JP2006282561A - Matrix metalloprotease inhibitor compounded with emblica officinalis gaertn. extract component - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new matrix metalloprotease(MMP) inhibitor excellent in the inhibitory effect on MMP activity. <P>SOLUTION: The new MMP inhibitor comprises an isolated component obtained by subjecting fruits of Emblica officinalis Gaertn. to extraction with a solvent. Compositions for the oral cavity, foods and cosmetics each containing the inhibitor are also provided, respectively. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel matrix metalloprotease inhibitor.

  The extracellular matrix is a biopolymer mainly composed of collagen, proteoglycan, elastin, and the like, and plays a role in filling gaps existing between cells constituting a living body tissue of a mammal. A group of metalloenzymes that act on the extracellular matrix and promote its degradation is generally called matrix metalloprotease (hereinafter simply referred to as “MMP”). The metabolic activity of the extracellular matrix is governed by the interaction between the MMP-specific tissue-derived metalloprotease inhibitor (hereinafter simply referred to as “TIMP”) and MMP.

  MMP is a substance that plays a central role in the degradation of extracellular matrix in various pathologies such as cancer (cancer) disease, ulceration, rheumatoid arthritis, osteoporosis, periodontitis, gingivitis (non-patent document). 1). Further, since MMP whose activity is enhanced by external stimuli such as ultraviolet rays decomposes components important for maintaining the structure of the skin, it also has an aspect as a skin aging promoting factor activated by ultraviolet rays.

  MMPs are produced by a wide variety of cells, including more than 10 types of collagenases (MMP-1 and MMP-8), stromelysin (MMP-3), gelatinases (MMP-2 and MMP-9), etc. Enzyme molecular species have been reported so far (see Non-Patent Document 2).

  Among these, collagenase is not normally produced from epithelial cells, but it has been reported that it is expressed around the adhesion epithelium and periodontal pocket epithelium of periodontal tissue (see Non-Patent Document 3). Gelatinase has been reported to degrade type IV collagen, which is produced from epithelial cells due to stimulation by lipopolysaccharide derived from periodontal disease-causing bacteria and is a constituent of basement membrane. That is, this is considered to suggest that the increase in MMP activity is closely correlated with the progression of periodontal disease.

  Actually, the amount of MMP contained in the mouth washes, gingival crevicular fluid, and saliva of periodontal disease patients reflects the pathology of the periodontal disease patients. It has also been reported that the amount has decreased (see Non-Patent Document 4). In addition, when the amount of MMP and TIMP held by gingivitis patients and healthy subjects were measured, the amount of MMP in gingivitis patients was very large, and the amount of MMP was significant for periodontitis patients. It has also been reported that the amount was (see Non-Patent Document 5). Therefore, a substance that inhibits MMP enzyme activity or MMP production is considered to be useful in promoting suppression of the progression of periodontal disease associated with worsening local inflammation.

  As MMP inhibitors, substances such as hydroxamic acid derivatives (see Patent Documents 1 and 2) and esculetin derivatives (see Patent Document 3) have been synthesized so far, and flavones and anthocyanidins derived from natural products have been synthesized. Inhibitors containing as an active ingredient have also been proposed (see Patent Document 4). Moreover, dexamethasone, which is one kind of glucocorticoids, has been synthesized as a substance that inhibits MMP production in cells (see Non-Patent Document 6).

  And as an inhibitor / substance derived from a natural product for the above collagenase, an inhibitor containing a koji extract or a thin load extract as an active ingredient (see Patent Document 5), an inhibitor containing nordihydroguaiaretec acid as an active ingredient ( Patent Document 6), an inhibitor containing polypolenic acid C as an active ingredient (see Patent Document 7), and a tetracyclic triterpene 20-carboxy-16-hydroxy-21- nor-5 α-7,9 (11) -lanostadien-3,24-dione (see Patent Document 8) has been reported so far.

Furthermore, an inhibitor containing grape seed extract as an active ingredient has been reported as an inhibitor of not only the activity of MMP but also the ability to produce MMP in cells (see Patent Document 9). In addition, recently, it has been clarified that the solvent extract of Mannentake exhibits inhibitory activity against MMP produced by cancer cells and also suppresses cancer metastasis involving MMP (Patent Literature). 10).
JP 7-505387 JP-A-8-81443 JP-A-8-183785 JP-A-8-104628 JP-A-6-183990 JP-A-4-217626 Japanese Patent Laid-Open No. 9-40552 JP 9-235293 A JP 2000-191487 A JP 2003-171307 A Nakata, Okada, "Respiration", 18, 4, pp.365-371 (1999) Yoshihara, Shinna, "Inflammation and Immunity", 2, pp.177-185 (1994) M. Kylmaniemi, et al: J. Dent Res., 75, pp.919-926 (1996) M. Makela, et al: J.M. Dent Res., 73, pp.1397-1406 (1994) A. Haerian, et al: J. Clin Periodontol., 22, pp.505-509 (1995) M. Kylmaniemi: Dent. Res., 75, pp.919-926 (1996)

  The object of the present invention is to provide a novel MMP inhibitor that has an excellent inhibitory effect on MMP activity and improves the preventive and therapeutic effects of oral diseases such as gingivitis and periodontitis.

  That is, the gist of the present invention is a medicinal plant that has been known since ancient times, and has been confirmed to be safe for humans, Amla (Latin language name: Emblica officinalis Gaertn., English scientific name: Emblic Myrobalan) in a solvent. It exists in the MMP inhibitor containing the isolation | separation component obtained by extraction. Moreover, according to the other aspect of this invention, the composition for oral cavity, foodstuffs, cosmetics, etc. which use the MMP inhibitor of this invention as an active ingredient are also provided.

  As a result of intensive research conducted by the present inventor, it has been found that the separated component obtained by extracting Amla with a solvent has an MMP inhibitory action, resulting in gingivitis and periodontitis (periodontal disease). And found applicability to uses such as prevention and treatment of oral diseases.

  According to the present invention, a novel MMP inhibitor that has an excellent inhibitory effect on MMP activity and is suitable for use in the prevention and treatment of oral diseases such as gingivitis and periodontitis is realized. In addition, oral compositions, foods, cosmetics and the like incorporating these MMP inhibitors as active ingredients are prepared, and by eating or using them, the effects of MMP inhibitors can be easily and continuously enjoyed. it can.

  The structure of the MMP inhibitor of the present invention will be described in detail below.

  According to one aspect of the present invention, there is provided an MMP inhibitor comprising a separation component obtained by extracting amla with a solvent. As used herein, the expression “MMP inhibition” refers to inhibition of enzyme activity of MMP and / or inhibition of production of MMP in cells. In other words, inhibition of intracellular MMP production means that MMP produced by cells constituting periodontal tissues of mammals suffering from diseases such as gingivitis and periodontitis are extracellular cells present between periodontal tissues. It is to inhibit a series of actions that break down the matrix and lead to destruction of the tissue. Inhibition of MMP production means that cells constituting the periodontal tissue of mammals suffering from diseases such as gingivitis and periodontitis inhibit MMP production and accumulate MMP in the same tissue. By suppressing, it means avoiding the destruction of periodontal tissue.

  Amla is a medicinal plant that has been used as a tonic in India since ancient times. The fruit contains about 3,000 mg of heat-stable vitamin C, which is very well absorbed by the body, and this content is comparable to the total amount of vitamin C contained in 12 oranges. And Amla fruit activates living tissue, increases red blood cell count, cleansing action in the mouth, hemostasis of gum bleeding, visual acuity improvement, bone regeneration, promotion of hair and nail growth, prevention of white hair formation, blood sugar level It is said to act on regulation and reduction of gastrointestinal inflammation.

  First, Amla fruit is applied to an extraction process using a solvent. The fruit applied to the extraction process may be applied to the extraction process as it is or after being physically crushed, or, if necessary, dried and crushed into a powder. it can.

  As the solvent used in this extraction step, a solvent well known in the technical field can be used. For example, a lower alcohol, a polyhydric alcohol, a nonpolar solvent, a polar solvent, and the like can be used.

  Examples of lower alcohols include alcohols having 1 to 4 carbon atoms such as methanol, ethanol, propanol, isopropyl alcohol and butanol; examples of polyhydric alcohols include glycerin and polyethylene glycol; examples of nonpolar solvents include pentane, hexane, heptane, Saturated hydrocarbons such as octane, nonane, decane; polar solvents include water, warm water (hot water), acetone, ethyl acetate, and methyl acetate. Of course, only one of the above-mentioned solvents can be selected and used, and two or more solvents can be used in combination. For example, in the case of using a raw material containing a large amount of fat, after performing a degreasing extraction treatment with a nonpolar solvent, the extraction treatment may be performed with any solvent, or the extraction treatment may be performed with a water-containing organic solvent.

  As a method for extracting Amla fruit, methods well known in the art can be used.

  For example, Amla fruits themselves, coarsely pulverized or cut products, or dried crushed materials (powder) are immersed in a solvent by immersion, digestion, etc. A method of obtaining an extract containing a desired separation component through filtration, a percolation method, and the like can also be used.

  The extract thus obtained is used as it is, or after removing unnecessary solid matter (solid content) by filtration or centrifugation, if necessary, depending on the use mode, or the solvent is distilled off. And partially concentrated or dried. The separated product obtained by concentrating or drying may be used after being purified by washing with an insoluble solvent, or may be used by further dissolving or suspending the product in an appropriate solvent.

  Alternatively, the obtained extract is used by obtaining and purifying a fraction having a desired inhibitory activity using a conventional purification method such as a countercurrent distribution method or liquid chromatography. Is also possible.

  Furthermore, the solvent extract obtained as described above can be processed into a dry concentrated extract form by a normal drying means such as drying under reduced pressure or freeze drying.

  Moreover, according to the preferable embodiment of this invention, the composition for oral cavity which uses the MMP inhibitor of this invention as an active ingredient is also provided. The amount of the separated component obtained by extracting Amla in the oral composition of the present invention with a solvent is usually about 0.0001 to about 20% by weight, preferably about 0.0001 to about 0.001% by dry weight. It is prepared in the range of 10% by weight. That is, if the blending amount of the separated component is less than about 0.0001% by weight, the corresponding MMP inhibitory effect is not expressed. On the other hand, if it exceeds about 20% by weight, the composition for oral cavity at the time of taking This results in an unpleasant taste, and a desired inhibition effect cannot be obtained.

  And unless it adversely affects the MMP inhibitory effect of the present invention, other components generally used in oral compositions, foods and cosmetics, such as pressure-sensitive adhesives, cooling agents, binders, sweeteners , Flavoring agents, disintegrating agents, lubricants, coloring agents, sustained release adjusting agents, surfactants, solubilizers, wetting agents, pH adjusting agents and the like can be optionally added.

  As the adhesive, water-soluble polymer materials derived from polysaccharides, cellulose polymer materials, synthetic polymer materials, natural polymer materials, amino acid polymer materials, rubber polymer materials, and the like can be used. For example, pullulan, pullulan derivatives and polysaccharides such as starch; hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, hydroxyethylmethylcellulose, carboxymethylcellulose salts (such as carboxymethylcellulose, sodium carboxymethylcellulose and potassium carboxymethylcellulose), methylcellulose, ethylcellulose, poly Acrylic acid, polyacrylate (sodium polyacrylate and acrylic acid / octyl acrylate copolymer), methacrylic acid copolymer (methacrylic acid and n-butyl acrylate polymer, methacrylic acid) Such as polymers of methyl methacrylate and methyl methacrylate and polymers of methacrylate and ethyl acrylate) Synthetic polymer materials such as carboxyvinyl polymer, polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol; lectin, alginic acid, alginate (sodium alginate, potassium alginate, magnesium alginate, propylene glycol alginate, triethanol alginate) Natural polymer substances such as amine, triisopropanolamine alginate, ammonium alginate, butylamine alginate and diamylamine alginate), sodium chondroitin sulfate, agar, chitosan, carrageenan; amino acid polymer substances such as collagen and gelatin; and gum arabic , Karaya gum, tragacanth gum, xanthan gum, locust bingham, guar gum, tamari Rubber polymer materials such as gum and gellan gum can be suitably used in the present invention.

  As the refreshing agent, l-menthol, dl-menthol, peppermint oil, camphor, peppermint water, borneol, peppermint essential oil, spearmint essential oil and the like can be used in the present invention, but are not limited thereto. Only one of them can be used, or two or more can be used in combination.

  Binders include sugars (such as glucose), sugar alcohols (such as sorbitol, xylitol, erythritol, mannitol), polyvinylpyrrolidone, starches, macrogol, dextrin, tragacanth, gelatin, polyvinyl alcohol, shellac, gum arabic, sodium alginate Hydroxypropyl cellulose and the like can be used in the present invention, but are not limited to these, and any one of them can be used, or two or more can be used in combination.

  As sweeteners, saccharin sodium, stevioside, stevia extract, aspartame, xylitol, starch syrup, honey, sorbitol, maltitol, mannitol, saccharides (lactose, sucrose, fructose, glucose etc.) can be used in the present invention. It is not limited to these, Only any one of these may be used, or two or more types may be used in combination.

  Natural flavors (spearmint oil, anise oil, eucalyptus oil, winter green oil, cassia oil, clove oil, thyme oil, sage oil, lemon oil, orange oil, peppermint oil, cardamom oil, coriander oil, mandarin oil are used as flavorings. , Lime oil, lavender oil, rosemary oil, laurel oil, camomil oil, caraway oil, marjoram oil, bay oil, lemongrass oil, orina gum oil, pine needle oil, etc., synthetic flavors (carvone, anethole, cineol, salicylic acid) Methyl, cinnamic aldehyde, eugenol, thymol, linalool, linalyl acetate, limonene, menthone, menthyl acetate, pinene, octylaldehyde, citral, pregon, carbyl acetate, anisaldehyde, etc.), natural fragrances listed above and / or A blended fragrance obtained by mixing a fragrance arbitrarily selected from synthetic fragrances can be used in the present invention. However, the present invention is not limited to these, and only one of these may be used, or 2 More than one type can be used in combination.

  As the disintegrant, starches, methylcellulose, crystalline cellulose, cellulose derivatives (such as sodium carboxymethylcellulose), alginic acid, alginate, carbonate, organic acid, polyvinylpyrrolidone, cross-linked polyvinylpyrrolidone and the like can be used in the present invention. It is not limited to these, Only any one of these can be used, or two or more can be used in combination.

  Lubricants include talc, metal soaps, fatty acids (such as stearic acid), stearates (such as magnesium stearate), talc, sucrose fatty acid esters, hydrous silicon dioxide, light anhydrous silicic acid, dry aluminum hydroxide gel, Macrogol and the like can be used in the present invention, but are not limited to these, and any one of them can be used, or two or more can be used in combination.

  As the colorant, blue No. 1, yellow No. 4, titanium dioxide, copper chlorophyllin sodium and the like can be used in the present invention. However, the present invention is not limited to these, and only one of these may be used. Alternatively, two or more types can be used in combination.

  Examples of preservatives include benzoic acids, salicylic acids, sorbic acids, parabens, cetylpyridinium chloride, decalinium chloride, benzethonium chloride, benzalkonium chloride, chlorhexidine chloride, chlorhexidine gluconate, isopropylmethylphenol, triclosan, hinokitiol, and phenol. Although it can utilize in this invention, it is not limited to these, Any one of these can also be used, or 2 or more types can also be used together.

  As the sustained release regulator, polyvinyl acetate, ethyl cellulose, aminoalkyl methacrylate copolymer and the like can be used in the present invention, but the invention is not limited thereto, and it is also possible to use only one of these, Or both can be used together.

  As the surfactant, sucrose fatty acid ester, polyoxyethylene hydrogenated castor oil, sorbitan monostearate, polysorbate, glyceryl monostearate, sodium lauryl sulfate, lauromacrogol and the like can be used in the present invention. There is no limitation, and only one of these can be used, or two or more can be used in combination.

  As the solubilizer, glycerin, concentrated glycerin, propylene glycol, ethanol, liquid paraffin, purified water, macrogol, polysorbate, and the like can be used in the present invention, but are not limited thereto, and any one of these can be used. Only one type can be used, or two or more types can be used in combination.

  As the wetting agent, glycerin, propylene glycol, sorbitol liquid, water, ethanol, dilute ethanol and the like can be used in the present invention, but are not limited to these, and only one of these may be used. Alternatively, two or more types can be used in combination.

  As the pH adjuster, lactic acid, pantothenic acid, phosphate, malic acid, citric acid, and the like can be used in the present invention, but are not limited thereto, and only one of these may be used. Alternatively, two or more types can be used in combination.

  As the dosage form of the oral composition of the present invention, any of tablets, pills, granules, liquids, sheets, gels, pastes and the like can be used. In particular, when the oral composition of the present invention is used as a dental preparation, periodontitis, gingivitis, periodontitis, wisdom teeth can be obtained in the form of toothpaste, mouthwash, oral paste, troche, etc. It can effectively cope with prevention and treatment of oral diseases caused by destruction of periodontal tissues such as peritonitis and peri-implantitis.

  The present invention will be described below with reference to examples thereof, but it is needless to say that the present invention should not be construed in a limited manner based on the disclosure of the examples.

A: Preparation of MMP inhibitor 900 ml of 50% ethanol was added to 100 g of dried Amla (Emblica officinalis Gaertn.) Fruit, and extraction was performed at room temperature for 7 days. Thereafter, the extract was filtered, and the obtained filtrate was concentrated to dryness to obtain 1.9 g of Amla dry extract.

  As a control sample, 20 g of dried fennel (Foeniculum vulgare Mill.) Fruit was selected, 400 ml of purified water was added thereto, and extraction was performed at 90 to 100 ° C. for 2 hours. Thereafter, the extract was filtered, and the obtained filtrate was concentrated to dryness to obtain 1.4 g of a dried fennel extract.

  A solution prepared by dissolving 0.02 g / ml (2% W / V) of each dry extract thus obtained in the solvent used in the extraction step was used in the subsequent Examples.

B: Assay of MMP inhibitory effect Regarding the MMP inhibitor prepared in Example A, the effect on the enzyme activity of gelatinase (MMP-2 and MMP-9) was assayed using gelatin zymography.

  As an MMP-2 sample, “MMP-2, Active, Human” (5 μg, CALBIOCHEM, catalog # PF023), 30 mM HEPES (pH 7.4) [contains 5 mM calcium chloride, 0.2 M sodium chloride, 0.02% sodium azide] Adjusted to 2 ml and stored at -20 ° C.

  Similarly, as an MMP-9 sample, “MMP-9, Recombinant, Active (67 kD)” (5 μg, CALBIOCHEM, catalog # PF140), 30 mM HEPES (pH 7.4) [5 mM calcium chloride, 0.2 M sodium chloride, 0.02% Containing sodium azide], adjusted to 2 ml and stored at -20 ° C.

  Next, 10% SDS-PAGE gel [1 mm separation gel; 0.3 mg / ml gelatin, 10% acrylamide / 0.8% bisacrylamide, 0.100% SDS (sodium dodecyl sulfate), 0.085% APS (adenylyl sulfate), 0.130% TEMED (containing N, N, N ′, N′-tetramethylethylenediamine)] was prepared. Next, the MMP-2 sample and MMP-9 sample converted to SDS with 250 mM Tris-HCl (pH 6.8) [containing 10% SDS and 50% glycerol] were placed in a groove for 2.0 μl and 1.5 μl sample application, respectively. According to the method, gelatin zymography was performed under an electric load condition of 250 V and 20 mA.

  As a buffer for the electrophoresis phase of SDS-PAGE, a buffer containing 25 mM Tris, 192 mM glycine, and 0.1% SDS was used. After the electrophoresis was completed, the separation gel was immersed twice in a 2.5% Triton X-100 (SIGMA) solution at room temperature for 30 minutes to remove SDS. Then, 50 mM Tris-HCl (pH 7.4) containing 200 mM sodium chloride, 5 mM calcium chloride, 0.02% sodium azide was added to the gel for concentration [3% acrylamide / 0.8% bisacrylamide, 0.125 M Tris-HCl (pH 6.8). , 0.100% SDS, 0.095% APS, 0.250% TEMED], and incubated at 37 ° C. for 19 hours. At this time, the sample solution of each dry extract (concentration of 1% and 0.1%) prepared in Example A was added to the Tris-HCl buffer.

  After the reaction was completed, the gel for concentration was immersed in a solution of 0.2% GelCode Blue Stain Reagent and shaken for 1 hour. Then, it was washed with distilled water for decolorization, and gelatinase activity appeared as a non-stained band on the blue gel (FIG. 1).

  1, FIG. 1 (a) is a test plot using a separated component obtained by extracting Amla (Emblica officinalis Gaertn.) With a solvent, and FIG. 1 (b) is a fennel (Foeniculum vulgare). Mill.) Is a test section using a separated component obtained by extraction with a solvent. The MMP-2 sample is placed in the left lane and the MMP-9 sample is placed in the right lane toward the center line of the broken line written on each gel, and subjected to electrophoresis. A gel containing no MMP inhibitory component was used as a negative control, and a gel containing o-phenanthroline (1 mM) was used as a positive control.

  By visually observing a non-stained band (outlined part) appearing on the gel, the MMP inhibitory effect of the MMP inhibitor prepared in Example A was significantly inhibited (+++) and strongly inhibited (++ ), And the normal inhibitory effect (+) and non-inhibitory effect (−), were graded according to a four-step evaluation. The evaluation results are summarized in Table 1 below.

As is apparent from the results shown in FIG. 1 and Table 1, the MMP inhibitor of the present invention containing a separation component obtained by extracting Amla (Emblica officinalis Gaertn.) With a solvent can exhibit an MMP inhibitory effect. confirmed. In addition, the MMP inhibitor of the present invention had sufficient MMP inhibitory activity for practical use despite its low concentration.

C: Preparation of oral composition Various oral compositions containing Amla extract (1.0%) were prepared.

  In addition, the unit of the component compounding amount in a present Example is displayed by weight%, unless there is particular notice.

1: Dentifrice Toothpaste was prepared by mixing and kneading the materials shown in Table 2 below at room temperature.

2: Dentifrice Toothpaste was prepared by mixing and kneading the materials shown in Table 3 below at room temperature.

3: Mouthwash The materials shown in Table 4 below were mixed at room temperature to prepare a mouthwash.

D. Preparation of Food Various foods containing Amla extract (1.0%) were prepared.

  In addition, the unit of the component compounding amount in a present Example is displayed by weight%, unless there is particular notice.

1: Chewing gum The ingredients listed in Table 5 below were mixed at room temperature to prepare chewing gum.

2: Solid formulation (tablet)
The materials listed in Table 6 below were mixed and tableted at room temperature to prepare tablets.

3: The material according confectionery in Table 7 below, at room temperature, was prepared by mixing the candy confectionery (candy).

E: Preparation of cosmetics Various cosmetics containing Amla extract (1.0%) were prepared.

  In addition, the unit of the component compounding amount in a present Example is displayed by weight%, unless there is particular notice.

1: Lotion Toner lotion was prepared by mixing the materials shown in Table 8 below at room temperature.

2: Cosmetic cream The ingredients listed in Table 9 below were mixed at room temperature to prepare a cosmetic cream.

3: Emulsion The materials described in Table 10 below were mixed at room temperature to prepare an emulsion.

  The MMP inhibitor of the present invention is useful as a means for preventing and treating oral diseases such as gingivitis and periodontitis.

It is a figure which shows the result of the electrophoresis implemented regarding the sample containing the separation component obtained by carrying out solvent extraction of (a) Amla (Emblica officinalis Gaertn.) and (b) Fennel (Foeniculum vulgare Mill.).

Claims (4)

  A matrix metalloprotease inhibitor characterized by comprising a separation component obtained by extracting Ammura (Emblica officinalis Gaertn.) With a solvent.   An oral composition comprising the matrix metalloprotease inhibitor according to claim 1.   A food comprising the matrix metalloprotease inhibitor according to claim 1. A cosmetic comprising the matrix metalloprotease inhibitor according to claim 1.