JP3509891B2 - Flavonoid polymer and glucosyltransferase inhibitor containing the same as active ingredient - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a flavonoid polymer produced by treating a flavonoid with peroxidase (EC 1.11.1.7) and its use. More specifically, the present invention relates to a flavonoid polymer produced by the method. The present invention relates to a glucosyltransferase inhibitor such as an anticaries agent and an oral hygiene agent containing the same as an active ingredient, and a food containing the same.

[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
Mutants (Streptococcus mutans) produce an enzyme called glucosyltransferase. This enzyme produces a sticky, insoluble polysaccharide (glucan) using sucrose in the mouth as a substrate. Due to this glucan, bacterial cells adhere to the tooth surface and form plaque.

Various microorganisms coexist and propagate in this dental plaque. When the pH of the tooth surface is lowered by the organic acid produced by the metabolism of these microorganisms, demineralization occurs on the enamel surface, and Erosion occurs and progresses. In addition to dental caries, plaque formation is said to cause periodontal disease and bad breath.

As described above, plaque formed by streptococci of the oral cavity centering on Streptococcus mutans (hereinafter abbreviated as "S. mutans") causes dental caries. Suppressing formation can eventually be an effective means of preventing the development of caries.

Conventionally, as a method for preventing caries, an anti-cariogenic substance such as an antibacterial agent against oral microorganisms and an enzyme that decomposes glucan formed using sucrose as a substrate, and a non-cariogenic substance that does not serve as a substrate for plaque formation. Various studies have been conducted on sugars.

Of these, polyphenols contained in plants have recently been attracting attention as one of the caries-preventing substances.

For example, Japanese Unexamined Patent Publication (Kokai) No. 59-152311 discloses an oral composition containing a condensed tannin which is a condensate of flavan-3-ol. The glucosyltransferase inhibitory activity was not sufficient.

On the other hand, as a flavonoid polymer, a dimer such as theaflavin produced in the fermentation process of tea is well known. They are polyphenol oxidase (EC 1.14.18.1) acting on catechins. And the peroxidase is not involved in their production.

[0009]

As described above, in the present situation that an anti-caries agent having a satisfactory effect has not yet been found, it has a sufficiently strong effect and is safe to the human body. The development of an anticaries agent that does not cause any problems was left as an issue.

[0010]

[Means for Solving the Problems] Hiroyuki Ono, one of the present inventors, first found a component that strongly inhibits glucosyltransferase in oolong tea extract, and revealed that it is a plant polyphenol having a molecular weight of about 2,000. [Hiroyuki Ono; Food Industry, Vol. 35, 18
No. 34-39 (1992)].

[0011] The inventors have been earnestly researching the industrial synthesis method of this plant polyphenol, and in the process, the flavonoid polymer obtained by accidentally treating the flavonoid with peroxidase is the oolong tea extract. Although it is different from polyphenol which is an active ingredient, it has been found that it has a sufficiently strong anti-caries activity by itself.

That is, the first object of the present invention is to provide a flavonoid polymer having a molecular weight of 800-5,000, which is produced by treating a flavonoid with peroxidase (EC 1.11.1.7). . Also,
A second object of the present invention is to provide a glucosyltransferase inhibitor containing a flavonoid polymer produced by treating a flavonoid with peroxidase (EC 1.11.1.7).

A third object of the present invention is to provide a food containing the above flavonoid polymer. Furthermore, a fourth object of the present invention is to provide a method for producing an anticaries substance characterized by treating flavonoids with peroxidase.

The flavonoid polymer of the present invention is produced by treating a flavonoid with peroxidase (EC 1.11.1.7) according to a conventional method.

Regarding this flavonoid polymer produced by peroxidase treatment (hereinafter referred to as "flavonoid polymer"), only a dimer is known [Klaus Weiniges, Hans Mattauc.
h, Cornelius Wilkins and David Frost; Liebigs Ann.
Chem. 754, 124-136 (1971)], and nothing is taught about the physiological activity of the dimer. Further, none of the flavonoid polymers having a molecular weight of 800 to 5,000 have been described in the literature and are novel compounds.

The flavonoid that can be used as a raw material for the preparation of the flavonoid polymer is not particularly limited, and all flavonoids can be used, but more preferably catechins, chalcones, dihydroflavonols, flavanones, These are flavones, flavonols and isoflavones, and catechins can be preferably used. Further, a ground product of a plant rich in flavonoids such as tea extract or the extract itself may be used as a raw material.

On the other hand, the enzyme peroxidase is not particularly limited, but microorganisms such as Arthromyces ramosus and horseradish can be used.

The peroxidase treatment of flavonoids
For example, it can be obtained by polymerizing flavonoids as raw materials with peroxidase in the presence of hydrogen peroxide to obtain a mixture of polymers having different degrees of polymerization.

The temperature of this reaction and the pH of the buffer used are
It is preferable that the range of the enzyme used is in the optimum range. The reaction time is preferably in the range of 1 minute to 24 hours, particularly preferably 1 to 3 hours.

The flavonoid polymer of the present invention thus obtained is produced by a peroxidase treatment, whereas the plant polyphenol which is the active ingredient of the oolong tea extract is treated by the action of polyphenol oxidase of tea leaves and heat treatment. Since they are produced, the production methods are different, and since peaks having the same retention time are not detected in the HPLC analysis, it is judged that they are different substances.

[0021] In addition, Hiroyuki Ono and two others who are one of the inventors of the present invention conducted extensive research into a method for enhancing the glucosyltransferase inhibitory activity of catechins, which is characterized in that catechins are heat-treated, and the results were characterized. Kaihei 3
It is disclosed in Japanese Patent No. 284671.

According to this, the increase in the glucosyltransferase inhibitory activity by heat treatment of catechin was about 4 times, but the increase in the inhibitory activity by the peroxidase treatment of the present invention was about 40 times or more in the case of catechins. Since it has been dramatically improved, it is clear that the flavonoid polymer of the present invention is different from that produced by the above method.

Although the reaction product obtained as described above has sufficient glucosyltransferase inhibitory activity by itself, it does not affect the activity of Develosil ODS.
(Nomura Kagaku) using a reversed-phase column and fractionating by high performance liquid chromatography (hereinafter referred to as “HPLC”) to further purify the compound to obtain a substance having a stronger inhibitory activity.

The mobile phase used in this purification operation is preferably water and methanol or acetonitrile. Excess hydrogen peroxide that has not been used in the reaction remaining in the system can be completely removed by column treatment using Diaion HP-20 (Mitsubishi Kasei) or HPLC treatment, but is removed by catalase treatment or the like. You can also

The above-mentioned peroxidase reaction product or the flavonoid polymer of the present invention obtained by preparative HPLC of the peroxidase reaction product can be used in any state such as a raw product, a concentrated product or a dried product without solvent. However, it is preferable to use a dried product in terms of storage stability and safety of organic solvent.

The glucosyltransferase inhibitor of the present invention is prepared by using the flavonoid polymer obtained as described above as an active ingredient and combining this with a known pharmaceutically acceptable carrier. Since the flavonoid polymer of the present invention has a strong glucosyltransferase inhibitory activity, it exhibits sufficient inhibitory activity due to the presence of an effective amount of flavonoid polymer, and as long as hydrogen peroxide is completely removed, Both the crude reaction product of the remaining flavonoids and the mixture of flavonoid polymers with various degrees of polymerization can be included as active ingredients. Of course, the flavonoid polymer of the present invention purified by HPLC fractionation can be contained as an active ingredient.

This glucosyltransferase inhibitor can be used mainly as an anti-caries agent for the purpose of anti-caries and as an oral hygiene agent, as well as for anti-fungal purposes.

Examples of the anticaries agent and oral hygiene agent in the present invention include toothpaste, mouthwash, troche and the like.

The flavonoid polymer of the present invention can be added to various foods for the purpose of anti-caries.
Examples of these foods include juice, gum, candy and the like, and appropriate components generally used depending on the type can be used for the production thereof.

The flavonoid polymer of the present invention is safe in view of the fact that the flavonoid, which is a raw material, is contained in tea etc. However, when the polymer of the present invention is blended into oral preparations, foods, etc., The concentration range of 0.0001-0.5% is preferable in terms of taste, color, scent and the like.

[0031]

FUNCTION AND EFFECT OF THE INVENTION Since the flavonoid polymer of the present invention has a glucosyltransferase inhibitory activity which is about 50 times or more that of the starting flavonoid, a glucosyltransferase inhibitor such as an anticaries agent containing this as an active ingredient is It is extremely strong and exhibits a sufficient effect in preventing caries.

In particular, since the flavonoid polymer of the present invention itself does not have a peculiar taste, odor, etc., when a flavonoid polymer obtained by sufficiently removing raw material flavonoids mixed as impurities is used as an active ingredient, an arbitrary amount is obtained. It is extremely excellent because it can be added to an anti-caries agent or food.

[0033]

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 a purification method using a reversed-phase carrier of a highly active substance, examples and reference examples relating to assay tests of glucosyltransferase inhibitory activity, but the present invention is not limited to these examples and the like. Not something.

Example 1 Production of flavonoid polymer: 1.6 g of (+)-catechin, which is a raw material, was dissolved in 5 ml of 40% (v / v) ethanol, and the solution was dissolved in 0.1 M phosphate buffer. (PH 6.0) 155
(+)-catechin solution (1
7.2 mM; final concentration, the same below), 0.16 M hydrogen peroxide solution 10 ml (8.8 mM), 0.1 M phosphate buffer (pH 6.0) 20 ml (15.0 mM) and 1 mg /
ml horseradish peroxidase (Worthington biochem., USA) 0.1M
Phosphate buffer solution (pH 6.0) 10 ml (50 μg /
ml) was used for peroxidase treatment at 37 ° C.

After reacting for 2 hours, distilled water (4.8 liters)
Was added, and this was filtered through Celite. This filtrate was adsorbed on a Diaion HP-20 (Mitsubishi Kasei) column,
After washing the column with distilled water, the adsorbed component was eluted with 40% ethanol, and this fraction was concentrated under reduced pressure to give 0.5 g of a powdery residue.
Got

Example 2 Purification of flavonoid polymer: The reaction product obtained in Example 1 was subjected to HPLC fractionation in a water-methanol system using a reverse phase Develocyl ODS-10 (Nomura Kagaku) column.
The mobile phase is 30% methanol water (flow rate 32 ml / mi
n.) and detected by UV at 210 nm. HPLC
Four peaks (1) (2) as the main peaks above
(3) and (4) were recognized. The respective molecular weights were measured using FAB-MS (Fast Atom Bombardment Mass Spectrometer). As a result, (1) was 290, (2) was 1440, 1152,
864 mixture, (3) is a mixture of 864 and 576,
(4) was 576.

From this, a mixture of (1) raw material (+)-catechin, (2) (+)-catechin trimer, tetramer and pentamer (hereinafter referred to as "catechin-3, 4"). , 5-m
abbreviated as "er"), a mixture of dimers and trimers of (3) (+)-catechin (hereinafter, referred to as "catechin-2, 3-
abbreviated as “mer”), and was presumed to be a dimer of (4) (+)-catechin. Each of these contents is (1) 6
1.0%, (2) 1.0%, (3) 9.0%, (4) 5.2
%Met.

Of the above-mentioned peaks, the structure of the substance showing the peak of (4) was analyzed from its physicochemical properties and various spectral data of the methylated product of this substance. As a result, it was found that dehydro-dicatechin A (dehydrodicatechin A) was obtained. A) 〔K
laus Weiniges, Hans Mattauch, Cornelius Wilkins and
David Frost; Liebigs Ann. Chem. 754,124-136 (197
1)] was identified.

The MS and ¹ H-NMR spectra of catechin-3,4,5-mer are shown in FIGS. 1 and 2, and the MS and ¹ H-NMR spectra of catechin-2,3-mer are shown in FIGS. 3 and 4. Show.

The peak found in the oolong tea extract, which has the same retention time as the polyphenol having a strong glucosyltransferase inhibitory activity and having a molecular weight of about 2,000, is the HPLC of the reaction product obtained in Example 1.
Not found in analysis.

Example 3 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, 5.0%
0.6 ml of 500 mM sodium phosphate buffer (pH 6.0) containing sucrose, 0.5% dextran T10 and 0.5% sodium azide, an aqueous solution of a test sample 0.15
ml, glucosyltransferase enzyme solution (S. Hamada et al., J. Gen. Microbiol., 135, 335-, extracted from bacterial cells with 8 M urea from S. mutans MT8148 strain cultured in Todd-Hewitt medium. 344 (198
9)) and a total amount of 3 ml of water are added to make a reaction system, and the reaction is carried out in a glass test tube. At this time, the amount of enzyme is set so that the absorbance at 550 becomes 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. In addition, the inhibition rate (%) was determined by the following calculation formula, using the absorbance when water was used instead of the sample solution as a control (B).

Inhibition rate = (B−A) × 100 / B

As a comparative sample, Japanese Patent Laid-Open No. 59-1523 is used.
Crude extract of crude drug and condensed tannin, which were prepared according to the method of Japanese Patent No. 11, were used. Table 1 shows the ID ₅₀ of glucosyltransferase inhibitory activity calculated from the inhibition rate obtained by the above method.

[0045]

As is apparent from these results, the glucosyltransferase activity of the flavonoid polymer of the present invention is several tens to hundreds of times higher than that of the raw flavonoid (control), and several times higher than that of the condensed tannin compound. It was several tens of times stronger.

Example 4 Toothpaste: Toothpaste having the following composition was prepared according to a conventional method.

Example 5 Mouth rinsing liquid: A mouth rinse was prepared according to a conventional method with the following composition. ** Catechin trimer, tetramer and 5 obtained in Example 2
A mixture of monomers.

Example 6 Chewing gum: A chewing gum having the following composition was prepared by a conventional method.

[Brief description of drawings]

FIG. 1: Trimer, tetramer and 5 of (+)-catechin
The mass spectrometry (MS) spectrum figure of the mixture which consists of a monomer.

FIG. 2: (+)-Catechin trimer, tetramer and 5
The < ¹ > H-NMR spectrum figure of the mixture which consists of a monomer.

FIG. 3 is an MS spectrum diagram of a mixture of a (+)-catechin dimer and a trimer.

FIG. 4 is a ¹ H-NMR spectrum diagram of a mixture of a (+)-catechin dimer and a trimer. that's all

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ryuji Tanaka 1-1-1, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka Suntory Ltd. Biomedical Research Institute (56) Reference JP-A-4-182480 (JP, A) J. Agric. Food Ch em. , Vol. 38, p. 1202-1204 (1990) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12P 17/06 A23L 1/28 A61K 7/16 C07D 311/62 C12N 9/99 CA / BIOSIS / MEDLINE / W PIDS (STN)

Claims (9)

(57) [Claims] 1. A catechin is a peroxidase (EC1.
11.1.7) 800 to 5, produced by processing
Polymers of catechins having a molecular weight of 000. 2. The polymer of catechins according to claim 1, which has a degree of polymerization of 3 to 10. 3. Catechins are treated with peroxidase (EC1.
11.1.7) Weight of catechins produced by treatment
A glucosyltransferase inhibitor containing a compound as an active ingredient. 4. The glucosyltransferase inhibitor according to claim 3 , wherein the polymer of catechins is a catechin dimer. 5. The glucosyltransferase inhibitor according to claim 3 or 4, which is used as an anticaries agent. 6. The glucosyltransferase inhibitor according to claim 3 or 4, which is used as an oral hygiene agent. 7. A catechin is a peroxidase (EC1.
11.1.7) Weight of catechins produced by treatment
A food characterized by containing a compound . 8. The food according to claim 7 , wherein the polymer of catechins is a catechin dimer. 9. A method for producing an anticaries agent , which comprises treating catechins with peroxidase.