JP6918529B2 - Oral composition - Google Patents

Description

The present invention relates to an oral composition, and more particularly to an oral composition effective in inhibiting the adhesion of periodontal disease bacteria to animal cells and suppressing the expression of pili of periodontal disease bacteria.

Dental diseases such as dental caries and periodontal disease are said to interfere with eating habits and social life because the degree of progression leads to tooth loss, which in turn affects general health. Of these, periodontal disease is a disease of the periodontal tissue caused by periodontal disease bacteria such as Porphyromonas gingivalis. The plaque formed by periodontal disease bacteria causes inflammation of the periodontal tissue, eventually melting the bones that support the teeth and eventually causing tooth loss. In the case of mild periodontal disease, it is known that inflammation is improved by sterilizing and removing periodontal disease bacteria and removing plaque, and the periodontal tissue returns to a healthy state. Therefore, it can be said that prevention and initial treatment of periodontal disease are important, and various bactericidal agents and antibacterial agents effective for prevention and initial treatment of periodontal disease have been developed.

Among the bactericidal and antibacterial components developed so far against periodontal disease bacteria, naturally derived components include cocoa extract and cacao mass extract (Patent Document 1) made from fermented cacao beans, and crushed outer shell of cacao beans. Cacao husk extracts derived from substances (Non-Patent Documents 1 to 3) are known. It is known that various components contained in cacao beans are changed by fermentation (Non-Patent Document 4), but there are reports of bactericidal effects of periodontal disease bacteria on cacao mass and cocoa extracts made from fermented cacao beans. On the other hand, no bactericidal effect of periodontal disease bacteria has been reported for the extract from unfermented cacao beans.

In recent years, the importance of pili possessed by periodontal disease bacteria has been shown when periodontal disease bacteria invade animal epithelial cells (Non-Patent Document 5). No inhibitory effect on pili expression or attachment to animal epithelial cells has been reported so far.

International Publication No. 2003/099304

Haruhiro Kuwashima et al., Medicine and Biology, Vol. 123, No. 1, pp. 33-38, 1991 Osawa K et al., J DENT RES 80: pp2000-2004 (2001) Osawa K et al., Bull Tokyo Dent Coll. 31: pp125-128 (1990) Yasunobu Goto et al., Journal of Japan Society for Food Science and Technology, Vol. 49, No. 11, 731-735 (2002) Chinmay K. Mantri et al., Microbiology Open 4 (1), 53-65 (2015)

An object of the present invention is to provide a novel oral composition, particularly an oral composition effective for inhibiting the adhesion of periodontal disease bacteria to animal cells and suppressing the expression of pili of periodontal disease bacteria. Another object of the present invention is to provide an oral composition effective against periodontal disease bacteria and halitosis-causing bacteria. Another object of the present invention is to provide an oral composition capable of preventing or alleviating various diseases such as angiopathy, premature birth, and diabetes caused by invasion of oral bacteria such as periodontal disease bacteria into the living body. do.

The present inventors have now announced that the unfermented cocoa bean extract suppresses the expression of pili of periodontal disease bacteria, and the unfermented cocoa bean extract and the fermented cocoa bean extract adhere to the animal cells of the periodontal disease bacterium. Was found to inhibit. The present inventors have also found that the unfermented cocoa bean extract has excellent antibacterial activity against periodontal disease bacteria and has excellent antibacterial activity against biofilms formed by periodontal disease bacteria. .. The present inventors further found that administration of unfermented cocoa bean extract to periodontitis model rats improved periodontitis. The present invention is based on these findings.

According to the present invention, the following inventions are provided.
[1] An oral composition comprising a cacao bean extract.
[2] The composition according to the above [1], which is an inhibitor for adhesion of periodontal disease bacteria to animal cells.
[3] The composition according to the above [1], which is an agent for suppressing the expression of cilia of periodontal disease bacteria.
[4] The composition according to the above [1], wherein the cacao bean extract is an unfermented cacao bean extract.
[5] The composition according to the above [4], which is an antibacterial agent against periodontal disease bacteria and / or halitosis-causing bacteria.
[6] The composition according to the above [4] or [5] for use in the treatment, prevention or amelioration of periodontal disease and related diseases.
[7] The composition according to the above [4] or [5] for use in suppressing halitosis.
[8] Periodontal disease bacteria are Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigresense, Treponema denticola, Tannerella forsysensis, Aggregatibacter actinomycetemcomitans, Campylobacter rectus and Frobacterium. The composition according to the above [2], [3] or [5], which is one kind or two or more kinds selected from the group consisting of um nucrateum.
[9] The above-mentioned [5], wherein the halitosis-causing bacteria is one or more selected from the group consisting of Fusobacterium nucleatum and Veillonella dispar and the periodontal disease bacteria described in the above [8]. Composition.
[10] The composition according to any one of the above [1] to [9], which is a food composition.

The oral composition of the present invention contains a cacao bean extract as an active ingredient. Since the cacao bean extract is a natural material that has been used as a raw material for foods by human beings for many years, according to the present invention, it is possible to provide a safe oral composition without fear of side effects.

The figure showing the inhibitory effect of unfermented cacao bean extract (CBP, HP-PW) on the expression of pili (Mfa1, FimaA) of Porphyromonas gingivalis ATCC 33277 strain (SDS polyacrylamide gel electrophoresis). Photo). “Pg” in the figure indicates the result of the control group in which Porphyromonas gingivalis was cultured in a medium without fermented cacao bean extract. It is a figure which showed the inhibitory effect of the cacao bean extract on the adhesion to the animal cell of Porphyromonas gingivalis ATCC 33277 strain. It is a figure which showed the effect of the unfermented cocoa bean extract against periodontal disease bacteria from the relationship between the CBP concentration and HP-PW concentration, and the number of surviving bacteria of Porphyromonas gingivalis ATCC 33277 strain. .. It is a figure which showed the effect of the unfermented cocoa bean extract against periodontal disease bacteria from the relationship between the CBP concentration and HP-PW concentration, and the survival rate of Porphyromonas gingivalis ATCC 33277 strain. It is a figure (micrograph) which showed the bactericidal effect of the unfermented cocoa bean extract (CBP and HP-PW) on the biofilm formed by Porphyromonas gingivalis ATCC 33277 strain. The number (%) in the photograph indicates the proportion of viable bacteria. In addition, "PW-VE-R" in FIG. 3 is synonymous with "HP-PW". It is a figure which showed the inhibitory effect of the unfermented cacao bean extract on the alveolar bone resorption of Porphyromonas gingivalis ATCC 33277 strain (* P <0.05). It is a figure (photograph) which showed the result of detecting Porphyromonas gingivalis ATCC 33277 strain in a rat oral cavity by PCR.

Specific description of the invention

The oral composition of the present invention comprises a cocoa bean extract. In the present invention, the term "cacao bean extract" is used to include not only fermented cacao bean extract but also unfermented cacao bean extract.

The oral composition of the present invention comprises a fermented cocoa bean extract. As the fermented cacao bean extract, the material obtained by processing the fermented cacao beans can be used as it is, or can be used as a raw material for the extraction treatment described later. Examples of the processed material include cocoa beans from which the skin has been removed with a separator, such as cocoa nibs, cocoa mass, defatted cocoa mass and cocoa powder. That is, in the present invention, cacao nibs, cacao mass, defatted cacao mass and cocoa powder, and extracts thereof can be used as fermented cacao bean extracts. In addition, the fermentation treatment of cacao beans can be carried out by natural fermentation according to a conventional method.

The fermented cacao bean extract can be prepared by solvent-extracting fermented cacao beans (including those processed above). Examples of the solvent that can be used include water, alcohols having 1 to 6 carbon atoms (for example, ethanol), aldehydes, and ketones, and a solvent suitable for the varieties of cocoa beans may be used as the extraction solvent in combination of each. An oil extraction (expeller) step may be carried out prior to the solvent extraction step. A defatted extract can be obtained by carrying out the oil extraction step. The extract obtained by the solvent extraction step may be in the form of a dry powder by, for example, filtering and concentrating, and then drying by a drying means such as a spray-drying method or a freeze-drying method. The method for preparing the fermented cacao bean extract is known, and for example, it can be prepared according to the description in JP-A-2001-69946.

The oral composition of the present invention comprises an unfermented cocoa bean extract. The unfermented cocoa bean extract can be prepared by solvent-extracting cocoa beans (raw beans). Examples of the solvent that can be used include water, alcohols having 1 to 6 carbon atoms (for example, ethanol), aldehydes, and ketones, and a solvent suitable for the varieties of cocoa beans may be used as the extraction solvent in combination of each. Prior to the solvent extraction step, a step of crushing the cocoa beans or an oil squeezing (expeller) step may be carried out. A defatted extract can be obtained by carrying out the oil extraction step. The extract obtained by the solvent extraction step may be in the form of a dry powder by, for example, filtering and concentrating, and then drying by a drying means such as a spray-drying method or a freeze-drying method.

A method for preparing an unfermented cacao bean extract is known, and for example, it can be prepared according to the description in JP-A-2014-118369. In addition, unfermented cacao bean extract is commercially available, and for example, "cacao bean extract CBP" and "HP cacao extract PW" manufactured by Meiji Co., Ltd. can be used in the present invention.

The content of the cocoa bean extract in the composition of the present invention (based on solid content conversion) is the activity of inhibiting the adhesion of periodontal disease bacteria to animal cells, the activity of suppressing the expression of pili of periodontal disease bacteria, the periodontal disease bacteria and halitosis. The upper limit can be 100% by mass, 30% by mass or 10% by mass, and the lower limit can be 0.01% by mass or 0.1% by mass, although the effect is not particularly limited as long as the effect such as antibacterial activity against the causative bacteria is exhibited. It can be 1% by mass or 5% by mass.

The composition of the present invention can be used as an oral composition, and specifically, an agent for adhering periodontal disease bacteria to animal cells (particularly epithelial cells) and pili of periodontal disease bacteria (particularly Mfa1 line). It can be used as an expression inhibitor for hair and Fima pili. The composition according to the present invention can also be used as an antibacterial agent against periodontal disease bacteria and / or halitosis-causing bacteria.

Here, as periodontal disease bacteria, Porphyromonas gingivalis and other Porphyromonas gingivalis bacteria, Prevotella intermedia , Prevotella nigrescens and other Prevotella bacteria, and Treponema Dentikora (Treponema denticola), Tan'nera forsythensis (Tannella forsythensis), Aggregatibacter actinomycetemcomitans (Aggregatibacter actinomycetemcomitans), Campylobacter rectus (Campylobacter rectus), and the like Fusobacterium nucleatum (Fusobacterium nucleatum) ..

Examples of the bacteria that cause mouth odor include Fusobacterium nucleatum and other Fusobacterium nucleatum bacteria, Veillonella dispar and other Veillonella bacteria, and the above-mentioned various periodontal disease bacteria.

According to the composition of the present invention, the adhesion of periodontal disease bacteria to animal cells (particularly epithelial cells) can be inhibited, and the pili of periodontal disease bacteria (particularly Mfa1 pili and Fima pili) can be inhibited. Expression can be suppressed. The colonization of periodontal disease bacteria in the oral cavity is based on the adhesion to cells, and the pili expressed on the cells is important for the colonization of periodontal disease bacteria in the oral cavity and the formation of biofilms. Playing a role. Therefore, according to the composition of the present invention, periodontal disease and related diseases can be treated, prevented and ameliorated by inhibiting the adhesion of periodontal disease bacteria to animal cells and suppressing the expression of pili of periodontal disease bacteria. ..

According to the composition of the present invention, the total number of oral pathogenic bacteria such as periodontal disease bacteria and halitosis-causing bacteria can be reduced or the growth can be suppressed, and eventually, the oral cavity such as periodontal disease and halitosis can be suppressed. The disease can be treated, prevented or ameliorated. That is, the composition of the present invention can be used for suppressing halitosis and can be used as a halitosis suppressant. Here, "halitosis" refers to a person who feels the halitosis component contained in the exhaled breath, and is a general term for the malodor emitted from the oral cavity together with the exhaled breath. Volatile sulfur-containing compounds such as hydrogen sulfide and methyl mercaptan can be mentioned as typical components of the malodor.

The compositions of the present invention can be used for the treatment, prevention or amelioration of periodontal disease and related diseases. That is, according to the present invention, there is provided an agent for treating, preventing or ameliorating periodontal disease and related diseases, which comprises an unfermented cocoa bean extract as an active ingredient.

In the present invention, "periodontal disease" is used in the sense of including gingival inflammation and periodontitis. In addition, periodontal disease-related diseases mean secondary diseases that can be caused by periodontal disease, such as aspiration pneumonia and vascular disorders (eg, invasion of oral bacteria in vivo). , Angina, myocardial infarction), premature birth, diabetes, etc.

The composition of the present invention is not particularly limited in its form and shape as long as it exhibits antibacterial activity against periodontal disease bacteria and halitosis-causing bacteria, and even if it is in a solid form, it may be in the form of a liquid, semi-liquid, gel, or the like. It may be in the form of a paste or powder. The compositions of the present invention include, for example, food compositions such as tablets (for example, troches, chewable tablets), gums, gums, candies, candy, beverages, jellies, edible films, dentifrices, mouthwashes, etc. Oral care compositions such as mouthwashes, dentifrices, orally disintegrating tablets, and gels are mentioned, and tablets, gums, gummy, and candy are preferable from the viewpoint of portability and retention in the oral cavity. The composition of the present invention may contain other known additives as optional components in addition to the cacao bean extract, depending on the dosage form.

When the composition of the present invention is provided as a tablet, in addition to the cocoa bean extract, sugars, excipients, sweeteners, acidulants, flavors, colorants, preservatives, stabilizers, emulsifiers, and plant extracts It can be produced by a conventional method by blending one or more kinds of ingredients selected from products, fruit juice powder and the like.

The cocoa bean extract is an edible material by itself, and has antibacterial activity against periodontal disease bacteria and halitosis-causing bacteria as shown in Examples below. You can also do it. That is, the composition of the present invention can be provided as a food composition. When the composition of the present invention is provided as a food composition, the composition of the present invention can be produced according to the usual production procedure of the food except that the cocoa bean extract as an active ingredient is blended.

When the cacao bean extract which is the active ingredient of the present invention is provided as a food composition, it can be prepared by incorporating the cacao bean extract into the food as it is, and the food composition contains the cacao bean extract. It is a food containing an effective amount. Here, "containing an effective amount" of the cacao bean extract means a content such that the cacao bean extract is ingested within a predetermined range when the amount normally eaten in each food is ingested. The scope of the present invention also includes an embodiment in which the cocoa bean extract itself, which is the active ingredient of the present invention, or a composition containing the same is added to a food (for example, a beverage) by an ingestor to obtain a food composition. Shall be.

The composition of the present invention is a single-dose cocoa bean extract effective for the activity of inhibiting the adhesion of periodontal disease bacteria to animal cells, the activity of suppressing the expression of pili of periodontal disease bacteria, and the antibacterial activity against periodontal disease bacteria. It can be provided as a composition comprising. The amount of one-time intake of cacao bean extract effective for these activities and the number of times of intake per day can be determined depending on the age, symptoms, and sensation of the ingestor. For example, the intake of a single dose of cocoa bean extract, which is effective for the activity of inhibiting the adhesion of periodontal disease bacteria to animal cells, the activity of suppressing the expression of pili of periodontal disease bacteria, and the antibacterial activity against periodontal disease bacteria, is the solid content. The reduced mass is 0.1 mg to 10 mg / time, preferably 1.0 mg to 10 mg / time. In addition, the number of times of ingestion of the cacao bean extract effective for these activities per day is 1 to 10 times / day, preferably 3 to 5 times / day.

According to another aspect of the present invention, a method for inhibiting the adhesion of periodontal disease bacteria to animal cells and teeth, which comprises ingesting or administering an effective amount of cocoa bean extract to human or non-human animals. A method for suppressing the expression of pili of periodontal disease bacteria is provided. According to the present invention, there is also a method for sterilizing periodontal disease bacteria and / or halitosis-causing bacteria, which comprises ingesting or administering an effective amount of unfermented cocoa bean extract to humans or non-human animals. Provided. The sterilization method, inhibition method and suppression method of the present invention can be carried out according to the description regarding the composition of the present invention.

According to yet another aspect of the present invention, treatment of periodontal disease and related diseases comprising ingesting or administering an effective amount of unfermented cocoa bean extract to human or non-human animals. , Prevention or improvement methods are provided. The method of treatment, prevention or amelioration of the present invention can be carried out according to the description regarding the composition of the present invention.

Here, both therapeutic and non-therapeutic use is intended for the use of the cocoa bean extract in humans and non-human animals in the present invention. Here, "non-therapeutic" means that it does not include the act of surgery, treatment or diagnosis of a human (that is, medical practice for a human), and specifically, a doctor or a person who has been instructed by a doctor. It means that it does not include a method of performing surgery, treatment or diagnosis on humans.

According to yet another aspect of the present invention, the use of cocoa bean extract and the use of periodontal disease bacteria for the production of an agent for inhibiting the adhesion of periodontal disease bacteria to animal cells and an agent for suppressing the expression of pili of periodontal disease bacteria. The use of cocoa bean extract as an inhibitor of adherence to animal cells and an agent for suppressing the expression of cilia of periodontal disease bacteria is provided. According to the present invention, the use of an unfermented cocoa bean extract for the production of an antibacterial agent against periodontal disease bacteria and / or halitosis-causing bacteria, and as an antibacterial agent against periodontal disease bacteria and / or halitosis-causing bacteria. , The use of unfermented cacao bean extract is provided. The use of the present invention can be carried out in accordance with the description regarding the composition of the present invention.

The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.

Example 1: Effect of unfermented cocoa bean extract on the expression of hairline of periodontal disease bacteria (1) Preparation of medium containing unfermented cocoa bean extract As a test sample, cocoa bean extract (polyphenol concentration 82% by mass, below "CBP") (manufactured by Meiji Co., Ltd.) and HP cacao extract PW (polyphenol concentration 17% by mass, hereinafter referred to as "HP-PW") (manufactured by Meiji Co., Ltd.) were used. CBP and HP-PW are unfermented cacao bean extracts having different extraction methods. The CBP powder and the HP-PW powder were each dissolved in a 50% ethanol solution to prepare a CBP-containing 50% ethanol solution and an HP-PW-containing 50% ethanol solution. BHI-YHK medium is a brain heart infusion medium (manufactured by Becton Dickinson, hereinafter referred to as "BHI medium") with 5 mg / mL yeast extract (manufactured by Becton Dickinson) and 5 μg / mL hemin (manufactured by Wako Pure Chemical Industries, Ltd.). And 0.5 μg / mL of Vitamin K ₁ (manufactured by Wako Pure Chemical Industries, Ltd.) was added to prepare the medium (hereinafter, the same applies). Next, CBP is contained by adding a CBP-containing 50% ethanol solution and an HP-PW-containing 50% ethanol solution to the BHI-YHK medium so as to have the CBP and HP-PW concentrations shown in Table 1, respectively. BHI-YHK medium containing BHI-YHK medium and HP-PW was prepared. In addition, as a control group, a test group was provided in which neither the CBP-containing 50% ethanol solution nor the HP-PW-containing 50% ethanol solution was added to the BHI-YHK medium.

(2) Preparation of periodontal disease bacteria Porphyromonas gingivalis ATCC 33277 strain (the same applies hereinafter) was used as the periodontal disease bacteria. Anaerobox (Teha type Anaero box ANX-3 type, Co., Ltd.) filled _{with anaerobic gas (70% N 2} , 15% H ₂ , 15% CO ₂ ) by inoculating Porphyromonas gingivalis in BHI-YHK medium. Inoculated at 37 ° C. for 18 hours in (Hirasawa Co., Ltd.). After culturing, the cells were collected by centrifugation to prepare a Porphyromonas gingivalis solution having an absorbance of 1.0 at a wavelength of 550 nm.

(3) Measurement of Inhibition of Fimbria Expression In the CBP-containing BHI-YHK medium and HP-PW-containing BHI-YHK medium prepared in (1) above, 10 μL of the Porphyromonas gingivalis solution prepared in (2) above was added. After inoculation and allowing to act at room temperature for 1 hour or 24 hours under anaerobic conditions, each BHI-YHK medium after culturing was subjected to a pili expression inhibition measurement test. Specifically, 1 mL of SDS sample buffer was added to each BHI-YHK medium after culturing and kept at 100 ° C. for 5 minutes to prepare each lysate sample. Each lysate sample was subjected to SDS polyacrylamide gel electrophoresis to examine the expression of each hair protein of Fima (41 kDa) and Mfa1 (67 kDa).

(4) Results The results were as shown in FIG. From the results shown in FIG. 1, it was confirmed that the CBP-containing BHI-YHK medium having a concentration of 0.01 mg / mL and higher concentrations had an inhibitory effect on the expression of Fima and Mfa1 pili. In addition, BHI-YHK medium containing 0.01 mg / mL and higher concentrations of HP-PW has an inhibitory effect on the expression of Mfa1 pili, and 0.1 mg / mL and higher concentrations of HP. It was confirmed that the -PW-containing BHI-YHK medium had an inhibitory effect on the expression of Fima pili.

Example 2: Effect of cocoa bean extract on adhesion of periodontal disease bacteria to animal cells (1) Preparation of cocoa bean extract-containing medium CBP-containing BHI-YHK medium and HP-PW-containing BHI-YHK medium are Example 1. According to the procedure described in (1), BHI-YHK medium having final medium concentrations of CBP and HP-PW of 0.001, 0.01 and 0.1 mg / mL, respectively, and CBP and HP-PW as control groups. A BHI-YHK medium to which neither was added was prepared. In addition, as a test sample, CLPr (polyphenol concentration 71% by mass), which is a fermented cacao bean extract prepared by the procedure described in Natsume et al., Biosci Biotechnol Biochem., 64, 2581-2587 (2000), was used. , A 50% ethanol solution containing CLPr is prepared according to the procedure described in Example 1 (1), and then BHI-YHK containing CLPr having final medium concentrations of CLPr of 0.001, 0.01 and 0.1 mg / mL, respectively. The medium was prepared.

(2) Preparation periodontal bacteria of periodontal bacteria, except that P. gingivalis bacterium solution the absorbance 0.3 at a wavelength of 550nm and ⁽¹⁰ 9 CFU / mL), Example 1 according to (2) Prepared according to the procedure.

(3) Measurement of Inhibitory Effect of Periodontal Disease Bacteria on Cell Adhesion Human gingival epithelial cells (HGEC cells) were used as animal cells. Human gingival epithelial cells were cultured for 48 hours under the conditions of 5% _CO 2 · 37 ℃ with KGM media 1mL per well in 24-well plates (LONZA Co., Ltd.). CBP-containing BHI-YHK medium, HP-PW-containing BHI-YHK medium, and CLPr-containing BHI-YHK medium prepared in (1) above were added to the cultured human gingival epithelial cells at 0.0001 mg / mL and 0.001 mg / mL. , 0.01 mg / mL, 0.1 mg / mL of cocoa bean extract concentration, then infused with 10 μL of Porphyromonas gingivalis bacterium solution prepared in (2) above, and 5% CO _2. The treatment was carried out under the condition of 37 ° C. for 1 hour. After washing the treated human gingival epithelial cells with PBS three times, the porphyromonas gingivalis solution prepared by destroying the epithelial cells by leaving it in 1 mL of water at room temperature for 20 minutes is appropriately diluted, and the diluted solution 0 .1 mL was smeared on BHI blood agar medium, cultured at 37 ° C. for 5 days, and then the viable cell count was measured. The BHI blood agar medium was prepared by adding 1.5% Bactagar (manufactured by Becton Dickinson) to BHI-YHK medium, sterilizing it, and then adding 5% sheep defibrated blood (Japan Biotest Research Institute).

(4) Results The results were as shown in FIG. From the results of FIG. 2, it was confirmed that CBP, HP-PW and CLPr have an inhibitory effect on the attachment of Porphyromonas gingivalis, which is a periodontal disease bacterium, to animal cells.

Example 3: Effect of unfermented cocoa bean extract on periodontal disease bacteria (1) Preparation of medium containing unfermented cocoa bean extract CBP-containing BHI-YHK medium and HP-PW-containing BHI-YHK medium are Example 1 (1). ), The final medium concentrations of CBP and HP-PW are 0.01, 0.1, 1.0 and 10 mg / mL, respectively, and BHI-YHK medium and CBP and HP-PW as control groups. A BHI-YHK medium to which neither was added was prepared.

(2) Preparation of periodontal disease bacteria The periodontal disease bacteria were prepared according to the procedure described in Example 1 (2).

(3) Measurement of bactericidal effect 10 μL of Porphyromonas gingivalis solution prepared in (2) above was added to the CBP-containing BHI-YHK medium and HP-PW-containing BHI-YHK medium prepared in (1) above and anaerobic. Under the conditions, after treating at room temperature for 1 hour, each BHI-YHK medium after the treatment was subjected to a viable cell count measurement test. Specifically, 0.1 mL of a diluted solution obtained by appropriately diluting each treated BHI-YHK medium was smeared on the BHI blood agar medium used in Example 2, cultured at 37 ° C. for 5 days, and then the viable cell count. Was measured, and the survival rate was determined according to the following formula (1).

(4) Results The results were as shown in FIGS. 3 and 4. From the results of FIGS. 3 and 4, it was confirmed that CPB and HP-PW have a bactericidal effect on the periodontal disease bacterium Porphyromonas gingivalis.

Example 4: Effect of unfermented cocoa bean extract on biofilm (1) Preparation of medium containing unfermented cocoa bean extract CBP-containing BHI-YHK medium and HP-PW-containing BHI-YHK medium are Example 1 (1). ), The BHI-YHK medium in which the final medium concentrations of CBP and HP-PW are 0.1, 1.0, and 10 mg / mL, respectively, and neither CBP nor HP-PW as a control group are added. BHI-YHK medium was prepared.

(2) Biofilm formation A circular cover glass was placed in a 24-well microplate containing 1 mL of BHI-YHK medium, and 10 μL of the Porphyromonas gingivalis solution prepared in Example 1 (2) was inoculated into the circular cover glass under anaerobic conditions. , 37 ° C. for 16.5 hours. After culturing, remove airborne bacteria in the wells with phosphate buffer (pH 7.2), replace with fresh BHI-YHK medium, incubate at 37 ° C. for 24 hours, and porphyromonas gingivalis on a cover glass. Biofilm was formed.

The CBP-containing BHI-YHK medium and HP-PW-containing BHI-YHK medium obtained in (1) above were added to the formed biofilm, allowed to act at room temperature for 1 hour under anaerobic conditions, and then LIVE / DEAD. (Brand name) BacLight (trade name) Bacterial Visibility Kit (manufactured by Thermo Fisher Scientific Co., Ltd.) was used to stain the biofilm, and the biofilm was observed with a fluorescence microscope (BZ-X700, manufactured by Keyence Co., Ltd.). Live bacteria are labeled in green and dead bacteria are labeled in red, so they can be distinguished under microscopic observation. In addition, the fluorescence micrograph was image-analyzed, and the ratio of the green area to the total area of the photograph (the ratio of viable bacteria) was calculated. Note that green corresponds to the portion shown in white to gray in FIG. 5, and red corresponds to the portion shown in dark gray to black in FIG.

(3) Results The results were as shown in FIG. From the results of FIG. 5, in the biofilm of Porphyromonas gingivalis on which CBP and HP-PW (denoted as "PW-VE-R" in FIG. 5) were allowed to act, it depends on the concentration of CBP and HP-PW. The death of biofilm-forming bacteria was observed. That is, in "Control" in which CBP and HP-PW are not added, the entire observation surface is stained green, and when 0.1 mg / mL of CBP and HP-PW are added, a portion slightly stained in red appears on the observation surface. When 1.0 mg / mL of CBP and HP-PW are added, multiple red-stained areas appear on the observation surface, and when 1.0 mg / mL of CBP and HP-PW are added, the entire observation surface turns red. It was dyed. In addition, the ratio (ratio of viable bacteria) of the green area (the part shown in white to gray in FIG. 5) to the total area of the photograph also showed a decreasing tendency as the addition concentration of the unfermented cocoa bean extract increased. From the above, it was confirmed that CBP and HP-PW have a bactericidal effect on the biofilm of Porphyromonas gingivalis, which is a periodontal disease bacterium.

Example 5: Effect of unfermented cacao bean extract on periodontal disease (1) Preparation of experimental periodontitis model Three-week-old Sprague-Dawley male rats (obtained from Claire Japan) were grouped. Specifically, the control group (hereinafter referred to as "Group I"), the Porphyromonas gingivalis infected group (hereinafter referred to as "Group II"), and the unfermented cacao bean extract intake group (hereinafter referred to as "Group III"). ) And the porphyromonas gingivalis-infected group (hereinafter referred to as "IV group") with unfermented cocoa bean extract intake and divided into 4 groups (each group n = 6). CBP was used as the unfermented cocoa bean extract. For the purpose of reducing indigenous bacteria in the oral cavity, each group was given a mixture of sulfamethoxazole with a final concentration of 1 mg / mL and trimethoprim with 200 μg / mL in ion-exchanged water for 4 days as drinking water, and then antibiotics for 3 days. After giving ion-exchanged water containing no substance, the following treatments were carried out on the 1st, 3rd and 5th days. That, I group 5% carboxymethyl cellulose solution (hereinafter "CMC") is called), II group P. gingivalis bacterium solution prepared in 5% CMC ⁽¹⁰ 9 CFU / mL) and, respectively in the rat oral Was administered to. Two hours after the administration of Group I and Group II, Group III administered a CBP solution prepared with 5% CMC so that the final concentration was 10 mg / mL into the oral cavity of the treated rats of Group I. In Group IV, a CBP solution prepared with 5% CMC so as to have a final concentration of 10 mg / mL was administered into the oral cavity of rats treated with Group II. All rats were fed in a cycle of temperature 23 ° C., humidity 60% and light and dark 12 hours with free access to food and drinking water. In addition, the body weight was measured immediately after infection with Porphyromonas gingivalis (4 weeks old), 1 week after infection with Porphyromonas gingivalis (5 weeks old), and at the time of slaughter (11 weeks old), and the average value of the body weight of each group. Asked. On the 40th day from the last administration day, all rats were sacrificed by decapitation lagoon blood under ether anesthesia. This animal infection experiment was carried out with the approval of the Kanagawa Dental University Experimental Animal Ethics Committee.

(2) Measurement of alveolar bone resorption amount The alveolar bone resorption amount was measured at 7 points from the cement enamel boundary of the upper jaw tooth portion of the rat slaughtered in (1) above to the alveolar bone apex. Specifically, the skull was heated at 2 atm for 10 minutes, immersed in a 3% sodium hypochlorite solution to remove soft tissues, and the alveolar bone was stained and dried with a 1% methylene blue solution as a sample. It was measured under a stereomicroscope at a magnification of 40 times. The average value of the measured values at 7 points was taken as the bone resorption amount per individual, and the average value of 6 animals in each group was expressed as the bone resorption amount of each experimental group in millimeters to obtain the standard deviation (SD). Statistical analysis was performed using Tukey's method.

(3) Detection of periodontal disease bacteria in the rat oral cavity by PCR To collect plaque bacteria in the rat oral cavity, wipe the rat oral cavity with a cotton swab moistened with water, and remove the bacteria attached to the cotton swab in 0.5 ml of water. The precipitate fraction obtained by suspending and centrifuging (15000 rpm, 21,500 × g for 3 minutes) was used as a plaque bacterium. DNA extraction from the Porphyromonas gingivalis standard strain and the collected plaque bacteria was carried out using ISPLANT (manufactured by Nippon Gene Co., Ltd.) according to the attached procedure manual. The extracted DNA was dissolved in 30 μL of T ₁₀ E ₁ solution and stored. For the PCR reaction, primers (5'-) reported by Ashimoto et al. (Ashimoto A. et al., Oral Microbiol Immunol. 1996; 11 (4): 266-73) prepared from the Porphyromonas gingivalis 16S rRNA gene. AGG CAG CTT GCC ATA CTG CG-3'(SEQ ID NO: 1) and 5'-ACT GTT AGC AAC TAC CGA TGT-3' (SEQ ID NO: 2), amplified fragment length 404 bp) were used. DNA was amplified by heating at 95 ° C. for 5 minutes, followed by 36 cycles of 95 ° C. for 30 seconds, 60 ° C. for 1 minute, and 72 ° C. for 1 minute. As the PCR reagent, GoTaq (trade name) Green Master Mix Gene (manufactured by Promega) was used, and gene amplification was performed using iCyller (manufactured by Bio-Rad). After completion of the PCR reaction, Smart Ladder (manufactured by Wako Pure Chemical Industries, Ltd.) was electrophoresed on a 1.5% agarose gel as a DNA sample and a molecular weight marker, and then photographed to detect an amplified DNA fragment.

(4) Results The results were as shown in FIGS. 6 and 7. From the results shown in FIG. 6, significant alveolar bone resorption was confirmed in the periodontal disease bacteria-infected group (group II) as compared with the periodontal disease bacteria-unadministered group (group I), but unfermented cacao beans after periodontal disease bacteria infection. It was confirmed that the amount of alveolar bone resorption was suppressed in the group to which the extract was administered (Group IV), and the amount of bone resorption was almost the same as that in the group to which the unfermented cacao bean extract was administered (Group III). In addition, from the results shown in FIG. 7, the periodontal disease bacteria in the oral cavity were reduced in the group (Group IV) to which the unfermented cacao bean extract was administered after the periodontal disease bacteria infection as compared with the periodontal disease bacteria-infected group (Group II). It was confirmed that it would be done. No difference was observed in the body weight of each group during the experiment period (data not shown).

Claims (9)

A composition for the oral cavity containing a cocoa bean extract, which is an inhibitor of the adhesion of periodontal disease bacteria to animal cells. A composition for the oral cavity containing a cocoa bean extract, which is an agent for suppressing the expression of pili of periodontal disease bacteria. The composition according to claim 1 or 2 , wherein the cocoa bean extract is an unfermented cocoa bean extract. The composition according to claim 3 , which is an antibacterial agent against periodontal disease bacteria and / or halitosis-causing bacteria. The composition according to claim 3 or 4 , for use in the treatment, prevention or amelioration of periodontal disease and related diseases. The composition according to claim 3 or 4 , for use in suppressing bad breath. Periodontal disease bacteria include Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigresens, Treponema denticola, Tannerella forsysensis, Aggregatibacter actinomycetemcomitans, Campylobacter rectus and Frobacterium nucrateum. The composition according to claim 1, 2 or 4 , which is one or more selected from the group consisting of. Halitosis causing bacteria is one or more compounds selected from the group consisting of periodontal bacteria described in Fusobacterium nucleatum and Beironera-disperser and 7. The composition of claim 4. The composition according to any one of claims 1 to 8 , which is a food composition.

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