JPH11269075A - Ige-ige receptor bonding inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject inhibitor capable of inhibiting bonding of IgE to an IgE receptor and useful for medicaments, cosmetics, foods, etc., and comprehensively improving allergic symptoms by using eugenin as an effective ingredient. SOLUTION: This IgE-IgE receptor bonding inhibitor contains eugenin, e.g. a compound of the formula, etc., as an effective ingredient. Eugenin is preferably obtained by extracting rose of the genus Rosa, the family Rosaceae, isolating and identifying, e.g. crushing a dried product of rose min buds pink, a plant of the genus Rosa, extracting with hot water at 50-70 deg.C for 30 minutes, separating the extracted solution by filtration, lyophilizing the solution and separating and purifying through column chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an IgE-IgE receptor binding inhibitor suitably used as a medicine, cosmetics, foods, and the like.

[0002]

2. Description of the Related Art Eugenin (1,2,3-triga)
lloyl-4,6-hexahydroxydiph
enyl-β-D-glucopyranose)
A compound represented by the following formula (I), which is one of tannins known to have an activity such as anti-platelet aggregation.

[0003]

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[0004] On the other hand, various compounds having an anti-allergic activity have hitherto been known.
Examples thereof include sodium cromoglycate and ibudilast.

[0005] On the other hand, an allergic reaction occurs as follows. Human IgE antibodies are produced when pollen and mite allergens enter the body, and bind to high-affinity IgE receptors (FcεRI) on the surface of basophils, mast cells, etc., which are abundant in blood and mucous membranes, and become sensitized. Become. When the same antigen enters again and binds to the human IgE antibody on the sensitized cells, this triggers the release of chemical mediators such as histamine and leukotriene from the cells,
These substances cause a large amount of tears and nasal secretion, cause coughing, and itchy skin. Therefore, the human IgE antibody I
If the binding to the gE receptor can be inhibited, the release of these chemical messengers can be prevented, and this can be an effective treatment for allergic diseases (Japanese medical journal,
Vol. 114, No. 9 (1995), Medical Society of Japan; Special Issue of Experimental Medicine, Vol. 12, No. 17, (1994), Yodosha).

[0006]

However, the above-mentioned sodium cromoglycate exerts an anti-allergic effect through antihistamine activity, and ibudilast exerts an anti-allergic effect through suppression of release of leukotriene. Therefore, it was insufficient from the viewpoint of comprehensive improvement of allergic symptoms.

[0007] Japanese Patent Application Laid-Open No. 2-53717 discloses that
It has been suggested that pseudofruits or fruits (Rosaceae, fruit) of Rosaceae or related plants have hyaluronidase inhibitory activity, but there is no mention of the antiallergic effect by inhibiting the binding of IgE to the IgE receptor.
Further, when actually examined, there was no inhibitory activity. Furthermore, Japanese Patent Application Laid-Open No. 9-124498 suggests that ellagitannin of Rosaceae has an antiallergic effect, but does not mention the inhibition of IgE binding to the IgE receptor.

[0008] With the recent increase in allergic diseases related to type I allergic reactions such as atopic dermatitis and hay fever, attempts have been made to impart an antiallergic effect to cosmetics and foods. .

Accordingly, there is a long-felt need for pharmaceuticals, cosmetics, foods and the like that can comprehensively improve allergic symptoms by inhibiting the binding of IgE to the IgE receptor.

[0010]

Means for Solving the Problems The present inventors have found that eugenin inhibits the binding between IgE and IgE receptor, and have completed the present invention.

That is, according to the present invention, there is provided an IgE-IgE receptor binding inhibitor comprising eugenin as an active ingredient.

Further, according to the present invention, the above IgE-Ig
An antiallergic drug containing an E receptor binding inhibitor is provided, and cosmetics, quasi-drugs, and foods containing the above IgE-IgE receptor binding inhibitor are provided.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The IgE-IgE receptor binding inhibitor of the present invention contains eugeniin represented by the following formula (I) as an active ingredient.

[0014]

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Eugeniin is a rose of the genus Rosaceae ( R)
osa. spp ), isolated and identified, but may be any other than these plants as long as they have eugeniin.

For the extraction, water alone or a mixed solvent of water and a polar solvent such as methanol, ethanol or acetone is used. The extraction temperature is appropriately selected from the range from room temperature to the boiling point of the solvent. It is desirable to use hot water of about 70 ° C. As an extraction method, the raw material that has been washed, dried, and shredded is mixed with about 5 to 50 times, preferably about 20 times, the amount of hot water, and the mixture is directly used for 30 times.
After immersion for 1 minute to 1 day, the mixture is filtered by membrane treatment and the like, and water is distilled off by concentration under reduced pressure to obtain a freeze-dried product.

As a purification method, this hot water extract is converted into an aqueous solution, and then a solvent fraction such as ethyl acetate or n-butanol, or Sephadex LH20, HW40F is used.
Gels such as SP850, SP825, HP2
Fractions having a high inhibitory activity were collected using synthetic resins such as HP21 and HP21, and several times with Inertsil PREP-OD.
A desired eugenin is obtained using a reversed-phase column for HPLC such as S (inner diameter 20 × 250 mm).

The IgE-IgE receptor binding inhibitor of the present invention can be suitably used as a pharmaceutical ingredient, and when added to foods, cosmetics, etc., can impart an antiallergic function thereto.

The antiallergic drug containing the IgE-IgE receptor binding inhibitor of the present invention is prepared by a commonly used method using pharmaceutical carriers, excipients and the like usually used in the art. be able to. The IgE-IgE receptor binding inhibitor of the present invention comprises a tablet,
Oral preparations such as powders, fine granules, granules, capsules, syrups and the like, and parenteral preparations such as suppositories, ointments, sprays and injections may be used.

The IgE-IgE receptor binding inhibitor of the present invention can be used by adding it to a wide range of foods, including beverages. Specific examples include alcohol, carbonated beverages, fruit beverages, coffee, black tea, and tea. Lactic acid bacteria drink, yogurt, ice cream, candy, gum, confectionery, bread, noodles and the like.

Examples of cosmetics to which the IgE-IgE receptor binding inhibitor of the present invention is added include soap,
Facial cleanser, cream, milky lotion, lotion, cologne, shaving cream, shaving lotion, cosmetic oil, tanning / sunscreen lotion, tanning / sunscreen oil, whitening powder, foundation, perfume, pack, nail cream, enamel, enamel Removal cosmetics, eyebrows, blushers, eye creams, eye shadows, mascaras, eye liners, lipsticks, lip balms, bath cosmetics, and other skin cosmetics; shampoos, rinses, hair dyes, hair cosmetics, and other cosmetics; Is mentioned. or,
It can also be suitably used for quasi-drugs such as medicated cosmetics, medicated dentifrices and bath preparations.

[0022]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Eugeniin was produced as follows. 100 g of dried rose mini bats pink, a plant belonging to the genus Rosaceae, crushed to 50 to 7
Extracted with 2 l of hot water at 0 ° C. for 30 minutes. Next, the extract was separated by filtration and freeze-dried to obtain about 30 g of a hot water extract. Next, 5% (W /
After preparing the aqueous solution of V), ethyl acetate was added, and the mixture was partitioned between an ethyl acetate layer and an aqueous layer, and the aqueous layer was extracted with n-butanol.

After obtaining about 4.3 g of a lyophilized sample (butanol-treated fraction) of the n-butanol layer, 5% (W /
V) was re-dissolved in methanol, and fractionation was repeated by the following column chromatography. The column is Sephadex LH with an inner diameter of 15 mm and a length of 250 mm.
20 (manufactured by Pharmacia) and methanol as an elution solvent at a flow rate of 2 ml / min.

The respective fractions were examined for IgE receptor inhibitory activity by ELISA. About 0.6 g of a fraction having high inhibitory activity was obtained, and then subjected to HPLC fractionation and purification.

The measurement of the IgE receptor inhibitory activity by the ELISA method was carried out by the following method. The final concentration of 0.1, 0. 0, 0, 1 was added to the well to which the IgE receptor was fixed.
After a sample prepared to be 01% (w / v) and human IgE so as to have a final concentration of 0.4 μg / ml, incubation was performed, followed by washing to remove a free sample and human IgE. did. Next, an anti-human IgE antibody conjugated with horseradish peroxidase (HRP) was added at a concentration of 0.4 μg / ml, incubation was performed, and then washing was performed to remove free anti-human IgE antibody. . Next, o-phenylenediamine dihydrochloride (OPD) was poured into each well as a substrate for HRP, and HR was added.
After reaction with P and color development, the absorbance of each well was measured with a plate reader. The IgE receptor inhibitory activity was calculated based on the obtained absorbance value. Human IgE
Absorbance when no was added was measured using a coloration rate of 0% and human Ig.
The color development rate of each sample was calculated with the absorbance when only E was added and the sample was not added as the color development rate of 100%, and the value obtained by subtracting the color development rate of each sample from 100 was defined as the IgE receptor inhibitory activity (%). .

In fractionation and purification by HPLC, the Sephadex LH20 fraction sample (LH20-treated fraction) was lyophilized and then adjusted to a 2% sample concentration with distilled water before use. The column has an inner diameter of 20 mm and a length of 250 mm.
ertsil PREP-ODS (manufactured by GL Sciences Inc.), and the mobile phase was A: 20% methanol + 0.1%.
1% acetic acid and B: 60% methanol + 0.1% acetic acid were used. The injection volume was 500 μl and the flow rate was 5 ml / min. Detection was performed by measuring ultraviolet absorption at 250 nm. Elution was performed according to the time program shown in Table 1.

[0028]

[Table 1]

Under the above elution conditions, the crude eugenin fraction peak 1 eluted at about 47 minutes after the start of elution in the chromatogram shown in FIG.
It was freeze-dried to obtain about 34 mg of a freeze-dried sample.

The lyophilized sample of the preparative peak fraction was distilled water
% (W / V) sample concentration and after HPLC injection
The peak 1-1 of the eugeniin fraction in the chromatogram shown in Table 2 was repeatedly collected. The peak 1-1 was lyophilized to obtain about 13 mg, which was then subjected to measurement of IgE receptor inhibitory activity by ELISA. In addition, HPL
For elution with C, the column was 20 mm in inner diameter and 2 mm in length.
Using a 50 mm Inertsil PREP-ODS (manufactured by GL Sciences Inc.), the mobile phase was A: 20% methanol + 0.1% acetic acid, B: 40% methanol + 0.
1% acetic acid was used. Flow rate 5 ml / min, injection volume 500
μl, and detection was performed at 250 nm UV. Elution was performed according to the time program shown in Table 2.

[0031]

[Table 2]

The above hot water extract, butanol-treated fraction, LH20-treated fraction, and eugenin fraction peak 1-1
Are shown in Table 3. As a control, the inhibitory activity of various plant extracts such as ketotifen fumarate, which is a histamine release inhibitor, is also shown.

[0033]

[Table 3]

FIG. 3 shows the outline of the production / purification process of eugeniin, and FIG. 3 shows the ¹ H nuclear magnetic resonance spectrum (CDCl ₃ +
CD ₃ OD) is shown in FIG.

Example 2 Eugenin obtained in Example 1 was examined for its inhibitory effect on histamine release. According to a conventional method, basophils separated from human peripheral blood were subjected to lactic acid treatment and washing to remove human IgE antibodies bound to IgE receptors. Next, the sample dissolved in TBS-HSA was added to 0.1 mL.
1. After adding to a concentration of 0.01% (W / V), human IgE was newly added to a concentration of 1 μg / ml.
The antibody was added, and basophils were sensitized at room temperature for 1 hour. T
After washing with BS-HSA to remove free human IgE antibody, anti-human IgE was adjusted to a concentration of 3 μg / ml.
The antibody was added and incubation was performed at 37 ° C. for 40 minutes. After collecting the supernatant and treating it with perchloric acid, HPL
C, the amount of histamine in the supernatant was measured.

The histamine release inhibition rate (%) was calculated by the following equation. {1- (SR-C) / (RC)} × 100 where C is the amount of histamine released when neither human IgE antibody nor anti-human IgE antibody is added, and R is the sample. SR represents the amount of histamine released when stimulated with human IgE antibody and anti-human IgE antibody without addition, and SR represents the amount of histamine released when stimulated with human IgE antibody and anti-human IgE antibody in the presence of the sample. . Table 4 shows the results.

[0037]

[Table 4]

From Table 4, it can be seen that eugenin significantly suppresses the release of histamine from basophils.

Example 3 A mixture obtained by mixing the following components in a conventional manner was tableted with a tableting machine to produce one tablet containing eugenin obtained in Example 1. Eugeniin 150mg D-mannitol 145mg Magnesium stearate 5mg

Example 4 The following components were mixed in a conventional manner to produce a bath agent containing eugeniin obtained in Example 1. Eugenin 3.0% by weight Sodium bicarbonate 55.0% by weight Sodium sulfate 40.0% by weight Pigment 1.0% by weight Fragrance 1.0% by weight

Example 5 The following components were mixed in a conventional manner to produce the eugeniin-containing cream obtained in Example 1. Eugenin 0.2% by weight Vaseline 30.0% by weight Liquid paraffin 20.0% by weight Paraffin 7.0% by weight Lanolin 4.0% by weight Sorbitan sesquioleate 4.0% by weight Propylene glycol 2.5% by weight Magnesium sulfate 0 0.2% by weight Methyl paraoxybenzoate 0.2% by weight Water 31.7% by weight Fragrance 0.2% by weight

Example 6 The following components were mixed by a conventional method to produce a eugeniin-containing lotion obtained in Example 1. Eugeniin 0.2% by weight Ethyl alcohol 10.0% by weight 1,3-butylene glycol 6.0% by weight Glycerin 5.0% by weight Polyoxyethylene sorbitan monolaurate (20EO) 1.00% by weight Paraoxybenzoic acid Methyl 0.20% by weight Citric acid 0.01% by weight Fragrance 0.20% by weight Water 77.39% by weight

Example 7 The following components were mixed by a conventional method to produce a beverage containing eugeniin obtained in Example 1. Eugeniin 0.20% by weight Fruit juice 20.0% by weight Sucrose 6.0% by weight Honey 5.0% by weight L-ascorbic acid 0.1% by weight Flavor 0.1% by weight Pigment 0.1% by weight Water 68.5 weight%

Example 8 The following components were mixed in a conventional manner to produce a candy containing eugeniin obtained in Example 1. Eugeniin 0.2% by weight Sucrose 52.0% by weight Syrup 46.4% by weight Citric acid 1.0% by weight Fragrance 0.2% by weight Pigment 0.2% by weight

[0045]

The IgE-IgE receptor binding inhibitor of the present invention has a high affinity for the IgE receptor.
It exhibits an anti-allergic effect by competitively inhibiting the binding of IgE to the IgE receptor. Therefore, release of histamine and the like from basophils and mast cells can be prevented, and allergic symptoms can be comprehensively improved.

Accordingly, the IgE-IgE receptor binding inhibitor of the present invention can be suitably used not only as a pharmaceutical ingredient but also in cosmetics, foods, bath preparations and the like, and imparts an antiallergic effect thereto. Can be. Also I
To exert an anti-allergic effect by inhibiting the binding of gE to the IgE receptor, atopic dermatitis,
Comprehensive improvement of allergic symptoms associated with type I allergic reactions such as hay fever is possible.

[Brief description of the drawings]

FIG. 1 is a graph showing a peak of crude eugenin by HPLC.

FIG. 2 is a graph showing the peak of eugenin by HPLC.

FIG. 3 is a process chart showing an outline of a production / purification process of eugeniin.

FIG. 4 is a ¹ H-NMR spectrum diagram of eugeniin.

[Explanation of symbols]

1: Crude eugenin peak; 1-1: Eugeniin peak.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 7/00 A61K 7/00 K W 7/48 7/48 35/78 35/78 H // A23G 3/00 101 A23G 3 / 00 101 A23L 2/52 A23L 2/38 C 2/38 A61K 7/50 A61K 7/50 C07H 13/02 C07H 13/02 A23L 2/00 F (72) Inventor Mitsuo Hirai Masuo Matsuyama, Kashiwa-shi, Chiba 967 Nikkau Pesky Co., Ltd., Production Technology Research Laboratories (72) Inventor Chiyasu Lu 2-14-13 Hanazono-ku Hanamigawa-ku, Chiba-shi

Claims (5)

[Claims] 1. An IgE-IgE receptor binding inhibitor comprising eugenin as an active ingredient. 2. An anti-allergic medicament comprising the IgE-IgE receptor binding inhibitor according to claim 1. 3. A cosmetic comprising the IgE-IgE receptor binding inhibitor according to claim 1. 4. A quasi-drug comprising the IgE-IgE receptor binding inhibitor according to claim 1. 5. A food comprising the IgE-IgE receptor binding inhibitor according to claim 1.