WO2014118829A1 - Composition for oral cavity - Google Patents
Composition for oral cavity Download PDFInfo
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- WO2014118829A1 WO2014118829A1 PCT/JP2013/005282 JP2013005282W WO2014118829A1 WO 2014118829 A1 WO2014118829 A1 WO 2014118829A1 JP 2013005282 W JP2013005282 W JP 2013005282W WO 2014118829 A1 WO2014118829 A1 WO 2014118829A1
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- mizuna
- cold water
- water extract
- biofilm
- extract
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/005—Antimicrobial preparations
Definitions
- the present invention relates to an oral composition excellent in periodontal disease improving effect on an oral biofilm which is a cause of oral diseases.
- Periodontal disease is a general term for a group of diseases found in periodontal tissues, and in a narrow sense, it is a disease to which gingivitis, periodontitis and occlusal trauma correspond.
- Periodontal disease is an oral infection caused mainly by dental plaque.
- Actinomyces naeslundii is said to be a causative bacterium of hemorrhagic gingivitis, and A.
- periodontopathic bacteria such as P. gingivalis
- periodontopathic bacteria are biofilms deep in the periodontal pocket.
- antibacterial substances are difficult to penetrate, and the desired effect is often not obtained.
- Patent Document 1 includes (A) N-acyl sarcosine or a salt thereof and (B) benzyl isothiocyanate, and the mass ratio of (A) / (B) is 0. It is disclosed that an oral biofilm antibacterial effect and a gingivitis improving effect are exhibited by being .5-20. However, even in Patent Document 1, the risk of appearance of resistant bacteria is still high.
- Periodontal disease progresses from the onset of gingivitis, so preventing gingivitis can prevent periodontal disease more effectively. Therefore, an effective periodontal disease prevention effect can be expected from a material that suppresses biofilm formation of A. naeslundii.
- the present inventors confirmed that the biofilm formation of A. naeslundii is promoted by acid stress, and the acid-inducibility of A. naeslundii in extracts of five kinds of cruciferous plants such as mizuna and komatsuna and ice plant The activity of reducing the amount of biofilm formed by 50 to 90% has been confirmed (Patent Document 2).
- the most active and easily available mizuna (mizuna, Brassica rapa var. Nipposinica) is used as a candidate material, and detailed activity evaluation and its activity are performed. The properties of the ingredients were examined in detail.
- Periodontopathic bacteria are present in the deep part of the periodontal pocket along with biofilms.
- oral biofilms are antibacterial agents. It was difficult to prevent periodontal disease as intended and prevent the penetration of Also, the use of antibacterial agents is not preferred because of the high risk of resistant bacteria appearing. Therefore, it is considered that the control of the initial periodontal pathogenic bacteria biofilm formation is a safer and more effective periodontal disease prevention method than the control of periodontopathic bacteria with antibacterial agents.
- the extract of mizuna has an inhibitory effect on acid-induced biofilm formation of Actinomyces naeslundii. This inhibitory effect was higher as the extraction temperature was lower.
- the active ingredient is estimated to be a component having a molecular weight of 10 kDa or more, and it is found that 80% or more of the components contained in the active fraction are proteins, and the present invention is completed. did.
- the mizuna extract had no effect on the growth of A. naeslundii, so the mechanism of action was considered to be different from the antibacterial action.
- Actinomyces naeslundii is a Gram-positive gonococcus found from gingivitis and root caries sites, and is said to be an early periodontopathogenic bacterium. Because it coaggregates with streptococci and periodontopathic bacteria, it is the key to the transition of the flora to periodontal disease plaques, and the control of A. naeslundii is thought to lead to periodontal disease prevention. In our study, we have confirmed that biofilm formation is increased by acids such as butyric acid produced by periodontopathic bacteria.
- composition for oral cavity containing the mizuna extract of the present invention remarkably suppresses biofilm formation of early periodontal pathogenic bacteria, and is useful as a safer and more effective periodontal disease prevention method than antibacterial agents. It is.
- This invention relates to the composition for oral cavity containing a mizuna extract. Furthermore, this invention relates to the composition for oral cavity whose said mizuna extract is a cold water extract. Furthermore, this invention relates to the periodontal disease biofilm formation inhibitor containing a mizuna extract. The present invention further relates to an acid-induced biofilm formation inhibitor, wherein the mizuna extract is a cold water extract. Furthermore, the present invention relates to a mouthwash, a toothpaste, an inhalant, a troche, and a food comprising the oral composition.
- Example 1 Preparation method of mizuna extract: Commercially available mizuna (produced in Ibaraki Prefecture) was purchased and freeze-dried to prepare dried mizuna leaves. The dried mizuna leaf was finely pulverized, and 1 g of the crushed dried mizuna leaf was extracted 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 and used for the test as a mizuna extract.
- Example 2-1 Biofilm formation using 96-well microtiter plates
- BHI Brain Heart Infusion
- TLB Trypticase Soy Broth
- Example 2-2 Quantification of amount of biofilm formed in 96-well microtiter plate Decant the culture supernatant cultured according to Example 2-1, wash each well with 200 ⁇ l of PBS, and then add 100 ⁇ l of 0.25% safranin solution (Nissui Pharmaceutical). The biofilm was stained by adding and allowing to stand for 15 minutes. The safranine solution was decanted and washed twice with deionized distilled water. After drying, 100 ⁇ l of 70% ethanol was added, and the mixture was shaken for 30 minutes to elute safranin, using a microplate reader at an absorbance of 492 nm. The amount of biofilm was quantified.
- HA Hydroxyapatite
- the HA disk used was a bovine tooth molded into a 7 mm x 7 mm x 1.5 mm shape so that the surface was covered with enamel.
- the HA disk was sterilized in an autoclave, equilibrated with PBS for 1 hour at room temperature, and 400 ⁇ l of aseptically collected human saliva was allowed to stand at room temperature for 1 hour to form a pellicle. After washing with PBS, 5 mg / ml A BSA solution blocked at room temperature for 30 minutes was used for the test.
- Biofilm formation was performed using 24-well microtiter plates.
- Example 2-1 Incubated according to
- Example 2-4 Quantification of amount of biofilm formed on HA After culturing according to Example 2-3, the HA disk was taken out with tweezers and washed by immersing it once in PBS. The washed HA disk was placed in a new well, 600 ⁇ l of safranin solution was added, and the biofilm was stained by allowing to stand for 15 minutes. Remove HA with tweezers, place it in a new well after washing with deionized distilled water, add 600 ⁇ l of 70% ethanol, shake for 30 minutes to elute safranin, and add 300 ⁇ l of the eluate to a 96-well microtiter plate. The amount of biofilm was quantified according to Example 2-2. FIG.
- FIG. 3 shows the results of evaluating the biofilm formation inhibitory activity of the mizuna cold water extract on the hydroxyapatite on which the pellicle was formed according to the above example.
- the mizuna cold water extract showed A. naeslundii biofilm formation inhibitory activity on hydroxyapatite as well as on 96 wells.
- Example 3-1 PCR / multiplex PCR PCR was performed by adding 2.5 ⁇ l of 10 ⁇ Ex Taq buffer, 1 ⁇ l of DNA template, 2 ⁇ l of dNTP, each primer 0.025 ⁇ l, Ex taq 0.125 ⁇ l, MgCl 2 2 ⁇ l, H 2 O 16.88 ⁇ l to the PCR tube.
- Multiplex PCR was performed by changing the above composition to three 50 ⁇ M Forward primers and three Reverse primers to 0.1 ⁇ l each and 16.75 ⁇ l H 2 O.
- the reaction conditions were 95 ° C for 30 minutes, 95 ° C for 30 seconds, 50 ° C for 30 seconds, 72 ° C for 30 seconds, and finally the extension reaction was completed completely at 72 ° C for 7 minutes. It was.
- Multiplex PCR was performed at an annealing temperature of 53 ° C.
- Example 3-2 Separation of clinical isolates Plaques were collected from 7 healthy males and females (man: 3 and female: 4) selected arbitrarily, cultured in Actinomyces selective medium (CFAT agar medium), and colonies formed were gram-stained Thereafter, Gram-positive rods were selected by microscopic observation. A genome was extracted from each Gram-positive bacillus with a genome extraction kit (sigma), and PCR was performed according to Example 3-1, to amplify about 500 bp upstream of the 16S rRNA gene. The primer sequences used for PCR are shown in Table 1. The PCR product was purified with a PCR Clean-Up kit (promega), and nucleotide sequence analysis was outsourced to Macrogen Japan.
- Bacteria were estimated by homology search of the obtained 16S rRNA gene base sequence with a database on GenBank.
- the bacteria presumed to belong to the genus Actinomyces were amplified atpA by multiplex PCR according to Example 3-1, and were similarly outsourced to Macrogen Japan Co., Ltd. for base sequence analysis.
- the primer sequences used are shown in Table 1. Species were identified by systematic analysis of the obtained nucleotide sequence together with the nucleotide sequence of atpA in the database, and the obtained A. naeslundii and A. oris were used as Actinomyces spp. Clinical isolates.
- Example 4 Evaluation of Biofilm Inhibition of Mizuna Cold Water Extract Using Flow Cell
- Mizuna cold water extract has Actinomyces naeslundii biofilm suppression activity in a static system evaluation using a 96-well plate.
- saliva is constantly secreted, and there is a flow of saliva in the oral cavity. Therefore, in order to evaluate whether the mizuna cold water extract shows the biofilm inhibitory activity of A. naeslundii even in the oral cavity, the A. naeslundii biofilm inhibitory activity of the mizuna cold water extract was evaluated using a flow cell system that mimics the oral environment. evaluated.
- Example 4-1 Evaluation strain Actinomyces naeslundii ATCC19039 strain was used.
- Example 4-2 Preparation of mizuna extract
- Commercially available mizuna was purchased, and dried mizuna leaves were prepared by freeze-drying. Extraction was performed for 2 hours at 4 ° C. with 50 ml of deionized distilled water per 1 g of finely ground dried mizuna leaves. The supernatant from which the mizuna residue was removed by suction filtration and centrifugation was lyophilized to recover the mizuna cold water extract.
- Example 4-3 Biofilm formation test using flow cell A.
- biofilm After standing, the biofilm was cultured for 48 hours while flowing the TSB medium with 0.25% sucrose and 60 mM butyric acid added at a flow rate of 3 ml / hour with a peristaltic pump. Formed.
- the biofilm inhibitory activity of the mizuna cold water extract was evaluated by adding the mizuna cold water extract having a final concentration of 1 mg / ml to the medium and culturing in the same manner. (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), followed by a confocal laser microscope. The biofilm was observed.
- the mizuna cold water extract suppressed the biofilm formation of A. naeslundii (FIG. 6, 7). Moreover, from the observation with a confocal laser microscope, in the biofilm formed under the mizuna cold water extract addition conditions, the ratio for which dead bacteria occupied decreased (FIG. 7). More specifically, in the evaluation using the flow cell, the addition of butyric acid increased the biofilm formation of A. naeslundii, and the biofilm was present with the same number of live and dead bacteria.
- the proportion of dead bacteria constituting the biofilm was higher than when no butyric acid was added.
- the amount of A. naeslundii biofilm formed was suppressed, especially the amount of dead bacteria attached. Therefore, it was revealed that the mizuna cold water extract suppresses biofilm formation of A. naeslundii depending on butyric acid.
- Example 5-1 Dialysis Mizuna cold water extract is dissolved in deionized distilled water, centrifuged at 13,000 xg for 10 minutes, the supernatant is recovered, put into a cellulose tube for dialysis with a molecular weight cut off of 10 kDa (As One), and against deionized distilled water Dialysis was performed in a cold room for 2 days. A portion of the dialyzed solution and the dialyzed solution were lyophilized and collected.
- the mizuna cold water extract was dialyzed with a cellulose tube for dialysis with a molecular weight cut off of 10 kDa, and the biofilm formation inhibitory activity of the dialysis internal solution and external solution was evaluated. The activity of the dialysis internal solution was higher. It was suggested that the molecular weight of the active ingredient is 10 kDa or more (FIG. 8).
- Example 5-2 Ion exchange chromatography of dialysis internal solution of mizuna cold water extract The sample was dissolved in 10 mM potassium phosphate buffer (pH 7.4), and the supernatant after centrifugation was applied to DEAE-TOYOPEARL 650M ( ⁇ 1.6 ⁇ 70 cm). . After washing with the same buffer, elution was performed with a 0-0.5M NaCl linear gradient, and 5 ml was fractionated into each fraction. All operations were performed at a flow rate of 1 ml / min. The dialysis internal solution of the mizuna cold water extract was fractionated by anion exchange chromatography, and it was evaluated which component shows the activity depending on the elution (FIG. 9). As a result, the activity was shown to depend on the protein elution pattern from the first peak to the second peak of the protein. These facts suggested that the active ingredient may be a protein.
- Example 5-3 Ammonium sulfate fraction A dialysis internal liquid sample of a mizuna cold water extract prepared according to Example 5-1 was dissolved in PBS, and the ammonium sulfate concentration was increased stepwise to 30%, 45%, 60%, and 75%. The precipitate was collected by centrifugation at 13,000 ⁇ g for 15 minutes. The collected precipitate fraction was dissolved in deionized distilled water, lyophilized after dialysis, and collected. The 75% unprecipitated fraction was also collected by lyophilization after dialysis.
- biofilm formation inhibitory active ingredient is protein
- ammonium sulfate fractionation is considered to be effective
- the dialysis internal solution of mizuna cold water extract is fractionated with ammonium sulfate, and the precipitate fraction bio at each concentration is separated.
- the film formation inhibitory activity was evaluated (FIG. 10). The highest biofilm formation inhibitory activity was observed in the precipitate fraction at an ammonium sulfate concentration of 45-60%.
- Example 5-4 Ion Exchange Chromatography of Ammonium Sulfate Fraction of Mizuna Cold Water Extract Precipitate fraction prepared at 45 to 60% ammonium sulfate concentration according to Example 5-3 was fractionated by anion exchange chromatography according to Example 5-2. As a result, two biofilm formation inhibitory activity peaks were observed (FIG. 11).
- Example 6-1 Protein quantification Protein quantification was performed using the BCA method.
- Example 6-3 Determination of polyphenols Polyphenols were determined using the Folin-ciocalteu method. Regarding the above-mentioned purification, Table 2 shows a summary of the results of fractionation of mizuna cold water extract by dialysis, ammonium sulfate fractionation and anion exchange chromatography. As the purification step progressed, the specific activity per weight increased, and the purification of the active ingredient progressed. In addition, 80% or more of the components contained in the active fraction of ion exchange chromatography are proteins, suggesting the possibility that the active components are proteins.
- Example 7-1 Preparation of Mizuna Mixed Chewing Gum
- Mizuna extract mixed chewing gum was prepared with the composition shown in Table 3.
- BFI-01 Mizuna cold water extract was added and kneaded for 12 minutes.
- BFI-02 After brewing for 12 minutes, the mizuna cold water extract was added, and then kneaded for about 3 minutes.
- Example 7-2 Preparation of chewing gum extract solution 25 ml of PBS heated to 37 ° C is added to 5 g of chewing gum, extracted by crushing in a mortar for 5 minutes, centrifuged at 1,100 xg for 15 minutes, the supernatant is recovered, and a sterile filter The product sterilized with (0.2 ⁇ m) was used as a gum extract.
- Example 7-3 Evaluation of biofilm inhibitory activity of chewing gum extract
- a 96-well microtiter plate was used. To each well, 50 ⁇ l of 4 ⁇ TSB medium supplemented with 1% sucrose, 100 ⁇ l of chewing gum extract, 20 ⁇ l of 125 mM butyric acid, 20 ⁇ l of the test bacterial suspension, and 10 ⁇ l of PBS were added at 37 ° C. under 5% CO 2 condition. Culture was performed for 20 hours. The amount of biofilm formed was determined according to Example 2-2.
- the biofilm formation inhibitory activity of the mizuna cold water extract-containing tune gum extract was examined.
- the mizuna cold water extract-containing chewing gum extract suppressed the biofilm formation of A. naeslundii potato (FIG. 12).
- blending a mizuna cold water extract with chewing gum was not seen.
- the elution rate of Mizuna cold water extract was 83.0% for BFI-01 and 85.7% for BFI-02.
- Mizuna cold water extract also showed biofilm formation inhibitory activity against Actinomyces clinical strains, and also suppressed biofilm formation on hydroxyapatite and flow cell evaluation. The possibility of exhibiting biofilm formation inhibitory activity was suggested. The fractionation of the active ingredient in the extract of cold water extract of mizuna suggested that protein may be the active ingredient. On the other hand, although not shown in the data, since BSA and milk protein preparations did not show biofilm inhibitory activity, not all proteins showed activity, and they were specific to the protein contained in mizuna extract. Actinomyces Biofilm formation inhibitory activity was considered.
- the mizuna cold water extract showed the activity of inhibiting the formation of Actinomyces biofilm and was also effective against Actinomyces clinical isolates.
- Mizuna cold water extract showed biofilm formation inhibitory activity on hydroxyapatite and in evaluation using a flow cell, suggesting the possibility of activity in the human oral cavity.
- chewing gum containing mizuna cold water extract showed A. naeslundii biofilm formation inhibitory activity.
- the mizuna cold water extract was fractionated by dialysis, ammonium sulfate fractionation, and anion exchange chromatography, suggesting that the active ingredient is a protein having a molecular weight of 10 kDa or more.
- Example 8 A mouthwash was prepared according to the following formulation. Ethanol 2.0% by weight Mizuna cold water extract 1.0 Fragrance 1.0 Water remaining 100.0
- Example 9 A toothpaste was produced according to the following formulation. Calcium carbonate 50.0% by weight Glycerin 19.0 Mizuna cold water extract 1.0 Carboxymethylcellulose 2.0 Sodium ralyl sulfate 2.0 Fragrance 1.0 Saccharin 0.1 Chlorhexidine 0.01 Water remaining 100.0
- Example 10 A spray for halitosis was produced according to the following formulation. Ethanol 10.0% by weight Glycerin 5.0 Mizuna cold water extract 1.0 Fragrance 0.05 Coloring 0.001 Water remaining 100.0
- Example 11 A lozenge was produced according to the following formulation. Mizuna cold water extract 92.3% by weight Gum arabic 6.0 Fragrance 1.0 Sodium monofluorophosphate 0.7 100.0
- Example 12 Candy was manufactured according to the following prescription. 51.0% by weight sugar Reduced water candy 32.0 Citric acid 1.0 Fragrance 0.2 L-Menthol 1.0 Mizuna cold water extract 0.4 Water remaining 100.0
- Example 13 Tablet confectionery was produced according to the following prescription. 74.7% by weight sugar Lactose 18.9 Mizuna cold water extract 2.0 Sucrose fatty acid ester 0.15 Water 4.25 100.0
- Example 14 Gummy jelly was manufactured according to the following prescription. Gelatin 60.0% by weight Reduced water candy 32.4 Mizuna cold water extract 0.5 Vegetable oil 4.5 Malic acid 2.0 Fragrance 0.5 100.0
- Example 15 A beverage was produced according to the following formulation. Orange juice 30.0% by weight Mizuna cold water extract 0.5 Citric acid 0.1 Vitamin C 0.04 Fragrance 0.1 Water remaining 100.0
- the composition for oral cavity containing the mizuna cold water extract of the present invention has a periodontal disease preventive action showing a biofilm formation inhibitory action, which is an action different from that of the conventional antibacterial agent against periodontal pathogenic bacteria. It is a preventive agent for periodontal disease in terms of eyes. Therefore, there are merits such that the risk of appearance of resistant bacteria is low compared to existing antibacterial agents and the like, and application to various products is possible.
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Abstract
Description
さらに、本願発明は、前記水菜抽出物が、冷水抽出物である口腔用組成物に関する。
さらにまた、本願発明は、水菜抽出物を含有する歯周病バイオフィルム形成抑制剤に関する。
さらに、本願発明は、前記水菜抽出物が、冷水抽出物である酸誘導バイオフィルム形成抑制剤に関する。
さらにまた、本願発明は、上記口腔用組成物からなる含そう剤、練り歯磨き剤、吸入剤、トローチ剤、及び食品に関する。 This invention relates to the composition for oral cavity containing a mizuna extract.
Furthermore, this invention relates to the composition for oral cavity whose said mizuna extract is a cold water extract.
Furthermore, this invention relates to the periodontal disease biofilm formation inhibitor containing a mizuna extract.
The present invention further relates to an acid-induced biofilm formation inhibitor, wherein the mizuna extract is a cold water extract.
Furthermore, the present invention relates to a mouthwash, a toothpaste, an inhalant, a troche, and a food comprising the oral composition.
水菜抽出物の調製法:
市販されている水菜(茨城県産)を購入し、凍結乾燥することで水菜乾燥葉を調製した。水菜乾燥葉を細かく粉砕し、この粉砕した水菜乾燥葉1gに対して脱イオン蒸留水50mlの割合で70℃、室温、及び4℃にて2時間抽出を行った。得られた抽出液を吸引ろ過し、13,000×g・10分間で遠心し、その上清を凍結乾燥したものを水菜抽出物として試験に供した。 (Example 1)
Preparation method of mizuna extract:
Commercially available mizuna (produced in Ibaraki Prefecture) was purchased and freeze-dried to prepare dried mizuna leaves. The dried mizuna leaf was finely pulverized, and 1 g of the crushed dried mizuna leaf was extracted 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 and used for the test as a mizuna extract.
バイオフィルム形成試験 (Example 2)
Biofilm formation test
96ウェルマイクロタイタープレートを用いたバイオフィルム形成
A. naeslundii ATCC19039またはActinomyces spp.臨床分離株を5mlのBrain Heart Infusion(BHI)液体培地にて37℃の嫌気条件下で一晩定常期まで培養し、1,100×g・10分間遠心集菌した。同条件にてPBSで3回遠心洗浄し、PBSでO.D.660nm=0.3に調製したものを供試菌懸濁液として試験系に供した。
バイオフィルム形成は96ウェルマイクロタイタープレートを用いて行った。各ウェルに0. 5%スクロース添加2×Trypticase Soy Broth(TSB)培地100μl、試験サンプル50μl、125 mM酪酸20μl、供試菌懸濁液20μl、PBS 10μlを添加し、37℃、5%CO2条件下にて16~20時間培養を行った。 Example 2-1
Biofilm formation using 96-well microtiter plates A. naeslundii ATCC19039 or Actinomyces spp. Clinical isolates were cultured in 5 ml Brain Heart Infusion (BHI) liquid medium under anaerobic conditions at 37 ° C. overnight until stationary phase, The cells were collected by centrifugation at 1,100 × g for 10 minutes. Under the same conditions, the plate was centrifuged 3 times with PBS, and prepared with PBS to an OD 660 nm of 0.3 was used as a test bacterial suspension in the test system.
Biofilm formation was performed using 96 well microtiter plates. To each well, 100 μl of 2 × Trypticase Soy Broth (TSB) medium supplemented with 0.5% sucrose, 50 μl of test sample, 20 μl of 125 mM butyric acid, 20 μl of the test bacterial suspension, 10 μl of PBS, 37 ° C., 5% CO 2 Culturing was performed for 16 to 20 hours under the conditions.
96ウェルマイクロタイタープレートでのバイオフィルム形成量の定量
実施例2-1に従い培養した培養上清をデカントし、PBS 200μlにて各ウェルを洗浄後に0.25%サフラニン溶液(日水製薬)100μlを添加し15分間静置することでバイオフィルムを染色した。サフラニン溶液をデカント後に脱イオン蒸留水にて2回洗浄し、乾燥後に70%エタノールを100μl添加し、30分間振とうすることで、サフラニンを溶出させ、マイクロプレートリーダーを用いて492nmの吸光度にてバイオフィルム量を定量した。
上記した実施例により、水菜から70℃、室温、4℃の各条件下で抽出したそれぞれの水菜抽出物の重量辺りの比活性を評価したところ、4℃の条件下で抽出した冷水抽出物の比活性が最も高かった(図1及び図2)。また、水菜冷水抽出物はA. naeslundii の増殖には影響を示さなかった。
そのため、水菜冷水抽出物をActinomyces バイオフィルム抑制素材の候補材料として、ヒト口腔内で活性を示す可能性があるのか更なる検討を行った。 (Example 2-2)
Quantification of amount of biofilm formed in 96-well microtiter plate Decant the culture supernatant cultured according to Example 2-1, wash each well with 200 μl of PBS, and then add 100 μl of 0.25% safranin solution (Nissui Pharmaceutical). The biofilm was stained by adding and allowing to stand for 15 minutes. The safranine solution was decanted and washed twice with deionized distilled water. After drying, 100 μl of 70% ethanol was added, and the mixture was shaken for 30 minutes to elute safranin, using a microplate reader at an absorbance of 492 nm. The amount of biofilm was quantified.
When the specific activity per weight of each mizuna extract extracted from mizuna under the conditions of 70 ° C., room temperature, and 4 ° C. was evaluated according to the above-mentioned examples, the cold water extract extracted under the condition of 4 ° C. Specific activity was the highest (FIGS. 1 and 2). Mizuna cold water extract did not affect the growth of A. naeslundii.
Therefore, we investigated further whether mizuna cold water extract may be active in human oral cavity as a candidate material for Actinomyces biofilm suppression material.
ハイドロキシアパタイト(HA)上でのバイオフィルム形成
HAディスクはウシ歯を表面がエナメル質で覆われるように7mm×7mm×1.5mmの形に成形したものを用いた。HAディスクをオートクレーブにて滅菌した後、PBSにより室温で1時間平衡化し、無菌的に採取したヒト唾液400μlを室温で1時間静置することでペリクルを形成させ、PBSにて洗浄後に5mg/ml BSA溶液で室温で30分間ブロッキングしたものを試験に用いた。
バイオフィルム形成は24ウェルマイクロタイタープレートを用いて行った。各ウェルに0.5%スクロース添加2×TSB培地400μl、試験サンプル200μl、125mM酪酸 80μl、供試菌懸濁液 80μl、PBS 40μlを添加し、HAディスクを各ウェルに置き、実施例2-1に従い培養した。 (Example 2-3)
Biofilm Formation on Hydroxyapatite (HA) The HA disk used was a bovine tooth molded into a 7 mm x 7 mm x 1.5 mm shape so that the surface was covered with enamel. The HA disk was sterilized in an autoclave, equilibrated with PBS for 1 hour at room temperature, and 400 μl of aseptically collected human saliva was allowed to stand at room temperature for 1 hour to form a pellicle. After washing with PBS, 5 mg / ml A BSA solution blocked at room temperature for 30 minutes was used for the test.
Biofilm formation was performed using 24-well microtiter plates. To each well was added 400 μl of 2 × TSB medium supplemented with 0.5% sucrose, 200 μl of test sample, 80 μl of 125 mM butyric acid, 80 μl of the test bacterial suspension, and 40 μl of PBS, and the HA disk was placed in each well. Example 2-1 Incubated according to
HA上のバイオフィルム形成量の定量
実施例2-3に従い培養した後にHAディスクをピンセットにて取り出し、PBSに一度浸すことで洗浄した。洗浄したHAディスクを新しいウェルに入れ、サフラニン溶液600μlを添加し15分間静置することでバイオフィルムを染色した。HAをピンセットにて取り出し、脱イオン蒸留水にて洗浄後に新しいウェルに置き70%エタノールを600μl添加し、30分間振とうすることでサフラニンを溶出させ、その溶出液300μlを96ウェルマイクロタイタープレートに移して実施例2-2に従いバイオフィルム量を定量した。
上記実施例によりペリクルを形成させたハイドロキシアパタイト上において、水菜冷水抽出物のバイオフィルム形成抑制活性を評価した結果を図3に示した。水菜冷水抽出物はハイドロキシアパタイト上においても、96ウェル上と同様にA. naeslundiiバイオフィルム形成抑制活性を示した。 (Example 2-4)
Quantification of amount of biofilm formed on HA After culturing according to Example 2-3, the HA disk was taken out with tweezers and washed by immersing it once in PBS. The washed HA disk was placed in a new well, 600 μl of safranin solution was added, and the biofilm was stained by allowing to stand for 15 minutes. Remove HA with tweezers, place it in a new well after washing with deionized distilled water, add 600 μl of 70% ethanol, shake for 30 minutes to elute safranin, and add 300 μl of the eluate 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 cold water extract on the hydroxyapatite on which the pellicle was formed according to the above example. The mizuna cold water extract showed A. naeslundii biofilm formation inhibitory activity on hydroxyapatite as well as on 96 wells.
Actinomyces 臨床分離株の分離 (Example 3)
Actinomyces clinical isolate isolation
PCR/マルチプレックスPCR
PCRは10×Ex Taq buffer 2.5μl、DNAテンプレート1μl、dNTP 2μl、プライマー 各0.025μl、Ex taq 0.125μl、MgCl2 2μl、H2O 16.88μl、を PCRチューブに加えて行った。マルチプレックスPCRは上記の組成を50μM Forward プライマー 3 つ、Reverse プライマー 3つを各 0. 1μl、H2O 16.75μlへと変更して行った。反応条件は95℃で10分間にて熱処理後に95℃・30秒、50℃・30秒、72℃・30秒を30サイクル行ない、最後に72℃で7分間にて完全に伸長反応を完了させた。マルチプレックスPCRはアニーリング温度を53℃にて行った。 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, each primer 0.025 μl, Ex taq 0.125 μl,
臨床分離株の分離
任意選択した健常人男女7人(男:3人、女:4人)よりプラークを採取し、Actinomyces 選択培地(CFAT寒天培地)にて培養し、形成されたコロニーをグラム染色後、顕微鏡観察によりグラム陽性桿菌を選別した。
各グラム陽性桿菌からゲノム抽出キット(sigma)にてゲノムを抽出し、実施例3-1に従いPCRを行い 16SrRNA遺伝子の上流の約500bpを増幅させた。PCRに用いたプライマー配列は表1に示した。
PCR産物は PCR Clean-Up キット(promega)にて精製し、塩基配列分析を(株)マクロジェンジャパン社に外部委託して行った。取得した16SrRNA遺伝子の塩基配列をGenBank上のデータベースとの相同性検索にて菌を推定した。上記にてActinomyces 属と推定された菌はatpAを実施例3-1に従いマルチプレックPCRにて増幅させ、同様に塩基配列分析を(株)マクロジェンジャパン社に外部委託して行った。用いたプライマー配列は表1に示した。取得した塩基配列をデータベース上のatpAの塩基配列と共に系統解析することで、種の同定を行ない、得られた A. naeslundii、A. oris を Actinomyces spp. 臨床分離株とした。 (Example 3-2)
Separation of clinical isolates Plaques were collected from 7 healthy males and females (man: 3 and female: 4) selected arbitrarily, cultured in Actinomyces selective medium (CFAT agar medium), and colonies formed were gram-stained Thereafter, Gram-positive rods were selected by microscopic observation.
A genome was extracted from each Gram-positive bacillus with a genome extraction kit (sigma), and PCR was performed according to Example 3-1, to amplify about 500 bp upstream of the 16S rRNA gene. The primer sequences used for PCR are shown in Table 1.
The PCR product was purified with a PCR Clean-Up kit (promega), and nucleotide sequence analysis was outsourced to Macrogen Japan. Bacteria were estimated by homology search of the obtained 16S rRNA gene base sequence with a database on GenBank. The bacteria presumed to belong to the genus Actinomyces were amplified atpA by multiplex PCR according to Example 3-1, and were similarly outsourced to Macrogen Japan Co., Ltd. for base sequence analysis. The primer sequences used are shown in Table 1. Species were identified by systematic analysis of the obtained nucleotide sequence together with the nucleotide sequence of atpA in the database, and the obtained A. naeslundii and A. oris were used as Actinomyces spp. Clinical isolates.
フローセルを用いた水菜冷水抽出物のバイオフィルム抑制評価
上記の実施例は、96ウェルプレートを用いた静止系による評価において、水菜冷水抽出物にActinomyces naeslundiiバイオフィルム抑制活性があることを示した。しかしながら実際の口腔内を考えると、唾液が絶えず分泌されており、口腔内に唾液の流動が存在する。そこで、水菜冷水抽出物が口腔内においてもA. naeslundiiのバイオフィルム抑制活性を示すか評価するため、口腔環境を模したフローセルシステムを用いて、水菜冷水抽出物のA. naeslundiiバイオフィルム抑制活性を評価した。
評価方法
(実施例4-1)評価菌株
Actinomyces naeslundii ATCC19039株を使用した。
(実施例4-2)水菜抽出物の調製
市販されている水菜を購入し、凍結乾燥にて水菜の乾燥葉を調製した。細かく粉砕した水菜乾燥葉1gに対して50mlの脱イオン蒸留水により、4℃条件下にて2時間抽出を行った。吸引濾過および遠心により水菜残渣を取り除いた上清を凍結乾燥し、水菜冷水抽出物を回収した。
(実施例4-3)フローセルを用いたバイオフィルム形成試験
A. naeslundiiを5mlのBHI培地にて一晩培養し、遠心集菌した後、PBSにて遠心洗浄を行ない、BHI培地にてO.D.660nm=0.4に調製し、これを供試菌液とした。供試菌液400μlをフローセルチャンバー(ACCFL0001:STOVALL LIFE SCIENCE社)に接種し、チャンバーの向きをバイオフィルム形成面を下側にした後、37℃条件下にて3時間静置し、バイオフィルム形成面に菌を付着させた。静置後、チャンバーの向きをバイオフィルム形成面を上側にし、0.25%スクロース・60mM酪酸を添加したTSB培地をペリスタポンプにより3ml/時間の流速にて流しながら48時間培養を行ない、バイオフィルムを形成させた。水菜冷水抽出物のバイオフィルム抑制活性は、終濃度1mg/mlの水菜冷水抽出物を上記培地に添加し、同様に培養することで評価した。
(実施例4-4)バイオフィルムの観察
形成されたバイオフィルムを脱イオン蒸留水にて洗浄し、LIVE/DEAE BIOFILM VIABILITY KIT(invitrogen社)によりLive/Dead染色を行った後、共焦点レーザー顕微鏡にてバイオフィルムを観察した。
上記した実施例により、フローセルを用いた流動系条件下にて水菜冷水抽出物のバイオフィルム形成抑制活性を評価したところ、水菜冷水抽出物はA. naeslundii のバイオフィルム形成を抑制した(図6、7)。また、共焦点レーザー顕微鏡による観察から、水菜冷水抽出物添加条件下にて形成されたバイオフィルムでは、死菌の占める割合が減少していた(図7)。
より詳細には、フローセルを用いた評価においても、酪酸を添加するとA. naeslundiiのバイオフィルム形成が増加し、そのバイオフィルムには生菌と死菌が同程度存在していた。バイオフィルムを構成する死菌の割合は、酪酸を添加しない場合に比べて高かった。1mg/mlの水菜冷水抽出物の存在下では、A. naeslundiiのバイオフィルム形成量が抑制され、特に死菌の付着量が減少した。従って、水菜冷水抽出物は、酪酸に依存したA. naeslundiiのバイオフィルム形成を抑制することが明らかとなった。 Example 4
Evaluation of Biofilm Inhibition of Mizuna Cold Water Extract Using Flow Cell The above examples showed that Mizuna cold water extract has Actinomyces naeslundii biofilm suppression activity in a static system evaluation using a 96-well plate. However, considering the actual oral cavity, saliva is constantly secreted, and there is a flow of saliva in the oral cavity. Therefore, in order to evaluate whether the mizuna cold water extract shows the biofilm inhibitory activity of A. naeslundii even in the oral cavity, the A. naeslundii biofilm inhibitory activity of the mizuna cold water extract was evaluated using a flow cell system that mimics the oral environment. evaluated.
Evaluation method (Example 4-1) Evaluation strain Actinomyces naeslundii ATCC19039 strain was used.
(Example 4-2) Preparation of mizuna extract Commercially available mizuna was purchased, and dried mizuna leaves were prepared by freeze-drying. Extraction was performed for 2 hours at 4 ° C. with 50 ml of deionized distilled water per 1 g of finely ground dried mizuna leaves. The supernatant from which the mizuna residue was removed by suction filtration and centrifugation was lyophilized to recover the mizuna cold water extract.
(Example 4-3) Biofilm formation test using flow cell A. naeslundii was cultured overnight in 5 ml of BHI medium, collected by centrifugation, centrifuged and washed with PBS, and O. D. It adjusted to 660nm = 0.4, and this was made into the test microbe liquid.
(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), followed by a confocal laser microscope. The biofilm was observed.
When the biofilm formation inhibitory activity of the mizuna cold water extract was evaluated under the flow system conditions using the flow cell according to the above-described examples, the mizuna cold water extract suppressed the biofilm formation of A. naeslundii (FIG. 6, 7). Moreover, from the observation with a confocal laser microscope, in the biofilm formed under the mizuna cold water extract addition conditions, the ratio for which dead bacteria occupied decreased (FIG. 7).
More specifically, in the evaluation using the flow cell, the addition of butyric acid increased the biofilm formation of A. naeslundii, and the biofilm was present with the same number of live and dead bacteria. The proportion of dead bacteria constituting the biofilm was higher than when no butyric acid was added. In the presence of 1 mg / ml mizuna cold water extract, the amount of A. naeslundii biofilm formed was suppressed, especially the amount of dead bacteria attached. Therefore, it was revealed that the mizuna cold water extract suppresses biofilm formation of A. naeslundii depending on butyric acid.
水菜冷水抽出物の分画 (Example 5)
Fractionation of mizuna cold water extract
透析
水菜冷水抽出物を脱イオン蒸留水に溶解し、13,000×g、10分間遠心して上清を回収し、分画分子量10kDaの透析用セルロースチューブ(アズワン)に入れ、脱イオン蒸留水に対して低温室内で透析を2日間行った。透析内液と透析外液の一部を凍結乾燥し回収した。
上記のように水菜冷水抽出物の分子量を推定するため、分画分子量10kDaの透析用セルロースチューブにより水菜冷水抽出物を透析し、透析内液と外液のバイオフィルム形成抑制活性を評価したところ、透析内液の活性の方が高かった。活性成分の分子量は10kDa以上であることが示唆された(図8)。 Example 5-1
Dialysis Mizuna cold water extract is dissolved in deionized distilled water, centrifuged at 13,000 xg for 10 minutes, the supernatant is recovered, put into a cellulose tube for dialysis with a molecular weight cut off of 10 kDa (As One), and against deionized distilled water Dialysis was performed in a cold room for 2 days. A portion of the dialyzed solution and the dialyzed solution were lyophilized and collected.
In order to estimate the molecular weight of the mizuna cold water extract as described above, the mizuna cold water extract was dialyzed with a cellulose tube for dialysis with a molecular weight cut off of 10 kDa, and the biofilm formation inhibitory activity of the dialysis internal solution and external solution was evaluated. The activity of the dialysis internal solution was higher. It was suggested that the molecular weight of the active ingredient is 10 kDa or more (FIG. 8).
水菜冷水抽出物の透析内液のイオン交換クロマトグラフィー
サンプルを10mMリン酸カリウムバッファー(pH7.4)に溶解し、遠心分離後の上清を DEAE-TOYOPEARL 650M ( ψ1.6×70cm)にアプライした。同バッファーにて洗浄後、0-0.5M NaClリニアグラジエントにより溶出し、各フラクションに5mlずつ分取した。なお、すべての操作は流速1ml/分にて行った。
水菜冷水抽出物の透析内液を陰イオン交換クロマトグラフィーにより分画し、どの成分の溶出に依存して活性を示すか評価した(図9)。その結果、活性はタンパク質の第1ピークから第2ピークにかけてタンパク質の溶出パターンに依存して活性を示した。これらのことから活性成分はタンパク質である可能性が示唆された。 (Example 5-2)
Ion exchange chromatography of dialysis internal solution of mizuna cold water extract The sample was dissolved in 10 mM potassium phosphate buffer (pH 7.4), and the supernatant after centrifugation was applied to DEAE-TOYOPEARL 650M (ψ1.6 × 70 cm). . After washing with the same buffer, elution was performed with a 0-0.5M NaCl linear gradient, and 5 ml was fractionated into each fraction. All operations were performed at a flow rate of 1 ml / min.
The dialysis internal solution of the mizuna cold water extract was fractionated by anion exchange chromatography, and it was evaluated which component shows the activity depending on the elution (FIG. 9). As a result, the activity was shown to depend on the protein elution pattern from the first peak to the second peak of the protein. These facts suggested that the active ingredient may be a protein.
硫安分画
実施例5-1に従って調製した水菜冷水抽出物の透析内液サンプルをPBSに溶解し、硫酸アンモニウムの濃度を30%、45%、60%、75%と段階的に高め、各濃度での沈殿物を13,000×g・15分間遠心し回収した。回収した沈殿画分を脱イオン蒸留水に溶解し、透析後に凍結乾燥し回収した。また、75%未沈殿画分も透析後に凍結乾燥にて回収した。
上記のように、バイオフィルム形成抑制活性成分がタンパク質であれば硫安分画が有効であると考え、水菜冷水抽出物の透析内液を硫安により分画し、各濃度での沈殿画分のバイオフィルム形成抑制活性を評価した(図10)。硫安濃度45~60%での沈殿画分に最も高いバイオフィルム形成抑制活性がみられた。 (Example 5-3)
Ammonium sulfate fraction A dialysis internal liquid sample of a mizuna cold water extract prepared according to Example 5-1 was dissolved in PBS, and the ammonium sulfate concentration was increased stepwise to 30%, 45%, 60%, and 75%. The precipitate was collected by centrifugation at 13,000 × g for 15 minutes. The collected precipitate fraction was dissolved in deionized distilled water, lyophilized after dialysis, and collected. The 75% unprecipitated fraction was also collected by lyophilization after dialysis.
As described above, if the biofilm formation inhibitory active ingredient is protein, ammonium sulfate fractionation is considered to be effective, and the dialysis internal solution of mizuna cold water extract is fractionated with ammonium sulfate, and the precipitate fraction bio at each concentration is separated. The film formation inhibitory activity was evaluated (FIG. 10). The highest biofilm formation inhibitory activity was observed in the precipitate fraction at an ammonium sulfate concentration of 45-60%.
水菜冷水抽出物の硫安分画物のイオン交換クロマトグラフィー
実施例5-3に従い調製した硫安濃度45~60%での沈殿画分を、実施例5-2に従い、陰イオン交換クロマトグラフィーにより分画した結果、2つのバイオフィルム形成抑制活性ピークがみられた(図11)。 (Example 5-4)
Ion Exchange Chromatography of Ammonium Sulfate Fraction of Mizuna Cold Water Extract Precipitate fraction prepared at 45 to 60% ammonium sulfate concentration according to Example 5-3 was fractionated by anion exchange chromatography according to Example 5-2. As a result, two biofilm formation inhibitory activity peaks were observed (FIG. 11).
含有成分定量 (Example 6)
Content determination
タンパク質定量
タンパク質の定量はBCA法を用いて行った。 Example 6-1
Protein quantification Protein quantification was performed using the BCA method.
糖定量
糖の定量はフェノール硫酸法を用いて行った。 (Example 6-2)
Sugar quantification Sugar was quantified using the phenol-sulfuric acid method.
ポリフェノール定量
ポリフェノールの定量はFolin-ciocalteu法を用いて行った。
上記、精製に関して、水菜冷水抽出物を透析、硫安分画および陰イオン交換クロマトグラフィーにより分画した結果のまとめを表2に示した。精製段階を進めるに従い、重量辺りの比活性が高くなっており、活性成分の精製は進んでいた。またイオン交換クロマトグラフィーの活性画分の含有成分は80%以上がタンパク質であり、活性成分がタンパク質である可能性が示唆された。 (Example 6-3)
Determination of polyphenols Polyphenols were determined using the Folin-ciocalteu method.
Regarding the above-mentioned purification, Table 2 shows a summary of the results of fractionation of mizuna cold water extract by dialysis, ammonium sulfate fractionation and anion exchange chromatography. As the purification step progressed, the specific activity per weight increased, and the purification of the active ingredient progressed. In addition, 80% or more of the components contained in the active fraction of ion exchange chromatography are proteins, suggesting the possibility that the active components are proteins.
水菜抽出物配合チューインガムのバイオフィルム形成抑制活性評価 (Example 7)
Evaluation of biofilm formation inhibitory activity of chewing gum with mizuna extract
水菜配合チューインガムの作成
表3の組成で水菜抽出物配合チューインガムを作成した。 Example 7-1
Preparation of Mizuna Mixed Chewing Gum Mizuna extract mixed chewing gum was prepared with the composition shown in Table 3.
BFI - 02:12分間練成後に水菜冷水抽出物を投入し、その後3分間程度混練。
BFI-02: After brewing for 12 minutes, the mizuna cold water extract was added, and then kneaded for about 3 minutes.
チューインガム抽出液の調製
5gのチューインガムに37℃に加温したPBSを25ml加え、乳鉢内で5分間押しつぶすことで抽出を行い、1,100×g ・15分間遠心し、その上清を回収し、滅菌フィルター( 0.2μm ) にて滅菌処理を行った物をガム抽出液とした。 (Example 7-2)
Preparation of chewing gum extract solution 25 ml of PBS heated to 37 ° C is added to 5 g of chewing gum, extracted by crushing in a mortar for 5 minutes, centrifuged at 1,100 xg for 15 minutes, the supernatant is recovered, and a sterile filter The product sterilized with (0.2 μm) was used as a gum extract.
チューインガム抽出液のバイオフィルム抑制活性評価
96ウェルマイクロタイタープレートを用いて行った。各ウェルに1%スクロース添加4×TSB培地50μl、チューインガム抽出液100μl、125 mM酪酸20μl、供試菌懸濁液20μl、PBS 10μlを添加し、37℃で5%CO2条件下にて16~20時間培養を行った。バイオフィルム形成量の定量は実施例2-2に従い行った。 (Example 7-3)
Evaluation of biofilm inhibitory activity of chewing gum extract A 96-well microtiter plate was used. To each well, 50 μl of 4 × TSB medium supplemented with 1% sucrose, 100 μl of chewing gum extract, 20 μl of 125 mM butyric acid, 20 μl of the test bacterial suspension, and 10 μl of PBS were added at 37 ° C. under 5% CO 2 condition. Culture was performed for 20 hours. The amount of biofilm formed was determined according to Example 2-2.
水菜抽出物のチューインガムからの溶出率評価
280nmの波長の吸光度 (Abs280nm) 測定を行い、以下の計算式により水菜抽出物の溶出率を評価した。
(A1)水菜抽出物を2000ppmの濃度でコントロールガム抽出液に溶解させたもののAbs280nm
(A2)水菜冷水抽出物配合チューインガム抽出液のAbs280nm
(A3)コントロールガムのAbs280nm
溶出率 (%)= ((A2-A3)/(A1-A3))×100 (Example 7-4)
Evaluation of dissolution rate of mizuna extract from chewing gum Absorbance at a wavelength of 280 nm (Abs 280 nm) was measured, and the dissolution rate of mizuna extract was evaluated by the following formula.
(A1) Abs 280 nm of mizuna extract dissolved in control gum extract at a concentration of 2000 ppm
(A2) Abs 280 nm of mizuna cold water extract mixed chewing gum extract
(A3) Abs 280 nm of control gum
Elution rate (%) = ((A2-A3) / (A1-A3)) × 100
水菜冷水抽出物の活性成分の分画によりタンパク質が活性成分である可能性が示唆された。一方、データでは示していないが、BSAや乳タンパク製剤にバイオフィルム抑制活性がみられなかったことから、タンパク質すべてに活性がみられるわけではなく、水菜抽出物中に含まれるタンパク質に特異的な Actinomyces バイオフィルム形成抑制活性がみられると考えられた。 Mizuna cold water extract also showed biofilm formation inhibitory activity against Actinomyces clinical strains, and also suppressed biofilm formation on hydroxyapatite and flow cell evaluation. The possibility of exhibiting biofilm formation inhibitory activity was suggested.
The fractionation of the active ingredient in the extract of cold water extract of mizuna suggested that protein may be the active ingredient. On the other hand, although not shown in the data, since BSA and milk protein preparations did not show biofilm inhibitory activity, not all proteins showed activity, and they were specific to the protein contained in mizuna extract. Actinomyces Biofilm formation inhibitory activity was considered.
水菜冷水抽出物を透析・硫安分画・陰イオン交換クロマトグラフィーにより分画したところ、活性成分は分子量10kDa以上のタンパク質であることが示唆された。 The mizuna cold water extract showed the activity of inhibiting the formation of Actinomyces biofilm and was also effective against Actinomyces clinical isolates. Mizuna cold water extract showed biofilm formation inhibitory activity on hydroxyapatite and in evaluation using a flow cell, suggesting the possibility of activity in the human oral cavity. In addition, chewing gum containing mizuna cold water extract showed A. naeslundii biofilm formation inhibitory activity.
The mizuna cold water extract was fractionated by dialysis, ammonium sulfate fractionation, and anion exchange chromatography, suggesting that the active ingredient is a protein having a molecular weight of 10 kDa or more.
下記処方に従って含そう剤を製造した。
エタノール 2.0重量%
水菜冷水抽出物 1.0
香料 1.0
水 残
100.0 (Example 8)
A mouthwash was prepared according to the following formulation.
Ethanol 2.0% by weight
Mizuna cold water extract 1.0
Fragrance 1.0
Water remaining
100.0
下記処方に従って練り歯磨きを製造した。
炭酸カルシウム 50.0重量%
グリセリン 19.0
水菜冷水抽出物 1.0
カルボオキシメチルセルロース 2.0
ラルリル硫酸ナトリウム 2.0
香料 1.0
サッカリン 0.1
クロルヘキシジン 0.01
水 残
100.0 Example 9
A toothpaste was produced according to the following formulation.
Calcium carbonate 50.0% by weight
Glycerin 19.0
Mizuna cold water extract 1.0
Carboxymethylcellulose 2.0
Sodium ralyl sulfate 2.0
Fragrance 1.0
Saccharin 0.1
Chlorhexidine 0.01
Water remaining
100.0
下記処方に従って口臭用スプレーを製造した。
エタノール 10.0重量%
グリセリン 5.0
水菜冷水抽出物 1.0
香料 0.05
着色料 0.001
水 残
100.0 (Example 10)
A spray for halitosis was produced according to the following formulation.
Ethanol 10.0% by weight
Glycerin 5.0
Mizuna cold water extract 1.0
Fragrance 0.05
Coloring 0.001
Water remaining
100.0
下記処方に従ってトローチを製造した。
水菜冷水抽出物 92.3重量%
アラビアガム 6.0
香料 1.0
モノフルオロリン酸ナトリウム 0.7
100.0 (Example 11)
A lozenge was produced according to the following formulation.
Mizuna cold water extract 92.3% by weight
Gum arabic 6.0
Fragrance 1.0
Sodium monofluorophosphate 0.7
100.0
下記処方に従ってキャンディを製造した。
砂糖 51.0重量%
還元水あめ 32.0
クエン酸 1.0
香料 0.2
L-メントール 1.0
水菜冷水抽出物 0.4
水 残
100.0 Example 12
Candy was manufactured according to the following prescription.
51.0% by weight sugar
Reduced water candy 32.0
Citric acid 1.0
Fragrance 0.2
L-Menthol 1.0
Mizuna cold water extract 0.4
Water remaining
100.0
下記処方に従って錠菓を製造した。
砂糖 74.7重量%
乳糖 18.9
水菜冷水抽出物 2.0
ショ糖脂肪酸エステル 0.15
水 4.25
100.0 (Example 13)
Tablet confectionery was produced according to the following prescription.
74.7% by weight sugar
Lactose 18.9
Mizuna cold water extract 2.0
Sucrose fatty acid ester 0.15
Water 4.25
100.0
下記処方に従ってグミゼリーを製造した。
ゼラチン 60.0重量%
還元水あめ 32.4
水菜冷水抽出物 0.5
植物油脂 4.5
リンゴ酸 2.0
香料 0.5
100.0 (Example 14)
Gummy jelly was manufactured according to the following prescription.
Gelatin 60.0% by weight
Reduced water candy 32.4
Mizuna cold water extract 0.5
Vegetable oil 4.5
Malic acid 2.0
Fragrance 0.5
100.0
下記処方に従って飲料を製造した。
オレンジ果汁 30.0重量%
水菜冷水抽出物 0.5
クエン酸 0.1
ビタミンC 0.04
香料 0.1
水 残
100.0 (Example 15)
A beverage was produced according to the following formulation.
Orange juice 30.0% by weight
Mizuna cold water extract 0.5
Citric acid 0.1
Vitamin C 0.04
Fragrance 0.1
Water remaining
100.0
This application claims priority from Japanese Patent Application No. 2013-018728 filed on Feb. 1, 2013, the contents of which are incorporated herein by reference.
Claims (11)
- アブラナ科植物の冷水抽出物を含有する口腔用組成物。 Oral composition containing cold water extract of cruciferous plant.
- 前記アブラナ科植物の冷水抽出物が、水菜冷水抽出物である請求項1に記載の口腔用組成物。 The composition for oral cavity according to claim 1, wherein the cold water extract of the cruciferous plant is a mizuna cold water extract.
- アブラナ科植物の冷水抽出物を含有するバイオフィルム形成抑制剤。 Biofilm formation inhibitor containing cold water extract of cruciferous plants.
- 前記アブラナ科植物の抽出物が、水菜冷水抽出物である請求項3に記載のバイオフィルム形成抑制剤。 The biofilm formation inhibitor according to claim 3, wherein the cruciferous plant extract is a mizuna cold water extract.
- 請求項3記載のバイオフィルム形成抑制剤が歯周病バイオフィルム抑制剤 The biofilm formation inhibitor according to claim 3 is a periodontal disease biofilm inhibitor.
- 請求項3記載のバイオフィルム形成抑制剤が酸誘導バイオフィルム抑制剤 The biofilm formation inhibitor according to claim 3 is an acid-derived biofilm inhibitor.
- 請求項1または2に記載の口腔用組成物からなる含そう剤。 A mouthwash comprising the oral composition according to claim 1 or 2.
- 請求項1または2に記載の口腔用組成物からなる練り歯磨き剤。 A toothpaste comprising the oral composition according to claim 1 or 2.
- 請求項1または2に記載の口腔用組成物からなる吸入剤。 An inhalant comprising the oral composition according to claim 1 or 2.
- 請求項1または2に記載の口腔用組成物からなるトローチ剤。 A lozenge comprising the oral composition according to claim 1 or 2.
- 請求項1または2に記載の口腔用組成物を含有する食品。
A food containing the composition for oral cavity according to claim 1 or 2.
Priority Applications (3)
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CN201380072053.8A CN104968327A (en) | 2013-02-01 | 2013-09-05 | Composition for oral cavity |
KR1020157023340A KR20150114521A (en) | 2013-02-01 | 2013-09-05 | Composition for oral cavity |
JP2014559359A JP6300736B2 (en) | 2013-02-01 | 2013-09-05 | Oral composition |
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JP2013-018728 | 2013-02-01 | ||
JP2013018728 | 2013-02-01 |
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PCT/JP2013/005282 WO2014118829A1 (en) | 2013-02-01 | 2013-09-05 | Composition for oral cavity |
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JP (2) | JP6300736B2 (en) |
KR (1) | KR20150114521A (en) |
CN (1) | CN104968327A (en) |
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JP6300736B2 (en) * | 2013-02-01 | 2018-03-28 | 株式会社ロッテ | Oral composition |
TWI702224B (en) * | 2018-11-30 | 2020-08-21 | 國立成功大學 | Aptamers of surface proteins of periodontal pathogens and uses thereof |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH10259136A (en) * | 1997-03-17 | 1998-09-29 | Taiyo Koryo Kk | Virus proliferation suppressor and pharmaceutical composition containing the same virus proliferation suppressor |
JP2007320926A (en) * | 2006-06-02 | 2007-12-13 | Sanei Gen Ffi Inc | Plaque formation inhibitor, or cariostatic agent |
JP2009027926A (en) * | 2007-07-24 | 2009-02-12 | Sunstar Inc | Powdery beverage containing plant as raw material |
JP2013056855A (en) * | 2011-09-08 | 2013-03-28 | Lotte Co Ltd | Oral composition |
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EP2397483A1 (en) * | 2009-02-13 | 2011-12-21 | Kaneka Corporation | Plant extract containing antifreeze substance and method for producing same |
TWI445671B (en) * | 2010-03-24 | 2014-07-21 | Sony Corp | Cation exchanger and method of removing heavy metal ions in wastewater |
JP6300736B2 (en) * | 2013-02-01 | 2018-03-28 | 株式会社ロッテ | Oral composition |
-
2013
- 2013-09-05 JP JP2014559359A patent/JP6300736B2/en active Active
- 2013-09-05 CN CN201380072053.8A patent/CN104968327A/en active Pending
- 2013-09-05 KR KR1020157023340A patent/KR20150114521A/en not_active Application Discontinuation
- 2013-09-05 WO PCT/JP2013/005282 patent/WO2014118829A1/en active Application Filing
- 2013-09-06 TW TW102132187A patent/TWI620576B/en active
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2018
- 2018-02-26 JP JP2018031798A patent/JP6564084B2/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH10259136A (en) * | 1997-03-17 | 1998-09-29 | Taiyo Koryo Kk | Virus proliferation suppressor and pharmaceutical composition containing the same virus proliferation suppressor |
JP2007320926A (en) * | 2006-06-02 | 2007-12-13 | Sanei Gen Ffi Inc | Plaque formation inhibitor, or cariostatic agent |
JP2009027926A (en) * | 2007-07-24 | 2009-02-12 | Sunstar Inc | Powdery beverage containing plant as raw material |
JP2013056855A (en) * | 2011-09-08 | 2013-03-28 | Lotte Co Ltd | Oral composition |
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JP6300736B2 (en) | 2018-03-28 |
JP2018127455A (en) | 2018-08-16 |
TWI620576B (en) | 2018-04-11 |
CN104968327A (en) | 2015-10-07 |
JP6564084B2 (en) | 2019-08-21 |
KR20150114521A (en) | 2015-10-12 |
TW201431565A (en) | 2014-08-16 |
JPWO2014118829A1 (en) | 2017-01-26 |
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