KR20190124074A - Composition containing Berberis amurensis extracts for preventing the formation of biofilm of microorganism - Google Patents

Abstract

The present invention relates to a composition having an inhibitory effect on biofilm formation, which comprises a Berberis amurensis extract, and more specifically, to a composition for preventing and alleviating tartar deposition and periodontal diseases. In addition, the present invention relates to a composition for preventing and alleviating oral diseases such as plaque, tartar, gingivitis or periodontitis, which comprises a Berberis amurensis extract having an inhibitory effect on the biofilm formation of oral bacteria as an active ingredient.

Description

Composition containing Berberis amurensis extracts for preventing the formation of biofilm of microorganism}

The present invention relates to a composition having an inhibitory effect on biofilm formation including barberry extract, and more particularly plaque, tartar, gingivitis or periodontitis comprising barberry extract having an inhibitory effect on biofilm formation of oral bacteria as an active ingredient. It relates to a composition for the prevention and improvement of oral diseases such as.

Biofilm is a microbial complex wrapped with various polymer materials formed by microorganisms, also called biofilm or biofilm. These biofilms serve as protective barriers to help microorganisms adapt to various environments, and the cells in the biofilm share metabolism with each other, and acquire properties for survival through gene transfer. . Therefore, the biofilm-forming cells have a much stronger resistance to harsh environments and antibiotics than when they are present in the form of ordinary suspended bacteria, and the biofilms formed in the organs cause various conditions. Examples of such conditions or locations include dental caries, gingivitis, periodontitis, otitis media, voice prostheses, hydrocephalus hunts, and cystic fibrosis. ), Valvular endocarditis, prosthetic heart valves, central venous catheter, prosthetic hip joint, prosthetic knee joint, chronic bacterial prostatitis , Intrauterine devices, urinary catheter, and the like.

On the other hand, oral cavity is known to exist about 1,000 kinds of bacteria, many bacteria inhabiting it forms the oral biofilm. Among the bacteria associated with the early oral biofilm formation is known as Streptococcus spp . These Streptococcus species are early adherent bacteria on the tooth surface, especially Streptococcus gordonii and Streptococcus oralis play an important role in the formation of oral biofilms that cause tartar. Porphyromonas gingivalis is also known as a major cause of gum disease such as periodontitis and gingivitis. Therefore, efforts to control these bacteria in suppressing tartar formation have continued.

When metal ions such as calcium or magnesium in saliva bind to the biofilm formed on the tooth surface, calcified calculus is formed. If tartar is not removed properly, it can cause gum disease such as gingivitis and periodontitis, and seriously loss of teeth. In an effort to prevent the development of periodontal disease, antibacterial agents such as chlorohexidine gluconate, cetylpyridium chloride, guinealine and triclosan have been developed and applied to oral products such as toothpaste and toothpaste. Outbreaks are not fundamentally prevented. In addition, these synthetic antibiotics, when used for a long time, not only irritate the oral mucosa, but also destroy normal bacteria, but rather increase the resistance to oral pathogens, and have a great side effect on the human body such as hurting tooth coloring and taste.

Therefore, there is a need for oral products for preventing periodontal disease, including natural antimicrobial agents, which can overcome the problems of existing products using synthetic compounds.

Barberry ( Berberis amurensis ) is an evergreen tree of the genus Yew, which lives in the mountains along the coast of southern Korea. Use fruit, root bark, and flowers for medicinal purposes. Efficacy of skin moisturizing and inflammation alleviation (Korea Patent Registration 10-1648466), anti-stress (Korea Patent Registration 10-1434449), etc. has been reported, but there is no report on the efficacy of barberry on oral periodontal disease.

Accordingly, the present inventors have devised the present invention to provide an oral composition containing barberry extract as an active ingredient, which is effective in preventing or improving tartar deposition and periodontal disease.

The problem to be solved by the present invention is to improve the disadvantages and problems of the prior art as described above, one aspect of the present invention is to provide a composition for inhibiting biofilm formation containing barberry extract.

Still another object of the present invention is to provide an oral composition which is effective in preventing and improving tartar deposition and periodontal disease, including barberry extract.

However, the problem to be solved by the present invention is not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

One aspect of the present invention to achieve the above object is at least one barberry extract selected from the group consisting of barberry extract, barberry leaf extract, barberry flower extract, barberry fruit extract and barberry seed extract as an active ingredient It provides a composition for inhibiting biofilm formation of microorganisms comprising.

In the present invention, the microorganisms are Streptococcus gordonii , Streptococcus oralis , Porphyromonas gingivalis , Fusobacterium nucleatum , Prebotela But not limited to at least one of the groups consisting of Prevotella intermedia , Parvimonasmicra; Peptostreptococcus micros , and Aggregatibacter actinomycetemcomitans; Actinobacillus actinomycetemcomitans .

In order to achieve the above object, the present invention provides a functional food composition for preventing and improving periodontal disease.

In another aspect of the present invention, the oral composition is effective in inhibiting biofilm formation, thereby providing an oral composition for preventing and improving periodontal diseases such as tartar, gingivitis and periodontitis. The oral composition may have any one or more formulations selected from toothpaste, chewing gum, mouthwash, mouthwash and mouth spray.

According to the present invention, the composition for inhibiting biofilm formation of microorganisms comprising barberry extract has antimicrobial activity and biofilm formation inhibitory effect on oral bacteria, thereby preventing and improving periodontal diseases such as tartar, gingivitis and periodontitis. can do.

In addition, it is possible to provide a composition that is safe for the human body, which is made of natural materials and has no side effects or risks caused by the synthetic material.

In addition, because it is safe for the human body, such as functional food composition, toothpaste, oral gargle can be applied to a variety of products that directly touch or even enter the human body.

The technical effects of the present invention are not limited to those mentioned above, and other technical effects that are not mentioned will be clearly understood by those skilled in the art from the following description.

1 is a photograph showing a biofilm formation aspect of the barberry leaf extract not treated and treated with barberry leaf extract.
FIG. 2 is a graph showing the biofilm formation of hydroxyapatite discs without treatment with barberry leaf extract and with treatment with barberry leaf extract.
Figure 3 is a graph showing the water-insoluble glucan synthesis aspect of not treated with barberry extract and treated with barberry leaf extract.
Figure 4 is a graph showing the cytotoxicity test results of barberry extract on oral mucosa cells.

Hereinafter, the present invention will be described in more detail. However, this is only specifically described to help the understanding of the present invention, the scope of the present invention is interpreted based on the claims.

The present invention is a microorganism comprising at least one barberry extract selected from the group consisting of barberry stem extract, barberry leaf extract, barberry flower extract, barberry fruit extract and barberry seed extract as an active ingredient It provides a composition for inhibiting biofilm formation.

The barberry extract may be divided into barberry stem extract, barberry leaf extract, barberry flower extract, barberry fruit extract and barberry seed extract depending on the portion used.

In the present invention, "barberry stem extract" is an extract of all forms obtained by extracting barberry trunks, for example, extracts in the form of essences obtained by applying heat directly or indirectly to the plant stem, the upper part of the living plant stem Extracts in the form of extracts from the essence, and extracts obtained by extracting the plant with an organic solvent or water, extracts obtained by cooling the gas when burning the plant stem, and the like.

"Barberry fruit extract" in the present invention includes an extract obtained by using an organic solvent or water and the extract of the progressive form obtained by pressing the barberry fruit, oil extract obtained by depressurizing the solvent extract.

"Barberry seed extract" in the present invention includes an extract obtained by using an organic solvent or water, a essence extract obtained by pressing the barberry seeds, or an oil extract obtained by depressurizing a solvent extract.

"Barberry leaf extract" in the present invention means an organic solvent or water extract.

"Barberry flower extract" in the present invention means an organic solvent or water extract.

For example, the barberry extract is pulverized each stem, leaf, flower, fruit and seed, and then extracted with an organic solvent such as alcohol having 1 to 4 carbon atoms, ethyl acetate, hexane, chloroform and water or a mixed solvent thereof. May be an extract.

The most preferred extract may be an ethanol extract of barberry leaves.

As the preparation method of the extract may be used, such as cold needle extraction method, hot needle extraction method or heat extraction method, it may be used a conventional extraction device, ultrasonic grinding extractor or fractionator.

According to another example, the barberry extract may be made of oil by reducing the organic solvent extract of the fruit and / or seed.

The prepared extract may be filtered, concentrated or dried to remove the solvent. Specifically, the filtration may be performed using a filter paper or a vacuum filter, and the concentration may be concentrated under reduced pressure using a vacuum evaporator, for example, a rotary evaporator. The drying may be performed by freeze drying, for example.

The composition according to the present invention is effective in inhibiting biofilm formation of microorganisms.

The term "microorganism" is a concept in which the composition of the present invention includes all bacteria, fungi, yeast and algae that are particularly effective in inhibiting biofilm formation of microorganisms. Thus, in addition to the microorganisms directly inhibited biofilm formation in the following Examples and Experimental Examples all those skilled in the art can be expected and confirmed to inhibit biofilm formation based on the disclosure herein within the scope of ordinary skill It is to be understood that microorganisms are included.

According to one embodiment of the invention, in particular Streptococcus gordonii, Streptococcus oralis , Porphyromonas gingivalis , Fusobacterium nucleatum , Prevotella intermedia , Parvimonas micra; Peptostreptococcus micros , and Aggregatibacter actinomycetemcomitans; Actinobacillus actinomycetemcomitans; It is effective in suppressing biofilm formation.

The barberry extract according to the present invention may be suitably used depending on the product used as long as it can suppress the biofilm formation. Typically, barberry extract according to the present invention is 0.001 to 5.0% by weight, preferably 0.2 to 2% by weight, based on the total weight of the composition with respect to the composition including the composition exhibiting the inhibitory effect of biofilm formation May be included.

The composition has an antimicrobial activity against periodontal disease-causing bacteria and inhibits the formation of tartar by inhibiting the formation of biofilm on the surface of the tooth, and is excellent in preventing and improving gingivitis or periodontitis disease.

In the present invention, gingivitis is an inflammation of the gum which is a part of the oral mucosa, and symptoms of swelling, redness, pain, or localized heat appear, and in severe cases, a disease in which an ulcer or necrosis may occur.

In the present invention, periodontitis (periodontitis) is also referred to as periodontitis, a disease mainly invented in the gums and bone tissue when chronic infection, cell tissue is destroyed, means a periodontal disease that loses teeth.

The composition for inhibiting biofilm formation of microorganisms according to the present invention can be directly applied to animals including humans.

The composition for inhibiting biofilm formation of microorganisms according to the present invention may further include various components depending on the method of use and purpose of use, besides the barberry extract.

According to one embodiment of the present invention, it is possible to provide a functional food composition comprising barberry extract, for example, food or drink.

In the present specification, the food means a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through a processing process to some extent, and as a general meaning, food, food additives, health functional foods. And it is intended to include all drinks, there are various foods, beverages, gum, candy, tea, vitamin complex, functional food and the like.

According to one embodiment of the present invention, a composition for preventing and treating periodontitis or gingivitis is provided. Examples of such compositions include toothpaste, toothpaste solution, mouthwash, toothpaste cream and the like.

When the composition according to the present invention is a toothpaste, in addition to the barberry extract, it may further include an abrasive, a binder, a moisturizer, a foaming agent, a sweetener, a whitening agent or a flavoring agent.

When the composition according to the present invention is a paste-like composition, the caking agent is a tarpaulin, such as tarpaulin, various cellulose derivatives, xanthan gum, and tragacanth gum, polyvinyl alcohol, sodium polyacrylate, and polyacrylic acid / maleic acid copolymer. And organic binders such as synthetic polymer derivatives such as carboxyvinyl polymer, and inorganic binders such as silica and laponite can be used alone or in combination.

The moisturizing agent may be used such as sorbitol, glycerin, ethylene glycol, propylene glycol, polyether glycol, polypropylene glycol and the like.

The sweetener may be used sucrose, lactose, maltose, xylitol, sodium cyclate, sodium saccharin, stevioside, aspartame and the like.

The fragrance is peppermint, menthol, anetol, eugenol, limonene, citronenol, alpha terpineol, salicylmethyl, cineol, linalol, italylinaol, vanillin, thymol, spearmint oil, sage oil, Rosemary oil and cinnamon oil can be used.

When the composition according to the present invention is a fern solution, it can be prepared by mixing the barberry extract in a conventional solvent, formulated as a fern solution, and washes the mouth 2 to 10 times a day for periodontal disease such as periodontitis or gingivitis. Can be booked and treated.

When the composition according to the present invention is a toothpaste cream, it may further include a liquid, a gelling or water-insoluble varnish, sweeteners, flavoring and the like containing water and a moisturizing agent.

When the composition according to the present invention is a mouthwash, it may further comprise a toothpaste carrier, in particular a nontoxic alcohol.

In addition to the above components, the composition may be suitably used according to the purpose to be used, nutritional supplements, vitamins, electrolytes, flavoring agents, coloring agents, fillers, alginic acid and salts thereof, organic acids, thickeners, pH adjusting agents, flavoring agents, stabilizers, preservatives, glycerin , Alcohol, carbonation agent, and the like may be further included.

According to an embodiment of the present invention, the composition of the present invention containing the barberry extract may be prepared by using a formulation such as toothpaste, chewing gum, mouthwash, mouthwash and mouth spray, but is not limited thereto.

Hereinafter, the present invention will be described in detail with reference to experimental examples and examples. However, the experimental examples according to the present invention may be modified in various other forms, and the scope of the present invention should not be construed as being limited to the experimental examples and examples described below. Experimental examples and examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

Preparation Example 1: Preparation of Barberry Extract

The dried barberry leaves were crushed and exposed to an ultrasonic apparatus for 30 minutes while being immersed in twice the amount of ethanol. After immersion for 24 hours, the filter was filtered and then extract was prepared by removing the solvent with a rotary pressure reducer.

Experimental Example 1: Oral Strains and Culture Conditions

Strains were used to obtain Streptococcus gordonii , Streptococcus oralis , and Porphyromonas gingivalis from KCTC BRC.

Streptococcus gordonii , Streptococcus oralis , and Porphyromonas gingivalis fungi ( Brep ) containing 5 ug / mL hemin and 1 ug / ml vitamin K brain heart infusion) and then inoculated in broth medium was used in the experiment by stationary culture at 37 ℃, anaerobic conditions.

Experimental Example 2: Confirmation of antimicrobial activity

Minimal inhibitory concentration (MIC) tests were conducted to determine the antimicrobial activity of barberry extracts against oral bacteria.

Extracts according to each concentration were treated in a bacterial culture solution at a concentration of about 5x10 ^ 5 CFU / mL so that the final mixture was 200 uL on a 96 well-plate. In the case of the extract, the barberry extract concentrated under reduced pressure was dissolved in DMSO at a concentration of 50 mg / mL, and diluted to a concentration of 500, 250, 125, 62.5, and 31.25 ug / mL in a sterilized liquid medium.

The 96-well plate treated with the mixed solution containing the bacterial culture solution and the extract was incubated at 37 ° C. under anaerobic conditions for 24 to 72 hours.

The antimicrobial activity was determined by comparing the absorbance of the experimental group with the extract-treated control group immediately after sample treatment and incubation at 600 nm using a microplate reader after incubation for 24 to 72 hours.

S. gordonii S. oralis P. gingivalis S. gordonii + S. oralis + P. gingivalis MIC
(ug / mL) 250 62.5 > 1,000 > 1,000

Table 1 shows the MIC values for Streptococcus gornea, Streptococcus oralis, Popiromonas jinjivalis single strain, and multiple strains, and the barberry extract was 250 for Streptococcus gornea and Streptococcus oralis, respectively. , 62.5 ug / mL or more was confirmed to exhibit an antimicrobial activity.

Experimental Example 3: Measurement of inhibitory ability of biofilm formation-Microplate

In order to confirm the inhibitory ability of biofilm formation of barberry extract, an experiment using a microplate was conducted.

Streptococcus gordon, Streptococcus oralis, Poppyromonas jinbaris complex, about 5x10 ^ 5 CFU / mL, barberry extract 200, 100, 50, 25, 10 ug / mL 200 uL of liquid medium containing% sucrose was treated. The 96 well plate was incubated for 72 hours at 37 ° C. under anaerobic conditions.

After incubation for 72 hours, the medium was removed and 200 uL of purified water was added to each well to remove suspended bacteria and bacteria that are weakly bound to the biofilm. Thereafter, 200 uL of 0.1% crystal violet staining reagent was added to each well, followed by reaction at room temperature for 15 minutes. After 15 minutes, the resultant was washed three times using purified water to remove residual dye. After drying the dyed biofilm at room temperature, the degree of biofilm formation in each well was confirmed.

As shown in Figure 1, it was confirmed that the experimental group treated with the barberry extract of the present invention inhibited biofilm formation at a concentration of 200 ppm or more compared to the control.

Experimental Example 4: Measurement of inhibitory ability of biofilm formation-Hydroxyyapatite disc

A hydroxyapatite disc experiment was conducted to confirm the inhibitory ability of biofilm formation of barberry extract.

In a well-plate, Streptococcus gordon, Streptococcus oralis, Poppyromonas jinjivalis complex, about 5x10 ^ 5 CFU / ml, each of the barberry extracts 200, 100, 50, 25 ug / mL, 1% sucrose Treating 1 mL of the liquid medium containing, and immersed hydroxyapatite disk coated with artificial saliva (artificial saliva). The 24 well-plates were incubated for 3 days at 37 ° C. under anaerobic conditions.

After 72 hours of incubation, the medium was removed, and 1 mL of purified water was added to each well to remove the bacteria that are weakly bound to the floating bacteria and the biofilm. Thereafter, 1 mL of 0.1% crystal violet staining reagent was added to each well, followed by reaction at room temperature for 15 minutes. After 15 minutes, the resultant was washed three times with purified water to remove residual staining reagent. The dyed biofilm was dried at room temperature and then reacted sufficiently by adding 95% ethanol. 100 uL of ethanol from which the dye was dissolved was transferred to a 96-well plate, and the absorbance was measured at 550 nm using a microplate reader.

As shown in Figure 2, it was confirmed that the experimental group treated with the extract of the present invention inhibited biofilm formation at a concentration of 50 ug / mL or more compared to the control.

Experimental Example 5: Determination of Insoluble Glucan Synthesis Inhibition

Streptococcus goni was incubated in BHI liquid medium for 24 hours, followed by centrifugation (6,000 g, 4 ° C, 15 minutes) to obtain a supernatant. The extracts were treated with each concentration of Streptococcus goni supernatant and the concentration of sucrose was 1%.

After 18 hours of incubation at 37 ° C, the reaction was stopped by addition of the same amount of methanol, followed by centrifugation (6,000 g, 4 ° C, 15 minutes), washing twice with purified water, washing once with 70% ethanol, and water-insoluble glucan. A pellet was obtained.

After drying the residual solvent, the pellet was treated with a reagent in which anthrone was dissolved at 2 mg / mL in 75% H ₂ SO ₄ , and reacted at 100 ° C. for 15 minutes. After waiting until the temperature of the reactant drops to room temperature, the absorbance was measured at 620 nm using a microplate reader.

As a result, as shown in FIG. 3, the water-insoluble glucan synthesis inhibitory activity of barberry extract was 29.7%, 29.7%, 36.1%, and 78.2 at 100, 50, 25, 10 ug / mL, respectively, compared to the untreated barberry extract. A water insoluble glucan production rate of% is shown.

Experimental Example 6: Cytotoxicity to Oral Mucosa Cells

MTT experiments were conducted to determine the toxicity of barberry extracts on oral mucosa cells.

hTERT-OME (immortalized human oral mucosal epithelial cells) were treated in 96 well plates at approximately 1x10 ^ 4 cells / well, then at 37 ° C and 5% CO2 in RPMI-1640, 10% FBS, 1% penicillin-streptomycin medium. Incubated for 24 hours. After 24 hours, the medium used for the previous culture was removed, and the barberry extract diluted to 200, 100, 50, 25, and 12.5 ug / mL was treated in the medium and reacted at 37 ° C. and 5% CO 2 for 24 hours. After treatment, 20 uL of 5 mg / mL MTT solution was added to each well, followed by reaction at 37 ° C. and 5% CO 2 for 3 hours. After the reaction, all of the supernatant was removed, and 150 μL of DMSO was added thereto, followed by reaction at room temperature for 10 minutes. The absorbance was measured at 540 nm using a microplate reader.

As a result, as shown in Figure 4, barberry extract did not show cytotoxicity to oral mucosal cells at 200 ug / mL or less.

Example 1 Preparation of Toothpaste Composition Containing Barberry Extract

Example-Toothpaste composition was prepared with the components and composition ratios shown in Table 2.

The procedure for preparing the toothpaste composition in the oral composition is to disperse a small amount of powder components such as sodium carboxymethylcellulose and sodium saccharin in an amorphous sorbitol liquid as a humectant, dilute with purified water, and then mix in a mixer first, and then, such as silica and calcium carbonate, etc. The abrasive was added and mixed secondly. And finally, a toothpaste composition was prepared by adding a foaming agent, a stabilizer, a perfume, and the like, and tertiary mixing.

Name Example (% by weight) Silica 20.0 Concentrated glycerin (98%) 30.0 Amorphous Sorbitol Liquid (70%) 30.0 Sodium Lauryl Sulfate 1.0 Carboxymethyl Cellulose Sodium 0.5 Xanthan Gum 0.3 Sodium fluoride 0.22 Spices 1.0 Saccharin Sodium 1.0 Citric acid Quantity Sodium bicarbonate 2.0 Barberry Extract 0.02 Purified water To 100

Example toothpaste composition was prepared containing 200 ug / mL barberry extract, the minimum concentration that inhibits the production of biofilm.

From these experiments, barberry extracts play an important role in the early stages of biofilm formation, the root cause of periodontal disease, Streptococcus gordonii , Streptococcus oralis and foreskin causing gum disease. By inhibiting the formation of bacterial membranes of Porphyromonas gingivalis , it was confirmed that it is a natural material that prevents or improves gum disease such as periodontitis and gingivitis.

Claims (7)

Biofilm formation of microorganisms comprising at least one barberry extract selected from the group consisting of barberry stem extract, barberry leaf extract, barberry flower extract, barberry fruit extract and barberry seed extract as active ingredients Inhibitory composition. The method of claim 1, wherein the barberry extract is a composition for inhibiting biofilm formation, characterized in that the extract is extracted with alcohol having 1 to 4 carbon atoms, ethyl acetate, hexane, chloroform, or a mixture thereof, or extracted with water. The method of claim 1, wherein the microorganism is Streptococcus gordonii, Streptococcus oralis , Porphyromonas gingivalis , Fusobacterium nucleatum , Prevotella intermedia , Parvimonas micra (Peptosterreptococcus micros ) and Aggregatibacter actinomycetemcomitans; Actinobacillus actinomycetemcomitans selected from the group consisting of at least one species selected from the group Composition for inhibiting biofilm formation of microorganisms, characterized in that. According to claim 1, The barberry extract is a composition for inhibiting the biofilm formation of the microorganism, characterized in that it comprises 0.001 to 5.0% by weight relative to the total content of the composition for inhibiting the biofilm of the microorganism. The method according to any one of claims 1 to 4,
The composition for inhibiting biofilm formation of a microorganism, wherein the composition for inhibiting biofilm of the microorganism is a health functional food composition for preventing or improving periodontitis or gingivitis. The method according to any one of claims 1 to 4,
Composition for inhibiting biofilm formation of the microorganism, characterized in that the composition for inhibiting the biofilm of the microorganism is an oral composition for preventing and improving periodontal disease. The composition for inhibiting biofilm formation of microorganisms according to claim 6, wherein the composition has at least one formulation selected from toothpaste, chewing gum, mouthwash, mouthwash and mouthwash.

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