JP2018083764A - Immunoregulatory composition containing cirsium maritimum makino extract - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an excellent immunoregulatory composition that is a natural product, has high safety, and can be applied to food and drink.SOLUTION: An immunoregulatory composition is prepared that contains Cirsium maritimum Makino, Cirsium, Asteraceae, or extract thereof.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a natural product having an immunoregulatory function such as an allergy prevention and / or improvement effect and useful as a food or drink, a pharmaceutical product, or a quasi drug.

  Currently, it is said that about one-third of the Japanese population develops allergic symptoms. Allergies are categorized from immediate allergy (type I) to delayed allergy (type IV), depending on the onset mechanism. The number is increasing rapidly. Mast cells and basophils are involved in the type I allergic reaction, and IgE bound to the cell surface is activated by the antigen to transmit signals downstream, and cells such as histamine and proteolytic enzymes The inner granule contents are released. This phenomenon is called degranulation. In order to suppress the onset of such type I allergy, suppression of degranulation from mast cells and basophils is important.

  In addition, recent studies have shown that there is a strong relationship between intestinal immunity and allergies. In particular, IgA secreted from Peyer's patch, a typical lymphatic tissue of the intestinal tract, prevents bacteria and viruses from adhering to the mucosal surface, eliminates foreign substances that capture foreign antigens and discharges them from the body, and prevents allergic development from foreign proteins. And plays an important role in mucosal immune function. Therefore, since promoting the secretion of IgA can be expected to enhance mucosal immune functions and prevent infections and allergic diseases, development of food materials having an IgA secretion promoting action is desired.

  So far, allergy-suppressing ingredients have been found in tea catechins, perilla extracts, and tea teas. Taking foods with these functions every day can not only reduce allergic symptoms, but also improve allergic predisposition. Expected (Non-Patent Documents 1 to 3).

Effect of Concentrated Tea Catechin Drink on Allergic Nasal Inflammation (Medicine and Pharmacy 59 (2), 207-211, 2008) Anti-allergic activity and anti-allergic activity of perilla extract (The Japanese Society of Allergology, Allergy 41 (8), 1035, 1992-08-30) Anti-inflammatory and anti-allergic active ingredients of strawberry tea extract: Food (Agricultural Chemical Society of Japan Journal of Agricultural Chemistry of Japan 69 (Special Issue), 165, 1995-07-05)

  Development of a highly safe and effective composition for immunomodulation is desired.

  Accordingly, an object of the present invention is to provide an excellent immunomodulating composition that is derived from a natural product, has high safety, and can be applied to food and drink products.

  As a result of intensive studies to solve the above problems, the present inventors have found that the compositae thistle genus, thistle extract, or the purified product of thistle extract has an allergy prevention and / or improvement effect and mucosal immunostimulatory action. The present inventors have found that it has an immunomodulating function such as the above, and have completed the present invention.

That is, the present invention
[1] A composition for immunomodulation containing a tooth thistle or a tooth thistle extract;
[2] The immunoregulatory composition according to item 1, which is a composition for preventing and / or improving allergy;
[3] The immunomodulating composition according to item 1, which is a mucosal immune stimulating composition;
[4] The composition for immunoregulation according to any one of Items 1 to 3, wherein the Hazel thistle or Hazel thistle extract contains silsimarin and / or silsimaritin;
[5] The item of any one of items 1 to 4, wherein the thistle thistle is derived from one or more selected from the group consisting of the whole plant or leaf part, stem part, root part, seed, and flower part of the thistle. The composition for immunomodulation described;
[6] The composition for immunoregulation according to any one of Items 1 to 5, wherein the extract of Hazel Thistle is an ethyl acetate extract of Hazel Thistle.
[7] A composition for immunomodulation containing silsimarin and / or silsimaritin.
[8] The composition for immunomodulation according to any one of items 1 to 7, which is administered orally, intravenously, by inhalation or transdermally;
[9] The composition for immunoregulation according to any one of claims 1 to 8, which is a food and drink product, a quasi drug, or a pharmaceutical product;
[10] The composition for immunoregulation according to any one of Items 1 to 9, wherein the food or drink is a tea beverage;
About.

  The composition for immunomodulation of the present invention contains an active ingredient obtained from naturally-occurring cattle thistle and has high safety. Such a safe composition for immunomodulation can be used, for example, for foods and drinks, pharmaceuticals or quasi-drugs that have anti-allergic effects and have few side effects.

FIG. 1 is a diagram schematically showing that an extract of the sea thistle was separated by chromatography. FIG. 2 is a diagram showing β-hexosaminidase release-inhibiting activity of leaf or flower extracts of cattle. FIG. 3 is a graph showing the concentration-dependent β-hexosaminidase release inhibitory activity of the extract of leaf or flower of thistle. FIG. 4 is a graph showing the β-hexosaminidase release inhibitory activity of a sample obtained by fractionating an ethyl acetate extract of the leaf of thistle by column chromatography. FIG. 5A is a diagram showing the concentration-dependent β-hexosaminidase release inhibitory activity of a purified product of the sea thistle extract. FIG. 5B is a graph showing the concentration-dependent β-hexosaminidase release inhibitory activity of commercially available silsimaritin. FIG. 6A is a view showing the amount of egg white lysozyme (LHE) -specific IgE in mouse serum. FIG. 6B is a diagram showing the amount of LHE-specific IgA in mouse serum. FIG. 6C is a diagram showing the amount of LHE-specific IgG1 in mouse serum. FIG. 6D is a graph showing the amount of LHE-specific IgG2 in mouse serum. FIG. 7A is a graph showing the total amount of IgE in mouse serum. FIG. 7B is a graph showing the total amount of IgA in mouse serum. FIG. 7C shows the total IgG amount in mouse serum. FIG. 8A is a diagram showing the amount of LHE-specific IgA in mouse feces. FIG. 8B is a graph showing the total amount of IgA in mouse feces. FIG. 9A is a graph showing the amount of egg white ovalbumin (OVA) -specific IgE in spleen lymphocytes. FIG. 9B is a diagram showing the amount of OVA-specific IgG1 in spleen lymphocytes. FIG. 9C is a diagram showing the amount of OVA-specific IgG2a in spleen lymphocytes. FIG. 10 is a graph showing the relationship between the balance of the amount of OVA-specific IgG2a / IgG1 in spleen lymphocytes and the concentration of thistle. FIG. 11 is a diagram showing the results of measuring the activity of inhibiting calcium influx into cells of an extract of leaf or flower of thistle. FIG. 12 is a diagram showing changes when a DNP-HSA antibody is driven into a mouse auricle by PCA evaluation. FIG. 12A is a diagram showing the state of the mouse pinna after the PCA evaluation. FIG. 12B is a graph showing the results of measuring the pinna thickness. FIG. 12C is a graph showing the amount of pigment in the pinna.

  The composition for immunomodulation of the present invention contains a Cirsium maritimum Makino or an extract thereof. Here, the extract includes a purified product purified to a single component or a fraction close to a single component.

  Thistle is a perennial that grows on temperate coasts, and in Japan, it is distributed from southwest of Chiba Prefecture to Kyushu. It is often found in sand, gravel and grasslands. The roots are used for food.

  In the present invention, thistle can be used as it is, but in that case, the preparation method is not particularly limited. Thistle is the whole or part of the thistle, especially leaves, stems, roots, seeds, and flower parts, one each separately or a combination of two or more thereof, or the dried product is appropriately cut. It can be prepared as a crushed or pulverized powder. The dried product of thistle or pulverized product thereof can be used after boiling in hot water, for example. The crushed or crushed product of thistle can be prepared by roasting if necessary.

  Roasting of thistle is performed by a known method using a rotary roasting machine, and is not particularly limited. The roasting temperature can be, for example, 150 to 400 ° C, preferably 180 to 350 ° C, and more preferably 200 to 330 ° C. The roasting time is appropriately determined depending on conditions such as the roasting temperature, and can be, for example, 5 to 120 minutes, preferably 10 to 90 minutes, and more preferably 15 to 60 minutes.

  In the present invention, when using the Hazel Thistle extract, the extraction method is not particularly limited. The extract of thistle is the whole or part of the thistle, especially the leaves, stems, roots, seeds, and flower parts separately one by one or a combination of two or more thereof, or the dried product as appropriate. From the cut, crushed or ground powder, it is extracted with a solvent. Thistle can be easily obtained from the ones that grow naturally, and commercial products can also be used.

  When drying the whole or part of thistle, it is not limited, but it can be performed by sun drying, air drying or using a dryer. When performing sun-drying, the time required for drying depends on the weather and the like, but can be, for example, 6 hours or more, preferably 1 day or more, more preferably 2 days or more. When using a dryer, it is generally 100 ° C. or lower, preferably 40 ° C. or lower, for example, a rotary dryer, hot air dryer, heat transfer dryer, vacuum dryer, vacuum freeze dryer, A cold air dryer, a vibration dryer, a filter dryer, a vacuum stirring dryer, or the like can be used.

  When using a hazel thistle extract as an immunomodulating composition, an extract obtained by extracting hazel thistle from a solvent may be used as it is, or a solution diluted or concentrated appropriately may be used as the extract. In order to obtain an extract of the sea thistle, it is possible to use a fresh plant body, such as using a coldly stored, frozen, or dry-stored thistle, or using a concentrate of the extract of thistle in an appropriate solvent such as water. It can also be used by dissolving or diluting. The concentrate of thistle extract is not limited, but a liquid, paste, or mud can be used.

  Examples of the solvent used for the extraction of thistle include water, an organic solvent, and a water-containing organic solvent. By using an appropriate extraction solvent, a water extract, organic solvent extract, or water-containing organic solvent extract of thistle can be obtained. These extraction solvents are not limited, but water, methanol, ethanol, ethylene glycol, 1,3-butylene glycol, isopropylene glycol, propylene glycol, glycerin, ethyl acetate, isopropyl alcohol, tetrahydrofuran, n-propanol, methyl ethyl ketone, Dioxane, acetone, acetonitrile, acetic acid, dimethylformamide, n-hexane or mixtures thereof are preferred. Of these extraction solvents, ethyl acetate is particularly preferable from the viewpoint of obtaining higher effects of the present invention. The concentration in the case of using ethyl acetate is not limited, but from the viewpoint of obtaining higher extraction efficiency, for example, 100% to 40%, preferably 100% to 60%, more preferably 100% to 80%. Can be used in concentration.

  The site of thistle used for extraction is not limited, but examples include leaves, stems, roots, seeds, flowers, etc., from the viewpoint of obtaining higher effects of the present invention, leaves, flowers or These combinations are preferable, and the leaf portion is more preferable.

  The extraction method is not particularly limited, but for example, whole or part of thistle is cut, crushed by a known method such as a mixer, added with an extraction solvent, stirred, and treated at room temperature or heating for a certain extraction time. Thereafter, a method of separating and extracting the extract of the sea thistle from the extracted supernatant is mentioned. Alternatively, the extraction method may be a method in which the thistle is immersed in an extraction solvent and then allowed to stand at room temperature or under heating.

  The crushing conditions are not limited, but when the whole thistle or part of the cuttlefish is a large scale such as 1 kg to 10 kg, a large vertical cutter mixer or the like can be used. In the case of a small scale of g, a home mixer or the like can be used. For example, when applying crushing conditions on a large scale, it is appropriately changed depending on the hardness of the raw material, moisture content, etc. For example, the crushing time is several tens of seconds to several minutes, and the rotation speed of the mixer is 10 to 3000 rpm. Is possible.

  Moreover, normal conditions can be applied as the extraction conditions, and the extraction conditions are not limited, but it is possible to employ a method of immersing a dried product of thistle in a solvent at 3 to 100 ° C., heating to reflux or microwave heating, for example. it can. The extraction temperature is not limited as long as an appropriate extraction amount can be obtained. For example, when ethyl acetate is used, it can be 20 to 100 ° C, preferably 22 to 80 ° C, and more preferably 25 to 60 ° C. preferable. Moreover, when water is used, extraction temperature can be 20-100 degreeC, for example, 50-100 degreeC is preferable and 70-100 degreeC is more preferable. The extraction time is not limited as long as an appropriate extraction amount can be obtained. For example, the extraction time can be 5 minutes to 14 days, preferably 10 minutes to 7 days, and more preferably 15 minutes to 5 days. The extraction is usually performed under normal pressure, but can be performed under pressure. About 1L-100L can be used for the addition amount of a solvent with respect to 1 kg of dry weight of thistle.

  It is also possible to increase extraction efficiency by adding acid, alkali, or enzyme to the cattle before adding the extraction solvent.

  A known method can be used for the operation of adding the extraction solvent and stirring, for example, a method of rotating the extraction container containing the cut or crushed product of the sea thistle and the extraction solvent, and the extraction container magnetically. Or the method of installing in a mechanical stirring apparatus and mixing, the method of shaking the said container for extraction, etc. are mentioned.

  In the stirring operation, vibration can be caused by ultrasonic treatment to increase the extraction efficiency. In the case of performing sonication, there is no limitation. For example, when the whole or part of cuttlefish is a large scale such as 1 kg to 10 kg, cut pieces or crushed cuticles on a commercially available ultrasonic generator An ultrasonic container can be obtained by installing an extraction container containing a product and an extraction solvent and treating with 26 kHz ultrasonic waves for 60 minutes. In the case of a small scale of several tens to several hundreds of grams, 40 kHz An ultrasonic treated product can be obtained by treating with the ultrasonic wave for 60 minutes. The temperature condition of the sonication is not limited as long as an appropriate extraction amount is obtained, but can be 2 ° C to 100 ° C, preferably 10 ° C to 70 ° C. As an ultrasonic generator for large scale, for example, UT-12 manufactured by Shinmeidai Kogyo Co., Ltd. can be used. As an ultrasonic generator for small scale, for example, US-3R manufactured by AS ONE Co., Ltd. is used. Can be used.

  In order to increase the extraction efficiency, it is possible to perform multi-stage extraction using the same type or multiple types of extraction solvents. When performing multi-stage extraction, the same or more types of extraction solvents are added to the residue obtained in the first stage extraction, followed by heating at room temperature or after heating, and separating and extracting the extract of thistle from the extracted supernatant. be able to.

  In the microwave heating, it is possible to appropriately set conditions such as the output of the microwave irradiation apparatus, the wavelength of the microwave, the irradiation time, and the like as long as the activity of the active ingredient of the present invention is not lost. For example, when a microwave of 2450 MHz and 500 W is applied to 100 g of dried thistle, it can be processed in 10 seconds to 10 minutes, preferably 30 seconds to 5 minutes.

  The extracted supernatant can be used as it is as a hazel thistle extract. In addition, it is possible to separate and extract the Hazel Thistle extract from the extracted supernatant. In this separation and extraction step, a known method can be employed, and for example, filtration, centrifugation, suction, squeezing, etc. can be performed.

  In the case of performing separation and extraction by filtration, for example, membrane filtration can be performed without limitation. When membrane filtration is performed, for example, the temperature condition can be set to 2 ° C. to 70 ° C., preferably 10 ° C. to 40 ° C., and the membrane pore diameter can be set to 0.1 μm to 10 μm, preferably 0.1 μm to 5 μm. Is possible.

  In the case of separation and extraction by centrifugation, a known device can be used, and examples of the centrifuge include a separation plate type, a cylindrical type, and a decanter type. In the case of performing centrifugation, for example, the temperature condition can be 2 ° C. to 70 ° C., preferably 10 ° C. to 40 ° C., and the number of rotations is 1000 rpm to 10,000 rpm, preferably 1500 rpm to 8000 rpm, more preferably 2000 rpm to 6000 rpm. The centrifugation time can be 10 seconds to 30 minutes, preferably 20 seconds to 20 minutes, and more preferably 30 seconds to 15 minutes.

  For the separation and extraction by pressing, a pressing machine can be used, and a known device can be used as the pressing machine, and examples thereof include a pneumatic pressing machine and a screw pressing machine.

  The thistle extract can be made into a purified product by subjecting it to further purification treatment before and after dilution or concentration. For the purification treatment, chromatographic methods, ion-exchange chromatographic methods, etc., which are methods known to those skilled in the art, can be used alone or in combination, in addition to the extraction with the above-mentioned solvent.

  In the case of using a chromatographic method, for example, any of column chromatography, high performance liquid chromatography, thin layer chromatography, centrifugal liquid chromatography, etc. using a normal phase or reverse phase carrier or ion exchange resin, or The method of using them in combination is mentioned. The purification conditions such as the carrier and the elution solvent when using the chromatographic method can be appropriately selected in accordance with various chromatographic methods.

  From the viewpoint of obtaining higher extraction efficiency, for example, when obtaining a water-containing organic solvent extract or a water-containing extract, the ratio of mixing the cut or crushed product of thistle and the extraction solvent is, for example, when obtaining a water-containing organic solvent extract or a water-containing extract. The cut or crushed product of thistle can be 5 g to 1 kg, preferably 10 g to 500 g, more preferably 20 g to 200 g. In the case of obtaining an ethyl acetate extract, for example, 5 g to 300 g, preferably 10 g to 200 g, and more preferably 20 g to 100 g of a cut product or a pulverized product of a dried thistle can be used per 1 L of ethyl acetate.

  In the present invention, from the viewpoint of obtaining higher effects of the present invention, the content of the sea thistle and the thistle extract is 0.000001 to 100 w / w% in terms of solid content, preferably based on the total amount of the composition for immunomodulation, preferably Can be 0.00001 to 90 w / w%, more preferably 0.0001 to 80 w / w%, and most preferably 0.0001 to 50 w / w%. (W / w% represents weight%. The same shall apply hereinafter.)

  Further, in the present invention, the content of the sea thistle and the thistle extract is 0.000001 to 100 w / v% in terms of solid content, based on the total amount of the composition for immunomodulation, from the viewpoint of obtaining the higher effect of the present invention. , Preferably 0.00001 to 90 w / v%, more preferably 0.0001 to 80 w / v%, and most preferably 0.0001 to 50 w / v%. Here, w / v indicates g / ml.

  In the present invention, the content of the purified product of thistle is 0.000000001 to 5 w / w% in terms of solid content, preferably from the viewpoint of obtaining a higher effect of the present invention, based on the total amount of the composition for immunomodulation, preferably It can be 0.00000001 to 3 w / w%, more preferably 0.0000001 to 2 w / w%.

  Here, in the present invention, the thistle extract or the further purified product may contain cilsimaritin (CAS Number: 6601-62-3) or cilsimarin (CAS Number: 13020-19-4) having the following chemical formula. More preferably, the product obtained by purifying from the thistle extract is silsimaritin and / or silsimalin.

[Usage]
The present inventors have newly found out that thistle, or an extract or purified product thereof, silsimaritin and / or silsimalin, has an excellent immunomodulatory action. Thistle is derived from nature and has been edible since ancient times, so it is suitable for safe and effective use of immunomodulation.

  Here, in this specification, “immunomodulation” is a concept including antiallergy and immunostimulation. Immunostimulation includes, but is not limited to, mucosal immunostimulation.

  Here, in the present specification, “allergy prevention and / or improvement” and anti-allergy are terms used interchangeably. The anti-allergic action including the thistle or the extract or purified product of the present invention is not particularly limited, but can be particularly immediate type allergy suppression. Type I allergies, represented by food allergies and pollen allergies, are defined as hypersensitivity reactions. Type I allergy is said to occur when an IgE receptor (FcεRI) expressed in mast cells and basophils is activated by an antigen. This is the first step in inducing extracellular release of many chemical mediators including histamine, prostaglandins and leukotrienes, and these mediators cause immediate allergic reactions.

  The anti-allergy prevention and / or amelioration composition as used in the present invention is particularly useful for the prevention and prevention of allergic symptoms such as sneezing, itching, runny nose, tears, dermatitis, urticaria, asthma, and inflammatory arthritis. / Has an effect on improvement. The antiallergic prevention and / or improvement of the present invention is particularly effective for type I allergy. Alternatively, it can be said that the prevention and / or improvement of the present invention is effective for IgE-dependent diseases.

  Such an antiallergic effect can be verified, for example, as a β-hexosaminisase release inhibitory effect from cells, that is, a basophil degranulation inhibitory effect.

  Moreover, in this specification, "immunostimulation" is meant to include general immune activation and mucosal immunity activation in addition to antiallergic action.

  The composition for enhancing mucosal immunity referred to in the present invention is effective in preventing infection or adverse effects caused by pathogenic bacteria, parasites, pathogenic antigens, viruses, or improving such infections or adverse effects. In particular, it is useful for alleviating indirect effects as well as direct effects due to a decrease in intestinal tract immune function such as intestinal tract and bacterial digestive tract infection.

  By activating mucosal immunity, for example, it is possible to prevent a decrease in the immune function of the defense system in the gastrointestinal mucosa. This may include symptoms such as fever, vomiting, diarrhea, and abdominal pain due to infection of the gastrointestinal tract due to colonization and growth of microorganisms in the gastrointestinal tract, and includes prevention and alleviation of these symptoms. Furthermore, mucosal immunity activation is useful for prevention and alleviation of bacterial diarrhea and the like. It is also useful for strengthening resistance in infants, the elderly, and those with weak physical strength. Thus, mucosal immunity activation enables neutralization of bacteria and viruses, suppression of bacterial adhesion to tissues, and suppression of allergy due to food antigens.

  Such an antiallergic action and immunostimulatory action based on the immunostimulatory action can be verified as a decreasing tendency of the IgE amount in the serum or an increase of the IgA quantity in the feces.

[Beverages]
The composition for immunomodulation of the present invention can be blended as one component of a food or drink product. These foods and drinks are not limited, but can also be used as foods and drinks having antiallergic action and immunostimulatory action, particularly mucosal immunostimulatory action, for example, for patients at medical institutions such as hospitals. Can be provided. These foods and beverages are not limited, but can be provided as functional beverages or functional foods, nutritional functional beverages or nutritional functional foods. , Drug stores, convenience stores, supermarkets, department stores and the like. Functional foods and drinks are foods and drinks with medicinal properties that are approved or designated by the government or public organizations, or foods and drinks that display functionality based on the content that the company has reported to the country or the like. Means. Examples of functional foods include foods for specified health use, functional foods for nutrition, foods for functional indications, foods for the elderly, health supplements (balanced nutritional foods, supplements) and the like. This includes foods and beverages including packages and containers that display pharmaceutical efficacy or functionality, package inserts, and instruction manuals. Food / beverage products that display the same efficacy or functionality as pharmaceutical products are included in the application form to the national government.

  In the case of the display for the purpose of allergy prevention or improvement, it is not limited, but for example, for allergic constitution, for those who are worried about pollen, adjust the nose, adjust the eye, Indications such as alleviating eye and nose discomfort, eye health, nose health, and eye fatigue are included.

  In the case of the display for the purpose of immunostimulation, the display is not limited, and examples thereof include a display for a person without physical strength, a weak constitution, and a condition of the stomach.

  Examples of food include all foods, such as cereals, potatoes, seafood, meats, eggs, fats and oils, milk, vegetables, beans, fruits, sugars, seaweeds, confectionery, seasonings And cooked processed foods.

  The seasoned processed food is not limited, but for example, processed fishery products such as chikuwa and kamaboko; processed livestock products such as ham and sausage; confectionery such as cookies, biscuits, snacks, chocolate, cakes; soba, udon, raw noodles Noodles such as Chinese noodles and pasta; bread such as bread and confectionery bread; fermented processed foods such as natto and miso; soybean foods such as tofu and okara; pickles such as pickles and pickles; marine products; processed meats and vegetables; Canned fruits and the like; dairy products such as butter, margarine, yogurt, cheese and milk; frozen dessert foods such as ice cream and sherbet.

  Examples of beverages include, but are not limited to, all beverages, for example, beverages that use teacups as tea, roasted dried products as substitutes for tea leaves, fruit juice beverages, 100% fruit juice beverages, low fruit juice beverages, and pulp Fruit beverages such as beverages, vegetable juices, flavored beverages, fruit beverages for dilution; carbonated beverages; coffee, coffee beverages, soft drinks with coffee, cocoa beverages, tea, green tea, matcha tea, oolong tea, barley tea, roasted tea, etc .; Vinegar drinks; soft drinks such as sports drinks; milk; milk drinks; dairy drinks; lactic acid drinks; lactic acid bacteria drinks; soy drinks such as soy milk and prepared soy milk; alcoholic drinks such as beer, sake, shochu, liqueur and wine; taurine , Nutritional drinks containing royal jelly, amino acids, vitamins, minerals, iron, and the like.

  Although there is no restriction | limiting in the time which applies this invention to food / beverage products, For example, in the manufacturing process of food / beverage products, it can apply before and after a process process, a cooking process, a heating process, a preservation | save process, etc. For example, in the processing step and cooking step, the composition for immunoregulation of the present invention can be included in the raw material. The application method can be appropriately changed according to the type of food and drink, the form of the raw material, and the like, and various methods such as mixing, addition, application, spraying, and immersion can be adopted.

  When the composition for immunomodulation of the present invention is applied to a beverage product, in one embodiment, a tea beverage is particularly preferable. Although not limited, the leaf part and / or stem part of the dried cattle thistle are cut | disconnected by a well-known method, Then, it crushes or grind | pulverizes and it is made into a powdery material. It is possible to sell it as a tea beverage in the state of a powder of a hammer thistle. If necessary, the powder of thistle is roasted. The powder of thistle can be sold as a tea drink in a roasted state. Roasted thistle is drunk by boiling it in hot water.

  Moreover, the said drink, other tea drinks, and thistle extract can be used as a mixture, or it can be diluted with water etc. as juice, a drink agent, etc. Furthermore, it can be used as a concentrated product of the above mixture, powdered tea powdered by spray drying, lyophilized instant tea, or the like. In that case, hot water can be poured into, for example, powdered tea or instant tea. Also, for example, a mixture of a tea preparation such as green tea or black tea and a scissors preparation can be used as it is as a tea leaf for beverages. In that case, it is possible to pour hot water into a mixed tea leaf of a tea preparation and a scissors preparation in the same manner as a general Japanese tea or black tea drink.

  When the composition for immunomodulation of the present invention is applied to food, in one embodiment, it is desirable to make a green juice food. Although it is not limited, the green juice food here is a refined treatment such as chopping, crushing, grinding, etc. of the leaf part and / or stem part of the fresh sea thistle or the leaf part and / or stem part of the dried cattle thistle Or a juice obtained by squeezing the raw material or the refined product, and the shape thereof is fine solid, semi-solid, paste, or liquid. Further, it may be in the form of a powder obtained by drying these shapes and then pulverizing them. As the powder, a raw material that has been dried as it is without being chopped may be pulverized. Examples of the drying method include natural drying, heat drying, spray drying, and the like. However, when the freeze drying method is used, there is no change in quality due to heat, pulverization is easy, and excellent palatability can be obtained. Therefore, it is preferable. It is also preferable from the viewpoint of storage stability.

  In addition, the leaves and stems of thistle can be used in combination with other green juice ingredients. These green juice raw materials are processed in the same manner as described above to obtain green juice food. The treatment may be carried out by mixing from the stage of the leaves and stems of the thistle and the raw material, mixing at the middle stage, or mixing separately prepared green juice food. The green juice material other than the leaves and stems in the present invention is not particularly limited. For example, kale, broccoli, cabbage, komatsuna, radish leaves, watercress, nazuna, young barley leaves, tomorrow leaves, seri , Parsley, carrot, celery, asparagus, spinach, bitter gourd, green tea, perilla, spring chrysanthemum, elderberry, chickweed, mugwort, mulberry, simmon leaf, kumazasa, moroheiya, etc., one or more of these Can be combined with leaves and / or stems. In the present invention, when a green juice product is prepared by combining the leaf and stem portion of the thistle and the other green juice ingredients, the components derived from the leaf and / or stem portion of the green juice as the dry residue, It is desirable that it is 10 w / w% or more of the dry residue amount. Within this range, the nutritional component derived from the sea thistle is effectively ingested, and the effect of improving the palatability of the green juice food is excellent.

  When the composition for immunomodulation of the present invention is applied to foods and drinks, it is possible to appropriately mix auxiliary agents used in ordinary foods and beverages as long as the effects of the present invention are not impaired. Examples of such auxiliaries include glucose, sucrose, fructose, oligosaccharide, starch syrup, maltose, maltose, xylitol, sorbitol, aspartame, sucralose, stevioside, rubusoside, corn syrup, lactose, citric acid, tartaric acid, malic acid Succinic acid, lactic acid, L-ascorbic acid, dL-α-tocophenol, sodium erythorbate, glycerin, glycerin fatty acid ester, propylene glycol, polyglycerin fatty acid ester, sucrose fatty acid ester, propylene glycol fatty acid, sorbitan fatty acid ester, ester Gum arabic, casein, pectin, gelatin, agar, carrageenan, vitamins B, vitamins C, nicotinamide, calcium pantothenate, calcium salts, amino acids, pigments, Fee, preservatives, and the like.

  The content of the sea thistle or the extract thereof when the composition for immunomodulation of the present invention is applied to foods and drinks is not limited. However, in terms of solid content, the content is generally 0.00 per day for humans and animals. It is 00001 to 5000 mg / kg body weight, preferably 0.01 to 200 mg / kg body weight, more preferably 0.1 to 100 mg / kg body weight.

  The pH when the composition for immunomodulation of the present invention is applied to a beverage is appropriately adjusted depending on the type of the beverage. For example, in the case of a tea beverage, the pH can be 3.5 to 7.5. 3.8 to 7.2 are preferable, 4.0 to 7.0 are more preferable, and 4.5 to 6.5 are even more preferable. The pH of the beverage can be adjusted, for example, by appropriately blending sodium bicarbonate, citric acid and the like.

[Pharmaceuticals, Quasi-drugs]
When the composition for immunomodulation of the present invention is used as a medicine or quasi-drug, it can be formulated into an appropriate form and administered to humans or animals in any dosage form. Examples of the dosage form include, but are not limited to, oral, transdermal, enteral, transmucosal, and injection. From the viewpoint of remarkably exhibiting the effects of the present invention, the administration form is preferably oral administration.

  When the composition for immunomodulation of the present invention is orally administered, the composition for immunomodulation of the present invention is, for example, a tablet, capsule, granule, syrup, powder, film, drop, jelly, It is formulated into a semi-solid purine-like dosage form by a known method. In the case of oral administration in the form of a film, drop, jelly, semi-solid purine or the like, the composition for immunomodulation of the present invention can be taken without water. Alternatively, it is also possible to orally administer a dry thistle of thistle or its extract or purified product as a herbal medicine or a herbal medicine preparation.

  For parenteral administration, for example, subcutaneous injection, intravenous injection, intramuscular injection, intrathecal injection, transdermal absorption agent, inhalant, suppository, eye drop, nasal drop and the like by a known method. It is possible to formulate.

  As one aspect of the dosage form, known drug delivery for the purpose of enhancing the stability and bioavailability of the thistle or its extract or purified product, or improving patient compliance, or a combination thereof The system can be used to deliver the immunomodulatory composition of the invention to the site of absorption.

  Examples of drug delivery systems include, but are not limited to, polymers such as cellulose, dextran, starch, polyvinyl alcohol, acetylated or methacrylated polymers, polylactic acid and polyglycolic acid and block copolymers thereof, and polyethylene glycol. Examples thereof include a method using a transport protein such as albumin, and other methods using micelles, liposomes, microspheres, nanoparticles, composite emulsions, dendrimers and the like. These drug delivery systems are used not only for the purpose of transporting the composition for immunomodulation of the present invention to a target site such as an absorption site but also for the purpose of controlled release for releasing the composition for immunomodulation of the present invention in a timely manner. Can be used.

  In the composition for immunomodulation of the present invention, an excipient, a lubricant, a binder, a disintegrant and the like are used in a solid preparation and a solvent in a liquid preparation, as long as the effects of the present invention are not impaired. Solubilizing agents, suspending agents, tonicity agents, buffers, soothing agents, and the like can be appropriately blended.

  Examples of the excipient include lactose, sucrose, D-mannitol, glucose, starch, calcium carbonate, kaolin, crystalline cellulose, and light anhydrous silicic acid.

  Preferable examples of the lubricant include magnesium stearate, calcium stearate, talc, purified talc, colloidal silica, borax, polyethylene glycol and the like.

  Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, bound cellulose, sucrose, D-mannitol, dextrin, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropyl starch. , Methylcellulose, ethylcellulose, shellac, calcium phosphate, polyvinylpyrrolidone and the like.

  Examples of the disintegrant include starch, dry starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, carmellose calcium, croscarmellose sodium, carboxymethyl starch sodium, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, Examples include stearic acid monoglyceride and lactose.

  Examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil and the like.

  Examples of the solubilizer include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.

  Examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate, for example, polyvinyl alcohol, polyvinyl Examples thereof include hydrophilic polymers such as pyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose.

  Examples of the isotonic agent include sodium chloride, glycerin, D-mannitol and the like.

  Examples of the buffer include buffer solutions such as phosphate, acetate, carbonate, citrate, and the like.

  Examples of soothing agents include benzyl alcohol.

  Examples of the preservative include p-hydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, and the like.

  The effective dose of the thistle or extract thereof when orally administering the composition for immunomodulation of the present invention is not limited, but generally 0.00001 per day for humans and animals in terms of solid content. -5000 mg / kg body weight, preferably 0.01-200 mg / kg body weight, more preferably 0.1-100 mg / kg body weight. The effective dose of the thistle or its extract when parenterally administering the composition for immunomodulation of the present invention is not limited, but generally 0.000001 per day for humans and animals in terms of solid content. -50 mg / kg body weight, preferably 0.00001-5 mg / kg body weight, more preferably 0.00001-1 mg / kg body weight. The number of administrations is usually about 1 to 4 times a day, but can be appropriately adjusted depending on the administration route.

  EXAMPLES Next, the present invention will be specifically described with reference to examples, but the present invention is not limited to the following examples.

(Example 1) Extraction method from dried pulverized product with ethyl acetate 100 g of dried thistle leaves and flowers were dispersed and stirred in 1000 ml of 100% ethyl acetate, respectively, and extracted at room temperature for 1 week. After the extraction supernatant was filtered off, the residue was dispersed again in ethyl acetate. After heating to reflux, the extraction supernatant was filtered off. The supernatant was successively concentrated under reduced pressure, dried under reduced pressure in a nitrogen gas atmosphere, and freeze-dried to obtain extract 1 (4 g) with leaves of cattle thistle and extract 2 (4 g) with flowers of thistle.

(Example 2) Extraction method with water from dry pulverized product Add 1.5 L of water to 100 g of dry pulverized leaf of thistle, pulverize with a mixer, agitate and apply ultrasonic waves, filter the extracted supernatant and By dehydrating, an extract (2 L) was obtained together with water. Further, the extract 3 (15 g) was obtained by successively performing vacuum concentration, vacuum drying and lyophilization under a nitrogen gas atmosphere.

(Example 3) Extraction method by squeezing Add 1 L of water to a mixture (1 kg) of fresh leaves and stalks of thistle, pulverize with a mixer, stir and apply ultrasonic waves, filter the extracted supernatant, wash, and By dehydrating, an extract (2 L) was obtained together with water. Further, the extract 4 (15 g) was obtained by successively performing vacuum concentration, vacuum drying and lyophilization under a nitrogen gas atmosphere.

(Example 4) Extraction method by pressing 1 L of water is added to a mixture of fresh leaves and stems (1 kg) of thistle, pulverized with a mixer, stirred and ultrasonically applied, and the extracted supernatant is filtered and dehydrated, then pressed. As a result, an extract (1.5 L) was obtained. Further, the extract was successively concentrated under reduced pressure, dried under reduced pressure and lyophilized in a nitrogen gas atmosphere to obtain an extract (20 g).

(Example 5) Production method of dry powder Fresh leaves and fresh stems (1 kg) of thistle were dried overnight in a 40 ° C cold air dryer and then finely pulverized with a pulverizer to obtain dry thistle powder (100 g).

(Example 6) Extraction method with hot water from dried pulverized product By adding 1.5 L of hot water to 100 g of dried pulverized leaf and stem leaves obtained in Example 5, and filtering the extract supernatant. The extract (2 L) was obtained together with water. Further, the extract 3 (20 g) was obtained by successively performing vacuum concentration, vacuum drying and lyophilization under a nitrogen gas atmosphere.

(Example 7) Extraction method by hot water from fresh leaves and fresh stems Add 1 L of hot water to a mixture (1 kg) of fresh leaves and fresh stems of cattle thistle and filter the extract supernatant to extract together with water. (1.5 L) was obtained. Further, the extract 3 (15 g) was obtained by successively performing vacuum concentration, vacuum drying and lyophilization under a nitrogen gas atmosphere.

(Example 8) Separation of French Thistle Extract 500 mg of the ethyl acetate extract obtained in the same manner as in Example 1 from the leaf of thistle was subjected to silica gel chromatography. The conditions are as follows.
・ Silica gel: Wako Pure Chemicals, Wakosil C-200, 25 g
・ Column size: 20mmφ × 100cm
Eluent: acetone / hexane + methanol (concentration 0 w / w% to 100 w / w%), 500 mL each
Under these conditions, samples of a total of 9 fractions (FIG. 1) of 0, 5-1, 5-2, 10, 20, 30, 50, 100% acetone fraction and 100% methanol fraction were collected. Each was analyzed according to Test Example 1.

Example 9
Furthermore, among the fractions obtained in Example 8, purification of the 30% acetone fraction, which had a high evaluation result in Test Example 1, was advanced.
That is, 70 mg of the 30% acetone fraction obtained in Example 8 was subjected to HPLC under the following conditions.
・ Analyzer: LC-10A, manufactured by Shimadzu Corporation
Column: Cosmosil C18-AR-II manufactured by Nacalai Tesque
・ Column size: 10mmφ × 250mm
・ Eluent: 80% methanol ・ Flow rate: 1.5 ml / min
・ Inlet pressure: MPa
・ Detector: Ultraviolet spectroscopic detector (UV)
-Column temperature: 35 ° C
・ Injection volume: 50 μl

HPLC was performed under the above conditions, and a peak with ultraviolet absorption was collected to obtain 12 mg of an active ingredient (silsimaritin).
¹ H NMR: (500 MHz, CDCl ₃ ): dH 7.90 (1H, d), 7.81 (1H, d), 6.97 (1H, m), 6.56, dd), 3.96 (3H, s) 3.92 (3H, s )

(Test Example 1) Evaluation of β-hexosaminidase release inhibition Cell Culture RBL-2H3 cells were treated with 10% (v / v) fetal bovine serum (FBS: Sigma-Aldrich), 100 U / ml penicillin (Nacalai Tesque), and 100 μg / ml streptomycin (Nacalai Tesque). And Dulbecco's modified Eagle medium (manufactured by Nacalai Tesque, DMEM). At this time, the temperature was 37 ° C. in a humidified atmosphere containing 5% CO ₂ .

2. β-hexosaminidase-releasing activity RBL-2H3 cells cultured in 1 above were seeded in a 24-well plate at 2.5 × 10 ⁵ cells / well in DMEM containing 10% FBS, and the cells were incubated at 37 ° C. Cultured overnight. Cells were then washed twice with PBS. The cells were sensitized with 50 ng / mL DNP-specific IgE (Sigma-Aldrich) for 2 hours. The cells were washed with MT buffer, and then a striped extract or purified product diluted in MT buffer was added. Here, the extracted sample of Hazel thistle was extracted with 100% ethyl acetate in the same manner as in Example 1, and then the concentrated sample was dimethyl sulfoxide (DMSO) to a concentration of 10 mg / mL. ) And diluted to 0.5% concentration in MT buffer. The silcymaritin obtained by purifying from the extract of the scissors obtained in Examples 8 and 9 was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mg / mL in a concentrated sample, and dissolved in MT buffer. Diluted to a concentration of 0.5%. Similarly, commercially available silsimaritin was also used for reference. After 10 minutes incubation, DNP-HSA (final concentration 50 ng / mL) was added and the culture was incubated for 30 minutes. The supernatant was collected and the cells were lysed with MT buffer containing 0.1% polyethylene glycol mono-p-isooctylphenyl ether (Triton X100, Nacalai Tesque). An aliquot of each supernatant and cell lysate was solubilized in 0.1 M citrate buffer (pH 4.5) for 30 minutes at 37 ° C. with 1 mM p-nitrophenyl N-acetyl-D-glucosamide ( Incubated with Wako Pure Chemical Industries, Ltd. The enzyme reaction was stopped by adding 2M glycine buffer (pH 10.4) and the absorbance was measured at 405 nm. The ratio of β-hexosaminidase release activity from RBL-2H3 cells by the sample of thistle was calculated using the following formula.
Enzyme releasing activity (%) = {(absorption of cell supernatant / absorption of cell supernatant + absorption of cell lysate) × 100}

  The results are shown in FIGS. Data are shown as the mean ± standard deviation of three experiments.

  As can be seen from FIG. 2 and FIG. 3, particularly excellent β-hexosaminisase release inhibitory action was found in the leaf and flower extracts.

  Moreover, about the sample of Example 8 fractionated by column acetate chromatography of the leaves of thistle, several fractions (DCC 30, 50, 100) were obtained as shown in FIG. It was found that it significantly attenuated the release of β-hexosaminidase. In particular, D.C. C. C. 30 and D.E. C. C. 50 was 50 μg / mL, 86.1 to 88.6 percent inhibition, and was found to have a potent inhibitory effect on β-hexosaminidase release compared to the positive control.

  Furthermore, tests using the purified thistle from Example 9 or commercially available silsimaritin showed a strong inhibitory effect on the concentration-dependent release of β-hexosaminidase as shown in FIGS. 5A and 5B. It was. Here, FIG. 5A shows the result of using a purified product of thistle, and FIG. 5B shows the result of using commercially available silsimaritin.

(Test Example 2) Antibody production evaluation using a living body Animals and antigens 5-week-old female C3H / HeJ mice (body weight 15-20 g) were purchased from Japan SLC (Hamamatsu, Shizuoka Prefecture, Japan). The animals were housed under conditions of a light-dark cycle of room temperature 24 ± 3 ° C., 12/12 h (AM.7: 00-PM.7: 00). The experimental design followed the guidelines for animal experiments approved by the Animal Experiment Committee of Kochi Prefectural University (approval number: 2015-006). The antigen was LHE and was purchased from Wako Pure Chemical.

2. Feeding, sensitization and sampling Mice were given free access to water and AIN-93G diet. The mice were then assigned to 3 groups of 10 animals according to their body weight, and were freely fed the following diet for 4 weeks.
Non-sensitized group: AIN-93G
LHE sensitization group: AIN-93G
LHE sensitization-Hazel thistle group: AIN-93G supplemented with 1% Hazel thistle (CMM).

  The body weight of mice was recorded 3 times / week throughout the experimental period. One sensitized group of mice received 0.2 mL PBS containing 50 μg LHE and 4 mg aluminum hydroxide intraperitoneally on days 14 and 21 (LHE sensitized group and LHE sensitized-cattle group). Both). The non-sensitized group received only 0.2 mL PBS containing 4 mg aluminum hydroxide intraperitoneally. To assess antibody levels, blood was obtained from the orbital vein of mice on day 27 under light anesthesia. Fecal pellets were collected on day 31. Next, the feces pellets were dried with ALPHA 2-4 LDplus (Christo, Osterode, Germany). These samples were subjected to enzyme-linked immunosorbent assay (ELISA) to measure total antibodies and specific antibodies to LHE.

3. Measurement of serum LHE-specific IgE, IgA, IgG1, and IgG2a The levels of LHE-specific IgE, IgA, IgG1, and IgG2a were measured using an enzyme linked immunosorbent assay (ELISA). Specifically, a Nung-Immunoplate (Thermo Scientific, Roskilde, Denmark) is coated with 100 μL of 0.1 M carbonate buffer (pH 9.6) containing LHE and incubated at 4 ° C. overnight. did. The wells were washed with PBS (PBST) containing 0.05% polyoxyethylene (20) sorbitan monolaurate (Tween 20, Wako Pure Chemicals, Osaka, Japan) and then 37 ° C. with 1% BSA / PBST solution. And incubated for 1 hour. A 100 μL volume of each serum sample (1:50 for IgE and IgA, 1: 250 for IgG1, IgG2a, diluted in PBST containing 1% BSA) is applied to each well and the mixture is brought to 37 ° C. And incubated for 1 hour. After washing each well 5 times with 200 μL PBST, HRP-conjugated rabbit anti-mouse IgE (1: 1000 diluted with PBST containing 1% BSA, manufactured by Santa Cruz Biotechnology) 100 μL, HRP-conjugated rabbit anti-mouse IgA (1 1: 1000 diluted with PBST containing% BSA, manufactured by Santa Cruz Biotechnology), HRP-conjugated rabbit anti-mouse IgG1 (1: 5000 diluted with PBST containing 1% BSA, manufactured by Santa Cruz Biotechnology), And HRP-conjugated rabbit anti-mouse IgG2a (1: 5000 diluted in PBST containing 1% BSA, Santa Cruz Biotechnology) was added to each well and incubated at 37 ° C. for 1 hour. After washing each well 5 times with 200 μL PBST, 2 mg of synthetic substrate ο-phenylenediamine (first grade; Wako Pure Chemical Industries) was added to 5 ml citrate buffer, and H2O2 (special grade; Wako Pure Chemical Industries) was further added to 0. 100 μl of 006% added was placed in each well, and after the reaction within 1-5 minutes at room temperature, the reaction was terminated with 50 μL of 2.5 MH ₂ SO ₄ (Nacalai Tesque), and at 490 nm Absorbance was measured with a xMark ™ microplate reader (BioRad Laboratories).

4). Measurement of total serum IgE, IgA, IgG Total IgE, IgG and IgA levels were quantified using an ELISA kit (manufactured by eBioscience) according to the manufacturer's instructions.

5. Measurement of total IgA and LHE-specific IgA in feces Fecal pellet extracts were prepared as follows. That is, 100 mg of fecal pellet was mixed with 1 mL of PBS and incubated overnight at 4 ° C. The pellet was then vortexed for 5 minutes. After centrifuging the mixture (4,000 × g, 15 minutes), the supernatant was collected and stored at −30 ° C. Procedures for detection of total IgA and LHE specific IgA were performed as described above.

  There was no difference in body weight between the three groups.

  As a result of confirming the production of LHE-specific IgE, IgA, IgG1, and IgG2a in serum (FIGS. 6 to 8), the following was found. In FIGS. 6 and 7, the data is shown as the mean ± standard error of 10 mice, the asterisk represents p <0.05, and the cross represents p <0.10. In FIG. 8, the data is shown as the mean ± standard error of 7 mice, the asterisk represents p <0.05, and the cross represents p <0.10.

  It was suggested that when a 1% boll thistle extract was given by diet, there was a tendency to suppress the production of antigen-specific IgE. (FIG. 6A)

  It was suggested that when a 1% bollworm extract was given by diet, the production of antigen-specific IgA was promoted (FIG. 6B).

  It was considered that the production of the antigen-specific IgG1 was not affected when the diet was fed with 1% boar thistle extract (FIG. 6C).

  It was considered that the production of the antigen-specific IgG2a was not affected when the diet was given a 1% bollworm extract (FIG. 6D).

  In terms of Th balance, no change in the IgG1 / IgG2a ratio was observed, suggesting that IgG does not shift to Th2 (immunosuppression).

In addition, it has been clarified that administration of 1% Hazel Thistle extract tends to suppress the production of antigen-specific IgE, but does not suppress total IgE. It was revealed that administration of 1% bollworm extract promotes antigen-specific IgA production in serum but does not promote total IgA. The total IgG1 in the serum of the group of thistle was comparable to that of the positive control LHE sensitized group and the negative control non-sensitized group. On the other hand, a significant increase was observed in the LHE-sensitized group compared to the non-sensitized group. (FIGS. 6 and 7)
This is considered to mean that thistle has an action of suppressing allergy.

  It was suggested that administration of the 1% boll thistle extract promotes the production of fecal antigen-specific IgA. It was revealed that administration of the 1% boll thistle extract promotes the production of fecal antigen-specific IgA, but does not promote total IgA. (FIG. 8). This is considered to mean that thistle has an effect of increasing mucosal immunity.

(Test Example 3) Antibody production evaluation using spleen lymphocytes Using a mouse sensitized with egg white ovalbumin (OVA), lymphocytes were isolated from the spleen, which is an antibody-producing tissue, and extracted with ethyl acetate. The effect on antibody production was evaluated. Furthermore, the Th1 / Th2 balance was examined for antiallergic effects.
1. Animals and Antigen Five-week-old female BALB / c mice (15-20 g body weight) were purchased from Japan SLC (Hamamatsu, Shizuoka Prefecture, Japan). Animals were maintained at 24 ± 3 ° C. and housed in a room with a 12 hour light / dark cycle. The experimental design followed the guidelines for animal experiments approved by the Animal Experiment Committee of Kochi Prefectural University. The antigen was egg white ovalbumin (OVA), which was purchased from Wako Pure Chemical.

2. Feeding, sensitization and sampling Mice were given free access to water and MF diet (Oriental Yeast Industry).

  Mouse sensitization was performed by performing a total of 3 celiac immunizations at weekly intervals. Specifically, 0.2 mL of PBS containing 50 μg of OVA and 4 mg of aluminum hydroxide was intraperitoneally administered. To assess the status of antibody production, blood was obtained from the orbital vein of mice on day 27 under light anesthesia. The spleen was taken out from the mice where antibody production was confirmed.

  First, 3 mL of RPMI 1640 medium was placed in a 5 mL tube, and the removed spleen was immersed. Thereafter, 5 mL of RPMI 1640 medium was placed in a 5 mL dish, and the spleen was ground with two frosted glasses. A cell suspension was made in a 50 mL centrifuge tube using a nylon mesh (Gliner: 70 μm mesh), and centrifuged at 400 × g for 5 minutes. The supernatant after centrifugation was removed, and 10 mL of fresh RPMI 1640 medium was added and suspended. This operation was repeated twice. Thereafter, 10 mL of RPMI 1640 medium was added to make a cell suspension.

  4 mL of Lympolyte (Cosmo Bio) was placed in another 15 mL centrifuge tube, and the cell suspension was gently overlaid thereon, and centrifuged at 1000 × g for 30 minutes. Lymphocytes collected in the intermediate layer were removed with a pipette and transferred to a 15 mL centrifuge tube, and then 10 mL of fresh RPMI 1640 medium was added and suspended. Further, it was centrifuged at 350 × g for 5 minutes. The supernatant after centrifugation was removed, and 10 mL of fresh RPMI 1640 medium was added and suspended. This operation was repeated twice. Thereafter, 10 mL of RPMI 1640 medium was added to make a cell suspension.

The thus obtained spleen lymphocyte solution (2 × 10 ⁶ cells / ml) was seeded in a 24-well plate by 0.5 ml each of 4 × 10 ⁶ cells / ml spleen lymphocyte solution. Add 0.5 ml of double thistle extract (0.2, 2, 10, 20, 100, 200, 1000 mg / ml) of the concentration added in the medium and add 2.5 mg / mL allergen (OVA) 10 μL (25 μg of allergen) was added to stimulate the antigen. The spleen lymphocytes were cultured for 72 hours.

  Thereafter, the amounts of OVA-specific antibodies IgE, IgG1, and IgG2a contained in the culture supernatant were measured. The measurement results are shown in FIG. Furthermore, the ratio of Ig2a / IgG1 obtained from the measurement results is shown in FIG.

  From this result, it was found that the antibody titers of IgE and IgG1 decreased in a concentration-dependent manner. Since the antibody titer related to allergy has decreased in the spleen where antibody production is active, the Hazel Thistle extract may suppress allergies in the spleen lymphocytes, which are the center of antibody production, in addition to the amount of antibody in the serum. It was suggested.

  With respect to the Th1 / Th2 balance, it can be seen that the Th1 value increases depending on the concentration of the thistle extract, and Th1 / Th2 is in a well-balanced and good state. This also suggests that thistle extract may suppress allergies.

(Test Example 4)
RBL-2H3 cells prepared in the same manner as in Test Example 1 were seeded at a density of 6.0 × 10 ⁴ cells / well in a 96-well black microplate and cultured at 37 ° C. overnight. Cells were then washed twice with PBS. The cells were sensitized with 50 ng / mL DNP-specific IgE (anti-DNP-IgE antibody, Sigma-Aldrich) for 2 hours. Thereafter, the cells were washed twice with PBS and then reacted with Fluo-3AM (acetoxymethyl) for 1 hour. After the reaction, the plate was further washed twice with PBS, 100 μl sample dissolved in PBS was added and allowed to react for 10 minutes. Thereafter, 10 μl of DNP-HSA adjusted to 0.625 μg / ml with MT buffer was added, and immediately, absorbance at 485 nm and 538 nm was measured with a fluorescence / luminescence microplate reader (Thermo).
Here, as samples, the extract of the leaflet prepared in Example 1 (the concentration was adjusted to 1 μg / ml and 10 μg / ml), the blank (MT buffer not containing DNP-HSA), and the control (therelet of thistle) PBS without) was used.

  Data are expressed as mean ± standard deviation (n = 4). Compared with the control, when a cattle thistle was added, a remarkable inhibitory effect was observed (FIG. 11).

(Test Example 5)
Anti-DNP IgE antibody (Sigma) adjusted to 1 μg / 20 μL (50 μg / mL) with PBS was intradermally administered to the auricle of an anesthetized BALB / c mouse. After 23 hours, the sample solution adjusted to 5% with PBS was forcibly administered orally (5 mg / 100 μg / animal (20 g); 250 mg / 5 mL / kg). As a control, PBS was forcibly administered orally in the same manner. One hour later, DNP-HSA (Sigma) adjusted to 100 μg / 200 μL (500 μg / mL) with PBS containing 1% Evans blue was intravenously administered. Thirty minutes after administration of DNP-HAS, the thickness of both ears was measured three times with a thickness gauge, and then the mice were sacrificed by cervical dislocation and the ears were collected. The collected pinna was incubated with 1 mL of formamide at 63 ° C. for 48 hours to confirm that the pigment was extracted from the ear, the ear was removed from the test tube, and the absorbance of the supernatant was measured at 610 nm.

The result is shown in FIG. The description in FIG. 12 means the following.
Uncontrollable control: No antibody sensitization-Antigen administration Anti DNP-HSA: Antibody sensitization Yes-Antigen administration Anti DNP-HSA-CMM: Antibody sensitization Yes-Hazel thistle administration-Antigen administration
Ear swelling refers to the result of pinna thickness measurement.
Ear dye intensity is expressed as a relative value when the dye amount of the unified control is 1.

  It can be seen that the oral administration of the sea thistle suppresses the swelling of the auricle and the amount of pigment.

(Prescription example)
The unit of the numerical value in each of the following prescription examples is indicated by “w / w%” where the total amount is 100.

Formulation Example 1 Composition (Tablet) for Allergy Prevention or Treatment
Using the extracts obtained in the above-mentioned Examples, tablets for preventing or treating allergies having the following composition can be produced by a conventional method. Here, a sample such as that obtained in Example 1 is used as the extract of the thistle.

(Prescription Example 2) Composition for preventing or treating allergy (granule)
Using the extract obtained in Example 1 above, the following composition is mixed and stirred to prepare uniformly, and granules can be obtained with a fluidized bed granulator. Granules can be taken with beverages.

(Prescription Example 3) Beverage for preventing or treating allergy Using the extract obtained in Example 1 above, a beverage for preventing or treating allergy having the following composition can be produced by a conventional method.

(Formulation Example 5) Tea (Formulation Example): Powdered tea
Ingredients Blending amount Thistle extract (powder) 22
Fragrance 3
Dextrin 75
Total 100

(Prescription example): Powdered tea
Ingredients Blending amount Thistle extract from hot water (powder) 22
Fragrance 3
Dextrin 75
Total 100

(Prescription example)
900 ml of water was heated to 60 ° C., and 30 g of thistle leaves or stems or a mixture of leaves and stems was added thereto and extracted for 6 minutes. The residue was removed with a 30-mesh strainer and clarified by filter paper filtration (industrial filter paper No. 26: manufactured by ADVANTEC, collected particle size = 3 μm) to obtain 760 ml of tea.

(Prescription example)
A chopped portion of 100g of thistle leaf and stem portion was stirred and roasted at 100 ° C for 1 hour by a conventional method.
This composition was packed in a thin paper bag having good permeability to obtain a bag portion such as a tea pack. The senna stem part contained per one bag part is 1.2 g of a pulverized product.

Claims (10)

  A composition for immunomodulation containing a toothpaste or a toothpaste extract.   The composition for immunoregulation according to claim 1, which is a composition for preventing and / or improving allergy.   The composition for immunoregulation according to claim 1, which is a mucosal immunostimulatory composition.   The composition for immunomodulation according to any one of claims 1 to 3, wherein the sea thistle or the sea thistle extract contains silsimarin and / or silsimaritin.   The said thistle is derived from 1 or 2 or more selected from the group which consists of the whole grass or leaf part, stem part, root part, seed, and flower part of a sea thistle. Composition for immunomodulation.   The composition for immunoregulation according to any one of claims 1 to 5, wherein the extract of the sea thistle is an ethyl acetate extract of the sea thistle.   A composition for immunomodulation comprising silsimarin and / or silsimaritin.   The composition for immunomodulation according to any one of claims 1 to 7, which is administered orally, intravenously, by inhalation or transdermally.   The composition for immunoregulation of any one of Claims 1-8 which is a food / beverage product, a quasi-drug, or a pharmaceutical.   The composition for immune regulation according to any one of claims 1 to 9, wherein the food or drink is a tea beverage.