KR20150114521A - Composition for oral cavity - Google Patents

Abstract

Provision of a periodontal disease prevention agent in a new point of merit, such as the fact that the risk of emergence of resistant bacteria is low compared to existing antimicrobial agents. A composition for oral use which contains a mizuna or cold water extract and has a biofilm formation inhibiting action.

Description

COMPOSITION FOR ORAL CAVITY [0002]

The present invention relates to an oral composition having an excellent periodontal disease-improving effect on oral biofilm which is a cause of oral disease.

Periodontal disease is a general term for the group of diseases seen in periodontal tissue. In a narrow sense, it is a disease equivalent to gingivitis, periodontitis, and trauma. Periodontal disease is an oral infection caused by dental plaque. There are more than 700 kinds of bacteria in the human oral cavity. In healthy oral cavity, early attachment bacteria such as Streptococcus or Actinomyces are attached to the tooth surface. Among them, Actinomyces naeslundii is called a hemorrhagic gingivitis causative organism, and the early attachment of actinomiasesinae to A.naeslundii forms a biofilm, Inflammation. It has been reported that gingival inflammation caused by Actinomyces is caused by IL-8 or TNF-α produced by TLR2 of gingival epithelial cells or macrophages by lipoproteins on the cell membrane. If the gums are inflamed, periodontal pockets are formed, and bleeding and exudation of gingival exudates are accompanied by the release of Porphyromonas gingivalis, known as periodontal pathogenic bacteria, Aggregatibacter actinomycetemcomitans, Treponema denticola, etc. are placed in periodontal pockets. In addition, Actinomyces sinae E slunidis not only adheres to the tooth surface but also co-aggregates with many oral bacteria, thereby providing a basis for the plaque of periodontal pathogenic bacteria. From these facts, actinomiasensea erosundi migrates from early plaques to late plaques (periodontal biofilms), and thus attracts attention as an important bacterium related to the development of periodontal disease.

Conventional prevention of periodontal disease has been mainly based on the idea of inhibiting periodontal disease by sterilizing periodontal pathogenic bacteria such as P. gingivalis. However, periodontal pathogenic bacteria have been found in the deep part of the periodontal pocket with biofilm The antimicrobial substance is difficult to permeate, and the effect as expected is often not obtained.

In order to solve this problem, Patent Document 1 discloses a process for producing a polylactic acid resin composition which comprises blending (A) N-acyl sarcosine or a salt thereof and (B) benzyl isothiocyanate and further having a mass ratio of (A) / 20, the oral biofilm exhibits an antibacterial effect and an effect of improving gingivitis. However, also in Patent Document 1, the risk of the appearance of resistant bacteria is still high.

Because periodontal disease progresses from promoting gingivitis, it is possible to prevent periodontal disease more effectively by preventing gingivitis. Therefore, it is expected that an effective periodontal disease-preventing effect can be expected in a material that inhibits the biofilm formation of Actinoisethnae ashundi.

The inventors of the present invention confirmed that the formation of biofilm of Actinomyces nasi schlenyi is promoted by acid stress and that actinomycin is added to the extracts of five kinds of cress plants and ice plant such as mizuna, The activity of reducing the amount of the acid-inducible biofilm of Sessnae slunidis by 50 to 90% is confirmed (Patent Document 2).

In this application, among the plant extracts in which the biofilm formation inhibitory activity is confirmed, the most active and easily obtainable mizuna (Brassica rapa var. Nipposinica) is used as a candidate material, and detailed activity evaluation and properties of the active ingredient Characteristics were examined in detail.

Japanese Patent Application Laid-Open No. 2008-174542 Japanese Laid-Open Patent Publication No. 2013-056855

Periodontal pathogenic bacteria are present along with the biofilm in the deep part of the periodontal pocket. In the conventional method of inhibiting periodontal disease using an antimicrobial agent targeting pathogenic bacteria, oral biofilm interferes with the penetration of the antibacterial agent, It was difficult to achieve the same periodontal disease inhibiting effect. In addition, the use of an antimicrobial agent is not preferable because of the high risk of occurrence of resistant bacteria. Therefore, it is considered safer and more effective prevention of periodontal disease by controlling early biofilm formation of periodontal pathogenic bacteria than control by periodontal pathogenic bacteria.

As a result of intensive study by the present inventors, it has been found that the extract of Mizuna has an inhibitory effect on the formation of actinomycetes and esophagus biofilms induced by acid. The inhibitory effect was higher at low temperature. As a result of fractionation of the active ingredient of Mizuna extract, it is estimated that the active ingredient is a component having a molecular weight of 10 kDa or more, and that more than 80% of the components contained in the active fraction are proteins, thereby completing the present invention. In addition, since the mizuna extract did not affect the proliferation of actinomycetes and asthmaticus, it was thought that the action mechanism is different from the antimicrobial action.

Actinomiasse Nae Slunid is a gram-positive bacterium found in gingivitis and root cavities, and is known as an early periodontal pathogenic bacterium. It is conceivable that the control of actinomesesinae and slunidia leads to the prevention of periodontal disease because it is coagulated with streptococci and periodontal pathogenic bacteria and thus holds clues to the transition of the fungus to periodontal disease plaques. The inventors of the present invention have confirmed that biofilm formation is increased by acids such as butyric acid, in which periodontal pathogenic bacteria are produced.

The oral composition containing the mizuna extract of the present invention is useful as a method for preventing periodontal disease which is safer and more effective than the antibacterial agent by remarkably suppressing the formation of biofilm of early periodontal pathogenic bacteria.

1 is a graph comparing the inhibition activity of biofilm formation by the difference in extraction temperature
Fig. 2 shows the comparison of the biofilm formation inhibiting activity by the difference of the extraction temperature
Fig. 3 is a graph showing the biofilm formation inhibitory activity on hydroxyapatite of mizuna or cold water extract
FIG. 4A is a graph showing the results of analysis
FIG. 4B is a schematic view of the oral dissection analysis system
FIG. 5 shows the biofilm formation inhibitory activity on the clinical isolates of mizuna or cold water extracts
6 is a graph showing the biofilm formation inhibitory activity of mizuna or cold water extract in a flow cell
7 is a graph showing the biofilm observation by a confocal laser microscope
Fig. 8 is a graph comparing the inhibitory activity of biofilm formation on the inner dialysate solution and dialysis external solution by dialysis treatment of mizuna or cold water extract, mizuna or cold water extract
9 shows the results of anion exchange chromatography of the inner dialysate solution of Mizuna or cold water extract
10 shows the biofilm formation inhibitory activity of the ammonium sulfate fraction of the dialyzed inner solution of mizuna or cold water extract
11 shows the results of the anion exchange chromatography of the ammonium sulfate fraction of the inner dialysate solution of mizuna or cold water extract
12 is a graph showing the biofilm formation inhibitory activity of the chewing gum containing mizuna or cold water extract

The present invention relates to an oral composition containing mizuna extract.

The present invention also relates to an oral composition wherein the mizuna extract is a cold water extract.

Further, the present invention relates to a periodontal disease biofilm formation inhibitor containing mizuna or an extract.

Further, the present invention relates to an acid-induced biofilm formation inhibitor wherein the mizuna extract is a cold water extract.

Furthermore, the present invention relates to a drenching agent, a dentifrice, an inhalant, a troche, and a food comprising the oral composition.

Hereinafter, the present invention will be described in detail with reference to examples. The present invention is not limited by these examples.

Example

(Example 1)

Preparation of Mizuna Extract:

Mizuna (from Ibaraki Prefecture), which is commercially available, was purchased and freeze-dried to prepare mizuna or dry leaves. Mizuna or dry leaves were finely pulverized, and 1 g of the pulverized mizuna or dried leaves was subjected to extraction at a rate of 50 ml of deionized distilled water at 70 ° C, room temperature, and 4 ° C for 2 hours. The obtained extract was subjected to suction filtration, centrifuged at 13,000 × g for 10 minutes, and the supernatant was freeze-dried to give a test as a mizuna extract.

(Example 2)

Biofilm formation test

(Example 2-1)

Bio-film formation using 96-well microtiter plates

Actinomyces nasi slunidis ATCC19039 or Actinomyces spp. Clinical isolates were cultured in 5 ml of BHI (Brain Heart Infusion) liquid medium under anaerobic conditions at 37 ° C overnight until steady state and cultured at 1,100 × g Centrifugation for 10 minutes. Centrifuged three times with PBS under the same conditions, and _adjusted to an OD ₆₆₀ nm = 0.3 with PBS, to give a test system as a culture suspension.

Biofilm formation was carried out using a 96 well microtiter plate. 0.5% sucrose was added 2 × TSB (Trypticase Soy Broth) culture medium 100 ㎕, the test sample 50 ㎕, 125 mM butyric acid 20 ㎕, disclose fungal suspension 20 ㎕, the addition of PBS 10 ㎕ and, 37 ℃, 5% CO ₂ to each well Lt; / RTI > for 16-20 hours.

(Example 2-2)

Quantification of biofilm formation on 96 well microtiter plates

The culture supernatant cultured in accordance with Example 2-1 was decanted and 100 μl of 0.25% sapranin solution (Nissui Pharmaceutical Co., Ltd.) was added after washing each well with 200 μl of PBS, and the mixture was allowed to stand for 15 minutes to stain the biofilm . The sapranin solution was decanted and then washed twice with deionized distilled water. After drying, 100 쨉 l of 70% ethanol was added and the shrapanin was eluted by shaking for 30 minutes. The sapranin was eluted with an absorbance of 492 nm The amount of biofilm was quantified.

According to the above examples, the inactivity of each of the mizuna and extracts extracted under the conditions of 70 ° C, room temperature, and 4 ° C from Mizuna was evaluated. As a result, the inactivity of the cold water extract extracted under the condition of 4 ° C was the highest 1 and Fig. 2). In addition, the mizuna or cold water extract did not affect the proliferation of actinomycetes nasoluni.

For this reason, additional studies were conducted to examine whether there is a possibility that the mizuna or cold water extract may exhibit activity in the human oral cavity as a candidate material for actinomiasis biofilm inhibiting material.

(Example 2-3)

Biofilm formation on hydroxyapatite (HA)

The HA disk was molded in a shape of 7 mm x 7 mm x 1.5 mm so that its surface was covered with enamel. The HA disk was sterilized in an autoclave, equilibrated at room temperature for 1 hour with PBS, and 400 쨉 l of human saliva collected aseptically was allowed to stand at room temperature for 1 hour to form a pellicle. After washing with PBS, 5 mg / ml BSA Solution for 30 minutes at room temperature was used for the test.

The biofilm formation was carried out using a 24-well microtiter plate. 400 μl of 2 × TSB medium supplemented with 0.5% sucrose, 80 μl of 125 mM butyric acid, 80 μl of 125 mM butyric acid, 40 μl of PBS suspension, and 40 μl of PBS were added to each well. 1 < / RTI >

(Example 2-4)

Quantification of biofilm formation amount on HA

After incubation according to Example 2-3, the HA disk was taken out with tweezers and washed with immersion once in PBS. The cleaned HA disk was placed in a new well, 600 사 of a saponin solution was added, and the suspension was allowed to stand for 15 minutes to stain the biofilm. HA was removed with tweezers, washed with deionized distilled water, placed in a new well, 600 70 of 70% ethanol added, and shaken for 30 minutes to elute the sapranin. 300 ㎕ of the eluate was transferred to a 96-well microtiter plate The amount of biofilm was quantified according to Example 2-2.

Fig. 3 shows the results of evaluating the biofilm formation inhibitory activity of the mizuna or cold water extract on the hydroxyapatite in which the pellicle was formed by the above example. Mizuna or cold water extracts showed activity to inhibit the formation of actinomycetes and eosin di-biotubes in the hydroxyapatite phase as in the case of 96 wells.

(Example 3)

Isolation of Actinomyces clinical isolate

(Example 3-1)

PCR / multiplex PCR

PCR was performed by adding 2.5 μl of 10 × Ex Taq buffer, 1 μl of DNA template, 2 μl of dNTP, 0.025 μl of each primer, 0.125 μl of Ex taq, ₂ μl of MgCl _{2 and} 16.88 μl of H ₂ O to the PCR tube. Multiplex PCR was performed by changing the above composition to three forward primers of 50 μM and three reverse primers of 0.1 μl and 16.75 μl of H ₂ O, respectively. The reaction was carried out at 95 ° C for 10 minutes, followed by 30 cycles of 95 ° C for 30 seconds, 50 ° C for 30 seconds and 72 ° C for 30 seconds, and finally complete extension reaction at 72 ° C for 7 minutes. Multiplex PCR was carried out at an annealing temperature of 53 ° C.

(Example 3-2)

Separation of Clinical Separation

Plaques were taken from 7 randomly selected healthy men and women (male: 3, female: 4), cultured in actinomycet selective medium (CFAT agar medium), colonies formed were stained with Gram Gram positive bacilli were selected.

The genome was extracted from each gram-positive bacterium with a genome extraction kit (sigma), and PCR was performed according to Example 3-1 to amplify about 500 bp upstream of the 16 SrRNA gene. The primer sequences used in the PCR are shown in Table 1.

The PCR products were purified with a PCR Clean-Up kit (promega), and the nucleotide sequence analysis was performed by externally entrusting to Macrogen Japan Co., The nucleotide sequences of the obtained 16 SrRNA genes were estimated by homology search with databases on GenBank. The bacteria suspected of being actinomycetes were amplified by multiplex PCR according to Example 3-1 and subjected to the same base sequence analysis as that of Macrogen Japan. The primer sequences used are shown in Table 1. The obtained nucleotide sequence was systematically analyzed along with the nucleotide sequence of aTPa on the database to identify the species and the obtained Actinomiasse nasluni and Actinomyces oris (A. oris) The clinical isolates were classified as Seth.

According to the above examples, clinical isolates were separated from human oral cavity. As a result, a total of 9 cases of actinomycetes clinical isolates (Actinomyces sinae slunidis: 4 weeks, Actinomyces orris: 5 The separation succeeded. As a result of evaluating the inhibition activity of biofilm formation of mizuna or cold water extracts on 7 out of the active isolates of actinomycetes, the amount of biofilm formation was different according to strains, (Fig. 5). 3, it was suggested that the mizuna or cold water extract had a high possibility of exhibiting biofilm formation inhibitory activity even in the human oral cavity.

(Example 4)

Assessment of biofilm inhibition of mizuna or cold water extracts using flow cells

The above example shows that the mizuna or cold water extract has activity inhibitory activity against actinomycetes and sulindi biofilms in the evaluation by a static system using a 96-well plate. However, considering the actual oral cavity, the saliva is constantly being secreted, and the flow of saliva in the oral cavity is present. Therefore, in order to evaluate whether the mizuna or cold water extract exhibits the biofilm inhibitory activity of actinomycetes and ehrundi even in the oral cavity, a flow cell system simulating the oral environment is used to measure the activity of the actinomycetes in the mizuna or cold water extract The slunei biofilm inhibitory activity was evaluated.

Assessment Methods

(Example 4-1) Evaluation strain

Actinoomesesnae slunidis ATCC19039 strain was used.

(Example 4-2) Preparation of Mizuna extract

A commercially available mizuna was purchased, and dried leaves of mizuna were prepared by freeze-drying. 1 g of finely pulverized mizuna or dried leaves was extracted with 50 ml of deionized distilled water for 2 hours under the condition of 4 캜. The supernatant, from which mizuna or residues were removed by suction filtration and centrifugation, was freeze-dried to recover mizuna or cold water extract.

(Example 4-3) Biofilm formation test using flow cell

Actinomiasensea slunidium was cultured overnight in 5 ml of BHI medium, centrifuged and centrifuged in PBS to prepare OD ₆₆₀ nm = 0.4 in BHI medium, and this was used as a test strain. (ACCFL0001: STOVALL LIFE SCIENCE), and the direction of the chamber was set to be the lower side of the biofilm formation surface, and the mixture was allowed to stand at 37 DEG C for 3 hours. On the surface of the biofilm formation surface, Respectively. After the incubation, the chamber was cultured for 48 hours while flowing the TSB medium supplemented with 0.25% sucrose and 60 mM butyric acid at a flow rate of 3 ml / hour by a peristaltic pump with the biofilm formation side as the upper side, To form a film. The biofilm inhibitory activity of the mizuna or cold water extract was evaluated by adding the mizu or cold water extract having a final concentration of 1 mg / ml to the medium and culturing the same.

(Example 4-4) Observation of biofilm

The formed biofilm was washed with deionized distilled water and subjected to Live / Dead staining with LIVE / DEAE BIOFILM VIABILITY KIT (Invitrogen). The biofilm was observed with a confocal laser microscope.

As a result of evaluating the biofilm formation inhibitory activity of mizuna or cold water extract under flow condition conditions using a flow cell, the mizuna or cold water extract inhibited the formation of actinomiasse and eustodian biofilm 6, 7). Further, from the observation by the confocal laser microscope, the proportion of dead cells in the biofilm formed under the condition of adding Mizu or cold water extract was decreased (Fig. 7).

More specifically, in the evaluation using the floccel, addition of butyric acid also increased the biofilm formation of actinomycetes and esendu dii, and the biofilm had viable bacteria and dead cells in the same degree. The proportion of dead cells constituting the biofilm was higher than that in the case of no addition of butyric acid. In the presence of 1 mg / ml of mizuna or cold water extract, the amount of biofilm formation of actinomycetes nasi schistoside was suppressed, and particularly the amount of adhered dead cells was reduced. Therefore, it has become clear that the mizuna or cold water extract inhibits the formation of biofilm of actinomycetes and eosinhodis depending on butyric acid.

(Example 5)

Fraction of mizuna or cold water extract

(Example 5-1)

dialysis

The mizuna or cold water extract was dissolved in deionized distilled water and centrifuged at 13,000 xg for 10 minutes to recover the supernatant. The extract was placed in a dialysis cellulosic tube (Asuwon) having a cutoff molecular weight of 10 kDa and dialyzed in deionized distilled water for 2 Day. Some of the dialysate internal solution and dialysate external solution were recovered by freeze drying.

In order to estimate the molecular weight of the mizu or cold water extract as described above, the mizu or cold water extract was dialyzed by a dialysis cellulosic tube having a molecular cutoff of 10 kDa, and the activity of inhibiting biofilm formation of the dialyzed inner solution and the outer solution was evaluated. As a result, . The molecular weight of the active ingredient was suggested to be greater than 10 kDa (Figure 8).

(Example 5-2)

The ion exchange chromatography of the inner dialysis liquid of mizuna or cold water extract

Samples were dissolved in 10 mM potassium phosphate buffer (pH 7.4) and the supernatant after centrifugation was applied to DEAE-TOYOPEARL 650 M (? 1.6 x 70 cm). After washing with the same buffer, elution was carried out with a 0 - 0.5 M NaCl linear gradient, and 5 ml of each fraction was separated and collected. All operations were carried out at a flow rate of 1 ml / min.

The dialyzed inner solution of Mizuna or cold water extract was fractionated by anion exchange chromatography to evaluate the activity depending on which component eluted (FIG. 9). As a result, the activity was dependent on the elution pattern of the protein from the first peak to the second peak of the activity. From these, it is suggested that the active ingredient may be a protein.

(Example 5-3)

Ammonium sulfate fraction

The dialysate internal solution samples of the mizuna or cold water extract prepared according to Example 5-1 were dissolved in PBS and the concentration of ammonium sulfate was gradually increased to 30%, 45%, 60%, and 75% g < / RTI > for 15 minutes. The precipitated fraction recovered was dissolved in deionized distilled water, and dialyzed and lyophilized to recover. The 75% uncompleted fraction was also recovered by lyophilization after dialysis.

As described above, when the biofilm formation inhibiting active ingredient is a protein, it is considered that the ammonium sulfate fraction is effective. The inner dialysis liquid of the mizuna or cold water extract is fractionated by ammonium sulfate, and the biofilm formation inhibitory activity of the precipitated fraction at each concentration is evaluated (Fig. 10). The highest biofilm formation inhibitory activity was confirmed in the precipitate fraction at an ammonium sulfate concentration of 45 to 60%.

(Example 5-4)

Ion exchange chromatography of ammonium sulfate fractions of mizuna or cold water extracts

The precipitated fraction at an ammonium sulfate concentration of 45 to 60% prepared according to Example 5-3 was fractionated by anion exchange chromatography according to Example 5-2, and as a result, two peaks indicating the formation inhibition of biofilm formation were confirmed (Fig. 11).

(Example 6)

Quantity of ingredients contained

(Example 6-1)

Protein quantification

Protein quantification was performed using the BCA method.

(Example 6-2)

Per dose

Quantification of sugar was carried out using the phenol sulfate method.

(Example 6-3)

Quantity of polyphenol

The determination of polyphenol was carried out using the Folin-ciocalteu method.

Regarding the above-mentioned tablets, the summary of the result of fractionation of mizuna or cold water extract by dialysis, ammonium sulfate fraction and anion exchange chromatography is shown in Table 2. [ As the purification step was carried out, the specific activity in the vicinity of the weight was increased, and purification of the active ingredient was proceeding. It was suggested that more than 80% of the components contained in the active fraction of ion exchange chromatography were proteins, and that the active component was a protein.

(Example 7)

Evaluation of biofilm formation inhibitory activity of chewing gum containing Mizuna extract

(Example 7-1)

Manufacture of mizuna or blended chewing gum

The chewing gum containing mizuna extract was prepared from the composition shown in Table 3.

BFI - 01: Mizuna or cold water extract is added and kneaded for 12 minutes.

BFI - 02: After kneading for 12 minutes, add mizuna or cold water extract, then knead for about 3 minutes.

(Example 7-2)

Preparation of chewing gum extract

25 ml of warmed PBS at 37 占 폚 was added to 5 g of chewing gum and extracted by crushing for 5 minutes in a mortar. The mixture was centrifuged at 1,100 xg for 15 minutes and the supernatant was recovered and sterilized (0.2 Mu m) as a gum extract.

(Example 7-3)

Evaluation of biofilm inhibitory activity of chewing gum extract

96 well microtiter plate. 1% sucrose was added 4 × TSB medium 50 ㎕, chewing gum extract 100 ㎕, 125 mM butyric acid 20 ㎕, disclose fungal suspension 20 ㎕, the addition of PBS 10 ㎕ and, under 5% CO ₂ conditions at 37 ℃ 16 ~ 20 in each well Time culture. Quantification of the biofilm formation amount was carried out in accordance with Example 2-2.

(Example 7-4)

Evaluation of dissolution rate of mizuna extract from chewing gum

The absorbance at 280 nm wavelength (Abs 280 nm) was measured, and the dissolution rate of Mizuna extract was evaluated by the following calculation formula.

(A1) Abs 280 nm of the mizuna extract dissolved in the control gum extract at a concentration of 2000 ppm

(A2) Abs 280 ㎚ of chewing gum extract mixed with mizuna or cold water extract

(A3) Abs 280 nm of control gum

(%) = ((A2 - A3) / (A1 - A3)) 100

As a result of examining the biofilm formation inhibitory activity of the extract of chewable gum containing mizuna or cold water extract, the extract of chewing gum containing mizuna or cold water extract inhibited the formation of actinomiasensee and ashundii biofilms (Fig. 12) . In addition, the decrease in activity by mixing mizu or cold water extract with chewing gum was not confirmed. In addition, the dissolution rates of the mizuna and cold water extracts were 83.0% for BFI-01 and 85.7% for BFI-02.

Since the mizuna or cold water extract exhibits biofilm formation inhibitory activity against Actinomycetes clinical trials and inhibits the biofilm formation in the evaluation using hydroxyapatite phase and flocell, Suggesting the possibility of exhibiting biofilm formation inhibitory activity.

It is suggested that the protein may be the active component by the fraction of active ingredient of mizuna or cold water extract. On the other hand, although not shown in the data, activity of inhibiting the biofilm was not confirmed in BSA or milk protein preparations. Therefore, the activity is not confirmed in all of the protein, but the activity of the actinomycese biofilm Activity was thought to be confirmed.

The actinomycet biofilm formation inhibitory activity was confirmed in Mizuna or cold water extract, and the effect was also confirmed for the actinomycetes clinical isolate. Mizuna or cold water extracts exhibited biofilm formation inhibitory activity in the hydroxyapatite phase and also in the evaluation using the flow cells, suggesting the possibility of exhibiting activity in human oral cavity. In addition, the chewing gum containing mizuna or cold water extract showed an activity inhibition of actinomycetes and eosin di-biofilm formation.

Mizuna or cold water extracts were fractionated by dialysis, ammonium sulfate fraction and anion exchange chromatography. As a result, it was suggested that the active ingredient was a protein having a molecular weight of 10 kDa or more.

Next, a functional agent, a toothpaste, a bad breath spray, a troki, a candy, a confectionery, a gummy jelly, a beverage containing a biofilm formation inhibitor containing a mizu or cold water extract of the present invention, which is a product other than the above- Were prepared in a conventional manner. Their prescriptions are shown below. Further, the scope of the present invention is not limited by these.

(Example 8)

The hydraulic agent was prepared according to the following formulation.

ethanol   2.0 wt%

Mizuna or cold water extract   1.0

Spices   1.0

Water balance

100.0

(Example 9)

The toothpaste was prepared according to the following prescription.

Calcium carbonate   50.0 wt%

glycerin   19.0

Mizuna or cold water extract    1.0

Carboxymethylcellulose    2.0

Sodium lauryl sulfate    2.0

Spices    1.0

saccharin    0.1

Chlorhexidine    0.01

Water balance

100.0

(Example 10)

A breath spray was prepared according to the following prescription.

ethanol   10.0 wt%

glycerin    5.0

Mizuna or cold water extract    1.0

Spices    0.05

flash    0.001

Water balance

 100.0

(Example 11)

Troches were prepared according to the following prescription.

Mizuna or cold water extract   92.3 wt%

Arabian sword    6.0

Spices    1.0

Sodium monofluorophosphate 0.7

 100.0

(Example 12)

Candies were prepared according to the following prescription.

Sugar    51.0 wt%

Reduced syrup    32.0

Citric acid     1.0

Spices     0.2

L-menthol     1.0

Mizuna or cold water extract     0.4

Water balance

  100.0

(Example 13)

The tablets were prepared according to the following prescription.

Sugar   74.7 wt%

Lactose   18.9

Mizuna or cold water extract    2.0

Sucrose fatty acid ester    0.15

Water 4.25

100.0

(Example 14)

Gumi jelly was prepared according to the following prescription.

gelatin   60.0 wt%

Reduced syrup   32.4

Mizuna or cold water extract    0.5

Plant maintenance    4.5

Malian    2.0

Fragrance 0.5

100.0

(Example 15)

The beverage was prepared according to the following prescription.

Orange juice   30.0 wt%

Mizuna or cold water extract    0.5

Citric acid    0.1

Vitamin C    0.04

Spices    0.1

Water balance

        100.0

Industrial availability

Since the oral composition containing the mizu or cold water extract of the present invention has a periodontal disease-preventing action, which is an action different from that of the conventional antimicrobial agent against periodontal pathogenic bacteria and exhibits a biofilm formation inhibiting action, to be. Therefore, compared with existing antimicrobial agents, it is possible to consider the merit that the risk of occurrence of resistant bacteria is low, and the application to various products is possible.

This application claims priority from Japanese Patent Application No. 2013-018728 filed on February 1, 2013, the contents of which are incorporated herein by reference.

Claims (11)

A composition for oral use containing cold water extract of cetacea. The method according to claim 1,
Wherein the cold water extract of the cress plant is a mizuna or cold water extract. A biofilm formation inhibitor containing cold water extracts of cress plants. The method of claim 3,
Wherein the extract of the cress plant is a mizuna or cold water extract. The biofilm inhibitor of claim 3, wherein the biofilm formation inhibitor is a periodontal biofilm inhibitor. 4. The acid-derived biofilm inhibitor according to claim 3, wherein the biofilm formation inhibitor is an acid-derived biofilm inhibitor. A functional agent comprising the oral composition according to any one of claims 1 to 3. A toothpaste preparation comprising the oral composition according to any one of claims 1 to 3. An inhalant comprising the oral composition according to claim 1 or 2. A troche agent comprising the oral composition according to claim 1 or 2. A food containing the oral composition according to claim 1 or 2.

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