KR101202302B1 - Composition for treating and preventing periodontal disease and caries comprising bee venom - Google Patents

Abstract

The present invention relates to a composition for preventing and treating periodontal disease and tooth decay, which contains bee venom as an active ingredient, and more specifically, has antibacterial activity against caries-causing bacteria, gum inflammation-causing bacteria and periodontal disease-causing bacteria, and inflammation in the oral cavity. It relates to a composition for preventing and treating periodontal disease and caries comprising bee venom as an active ingredient to inhibit the.

Description

Composition for treating and preventing periodontal disease and caries comprising bee venom}

The present invention relates to a composition for preventing and treating periodontal disease and caries, which contains bee venom as an active ingredient.

The causes of periodontal disease and tooth decay are diverse, but lactic acid produced by the breakdown of carbohydrates such as sugar and starch attached to teeth by the fermentation of bacteria in the oral cavity is known to cause dental caries by demineralizing the dental hard tissue. have. In addition, periodontal disease refers to the loss of teeth due to gingival inflammation and bleeding, periodontal pocket formation and destruction of alveolar bone. The periodontal disease is a series of processes consisting of colonization of bacteria, infiltration of periodontal tissues of bacteria, and destruction of periodontal tissues. First, saliva proteins in the oral saliva are adsorbed to dentin and chalky surfaces to form a film. As the bacteria grow on the surface of the film, they form plaques, and as time passes, the plaques move toward the root end and at the same time, anaerobic bacteria grow, and these bacteria, bacterial components, and bacterial products penetrate into the gingival connective tissue through the gingival epithelium. Periodontal pockets are formed. As a result of metabolism of these bacteria, it secretes toxic toxins such as hydrogen sulfide, ammonia and amine into the periodontal tissue, and the cell wall components are directly destroyed by endotoxins such as lipopolysaccharide, or at the same time the biological immune system Inflammation of gums is caused by several kinds of cytokines such as free radicals secreted extracellularly and interleukin by various actions of the humoral and cellular immune system stimulated, and collagenase secreted from bacteria and leukocytes. Collagen, which is a substrate of periodontal tissue, is degraded by the same enzyme, and gum retraction occurs. If left untreated, it progresses to periodontal disease. In an effort to prevent the occurrence of periodontal disease, various antibacterial and anti-inflammatory agents have been developed that can be killed by short-term exposure of periodontal disease bacteria, and have been applied to oral products such as toothpaste and ointment. The situation is not prevented.

As another method, a method of inhibiting pathogenic bacteria by organic or inorganic fluorine may be considered, but it may be resistant to prolonged use and may cause side effects such as diarrhea and vomiting. There is a problem that growth is inhibited.

Therefore, there is a need for the development of a new drug that has a selective antimicrobial activity against periodontal pathogens rather than a synthetic compound, and excellent early regeneration of damaged periodontal period.

Bee venom of domestic honey bee ( Apis mellifera ) can be obtained by collecting only pure bee venom without bee damage by using bee venom gathering device using electric shock method, freeze drying after removing foreign substances to obtain purified bee venom. Thus obtained bee venom is composed of various components, but the effect of bee venom on periodontal disease, tooth decay has not been studied yet.

Thus, the present inventors have conducted research to solve the above problems, by confirming that bee venom has antibacterial activity against caries-causing bacteria, gum inflammation-causing bacteria and periodontal disease-causing bacteria, and can suppress inflammation in the oral cavity The present invention has been completed.

Accordingly, an object of the present invention is to provide a composition for the prevention and treatment of periodontal disease and tooth decay, oral cleaning composition and functional food containing bee venom as an active ingredient.

The present invention is characterized by a composition composition for periodontal disease, caries prevention and treatment containing bee venom as an active ingredient.

In addition, the present invention also includes the composition for oral cleaning and functional food containing bee venom as an active ingredient in the scope of rights.

It is thought to be able to improve the quality of life of the people by developing into a periodontal disease containing bee venom according to the present invention, oral cleaning agent, toothpaste, functional foods and the like containing bee venom having the effect of preventing and treating tooth decay. It will contribute to the income increase of bee farms by developing high value-added uses.

Figure 1 shows the cytotoxicity of bee venom on activated periodontal ligament fibroblasts.
Figure 2a shows the inhibitory effect of IL-6 production after bee venom treatment in activated periodontal ligament fibroblasts.
Figure 2b shows the inhibitory effect of IL-8 production after bee venom treatment in activated periodontal ligament fibroblasts.
Figure 2c shows the inhibition of COX-2 protein expression after bee venom treatment in activated periodontal ligament fibroblasts.
Figure 3a confirms the inhibition of IL-6 production by performing RT-PCR after bee venom treatment in activated periodontal ligament fibroblasts.
Figure 3b confirms the inhibition of IL-8 production by performing RT-PCR after bee venom treatment in activated periodontal ligament fibroblasts.
Figure 3c confirms the inhibition of COX-2 protein expression by RT-PCR after bee venom treatment in activated periodontal ligament fibroblasts.

Hereinafter, the present invention will be described in detail.

The present invention contains bee venom as an active ingredient, has an inhibitory effect on caries-causing bacteria, gum inflammation-causing bacteria, periodontal disease-causing bacteria and anaerobic bacteria in the oral cavity, inhibits the production of inflammation-related cytokines, COX-2 The present invention relates to a composition that can be suppressed and effective for the prevention and treatment of periodontal disease, tooth decay.

Bee venom in the present invention are bacteria of Streptococcus mutans causing dental caries (Streptococcus mutans), a Streptococcus sanggwi switch (Streptococcus gum inflammation bacteria sanguis ) Porphyromonas , a periodontal disease-causing bacterium Gingivalis ) and oral anaerobic bacteria Fusobacterium nucleatum .

The bee venom may also inhibit the production of inflammatory cytokines IL-6 and IL-8, and may inhibit COX-2, which is involved in the production of prostaglandin E2 (PGE2).

Therefore, bee venom may be applied to oral cleaning compositions or toothpastes as well as compositions for the prevention and treatment of periodontal disease and tooth decay, as well as to be developed as a variety of functional foods such as gum, candy, jelly, chocolate, beverages, tea, and the like. have.

In the bee venom-containing composition according to the present invention, the content of bee venom is preferably 0.0001 to 10% (w / v) based on purified bee venom, and more preferably 0.01 to 1% (w / v). Do. If less than 0.0001% (w / v) is used, the antimicrobial and anti-inflammatory effects are low, the efficacy may be halved, and when used more than 10% (w / v) may cause cytotoxicity.

The composition according to the present invention may include, in addition to the active ingredient, a pharmaceutically acceptable carrier, excipient or diluent.

The compositions of the present invention may be in various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have.

The composition of the present invention may be administered orally or parenterally, and may be used as an external skin preparation or injection such as a dressing agent, a patch, a bandage, an ointment, and the like during parenteral administration.

The dosage of the composition of the present invention varies depending on the weight, age, sex, health condition, diet, time of administration, method of administration, excretion rate and severity of the disease of the patient, the daily dosage is gelatin degrading product 0.1 to 100 mg / kg, preferably 30 to 80 mg / kg, more preferably 50 to 60 mg / kg, based on the amount, can be administered 1 to 6 times per day.

And the composition of the present invention can also be used in combination with existing periodontal disease, caries treatment.

In addition, the present invention includes a composition for oral cleaning containing bee venom as an active ingredient. The oral cleaning composition may be formulated as a toothpaste, mouthwash solution, ointment, spray, dressing solution, coating, floss or periodontal bag for the prevention or treatment of periodontal disease. In order to formulate the oral cleaning composition, according to the formulation or the desired effect according to the desired effect should be used, and additionally, abrasives, wetting agents, foaming agents, solubilizers, binders, sweeteners, pH adjusting agents, preservatives, caries preventive agents, active ingredients , Fragrances, brighteners, pigments, astringents, solvents and the like. Examples of the abrasive include calcium carbonate, precipitated silica, aluminum hydroxide, calcium monohydrogen phosphate, insoluble sodium metaphosphate, and these abrasives may be used alone or in combination of two or more thereof, preferably 1 to 60% by weight, preferably 10 to 50% by weight. use. As the humectant, glycerin, sorbitol solution, polyethylene glycol, propylene glycol, or the like is used alone or in combination of two or more, and 10 to 60% by weight, preferably 20 to 50% by weight is used. As the foaming agent, anionic and nonionic surfactants such as sodium lauryl sulfate, sodium lauryl sarcosinate, sucrose fatty acid ester, polyoxyethylene hardened castor oil, and polyoxyethylene polyoxypropylene copolymer may be used alone or in combination of two or more. To 5% by weight, preferably 0.5 to 3% by weight. As the binder, 0.1 to 5% by weight, preferably 0.1 to 2% by weight, of carboxymethyl cellulose sodium, hydroxymethyl cellulose, carrageenan, xanthan gum, sodium alginate, etc. alone or in combination of two or more thereof is used. As a sweetening agent, it is preferable to use 0.05-5 weight% of saccharin sodium, aspartame, stevioside, ziitol, and licorice acid individually or in mixture of 2 or more types. The pH adjusting agent includes sodium phosphate, disodium phosphate, trisodium phosphate, sodium pyrophosphate, citric acid, sodium citrate, tartaric acid, and the like, and preservatives alone or two or more of methyl paraoxybenzoate, propyl paraoxybenzoate, and sodium benzoate. Use by mixing. The active ingredients include sodium fluoride, sodium monofluorophosphate, tin fluoride, chlorohexidine, allantoin chlorohydroxy aluminate, aminocaproic acid, tranexamic acid, triclosan, cetylpyridium chloride, zinc chloride, pyridoxine hydrochloride, acetic acid Tocopherol or the like may be used alone or in combination of two or more thereof.As a fragrance, peppermint oil, spearmint oil, menthol, and anetol may be mixed in an appropriate amount.Titanium oxide is used as a brightener, food coloring is used as a solvent, Mixes purified water and ethanol.

The present invention also includes functional foods for the prevention and improvement of periodontal disease and tooth decay containing bee venom as an active ingredient.

Functional foods according to the present invention, for example, chewing gum, caramel products, candy, ice cream, confectionery, such as various food products, soft drinks, mineral water, alcoholic beverages, beverage products such as vitamins and minerals Can be.

The bee venom of the present invention may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The blending amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or hygiene).

The functional food of the present invention may contain various flavors, natural carbohydrates, and the like as additional ingredients. The above-mentioned natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is preferably selected in the range of 0.01 to 0.04 parts by weight, preferably about 0.02 to 0.03 parts by weight, per 100 parts by weight of the health food of the present invention.

In addition to the above, the functional food of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols. And carbonation agents used in carbonated beverages. In addition, the functional food of the present invention may contain fruit flesh for the production of natural fruit juice, fruit juice drink and vegetable drink. These components can be used independently or in combination.

Hereinafter, the present invention will be described in detail with reference to the following examples, but the present invention is not limited thereto.

Example 1 Separation of Active Ingredients of Bee Venom

Western bee ( Apis meliffera L.) was collected by bee venom using the bee venom collection device, and the collected dried bee venom was dissolved in distilled water, centrifuged and filtered, and then lyophilized to obtain pure bee venom.

Example 2: Determination of antimicrobial activity against caries-causing bacteria and gum inflammation-causing bacteria of bee venom

Streptococcus mutans (Streptococcus bacteria known to cause tooth decay mutans, ATCC 27175), the Streptococcus bacteria cause inflammation of the gums sanggwi's (Streptococcus sanguis , ATCC 10556) to determine the antimicrobial effect, the minimum growth inhibition concentration (MIC) by liquid medium dilution method was confirmed by measuring.

Dry bee venom was diluted with sterile distilled water and then sterile filtered and diluted stepwise according to the liquid medium dilution method. Streptococcus mutans were incubated in BHI broth (Brain heart infusion broth) medium and Streptococcus sangwis were trypticase soy broth (added 5% sheep blood) and incubated at 37 ° C. for 18 hours at aerobic conditions. Used for post test. Each inoculation bacterium was adjusted to 2 × 10 ⁶ CFU / well, incubated with the diluted bee venom sample for 18 hours, and the growth of the bacteria was visually observed under a microscope and absorbed according to the dilution concentration of bee venom. The absorbance was measured at a wavelength of 540 nm to obtain the same result as the absorbance value of the pure culture solution. The results are shown in Table 1 below.

Strain Minimum growth inhibitory concentration
(Μg / ml) S. mutans 0.68 ± 0.029 S. sanguis 0.85 ± 0.042

As shown in Table 1, bee venom was confirmed to have excellent antimicrobial activity against the strain.

Example 3: Determination of antimicrobial activity against bee venom causing periodontal disease-causing bacteria and oral anaerobic bacteria

To measure the antimicrobial effect against Porphyromonas gingivalis ( ATCC 33277) , known as periodontal disease-causing bacteria, and Fusobacterium nucleatum ( ATCC 25286), an oral anaerobic bacterium that causes bad breath, Minimal Inhibitory Concentration (MIC) was determined by the liquid medium dilution method.

Porphyromonas gingivalis and Fusobacterium nucleatum were incubated in a CO ₂ incubator at 37 ° C. for 24 hours using Chopped meat medium. Each inoculation was adjusted to 2 × 10 ⁶ CFU / well and incubated with the diluted bee venom sample for 24 hours, and then the growth of the bacteria was visually observed under a microscope and under the dilution concentration of bee venom. The absorbance was measured at 540 nm to obtain the same result as the absorbance value of the pure culture. The results are shown in Table 2 below.

Strain Minimum growth inhibitory concentration
(Μg / ml) P. gingivalis 3.49 ± 0.13 F. nucleatum 2.79 ± 0.28

As shown in Table 2, bee venom was confirmed to have excellent antimicrobial activity against the strain.

Example 4 Determination of Antimicrobial Duration of Bee Venom

The bactericidal duration (PAE, postantibiotic effect) of bee venom on the tested strains was measured by the method of Pankuch and Appelbaum (2006). The strains of Examples 2 and 3 were adjusted to 2.5 × 10 ⁵ CFU / mL, incubated with bee venom samples at a concentration of 2 times the minimum inhibitory concentration, and then cultured for 1 hour using centrifugation and media dilution. Bee venom was removed. Subsequently, the cells were replaced with fresh medium, incubated in a 37 ° C. incubator, and the bacterial counts were measured at 2 hour intervals. The higher the PAE, the better the antimicrobial effect. The formula for obtaining the PAE is shown in Equation 1 below, and the measurement results are shown in Table 3 below.

[Equation 1]

PAE = T-C

T: Time taken for growth up to 1 log 10 in test bee treated

C: Time taken to grow up to 1 log 10 in the untreated plot

Strain PAE (hour) S. mutans 3.30 S. sanguis 3.45 P. gingivalis 2.0 F. nucleatum 2.0

As shown in Table 3, bee venom was confirmed to have excellent antimicrobial sustaining activity against the strain.

Example 5 Measurement of IL-6, IL-8 and COX-2 Expression in Periodontal Ligament Fibroblasts

Lipopolysaccharide (LPS) was applied to primary cultured periodontal ligament fibroblasts to determine whether it can inhibit the expression of IL-6 and IL-8, which cause bee venom, and COX-2, which mediates PGE2 production. Treatment caused inflammation. Periodontal ligament fibroblasts were passaged in 37%, 5% CO ₂ incubator using DMEM medium containing 10% FBS was used in the experiment. When cultured cells were grown to 90% of the floor area, subcultures were used and the cells of logarithmic growth phase were used for the experiment. Periodontal ligament fibroblasts were aliquoted at 5 × 10 ⁵ cells / ml in a 24 well plate, treated with LPS (1 μg / ml), and the bee venom of Example 1 was 0.01, 0.1, 1, 5 and 10 μg. Treatment was diluted to a concentration of / ml. After 48 hours of incubation in a 37 ° C., 5% CO ₂ incubator, cell viability was treated with MTT (methylthiazol-2-yl-2.5-diphenyl tetrazolium bromide) reagent and the absorbance at 540 nm was measured with ELISA reader. Hair cell toxicity was evaluated and the results are shown in FIG. 1. When bee venom was treated within 10 μg / ml, it was confirmed that the bee venom was not toxic to the periodontal ligament fibroblasts.

Expression of IL-6, IL-8 and COX-2 from the cell suspension treated with LPS and bee venom was measured using an ELISA kit, and the results are shown in FIG. As shown in FIG. 2, bee venom was found to significantly inhibit the expression of IL-6, IL-8 and COX-2 in periodontal ligament fibroblasts.

Example 6 Measurement of IL-6, IL-8 and COX-2 Expression in Periodontal Ligament Fibroblasts at RNA Level

Cell culture In a 100 mm dish, primary cultured periodontal ligament fibroblasts were dispensed at 5 × 10 ⁵ cells / ml, and treated with LPS (1 μg / ml) and bee venom (100 ng / ml), followed by 37 ° C. and 5%. 48 hours incubation in a CO ₂ incubator. RNA was extracted from the cultured periodontal ligament fibroblasts and RT-PCR was performed using the primers of Table 4, and the results are shown in FIG. 3.

Forward primer Reverse primer IL-6 5'-ATGAACTCCTTCTCCACAAGCGC-3 '
(SEQ ID NO 1) 5'-GAAGAGCCCTCAGGCTGGACTG-3 '
(SEQ ID NO: 2) IL-8 5'-ATGACTTCCAAGCTGGCCGTGGCT-3 '
(SEQ ID NO: 3) 5'-TCTCAGCCCRCRRCAAAAACTTCTC-3 '
(SEQ ID NO: 4) COX-2 5'-TTCAAATGAGATTGTGGGAAAATTG-3 '
(SEQ ID NO: 5) 5'-AGATCATCTCTGCCTGAGTATCTT-3 '
(SEQ ID NO: 6)

As confirmed in FIG. 3, it was confirmed that RT-PCR using the primers significantly inhibited the expression of IL-6, IL-8, and COX-2 in the periodontal ligament fibroblasts at the RNA level.

Formulation Example 1 Preparation of Toothpaste

The components belonging to a of Table 5 were added to the preparation unit at a temperature of 70 ° C., and stirred for 20 minutes to completely dissolve the powder, and then cooled to room temperature. Subsequently, as a dissolution step of the wetting agent, the sweetening agent and the active ingredient, the components belonging to b were added to the production unit at 25 ° C., followed by stirring for 15 minutes to dissolve. Next, the ingredients belonging to c were mixed in steps of 25 to 35 ° C. The next process is the foaming agent mixing and primary degassing process, adding the component belonging to d to complete the first defoaming for 40 minutes at 35 ° C, finally adding the component belonging to e and performing the second defoaming at 35 ° C to remove the toothpaste. I did gymnastics.

Ingredients Content (g) Remarks Purified water Suitable amount a Dissorbitol solution 33.00 Propylene glycol 5.10 Concentrated glycerine 8.25 Tim Calcium Carbonate 30.00 b Amino acid capronic acid 0.01 Calcium dihydrogen phosphate 0.01 Silicon dioxide 18.84 Sodium benzoate 0.18 Green tea extract 0.01 Zaccarin Sodium 0.01 Xylitol 0.01 Chitosan 0.01 Propolis Extract 0.01 Aloe extract 0.01 Bee venom 0.01 Polyethylene glycol 1500 0.48 c Carboxymethyl Cellulose Sodium 0.72 Sodium Lauryl Phenate 2.44 d DL-menthol Suitable amount Peppermint Suitable amount e

Formulation example  2: Preparation of functional food

< Production of Drinks>

Bee Venom 100 mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components according to the conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored Used to prepare beverage compositions of the invention.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

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Claims (5)

A composition for treating periodontal disease caused by Porphyromonas jinjibilis, which is a periodontal disease-causing bacterium, characterized in that it contains purified venom as an active ingredient 0.01 to 1% (w / v).
delete Oral cleaning composition having an antimicrobial activity against fusobacterium nucleatum, which is an oral anaerobic bacterium, characterized in that it contains 0.01 to 1% (w / v) as an active ingredient.
Composition for the treatment of gum inflammation induced from Streptococcus sangwicus, which is a gum inflammation-inducing bacterium, characterized in that it contains tablet bee venom as 0.01 to 1% (w / v) as an active ingredient. delete

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