aggrecan degradation inhibitor capable of inhibiting degradation of aggrecan.SOLUTION: An aggrecan degradation inhibitor includes at least one kind selected from a group comprising ellagic acid and ellagitannin as an active ingredient.

Description

  The present invention relates to an aggrecan degradation inhibitor.

In recent years, the population affected by bone and joint disease, which is one of the factors that impair the healthy life expectancy of the elderly, has been increasing. Many osteoarthritis diseases tend to increase in number with the onset of aging, and with the recent aging of the population, a rapid increase in the affected population is expected in the future.
Osteoarthritis, a bone and joint disease, is a progressive disease characterized by destruction, degeneration, and disappearance of articular cartilage. For example, knee osteoarthritis is a disease in which cartilage and meniscus are gradually worn down, making joint deformation, pain generation, and bending and stretching of joints difficult. The total number of patients with knee osteoarthritis in Japan is estimated to be about 8 million, which is far greater than rheumatoid arthritis, which is one of the same bone and joint diseases.
At present, pharmacotherapy for osteoarthritis is a symptomatic treatment for the purpose of pain relief, and there are few established treatments for suppressing the progression of the disease itself. Further, many conventional therapeutic drugs have problems such as side effects, and safer and more effective preventive / ameliorating drugs are desired.

  Osteoarthritis is caused by the destruction of cartilage tissue. Cartilage tissue is composed of chondrocytes and the extracellular matrix surrounding them, and the main components of the articular cartilage extracellular matrix (hereinafter also simply referred to as the cartilage matrix) are type II collagen and aggrecan (proteoglycan). Collagen contributes to cartilage morphology maintenance, while aggrecan contributes to cartilage viscoelasticity. In a healthy person's cartilage tissue, the metabolic balance between collagen and aggrecan production and cartilage matrix degradation by cartilage matrix degrading enzymes is maintained appropriately. On the other hand, in osteoarthritis patients, since the degradation and degeneration of articular cartilage matrix is observed in the early stage of onset, it is considered that the metabolic balance of cartilage matrix tends to be degraded.

As enzymes that degrade cartilage matrix, metalloproteases belonging to the ADAMTS (A Disintegrin And Metalloproteinase with ThoromboSpondin motifs) family and metalloproteases belonging to the MMP (Matrix Metalloproteinase) family are known. These families include the enzyme aggrecanase that degrades aggrecan and the enzyme MMP that degrades collagen and aggrecan.
The onset and progression of osteoarthritis is thought to involve proteolytic enzymes responsible for the degradation of cartilage matrix centered on aggrecan and collagen. Recent research suggests that cartilage matrix destruction is first protected by enzymatic degradation and disappearance of aggrecan, followed by enzymatic degradation of collagen, and is protected from collagen degradation by the presence of aggrecan. (For example, refer nonpatent literature 1). Furthermore, it has been reported that many aggrecan degradation products are observed in synovial fluid collected from osteoarthritis patients, and it seems that aggrecanase plays an important role in cartilage destruction in osteoarticular diseases. (For example, see Non-Patent Documents 2 and 3). It has also been reported that aggrecan degradation in osteoarthritic cartilage is suppressed by an aggrecanase inhibitor but not by an MMP inhibitor (see Non-Patent Document 4).

  On the other hand, an arthritis preventive or ameliorating agent containing ellagic acid as an active ingredient has been proposed (see, for example, Patent Document 1). Patent Document 1 reports on the inhibitory activity of ellagic acid on gelatinase, which is one of the enzymes that degrade collagen (gelatin) that constitutes the extracellular matrix.

JP 2005-47817 A

Linda Troeberg, et al., Biochimica et Biophysica Acta 1824, 2012, p. 133-145 Glasson SS, et al., Nature, 2005, vol. 434, p. 644-648 Stanton H, et al., Nature, 2005, vol. 434, p. 648-652 Ann-Marie, et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY, 2002, vol. 277, p. 22201-22208

  An object of the present invention is to provide an aggrecan degradation inhibitor that suppresses the degradation of aggrecan. Another object of the present invention is to provide an aggrecanase activity inhibitor that inhibits aggrecanase activity. Another object of the present invention is to provide an articular cartilage extracellular matrix degradation inhibitor that inhibits the degradation of articular cartilage extracellular matrix. Furthermore, this invention makes it a subject to provide the prevention or improvement agent which prevents or improves the symptom or disease resulting from a decomposition | disassembly of an articular cartilage extracellular matrix.

  In view of the above problems, the present inventors have searched for new materials that suppress aggrecan degradation. As a result, it was found that ellagic acid inhibits the activity of aggrecanase, which is an aggrecan-degrading enzyme, and effectively suppresses aggrecan degradation of the cartilage matrix. The present invention has been completed based on this finding.

The present invention relates to an aggrecan decomposition inhibitor comprising at least one selected from the group consisting of ellagic acid and ellagitannin as an active ingredient.
The present invention also relates to an aggrecanase activity inhibitor comprising as an active ingredient at least one selected from the group consisting of ellagic acid and ellagitannin.
The present invention also relates to an articular cartilage matrix decomposition inhibitor comprising at least one selected from the group consisting of ellagic acid and ellagitannin as an active ingredient.
Furthermore, the present invention relates to a prophylactic or improving agent for preventing or ameliorating a symptom or disease caused by degradation of the articular cartilage extracellular matrix, comprising at least one selected from the group consisting of ellagic acid and ellagitannin as an active ingredient. .

  The aggrecan degradation inhibitor of the present invention can inhibit aggrecan degradation. Moreover, the aggrecanase activity inhibitor of this invention can inhibit aggrecanase activity. Moreover, the joint cartilage extracellular matrix degradation inhibitor of the present invention can inhibit the degradation of the articular cartilage extracellular matrix. In addition, the preventive or ameliorating agent of the present invention can prevent or ameliorate symptoms or diseases caused by degradation of the articular cartilage extracellular matrix.

The photomicrograph of the knee joint part of the osteoarthritis disease model which ingested the test meal containing ellagic acid is shown. The microscope picture of the knee joint part of the osteoarthritis disease model which ingested the test meal which does not contain ellagic acid is shown.

The aggrecan degradation inhibitor and aggrecanase activity inhibitor of the present invention contain at least one selected from the group consisting of ellagic acid and ellagitannin as an active ingredient. As shown in Examples described later, ellagic acid significantly reduces aggrecanase activity and suppresses aggrecan degradation by aggrecanase. Elazitanin is hydrolyzed into ellagic acid in the body, and is absorbed and metabolized. (For example, see Espin J.C., et al., JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 2007, vol. 55, p. 10476-10485). Therefore, ellagitannin has the same effect.
Aggrecan is a major proteoglycan present in the extracellular matrix of articular cartilage, and consists of a core protein and glycosaminoglycans such as chondroitin sulfate and keratan sulfate that modify the core protein. Aggrecanase is an enzyme that cleaves a specific site in the core protein of aggrecan to degrade aggrecan. Representative aggrecanases include ADAMTS-4 and ADAMTS-5, which have been confirmed to express proteins and genes in the cartilage of osteoarthritis patients (see Non-Patent Document 4). In particular, ADAMTS-5 is considered to play a central role in aggrecan destruction, and it has been reported that cartilage matrix destruction is significantly suppressed in ADAMTS-5 gene-deficient mice (Non-patent Document 2). And 3).
Especially the inhibitor and inhibitor of this invention inhibit the activity of aggrecanase ADAMTS-5 suitably.
As described above, it is suggested that the destruction of the cartilage matrix is first preceded by the enzymatic degradation and disappearance of aggrecan, and then the enzymatic degradation of collagen. Therefore, in the present invention, the degradation of aggrecan by aggrecanase and the enzymatic degradation of collagen by collagenase or the like are completely different from the viewpoint of mechanism.

Furthermore, the present invention provides an articular cartilage extracellular matrix degradation inhibitor comprising at least one selected from the group consisting of ellagic acid and ellagitannin as an active ingredient. As described above, the cartilage matrix includes aggrecan. By suppressing the degradation of aggrecan involving aggrecanase by the action of ellagic acid, the degradation of the articular cartilage extracellular matrix can be suppressed. Further, according to the articular cartilage extracellular matrix degradation inhibitor of the present invention, the metabolic balance of aggrecan in the cartilage tissue inclined to degradation is restored to the normal state, or the aggrecan in the decreased cartilage matrix is increased to increase the viscosity. The elastic cartilage matrix can be recovered.
It has never been known that ellagic acid has an aggrecan degradation inhibitory action, an aggrecanase activity inhibitory action, and an articular cartilage matrix degradation inhibitory action, and is a finding newly obtained by the present inventors.
As used herein, “articular cartilage extracellular matrix degradation” means that aggrecan constituting the articular cartilage extracellular matrix is degraded by cartilage matrix degrading enzymes such as MMP and aggrecanase. Characteristically refers to being degraded by aggrecanases such as 5.

The present invention also provides a prophylactic or improving agent for preventing / ameliorating various symptoms or diseases caused by degradation of the articular cartilage extracellular matrix, comprising at least one selected from the group consisting of ellagic acid and ellagitannin as an active ingredient I will provide a. By suppressing the excessive aggrecan degradation by the action of ellagic acid, the cartilage matrix can be returned to the normal state by returning the metabolic balance of the cartilage matrix leaning to the normal state or increasing the decreased aggrecan. .
Examples of symptoms or diseases caused by degradation of the articular cartilage extracellular matrix include various bone and joint diseases. Examples of osteoarthritis include osteoarthritis, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, inflammatory bowel disease, periodontal disease, osteoporosis and the like. In particular, the preventive agent or ameliorating agent of the present invention is preferably used for the prevention or amelioration of osteoarthritis. Osteoarthritis refers to a disease characterized by articular cartilage degeneration, wear, subchondral bone sclerosis, and proliferative changes.

In the aggrecan degradation inhibitor, aggrecanase activity inhibitor, articular cartilage extracellular matrix degradation inhibitor, and preventive or ameliorating agent of the present invention, at least one selected from the group consisting of ellagic acid and ellagitannin is used as an active ingredient. In the present invention, ellagic acid, ellagitannin, or ellagic acid and ellagitannin are preferably used as active ingredients, and ellagic acid is more preferably used as an active ingredient.
In the present specification, “elagitannin” is a kind of hydrolyzable tannin and is a general term for tannin that generates ellagic acid during acid hydrolysis. The ellagitannin used in the present specification is not particularly limited as long as it produces ellagic acid by metabolism.

  Ellagic acid and ellagitannin used in the present invention are widely available. For example, it may be chemically synthesized by an ordinary synthesis method, extracted from a natural product, or a commercially available product may be used.

  In the present invention, the active ingredient may be dissolved or dispersed in a solvent and used as an aggrecan degradation inhibitor, an aggrecanase activity inhibitor, an articular cartilage extracellular matrix degradation inhibitor, or a preventive or ameliorating agent. There is no restriction | limiting in particular as a solvent which can be used by this invention, Water, ethanol, propylene glycol, dimethylsulfoxide, etc. are mentioned. The solvent that can be used in the present invention is preferably one or more selected from the group consisting of water, ethanol, propylene glycol, and dimethyl sulfoxide, and one or more selected from the group consisting of water and ethanol. Two types are more preferable, and ethanol is more preferable.

  In another aspect, the present invention provides use of at least one selected from the group consisting of ellagic acid and ellagitannin as an aggrecan degradation inhibitor, aggrecanase activity inhibitor, or articular cartilage extracellular matrix degradation inhibitor. . Furthermore, the present invention provides, as yet another aspect, at least one member selected from the group consisting of ellagic acid and ellagitannin for producing an aggrecan degradation inhibitor, an aggrecanase activity inhibitor, or an articular cartilage extracellular matrix degradation inhibitor. Provide use. In still another aspect, the present invention provides at least one selected from the group consisting of ellagic acid and ellagitannin, an aggrecan degradation inhibitor, an aggrecanase activity inhibitor, an articular cartilage extracellular matrix degradation inhibitor, or an articular cartilage extracellular Provided is a method for use as a preventive or ameliorating agent for preventing or ameliorating a symptom or disease caused by degradation of a substrate. Further, the present invention provides, as yet another aspect, a method for treating a symptom or disease caused by degradation of an articular cartilage extracellular matrix, or a non-medical treatment method for a symptom or disease caused by degradation of an articular cartilage extracellular matrix And at least one selected from the group consisting of ellagic acid and ellagitannin. In another aspect, the present invention provides at least one selected from the group consisting of ellagic acid and ellagitannin for the prevention or improvement of symptoms or diseases caused by degradation of the extracellular matrix of articular cartilage. I will provide a. Furthermore, the present invention provides, as another aspect, an aggrecan degradation inhibition method, an aggrecanase activity inhibition method, an articular cartilage extracellular matrix degradation inhibition method, and an articular chondrocyte using at least one selected from the group consisting of ellagic acid and ellagitannin. A method for preventing or ameliorating a symptom or disease caused by degradation of an outer matrix is provided.

  The aggrecan degradation inhibitor, aggrecanase activity inhibitor, articular cartilage matrix degradation inhibitor, or preventive or ameliorating agent of the present invention is used for pharmaceutical compositions, quasi-drug compositions, foods, beverages, feed / pet foods, etc. Can be applied to. Furthermore, there are no particular limitations on the dosage form of the aggrecan degradation inhibitor, aggrecanase activity inhibitor, articular cartilage matrix degradation inhibitor, or preventive or ameliorating agent of the present invention, liquid formulations (water solutions, suspensions, emulsions, etc.), solids Formulations (powder, granules, capsules, pills, tablets, etc.), pastes, gels, powders (powder), pellets, sticks, etc. can be applied to humans and non-human animals Various dosage forms can be taken.

  When applying the aggrecan degradation inhibitor, aggrecanase activity inhibitor, articular cartilage matrix degradation inhibitor, or preventive or ameliorating agent of the present invention to a pharmaceutical composition or quasi-drug composition, various additives are blended as necessary. It can be prepared as a pharmaceutical composition or a quasi-drug composition in various dosage forms by containing an appropriate amount of the active ingredient. For example, as an oral medicine such as tablets, coated tablets, capsules, granules, powders, syrups, enteric solvents, troches, drinks, etc., or non-injectables such as injections, suppositories, transdermal absorption agents, external preparations, etc. It can be prepared as an oral medicine. Of these forms, the preferred form is oral medicine. In order to prepare pharmaceutical preparations of such various dosage forms, they may be produced according to conventional methods using various additives. There is no restriction | limiting in particular in the additive to be used, The normally used thing can be used. Examples thereof include pharmaceutically acceptable excipients (sugars such as sorbitol, glucose, lactose, dextrin, starch, inorganic substances such as titanium oxide and calcium carbonate, crystalline cellulose, distilled water, sesame oil, corn oil, olive oil, Rapeseed oil, etc.), liquid carriers (distilled water, physiological saline, aqueous glucose solution, alcohol such as ethanol, propylene glycol, polyethylene glycol, etc.), oily carriers (various animal and vegetable oils, white petrolatum, paraffin, waxes, etc.), absorption promotion Agent, stabilizer, wetting agent, emulsifier, binder, tonicity agent, disintegrant, lubricant, extender, surfactant, dispersant, suspending agent, diluent, osmotic pressure adjusting agent, pH adjustment Agents, preservatives, antioxidants, colorants, UV absorbers, moisturizers, thickeners, brighteners, buffers, preservatives, flavoring agents, fragrances, film agents, flavoring agents, bacterial inhibitors, etc. It is.

  When the aggrecan degradation inhibitor, aggrecanase activity inhibitor, articular cartilage matrix degradation inhibitor, or preventive or ameliorating agent of the present invention is applied to food, drink, feed / pet food, etc., it is suitable for food, drink, feed / pet food For example, it can be provided in the form of granules, granules, tablets, capsules, pastes and the like. Furthermore, the food and drink is a concept of the prevention or improvement of symptoms or diseases caused by the degradation of the articular cartilage extracellular matrix in addition to the general food and drink, and if necessary, beauty foods, foods for the sick It can be made into the form of functional food-drinks, such as a nutritive functional food or a food for specified health.

There are no particular restrictions on the form of food and drink, such as processed foods such as bread and noodles, processed rice foods such as rice cakes and cooked rice, biscuits, cakes, jelly, chocolate, rice crackers, ice cream, etc. Processed soy foods such as confectionery, tofu, processed foods thereof, soft drinks, fruit juice drinks, milk drinks, carbonated drinks and other dairy products such as yogurt, cheese, butter and milk, soy sauce, sauce, miso, mayonnaise, Examples include seasonings such as dressings, meat storage such as ham, bacon and sausage, processed meat processed foods, marine processed foods such as hampen, chikuwa and canned fish, cooking oil and frying oil. In addition, tablets, tablets, and other tablet foods, concentrated liquid foods, natural liquid foods, semi-digested nutritional foods, ingredient nutritional foods, drink nutritional foods, etc. Good.
There is no restriction | limiting in particular as a form of feed, The pet food etc. which are used for the feed for small animals used for a rabbit, a rat, a mouse | mouth, a dog, a cat, a small bird, a squirrel, etc. are mentioned.
These foods and drinks use, for example, an appropriate combination of food ingredients such as sweeteners, colorants, antioxidants, vitamins, fragrances, minerals, etc., proteins, lipids, carbohydrates, carbohydrates, dietary fibers, etc. This and the aggrecan degradation inhibitor, aggrecanase activity inhibitor, articular cartilage matrix degradation inhibitor, or preventive or ameliorating agent of the present invention are contained, and in the form of various foods and beverages (for example, granular, granular, tablet, Capsules, pastes, and liquids).

  The aggrecan degradation inhibitor, aggrecanase activity inhibitor, articular cartilage matrix degradation inhibitor, or preventive or ameliorating agent of the present invention may be used as it is as a pharmaceutical composition, quasi-drug composition, food or beverage, You may use as additives or compounding agents, such as a pharmaceutical composition, a quasi-drug composition, a foodstuff, or a drink.

  The compounding amount of the active ingredient in the aggrecan degradation inhibitor, aggrecanase activity inhibitor, articular cartilage matrix degradation inhibitor, or preventive or ameliorating agent of the present invention varies depending on the form of use and can be appropriately set. For example, the content of the active ingredient is preferably 0.0001% by mass or more, more preferably 0.01% by mass or more, further preferably 0.1% by mass or more, and preferably 100% by mass or less in terms of solid content. 80% by mass is more preferable. Or 0.0001-100 mass% is preferable in conversion of solid content, as for content of the said active ingredient, 0.01-80 mass% is more preferable, 0.1-80 mass% is further more preferable.

  The administration or intake of the aggrecan degradation inhibitor, aggrecanase activity inhibitor, articular cartilage matrix degradation inhibitor, or preventive or ameliorating agent of the present invention is the individual's state, body weight, sex, age, material activity, administration or intake route. It is appropriately selected and determined according to the administration or intake schedule, formulation form or other factors. For example, the daily dose or intake for adults (60 kg) is generally preferably 0.001 g / kg body weight or more, and more preferably 10 g / kg body weight or less. In addition, the aggrecan degradation inhibitor, aggrecanase activity inhibitor, articular cartilage matrix degradation inhibitor, or preventive or ameliorating agent of the present invention is divided into once to several times a day, or is taken and administered at an arbitrary period and interval. obtain.

  Although not particularly limited as a subject of ingestion or administration of the above pharmaceutical composition, quasi-drug composition, food or beverage, a human for the purpose of prevention, improvement or amelioration of symptoms or diseases caused by degradation of the articular cartilage extracellular matrix And non-human animals such as mammals are preferred. In addition, the subject of ingestion or administration includes humans and non-human mammals in which symptoms or diseases caused by degradation of the articular cartilage extracellular matrix are observed, and humans and non-human animals that may be at risk, and prevention of the symptoms or diseases. It includes humans and non-human animals that expect

Next, test methods for the aggrecanase activity inhibition test and the aggrecan degradation inhibition test will be described. However, the present invention is not limited to this.
Aggrecan degradation at the cartilage matrix occurs by cleaving two specific sites in the core protein of aggrecan. Here, the cleavage site will be described using the amino acid sequence of human aggrecan as an example (for the cleavage site of human aggrecan, see Torotorella MD, et al., MATRIX BIOLOGY, 2002, vol. 21, p. 499-511). . The two cleavage sites are between the 341st asparagine residue and the 342nd phenylalanine residue (hereinafter referred to as the Asn341-Phe342 site), the 373rd glutamic acid residue, and the 374th residue. Between the alanine residues (hereinafter referred to as Glu373-Ala374 site), and the former Asn341-Phe342 site is cleaved by MMP-3, which is one of the metalloproteases belonging to the MMP family, and the latter Glu373- The Ala374 site is cleaved by aggrecanase (ADAMTS-4, ADAMTS-5, etc.), which is one of the metalloproteases belonging to the ADAMTS family, and aggrecan is degraded. When the degradation products of aggrecan in the synovial fluid of patients with degenerative bone joint diseases and the like are examined, since a large number of fragments cleaved at the Glu373-Ala374 site are included, aggrecanase plays an important role in cartilage matrix destruction. Conceivable. In this specification, focusing on the fact that the site cleaved by ADAMTS-5 is only the Glu373-Ala374 site, an aggrecan degradation inhibition test was conducted.

Details of the test methods for the aggrecanase activity inhibition test and the aggrecan degradation inhibition test are shown below. However, the present invention is not limited to this.
An aggrecan of an interglobular domain (IGD) region containing ADAMTS-5, which is a kind of aggrecanase, and Glu373-Ala374 as a substrate is used as an enzyme. The aggrecan core protein has two globular domains G1 and G2 on the N-terminal side, a globular domain G3 on the C-terminal side, an interglobular domain (IGD) between G1 and G2, and G2 and G3. It has a glycosaminoglycan binding region in between. The Glu373-Ala374 site is present on the intersphere domain.
First, an extract sample is added to the enzyme and the substrate to perform an enzymatic decomposition reaction. By the action of ADAMTS-5, cleavage is performed at the Glu373-Ala374 site in aggrecan, and two types of aggrecan degradation products are produced. Of these, one has Glu373 at the C-terminus and the other has Ala374 at the N-terminus. These degradation products are produced quantitatively depending on the intensity of the enzyme activity.
An aggrecan degradation product (N antibody that specifically binds to N-terminal Ala374, an antibody that specifically binds to C-terminal Glu373, or both of these antibodies) is then used to specifically bind to the aggrecan degradation product. An aggrecan degradation product having Ala374 at the end, an aggrecan degradation product having Glu373 at the C-terminus, or both of these degradation products) is detected and quantified. Alternatively, detection and quantification can be performed using an antibody that specifically binds to an aggrecan degradation product as a primary antibody and further using a secondary antibody that binds to the primary antibody.
By comparing the obtained results with the results obtained in a system in which the test sample is not added, the aggrecan degradation inhibitory effect and the aggrecanase activity inhibitory effect due to the addition of the test sample can be confirmed.

The aggrecan used in the test method is not limited to the above-mentioned IGD region, and may be any one that can be specifically decomposed by the aggrecan-degrading enzyme to be used and the obtained degradation product can be quantified. The antibody used in the test method is not particularly limited as long as it can specifically detect and quantify the aggrecan degradation product cleaved by the aggrecan degrading enzyme.
In the present specification, “inhibiting aggrecanase activity” means that aggrecanase inhibits the action of degrading aggrecan. For example, when the aggrecanase is ADAMTS-5, it means that cleavage of the Glu373-Ala374 site of the aggrecan core protein is inhibited.

  The present invention further discloses the following aggrecan degradation inhibitor, aggrecanase activity inhibitor, articular cartilage extracellular matrix degradation inhibitor, preventive or ameliorating agent, production method, and use relating to the above-described embodiment.

<1> An aggrecan decomposition inhibitor comprising at least one selected from the group consisting of ellagic acid and ellagitannin as an active ingredient.

<2> The aggrecan degradation inhibitor according to <1>, wherein the degradation of aggrecan is suppressed by inhibiting the activity of aggrecanase.
<3> The aggrecan degradation inhibitor according to <2>, wherein the aggrecanase is ADAMTS-5.
<4> The aggrecan decomposition inhibitor according to any one of <1> to <3>, wherein the active ingredient is ellagic acid.
<5> The aggrecan degradation inhibitor according to any one of <1> to <4>, wherein the degradation of aggrecan in the cartilage tissue of the knee joint is suppressed.
<6> The aggrecan decomposition inhibitor according to any one of <1> to <5>, wherein the content of the active ingredient is 0.0001% by mass to 100% by mass in terms of solid content.

<7> An aggrecanase activity inhibitor comprising as an active ingredient at least one selected from the group consisting of ellagic acid and ellagitannin.

<8> The aggrecanase activity inhibitor according to <7>, wherein the aggrecanase is ADAMTS-5.
<9> The aggrecanase activity inhibitor according to <7> or <8>, wherein the active ingredient is ellagic acid.
<10> The aggrecanase activity inhibitor according to any one of <7> to <9>, wherein the content of the active ingredient is 0.0001% by mass to 100% by mass in terms of solid content.

<11> An articular cartilage extracellular matrix degradation inhibitor comprising at least one selected from the group consisting of ellagic acid and ellagitannin as an active ingredient.

<12> The articular cartilage extracellular matrix degradation inhibitor according to <11>, wherein the articular cartilage extracellular matrix is aggrecan.
<13> The articular cartilage extracellular matrix degradation inhibitor according to <12>, wherein the degradation of the aggrecan is inhibited by inhibiting the activity of aggrecanase.
<14> The articular cartilage extracellular matrix degradation inhibitor according to <13>, wherein the aggrecanase is ADAMTS-5.
<15> The articular cartilage extracellular matrix degradation inhibitor according to any one of <11> to <14>, wherein the active ingredient is ellagic acid.
<16> The articular cartilage extracellular matrix degradation inhibitor according to any one of <11> to <15>, wherein the content of the active ingredient is 0.0001% by mass to 100% by mass in terms of solid content.

<17> A preventive or ameliorating agent for preventing or ameliorating a symptom or disease caused by degradation of an articular cartilage extracellular matrix, comprising at least one selected from the group consisting of ellagic acid and ellagitannin as an active ingredient.

<18> The preventive or ameliorating agent according to <17>, wherein the symptom or disease caused by degradation of the articular cartilage extracellular matrix is osteoarthritis.
<19> The preventive or ameliorating agent according to <17> or <18>, wherein the articular cartilage extracellular matrix is aggrecan.
<20> The preventive or ameliorating agent according to <19>, wherein the degradation of the aggrecan is suppressed by inhibiting the activity of aggrecanase.
<21> The preventive or ameliorating agent according to <20>, wherein the aggrecanase is ADAMTS-5.
<22> The preventive or improving agent according to any one of <17> to <21>, wherein the active ingredient is ellagic acid.
<23> The preventive or improving agent according to any one of <17> to <22>, wherein the content of the active ingredient is 0.0001% by mass or more and 100% by mass or less in terms of solid content.

<24> Use of ellagic acid, ellagitannin, or ellagic acid and ellagitannin as an aggrecan degradation inhibitor.

<25> Use of ellagic acid, ellagitannin, or ellagic acid and ellagitannin for the production of an aggrecan degradation inhibitor.

<26> A method of using ellagic acid, ellagitannin, or ellagic acid and ellagitannin as an aggrecan degradation inhibitor.

<27> A method for inhibiting aggrecan degradation using ellagic acid, ellagitannin, or ellagic acid and ellagitannin.

<28> The use or method according to any one of <24> to <27>, wherein the degradation of aggrecan is suppressed by inhibiting the activity of aggrecanase.
<29> The use or method according to <28>, wherein the aggrecanase is ADAMTS-5.
<30> The use or method according to any one of <24> to <29>, for suppressing degradation of aggrecan in the cartilage tissue of the knee joint.
<31> In the aggrecan degradation inhibitor, the content of ellagic acid, ellagitannin, or ellagic acid and ellagitannin is 0.0001% by mass to 100% by mass in terms of solid content, <24> to <30> Use or method according to any of paragraphs 30>.

<32> Use of ellagic acid, ellagitannin, or ellagic acid and ellagitannin as an aggrecanase activity inhibitor.

<33> Use of ellagic acid, ellagitannin, or ellagic acid and ellagitannin for the production of an aggrecanase activity inhibitor.

<34> A method of using ellagic acid, ellagitannin, or ellagic acid and ellagitannin as an aggrecanase activity inhibitor.

<35> A method for inhibiting aggrecanase activity using ellagic acid, ellagitannin, or ellagic acid and ellagitannin.

<36> The use or method according to any one of <32> to <35>, wherein the aggrecanase is ADAMTS-5.
<37> In the aggrecanase activity inhibitor, the content of ellagic acid, ellagitannin, or ellagic acid and ellagitannin is 0.0001% by mass or more and 100% by mass or less in terms of solid content. 36. Use or method according to any of paragraphs 36>.

<38> Use of ellagic acid, ellagitannin, or ellagic acid and ellagitannin as an articular cartilage extracellular matrix degradation inhibitor.

<39> Use of ellagic acid, ellagitannin, or ellagic acid and ellagitannin for the production of an articular cartilage extracellular matrix degradation inhibitor.

<40> A method of using ellagic acid, ellagitannin, or ellagic acid and ellagitannin as an articular cartilage extracellular matrix degradation inhibitor.

<41> A method for inhibiting degradation of articular cartilage extracellular matrix using ellagic acid, ellagitannin, or ellagic acid and ellagitannin.

<42> The use or method according to any one of <38> to <41>, wherein the articular cartilage extracellular matrix is aggrecan.
<43> The use or method according to <42>, wherein the degradation of the aggrecan is suppressed by inhibiting the activity of aggrecanase.
<44> The use or method according to <43>, wherein the aggrecanase is ADAMTS-5.
<45> In the articular cartilage extracellular matrix degradation inhibitor, the content of ellagic acid, ellagitannin, or ellagic acid and ellagitannin is 0.0001 mass% or more and 100 mass% or less in terms of solid content, <38 The use or method according to any one of items> to <44>.

<46> A method of using ellagic acid, ellagitannin, or ellagic acid and ellagitannin as a preventive or ameliorating agent for preventing or ameliorating a symptom or disease caused by degradation of an articular cartilage extracellular matrix.

<47> Use of ellagic acid, ellagitannin, or ellagic acid and ellagitannin for the manufacture of an agent for preventing or ameliorating a symptom or disease caused by degradation of an articular cartilage extracellular matrix.

<48> A method for preventing or ameliorating a symptom or disease caused by degradation of an articular cartilage extracellular matrix using ellagic acid, ellagitannin, or ellagic acid and ellagitannin.

<49> The use or method according to any one of <46> to <48>, wherein the symptom or disease caused by the degradation of the articular cartilage extracellular matrix is osteoarthritis.
<50> The use or method according to any one of <46> to <49>, wherein the articular cartilage extracellular matrix is aggrecan.
<51> The use or method according to <50>, wherein the degradation of the aggrecan is suppressed by inhibiting the activity of aggrecanase.
<52> The use or method according to <51>, wherein the aggrecanase is ADAMTS-5.
<53> In the preventive or ameliorating agent, the content of ellagic acid, ellagitannin, or ellagic acid and ellagitannin is 0.0001% by mass or more and 100% by mass or less in terms of solid content, <46> to <52> The use or method of any of paragraphs 52>.

<53> Ellagic acid, ellagitannin, or ellagic acid and ellagitannin used for a method of treating a symptom or disease caused by degradation of the extracellular matrix of articular cartilage.

<54> Ellagic acid, ellagitannin, or ellagic acid and ellagitannin used for a non-pharmaceutical treatment method of symptoms or diseases caused by degradation of the articular cartilage extracellular matrix.
<55> The ellagic acid, ellagitannin, or ellagic acid and ellagitannin according to the above <54>, wherein the ellagic acid, ellagitannin, or ellagic acid and ellagitannin are applied in the form of food or beverage.

<56> The ellagic acid, ellagitannin, or ellagic acid and ellagitannin according to any one of the above <53> to <55>, wherein the symptom or disease caused by degradation of the articular cartilage extracellular matrix is osteoarthritis.
<57> The ellagic acid, ellagitannin, or ellagic acid and ellagitannin according to any one of the above <53> to <56>, wherein the articular cartilage extracellular matrix is aggrecan.
<58> Ellagic acid, ellagitannin, or ellagic acid and ellagitannin according to <57>, wherein the degradation of the aggrecan is suppressed by inhibiting the activity of aggrecanase.
<59> The ellagic acid, ellagitannin, or ellagic acid and ellagitannin according to <58>, wherein the aggrecanase is ADAMTS-5.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.

Preparation Example Pomegranate acid (a product obtained by subjecting pomegranate peel to solvent hydrolysis, acid hydrolysis, and filtration and purification of crystal components, obtained from Sabinsa Japan Corporation) to dimethyl sulfoxide so that the concentration becomes 0.3% (w / v) Dissolve and prepare a test sample of ellagic acid.

Test example 1
The following operation was performed using the test sample of ellagic acid prepared in the above Preparation Example.
In the following operations, Tris Buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM CaCl ₂ ) was used for dilution of the test sample, the substrate, the enzyme, and the reaction stop solution.

In each well of a 96-well protein high-binding microplate (CORNING, product number # 3361), 40 μL of aggrecan (trade name: RECOMBINANT INTERGLOBULAR DOMAIN T ₃₃₁ -G ₄₅₈ (Aggrecan-IGD1), CHEMICON) 40 mg, aggrecan 40 μL of ADAMTS-5 (trade name: RECOMBINANT HUMAN ADAMTS-5, manufactured by CHEMICON) as a degrading enzyme and 20 μL of a test sample of ellagic acid prepared in the above preparation example were added and mixed. The substrate aggrecan was prepared at a final concentration of 0.025 μM, the enzyme ADAMTS-5 at a final concentration of 1.5 nM, and the test sample was prepared at a final concentration of 0.0003% (w / v). After 3 hours of reaction at 37 ° C., 50 μL / well (30 mM) of EDTA was added to stop the reaction, and the mixture was allowed to stand at 4 ° C. overnight. The next day, the plate was washed three times using TBS-T (manufactured by BIO-RAD; 20 mM Tris-HCL (pH 7.6), 0.8% NaCl, 0.1% Tween-20) as a washing solution, and a blocking solution. After adding 250 μL / well of 1% BSA (Bovine Serum Albumin, manufactured by ROCKLAND), the mixture was allowed to stand at room temperature for 2 hours. After washing the plate 3 times with TBS-T, anti-aggrecan antibody (trade name: Aggrecan ARGxx antibody [BC-3], manufactured by abcam) as a primary antibody was increased 500 times with blocking solution (1% BSA, TBS-T). Diluted 100 μL / well was added and allowed to react at room temperature for 1.5 hours. After washing the plate 3 times with TBS-T, a horseradish peroxidase-conjugated anti-mouse IgG antibody (trade name: ECL ^™ Anti-mouse IgG, Horseradish Peroxidase, manufactured by GE Healthcare UK) was used as a secondary antibody in a blocking solution (1% BSA). , TBS-T) was diluted 1000 times with 100 μL / well and allowed to react at room temperature for 1.5 hours. After washing 3 times with TBS-T, 100 μL / well of TMB-solution (TMB-solution Kit (manufactured by BIO-RAD)) was added as a color former, reacted for 30 minutes in the dark, and then 1N H ₂ SO ₄ The reaction was stopped by adding 100 μL / well.
Moreover, the control sample which performed operation similar to the above except not adding the test sample of ellagic acid was produced.
Further, in place of the ellagic acid test sample, a reference sample (Actinonin (manufactured by SIGMA) known as an aggrecanase activity inhibitor was added in the same manner as above except that a final concentration of 10 μM was added. Positive control) was prepared.

  For each sample, the absorbance at a maximum wavelength of 450 nm was measured with a microplate reader (manufactured by BIO-RAD). When the absorbance of the control sample was 100, the absorbance of each sample was expressed as a relative value, which was defined as aggrecan degradation activity (ADAMTS-5 enzyme activity). The results are shown in Table 1.

  As shown in Table 1, the aggrecan decomposition activity was very low in the system to which ellagic acid was added as compared with the control. That is, ellagic acid was found to inhibit aggrecanase activity and suppress aggrecan degradation.

Test example 2
SD rats (male, 7 weeks old) were divided into 7 groups (10 per group) according to body weight. Among these, six groups were subjected to ACLT (anterior cruciate ligament amputation) operation to produce osteoarthritis disease models. The remaining group was subjected to sham surgery (cutting only the skin) to form a sham group.
The SD rats were given 18 g of the test diet shown in Table 2 per day, and were reared for 10 weeks by restricted feeding.

The right limb of the SD rat was removed from the thigh, the excess tissue was removed so as not to damage the knee joint, and fixed with 10% neutral buffered formalin. This was decalcified with 10% EDTA and embedded in paraffin to prepare a paraffin block. Samples were prepared with a forehead cross section, and three continuous sections were prepared with a thickness of 4 to 5 μm at the same position. The prepared slices were each subjected to hematoxylin-eosin staining and safranin 0 staining, histological examination of the knee joint portion with an optical microscope, and scored based on the Mankin score.
Evaluation items and evaluation criteria based on the Mankin score are shown below.

(1) Cartilage tissue The destruction state of the cartilage tissue was evaluated in seven stages from score 0 to score 6.
Score 0: Normal Score 1: Irregular surface (Tangential zone) Score 2: Irregular pannus and surface Score 3: Cracks on the surface can be seen Score 4: Cracks in the radial zone can be seen Yes Score 5: Cracks in the Calcified zone are observed Score 6: Complete disappearance of the tissue (2) Chondrocytes The state of chondrocytes was evaluated in four stages from score 0 to score 3.
Score 0: Normal Score 1: Increased diffuse cell assembly Score 2: Cell division Score 3: Decreased cell assembly (3) Substrate staining by safranin 0 staining Decrease in proteoglycan in cartilage observed by safranin 0 staining Was evaluated in five stages from 0 to 4.
Score 0: Normal Score 1: Slightly decreased Score 2: Moderately decreased Score 3: Severely decreased Score 4: Not stained (4) Tide mark state Observed Tid mark state of articular cartilage, subchondral bone The progress of calcification from the side was evaluated in two stages from 0 to 1.
Score 0: intact Score 1: Crossing of blood vessels

  The results are shown in Table 3. Moreover, the microscope picture of the knee joint part of the SD rat of an ellagic acid group is shown in FIG. 1, and the microscope picture of the SD rat of a control group is shown to FIG. 2 (A) and (B), respectively.

As is clear from the results in Table 3, the osteoarthritis disease model of the ellagic acid group has a low Mankin score, suppresses the degradation of aggrecan that constitutes the articular cartilage extracellular matrix, and improves osteoarthritis You can see that.
In addition, in the osteoarthritis disease model in the control group as shown in FIG. 2, cracks on the cartilage surface and a decrease in substrate staining due to safranin O staining were observed, whereas ellagic acid was shown in FIG. These findings were not observed in the group of osteoarthritis disease models.

  From the above test examples, ellagic acid and ellagitannin metabolized to ellagic acid in the body inhibit aggrecanase activity, suppress aggrecan degradation, and suppress degradation of the articular cartilage extracellular matrix. Therefore, ellagic acid and ellagitannin can be suitably used as a preventive or ameliorating agent for preventing or ameliorating a symptom or disease caused by degradation of the articular cartilage extracellular matrix.

Formulation Example As the preventive or ameliorating agent of the present invention, the following preventive or ameliorating agents having the compositions shown below were prepared by conventional methods.

1. Production of Soft Capsule Soft capsule capsules having the following composition were filled with 400 mg of soybean oil and 100 mg of ellagic acid by a conventional method to produce soft capsules.
(Composition) (Blending amount)
Gelatin 70.0% by mass
Glycerin 22.9% by mass
Methyl paraoxybenzoate 0.15% by mass
Propyl paraoxybenzoate 0.51% by mass
6.44% by mass of water

2. Production of Flour Food (Cupcake) After preparing a dough having the following composition in the manner of preparing cupcake, it was divided into 10 molds, baked and produced by a conventional method, and a cupcake with good flavor was obtained.
(Composition) (Blending amount)
Soft flour 100g
100g chicken egg
100g sugar
Shortening 35g
Margarine 35g
Baking powder 2.5g
Western liquor 6g
Ellagic acid 15g
Appropriate amount of water

3. Manufacture of a powdered beverage After sufficiently mixing powders having the following composition, a 10-gram sachet was obtained, and a powdered beverage with a good flavor was obtained by dissolving in water or hot water for ingestion once a day.
(Composition) (Blending amount)
Glucose 60% by mass
Dextrin 15% by mass
Sodium citrate 5% by mass
Vitamin C 1% by mass
Ellagic acid 15% by mass
Fragrance (lemon flavor) 4% by mass

Claims (14)

  An aggrecan degradation inhibitor comprising at least one selected from the group consisting of ellagic acid and ellagitannin as an active ingredient.   The aggrecan degradation inhibitor according to claim 1, wherein the degradation of aggrecan is suppressed by inhibiting the activity of aggrecanase.   The aggrecan degradation inhibitor according to claim 2, wherein the aggrecanase is ADAMTS-5.   An aggrecanase activity inhibitor comprising at least one selected from the group consisting of ellagic acid and ellagitannin as an active ingredient.   The aggrecanase activity inhibitor according to claim 4, wherein the aggrecanase is ADAMTS-5.   An articular cartilage extracellular matrix degradation inhibitor comprising at least one selected from the group consisting of ellagic acid and ellagitannin as an active ingredient.   The joint cartilage extracellular matrix degradation inhibitor according to claim 6, wherein the articular cartilage extracellular matrix is aggrecan.   The joint cartilage extracellular matrix degradation inhibitor of Claim 7 which suppresses decomposition | disassembly of the said aggrecan by inhibiting the activity of aggrecanase.   The articular cartilage extracellular matrix degradation inhibitor according to claim 8, wherein the aggrecanase is ADAMTS-5.   A preventive or ameliorating agent for preventing or ameliorating a symptom or disease caused by degradation of an articular cartilage extracellular matrix, comprising at least one selected from the group consisting of ellagic acid and ellagitannin as an active ingredient.   The preventive or ameliorating agent according to claim 10, wherein the symptom or disease caused by degradation of the articular cartilage extracellular matrix is osteoarthritis.   The preventive or ameliorating agent according to claim 10 or 11, wherein the articular cartilage extracellular matrix is aggrecan.   The preventive or ameliorating agent according to claim 12, wherein the degradation of the aggrecan is suppressed by inhibiting the activity of aggrecanase.   The preventive or ameliorating agent according to claim 13, wherein the aggrecanase is ADAMTS-5.