WO2012117969A1 - Anti-inflammatory agent and process of producing same - Google Patents

Abstract

Provided is a material which is safe and inexpensive, can be ingested easily and continuously on a daily basis, and has a significant anti-inflammatory activity. An anti-inflammatory agent comprising a mixture of: a soybean seed or an extract thereof each of which has been activated by a light irradiation treatment and/or a heating treatment; and chlorophyll.

Description

Anti-inflammatory agent and method for producing the same

The present invention relates to an anti-inflammatory agent, a method for producing the same, a pharmaceutical, a cosmetic, a food and drink, and a feed containing the anti-inflammatory agent.

Anti-inflammatory agents treat inflammatory diseases such as gastritis and ulcerative colitis, arthritis such as rheumatoid arthritis and degenerative osteoarthritis, and inflammatory symptoms such as hay fever, allergic rhinitis, allergic conjunctivitis and atopic dermatitis Useful for. As anti-inflammatory agents as pharmaceuticals, steroidal anti-inflammatory agents, non-steroidal anti-inflammatory agents, immunosuppressive agents and the like are known so far. However, since all have strong side effects, there is a problem that careful administration under the guidance and management of a doctor is required.

On the other hand, attempts have been made to improve inflammatory diseases and symptoms by ingesting so-called health foods exhibiting anti-inflammatory action, such as health functional foods, instead of these pharmaceuticals. For example, strawberry tea, glucosamine, chondroitin, astaxanthin, catechin and the like or foods to which it is added. However, although these health foods have fewer side effects than pharmaceuticals, it is difficult to say that these health foods have sufficient anti-inflammatory effects due to weak inflammation-improving effects.

Therefore, it is desired to develop an anti-inflammatory agent that can be taken on a daily basis and that can effectively relieve inflammatory diseases and symptoms.

Soybean is well known as a raw material for tofu, soy sauce, natto and the like, and many attempts have been made to obtain functional ingredients from soybean seeds. For example, Patent Document 1 discloses an antiallergic composition that improves inflammatory symptoms in an atopic dermatitis model animal containing stachyose as an active ingredient, and describes that stachyose can be extracted from soybean seeds. However, the amount of stachyose contained in soybean seeds is extremely small, and a sufficient amount cannot be obtained only by performing a normal extraction operation. Therefore, in order to concentrate to a concentration of 98% or more where the effect is recognized, it is necessary to perform a complicated purification step such as chromatography, and as a result, there is a problem that it is too costly. In addition, stachyose is indigestible, and there is a problem that diarrhea and the like may occur when ingested at a high concentration.

On the other hand, chlorophyll is known as a molecule involved in the energy production system of plants and is also used as an additive for foods and cosmetics. It is known that chlorophyll has a wound healing effect that promotes granulation of ulcers and the like, a deodorizing effect, and an inhibitory action of the complement system (Patent Document 2), but a small amount of chlorophyll can contribute to anti-inflammation. That was not known.

JP 2003-321372 A JP 2004-256423 A

The present invention provides an anti-inflammatory agent containing a mixture of soybean seeds or an extract thereof and chlorophyll activated by light irradiation treatment and / or heat treatment.
The present invention also provides a method for producing an anti-inflammatory agent, comprising a step of obtaining a mixture of soybean seeds or an extract thereof and chlorophyll, and a step of subjecting the mixture to activation treatment by light irradiation and / or heating. .
The present invention also provides a method for enhancing the anti-inflammatory effect of soybean, comprising the steps of obtaining a mixture of soybean seeds or an extract thereof and chlorophyll, and a step of subjecting the mixture to activation treatment by light irradiation and / or heating. provide.
Furthermore, this invention provides the pharmaceutical, cosmetics, food-drinks, and feed containing the mixture of the soybean seed or its extract, and chlorophyll activated by the light irradiation process and / or heat processing.
Furthermore, the present invention provides the use of a mixture of soybean seeds activated by light irradiation treatment and / or heat treatment or an extract thereof and chlorophyll for producing an anti-inflammatory agent.
Furthermore, the present invention provides a method for treating inflammatory diseases, comprising administering to a subject a soybean seed activated by light irradiation treatment and / or heat treatment or a mixture of the extract thereof and chlorophyll.

Inhibitory effect of NO production in macrophages. Inhibitory effect of IL-2 production in T cells. Inhibitory effect of NO production in macrophages.

The present invention is a natural substance that can be continuously ingested safely, inexpensively, and easily on a daily basis, has a remarkable inflammation-suppressing action, and is effective in preventing and / or improving various inflammatory diseases and inflammatory symptoms. It relates to providing materials of origin.

As a result of diligent research to solve the above problems, the present inventors have found that a composition obtained by activating a mixture of soybean seeds and chlorophyll by light irradiation and / or heating has excellent macrophage activation. It has been found that it has an inhibitory action and an IL-2 production inhibitory action and is useful for anti-inflammation.

According to the present invention, it can be ingested safely, inexpensively and easily on a daily basis, has a remarkable anti-inflammatory action, and is effective in preventing and / or improving various inflammatory diseases and inflammatory symptoms. Inflammatory agents are provided.

The anti-inflammatory agent of the present invention can be produced by obtaining a mixture of soybean seeds or an extract thereof and chlorophyll, and subjecting the mixture to an activation treatment by light irradiation and / or heating. Therefore, the present invention provides an anti-inflammatory agent containing a mixture of soybean seeds or an extract thereof and chlorophyll activated by light irradiation treatment and / or heat treatment.
The present invention also provides a method for producing an anti-inflammatory agent, comprising a step of obtaining a mixture of soybean seeds or an extract thereof and chlorophyll, and a step of subjecting the mixture to activation treatment by light irradiation and / or heating. .
The present invention also provides the use of a mixture of soybean seeds activated by light irradiation treatment and / or heat treatment or an extract thereof and chlorophyll for producing an anti-inflammatory agent.

For example, the anti-inflammatory agent of the present invention can reduce IL-2 production by T cells, as shown in Examples below. IL-2, a kind of cytokine, is also called inflammatory cytokine, is produced by T cells and NK cells (natural killer cells), and proliferates and activates T cells, B cells, NK cells, macrophages, etc., in B cells Functions to enhance antibody production, and enhance cytotoxic activity, resulting in local or systemic inflammatory symptoms, and various secondary symptoms induced by them; for example, inflammatory dermatitis, gastritis and Can cause inflammatory symptoms of inflammatory diseases such as ulcerative colitis, arthritis such as rheumatoid arthritis and degenerative osteoarthritis, and various allergic diseases such as hay fever, allergic rhinitis, allergic conjunctivitis and atopic dermatitis . Therefore, the anti-inflammatory agent of the present invention can prevent, treat, ameliorate or alleviate the inflammatory symptoms of these diseases caused by IL-2.

For example, the anti-inflammatory agent of the present invention can reduce NO production by macrophages, as shown in Examples below. Macrophages take in pathogens that invade while circulating in the body by phagocytosis and produce and kill and destroy various cytotoxic factors such as digestive enzymes, active oxygen, and nitric oxide (NO). Production of NO is known as one of macrophage activation markers. The NO produced by activated macrophages acts as a host defense, while exacerbating inflammation when produced in excess. By suppressing NO production by macrophages, anti-inflammatory action can be expected. Therefore, the anti-inflammatory agent of the present invention can exert an anti-inflammatory action by suppressing the activation of macrophages and suppressing its NO production.

Therefore, the “anti-inflammatory” action according to the present invention is different from the immediate allergic reaction, ie, the suppression of Th2 bias due to IL-4 reduction or IFNγ enhancement, and the suppression of type I allergy reaction due to IgE reduction action. It is an action. Therefore, the allergic disease that is prevented, treated, ameliorated or alleviated by the anti-inflammatory agent of the present invention is preferably a disease caused by type II to type V allergic reaction excluding diseases caused by type I allergic reaction. For example, when the allergic disease is atopic dermatitis, symptoms that are prevented, treated, ameliorated or alleviated by the anti-inflammatory agent of the present invention include papules and pigmentation, but pruritus caused by IgE. Improvements are not included. Also, for example, symptoms that are prevented, treated, ameliorated or alleviated by the anti-inflammatory agent of the present invention include pain, redness, thermal sensation and swelling, which are the four major signs of inflammation. It does not include edema caused by increased vascular permeability by chemical mediators released from mast cells.

Accordingly, in an exemplary embodiment, the anti-inflammatory agent of the present invention comprises an IL-2 production inhibitor; a macrophage activation inhibitor; a macrophage NO production inhibitor; a type II to type V allergic reaction, IL-2 production or macrophage It can be a treatment, prevention, treatment, amelioration or alleviation of various inflammatory diseases, symptoms or conditions resulting from activation.
Examples of inflammatory diseases, symptoms or conditions resulting from IL-2 production include local or systemic inflammatory symptoms and various secondary symptoms induced by them; for example, inflammatory dermatitis, Inflammatory diseases such as gastritis and ulcerative colitis, arthritis such as rheumatoid arthritis and degenerative osteoarthritis, and inflammatory symptoms of various allergic diseases such as hay fever, allergic rhinitis, allergic conjunctivitis and atopic dermatitis Can be mentioned. Examples of inflammatory diseases, symptoms or conditions resulting from macrophage activation include exacerbation of inflammation due to NO produced by macrophages.

As shown in the Examples below, the composition obtained by activating the mixture of soybean seeds or an extract thereof and chlorophyll by light irradiation and / or heating is the light irradiation and / or heat treatment of the soybean extract. The anti-inflammatory activity is enhanced compared to the product. It was shown that the anti-inflammatory action of soybean can be enhanced by obtaining a mixture of soybean seeds or an extract thereof and chlorophyll, and subjecting the mixture to activation treatment by light irradiation and / or heating.
Therefore, the present invention also enhances the anti-inflammatory effect of soybean, comprising the steps of obtaining a mixture of soybean seeds or an extract thereof and chlorophyll, and subjecting the mixture to an activation treatment by light irradiation and / or heating. Provide a method.

In the present specification, the term “soybean seed” refers to a mature seed of soybean (Glycine max). The variety of soybean seeds used in the present invention is not particularly limited, but the most commonly eaten soybeans, for example, Axylome (Certified No. No. 69), Ayakogane (Certified No. No. 114, registered varieties) Number: 10475), Iwakuro (Certified number agriculture forest 107, Variety registration number 9796), Elster (Certification number agriculture forest 115, Variety registration number 8646), Enrei (Certification number agriculture forest 57), Osuzu ( Accreditation No. No. 109, Variety Registration No. 9795), Otsuru (Author No. No. 91, Variety Registration No. 2044), Ochirome (Author No. No. 59), Kariyutaka (Author No. No. 95, Variety Registration No. 3293) ), Kitasumume (Certification No. 49), Ginrei (Certification No. 102, Variety Registration No. 5860), Kurodamaru (Certification No.) Agricultural forest 128, variety registration number 15130), Kozus (certification number agriculture forest 87, variety registration number 2397), Kotoyuka (authorization number agriculture forest 132, variety registration number 19477), Sachiyutaka (accreditation number agriculture forest 116, Variety registration number 11367), Sayanami (certification number agriculture forest 104, variety registration number 8644), Suzumaru (authorization number agriculture forest 89, variety registration 2043), Suzukari (certification number agriculture forest 83, variety registration number 1218) ), Suzukogane (Certified No. 111, Variety Registration No. 9793), Suzukomachi (Certified No. No. 119, Variety Registration No. 12278), Sayaka Suzuki (Certified No. No. 125, Variety Registration No. 14042), Tachinagaha (Certified No. No. 85, Variety Registration No. 1509), Tachi Homare (Certified No. 134, Item Registration number 19476), Tachiyutaka (certification number agriculture forest 86, variety registration number 1818), tamahomare (authorization number agriculture forest 72, variety registration number 201), Tamaura (certification number agriculture forest 112, variety registration number 10622) ), Daikoku Tamaki (Authorized No. Norin 106, Variety Registration No. 8835), Tamamasari (No. No. No. 110, Variety Registration No. 9792), Tanrei (No. No. 65 No. 65), Mitsukuro Nakasei (963) No., Hokkaido), Tomoyutaka (Authorized No. No. 92, Variety Registration No. 2878), Toyo Komachi (No. No. No. 90, Variety Registration No. 2042), Toyomusume or Toyomasari (No. No. No. 81, Variety Registration No. No. 1216), Nakasennari (Authorized No. Norin 66), Natto small grain, Nanahomare (variety registration No. 20592), Nishimus Me (certification number agriculture forest No. 93, variety registration number 2877), Hata Yutaka (authorization number agriculture forest 113, variety registration number 10623), Hayahikari (certification number agriculture forest 108, variety registration number 9797), Fukubuki (certification number) Agricultural forest 122, variety registration number 13191), Fukuminori (variety registration application number 24879), Fukuyutaka (certification number agriculture forest 73, variety registration number 202), Hoen (certification number agriculture forest 105, variety registration number 8645) ), Miyagi Shiromome (Iwanuma 1, Miyagi Prefecture), Yutaka Mura (variety registration No. 2156), Yuzuru (certification number Norin 55), Yukizuka (certification No. 124, variety registration No. 13193), Yuki Pirika (certification number) Agriculture and Forestry 133, Variety Registration No. 19475), Yukihomare (Certification Number Agriculture and Forestry 118, Variety Registration Number 1227) No.), dream Minori (certification number Agriculture and Forestry 117 No., Variety Registration No. 12280), Liu Bang (certification number Agriculture and Forestry No.100, mature seeds of variety registration No. 5859) and the like are preferable. The “certification number” in the above is the certification number of the certified variety of agriculture and forestry certified by the Ministry of Agriculture, Forestry and Fisheries, and the “variety registration number” means the variety registration number registered based on the seedling method. Information on these soybean varieties can be found at the Ministry of Agriculture, Forestry and Fisheries Variety Registration Homepage [www. Hinsyu. maff. go. It can be searched from the product registration data search system of jp /]. Alternatively, “Characteristic Table for Encouraged Variety Rice, Wheat, and Soybean” (Production Bureau, Ministry of Agriculture, Forestry and Fisheries, March 2008), or “Dictionary 2010 for Domestic Soybean Varieties” (Production and Distribution Promotion Division, Production Bureau, Ministry of Agriculture, Forestry and Fisheries, February 23). The “seed” includes a seed coat and / or an embryo, and the embryo includes a cotyledon and a hypocotyl. Preferably, the seed is a molting seed.

In the present specification, the soybean seeds may be any form of soybean seeds, such as soybean seeds (preferably molted seeds) themselves; those soybean seeds that have been cut, ground or powdered; It may be in the form of a dried product; a soy seed that has been dried and pulverized or powdered; a juice obtained by pressing and extracting the soybean seed or a residue thereof; or a solution, suspension, or the like thereof. Alternatively, the soybean seeds used in the present invention include, for example, concentrated soy protein, soy milk, okara, soy whey that is replicated during the production of the isolated soy protein, acid washing solution or alcohol that is replicated during the production of the concentrated soy protein. A washing liquid, a soaking solution of soybean that is replicated during production of tofu, soy milk, and the like may be included.
In the present invention, the form of soybean seed to be used may be selected based on the desired activity strength, cost, quality, degree of flavor as soybean, and the like.

In the present specification, the soybean seed extract refers to soybean seeds of various forms listed above, such as soybean seeds themselves, or those obtained by cutting or pulverizing soybean seeds, dried, pulverized after drying, compressed extraction It can be an extract obtained by solvent extraction from the squeezed juice or the like by a known method. The extraction method is not particularly limited, and examples thereof include a method of immersing, stirring or refluxing the soybean seeds of the above-mentioned various forms in a solvent, a supercritical fluid extraction method, and the like.

The solvent that can be used for the extraction is preferably water or an organic solvent, and is preferably a polar solvent. Specifically, the water includes pure water, distilled water, tap water, acidic water, alkaline water, neutral water, and the like, and the organic solvent includes methanol, ethanol, n-propanol, isopropanol, n-butanol. And alcohols which are liquid at room temperature such as polyhydric alcohols such as 1,3-butylene glycol, propylene glycol and glycerin; chloroform; dimethyl sulfoxide (DMSO) and the like. These solvents may be used alone or in combination of two or more.
Among the above organic solvents, alcohols that are liquid at room temperature, for example, lower alcohols having 1 to 4 carbon atoms, are preferably used from the viewpoint of operability and environmental performance. Ethanol is more preferred. The organic solvent also includes a water-containing organic solvent in which an aqueous component is further contained in the organic solvent. As the water-containing organic solvent, a water-containing alcohol in which an aqueous component is further contained in the alcohol as described above, more preferably water-containing ethanol, can be used.

Preferable extraction solvents include water, alcohols such as ethanol, and water-containing alcohols such as water-containing ethanol. Among these, water, ethanol, and water-containing ethanol are more preferable.
The content of the organic solvent in the water-containing organic solvent is usually 50% by volume or higher, preferably 80% by volume or higher, more preferably 90% by volume or higher.

Specific methods for extracting the soybean seed extract include, for example, a method in which soybean seeds are added to a room temperature or warmed solvent under reduced pressure, normal pressure, or increased pressure, and extracted while being immersed or stirred. And a method of extraction while refluxing. At that time, it is appropriate that the extraction temperature is 5 ° C. to the boiling point of the solvent or less, and the extraction time varies depending on the type of solvent used, the extraction conditions, and the content of aqueous components in the case of a water-containing organic solvent. However, about 30 minutes to 72 hours is appropriate. When extraction is performed by refluxing, it is preferable to use a low-boiling solvent so that the soybean seed extract does not denature or undergo thermal decomposition. Further, the extraction operation can also be performed by a supercritical fluid extraction method using carbon dioxide or the like.

Next, the mixture containing the extract and the residue is subjected to filtration or centrifugation, if necessary, to remove the solid component as a residue to obtain an extract. The removed solid component can be used again for the extraction operation, and this operation may be repeated several times.

The extract thus obtained may be used in the present invention as it is as an extract of soybean seeds, or, if necessary, further dried or powdered by a method such as concentration, freeze drying or spray drying. Or prepared as a liquid, powder or paste.

In the case of drying the extract, any specific drying method may be used as long as it can be performed under conditions where the soybean seed extract does not undergo denaturation or thermal decomposition. Examples include a method in which a form is added and subjected to filtration, centrifugation, centrifugal filtration, spray drying, spray cool, drum drying, vacuum drying, freeze drying, and the like. These methods can be used alone or in combination.

In this specification, examples of chlorophyll include not only chlorophyll (chlorophyll a, chlorophyll b) contained in green plants, but also chlorophyll c1, chlorophyll c2, chlorophyll d, chlorophyllin and the like contained in algae. The chlorophyll may be purified chlorophyll, or may be a plant or algae processed by a known method so that chlorophyll can be used (for example, pulverized or extracted). Alternatively, commercially available reagents such as chlorophyll a and chlorophyll b (manufactured by SIGMA) may be used.

The mixture of soybean seeds or an extract thereof and chlorophyll in the present invention may be a mixture obtained by mixing soybean seeds of any form described above or an extract thereof and the above-described chlorophyll in any combination. . Preferable examples of the mixture include a mixture of soybean extract and purified chlorophyll, a mixture of soybean seed powder and plant or algal powder, and the like.

The ratio of soybean seeds or an extract thereof and chlorophyll in the mixture may be, for example, 50000: 1 to 10: 1 as a mass ratio of soybean extract (dry solid mass) and purified chlorophyll, and 20000: 1 to 50: 1 is preferred. Further, for example, the mass ratio of soybean seed powder to plant or algal powder may be 5000: 1 to 1:10, and preferably 1000: 1 to 1: 5.

The anti-inflammatory agent of the present invention can be produced by subjecting the above mixture to an activation treatment by light irradiation and / or heating. In the present invention, the “activation process” refers to a process of converting the active substance precursor contained in the mixture into an active substance by irradiating the mixture with light and / or heating.

In this specification, “light” in light irradiation includes not only visible light but also invisible light such as infrared light and ultraviolet light, and has a wavelength of, for example, 200 nm to 800 nm, preferably 280 to 700 nm, more preferably 360 to 650 nm. It may be a light beam having The specific procedure of the light irradiation is not particularly limited, but preferably the product of the illuminance (kilux: klx) and the irradiation time (hr) for the mixture is 40 klx · hr or more, preferably 100 klx · hr or more. This is a process of irradiating light.

In the present specification, the heating can be, for example, heating the above mixture at a temperature of 40 to 105 ° C., preferably 70 to 105 ° C., for 0.5 to 144 hours, preferably 2 to 72 hours. . The specific procedure for heating is not particularly limited, and any heating means known in the art can be used.

In this specification, when the light irradiation treatment and the heat treatment are combined, each treatment may be performed simultaneously and / or sequentially based on each treatment condition. It is only necessary to achieve a processing amount necessary for activation by combining the light irradiation treatment and the heat treatment. For example, the light irradiation treatment and the heat treatment are each 50% of the amount set above, that is, a light treatment of 20 klx · hr or more and a heat treatment of 0.25 hours or more, or a light treatment of 50 klx · hr or more and 1 hour or more. Heat treatment and the like may be performed simultaneously or sequentially. Further, for example, the light irradiation treatment and the heat treatment may be performed at a ratio of 5%: 95% to 95%: 5% of the processing amount set above. Alternatively, the total processing amount of the light irradiation treatment and the heat treatment may exceed the processing amount set above. For example, a combination of a light treatment of 100 klx · hr or more and a heat treatment of 1 day or more may be used.

With the above procedure, the anti-inflammatory agent of the present invention can be produced by obtaining a mixture of soybean seeds or an extract thereof and chlorophyll, and subjecting the mixture to activation treatment by light irradiation and / or heating.
That is, the “activation treatment” means that the amount of IL-2 produced by T cells is 70% or less, preferably the amount of IL-2 produced is 60% or less, compared to the case where the mixture is not applied. More preferably, it is a treatment that gives the mixture an activity that reduces it to 50% or less. Alternatively, the “activation treatment” means that the amount of NO produced by macrophages is 70% or less, preferably the amount of NO produced is 60% or less, more preferably 50, compared to the case where the mixture is not applied. % Is a treatment that gives the mixture an activity that reduces it to a percentage or less.

Therefore, the anti-inflammatory agent of the present invention preferably reduces the production amount of IL-2 by T cells to 70% or less, more preferably to 60% or less of the case where the agent of the present invention is not given. More preferably, it is reduced to 50% or less. Alternatively, the agent of the present invention preferably reduces the NO production amount by macrophages to 70% or less, more preferably 60% or less, more preferably 50% when the agent of the present invention is not given. % Or less.

The anti-inflammatory agent of the present invention contains, as an active ingredient, a mixture of soybean seeds or an extract thereof and chlorophyll activated by light irradiation treatment and / or heat treatment. The anti-inflammatory agent of this invention contains what is comprised only from the said mixture substantially.
In an exemplary embodiment, the anti-inflammatory agent is an IL-2 production inhibitor, a macrophage activation inhibitor, a NO production inhibitor by macrophages, or a type II-V allergic reaction, IL-2 production or macrophage activation. It can be the treatment, prevention, treatment, amelioration or alleviation of various inflammatory diseases, symptoms or conditions caused by it.

Soybean seed is a substance that has been used for food for a long time and has been confirmed to be safe. Chlorophyll is also a component that is regularly consumed in vegetables and algae. Therefore, the anti-inflammatory agent of the present invention using these as a raw material does not cause side effects even when administered or ingested for a long time, and is safe and continuous not only for healthy and adults but also for children, the elderly and the sick. Can be administered or ingested. Moreover, it is known that chlorophyll is decomposed into pheophorbide and causes health damage, and there is a problem in taking a large amount of chlorophyll containing pheophorbide. However, since the anti-inflammatory agent of the present invention is activated by chlorophyll, the content of chlorophyll is very small, so there is no concern of taking a large amount of pheophorbide even if the anti-inflammatory agent of the present invention is administered or ingested. .

Therefore, the mixture of soybean seeds or an extract thereof and chlorophyll activated by the light irradiation treatment and / or heat treatment (hereinafter referred to as an activated mixture) is a pharmaceutical, cosmetics, food or drink, feed for humans or animals. Etc. or for producing them.

Therefore, the present invention also provides a medicine, cosmetics, food and drink, and feed containing the activated mixture. The pharmaceuticals, cosmetics, foods and drinks, and feeds are anti-inflammatory, for suppressing IL-2 production, for suppressing macrophage activation, or for various inflammatory diseases resulting from IL-2 production or macrophage activation, It can be used for prevention, treatment, amelioration or alleviation of symptoms or conditions.

The medicament of the present invention comprises an anti-inflammatory agent containing the above-mentioned activation mixture as an active ingredient; an IL-2 production inhibitor; a macrophage activation inhibitor; or various inflammatory diseases caused by IL-2 production or macrophage activation , Prevention, treatment, amelioration or alleviation of symptoms or conditions, and the like.
The pharmaceutical dosage form of the present invention includes, for example, oral preparations such as tablets, capsules, granules, powders, syrups, dry syrups, liquids and suspensions; enteral preparations such as inhalants and suppositories; Agents; injections; external preparations; transdermal, transmucosal and nasal agents; inhalants;
Liquid preparations such as liquids and suspensions may be dissolved or suspended in water or other appropriate medium immediately before taking. In the case of tablets and granules, the preparations may be prepared by well-known methods. The surface may be coated.

The cosmetic of the present invention contains the above activated mixture as an active ingredient. Examples of the cosmetics include any form that cosmetics can usually take, such as creams, emulsions, lotions, suspensions, gels, powders, packs, sheets, patches, sticks, cakes, and the like.

The medicament and cosmetic of the present invention may contain the above activated mixture alone, or each may contain a combination of a pharmaceutically acceptable carrier and a cosmetically acceptable carrier. The pharmaceuticals and cosmetics of the present invention are prepared by adding a conventional carrier such as an excipient, a disintegrant, a binder, a lubricant, a surfactant, a pH adjuster, a dispersant, an emulsifier, an antiseptic to the above activated mixture. An agent, an antioxidant, a colorant, alcohol, water, a water-soluble polymer, a fragrance, a sweetener, a corrigent, an acidulant and the like can be blended according to the dosage form, and can be produced according to a conventional method. If necessary, other active ingredients or medicinal ingredients may be further blended.

The content of the activated mixture in the pharmaceutical and cosmetic products of the present invention varies depending on the dosage form, but is usually from 0.001 to 99% by mass, preferably from 0.01 to 80% by mass, based on the dry mass. Range.

The food / beverage product or feed of the present invention contains the above-mentioned activated mixture as an active ingredient, and is anti-inflammatory; IL-2 production inhibition; macrophage activation inhibition; or various inflammatory properties resulting from IL-2 production or macrophage activation Health foods, functional foods, foods and drinks for specific health use, foods and drinks for sick people, livestock, and races that are intended to prevent, treat, improve, or alleviate diseases, symptoms or conditions It can be a feed for horses, appreciation animals, etc., a pet food, and the like.

The form of the food / beverage products and feed of the present invention is not particularly limited, and includes all forms in which the activated mixture can be blended. For example, the form may be solid, semi-solid or liquid, or various forms such as tablets, chewable tablets, powders, capsules, granules, drinks, gels, syrups, liquid foods for enteral nutrition. .
Specific examples of the form of food and drink include tea drinks such as green tea, oolong tea and black tea, coffee drinks, soft drinks, jelly drinks, sports drinks, milk drinks, carbonated drinks, fruit juice drinks, lactic acid bacteria drinks, fermented milk drinks, Powdered beverages, cocoa beverages, alcoholic beverages, beverages such as purified water, butter, jam, sprinkles, margarine spreads, mayonnaise, shortening, custard cream, dressings, breads, cooked rice, noodles, pasta, miso soup, tofu , Milk, yogurt, soup or sauce, confectionery (for example, biscuits and cookies, chocolate, candy, cake, ice cream, chewing gum, tablet) and the like.
Since the feed of the present invention can be used in almost the same composition and form as foods and drinks, the description relating to the foods and drinks in this specification can be applied to feeds as well.

The above-mentioned food and drink and feed are added to the above activated mixture, other food and drink materials used for the production of food and drink and feed, various nutrients, various vitamins, minerals, amino acids, various fats and oils, and various additives (for example, taste ingredients) , Sweeteners, acidulants such as organic acids, surfactants, pH adjusters, stabilizers, antioxidants, dyes, flavors) and the like, and the like, and can be produced according to conventional methods. Or the food / beverage products or feed which concerns on this invention can be manufactured by mix | blending the said activated mixture with the food / beverage products or feed currently eaten normally.

The content of the activated mixture in the food and drink and the feed of the present invention varies depending on the form of the food, but is usually 0.001 to 80% by mass, preferably 0.01 to 50% by mass based on the dry mass. %, More preferably in the range of 1 to 50% by mass.

The pharmaceuticals, cosmetics, foods and drinks, and feeds are administered or ingested in the range of 0.01 to 100 g per day for adults based on the dry mass of the activated mixture. In the case of oral administration or ingestion, the general daily dose is 0.1 to 50 g. However, since the activated mixture is highly safe, the amount can be further increased. The daily administration or intake may be administered or ingested once, but may be administered or ingested in several times.
It is desirable that the pharmaceutical dosage form or dosage regimen of the present invention, or the form of food, drink, and feed can be controlled so that the daily dosage or intake can be managed so that the above daily dosage can be appropriately administered or consumed. .

The invention further provides the use of the activated mixture for the treatment, prevention, treatment, amelioration or alleviation of inflammatory diseases. Alternatively, the present invention provides a method for treating, preventing, treating, ameliorating or alleviating an inflammatory disease, comprising administering the activation mixture to a subject. Examples of the inflammatory disease include various inflammatory diseases, symptoms or conditions caused by type II to type V allergic reaction, IL-2 production or macrophage activation.
The method for preparing the activated mixture is as described above. Administration targets include humans or animals with inflammatory diseases; humans or animals that require anti-inflammation, IL-2 production inhibition, macrophage activation inhibition, or macrophage NO production inhibition; type II to type V allergic reactions And humans or animals in need of treatment, prevention, treatment, amelioration or alleviation of various inflammatory diseases, symptoms or conditions resulting from IL-2 production or macrophage activation. Examples of inflammatory diseases, symptoms or conditions resulting from IL-2 production, and examples of inflammatory diseases, symptoms or conditions resulting from macrophage activation are as described above.

The route of administration of the activated mixture may be oral or parenteral. The dose of the activation mixture may be in the range of 0.01 to 100 g per adult day based on the dry mass. The amount and method of administration can be appropriately determined by those skilled in the art depending on the condition of the subject. For example, the activation mixture is administered to a subject once a day, twice a day, three times a day or more, once every two days, once every two days, or once every few days or more. obtain.

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

(Examples 1 and 2)
(1) 5600 g of water was added to 800 g of soybean powder (Fukuyutaka, manufactured by Nishio Flour Milling) and heated at 85 to 90 ° C. for 1 hour. The heat treatment liquid was dried with a spray dryer (L-8 type, manufactured by Okawara Processing Machine Co., Ltd.) to obtain 533 g of powder.
(2) After adding 50 mL of ethanol to 10 g of the powder obtained in (1) and stirring and extracting at 25 ° C. and 300 rpm for 2 hours, the mixture was filtered using glass fiber filter paper (manufactured by Kiriyama Seisakusho) to remove insoluble matters. The extract was collected. Ethanol was distilled off to obtain 230 mg (yield 2.3%) of soybean extract.
(3) Next, 63 mg of this soybean extract was dissolved in 30 mL of ethanol. Chlorophyll (chlorophyll b: manufactured by SIGMA) was added thereto at 1 mg (1/60 mass ratio of soybean extract), and the petri dish was sealed with a lid. Each petri dish was irradiated with light (fluorescent lamp: illuminance 2 klx / h) while being stirred (300 rpm) at 25 ° C. to perform an activation treatment.
(4) Three and six days after the start of light irradiation, 10 mL of ethanol solution was collected from the petri dish and concentrated to dryness to prepare Examples 1 and 2, respectively (Yield of Example 1: 26.7 mg, Yield of Example 2: 15.9 mg).

(Comparative Examples 1 and 2)
Comparative Examples 1 and 2 were prepared in the same procedure as in Steps (1) to (4) of Examples 1 and 2, except that chlorophyll was not added in Step (3) (Yield of Comparative Example 1: 22 0.7 mg, yield of Comparative Example 2: 35.4 mg).

(Comparative Example 3)
Comparative Example 3 was obtained by taking 63 mg of the soybean extract obtained by the same procedure as in Step (2) of Examples 1 and 2 and dissolving it in 30 mL of ethanol.

(Comparative Examples 4 to 5)
Only 30 mg of chlorophyll was added to 30 mL of ethanol without adding the soybean extract. This was subjected to light treatment in the same procedure as steps (3) to (4) in Examples 1 and 2, and Comparative Example 4 was obtained.

(Comparative Examples 6-7)
63 mg of soybean extract obtained in the same procedure as in steps (1) to (2) of Examples 1 and 2 was placed in a petri dish, dissolved in 30 mL of ethanol, and further 1 mg of chlorophyll (chlorophyll b: manufactured by SIGMA) (soy extract) The petri dish was sealed with a lid. Stirring was performed at 25 ° C. in the dark (300 rpm), 2 mL was taken after 3 days and 6 days, and concentrated to dryness to give Comparative Examples 6 and 7, respectively (Yield of Comparative Example 6: 1.7 mg, Comparative Example 7) Yield: 4.4 mg).

(Examples 3 to 4)
(1) Take 20 g of soybean extract obtained in the same procedure as in step (2) of Examples 1 and 2 in a flask and mix 3 g of green juice powder (manufactured by Nisshin Pharma; chlorophyll content = 4 mg / g) (soybean) The extract dry solid mass and chlorophyll mass ratio = 1667: 1) were dissolved in 115 mL of methanol and subjected to heat treatment with stirring (300 rpm) at 40 ° C. under light shielding.
(2) One day and three days after the start of heating, 10 mL of methanol solution was recovered from the flask and concentrated to dryness to prepare Examples 3 and 4 respectively (Yield of Example 3: 250.1 mg, implementation) Yield of example 4: 220.9 mg).

(Examples 5 to 6)
(1) A heat treatment was performed in the same procedure as in step (1) of Examples 3 to 4, except that the temperature was 75 ° C.
(2) One and three days after the start of heating, 10 mL of a methanol solution was recovered from the flask and concentrated to dryness to prepare Examples 5 and 6, respectively (Yield of Example 5: 210.8 mg, implementation) Yield of example 6: 180.4 mg).

(Test Example 1: NO production inhibitory action)
Macrophages were activated with lipopolysaccharide (LPS) to enhance NO production, and the effects of the samples prepared above (Examples 1-2 and Comparative Examples 1-7) on this NO production were examined.
RAW264.7 cells (DS Pharma Biomedical Co., Ltd.) were maintained in 10% FCS + DMEM medium. A cell suspension prepared by preparing RAW264.7 cells at 2 × 10 ⁵ cells / mL (10% FCS + DMEM medium) was seeded on a 24-well plate by 1 mL and pre-cultured for 24 hours. The medium was replaced with 10% FCS + DMEM medium containing 500 ng / ml LPS and various concentrations of samples (Examples 1-2 and Comparative Examples 1-7), and cultured for 18 hours. As a control, the medium was exchanged with LPS alone and no sample, and cultured for the same time. After completion of the culture, NO released into the medium was induced into NO ₂ ions for quantification.
The quantification of NO ₂ ions was performed using a measurement kit (Gries Reagent System: Promega) according to the operating instructions. Each sample was dissolved in DMSO and added to the medium at 0.2% by volume. The final concentrations were 0.05 mg / mL for Examples 1 to 6 and Comparative Examples 1 to 3 and 6 to 7, and 0.00083 mg / mL for Comparative Examples 4 to 5 (0.05 mg / mL ÷ 60: Example 1). ˜2 and chlorophyll concentration).

The results are shown in FIG. Examples 1-2 obtained by subjecting a mixture of soybean extract and chlorophyll to light irradiation and Examples 3-6 obtained by subjecting a mixture of soybean extract and chlorophyll to heat treatment are macrophages Production of NO was significantly suppressed. On the other hand, in the case where only the soybean extract was used, neither Comparative Examples 1 and 2 subjected to the light treatment and Comparative Example 3 not subjected to the light treatment did not affect the production of NO. Also, Comparative Example 4 (with light treatment) and Comparative Example 5 (without light treatment) containing only chlorophyll did not affect NO production. Further, the mixture of soybean extract and chlorophyll (Comparative Examples 6 to 7) that was not subjected to light irradiation treatment or heat treatment had a slight effect on NO production.

(Test Example 2: IL-2 production inhibitory action)
The production inhibitory effect of IL-2 which is an inflammatory cytokine was examined. Jurkat cells used a working stock that had been stored frozen. This stock cell was used in a range where the number of passages did not exceed 10. As a medium, RPMI1640 was used as a basal medium, and 10% of fetal bovine serum inactivated by heating at 56 ° C. for 30 minutes was added thereto. Cultivation was carried out in a carbon dioxide incubator under 100% humidity at 5% carbon dioxide gas concentration at 37 ° C. For the test, a culture in a semi-confluent state was used. The cells were prepared and suspended in 25 mL at a concentration of 2 × 10 ⁵ cells / mL, and 12.5 μL of 10 μg / mL concentration of Phorbol myristate (PMA) and 7.5 μL of 500 μg / mL concentration of Ca ²⁺ ionophore A23187. Added and resuspended. This cell suspension was dispensed into a 24-well culture plate (manufactured by BDFalcon) at 1 mL / well, and each test sample (Example 2 and Comparative Examples 2 to 3) was added thereto, and about 24 in a carbon dioxide incubator. After incubation for a period of time, the amount of IL-2 contained in the culture supernatant was measured by ELISA (DuoSet ELISA Development system human IL-2; manufactured by R & D Systems). Each sample was dissolved in DMSO and added to the medium at 0.2% by volume, and the final concentration was 0.05 mg / mL.

The results are shown in FIG.
Example 2 obtained by subjecting a mixture of soybean extract and chlorophyll to light irradiation treatment markedly suppressed the production of IL-2. On the other hand, Comparative Examples 2 to 3 to which chlorophyll was not added did not affect the production of IL-2 regardless of the presence or absence of light treatment.

(Example 7)
The anti-inflammatory agent of the present invention was produced using spinach as the chlorophyll raw material.
16 g of spinach powder (manufactured by Nippon Powder Co., Ltd .; chlorophyll content = 1 mg / g) was mixed with 200 g of soybean powder (manufactured by Nishio Milling) (mass ratio of soybean and chlorophyll = 12,500: 1). After adding 500 ml of ethanol to 100 g of this mixture and stirring at 75 ° C. at 300 rpm for 3 hours, the mixture was filtered using a glass fiber filter paper (manufactured by Kiriyama Seisakusho) to remove insoluble matters, and ethanol was distilled off to obtain 4.0 g of powder ( Yield 4%). Next, this powder was subjected to a light irradiation treatment at 25 ° C. and an illuminance of 2 klx / h for 6 days to obtain the anti-inflammatory agent of the present invention.

(Example 8)
The anti-inflammatory agent of the present invention using green juice as a chlorophyll raw was produced.
Soybean defatted powder soya flour (Nisshin Oilio Group) 25g mixed with 3.5g green juice powder (Nisshin Pharma; chlorophyll content = 4mg / g) (mass ratio of soybean and chlorophyll = 1786: 1), ethanol After adding 125 ml and stirring at 85 ° C. and 300 rpm for 3 hours, the mixture was filtered using glass fiber filter paper (manufactured by Kiriyama Seisakusho) to remove insoluble matter, and ethanol was distilled off to obtain 1.5 g of powder (yield 5%). Got. Next, this powder was subjected to a light irradiation treatment at 25 ° C. and an illuminance of 2 klx / h for 6 days to obtain the anti-inflammatory agent of the present invention.

(Comparative Examples 8-9)
Comparative Examples 8 and 9 were prepared in the same procedure as in Examples 7 and 8, but at 20 ° C. and protected from light.

(Test Example 3: NO production inhibitory effect)
Using the compositions of Examples 7 to 8 and Comparative Examples 8 to 9 as samples, the NO production inhibitory activity was measured in the same manner as in Test Example 1. Each sample was dissolved in DMSO and added to the medium at 0.2% by volume. The final concentration was 0.05 mg / mL for the compositions of Example 7 and Comparative Example 8, and the compositions of Example 8 and Comparative Example 9 were used. The product was 0.1 mg / mL.

The results are shown in FIG. Even when spinach or green juice was used as the chlorophyll raw material, remarkable NO production inhibitory activity was observed in the mixture obtained by combining the light irradiation treatment and the heat treatment. On the other hand, when neither light irradiation treatment nor heat treatment was performed, NO production inhibitory activity was not observed in the obtained mixture.

Claims (36)

1. An anti-inflammatory agent containing a mixture of soybean seeds or an extract thereof and chlorophyll activated by light irradiation treatment and / or heat treatment.
2. The anti-inflammatory agent according to claim 1, wherein the light irradiation treatment is a treatment of irradiating light so that a product of illuminance (kilux: klx) and irradiation time (hr) is 40 klx · hr or more.
3. The anti-inflammatory agent according to claim 1, wherein the heat treatment is a treatment of heating at a temperature of 40 to 105 ° C for 0.5 to 144 hours.
4. The anti-inflammatory agent according to any one of claims 1 to 3, wherein the mixture is a mixture of soybean seed extract and chlorophyll.
5. The anti-inflammatory agent according to claim 4, wherein the mass ratio of soybean seed extract to chlorophyll in the mixture is 50,000: 1 to 10: 1.
6. The anti-inflammatory agent according to any one of claims 1 to 3, wherein the mixture is a mixture of soybean seed powder and plant or algal powder.
7. The anti-inflammatory agent according to claim 6, wherein the mass ratio of the soybean seed powder to the plant or algae powder in the mixture is 5000: 1 to 1:10.
8. The anti-inflammatory agent according to claim 1, which is prepared by activating a mixture obtained by mixing soybean seeds or an extract thereof and chlorophyll by light irradiation and / or heat treatment.
9. A method for producing an anti-inflammatory agent, comprising a step of obtaining a mixture of soybean seeds or an extract thereof and chlorophyll, and a step of subjecting the mixture to an activation treatment by light irradiation and / or heating.
10. The method according to claim 9, wherein the activation process by light irradiation is a process of irradiating light so that a product of illuminance (kilux: klx) and irradiation time (hr) is 40 klx · hr or more.
11. The method according to claim 9, wherein the activation treatment by heating is a treatment of heating at a temperature of 40 to 105 ° C for 0.5 to 144 hours.
12. The method according to any one of claims 9 to 11, wherein the step of obtaining the mixture is a step of obtaining a mixture of soybean seed extract and chlorophyll.
13. The method according to claim 12, wherein the mass ratio of soybean seed extract to chlorophyll in the mixture is 50,000: 1 to 10: 1.
14. The method according to any one of claims 9 to 11, wherein the step of obtaining the mixture is a step of obtaining a mixture of soybean seed powder and plant or algal powder.
15. The method according to claim 14, wherein a mass ratio of the soybean seed powder to the plant or algal powder in the mixture is 5000: 1 to 1:10.
16. A method for enhancing the anti-inflammatory effect of soybean, comprising a step of obtaining a mixture of soybean seeds or an extract thereof and chlorophyll, and a step of subjecting the mixture to an activation treatment by light irradiation and / or heating.
17. A pharmaceutical containing a mixture of soybean seeds or an extract thereof and chlorophyll activated by light irradiation treatment and / or heat treatment.
18. Cosmetics containing a mixture of soybean seeds or an extract thereof and chlorophyll activated by light irradiation treatment and / or heat treatment.
19. Food and drink containing a mixture of soybean seeds or an extract thereof and chlorophyll activated by light irradiation treatment and / or heat treatment.
20. A feed containing a mixture of soybean seeds or an extract thereof and chlorophyll activated by light irradiation treatment and / or heat treatment.
21. Use of a mixture of soybean seeds activated by light irradiation treatment and / or heat treatment or an extract thereof and chlorophyll for the production of an anti-inflammatory agent.
22. The use according to claim 21, wherein the light irradiation process is a process of irradiating light so that a product of illuminance (kilux: klx) and irradiation time (hr) is 40 klx · hr or more.
23. The use according to claim 21, wherein the heat treatment is a treatment of heating at a temperature of 40 to 105 ° C for 0.5 to 144 hours.
24. The use according to any one of claims 21 to 23, wherein the mixture is a mixture of soybean seed extract and chlorophyll.
25. The use according to claim 24, wherein the mass ratio of soybean seed extract to chlorophyll in the mixture is 50,000: 1 to 10: 1.
26. The use according to any one of claims 21 to 23, wherein the mixture is a mixture of soybean seed powder and plant or algal powder.
27. 27. The use according to claim 26, wherein the mass ratio of soybean seed powder to plant or algal powder in the mixture is 5000: 1 to 1:10.
28. The anti-inflammatory agent is an IL-2 production inhibitor, a macrophage activation inhibitor, a NO production inhibitor by macrophages, or various inflammatory effects caused by type II to type V allergic reaction, IL-2 production or macrophage activation. The method according to claims 9 to 15, which is a treatment, prevention, treatment, amelioration or alleviation of a disease, symptom or condition.
29. The anti-inflammatory agent is an IL-2 production inhibitor, a macrophage activation inhibitor, a NO production inhibitor by macrophages, or various inflammatory effects caused by type II to type V allergic reaction, IL-2 production or macrophage activation. Use according to claims 21-27, which is a treatment, prevention, treatment, amelioration or alleviation of a disease, symptom or condition.
30. A method for treating inflammatory diseases, comprising administering to a subject a soybean seed activated by light irradiation treatment and / or heat treatment or a mixture of the extract thereof and chlorophyll.
31. The method according to claim 30, wherein the light irradiation process is a process of irradiating light so that a product of illuminance (kilux: klx) and irradiation time (hr) is 40 klx · hr or more.
32. The method according to claim 30, wherein the heat treatment is a heat treatment at a temperature of 40 to 105 ° C for 0.5 to 144 hours.
33. The method according to claim 30, wherein the mixture is a mixture of soybean seed extract and chlorophyll.
34. The method according to claim 33, wherein a mass ratio of soybean seed extract to chlorophyll in the mixture is 50,000: 1 to 10: 1.
35. The method according to claim 30, wherein the mixture is a mixture of soybean seed powder and plant or algal powder.
36. The method according to claim 34, wherein a mass ratio of the soybean seed powder to the plant or algal powder in the mixture is 5000: 1 to 1:10.

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