JP6882730B2 - Immunoregulatory composition containing Cirsium maritimum extract - Google Patents

Description

The present invention relates to a naturally derived product which has an immunomodulatory function such as an allergy prevention and / or ameliorating effect and is useful as a food or drink, a drug, or a quasi drug.

Currently, it is said that about one-third of the Japanese people develop some allergic symptoms. Allergies are classified from immediate type allergy (type I) to delayed type allergy (type IV) according to the onset mechanism, but in recent years, cases of immediate type allergy represented by pollinosis, bronchial asthma, atopic dermatitis, etc. The number is skyrocketing. Mast cells and basophils are involved in the type I allergic reaction, and IgE bound to the cell surface is activated by an antigen to transmit a signal downstream, and cells such as histamine and proteolytic enzymes The contents of the inner granules are released. This phenomenon is called degranulation. In order to suppress the onset of such type I allergy, it is important to suppress degranulation from mast cells and basophils.

In addition, recent studies have revealed that there is a strong relationship between intestinal immunity and allergies. In particular, IgA secreted from Peyer's patches, which is a typical lymphoid tissue of the intestinal tract, prevents the adhesion of bacteria and viruses to the mucosal surface, eliminates foreign substances that capture foreign antigens and excrete them from the body, and prevents the onset of allergies due to foreign proteins. It has such actions and plays an important role in mucosal immune function. Therefore, promoting IgA secretion can be expected to have effects such as enhancing mucosal immune function and preventing infectious diseases and allergic diseases. Therefore, development of a food material having an IgA secretion promoting action is desired.

So far, allergy-suppressing ingredients have been found in tea catechin, perilla extract, and sweet tea, and daily intake of foods with these functions can not only reduce allergic symptoms but also improve allergic constitution. It is expected (Non-Patent Documents 1 to 3).

Effect of concentrated tea catechin beverage on allergic rhinitis symptoms (medicine and pharmacy 59 (2), 207-21,2008) TNF production inhibitory effect and antiallergic activity of perilla extract (Japanese Society of Allergology Allergy 41 (8), 1035, 1992-08-30) About anti-inflammatory and anti-allergic active ingredients of sweet tea extract: Food (Japan Society for Bioscience and Biotechnology, Japan Society for Bioscience and Biotechnology 69 (extra edition), 165, 1995-07-05)

It is desired to develop a highly safe and effective immunomodulatory composition.

Therefore, an object of the present invention is to provide an excellent immunomodulatory composition derived from a natural product, which is highly safe and can be applied to foods and drinks.

As a result of diligent studies to solve the above problems, the present inventors have found that Cirsium maritimum, Cirsium maritimum extract, or purified product of Cirsium maritimum extract has an allergy prevention and / or ameliorating effect and a mucosal immunostimulatory effect. We have found that it has an immunomodulatory function such as, and have completed the present invention.

That is, the present invention
[1] An immunomodulatory composition containing Cirsium maritimum or Cirsium maritimum extract;
[2] The immunomodulatory composition according to Item 1, which is a composition for preventing and / or improving allergies;
[3] The immunomodulatory composition according to Item 1, which is a mucosal immunostimulatory composition;
[4] The immunomodulatory composition according to any one of Items 1 to 3, wherein the Cirsium maritimum or Cirsium maritimum extract contains silcimarin and / or silcimarintin;
[5] Item 1 to any one of Items 1 to 4, wherein the Cirsium maritimum is derived from one or more selected from the group consisting of whole grass or leaves, stems, roots, seeds, and flowers of Cirsium maritimum. The immunomodulatory composition described;
[6] The composition for immunoregulation according to any one of Items 1 to 5, wherein the Cirsium maritimum extract is an ethyl acetate extract of Cirsium maritimum.
[7] An immunomodulatory composition containing silcymarin and / or silcimarintin.
[8] The immunomodulatory composition according to any one of Items 1 to 7, which is administered orally, intravenously, inhaled or transdermally;
[9] The immunomodulatory composition according to any one of claims 1 to 8, which is a food or drink, a quasi-drug, or a pharmaceutical product;
[10] The immunomodulatory composition according to any one of Items 1 to 9, wherein the food or drink is a tea beverage;
Regarding.

The immunomodulatory composition of the present invention contains an active ingredient obtained from naturally occurring Cirsium maritimum and is highly safe. Such a safe immunomodulatory composition can be used for foods and drinks, pharmaceuticals or quasi-drugs having, for example, an antiallergic effect and few side effects.

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

The immunomodulatory composition of the present invention contains Cirsium maritimum Makino of the Asteraceae family or an extract thereof. Here, the extract also includes a purified product that has been purified to a single component or a fraction close to a single component.

Cirsium maritimum is a perennial plant that grows on temperate coasts. In Japan, it is distributed west of Chiba prefecture to Kyushu. It is often found in sandy areas, gravel areas, and grasslands. The roots are used for food.

In the present invention, Cirsium maritimum can be used as it is, but in that case, the preparation method thereof is not particularly limited. Cirsium maritimum is a whole plant or part of Cirsium maritimum, especially leaves, stems, roots, seeds, and flowers, one type at a time or a combination of two or more of them as a dried product, or the dried product is cut as appropriate. , Can be prepared as a crushed or crushed powder. The dried product of Cirsium maritimum or the crushed product thereof can be used, for example, by boiling in boiling water. The crushed or crushed Cirsium maritimum can also be prepared by roasting if necessary.

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

When Cirsium maritimum extract is used in the present invention, the extraction method is not particularly limited. Cirsium maritimum extract is a whole plant or part of Cirsium maritimum, especially leaves, stems, roots, seeds, flowers separately from a dried product of one type or a combination of two or more thereof, or a dried product thereof as appropriate. It is extracted with a solvent from the powder that has been cut, crushed or crushed. Cirsium maritimum is easily available from native ones, and commercial products can also be used.

When the whole plant or a part of Cirsium maritimum is dried, it can be carried out by using a sun-drying, air-drying or a dryer, but not limited to. When sun-drying is performed, 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, and more preferably 2 days or more. When a dryer is used, it is generally under drying conditions of 100 ° C. or lower, preferably 40 ° C. or lower, for example, a rotary dryer, a hot air dryer, a heat transfer dryer, a vacuum dryer, a vacuum freeze dryer, etc. It is possible to use a cold air dryer, a vibration dryer, a filtration dryer, a vacuum stirring dryer, or the like.

When Cirsium maritimum extract is used as an immunomodulatory composition, the extract obtained by extracting Cirsium maritimum from a solvent may be used as it is, or an appropriately diluted or concentrated solution may be used as the extract. When obtaining Cirsium maritimum extract, fresh plants can be used, and Cirsium maritimum that has been refrigerated, frozen, or dried and stored can be used, or the concentrate of Cirsium maritimum extract can be used in an appropriate solvent such as water. It can also be used after being dissolved or diluted. The concentrate of Cirsium maritimum extract is not limited, but liquid, paste-like, or muddy-like ones can be used.

Examples of the solvent used for extracting Cirsium maritimum include water, an organic solvent, and a hydrous organic solvent. By using an appropriate extraction solvent, a water extract, an organic solvent extract or a water-containing organic solvent extract of Cirsium maritimum can be obtained. These extraction solvents are, but are not limited to, 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 a higher effect of the present invention. When ethyl acetate is used, the concentration 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 Cirsium maritimum used for extraction is not limited, but includes, for example, leaves, stems, roots, seeds, flowers, etc., and from the viewpoint of obtaining a higher effect of the present invention, the leaves, flowers, or the like. A combination of these is preferable, and a leaf portion is more preferable.

The extraction method is not particularly limited, but for example, whole or a part of Cirsium maritimum is cut, crushed by a known method such as a mixer, added with an extraction solvent and stirred, and treated at room temperature or heating at a constant extraction time. After that, a method of separating and extracting Cirsium maritimum extract from the extraction supernatant can be mentioned. Further, the extraction method may be a method in which Cirsium maritimum is immersed in an extraction solvent and then allowed to stand at room temperature or under heating conditions.

The crushing conditions are not limited, but if the whole plant of Cirsium maritimum or a part of the cut material is a large scale such as 1 kg to 10 kg, a large vertical cutter mixer or the like can be used, and tens of g to several hundreds can be used. In the case of a small scale of g, a household mixer or the like can be used. For example, when applying crushing conditions on a large scale, the crushing time is tens of seconds to several minutes, and the rotation speed of the mixer is 10 to 3000 rpm, although it is appropriately changed depending on the hardness of the raw material, the water content, etc. It is possible to do.

Further, the extraction conditions can be applied to ordinary conditions, and the extraction conditions are not limited, but a method of immersing the dried product of Cirsium maritimum in a solvent at, for example, 3 to 100 ° C., heating at reflux or microwave heating can be adopted. it can. The extraction temperature is not limited as long as an appropriate extraction amount can be obtained, but for example, when ethyl acetate is used, it can be 20 to 100 ° C., preferably 22 to 80 ° C., more preferably 25 to 60 ° C. preferable. The extraction temperature can be, for example, 20 to 100 ° C., preferably 50 to 100 ° C., more preferably 70 to 100 ° C. when water is used. The extraction time is not limited as long as an appropriate extraction amount can be obtained, but can be, for example, 5 minutes or more and 14 days or less, preferably 10 minutes or more and 7 days or less, and more preferably 15 minutes or more and 5 days or less. The extraction is usually carried out under normal pressure, but it can also be carried out under pressure. The amount of the solvent added can be about 1 L to 100 L with respect to 1 kg of the dry weight of Cirsium maritimum.

It is also possible to increase the extraction efficiency by adding an acid, alkali, or enzyme to Cirsium maritimum 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 an extraction container containing a cut or crushed product of Cirsium maritimum and an extraction solvent, the method of rotating the extraction container is a magnetic method. Alternatively, a method of installing the mixture in a mechanical stirring device for mixing, a method of shaking the extraction container, and the like can be mentioned.

In the stirring operation, it is possible to increase the extraction efficiency by causing vibration by ultrasonic treatment. When performing ultrasonic treatment, there is no limitation, but for example, when the whole plant or a part of the Hama-Zami is cut on a large scale such as 1 kg to 10 kg, a commercially available ultrasonic generator is used to cut or crush the Hama-Zami. An ultrasonically treated product can be obtained by installing an extraction container containing the 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 of g to several hundred g, 40 kHz. An ultrasonically treated product can be obtained by treating with the ultrasonic waves of the above for 60 minutes. The temperature condition of the ultrasonic treatment is not limited as long as an appropriate extraction amount can be obtained, but can be 2 ° C to 100 ° C, preferably 10 ° C to 70 ° C. As the ultrasonic generator for large scale, for example, UT-12 manufactured by Shinmeidai Kogyo Co., Ltd. can be used, and as the ultrasonic generator for small scale, for example, US-3R manufactured by AS ONE Co., Ltd. can be used. Can be used.

In order to increase the extraction efficiency, it is also possible to perform multi-step extraction using the same type or a plurality of types of extraction solvents. In the case of multi-step extraction, the same or more kinds of extraction solvents are further added to the residue obtained in the first step extraction, and after heating at room temperature or heating, the extract of Cirsium maritimum is separated and extracted from the extraction supernatant. be able to.

In the microwave heating, conditions such as the output of the microwave irradiation device, the wavelength of the microwave, and the irradiation time can be appropriately set within a range in which 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 a dried product of Cirsium maritimum, it can be processed in 10 seconds to 10 minutes, preferably 30 seconds to 5 minutes.

The extract supernatant can be used as it is as a Cirsium maritimum extract. Furthermore, Cirsium maritimum extract can be separated and extracted from the extract supernatant. In this separation / extraction step, a known method can be adopted, and for example, filtration, centrifugation, suction, squeezing, etc. can be performed.

In the case of separation and extraction by filtration, for example, membrane filtration can be performed, but not limited to. When performing membrane filtration, for example, the temperature condition can be 2 ° C. to 70 ° C., preferably 10 ° C. to 40 ° C., and the membrane pore diameter can be 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. When centrifuging, for example, the temperature condition can be 2 ° C. to 70 ° C., preferably 10 ° C. to 40 ° C., and the rotation speed is 1000 rpm to 10000 rpm, preferably 1500 rpm to 8000 rpm, and 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 squeezing, a squeezing machine can also be used, and a known device can be used as the squeezing machine, and examples thereof include a pneumatic squeezing machine and a screw type squeezing machine.

Cirsium maritimum extract can be refined by further purifying it before and after dilution or concentration. In addition to the above-mentioned extraction with a solvent, a chromatographic method, an ion exchange chromatograph method, or the like known to those skilled in the art can be used alone or in combination for the purification treatment.

When the chromatograph method is used, for example, either column chromatography using a normal phase or reverse phase carrier or an ion exchange resin, high performance liquid chromatography, thin layer chromatography, centrifugal liquid chromatography, or the like, or A method of using them in combination can be mentioned. When the chromatographic method is used, the purification conditions such as the carrier and the elution solvent can be appropriately selected according to various chromatographic methods.

The ratio of the cut or ground product of Cirsium maritimum to the extraction solvent is such that, from the viewpoint of obtaining higher extraction efficiency, for example, when a hydrous organic solvent extract or a hydrous extract is obtained, the ratio is relative to 1 L of the solvent. The cut or ground product of Cirsium maritimum can be 5 g to 1 kg, preferably 10 g to 500 g, and more preferably 20 g to 200 g. When the ethyl acetate extract is obtained, for example, the amount of the cut or ground product of dried Cirsium maritimum can be 5 g to 300 g, preferably 10 g to 200 g, and more preferably 20 g to 100 g with respect to 1 L of ethyl acetate.

In the present invention, the content of Cirsium maritimum and Cirsium maritimum extract is preferably 0.000001 to 100 w / w% in terms of solid content, based on the total amount of the immunomodulatory composition, from the viewpoint of obtaining a higher effect of the present invention. 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 Cirsium maritimum and Cirsium maritimum extract is 0.000001 to 100 w / v% in terms of solid content based on the total amount of the immunomodulatory composition from the viewpoint of obtaining a higher effect of the present invention. It can be 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 Cirsium maritimum is 0.000000001 to 5 w / w% in terms of solid content, preferably 0.000000001 to 5 w / w%, based on the total amount of the immunomodulatory composition, from the viewpoint of obtaining a higher effect of the present invention. It can be 0.00000001-3w / w%, more preferably 0.000000001-2w / w%.

Here, in the present invention, Cirsium maritimum extract or a further purified product may contain silcimarintin (CAS Number: 6601-62-3) or silcimarin (CAS Number: 13020-19-4) having the following chemical formula. More preferably, the product obtained by purification from Cirsium maritimum extract is silcimarintin and / or silcimarin.

[Use]
The present inventors have newly found that Cirsium maritimum or its extract or purified product, silcimaritin and / or silcimarin, has an excellent immunomodulatory effect. Cirsium maritimum is naturally derived and has been edible since ancient times, making it suitable for safe and effective immunomodulatory applications.

Here, in the present specification, "immunomodulation" is a remorse including anti-allergy and immunostimulation. Immunostimulation includes, but is not limited to, mucosal immunostimulation.

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

The antiallergic preventive and / or ameliorating composition referred to in the present invention particularly prevents and prevents allergic symptoms such as sneezing, itchiness, runny nose, tears, dermatitis, urticaria, asthma, and diseases such as inflammatory arthritis. / Has an effect on improvement. The antiallergic prevention and / or amelioration of the present invention is particularly effective for type I allergies. 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 an effect of suppressing β-hexosaminize release from cells, that is, an effect of suppressing basophil degranulation.

Further, in the present specification, "immune activation" means to include general immunostimulation and mucosal immunity activation in addition to antiallergic action.

The mucosal immunostimulatory composition referred to in the present invention is effective in preventing infection or adverse effects caused by pathogenic bacteria, parasites, pathogenic antigens, viruses, or ameliorating such infectious diseases or adverse effects. In particular, it is useful for alleviating indirect effects as well as direct effects due to deterioration of gastrointestinal immune function such as intestinal tract and bacterial gastrointestinal infection.

Mucosal immune activation can prevent, for example, a decrease in the immune function of the defense system in the gastrointestinal mucosa. This includes prevention and alleviation of symptoms such as fever, vomiting, diarrhea, and abdominal pain due to gastrointestinal infection due to colonization of microorganisms in the gastrointestinal tract and proliferation. Furthermore, mucosal immunostimulation is also useful for prevention and alleviation of bacterial diarrhea and the like. It also helps to strengthen resistance in infants, the elderly, and those who are weak. As described above, mucosal immunostimulation makes it possible to neutralize bacteria and viruses, suppress bacterial adhesion to tissues, and suppress allergies caused by food antigens.

Such anti-allergic action and immunostimulatory action based on the immunostimulatory action can be verified as a tendency of a decrease in the amount of IgE in serum and an increase in the amount of IgA in feces.

[Food and drink]
The immunomodulatory composition of the present invention can be blended as one component of foods and drinks. These foods and drinks are not limited, but can also be used as foods and drinks having an antiallergic action and an immunostimulatory action, particularly a mucosal immunostimulatory action. Can be provided to. Alternatively, these foods and drinks can be provided as functional beverages or functional foods, nutritional functional beverages or nutritional functional foods, and these functional foods and drinks can be provided by other medical institutions. , Drug stores, convenience stores, supermarkets, department stores, etc. Functional foods and drinks are foods and drinks that have medicinal properties permitted and designated by the national government and public organizations, or foods and drinks that display functionality based on the content that the company has notified the national government of the prescribed effects. Means. Examples of functional foods include foods for specified health use, foods with nutritional function, foods with functional claims, foods for the elderly, health supplements (balanced nutritional foods, supplements) and the like. It also includes foods and drinks including packages and containers, package inserts, instruction manuals, etc. that indicate pharmaceutical efficacy or functionality. Foods and drinks that have the same efficacy or functionality as pharmaceuticals are also included in the application form to the national government.

In the case of labeling for the purpose of preventing or improving allergies, there is no limitation, but for example, in addition to labeling for those with allergies and those who are concerned about pollen, the nose is toned, the eyes are toned, etc. Labels such as alleviating discomfort in the eyes and nose, health of the eyes, health of the nose, and alleviation of tiredness of the eyes can be mentioned.

In the case of a display for the purpose of immunostimulation, the display is not limited, and examples thereof include a display for a person with weak physical strength, a display for a weak constitution, and a display for adjusting the condition of the abdomen.

Foods include all kinds of foods, such as grains, potatoes, seafood, meats, eggs, fats and oils, milks, vegetables, legumes, fruits, sugars, seaweeds, confectionery, seasonings. Kind, cooked processed foods and the like.

The seasoned processed foods are not limited, but for example, processed marine products such as chikuwa and kamaboko; processed livestock products such as ham and sausage; sweets such as cookies, biscuits, snacks, chocolates and cakes; buckwheat noodles, udon noodles and raw noodles. , Chinese noodles, noodles such as pasta; bread such as bread and sweet bread; fermented processed foods such as natto and miso; soybean foods such as tofu and okara; pickles such as lightly pickled and rice bran pickles, marine products, processed meats, 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.

Beverages include, but are not limited to, all beverages, such as beverages made from Hama-Zami itself, roasted dried products as a substitute for tea leaves, fruit juice beverages, 100% fruit juice beverages, low fruit juice beverages, and fruit meat. Beverages, vegetable juices, flavored beverages, fruit beverages for dilution, and other fruit beverages; carbonated beverages; coffee, coffee beverages, soft beverages with coffee, cocoa beverages, tea, green tea, matcha, Karyu tea, barley tea, roasted tea, and other favorite beverages; Vinegar drinks; soft drinks such as sports drinks; milk; milk drinks; milk drinks; lactic acid drinks; lactic acid bacteria drinks; soybean drinks such as soymilk and prepared soymilk; alcoholic drinks such as beer, sake, shochu, liqueurs, and wine; taurine , Royal jelly, nutritional beverages containing amino acids, vitamins, minerals, iron and the like.

There is no limitation on the time when the present invention is applied to foods and drinks, but for example, it can be applied before and after a processing step, a cooking step, a heating step, a storage step and the like in a food and drink manufacturing process. For example, in a processing step or a cooking step, the raw material may contain the immunomodulatory composition of the present invention. The application method can be appropriately changed depending on the type of food and drink, the form of the raw material, and the like, and various methods such as mixing, addition, coating, spraying, and dipping can be adopted.

When the immunomodulatory composition of the present invention is applied to a beverage product, it is particularly preferable to use a tea beverage in one embodiment. Although not limited, the dried leaf and / or stem of Cirsium maritimum is cut by a known method and then crushed or crushed into a powder. It is possible to sell it as a tea beverage in the form of powdered Cirsium maritimum. If necessary, Cirsium maritimum powder is roasted. Cirsium maritimum powder can also be sold as a tea beverage in a roasted state. Roasted Cirsium maritimum is drunk by boiling it in boiling water.

In addition, the beverage can be used as a mixture of other tea beverages and Cirsium maritimum extract, or diluted with water or the like and used as a juice, a drink, or the like. Further, it can be used as a concentrated product of the mixture, powdered tea powdered by spray drying or the like, freeze-dried instant tea or the like. In that case, for example, hot water can be poured into powdered tea or instant tea to drink. Further, for example, a mixture of a tea preparation such as green tea or black tea and a cirsium maritimum preparation can be used as it is as tea leaves for beverages. In that case, hot water can be poured into the mixed tea leaves of the tea preparation and the cirsium maritimum preparation in the same manner as general Japanese tea and black tea drinks.

When the immunomodulatory composition of the present invention is applied to a food, it is desirable to use a green juice food in one embodiment. Although not limited, the green juice food here is a fine treatment such as shredding, crushing, and grinding the leaves and / or stems of raw Cirsium maritimum or dried Cirsium maritimum. The juice is obtained by pressing the raw material or the micronized product, and its shape 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 powdery material, the raw material may be crushed as it is dried without being shredded. Examples of the drying method include natural drying, heat drying, spray drying, etc. However, when the freeze-drying method is used, it is not deteriorated by heat, is easily crushed, and has excellent palatability. Therefore, it is preferable. It is also preferable from the viewpoint of storage stability.

In addition, the leaves and stems of Cirsium maritimum can be used in combination with other green juice raw materials. These green juice raw materials are processed in the same manner as in the above case to obtain green juice foods. The treatment may be carried out by mixing the leaves and stems of Cirsium maritimum from the raw material stage, in the middle stage, or by mixing separately prepared green juice foods. The material of green juice other than the leaves and stems of Hama-Azami in the present invention is not particularly limited, but for example, kale, broccoli, cabbage, Japanese mustard spinach, radish leaf, cresson, nazuna, young barley leaf, tomorrow's leaf, and auction. , Parsley, carrot, celery, asparagus, spinach, Japanese mustard spinach, green tea, perilla, spring chrysanthemum, Japanese mustard spinach, Japanese mustard spinach, Japanese mustard spinach, mulberry, Japanese mustard spinach, Japanese mustard spinach, Japanese mustard spinach, etc. Can be combined with leaves and / or stems. In the present invention, when the leaves and stems of Cirsium maritimum are combined with the other green juice raw materials to prepare a green juice food, the components derived from the leaves and / or stems of Cirsium maritimum in the green juice food are all as a dry residue. It is desirable that the amount of dry residue is 10 w / w% or more. Within this range, nutritional components derived from Cirsium maritimum are effectively ingested, and the effect of improving the palatability of green juice foods is excellent.

When the immunomodulatory composition of the present invention is applied to foods and drinks, an auxiliary agent used in ordinary foods and beverages can be appropriately blended as long as the effects of the present invention are not impaired. Such auxiliaries include, for example, glucose, sucrose, fructose, oligosaccharide, candy, maltose, multitose, xylitol, sorbitol, aspartame, sclarose, stebioside, rubusoside, corn syrup, lactose, citric acid, tartrate, malic acid. , Citric acid, lactic acid, L-ascorbic acid, dL-α-tocophenol, sodium erythorbicate, glycerin, glycerin fatty acid ester, propylene glycol, polyglycerin fatty acid ester, sucrose fatty acid ester, propylene glycol fatty acid, sorbitan fatty acid ester, ester Examples include arabic gum, casein, pectin, gelatin, agar, carrageenan, vitamin Bs, vitamin Cs, nicotinic acid amide, calcium pantothenate, calcium salts, amino acids, pigments, fragrances, preservatives and the like.

The content of Cirsium maritimum or its extract when the immunomodulatory composition of the present invention is applied to foods and drinks is not limited, but in terms of solid content, it is generally 0. per day for humans and animals. It is 0000001 to 5000 mg / kg body weight, preferably 0.01 to 200 mg / kg body weight, and more preferably 0.1 to 100 mg / kg body weight.

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

[Pharmaceuticals, quasi-drugs]
When the composition for immunomodulation of the present invention is a drug or a quasi-drug, it can be formulated into an appropriate form and administered to humans or animals in any administration form. The administration form is not limited, and examples thereof include oral, transdermal, enteral, transmucosal, and injection. From the viewpoint of remarkably exerting the effect of the present invention, the administration form is preferably oral administration.

When the immunomodulatory composition of the present invention is orally administered, the immunomodulatory composition of the present invention may be, for example, tablets, capsules, granules, syrups, powders, films, drops, jellies, etc. It is formulated into a semi-solid syrup-like dosage form by a known method. The immunomodulatory composition of the present invention can be ingested without water in the case of oral administration due to a film-like, drop-like, jelly-like, semi-solid pudding-like dosage form or the like. Alternatively, the dried powder of Cirsium maritimum or its extract or purified product can be orally administered as a crude drug or a Chinese herbal medicine preparation.

In the case of parenteral administration, for example, by a method known for the dosage form such as subcutaneous injection, intravenous injection, intramuscular injection, intrathecal injection, transdermal absorbent, inhalant, suppository, eye drops, nasal drops, etc. It can be formulated.

As one aspect of the dosage form, known drug delivery for the purpose of enhancing the stability and bioavailability of Cirsium maritimum or its extract or its purified product, or improving the patient's medication compliance, or for a combined purpose. The system can be used to deliver the immunomodulatory compositions of the invention to the site of absorption.

The drug delivery system utilizes, for example, but not limited to, polymers such as cellulose, dextran, starch, polyvinyl alcohol, acetylated or methacrylicated polymers, polylactic acid and polyglycolic acid and their block copolymers, polyethylene glycol and the like. Methods, methods using transport proteins such as albumin, and other methods using micelles, liposomes, microspheres, nanoparticles, composite emulsions, dendrimers and the like can be mentioned. These drug delivery systems are used not only for the purpose of transporting the immunomodulatory composition of the present invention to a target site such as an absorption site, but also for the purpose of release control for releasing the immunomodulatory composition of the present invention in a timely manner. Can be used.

The immunomodulatory composition of the present invention contains excipients, lubricants, binders, disintegrants, etc. in solid formulations and solvents in liquid formulations, as long as the effects of the present invention are not impaired. It is possible to appropriately add a solubilizing agent, a suspending agent, an isotonic agent, a buffering agent, a pain-relieving agent and the like.

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

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, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and hydroxypropyl starch. , Methyl cellulose, ethyl cellulose, shelac, calcium phosphate, polyvinylpyrrolidone and the like.

Examples of the disintegrant include starch, dried starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, carmellose calcium, croscarmellose sodium, carboxymethyl starch sodium, sodium alginate, canten powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, and the like. 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 solubilizing agent 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 glycerin monostearate, and for example, polyvinyl alcohol and polyvinyl. Examples thereof include hydrophilic polymers such as pyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose and hydroxypropyl cellulose.

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

Examples of the buffer include a buffer solution such as phosphate, acetate, carbonate, and citrate.

Examples of the soothing agent include benzyl alcohol and the like.

Examples of preservatives include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.

The effective dose of Cirsium maritimum or its extract when the immunomodulatory composition of the present invention is orally administered is not limited, but is generally 0.00001 per day for humans and animals in terms of solid content. It has a body weight of ~ 5000 mg / kg, preferably 0.01 to 200 mg / kg body weight, and more preferably 0.1 to 100 mg / kg body weight. The effective dose of Cirsium maritimum or its extract when the immunomodulatory composition of the present invention is orally administered is not limited, but is 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.

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 with ethyl acetate from dried ground product 100 g of each dried ground product of Cirsium maritimum leaves and flowers was dispersed in 1000 ml of 100% ethyl acetate, stirred, and extracted at room temperature for 1 week. The extract supernatant was filtered off, the residue was dispersed again in ethyl acetate, heated under reflux, and the extract supernatant was filtered off. The supernatant was sequentially concentrated under reduced pressure, dried under reduced pressure and freeze-dried under a nitrogen gas atmosphere to obtain Extract 1 (4 g) from Cirsium maritimum leaves and Extract 2 (4 g) from Cirsium maritimum flowers.

(Example 2) Extraction method with water from dried crushed product 1.5 L of water was added to 100 g of dried crushed product of Cirsium maritimum leaf, crushed with a mixer, stirred and ultrasonically applied, and the extraction supernatant was filtered and separated. By dehydration, an extract (2 L) was obtained together with water. Further, the mixture was sequentially concentrated under reduced pressure, dried under reduced pressure and freeze-dried under a nitrogen gas atmosphere to obtain Extract 3 (15 g).

(Example 3) Extraction method by squeezing 1 L of water is added to a mixture (1 kg) of fresh leaves and fresh stems of Cirsium maritimum, crushed with a mixer, stirred and ultrasonically applied, and the extracted supernatant is filtered, washed, and subjected to ultrasonic waves. By dehydration, an extract (2 L) was obtained together with water. Further, the mixture was sequentially concentrated under reduced pressure, dried under reduced pressure and freeze-dried under a nitrogen gas atmosphere to obtain Extract 4 (15 g).

(Example 4) Extraction method by squeezing 1 L of water is added to a mixture (1 kg) of fresh leaves and stalks of Cirsium maritimum, crushed with a mixer, stirred and ultrasonically applied, and the extracted supernatant is filtered and dehydrated and then squeezed. By doing so, an extract (1.5 L) was obtained. Further, the mixture was sequentially concentrated under reduced pressure, dried under reduced pressure and freeze-dried under a nitrogen gas atmosphere to obtain an extract (20 g).

(Example 5) Method for producing dry powder Cirsium maritimum leaves and stems (1 kg) were dried overnight in a 40 ° C. cold air dryer and then finely pulverized in a crusher to obtain dried Cirsium maritimum powder (100 g).

(Example 6) Extraction method with hot water from dried crushed product By adding 1.5 L of hot water to 100 g of dried crushed product of Cirsium maritimum leaves and stems obtained in Example 5, and filtering the extraction supernatant. , An extract (2 L) was obtained together with water. Further, the mixture was sequentially concentrated under reduced pressure, dried under reduced pressure and freeze-dried under a nitrogen gas atmosphere to obtain Extract 3 (20 g).

(Example 7) Extraction method from fresh leaves and fresh stems with hot water 1 L of hot water is added to a mixture (1 kg) of fresh leaves and fresh stems of Cirsium maritimum, and the extraction supernatant is filtered to extract the extract together with water. (1.5 L) was obtained. Further, the mixture was sequentially concentrated under reduced pressure, dried under reduced pressure and freeze-dried under a nitrogen gas atmosphere to obtain Extract 3 (15 g).

(Example 8) Fraction separation of Cirsium maritimum extract 500 mg of ethyl acetate extract obtained from the leaves of Cirsium maritimum as in Example 1 was subjected to silica gel chromatography. The conditions are as follows.
-Silica gel: Wakosil C-200, 25g, manufactured by Wako Pure Chemical Industries, Ltd.
-Column size: 20 mmφ x 100 cm
-Eluent: Acetone / Hexane + Methanol (concentration 0w / w% -100w / w%), 500mL each
Under these conditions, 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 sampled. Each was analyzed according to Test Example 1.

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

HPLC was performed under the above conditions, and the peaks that had absorbed ultraviolet rays were separated to obtain 12 mg of the active ingredient (silcimaritin).
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 1. Cell culture RBL-2H3 cells are mixed with 10% (v / v) fetal bovine serum (FBS: Sigma-Aldrich), 100 U / ml penicillin (Nacalai Tesque), and 100 μg / ml streptomycin (Nacalai Tesque). Cultivated in Dalveco modified Eagle's medium (manufactured by Nacalai Tesque, DMEM) containing (manufactured by Nakarai Tesque). At this time, the condition was 37 ° C. in a humidified atmosphere containing _{5% CO 2.}

2. β-hexosaminidase free activity The 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 1 at 37 ° C.} It was cultured in the evening. The cells were then washed twice with PBS. The cells were sensitized with 50 ng / mL DNP-specific IgE (manufactured by Sigma-Aldrich) for 2 hours. After washing the cells with MT buffer, diluted Cirsium maritimum extract sample or purified product was added to MT buffer. Here, the Cirsium maritimum extract sample is prepared by extracting the leaves or flowers of Cirsium maritimum with 100% ethyl acetate in the same manner as in Example 1, and then the concentrated sample is dimethylsulfoxide (DMSO) so as to have a concentration of 10 mg / mL. ), Diluted in MT buffer to a concentration of 0.5% and used. Silcimaritin obtained by purifying from the Cirsium maritimum extract obtained in Examples 8 and 9 was prepared by dissolving the concentrated sample in dimethyl sulfoxide (DMSO) to a concentration of 10 mg / mL in MT buffer. It was diluted to a concentration of 0.5% and used. Similarly, commercially available silcimaritin was also used in the test for reference. After 10 minutes of 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-isooctyl phenyl ether (Triton X100, manufactured by Nacalai Tesque). 1 mM p-nitrophenyl N-acetyl-D-glucosamide (1 mM p-nitrophenyl N-acetyl-D-glucosamide) in which an aliquot of each supernatant and cell lysate was solubilized in 0.1 M citrate buffer (pH 4.5) at 37 ° C. for 30 minutes. Incubated with Wako Junyakusha). The enzymatic reaction was stopped by adding 2M glycine buffer (pH 10.4) and the absorbance was measured at 405 nm. The percentage of β-hexosaminidase release activity from RBL-2H3 cells from Cirsium maritimum extract was calculated using the following formula.
Enzyme release activity (%) = {(absorption of cell supernatant / absorption of cell supernatant + absorption of cell lysate) x 100}

The results are shown in FIGS. 2 to 5, respectively. The data are shown as the mean ± standard deviation of the three experiments.

As can be seen from FIGS. 2 and 3, a particularly excellent β-hexosaminize release inhibitory effect was found in the leaf and flower extracts of Cirsium maritimum.

In addition, as shown in FIG. 4, some fractions (DCC 30, 50, 100) of the sample of Example 8 fractionated by the ethyl acetate extract column chromatography of Cirsium maritimum leaves were shown. It was found that the release of β-hexosaminidase was significantly attenuated. In particular, D. C. C. 30 and D. C. C. 50 was found to have a strong inhibitory effect on the release of β-hexosaminidase, at 50 μg / mL, 86.1 to 88.6 percent inhibitory compared to positive control.

Furthermore, tests using the purified Cirsium maritimum obtained in Example 9 or commercially available silcimaritin showed a strong inhibitory effect on the release of β-hexosaminidase in a concentration-dependent manner, as shown in FIGS. 5A and 5B. Was done. Here, FIG. 5A shows the result of using the purified Cirsium maritimum product, and FIG. 5B shows the result of using the commercially available cirsium maritimum.

(Test Example 2) Evaluation of antibody production using a living body 1. Animals and Antigens 5-week-old female C3H / HeJ mice (body weight 15-20 g) were purchased from Japan SLC (Hamamatsu, Shizuoka Prefecture, Japan). Animals were bred at room temperature of 24 ± 3 ° C. under the conditions of a light-dark cycle of 12 / 12h (AM7: 00-PM7: 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 Junyaku.

2. Feeding, Sensitization and Sampling Mice were given free access to water and an AIN-93G diet. Then, the mice were assigned to 3 groups consisting of 10 mice according to their body weights, and the following diets were freely supplied for 4 weeks.
Non-sensitized group: AIN-93G
LHE Sensitization Group: AIN-93G
LHE Sensitization-Cirsium maritimum group: AIN-93G with 1% Cirsium maritimum (CMM) added.

Mice body weight was recorded 3 times / week throughout the experiment. Mice in one sensitization group received intraperitoneally 0.2 mL PBS containing 50 μg LHE and 4 mg aluminum hydroxide on days 14 and 21 (LHE sensitization group and LHE sensitization-thistle 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 veins of mice on day 27 under light anesthesia. Fecal pellets were collected on day 31. The fecal pellets were then dried with ALPHA 2-4 LDplus (Cristo, Osterode, Germany). These samples were subjected to an enzyme-linked immunosorbent assay (ELISA) to measure total antibody and specific antibody against LHE.

3. 3. Serum LHE-Specific IgE, IgA, IgG1, and IgG2a Measurements LHE-specific IgE, IgA, IgG1, and IgG2a levels were measured using an enzyme-linked immunosorbent assay (ELISA). Specifically, Nunk-Imnoplate (Thermo Scientific, Roskilde, Denmark) is coated with 100 μL of 0.1 M carbonate buffer (pH 9.6) containing LHE and incubated overnight at 4 ° C. did. Wells are washed with PBS (PBST) containing 0.05% polyoxyethylene (20) sorbitan monolaurate (Tween 20, Wako Pure Chemical Industries, Osaka, Japan) and then in 1% BSA / PBST solution at 37 ° C. Incubated for 1 hour. A 100 μL amount of each serum sample (diluted with PBST containing 1% BSA at a ratio of 1:50 for IgE and IgA and 1:250 for IgG1 and IgG2a) was applied to each well and the mixture was brought to 37 ° C. Incubated for 1 hour. After washing each well with 200 μL PBST 5 times, 100 μL of HRP-conjugated rabbit anti-mouse IgE (diluted 1: 1000 with PBST containing 1% BSA, manufactured by Santa Cruz Biotechnology, Inc.), HRP-conjugated rabbit anti-mouse IgA (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 (diluted 1: 5000 with PBST containing 1% BSA, manufactured by Santa Cruz Biotechnology, Inc.) was added to each well and incubated at 37 ° C. for 1 hour. After washing each well with 200 μL PBST 5 times, 2 mg of synthetic substrate ο-phenylendiamine (first grade; Wako Pure Chemical Industries, Ltd.) was added to 5 ml of citric acid buffer, and H2O2 (special grade; Wako Pure Chemical Industries, Ltd.) was added to 0. Add 100 μl of 006% to each well, react within 1-5 minutes at room temperature _{, terminate the reaction with 50 μL of 2.5 MH 2} SO ₄ (manufactured by Nacalai Tesque), and at 490 nm. Absorbance was measured with an xMarkTM microplate reader (manufactured by Bio-Rad Laboratories).

4. Measurement of Serum Total 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 Extracts of fecal pellets were prepared as follows. That is, 100 mg of fecal pellets were mixed with 1 mL of PBS and incubated overnight at 4 ° C. The pellet was then vortexed for 5 minutes. The mixture was centrifuged (4,000 xg for 15 minutes), the supernatant was collected and stored at -30 ° C. The procedure for detecting total IgA and LHE-specific IgA was the same as described above.

No weight difference was observed 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 addition, in FIG. 6 and FIG. 7, the data is shown by the average ± standard error of 10 mice, the star mark represents p <0.05, and the cross mark represents p <0.10. Also, in FIG. 8, the data are shown as mean ± standard error of 7 mice, where the stars represent p <0.05 and the crosses represent p <0.10.

It was suggested that when 1% Cirsium maritimum extract was fed by diet, it tended to suppress the production of antigen-specific IgE. (Fig. 6A)

It was suggested that feeding 1% Cirsium maritimum extract by diet promotes the production of antigen-specific IgA (Fig. 6B).

It was considered that feeding 1% Cirsium maritimum extract by diet did not affect the production of antigen-specific IgG1 (Fig. 6C).

It was considered that feeding 1% Cirsium maritimum extract by diet did not affect the production of antigen-specific IgG2a (Fig. 6D).

No change in the IgG1 / IgG2a ratio was observed in the Th balance, suggesting that IgG does not shift to Th2 (immunosuppressive drug).

It was also revealed that administration of 1% Cirsium maritimum extract tends to suppress the production of antigen-specific IgE, but does not suppress the total IgE. It was revealed that administration of 1% Cirsium maritimum extract promotes antigen-specific IgA production in serum, but does not promote total IgA. The total IgG1 in the serum of the Cirsium maritimum group 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 as compared with the non-sensitized group. (Fig. 6 and Fig. 7)
This is considered to mean that Cirsium maritimum has an effect of suppressing allergies.

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

(Test Example 3) Evaluation of antibody production using spleen lymphocytes Lymphocytes were isolated from the spleen, which is an antibody-producing tissue, using egg white ovoalbumin (OVA) -sensitized mice, and ethyl acetate-extracted Hama thistle The effect on antibody production was evaluated. Furthermore, the Th1 / Th2 balance was examined for the anti-allergic effect.
1. 1. Animals and Antigens 5-week-old female BALB / c mice (body weight 15-20 g) were purchased from Japan SLC (Hamamatsu, Shizuoka Prefecture, Japan). Animals were maintained at 24 ± 3 ° C. and housed in the 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 Junyaku.

2. Feeding, Sensitization and Sampling Mice were given free access to water and MF feed (Oriental Yeast Co., Ltd.).

Mice sensitization was performed by performing a total of 3 peritoneal immunizations at weekly intervals. That is, 0.2 mL of PBS containing 50 μg of OVA and 4 mg of aluminum hydroxide was intraperitoneally administered, respectively. To assess the status of antibody production, blood was obtained from the orbital veins of mice on day 27 under light anesthesia. The spleen was taken out from the mouse in which antibody production was confirmed.

First, 3 mL of RPMI1640 medium was placed in a 5 mL tube, and the removed spleen was immersed. Then, 5 mL of RPMI1640 medium was placed in a 5 mL dish, and the spleen was ground with two frosted glasses. Using a nylon mesh (Gliner: 70 μm mesh), a cell suspension was prepared in a 50 mL centrifuge tube and centrifuged at 400 × g for 5 minutes. The supernatant after centrifugation was removed, and 10 mL of fresh RPMI1640 medium was added and suspended. This operation was repeated twice. Then, 10 mL of RPMI1640 medium was added to prepare a cell suspension.

4 mL of Lymfluorite (Cosmo Bio) was placed in another 15 mL centrifuge tube, and the cell suspension was gently layered on this and centrifuged at 1000 × g for 30 minutes. Lymphocytes collected in the middle layer were pipetted out, transferred to a 15 mL centrifuge tube, and then 10 mL of fresh RPMI1640 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 RPMI1640 medium was added and suspended. This operation was repeated twice. Then, 10 mL of RPMI1640 medium was added to prepare a cell suspension.

The spleen lymphocyte solution thus obtained (2 × 10 ⁶ cells / ml) was ^{seeded on a 24-well plate with 0.5 ml each of 4 × 10 6} cells / ml of the spleen lymphocyte solution. Add 0.5 ml of Cirsium maritimum extract (0.2, 2, 10, 20, 100, 200, 1000 mg / ml) at twice the concentration added in the medium, and add 2.5 mg / mL allergen (OVA). Was added in an amount of 10 μL (25 μg of allergen) and stimulated with an antigen. The spleen lymphocytes were cultured as they were for 72 hours.

After that, the amounts of OVA-specific antibodies IgE, IgG1, and IgG2a contained in the culture medium 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 decreased in the spleen where antibody production is active, the Hama thistle extract may suppress allergies not only in the amount of antibody in serum but also in spleen lymphocytes, which are the center of antibody production. It was suggested.

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

(Test Example 4)
The RBL-2H3 cells prepared in the same manner as in Test Example 1, were inoculated to a 96-well black microplate 6.0 × 10 ⁴ cells / well and cultured overnight at 37 ° C.. The cells were then washed twice with PBS. The cells were sensitized with 50 ng / mL DNP-specific IgE (anti-DNP-IgE antibody, manufactured by Sigma-Aldrich) for 2 hours. The cells were then washed twice with PBS and then reacted with Fluo-3AM (acetoxymethyl) for 1 hour. After the reaction, the mixture was further washed twice with PBS, 100 μl of the sample dissolved in PBS was added, and the reaction was carried out for 10 minutes. Then, 10 μl of DNP-HSA adjusted to 0.625 μg / ml with MT buffer was added, and the absorbance at 485 nm and 538 nm was immediately measured with a fluorescence / emission microplate reader (Thermo).
Here, as samples, the extract of Cirsium maritimum leaf prepared in Example 1 (concentration was adjusted to 1 μg / ml and 10 μg / ml), blank (MT buffer containing no DNP-HSA), and control (Cirsium maritimum) were used. PBS) not included was used.

Data are represented by mean ± standard deviation (n = 4). Compared with the control, when Cirsium maritimum 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 anesthetized BALB / c mice. After 23 hours, the sample solution adjusted to 5% with PBS was orally administered by gavage (5 mg / 100 μg / animal (20 g); 250 mg / 5 mL / kg). Controls were similarly gavage orally administered PBS. 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 auricles of the mice was measured three times with a tickness gauge, and then the mice were killed by cervical dislocation and the auricles were collected. The collected auricle 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.
United control: No antibody sensitization-Antigen administration Anti DNP-HSA: Antibody sensitization Yes-Antigen administration Anti DNP-HSA-CMM: Antibodies sensitization-Cirsium maritimum solution administration-Antigen administration
Ear swelling refers to the result of auricle thickness measurement.
Ear dye intensity is shown as a relative value when the amount of dye in the United control is 1.

It can be seen that oral administration of Cirsium maritimum 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%" with the total amount as 100.

(Prescription Example 1) Composition (tablet) for prevention or treatment of allergies
Using the extract obtained in the above example, tablets having the following composition for prevention or treatment of allergies can be produced by a conventional method. Here, as the Cirsium maritimum extract, a sample as obtained in Example 1 is used.

(Prescription example 2) Composition for prevention or treatment of allergies (granule)
Using the extract obtained in Example 1 above, the following composition can be mixed and stirred to uniformly prepare the granules, and granules can be obtained by a fluidized bed granulator. Granules can be taken with beverages.

(Prescription Example 3) Beverage for Allergy Prevention or Treatment A beverage for allergy prevention or treatment having the following composition can be produced by a conventional method using the extract obtained in Example 1 above.

(Prescription example 5) Tea (Prescription example): Powdered tea
Cirsium maritimum water extract (powder) 22
Fragrance 3
Dextrin 75
Total 100

(Prescription example): Powdered tea
Cirsium maritimum hot water extract (powder) 22
Fragrance 3
Dextrin 75
Total 100

(Prescription example)
900 ml of water was heated to 60 ° C., and 30 g of Cirsium maritimum leaves or stems or a mixture of leaves and stems was added thereto, and the mixture was 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)
100 g of Cirsium maritimum leaves and stems were chopped to a length of about 3 mm and roasted by stirring 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 amount of Cirsium maritimum leaf and stalk crushed product contained in each bag is 1.2 g.

Claims (10)

A composition for preventing and / or ameliorating type 1 allergy containing Cirsium maritimum or Cirsium maritimum derived from Cirsium maritimum and / or cirsium maritin. A composition for suppressing the release of β-hexosaminidase, which contains Cirsium maritimum or Cirsium maritimum derived from Cirsium maritimum and / or Cirsium maritimum. A mucosal immunostimulatory composition containing Cirsium maritimum or Cirsium maritimum derived from Cirsium maritimum and / or cirsium maritin . A composition for preventing and / or ameliorating type 1 allergy containing an ethyl acetate extract of Cirsium maritimum. A composition for suppressing β-hexosaminidase release, which contains an ethyl acetate extract of Cirsium maritimum. A mucosal immunostimulatory composition containing an ethyl acetate extract of Cirsium maritimum . The item according to any one of claims 1 to 6 , wherein the Cirsium maritimum is derived from one or more selected from the group consisting of whole plants or leaves, stems, roots, seeds, and flowers of Cirsium maritimum. Composition. A composition for suppressing the release of β-hexosaminidase, which contains silcimarin and / or silcimarintin. The composition according to any one of claims 1 to 8, for administration by oral, intravenous injection, inhalation, or transdermal. The composition according to any one of claims 1 to 9, which is a food or drink, a quasi-drug, or a pharmaceutical product.

Images