JP3254553B2 - Anti-caries material, its production method and use - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an anti-cariogenic material obtained from hop bracts, a method for producing the same and a use thereof.

[0002]

2. Description of the Related Art Hops are perennial plants of the mulberry family, and their cones (mature of unfertilized female flowers) are generally called hops. The lupulin portion of this hop (yellow granules formed at the root of the inner bract of the cone) is the main component of the bitterness and aroma of the hop, and is an important beer raw material along with yeast and malt in beer brewing. Hops are also used in folk medicine as a sedative and anti-aphrodisiac. It is said that the main components of the various actions of these hoppurine moieties are bitter acids such as α-acids and β-acids. 1988
No. 219), antibacterial action (JP-A-06-98738),
Inhibition of protease action useful for prevention or treatment of periodontal disease (JP-A-06-25000), antioxidant action (JP-A-04-202138, JP-A-06-025081, JP-A-06-313924), superoxide elimination The function (JP-A-04-5237) has already been clarified.

On the other hand, hop bracts are obtained by removing lupulin from hop cones, and are not useful for beer brewing. In some cases, hop bracts are removed during beer brewing and are produced as by-products. At that time, hop bracts have not been found to be particularly effective in use other than being used as a fertilizer for soil improvement, and it has been desired to develop a use method with higher added value. It should be noted that Japanese Patent Application No.
No. 7-173931, it has been confirmed that polyphenol preparations derived from hops, especially hop bracts, have an antioxidant effect. In addition, Japanese Patent Application No.
In No. 7-347151, an extract derived from hops, particularly hop bracts, is used as a foam stabilizing agent in a sparkling malt beverage.

[0004] Green tea extracts (Japanese Unexamined Patent Publication No. 01-9922, Japanese Unexamined Patent Publication No. 01-90124, Japanese Unexamined Patent Publication No.
265010), oolong tea extract (JP-A-03-284625), apple polyphenol (JP-A-07-285876), and the like are commercially available.

[0005]

[Problems to be Solved by the Invention] Dental caries, so-called caries, are said to have existed before 2,000 B.C., but they are often not directly related to life. Not in. However, according to the Ministry of Health and Welfare Survey on Dental Diseases, about 85% of Japanese people have dental caries, which is a so-called national disease.

[0006] Dental caries is considered to be a disease caused by bacteria. That is, so-called "caries bacteria" such as S. mutans and S. sobrinus existing in the oral cavity
However, it produces a glucan that is more sticky and insoluble than sucrose, and adheres to the tooth surface together with the bacterial cells to form plaque. Next, various bacteria such as dental caries grow in this plaque, the pH of the tooth surface decreases due to the organic acid generated by the metabolism of the bacteria, and the enamel of the teeth dissolves out (demineralization). It is said that a hole is formed and a tooth is formed.

[0007] Methods for preventing dental caries include a method for strengthening the dentin using fluorine, a method using an antibacterial substance against cariogenic bacteria, a method using a sugar substitute that does not produce glucan, instead of sucrose, A method using an inhibitor of an enzyme that produces lipase is already known. However, each method has advantages and disadvantages in terms of effect, cost, nutritional effects, etc., and it is hard to say that it is a perfect method considering that the incidence of caries has not actually decreased. . Therefore, if an excellent method for preventing dental caries was developed, it would have great industrial significance.

Accordingly, an object of the present invention is to provide an anti-cariogenic material having a higher activity than an anti-cariogenic material currently available on the market, which contains naturally occurring polyphenols as an active ingredient. More specifically, the present invention is used in foods and drinks and oral preparations.

[0009]

Means for Solving the Problems As a result of intensive studies on the above problems, the present inventors have obtained polyphenol-like substances contained in hop bracts by treating them with a gel-type synthetic resin or an ultrafiltration membrane. Has been found to be a substance that strongly inhibits adhesion of glucan and dental caries to the tooth surface, that is, a substance that can be used as an anti-cariogenic material. Further, the present invention has been completed by using this substance as an anti-cariogenic material in foods and drinks and oral preparations.

[0010] That is, the first aspect of the present invention relates to a polyphenol-like substance contained in hop bracts, which is a substance which is adsorbed to a gel-type synthetic resin, and the second is a polyphenol contained in hop bracts. With similar molecular weight cutoff
The third is a polyphenol-like substance contained in hop bracts, which is a substance that adsorbs to a gel-type synthetic resin, with respect to an anti-cariogenic material that is a substance that does not pass through the membrane when treated with an ultrafiltration membrane of 1,000 or more, The present invention also relates to an anti-cariogenic material that is a substance that does not pass through a membrane when treated with an ultrafiltration membrane having a molecular weight cut-off of 1,000 or more.

[0011] The anti-cariogenic material in the first invention is:
Hop bracts are extracted with water or an aqueous solution of an organic solvent miscible with water, and the extract is passed through a gel-type synthetic adsorbent, washed with an aqueous solution of water or an organic solvent miscible with water, and then mixed with water. It is produced by eluting a fraction adsorbed on the adsorbent with an aqueous solution of an organic solvent (a method using a gel-type synthetic adsorbent).

The anticariogenic material according to the second invention is characterized in that hop bracts are extracted with water or an aqueous solution of an organic solvent miscible with water, and the extract is treated with an ultrafiltration membrane having a molecular weight cut-off of 1,000 or more. In addition, it is manufactured by removing the solvent from the remaining liquid (method using an ultrafiltration membrane). The anti-caries material of the third invention is characterized by being obtained by combining the method using the gel-type synthetic adsorbent and the method using the ultrafiltration membrane. The present invention also relates to foods and drinks containing the above-mentioned anti-cariogenic material, and oral preparations.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The hop bract, which is a raw material of the present invention, is obtained by removing the lupulin part from hop cones. Generally, hop cones are pulverized and then sieved to remove the lupulin part. Get hop bracts. However, in recent beer brewing, hop bracts are formed into pellets without removing hop bracts that are not useful for beer brewing, so as to eliminate the trouble of sieving and removing hop bracts. Tend to use it. Therefore, the raw material of the present invention is not particularly limited as long as it contains hop bracts, and there is no problem even if hop cones or hop pellets containing hop bracts are used as raw materials.

As a method for producing an anti-cariogenic material, first, hop bracts or hop cones or hop pellets containing hop bracts are mixed with water or water such as alcohol, acetone, acetonitrile or the like at 50 v / v% or less. Extract with an aqueous solution of a miscible organic solvent. Preferred examples include water or ethanol containing 50 v / v% or less of ethanol.
The ratio between the raw material and the extraction solvent is 1:20 to 100 (weight ratio).
The extraction is preferably carried out at a temperature of 4 to 95 ° C. under stirring, and the
It is desirable to perform for about 60 minutes. An extract is obtained by filtration. If necessary, a filter aid such as pearlite can be used.

[0015] The extract thus obtained is treated with a gel-type synthetic adsorbent and / or an ultrafiltration membrane. Treatment with gel-type synthetic adsorbent For the extract of the raw material obtained in the above extraction step, an adsorption step of adsorbing the anti-cariogenic material to the gel-type synthetic adsorbent, water or ethanol aqueous solution, preferably 1 to 10 v / v% A washing step of washing the gel-type synthetic adsorbent with an aqueous ethanol solution, and an elution step of eluting an adsorbed fraction from the gel-type synthetic adsorbent with an aqueous ethanol solution or ethanol of 30 v / v% or more are performed to obtain an anti-carious material.

In the adsorption step, the extraction solution is heated at 15 to 30 ° C.
After cooling to about room temperature, the solution is passed through a column filled with a gel-type synthetic adsorbent, and the anti-cariogenic material is adsorbed on the adsorbent. At this time, if necessary, it is desirable to lower the organic solvent concentration of the extract to 10% or less in advance by concentration under reduced pressure or the like in order to increase the adsorption efficiency. Materials for the gel-type synthetic adsorbent include hydrophilic vinyl polymers,
Examples thereof include hydroxypropylated dextran and styrene-divinylbenzene polymer. The liquid passing time is preferably set so that the SV value is between 0.5 and 100. The SV value here is a value defined by the following equation.

SV value = (flowing amount (L)) / ｛(resin amount (L))
× (Pouring time (h)) 洗浄 The washing step is a step of washing the gel-type synthetic adsorbent holding the anti-cariogenic material. This step removes contaminants and improves the degree of purification of the anti-cariogenic material. It is possible to give. As a solvent used for washing, water to 1 to 10 v / v%
Is preferable, and it is desirable to wash by passing a solvent amount of about 1 to 10 times the resin amount.

The elution step is a step of desorbing and eluting the anti-cariogenic material from the gel-type synthetic adsorbent holding the anti-cariogenic material. As the solvent used for elution, use is made of hydroalcohol, hydroacetone, hydroacetonitrile and the like. It is possible,
Particularly preferred examples include 30 v / v% or more aqueous ethanol solution or ethanol. The flow rate of the elution solvent is preferably about 1 to 6 times the amount of the resin.

The obtained eluate is concentrated under reduced pressure, lyophilized,
The anti-caries material can be obtained as a powder by removing the solvent by a usual method such as spray drying. At the time of concentration under reduced pressure, a solvent such as alcohol, acetone, or acetonitrile can be recovered and reused. The anti-cariogenic material thus obtained is a slightly bitter, odorless flesh-colored, brown or pale yellow powder, the anti-cariogenic body of which is adsorbed on the gel-type synthetic resin and fractionated. Molecular weight
It is a polyphenol-like substance that does not permeate through a membrane when treated with 1,000 or more ultrafiltration membranes.

The yield is 0.5 to 100% in terms of hop bract weight.
20.0 w / w%, 0.5 to 15.
0 w / w%. The gel-type synthetic adsorbent used was 80 v / v
It can be used repeatedly after washing with an aqueous alcohol solution of at least%, an aqueous solution of sodium hydroxide of about 0.05N or the like.

Treatment with Ultrafiltration Membrane The hop bract extract obtained in the above extraction step is treated with an ultrafiltration membrane having a molecular weight cut-off of 1,000 or more. At that time, if necessary, the extract can be concentrated under reduced pressure to reduce the concentration of the organic solvent. The recovered organic solvent can be reused. As a material for the membrane, any material that is usually used as a material for an ultrafiltration membrane, such as cellulose, cellulose acetate, polysulfone, polypropylene, polyester, polyethersulfone, and PVDF, can be used without any particular limitation. In addition, if the molecular weight cut-off is 1,000 or more, it can be used without any problem.However, if a membrane having a too high molecular weight cut-off is used, the yield is extremely reduced,
When the molecular weight cut-off is small, the time required for the treatment is prolonged. Therefore, an ultrafiltration membrane having a molecular weight cut-off of 10,000 to 20,000 is suitable. The treatment is preferably performed until the amount of the remaining liquid is about 1/10 to 1/100 of the time when the treatment is started, although it depends on the type of the extraction solvent and the ratio of the extraction solvent to the hop or hop bract. The pressure at that time depends on the ultrafiltration membrane and the filtration device, but is preferably about 0.1 to 10.0 kg / cm ² . If necessary, after the treatment, the remaining liquid can be diluted again with a suitable solvent such as water, and then re-treated in the same manner to increase the degree of purification.

The anti-caries material can be obtained as a powder by removing the solvent of the obtained upper residue by a conventional method such as concentration, freeze-drying and spray-drying. At the time of concentration under reduced pressure, a solvent such as alcohol, acetone, or acetonitrile can be recovered and reused. The anti-cariogenic material thus obtained is a slightly bitter, odorless flesh-colored, brown or pale yellow powder, the anti-cariogenic body of which is adsorbed on the gel-type synthetic resin and fractionated. Molecular weight 1,
It is a polyphenol-like substance that does not permeate through a membrane when treated with 000 or more ultrafiltration membranes.

The yield is 0.5 to 100% in terms of hop bract weight.
20.0 w / w%, 0.5 to 15.
0 w / w%. Treatment with the gel-type synthetic adsorbent,
Alternatively, since the active substance of the anti-cariogenic material obtained by the treatment with the ultrafiltration membrane is the same polyphenol, the anti-cariogenic material obtained by the treatment with the gel-type synthetic adsorbent is appropriately treated with an aqueous alcohol solution or the like. It can be dissolved in a solvent and treated with an ultrafiltration membrane to further enhance the degree of purification of the polyphenols that are the active substance. The reverse is also possible, and a sufficiently useful anti-cariogenic material can be obtained by or of course.

[0024] The obtained anti-cariogenic materials are foods and drinks such as confectionery, foods and beverages, particularly preferably candy,
It can be used for foods and drinks having a relatively long oral residence time, such as chocolate, caramel and chewing gum. It can also be used by adding it to oral preparations such as gargles and dentifrices. When adding an anti-cariogenic material to these foods and drinks and oral preparations, the anti-cariogenic material may be added as powder, but preferably, the anti-cariogenic material is 1 to 2% aqueous solution or A solution of alcohol aqueous solution or alcohol solution, the final concentration is 1 to food and drink or oral agent
It is desirable to add so as to be 500 ppm, preferably 10 to 100 ppm.

As described above, the use of a hop extract as an anticariogenic material has already been disclosed in Japanese Patent Application Laid-Open No. 62-211219. According to the publication, "S.mutans
A hop extract, an isomerized product, and a metal salt thereof, which can effectively suppress the growth of bacteria and suppress the production of insoluble glucan, can be provided as an agent for preventing dental caries. " However, unlike the antibiotics, the product of the present invention has no antibacterial property and bacteriostatic property against S. mutans bacteria, and thus prevents caries without affecting the state of the natural flora in the oral cavity. It is possible. Further, α-acids (humulones), β-acids, which are mentioned as examples of hop extracts in JP-A-62-211219,
Acids (lupulones) are the main components of the hop lupulin part of the hop, and are not contained in the hop bract part, which is the raw material of the product of the present invention. Further, Japanese Patent Application Laid-Open No. 62-211219 states that the compounding amount of the caries preventive agent is "effectively exhibits a caries preventive effect at 0.01 to 20% by weight, preferably 0.1 to 10% by weight". (0.1 wt% is 1000 ppm
), The product of the present invention is 1/10 to 100
The effect is shown to be effective with a 1/1 compounding amount.

[0026]

The present invention is not limited to the following examples. Example 1 (Preparation of anti-cariogenic material from hop cones using gel-type synthetic adsorbent) 20 g of hop cones was ground in a mortar and extracted with 2 liters of water at 95 ° C for 40 minutes with stirring. After filtration, the mixture is allowed to cool, and the extract is subjected to hydrophilic vinyl polymer resin (Toyopearl HW4 manufactured by Tosoh Corporation).
0) Pass the solution through a column packed with 80 ml for 2 hours (SV
= 12.5) and then washed with 400 ml of 5% aqueous ethanol. Further, 400 ml of an 80% aqueous ethanol solution was passed through the column, and the eluate was collected and freeze-dried to obtain 800 mg of an anticariogenic material as a pale yellow powder having an odorless and slightly bitter taste. The yield from hops was 4%.

Example 2 (Preparation of anti-cariogenic material from hop bract using gel-type synthetic adsorbent) 20 g of hop bract was stirred with 600 ml of 50% ethanol aqueous solution.
Extraction was performed at 80 ° C. for 40 minutes. After filtration, the solution was concentrated under reduced pressure to a volume of 300 ml, and the concentrated solution was passed through a column filled with 80 ml of styrene-divinylbenzene resin (Sepabeads 825, manufactured by Mitsubishi Chemical Corporation) for 1 hour (SV = 3.75). 40
Washed with 0 ml water. Further, 400 ml of an 80% aqueous ethanol solution was passed through the column, and the eluate was collected and freeze-dried to obtain 1.6 g of an anti-cariogenic material as a pale yellow powder having an odorless and slightly bitter taste. The yield from hop bracts was 8%.

Example 3 (Preparation of an anti-cariogenic material from hop cones using an ultrafiltration membrane) 20 g of hop cones was ground in a mortar and extracted with 2 liters of water at 95 ° C for 40 minutes with stirring. After filtration, the mixture is allowed to cool, and the extract is subjected to ultrafiltration membrane having a molecular weight cutoff of 50,000 (XM50 manufactured by Amicon).
To 1.0 kg / cm ² at room temperature until the volume became 20 ml. The obtained upper remaining liquid was dried under reduced pressure, and the anti-caries material 200m
g was obtained as an odorless, slightly bitter, pale yellow powder. The yield from hops was 1%.

Example 4 (Preparation of an anti-cariogenic material from hop bracts using an ultrafiltration membrane) 20 g of hop bracts were stirred with 600 ml of a 50% aqueous ethanol solution.
Extraction was performed at 80 ° C. for 40 minutes. After filtration, the extract has a molecular weight cut off.
3.0k by ultrafiltration membrane of 10,000 (YM10 manufactured by Amicon)
The treatment was performed at 60 g / cm ² at room temperature until the volume reached 60 ml. The obtained upper residue was freeze-dried to obtain 0.8 g of an anti-cariogenic material as a pale yellow powder having an odorless and slightly bitter taste. The yield from hop bracts was 4%.

Example 5 (Preparation of an anti-cariogenic material from Example 2 using an ultrafiltration membrane) 0.8 g of the anti-cariogenic material obtained in Example 2 was dissolved in 500 ml of a 10% aqueous ethanol solution and fractionated. With an ultrafiltration membrane with a molecular weight of 10,000 (YM10 manufactured by Amicon), 1.0 kg / cm ² ,
Processed until 0 ml. The obtained upper residue was freeze-dried to obtain 0.4 g of an anti-cariogenic material as a pale yellow powder having an odorless and slightly bitter taste. When this powder was analyzed by HPLC, a characteristic chromatogram as shown in FIG. 1 was obtained.
When catechin was quantified by the method described in Official Food Analysis, it was converted to catechin content to give a value of 40.6%. (HPLC conditions) Apparatus: Shimadzu LC-10A system, column: ODS-80TM (Tosoh, 4.6 mm ID × 25 cm), mobile phase:
(Solution A: Solution B) = Linear gradient for 30 minutes from (100: 0) to (50:50), Solution A: 5% acetonitrile (containing 0.1% HCl), Solution B: acetonitrile, Flow rate: 1.0 m
l / min, sample injection amount: 20 μg, detection: multi-wavelength detection at 200 to 300 nm.

Example 6 (Estimation of Molecular Weight of Example 5 by Gel Filtration) Gel filtration was performed under the following HPLC conditions, and the molecular weight of the anti-caries material obtained in Example 5 was estimated. Comparing the retention time with the calibration curve, the molecular weight is approximately 36,000 to 40,000. (HPLC conditions) Apparatus: Shimadzu LC-8A system, column: Tosoh TSK-GEL G4000PWXL, G5000PWXL, mobile phase:
0.1 M Tris-HCl buffer (pH 10.0): 2-propanol = 1: 1
, Detection: UV220nm, Marker: LMW GEL FILTRATION KIT
(Pharmacia Biotech), flow rate: 0.3 ml / min. Example 7 (Dentifrice) Dibasic calcium phosphate 42.0 Glycerin 18.0 Carrageenan 0.7 Sodium lauryl sulfate 1.2 Saccharin sodium 0.09 Butyl p-hydroxybenzoate 0.005 Material obtained in Example 1 0.005 Fragrance 1.0 Water 37.0 Total 100.0 A dentifrice was prepared using the above-mentioned components in each part by weight in accordance with a conventional method. In addition, instead of the raw material obtained in Example 1, dentifrices to which the raw materials obtained in Examples 2, 3, 4, and 5 were respectively added were similarly obtained.

Example 8 (Mouthwash) Glycerin 7.0 Sorbitol 5.0 Ethyl alcohol 15.0 Sodium lauryl sulfate 0.8 Saccharin sodium 0.1 1-Menthol 0.05 Fragrance 0.045 Obtained in Example 1 Material 0.005 Water 72.0 Total 100.0 The mouthwash was used according to a conventional method using the above components of each part by weight. Note that, instead of the material obtained in Example 1, mouthwashes to which the materials obtained in Examples 2, 3, 4, and 5 were added were also obtained in the same manner.

Example 9 (troche) Gum arabic 6.0 Magnesium stearate 3.0 Glucose 73.0 Lactose 17.6 Dipotassium phosphate 0.2 Potassium potassium 0.1 Spice 0 095 Raw material obtained in Example 1 0.005 Total 100.0 Using the above components in each part by weight, a troche was prepared according to a conventional method. In addition, instead of the raw material obtained in Example 1, troches prepared by adding the raw materials obtained in Examples 2, 3, 4, and 5 were obtained in the same manner.

Example 10 (candy) sucrose 20.0 starch syrup (75% solid content) 70.0 water 9.5 coloring agent 0.45 fragrance 0.045 material obtained in Example 1 0.005 total 100.0 A candy was prepared according to a conventional method using each of the above parts by weight. In addition, instead of the raw material obtained in Example 1, candy to which the raw materials obtained in Examples 2, 3, 4, and 5 were respectively added was similarly obtained.

Example 11 (Chewing gum) Gum base 20.0 Calcium carbonate 2.0 Lactose 77.0 Stevioside 0.095 Material obtained in Example 1 0.005 Fragrance 0.9 Total 100.0 Each of the above Chewing gum was prepared according to a conventional method using parts by weight of each component. In addition, instead of Example 1, chewing gums to which Examples 2, 3, 4, and 5 were respectively added were similarly obtained.

Example 12 (Juice) Concentrated tangerine juice 15.0 Fructose 5.0 Citric acid 0.2 Flavor 0.1 Color pigment 0.15 Sodium ascorbate 0.048 Material obtained in Example 1 0.0 002 Water 79.5 Total 100.0 Using the above parts by weight of each component, a juice was prepared according to a conventional method. Instead of the raw material obtained in Example 1, juices to which the raw materials obtained in Examples 2, 3, 4, and 5 were respectively added were obtained in the same manner.

Example 13 (Cookies) Soft flour 32.0 Whole egg 16.0 Butter 16.0 Sugar 25.0 Water 10.8 Baking powder 0.198 Material obtained in Example 1 0.002 Total 100. 0 Using the above parts by weight of each component, a cookie was prepared according to a conventional method. In addition, instead of the material obtained in Example 1, cookies to which the materials obtained in Examples 2, 3, 4, and 5 were added were obtained in the same manner.

Example 14 (Caramel) Granulated sugar 31.0 starch syrup (75% solid content) 20.0 Powdered milk 40.0 Hardened oil 5.0 Food salt 0.6 Flavor 0.025 Obtained in Example 1. Material 0.005 Water 3.37 Total 100.0 Using the above components in each part by weight, caramel was prepared according to a conventional method. Note that, instead of the material obtained in Example 1, caramels to which the materials obtained in Examples 2, 3, 4, and 5 were respectively added were similarly obtained.

Comparative Example 1 (Preparation of Hop Pellet Extract) 20 g of hop pellets (produced by Halatau, Germany) were ground in a mortar and extracted with 600 ml of 50% ethanol at 80 ° C. for 40 minutes.
After extraction, the mixture was filtered, and the extract was centrifuged at 3,000 rpm for 15 minutes.
The supernatant was freeze-dried to obtain 6.3 g of a hop pellet extract as a pale green powder.

Comparative Example 2 (Purification of α-acid and β-acid) The peaks of α-acid (Humuron) and β-acid (Lupron) separated from the mixture of hop bitter components purchased from the Swiss Brewing Institute under the following HPLC conditions were analyzed. Taked and purified. The yield of hop bitter component from the mixture is 8% for α-acid and 5.5% for β-acid
Met. (HPLC conditions) Apparatus: Shimadzu LC-8A system, Column: Shim-pack PREP-ODS (20 mm ID × 25 cm), mobile phase: 80
% Acetonitrile aqueous solution, flow rate: 10 ml / min.

Example 15 (Anti-cariogenic activity: effect of suppressing the adhesion of dental caries) The commercially available oolong tea-derived material (san oolong: Suntory), green tea-derived material (Sunflavon HV:
Taiyo Kagaku, Polyphenon 60: Mitsui Norin), apple-derived material (Applephenone: Nikka Whiskey), epigallocatechin gallate (Kurita Kogyo), a typical natural product having anti-carious properties, hop pellets obtained in Comparative Example 1 The extract, α-acid (humulone) and β-acid (lupulone) obtained in Comparative Example 2 were analyzed by the method of Hamada et al. (S. Hamada & M. Torii,
Infect. Immn., 20, 592-596). S. mutans (RIKEN preserved strain 5175 MT8148) and S. sobrinus (A.
TCC stock strain 33478) was used. 0.5mg / ml caries 2m
l, 0.2 ml of 10% sucrose (1% for S. sobrinus) and 0.2 ml of each concentration of test substance (all 50 mM phosphate buffer (pH 6.8)
In a test tube inclined at 30 ° C, at 37 ° C,
Incubated for 18 hours. After gently removing the mixture,
The tubes were gently rinsed with 3 ml of phosphate buffer.
The amount of bacterial cells adhering to the mixed solution, the washing solution and to the test tube wall was measured by turbidity at 550 nm.
₅₀ : μg / ml). Table 1 shows the results. The material obtained in Comparative Example 1, the α-acid and the β-acid show no activity, and also shows the IC _{50 of} epigallocatechin gallate and a commercially available anti-cariogenic material.
There compared to was 20~200μg / ml, anti-cariogenic material obtained in Examples 1 to 5 showed a very strong anti-caries activity of an IC ₅₀ of approximately 2~15μg / ml.

Table 1 Sample of S. mutans S. sobrinus Adhesion Suppressing Activity (IC ₅₀ : μg / ml) of Caries Fungi Material obtained in Example 1 3.2 11.1 Material obtained in Example 2 2.6 10.6 Material obtained in Example 3 Material 2.5 14.1 Material obtained in Example 4 10.4 13.9 Material obtained in Example 5 1.5 8.5 Oolong tea-derived material 20.2 118.5 Green tea-derived material 1 26.6 170.6 Green tea-derived material 2 42.0 208.5 Apple-derived material 64.3 148.3 Epigallocatechin gallate 22.4 69.7 Material obtained in Comparative Example 1>200> 400 α-acid (humulone)>200> 400 β-acid (Lupron)>200> 400

Example 16 (Anti-cariogenic growth inhibiting activity) The anti-cariogenic material obtained in Examples 1 to 5, the hop pellet extract obtained in Comparative Example 1, the α-acid (humulone) obtained in Comparative Example 2,
Regarding β-acid (Lupron) and streptomycin (Streptomycin sulfate: Meiji Seika Co., Ltd.), a typical antibacterial substance, S. mutans, a typical strain of caries bacteria,
(RIKEN preserved strain 5175 MT8148) was examined for its effect on growth. That is, 1% sucrose added Todd He
0.1% (1000 ppm) of each test substance was added to the witte broth medium, and S. mutans was propagated with a platinum loop and cultured at 37 ° C. for 20 hours. Water was used as a control. As the bacteria grow, the turbidity of the medium increases, and the metabolism of the bacteria produces organic acids,
The pH of the medium drops. Changes in turbidity (OD660nm) and pH before and after culture were measured and used as an index of bacterial growth. Table 2 shows the results. The materials obtained in Comparative Example 1, the α-acid and the β-acid completely inhibited the growth of bacteria, but the materials obtained in Examples 1 to 5 did not affect the growth of caries.

Table 2 Influence samples on growth of S. mutans Turbidity increase value pH before culture pH control after culture (water) 0.302 7.67 4.28 Material obtained in Example 1 0.288 7.73 4.55 Obtained in Example 2 Material 0.305 7.68 4.37 Material obtained in Example 3 0.297 7.71 4.26 Material obtained in Example 4 0.300 7.66 4.58 Material obtained in Example 5 0.292 7.69 4.65 Material obtained in Comparative Example 1 0.001 7.68 7.69 α-acid (humulone) 0.002 7.66 7.62 β-acid (Lupron) 0.000 7.71 7.71 Streptomycin 0.001 7.68 7.66

Example 17 (Antibacterial activity by MIC measurement) The anti-cariogenic material obtained in Examples 1 to 5, the hop pellet extract obtained in Comparative Example 1, the α-acid (humulone) obtained in Comparative Example 2,
Regarding β-acid (Lupron) and streptomycin (Streptomycin sulfate: Meiji Seika Co., Ltd.), a typical antibacterial substance, S. mutans, a typical strain of caries bacteria,
The minimum effective inhibitory concentration (MIC) for (RIKEN stock strain 5175 MT8148) was measured. The medium is Todd Hewitte B
Culture was performed at 37 ° C. for 2 days using a roth medium. Table 3 shows the results. The material obtained in Comparative Example 1, α acid and β acid
While the MIC of 〜100 μg / ml was shown, the materials obtained in Examples 1 to 5 did not inhibit the growth of caries even at 100 to 1000 times the amount. Table 3 Antibacterial activity (μg / ml) sample against carious bacteria MIC Materials obtained in Example 1> 30 Materials obtained in Example 2> 30 Materials obtained in Example 3> 30 Materials obtained in Example 4> 30 Material obtained in Example 5> 30 Material obtained in Comparative Example 1 5.0 α-acid (humulone) 1.0 β-acid (Lupron) 1.0 streptomycin 0.5

Example 18 (Inhibitory Effect on Insoluble Glucan-forming Enzymes of Dental Caries) The anti-cariogenic material obtained in Examples 2 and 5, a commercially available oolong tea-derived material (Sunoolon: Suntory), and a representative anti-cariogenic material Nakahara et al.'S method for epigallocatechin gallate (Kurita Kogyo Co., Ltd.)
S. Kawabata, H. Ono, K. Ogura, T. Tanaka, T. Ooshim
a, and S. Hamada, Appl.Environ.Microbiol., 59, 9
68-973 (1993)), the inhibitory effect of caries on the insoluble glucan-forming enzyme was examined. S. mutans, a typical caries fungus (RIKEN preserved strain 5175 MT814)
8) and S. sobrinus (ATCC stock strain 33478) were respectively cultured, and the cells and the culture supernatant were separated by centrifugation. For S. mutans, the cells were treated with 8M urea, and the treated solution was dialyzed to obtain a crude solution of an insoluble glucan-forming enzyme.
For S. sobrinus, the culture supernatant was salted out with 50% ammonium sulfate, and the precipitate was dialyzed to obtain a crude solution of an insoluble glucan synthase. These enzyme crude solutions, test samples, sucrose (final concentration 50
mM) in a total volume of 750 μl of 100 mM potassium phosphate buffer (pH 6.5)
And incubated at 25 ° C. for 16 hours. After incubation, the mixture was centrifuged at 15,000 rpm for 10 minutes, and after removing the supernatant, the precipitate was washed with 100 mM potassium phosphate buffer.
It was washed three times with a 1: 1 mixture of (pH 6.5) and ethanol. The precipitate was dissolved in a 1N aqueous solution of sodium hydroxide, and saccharide was quantified by the phenol-sulfuric acid method. S.mutans and S.soburi
Figure 2 (a) shows the inhibition rate of each material against nus.
This is shown in (b). As a result, the anti-cariogenic materials obtained in Example 2 and Example 5 exhibited stronger enzyme inhibitory activities than the oolong tea-derived material and epigallocatechin gallate. (In the figure, Δ: Example 5, Δ: Example 2, ▲: Epigallocatechin gallate, ×: Material derived from oolong tea.) As a result of Examples 14 to 16, the product of the present invention was antibacterial against carious bacteria It has no bacteriostatic and bacteriostatic properties and strongly inhibits the adhesion of dental caries to the tooth surface. Example 1
According to 7, the effect is considered to be an inhibitory effect on the insoluble glucan-forming enzyme.

[0047]

According to the present invention, an anti-cariogenic material which is much more active than a commercially available anti-cariogenic material can be obtained from hop bracts and hop cones containing the hop bracts as raw materials. Furthermore, it could be easily used as a plant-derived anti-cariogenic material, food and drink, or oral agent.

[Brief description of the drawings]

FIG. 1 is a chromatogram of the material obtained in Example 5 by HPLC analysis.

FIG. 2 is a graph showing the difference in enzyme inhibitory activity between a product of the present invention and a commercial product in the relationship between the inhibition rate (%) and the concentration (μ / ml).

Continued on the front page (51) Int.Cl. ⁷ Identification code FI A23L 2/02 A23L 2/02 B A61K 7/26 A61K 7/26 9/20 9/20 A61P 1/02 A61P 1/02 (58) Survey Field (Int.Cl. ⁷ , DB name) A61K 35/78 A21D 2/36 A21D 13/08 A23G 3/00 101 A23G 3/30 A23L 2/02 A61K 7/26 A61K 9/20 BIOSIS (DIALOG) CA (STN)

Claims (12)

(57) [Claims] An anti-cariogenic material which is a polyphenol-like substance contained in hop bracts and which is adsorbed on a gel type synthetic resin. 2. An anti-cariogenic material which is a polyphenol-like substance contained in hop bracts and which does not permeate through an ultrafiltration membrane having a molecular weight cut-off of 1,000 or more. 3. A polyphenol-like substance contained in hop bracts, which is a substance adsorbed on a gel-type synthetic resin and which does not permeate through an ultrafiltration membrane having a molecular weight cut-off of 1,000 or more. Certain anti-caries material. 4. The hop bract is extracted with water or an aqueous solution of an organic solvent miscible with water, and the extract is passed through a gel-type synthetic adsorbent and washed with water or an aqueous solution of an organic solvent miscible with water. A method for producing an anti-caries material, characterized in that the fraction adsorbed on the adsorbent is eluted with an aqueous solution of an organic solvent miscible with water to obtain the anti-caries material according to claim 1. 5. A hop bract is extracted with water or an aqueous solution of an organic solvent miscible with water, and the extracted solution has a molecular weight cut-off of 1,00.
A method for producing an anti-cariogenic material, characterized in that the material is treated with zero or more ultrafiltration membranes and the solvent is removed from the remaining solution to obtain the anti-cariogenic material according to claim 2. 6. A method for producing an anti-caries material, comprising obtaining the anti-caries material according to claim 3 by combining the method according to claim 4 and the method according to claim 5. 7. An active ingredient comprising the anti-cariogenic material according to claim 1.
Foods and beverages for caries including as . 8. An anti-cariogenic material according to claim 2 as an active ingredient.
Foods and beverages for caries including as . 9. An anti-cariogenic material according to claim 3, which is an active ingredient.
Foods and beverages for caries including as . 10. An oral preparation comprising the anti-cariogenic material according to claim 1. An oral agent comprising the anti-cariogenic material according to claim 2. 12. An oral preparation comprising the anticariogenic material according to claim 3.