JP2020019800A - Composition for inhibiting periodontal disease bacteria from invading cells - Google Patents

Abstract

To provide means of inhibiting periodontal disease bacteria from invading cells.SOLUTION: A composition for inhibiting periodontal disease bacteria from invading cells contains at least one selected from the group consisting of α-cyclodextrin, β-cyclodextrin, sodium copper chlorophylline, flavone and resveratrol.SELECTED DRAWING: None

Description

  The present invention relates to a composition for suppressing periodontal disease cell invasion (composition for suppressing periodontal disease cell invasion of cells) and the like.

  Periodontal disease is an inflammatory disease caused by the infection of bacteria (periodontal bacteria). Therefore, as one of the important factors for prevention or treatment of periodontal disease, suppressing the activity of periodontal disease bacteria Is mentioned. For this reason, many oral compositions have been researched and developed for the purpose of sterilizing or suppressing periodontal disease bacteria (for example, Patent Document 1).

JP 2012-111732 A

FEMS Microbiology Letters 216 (2002) 217-222

P. gingivalis, a periodontal disease bacterium, is known to invade cells (especially gingival cells). The invading bacterium exerts strong cytotoxicity. Although some of the invading bacteria are degraded by lysosomes, P. gingivalis, even when destined to be degraded, persists in autolysosomes for a fairly long time, exerting cytotoxicity. Furthermore, nearly half of the bacteria that have entered the cell exit the cell from the early endosome via the recycling pathway and re-enter the surrounding cells. In short, P. gingivalis moves between cells, survives, proliferates,
The infection continues (for example, see the following web page: http://web.dent.osaka-u.ac.jp/~prevent/research01.html). Moreover, when the periodontal disease bacteria enter the gingival cells, the active ingredient reaches the periodontal disease bacteria even when an oral composition containing an active ingredient that exerts a sterilizing or suppressing effect on the periodontal disease bacteria is applied in the oral cavity. , It is difficult to sterilize or control periodontal disease bacteria. From the above, the inventors of the present invention have studied since finding a means for suppressing the invasion of periodontal disease bacteria into cells is considered to greatly contribute to the prevention or treatment of periodontal disease.

  The present inventors have found that a specific compound has an effect of suppressing the invasion of periodontal disease bacteria into cells, and have further studied to complete the present invention.

The invention includes, for example, the subject matter described in the following section.
Item 1.
A composition for inhibiting cell invasion of periodontal disease cells, comprising at least one selected from the group consisting of α-cyclodextrin, β-cyclodextrin, sodium copper chlorophyllin, flavone, and resveratrol.
Item 2.
Periodontal disease bacteria are P. Item 1. The composition according to Item 1, which is gingivalis.
Item 3.
Item 3. The composition according to Item 1 or 2, wherein the cells are gingival epithelial cells.
Item 4.
Item 4. The composition according to any one of Items 1 to 3, which is an oral composition.

  ADVANTAGE OF THE INVENTION According to this invention, it can suppress efficiently that periodontal disease bacteria invade a cell.

FIG. 1 shows a confocal laser microscope observation image when examining the degree of cell invasion of vesicle beads (pseudo-P. Gingivalis) using 1 mM copper chloroferrin sodium as a test substance. The results when no test substance was added are shown on the left side of FIG. 1, and the results when the test substance was added are shown on the right side of FIG. 1, respectively.

  Hereinafter, each embodiment included in the present invention will be described in more detail.

  The composition included in the present invention is used for suppressing periodontal disease bacteria from invading cells. Therefore, the composition included in the present invention can be referred to as a composition for inhibiting cell invasion of periodontal disease cells. Hereinafter, the composition included in the present invention may be referred to as “the composition of the present invention”.

  The composition of the present invention comprises at least one selected from the group consisting of α-cyclodextrin, β-cyclodextrin, sodium copper chlorophyllin, flavone (2,3-didehydroflavan-4-one), and resveratrol. It contains. In addition, these components contained in the composition of the present invention may be collectively referred to as an active ingredient. The active ingredients may be included in the composition of the present invention alone or in combination of two or more.

  The amount of the active ingredient contained in the present invention is not particularly limited as long as the effect of the present invention is exhibited, and can be appropriately set. For example, it can be 0.1 to 100% by mass. If the content is 100% by mass and only one type of active ingredient is contained, the active ingredient itself is used. This state is also included in the composition of the present invention. In this case, it can also be referred to as a periodontal disease cell invasion inhibitor.

  In addition, about (alpha) -cyclodextrin and (beta) -cyclodextrin, respectively, it is preferable to contain 3 mM or more, for example, It is more preferable that it contains 4 mM or more or 5 mM or more, and it is 6 mM or more, 7 mM or more, 8 mM or more, 9 mM or more, or More preferably, it is contained at 10 mM or more. The upper limit is not particularly limited as long as the effect of the present invention is exerted. Examples thereof include 30 mM or less, 25 mM or less, 20 mM or less, 15 mM or less, or 10 mM or less.

  For sodium copper chlorophyllin, flavone, and resveratrol, for example, it is preferably contained, for example, at least 0.3 mM, more preferably at least 0.4 mM or at least 0.5 mM, more preferably at least 0.6 mM, and at least 0.5 mM. More preferably, the content is 7 mM or more, 0.8 mM or more, 0.9 mM or more, or 1 mM or more. The upper limit is not particularly limited as long as the effect of the present invention is exerted. For example, 5 mM or less, 4.5 mM or less, 4 mM or less, 3.5 mM or less, 3 mM or less, 2.5 mM or less, 2 mM or less, 1.5 mM or less. Or 1 mM or less.

  The periodontal disease bacterium in which the composition of the present invention suppresses cell invasion is not particularly limited as long as the effects of the present invention are not impaired. Among the periodontal disease bacterium, Porphyromonas gingivalis (hereinafter sometimes referred to as “P. gingivalis”). Is preferred.

  The composition of the present invention can be a solid composition, a liquid composition. Further, for example, it can be preferably used as an oral composition. The oral composition can be used, for example, as a drug or a quasi drug. In addition, the form of the oral composition of the present invention is not particularly limited, but, for example, ointments, pastes, pasta, gels, liquids, sprays, mouthwashes, liquid dentifrices, and the like in accordance with ordinary methods. It can be in the form (dosage form) of toothpaste, gum and the like. Of these, mouthwash, liquid dentifrice, toothpaste, ointment, paste, liquid, and gel are preferred.

  The oral composition of the present invention may further contain one or more optional components that can be added to the oral composition as long as the effects of the present invention are not impaired.

  For example, a nonionic surfactant, an anionic surfactant or an amphoteric surfactant can be blended as the surfactant. Specifically, nonionic surfactants include sugar fatty acid esters such as sucrose fatty acid esters, maltose fatty acid esters, and lactose fatty acid esters; fatty acid alkanolamides; sorbitan fatty acid esters; fatty acid monoglycerides; A polyoxyethylene alkyl ether having an alkyl group having 13 to 15 carbon atoms; a polyoxyethylene alkylphenyl ether having a polyoxyethylene addition coefficient of 10 to 18 and an alkyl group having 9 carbon atoms; diethyl sebacate; Ethylene-hardened castor oil; fatty acid polyoxyethylene sorbitan and the like. Examples of the anionic surfactant include sulfates such as sodium lauryl sulfate and sodium polyoxyethylene lauryl ether sulfate; sulfosuccinates such as sodium lauryl sulfosuccinate and sodium polyoxyethylene lauryl ether sulfosuccinate; cocoyl sarcosine sodium and lauroylmethylalanine Acyl amino acid salts such as sodium; sodium cocoylmethyltaurine; Examples of the amphoteric surfactant include betaine acetate-type surfactants such as betaine lauryl dimethylaminoacetate and betaine coconut fatty acid amidopropyldimethylaminoacetate; and imidazoline-type surfactants such as N-cocoyl-N-carboxymethyl-N-hydroxyethylethylenediamine sodium. Activator; Amino acid-type activators such as N-lauryldiaminoethylglycine and the like. These surfactants may be used alone or in combination of two or more. The compounding amount is usually 0.1 to 5% by mass based on the total amount of the composition.

  Further, as a flavoring agent, menthol, carboxylic acid, anethole, eugenol, methyl salicylate, limonene, ocimene, n-decyl alcohol, citronell, α-terpineol, methyl acetate, citronenyl acetate, methyl eugenol, cineol, linalool, Ethyl linalool, thymol, spearmint oil, peppermint oil, lemon oil, orange oil, sage oil, rosemary oil, cinnamon oil, perilla oil, winter green oil, clove oil, eucalyptus oil, pimento oil, d-camphor, d- Perfume such as borneol, fennel oil, cinnamon oil, cinnamaldehyde, peppermint oil, vanillin and the like can be used alone or in combination of two or more kinds thereof in an amount of 0.001 to 1.5% by mass based on the total amount of the composition.

  In addition, sweeteners such as saccharin sodium, acesulfame potassium, stevioside, neohesperidyl dihydrochalcone, perillartin, thaumatin, asparatylphenylalanyl methyl ester, p-methoxycinnamic aldehyde, and the like are used in an amount of 0.01 to the total amount of the composition. １1% by mass.

  Further, as a wetting agent, sorbitol, ethylene glycol, propylene glycol, glycerin, 1,3-butylene glycol, polypropylene glycol, xylit, maltitol, lactit, polyoxyethylene glycol, or the like may be used alone or in combination of two or more. it can.

  As preservatives, parabens such as methylparaben, ethylparaben, propylparaben and butylparaben, sodium benzoate, phenoxyethanol, alkyldiaminoethylglycine hydrochloride and the like can be blended.

  As a coloring agent, legal dyes such as Blue No. 1, Yellow No. 4, Red No. 202, and Green No. 3, mineral dyes such as ultramarine, enhanced ultramarine, and navy blue, and titanium oxide may be blended.

  As a pH adjuster, citric acid, phosphoric acid, malic acid, pyrophosphoric acid, lactic acid, tartaric acid, glycerophosphoric acid, acetic acid, nitric acid, or a chemically possible salt thereof, sodium hydroxide, or the like may be blended. These may be used alone or in combination of two or more such that the pH of the composition is in the range of 4 to 8, preferably 5 to 7. The usual compounding amount of the pH adjuster is 0.01 to 2% by weight.

  In addition, the oral composition of the present invention further includes, as a medicinal component, a bactericide such as cetylpyridinium chloride, benzalkonium chloride, benzethonium chloride, chlorhexidine hydrochloride, chlorhexidine gluconate, or dl-α-acetate. Vitamin E such as tocopherol, tocopherol succinate, or tocopherol nicotinate; amphoteric fungicides such as dodecyldiaminoethylglycine; nonionic fungicides such as triclosan and isopropylmethylphenol; dextranase, amylase, protease, mutanase, lysozyme , Enzymes such as lytic enzymes (Litec enzyme), fluorides such as sodium monofluorophosphate, sodium fluoride, stannous fluoride, tranexamic acid and epsilon aminocaproic acid, aluminum chlorohydroxyl Allantoin, dihydrocholesterol, glycyrrhetinic acid, glycerophosphate, chlorophyll, sodium chloride, Karopeputaido, dipotassium glycyrrhizinate, allantoin, hinokitiol, potassium nitrate, etc., may be blended alone or in combination of two or more.

  As a base, it is also possible to add alcohols, silicon, apatite, white petrolatum, paraffin, liquid paraffin, microcrystalline wax, squalane, plastibase and the like.

  The oral composition can be prepared according to a known method (for example, a conventional method).

  In this specification, the term "comprising" includes "consisting essentially of" and "consisting essentially of" and "consisting of." In addition, the present invention encompasses any combination of the constituent features described in this specification.

  In addition, various characteristics (properties, structures, functions, and the like) described in each embodiment of the present invention described above may be combined in any way when specifying a subject included in the present invention. That is, the present invention encompasses all subjects including any combination of the characteristics that can be combined as described in this specification.

  Hereinafter, the present invention will be described more specifically, but the present invention is not limited to the following examples.

Preparation of vesicle beads gingivalis is known to constantly secrete outer membrane vesicles Outer membrane vesicles (OMVs). OMVs include P.I. gingivalis contains almost all cytotoxic factors, and even OMVs alone enter cells. When OMVs are added to cultured cells, they are taken up by endocytosis. For this reason, P. gingivalis by coating the particles (eg, fluorescent beads) with OMVs purified from P. gingivalis. gingivalis can be prepared.

  Therefore, the pseudo P.P. gingivalis (OMVs coated fluorescent beads) was prepared. Hereinafter, OMVs may be simply referred to as “vesicles”.

The 50 μl fluorescent bead solution (containing 1.35 × 10 ⁹ beads) was washed with PBS (phosphate buffered saline), and then washed with 200 μl of 0.1 M MES monohydrate buffer (2-morpholinoethanesulfonic acid monohydrate buffer). Washed. After washing, the suspension was centrifuged at 10,000 rpm. After removing the supernatant, the suspension was resuspended in 73.8 μl 0.1 M MES monohydrate buffer.

To the resuspended bead solution, 86.2 μl (adjusted so that the total protein amount of the vesicle becomes 100 μg) was added, and the mixture was incubated at room temperature while shaking with a seesaw shaker. The vesicle liquid was prepared with reference to Non-Patent Document 1 (FEMS Microbiology Letters 216 (2002) 217 to 222). After the incubation, 40 μl of 2 g / ml EDAC was added and incubated at room temperature in the dark. 25 μl of a 1 M glycine aqueous solution and 25 μl of a 1 g / ml EDAC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) aqueous solution were added (reaction stopped), incubated at room temperature in the absence of light, and then centrifuged at 13,000 rpm. After centrifugation, the supernatant was removed and washed twice with PBS (-). After washing, the cells were suspended in 1 ml of PBS. 74 μl (for 8 samples) of the suspension was transferred to a tube and centrifuged at 12,000 rpm. After centrifugation, the supernatant was removed and resuspended in 450 μl of MEM. This was used as a vesicle bead solution in the following studies.

  The 1M glycine aqueous solution and the EDAC aqueous solution used were prepared using PBS (-).

Cell Invasion Inhibition Test of Vesicle Beads 300 μl of Ca9-22 cells (derived from human gingival epithelial cells) were subcultured into each well (inoculation amount: 5 × 10 ⁴ cells / 300 μl) and cultured for 1 day. Discard the old medium cultured for one day, replace with 200 μl of MEM medium in which various concentrations of the test substance (10 mM, 1 mM, or 0.1 mM) are dissolved, and vesicle beads liquid in which vesicle beads are well dispersed by ultrasonic waves in advance of 50 μl. Added to each well and incubated for 1 hour at 37 ° C., 5% CO ₂ . (Accordingly, the concentration (treatment concentration) of each test substance applied to the cells is 0.8 times the initial concentration and is 8 mM, 0.8 mM, or 0.08 mM.)

  After incubation, slides were placed on ice and washed three times with 250 μl of PBS (+). After washing, PBS (+) was removed, 200 mg of 0.3 mg / ml biotin was added, and the mixture was incubated on ice and protected from light to label the cell membrane. After the biotin treatment, the plate was washed once with PBS (+) containing 0.1 M glycine, and further washed twice with PBS (+).

The PBS (+) was removed and 200 μl of 4% PFA (paraformaldehyde) was added to fix the cells. After the cells were fixed, the cells were washed once with PBS (−) containing 0.1 M glycine, and further washed twice with PBS (−). The PBS was removed, 200 μl of PBS (−) containing 1% BSA (bovine serum albumin) was added, and the mixture was incubated at 25 ° C. for 30 minutes. After completion of the incubation, the BSA was removed, and 200 μl Alexa Fluor 4
88 Streptavidin (1: 1000 in 1% BSA / PBS (-)) was added and incubated at room temperature to stain the cell membrane-labeled biotin. After the cell membrane staining, the cells were washed three times with PBS (-) and observed with a confocal laser microscope (cell membrane staining: excitation light 488 nm, emission light 519 nm; vesicle beads: excitation light 580 nm, emission light 605 nm). FIG. 1 shows an image observed with a confocal laser microscope when examined using 1 mM of copper chloroferrin sodium as a test substance. The results when no test substance was added are shown on the left side of FIG. 1, and the results when the test substance was added are shown on the right side of FIG. 1, respectively.

In confocal laser microscopy, a 60x oil lens was used and the number of cells in the observation field was 20
Three regions of up to 40 regions were randomly selected and observed (N = 3). Then, the number of vesicle beads per cell (beads / cell) was calculated by counting the number of beads present in the cells included in the region. In addition, in order to examine the cell entry inhibition effect of each test substance, the cell entry rate of vesicle beads was calculated by the following equation.

Vesicle beads cell penetration rate (%) =
The number of vesicles per cell treated with the test substance-containing medium / the number of vesicles per cell treated with the test substance-free medium (MEM medium only) x 100

  Furthermore, the average value and standard deviation (SD) of the vesicle bead cell penetration rates at three locations (N = 3) were also calculated.

  The examination results when each test substance was used are shown in Tables 1, 2 and 3. In addition, the value indicating that the cell invasion inhibitory effect is particularly high (average cell invasion rate) is underlined. Further, in Tables 2 and 3, the results obtained when only the positive control methyl-β-cyclodextrin was used at a concentration of 10 mM as in Table 1 are shown.

Claims (4)

A composition for inhibiting periodontal disease cell invasion, comprising at least one member selected from the group consisting of resveratrol and flavone. Periodontal disease bacteria are P. The composition of claim 1, wherein the composition is gingivalis. 3. The composition according to claim 1, wherein the cells are gingival epithelial cells. The composition according to any one of claims 1 to 3, which is an oral composition.