JP5294536B2 - Gingival epithelial cell spreading inhibitor - Google Patents

Description

  The present invention relates to a novel gingival epithelial cell extension inhibitor that inhibits abnormal extension of gingival epithelial tissue involved in the cause of oral diseases such as gingivitis and periodontitis (periodontal disease).

  Periodontal tissue surrounding the tooth neck and root of a mammalian tooth is composed of gingiva (gum), periodontal ligament, cementum and alveolar bone, and plays a role of supporting the tooth. Inflammatory symptoms appear in this periodontal tissue, the state where the inflamed area stays in the gingiva is "gingivitis", and the inflamed area extends beyond the gingiva to the periodontal ligament and alveolar bone, leading to tissue destruction Is distinguished from "periodontitis". In both gingivitis and periodontitis, inflammation in the periodontal tissue is affected by plaques formed by the growth of bacteria attached to food residues in the oral cavity and other environmental factors. Due to For gingivitis, the site of inflammation is limited to the gingiva, and inflammation has not spread to the periodontal ligament and alveolar bone, so the symptoms are generally alleviated by physically cleaning the mouth (for example, brushing teeth). Or can be cured. On the other hand, if the symptom progresses to periodontitis, the connective tissue adhesion between the gingiva and the tooth is lost, so that not only the tooth swings but also the periodontal ligament and alveolar bone. It leads to damage and destruction, and it is difficult to expect periodontitis to recover even if the oral cavity is simply physically cleaned. In addition, as the periodontal tissue is destroyed due to the development of periodontitis, the gingival epithelial cells expand toward the root of the tooth (downgrowth). Grooves, so-called periodontal pockets, are formed.

  As a method of chemically treating periodontal inflammation involved in the development of periodontitis with a pharmaceutical compound, gingivitis is achieved by using ε-aminocaproic acid, which has an excellent anti-inflammatory effect, and its antiplasmin action. And methods for suppressing the onset of periodontitis symptoms. Alternatively, there is a technique using a drug for removing Tetracycline-resistant staphylococci that is a causative agent of periodontal tissue inflammation, that is, a tetracycline-based minocycline hydrochloride.

  In addition, macrolide antibiotics, specifically the number of atoms that make up the lactone ring, are used to remove the causative bacteria that have settled in the periodontal pockets and their staying phase (biofilm). Depending on the case, there are 14-membered to 16-membered substances such as erythromycins, 16-membered macrolide josamycins, clarithromycins and the like. Among these, 14-membered macrolide antibiotics are useful because they can effectively prevent and eliminate biofilm formation.

  By the way, since about 60% of the components of the periodontal tissue is collagen, when the degradation of collagen proceeds, a phenomenon of gingival shrinkage and receding is induced. A group of metalloenzymes that act on the extracellular matrix such as collagen to promote its degradation is generally called matrix metalloprotease (hereinafter simply referred to as “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 in addition to periodontitis and gingivitis. (Refer nonpatent literature 1). MMP is produced by a wide variety of cells, and there are 10 types such as collagenase (MMP-1 and MMP-8), stromelysin (MMP-3), gelatinase (MMP-2 and MMP-9). The above 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.
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)

  The present invention relates to a novel method for inhibiting abnormal extension of gingival epithelial tissue in mammals, thereby effectively suppressing the progression of oral diseases such as periodontitis and maintaining healthy gingival tissue It is intended to provide a gingival epithelial cell spreading inhibitor.

  That is, the gist of the present invention is not only a medicinal plant known from ancient times, but also safety for humans, red grape (Vitis vinifera L.), rosemary (Rosmarinus officinalis L. ), Ginkgo biloba L., St. John's wort (Hypericum perforatum L.) and Amla (Emblic officinalis Gaertn.) And an inhibitor of gingival epithelial cell extension that inhibits abnormal extension of gingival epithelial tissue in mammals. Moreover, according to the other aspect of this invention, the composition for oral cavity containing the gingival epithelial cell extension inhibitor of this invention is also provided.

  As a result of intensive research conducted by the present inventor, the separated components obtained by extracting red grape, rosemary, ginkgo, St. John's wort, and Amla plants with a solvent are gingival epithelial tissues, particularly in mammals. As a result, the present inventors have found that the abnormal extension of gingival epithelial tissue is significantly inhibited, and found applicability to the prevention and treatment of oral diseases such as periodontitis.

  In addition, as will be described later, in order to efficiently obtain the required separated components from the raw plant, the leaves, branches, trunks, bark, fruits or roots of the raw plant are extracted with a solvent composed of water and / or alcohol. It is preferable to do.

  According to the present invention, a novel gingival epithelial cell extension inhibitor that inhibits abnormal extension of gingival epithelial tissue and contributes to effective suppression of the formation of periodontal pockets is realized.

  The structure of the gingival epithelial cell extension inhibitor of the present invention will be described in detail below.

  According to one aspect of the present invention, red grape (Vitis vinifera L.), rosemary (Rosmarinus officinalis L.), ginkgo (Ginkgo biloba L.), St. John's wort (Hypericum perforatum L.) and Amla (Emblic officinalis Gaertn) And a gingival epithelial cell extension inhibitor containing a separated component obtained by extracting a raw material plant selected from the group consisting of a solvent with a solvent and inhibiting abnormal extension of gingival epithelial tissue in a mammal. The

  In the present specification, the applicant of the present application includes a substance or composition that has been confirmed to have an inhibitory effect on abnormal extension of gingival epithelial tissue, at least in an in vitro test system, and is referred to as a “gingival epithelial cell extension inhibitor”. It is called.

  As used herein, the term “gingival epithelial cells” refers to epithelial cells of the oral marginal mucosa (oral marginal mucosal epithelial cells) that surround the tooth neck and root and serve as supporting structures for adjacent tissues. The epithelial cells attach to the surface of the tooth enamel layer and form periodontal pockets as periodontitis progresses. In addition, the oral mucosal epithelial cells are always placed in an environment that is exposed to the risk of infection by bacteria, so that the oral mucosal epithelial cells in other parts of the oral cavity have, for example, superior cell division The nature is a clear line.

  The outline of the raw material plant used in the gingival epithelial cell spreading inhibitor of the present invention is as follows.

  Red grapes (Vitis vinifera L.), especially the dried leaves of red grapes for wine, contain more than 4.0% total polyphenols and more than 0.2% anthocyan glycosides. The extract contains 10-15% total polyphenols, the amount of which is comparable to 100-300 times that of red wine. Since polyphenols are thought to have a vascular protective effect, they are said to act significantly on symptoms such as venous insufficiency and superficial capillary syndrome.

  Rosemary (Rosmarinus officinalis L.) is a plant with a unique aroma similar to camphor that originated in Spain and the Mediterranean region, and its leaves include monoterpene hydrocarbons, camphor, borneol, cinerol, linalool, It contains components such as berbenol, phenolic acid, carnosic acid (rosmarinesin), triterpenic acid, saponoside, and tannin. Rosemary extract is also used as a component of warm bath, and by bathing in a bath containing this warm bath, activity of the body's circulatory system is promoted, and it is also effective for cooling the body. is there. In addition, fatigue recovery can be achieved by taking rosemary extract.

  Furthermore, the bactericidal effect exhibited by the rosemary extract is also effective for preventing dandruff.

  Ginkgo biloba L. is a deciduous tree native to China, and its leaves contain medicinal ingredients of flavonoids and ginkgolides, and it has been reported to have the effect of improving thinking ability. In addition to improving blood circulation and activating brain cell functions, flavonoids, which are plant pigments, are said to exert various effects on symptoms such as lifestyle-related diseases, stiff shoulders, and coldness. On the other hand, ginkgolide has been reported to be effective in preventing dementia because it has been reported to prevent active oxygen in the brain and protect brain cells.

  St. John's wort (Hypericum perforatum L.) is a plant with long stems and small yellow flowers, and has a vast vegetation distribution in Europe, Asia, North Africa, North America, and so on. The oily extract of the above-ground part of the plant has a high anti-inflammatory effect and sedative effect, and has been used for the treatment of depression and insomnia for a long time.

  Amla (Emblic officinalis Gaertn.) 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. In addition to hemostasis of gum bleeding and cleansing action in the oral cavity, Amla fruit is also vitalized tissue, increased red blood cell count, improved vision, bone regeneration, promotion of hair and nail extension, prevention of white hair formation, It is said to act on blood glucose level adjustment and gastrointestinal inflammation reduction.

  Next, these raw material plants are applied to an extraction process using a solvent. Plants or any part of them (eg branches, branches, trunks, bark, fruits or roots) that are applied in the extraction process can be left as they are, or after being physically crushed, or as needed In addition, it can be applied to the extraction process after being dried and pulverized to be processed into a powder.

  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 a raw material plant, a method well known in the art can be used. For example, the raw plant itself, its coarsely pulverized or cut product, or its dry crushed product (powder) is immersed in a solvent by immersion, digestion, etc., or extracted while heating and stirring 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, after removing unnecessary solids by filtration or centrifugation, if necessary, or used as it is, or partially concentrated by distilling off the solvent. Alternatively, a dried product can be used. 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 gingival epithelial cell extension inhibitor of this invention as an active ingredient is also provided. As long as the inhibitory effect of the gingival epithelial cell extension inhibitor of the present invention is not adversely affected, other components generally used in oral compositions, such as adhesives, cooling agents, binders, etc. , 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) Cells such as polymers of methacrylic acid and methyl methacrylate and polymers of methacrylic acid 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, octyl aldehyde, citral, pregon, carbyl acetate, anisaldehyde), 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, methyl cellulose, crystalline cellulose, cellulose derivatives (such as sodium carboxymethyl cellulose), alginic acid, alginates, carbonates, organic acids, polyvinyl pyrrolidone, cross-linked polyvinyl pyrrolidone, etc. 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.

  In addition, for the purpose of further promoting inhibition of gingival epithelial tissue extension, the gingival epithelial cell extension inhibitor or oral composition of the present invention may be applied to a conventionally known plant extract or medicinal compound having an effect of inhibiting gingival epithelial tissue extension ( It is also possible to arbitrarily add a composition), and the amount of addition thereof can be easily adjusted by those skilled in the art.

  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: Culture of gingival epithelial cells Gingival epithelial tissue (about 5.0 mm × about 5.0 mm slice) was collected from the oral cavity of a healthy human gingival tissue. The collected tissue pieces are immersed in phosphate buffer (containing 1 × 10 ⁵ U / ml penicillin, 100 μg / ml streptomycin, 80 μg / ml kanamycin) and within about 24 hours with a scalpel about 0.5 mm. X Cut into small sections of about 0.5 mm. This small section was allowed to stand in a well of a type I collagen-coated 6-well plate. After confirming the adhesion between the small section and collagen, the primary culture medium (DMEM / Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd.) containing L-glutamine, 10% FBS, 10pg / ml EGF, 80μg / ml kanamycin) was added to the well. I poured it quietly. The culture system thus prepared was cultured under the conditions of 37 ° C. and 5% carbon dioxide, and the primary culture medium was replaced with a fresh one every two days.

After confirming that the epithelial cells are proliferating from the small section, that is, confirming that the epithelial cells are proliferating to an extent that covers approximately 30% of the wells, the culture medium is subcultured (EDGS, It was replaced with 80 μg / ml kanamycin-containing EpiLife medium (casdade). The culture solution is transferred to a type I collagen-coated T75 flask, and a confluence of 2500 cells / cm ² is used as a medium, and subculture is performed in accordance with the cell growth rate to obtain gingival epithelial cells used in the following examples. It was.

B: Assay for the effect of inhibiting the growth of gingival epithelial cells First, an inflammation-inducing medium for artificially inducing periodontitis was prepared. Recombinant human TNF-α, an inflammation inducer, was purchased from PEPROTECH (Size B vial: 50 μg, 300-01A; Lot No. 121CY25). 50 μg of recombinant human TNF-α was allowed to stand at room temperature for 1 hour and then applied to a tabletop centrifuge. The precipitate obtained by centrifugation was dissolved in 500 μl of distilled water, 0.1 mg / ml Tris buffer was added, and 36 μl of the obtained aqueous solution was then added to 3.564 ml of physiological saline (PBS (−) ) Was further dissolved. The PBS solution thus obtained was dispensed into a serum tube in an amount of 1.2 ml and stored at a temperature of −20 ° C. The remaining aqueous solution was dispensed at 100 μl each into a serum tube and stored at a temperature of −20 ° C. 1.2 ml of PBS solution (containing 1 μg / ml TNF-α) is dissolved in 10.8 ml EpiLife medium (without serum and antibiotics) and filtered and sterilized using a 0.2 μm pore size filter. Thus, a medium for inducing inflammation was obtained.

  Next, a solution in which a component concentration of 0.02 g / ml (2% w / v) was dissolved was obtained using the raw material plants and solvents described in Table 1 below.

The solution containing the extracted component from each raw plant was filtered and sterilized using a 0.2 μm pore size filter, and the component concentration was adjusted using an inflammation induction medium.

Inhibition of gingival epithelial cell spreading according to the present invention with respect to a solution containing an extract component derived from each raw material plant is Masami Yoshioka, et al., `Effect of Hydroxamic Acid Based Matrix Metalloproteinase Inhibitors on Human Gingival Cells and Porphyromonas gingivalis`, J. Periodontol. , vol. 74, pp. 1219-1224 (2003).

First, gingival epithelial cells are placed at a rate of 2 × 10 ⁴ cells / well in a 24-well plate (1.8 cm ² / well). Once every two days, the subculture medium was replaced with fresh one and the culture was continued until the cells covered the entire well. After confirming that the cells covered the entire well, half of the cells in the well were scraped with the tip of the pipette tip. After washing the wells with PBS, a medium for inflammation induction and a solution containing an extraction component (concentration of 1.0% or 0.1%) derived from each raw plant were poured, and conditions of 37 ° C. and 5% carbon dioxide The cells were cultured below, and at 0, 24 and 48 hours after the start of the culture, wells were photographed to verify the degree of cell extension (FIG. 1 (b)). In addition, the test group which followed the same procedure except having poured only the culture medium for inflammation induction after wash | cleaning a well with PBS was set as the control | contrast (FIG. 1 (a)).

  Based on the photograph which image | photographed the cell which appeared on the well, the moving distance of the cell was determined visually.

  And based on the comparison with the movement distance in the control, the gingival epithelial cell extension inhibitory effect by the extracted components derived from each raw material plant, the remarkable inhibitory effect (+++), the strength inhibitory effect (++), The grading was performed according to a four-step evaluation of normal inhibitory effect (+) and no inhibitory effect (−).

  The evaluation results are summarized in Table 2 below.

As is apparent from the results shown in Table 2, the separated components obtained by extracting red grape, rosemary, ginkgo, St. John's wort and amla with a solvent significantly inhibited the extension of gingival epithelial cells. It was confirmed that the inhibitory effect was concentration-dependent.

C: Examination of Gingival Epithelial Cell Extension Inhibitory Effect in Living Body Next, the in vivo activity of the gingival epithelial cell spreading inhibitor of the present invention was verified with respect to a separated component obtained by extracting Amla with a solvent.

First, male Wistar rats (15 animals) 6 weeks old were prepared. The growth inhibition effect of gingival epithelial cells in these rats is described in D. Ekuni, et al., `Protease augment the effects of lipopolysaccharide in rat gingiva`, J. Periodont Res , vol.38, pp.591-596 (2003). Assays were performed according to procedures reported in the literature. That is, along with the progress of periodontal tissue destruction due to the development of periodontitis, healthy gingival epithelial cells (Fig. 2 (a)) downgrow toward the tooth root (Fig. 2 (b)). As a result, focusing on the unique phenomenon that a gingival crevice (periodontal pocket) peculiar to periodontitis is formed between the gingiva and the tooth, the effect of inhibiting the growth of gingival epithelial cells in the living body was tested.

  Specifically, first, general anesthesia was performed by administering 1 ml / kg of Nembutal Injection (Dainippon Pharmaceutical) into the abdominal cavity of rats. Thereafter, 0.5 μl of actinomycete Streptomyces griseus-derived protease (Sigma) 2.25 units / μl was administered to the maxillary first molar palatal gingival sulcus and allowed to act for 5 minutes, and immediately thereafter LPS (E. coli-derived toxin, Sigma Company) 0.5 μl of 25 mg / ml aqueous solution was administered to the same site and allowed to act for 5 minutes. This procedure was repeated 3 times to induce periodontitis. After administration of LPS, 5% aqueous solution of dipotassium glycyrrhizinate (positive control: 5 animals), 5% aqueous solution of amla extract (5 inventions of this application), and purified water (negative control: 5 animals) were administered. It was allowed to act for another 5 minutes. This series of work was performed daily for 4 weeks.

  Four weeks later, the rats were sacrificed and their upper jaws were removed. The extracted upper jaw was subjected to immobilization treatment at 4 ° C. overnight using a 10% formaldehyde aqueous solution (0.1 M phosphate buffer, pH 7.4), and then K-CX (Pharmacia) Was used for deashing at 4 ° C. overnight. Thereafter, the treated upper jaw was immersed in a 30% aqueous sucrose solution (0.1 M phosphate buffer, pH 7.4) at 4 ° C. overnight. The sample thus obtained was embedded in an OCT compound (Sakura Seiki), and then a section in the buccal tongue direction having a thickness of 8 μm was prepared with a cryostat (Leica). Subsequently, the sections were stained with hematoxylin and eosin and microscopically observed to measure the downgrowth (μm) of the gingival epithelial tissue. As shown in FIG. 3 (a), the gingival epithelial tissue downgrowth can be seen from the position where the gingival epithelium and teeth contact (Central Enamel Junction (CEJ): position X). The distance to Y) (μm: XY distance α) was measured with an eyepiece micrometer. The measurement results are summarized in Table 2 below. The number of rats (the number of non-extending individuals) of rats having a distance α of zero, that is, rats in which no downgrowth was observed was also shown in Table 3 below.

As is apparent from the results shown in Table 3 and FIG. 3, in the rats treated with purified water, clear downgrowth of gingiva was confirmed 4 weeks after the start of the test (FIG. 3 (a)). . In contrast, Amla extract showed almost no gingival downgrowth even 4 weeks after the start of the test (FIG. 3 (b)), and dipotassium glycyrrhizinate (FIG. 3 (c)), which is an anti-inflammatory agent. The healthy tissue was substantially maintained with the same inhibitory effect as in)).

  From the results of this Example, it was confirmed that the separated component obtained by extracting Amla with a solvent significantly inhibits the extension of gingival epithelial cells even in vivo.

  In addition, the results of this example also show that the separated components from other raw material plants (red grapes, rosemary, ginkgo, St. John's wort) that have been demonstrated to have an inhibitory effect on the extension of gingival epithelial cells in Example B are also in vivo. This suggests a substantial growth inhibitory effect on gingival epithelial cells, and its usefulness can be greatly expected.

D: Preparation of oral composition An oral composition for various uses was prepared by blending a plant extract (0.5%) whose gingival epithelial cell spreading inhibitory effect was demonstrated in the previous examples. . In addition, the unit of the component compounding amount in a present Example is displayed by weight%, unless there is particular notice.

D-1: Dentifrice Toothpaste was prepared by mixing and kneading the materials listed in Table 4 below at room temperature.

D-2: Dentifrice Toothpaste was prepared by mixing and kneading the materials shown in Table 5 below at room temperature.

D-3: Mouthwash The materials shown in Table 6 below were mixed at room temperature to prepare a mouthwash.

D-4: Oral pasta The materials described in Table 7 below were mixed at room temperature to prepare oral pasta.

D-5: troche To a granule obtained by mixing maltitol, gum arabic, citric acid, plant extract and xylitol described in Table 8 below at room temperature, sucrose fatty acid ester and powder flavor are added. And troche was prepared.

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

It is a schematic diagram explaining the principle of the gingival epithelial cell extension inhibition test. It is a schematic diagram explaining the principle of extension of a gingival epithelial tissue. It is a photograph which shows the extension state of the gingival epithelial tissue in the rat treated with (a) purified water, (b) amla extract and (c) dipotassium glycyrrhizinate.

Claims (1)

Cents John's wort (Hypericum perforatum L.) and comprising a separating component obtained by extraction with a solvent, inhibitor compositions not normal extension of gingival epithelium.