JP2007210920A - Anti-allergic substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an anti-allergic substance obtained from a natural material, especially a food stuff raw material taken daily, also available stably in a large amount and inexpensively, excellent in anti-allergic activity and having a high safety, and an antiallergic agent, a food, a beverage and a cosmetic containing such the anti-allergic substance. <P>SOLUTION: This anti-allergic substance consisting of a lipid soluble fraction contained in an extract is obtained by extracting a wheat-crushed material with a basic solvent. The anti-allergic agent is obtained by blending such the anti-allergic substance as an active ingredient. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an antiallergic substance, an antiallergic agent containing the antiallergic substance, food and drink, and cosmetics, and a method for producing the antiallergic substance.

  In general, allergic symptoms are called "degranulation" in which antibodies are produced in the body by stimulation of the invasion of antigens such as pollen, the antibodies bind to specific lymphocyte cells, and granules are released from these cells. It is thought that this phenomenon occurs when an inflammatory substance such as histamine is released from the granule. Therefore, in order to suppress allergy, it is necessary to suppress the release of inflammatory substances such as histamine.

  Conventionally, in order to treat diseases caused by allergic reactions, antihistamines composed of chemically synthesized substances have been applied as therapeutic agents. On the other hand, since safety is high, various proposals have been made to use natural products-derived antiallergic substances. For example, pectin or a degradation product thereof (see Patent Document 1), tea leaf extract mainly composed of polyphenols (see Patent Document 2), amaranth seed extract (see Patent Document 3), perilla seed or egoma seed alcohol Extracts (see Patent Document 4), apples, pears, peaches, grapes, barley, guava, hops, red beans, or pine bark-derived polyphenols (see Patent Document 5) have been proposed. However, antiallergic substances derived from natural products are inferior in antiallergic action to antihistamines made of chemically synthesized substances, and in order to obtain sufficient antiallergic action, they must be ingested in large amounts. Therefore, it is not easy to take for a long time, and there are some natural products that are stable and difficult to obtain in large quantities at low cost.

JP 2004-107295 A JP 2002-12545 A JP 2003-63972 A JP 2000-86510 A JP-A-2005-82497

  An object of the present invention is to provide an antiallergic substance having excellent antiallergic action and high safety obtained from a natural product, particularly a food material that is ingested on a daily basis and can be obtained stably and in large quantities at low cost. The object is to provide antiallergic agents, foods and drinks and cosmetics containing allergic substances.

As a result of various studies to achieve the above object, the present inventors have found that the wheat pulverized extract effectively suppresses histamine release.
The present invention has been made on the basis of the above-mentioned findings, and “an antiallergic substance consisting of a fat-soluble fraction contained in an extract obtained by extracting a wheat pulverized product with a basic solvent”, “the above-mentioned antiallergic properties” The object is achieved by providing an “antiallergic agent containing a substance as an active ingredient” and “a food and drink and a cosmetic containing the antiallergic substance”.
In addition, the present invention provides a method for producing the above-mentioned antiallergic substance, “extracting a wheat pulverized product with a basic solvent, neutralizing the extract, and then separating and collecting a fat-soluble fraction from the extract. A method for producing an antiallergic substance characterized by the above.

  ADVANTAGE OF THE INVENTION According to this invention, the antiallergic substance which can suppress histamine release effectively, and is high safety, and the antiallergic agent, food / beverage products, and cosmetics containing this are provided. The allergenic substance of the present invention is excellent in safety because it is obtained from pulverized wheat that is taken daily as a food raw material.

  The wheat pulverized product used in the present invention is not particularly limited, but wheat grains that are crushed wheat grains, wheat bran, wheat flour belonging to the grade of powdered powder among commercially available flours, milling mills Among the fractions collected in each step such as sieving, finishing, etc., the fraction containing 1.0% by mass or more of ash, and the peeled part of the skin peeled off from the wheat grain by using a wheat mill or the like were crushed. A thing etc. are used suitably. These pulverized wheat products preferably contain 1 to 2 to 3 cell layers on the outermost periphery of the endosperm portion of wheat, called the Aleurone layer. These pulverized wheat products may be used alone or in admixture of two or more. Moreover, the kind and production area of the wheat which is a raw material are not restrict | limited.

  In order to efficiently extract the antiallergic substance of the present invention from the pulverized wheat product, the pulverized wheat product is preferably finely pulverized, particularly preferably pulverized to a median diameter of 50 μm or less. The median diameter of 50 μm or less means that the particle diameter when the cumulative frequency of the pulverized product reaches 50% is 50 μm or less.

The antiallergic substance of the present invention is extracted from the above pulverized wheat flour using a basic solvent, and is obtained, for example, by a production method comprising the following steps (1) and (2), and further if necessary The following steps (3) and / or (4) may be applied.
(1) A step of extracting a pulverized wheat product with a basic solvent and neutralizing the extract.
(2) A step of separating and collecting a fat-soluble fraction from the extract obtained in step (1).
(3) A step of desalting and / or concentrating the fat-soluble fraction obtained in step (2).
(4) A step of recovering polyphenol from the fat-soluble fraction obtained in step (2) or (3).

The steps (1) to (4) will be described in detail below.
Step (1):
Examples of the solvent used for the basic solvent include water, alcohols such as methanol, ethanol, isopropanol, butanol, and isoamyl alcohol, and ethers such as diethyl ether, propyl ether, butyl ether, and isoamyl ether. Examples of the alkali agent for making the solvent basic include sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate (sodium bicarbonate), sodium carbonate, disodium hydrogen phosphate, ammonia and the like. Examples of the basic solvent include sodium hydroxide aqueous solution, aqueous ammonia, sodium hydroxide aqueous solution / methanol mixed solvent, potassium hydroxide aqueous solution / methanol mixed solvent, sodium hydroxide aqueous solution / ethanol mixed solvent, potassium hydroxide. An aqueous solution / ethanol mixed solvent and the like can be mentioned, and among these, a sodium hydroxide aqueous solution / methanol mixed solvent is particularly preferable.

  Examples of the extraction method include a method in which a basic solvent is added to a pulverized wheat product and the mixture is stirred or homogenized. In this case, the basic solvent is usually used in an amount of 50 to 500 parts by mass with respect to 10 parts by mass of the crushed wheat. Although extraction time can be suitably determined according to the quantity of the wheat ground material to be used and the kind of basic solvent, it is the range of 10 minutes-5 hours normally, for example, is 30-60 minutes. Moreover, although extraction temperature can be suitably determined according to the quantity of the wheat ground material to be used, the kind of basic solvent, and extraction time, it is 0-60 degreeC normally, for example, room temperature.

After the extraction, it is preferable to remove the residue by a technique such as filtration, membrane separation, and centrifugation, and then neutralize the extract, or neutralize the extract and then remove the residue by the technique. Here, when an organic solvent is used as the basic solvent, the organic solvent may be removed from the neutralized solution by a technique such as evaporation.
The neutralization may be performed using, for example, hydrochloric acid, sulfuric acid, nitric acid, acetic acid and the like.

Step (2):
The extract obtained in the step (1) contains a water-soluble component in addition to the fat-soluble component. In the present invention, the fat-soluble fraction is extracted from the extract obtained in the step (1). Separate and collect. Separation and collection of this fat-soluble fraction may be carried out by means of membrane separation, centrifugation, decantation, etc., but it is distributed between an organic solvent and water to remove water-soluble components and intermediate layer emulsion, The fat-soluble fraction may be separated. In this case, examples of the organic solvent to be used include ethyl acetate, butyl acetate, butanol, hexane, ethers such as diethyl ether and petroleum ether, and among these, ethyl acetate is particularly preferable.

Step (3):
The fat-soluble fraction obtained in step (2) contains the desired antiallergic substance and may be used as it is. However, in order to increase the concentration of the desired antiallergic substance, It is preferable to remove (desalinate) the salt produced by the sum. The desalting and / or concentration of this fat-soluble fraction can be performed according to a conventional method by a dialysis method such as electrodialysis, or a membrane separation method such as ultrafiltration or reverse osmosis. Furthermore, the fat-soluble fraction can be solidified by means such as freeze drying and spray drying.

Step (4):
As a method for recovering polyphenol from the fat-soluble fraction obtained in step (2) or (3), for example, a fraction containing polyphenol is collected by gel filtration chromatography, ultrafiltration, or other membrane separation methods. The method of doing is mentioned. If necessary, the fraction containing polyphenols may be concentrated by a dialysis method such as electrodialysis or by means such as reverse osmosis. Further, it can be solidified by means of freeze drying, spray drying or the like.
The polyphenol preferably has a molecular weight of 100 to 10,000,000, and more preferably has a molecular weight of 200 to 1,000,000. The molecular weight of the polyphenol can be measured by a method such as HPLC or electrophoresis (including capillary electrophoresis).

  The antiallergic substance of the present invention is prepared as an antiallergic agent by blending various pharmaceutically acceptable carriers, excipients, other additives, and other ingredients as they are or as necessary. It can be. The dosage form of this antiallergic agent is not particularly limited, and includes oral preparations such as tablets, powders, granules, capsules, and parenteral preparations such as ointments, lotions, gels, eye drops, and nasal drops. And can be formulated by a conventional method. As other components, other antiallergic components, anti-inflammatory drugs, various vitamins, herbal medicines, and minerals can be appropriately blended.

  Moreover, the antiallergic substance of this invention can provide the food-drinks which have antiallergenicity by adding to food-drinks. Examples of the food and drink to be added include beverages such as soft drinks, juices, and nutritional drinks, and confectionery such as breads, noodles, tablets, and candy, but are not limited thereto. The timing of addition to the food or drink is not particularly limited, and may be added during the production process of the food or drink, or may be added to the manufactured food or drink.

  Moreover, the antiallergic substance of this invention can provide cosmetics which have antiallergenicity by adding to cosmetics, such as a cream, a lotion, an essence, and a bath agent. In the cosmetics of the present invention, ingredients usually used in cosmetics (for example, various herbal extracts, oily ingredients, moisturizing ingredients, thickeners, emulsifiers, antioxidants, stratum corneum release agents, preservatives, chelating agents, etc. ) Can be blended. For example, examples of the oil component include hydrocarbons, various synthetic esters, waxes, fats and oils, higher fatty acids, higher alcohols, and silicones.

The content of the antiallergic substance of the present invention in the above-mentioned antiallergic agent, food and drink and cosmetics may be any amount as long as it can suppress histamine release. It can be appropriately changed depending on the dosage form, the type of food and drink and cosmetics, the symptom and age sex of the person who administers or ingests. When the antiallergic substance of the present invention is orally administered or ingested, it is preferably contained so that the daily dose or intake per person is 0.1 mg to 2500 mg. In the case where the non-orally or ingested, dropping to the skin or mucosa an anti-allergic agent in the range of 0. 01mg~250mg / cm ² of the present invention, it is preferable to apply or spray.

  EXAMPLES Next, examples are given to describe the present invention more specifically, but the present invention is not limited to the following examples.

Example 1
100 g of powdered powder (Nisshin Flour Mills “Kougame”) was homogenized for 60 minutes in 1000 ml of a mixed solvent of 10% by volume of 1N aqueous sodium hydroxide / 90% by volume of methanol, and the resulting extract was neutralized with hydrochloric acid. After that, the residue was removed. Methanol was removed from the resulting solution, and solvent fractionation was performed with water and ethyl acetate to obtain a fat-soluble fraction.

Example 2
In the same manner as in Example 1, except that a mixed solvent of 10% by volume of 1N sodium hydroxide / 90% by volume of ethanol was used instead of the mixed solvent of 10% by volume of 1N sodium hydroxide / 90% by volume of methanol. A soluble fraction was obtained.

Example 3
Whole wheat flour was pulverized to a median diameter of 15 μm. 100 g of the finely pulverized powder was homogenized for 60 minutes in 1000 ml of a mixed solvent of 10% by volume of 1N sodium hydroxide / 90% by volume of methanol, and the residue was removed from the resulting extract, followed by neutralization with hydrochloric acid. Methanol was removed from the neutralized solution, and the solvent was fractionated with water and ethyl acetate to obtain a fat-soluble fraction.

Comparative Example 1
The same operation as in Example 1 was performed, and the solvent fractionation was performed with water and ethyl acetate, and then the water-soluble fraction was recovered.

Comparative Example 2
A polyvinylpyrrolidone polymer (PVPP) having strong polyphenol adsorption activity was added to the fat-soluble fraction obtained in Example 1 and mixed well, and then the upper layer obtained by centrifugation was collected.

Comparative Example 3
After adding PVPP to the fat-soluble fraction obtained in Example 2 and mixing well, the upper layer obtained by centrifugation was collected.

Comparative Example 4
After adding PVPP to the fat-soluble fraction obtained in Example 3 and mixing well, the upper layer obtained by centrifugation was collected.

Test example 1 (antiallergic test)
The fat-soluble fraction obtained in Examples 1 to 3, the water-soluble fraction obtained in Comparative Example 1 and the upper layer (test sample) obtained in Comparative Examples 2 to 4 were subjected to the following test methods. An antiallergic test was performed using the derived basophil leukemia cells (RBL-2H3).

Test method
(1) Degranulation RBL-2H3 cells are precultured in Dulbecco's modified Eagle medium (manufactured by Sigma) containing 10% fetal bovine serum (manufactured by JRH Bioscience) at a CO ₂ concentration of 5% and 37 ° C.
After pre-culture, the cells are detached using an EDTA / trypsin solution according to a conventional method, and the cells are collected by centrifugation.
RBL-2H3 cells collected in a 24-well culture plate are seeded at 2.5 × 10 ⁵ cells / ml and cultured at 37 ° C. for 12 hours. After culturing, the medium is removed, 500 μl of 50 ng / ml anti-DNP-IgE antibody is added per well, and the cells are sensitized at 37 ° C. for 2 hours.
The wells are washed twice with a modified Tyrode solution (hereinafter referred to as MT), 500 μl of MT containing a test sample at a concentration of 600 μg / ml is added thereto, and incubated at 37 ° C. for 10 minutes. As a control, 500 μl of MT containing no test sample is added and incubated at 37 ° C. for 10 minutes.
DNP-HAS antigen is added to these so that it may become 50 ng / ml, and also culture | cultivation supernatant is collect | recovered after culture | cultivating for 30 minutes at 37 degreeC (henceforth a culture supernatant is called a supernatant liquid).
In addition, 500 μl of MT containing 0.1% Triton X-100 is added to the cells in the well to lyse the cells (hereinafter, this lysate is referred to as a cell solution).

(2) Measurement of the amount of histamine Add 100 μl of 0.1M hydrochloric acid to 100 μl each of the supernatant and the cell solution, and centrifuge at 15,000 rpm for 10 minutes at room temperature.
The obtained centrifuge supernatant is separated by HPLC, and the peak area of the obtained histamine is measured (the position of the histamine peak is confirmed by running a histamine sample).
The conditions of HPLC are as follows.
Column: Guard column on shodex Asahipak ODP50 4E (4.6 × 250mm, Showa Denko)
Shodex Asahipak ODP50G 4A (Showa Denko) was connected Mobile phase: Acetonitrile (180 ml), 50 mM sodium boride (820 ml),
o-Phthalaldehyde (134mg), N-acetylcysteine (215mg)
Flow rate: 0.5 ml / min Column temperature: 40 ° C
Detection: excitation wavelength 330 nm, fluorescence wavelength 430 nm

From the measured peak area of histamine, the histamine release rate (%) is calculated by the following formula.
[Supernatant liquid peak area / (cell liquid peak area + supernatant liquid peak area)] × 100
Based on the obtained histamine release rate, the histamine release inhibition rate (%) is calculated by the following formula.
[(Control release rate−Test product release rate) / Control release rate] × 100
The histamine release inhibition rate (%) thus obtained is shown in Table 1.

Example 4
Ethyl acetate was removed from the fat-soluble fraction obtained in Example 1 using a rotary evaporator. To this, about 20 ml of 50 volume% methanol solution containing 0.1 volume% formic acid is added and then washed with hexane. The methanol layer was collected and applied to a column packed with Toyopearl HW40C (gel filtration resin manufactured by Tosoh Corporation). The components were eluted with an eluent that had been subjected to a gradient from 50 volume% methanol containing 0.1 volume% formic acid to 100 volume% methanol to obtain 15 fractions (Nos. 4-1 to 4-15).

Comparative Example 5
Among the 15 fractions obtained in Example 4, PVPP was added to the 9th fraction (No. 4-9) and the 14th fraction (No. 4-14) and mixed well, followed by centrifugation. The upper layers (Nos. 5-9 and 5-14) obtained by the above were collected respectively.

Test example 2 (antiallergic test)
The 15 fractions (No. 4-1 to 4-15) obtained in Example 4 and the upper layers (No. 5-9 and 5-14) obtained in Comparative Example 5 were the same as in Test Example 1. An antiallergic test was conducted. The results (histamine release inhibition rate) are shown in Table 2.

  As a result of the antiallergic test of Test Examples 1 and 2, the antiallergic substance of the present invention [the fat-soluble fraction obtained in Examples 1 to 3 and the ninth fraction obtained in Example 4 ( No. 4-9) and the 14th fraction (No. 4-14)] showed significant antiallergic effects. Moreover, it turns out that the main body of the activity of the antiallergic substance of this invention is a polyphenol from the comparison with Examples 1-4 and Comparative Examples 2-5.

Next, production examples of the antiallergic agent, food and drink, and cosmetics of the present invention are shown.
Example 5 (tablets)
Fat-soluble fraction obtained in Example 1 5 g
Corn starch 10 g
Lactose 40 g
Carboxymethylcellulose calcium 8 g
Microcrystalline cellulose 27 g
Polyvinylpyrrolidone 7 g
Magnesium stearate 3 g
Total 100 g
The fat-soluble fraction obtained in Example 1 is dissolved in ethanol, then adsorbed onto microcrystalline cellulose, and then dried. Corn starch, lactose and carboxymethylcellulose calcium are mixed with this, and then an aqueous solution of polyvinylpyrrolidone is added as a binder and granulated by a conventional method. To this, magnesium stearate as a lubricant is added and mixed, and then tableted into 100 mg tablets.

Example 6 (hard capsule)
10 g of fat-soluble fraction obtained in Example 1
Microcrystalline cellulose 55 g
Corn starch 25 g
Lactose 30 g
Polyvinylpyrrolidone 4 g
Magnesium stearate 1 g
Total 125 g
The above ingredients are granulated by a conventional method and then filled into gelatin hard capsules.

Example 7 (powder)
50 g of fat-soluble fraction obtained in Example 1
Microcrystalline cellulose 600 g
Corn starch 300 g
Polyvinylpyrrolidone 50 g
Total 1000 g
The fat-soluble fraction obtained in Example 1 is dissolved in ethanol, then adsorbed onto microcrystalline cellulose, dried and crushed. This is mixed with corn starch and polyvinylpyrrolidone and powdered by conventional methods.

Example 8 (granule)
10 g of fat-soluble fraction obtained in Example 1
Lactose 130 g
Corn starch 87 g
Polyvinylpyrrolidone 8 g
L-menthol 15 g
Light anhydrous silicic acid 5 g
Total 255g
In the above formulation, the fat-soluble fraction obtained in Example 1, lactose, corn starch and an aqueous polyvinylpyrrolidone solution are mixed, and heated and granulated with stirring in a granulator. After cooling, it is sieved to a particle size of 500 μm or less, L-menthol is added, silicic anhydride is added, mixed and packaged (1.0 g) to give granules.

Example 9 (candy)
50 g sugar
Minamata 33 g
Citric acid 2 g
Fragrance 0.2g
1.5 g of the fat-soluble fraction obtained in Example 1
Water remaining Total 100.0 g
Sugar, starch syrup and water are put in a pan to boil and dissolve, and after the boiling temperature reaches 125 ° C., it is removed from the fire, and the fragrance and the fat-soluble fraction obtained in Example 1 are added. The mixture is poured into a cooling plate with stirring, cooled to 80 ° C., then cut into an appropriate length in a bar shape, and 3.33 g of candy is produced per grain.

Example 10 (roll bread)
Mix 150g of flour (strong flour) and 2g of dry yeast. Separately, 2 g of the fat-soluble fraction obtained in Example 1, 20 g of sugar, 3 g of sodium chloride and 6 g of skim milk powder are dissolved in 70 g of hot water, and 1 egg is added and mixed well. Add this to the mixture of flour and dry yeast and knead well by hand, then add 40 g of butter and knead well by hand to make 8 dough rolls. Next, after fermenting, a beaten egg is applied to the surface and baked in an oven at 180 ° C. for about 15 minutes to produce a roll. This roll contains about 250 mg of the fat-soluble fraction obtained in Example 1 per piece.

Example 11 (Udon)
A solution obtained by dispersing 10 g of the fat-soluble fraction obtained in Example 1 and 15 g of sodium chloride in 150 g of water is mixed well with 300 g of wheat flour (medium flour) and then laid down. Thereafter, the dough is stretched and cut into widths of about 5 mm to produce udon. When this was boiled with boiling water for about 10 minutes, the appearance, taste and texture were good. This udon contains about 1.2 g of the fat-soluble fraction obtained in Example 1 per serving.

Example 12 (fruit juice jelly)
To 200 ml of orange juice, take 4.5 g of gelatin, 45 g of sugar, and 15 g of water, and put on a fire to dissolve the gelatin completely. After confirming that the gelatin is dissolved, 3 g of the fat-soluble fraction obtained in Example 2 is mixed well and dissolved. Then, it is poured into 4 cups, cooled in a refrigerator for 2 hours or more and hardened to obtain orange juice jelly.

Example 13 (green juice)
15 g of the fat-soluble fraction obtained in Example 2 and 585 g of organic green juice (manufactured by Nisshin Pharma) are mixed well, and thereafter, about 3 g of green juice in stick packaging per serving is obtained.

Example 14 (Cream)
A Squalane 20 g
8 g olive oil
Refined beeswax 5 g
Glycerol monostearate 3 g
Cetostearyl alcohol 2 g
B Polyoxyethylene hydrogenated castor oil 3 g
Fat-soluble fraction obtained in Example 1 2 g
Glycerin 10 g
Purified water Appropriate amount Solution A and solution B are heated to 80 ° C. Add B liquid to A liquid while stirring and emulsify until uniform to obtain a cream.

Example 15 (bathing agent)
Glycerol monostearate 10 g
Glycerin 50 g
Rice bran extract 10 g
10 g of fat-soluble fraction obtained in Example 1
Fragrance 0.1g
0.1g of dye
20 g of purified water
Total 100.2g
All components are mixed and emulsified with a homomixer to prepare a bath agent.

Claims (6)

  An antiallergic substance comprising a fat-soluble fraction contained in an extract obtained by extracting wheat pulverized material with a basic solvent.   The antiallergic substance according to claim 1, wherein the fat-soluble fraction contains polyphenol.   An antiallergic agent comprising the antiallergic substance according to claim 1 or 2 as an active ingredient.   A food or drink or cosmetic comprising the antiallergic substance according to claim 1 or 2.   A method for producing an antiallergic substance, comprising extracting a ground wheat product with a basic solvent, neutralizing the extract, and then separating and collecting a fat-soluble fraction from the extract. The method for producing an antiallergic substance according to claim 5, wherein polyphenol is separated and collected from the fat-soluble fraction.