JP4558294B2 - Interleukin 4 production inhibitor, antiallergic composition and anti-inflammatory composition - Google Patents

Description

  The present invention relates to an interleukin 4 production inhibitor, an antiallergic composition containing the same, an antiinflammatory composition, an oral composition thereof, and an external composition for skin.

  In recent years, various allergies such as hay fever, food allergies and bronchial asthma have become problems. Hormonal agents with high pharmacological effects are generally used for the treatment of such symptoms, but there is a problem that side effects such as adrenocortical dysfunction and gastrointestinal ulcers are large. In addition, nonsteroidal histamine, leukotriene release inhibitors, IgE antibody production inhibitors, and the like are also used, but their effects are not sufficient.

Interleukin 4 (hereinafter referred to as “IL-4”) is a glycoprotein produced by antigen-stimulated T lymphocytes. It is known to act on B lymphocytes to enhance the production of antibodies such as IgE and to promote the infiltration of inflammatory cells into the inflammatory site. In recent years, it is considered that allergic diseases and inflammation can be treated in a point different from conventional treatment methods and prevention methods if IL-4 production related to this allergic disease can be suppressed.
As an IL-4 production inhibitor, a sulfonium derivative (see, for example, Non-Patent Document 1) is known, and as a plant having an IL-4 production inhibitory action, an extract (eg, nettle, thyme, or yarrow) , Patent Document 1) and Inchinkou extract (see, for example, Patent Document 2) have been reported, but these effects are not sufficient. Therefore, there is a demand for an IL-4 production inhibitor that is excellent in stability and safety and has no problem in price.

  So far, an inhibitory effect on IgE increase has been reported to broccoli, cauliflower, nazuna, suzushiro, cabbage, apples and the like (see, for example, Patent Document 3), but the inhibitory effect on IL-4 production in vegetables is known. Absent. Further, the present inventor found that kale, which is a cruciferous vegetable used in vegetable beverages, or the like, has an IL-4 production inhibitory activity, and filed a patent application (see Patent Document 4).

JP 10-279491 A JP-A-10-298090 JP 2000-169382 A Patent application for Japanese Patent Application No. 2002-71269 Japan. J. et al. Pharmacol. 61, 27-30, 1993.

  The present invention provides an IL-4 production inhibitor excellent in stability, safety and price, an antiallergic composition containing them, an anti-inflammatory composition or an oral composition thereof, and an external composition for skin. It is intended to do.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor has identified IL-, which is considered to be one of the causes of particularly excellent allergic diseases, in a specific fraction of kale's ethanol extract. 4 has been found to have a production inhibitory action, and the present invention was completed.

That is, the gist of the present invention is as follows.
(1) When the extract obtained by extracting the dried kale with 70% by mass of ethanol was concentrated, and the fat-soluble fraction of the obtained concentrate was dissolved in 50% by mass of ethanol, An interleukin 4 production inhibitor comprising, as an active ingredient, a fraction dissolved in 50% by mass of ethanol obtained as a lower layer separated into two layers .
(2) An antiallergic composition comprising the interleukin 4 production inhibitor described in (1) above .
(3) An anti-inflammatory composition comprising the interleukin 4 production inhibitor described in (1) above .
(4) The interleukin 4 production-suppressing agent according to (1), the antiallergic composition according to (2), or the anti-inflammatory composition according to (3), which is an oral composition.
(5) The interleukin 4 production-suppressing agent according to (1), the antiallergic composition according to (2), or the anti-inflammatory composition according to (3), which is a skin external preparation composition.

According to the present invention, an interleukin production inhibitor having an action of suppressing the production of IL-4, which is one of inflammatory cytokines, can be provided, and further, allergic rhinitis containing this interleukin production inhibitor, It is possible to provide a composition useful for anti-allergy and anti-inflammation, which has an excellent effect on allergies such as hay fever and various inflammations.
In addition, the present invention uses a specific fraction of an ethanol extract of kale, which is a plant, as its active ingredient, and since this is a natural product, its toxicity is low and no harmful side effects have been reported. Is. Furthermore, since kale is a vegetable that can be easily obtained by cultivation, the composition of the present invention containing a specific fraction of the ethanol extract of kale as its active ingredient is highly safe and stable and inexpensive. In addition, it can be obtained in large quantities and is excellent with no problems in price.

  Kale, which is a raw material for the ethanol extract used in the present invention, is a cruciferous vegetable native to Southern Europe and is said to be the original species of cabbage. Its scientific name is Brassicca Oleracea L. var. There is acephala DC. There are various types of kale such as kitchen kale, mallow kale, bush kale, tree kale, collard, green alga kanran, and any of these can be used. As the use part of the kale for obtaining the kale extract of the present invention, kale stems, flowers, roots, etc. can be used in addition to those usually used for food such as leaves, and are not particularly limited. The cultivation method and cultivation place are not particularly limited.

  The ethanol extract of kale used in the present invention was obtained by extracting kale leaves, stems, flowers, roots and the like as described above with ethanol having a concentration of 70 to 85% by mass, preferably 75 to 80% by mass. It is a fat-soluble fraction of the extract. Specifically, a concentration of 70 to 85 mass%, preferably 75 to 80 mass% ethanol is added to dry matter such as kale leaves, stems, flowers and roots, and the mixture is immersed and stirred at room temperature for several hours to several tens of hours. The extract was obtained, and the extract was concentrated to about 10 to 20 times by heat evaporation or the like, and separated into a water-soluble fraction and a fat-soluble fraction by a separatory funnel. It is a fat-soluble fraction. The fat-soluble fraction obtained here can be extracted again with ethanol having a concentration of 50% by mass and separated into two layers by a separating funnel. This water-soluble fraction is mainly composed of highly water-soluble saccharides, dietary fiber, etc., and the fraction not soluble in 50% by mass of ethanol (upper layer, fat-soluble fraction 1) is mainly chlorophyll or The fraction that is phytosterol or the like and is soluble in 50% by mass ethanol (lower layer, fat-soluble fraction 2) is mainly composed of polyphenols and the like. The interleukin production inhibitor of the present invention, and the antiallergic composition and anti-inflammatory composition containing the same include the upper fat-soluble fraction 1 and the lower fat-soluble fraction 2 which are the fat-soluble fractions. Is effective.

  In the IL-4 production inhibitor of the present invention, the fat-soluble fraction of the ethanol extract in the specific concentration range of kale thus obtained may be used as the active ingredient as it is, It is good also as a composition containing a fat-soluble fraction. The present invention is also an antiallergic composition or anti-inflammatory composition containing such an IL-4 production inhibitor. As such a composition, any of various components that do not impair the effects of the present invention and do not adversely affect them as an auxiliary, medium or carrier, and contain the IL-4 production inhibitor of the present invention. Can constitute the composition.

  The fat-soluble fraction of the Kale ethanol extract described above has an excellent IL-4 production inhibitory action. IL-4 has the effect of enhancing the production of antibodies such as IgE and IgG4, and also has the effect of promoting the infiltration of inflammatory cells into the inflammatory site. Furthermore, IL-4 also has a function of differentiating immature T lymphocytes into mature T lymphocytes, and mature T lymphocytes produce more IL-4 than immature T lymphocytes. Moreover, IL-4 is also involved in skin troubles, particularly in skin inflammation. The IL-4 production inhibitor of the present invention exhibits such an excellent effect in suppressing IL-4 production, and is effective in the prevention and treatment of allergic diseases such as atopic dermatitis. Furthermore, the IL-4 production inhibitor of the present invention can also be used effectively for suppressing wrinkles, sagging, itching, etc., which are skin troubles involving IL-4.

  In the IL-4 production inhibitor, anti-allergic composition, or anti-inflammatory composition of the present invention, the application amount is appropriately determined depending on the age, weight, symptoms, application route, application schedule, formulation form, etc. of the intaker. For example, in the case of oral administration, the weight standard of the fat-soluble fraction is usually about 0.0001 to 0.5 g / kg, more preferably 0.001 to 0.2 g / kg in terms of an adult. The dosage may be divided into several times a day.

The IL-4 production inhibitor, anti-allergy composition, and anti-inflammatory composition of the present invention can be produced as an oral composition for food or medicine, and used externally for cosmetics or medicine. It can also be produced as a composition.
As a method for applying these compositions of the present invention for pharmaceutical use, either oral administration or parenteral administration can be employed. In administration, the active ingredient can be mixed with a solid or liquid nontoxic pharmaceutical carrier suitable for administration methods such as oral administration, rectal administration, and injection, and administered in the form of a conventional pharmaceutical preparation. Examples of such preparations include solid preparations such as tablets, granules, powders and capsules, solutions such as solutions, suspensions and emulsions, freeze-dried preparations, and the like. It can be prepared by conventional means. Examples of the non-toxic pharmaceutical carrier include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin , Water, physiological saline and the like. Further, if necessary, conventional additives such as a stabilizer, a wetting agent, an emulsifier, a binder, and an isotonic agent can be appropriately added.

  For food use, these compositions of the present invention are used as health foods or food materials useful for the treatment and prevention of allergic symptoms as they are, or with various nutritional components added or contained in foods and drinks. it can. For example, after adding a suitable auxiliary agent such as starch, lactose, maltose, vegetable oil powder, cocoa butter powder, stearic acid, etc., using conventional means, a form suitable for oral use, such as granules, granules, tablets It may be formed into capsules, pastes, etc. and used for food, and various foods such as processed foods such as ham and sausage, fishery processed foods such as kamaboko and chikuwa, bread, confectionery, butter, milk powder, ferment It may be used by adding to dairy products. The blending amount of these compositions of the present invention is appropriately set depending on the type and state of the food or food material.

Moreover, the above-mentioned IL-4 production inhibitor, anti-allergic composition or anti-inflammatory composition of the present invention can be produced in the form of a cosmetic material or cosmetic containing these compositions. For example, these compositions can be added to wheat germ oil or olive oil and used as a cosmetic material.
Moreover, these compositions can be directly used as cosmetic ingredients to produce cosmetics. Such cosmetics include oils and fats such as vegetable oils, higher fatty acids, higher alcohols, silicones, anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, preservatives, sugars, metals Contains sequestering agents, polymers such as water-soluble polymers, thickeners, powder components, UV absorbers, UV blockers, moisturizers such as hyaluronic acid, perfumes, pH adjusters, desiccants, etc. be able to. In addition, vitamins, skin activators, blood circulation promoters, resident bacteria control agents, active oxygen scavengers, anti-inflammatory agents, anticancer agents, whitening agents, bactericides, and other medicinal and physiologically active ingredients may be included. it can.

The types of cosmetics include skin cosmetics such as lotions, emulsions, creams, packs, makeup base lotions, makeup creams, emulsions, creams or ointment foundations, lipsticks, eye colors, and cheek colors. Cosmetics for the body such as up cosmetics, hand creams, leg creams, body lotions, and the like, and bathing agents, oral cosmetics, hair cosmetics, and the like. The cosmetics containing the IL-4 production inhibitor, anti-allergic composition or anti-inflammatory composition of the present invention include, for example, milky lotion, cosmetic liquid, face cream, hand cream, lotion, essence from the functional aspect. Etc. are preferable.
Such a cosmetic containing the composition of the present invention can be produced according to a conventional method. The addition amount of the composition of the present invention in the cosmetic is not particularly limited, but as an example, 0.01 to 20 of the total weight of the cosmetic as a weight standard of the fat-soluble fraction of the ethanol extract. A mass%, preferably 0.01 to 10 mass% is appropriate.

  Next, the present invention will be described in more detail with reference to examples. The following production examples, examples and formulation examples show preferred examples of the present invention and are not limited thereto. Further, “%” in each example is based on mass.

Production Example 1: Production of Kale extract with 70% ethanol The dried kale leaf was pulverized, and 100 g of the kale pulverized product was extracted with 2000 mL of 70% ethanol using a 70% ethanol column (column size 45 mmΦ × 415 mm). The extract was concentrated to 100 g using an evaporator. 100 g of the obtained concentrate was separated into a water-soluble fraction and a fat-soluble fraction by a separating funnel, and the fat-soluble fraction was further dissolved in 200 mL of 50% ethanol. This ethanol solution was further divided into two layers by separating with a separatory funnel, and the upper layer was concentrated to dryness to obtain 5.93 g as the “lipid soluble fraction 1” of the kale ethanol extract, and the lower layer was concentrated to dryness. As a result, 4.50 g was obtained as “Fat-soluble fraction 2” of the kale ethanol extract.

Production Example 2: Production of Kale extract with 80% ethanol The dried kale leaf was pulverized, and 100 g of the kale pulverized product was extracted with 2000 mL of 80% ethanol using an 80% ethanol column (column size 45 mmΦ × 415 mm). The extract was concentrated to 100 g using an evaporator. 100 g of the obtained concentrate was separated into a water-soluble fraction and a fat-soluble fraction by a separating funnel, and the fat-soluble fraction was further dissolved in 200 mL of 50% ethanol. This ethanol solution was further divided by a separating funnel and separated into layers, and the upper layer was concentrated to dryness to give 5.22 g as the “fat-soluble fraction 3” of the kale ethanol extract, and the lower layer was concentrated to dryness. In the same manner, 3.71 g was obtained as “Fat-soluble fraction 4”.

Production Example 3: Production of Kale Extract with Water The dried kale leaf was pulverized, and 100 g of the kale pulverized product was added to 2000 mL of ion-exchanged water at 80 ° C. and stirred for 2 hours for extraction treatment. The extract thus obtained was filtered, and 2083 g of the filtrate was concentrated 5-fold with an evaporator, and then fractionated using an ion exchange resin HP-20 column. This ion exchange resin is a porous adsorption resin and adsorbs sugars and polyphenols. 21.41 g as the “HP-20 non-adsorbing part” of the kale water extract obtained by concentrating and drying the non-adsorbing part flowing out from the ion exchange resin, and 95% ethanol as the adsorption part adsorbed on the ion exchange resin HP-20. 3.18 g was obtained as “HP-20 adsorbing part” of the kale water extract which was desorbed and concentrated to dryness.

Production Example 4 Production of Kale Extract with 99.5% Ethanol The dried kale leaf was pulverized, 4 kg of the kale pulverized product was used, and 2000 mL of 99.5 using an ethanol column (column size 45 mmΦ × 415 mm). Extraction with% ethanol gave 80 g of an extract with 99.5% ethanol.

Next, the various kale extracts obtained as described above were subjected to an IL-4 production inhibition evaluation test using peripheral blood mononuclear cells (PBMC) by the following method.
(i) Preparation of culture supernatant sample for evaluation of IL-4 production inhibition The IL-4 production inhibition evaluation test was based on the method of Endo et al. (Int. arch. Allergy Immunol. Vol. 1, p425-430, 1993). ). RPMI 1640 medium (Dainippon Pharmaceutical) supplemented with 10% fetal bovine serum, 300 mg / L glutamine (SIGMA), 100 U / ml penicillin, 100 μg / mL streptomycin (Dainippon Pharmaceutical) And 2 × 10 ⁵ cells / well were seeded on a 96 microplate (Nunc). After seeding, concanavalin A (manufactured by SIGMA) is added to each well so as to be 20 μg / ml, and dexamethasone (hereinafter referred to as “DXM”), which is known to have a strong IL-4 production inhibitory effect, in the positive control group 0.01 μM was added, and only the medium was added to the negative control group, and each extract obtained in Production Examples 1 to 4 of 100 μg / mL was added to the test group, followed by culturing for 2 days. After completion of the culture, each culture solution containing PBMC was recovered from each well, and the culture supernatant obtained by centrifugation (400 × g, 8 min) was subjected to an IL-4 production inhibition evaluation test by ELISA.

(ii) “IL-4 ELISA Kit” (manufactured by BIO SOURCE) was used for the evaluation test of the IL-4 production inhibition evaluation test by ELISA. To create a standard curve, use the standard diluent supplied with the kit as the standard sample so that the IL-4 concentrations are 0, 7.8, 15.6, 31.2, 62.5, 125, 250, and 500 pg / ml, respectively. Diluted and prepared.
Next, 100 μL each of the culture supernatant sample and the standard sample obtained in (i) above was put into each well of a 98-well plate (manufactured by Nunc), and then 50 μL each of biotin-labeled anti-human IL-4 antibody. In addition, the plate was covered and incubated at room temperature (20-25 ° C.) for 2 hours. The bottom of each well is pre-coated with an anti-IL-4 antibody that binds to the IL-4 antibody, binds to the IL-4 produced by the cells, and the IL-4 antibody attached to the kit is firmly attached. And fixed in the well (sandwich ELISA). After the incubation, the liquid in these wells was removed by aspiration, and each well was washed 4 times with 150 μL of washing buffer. After washing, 100 μL of Streptavidin-Peroxidase (HRP) solution was applied to each well, covered with a plate, and incubated at room temperature (20-25 ° C.) for 30 minutes. After completion of the incubation, the liquid in the wells was removed by aspiration, and each well was washed 4 times with 150 μL of washing buffer. Tetramethylbenzidine (TMB) substrate solution (100 μL) was placed in each well and incubated in the dark at room temperature (20-25 ° C.) for 30 minutes. Immediately after the incubation, 100 μL of the reaction stop solution was applied to each well, and the absorbance at a wavelength of 450 nm was measured using a microplate reader to determine the amount of IL-4 produced immobilized in each well. That is, the amount of IL-4 produced was calculated from the measured absorbance value of each sample under the standard curve obtained from the absorbance of the standard sample and the IL-4 concentration.
The results obtained for each sample are shown in Table 1 and FIG.

  From these results, it was found that the fat-soluble fraction obtained by fractionating a 70-85% ethanol extract of kale exhibits a very excellent IL-4 production inhibitory effect comparable to that of dexamethasone. In contrast, the water-soluble fraction of 70-85% ethanol extract of Kale also has an IL-4 production inhibitory effect, but the degree is similar to that of Kale's water extract. Moreover, although the extract (crude extract) of the kale obtained by extracting kale with 99.5% ethanol also has an IL-4 production inhibitory effect, the extent was the same as the water extract.

Next, formulation examples as pharmaceuticals and foods, which are specific usage forms of the IL-4 production inhibitor, anti-allergic composition or anti-inflammatory composition of the present invention, are shown below.
Formulation Example 1: Antiallergic composition (tablet)
Using the fat-soluble fraction 2 of kale 70% ethanol extract obtained in Production Example 1 above, tablets of the antiallergic composition having the following composition were produced by a conventional method.
(Composition) (mass%)
Kale extract (Fat-soluble fraction 2) 24.0
Lactose 63.0
Cornstarch 12.0
Guar gum 1.0

Formulation Example 2: Juice Using the fat-soluble fraction 4 of the 80% ethanol extract of kale obtained in Production Example 2 above, a juice having the following composition was produced by a conventional method.
(Composition) (mass%)
Frozen and concentrated Wenzhou orange juice 5.0
Fructose glucose liquid sugar 11.0
Citric acid 0.2
L-ascorbic acid 0.02
Perfume 0.2
Color element 0.1
Kale extract (Fat-soluble fraction 4) 0.2
Water 83.28

Formulation Example 3: Lotion Toner lotion having the following composition was produced by a conventional method using the fat-soluble fraction 2 of Kale 70% ethanol extract obtained in Production Example 1 above.
(Composition) (mass%)
Glycerin 8.0
1,3-butylene glycol 2.0
Citric acid 0.1
L-serine 0.05
P-Hydroxybenzoate ester 0.2
Fragrance 0.05
Kale extract (Fat-soluble fraction 2) 0.5
Purified water 89.1

  According to the present invention, using kale that is widely used for eating and drinking, based on the knowledge that a specific ethanol extract has an IL-4 production inhibitory action, it has high safety and is excellent. Compositions such as foods, pharmaceuticals, and cosmetics having IL-4 production inhibitory action, antiallergic action, and anti-inflammatory action can be obtained.

It is a graph which shows the result of the evaluation test of IL-4 production suppression of various fractions of an ethanol extract.

Claims (5)

The extract obtained by extracting the dried kale with 70% by mass of ethanol was concentrated, and when the fat-soluble fraction of the obtained concentrate was dissolved in 50% by mass of ethanol, it was separated into two layers. An interleukin 4 production inhibitor comprising, as an active ingredient, a fraction dissolved in 50% by mass of ethanol obtained as a separated lower layer . An antiallergic composition comprising the interleukin 4 production inhibitor according to claim 1 . An anti-inflammatory composition comprising the interleukin 4 production inhibitor according to claim 1 . The composition for interleukin 4 production suppression according to claim 1, which is an oral composition, the composition for anti-allergy according to claim 2, or the composition for anti-inflammation according to claim 3. The composition for interleukin 4 production suppression according to claim 1, the composition for antiallergy according to claim 2, or the composition for anti-inflammation according to claim 3, which is an external preparation composition for skin.

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