JPH0217525B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a caries preventive agent, and more particularly to a caries preventive agent containing a herbal medicine extract having a glucosyltransferase inhibitory effect as an essential ingredient. Many researchers have proposed many theories regarding the cause of dental caries in the past, but today, the bacterial infection theory, developed from the chemical-bacteria theory of Miller et al., is the prevailing theory. According to this theory, caries occurs by the mechanism described below. That is, microorganisms in the oral cavity, especially Streptococcus mutans (hereinafter referred to as S.mutans),
Sucrose in food is converted into sticky polysaccharide (glucan) by glucosyltransferase (hereinafter referred to as GTase), which is an extracellular enzyme of bacterial cells. form agglomerates, that is, dental plaque. Microorganisms proliferate in this dental plaque, and organic acids such as lactic acid are produced through glycolysis. When the pH of the tooth surface drops below 5.4 due to this organic acid, demineralization occurs in the etemel, causing caries.
proceed. As is clear from the above caries development mechanism, three major factors are necessary for the development of caries: 1) food components, especially sucrose, 2) tooth structure that is easily attacked by organic acids, and 3) oral microorganisms. be. Therefore, to prevent dental caries, it is best to avoid sucrose intake, strengthen tooth structure, or kill oral microorganisms. For this purpose,
Various caries prevention measures have been taken in the past, but
None of them have reached the point where they have achieved any noticeable effects. In other words, it is practically impossible to completely remove sucrose from food, and there are also methods to strengthen tooth structure by using fluoride-containing toothpaste or adding fluoride to tap water. It is not very effective and has the side effect of causing mottled teeth, so it is by no means satisfactory. On the other hand, enzyme-containing toothpastes and oral disinfectants that decompose formed dental plaque are also commercially available, but their effectiveness is currently far from what was expected. The present inventors inhibited the insoluble glucan biosynthesis action of GTase, thereby preventing bacterial cells from adhering to the tooth surface, preventing the formation of dental plaque itself, and thereby preventing the development of dental caries. Focusing on the fact that this is one of the most effective methods, and as a result of repeated research in search of such inhibitors, we surprisingly found that GTase is present in many crude drug extracts currently used for other purposes. They discovered that it has an inhibitory effect and completed the present invention. That is, an object of the present invention is to provide an anti-caries agent containing as an essential ingredient a crude drug extract that inhibits GTase, which is an extracellular enzyme of S. Mutans. In this specification, unless otherwise specified, the crude drug extract means a crude drug extract obtained by the method described below, a diluted solution thereof, a concentrated extract thereof, or a dried powder thereof. Typical herbal medicines that can be used for the above purposes include daisy, peonies, gentian, senso, white ho,
These include Longtan, Huanglian, Oriental chinensis, and Huangli. If these crude drugs are extracted using a suitable extraction solvent using a conventional method, crude drug extracts having GTase inhibitory activity can be obtained. As the extraction solvent, water, water-miscible organic solvents such as methanol, ethanol, or acetone, or mixtures of water and these organic solvents can be used, and hydrocarbon-based organic solvents such as ether and hexane can be used. can also be used. If the crude drug extracts obtained in this way are used alone or in a mixture of two or more, or if applied to the oral cavity after forming into an appropriate formulation as described below, S. Mutans GTase, an extracellular enzyme of bacteria, is inactivated and the formation of dental plaque is prevented. If the herbal medicine extract obtained from one or more herbal medicines according to the present invention is an aqueous extract, it can be used as it is or diluted appropriately, but if necessary, it can be concentrated or lyophilized. It is preferable to formulate it into a powder or paste form and then formulate it into a suitable oral composition before use. Such oral compositions may be in the form of liquids, solids, or semi-solids, and preferred compositions include toothpastes, gargles, lozenges, gargles, liniments, and chewing gums. It will be done. The excipients or auxiliary agents used to produce these oral compositions may be appropriately selected from those normally used for this type of purpose depending on the dosage form, and are not particularly limited. However, for example, lactose, starch, corn starch, magnesium stearate, dicalcium phosphate dihydrate, sorbitol, carboxymethylcellulose, saccharin, sodium lauryl sulfate, glycerin, sodium carboxymethylcellulose, silicic anhydride, gelatin, titanium dioxide , menthol, fatty acids, citric acid, polyethylene glycol, sodium lauroyl sarcosinate, calcium carbonate,
Alcohol, carrageenan, sodium acyl taurate, peptone, gum arabic, lauryl diethanolamide and the like are preferably used. A bactericidal agent such as chlorhexidine and benzalkonium chloride can be added to the above composition to further enhance its caries prevention effect, and a preservative, fragrance, coloring agent, etc. can also be added as appropriate. The amount of the herbal medicine extract in the oral composition for caries prevention produced in this way varies depending on the dosage form, but it is usually desirable that it be approximately 0.001% (weight/weight) or more by weight of the concentrated extract. . Measurement of GTase inhibitory effect of various crude drug extracts 1 Preparation of GTase enzyme Cariogenic streptococcus Streptococcus miutans OMZ176 (serotype, type d) was grown in slant culture medium.
After pre-culture, the cells were cultured in brain heart infusion broth for 24 hours at 37°C and centrifuged at 5000 rpm for 10 minutes to obtain a culture supernatant. Under ice-cooling, (NH ₄ ) ₂ SO ₄ was added to the supernatant until it reached 50% saturation for salting out, and the precipitate was collected by centrifugation at 5000 rpm for 10 minutes. This precipitate was dissolved in 1/1000M acetate buffer and dialyzed against the same buffer at 4°C for 18 hours to obtain a GTase enzyme preparation solution. 2. Preparation of crude drug extract Add about 10 times the amount of extract liquid such as water, methanol, petroleum ether or water-methanol mixture to about 1 g to 10 g of crude drug and extract by shaking for 1 hour or in a mantle heater for 1 hour. After extraction under heating and reflux, centrifugation is performed at 2500 rpm for 15 minutes, the supernatant liquid is filtered through paper, and concentrated to dryness under reduced pressure. The yield of the obtained extract varies depending on the type of crude drug and extraction solvent, but an example is shown in Table 1.

[Table] 3 Measurement of GTase inhibitory activity 500 μl of 20% sucrose solution in 0.04% NaN ₂ aqueous solution,
200 μl of 200 mM acetate buffer (PH7.5), crude drug extract (undiluted solution, ×10, ×100, or ×1000 solution)
Mix 200 μl and 100 μl of GTase enzyme standard solution,
Incubate for 18 hours at °C. 100 μl of 10% trichloroacetic acid
to terminate the reaction, and centrifuged at 13,000 rpm for 5 minutes. The supernatant was decanted and discarded, and the precipitate was washed three times with 1 ml of water and then three times with 1 ml of acetone, dried, added with 1 ml of 1M NaOH, and left overnight.
Dissolve completely. Add 100μl of this solution to 1M NaOH.
Dilute 20-30 times and quantify insoluble glucan produced by the action of GTase using the phenol-sulfuric acid method*. A similar operation was performed using distilled water instead of the herbal medicine extract as a control, and from the following formula:
Calculate the GTase inhibition rate. GTase inhibition rate (%) = (1-Sample OD490nm/Control OD490nm
)×100 Phenol sulfuric acid method: Glucan-containing NaOH solution
A mixture of 200 μl, 200 μl of 5% aqueous phenol solution, and 1 ml of concentrated sulfuric acid is stirred well, and the absorbance at 490 nm (OD490 nm) is measured. The results of experiments conducted using various crude drug extracts are shown in Table 2 below.

【table】

[Table] *Concentration of concentrated extract in enzyme reaction solution From Table 2, it can be seen that Japanese jujube, fenugreek, peony, and gentian have strong inhibitory activity. These were found to retain significant activity even at low concentrations of 0.0001% to 0.001%. In this way, crude drug extracts that exhibit activity over a wide concentration range are
It is particularly useful for producing anti-caries oral compositions. Examples of methods for extracting crude drug extracts and methods for producing caries preventive agents containing the extracts as an essential ingredient are listed below. Example 1 Finely crush a large jujube, put 10 g of this into a 200 ml Tess-type flask, add 100 ml of water, attach a reflux condenser and reflux for 1 hour over an open flame, and then concentrate under reduced pressure to form a large jujube. 3.4 g of water-extracted concentrated extract was obtained. Almost the same amount of concentrated extract was obtained when shaking and extracting with cold water. After pulverizing Senso, herbal medicine powders were used as they were for Paeonia japonica, Urticaria, gentian, Baibo, Longtan, Huanglian, Oriental japonica, and Huanglin, and the water extracts of herbal medicines were obtained in the same manner as above. Example 2 Example 1 except that methanol, a mixed solvent of water and ethanol, and petroleum ether are used instead of water.
The same operation was performed. The amount of concentrated extract obtained was 2.8 g for methanol, 3.0 g for water/ethanol, and 0.4 g for petroleum ether. Example 3 Dentifrice Dibasic calcium phosphate 42 Glycerin 18 Carrageenan 0.9 Sodium lauryl sulfate 1.2 Saccharin 1.0 Jujube extract* 0.005 Butyl paraoxybenzoate 0.005 Fragrance 1.1 Water Total remaining amount 100 (wt%) Herbal extract is water, MeOH or petroleum ether Concentrated extract. The same applies to the following examples. Manufactured according to conventional methods for manufacturing toothpaste. That is, the prescribed amounts of water, glycerin, carrageenan, saccharin, jujube extract, butyl paraoxybenzoate, and fragrance are measured and mixed to swell the binder, and then dibasic calcium phosphate and sodium lauryl sulfate are added and further mixed. do. After thoroughly defoaming and mixing, fill the tube. Example 4 Mouthwash A mouthwash having the following formulation is manufactured according to a conventional method. Ethanol (90%) 20 Satucalin 0.3 Sodium acyl taurate 0.5 Gelatin 0.5 Wickerberry extract 0.005 Flavor 1.0 Chlorhexidine 0.005 Water Total remaining amount 100 (wt%) Example 5 Lozenge Gum Arabic 8 White sugar 70 Paeonia extract 0.005 Jujube extract 0.00 5 Flavoring 1.0 Lactose 17.0 Magnesium stearate Appropriate amount Total amount 100 (wt%) Add water to gum arabic, white sugar, lactose, peonies extract, jujube extract, fragrance, mix, then granulate with a suitable granulator and dry. . 12 After adjusting the grains through a mesh filter, add magnesium stearate and tablet into an appropriate shape. Example 6 Chewing Gum Chewing gum having the following formulation is manufactured according to a conventional method. Gum base 20 Calcium carbonate 2 Saccharin 0.1 Jujube extract 0.005 Lactose 74.9 Flavor 1 Total amount 100 (wt%) Example 7 Toothpaste A toothpaste with the following formulation is manufactured according to a conventional method. Dicalcium phosphate dihydrate 50 Calcium carbonate 25 Glycerin 10 Sodium lauryl sulfate 1.3 Chlorhexidine digluconate 0.5 Gentiana extract 0.01 Satucharin 0.1 Flavoring 1 Water Total amount remaining 100 (wt%) Example 8 Lozenge The following formulation according to the conventional method Manufacture lozenges. Gum arabic 6 Glucose 72 Wickerberry extract 0.05 Jujube extract 0.005 Dipotassium phosphate 0.2 Monopotassium phosphate 0.1 Flavoring 1.0 Lactose 17.0 Magnesium stearate Appropriate total amount 100 (% by weight) Example 9 Dentifrice containing two types of crude drug extracts A toothpaste with the following formulation is manufactured according to a conventional method. Calcium pyrophosphate 42 Sodium lauryl sulfate 1.2 Sodium lauroyl sarcosinate 0.2 Glycerin 20 Carrageenan 1.1 Sucrose fatty acid ester 2.0 Paeonia extract 0.06 Jujube extract 0.06 Chlorhexidine digluconate 0.5 Satucalin 0.1 Flavor 1.2 Water Total remaining amount 100 (wt%)

Claims (1)

[Scope of Claims] 1. An anti-caries agent containing a crude drug extract having a glucosyltransferase inhibitory effect as an essential ingredient, wherein the crude drug extract is a water extract of large jujube,
One or more selected from the group consisting of methanol extract, petroleum ether extract, fenugreek water extract, methanol extract, petroleum ether extract, gentian methanol extract, petroleum ether extract, and water extracts of each of Senso, Longtan, and this drug. A caries prevention agent characterized by being an extract of the above herbal medicine. 2 The content of herbal medicine extract is 0.001 as a concentrated extract.
The caries preventive agent according to claim 1, which is at least % by weight.