KR102260939B1 - Composition for prevention or treatment of dental disease comprising an extract of Matteuccia struthiopteris - Google Patents

Abstract

The present invention relates to a composition for preventing or treating oral diseases comprising an extract of Blue fern ( Matteuccia struthiopteris ), and more specifically, to a composition for preventing or treating oral diseases, including a blue fern having antibacterial activity against harmful bacteria that cause periodontal disease and tooth decay in the oral cavity. It relates to a composition for oral use, including. The composition for oral use containing the extract of the blueberry fern of the present invention has an antibacterial effect on the causative bacteria causing oral diseases, and at the same time has an effect of alleviating inflammation by suppressing inflammatory cytokines. The composition has the advantage of few side effects including natural ingredients, and can be used as a variety of oral products and pharmaceuticals.

Description

Composition for prevention or treatment of dental disease comprising an extract of Matteuccia struthiopteris

The present invention relates to a composition for preventing or treating oral diseases comprising an extract of Blue fern ( Matteuccia struthiopteris ), and more specifically, to a composition for preventing or treating oral diseases, including a blue fern having antibacterial activity against harmful bacteria that cause periodontal disease and tooth decay in the oral cavity. It relates to a composition for oral use, including.

Oral diseases such as periodontitis and dental caries occurring in the oral cavity are caused by various oral bacteria. More than 600 to 800 types of microorganisms exist in the oral cavity, and more than 200 million bacteria inhabit. Most of these are controlled by lysozyme contained in saliva, but the oral environment provides a good habitat for bacteria that cause oral diseases to thrive.

Periodontal disease, also known as periodontal disease, refers to an inflammatory disease accompanied by destruction of periodontal tissue due to a mixed infection of bacteria, gingival bleeding and swelling, formation of periodontal pockets, loss of attached gingiva, destruction of alveolar bone, and bad breath. It exhibits various clinical symptoms such as, and is the main cause of tooth loss and implant failure.

Periodontal disease is divided into gingivitis and periodontitis, depending on the severity of the disease. Advanced cases are called periodontitis.

Dental caries is often referred to as tooth decay, and minerals such as calcium and phosphorus on the tooth surface are drained by acid excreted by Streptococcus mutans present in the dental plaque, and organic substances such as proteins are dissolved in it. Symptoms that lead to tooth destruction. When the pH is lowered by the acid secreted by mutans bacteria, demineralization of the tooth surface occurs. This phenomenon is called dental caries. Dental caries is caused by various personal factors such as tooth position, amount and consistency of saliva, heredity, pregnancy, and lactation, bacterial factors due to the type and amount and activity of acid bacteria in the oral cavity, and environmental factors such as oral hygiene conditions and food types. proceeds

As described above, various compositions, toothpastes, etc. exist for the prevention and treatment of oral diseases, and various components whose efficacy is known, such as antibiotics and fluorine compounds, are used. In particular, an adjuvant antimicrobial agent may be used to treat oral diseases. In general, in the case of dental caries, 0.2% chlorhexidine gargle is used for plaque control, but it is only used for short-term therapy due to the side effects of dental stains and unpleasant taste. In periodontal disease, tartar removal is primarily performed, but as an adjuvant treatment with antimicrobial agents, more diverse microorganisms than caries are involved, so tetracycline and metronidazole are used in addition to chlorhexidine mouthwash. have. However, these antimicrobial agents may cause side effects such as generation of resistant bacteria and non-selective sterilization, so it is necessary to find an effective substance that can replace existing antimicrobial agents.

In Korea, oral treatments using natural products are attracting attention in order to reduce side effects and improve treatment effects. In the case of a treatment using a natural product, it is possible to overcome the tolerance or safety problem caused by the existing treatment. In addition, since oral hygiene is important in the case of tooth decay, a toothpaste or mouthwash having antibacterial activity against tooth decay bacteria will be effective in preventing tooth decay. In addition, in the case of periodontal disease, since it accompanies an inflammatory response, it should be considered to have an anti-inflammatory effect including a component having an anti-inflammatory effect.

Accordingly, the present inventors have been studying for a naturally-derived active substance that has antibacterial activity against the causative bacteria that causes oral diseases as described above and has fewer side effects, and the blue fern ( Matteuccia struthiopteris ) extract is Porphinomonas long genitalia. It was confirmed that it has antibacterial activity against lys and Streptococcus mutans, and the present invention was completed.

Republic of Korea Patent No. 10-0573631 Republic of Korea Patent Registration No. 10-1592549

Accordingly, the present invention is to solve the above problems, and to provide a useful use of the extract of chrysanthemum bracken as a novel active ingredient derived from a natural product that is safe with few side effects and has antibacterial activity against oral bacteria.

That is, it is an object of the present invention to provide a composition for oral use containing the blue fern extract, which has an oral disease prevention and treatment effect.

Another object of the present invention is to provide a variety of oral products comprising a composition for oral use comprising the extract of the blueberry bracken.

Another object of the present invention is to provide a pharmaceutical composition for the treatment of oral diseases, including a composition for oral use comprising the extract of the blueberry bracken.

In order to solve the above problems, the present invention provides a composition for oral use comprising an extract of blueberry bracken.

The composition for oral use is not limited thereto, but it is characterized in that it has an effect of preventing and treating oral diseases such as bad breath, dental caries, periodontitis, and gingivitis.

In addition, the composition for oral use of the present invention is characterized in that it has an antibacterial effect against bacteria present in the oral cavity, such as Streptococcus genus or Porphyromonas genus, wherein the bacterium is Streptococcus radish Tans ( Streptococcus mutans ), Streptococcus sanguinis ) and Porphyromonas gingivalis ) At least one selected from the group consisting of.

In addition, the composition for oral use of the present invention is characterized in that it has an anti-inflammatory effect on inflammatory diseases in the oral cavity. With respect to the anti-inflammatory effect, in one embodiment of the present invention, the inflammatory cytokine inhibitory effect such as TNFα, IL-1β and IL-6 was confirmed by using the blue fern extract.

In addition, the extract of the blueberry bracken of the present invention is characterized in that the effective concentration is 0.01 to 0.1%.

In another aspect, the present invention includes oral products and pharmaceutical compositions for oral use using the composition for oral use comprising the extract of Blueberry bracken. The formulation of the product and pharmaceutical is not particularly limited, but one or more may be selected from the group consisting of toothpaste, mouthwash, gum, oral spray, oral gel, oral ointment, and oral patch.

The composition for oral use containing the extract of the blueberry fern of the present invention has an antibacterial effect on the causative bacteria causing oral diseases, and at the same time has an effect of alleviating inflammation by suppressing inflammatory cytokines. The composition has the advantage of few side effects including natural ingredients, and can be used as a variety of oral products and pharmaceuticals.

Figure 1 shows the result of measuring the minimum growth inhibitory concentration (MIC) of the Porphyromonas gingivalis (P. gingivalis) of the extract of the present invention.
Figure 2 shows the results of measuring the minimum growth inhibitory concentration (MIC) for Streptococcus mutans (S. mutans) bacteria of the extract of the blueberry bracken of the present invention.
Figure 3 shows the result of measuring the minimum growth inhibitory concentration (MIC) for Streptococcus sanguinis (S. sanguinis) of the blue fern extract of the present invention.
Figure 4 shows the growth retardation effect on P. gingivalis bacteria of the extract of the blueberry bracken of the present invention.
Figure 5 shows the growth retardation effect on S. sanguinis bacteria of the extract of the blueberry bracken of the present invention.
Figure 6 shows the results of experiments for the concentration having a cytotoxicity of the extract of the present invention.
Figure 7 shows the results of measurements with respect to the concentration having NO inhibitory activity of the extract of the blue leaf bracken of the present invention.
Figure 8 shows the results of measuring the cytokine production inhibitory ability of the extract of the blueberry bracken of the present invention.
Figure 9 shows the results of measuring the cytotoxicity and TNFα production inhibitory ability of the cytokines of the extract of the blue ragweed bracken extracted by various collection papers and parts of the present invention.
FIG. 10 shows the results of measuring the cytotoxicity and the inhibitory ability of IL-6 production among cytokines of the extract of blue ragweed bracken extracted by various collection papers and parts of the present invention.

The present invention relates to a composition for oral use containing an extract of blue fern ( Matteuccia struthiopteris ), which has antibacterial activity and anti-inflammatory effect on oral bacteria.

Blue fern ( Matteuccia struthiopteris ), which is an active ingredient of the oral composition of the present invention, is a perennial grass of the fern phylum Bracken and is distributed in Korea, Japan, China, Siberia, Sakhalin or Europe. It is about 10-20cm long and has a flat triangular cross section, and there are scales at the bottom. The leaves are 40-80cm long and 15-30cm wide, and the tip is sharply pointed, the lower part is gradually narrowed, and it is bright green. The right side has a wide linear shape, and it has no stem with a sharp tip, and is divided into an idol. Lobes are long oval or narrow egg-shaped, open at an angle, and have fine serrations with a width of 2.5-5mm. The leaf comes out in autumn and is about 30-60cm in height. The petiole is shorter than the leaf, and there is a deep groove on the surface. It grows in shady, moist places in forests of forests. It mainly grows in shady valleys or lower deciduous forests at low elevations. It is known to have anti-parasitic properties.

The method of using the blueberry bracken is not particularly limited, and may be included in the oral composition in the form of a powder pulverized after drying, more preferably, an extract.

As used herein, the term "extract" refers to an extract obtained by extraction treatment of blueberry bracken, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, a prepared or purified product of the extract, or a mixture thereof, etc. , including the extract itself and extracts of all formulations that can be formed using the extract.

The extract of the blueberry bracken of the present invention can be extracted from various organs of natural, hybrid or mutated plants of the blueberry bracken, for example, seeds (seeds), roots, above-ground parts, stems, leaves, Extraction is possible not only from flowers, fruit bodies, and fruit skins, but also from plant tissue cultures.

In the extract of the blue ragweed bracken of the present invention, the method of extracting the blue lardly bracken is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include hot water extraction method, ultrasonic extraction method, filtration method, reflux extraction method, etc., which may be performed alone or in combination of two or more methods, preferably using a cold extraction method can be done by

The type of extraction solvent used for extracting the blue fern of the present invention is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water, distilled water, ethanol, methanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, n-hexane, hydrochloric acid, acetic acid, formic acid, diethyl ether and one or more solvents selected from the group consisting of cyclohexane, and the like. When alcohol is used as the solvent, preferably C1 to C4 alcohols (methanol, ethanol, propanol, butanol) may be used. More preferably, water, ethanol, or a mixture thereof may be used as the extraction solvent of the blue fern in the present invention.

In the present invention, the composition may be for preventing or treating oral diseases.

As used herein, the term “treatment” is an approach for obtaining beneficial or desirable clinical results, whether for the purposes of the present invention beneficial or desirable clinical results, whether appreciable or impossible, and whether partial or total. including, but not limited to, alleviation of symptoms, reduction of the severity of the disease, a stabilized (i.e., not getting worse) state of the disease, delaying or reducing the rate of disease progression, amelioration or temporary alleviation and amelioration of the disease state without . Accordingly, treatment refers to both therapeutic treatment and prophylactic dimensions, and those that need to be treated include those already having the disease as well as those in which the disease is to be prevented. In addition, it may refer to any action to improve or benefit the symptoms of oral disease by administering the pharmaceutical composition of the present invention to an individual.

As used herein, the term "prevention" refers to any action that inhibits or delays a disease by using the composition for oral use, and the composition for oral use may be used in the form of a pharmaceutical composition, a composition for oral hygiene, It is not limited.

In the present invention, the oral disease is not limited thereto, but is a comprehensive concept including bad breath, dental caries, periodontitis, gingivitis and mucosal ulcers in the oral cavity caused by bacteria in the oral cavity, more preferably P. gingivalis or S. mutans -induced periodontitis, gingivitis or dental caries.

That is, the oral composition of the present invention is characterized in that it has an antibacterial effect against bacteria present in the oral cavity, such as Streptococcus genus or Porphyromonas genus, and more specifically, Streptococcus mutans ( S. mutans ), Streptococcus sanguinis ( S. sanguinis ) and Porphyromonas gingivalis ( P. gingivalis ) Excellent antibacterial effect against one or more bacteria selected from the group consisting of preventing or treating oral diseases may have an effect on

More preferably, the composition for oral use of the present invention may exhibit an antibacterial effect on Streptococcus mutans ( S. mutans ) or Porphyromonas gingivalis ( P. gingivalis ) bacteria.

The composition for oral use of the present invention may include the blueberry bracken extract alone as an active ingredient, but may further include a pharmaceutically acceptable carrier, excipient, diluent, etc. depending on the formulation and purpose of use.

In more detail, the composition for oral use, in addition to the blueberry bracken extract, additionally nutrients, vitamins, electrolytes, flavoring agents, coloring agents, neutralizers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH It may further contain a regulator, a stabilizer, a preservative, glycerin, alcohol, a carbonation agent used in carbonated beverages, and the like. In addition, the carrier, excipient or diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline From the group consisting of cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, physiological saline It may be one or more selected, but the present invention is not limited thereto, and any conventional carrier, excipient or diluent may be used.

In addition, in the case of a pharmaceutical composition comprising the composition for oral use of the present invention, conventional fillers, weight agents, binders, disintegrants, surfactants, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, etc. may be further included, Both parenteral and oral can be used, but more preferably parenteral.

Forms for parenteral administration include toothpaste, mouthwash, topical administration agents (creams, ointments, dressing solutions, sprays, other coating agents, etc.). As an example of the formulation of the topical administration agent, a composition for preventing or treating oral diseases, including an active ingredient, a blueberry bracken extract, is fixed to a film or patch containing a water-soluble polymer so that it can be attached to teeth and surrounding areas. it may be angry

The dosage of the composition for preventing and treating oral diseases according to the present invention may be appropriately selected by those skilled in the art in consideration of the administration method, the age, sex and weight of the user, and the severity of the disease. As an example, the composition for preventing or treating oral diseases of the present invention may be administered in an amount of 0.0001 mg/kg to 1000 mg/kg, and 0.01 mg/kg to 100 mg/kg to be more effective. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.

In addition, it is preferred that the extract of the present invention is contained in the composition at an effective concentration of 0.01 to 0.1% of the composition. Specifically, the extract of blue fern is contained in the composition in an amount of 1 μg/mL or more, preferably 1 to 100 μg/mL, and more preferably 1 to 10 μg/mL in the composition to minimize cytotoxicity and anti-inflammatory activity. good for promotion.

The present invention provides a product for oral use comprising a composition for oral use containing a blueberry bracken extract as an active ingredient, and in particular, it provides use as a composition for oral hygiene. The oral hygiene products include all types and formulations that can be used for oral hygiene. Although not limited thereto, examples of the hygiene composition include toothpaste, mouthwash, gum, oral spray, oral gel, oral ointment, oral patch, and the like.

When the oral product of the present invention is a toothpaste, in addition to including the blueberry bracken extract as an active ingredient, an abrasive, a binder, a moisturizing agent, a foaming agent, a sweetener, a whitening agent, or a flavoring agent may be additionally included.

Examples of the abrasive include aluminum hydroxide, silicic anhydride, aluminum silicate, dicalcium phosphate dihydrate and anhydride, tricalcium phosphate, calcium carbonate, calcium pyrophosphate, insoluble sodium metaphosphate, magnesium triphosphate, magnesium carbonate, calcium sulfate, Polymethyl methacrylate, etc. can be used individually or in combination. The content of the abrasive may be generally 20 wt% to 90 wt% based on the total composition, but is not limited by the content.

When the oral hygiene composition is a paste-like composition, the binder is garrageenin, various cellulose derivatives for thickening, xanthan gum, gums such as tragacanth gum, polyvinyl alcohol, sodium polyacrylate, polyacrylic acid / An organic binder such as a synthetic polymer derivative such as a maleic acid copolymer or a carboxyvinyl polymer and an inorganic binder such as silica or laponite may be used alone or in combination. The content of the binder may be usually 0.3 wt% to 5 wt% based on the total composition, but is not limited by the content.

In addition, the oral hygiene composition may further include sorbitol, glycerin, ethylene glycol, propylene glycol, polyether glycol, polypropylene glycol, and the like as a moisturizing agent during the preparation thereof.

In addition, the oral hygiene composition may further include a flavoring agent or a sweetener. The sweetening agent may be sucrose, lactose, maltose, sorbitol, xylitol, sodium cyclamate, glycerin, sodium saccharin, stevioside, aspartame, and the like, and the flavoring agent is peppermint, menthol, anethol, eugenol, Limonene, citronenol, alphaterpineol, salicylmethyl, cineol, linalol, ethylinarol, vanillin, thymol, spearmint oil, sesame oil, rosemary oil, cinnamon oil, etc., and the sweetener or flavoring agent is alone may be used as or in combination.

In addition, the oral hygiene composition of the present invention may contain a surfactant or additional effective component used as a foaming component alone or in combination, and the surfactant used as the foaming component is an anionic surfactant, a nonionic surfactant and a cationic surfactant. It may be any one selected from the group consisting of active agents, and more specifically, sodium lauryl sulfate as an anionic surfactant, a copolymer of polyoxyethylene polyoxypropylene as a nonionic surfactant (poloxamer), and polyoxyethylene hydrogenated castor oil , polyoxyethylene sorbitan fatty acid ester, etc. may be used without limitation.

When the oral hygiene composition of the present invention is a toothpaste, it may be prepared by a conventional toothpaste manufacturing method except for including the extract of blueberry bracken as an active ingredient, and when the oral hygiene composition of the present invention is a toothbrushing solution, it is conventional It can be prepared by mixing the blueberry bracken extract with a phosphorus solution and formulating it as a toothbrushing solution, and can prevent or treat oral diseases by washing the oral cavity 2 to 10 times a day.

In addition, when the oral care composition of the present invention is a mouthwash (cleansing agent), it may further include a toothpaste carrier, more specifically, a non-toxic alcohol.

In the present invention, the extract may be from 0.00001 to 10% by weight based on the total weight of the composition, preferably from 0.0005 to 5% by weight based on the total weight of the composition, more preferably from 0.005 to 10% by weight based on the total weight of the composition. It may be 1% by weight. If the amount of the blue fern extract is less than 0.00001% by weight relative to the total weight of the composition, the effect on oral diseases may be insignificant, and if it is more than 10% by weight, cytotoxicity may occur and irritation or inflammation may be rather induced. can

Hereinafter, the present invention will be described in more detail by way of Examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited to these examples.

<Example 1> Preparation of extract of blue narcissus bracken

100 g of blueberry bracken outpost was weighed and extracted by cold extraction at room temperature for 24 hours using 1 L of 70% ethanol, and the extract was filtered, concentrated with a vacuum concentrator, and then freeze-dried.

<Example 2> Analysis of Minimum Bacterial Growth Inhibitory Concentration (MIC) of Blue ragweed extract

In order to measure the antibacterial activity of the extract of the present invention, the minimum inhibitory concentration (MIC) for each oral bacterium was measured in vitro.

Streptococcus mutans (S. mutans ) or Streptococcus sanguinis (S. sanguinis ) was cultured in Brain Heart Infusion ( BHI ) broth, Porphyromonas gingivalis (P. gingivalis ) Tryptic soy broth ( 30g/l) supplemented 0.5% yeast extract, 0.05% L-cystein hydrochloride monohydrate, resazurin (final 10ug/ml), Hemin Solution (final 50 ug/ml), Vitamin K1 solution (final 10 ug/ml) did. Among oral bacteria, periodontal disease causative bacteria Porphyromonas gingivalis ( P. gingivalis ) , dental caries causative bacteria Streptococcus mutans ( S. mutans ) , non-pathogenic strains After centrifuging the bacterial culture medium of Streptococcus sanguinis at 4° C. for 15 minutes at 4000 rpm, the supernatant was removed and the viable cell concentration at the time of measurement was 5×10 ⁵ CFU/ml ( P. gingivalis , 1 × 10 ⁶ CFU/ml) was diluted with fresh culture medium. 100 μl of the diluted culture solution was inoculated into 96 wells.

After preparing a stock solution by dissolving the dried blue fern extract obtained through extraction in 70% ethanol, dilute it so that the final treatment concentration is 0.1%, 0.05%, 0.025%, 0.0125%, and each inoculated with the bacteria Each well was treated with 100 μl.

After culturing for 48 hours, the absorbance was measured at a wavelength of 660 nm by putting the bacterial culture solution in a spectrophotometer. At this time, the lowest concentration at which the absorbance was 0.05 or less was used as the minimum bacterial growth inhibitory concentration (MIC), and the MIC was finally determined by simultaneously performing the drop plating method (10 μl).

As a result, as shown in Table 1 below, the minimum bacterial growth inhibitory concentration of each strain is P. gingivalis was measured to be 0.0063%, S. mutans was 0.1%, and S. sanguinis was 0.025%. That is, it was confirmed that the extract of the blueberry bracken of the present invention has antibacterial activity against all oral bacteria ( FIGS. 1 to 3 ).

strain name MIC Porphyromonas gingivalis ( Porphyromonas gingivalis ) 0.0063% Streptococcus mutans 0.1% Streptococcus sanguinis 0.025%

<Example 3> Analysis of growth delay effect through short-term exposure

In order to measure the antibacterial activity of the extract of the present invention, the growth retardation effect of the extract of the blue ragweed bracken compared to the control sample was measured and compared through a short-term exposure model. For the control sample, NC (Negative control: bacterial culture solution + ethanol of the same concentration contained in the treated natural product) was used.

First, the bacterial culture medium of oral bacteria P. gingivalis, S. mutans, and S. sanguinis was centrifuged (4000rpm, 4℃, 15 minutes), the supernatant was removed, and the live cell concentration S. mutans and S. sanguinis were 5×10 ⁵ CFU/ml, and P. gingivalis was diluted by adding to the culture medium to 1×10 ^{6 CFU/ml.} The live cell concentration was determined by measuring the absorbance at 660 nm using a spectrophotometer (multiscan GO microplate spectrophotometer, Thermo Fisher scientific, Inc.), and then using a plate medium plating method.

A sample of 4 to 8 times the minimum bacterial growth inhibitory concentration (MIC) for each bacteria and 500 μl of chlorhexidine and distilled water showing growth inhibition were mixed with 500 μl of medium and 10 μl of bacterial culture. , placed in appropriate culture conditions for each strain for 5 minutes, centrifuged to remove the supernatant, and then dispensed into 20 ml of fresh culture solution.

After culturing each of the prepared strain cultures under appropriate conditions, absorbance was measured over at least 5 times while culturing for 48 hours.

As a result, it was confirmed that there was a difference in the growth retardation effect depending on the type of strain. If you look at the negative control (NC) as a normal growth curve of the fungus, you can see that it enters the logarithmic growth phase after about 4 hours and enters the stationary phase after about 20 hours. However, when the growth curve was drawn after short-term exposure to the algae fern sample, it was confirmed that growth was inhibited like hexamedin, a condition in which growth was completely inhibited.

In general, the concentration of mixing functional ingredients in oral care products is 0.1%-1%. At this time, the concentration should be at least 10 times higher than the MIC concentration in order to show the actual clinical effect on the strain. Therefore, data with a MIC of less than 0.05% is meaningful, and only the strains P. gingivalis and S. sanguinis whose MIC was confirmed to be less than 0.05% were tested for the growth delay effect ( FIGS. 4 and 5 ).

<Example 4> Confirmation of anti-inflammatory effect

<4-1> Cytotoxicity analysis

RAW 264.7 cells were treated with the extract of the present invention and cultured for 24 hours (37° C., 5% CO ₂ incubator, RPMI1640 medium supplemented with 10% FBS), and then MTT reagent was additionally administered.

After formazan was formed, it was dissolved in DMSO and absorbance was measured at a wavelength of 595 nm. Cytotoxicity was confirmed by comparing the absorbance with normal cells that were not treated with the blueberry bracken extract.

As a result, it was determined that there was cytotoxicity at a concentration of 0.01% or more. In other words, cytotoxicity was not confirmed in less than 0.01% of the extract of the blueberry bracken (FIG. 6).

Consistent with the above results, cytotoxicity was not observed even in an experiment in which blue fern extracts (No. 37, 40, 42) were obtained from various collection sites and parts and treated at a concentration of 0.01% to confirm cytotoxicity (Fig. 9, 10).

<4-2> Anti-inflammatory activity assay (NO assay)

RAW 264.7 cells were treated with blue fern extract for 30 minutes or 180 minutes, and then LPS (100 or 250 ng/mL) was additionally treated. After 24 hours, the supernatant and the grease (Griess) reagent were added and reacted, and the absorbance was measured.

Specifically, NO was not generated in the group not treated with LPS and blue fern extract. When only LPS was treated, the amount of NO generated when treated with LPS at each concentration (0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%) with LPS was compared in %.

As a result, it was confirmed that the amount of NO generation was reduced in a concentration-dependent manner when the blue nae fern extract was treated, compared to that not treated with the cheongnae bracken extract. In addition, the IC50 value for NO generation was confirmed that the IC50 value was 0.00138% of the blue fern extract (Fig. 7).

<4-3> Cytokine assay

RAW 264.7 cells were treated with the blue fern extract for 30 minutes, and then LPS (250 ng/mL) was additionally treated. After 6 hours, the supernatant was separated, and the production amount of cytokines (TNFα, IL-1β, IL-6) was measured according to the ELISA test method.

Specifically, after 6 hours, the supernatant was separated and reacted according to the manufacturer's manual using TNFα, IL-1β, and IL-6 ELISA kit (BD Biosciences, USA), and absorbance was measured at 450 nm, and a standard curve was used to quantify the production amount of each cytokine.

As a result, it was confirmed that the production of inflammatory cytokines was increased by the stimulation of LPS, and although there was a difference in degree, it was confirmed that the production of these cytokines was decreased by treatment with the blue fern extract. That is, as shown in FIG. 8 , in the case of TNFα, it was confirmed that the production amount was reduced in the group treated with the blue ragweed extract, and in particular, it was confirmed that the inhibitory activity was the greatest when 0.00001% of the blue fern extract was treated.

Even when the extracts obtained from various collection sites and extracted from each part were treated with 0.01%, it was confirmed that the production of TNFα was significantly inhibited compared to the vehicle-treated group (FIG. 9).

In addition, in the case of IL-1β, as shown in FIG. 8 , the production amount was reduced in the group treated with the blueberry bracken extract, and when it was treated with 0.00001%, it was confirmed that the inhibitory activity was the greatest.

Even when the extracts obtained from various collection sites and extracted from each part were treated with 0.01%, it was confirmed that the production of IL-1β was significantly inhibited compared to the vehicle-treated group (FIG. 10).

As a result, in the case of cytotoxicity experiments, the concentration that does not show cytotoxicity to macrophages, which are RAW 264.7 cells, was identified, and at the same time, whether or not NO expression was inhibited due to cell death, or whether cells died and affected cytokine expression at the same time This is an experiment to verify.

In addition, in the case of the cytokine inhibition test and the cytotoxicity test, the optimal experimental conditions were set to show the anti-inflammatory effect in vitro, whereas in the case of the MIC test, the concentration acts directly on the bacteria. It was confirmed that the concentration range of the optimized extract was 0.0063% or more for P. gingivalis , 0.1% or more for S. mutans , and 0.025% or more for S. sanguinis.

So far, the present invention has been looked at with respect to preferred embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (13)

Contains blueberry bracken extract as an active ingredient,
The blue fern extract is Streptococcus ( Streptococcus ) or Porphyromonas ( Porphyromonas ) The composition for preventing or treating oral diseases, characterized in that it has an antibacterial effect against bacteria. According to claim 1,
The oral disease is dental caries, periodontitis, and a composition for preventing or treating oral diseases, characterized in that it has an effect of preventing or treating one or more oral diseases of gingivitis and periodontitis. According to claim 1,
The composition for preventing or treating oral diseases, characterized in that the extract of the blueberry bracken is comprised in an amount of 0.00001 to 0.1% by weight based on the total weight of the composition. delete According to claim 1,
The Streptococcus ( Streptococcus ) genus or Porphyromonas genus Bacteria, Streptococcus mutans ( S. mutans ), Streptococcus sanguinis ( S. sanguinis ) and Porphyromonas gingivalis ( P gingivalis ) composition for preventing or treating oral diseases, characterized in that at least one selected from the group consisting of. According to claim 1,
The composition is a composition for preventing or treating oral diseases, characterized in that it has an anti-inflammatory effect. According to claim 1,
The composition is a composition for preventing or treating oral diseases, characterized in that it has an inhibitory effect on inflammatory cytokines TNFα and IL-1β. According to claim 1,
The blue naraeus bracken extract is a composition for preventing or treating oral diseases, characterized in that the extract containing the leaves, stems and roots of the blue ragweed bracken is extracted using ethanol as an extraction solvent. According to claim 1,
The composition for preventing or treating oral diseases, characterized in that the extract of the blueberry bracken has an effective concentration of 0.01 to 0.1%. A product for preventing or treating oral diseases comprising the composition for preventing or treating oral diseases of any one of claims 1 to 3, 5 to 9. 11. The method of claim 10, wherein the oral disease prevention or treatment product is toothpaste, mouthwash, gum, oral spray, oral gel, oral ointment, oral disease prevention or characterized in that at least one selected from the group consisting of oral patches therapeutic products. A composition for preventing or improving oral disease, comprising the composition for preventing or treating oral disease according to any one of claims 1 to 3, 5 to 9. 13. The method of claim 12,
The oral disease is a composition for preventing or improving oral diseases, characterized in that at least one of dental caries, periodontitis, and gingivitis.

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