JPH04290831A - New substance af90-nf and glucosyltransferase inhibitor - Google Patents

Abstract

PURPOSE:To obtain new water-soluble substance Af90-NF effective as a dental caries preventing agent, having glycosyltransferase inhibitory activity. CONSTITUTION:New substance Af90-NF having physical and chemical properties of a brown cotton shape, not showing a melting point, decomposing at >=270 deg.C, having an elemental analysis value of C 42.5%, H 3.1%, N 0% ash 5.1%, 1,060 weight-average molecular weight using N,N'-dimethylformamide (0.05N LiCl, 0.05 M phosphoric acid) as a mobile phase based on polyethylene glycol as molecular weight standard by gel permeation chromatography, having infrared absorption spectrum and ultraviolet absorption spectrum shown by the table, color reaction positive in ferric chloride reaction and negative in aniline and diphenylamine reaction, etc., solubility: insoluble in hexane, slightly soluble in methanol, soluble in water. The compound is obtained by extracting fruits of Alnus firma with hot water, can prevent glucan to cause bacterial plaque from forming and has not side effects at all.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to a novel substance Af90-NF having a glucosyltransferase inhibitory effect and a glucosyltransferase inhibitor containing it as an active ingredient.

0002

BACKGROUND OF THE INVENTION Dental caries is induced and progresses by the formation of dental plaque. This dental plaque is, so to speak, bacterial flora that adheres to the tooth surface. Among these caries-causing bacteria, the main one is Streptococcus mutans (Streptococcus mutans).
ococcusmutans, hereinafter referred to as S. ococcusmutans. mutan
(abbreviated as s), which is classified by its serotype from a to h
It is classified into eight types, and among these, type C is said to be the predominant type in the human oral cavity.

[0003]S. mutans secretes glucosyltransferase outside the bacterial body and synthesizes water-insoluble sticky glucan from sucrose. The synthesized glucans form dental plaque, and within the plaque, bacteria produce organic acids that demineralize the tooth enamel surface and promote caries. Therefore,
Suppressing the production of sticky glucan, which causes plaque formation, is considered to be an important means for preventing dental caries. Since glucans are not produced if glucosyltransferase is inhibited, various studies have been conducted on glucosyltransferase inhibitors.

[0004] For example, JP-A-58-121218 discloses a caries preventive agent (Claim 1 of the patent) which contains a herbal medicine extract having a glucosyltransferase inhibitory effect as an essential ingredient. , fennel,
The following are mentioned: peony, gentian, senso, white lily, long gall, yellow lotus, common lily, and yellow quince. Other glucosyltransferase inhibitors include JP-A-59-1
Extracts from Casuarina and Obayashi Shabushi disclosed in Japanese Patent Publication No. 52313, and Japanese Patent Application Laid-Open No. 1523-1983
Examples include condensed tannins disclosed in Japanese Patent No. 11.

However, none of the conventional glucosyltransferase inhibitors is effective against c-type glucosyltransferase, and no one has yet been found to have a satisfactory effect.

[0006]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a substance that is soluble in water and has inhibitory activity against c-type glucosyltransferase, and a glucosyltransferase inhibitor containing the same as an active ingredient. .

[0007]

[Means for Solving the Problems] The present inventors conducted intensive research to find a substance that effectively inhibits glucosyltransferase, and as a result, discovered that Af90-NF is extremely effective in inhibiting glucosyltransferase. However, the present invention was completed. The present invention provides a novel substance Af90-NF having the following physical and chemical properties. (i) Shape: Brown flocculent (ii) Melting point: No melting point, decomposes at 270°C or higher. (iii) Elemental analysis: carbon 42.5% hydrogen
3.1% Nitrogen 0.0% Ash 5.1% (iv) Molecular weight: 1060 (weight average molecular weight)
Measured using gel permeation chromatography using polyethylene glycol as the molecular weight standard and N,N-dimethylformamide (0.05NLiCl, 0.05M phosphoric acid) as the mobile phase.

[0008]

[Chemical formula 2] (vii) Color reaction: Ferrous chloride reaction
Positive aniline/diphenylamine reaction Negative ninhydrin reaction
Negative Dragendorff reaction
Negative p-anisidine phthalate reaction
Negative Lieberman-Burchard reaction
Negative hydrochloric acid-magnesium reaction
Negative (viii) Solubility: Insoluble in hexane, diethyl ether, chloroform. Slightly insoluble in methanol and pyridine, soluble in water and dimethyl sulfoxide.

The present invention also provides a glucosyltransferase inhibitor containing the above Af90-NF as an active ingredient. The present invention will be explained in detail below. i) Raw material Alnus firma, which is the raw material for extracting the glucosyltransferase inhibitor of the present invention, is
It is a deciduous tree belonging to the Birchaceae family, distributed in the mountains of the Japanese archipelago, and is also called Minebari.

The raw material used in the present invention is the fruit part of Yashabushi (commonly referred to as Yashajitsu). When using the fruit of the above-mentioned plants as an extraction raw material in the present invention, it is preferable to use one that has been subjected to treatments such as drying, coarse crushing, and pulverizing to facilitate extraction. ii) Extraction Af90-NF of the present invention is a phenolic substance that shows positivity in the ferric chloride reaction and is characterized by its glucosyltransferase inhibitory activity. Therefore, Af90-NF can be purified using appropriate purification methods such as extraction with organic solvents or water, centrifugation, filtration, etc., using these inhibitory activities as indicators. These methods can be applied singly or in combination in any order, or repeatedly, as necessary. An example of a method for extracting and purifying Af90-NF will be described below.

(a) The extraction raw material, Yashabushi fruit (Yasha fruit), is extracted with a highly polar solvent such as methanol, ethanol, or ethyl acetate. This operation allows removal of the polar solvent soluble portion. A known method can be used as the extraction method, and the extraction solvent is used at a ratio of 2 to 10 times the raw material.
Extraction is repeated once or several times at 65 to 85°C for about 1 to 3 hours.

(b) The extracted raw material that has been extracted in (a) is air-dried to remove the polar solvent and dry the raw material. (c) The extracted raw material that has been air-dried in (b) is further extracted with hot water. As the extraction method, a known method is used as in (a), and the extraction solvent is used in a ratio of 2 to 10 times the extraction raw material. Extraction is repeated one to several times in a water bath at 65 to 85°C for about 1 to 3 hours. Each time, the extract is filtered using filter paper. The obtained filtrates are combined and freeze-dried to obtain an extract.

(d) The extracted material obtained in (c) is further subjected to column chromatography for separation and purification. Although the column chromatography performed here is not particularly limited, gel permeation column chromatography is effective. Using water as the mobile phase, elute by adding acetone at appropriate times and changing the solvent mixture ratio until acetone is used. The desired active substance is eluted with water-acetone (1:1), and this eluted fraction is lyophilized to obtain a brown flocculent material.

[0014] The brown flocculent material obtained here was converted into Af90-
Name it NF. This extraction operation is optimal as a method for obtaining the desired glucosyltransferase inhibitor. The method for measuring glucosyltransferase activity and the amount of attached bacterial cells used in the present invention are as follows. I. Method for measuring glucosyltransferase activity i) Preparation of g-type glucosyltransferaseS.
Mutans 6715 strain (type G) is statically cultured in brain heart infusion (BHI) medium at 37° C. for 24 hours, and the culture filtrate is centrifuged at 6000 rpm for 15 minutes to obtain a culture supernatant. Under ice, ammonium sulfate was added to this supernatant until it became 50% saturated for salting out.
Centrifuge at 0 rpm for 15 minutes to collect the precipitate. This precipitate is dissolved in 50 mM phosphate buffer (pH 6.5) and dialyzed against the same buffer overnight at 4°C to obtain a glucosyltransferase enzyme preparation solution. This is used to measure enzyme activity.

ii) Preparation of c-type glucosyltransferase S. Mutans MT8148 strain (c type) is statically cultured in TTY medium at 37°C to obtain bacterial cells. The crude enzyme was extracted with 8M urea, dissolved in 50mM phosphate buffer (pH 6.5), and dialyzed against the same buffer at 4°C. This is used to measure enzyme activity.

iii) Measurement of glucosyltransferase activity A reaction solution consisting of 50mM phosphate buffer (pH 6.5), 1% sucrose and 0.2% sodium azide was prepared, and the enzyme and sample were added to it in a glass test tube. Enzyme reaction was carried out at 37°C for 18 hours. At this time, the amount of enzyme was 550n in the above reaction system.
Adjust so that the absorbance of m is 0.5.

[0017] The produced insoluble glucan is crushed by ultrasonication, and 5
Measure the absorbance at 50 nm. iv) Glucosyltransferase inhibition rate Af90
A similar operation was performed using distilled water instead of -NF as a control, and the glucosyltransferase inhibition rate was calculated from the following formula. II. Method for measuring the amount of bacteria attached To measure the amount of bacteria attached, S. mutans strain 6715 (
Type G) and S, which is said to be the mainstream of the human oral bacterial flora.
．． mutans MT8148 strain (type c) was used.

6715 in a dry heat sterilized test tube with a lid.
Brain heart infusion (BH) is used for strain (g type).
I) medium, and for MT8148 strain (type c), RP
1.5 ml each of MI medium (pH 7.0, sterilized by filtration with a 0.45 μm filter) (the following is common)
300 μl of sterile 10% sucrose, S. Add 200 μl of mutans overnight culture, sample, and water to make a total volume of 3 m
Prepare the reaction system so that the amount of

The test tube was tilted at 30° and cultured at 37°C for 22 hours, and the adhesion rate was determined as follows. i) After rotating the test tube three times, transfer the unattached bacterial cells to a new test tube (No. 1) with the attached side facing up. ii) Add 3 ml of deionized water with the attached side up, rotate the tube 3 times, and transfer the liquid with the unattached bacterial cells to a new test tube (No. 2) with the attached side up.

iii) Add 3 ml of deionized water with the attached side up, mix with a vortex mixer, and transfer the liquid with the unattached bacterial cells to a new test tube (No. 3) with the attached side up. Move. iv) Add 3 ml of deionized water to the test tube where steps i) to iii) have been completed. (NO.4) v) NO. 1~NO. Sonicate all 4 test tubes and measure the absorbance at 550 nm. Test tube no. 2~
4 is water, NO. 1, each medium is used as a blank.

[0021]NO. 1~NO. The absorbance of 4 is D
1 to D4, and the adhesion rate was calculated using the following formula. (1) Loose (total adhesion amount): (D3 +D4)/(D1 +D2 +D3
+D4 ) x 100 (%) (2) Firm (things that cannot be removed by mixing): (D4 ) / (D1 +D2
+D3 +D4)×100(%) The glucosyltransferase inhibitor of the present invention is the Af90-N of the present invention.
F is added to oral compositions, food and drinks, etc. by a known method, and caries can be prevented by using this.

[0022] Examples of oral compositions include toothpaste, powdered toothpaste, mouthwash, and the like. In addition, food and drink products include various confectionery products such as candy and chewing gum, as well as all other beverages and food products. The compound generally contains 0.01% of the total composition.
~10% (wt%, same below), preferably 0.1~
A sufficient effect can be achieved by blending at a ratio of 5%.

[0023]

EXAMPLES The present invention will be explained in more detail with reference to Examples below. Percentages are by weight unless otherwise specified. Example 1 Alnus firma as an extraction raw material
The glucosyltransferase inhibitor Af90-NF was produced using the fruit (Yakshami).

A) 2000 ml of methanol was added to 490 g of Yashami (purchased from Uchida Wakanyaku Co., Ltd.), a cooling tube was attached, and extraction was performed at 80° C. for 2 hours in a water bath. Extraction was performed twice in total, and the extracts were combined and concentrated to dryness under reduced pressure. (b) After the methanol extraction, the Yashami was air-dried at room temperature. c) After the air-drying, the Yasha seeds were extracted with 2000 ml of water at 80° C. for 2 hours in a water bath. Extraction was performed twice in total, and each extract was filtered and combined and freeze-dried. (Extract I, yield 27.2g) d)
After adding and dissolving 60 ml of water to 2.0 g of the water extracted portion, chromatography was performed using TOYOPEARL HW-40F. Elution was first performed using water, and then acetone was added at appropriate times to change the solvent mixture ratio until acetone was used. Water-acetone (1:1)
Strong activity was observed in the fraction eluted with. The active substance targeted by this operation (Af90-NF)264
mg (Extract II) was obtained.

Table 1 shows the yield and 50% inhibitory concentration (IC50) for glucosyltransferase in each extraction step.
Table 1 ━━━━━━━━
━━━━━━━━━━━━━━━━━━━━━━━━━

IC50
(μg/ml) Extract
Yield ━━━━━━━━
━━━━━━━
(g) g
Type c Type ━━━━━━━
━━━━━━━━━━━━━━━━━━━━━━━━━
━I
27.2 15.3
45.2 II
0.264 12.8
18.6 ━━━━━━━━━━━━━━━━
━━━━━━━━━━━━━━━━━Also, S. mut
ans6715 strain (G type) and MT8148 strain (C type)
The effect of inhibiting the bacterial adhesion rate was investigated for both loose and farmed types. The bacterial adhesion rate inhibition effect was calculated based on the adhesion inhibition rate when Af90-NF was not added as 0%. The results are shown in Table 2.

[0026]
Table 2 ━━━━━━━━━━
━━━━━━━━━━━━━━━━━━━━━━━

Adhesion rate inhibition effect (%)
Concentration ━━━━━━━━━━━━━━━
━━━━━━━━
MT 8148 shares (
type c) (μg/ml) ━━━━━━━
━━━━━━━━━━━━━━━━
Ruth
Farm ━━━━
━━━━━━━━━━━━━━━━━━━━━━━━━
━━━━ 0
0.0
0.0 10
3.7
26.0 20
0.0
27.3 40
2.2
86.9 80
74.1
100.0 ━━━━━━━━━━━━━━
━━━━━━━━━━━━━━━━━━━ ━━━━
━━━━━━━━━━━━━━━━━━━━━━━━━
━━━━
Adhesion rate inhibition effect (%)
Sample concentration ━━━━━━━━━
━━━━━━━━━━━━━━

6715 strains (G type) (μg/ml) ━
━━━━━━━━━━━━━━━━━━━━━━

Ruth Farm ━━━━━━━━━━━━━━━━━━━━━━━
━━━━━━━━━━ 0
0.0
0.0
10 0.0
0.0
25 0.7
0.0
50 2.4
2.3
100 3.
1 2.3 ━━━
━━━━━━━━━━━━━━━━━━━━━━━━━
━━━━━In addition, Comparative Example 1 is JP-A-58-121218
A water extract and a methanol extract of fennel were prepared according to the method for preparing crude drug extracts described in the above issue, and used as samples for measuring GTase inhibitory activity.

Comparative Example 1 ━━━━━━━━━━━━━━━━━━━━━━━
━━━━━━━━━━

IC50 (μg/ml) Extraction solvent ━━━━━━━
━━━━━━━━━━

G type C type ━━━━━
━━━━━━━━━━━━━━━━━━━━━━━━━
━━━ Water
>500
>500 MeOH
328.2
＞500 ━━━━━━━━━━━━━━━━━━
Example 2 A toothpaste having the following formulation was produced according to a conventional method.

Ingredients Dibasic calcium phosphate
45.0 (%) Carboxylmethyl
cellulose sodium
1.0 Glycerin
20
．． 0 Sodium lauryl sulfate
1.5 〃
fragrance
1.0
〃 Saccharin sodium
0.15 A
f90-NF
0.1 〃
water
Adjustment to 100% Example 3 A gargle with the following formulation was manufactured according to a conventional method.

Ingredients Ethanol
20.0 (%)
saccharin sodium
0.2 〃 Fragrance

1.0 Glycerin
5.0 Chlorhexidine
0.005〃 Af90-NF

0.01 Water

Adjustment to 100% Example 4 A chewing jelly was produced according to a conventional method with the following formulation.

Ingredients Granulated sugar
31.0 (%)
starch syrup
29.0 〃
gelatin
7.7 〃
Sorbitol
4.2 〃
citric acid
0.7 〃
Af90-NF
0.1 〃
Water (A)
11.8 〃
Water (B)
15.5 〃
Coloring agent Flavoring agent granulated sugar, starch syrup, sorbitol in water (A), 13
After boiling down to 0°C (sugar content 85°), it was cooled to 115°C. Gelatin swollen with water (B) is added and dissolved, and further citric acid, Af90-NF, coloring agent,
The flavoring material was combined, molded, and cooled (25° C., 18 hours). Example 5 A coffee beverage was produced using the following formulation according to a conventional method.

Ingredients Instant coffee
1.7 (%) Granulated sugar
5.0〃Af90
-NF
0.05 〃 Water (or hot water)
Adjust to 100%

[0032]

Effects of the Invention According to the present invention, a new substance Af90-NF, which is made from the fruit of the palm tree and has a glucosyltransferase inhibitory effect (particularly effective against c-type glucosyltransferase), and it is contained as an active ingredient. Glucosyltransferase inhibitors are provided.

[0033] This new substance Af90-NF and the glucosyltransferase inhibitor containing it have a glucosyltransferase inhibitory effect, so they can prevent the production of glucans that cause plaque formation, and in addition, they are Since it is soluble, it is effective as a caries preventive agent. Furthermore, since this new substance Af90-NF is made from plants, it can be used with confidence without worrying about side effects.

[Brief explanation of the drawing]

FIG. 1 shows the infrared absorption spectrum of Af90-NF.

FIG. 2 shows the ultraviolet absorption spectrum of Af90-NF.

Claims (2)

[Claims] Claims: 1. A new substance Af90-NF having the following physical and chemical properties: (i) Shape: Brown flocculent (ii) Melting point: No melting point and decomposes at 270°C or above. (iii) Elemental analysis: carbon 42.5% hydrogen
3.1% Nitrogen 0.0% Ash 5.1% (iv) Molecular weight: 1060 (weight average molecular weight)
Measured using gel permeation chromatography using polyethylene glycol as the molecular weight standard and N,N-dimethylformamide (0.05NLiCl, 0.05M phosphoric acid) as the mobile phase [Formula 1] (vii) Color reaction: Chloride II iron reaction
Positive aniline/diphenylamine reaction Negative ninhydrin reaction
Negative Dragendorff reaction
Negative p-anisidine phthalate reaction
Negative Lieberman-Burchard reaction
Negative hydrochloric acid-magnesium reaction
Negative (viii) Solubility: Insoluble in hexane, diethyl ether, chloroform. Slightly insoluble in methanol and pyridine, soluble in water and dimethyl sulfoxide. 2. A glucosyltransferase inhibitor containing Af90-NF according to claim 1 as an active ingredient.