KR20190095156A - Composition for preventing or treating periodontal disease and dental caries containing fluoride varnish and antibacterial agents as an active ingredient - Google Patents

Abstract

The present invention relates to a composition comprising a fluoride varnish and an antimicrobial material as active ingredients, which not only can prevent dental caries but also can prevent or treat periodontal disease by adding an antimicrobial material for Porphyromonas gingivalis to a fluoride varnish. Specifically, Chrysophanol, Emodin, Anthrarufin, Bavachalcone or Isobavachromene, and a fluoride varnish composition comprising the same of the present invention can be useful in prevention or treatment of periodontal disease by exhibiting antibacterial activities with respect to Porphyromonas gingivalis. Particularly, the fluoride varnish has an inhibition effect by itself with respect to Streptococcus mutans (S.mutans), which is bacteria causing dental caries. Further, Bavachalcone or Isobavachromene in the compounds has a significant antibacterial activity even with respect to S.mutans. Therefore, a fluoride varnish composition comprising Bavachalcone or Isobavachromene may be used in prevention or treatment of dental caries and periodontal disease at the same time.

Description

Composition for preventing or treating periodontal disease and dental caries containing fluoride varnish and antibacterial agents as an active ingredient

The present invention is a fluoride varnish ( porphyromonas gingivalis ) by adding an antimicrobial material for porphyromonas gingivalis , as well as preventing dental caries, including fluoride varnish and antimicrobial material that can prevent or treat periodontal disease. It relates to a composition.

Diseases that cause problems worldwide in oral disease are dental caries and periodontal disease (Board of Trustees of the American Academy of Periodontology, 2000). The most important cause of oral disease is dental plaque. There are about 700 species of bacteria in the dental plaque, which are the causative agents of dental caries and periodontal disease (Kolenbrander PE, 2000). Among the periodontal diseases, gingivitis and periodontitis are mainly related to Gram-negative anaerobes such as Porphyromonas gingivalis , Prevotella intermedia , Aggregatibacter actinomycetemcomitans , Fusobacterium nucleatum, Treponema denticola, etc. (Haffajee, Socransky, 1994; 1997) Darveau et al.

Periodontal disease is an inflammatory disease leading to the destruction of peripheral tissues including alveolar bone and is the most important cause of tooth loss. P. gingivalis , which causes periodontal disease, is known as a representative cause of periodontal disease (Ximenez-Fyvie LA et al., 2000). P. gingivalis expresses many adherents associated with the outer membrane or cilia and is known to promote adhesion to tooth surfaces, gingival epithelial cells, basement membrane components, erythrocytes and oral microorganisms (Lamont RJ et al 1998). When periodontal disease progresses, antibiotics are usually administered. Tetracycline, an antibiotic commonly used as a treatment for periodontitis, is not recommended for children under 9 years of age due to tooth discoloration (Mcculloch CA et al., 1990). In addition, Chlorhexidine is an oral solution used for periodontal treatment to inhibit gram-negative bacteria, causing coloration of teeth and prostheses, and causing side effects such as taste disorders and burning of the oral mucosa (Tredwin et al., 2005).

In addition, since antibiotics have side effects due to their resistance to humans (Eun et al., 2003), many studies are attempting to develop products using natural antimicrobial agents that have less side effects and have safety than antimicrobial agents used previously. Progression (Araghizadeh et al., 2013; Gonzalez et al., 2013; Labrecque et al., 2006).

Fluoride varnish, which prevents dental caries, was first introduced in Europe in 1964 and has been recognized as a safe and effective fluoride product through numerous clinical studies. Fluorine varnishes have excellent adhesion to tooth surfaces and harden when applied thinly on teeth. After rosin dissolves in saliva, the fluorine contained in the fluorine varnish slowly releases the fluorine concentration on the tooth surface for a long time, which acts as a barrier to prevent mineral exchange between the tooth and saliva, resulting in long-term dental caries prevention effects (Demito et al. , 2004; Todd et al., 1999). In addition, fluorine varnish itself has some effects on the inhibition of caries-causing bacteria, and contributes to the prevention of dental caries by replacing hydroxyapatite of tooth enamel with fluroapatite having high acid resistance. Prior invention of the present inventors has developed and registered a patent for fluorine varnish which can release fluorine for a long time compared to the existing fluorine varnish (Patent Registration Nos .: 10-1728459, 10-1728461, 10-1728463) ).

On the other hand, many studies are being conducted to effectively treat periodontal disease, but most of them only verify the antimicrobial effect of the natural antimicrobial agent itself, and there are no studies in which the natural antimicrobial agent was directly added to the fluorine varnish in relation to the periodontal disease. .

Accordingly, the present inventors have searched for an effective antimicrobial substance, and applied to fluorine varnish to develop a composition having a significant effect on periodontal disease. As a result, chrysophanol, emodin, and anthrafin ( It was confirmed that the composition to which Anthrarufin, Bavachalcone or Isobavachromene was added to the fluorine varnish exhibited antimicrobial activity against Porphyromonas gingivalis , in particular, the Babachalcon and iso Babacromine also exhibits significant antibacterial activity against Streptococcus mutans ( S.mutans ), a tooth caries-inducing bacterium, and prevents or treats periodontal disease and dental caries with fluoride varnishes. The present invention has been completed by revealing that it can be usefully used as a composition.

An object of the present invention is to add fluoride varnish to the antibacterial material for Porphyromonas gingivalis ( Pyphyromonas gingivalis ) to prevent dental caries, as well as to prevent or treat periodontal disease fluorine varnish and antimicrobial material It is to provide a composition comprising.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing and treating periodontal disease (periodontal disease) containing fluoride varnish and antibacterial substances as an active ingredient.

In addition, the present invention is an active ingredient any one or more selected from the group consisting of Chrysophanol (Chrysophanol), Emodin (Emodin), Anthrufin (Anthrarufin), Babachalcone and Isobavachromene (Isobavachromene) as an active ingredient It provides a pharmaceutical composition for preventing and treating periodontal disease containing, and health food for preventing and improving periodontal disease containing the compound as an active ingredient.

Chrysophanol, Emodin, Anthrarufin, Bavachalcone or Isobavachromene and fluorine varnish composition comprising the same according to the present invention are porphyromonas gingivavalis Porphyromonas gingivalis ) has an antimicrobial activity and can be useful for the prevention and treatment of periodontal disease. In particular, fluoride varnish suppresses itself against the dental caries-inducing Streptococcus mutans ( S.mutans ). It is effective, and also barbakalcon or isovabacromine of the compound has a significant antimicrobial activity against S.mutans , fluorine varnish composition comprising the same can be used to prevent or treat dental caries and periodontal disease at the same time .

1 is a diagram confirming the antimicrobial activity of Porphyromonas gingivalis of the compounds screened in the present invention:
Positive control: chlorohexidine stock solution;
Negative control: PBS;
Vehicle Control: DMSO
No. 1: Chrysophanol;
No. 2: Emodin;
3: anthrarufin;
4: Bavachalcone; And
Number 5: Isobavachromene.
2 is a view showing the antimicrobial activity of porphyromonas jinjivalis of fluoride varnish mixed with an antimicrobial material:
Positive control: chlorohexidine stock solution;
Negative control: PBS;
Vehicle Control: DMSO Mixed with Fluorine Varnish
No. 1: Chrysopanol mixed fluorine varnish;
No. 2: Fluorine varnish mixed with emodine;
3: fluorine varnish mixed with anthral lupine;
4: fluorine varnish mixed with babakalcon; And
Number 5: Fluorine varnish mixed with isobavacromine.
Figure 3 is a measure of cell activity using CCK-8 (Cell counting kit-8):
1: chrysopanol;
Number 2: emodine;
No. 3: anthrupine;
4: babakalcon; And
Number 5: isovabachromin.
Figure 4 is a measure of cytotoxicity using the LDH (Lactate dehydrogenase) assay:
1: chrysopanol;
Number 2: emodine;
No. 3: anthrupine;
4: babakalcon; And
Number 5: isovabachromin.
5 and 6 are diagrams showing the results of evaluation of antimicrobial persistence using an OHP film disc:
+ cont: treatment group of Chlorhexidine and fluorine varnish mixture;
v cont: DMSO and fluorine varnish mixture treatment group;
CHR: chrysopanol and fluorine varnish mixture treatment group;
EMO: Emodine and Fluorine Varnish Mixture Treatment Group;
ANT: anthral lupine and fluorine varnish mixture treatment group;
BCC: group of babakalcon and fluorine varnish mixture; And
IBC: group treated with isobavacromine and fluorine varnish mixture.
7 is a diagram showing the results of evaluation of antimicrobial persistence using Hydroxyapatite disc.

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

The present invention provides a pharmaceutical composition for the prevention and treatment of periodontal disease (periodontal disease) containing fluoride varnish and antibacterial substances as an active ingredient.

The fluorine varnish is preferably prepared by the following method, but is not limited thereto:

1) rosin; menstruum; And blending a fluorine compound; And

2) stirring the blend at 70-100 ° C. for 25-40 minutes with stirring.

Rosin of step 1) above; The mixing ratio of the solvent and the fluorine compound is 30 to 65% by weight: 35 to 65% by weight: 1 to 10% by weight, more specifically, 35 to 55% by weight: 40 to 60% by weight: 2 to 8% by weight of the total compound weight. %, Even more specifically 45%: 50%: 5% by weight.

The solvent of step 1) is at least one selected from the group consisting of distilled water, dimethyl sulfoxide (DMSO), isoamyl propionate, hydrogen fluoride (HF), ethanol, and ethyl acetate. And more specifically ethanol or ethyl acetate.

The fluorine compound of step 1) is HF, NH ₄ F, NaF, KF, Na ₂ FPO ₄ , K ₂ FPO ₄ , CaF ₂ , TiF ₄ , Ca ₅ (PO ₄ ) ₃ F, H ₂ F ₂ Si (Difluorosilane ), C ₁₆ H ₃₆ FN, SnF ₂ and Cetylamine hydrofluoride may be any one or more selected from the group consisting of, more specifically NaF. In addition, a fluorine compound can be used individually by 1 type and can be used in combination of multiple types.

The rosin may be hydrogenated rosin added with hydrogen (hydrogenated), compounded with hydrogen, or cured with hydrogen, but any rosin that may be used for the production of fluorine varnish may be used.

The antimicrobial material is Chrysophanol (Chrysophanol) described in the following [Formula 1], Emodin (Emodin) described in the following [Formula 2], Anthrufin (Anthrarufin) described in the following [Formula 3], It is preferable that it is Bavachalcone described in 4] or Isobavachromene described in the following Chemical Formula 5.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

In addition, the periodontal disease is a disease caused by Porphyromonas gingivalis , etc., preferably gingivitis or periodontitis (Periodontitis), but is not limited thereto.

On the other hand, when the alveolar bone by the periodontal disease absorbed and down when the gingiva is exposed tooth root, root, unlike crown covered in enamel, because cementum covered by a weaker (cementum]) on dental caries dental caries-related bacteria such as S.mutans Caries are easily generated by this, and it is called root caries. Therefore, the fluorine varnish itself of the present invention is also effective in inhibiting dental caries-inducing bacteria, especially barbakalcon or isobavacromine showed a significant antimicrobial activity against S.mutans composition of the present invention periodontal disease and It is obvious that dental caries, including root caries, can be simultaneously prevented or treated.

In addition, the composition of the present invention is Chrysophanol (Chrysophanol) described in [Formula 1], Emodin (Emodin) described in the following [Formula 2], Anthrufin (Anthrarufin) described in the following [Formula 3], Babachalcone described in [Formula 4] or isobavachromene (Isobavachromene) described in [Formula 5] is added to the fluorine varnish, and is characterized by increasing antibacterial activity.

In a specific embodiment of the present invention, the present inventors screened an antimicrobial agent having an antimicrobial activity against Porphyromonas gingivalis, out of a total of 100 substances, chrysopanol, emodin, anthrapine, babakalcon Or it was confirmed that isovaba cromine showed antimicrobial activity against P. gingivalis (see Fig. 1).

In addition, the present inventors confirmed the antimicrobial effect of the fluorine varnish mixed with the above-described antimicrobial substance, 10 mM chrysopanol fluoride varnish was 1.67 times higher than the antibacterial effect of the same concentration of chrysopanol itself, and emodine, Fluorine varnishes mixed with anthrupine, babakalcon, and isovaba cromine were also confirmed to exhibit antimicrobial activity against P. gingivalis (see FIG. 2).

In addition, the present inventors confirmed the lowest growth inhibitory concentration and the lowest bactericidal concentration of the antimicrobial substance using the P. gingivalis strain, MIC and MBC of the antimicrobial material itself is the lowest concentration of Babakalcon (MIC 0.0035 mM, MBC 0.007 mM ) Inhibited P. gingivalis , followed by low MIC and MBC of emodin, anthrapine, and isovabacromine, and the highest MIC and MBC of chrysophanol. On the other hand, in the case of the fluorine varnish mixed with the antimicrobial material, it was confirmed that significantly inhibit the growth of P. gingivalis bacteria even at a concentration 2 to 8 times lower than the antimicrobial itself (see Table 2).

In addition, the present inventors evaluated the cell activity and cytotoxicity of the antimicrobial substances identified in the present invention, all the antimicrobial substances in the cell activity test through CCK-8 showed a high cell activity, such as Vehicle Control up to 100 μM concentration In the cytotoxicity test through LDH, all antimicrobial substances were confirmed to be non-toxic up to 100 μM concentration. Therefore, it was confirmed that the concentration of MIC and MBC when the antimicrobial material of the present invention was mixed with the fluorine varnish was a safe concentration (see FIGS. 3 and 4).

In addition, the present inventors confirmed the persistence of the antimicrobial effect of the fluorine varnish mixed with the above-described antimicrobial material, the fluorine varnish composition containing the chrysopanol, emodine, anthrapine, babakalcon or isovaba cromine of the present invention The antimicrobial effect lasted up to 30 days, and in particular, the fluorine varnish mixed with Chrysophanol was confirmed to exhibit excellent antimicrobial persistence (see FIGS. 5 to 7).

Therefore, the fluorine varnish composition comprising chrysopanol, eminine, anthrapine, barbacalcon or isobavacromine of the present invention exhibits significant antimicrobial activity against porphyromonas gingivalis strains causing periodontal disease, in particular As a result of confirming the inhibitory effect of Porphyromonas gingivalis strains using the MIC method that can more accurately measure the oral clinical effects, emodin, anthrafin, isovabacromine and chrysopanol were mixed in fluorine varnish. In this case, it is possible to suppress the growth of P. gingivalis bacteria even at a significantly low concentration compared to the effect of the compound itself, thereby containing the chrysopanol, emodine, anthrapine, babakalcon or isovaba cromine Fluoride varnish may be useful for preventing or treating periodontal disease.

The composition of the present invention additionally includes polymerization inhibitors, pigments, colorants, fluorescent agents, ultraviolet absorbers, sweeteners, flavorings, waxes, xylitol for neutralizing pH, apatite and calcium phosphate for remineralization of teeth, phosphoric acid Sodium phosphate, Calcium sulfate dihydrate, Tri-Calcium phosphate (-TCP), TiF ₄ , Wax, Hexane (Catechol), Poly isocyanate with adhesive properties on teeth (polyisocyanate), glutaraldehyde, 10-MDP (10-Methacryloyloxydecyl dihydrogen phosphate), HEMA (2-hydroxylethyl methacrylate), HEMA-phosphate, phenyl-P (2- [methacryloyloxyethyl] phenyl hydrogen phosphate), 4- MET (4-methacryloyloxyethyl trimellitic acid), 4-META (4-methacryloyloxyethyl trimellitic anhydride), MAC-10 (11-methacryloyloxy-1,1-undec-anedicarboxylic acid), polyethylene glycol- dimethacrylate, Di- and tri-methacrylate resins, oxalic acid, maleic acid, methylmethacrylate p-styrenesulfonic acid, benzalkonium chloride, strontium chloride, potassium nitrate, potassium salt, etc., may be blended in such an amount as to not lose the effects of the present invention. .

In addition, the composition of the present invention is preferably administered parenterally (eg, applied), and is preferably applied to teeth of 5% fluoride varnish 4 mg / 1 day to 6 mg / 1 day.

In addition, the present invention is to prevent periodontal disease containing any one or more compounds selected from the group consisting of chrysopanol, emodin, anthrapine, babakalcon and isobavacromine or a pharmaceutically acceptable salt thereof as an active ingredient. And pharmaceutical compositions for treatment.

The composition includes oral solution, dental perceptual hypersensitivity agent, antimicrobial dental adhesive, dental filler, dental restorative material, dental root canal filling material, dental root canal sealer, dental vortex blocker, dental coating and It can be formulated and used through known methods for dental cement.

In a specific embodiment of the present invention, the present inventors screened an antimicrobial agent having an antimicrobial activity against Porphyromonas gingivalis, out of a total of 100 substances, chrysopanol, emodin, anthrapine, babakalcon Or it was confirmed that isovaba cromine showed antimicrobial activity against P. gingivalis (see Fig. 1).

Therefore, the compound of the present invention has a significant antimicrobial effect against P. gingivalis , it can be usefully used as a pharmaceutical composition for preventing or treating periodontal disease.

The present invention includes not only compounds, but also pharmaceutically acceptable salts thereof, and possible solvates, hydrates, racemates, or stereoisomers which may be prepared therefrom.

The compounds of the present invention can be used in the form of pharmaceutically acceptable salts, and acid salts formed by pharmaceutically acceptable free acids are useful as salts. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. Obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically nontoxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and iodide. Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suverate , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, meth Oxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesul Nate, Xylene Sulfonate, Phenyl Acetate, Phenylpropionate, Phenyl Butyrate, Citrate, Lactate, Hydroxybutyrate, Glycolate, Maleate, Tartrate, Methanesulfonate, Propanesulfonate, Naphthalene-1-sulfonate , Naphthalene-2-sulfonate or mandelate.

The acid addition salts according to the invention are dissolved in conventional methods, for example, by dissolving the compounds of the invention in an excess of aqueous acid solution and using the water miscible organic solvents such as methanol, ethanol, acetone or acetonitrile. It can be prepared by precipitation. The mixture may also be prepared by evaporation of a solvent or excess acid to evaporate or by precipitation filtration of the precipitated salt.

Bases can also be used to make pharmaceutically acceptable metal salts. An alkali metal or alkaline earth metal salt is obtained by, for example, dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. Corresponding silver salts are also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

When formulating the composition, it is prepared using commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants.

Solid form preparations for oral administration include tablets, patients, powders, granules, capsules, troches, and the like, which form at least one excipient such as starch, calcium carbonate, water, or the like. It is prepared by mixing cross, lactose or gelatin. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solvents, emulsions or syrups, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water, liquid paraffin, and waxes This may be included. In addition, the composition of the present invention can be applied to the gum composition by adding to a gum base consisting of vegetable resin, vinyl acetate resin, ester gum, polyisobutyrene, wax, emulsifier and the like.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like.

As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used.

The composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level means the type, severity, and activity of the patient's disease. , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of the drug, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

Specifically, the effective amount of the compound according to the present invention may vary depending on the age, sex, and weight of the patient, and in the present invention, it was confirmed that all the antimicrobial substances are not toxic up to 100 uM, and based on this, daily or every other day It may be administered by dividing 1 to 3 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.

In addition, the present invention is to prevent periodontal disease containing any one or more compounds selected from the group consisting of chrysopanol, emodin, anthrapine, babakalcon and isobavacromine or a pharmaceutically acceptable salt thereof as an active ingredient. And it provides a health food for improvement.

When the compound of the present invention is used as a food or beverage additive, the compound 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 mixed amount of the compound may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In the case of prolonged ingestion for health and hygiene purposes or for health control purposes, the compound can be taken for a long time since there is no problem in terms of safety. There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, dairy products including other noodles, gum, ice cream, various soups, drinks, tea, and drinks. Alcoholic beverages and vitamin complexes. The liquid component added in addition to the compound of the present invention when formulated into a beverage is not limited thereto, but may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in conventional beverages. Examples of the natural carbohydrates described above include monosaccharides (e.g. glucose, fructose, etc.), disaccharides (e.g. maltose, sucrose, etc.) and polysaccharides (e.g. conventional sugars such as dextrins, cyclodextrins, etc.), and xylitol Sugar alcohols such as sorbitol and erythritol. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention. As flavoring agents other than those described above, natural flavoring agents (taumarin, stevia extract (e.g., Rebaudioside A, glycyrzin, etc.)) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) can be used. .

In another embodiment, the food composition of the present invention is a nutrient, colorant and enhancer (cheese, chocolate, etc.), pectic acid and salts thereof, organic acids of various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, etc. , Protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. The food composition of the present invention may also contain pulp for the production of fruit and vegetable drinks. These components may be used alone or in combination, and the proportion of such additives is generally selected in the range of 0.001 to 50 parts by weight per total weight of the composition.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

Example 1 Preparation of Fluorine Varnish

Rosin (KR-610, ARAKAWA, Japan) 45 wt% (9 g) was used to prepare the fluorine varnish. 50 wt% (10 g) of ethanol (absolute ≧ 99.7%, Merck KGaA) was used as a solvent, and 5 wt% (1 g) of NaF was used. The above material was heated to 240 ° C. at 240 rpm with an overhead stirrer on a heated stirrer (RCH-3, Tokyo Rikakikai Co., LTD., Tokyo, Japan). Mixing was performed for minutes to prepare a fluorine varnish.

Example 2 Preparation of Fluorine Varnishes Mixed with Antimicrobial Substances

The antimicrobial powder was dissolved in DMSO and prepared and stored at 100 mM, and diluted with DMSO to produce 20 mM and 2 mM antimicrobial materials, respectively. Then, 1: 1 mixed with the fluorine varnish prepared in Example 1, to prepare a fluorine varnish mixed with 10 mM or 1 mM antimicrobial material.

Specifically, 10 μL of 20 mM antimicrobial material + 10 μL of fluorine varnish (mixing 20 mM antimicrobial material and fluorine varnish in a 1: 1 ratio) were mixed to prepare a fluorine varnish mixed with 10 mM antimicrobial material. In addition, 10 μL of 2 mM antimicrobial substance + 10 μL of fluorine varnish (1 mM mixed with 2 mM antimicrobial substance and fluorine varnish) were mixed to prepare a fluorine varnish mixed with 1 mM antimicrobial substance. In addition, the vehicle control was prepared by mixing fluorine varnish and DMSO 1: 1 in the same manner.

<Experimental Example 1> porphyromonas ginjivalis ( Porphyromonas gingivalis ; P. gingivalis Antimicrobial Screening with Antimicrobial Activity against

To develop an antimicrobial fluoride varnish that can be added to fluorine varnish to prevent or treat periodontal disease, P. gingivalis (ATCC33277, Korea Institute of Bioscience and Biotechnology) related to periodontal disease was obtained from Korea Compound Bank. Antimicrobial evaluation of 100 compounds was carried out using agar medium diffusion test.

Specifically, P. gingivalis was incubated in BHI medium for 72 hours at 37 ° C. CO ₂ incubator, and then OD value (mechanical: eppendorf Biophotometer plus) was measured, followed by inoculation onto agar medium coated with Base agar by mixing bacteria with Top agar. . After the top agar had solidified, a 6 mm diameter, 0.7 mm thick paper disc was placed on the agar plate. 1 mM and 10 mM of the compound to be evaluated were applied to the paper disc by 5 μL, respectively. As a positive control, chlorohexidine stock solution (hexamedin solution, bugwang medicine) was used, a negative control group was used as PBS, and vehicle control was used as DMSO 100%. After culturing in a 37 ℃ CO ₂ incubator for 72 hours, the diameters of the P. gingivalis inhibitors formed around the paper disc were measured in right angles to calculate the average. It was determined that there was an antimicrobial effect when showing a suppressor band larger than 6 mm, the diameter of the paper disc.

As a result, as shown in Figure 1 and Table 1, among the 100 compounds obtained from the Korea Compound Bank, Emodin (Emodin), Chrysophanol (Chrysophanol), Anthrarufin (Anthrarufin), Bavachalcone, Isobavachromene (Isobavachromene) showed a significant antimicrobial effect against P. gingivalis , in particular it was confirmed that the antimicrobial effect was higher at 10 mM than 1 mM. On the other hand, DMSO itself was confirmed that there is no antimicrobial effect against P. gingivalis like the negative control (Fig. 1 and Table 1).

Experimental Example 2 Confirmation of Antimicrobial Effects of Fluorine Varnishes Mixed with Antimicrobial Substances

By using the method disclosed in the <Example 2>, to prepare a fluorine varnish containing the antimicrobial material confirmed in the <Experimental Example 1>, the antimicrobial activity of the P. gingivalis was confirmed.

Specifically, antimicrobial evaluation was performed using the same agar medium diffusion test as in <Experimental Example 1>, Vehicle Control was used by mixing a fluorine varnish and DMSO in a ratio of 1: 1.

As a result, as shown in FIG. 2, as a result of mixing fluorine varnish with an antimicrobial substance, 10 mM chrysopanol-blended fluorine varnish (20 mM chrysopanol and fluorine varnish mixed 1: 1) of the same concentration of chrysopha It was found to be 1.67 times higher than the antimicrobial effect of Knoll itself and 2 times higher than fluorine varnish, which is a vehicle control. In addition, it was confirmed that the fluorine varnish mixed with emodin, anthrapine, babakalcon, and isovaba cromine also showed antimicrobial activity against P. gingivalis (FIG. 2).

Experimental Example 3 Confirmation of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

P. gingivalis (ATCC33277, Korea Research Institute of Bioscience and Biotechnology) strains associated with periodontal disease were used to identify the lowest growth inhibitory and minimum bactericidal concentrations.

Specifically, to measure the MIC was performed on a 96 well plate containing 100 μL of BHI broth per well. The antimicrobial material was added to the first well of 4 mM concentration, and diluted by 1/2 in steps to 100 μL. P. gingivalis is BHI broth was used as a medium to activate the bacteria, and after incubation for 72 hours in a 37 ° C CO ₂ incubator, 100 μL was added to a 96 well plate. After 72 hours of incubation, the lowest growth inhibitory concentration was determined by measuring the absorbance at 600 nm with an Enzyme-linked immuno-assay reader (Spectra MAX250, Molecular Devices Co.).

As a result, as shown in Table 2, MIC and MBC of the antimicrobial material itself inhibited P. gingivalis bacteria at the lowest concentrations of Babakalcon (MIC 0.0035 mM, MBC 0.007 mM), followed by emodin and anthrapine. It was confirmed that the MIC and MBC of and isovaba cromine was low, and the MIC and MBC of chrysophanol were the highest.

On the other hand, even in the fluoride varnish mixed with antimicrobial material, it was confirmed that MIC and MBC of Babakalcon inhibit the growth of P. gingivalis bacteria at the lowest concentration (0.05 mM for both MIC and MBC). However, it was confirmed that the emodin, anthrapine, isovaba cromine and chrysopanol inhibit the growth of P. gingivalis bacteria at a concentration of 2 to 8 times lower than that of the antimicrobial substance when mixed with fluorine varnish. .

In other words, unlike the agar diffusion test, which shows antimicrobial effect as it diffuses into agar, the MIC test is in direct contact with the antimicrobial agent. In fact, when the antimicrobial agent is applied to the teeth through fluoride varnish in the oral cavity, the MIC method is clinical. It is a test method reproduced more similarly to. Therefore, MIC test results show that emindine, anthrafin, isovabacromine and chrysopanol can inhibit the growth of P. gingivalis bacteria even when mixed with fluorine varnish at a significantly lower concentration than the antimicrobial material itself. It was.

Antibacterial substance alone Fluorine varnish mixed with antibacterial substance MIC (mM) MBC (mM) MIC (mM) MBC (mM) Chrysopanol 0.25 0.5 0.031 0.031 Emodin 0.062 0.125 0.031 0.031 Anthrupine 0.062 0.125 0.031 0.062 Babacalcon 0.0035 0.007 0.015 0.015 Isovaba cromine 0.062 0.125 0.031 0.31

Experimental Example 4 Evaluation of Cell Activity and Cytotoxicity

<4-1> Cell activity measurement

Cell activity was measured using CCK-8 (Cell counting kit-8) for compounds exhibiting an antimicrobial effect against P. gingivalis identified through Experimental Example 1.

Specifically, L-929 cells were cultured in RPMI medium containing 10% FBS and antibiotics (Penicillin Streptomycin, Life technologies corporation, NY, USA) added to RPMI (RPMI-1640, Life technologies corporation, NY, USA) for 96 wells. The plate was aliquoted at 90 μL to 1 10 ⁴ cells / well. Cells were attached by incubating for 24 hours in a 37 ° C., 5% CO ₂ incubator (MCO175, SANYO Electric Biomedical Co. Lit., Osaka, Japan). Each test substance was further incubated for 24 hours by adding 10 μL at 100 μM, 50 μM, 25 μM, 12.5 μM, 0 μM concentrations. 0 μM was added as 2% DMSO as vehicle control to 0.2% DMSO. 10 μL of CCK-8 (Dojindo) solution was added thereto, followed by further reaction in a CO ₂ incubator for 1-2 hours. After the reaction, the absorbance was measured at 450 nm with an Enzyme-linked immuno-assay reader (Spectra MAX250, Molecular Devices Co.).

As a result, as shown in Figure 3, all the antimicrobial material in the cell activity test through CCK-8 was confirmed to exhibit the same or similar cell activity to the vehicle control to 100 μM concentration (Fig. 3).

<4-2> Cytotoxicity Measurement

Cytotoxicity was measured by using LDH (Lactate dehydrogenase) assay for the compound exhibiting the antimicrobial effect against P. gingivalis confirmed through the <Experimental Example 1>.

Specifically, L-929 cells were cultured in a medium containing 10% FBS and antibiotics (Penicillin Streptomycin, Life technologies corporation, NY, USA) in RPMI (RPMI-1640, Life technologies corporation, NY, USA) to 96 well plate 90 μL was dispensed at 1 × 10 ⁴ cells / well. Cells were attached by incubating for 24 hours in a 37 ° C., 5% CO ₂ incubator (MCO175, SANYO Electric Biomedical Co. Lit., Osaka, Japan). Each test substance was further incubated for 24 hours by adding 10 μL at 100 μM, 50 μM, 25 μM, 12.5 μM, 0 μM concentrations. 0 μM was added as 2% DMSO as vehicle control to 0.2% DMSO. 45 minutes before the supernatant harvest, Lysis solution was added to the positive control group by 10 uL, and the cells were physically broken and killed, resulting in LDH, which was calculated as 100% toxicity. After 45 minutes, transfer 50 μL of supernatant from each well to a new plate. Add 50 μL of a mixture of LDH assay kit catalyst and pigment solution and react for 30 minutes at room temperature. 50 μL of stop solution was added and absorbance was measured at 490 nm of Enzyme-linked immuno-assay reader (Spectra MAX250, Molecular Devices Co.).

As a result, as shown in Fig. 4, the results of cytotoxicity test through LDH confirmed that all antimicrobial substances were not toxic until the concentration of 100 μM compared to the positive control group.

In conclusion, all the antimicrobial substances in the cell activity test with CCK-8 showed the same high cellular activity as Vehicle Control up to 100 μM concentration. In the cytotoxicity test with LDH, all the antimicrobial substances were toxic up to 100 μM concentration. It was confirmed that there was no. Therefore, it was confirmed that the concentration of MIC and MBC when the antimicrobial material of the present invention was mixed with the fluorine varnish was a safe concentration.

Experimental Example 5 Antimicrobial Sustainability Test

<5-1> Antimicrobial Persistence Evaluation Using OHP Film Disc

Agar diffusion test was performed using P. gingivalis as a strain related to dental caries.

Specifically, the experimental fluorine varnish prepared in Example 2 was mixed with natural antibacterial substances Chrysophanol, Emodin, Anthrarufin, Bavachalcone, Isobavachromene and proceeded to a concentration of 10 mM. Positive control group was evaluated using Chlorhexidine 20 μg / ml + fluorine varnish, negative control group using OHP (polyethylene terephthalate) film disc itself.

Four films (diameter 5 mm OHP film discs) required for each group were placed in Petri dishes, and 5 μL of 10 mM material was dropped. Thereafter, distilled water was put in a Petri dish so that the film was immersed and then stored at 80 rpm in a shaking water bath at 37 ° C. for measurement. Measurement time was set to 0 hours, 4 hours, 1 day, 2 days, 3 days, 5 days, 10 days, 20 days, 30 days.

As a result, the positive control and Experimental fluoride varnish (Vehicle Control), as shown in Figure 5 and 6, the constant persistence over time over 3 days, Chrysophanol, Emodin, Anthrarufin, Bavachalcone, Isobavachromenene of the present invention This mixed fluorine varnish lasted up to 30 days antimicrobial effect, in particular, it was confirmed that the fluorine varnish mixed with Chrysophanol exhibited excellent antimicrobial persistence (Figs. 5 and 6).

<5-2> Evaluation of Antimicrobial Sustainability Using Hydroxyapatite Disc

Apply 5 μL of each substance to Hydroxyapatite disc (3D Biotek, USA) with a diameter of 5 mm and a height of 1.7 mm, dry and sterilize for 30 minutes in UV MODE of clean bench, and place sterilized Hydroxyapatite disc in distilled water and store in shaking water bath. (37 ° C., 80 RPM). Then, Hydroxyapatite discs were taken out at 0 hours, 4 hours, 1 day, 2 days, 3 days, 5 days, 10 days, 20 days, and 30 days, and subjected to agar diffusion test. In addition, each group is shown in Table 3 below.

group code How to make Positive control
(Chlorhexidine) PC Chlorhexidine: Fluorine varnish = 1: 1 Negative control NC Hydroxyapatite Vehicle control VC Experimental Fluorine Varnish and DMSO = 1: 1 10 mM Chrysophanol One 20 mM material of Example 2: Fluorine varnish = 1: 1 10 mM Emodin 2 20 mM material of Example 2: Fluorine varnish = 1: 1 10 mM Anthrarufin 3 20 mM material of Example 2: Fluorine varnish = 1: 1 10 mM Bavachalcone 4 20 mM material of Example 2: Fluorine varnish = 1: 1 10 mM Isobavachromene 5 20 mM material of Example 2: Fluorine varnish = 1: 1

As a result, as shown in Figure 7, the fluorine varnish containing the chlorohexidine, a positive control group lasted for 5 days, while the antibacterial activity of the fluorine varnish containing the antimicrobial material of the present invention is significant until 30 days It was confirmed to persist as (Fig. 7).

Claims (12)

Pharmaceutical composition for the prevention and treatment of periodontal disease (fluoride varnish) and antiperiodontal disease (periodontal disease) containing as an active ingredient.
The pharmaceutical composition for preventing and treating periodontal disease according to claim 1, wherein the fluorine varnish comprises a rosin, a solvent, and a fluorine compound.
The pharmaceutical composition for preventing and treating periodontal disease according to claim 2, wherein the blending ratio of the rosin, the solvent and the fluorine compound is 30 to 65% by weight: 30 to 65% by weight: 1 to 10% by weight of the total compound weight. Composition.
The method of claim 2, wherein the solvent is any one selected from the group consisting of distilled water, dimethyl sulfoxide (DMSO), isoamyl propionate, hydrogen fluoride (HF), ethanol and ethyl acetate Pharmaceutical composition for preventing and treating periodontal disease, characterized in that above.
The method of claim 2, wherein the fluorine compound is HF, NH ₄ F, NaF, KF, Na ₂ FPO ₄ , K ₂ FPO ₄ , CaF ₂ , TiF ₄ , Ca ₅ (PO ₄ ) ₃ F, H ₂ F ₂ Si (Difluorosilane), C ₁₆ H ₃₆ FN, SnF ₂ and Cetylamine hydrofluoride characterized in that any one selected from the group consisting of periodontal disease prevention and treatment pharmaceutical composition.
The method of claim 1, wherein the antimicrobial agent is any one selected from the group consisting of Chrysophanol, Emodin, Anthrarufin, Bavachalcone, and Isobavachromene. Pharmaceutical composition for preventing and treating periodontal disease, characterized in that above.
According to claim 1, wherein the periodontal disease is gingivitis (gingivitis) or periodontitis (Periodontitis) pharmaceutical composition for preventing and treating periodontal disease, characterized in that.
The pharmaceutical composition for preventing and treating periodontal disease according to claim 1, wherein the composition has antibacterial activity against Porphyromonas gingivalis .
Any one or more compounds selected from the group consisting of Chrysophanol, Emodin, Antharufin, Anthrarufin, Bavachalcone and Isobavachromene, or a pharmaceutically acceptable thereof A pharmaceutical composition for preventing and treating periodontal disease, comprising a salt as an active ingredient.
The pharmaceutical composition for preventing and treating periodontal disease according to claim 9, wherein the composition has antimicrobial activity against Porphyromonas gingivalis.
The method of claim 9, wherein the periodontal disease is gingivitis or periodontitis, characterized in that the periodontal disease prevention and treatment pharmaceutical composition.
 10. The method of claim 9, wherein the composition is oral solution, dental perceptual hypersensitivity agent, antimicrobial dental adhesive, dental filler, dental restorative material, dental root canal filler, dental root canal sealer (sealer), subfoveal blockade A pharmaceutical composition for preventing and treating periodontal disease, characterized in that it is mixed with any one selected from the group consisting of ash, dental coatings and dental cement.

Images