JPH07242555A - Glucosyl transferase-inhibiting agent and dental caries-resistant agent and dental caries-resistant food containing the same as active ingredient - Google Patents

Abstract

PURPOSE:To provide the subject safe inhibiting agent containing a tree- originated tannin as an active ingredient, having a strong glucosyl transferase- inhibiting action, preventing the formation of bacterial plaques or the generation of dental caries, and useful as a dental caries-resistant agent for oral cavity hygienic agents or food additive agents. CONSTITUTION:This inhibiting agent contains as an active ingredient a condensed tannin originated from the core or bark of a tree, e.g. a condensed tannin having a weight-average mol.wt. of 800-10,000 and obtained by extracting the core or bark of a tree belonging to the Anacardaceae, the Pinaceae or the Leguminosae, especially acacia bark or the bark of Larix leptolepis, with a solvent such as methanol, ethanol, ethyl acetate or (hot) water at a temperature of 15-35 deg.C under shading and subsequently purifying the obtained extract with an adsorption column chromatography.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a glucosyltransferase inhibitor containing condensed tannin derived from heartwood and bark of trees as an active ingredient. More specifically, it is an anticaries agent such as an oral hygiene agent or an anti-caries food. The present invention relates to a glucosyltransferase inhibitor containing the above-mentioned tannin, which can be used as an active ingredient, and a method for producing the tannin.

[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 the 2- to 4-meric procyanidins isolated from areca nut [Sumitra Hada et al .; Phytotherapy Resea
rch 3 (4), 140-144 (1989)] have also been tested, but this too is less than adequate.

[0008]

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

[0009]

In view of such circumstances, the present inventors have found that a substance that effectively suppresses the formation of dental plaque or the occurrence of dental caries and that is harmless to the human body is used as a natural substance. As a result of earnest research to find out from the inside, it was found that the specific condensed tannin present in the tree effectively suppressed glucosyltransferase and was safe, and completed the present invention.

That is, an object of the present invention is to provide a glucosyltransferase inhibitor containing a condensed tannin derived from the heartwood or bark of trees as an active ingredient. Another object of the present invention is to provide an anticaries agent such as an oral hygiene agent and an anticaries food containing the above-mentioned glucosyltransferase inhibitor as an active ingredient. Still another object of the present invention is to provide a method for producing an anticaries agent having a glucosyltransferase inhibitory activity, which comprises subjecting a heartwood or a bark of a tree to solvent extraction and subjecting the extract to adsorption column chromatography. It is to be.

As the condensed tannin derived from the heartwood or bark of the tree in the present invention (hereinafter referred to as "tree-derived tannin"), the extract itself obtained by extracting the heartwood or bark of various trees with a solvent may be used. Alternatively, a product purified from this extract may be used.

The tree used as the extraction raw material is
It is not particularly limited, but is preferably a family of Alacaceae (Anacardaceae), a family of Pinaceae, and a family of legumes (Legu).
minosae) heartwood or bark, and more preferably Akashiya bark, larch bark, and the like.

Further, the solvent for extracting the heartwood and bark of the tree is not particularly limited, but methanol, ethanol, acetone, ethyl acetate, water, hot water, or a mixed solvent system thereof is used. I can. The extraction temperature at this time is also not particularly limited, but is 5 ° C to
It is preferably not higher than the boiling point of the solvent, particularly preferably 15 to 35 ° C. The extraction time is also not particularly limited, but it is preferably within 2 days. In addition, it is more preferable to perform the extraction under light shielding.

In place of extracting the heartwood and bark of the tree as described above, commercially available kebracotannin, wattle tannin, gambir tannin, and catatinnin may be used as the extract. Of these, kebraco tannin is the sumacaceae plant Schinopsis lorentzii Engl.
It is a condensed tannin of heartwood such as Schinopsis balanse Engl. And Wattle tannin is a legume, Acacia moli.
It is a condensed tannin of the bark of ssima.

Although the extract obtained as described above and commercially available kebracotannin, wattle tannin and the like have a tentative glucosyltransferase inhibitory action, these purified products are used in actual use. Is more preferable.

As a purification means for obtaining a purified product, Sephadex LH-20 (manufactured by Pharmacia, USA), Diaion HP-20 (manufactured by Mitsubishi Kasei Kogyo),
Column chromatography using synthetic adsorbent columns such as SepaBeads HP1MG (manufactured by Mitsubishi Kasei Co., Ltd.) and Toyopearl HW40F (manufactured by Toyo Soda Co., Ltd.) can be used.

Specifically, the extract, commercially available kebracotannin, wattle tannin and the like are dissolved in a small amount of water, a solvent such as methanol or ethanol, or a mixed solvent thereof,
After being adsorbed on the synthetic adsorbent column, it may be sufficiently washed with water and eluted with a hydrophilic organic solvent such as methanol or ethanol or a mixed solvent thereof.

The average molecular weight [number average molecular weight (Mn), weight average molecular weight (M
w)] can be calculated.

The obtained tree-derived tannin has a weight average molecular weight of about 800 to 10,000 and a degree of polymerization of 3 to 30 as judged from the molecular weight.
Among them, those having a particularly strong glucosyltransferase inhibitory action have a weight average molecular weight of 1,500 to 6,
And the degree of polymerization is 5 to 20. Furthermore, as a substance having a particularly strong glucosyltransferase inhibitory action, a weight average molecular weight of 1,500 to 4,50
It is 0 and the degree of polymerization is 5 to 15. In the case of tree-derived tannin, the minimum molecular weight value is about 1 of the weight average molecular weight value.
/ 4, the maximum molecular weight value is about twice the weight average molecular weight value.

Whether an extract is used as the tree-derived tannin of the present invention or a refined product obtained by further purifying the tannin is used, the obtained raw product, a concentrated product, a solvent-free dried product are used. Although it can be used in any state such as a product, it is preferable to make it in a dried state from the viewpoint of storability and safety of organic solvent.

The glucosyltransferase inhibitor of the present invention is produced by using the tree-derived tannin obtained as described above as an active ingredient and combining this with a known pharmaceutically acceptable carrier.

The glucosyltransferase inhibitor of the present invention can be used mainly as an anticaries agent for the purpose of anticaries, for example, an oral hygiene agent and an additive for anticaries foods. Examples of the oral hygiene agent include toothpaste, mouthwash, troches and the like, and in the production thereof, for example, calcium carbonate, dicalcium phosphate, silicic anhydride, magnesium carbonate, glycerin, sorbitol, Propylene glycol, polyethylene glycol, carboxymethylcellulose, methylcellulose, sodium alginate, carrageenan, carboxyvinyl polymer, sodium dioctylsulfosuccinate, sodium lauryl sulfate, sodium dodecylbenzenesulfonate, butyl paraoxybenzoate, hinokitiol, allantoin, glycyrrhizin, alcohol, gum arabic , Starch,
Corn starch, sodium saccharin, stevioside, glucose, lactose, magnesium stearate, monopotassium phosphate, dipotassium phosphate, menthol, eucalyptus oil, peppermint, spearmint, pigments, sodium fluoride, sodium monofluorophosphate, etc. Fluorides, anti-inflammatory agents such as lysozyme chloride and azulene, and commonly used components such as sodium chloride can be appropriately blended.

The anti-caries agent of the present invention may be in a dosage form to be added to various foods for the purpose of anti-caries. Examples of foods include paste, castella, water yokan, dorayaki peel, sponge cake, butter cake,
Bavaroa, custard cream, butter cream, custard pudding, cookies, sweet bread, steamed bread, jam, lactic acid bacteria beverage, carbonated beverage, coffee beverage, coffee jelly, caramel, ice cream, chewing gum, juice, candy, chocolate, and the like. In the production, appropriate components usually used can be used depending on the kind.

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 anti-caries agent of the present invention can be combined with sugar to form an anti-caries sugar, but when sugar and an anti-caries agent are added to food, the anti-caries agent is replaced with the anti-caries agent. It is also possible to use edible sugar.

The tannin of the present invention is a natural product derived from the heartwood or bark of trees, so there is no problem in terms of safety. However, when the tannin of the present invention is added to oral preparations, foods, etc., the taste, In terms of color and scent, 0.0001-0.
A concentration range of 5% is preferred, especially 0.001
It is preferable to set the concentration range to 0.2%.

[0027]

INDUSTRIAL APPLICABILITY The tree-derived tannin used in the present invention has an extremely strong glucosyltransferase inhibitory effect, remarkably suppresses the formation of plaque, and does not have a peculiar taste or odor. It can be added to drugs, foods, etc. in the amount of. Therefore, the glucosyltransferase inhibitor of the present invention containing the tannin derived from the tree as an active ingredient can be advantageously used as an additive ingredient of an anti-caries agent or an anti-caries food used for the prevention of caries and the like. Is.

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, the tree-derived tannin of the present invention can be obtained from the bark, and thus has an effect of effective use of resources.

[0029]

[Examples] Next, the present invention will be described in more detail with reference to Examples relating to a method for producing a tree-derived tannin of the present invention, a method for purifying a highly active substance, a molecular weight measurement method, and an assay test for glucosyltransferase inhibitory activity. The present invention is not limited to these examples.

Example 1 Production of tree-derived tannins: Tree-derived tannins were produced using acacia bark (A) and larch bark (K) as raw materials. That is, first 60 kg of each bark
It is crushed to about mesh, and this is acetone-water (7: 3v
/ V) extracted with 10 liters. The extraction was performed at 25 ° C. for 2 days, and this was filtered with a glass filter. The obtained filtrate was concentrated under reduced pressure to obtain powder residues of 107 g and 210 g, respectively.

Example 2 Purification of tree-derived tannins: The extracts from the acacia bark and larch bark obtained in Example 1, the commercially available wattle tannins and quebraco tannins were purified as follows. That is, 5 g of each extract or tannin was dissolved in 10 ml of water-ethanol (1: 1 v / v) and adsorbed on a column (φ3 cm × 100 cm) of Sephadex LH-20 (manufactured by Pharmacia, USA). After washing with 2 liters of water, eluting with 2 liters each of ethanol, methanol, and acetone in order, the obtained fractions were concentrated under reduced pressure, and the ethanol elution fractions (EE),
Methanol elution fraction (ME), acetone elution fraction (A
E).

As a result, from the acacia bark, the ethanol elution fraction (A-EE) of the acacia bark extract was 0.75.
g, the same methanol elution fraction (A-ME) 3.25 g, and the same acetone elution fraction (A-AE) 0.70 g from the larch bark, the ethanol elution fraction (K-EE) of the larch bark extract. 0.9 g, the same methanol elution fraction (KM
E) 1.50 g, same acetone elution fraction (K-AE) 2.2
5 g each was obtained.

From Wattle tannin, the ethanol elution fraction (W-EE) 0.25 g, the methanol elution fraction (W-ME) 2.90 g, and the acetone elution fraction (W-AE) 0 were obtained. .50 g from Kebraco Tannin
The ethanol elution fraction (Q-EE) was 0.60 g, the methanol elution fraction (Q-ME) was 1.75 g, and the acetone elution fraction (Q-AE) was 0.90 g.

Example 3 Measurement of average molecular weight of each fraction: Shodex
x) The column was used to measure the number average molecular weight and the weight average molecular weight of each fraction obtained in Example 2. That is, 2 mg of each sample was dissolved in 10 ml of tetrahydrofuran, and this was dissolved in Shodex KF-8.
02 and 804 (Showa Denko) columns after adsorption,
Elute with tetrahydrofuran to elute each fraction component with 28
The number average molecular weight and the weight average molecular weight of each fraction were measured by detecting at 0 nm. The calibration curve was created using polystyrene standards.

The degree of polymerization of each fraction 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.

[0036]

Example 4 Assay of glucosyltransferase inhibitory activity: The tannin of the present invention was tested for glucosyltransferase inhibitory activity according to the following method. That is, sucrose 5.0%, dextran T-10 0.5% and sodium azide 0.0.
500 mM sodium phosphate buffer containing 5% (pH
6.0) 0.6 ml and test sample aqueous solution 0.15 ml
And Streptococcus mutans cultured in Todd-Hewitt medium.
cus mutans) MT8148 strain glucosyltransferase enzyme solution extracted with 8M urea [S. Hamada et al.
al .; J. Gen. Microbiol., 135, 335-344 (1989)], and a total amount of 3 ml of water was added to prepare a reaction system. This reaction system was reacted in a glass test tube at 37 ° C. for 3 hours. The amount of enzyme added at this time is 37 ° C,
The absorbance at 550 nm was set to about 1.0 after the reaction for 3 hours.

The produced 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. Inhibition rate (%) = (BA) x 100 / B Table 2 shows the glucosyltransferase inhibition rate of the tannins derived from each tree obtained in Example 3.

[0039]

Example 5 Assay of Plaque Formation Inhibitory Action: The plaque formation inhibitory action of the tannin of the present invention was examined according to the following method. That is, sucrose
Streptococcus sobrinus (Stre) at 37 ° C. in an obliquely inclined test tube using an Eagle medium (Nissui Pharmaceutical Co., Ltd.) containing 5% and a test sample aqueous solution.
Strain 6715 of Ptococcus sobrinus was cultured. 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 3.

[0041]

Example 6 Toothpaste: Toothpaste having the following composition was prepared according to a conventional method. (Composition) Heavy part Dicalcium phosphate 42 Glycerin 18 Carrageenan 0.9 Sodium lauryl sulphate 1.2 Sodium saccharin 0.09 Butyl paraoxybenzoate 0.005 Flavor 1 A-AE ^* 0.05 Water remaining ─── ─────────────────── Total amount 100 * Acetone-eluted fraction of the acacia bark extract obtained in Example 2

Example 7 Mouthwash: A mouthwash having the following composition was prepared according to a conventional method. (Composition) Heavy part Sodium lauryl sulfate 0.8 Glycerin 7 Sorbitol 5 Ethyl alcohol 15 W-AE ^** 0.05 L-Menthol 0.05 Perfume 0.04 Saccharin sodium 0.1 Water residual amount ─── ────────────────── Total amount 100 ** Acetone elution fraction of wattle tannin obtained in Example 2

Example 8 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 Q-AE ^*** 0.01 Lactose 76.89 Flavor 1 1 ────────────────── ──── Total amount 100 *** Acetone elution fraction of kebracotannin obtained in Example 2 and above

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

Claims (11)

[Claims] 1. A glucosyltransferase inhibitor containing a condensed tannin derived from heartwood or bark of trees as an active ingredient. 2. An anti-caries agent which is derived from heartwood or bark of a tree and contains condensed tannin having a glucosyltransferase inhibitory activity as an active ingredient. 3. The anticaries agent according to claim 2, which is an oral hygiene agent. 4. The anticaries agent according to claim 2, which is a food additive. 5. An anti-cariogenic food containing a condensed tannin derived from heartwood or bark of a tree and having a glucosyltransferase inhibitory action. 6. An anti-cariogenic sugar consisting of sugar and condensed tannin derived from heartwood or bark of a tree and having a glucosyltransferase inhibitory action. 7. A method for producing an anticaries agent having a glucosyltransferase inhibitory activity, which comprises subjecting a heartwood or bark of a tree to solvent extraction and subjecting the extract to adsorption column chromatography. 8. The weight average molecular weight is 800 to 10,000.
8. The method for producing an anticaries agent according to claim 7, which is a condensed tannin. 9. A weight average molecular weight of 1,500 to 6,000.
8. The method for producing an anticaries agent according to claim 7, which is a condensed tannin. 10. The method for producing an anticaries agent according to claim 7, which is a condensed tannin having a degree of polymerization of 3 to 30. 11. The method for producing an anticaries agent according to claim 7, which is a condensed tannin having a degree of polymerization of 5 to 20.