JPH07242556A - Dental caries-resistant lavonoid polymer and glucosyl transferase-inhibiting agent and food containing the same active ingredient - Google Patents

Abstract

PURPOSE:To provide the subject safe polymer obtained by condensing a specific compound with a catechin compound, having a strong glucosyl transferase- inhibiting action, preventing the formation of bacterial plaques or the generation of dental caries, and useful for a dental caries-resistant agent as an oral cavity hygienic agent or a food additive agent. CONSTITUTION:This polymer is obtained by condensing (A) the reduction product of a flavonone compound, the reduction product of a flavonol compound, or a leucoanthocyanidin compound with (B) a catechin compound such as (+)-epi(gallo)catechin, (-)epi(gallo)catechin or (-)-epi(gallo)catechin gallate and has a weight-average mol.wt. of 1,000-10,000. The objective polymer is preferably obtained by condensing 1 pt.wt. of the component A with 0.1-0.5 pt.wt. of the component B in the presence of hydrochloric acid, sulfuric acid, boron trifluoride, etc.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an anti-caries flavonoid polymer produced by condensing a reduced product of flavanones or flavanonols or leucoanthocyanidins with catechins and a glucosyltransferase inhibitor containing the same. And related to food.

[0002]

2. Description of the Related Art Although various hypotheses have been proposed in the past regarding the cause of dental caries, it is now recognized as a kind of bacterial infectious disease based on Miller's theory of chemical bacteria.
The caries generation mechanism based on this theory is as follows. That is, oral streptococci, especially Streptococcus
First, an enzyme called glucosyltransferase produced by mutans (Streptococcus mutans) produces a sticky, insoluble polysaccharide (glucan) using sucrose in the mouth as a substrate. Streptococcus mutans (hereinafter referred to as "S. mutans") cells are
The generated glucan attaches to the tooth surface and forms a bacterial mass (plaque).

In this dental plaque, various microorganisms including S. mutans coexist and propagate. The metabolism of these microorganisms produces organic acids, and the action of these organic acids lowers the pH of the tooth surface. , Demineralization occurs on the enamel surface, causing caries to develop. In addition to dental caries, plaque formation
It is said to cause periodontal disease and bad breath.

As described above, plaque formed by S. mutans and other oral streptococci causes dental caries. Therefore, suppressing the formation of dental plaque prevents the occurrence of dental caries. Can be an effective means of

Conventionally, in order to prevent dental caries, an antibacterial agent for suppressing the growth of oral microorganisms, an inhibitor for suppressing the action of glucosyltransferase produced by oral microorganisms, sucrose as a substrate, and glucosyltransferase Various studies have been conducted on anti-carious substances such as enzymes that decompose the formed glucan, and various studies have also been conducted on non-cariogenic saccharides that do not serve as a substrate for plaque formation.

In recent years, plant components, especially tannins, have been attracting attention as anticaries substances. For example, JP-A-59-152311 discloses an oral composition containing a condensed tannin which is a condensate of flavan-3-ol.

[0007] In this publication, the glucosyltransferase inhibitory activity is actually tested for rhododendron tannin, swordbird tannin and yam tannin, but this is insufficient and cannot be put to practical use at all. It was In addition, the glucosyltransferase inhibitory activity of a 2- to 4-meric procyanidins isolated from areca nut [Sumitra Hada et al .; Phytotherapy Resear
ch 3 (4), 140-144 (1989)] have also been tested, but this too is less than satisfactory.

[0008]

Therefore, the development of a new anticaries agent which has a sufficiently strong effect and does not cause any problems in terms of safety for the human body has been left as a problem. .

[0009]

In view of such circumstances, the present inventors have effectively suppressed the formation of dental plaque or the occurrence of dental caries and have no harmful effect on the human body. As a result of diligent research to find a substance, it was found that a flavonoid polymer synthesized by polymerizing a reduced product of flavanones or flavanonols or leucoanthocyanidins with catechins has a strong anticaries activity, and the present invention. Has been completed.

That is, the first object of the present invention is to produce a reduced product of flavanones or flavanonols or a leucoanthocyanidin with catechins, which has a weight average molecular weight of 1,000 to 10,000.
To provide an anti-cariogenic flavonoid polymer of 0. A second object of the present invention is to provide a glucosyltransferase inhibitor containing a flavonoid polymer produced by condensing a reduced product of flavanones or flavanonols or leucoanthocyanidins with catechins as an active ingredient. Is. Further, a third object of the present invention is to provide a food containing the flavonoid polymer.

The anti-caries flavonoid polymer of the present invention (hereinafter referred to as "flavonoid polymer") is a reduced product of flavanones or flavanonols having a hydroxyl group at the 4-position or leucoanthocyanidins (hereinafter "4-hydroxy"). It is produced by condensing catechins with flavonoids)

More specifically, flavans or flavanonols may be subjected to reduction treatment according to a conventional method to reduce the carbonyl group at the 4-position to a hydroxyl group and then condensed with catechins. Leucoanthocyanidins having a hydroxyl group may be condensed with catechins.
This condensation is carried out by dehydration between 4-hydroxyflavonoid 4-position hydroxyl group and catechins or 4-hydroxyflavonoid 8-position hydrogen to form a 4-8'-carbon bond or 4-hydroxyflavonoid 4-position hydroxyl group. It proceeds by the formation of a carbon bond at the 4-6 'position by dehydration between hydrogens at the 6'position of catechins or 4-hydroxyflavonoid.

In preparing the anti-cariogenic flavonoid polymer of the present invention, flavanones or flavanonols can be used without particular limitation as a raw material for reducing the carbonyl group at the 4-position to a hydroxyl group. At this time, a ground product of the plant rich in flavanones or flavanonols or the extract itself may be used as a raw material.

The reducing agent used for the reduction treatment of flavanones or flavanonols is not particularly limited, but sodium borohydride, lithium aluminum hydride or the like can be preferably used.

On the other hand, as catechins to be polymerized to 4-hydroxyflavonoid, (+)-catechin, (-)
-Epicatechin, (+)-gallocatechin, (-)-epigallocatechin, (-)-epicatechin gallate, (-)
-Epigallocatechin gallate and the like.

To polymerize these catechins with 4-hydroxyflavonoid, they are reacted in the presence of hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, and Lewis acids such as boron trifluoride, antimony pentafluoride and aluminum chloride. Just go. Further, in this polymerization reaction, catechins in a weight ratio of 0.01 to 10 with respect to 4-hydroxyflavonoid 1 can be used.

The degree of polymerization of the resulting flavonoid polymer increases as the amount of catechins decreases. For example, if the weight ratio of catechins to 1 of 4-hydroxyflavonoid is 1, the degree of polymerization is about 2 and catechins is 0.3.
If so, the degree of polymerization is about 5; if catechins are around 0.2, the degree of polymerization is about 7 to 10; if catechins are around 0.1, the degree of polymerization is 1
A flavonoid polymer having a degree of polymerization of about 5 can be obtained.

The reaction temperature at this time is not particularly limited, but room temperature is preferable. The reaction time is not particularly limited, but is preferably about 24 hours.

The flavonoid polymer produced as described above can be obtained as various polymers depending on the 4-hydroxyflavonoid used. For example, when fustin (Fustin) is used as the raw material, prophycetinidine-type tannin is synthesized, and when taxifolin is used as the raw material, procyanidin-type flavonoid polymer is synthesized.

The reaction product obtained as described above has a glucosyltransferase-inhibiting ability by itself, but Sephadex LH-20 (manufactured by Pharmacia, USA), Diaion HP20 (manufactured by Mitsubishi Kasei Co., Ltd.), It can also be purified using a synthetic adsorbent column such as SepaBeads HP1MG (manufactured by Mitsubishi Kasei) or Toyopearl HW40F (manufactured by Toyo Soda Kogyo).

At this time, the average molecular weight [number average molecular weight (Mn), weight average molecular weight (Mw)] depends on each fraction.
Can be calculated.

The polymer obtained as described above can be used as it is, in a concentrated state, in a dried state after removing the solvent, in any state, but in storage stability and safety of organic solvent. From the point of, it is preferable to make it a dried product.

The applicant of the present application has previously filed an application for an anticaries agent containing a flavonoid polymer produced by treatment with peroxidase as an active ingredient (Japanese Patent Application No. 5-57991). Since this flavonoid polymer is a compound that oxidizes and polymerizes flavonoids, it has a different production method from the flavonoid polymer of the present invention, and is therefore considered to be a polymer having a different flavonoid polymerization mode and thus a different structure.

The glucosyltransferase inhibitor of the present invention is prepared by using the anti-cariogenic flavonoid polymer obtained as described above as an active ingredient and combining this with a known pharmaceutically acceptable carrier. The flavonoid polymer may be either a crude product of the polymerization reaction or a mixture of flavonoid polymers having various degrees of polymerization, but it is more preferable to use a flavonoid polymer purified by column fractionation or the like. .

The preferred flavonoid polymer as the active ingredient of the glucosyltransferase inhibitor of the present invention is
The flavonoid polymer having a weight average molecular weight of 1,000 to 10,000 and a degree of polymerization of 3.5 to 35 is more preferable, and the flavonoid polymer of the present invention has a weight average molecular weight of 1,300.
Up to 4,300 and a degree of polymerization of 4.6 to 15.0.
Among them, the flavonoid polymer having a weight average molecular weight of 1,700 to 3,500 and a degree of polymerization of 6.0 to 12.0 is most suitable. In the case of the flavonoid polymer of the present invention, the minimum molecular weight value is about ¼ of the weight average molecular weight value, and the maximum molecular weight value is about twice the weight average molecular weight value. Is.

The glucosyltransferase inhibitor of the present invention can be used mainly as an anti-caries agent for the purpose of anti-caries, an oral hygiene agent and the like, and examples of its dosage form include toothpaste, mouthwash, troche. Etc.

In producing an anticaries agent, an oral hygiene agent, etc., for example, calcium carbonate, dicalcium phosphate,
Silica anhydride, magnesium carbonate, glycerin, sorbitol, propylene glycol, polyethylene glycol, carboxymethyl cellulose, methyl cellulose,
Sodium alginate, carrageenan, carboxyvinyl polymer, sodium dioctyl sulfosuccinate, sodium lauryl sulfate, sodium dodecylbenzenesulfonate, butyl paraoxybenzoate, hinokitiol, allantoin, glycyrrhizin, alcohol, acacia, starch, corn starch, sodium saccharin, stevioside, glucose, Lactose, magnesium stearate, monopotassium phosphate, dipotassium phosphate, menthol, eucalyptus oil, peppermint, spearmint, pigments, etc., fluorides such as sodium fluoride, sodium monofluorophosphate, lysozyme chloride, azulene, etc. Ordinarily used ingredients such as anti-inflammatory agents, sodium chloride and the like can be appropriately blended.

The glucosyltransferase inhibitor of the present invention can be added to various foods for the purpose of caries prevention.

[0029] Examples of foods include paste bean paste, castella,
Water yokan, dorayaki peel, sponge cake, butter cake, bavarois, custard cream, butter cream, custard pudding, cookies, sweet bread, steamed bread, jam, lactic acid beverage, carbonated beverage, coffee beverage,
Coffee jelly, caramel, ice cream, chewing gum, juice, candy, chocolate and the like can be mentioned, and appropriate components generally used can be used for the production thereof depending on the type.

Examples of components used in foods include glucose, fructose, sucrose, maltose, sorbitol, stevioside, corn syrup, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester,
Sucrose fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, gum arabic, carrageenan, casein, gelatin, pectin, agar, vitamin Bs, nicotinic acid amide, calcium pantothenate, amino acids, calcium salts, pigments, flavors, preservation Examples include agents that are used as usual food materials, such as agents.

The glucosyltransferase inhibitor of the present invention can be combined with sugar to give an anti-cariogenic sugar. However, when sugar and a glucosyltransferase inhibitor are added to foods, the sugar is replaced with this. It is also possible to utilize this anti-caries sugar.

The flavonoid polymer of the present invention contains the flavonoid, which is the raw material, in edible plants, so there is no problem in terms of safety, but when blended in oral hygiene agents, foods, etc. From the perspective of color, scent, etc.
It is preferable to set the concentration range of 0001 to 0.5%,
It is more preferable that the concentration range is 0.001 to 0.2%.

[0033]

INDUSTRIAL APPLICABILITY The flavonoid polymer of the present invention has an extremely strong glucosyltransferase inhibitory action,
Since it notably suppresses the formation of plaque and does not have a peculiar taste or odor, it can be incorporated into a drug, a food or the like in an arbitrary amount. Therefore, the glucosyltransferase inhibitor of the present invention having the flavonoid polymer as an active ingredient can be advantageously used as an additive component of an anti-caries agent or an anti-caries food used for the prevention of caries, etc. is there.

At present, 120 million cubic meters of wood are consumed annually in Japan, 10 of which are consumed.
Although ~ 20% of the bark is industrial waste, in the present invention, since the ground product or extract of flavonoid-rich plants can be used as a raw material, it also has an effect of effectively utilizing this wood resource. .

[0035]

EXAMPLES Next, a method for producing the flavonoid polymer of the present invention,
The present invention will be described in more detail with reference to Examples relating to a method for purifying a highly active substance, a method for measuring a molecular weight, and an assay test for glucosyltransferase inhibitory activity, but the present invention is not limited to these Examples.

Example 1 Production Example of Flavonoid Polymer: As a raw material, fustine (Fu
stin) and taxifolin (300 mg each) were dissolved in ethanol (3.0 ml), and sodium borohydride (600 mg) was added for reduction treatment. 25 ° C,
After reacting for 2 hours, the reaction product was adsorbed on a column of Sephadex LH-20 (manufactured by Pharmacia, USA), and this was eluted with ethanol. An equal amount of distilled water was added to the obtained ethanol solution, which was concentrated under reduced pressure to remove ethanol and then freeze-dried. As a result, leucofisetinidin and leucocyanidin
Cyanidin) was obtained in an amount of 270 mg and 219 mg, respectively.

Further, the leukophycetinidine was divided into three (90 mg each), and 90 mg, 30 mg and 1 mg of D-(+)-catechin were added to each of them in 0.05N HCl.
5 mg was added, and a polymerization reaction was carried out at 25 ° C. for 24 hours. Similarly, leucocyanidin was divided into three (73 mg each), and 18 mg, 10 mg and 7 mg of D-(+)-catechin were added in the same manner to carry out the polymerization reaction.

Example 2 Purification Example of Flavonoid Polymer: Each reaction solution obtained in Example 1 was treated with Sephadex LH-2.
0 (manufactured by Pharmacia, USA) column, eluted with acetone-water (7: 3 v / v), and concentrated under reduced pressure.
Lyophilized. As a result, leucoficetinidine 90m
When 90 g, 30 mg, and 15 mg of D-(+)-catechin were reacted with g, 160 mg, 110 mg, and 95 mg of flavonoid polymer of prophycetinidine type were obtained, respectively.
When leucocyanidin (73 mg) was reacted with D-(+)-catechin (18, 10, 7 mg), procyanidin type flavonoid polymer was obtained (82, 7).
5,71 mg was obtained.

Example 3 Measurement of average molecular weight of each flavonoid polymer: The number average molecular weight and weight average molecular weight of each polymer obtained in Example 2 were measured using a Shodex column. That is, 2 mg of each sample was dissolved in 10 ml of tetrahydrofuran, and this was dissolved in Shodex KF.
After adsorbing to -802 and 804 (Showa Denko) columns, elution with tetrahydrofuran was performed to elute each polymer by 2 times.
The number average molecular weight and the weight average molecular weight of each polymer were measured by detecting at 80 nm. The calibration curve was created using polystyrene standards.

The degree of polymerization of each polymer was calculated for the maximum contained molecular species showing the maximum peak. That is, after calculating the molecular weight from the calibration curve, the degree of polymerization was calculated considering 1 unit 290. The results are summarized in Table 1. The relationship between the amounts of each polymer and the polymerization raw materials is as shown in Table 2.

[0041]

[0042]

Example 4 Assay of glucosyltransferase inhibitory activity: The glucosyltransferase inhibitory activity of the flavonoid polymer of the present invention was examined according to the following method. That is, 0.6 ml of a 500 mM sodium phosphate buffer (pH 6.0) containing 5.0% sucrose, 0.5% dextran T-10 and 0.5% sodium azide, and a test sample aqueous solution.
Todd-Hewit to 0.15 ml
t) 8M from Streptococcus mutans MT8148 strain cultured in medium
Glucosyltransferase enzyme solution extracted with urea [S. Hamada et al .; J. Gen. Microbiol., 135, 335-3
44 (1989)], and a total amount of 3 ml of water was added to prepare a reaction system. This reaction system was allowed to react in a glass test tube at 37 ° C. for 3 hours. The amount of enzyme added at this time was set so that the absorbance at 550 nm was about 1.0 in the reaction at 37 ° C. for 3 hours.

The resulting insoluble glucan is ultrasonically disrupted,
The absorbance (A) at 550 nm was measured, and the absorbance when water was used instead of the sample solution was used as a control (B).
The inhibition rate (%) was calculated by the following formula. From the inhibition rate (%) = (B-A ) × 100 / B obtained inhibition rate was measured profilin cell Hee Jin obtained in Example 2, procyanidin type of flavonoid polymer of glucosyltransferase inhibitory activity (IC ₅₀₎ . The results are shown in Table 3.

[0045]

Example 5 Assay of Plaque Formation Inhibitory Action: The flavonoid polymer of the present invention was examined for the plaque formation inhibitory action according to the following method. That is, using an Eagle medium (Nissui Pharmaceutical Co., Ltd.) containing 5% sucrose and an aqueous solution of a test sample, Streptococcus sobrinus 6715 strain was cultured at 37 ° C. in a test tube inclined at an angle.
After culturing for 2 days, the weight of the glucan-bacterial body complex (plaque) attached to the wall of the test tube was measured, and this was defined as the amount of plaque formation.
The results are shown in Table 4.

[0047]

Example 6 Toothpaste: A dentifrice having the following composition was prepared according to a conventional method. (Composition) Heavy part Dibasic calcium phosphate 42 Glycerin 18 Carrageenan 0.9 Sodium lauryl sulfate 1.2 Sodium saccharin 0.09 Butyl paraoxybenzoate 0.005 Flavor 1 Flavonoid polymer ^* 0.05 Water remaining amount ─── ───────────────── Total amount 100 * Proficetinidine type polymer obtained in Example 2 (degree of polymerization 7.2)

Example 7 Mouthwash: A mouthwash having the following composition was prepared by a conventional method. (Composition) Heavy part Sodium lauryl sulfate 0.8 Glycerin 7 Sorbitol 5 Ethyl alcohol 15 Flavonoid polymer ^** 0.05 L-Menthol 0.05 Perfume 0.04 Saccharin sodium 0.1 Water residual amount ─── ─────────────── Total amount 100 ** Procyanidin type polymer obtained in Example 2 (degree of polymerization 10.0)

Example 7 Chewing gum: A chewing gum having the following composition was prepared according to a conventional method. (Composition) Weight part Gum base 20 Calcium carbonate 2 Stevioside 0.1 Flavonoid polymer ^*** 0.01 Lactose 76.89 Flavor 1 1 ───────────────── ─── Total amount 100 *** Procyanidin type polymer obtained in Example 2 (degree of polymerization 7.2)

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C07G 17/00 Z

Claims (13)

[Claims] 1. An anti-caries flavonoid polymer having a weight average molecular weight of 1,000 to 10,000 produced by condensing flavanones or flavanonols or leucoanthocyanidins with catechins. 2. The cariostatic flavonoid polymer according to claim 1, which has a degree of polymerization of 4.6 to 15.0. 3. The cariogenic flavonoid polymer according to claim 1, having a degree of polymerization of 6.0 to 12.0. 4. A glucosyltransferase inhibitor containing a flavonoid polymer produced by condensing a reduced product of flavanones or flavanonols or leucoanthocyanidins with catechins as an active ingredient. 5. The glucosyltransferase inhibitor according to claim 4, which is used as an anticaries agent. 6. The glucosyltransferase inhibitor according to claim 4 or 5, which is used as an oral hygiene agent. 7. The glucosyltransferase inhibitor according to claim 4, which is used as a food additive. 8. An anti-cariogenic food containing a flavonoid polymer produced by condensing a reduced product of flavanones or flavanonols or leucoanthocyanidins with catechins. 9. The flavonoid polymer is 4.6 to 15.0.
A flavonoid polymer having a degree of polymerization of 8.
An anti-cariogenic food according to the item. 10. The flavonoid polymer is from 6.0 to 12.
The anti-cariogenic food according to claim 9, which is a flavonoid polymer having a degree of polymerization of 0. 11. An anti-cariogenic sugar consisting of sugar and a flavonoid polymer produced by condensing a reduced product of flavanones or flavanonols or leucoanthocyanidins with catechins. 12. A method for producing an anticaries agent, which comprises condensing a reduced product of flavanones or flavanonols or leucoanthocyanidins with catechins. 13. The catechins are added in an amount of 0.1 to 0.5 parts by weight to 1 part by weight of a reduced product of flavanones or flavanonols or leucoanthocyanidins to condense them. A method for producing the anti-carious substance described.