JPH0640871A - Composition for oral cavity application - Google Patents

Abstract

PURPOSE:To provide a composition for oral cavity application, effective in remarkably suppressing the colonization of periodontosis-causative bacteria in the oral cavity and capable of effectively preventing and treating periodontosis. CONSTITUTION:An animal is immunized with an antigen consisting of a polysaccharide originated from the surface layer of the cell of periodontosis-causative bacteria and the obtained antibody is compounded in the composition.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an oral composition capable of preventing colonization of periodontal disease-causing bacteria in the oral cavity and preventing or treating periodontal disease.

[0002]

BACKGROUND OF THE INVENTION In order to prevent periodontal diseases, it is necessary to prevent colonization of causative bacteria in the oral cavity,
It is effective to suppress proliferation. From such a point of view, conventionally, proposals have been made regarding an oral composition and an anti-periodontal disease composition containing an antibody against a periodontal disease-causing bacterium (JP-A-60-142915, JP-A-1-142915). 313
No. 438). However, the cross-reactivity of these antibodies with other bacterial species cannot be ruled out because the immune antigen is whole cells or a mixture such as a cell extract. Moreover, it was not completely satisfactory in terms of practical effectiveness.

Therefore, it has been desired to develop an active ingredient which can more effectively suppress the colonization of periodontal disease-causing bacteria in the oral cavity and effectively prevent periodontal disease.

[0004]

Means and Actions for Solving the Problems As a result of intensive studies to meet the above-mentioned demands, the present inventor has prepared a polysaccharide derived from the surface layer of periodontal disease-causing bacteria and used it as an antigen When immunizing animals or poultry), the obtained blood antibodies, milk antibodies, and egg antibodies significantly suppress the colonization of the bacterium in the oral cavity, and therefore, these antibodies should be incorporated into the oral composition. It was found that the effective prevention and treatment of periodontal disease can be achieved by the above, and the present invention has been completed.

Therefore, the present invention provides an oral composition comprising an antibody obtained by immunizing an animal with a polysaccharide derived from the surface layer of periodontal disease-causing bacteria as an antigen.

The present invention will be described in more detail below. The antigen used to obtain the antibody to be incorporated into the oral composition of the present invention is a polysaccharide derived from the surface layer of periodontal disease-causing bacteria.

Here, as the periodontal disease-causing bacterium, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella vulgaris, which are generally considered to have a deep causal relationship with periodontal disease, Spirochetes such as Intermedia, Fusobacterium nucleatum, Capnosite Faga spp., Eukenella colodens, Bolinella recta, Bacteroides forsysus, Treponema denticola, etc. can be used.

The polysaccharides derived from the surface layer of these periodontal disease-causing bacteria can be prepared by a well-known method. For example, a method of subjecting the cultured bacterial cells to an autoclave, a method of allowing the bacterial cells to act on nitrous acid for extraction, a method of extracting the bacterial cells with phenol / water, and the like can be used.

As a method of immunizing an animal with a polysaccharide derived from the surface layer of the periodontal disease-causing bacterium as an antigen, subcutaneous injection, intramuscular injection, buccal injection, intravenous injection, nasal administration, oral administration,
A method such as oral mucosal administration can be adopted. In addition, in order to increase the antibody titer, a method of immunizing with an adjuvant such as Freund's complete adjuvant, incomplete adjuvant, cholera toxin B subunit can be appropriately used. The animals to be immunized are rabbits,
Examples include mammals such as goats, sheep, cows, horses, and poultry such as chickens, ducks, ostriches, ducks, and quails. As the antigen to be immunized, one of the above-mentioned antigens may be used alone, or two or more of them may be used in combination.

[0010] The oral composition of the present invention uses the surface polysaccharide of the periodontal disease-causing bacterium as an antigen in this manner, and immunizes a mammal with the antibody in blood or antibody in milk, or poultry. It is intended to contain an antibody in the egg obtained by immunizing the., To which antiserum containing such antibody, milk, egg may be blended as it is, or according to the usual purification method, serum Alternatively, purified antibody separated from milk or egg may be added. Here, the antibody can be purified from serum, milk, or egg according to a conventional purification method. As the antibody purification method, salting out method, gel filtration method, ion exchange chromatography, affinity chromatography and the like can be used.

In the oral composition of the present invention, one type of the above-mentioned antibodies may be blended alone, or two or more types may be used in combination. The dose of the antibody is 0.0001 to 50.
The amount is preferably g / kg / day, and in this case, the amount of the antibody is preferably 0.0002 to 10% (% by weight, the same applies hereinafter) of the entire composition, and more preferably 0.002 to 5%.

The oral composition of the present invention comprises toothpaste, powdered toothpaste, liquid toothpaste and other toothpaste, liquid refreshing agent such as mouthwash, troche, oral pasta, gum cream, mouthwash, chewing gum, It is used as a candy, a dairy product or the like, and as the other components of the oral composition of the present invention, appropriate components depending on the type of the oral composition and the like are used.

For example, in the case of toothpaste, dibasic calcium phosphate, calcium carbonate, calcium pyrophosphate,
Abrasives such as insoluble sodium metaphosphate, amorphous silica, crystalline silica, aluminosilicate, aluminum oxide, aluminum hydroxide, resin, carboxymethyl cellulose, hydroxyethyl cellulose, alginate, carrageenan, gum arabic, polyvinyl alcohol, etc. Agent, polyethylene glycol, sorbitol,
Glycerin, thickener such as propylene glycol, sodium lauryl sulfate, sodium dodecylbenzene sulfonate, hydrogenated coconut fatty acid monoglyceride, sodium monosulfate, sodium lauryl sulfoacetate, N-
Foaming agents such as sodium lauroyl sarcosinate, N-acyl glutamate, sucrose fatty acid ester, essential oils such as peppermint and spearmint, 1-menthol,
Fragrances such as fragrance materials such as carvone, eugenol and anethole, saccharin sodium, stevioside, neohesperidyl dihydrochalcone, sweeteners such as glycyrrhizin, perillartin, p-methoxycinnamic aldehyde, preservatives and the like are mixed with water, It is manufactured according to the law. Further, in mouthwashes such as mouthwash and the like, components suitable for the properties of the product are appropriately mixed.

In the present invention, dextranase, mutanase, sorbic acid, chlorhexidine, hinokitiol, cetylpyridinium chloride, alkylglycine, alkyldiaminoethylglycine salt, allantoin, ε-aminocaproic acid, tranexamic acid, azulene, vitamin E. , A water-soluble primary or secondary phosphate,
An active ingredient such as a quaternary ammonium compound, sodium chloride or a crude drug extract can also be added.

[0015]

EFFECTS OF THE INVENTION The oral composition of the present invention contains an antibody obtained by immunizing an animal with a polysaccharide derived from the bacterial cell surface of periodontal disease-causing bacteria as an antigen, and It is possible to effectively prevent the colonization of E. coli and effectively prevent or treat periodontal diseases, and this antibody has a high specificity to periodontal disease-causing bacteria.

[0016]

[Experimental example]

(1) Preparation of bacterial cell surface surface polysaccharide Actinobacillus actinomycetemcomitans Y4
The strain was inoculated into Todd Hebit broth supplemented with 1% yeast extract and incubated in an incubator containing 5% CO _2.
Culturing was carried out at 0 ° C for 3 days. After collecting the cells, the cells were washed 3 times with physiological saline, suspended in physiological saline, and autoclaved at 121 ° C. for 15 minutes. After cooling, centrifugation was performed at 10,000 × g for 20 minutes to separate the supernatant, and physiological saline was added again to the precipitate, and the above extraction operation was repeated. The obtained supernatant was collected, dialyzed thoroughly against distilled water, and freeze-dried. This was designated as the bacterial cell surface polysaccharide.

In addition, Porphyromonas gingivalis 3
After culturing 81 strains in Todd Hebit Broth containing hemin and menadione for 2 days, the cells were collected, and bacterial cell surface polysaccharides were prepared in the same manner as in the case of Actinobacillus actinomycetemcomitans.

(2) Preparation of antibody Rabbits were immunized with the prepared polysaccharide antigen to obtain antibodies. The method of immunization was carried out according to a commonly used method. That is, for the primary immunization, 1 mg of the polysaccharide antigen was subcutaneously injected together with Freund's complete adjuvant. Then, every 7 days, 2 mg of the polysaccharide antigen was administered 4 times in total into the ear vein. The obtained antiserum was salted out twice with 50% ammonium sulfate and then dialyzed against physiological saline to prepare an antibody preparation.

(3) Evaluation of Antibodies (i) Cell Surface Aggregation Activity of Cell Surface Polysaccharide Immune Rabbit Serum Antibody The cell aggregation activity of the various antibody preparations prepared as described above was measured. Actinobacillus actinomycetemcomitans Y4 strain, Porphyromonas gingivalis 381
Strain, Streptococcus sanguis
occus sanguis) ATCC 10556 strain,
Streptococcus salivarius
occus salivarus) ATCC1341
Each of the 9 strains was cultured until the late logarithmic growth phase, and the cells were collected, washed three times with physiological saline, and suspended in physiological saline so that the turbidity was OD _550nm = 1.0. On the other hand, various antibody preparations were prepared with physiological saline so that the protein concentration was 1 mg / ml, and this was used as an assay antibody preparation for bacterial cell agglutination activity. Each assay antibody preparation was serially diluted 2 ⁿ times with physiological saline. Add an equal amount of cell suspension to each diluted solution, stir well,
The mixture was allowed to stand overnight at room temperature and observed for the presence or absence of bacterial aggregation. The results are shown in Table 1.

[0020]

[Table 1]

As is clear from the results shown in Table 1, the antibodies obtained by immunization with the bacterial cell surface polysaccharide as an antigen have low reactivity to Streptococcus sanguis, Streptococcus salivarius, etc. which are indigenous bacteria in the oral cavity, It was confirmed that the antibody has high specificity.

(Ii) Inhibitory effect of actinobacillus actinomycetemcomitans on the hamster oral colonization of Y4 bacterial surface polysaccharide immune rabbit serum antibody. Turbidity is OD in buffer solution
_The suspension was suspended at _{550 nm} = 1.0, mixed with the anti-Y4 surface polysaccharide antibody at a volume ratio of 1: 1 and reacted at 37 ° C for 30 minutes. As a control group, a mixture with a phosphate buffer solution was used instead of the antibody solution.

Eight hamsters in each county were inoculated with 0.1 ml each of the above treated bacterial solution for 5 consecutive days. One week later, the gingival margin was swabbed with a sterilized cotton ball, and the number of Actinobacillus actinomycetemcomitans and the total number of bacteria were measured. The ratio of the number of bacteria of Actinobacillus actinomycetemcomitans to the total number of bacteria was obtained and used as the colonization rate of Actinobacillus actinomycetemcomitans. The results are shown in Table 2.

[0024]

[Table 2]

As is clear from the results in Table 2, the anti-Y4 surface polysaccharide antibody which is the antibody of the present invention can significantly inhibit the oral colonization of Actinobacillus actinomycetemcomitans as compared with the control group. Admitted.

(Iii) Inhibitory effect of Porphyromonas gingivalis on the hamster oral colonization of 381 surface polysaccharide polysaccharide immune antibody of Porphyromonas gingivalis 381 cultured for 3 days
The strain was suspended in 1 mM phosphate buffer so that the turbidity was OD _550nm = 1.0, and the volume ratio of the anti-381 surface polysaccharide antibody to the anti-381 surface polysaccharide antibody was 1:
The mixture was mixed at 1 and reacted at 37 ° C. for 30 minutes. As a control group, a mixture with a phosphate buffer solution was used instead of the antibody solution.

A cotton thread was previously ligated to the lower first molars of 8 hamsters in each county, and 0.1 ml of each of the above treatment solutions was ligated.
Each was inoculated for 5 consecutive days. One week after that, the ligature was taken out, and the number of Porphyromonas gingivalis bacteria and the total number of anaerobic bacteria in the ligature were measured. The ratio of Porphyromonas gingivalis to the total number of anaerobic bacteria was determined and used as the colonization rate of Porphyromonas gingivalis. The results are shown in Table 3.

[0028]

[Table 3]

As is clear from the results in Table 3, the anti-381 surface polysaccharide antibody which is the antibody of the present invention was found to have an effect of significantly inhibiting oral colonization of Porphyromonas gingivalis as compared with the control group.

Examples will be shown below. In addition, all% show weight%. In addition, A. a. Actinobacillus actinomycetemcomitance, P. g. Porphyromonas gingivalis, P. i. Prevotella Intermedia, F.I. n. Is a Fusobacterium nucleatum, W. r. Bolinella Lecta, B. f. Bacteroides Forsythus, T. d. Indicates Treponema Denticola.

Example 1 Toothpaste Dibasic calcium phosphate dihydrate 50.0% Glycerin 20.0 Carboxymethyl cellulose 1.0 Sodium lauryl sulphate 1.5 Sodium lauroyl sarcosinate 0.5 Perfume 1. 0 saccharin 0.1 dextranase 0.01 rabbit anti-A. a. Cell surface polysaccharide serum 0.1 Water, total 100.0%

Example 2 Toothpaste Dicalcium Phosphate Dihydrate 47.0% Sorbit 10.0 Glycerin 10.0 Carboxymethyl Cellulose 1.0 Sodium Lauryl Sulfate 2.0 Perfume 1.0 Saccharin 0.1 Ethanol 2.0 Chlorhexidine gluconate 0.01 Mutanase 0.1 Chicken egg anti-P. g. Cell surface polysaccharide serum 0.1 Water, total 100.0%

[Example 3] Toothpaste Calcium carbonate 48.0% Glycerin 20.0 Carrageenan 0.5 Carboxymethyl cellulose 1.0 Lauryl diethanolamide 1.0 Sucrose monolaurate 2.0 Perfume 1.0 Tranexamic acid 0 .05 saccharin 0.1 bovine anti-A. a. Bacterial cell surface polysaccharide breast milk 0.05 Water balance 100.0%

[Example 4] Toothpaste Aluminum hydroxide 50.0% Glycerin 20.0 Carboxymethyl cellulose 2.0 Sodium lauryl sulfate 2.0 Fragrance 1.0 Saccharin 0.1 Goat anti-P. g. Cell surface polysaccharide serum 0.1 Water, total 100.0%

[Example 5] Toothpaste Silicic anhydride 30.0% Glycerin 30.0 Sorbitt 20.0 Carboxymethyl cellulose 1.0 Sodium lauryl sulfate 2.0 Fragrance 1.0 Saccharin 0.1 Sodium fluoride 0. 1 ethanol 2.0 chicken egg anti-P. i. Cell surface polysaccharide antibody 0.1 Water balance 100.0%

Example 6 Toothpaste Dicalcium phosphate dihydrate 50.0% Calcium carbonate 30.0 Glycerin 10.0 α-Olefin sulfonate 1.0 Fragrance 1.0 Saccharin 0.1 Dextran 0 .5 Horse Anti-F. n. Cell surface polysaccharide serum 0.05 Water residual 100.0%

[Example 7] Liquid toothpaste Sodium polyacrylate 50.0% Glycerin 30.0 Fragrance 0.9 Saccharin 0.1 Ethanol 3.0 Cetylpyridinium chloride 0.05 Linoleic acid 0.05 Goat anti-A. a. Bacterial cell surface polysaccharide breast milk 0.1 Water balance 100.0%

Example 8 Mouthwash Ethanol 20.0% Fragrance 1.0 Saccharin 0.05 Lauryldiethanolamide 0.3 Goat anti-W. r. Cell surface polysaccharide serum 0.1 Water, total 100.0%

Example 9 Mouthwash Tablet Sodium Bicarbonate 54.0% Dibasic Sodium Phosphate 10.0 Polyethylene Glycol 3.0 Citric Acid 17.0 Sodium Sulfate (Anhydrous) 13.6 Perfume 2.0 Chlorhexidine Glucon Acid salt 0.05 Oleic acid 0.1 Rabbit anti-B. f. Cell surface polysaccharide serum 0.1 Water, total 100.0%

Example 10 Gingival Massage Cream White Vaseline 8.0% Propylene Glycol 4.0 Stearyl Alcohol 8.0 Polyethylene Glycol 4000 25.0 Polyethylene Glycol 400 37.0 Tocopherol Acetate 0.1 Sucrose Stearate 0 .5 bovine anti-T. d. Cell surface polysaccharide serum 0.1 Water, total 100.0%

[Example 11] Chewing gum Gum base 43.85% Calcium carbonate 2.0 Water candy 15.0 Sugar 30.0 Sucrose palmitate 1.0 Fructose 4.0 Maltose 1.0 Perfume 1.0 Chicken egg anti-A. a. Cell surface polysaccharide antibody 0.1 Total 100.0%

Example 12 Lozenge Gum Arabic 6.0% Glucose 72.0 Gelatin 3.0 Fragrance 0.2 l-Menthol 0.1 Spearmint oil 0.1 Sodium ascorbate 0.1 Goat anti-P. g. Bacterial cell surface polysaccharide breast milk 0.05 Water balance 100.0%

Example 13 Oral Pasta Polyoxyethylene Monostearate 2.0% Sorbitan Monooleate 2.0 Cetyl Alcohol 2.0 Palmityl Alcohol 3.0 Propylene Glycol 15.0 Carboxymethyl Cellulose 5.0 Gelatin 1.0 Saccharin 0.2 Pehermint oil 0.5 Spearmint oil 0.5 Horse Anti-A. a. Cell surface polysaccharide serum 0.05 Lysozyme chloride 5000 units / g Leftovers 100.0% in total

Example 14 Oral Pasta Glyceryl Monolaurate 3.0% Oleyl Alcohol 5.0 Polyethylene Glycol 15.0 White Vaseline 3.0 N-Palmitoyl Glutamate Monosodium 0.5 Hydroxyethyl Cellulose 5.0 Tocopherol Acetate 0.1 Saccharin sodium 0.2 Japanese peppermint oil 0.7 Carvone 0.5 Anethole 0.3 Eugenol 0.1 Egg anti-P. g. Cell surface polysaccharide antibody 0.05 Water balance 100.0%

[Example 15] Ice cream Cream (fat ratio 50%) 16.84% Milk (fat ratio 3.7%) * 42.65 Sugar-free skimmed condensed milk 24.24 Sugar 11.25 Corn syrup 4.65 Stabilizer 0.35 Total 100.0% * Bovine anti-A. a. Containing 5% of surface polysaccharide polysaccharide milk

[Example 16] Ice cream cream (fat ratio 40%) 31.54% Milk (fat ratio 3.7%) ** 37.16 Sugar-free skimmed condensed milk 15.08 Sugar 11.25 Corn syrup 4. 67 Stabilizer 0.30 Total 100.0% ** Bovine anti-P. g. Containing 5% of surface polysaccharide polysaccharide milk

[Example 17] Yogurt skim milk 320 kg Sweetened condensed milk *** 56 Sugar 23 The above components are fermented by a conventional method. *** Cow anti-A. a. Containing 10% of the surface polysaccharide polysaccharide milk

Example 18 Bavarois Dough Milk **** cc 375cc Sugar 60g Vanilla Saya 1 Egg Yolk 5 Gelatin 20g Fresh Cream 75cc ******** Beef Anti-P. g. Containing 5% of surface polysaccharide polysaccharide milk

Claims (1)

[Claims] 1. An oral composition comprising an antibody obtained by immunizing an animal with a polysaccharide derived from the surface layer of periodontal disease-causing bacteria as an antigen.