JP2016084311A - Mouth odor inhibitor and oral composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a mouth odor inhibitor and an oral composition which can inhibit the production of methyl mercaptan, a mouth odor component.SOLUTION: The invention provides an oral composition for preventing mouth odor comprising one or more selected from magnolia bark and gobaishi. The magnolia bark and the gobaishi inhibit the expression of mgl mRNA of Porphyromonas gingivalis encoding the enzyme which produces methyl mercaptan by decomposition of methionine. Mouth odor can be improved by inhibiting the production of methyl mercaptan.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a bad breath suppressant and a composition for oral cavity.

  Bad breath is defined as a general term for exhaled breath that the person or a third party feels uncomfortable. In recent years, the percentage of people who suffer from bad breath has increased. Bad breath occurs mainly in the process in which bacteria break down proteins contained in exfoliated epithelial cells, secretions such as saliva, plasma components, and food residues. Examples of bad breath odor substances include volatile sulfur compounds, volatile nitrogen compounds, and lower fatty acids. Among them, volatile sulfur compounds such as hydrogen sulfide, methyl mercaptan, and dimethyl sulfide are the main bad breath components.

  Volatile sulfur compounds, the main halitosis component, are produced by oral gram-negative anaerobic bacteria and are believed to be associated with the progression of periodontitis. In particular, methyl mercaptan is detected at a high concentration in the oral cavity of periodontitis patients. Periodontitis is an infectious disease caused by bacteria in the oral cavity, and bad breath is reduced by appropriate treatment, but various mouthwash compositions such as various mouthwashes and dentifrices can be used to easily suppress bad breath. It is distributed in the market (Patent Documents 1, 2, etc.).

JP2013-129601A JP-A-11-217321

  However, the composition for oral cavity on the market is expected to have a deodorizing effect by deodorizing the produced methyl mercaptan, etc., and simply making a refreshing sensation. it dose not connect.

  This invention is made | formed in view of the said matter, The place made into the objective is providing the bad breath inhibitor and oral cavity composition which can suppress the production of methyl mercaptan which is a component of bad breath.

The bad breath suppressant according to the first aspect of the present invention,
Containing one or more selected from abundant and pentaploid,
It is characterized by that.

The composition for oral cavity according to the second aspect of the present invention,
Containing the bad breath suppressant according to the first aspect of the present invention,
It is characterized by that.

  The halitosis inhibitor according to the present invention suppresses the expression of mgl mRNA of Porphyromonas gingivalis (P. gingivalis) encoding an enzyme (METase) that degrades methionine to produce methyl mercaptan. Since the production of methyl mercaptan is suppressed, bad breath can be improved.

P. It is a graph which shows the difference in mgl mRNA expression level between gingivalis strains. P. of each herbal medicine. It is a graph which shows the methyl mercaptan production ability of gingivalis W38 strain | stump | stock. P. of each herbal medicine. It is a graph which shows the inhibitory effect with respect to mgl mRNA expression of gingivalis W38 strain | stump | stock.

  The halitosis suppressant according to the present embodiment contains one or more selected from the group consisting of abundant and pentaploid.

  Koboku is a dried magnoliaceae, dentaceae, cypress bark and root bark. The pentaploid is a dried insect bump formed on the root of the leaf of the Ursiaceae Nurde genus Rhus tree.

  Periodontal disease is a major cause of bad breath. Periodontal disease is an infection caused by bacterial infection. P. gingivalis is positioned. This P.I. Methyl mercaptan, which is one of the bad breath gases, is produced by the enzyme encoded by mgl mRNA, which is a gene of gingivalis (L-methionine-α-deamino-γ-mercaptomethane-lyase).

  The bad breath suppressant of the present application suppresses the production of methyl mercaptan, not the methyl mercaptan produced. The mechanism is unknown, but the expression of the gene mgl mRNA itself, which encodes an enzyme required for the production of methyl mercaptan, is suppressed by the amber or pentaploid. Thereby, since the production of methyl mercaptan is suppressed, it becomes possible to improve and prevent bad breath.

  Moreover, since both Kokuboku and Pentagon are herbal medicines used in traditional Chinese medicine, the bad breath suppressant has a low risk of side effects.

  The bad breath suppressant is used as an oral composition. Examples of oral compositions include toothpaste, gel, liquid dentifrice, mouthwash, spray, pasta, soft paste (cream formulation), ointment, tablet, granule, chewing gum, It can be used in the form (dosage form) of candy, troche, tablet, chewable tablet, granule capsule, beverage and the like.

  The oral composition may contain other components as appropriate depending on the purpose of use, dosage form, etc., in addition to the body and pentaploid. For example, abrasives such as calcium hydrogen phosphate, calcium carbonate, insoluble sodium metaphosphate, aluminosilicate, silicic anhydride, resin, etc .; interfaces with long-chain sodium alkyl sulfate, sodium lauryl sulfoacetate, lauryl diethanolamide, sucrose fatty acid ester, etc. Activating agents; binders such as hydroxyethyl cellulose, alginate, carrageenan, gum arabic, and polyvinyl alcohol; viscous agents such as polyethylene glycol, sorbitol, glycerin, and propylene glycol; sweeteners such as saccharin, steviosides, glycyrrhizic acid, and thaumatin Preservatives such as dehydroacetic acid and sodium dehydroacetate; perfumes such as menthol, carvone, eugenol, anethole, mint oil, spearmint oil, peppermint oil, eucalyptus oil; various pigments , A raw material normally used in the manufacture of the oral composition optionally selected depending on the product type and application, can be manufactured in a conventional manner.

  First, P.P. has high methyl mercaptan production ability. The expression level of mgl RNA among gingivalis strains was examined.

(Test strain, culture method and adjustment method)
P. used in this example. The gingivalis strains are shown in Table 1. ATCC33277, ATCC49417, W83 ATCC53978, S2 were purchased from American Type Culture Collection (ATCC) and used. HW24D1 and 6/26 were used after receiving a grant from Prof. Masao Amano of Osaka University School of Dentistry. P. As the gingivalis strain, one that had been cryopreserved at −80 ° C. was pre-cultured on a blood agar medium (Japan BD).

  P. The culture of gingivalis was carried out in TSBYE [Trypticase soy broth (TSB, Difco Laboratories)] supplemented with 1% Yeast extract (Difco Laboratories, Detroit, MI, USA) (5 μg / ml). It was performed in an anaerobic state at 37 ° C. for 2 days in a liquid medium supplemented with (1 μg / ml) (Katayama Chemical).

P. The gingivalis was prepared by centrifugation (5,800 × g, 4 ° C., 10 minutes) of the above bacterial culture, washing the cells twice with PBS (−), and phosphate buffer (pH 7.7) [ Suspended in 40 mM potassium phosphate (K ₂ HPO ₄ ) 7 g / l, 50 mM sodium chloride (NaCl) 3 g / l], the bacterial density was adjusted to absorbance OD ₆₆₀ = 1.0.

  Adjusted P.I. gingivalis was cultured as follows, total RNA was purified, and mgl mRNA was quantified.

In a 15 ml centrifuge tube containing 8700 μl of phosphate buffer, 1000 μl of each bacterial solution adjusted to the above OD ₆₆₀ = 1.0, 300 μl of 1 mM L-methionine, and sealed with parafilm, 37 ° C. Incubated for 60 minutes. Next, the reaction solution was centrifuged (20,400 × g, 4 ° C., 5 minutes).

  The supernatant was removed and the cells were collected. After adding 500 μl of RNAiso Plus (Takara Bio Inc.) and lysis, 200 μl of chloroform was added and mixed.

  The solution was separated into three layers by centrifugation (20,400 × g, 4 ° C., 15 minutes), and the upper aqueous layer was transferred to a tube containing 300 μl of isopropanol (Wako Pure Chemical Industries, Ltd.). (20,400 × g, 4 ° C., 10 minutes) to obtain RNA precipitates.

  This was washed with 75% ethanol, dried, and then dissolved in 50 μl of DEPC (Diethylpyrocarbonate) water.

  Next, DNase I treatment for removing DNA in total RNA was performed. DNase I (Roche, Germany) was diluted 50-fold with DEPC, added in the same amount as the RNA solution, and allowed to stand at 37 ° C. for 30 minutes.

  After adding 100 μl of a phenol / chloroform (Wako Pure Chemical Industries, Ltd.) solution, vortexed for 10 seconds and then centrifuged (20,400 × g, 4 ° C., 15 minutes) to separate into two layers.

  The upper aqueous layer was collected, transferred to a tube containing 250 μl of ethanol (99.5%), mixed and then centrifuged again (20,400 × g, 4 ° C., 15 minutes) to obtain an RNA precipitate.

  This was washed with 75% ethanol, dried, dissolved in 50 μl of DEPC water, and stored at −80 ° C.

The amount of RNA from the purified total RNA was measured using GeneQuant (Pharmacia LKB).
For real-time PCR, a two-step method was used in which cDNA was synthesized from total RNA using 1st Strand cDNA Synthesis Kit (Roche, Mannheim, Germany), and the cDNA was used as a template.

In reverse transcription reaction, first, template RNA (2 μg), 10 × reaction buffer [100 mM Tris, 500 mM KCL (pH 8.3)] 2 μl, 25 mM MgCl _{2 4} μl, 10 mM dNTPs 1 μl, random primer (0.02 units / μl) ), 1 μl, RNase inhibitor (50 units / μl) 0.5 μl, and AMV Reverse Transscriptase 0.2 μl were added, and this was performed using Gene Amp PCR system 9700 (Applied Biosystems). The reverse transcription reaction was performed at 42 ° C., 1 hour, 99 ° C., 5 minutes.

  A relative quantification method was used as a quantification method by real-time PCR. The relative quantification method analyzes the target gene and the reference gene at the same time, and compares the expression level of the target gene relative to the reference gene. 1 μl of cDNA, 4 μl of Core Reagent Fast SYBR (r) Master Mix system (Applied Biosystem, CA, USA), 0.5 μl of primer, and 4.5 μl of sterile ion-exchanged water were added. For analysis, ABI Step One Plus real-time PCR system was used. The base sequences of the primers used in this experiment are shown in Table 2, the base sequence of the mgl-F primer is SEQ ID NO: 1, the base sequence of the mgl-R primer is SEQ ID NO: 2, and the base sequence of the Pg16S rRNA-F primer Is shown in SEQ ID NO: 3, and the base sequence of the Pg16S rRNA-R primer is shown in SEQ ID NO: 4.

  Each P.I. The expression level of mgl RNA in gingivalis is shown in FIG. It can be seen that the expression level of mgl mRNA of the W83 strain is the largest.

  Subsequently, W83 with the highest expression level of mgl mRNA was used, and the influence on mgl mRNA expression and methyl mercaptan production ability was examined using various crude drugs.

  As herbal medicines, we used licorice, dodomi, auren, koboku, barley winter, chamomile, daiou, gardenia, ougon, seikyo, mandarin orange, gadgets, cucumber, fennel, yellow spirit, berry, pentaploid, keihi.

The production of CH ₃ SH was performed according to the method of Persson et al. (Persson S, Ed. MB., (1990) The formation of hydrogen sulfide and methylcapital by oral bacteria. It was. Each of the sterilized 13 ml glass test tubes was charged with 870 μl of phosphate buffer and further adjusted to OD ₆₆₀ = 1.0. gingivalis W83 (100 μl), L-methionine (10 nmol) and respective herbal medicines (100 μg) were added, sealed with parafilm, and allowed to stand at 37 ° C. and cultured. The culture time was 60 minutes. Further, as a reference example, the same culture as described above was performed except that no herbal medicine was added.

After culturing, the upper layer gas in each test tube was collected with a 1 ml airtight syringe (Top Corporation, Tokyo), and 0.9 ml was discarded. Then, 0.9 ml of air that was confirmed not to affect VSCs was sucked and diluted 10 times. Of these, 0.5 ml was injected into oral chroma (FIS Co., Ltd.), and the amount of CH ₃ SH produced was measured.

  Further, the expression level of mgl mRNA was quantified by the method described above.

  The influence of each herbal medicine on methyl mercaptan production ability is shown in FIG. 2, and the influence on mgl mRNA expression level is shown in FIG.

  When FIG. 2 is seen, it turns out that the production of methyl mercaptan is significantly suppressed by 69% in the Japanese apricot and 76.4% in the pentaploid. Moreover, when FIG. 3 is seen, it is understood that the expression of mgl mRNA is significantly suppressed by 78%.

As described above, it has been proved that the bad breath can be suppressed because Kokuboku and pentaploid suppress the ability to produce CH ₃ SH by PgW83.

  As described above, the bad breath suppressant is a P. aureus encoding an enzyme that degrades methionine to produce methyl mercaptan. Suppresses the expression of mgl mRNA of gingivalis. Since production of methyl mercaptan is suppressed, it is possible to improve bad breath and can be used as a composition for oral cavity in various forms.

Claims (2)

Containing one or more selected from abundant and pentaploid,
A bad breath suppressant characterized by that. Containing the bad breath suppressant according to claim 1;
The composition for oral cavity characterized by the above-mentioned.

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