KR102431182B1 - Oral composition comprising fermented green tea extract having excellent antibacterial effect on oral bacteria and anti-inflammatory effect - Google Patents

Abstract

The present invention relates to a composition for oral use containing a fermented green tea extract, and more particularly, to a tea tree leaf of the genus Aspergillus ( Aspergillus sp . ) strain and fermented green tea obtained by fermenting under specific fermentation conditions using a solvent and containing fermented green tea extract as an active ingredient to inhibit the growth of Streptococcus mutans , a bacterium that causes tooth decay, and It relates to a composition for oral use that suppresses the expression of inflammation-inducing cytokines, particularly IL-8, and provides excellent oral bacteria suppression and anti-inflammatory effects at the same time.

Description

An oral composition comprising fermented green tea extract having excellent antibacterial effect on oral bacteria and anti-inflammatory effect, comprising fermented green tea extract as an active ingredient

The present invention relates to a composition for oral use containing Aspergillus sp. strain fermented green tea extract as an active ingredient, which has excellent oral bacterial inhibition and anti-inflammatory effects.

Teeth are minerals made of calcium phosphate, and their diseases are largely tooth decay and periodontal disease. Among them, tooth decay accounts for the largest portion of dental diseases from children to adults, and the incidence is increasing. According to the report of the Korean Dental Association, more than 90% of children experience dental caries, and more than 80% of adults have gum disease. The main cause of these diseases is infection by microorganisms in the oral cavity, which is caused by the interaction of bacteria, food, and saliva. In other words, nutrients and moisture necessary for the growth of bacteria in the oral cavity are continuously supplied by food, saliva, and dental saliva, etc., and the environment in the oral cavity has a temperature (37° C.) and pH (near neutral) suitable for the growth of microorganisms.

Typical microorganisms that cause periodontal disease include Streptococcus mutans and Prevotella intermedia . These microorganisms cause microbial metabolism to produce sucrose in food into glucose and fructose, forming insoluble glucan, a polymer of glucose, on the tooth surface. In this process, glucan attaches to the tooth surface with other microorganisms in the oral cavity, forming a dental plaque, that is, dental plaque. Lactic acid accumulated by lactic acid bacteria including Streptococcus mutans in the formed plaque dissolves the enamel on the tooth surface, causing tooth decay. In addition, since the disgrace is dissolved by the products according to the proliferation of such various bacteria, periodontal disease occurs.

Periodontal disease, also called periodontal disease, is divided into gingivitis and periodontitis according to the severity of the disease. It is a relatively light and fast-recovering form of periodontal disease, which is limited to the gums, that is, soft tissue, and is called gingivitis, and when the inflammation progresses to the gums and around the gums, it is called periodontitis.

In addition, there is a V-shaped gap between the gingiva (gum) and the teeth. Periodontal disease refers to the damage to the periodontal ligament and adjacent tissues by bacteria attacking the lower part of the gum line of the sulcus. As inflammation progresses and more tissue is damaged, the groove develops into a periodontal pocket, and as periodontitis is severe, the depth of the pocket becomes deeper. As the periodontal pocket deepens, the periodontal ligament becomes inflamed and bone loss occurs.

In order to suppress the tooth decay and periodontal disease, antibacterial substances such as sodium bicarbonate (NaHCO ₃ ), triclosan (C ₁₂ H ₇ Cl ₃ O ₂ ), polyphosphate and sodium fluoride (NaF), vancomycin ), synthesis of antibiotics such as Chlorhexidine and Spiramycin, sodium chloride (NaCl), Allantoine Chlorohydroxy Aluminum, Aminocaproic Acid, and tocopherol acetate Raw materials, or organic/inorganic fluorine have been used. However, although these methods are effective in preventing tooth decay, their use is limited because of the disadvantages of vomiting, diarrhea, and resistance to antibiotics. In particular, the conventional use of antibiotics for bacterial infectious diseases such as tooth decay and periodontitis was common, but antibiotics not only harm all beneficial bacteria in the human body, but also cause the appearance of resistant bacteria, decrease the patient's immune ability, It causes side effects such as mycosis.

Therefore, it is necessary to develop a natural material that has no side effects and has high antibacterial activity selectively against harmful oral microorganisms. Recently, as interest in natural extracts increases, research on various plants is being actively conducted, and as the effects on the oral cavity are known, many extracts are being applied to oral compositions related to prevention of gum disease and suppression of bad breath.

However, many plant extracts have been formulated and used for the purpose of improving oral hygiene, but in most cases, they have poor antibacterial effects or simply prevent the occurrence of periodontal disease through abstract effects such as anti-inflammatory, astringent, hemostasis, and blood circulation promotion. It shows only the degree of prevention.

Green tea (Green tea/Thea sinensis Linnaeus) is an evergreen tree or shrub belonging to the family Theaceae. Green tea is the most widely consumed beverage after water, and its efficacy has been studied for a long time. Green tea, black tea, and oolong tea are produced from the leaves. Green tea is obtained by inactivating oxidation-related enzymes by steaming tea leaves, stems or flowers and stably maintaining polyphenol components. Polyphenols found in green tea are known as flavonols and catechins, and as disclosed in Patent Application No. 2010-0048323 and Patent Application No. 2010-0048323, various efficacy effects such as antioxidant, anticancer, anti-inflammatory, and antibacterial is known to have However, the literature [Yeo Saenggyu et al., Antibacterial effect of green tea, Oryong tea and black tea extracts, Journal of the Korean Society for Food and Nutrition, 1995. 4. 30., Vol. 24, No. 2 pp. 293~298], compared to fermented teas such as green tea, Olong tea, and black tea, steamed tea and roasted tea, which are non-fermented teas, were confirmed to have a greater antibacterial action, and in particular, fermented against Streptococcus mutans , a bacteria that causes tooth decay. It was found that the higher the degree of tea, the lower the antibacterial activity.

According to the results of such prior studies, research efforts to develop products capable of providing oral bacteria and inflammation inhibitory activity by utilizing fermented green tea and extracts have hardly been made.

Korean Patent Application No. 2010-0048323 Korean Patent Application No. 2014-0146449 Korean Patent Application No. 2011-0107337 Korean Patent Application No. 2015-0038323

Yeo Saeng-gyu and 5 others, Antibacterial effect of green tea, Oryong tea and black tea extracts, Journal of the Korean Society for Food and Nutrition, 1995. 4. 30., Vol. 24, No. 2, pp 293-298.

Among the natural extracts, green tea extract is known for its antibacterial and anti-inflammatory effects, but it has a problem with low oral bacteria inhibitory and anti-inflammatory properties. In addition, in the case of fermented green tea extract, effects such as whitening activity are known, but studies on antibacterial and anti-inflammatory effects applied to the oral cavity are virtually non-existent. Therefore, the present inventors recognized this problem and studied fermented green tea extract, as a result of studying fermented green tea obtained by using an Aspergillus sp. strain, preferably Aspergillus oryzae , as a fermented strain on green tea leaves, specifically fermented green tea. Extracts of green tea leaves (hereinafter referred to as 'extracts of fermented green tea obtained by fermentation with Aspergillus sp. strains' or 'Aspergillus sp. strains fermented green tea extracts') are produced using existing green tea, specifically green tea leaf extracts and other fermented strains. The present invention was completed by confirming that fermented green tea obtained by fermenting, more specifically, superior oral bacterial inhibition and anti-inflammatory effect than fermented green tea leaf extract.

Accordingly, an object of the present invention is to provide a composition for oral cavity excellent in inhibiting oral bacteria and oral inflammation containing Aspergillus sp. strain-fermented green tea extract as an active ingredient.

In order to achieve the above object, the present invention provides a composition for oral use excellent in inhibiting oral bacteria and oral inflammation containing Aspergillus sp. strain-fermented green tea extract as an active ingredient.

The present invention relates to a composition for oral use excellent in inhibiting oral bacteria and having anti-inflammatory effects, comprising an Aspergillus sp . strain-fermented green tea extract as an active ingredient, wherein the fermented green tea extract directly affects tooth decay. It has excellent proliferation inhibitory activity of oral inflammatory substances, and has excellent expression inhibitory activity of cytokines, particularly IL-8, which are substances that cause oral inflammation, so it can be used in various ways in the field of oral cleaning or cleaning products.

The present invention relates to a composition for inhibiting oral bacteria and oral inflammation containing Aspergillus sp. strain-fermented green tea extract as an active ingredient.

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

The Aspergillus sp. strain-fermented green tea extract of the present invention is obtained by drying the leaves of a tea tree in sunlight or hot air, then sieving, inoculating the Aspergillus spp. It can be prepared by extraction with an organic solvent.

Examples of the Aspergillus sp. strain used as the fermentation strain in the present invention include Aspergillus niger , Aspergillus duck material ( Aspergillus oryzae ), Aspergillus teraeus ( Aspergillus terreus ), Aspergillus material ( Aspergillus sojae ), and Aspergillus fumigatus ( Aspergillus fumigatus ) and the like, and Aspergillus duck material ( Aspergillus oryzae ) is preferably used.

The fermented green tea of the present invention can be prepared by inoculating green tea leaves with the Aspergillus sp. strain at a concentration of 1.0 x 10 ⁵ to 1.0 x 10 ⁹ CFU/g, and then fermenting it at 20 to 30°C for 3 to 15 days, preferably Preferably, fermentation is carried out at 25 to 30° C. for 5 to 10 days.

The extract of fermented green tea obtained by fermentation with the strain of Aspergillus sp. according to the present invention is inoculated with the fermented strain into tea leaves and fermented during the fermentation period. It is an extract obtained by adding and extracting a lower alcohol (eg, alcohol) or a mixture of water and a lower alcohol having 1 to 4 carbon atoms. The concentration of the lower alcohol used as the extraction solvent is preferably 10 to 100%, preferably 30 to 70%.

The extraction method of the extract of fermented green tea obtained by fermentation with the strain of Aspergillus sp. according to the present invention can use common extraction methods such as cold extraction method and alcohol extraction method. it doesn't happen

The extract of fermented green tea obtained by fermentation with the strain of Aspergillus sp. according to the present invention may be mixed in an amount of 1 to 90% by weight, preferably 1 to 30% by weight, more preferably 1 to 10% by weight based on the total weight of the composition. % can be mixed. When it contains less than 1% by weight, sufficient oral bacteria inhibition and anti-inflammatory effects cannot be expected, and when it contains more than 90% by weight, there is irritation when applied to the oral cavity, and thus the feeling of use is not good.

The composition for oral use containing an extract of fermented green tea obtained by fermenting the strain of Aspergillus sp. according to the present invention is not particularly limited in its formulation as long as the purpose is to inhibit oral bacteria and anti-inflammatory. Specifically, for example, it may have formulations such as toothpaste, mouthwash, and tooth whitening agent.

The composition for oral use containing an extract of fermented green tea obtained by fermentation with the Aspergillus sp. strain according to the present invention is an abrasive, a wetting agent, an auxiliary foaming agent, a binder, a sweetener, a pH adjuster, a preservative, and a medicinal effect commonly used depending on the formulation and purpose of use. It may contain ingredients, fragrances, brighteners, colorants, solvents, and the like.

Hereinafter, the configuration and effect of the present invention will be described in more detail by way of test examples and preparation examples. However, these test examples and preparation examples are provided only for the purpose of illustration to help the understanding of the present invention, and the scope and scope of the present invention are not limited by the following examples.

[Preparation Example 1] Preparation of extract of Aspergillus sp. strain fermented green tea

100 kg of green tea leaves collected from tea trees (obtained from: Jeju Osulloc Farm) were put in a culture container, 30-60 kg of water was added, and Aspergillus oryzae (Acquired: Korea Microbial Conservation Center (KCCM)) ))) was added in an amount of 0.1 to 5% by weight based on the weight of green tea, and the raw green tea leaves and fermented strains were stirred to mix well to prepare a raw material mixture. In an incubator, the mixture was fermented for about 15 days while maintaining a culture temperature of 30° C. and a pH of about 3.0.

5 L of 70% ethanol aqueous solution as an extraction solvent was added to 100 g of fermented green tea thus obtained, extracted under reflux three times, and then immersed at room temperature for 1 day. Thereafter, the residue and the filtrate were separated through filter cloth filtration and centrifugation, and the separated filtrate was concentrated under reduced pressure to obtain 35 g of fermented green tea extract.

[Comparative Preparation Example 1] Preparation of general green tea extract

To 100 g of green tea leaves (obtainer: Osulloc Farm, Jeju), 5 liters of 70% ethanol aqueous solution was added, extracted under reflux three times, and then immersed at room temperature for 1 day. Then, the residue and the filtrate were separated through filter cloth filtration and centrifugation, and the separated filtrate was concentrated under reduced pressure to obtain about 30 g of a general green tea extract.

[Comparative Preparation Example 2] Preparation of extract of fermented green tea extracted with purified water

About 33 g of fermented green tea extract was obtained by extraction in the same manner as in Preparation Example 1, except that purified water was used as an extraction solvent.

[Comparative Preparation Example 3] Preparation of extract of fermented green tea fermented with dry yeast

About 32 g of fermented green tea extract was obtained in the same manner as in Preparation Example 1, except that dry yeast ( Saccharomyces cerevisiae (obtainer: KCCM)) was used as a fermentation strain.

[Comparative Preparation Example 4] Preparation of extract of fermented green tea under different fermentation conditions

About 32 g of fermented green tea extract was obtained in the same manner as in Preparation Example 1, except that it was fermented at 25° C. for 24 hours.

[Comparative Preparation Example 5] Preparation of extract of fermented green tea under different fermentation conditions and fermented strains

About 32 g of fermented green tea extract was obtained in the same manner as in Preparation Example 1, except that dry yeast ( Saccharomyces cerevisiae (obtainer: KCCM)) was used as a fermentation strain and fermented at 25° C. for 24 hours.

[Test Example 1] Measurement of oral bacteria inhibition efficacy by extract of fermented green tea

Streptococcus mutans ( Streptococcus mutans ) was cultured for 24 hours in a constant temperature incubator at 35 ° C. when reaching the exponential phase (Exponential phase) was used in the test. In a 96 well plate, except for the first row, 100 μl of liquid medium (for 1 L of medium, Calf brains 7.7 g, Beef heart 9.8 g, Proteosepeptone 10.0 g, Dextrose 2.0 g, Sodium chloride 5.0 g, Disodium phosphate 2.5 g, contained as the remaining amount of deionized water). In the first row, 200 μl of a liquid medium in which Preparation Example 1 and Comparative Preparation Examples 1 to 5 were added at a concentration of 2% (w/w) was added, and serial dilution was performed so that the concentration was diluted by 1/2. It was run 7 times. The sample was inoculated with a strain of Streptococcus mutans cultured for 24 hours before the medium of various concentrations to which the sample was added at a concentration of 1 x 10 ⁶ CFU/ml and cultured in an incubator at 35°C. The suspension of the liquid medium was measured, and the minimum concentration at which the growth of Streptococcus mutans was inhibited was determined from this suspension. [Table 1] and [Table 2] show the average values of the minimum concentrations obtained by performing the experiment three times in total.

density(%) CPC Preparation Example 1 Comparative Preparation Example 1 Comparative Preparation Example 2 Comparative Preparation Example 3 Comparative Preparation Example 4 Comparative Preparation Example 5 2 - - - + + - + One - - + + + - + 0.5 - - + + + - + 0.25 - - + + + + + 0.125 - - + + + + + 0.0625 - - + + + + + 0.031 - + + + + + + 0.015 - + + + + + +

Note) CPC: Cephalosporin C; -: Bacteria do not grow, + Bacteria grow

antibacterial substances MIC concentration (%) for S. mutans CPC (synthetic antibacterial agent) <0.015625 Preparation Example 1 0.0625 Comparative Preparation Example 1 2 Comparative Preparation Example 2 >2 Comparative Preparation Example 3 >2 Comparative Preparation Example 4 0.5 Comparative Preparation Example 5 >2

Through [Table 1] and [Table 2], the fermented green tea extract of Preparation Example 1 according to the present invention was obtained by varying the presence or absence of fermentation of green tea leaves, extraction solvent, fermentation strain, fermentation conditions, or fermentation strains and fermentation conditions. It was found that the antibacterial effect against Streptococcus mutans was significantly superior to that of the green tea extract (ie, Comparative Preparation Examples 1 to 5). In particular, it was confirmed that the difference in antibacterial activity by the extraction solvent and the fermentation strain was very large.

[Test Example 2] Measurement of anti-inflammatory activity of each extract of fermented green tea

In order to confirm whether the extract of fermented green tea has the activity of inhibiting the production of inflammatory cytokines that cause inflammation, the following experiment was performed. YD 38 cells, an oral cell line (Korea Cell Line Bank), were diluted in RPMI-1640 medium (containing 10% FBS, 1% Penicillin-streptomycin) and seeded at a density of 2 x 10 ⁴ cells/well and 5 for 24 hours. % CO ₂ , and cultured at 37°C. After washing once with sterile PBS on the second day of the test, 200 μl of RPMI-1640 without serum and antibiotics was dispensed and cultured at 5% CO ₂ , 37° C. for 24 hours. On the 3rd day of the test, fresh serum and RPMI-1640 without antibiotics were dispensed, and then the fermented green tea extract of Preparation Example 1 and the extracts of Comparative Preparation Examples 1 to 5 were treated for each test concentration, and then LPS at a concentration of 5 μg/ml treated with 5% CO ₂ , and incubated for 6 hours at 37°C. After 6 hours of treatment with the test substance, the cell culture medium was recovered and the expression level of IL-8 was evaluated using an ELISA kit (BD PharMingen, CA, USA, R&D system, MN, USA). The degree of inhibition was quantified through the diagram below for the IL-8 expression levels measured in the untreated group, the LPS-only treatment group, and the LPS and test substance-treated group. The higher this number, the better the anti-inflammatory effect. The results are shown in [Table 3].

% of inhibition = [(cytokine secretion _LPS - cytokine secretion _CTL ) - (cytokine secretion _Test - cytokine secretion _CTL )/ (cytokine secretion _LPS - cytokine secretion _CTL )] X 100

anti-inflammatory % of inhibition 250ppm 125ppm 62ppm Aminocaproic acid (synthetic anti-inflammatory) 56.13 38.96 37.6 Preparation Example 1 72.09 62.23 12.07 Comparative Preparation Example 1 -25.78 54.71 7.14 Comparative Preparation Example 2 21.44 32.57 1.53 Comparative Preparation Example 3 5.31 30.31 32.21 Comparative Preparation Example 4 34.10 40.11 38.19 Comparative Preparation Example 5 10.53 4.41 5.27

Looking at [Table 3], it can be seen that the extract of fermented green tea of Preparation Example 1 according to the present invention has superior ability to inhibit the expression of IL-8 than aminocaproic acid, which is a synthetic anti-inflammatory substance. In particular, compared with the green tea extract of Comparative Preparation Example 1, it was confirmed that the fermented green tea extract of Preparation Example 1 exhibited significantly higher anti-inflammatory properties at all test concentrations.

By using the extract of Aspergillus sp. fermented green tea according to the present invention as an active ingredient, oral products having formulations and compositions as shown in Table 4 to Table 6 were prepared.

[Formulation Example 1] Formulation example of toothpaste Ingredient name Weight ratio (%) One Purified water Balance (to 100) 2 Sodium monofluorophosphate 0.76 3 Extract of fermented green tea (Preparation Example 1) 5.00 4 Sorbitol solution (70%) 50.00 5 Sodium Saccharin 0.40 6 silica 10.00 7 Cellulose Gum 1.00 8 combination spices 1.00 9 Sodium Lauryl Sulfate 2.00

[Formulation Example 2] Formulation example of oral rinse Ingredient name Weight ratio (%) One Purified water Balance (to 100) 2 sodium fluoride 0.03 3 Extract of fermented green tea (Preparation Example 1) 1.00 4 Sorbitol solution (70%) 10.00 5 Sodium Saccharin 0.01 6 combination spices 0.10

[Formulation Example 3] Formulation example of tooth whitening agent Ingredient name Weight ratio (%) One polyethylene glycol Balance (to 100) 2 glycerin 35.00 3 functional silicate 20.00 4 Extract of fermented green tea (Preparation Example 1) 10.00 5 Sodium monofluorophosphate 0.76 6 Sodium Saccharin 0.30 7 polyvinylpyrrolidone 1.00 8 combination spices 1.00 9 Sodium Lauryl Sulfate 2.00

Claims (12)

Aspergillus sp . ) A composition for inhibiting harmful oral bacteria containing an extract of fermented green tea obtained by fermentation with a strain as an active ingredient. The composition for inhibiting harmful oral bacteria according to claim 1, wherein the Aspergillus sp. strain is Aspergillus oryzae . The inhibition of oral harmful bacteria according to claim 1, wherein the extract of fermented green tea is obtained by extraction using an alcohol having 1 to 4 carbon atoms or a mixed solvent of water and an alcohol having 1 to 4 carbon atoms as an extraction solvent. for composition. The composition for inhibiting harmful oral bacteria according to claim 1, wherein the extract of the fermented green tea is contained in an amount of 1 to 90% by weight based on the total weight of the composition. The composition for inhibiting oral harmful bacteria according to claim 1, wherein the oral harmful bacteria are Streptococcus mutans . The composition for inhibiting harmful bacteria in the oral cavity according to any one of claims 1 to 5, having any one formulation selected from the group consisting of toothpaste, mouthwash, and tooth whitening agent. Aspergillus sp . ) A composition for inhibiting oral inflammation containing an extract of fermented green tea obtained by fermentation with a strain as an active ingredient. The composition for inhibiting oral inflammation according to claim 7, wherein the Aspergillus sp. strain is Aspergillus oryzae . The method according to claim 7, wherein the extract of fermented green tea is obtained by extraction using an alcohol having 1 to 4 carbon atoms or a mixed solvent of water and alcohol having 1 to 4 carbon atoms as an extraction solvent. composition. The composition for inhibiting oral inflammation according to claim 7, wherein the extract of the fermented green tea is contained in an amount of 1 to 90% by weight based on the total weight of the composition. The composition for inhibiting oral inflammation according to claim 7, wherein the oral inflammation is inflammation caused by IL-8. The composition for inhibiting oral inflammation according to any one of claims 7 to 11, having any one formulation selected from the group consisting of toothpaste, mouthwash, and tooth whitening agent.