JP2013014550A - Aggrecan degradation inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new aggrecan degradation inhibitor.SOLUTION: The aggrecan degradation inhibitor contains a litchi extract as an active ingredient.

Description

  The present invention relates to an aggrecan degradation inhibitor, an aggrecanase activity inhibitor, and an articular cartilage extracellular matrix degradation inhibitor. The present invention further relates to a preventive or therapeutic agent for symptoms or diseases caused by degradation of the articular cartilage extracellular matrix.

In recent years, the population affected by bone and joint diseases has increased. Many bone and joint diseases tend to increase in number with age, and with the recent aging, the affected population is expected to further increase.
Osteoarthritis, a bone and joint disease, is a progressive disease characterized by destruction, degeneration and loss of articular cartilage. The total number of patients in the country is estimated to be about 8 million, which is far larger 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 agents have problems such as side effects, and safer and more effective preventive / therapeutic agents 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 proteoglycan. In a healthy person's cartilage tissue, the metabolic balance between the production of cartilage matrix collagen and proteoglycan (aggrecan) and cartilage matrix degradation by cartilage matrix degrading enzymes is properly maintained. 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.

Known enzymes that degrade cartilage matrix include metalloproteases belonging to the ADAMTS (A Disintegrin And Metalloproteinase with ThoromboSpondin motifs) family and metalloproteases belonging to the MMP (Matrix Metalloproteinase) family. These families include the enzyme aggrecanase that degrades aggrecan and the enzyme MMP that degrades collagen and aggrecan.
In recent studies, cartilage matrix destruction has been preceded by enzymatic degradation and disappearance of aggrecan, followed by enzymatic degradation of collagen, and aggrecan degradation in joint fluid collected from osteoarthritis patients. It has been reported that many products are observed. Furthermore, 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 1).

Ann-Marie, et al .; THE JOURNAL OF BIOLOGICAL CHEMISTRY 277; 22201-22208, 2002

  An object of the present invention is to provide an aggrecan degradation inhibitor, an aggrecanase activity inhibitor, and an articular cartilage extracellular matrix degradation inhibitor. Another object of the present invention is to provide a preventive or therapeutic agent for symptoms or diseases caused by degradation of the 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 the lychee extract 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 degradation inhibitor containing a lychee extract as an active ingredient.
The present invention also relates to an aggrecanase activity inhibitor containing a lychee extract as an active ingredient.
The present invention also relates to an articular cartilage matrix degradation inhibitor containing a lychee extract as an active ingredient.
Furthermore, the present invention relates to a prophylactic / therapeutic agent for preventing or ameliorating a symptom or disease (for example, osteoarthritis) caused by degradation of an articular cartilage extracellular matrix, containing a lychee extract as an active ingredient.

  According to the present invention, an aggrecan degradation inhibitor, an aggrecanase activity inhibitor, and an articular cartilage extracellular matrix degradation inhibitor can be provided. Moreover, according to this invention, the preventive agent, therapeutic agent, foodstuff, and drink for preventing and improving the symptom or disease caused by decomposition | disassembly of an articular cartilage extracellular matrix can be provided.

Hereinafter, the present invention will be described in detail.
The aggrecan degradation inhibitor and aggrecanase activity inhibitor of the present invention contain a lychee extract as an active ingredient. As shown in the examples described below, lychee extract significantly reduces aggrecanase activity and suppresses aggrecan degradation by aggrecanase.
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 for protein and gene expression in the cartilage of osteoarthritis patients (Ann-Marie, et al .; THE JOURNAL OF BIOLOGICAL CHEMISTRY 277; 22201-22208, 2002). In particular, ADAMTS-5 is considered to play a central role in aggrecan destruction. In ADAMTS-5 gene-deficient mice, destruction of cartilage matrix is significantly suppressed, and ADAMTS-5 is involved in arthritis. (Glasson SS, et al .; Nature; vol. 434: 644-648, 2005 and Stanton H, et al .; Nature; vol. 434: 648-652, 2005).
In particular, the aggrecanase activity inhibitor of the present invention suitably inhibits the activity of aggrecanase ADAMTS-5.

Furthermore, the present invention provides an articular cartilage extracellular matrix degradation inhibitor containing a lychee extract as an active ingredient. As described above, the cartilage matrix contains aggrecan and collagen, and the degradation of the entire articular cartilage extracellular matrix can be suppressed by suppressing the degradation of aggrecan by aggrecanase by the action of the lychee extract.
The fact that lychee extract has an aggrecan degradation inhibitory action, an aggrecanase activity inhibitory action, and an articular cartilage matrix degradation inhibitory action has never been known so far, and is a knowledge newly obtained by the present inventors.

The present invention also provides a prophylactic / therapeutic agent containing lychee extract as an active ingredient for the purpose of preventing / ameliorating various symptoms and diseases caused by degradation of the articular cartilage extracellular matrix. The action of the lychee extract can suppress excessive aggrecan degradation, repair the metabolic balance of the cartilage matrix inclined to degradation, increase the decreased aggrecan, and normalize the cartilage matrix.
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 / therapeutic agent of the present invention is preferably used for the prevention / amelioration of osteoarthritis. Osteoarthritis refers to a disease characterized by degeneration and wear of articular cartilage, hardening of subchondral bone, and proliferative change.

The extract used in the present invention is made from lychee. Lychee is a plant belonging to the genus Litchiaceae , which has the scientific name: Litchi chinensis , and is also called litchi.
Any arbitrary part of the lychee can be used for the production of the extract. For example, the whole tree or any part (root, rhizome, trunk, branch, stem, leaf, bark, sap, resin, flower, fruit, seed, etc.) can be used, and a plurality of these may be used in combination. Especially, in this invention, it is preferable to extract using the fruit of a lychee. Lychee fruit has been used as an edible food, and has high safety.

For extraction of the lychee extract, a normal extraction method can be used. The lychee extract used in the present invention may be an extract obtained using a solvent or the like, or a fruit juice obtained by squeezing a fruit.
Although the extraction method is not particularly limited, it is preferably obtained by extracting the plant at room temperature or under heating, or by using an extraction tool such as a Soxhlet extractor.
For the preparation of the extract, the above plant can be used as it is or after dry pulverization. Moreover, the steam-distilled material or pressing material of the said plant can also be used, These can also use what refine | purified from essential oil etc., and can also use a commercial item. You may use the said plant or its steam distillate or pressing material individually or in combination of 2 or more types.

  When a solvent is used for extraction, the solvent is not particularly limited, but those usually used for extraction of plant components such as water, petroleum ether, n-hexane, toluene, dichloroethane, chloroform, ether, ethyl acetate, acetone, methanol, Ethanol, propanol, butanol, ethylene glycol, propylene glycol, butylene glycol, supercritical carbon dioxide and the like can be used. Of these, water, ethanol, and acetone are preferably used. These can be used alone or in combination of two or more. Further, acid or alkali may be added during extraction to adjust the pH of the extraction solvent.

  Extraction conditions can also apply normal conditions, for example, what is necessary is just to immerse or heat-reflux the said plant at 0-100 degreeC for 0.5 hour-30 days. The amount of the extraction solvent to be used is 1 to 50 times, preferably 5 to 20 times the weight of the plant.

In the present invention, the lychee extract may be used as it is, or a fraction having high activity may be fractionated and used by suitable separation means such as adsorption resin purification, gel filtration, chromatography, precision distillation and the like. it can. Further, the obtained lychee extract can be diluted, concentrated or lyophilized, and then prepared into a powder or paste form for use.
In the present invention, the lychee extract includes various solvent extracts obtained by the extraction method as described above, diluted solutions thereof, concentrated solutions thereof, purified fractions thereof or dried powder thereof.

Although the following method can be illustrated as a specific extraction method of a lychee extract, this invention is not limited to this.
Extraction method of lychee extract: First, 1 L of 50% ethanol water is added to 100 g of lychee fruit, and immersion extraction is performed at 20 ° C. for 7 days. Thereafter, the insoluble extraction residue is removed by filtration using filter paper to obtain an extract. The obtained extract is concentrated under reduced pressure and then freeze-dried to obtain an extract.

The lychee extract used in the present invention preferably contains lychee-derived polyphenol as a main component. In particular, the lychee extract preferably contains 40% by mass or more of lychee-derived polyphenol. The lychee-derived polyphenol can be obtained, for example, by purifying the lychee extract obtained by the above-described method using an adsorption resin or the like described later.
Furthermore, in the present invention, it is preferable to use a lychee extract in which the polyphenol component has a low molecular weight. The lychee extract in which the polyphenol component has been reduced in molecular weight is a lychee extract whose main component is a low-polymer polyphenol having a polymerization degree of catechin molecules of 5 or less (preferably 10% by mass or more is contained in the lychee extract). Say things.
Such a low molecular weight polyphenol can be obtained by decomposing and fragmenting a high polymer polyphenol in which 10 or more units of catechin molecules are polymerized (with a polymerization degree of 10 or more). As a method for reducing the molecular weight of the polyphenol component of the extract, for example, the method described in International Publication No. 2006/090830 can be referred to, and this method can also be preferably used in the present invention. Specifically, the lychee extract can be obtained by thermally decomposing in an acidic solution together with a substance having a phloroglucinol ring or resorcinol ring structure and then purifying with an adsorption resin or the like.

Examples of substances having a phloroglucinol ring or resorcinol ring structure used to lower the molecular weight of lychee extract include phloroglucinol, resveratrol, catechin, epicatechin, catechin gallate, epicatechin gallate, gallocatechin, epigallocatechin , Gallocatechin gallate, epigallocatechin gallate, green tea extract, grape skin extract and the like. Especially, in this invention, it is preferable to use a green tea extract.
A commercially available product can be used as the compound having the phloroglucinol ring or resorcinol ring structure. Further, it can be chemically synthesized by a usual method.
Extraction methods of plant extracts such as green tea extract and grape skin extract are not particularly limited, and general plant extraction methods can be used. Moreover, it can also carry out based on the extraction method of the said lychee extract. As the green tea extract, an extract obtained from the leaves of Camellia sinensis is preferably used, and water and ethanol are preferable as the extraction solvent, and water is particularly preferable. Moreover, 20-100 degreeC is preferable and, as for extraction temperature, 80-100 degreeC is especially preferable. The extraction time is preferably 30 seconds to 10 days, particularly preferably 1 to 5 minutes. An example of a specific extraction method is a method of adding 100 mL of hot water at 98 ° C. to 10 g of green tea leaves, extracting for 2 minutes, obtaining an extract by filtration, and then lyophilizing to obtain an extract. However, the present invention is not limited to this.

The amount of the substance having a phloroglucinol ring or resorcinol ring structure used together with the lychee extract is not particularly limited as long as it can reduce the molecular weight of the polyphenol derived from the lychee extract.
The reaction between the lychee extract and a substance having a phloroglucinol ring or resorcinol ring structure is carried out by heating in a solvent. As the reaction solvent, one kind or a mixture of two or more kinds of water, methanol, ethanol and the like are used. In consideration of the use of the agent of the present invention for applications such as pharmaceuticals and foods, it is preferable to use water and ethanol. The reaction is preferably performed under acidic conditions. The acid can be appropriately selected from inorganic acids such as hydrochloric acid, sulfuric acid and nitric acid, or organic acids such as acetic acid, citric acid, ascorbic acid and malic acid. These acids are used at a concentration of about 0.1N to 1.0N, preferably about 0.5N. The reaction between the lychee extract and the substance having a phloroglucinol ring or resorcinol ring structure is preferably carried out at room temperature to 100 ° C. for 0.5 hour to 1 week, more preferably at 90 to 100 ° C. for 1 to 4 hours. preferable.

After the above reaction, the solid content is removed and separated by a method such as filtration to obtain a lychee extract containing a polyphenol component having a reduced molecular weight. Moreover, you may perform processes, such as concentration, drying, fractionation, and purification, after filtration. For example, the target component can be separated, concentrated, and purified by filtering the residue from the reaction solution and treating the obtained filtrate with a membrane treatment (ultrafiltration, reverse osmosis, etc.) or an adsorbent.
As the adsorbent, styrene-divinylbenzene adsorbent, methacrylic acid adsorbent, hydrophilic vinyl polymer, modified dextran gel, polyacrylamide gel, reverse phase silica gel, ion exchange resin and the like are used. By eluting the fraction adsorbed on these adsorbents with hydrous alcohol, alcohol, acetone or the like, a lychee extract having a polyphenol component reduced in molecular weight can be obtained.

Another method for reducing the molecular weight of the polyphenol component of the lychee extract is, for example, a method in which the crude extract is heated using an inorganic acid such as hydrochloric acid or an organic acid.
A commercially available product can be used as the lychee extract in which the polyphenol component has been reduced in molecular weight. An example of a commercially available product is oligonol (trade name, manufactured by Amino Up Chemical Co., Ltd.).

Aggrecan degradation inhibitor, aggrecanase activity inhibitor, articular cartilage matrix degradation inhibitor, and prophylactic / therapeutic agent for preventing / ameliorating symptoms or diseases caused by degradation of the articular cartilage extracellular matrix (hereinafter collectively) For the “agent of the present invention” and “each agent of the present invention”, a lychee extract may be used as it is. Further, the composition may be obtained by adding other components. As other components, for example, a suitable liquid or solid excipient or extender such as titanium oxide, calcium carbonate, distilled water, lactose, starch and the like can be added. The amount of the lychee extract contained in the agent is not particularly limited, but the lychee extract is preferably contained in an amount of 0.0001 to 100% by mass, more preferably 0.01 to 80% by mass in terms of solid content. The content is preferably 0.1 to 80% by mass.
Although the dosage form of the agent of this invention is not specifically limited, For example, liquid preparations, such as solid preparations, such as a powder, a granule, a capsule, a pill, and a tablet, a liquid agent, a suspension agent, an emulsion, etc. are mentioned.

Each of the above-mentioned agents of the present invention is a pharmaceutical composition for humans or animals or a quasi-drug composition for the purpose of preventing or treating various symptoms or diseases caused by the degradation of articular cartilage extracellular matrix such as aggrecan. Can be applied to. For example, it can be used as an additive or a compounding agent for a pharmaceutical composition or a quasi-drug composition. Additives and compounding agents for such pharmaceutical compositions and quasi-drug compositions are additives or compounding agents usually used for pharmaceuticals such as fragrances, pigments and antioxidants, and the lychee extract as an active ingredient. Can be prepared by mixing.
For drugs and quasi-drugs, in addition to the agent of the present invention, pharmaceutically acceptable carriers such as auxiliaries, stabilizers, wetting agents, emulsifiers, absorption promoters and surfactants, activators, etc. Additives can be blended in any combination. In the case of a pharmaceutical composition for oral administration, in addition to the agent of the present invention, excipients, disintegrating agents, binders, lubricants, surfactants, alcohols generally used according to the form of the oral administration agent, Water, a water-soluble polymer, a sweetener, a corrigent, a sour agent, and the like can be added and produced according to a conventional method.
When used for pharmaceuticals, etc., the amount of the agent of the present invention is appropriately selected and determined according to the patient's age, symptoms, patient level, administration route, administration schedule, formulation form, strength of inhibitory activity of the material, etc. . For example, in the case of oral administration, generally 0.001 to 10 g / kg body weight per day is preferable, and the dose may be divided into several times a day.

Moreover, you may use each agent of this invention as an additive or compounding agent for foodstuffs or drinks. Such food or beverage additive or compounding agent is a mixture of the above-mentioned lychee extract as an active ingredient in an additive or compounding agent usually used for foods and beverages such as fragrances, pigments and antioxidants. Can be prepared.
The shape of the food and drink to which the agent of the present invention is applied is not particularly limited. For example, after adding an appropriate auxiliary agent to the agent of the present invention, it is provided by using ordinary means to form it into an edible form, for example, granular, granular, tablet, capsule, paste, liquid, etc. be able to. Food and beverages include, for example, glucose, fructose, sucrose, maltose, sorbitol, stevioside, rubusoside, corn syrup, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dL-α-toco Phenol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester gum arabic, carrageenan, casein, gelatin, pectin, agar, vitamin B, Nicotinamide, calcium pantothenate, amino acids, calcium salts, pigments, fragrances, preservatives, and other additives used in normal foods and beverages can be appropriately blended and manufactured according to conventional methods. Kill.
When used in foods, beverages, etc., the intake of the agent of the present invention is appropriately selected and determined according to the age, weight, symptoms, strength of the inhibitory activity of the material, and the like. In general, 0.001 to 10 g / kg body weight per day is preferable, and it may be taken in several divided doses per day.

  In another aspect, the present invention provides a method for inhibiting aggrecan degradation, a method for inhibiting aggrecanase activity, a method for inhibiting degradation of articular cartilage extracellular matrix, and a condition or disease caused by degradation of articular cartilage extracellular matrix. Prophylactic / therapeutic methods can be provided. The method is characterized in that the lychee extract is applied to the human or animal body. The application site and form are not particularly limited, and are the same as the application site and application form when the above-described agent of the present invention is used as a pharmaceutical product, quasi-drug, food, beverage or the like. Further, the application amount is the same as the use or intake when the above-described agent of the present invention is applied to pharmaceuticals, quasi drugs, foods, beverages and the like.

Next, a method for searching for a substance having an aggrecanase activity inhibitory action and an aggrecan degradation inhibitory action will be described.
Aggrecan degradation at the cartilage matrix occurs by cleaving two specific sites in the core protein of aggrecan. These two cleavage sites are between the 341st asparagine residue and the 342nd phenylalanine residue (hereinafter referred to as Asn341-Phe342), the 373rd glutamic acid residue, and the 374th alanine residue. Between the residues (hereinafter referred to as Glu373-Ala374), the former Asn341-Phe342 is cleaved by MMP-3 and the like, and the latter Glu373-Ala374 is cleaved by aggrecanase. 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 amount of cleavage fragments between Glu373-Ala374 are included, it is considered that aggrecanase plays an important role in cartilage matrix destruction . In the present invention, focusing on the cleavage between aggrecan Glu373-Ala374 by aggrecanase, an aggrecan degradation inhibitor was searched.

One aspect of the search method used in the present invention is shown below, but the present invention is not limited to this.
ADAMTS-5 which is one of aggrecanases is used as an enzyme, and an aggrecan in an interglobular domain (IGD) region containing Glu373-Ala374 as a substrate is used. 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 cleavage site Glu373-Ala374 by aggrecanase 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, the Glu373-Ala374 bond in aggrecan is cleaved, and an aggrecan degradation product is produced. This aggrecan degradation product contains two types of aggrecan fragments, one with Glu373 at the C-terminus and the other with Ala374 at the N-terminus, both of which are quantitatively produced according to the aggrecanase activity intensity. . In the IGD region on the aggrecan used as a substrate, the above-mentioned Glu373-Ala374 is the only site cleaved by the enzyme ADAMTS-5.
Next, an aggrecan fragment having Ala374 at the N-terminus is detected and quantified using an antibody that specifically binds to the N-terminus Ala374. Alternatively, an aggrecan fragment having Glu373 at the C-terminus may be detected and quantified using an antibody that specifically binds to C-terminal Glu373. Alternatively, detection and quantification can be performed using an antibody that specifically binds to the aggrecan fragment as the 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 no extract sample was added, the aggrecan degradation inhibitory effect and the aggrecanase activity inhibitory effect due to the addition of the sample can be confirmed.

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

Example 1. Preparation of lychee extract (1) Lychee extract sample 1
495 g of lychee ( Litchi chinensis fruit, made in China) was freeze-dried, pulverized in a mill, 1 L of 50% ethanol water was added, and immersion extraction was performed at room temperature for 1 day. The insoluble material was filtered off, concentrated under reduced pressure, and lyophilized to obtain 62.5 g of a lychee extract. The obtained lychee extract was dissolved in 50 v / v% ethanol water so as to have an extract concentration of 1 w / v%, and lychee extract sample 1 was obtained.

(2) Lychee extract samples 2, 3
100 mg of the lychee extract obtained above was adsorbed on a Sephadex LH-20 (trade name, 20 × 65 mm, manufactured by GE Healthcare) column, washed with 60 mL of 50% methanol water, and eluted with 60 mL of methanol. The obtained fraction was designated as W3 fraction (1.8 mg). Next, the fraction obtained by elution with 60 mL of 70% acetone water was designated as W4 fraction (2.4 mg). Each of the obtained W3 fraction and W4 fraction was dissolved in 50 v / v% ethanol water to a concentration of 1 w / v%, and lychee extract sample 2 (W3 fraction) and sample 3 (W4 fraction) ).

(3) Lychee extract sample 4
Oligonol (trade name, manufactured by Amino Up Chemical Co., Ltd.) obtained by extracting the fruit of lychee ( Litchi chinensis ), mixing with green tea extract, reducing the molecular weight by heating, and purifying with an adsorption resin Was dissolved in 50 v / v% ethanol water so as to have a concentration of 1 w / v% to obtain a lychee extract sample 4.

  The amount of polyphenol in each lychee extract obtained above was calculated by the foreign-dennis method. Ao, (+)-catechin was used as a standard substance. The results are shown in Table 1.

2. Verification of the effect of inhibiting aggrecan degradation For the lychee extract samples 1 to 4 prepared in Step 1, the aggrecan degradation inhibitory effect was verified by the following procedure. In the following operation, Tris Buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM CaCl ₂ ) was used for dilution of the lychee extract, 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 the lychee extract sample prepared above 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 lychee extract sample was prepared at a final concentration of 0.0001%. 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 a lychee extract sample was produced.

  About each obtained sample, the light absorbency of the maximum wavelength 450nm was measured with the microplate reader (made 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 2.

In addition, about the effectiveness of the said evaluation system, it verified using actinonin (Actinonin) known as an aggrecanase activity inhibitor as a positive control.
In the above system, a sample in which actinonin (manufactured by SIGMA) was added to a final concentration of 10 μM instead of the lychee extract was prepared, and aggrecan degradation activity was evaluated. As a result, aggrecanin-added sample significantly decreased the aggrecan degradation activity, confirming the effectiveness of the evaluation system.

  As is clear from Table 2, it was found that the system to which the lychee extract sample 1 was added had significantly lower aggrecan degradation activity than the control, and aggrecan degradation was suppressed. In particular, the lychee extract samples 2 to 4 having a polyphenol content of 40% by mass or more exhibited a further excellent aggrecan degradation inhibitory action. From the above results, it was confirmed that the lychee extract inhibits aggrecanase activity and effectively suppresses aggrecan degradation.

Claims (6)

  An aggrecan degradation inhibitor containing a lychee extract as an active ingredient.   An aggrecanase activity inhibitor containing lychee extract as an active ingredient.   The aggrecanase activity inhibitor according to claim 2, wherein the aggrecanase is ADAMTS-5.   Articular cartilage extracellular matrix degradation inhibitor containing lychee extract as an active ingredient.   A prophylactic / therapeutic agent for preventing or ameliorating a symptom or disease caused by degradation of an articular cartilage extracellular matrix, comprising a lychee extract as an active ingredient.   6. The preventive / therapeutic agent according to claim 5, wherein the symptom or disease caused by degradation of the articular cartilage extracellular matrix is osteoarthritis.