KR101506995B1 - Anticariogenic composition comprising extract of bark of alnus japonica (thunberg) steudel - Google Patents

Abstract

The present invention relates to an anticalculus pharmaceutical composition, a quasi-drug, and a food composition comprising an extract of a bark extract, a lactic acid bacterium, or a duck bark extract and a lactic acid bacterium. According to the present invention, the antibacterial activity against Streptococcus mutans , which is a causative organ of dental caries, can be inhibited by using biofilm extracts and lactic acid bacteria selected from the viewpoint of anti-wrinkle efficacy and inhibiting the biofilm which plays an important role in dental caries. It is possible to exert the inhibitory effect on glucan formation and to exert effective effects on the already formed biofilm removal. In particular, the extract of bark bark and the lactic acid bacterium used in the present invention exhibit excellent antimicrobial efficacy when used alone, and exhibit a remarkable synergistic effect on the antibacterial effect when used in combination, and are effectively used for prevention and treatment of dental caries . In addition, the present invention can be applied to various fields by securing safety without causing toxicity and side effect problems by using extracts of bark bark extract and lactic acid bacteria that have no problem in safety.

Description

TECHNICAL FIELD The present invention relates to an antibacterial composition comprising a bark extract of Aspergillus oryzae and a lactic acid bacterium.

The present invention relates to anti-wasting compositions, and more particularly to anticoagulant compositions comprising extracts of bark bark, lactic acid bacteria or bark extracts and lactic acid bacteria.

Dental caries is one of the most widespread cultural diseases in the world, and the morbidity rate in Korea is gradually increasing. Dental caries is clinically very frequent and is a major cause of tooth loss despite the development of treatment. The cause of dental caries is the dental plaque on the surface of the teeth. Many microorganisms in the tooth form metabolize carbohydrates to form organic acids, which dissolve the enamel, which is the outer membrane of teeth. The causative organism that plays a major role in plaque formation is known as Streptococcus mutans .

Streptococcus mutans forms colonies in the host after the first strikes and then attaches to the membrane of the tooth surface to secrete an enzyme called Glucosytransferase (GTase), and sucrose contained in the food is used as a substrate and glucose glucose and fructose are synthesized. At the same time, insoluble Glucan, a glucose polymer, is synthesized, and then other microorganisms in the oral cavity attach to the tooth surface by the glucan to form a plaque. After the plaque is formed, the resistance to physical force or antimicrobial component is increased, and the antibacterial effect of the antibacterial agent is deteriorated because it is not removed by brushing. Therefore, it is important to prevent plaque formation early by inhibiting the growth of Streptococcus mutans, which plays an important role in plaque formation, in order to prevent effective tooth decay.

Currently, chemical sterilizing agents and antibiotics are mainly used as methods for suppressing dental caries. However, antibiotics can cause systemic side effects to the whole body including diarrhea and vomiting, and it can cause the appearance of resistant bacteria in the oral cavity and cause a bacterial shift. Therefore, it is difficult to use for a long time, . For example, chlorohexidin, a widely used antibiotic, is used at high concentrations in the oral cavity or when used at low concentration for a long time, causing coloration of the tongue and teeth, peeling of the oral mucosa, There is a disadvantage in that side effects may occur and there is a side effect that can induce a bacterial shift, and there is a disadvantage in that the use of triclosan is limited in the case of pregnant women due to carcinogenicity, and the cytotoxicity of triclosan is high, There is a problem that a high and resistant microorganism appears. Therefore, it is very urgent to develop a safe dental caries inhibitor that can be used for a long period of time without side effects.

Recently, there have been very active researches on the mechanism of dental caries development and prevention of dental caries. In particular, exploration of dental caries prevention materials has continued to be explored and developed for various natural products such as microorganisms, green tea, and oriental herb.

Meanwhile, alder (Alnus japonica (Thunberg) Steudel) is a deciduous tree belonging to the Alnus of the Betulaceae. In one room, the bark of an alder tree is called a rape. Auckland is a safe medicinal substance registered in the KFDA food raw material circulation. It has 5 ~ 10% tannic acid, triterpenoid, taaracrol, and betulinic acid in the shell, about 10% tannic acid in the fruit, and flavonoid hiper Seed, and 0.2% refined oil. Conventional techniques using such a mushroom extract include anti-inflammatory and moisturizing cosmetic compositions, hangover cleansing agents, antioxidant and liver protecting compositions, compositions for treating cardiac circulatory system, and compositions for detoxifying smoking toxins. Technologies are reported.

On the other hand, Lactobacillus plantarum is a lactic acid bacterium isolated mainly from kimchi, which is a Korean traditional food, and its antibacterial activity against various pathogenic microorganisms has been reported. It is also known to produce various antimicrobial substances such as lactic acid, acetic acid, and organic acids, hydrogen peroxide, and bacteriocin, and they are known to be important biological preservatives of foods and feeds.

Lactobacillus is of particular interest because it is a generally regarded as safe microorganism, which is generally regarded as safe, since it has been widely consumed in foods, both east and west.

However, there has been no research on anticoagulant compositions having dental caries treatment or prevention efficacy, which can secure long-term use safety without side effects by using alder tree extract or lactic acid bacterium.

The inventors of the present invention found that the extracts of bark bark and lactic acid bacterium exhibit superior antimicrobial efficacy and, when used in combination, exhibit a synergistic effect of antibacterial activity, thus completing the present invention. The present invention relates to a method for inhibiting biofilm formation, having antibacterial activity against glucocorticoid-causing strains and inhibiting glucan formation, as well as extracting a bark extract, a lactic acid bacterium or a duck bark extract And an antibacterial composition comprising a lactic acid bacterium.

One aspect of the present invention for solving the above-mentioned problems is to provide a pharmaceutical composition comprising a duck bark extract, a lactic acid bacterium, or a duck bark extract and a lactic acid bacterium.

In another aspect of the present invention, there is provided an anti-widespread quasi-drug comprising an extract of Alnus bark, a lactic acid bacterium, or a duck bark extract and a lactic acid bacterium.

Further, another aspect of the present invention provides an anti-wrinkle food composition comprising a bark extract, a lactic acid bacterium, or a bark extract and a lactic acid bacterium.

In one aspect of the present invention, the lactic acid bacterium is L. plantarum VITA-L03 (KCTC 12177BP).

According to the present invention, the antibacterial activity against Streptococcus mutans , which is a causative organ of dental caries, can be inhibited by using biofilm extracts and lactic acid bacteria selected from the viewpoint of anti-wrinkle efficacy and inhibiting the biofilm which plays an important role in dental caries. It is possible to exert the inhibitory effect on glucan formation and to exert effective effects on the already formed biofilm removal.

In particular, the extracts of bark bark extract and lactic acid bacteria used in the present invention exhibit superior antimicrobial efficacy and, when used together, exhibit a remarkable synergistic effect in antibacterial efficacy and can be effectively used for prevention and treatment of dental caries.

In addition, the present invention can be applied to various fields by securing safety without causing toxicity and side effect problems by using extracts of bark bark extract and lactic acid bacteria that have no problem in safety.

FIG. 1 shows the results of measuring biofilm inhibition ability in vitro of herbal materials selected in the examples of the present invention.
FIG. 2 shows the result of measuring antimicrobial activity of bark bark extract according to an embodiment of the present invention.
Figure 3 shows the results of measuring biofilm inhibition ability of the selected lactic acid bacteria in vitro according to the embodiment of the present invention.
4 shows the results of measuring glucosyltransferase inhibitory activity of a selected lactic acid bacterium according to an embodiment of the present invention.
Figure 5 shows a phylogenetic analysis of the final selected strain ( L. plantarum VITA-L03 (KCTC 12177BP)) according to an embodiment of the present invention.
6 shows the results of the antimicrobial activity measurement of a mixture of the extract of bark bark and Lactobacillus plantarum VITA-L03 (KCTC 12177BP) according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

According to one embodiment of the present invention, there is provided a method for inhibiting glucocorticoid activity, which is effective in inhibiting biofilm, antibacterial activity against Streptococcus mutans , and glucan formation inhibitory activity, .

The bark bark extract used in the present invention exhibits excellent antibacterial activity against inhibition of biofilm formation in vitro, antimicrobial activity against Streptococcus mutans and inhibition of glucan production, LD ₅₀ Value can be secured and safety can be ensured.

These bark bark extracts exhibit superior antimicrobial efficacy when used alone, and can exhibit a remarkable synergistic effect on the anticancer efficacy when used in combination with the following lactic acid bacteria.

In the present invention, "extract of bark bark" is meant to encompass fractions obtained by addition of one or more "fractionated organic solvent" to bark bark extract of the duckwood bark extract prepared by using an extraction solvent .

The solvent used for the formation of the bark extract is not particularly limited and various solvents generally used for extracting natural products can be used. For example, the extract of bark bark can be formed by one or more methods selected from the group consisting of hot water extraction, alcohol extraction and alcohol extraction.

In addition, the fractionated organic solvent used for forming the fraction of the extract of the bark extract is not particularly limited, and any conventional organic solvent can be used without limitation.

In one form, the extract of bark bark comprises the steps of adding distilled water; Hydrothermal extraction at a temperature of 60 to 130 캜, preferably about 100 캜, for 30 to 240 minutes, preferably about 180 minutes; And filtering.

The concentration of the extract of bark bark may be 1 to 100% by weight, preferably 5 to 50% by weight, more preferably about 20% by weight. When the concentration of the extract of bark bark is less than 1% by weight, there is a fear that the sufficient anti-wrinkle efficacy may not be exhibited.

Meanwhile, according to another embodiment of the present invention, lactic acid bacteria exhibiting the biofilm inhibiting ability, antibacterial activity against Streptococcus mutans and glucuronylation-inhibiting ability are used, .

In one embodiment, the lactic acid bacterium used in the present invention is characterized by being L. plantarum VITA-L03 (KCTC 12177BP).

The present inventors selected L. plantarum VITA-L03 (KCTC 12177BP) lactic acid bacteria having excellent antimicrobial activity from kimchi, healthy Korean adults and infant feces and saliva samples collected from all over Japan. L. plantarum VITA-L03 (KCTC 12177BP) has superior antimicrobial activity against inhibiting biofilm formation in vitro, Streptococcus mutans and glucan production inhibition, appear.

In addition, when lactic acid bacteria of L. plantarum VITA-L03 (KCTC 12177BP) are used alone, they exhibit excellent antimicrobial activity and also exhibit a remarkable synergistic effect on the anticancer efficacy when they are used in combination with the aforementioned bark extract .

In addition, since these lactic acid bacteria are 'generally regarded as safe' microorganisms, they can exhibit excellent antimicrobial efficacy while securing safety.

In the composition of the present invention, the lactic acid bacterium can be used as a culture supernatant or as a cell itself.

According to another embodiment of the present invention, the combination of the above-mentioned extract of the almond bark extract and the lactic acid bacterium exhibits a synergistic effect which is effective in dental caries.

In one form, the weight ratio of the extract of bark bark to the lactic acid bacteria may range from 9.5: 0.5 to 5: 5, preferably from 9: 1 to 6: 4. When the weight ratio of the extract of bark bark and lactic acid bacterium is out of the above range, there is no significant difference in the antibacterial activity between the herbal medicine and the lactic acid bacterium alone.

According to the above example, biofilm inhibiting ability, antimicrobial activity against Streptococcus mutans and glucan formation inhibitory ability can be demonstrated by using the selected bark extract and lactic acid bacterium selected from the viewpoint of antibacterial activity, It can exhibit effective efficacy and can be effectively used for prevention and treatment of dental caries.

In addition, when a combination of the extract of bark bark extract and lactic acid bacterium is used in combination, it exhibits not only a remarkable synergistic effect on the anti-wrinkle effect, but also does not show any toxicity or side effects, and safety can be secured.

In the present invention, it is possible to broadly apply the extracts of bark bark extract and lactic acid bacterium, which exhibit such an effect, alone or in combination, to a pharmaceutical composition for anti-oozing pharmacology, anti-wormy quasi-drug or anti-wasting food composition.

One aspect of the present invention relates to a pharmaceutical composition comprising a duck bark extract, a lactic acid bacterium or a duck bark extract and a lactic acid bacterium.

In one embodiment, the anti-aging pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier, excipient or diluent.

The pharmaceutically acceptable carrier, excipient or diluent which can be used in the present invention is not particularly limited so long as the effect of the present invention is not impaired, and examples thereof include fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, Flavoring agents, fragrances, preservatives, and the like.

Representative examples of pharmaceutically acceptable carriers, excipients or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, gelatin, glycerin, acacia rubber, alginate, calcium phosphate, calcium carbonate, calcium Methylcellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, propylene glycol, polyethylene glycol, vegetable oil, injectable Ester, witepsol, macrogol, tween 61, cacao paper, and laurie paper.

The pharmacological pharmaceutical composition of the present invention may be in a form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and injections.

The preparation method of the pharmaceutical composition may be carried out according to a conventional method known in the art, and is not particularly limited.

The pharmaceutical composition of the present invention may be administered orally or parenterally. The dosage may be appropriately selected depending on the age, sex, weight, condition, degree of disease, drug form, route of administration, and duration of the subject to be administered , Generally about 5 to 500 mg / kg, preferably about 100 to 250 mg / kg, may be administered one to three times a day.

It will be apparent to those of ordinary skill in the art that the formulation, dosage, route of administration, composition, etc. of the pharmaceutical composition of the present invention can be appropriately selected from conventional techniques known in the art.

The anti-aging pharmaceutical composition of the present invention can be used for prevention and treatment of dental caries.

In one embodiment, the anti-aging pharmaceutical composition of the present invention is a pharmaceutical composition comprising, as an active ingredient, a pharmacologically active ingredient in addition to an extract of Oriental bark extract, a lactic acid bacterium or a duck bark extract and a lactic acid bacterium, Can be mixed and used.

Another aspect of the present invention relates to anti-widespread quasi-drugs including extracts of bark bark, lactic acid bacteria or bark extract of bark and lactic acid bacteria.

In one embodiment, the anti-widespread quasi-drug of the present invention may be an oral product.

Examples of oral products may include, but are not limited to, toothpaste, mouthwash, toothpaste, softener and chewing gum.

It should be clear to those skilled in the art that the formulation method, dosage, method of use, constituents, etc. of the anti-plague quasi-drug of the present invention can be appropriately selected from conventional techniques known in the art.

In one embodiment, the anti-inflammatory quasi-drug of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent, the details of which are as described above for the pharmaceutical composition.

The anti-plague quasi-drug of the present invention can be used for prevention or treatment of dental caries.

Another aspect of the present invention relates to an anti-wrinkle food composition comprising a bark extract, a lactic acid bacterium or a duck bark extract and a lactic acid bacterium.

In one embodiment, the anti-inflammatory food composition of the present invention further comprises a pharmaceutically acceptable food-aid additive.

Food-acceptable food supplementary additives that may be used in the present invention include sugars such as glucose, fructose, maltose, sucrose, dextrin, cyclodextrins, natural carbohydrates such as sugar alcohols such as xylitol, sorbitol, erythritol, Natural flavors such as martin and stevia extract, synthetic flavors such as saccharin and aspartame, colorants, pectic acid or its salts, alginic acid or its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin , Alcohols, carbonating agents, and the like.

The anti-spoilage food composition of the present invention can be applied to various food products such as flour products such as bread, cake, cookies, noodles, beverages, various dairy products, chewing gum, candy, jelly, powder, granule, Can be manufactured in various forms.

The content of the extract of Lilac bark extract, Lactobacillus acidus or Aspergillus bark extract and Lactobacillus can be suitably selected in consideration of the form, flavor, taste and the like of the food, for example, By weight to 50% by weight.

It is apparent to those of ordinary skill in the art that the form, composition and manufacturing method of the anti-wrinkle food composition of the present invention can be suitably selected from conventional techniques known in the art.

As described above, the anti-wasting composition according to the present invention exhibits biofilm inhibiting activity, antibacterial activity against Streptococcus mutans , and efficacy in inhibiting glucan formation, and can be applied to various pharmaceutical compositions, quasi-drugs, food compositions and the like.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

[ Example ]

1. Selection of herbal materials Biofilm  formation Inhibition  Measure

(1) Selection method

1) Collection of herbal medicine materials

Approximately 385 kinds of herbal medicine resources were collected based on the prescription described in the upper limit, the gold germination,

2) Production of pharmaceutical extract

385 kinds of herbal medicines were prepared by hot water extraction and methanol reflux cooling extraction. Lyophilized samples were prepared using this extract and used as a sample for selection of medicinal herbs. The lyophilized sample was suspended in DMSO at an appropriate concentration and stored at -20 ° C.

3) Method for measuring inhibition of biofilm formation

I) Adherence Buffer (KPO ₄ ) containing artificial saliva (1% Hog Gastric Mucin) in a 96-well microplate ₂₀₀ mM each of 10 mM / L, KCl 50 mM / L, CaCl ₂ 1 mM / L, and MgCl ₂ 0.1 mM / L, pH 7.0) was added thereto, followed by stirring at room temperature for 4 hours. After the reaction was completed, all the added solution was removed and dried.

Ⅱ) Streptococcus mutans (S. mutans) is BHI broth (Brain Heart Infusion broth) (BHI , Difco, USA KCTC3065 the strains received pre-sale to the Gram-positive facultative anaerobic bacteria from the Korea Research Institute of Bioscience and Biotechnology Microbial Resource Center) used in this experiment And agar medium for the experiment.

Iii) Add 180 B of BHI broth containing 3% (w / v) sucrose to a dried 96-well microplate and incubate with S. mutans Inoculation solution ( S. mutans was cultured in 3 ml of BHI broth at 37 ° C for 12 hours, centrifuged to remove supernatant, washed twice with PBS buffer, and then added with Adherence Buffer to obtain 2 × 10 ⁸ cfu / ml Concentration) was inoculated. The lyophilized herbal medicine sample was suspended in sterilized distilled water at a concentration of 25 mg / ml, and centrifuged to use 50 μl of the supernatant. For the control group, the same amount of sterilized distilled water was added instead of the treated material.

Iv) After incubation at 37 占 폚 for 24 hours, 200 占 퐇 of 0.2% (w / v) Crystal Violet Solution (dissolved in 2% EtOH) was added to each well and stained for 10 minutes Then, it was washed with tap water three times and dried.

V) 250 μl of a 0.5% (w / v) SDS solution (dissolved in 50% EtOH) was added and 200 μl of the solution was transferred to a new microplate for more than 1 hour. Absorbance at 595 nm Respectively. The biofilm formation inhibition rate can be calculated as follows.

(2) In vitro biofilm formation inhibition test results

The in vitro biofilm formation inhibiting ability of the selected herbal materials was measured, and the results are shown in Table 1 and FIG. As can be seen from Table 1 and FIG. 1, inhibition activity of the bark of Oriental pine bark was the highest at 89% in the same concentration.

Inhibition of biofilm formation in vitro Control group Schisandra Duck Duck bark 5 mg / ml 0% 46% 19% 89%

2. Antimicrobial activity analysis of herbal materials

(1) Antimicrobial activity analysis

1) In order to confirm the growth inhibition activity of the medicinal herb, Streptococcus mutans strains were inoculated into BHI broth and subcultured. BHI broth was added to each dilution sample diluted with the appropriate amount of hot water extract of bark sarkin bark 1: 1. The control group was mixed with sterilized distilled water instead of hot water extract of bark sarkin.

2) Two passages of S. mutans were inoculated 2% in the above mixed culture medium, stained with 0H and 12H on BHI solid medium, and incubated for 36 hours in anaerobic culture.

(2) Antimicrobial activity measurement result

Fig. 2 shows the results of the antimicrobial activity test of the bark of Duck. As shown in Fig. 2, only about 20% of the strains survived in the 5% duck bark and only less than 0.01% of the strains survived in the bark of 20% or more. From this, it can be confirmed that about 90% of the antimicrobial activity is exhibited by the treatment with 10% extract of Duck bark extract.

3. Chinese medicine material Glucan ( Glucan ) production Low performance  Measure

(1) Measurement method

1) S. mutans was inoculated into BHI broth and cultured in a 37 ° C incubator for 24 hours. Then, the supernatant was taken by centrifugation at 4300 rpm for 30 minutes at 4 ° C. The supernatant was filtered through a 0.2 μm syringe filter, and then a crude enzyme solution was prepared using an Amicon Ultra Centrifugal Filter (MWCO 30 kDa, Cat. No. UFC903008) manufactured by Millipore.

2) After suspending the hot water extract of herbal medicines in water, hexane (Hexane), ethyl acetate (Ethanol) and butanol were fractionated in a solvent with a low polarity and a solvent with a high polarity consecutively in consideration of the polarity of the solvent Each extract was obtained. The obtained solvent fractions were concentrated using a rotary evaporator under reduced pressure, dissolved in DMSO (Dimethyl Sulfoxide) at a concentration of 25 mg / ml, stored at -20 ° C, and diluted in the experiment.

3) The activity of glucosyltransferase (GTase) was measured by using a spectrophotometer with insoluble glucan produced from sucrose as a substrate. Substrate in a glass test tube, a solution (0.0625M Potassium Phosphate Buffer (pH 6.5 ) 1L of sucrose 12.5g, NaN ₃ containing 0.25g) 800㎕, crude enzyme solution, and medicinal plants 20㎕ 180㎕ solvent fraction is added to the final volume was adjusted to 1㎖ . In the control group, 180 占 퐇 of sterilized distilled water was added instead of the treatment substance. The test tube was allowed to react at 30 ° C for 30 hours in a 37 ° C incubator. After the reaction, the supernatant was discarded by centrifugation, 4 ml of distilled water was added, and ultrasonication was performed for 10 minutes to disperse the formed glucan. Immediately after dispersion, absorbance at 550 nm was measured with a spectrophotometer, and the inhibition rate of each sample against GTase was calculated as follows.

(2) Measurement results

The measurement results of the GTase inhibitory activity of the solvent fraction of the bark bark are shown in Table 2 below. As shown in Table 2, the GTase inhibitory activity of the bark fraction of Oriole bark was found to be about 35% inhibitory in the ethyl acetate layer of bark bark.

GTase inhibitory activity of solvent fraction of bark bark Ethyl acetate layer of bark Butanol layer of the bark of the duck GTase inhibitory activity 35.7 ± 0.19% 23.9 ± 0.16%

* Concentration of herbal medicines: 0.5 mg / ml

4. Stability test of bark of duck in vivo )

(1) Acute toxicity test of candidate herbal medicine materials using mouse

1) Experimental method

I) Single-dose toxicity studies were conducted in order to provide data on the safety of selected herbal medicinal herbs. Single dose toxicity tests were conducted using male and female ICR mice according to the "Nonclinical Test Control Standards" (Oct. 25, 2005) of the Toxicological Test Methods for Drug and Other Notification Notification No. 2005-60 of the Food and Drug Administration.

Ⅱ) The temperature of 23 ± 3 ℃, the relative humidity of 50 ± 10%, the number of times of ventilation 12 ~ 16 times per hour, lighting 12 hours of light intensity (7:00 on, 19:00 off) After quarantining and purifying under a 300 Lx rearing environment, the appearance was visually inspected and healthy animals were selected for the experiment. Male and female mice were set up in groups of 5 per cage and individually housed five cages per cage. Feeds and water were fed ad libitum and feeds were fed with animal feed (PMI nutrition, USA).

Ⅲ) The dose of hot water extract of bark bark was set at three doses of 1250, 2500 and 5000 mg / kg (mouse, BW) with an air ratio of 0.5, based on 5000 mg / kg. Were diluted to be used for the test. The route of administration was oral administration. The animals were fasted overnight and then orally administered after fixation of the body using a disposable sonde and a suture tube for oral administration.

Ⅳ) Clinical symptoms were observed every hour for 6 hours immediately after administration, and then general condition and mortality such as change of general condition, mobility, appearance, autonomic nerve were observed once a day for 14 days. Body weights were measured at the time of arrival, before separation of the group and at 1, 3, 7, and 14 days after the test substance administration, respectively. The animals were fasted for 18 hours the night before sacrifice, and anesthetized by intraperitoneal injection of Zoletil 50 (Virbac SA, France) and Xylazine Hydrochloride (Bayer Korea Co., Ltd., Kyonggi-do) Were observed.

2) Experimental results

I) The mortality and LD ₅₀ for the hot-water extract of bark sarka bark were checked, and the results are shown in Table 3 below. As a result of oral administration of oral extracts of bark bark extract at oral doses of 0, 1250, 2500 and 5000 mg / kg for 14 days, deaths were not observed in all male and female ICR mice. Therefore, LD ₅₀ values were not calculated for oral extracts of bark bark extract even after oral administration to the respective limit dose (5000 mg / kg / day, 2000 mg / kg / day).

Single-dose toxicity test results of bark bark extract Group Capacity (mg / kg) gender Number of animals Lethality (%) Control group 0 cock 5 0 female 5 0

Duck bark 1250 cock 5 0 female 5 0 2500 cock 5 0 female 5 0 5000 cock 5 0 female 5 0

Ii) The test animals were observed for 14 days after oral administration of the hot water extract of bark sarkin bark, and the results are shown in Table 4 below. As a result of observing the experimental animals for 14 days after oral administration of hot water extract of bark bark of 0, 1250, 2500, and 5000 mg / kg respectively, all the male and female ICR mice used for the experiment showed that walking disorders, No abnormal findings of clinical symptoms related to the administration of the test substance such as diarrhea, edema, shortness of breath, grooming, jumping, blushing, lethargy, vomiting, burning, paralysis,

Clinical symptoms of male and female ICR mice treated with hot water extract Group Capacity (mg / kg) gender Number of animals Number of abnormal findings Control group 0 cock 5 0 female 5 0

Duck bark 1250 cock 5 0 female 5 0 2500 cock 5 0 female 5 0 5000 cock 5 0 female 5 0

Iii) Weight gain of experimental animals was observed for 1, 3, 7, and 14 days after oral administration of hot water extract of bark sarkin bark, respectively, and the results are shown in Tables 5 and 6 below. As shown in Tables 5 and 6, the body weights of all experimental animals were increased by about 25% or more in males and by about 10% or more in females compared to the initial body weight. There was no statistically significant difference in body weight gain in the high concentration group of the bark (5000 ㎎ / ㎏). In addition, no significant weight loss or increase by test substance was observed before (1 day) and at 1, 3, 7, and 14 days after administration.

Mean weight of male ICR mice treated with duckwood hydrothermal extract Group
(cock) Volume
(Mg / kg) Weight (g) 0 days 1 day 3 days 7 days 14 days Control group 0 Average 25.136 28.210 29.070 31.264 33.030 Standard Deviation 1.984 2.271 2.008 2.419 0.466

Duck
fell 1250 Average 25.090 26.964 27.730 30.376 32.214 Standard Deviation 1.710 1.761 1.859 1.849 1.627 2500 Average 25.006 27.390 28.062 30.012 32.304 Standard Deviation 1.348 0.625 0.565 0.647 0.644 5000 Average 24.920 26.352 26.950 29.280 31.132 Standard Deviation 1.128 1.605 1.610 1.628 1.667

Mean weight of female ICR mice treated with duckwood hydrothermal extract Group
(female) Volume
(Mg / kg) Weight (g) 0 days 1 day 3 days 7 days 14 days Control group 0
Average 22.008 21.308 23.382 23.458 24.932 Standard Deviation 1.299 0.952 1.104 0.923 1.025

Duck
fell 1250 Average 21.618 22.358 22.152 23.412 25.240 Standard Deviation 1.162 1.438 1.453 1.483 1.783 2500 Average 21.616 22.778 22.878 23.430 25.250 Standard Deviation 0.875 0.889 0.891 1.172 1.433 5000 Average 21.330 22.026 22.046 23.882 25.354 Standard Deviation 0.668 1.918 1.937 1.536 2.414

Ⅳ) After the end of the experiment, all the experimental animals were autopsied and the gross appearance of the major internal organs was observed. As a result, it was found that all of the male and female ICR mice had a major internal defect such as heart, lung, thymus, liver, kidney, adrenal gland, No apparent abnormal lesions were found in the organ.

V) The above result suggests that the LD ₅₀ of hot water extract of Aspergillus oryzae is more than 5000 mg / kg in male and female ICR mice.

5. Collection of lactic acid bacteria and Biofilm  formation Inhibition  Selection by measurement

(1) Experimental method

1) Collection of sample

Eight kinds of feces and 24 saliva samples were obtained from 11 kinds of Kimchi and healthy Korean adults and infants from all over Japan.

2) Isolation of lactic acid bacteria

In order to isolate lactic acid bacteria having acid resistance and bile acid resistance, 4 ml of sterilized PBS buffer was added to 1 ml of each homogenized sample, and then 10 ml of pepsin 0.3 (0.1 ml) was added to MRS broth adjusted to pH 2.5 with artificial gastric juice %) And artificial bile juice (MRS broth supplemented with 0.3% bile) were sequentially exposed for 30 minutes. After exposure, the cells were washed once with PBS and decentralized using sterile saline. In order to isolate a variety of lactic acid bacteria, a dilution was applied to agar medium such as MRS, PES, and m-Enterococcus. A single colony formed after 36 hours of culture was determined to be an acid-resistant and bile-resistant bacterium, and the production of acid was confirmed in BCP agar medium to isolate it as a lactic acid Bacteria-like Strain.

3) Method for measuring inhibition of biofilm formation in vitro

I) Adherence Buffer (KPO _{4 10} mM / L, KCl 50 mM / L, CaCl ₂ 1 mM / L, MgCl ₂ 0.1 mM / L, pH 7.0) containing artificial saliva (1% Hog Gastric Mucin) ) Was added thereto in an amount of 200 쨉 l each, and the mixture was reacted at room temperature for 4 hours with stirring. After the reaction was completed, all the added solution was removed and dried.

Ii) Add 180 B of BHI Broth containing 3% (w / v) sucrose to a dried 96-well microplate and incubate with S. mutans Inoculation solution ( S. mutans was cultured in 3 ml of BHI broth at 37 ° C for 12 hours, centrifuged to remove supernatant, washed twice with PBS buffer, and then added with Adherence Buffer to obtain 2 × 10 ⁸ cfu / ml Concentration) was inoculated. Probiotic lactic acid bacteria were cultured by inoculating 2% on MRS broth, centrifuged to remove the pellet, and the culture supernatant was sterilized with a 0.2 쨉 syringe filter and concentrated by Speed Vac Was added in 50 mu l each. As a control, sterile distilled water was used instead of lactic acid bacteria culture supernatant.

Iii) After the incubation at 37 ° C for 24 hours, 200 μl of a 0.2% (w / v) crystal violet solution (dissolved in 2% EtOH) was added to each well, stained for 10 minutes, Washed three times and then dried.

Iv) 250 μl of a 0.5% (w / v) SDS solution (dissolved in 50% EtOH) was added and the sample was destained for 1 hour or more. Then, 200 μl was transferred to a new microplate and the absorbance at 595 nm Respectively. The biofilm formation inhibition rate can be calculated as follows.

(2) Experimental results

Fig. 3 shows the results of measuring the ability of the lactic acid bacteria-like strain to inhibit biofilm formation in vitro. As shown in FIG. 3, some strains showed excellent biofilm inhibiting ability, and VITA-L03, Feh3-8, Feh3-10, Feh3-14, SUP-8, SCE-11, SOE-1, SR-16-2, SR-10-3 and SR-23-1 were selected.

6. Analysis of antibacterial activity of antibacterial activity of lactic acid bacteria

(1) Experimental method

For the measurement of antibacterial activity, experiments were conducted according to the Paper Disc Method as described below.

After a paper disc was placed on a BHI agar medium coated with S. mutans , the culture broth of lactic acid bacteria (2% in MRS broth, inoculated with the cultured Lactobacillus cultures was centrifuged and sterilized using a 0.2 μm syringe filter, In this experiment, 50 μl of the solution was concentrated and diluted to an appropriate volume), and the solution was incubated for 12 hours.

 2) Experimental results

The results of the antimicrobial activity measurement of the selected lactic acid bacteria against Streptococcus mutans are shown in Table 7 below. As shown in Table 7, the antibacterial activity was confirmed in most of the selected strains.

Antimicrobial activity of each strain measured by paper disk method no. Name of sample Separation source Growth inhibition (mm) One VITA-L03 Kimchi 15 2 Feh3-8 Human feces 11 3 Feh3-10 Human feces 10 4 Feh3-14 Human feces 10 5 SUP-8 Human saliva 12 6 SCE-11 Human saliva 11 7 SOE-1 Human saliva 13 8 SR-16-2 Human saliva 13 9 SR-10-3 Human saliva 15 10 SR-23-1 Human saliva 14 11 L. brevis ^One Commercially available dairy products - 12 L. casei ^One Commercially available dairy products 9.5 13 L. acidophilus ^One Commercially available dairy products 10

¹ : positive control group

7. Lactic acid bacteria Glucan  production Low performance  analysis

(1) Experimental method

1) S. mutans was inoculated into BHI broth and cultured in a 37 ° C incubator for 24 hours, followed by centrifugation at 4300 rpm for 30 minutes at 4 ° C to take a supernatant. The supernatant was filtered through a 0.2 μm syringe filter, and then a crude enzyme solution was prepared using an Amicon Ultra Centrifugal Filter (MWCO 30 kDa, Cat. No. UFC903008) manufactured by Millipore.

2) The activity of glucosyltransferase was measured by using a spectrophotometer with insoluble glucan produced using sucrose as a substrate. Add 800 μl of the substrate solution (12.5 g of sucrose in 1 L of 0.0625M Potassium Phosphate Buffer (pH 6.5), 0.25 g of NaN ₃ ), 20 μl of crude enzyme solution and 180 μl of concentrated lactic acid bacteria culture supernatant to a final volume of 1 ml Respectively. In the control group, 180 占 퐇 of distilled water was added instead of the treatment substance. The test tube was allowed to react at 30 ° C for 30 hours in a 37 ° C incubator. After the reaction, the supernatant was discarded by centrifugation, 4 ml of distilled water was added, and ultrasonication was performed for 10 minutes to disperse the formed glucan. Immediately after dispersion, absorbance at 550 nm was measured with a spectrophotometer, and the inhibition rate of each sample against GTase was calculated as follows.

(2) Experimental results

The results of the GTase inhibition assay for the selected lactic acid bacteria are shown in FIG. As shown in Fig. 4, the activity of SOE-1 and VITA-L03 was the highest. In the case of some probiotic lactic acid bacteria isolates with excellent biofilm formation inhibition ability, it is considered that the low level of GTase inhibition is due to the inhibition of the biofilm formation due to the antimicrobial activity.

7. Selection of Lactic Acid Bacteria Systematic  analysis

(1) Systematic analysis method

: The phylogenetic analysis of the selected lactic acid bacteria was carried out using the 16S rDNA sequence as described below.

1) The cells were recovered and suspended in 100 μl of STES buffer (0.4 M NaCl, 0.2 M Tris-HCl (pH 7.6), 0.01 M EDTA, 1% SDS).

2) Glass beads were added to the suspension, and the cells were vigorously agitated for 5 minutes using a micro-tube mixer MT-360 (TOMY) in order to disrupt the cells.

3) After cell disruption, phenol extraction was carried out, and 5 ㎕ of RNase A was added to the supernatant, followed by reaction at 37 캜 for 1 hour.

4) After the reaction, phenol extraction was carried out again, and ethanol precipitation was performed to recover the DNA.

5) 16S rDNA was amplified by PCR method and the primers used were as follows

Forward primer 5'-GA GTT TGA TCC TGG CTC AG-3 '( E. coli numbering system, positions 9-27)

Reverse primer 5'-GGT TAC CTT GTT ACG ACT T-3 '( E. coli numbering system, positions 1,492-1,510)

6) Conditions for PCR reaction were as follows.

: A total of 30 cycles. Each cycle was performed at 94 ° C for 60 seconds for denaturation, 60 seconds at 50 ° C for annealing, and 72 seconds at 90 ° C for extension.

7) The 16S rDNA sequences of the isolates were sorted using the CLUSTAL W Software Program with the nucleotide sequences of the comparative strains representing the relevant Genus.

8) The Evolutionary Distance Materices were constructed based on the Kimura Two Parameter method and the phylogenetic tree was created using the Neighbor - Joining Method Tool provided by PHYLIP Package version 3.572.

(2) Experimental results

The results of systematic analysis using 16S rDNA are shown in Fig. From FIG. 5, it can be seen that the finally selected strain is in close relation with Lactobacillus plantarum .

8. Synergistic effect of antimicrobial efficacy of a mixture of extracts of bark bark and lactic acid bacteria

(1) Experimental method

1) Artificial biofilm assay (Alviano et al., 2008) was conducted to investigate the synergistic effect of extracts of bark bark extract and lactic acid bacteria composition on the antimicrobial activity. The extracts of 20% duck bark showing more than 99.9% antibacterial activity of antibacterial activity and VITA-L03, which had the highest antimicrobial activity, were mixed at an appropriate ratio.

2) 0.22 μm membrane disks (Millipore, 13 mm diameter) were placed on a BHI agar medium, and 20 μl of the S. mutans culture was spotted and incubated at 37 ° C. for 48 hours to form a biofilm.

3) A sample prepared by mixing duck bite extract and lactic acid supernatant in an appropriate ratio was prepared, and the sample was diluted with physiological saline at a proper ratio and 5 ml each was dispensed into a 50 ml tube. As a control, physiological saline was used.

4) The membrane disk with the biofilm formed was detached with sterilized tweezers and placed in the 50 ml tube, followed by shaking culture at 37 ° C for 1 hour.

5) Membrane discs were washed with distilled water to remove non-adherent bacteria and residues, 10 ml of sterile physiological saline was added, and the biofilm was dropped from the membrane disk by stirring with a vortex mixer.

6) The bacterial suspension was subjected to decentralization, and 100 μl was plated on a BHI agar medium and cultured at 37 ° C for 48 hours to measure viable cell count.

(2) Experimental results

Fig. 6 shows the results of the antimicrobial activity measurement of the extract of Duck Bark and the mixed composition of VITA-L03. As shown in FIG. 6, the artificial biofilm assay showed that 9: 1, 8: 2, 7: 3, and 6: 4 of the extracts of Duck Bark extract and VITA-L03 culture supernatant, , The biofilm removal rate was significantly increased by about 3 to 4 times, and it was confirmed that there was a synergistic effect of the antibacterial activity in the mixed composition. The survival rate of S. mutans was 7.0% and 7.7%, respectively, when the mixture was treated with 9: 1 and 8: 2 mixed samples. However, as the ratio of VITA-L03 culture supernatant was relatively increased, the synergistic effect of the antibacterial activity decreased, and when the sample was mixed at the ratio of 5: 5, the biofilm removal rate was significantly different from that of VITA-L03 alone There was no.

9. Manufacturing example

In the following preparations, the following three samples were used as "active ingredients"

I) extracts of bark bark prepared in 1. to 3. above;

Ii) VITA-L03 culture supernatant or cells prepared in 5 and 6 above; And

Iii) An 8: 2 mixed sample of bark bark extract and VITA-L03 culture supernatant or bacterium prepared in 8 above.

(1) Injection

Active ingredient: 100 mg

Sodium metabisulfite: 3.0 mg

Methylparaben: 0.8 mg

Propyl paraben 0.1 mg

Sterile sterilized distilled water for injection:

The above ingredients were mixed and made into a final volume of 2 ml by a conventional method, filled in an ampoule and sterilized to prepare an injection.

(2) Purification

Active ingredient: 200 mg

Potato starch: 100 mg

Lactose: 100 mg

Colloidal silicic acid: 16 mg

Magnesium stearate:

The ingredients were mixed and tableted according to a conventional tablet preparation method to prepare tablets.

(3) Capsules

Active ingredient: 100 mg

Lactose: 50 mg

Starch: 50 mg

Talc: 2 mg

Magnesium stearate:

The above components were mixed according to a conventional capsule manufacturing method and filled in gelatin capsules to prepare capsules.

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(4) pillars

Active ingredient: 120 mg

Corn starch: 100 mg

Sterilized distilled water: suitable amount

The above ingredients were mixed and pelletized to spheres of appropriate size according to conventional pellet manufacturing methods to produce pellets.

(5) Quasi-drugs for oral care

1) Toothpaste

Active Ingredient: 20 wt%

Glycerin: 6 wt%

Sodium saccharin: 0.1 wt%

Silica: 7 wt%

Precipitated calcium carbonate: 39 wt%

Sodium lauryl sulfate: 2 wt%

Menthol: 0.4 wt%

Peppermint: 0.1 wt%

Purified water: Remaining amount

The above ingredients were mixed and a toothpaste was prepared according to a conventional toothpaste manufacturing method.

2) Other

The above active ingredients (the bark bark extract prepared in the above 1. to 3., the VITA-L03 culture supernatant or the bacterium prepared in the above 5. and 6. and the bark bark extract prepared in the above 8. and the VITA- L03 culture supernatant or an 8: 2 mixed sample of cells) were added to oral care products such as various oral cleansers, mouthwashes, ointments or chewing gums to prepare quasi-drugs for oral care.

(6) Food

1) Manufacture of flour food

The above active ingredients (the bark bark extract prepared in the above 1. to 3., the VITA-L03 culture supernatant or the bacterium prepared in the above 5. and 6. and the bark bark extract prepared in the above 8. and the VITA- L03 culture supernatant or 8: 2 mixed sample of cells) Foods such as bread, cake, cookies and noodles were prepared using wheat flour added in an appropriate amount.

2) Manufacture of beverages

The above-mentioned active ingredients (the above-mentioned active ingredients (the ducklings prepared in the above 1. to 3. above) were added to beverage ingredients such as oligosaccharide (2%), liquid fructose (0.5%), sugar (2%), Bark extracts: VITA-L03 culture supernatant or cells prepared in the above 5. and 6. and the almond bark extract prepared in 8. above and 8: 2 mixed sample of VITA-L03 culture supernatant or microbial cells) And sterilized to prepare a beverage.

Korea Biotechnology Research Institute KCTC12177BP 20120327

Claims (23)

L. plantarum VITA-L03 (KCTC 12177BP), and exhibits antimicrobial activity against Streptococcus mutans, inhibition of biofilm formation, and inhibition of glucosyltransferase (GTase) activity.
Which is characterized by exhibiting antimicrobial activity against Streptococcus mutans, inhibition of biofilm formation inhibition and inhibition of glucosyltransferase (GTase) activity, including the extract of Duck Bark and L. plantarum VITA-L03 (KCTC 12177BP) A pharmaceutical composition.
3. The method of claim 2,
Wherein the weight ratio of the extract of bark bark to L. plantarum VITA-L03 (KCTC 12177BP) is in the range of 9.5: 0.5 to 5: 5
A pharmaceutical composition for pharmacopoeia.
3. The method according to claim 1 or 2,
Characterized in that it further comprises a pharmaceutically acceptable carrier, excipient or diluent
A pharmaceutical composition for pharmacopoeia.
3. The method according to claim 1 or 2,
Characterized in that it is used for the treatment or prevention of dental caries
A pharmaceutical composition for pharmacopoeia.
L. plantarum VITA-L03 (KCTC 12177BP), exhibiting antibacterial activity against Streptococcus mutans, inhibition of biofilm formation, and inhibition of glucosyltransferase (GTase) activity.
Which is characterized by exhibiting antimicrobial activity against Streptococcus mutans, inhibition of biofilm formation inhibition and inhibition of glucosyltransferase (GTase) activity, including the extract of Duck Bark and L. plantarum VITA-L03 (KCTC 12177BP) Quasi-drug composition.
8. The method of claim 7,
Wherein the weight ratio of the extract of bark bark to L. plantarum VITA-L03 (KCTC 12177BP) is in the range of 9.5: 0.5 to 5: 5
Antiepidemic quailty composition.
8. The method according to claim 6 or 7,
Wherein said anti-widespread quasi-drug is a form selected from the group consisting of toothpaste, oral cleanser, toothpaste, softening solution and chewing gum
Antiepidemic quailty composition.
8. The method according to claim 6 or 7,
Characterized in that it is used for the treatment or prevention of dental caries
Antiepidemic quailty composition.
L. plantarum VITA-L03 (KCTC 12177BP), exhibiting antibacterial activity against Streptococcus mutans, inhibition of biofilm formation, and inhibitory activity of glucosyltransferase (GTase).
Which is characterized by exhibiting antimicrobial activity against Streptococcus mutans, inhibition of biofilm formation inhibition and inhibition of glucosyltransferase (GTase) activity, including the extract of Duck Bark and L. plantarum VITA-L03 (KCTC 12177BP) Food composition.
13. The method of claim 12,
Wherein the weight ratio of the extract of bark bark to L. plantarum VITA-L03 (KCTC 12177BP) is in the range of 9.5: 0.5 to 5: 5
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13. The method according to claim 11 or 12,
Characterized in that it further comprises a food-acceptable food-aid additive
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