KR101490712B1 - Composition with antibacterial effect on oral bacteria containing Green Tea Polysaccharides - Google Patents

Abstract

The present invention relates to a composition for inhibiting oral bacteria containing a green tea polysaccharide as an active ingredient, and more particularly, to a composition for inhibiting oral antibiotics comprising a green tea polysaccharide as an active ingredient, And a composition for inhibiting oral bacteria which is excellent in antibacterial activity. The composition for inhibiting oral bacteria according to the present invention contains green tea polysaccharide extracted from a natural product and minimizes side effects and has excellent antibacterial activity, so that it can be applied to food, medicine and cosmetic industry in various ways.

Green tea polysaccharide, oral bacteria inhibition, Streptococcus mutans, Flebotella intermedia, antibacterial activity

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for inhibiting oral bacteria, which comprises a green tea polysaccharide as an active ingredient,

The present invention relates to a composition for inhibiting oral bacteria, which is excellent in antimicrobial activity against Streptococcus mutans, which is a carrot, or Plebitella intermedia, which is a periodontal disease, by containing a green tea polysaccharide as an active ingredient. The composition for inhibiting oral bacteria according to the present invention contains green tea polysaccharide extracted from a natural product and minimizes side effects and has excellent antibacterial activity, so that it can be applied to food, medicine and cosmetic industry in various ways.

The teeth are minerals made of calcium phosphate. The disease is mainly tooth decay and periodontal disease. Dental cavities are the largest part of dental disease from childhood to adult and are increasing in frequency. According to the Korean Dental Association, more than 90% of children experience dental caries and more than 80% of adults have gum disease. The major cause of this disease is infection by microorganisms in the mouth, which is caused by the interaction of bacteria, food, and saliva. That is, the nutrients and moisture required for the development of oral bacteria are continuously supplied by food, saliva, and saliva, and the environment within the oral cavity has a temperature (37 ° C) and pH (neutral vicinity) suitable for development of microorganisms.

Representative microorganisms that cause periodontal disease, it is Streptococcus mutans (Streptococcus mutans) and play beam telra Inter Media (Prevotella intermedia). These microorganisms cause microbial metabolism, which generates sucrose in food as glucose and fructose, forming insoluble glucan, a polymer of glucose, on the tooth surface. In this process, glucan adheres to the tooth surface with other microorganisms in the oral cavity to form a dental plaque. Lactic acid accumulated by lactic acid bacteria including Streptococcus mutans in the formed plaque dissolves the enamel on the tooth surface, and tooth decay is generated. In addition, since the discomfort is dissolved by the products resulting from the proliferation of such various bacteria, periodontal disease is caused.

To suppress the cavity and periodontal disease, antimicrobial substances such as sodium bicarbonate (NaHCO ₃ ), triclosan (C ₁₂ H ₇ Cl ₃ O ₂ ), polyphosphate and sodium fluoride (NaF), vancomycin , Antibiotics such as chlorhexidine and spiramycin, or organic / inorganic fluorine have been used. However, the above methods are effective for preventing tooth decay, but their use is limited due to the disadvantage of vomiting, diarrhea, and antibiotic resistance. In particular, antibiotics are generally used for bacterial infectious diseases such as tooth decay and periodontitis in the past, but antibiotics not only hurt all the beneficial bacteria of the human body but also cause the emergence of resistant bacteria, immunity of the patient, , And gynecomastia.

Therefore, it is necessary to develop a natural substance which has no side effects and selective antibacterial activity against harmful oral microorganisms. In recent years, a lot of studies have been conducted in the field of dentistry and medicine to find out and maximize the utility of traditional traditional medicine using natural plants.

The inventors of the present invention have conducted research to find a composition for inhibiting oral bacteria which has high antimicrobial activity against harmful oral microorganisms and has no side effects. As a result, it has been found that compositions for inhibiting oral bacteria containing green tea polysaccharide as an active ingredient are effective against Streptococcus mutans and Flavobacterium The antimicrobial activity against harmful oral microorganisms such as intermedia is excellent, and it is found that there is no side effect using natural products, and the present invention has been completed.

Accordingly, an object of the present invention is to provide a composition containing a green tea polysaccharide as an active ingredient and having a high antimicrobial activity against harmful oral microorganisms.

In order to accomplish the above object, the present invention provides a composition for inhibiting oral bacteria, which comprises a green tea polysaccharide as an active ingredient.

The green tea polysaccharide of the present invention is extracted from natural products to minimize adverse effects and has an excellent antimicrobial activity against harmful oral microorganisms, so that it can provide excellent oral bacteria inhibiting effect. Accordingly, the composition for inhibiting oral bacteria containing the green tea polysaccharide of the present invention as an active ingredient can be variously applied in the food, medicinal and cosmetic industries.

The present invention provides a composition for inhibiting oral bacteria containing Green Tea Polysaccharides as an active ingredient.

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

The Green Tea Polysaccharides of the present invention are plant-derived theales of Camellia sinensis, and belong to the family Camellia. They are immunocompetent, anti-radioactive, anti-coagulant, anti-cancer , Anti-HIV and hypoglycemic effect, and it has excellent antimicrobial activity and anti-cavitation function due to its high antimicrobial activity and is excellent in removing bad breath.

The green tea polysaccharide of the present invention comprises a) removing chlorophyll and low molecular weight polyphenol from a green tea powder using a solvent; b) hydrothermally extracting the water-soluble active ingredient from the green tea residue of step a); And c) separating the green tea polysaccharide from the hot water extract of step b) by ultrafiltration and ethanol precipitation.

Hereinafter, the production method of the green tea polysaccharide will be described in detail for each step.

a) removing chlorophyll and low molecular weight polyphenol from green tea powder using a solvent;

In order to remove chlorophyll and polyphenol from green tea powder, extraction is carried out with hexane, ethanol or methanol. Since the present invention is aimed at a food material, ethanol is most preferable in order to prevent problems such as toxicity due to a trace residual amount. The solvent is added to the green tea powder, the solution is sufficiently stirred at room temperature and centrifuged to remove the solvent in which the chlorophyll and polyphenol are dissolved and dried.

b) hydrothermally extracting the water-soluble active ingredient from the green tea residue of step a);

Extracted water extract is used to extract water soluble active ingredient from green tea leaves with chlorophyll and polyphenol removed. The hot water extraction is preferably carried out at a temperature of 30 to 40 ° C to prevent thermal denaturation. The extract obtained by hot water extraction is filtered and concentrated under reduced pressure.

c) separating the green tea polysaccharide from the hot water extract of step b) by ultrafiltration and ethanol precipitation;

Ultrafiltration and ethanol precipitation reaction are carried out in order to separate the polysaccharide from the hot water extract. The polysaccharide is present in about 60 to 65% of the polysaccharide portion and about 8 to 9% of the protein portion. These two moieties are linked by an amino acid-sugar bond, and the remaining components are present as free proteins. Thus, the polysaccharide has a molecular weight of about 100,000 to 300,000 daltons, which is much larger than the free protein. By performing ultrafiltration using the difference in molecular weight, only the green tea polysaccharide can be isolated. Ultrafiltration is a continuous process which not only enables simultaneous separation and concentration of low molecular weight but also eliminates the problem of heat deformation because the process is carried out at room temperature. Ethanol is slowly added to the ultrafiltration concentrate to conduct an ethanol precipitation reaction. At this time, the addition rate of ethanol is preferably from 100 to 200 ml / min, and if it exceeds 200 ml / min, the size of the final produced particles may become too large or clump. After the ethanol precipitation reaction is completed, the ethanol is removed, and the obtained green tea polysaccharide is vacuum dried at 40 to 50 ° C to obtain a powdery green tea polysaccharide.

The green tea polysaccharide of the present invention is preferably contained in an amount of 0.01 to 10% by weight based on the total weight of the composition. If the content is less than 0.01% by weight, the effect of inhibiting oral bacteria is hardly exhibited. If the content is more than 10% by weight, the foreign body sensation in the mouth increases and the intake is not good.

The oral bacteria in the present invention are Streptococcus mutans, a necrotic bacterium, or Prevotella intermedia, a periodontal disease bacterium.

The oral antimicrobial composition of the present invention may be formulated into oral or parenteral formulations in solid, semi-solid or liquid form by addition of an inorganic or organic carrier. In addition, temperature-sensitive sol-gel phase transfer can be made into oral formulations. The composition is characterized by being a liquid agent, a spray agent or a tooth paste formulation.

In the temperature-sensitive sol-gel phase transfer formulations of the present invention, when the polymer is dissolved in water at a low temperature, the solubility of the polymer is drastically reduced to a gel state when the sol is in a sol state and reaches a specific critical temperature or more, Is soluble in water. Therefore, the temperature-sensitive sol-gel phase transition oral formulation is in the form of a liquid sol before application to the oral cavity and is applied to the oral cavity and then transferred to the gel state due to the body temperature and the adhesive force to the teeth, The advantage is that the active ingredient can exert its effects for a long time in the oral cavity. In addition, when rinsed with cold water, it is transferred again to a liquid sol state and can be easily washed out in the oral cavity.

Poly (N-isopropylacrylamide) systems having a critical temperature of 33.2 DEG C have been extensively studied as the temperature responsive polymers, and poly (ethylene glycol) copolymers, polyethylene oxide copolymers, polyethylene oxide- (Acrylamide-vinyl) copolymer, a poly (acrylamide-acrylic) copolymer, and a polyphosphazene-based polymer Was reported. In order to control temperature sensitivity and biocompatibility, a polymer known as the thermosensitive polymer may be grafted onto a polysaccharide having excellent biocompatibility. The polysaccharide may be chitosan or cellulose, preferably chitosan. Polysaccharides such as cellulose or chitosan are natural polymeric materials that are degraded in vivo (Korean Patent Laid-Open No. 10-2008-0049177).

The temperature-sensitive sol-gel phase-transfer formulation containing the green tea polysaccharide of the present invention can be applied directly to the tooth surface using a tool such as a brush, a swab or a tip, or sprayed into the mouth by spraying or the like. Oral formulations include toothpastes, mouthwashes and mouthwashes.

Examples of the oral administration agent include tea powder, liquid, tablets, pills, granules, drinks, soft capsules, hard capsules, powders, granules, powders, emulsions Emulsion), syrups and pellets. Examples of the parenteral administration agents include injections, ointments, ointments, lotions, spray agents, suspensions, emulsions and suppositories. In addition, it may further contain surfactants, excipients, coloring agents, spices, preservatives, stabilizers, buffers, suspending agents and other adjuvants commonly used in the art for reconstituting the active ingredient.

Further, the dosage of the composition according to the present invention will vary depending on the age, sex, body weight and the specific disease or condition to be treated, the severity of the disease or pathological condition, the route of administration and the judgment of the prescriber. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage is from 0.001 mg / kg / day to 2000 mg / kg / day, more preferably from 0.5 mg / kg / day to 2.5 mg / kg / day to be.

In addition, the composition according to the invention contains a cosmetically acceptable medium or base. It may be in any form suitable for topical application, for example as a solution, a gel, a solid or a paste anhydrous product, an emulsion obtained by dispersing an oil phase in water, a suspension, a microemulsion, a microcapsule, a microgranule or an ionic form (liposome) and / In the form of a non-ionic follicle dispersing agent, or in the form of creams, skins, lotions, powders, ointments, sprays or conical sticks. These compositions may be prepared according to conventional methods in the art. The composition according to the invention can also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

The composition of the present invention can be used as a lubricant, a lubricant, a thickening agent and a gelling agent, a softener, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant, water, Ionic sequestering agents and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredient commonly used in cosmetics And may contain auxiliary agents used. These adjuvants are introduced in amounts commonly used in the cosmetics field.

Hereinafter, the present invention will be described more specifically by way of examples. However, the scope of the present invention is not limited to these examples.

[Example 1] Preparation of green tea polysaccharide

10 kg of green tea powder (d50 = 100 to 1000 μm) prepared by pulverizing dried green tea leaves and sieving and classification treatment was dispersed in 150 L of 95% (v / v) ethanol and stirred at room temperature. The prepared mixture was centrifuged to recover the green tea leaves from which chlorophyll and low molecular weight polyphenols were removed. The recovered residue was dried once after repeating the solvent extraction step once.

In the solvent extraction process, 125 l of water was added to the green tea powder from which chlorophyll and low molecular weight polyphenol had been removed, and the mixture was stirred at 35 ° C for 7 hours to conduct hot water extraction. The obtained green tea polysaccharide extract was filtered and recovered using a filter press machine, and then concentrated under reduced pressure at 62 ° C to be 1/10 of the initial volume.

Subsequently, the green tea polysaccharide concentrate was subjected to ultrafiltration (molecular weight cut off: 30,000 daltons) to remove low molecular weight free proteins, and ethanol of 5 times the volume of the final ultrafiltration concentrate was slowly added at a rate of 100 ml / min to effect ethanol precipitation . The precipitated green tea polysaccharide was vacuum dried at 45 캜 to obtain 250 g of powdery green tea polysaccharide.

[Test Example 1] Cytotoxicity test

Human keratinocyte HaCaT cells were seeded in each well of a 96-well plate at a density of 1 × 10 ⁴ cells for 24 hours. 100 ㎕ of phosphate buffered saline (PBS) was added to each well, and the plate was washed once, and the plate was changed to a medium containing 1, 10, 50, and 100 ppm of green tea polysaccharide, and cultured for 48 hours. WST-1 solution per well (Roche Diagnostic GmbH, Germany) was diluted to 1/10 concentration in the medium, treated at 37 ° C for 2 hours, and absorbance was measured at 450 nm. The control (CTL) used dimethyl sulfoxide as a solvent for dissolving the sample. Each data value was corrected to a control group, and the results are shown in Fig.

As can be seen from FIG. 1, the green tea polysaccharide of the present invention showed cell viability similar to that of the control group, and thus was not cytotoxic at the concentration range used.

[Test Example 2]

The microorganism test was conducted according to the broth dilution method using the sample of Example 1, and Streptococcus mutans ( NCTC 10449 (medium: BHI broth)), which is a nasal decongestant, Prevotella intermedia, KCTC6392 (medium: BHI + Calf serum 10% broth), which is a periodontal disease, was used. The Streptococcus mutans strain was inoculated on BHI medium and incubated at 37 ° C for 1 day. The diluted solution was diluted 1/100 with BHI broth, and the BHI + cap serum 10% medium was inoculated at 35 ° C After anaerobic cultivation, a dilution of 1/100 of the culture broth with BHI + serum 10% broth was prepared and used as a test strain. To each 15 ml of broth, 0.15 ml of each of the above-mentioned broths was added and well mixed. The sample was prepared by diluting 1 ml of the green tea with 1.8 ml of the medium (BHI broth, BHI + cap serum 10% broth) g, respectively. The common green tea used here is green tea produced by AMOREPACIFIC Co., Ltd. Green tea leaves are used as a green tea for fever - crude oil - oil - heavy oil - essential oil - dried green tea.

First, 180 쨉 l of each sample was added to the 96 well plate 1, and 20 쨉 l of the diluted solution of the test solution at 1/10 was added. Then, 100 μl of the diluted solution was added to the remaining wells of the well plate. The mixed solution of line 1 was mixed well and then 100 μl was taken and placed in line 2 and mixed well. Then, 100 μl of the mixed solution of line 2 was taken and double dilution was carried out in the same manner as in line 3 . In the case of Streptococcus mutans, the culture was incubated at 37 ° C for 24 hours, and the minimum inhibitory concentration (MIC) was determined as the minimum inhibitory concentration (MIC). When the mixture was opaque and it was difficult to judge the growth of the bacteria, it was confirmed by microscopic observation. In the case of Flebotella Intermedia, all operations were carried out in an anaerobic chamber and the culture was incubated in anaerobic condition at 35 ° C for 2 days, and the MIC value was determined. The results are shown in Table 1 below.

Name of sample Minimum inhibitory concentration (MIC) S. mutans P. intermedia Regular green tea 2.50% 1.25% Example 1 0.01% 0.1%

As shown in Table 1, the green tea polysaccharide (Example 1) showed an antibacterial activity of harmful oral microorganisms as compared with ordinary green tea. In addition, considering that the minimum inhibitory concentration of Streptococcus mutans of triclosan, which has been commercialized in the past, is 0.02%, the antimicrobial activity of the green tea polysaccharide (Example 1) was excellent and the efficacy was excellent in terms of economy.

Hereinafter, a formulation example of the composition will be described, but it is to be understood that the present invention is not intended to be limited thereto but is specifically described.

[Formulation Example 1]

50 mg of the green tea polysaccharide of Example 1, the sterilized distilled water suitable amount for injection, and the pH adjuster were mixed, and the contents were adjusted to the above contents in accordance with the usual injection preparation method (2 ml) per ampoule.

[Formulation Example 2]

100 mg of the green tea polysaccharide of Example 1, 10 g of isomerized sugar, 5 g of mannitol, and purified water were mixed, and each component was added to and dissolved in purified water according to the usual preparation method of the liquid agent, and the lemon flavor was added in an appropriate amount. And then purified water was added to adjust the total volume to 100 ml. The solution was filled in a brown bottle and sterilized to prepare a liquid preparation.

[Formulation Example 3] Soft capsule

A soft capsule was prepared by mixing 50 mg of the green tea polysaccharide of Example 1, 80 to 140 mg of L-carnitine, 180 mg of soybean oil, 2 mg of palm oil, 8 mg of vegetable hydrogenated oil, 4 mg of yellow radish and 6 mg of lecithin, Respectively.

[Formulation Example 4] Tablets

50 mg of the green tea polysaccharide of Example 1, 200 mg of galactooligosaccharide, 60 mg of lactose and 140 mg of maltose were mixed and granulated using a fluidized bed drier, followed by addition of 6 mg of sugar ester and tabletting to prepare tablets.

[Formulation Example 5]

50 mg of the green tea polysaccharide of Example 1, 250 mg of anhydrous crystalline glucose, and 550 mg of starch were mixed and granulated into granules using a fluidized bed granulator, and filled in a vial.

[Formulation Example 6] Drinking agent

50 mg of the green tea polysaccharide of Example 1, 10 g of glucose, 0.6 g of citric acid and 25 g of liquid oligosaccharide were mixed and then 300 ml of purified water was added to fill each bottle with 200 ml each. And sterilized at 130 DEG C for 4 to 5 seconds to prepare a drink.

[Formulation Example 7] Temperature-sensitive sol-gel phase transition polysaccharide oral formulation

30 mg of the green tea polysaccharide of Example 1 and chitosan-g-poly (N-isopropylacrylamide) (temperature-sensitive sol-gel phase transfer polysaccharide) were mixed and each component was added to purified water according to a conventional liquid preparation method After adding proper amount of lemon flavor or other fragrance, purified water was added to adjust the total volume to 100 ml, filled in a brown bottle and sterilized to prepare a liquid preparation.

[Formulation Example 8] Oral spray agent

10 mg of the green tea polysaccharide of Example 1 was added to purified water according to a conventional oral spray preparation method and the taste blocker and / or flavoring agent was added in an amount of 0.75 to 7.5% by weight of the total composition, and then the whole was adjusted to 300 ml, .

[Formulation Example 9] Toothpaste

The green tea polysaccharide of Example 1 was contained in an amount of 1 to 3% by weight of the total composition, and the total amount of the perfume, the sweetener, the thickening agent, the abrasive agent and the dental plaque removing agent was adjusted, and the total amount was adjusted.

[Formulation Example 10] Tea powder formulation

100 mg of the green tea polysaccharide of Example 1, 2 g of common green tea (powder) and 1 g of anhydrous crystalline glucose were mixed and charged into a tank of a hopper top plate of a slope paper wrapping machine and filled in a bag.

1 is a graph showing the cytotoxicity test results of green tea polysaccharide according to the present invention.

Claims (5)

A composition for inhibiting oral bacteria, comprising a green tea polysaccharide as an active ingredient, wherein the green tea polysaccharide a) removing chlorophyll and low molecular weight polyphenol from green tea powder using a solvent; b) hydrothermally extracting the water-soluble active ingredient from the green tea residue of step a); And c) separating the green tea polysaccharide from the hot water extract of step b) by ultrafiltration and ethanol precipitation; The composition for inhibiting oral bacteria according to claim 1, delete The composition for inhibiting oral bacteria according to claim 1, wherein the green tea polysaccharide is contained in an amount of 0.01 to 10% by weight based on the total weight of the composition. The composition for inhibiting oral bacteria according to claim 1, wherein the oral bacteria is Streptococcus mutans or Prevotella intermedia . The composition for inhibiting oral bacteria according to claim 1, wherein the composition for inhibiting oral bacteria is a temperature-sensitive sol-gel phase transfer oral formulation.

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