JP7474432B2 - Antiallergic - Google Patents

Description

The present invention relates to an antiallergic agent. The antiallergic agent of the present invention can prevent, alleviate, or treat allergic diseases. The present invention also relates to a food composition for preventing or treating allergic diseases.

Allergies refer to the immune system's response to normally harmless antigens, resulting in adverse effects on the human body. Allergic diseases include atopic dermatitis, bronchial asthma, allergic rhinitis, hay fever, and food allergies, and it is said that more than 30% of Japanese people have some kind of allergy.

Type I allergies are caused by a phenomenon called degranulation, in which substances such as histamine present inside mast cells and basophils are released. Specifically, when an antigen binds to IgE on the IgE receptors on the surface of mast cells and basophils, granules containing histamine are released from these cells, which causes an inflammatory reaction in the cells and induces allergic symptoms. Therefore, in order to prevent or treat allergic symptoms, it is important to inhibit the release of these granules.

Various research activities are being conducted to suppress and treat allergies. Among these, ingredients derived from food are known to be extremely safe, and various ingredients with anti-allergic effects have been reported. For example, Patent Document 1 describes an anti-allergic agent containing apolactoferrin. Also, Patent Document 2 describes an anti-allergic agent containing sake lees. However, the current situation is that no substance has yet been developed that fully possesses the desired anti-allergic effect and is safe enough to be actually used as a food or medicine.

JP 2013-236623 A JP 2009-292785 A

As a result of extensive research into safe anti-allergy agents, the inventors have newly discovered that cumin extract exhibits anti-allergy effects. It was completely unknown that cumin extract has anti-allergy effects, and the present invention is based on this finding.

Thus, the present invention provides
[1] An antiallergic agent containing ground cumin or an extract thereof;
[2] The antiallergic agent according to [1], which is an antiallergic agent for type I allergy.
[3] The antiallergic agent according to [1] or [2], wherein the ground product or extract of cumin is a ground product or extract of cumin seeds.
[4] The antiallergic agent according to any one of [1] to [3], wherein the extract is an extract obtained using an aqueous solvent or a mixture of an aqueous solvent and an organic solvent; and [5] a food composition for preventing or treating an allergic disease, comprising the antiallergic agent according to any one of [1] to [4] and a food or drink.
Regarding.

The antiallergic agent of the present invention can prevent the onset of allergic diseases or alleviate or treat their symptoms. Furthermore, the antiallergic agent of the present invention can provide foods and beverages that have an antiallergic effect by inhibiting degranulation and are highly safe.

1 is a graph showing the degranulation inhibitory effect of a water-soluble extract of cumin after inactivation of an endogenous enzyme (β-hexosaminidase). 1 is a graph showing the degranulation inhibitory effect of an ultrafiltration (3000 Da membrane) filtrate of a cumin water-soluble extract. 1 is a graph showing the cytotoxicity of the ultrafiltration (3000 Da membrane) filtrate of a cumin water-soluble extract. 1 is a graph showing the degranulation inhibitory effect after the cumin water-soluble extract ultrafiltration (3000 Da membrane) filtrate was further dialyzed (500 Da). 1 is a graph showing a comparison of the degranulation inhibitory effect depending on the heat treatment time of the ultrafiltration (3000 Da membrane) filtrate of a cumin water-soluble extract. 1 is a graph showing the effect of cumin water-soluble extract on degranulation signaling. 1 is a graph showing the effect of cumin water-soluble extract on degranulation signaling. 1 is a graph showing the inhibitory effect of a water-soluble extract of cumin in the antigen-antibody reaction. FIG. 1 is a schematic diagram showing the relationship between an increase in intracellular calcium ion concentration and degranulation. 1 is a graph showing the effect of ultrafiltration (3000 Da membrane) filtrate of a cuminaceous water-soluble extract on antigen-induced increase in intracellular calcium ion concentration. 1 is a graph showing the effect of cuminata water-soluble extract ultrafiltration (3000 Da membrane) filtrate on calcium ionophore-induced degranulation. 1 is a graph showing the effect of ultrafiltration (3000 Da membrane) filtrate of a cuminaceous water-soluble extract on calcium ionophore-induced increase in intracellular calcium ion concentration. 1 is a schematic diagram and graph showing the outline and results of an experiment in which a water-soluble extract of cumin was orally administered to mice.

[1] Antiallergic Agent The antiallergic agent of the present invention contains a ground cumin or an extract as an active ingredient. It may contain both the ground cumin and the extract.

(cumin)
Cumin (Cuminum cyminum) is an annual or biennial herb of the Apiaceae family native to the eastern Mediterranean coast, and is also called Umazeri (horse parsley) in Japan. The plant is 20-40 cm tall and hairless on the entire plant. The petiole is short, about 1 cm long, and has a needle-shaped sheath. The leaves are long and needle-shaped, and are pinnately lobed twice. The flowers are umbrella-shaped, with a diameter of 2-3 cm. The petals are pink or white. The petals are oblong, with slightly chipped tips. The seeds are oblong, narrow at both ends, about 6 mm long and 1.5 mm wide, and covered entirely with white bristles. They flower in April, and the seeds are produced in May.
Cumin seeds are generally recognized as seeds, but in botanical terms, they are fruits. These cumin seeds are often used as spices. In this specification, the term "seeds" refers to cumin seeds with respect to cumin. Cumin seeds are widely used in meat dishes, vegetable dishes, stews, stir-fries, bread, cheese, and the like.

The part of cumin used in the antiallergic agent of the present invention is not particularly limited, and may be the whole plant, roots, stems, leaves, flowers, fruits, or seeds, or a mixture of at least two of them, but preferably the seeds are used. When seeds are used, other parts may also be included. Cumin may be used fresh, but it is preferable to use dried cumin.

(Crushed material)
In the present invention, the ground product may be cumin in a ground state, and may be, for example, in the form of a powder, granules, or paste. The ground product of cumin is preferably cumin powder. In addition, a powdered product molded or granulated into, for example, a cube, block, or granule can also be preferably used. The process for processing into the ground product or powder is not particularly limited, and examples thereof include a process of grinding the plant body by any method commonly used by those skilled in the art using a grinding device or instrument such as a crusher, mill, blender, mixer, or stone mill. The plant body may be dried before grinding.

When the cumin contained in the antiallergic agent of the present invention is ground, the average longest diameter of the ground cumin may be, but is not limited to, 0.01 to 2 mm, preferably 0.01 to 1.5 mm, more preferably 0.01 to 1 mm, even more preferably 0.01 to 0.75 mm, and most preferably 0.01 to 0.5 mm. In addition, 90% or more by weight of the ground cumin may have a longest diameter of 0.01 to 2 mm, preferably 0.01 to 1.5 mm, more preferably 0.01 to 1 mm, even more preferably 0.01 to 0.75 mm, and most preferably 0.01 to 0.5 mm. In addition, 90% or more by weight of the ground cumin may pass through 8.6 mesh (2 mm), 10 mesh (1.7 mm), 16 mesh (1 mm), or 30 mesh (0.5 mm) in the JIS test sieve mesh conversion table. If the maximum diameter of the ground cumin is 2 mm or less, the anti-allergic effect of the present invention is improved, so it is preferable to use ground cumin with a maximum diameter of 2 mm or less.

The average longest diameter of cumin can be measured using a known device for measuring particle size. It can also be calculated by randomly selecting 100 particles from the ground cumin, measuring their longest diameters using a stereomicroscope, and calculating the average.

(Extract)
The cumin used for extracting the active ingredient of the antiallergic agent of the present invention may be used in a raw form or in a dried form. In addition, in order to improve the extraction efficiency, the cumin may be processed into a crushed or powdered form before extraction.

To extract the active ingredient, a conventional extraction method used for extracting ingredients derived from plants can be used, for example, but not limited to, solvent extraction, steam distillation, squeezing (direct, high temperature, or low temperature), or supercritical extraction. A combination of these extraction methods, for example squeezing followed by solvent extraction, may also be used. The cumin extract contained in the antiallergic agent of the present invention is preferably extracted by solvent extraction.

The extraction solvent used when extracting by the solvent extraction method is not limited as long as the active ingredient can be sufficiently extracted into the extract, but for example, an organic solvent, an aqueous solvent, or a mixture of an organic solvent and an aqueous solvent can be used.

The cumin extract contained in the antiallergic agent of the present invention can be extracted with an organic solvent, such as alcohol, acetone, benzene, ester, ethyl acetate, hexane, chloroform, and diethyl ether. The alcohol is not limited as long as the active ingredient can be sufficiently extracted into the extract, and for example, a monohydric alcohol having 1 to 5 carbon atoms, such as methanol, ethanol, propyl alcohol, isopropyl alcohol, and butyl alcohol, can be used. Polyhydric alcohols having 2 to 5 carbon atoms, such as 1,3-butylene glycol, propylene glycol, dipropylene glycol, and glycerin, can also be used.

The cumin extract contained in the antiallergic agent of the present invention can be extracted with an aqueous solvent. The aqueous solvent is not limited as long as it contains water, and for example, water, physiological saline, or a buffer solution can be used. Examples of the buffer solution include phosphate buffer, sodium phosphate buffer, sodium carbonate buffer, citrate buffer, citrate phosphate buffer, acetate buffer, and Tris buffer. A preferred aqueous solvent is sodium phosphate buffer. The pH of the aqueous solvent is not particularly limited.

The cumin extract contained in the antiallergic agent of the present invention can be extracted with a mixture of an organic solvent and an aqueous solvent. The amount of the aqueous solvent contained in the extraction solvent is not limited as long as the active ingredient can be sufficiently extracted into the extract, but it can be, for example, 50% by weight or more, 70% by weight or more, or 90% by weight or more based on the total amount of the extraction solvent.

When the cumin extract contained in the antiallergic agent of the present invention is extracted by a solvent extraction method, the extraction temperature is not particularly limited as long as it is a temperature at which the active ingredient can be sufficiently extracted into the extract, but is preferably -50°C to 100°C, more preferably -25°C to 50°C, even more preferably -25°C to 25°C, even more preferably -10°C to 10°C, and most preferably 0°C to 10°C.

In addition, it is preferable to carry out the extraction with stirring or shaking so as to improve the extraction efficiency. The extraction time can be appropriately determined depending on the part used, such as the root, stem, leaf, flower, fruit, or seed. The extraction time can also be appropriately determined depending on the state of cumin, that is, whether it is raw or dried, or the processed state when it is processed into a crushed or powdered state. Furthermore, the extraction time can be appropriately determined depending on the extraction conditions, such as the temperature of the extract or the presence or absence of stirring or shaking. The extraction time is usually 1 minute to 72 hours, preferably 1 hour to 48 hours, and most preferably 12 hours to 36 hours.

The cumin extract contained in the antiallergic agent of the present invention can be extracted by steam distillation. Steam distillation is a method in which steam is passed through a raw material packed in a column, and the aroma components distilled along with the steam are condensed together with the steam. As the distillation method, any of pressurized steam distillation, normal pressure steam distillation, and reduced pressure steam distillation can be used.

The pressing method is a method of extracting an extract by applying physical pressure to cumin. There are direct pressing at room temperature, high-temperature pressing at high temperature, and low-temperature pressing at low temperature. The extract contained in the anti-allergic agent of the present invention can be extracted using any of the pressing methods.

The extract contained in the antiallergic agent of the present invention can be extracted using supercritical extraction. Supercritical extraction is a method of extracting an extract from a specific plant using a substance in a supercritical state. Carbon dioxide, for example, can be used as a substance in a supercritical state. Carbon dioxide in a supercritical state has a strong dissolving power, so it is also commonly used for decaffeinating coffee, or for extracting flavorings and medicinal ingredients from natural raw materials such as plants.

The antiallergic agent of the present invention may contain a cumin extract, a fraction containing an active ingredient fractionated from a cumin extract, or a purified active ingredient. The fraction containing the active ingredient can be obtained, for example, by removing substances with large molecular weights through ultramembrane filtration. The fraction containing the active ingredient fractionated from a cumin extract is preferably, but not limited to, a fraction obtained when an aqueous solvent extract of cumin is subjected to ultramembrane filtration at 3000 Da.

The active ingredient contained in the antiallergic agent of the present invention is contained in an extract extracted from cumin. Therefore, cumin contains ingredients that have antiallergic effects, and ground cumin, for example in powder, granules, or paste form, also contains the same active ingredient as that contained in the extract. Therefore, ground cumin can also be used as an antiallergic agent.

(Antiallergic agent)
The antiallergic agent of the present invention can be used particularly for subjects who have developed or may develop allergic diseases such as type I allergy, specifically, atopic dermatitis, bronchial asthma, allergic rhinitis, hay fever, hives, food allergy, animal allergy, allergic conjunctivitis, anaphylactic shock, or allergic gastroenteritis. "Anti-allergic" means having an effect of preventing the development of an allergic disease or alleviating or treating an allergic disease that has developed in a subject who has developed or may develop an allergic disease.

Type I allergic reactions involve antigen-specific IgE antibodies and mast cells and basophils that have the IgE-specific high-affinity IgE receptor FcεRI. When IgE bound to the surface of mast cells or basophils via FcεRI is crosslinked by an antigen, FcεRI is activated, transmitting a signal downstream, and intracellular granule contents such as histamine, leukotriene C4, PAF, or eosinophil chemotactic factor are released. This phenomenon is called degranulation. In each tissue where degranulation occurs, smooth muscle contraction, increased vascular permeability, or increased glandular secretion occurs, resulting in the appearance of allergic symptoms. Degranulation also involves an increase in intracellular calcium ion concentration. The antiallergic agent of the present invention can inhibit the antigen-antibody reaction between IgE antibodies and antigens. The antiallergic agent of the present invention can suppress an increase in intracellular calcium ion concentration. The antiallergic agent of the present invention can also inhibit degranulation signaling. The antiallergic agent of the present invention may have one of these effects, or two or more of them.

The dosage form of the antiallergic agent of the present invention is not particularly limited, and may be oral or parenteral, with oral preparations being preferred. Examples of the oral preparation include solid or powdered preparations such as fine granules, granules, tablets, capsules, and pills, as well as liquid preparations such as suspensions, emulsions, syrups, and extracts. Examples of parenteral preparations include injections.

The antiallergic agent of the present invention may consist of ground cumin or cumin extract, or may contain ground cumin or cumin extract. When the antiallergic agent of the present invention contains ground cumin or cumin extract, it may contain other additives.

When the antiallergic agent of the present invention is an oral preparation, other additives may include excipients, binders, disintegrants, emulsifiers, lubricants, flow promoters, diluents, preservatives, colorants, flavorings, flavorings, stabilizers, moisturizers, preservatives, antioxidants, or suspending agents, and specific examples thereof include gelatin, sodium alginate, starch, corn starch, white sugar, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soybean lecithin, sucrose, fatty acid esters, talc, magnesium stearate, polyethylene glycol, magnesium silicate, anhydrous silicic acid, or synthetic aluminum silicate.

When the antiallergic agent of the present invention is a parenteral agent, other additives may include water-soluble solvents such as physiological saline or Ringer's solution, water-insoluble solvents such as vegetable oil or fatty acid ester, isotonicity agents such as glucose or sodium chloride, solubilizers, stabilizers, preservatives, suspending agents, or emulsifiers.

The antiallergic agent of the present invention may contain cumin powder or extract in an amount of 90% by weight or more, 50% by weight or more, 10% by weight or more, or 1% by weight or more.

The dosage or intake of the antiallergic agent of the present invention can be adjusted as appropriate depending on the formulation, and the age, sex, body weight, and level of allergic symptoms of the subject to be used, but it is preferable that the administration or intake of the antiallergic agent is an amount that can prevent the onset of allergic diseases or alleviate or treat an allergic disease that has already developed. Specifically, in terms of the amount of cumin aqueous solvent extract added, the amount can be 0.01 to 1000 mg protein/kg body weight/day, preferably 0.1 to 750 mg protein/kg body weight/day, more preferably 1 to 500 mg protein/kg body weight/day, even more preferably 5 to 400 mg protein/kg body weight/day, even more preferably 10 to 300 mg protein/kg body weight/day, even more preferably 15 to 200 mg protein/kg body weight/day, or most preferably 20 to 150 mg protein/kg body weight/day.

Of course, the above administration method is just one example, and other administration methods are also possible. It is desirable that the administration method, dosage, administration period, and administration interval of the antiallergic agent to humans be determined through controlled clinical trials.

The antiallergic agent of the present invention can be administered to humans, but may also be administered to animals other than humans, including pets such as dogs, cats, rabbits, hamsters, guinea pigs, and squirrels; livestock such as cows and pigs; laboratory animals such as mice and rats; and animals kept in zoos, etc.

The antiallergic agent of the present invention can be a pharmaceutical composition for preventing or treating allergies.

The pharmaceutical compositions include medicines and quasi-drugs. Examples of medicines include herbal medicine preparations and herbal medicine preparations. Examples of quasi-drugs include nutritional drinks and health medicines containing herbal medicines.

[2] Food composition In this specification, the food composition means a composition containing the antiallergic agent of the present invention and a food or beverage. The food composition of the present invention contains the antiallergic agent of the present invention and can therefore be used for preventing or treating allergies.

Specific examples of foods include fresh food such as salads; cooked food such as steak, pizza, and hamburger; fried food such as stir-fried vegetables; vegetables such as tomatoes, bell peppers, celery, bitter melon, carrots, potatoes, and asparagus, and cooked products made from these vegetables; confectioneries such as cookies, bread, biscuits, dry bread, cakes, rice crackers, yokan, puddings, jellies, ice creams, chewing gum, crackers, chips, chocolate, and candies; noodles such as udon, pasta, and soba; fish paste products such as kamaboko, ham, and fish sausage; dairy products such as cheese, cream, and butter; seasonings such as miso, soy sauce, dressing, ketchup, mayonnaise, soup base, noodle soup, curry powder, mirin, and roux; soy foods such as tofu; and konjac. The food is preferably a roux (for example, for curry or stew), a seasoning spice, or a supplement.

Examples of beverages include coffee beverages; cocoa beverages; vegetable juices obtained from the above-mentioned vegetables; fruit juice beverages such as grapefruit juice, orange juice, grape juice, and lemon juice; tea beverages such as green tea, black tea, green tea, and oolong tea; alcoholic beverages such as beer, wine (red wine, white wine, sparkling wine, etc.), sake, plum wine, happoshu, whiskey, brandy, shochu, rum, gin, and liqueurs; milk beverages; soy milk beverages; liquid diets; and sports drinks. The beverage is preferably spiced tea (e.g., herbal tea or chai).

Food or beverage includes feed for animals. Target animals include, for example, primates such as humans, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, rats, and mice.

If desired, these foods or beverages may contain food additives and food ingredients, such as antioxidants, flavorings, acidulants, colorings, emulsifiers, preservatives, seasonings, sweeteners, spices, pH adjusters, stabilizers, vegetable oils, animal oils, sugars and sugar alcohols, vitamins, organic acids, fruit juice extracts, vegetable extracts, grains, beans, vegetables, meat, and seafood, either alone or in combination of two or more. The amounts of these food ingredients and food additives may be appropriately determined within a range that does not impair the object of the present invention.

These foods or beverages can be subjected to common sterilization processes, such as heat-pressure sterilization using retort and autoclave, batch sterilization, plate sterilization, electric heating sterilization, microwave heating sterilization, and steam sterilization such as injection and infusion.

Foods and beverages include functional foods (beverages) and health foods (beverages). In this specification, "health foods (beverages)" refers to foods or beverages that have some effect on health or that can be expected to have an effect, and "functional foods (beverages)" refers to foods or beverages among the above-mentioned "health foods (beverages)" that are designed and processed to fully exert a bioregulatory function (i.e., the function of preventing the onset of allergic symptoms, or the function of alleviating or treating allergic symptoms). Functional foods and health foods can be in the form of granules, solids, liquids, capsules, gels, or tablets.

The present invention will be described in detail below with reference to examples, but these are not intended to limit the scope of the present invention.

Preparation Example 1: Preparation of water-soluble extract of cumin seed powder
A sample was prepared using 10 mM sodium phosphate buffer (NaPB: pH 7.4) as a solvent, as follows to obtain a cumin extract sample. Cumin seed powder was suspended in 10 mM NaPB to a concentration of 0.05 g/mL, and was subjected to suspension extraction for 24 hours using a super-small rotary incubator rotator (15 rpm). After extraction, the mixture was centrifuged at 4°C and 12,000 rpm for 20 minutes to recover the supernatant, and then ultracentrifuged at 4°C and 70,000 rpm for 30 minutes to recover the supernatant. The pH was adjusted to 7.4 using an aqueous sodium hydroxide solution. The mixture was sterilized by filter filtration (0.45 μm).

Example 1: Degranulation test
Rat basophil cell line RBL-2H3 cells were precultured in 5% FBS-DMEM medium in a 96-well culture plate at a cell concentration of ^2x105 cells/mL for 18 hours. The cells were washed once with phosphate-buffered saline (PBS). Next, the cells were sensitized with dinitrophenyl (DNP)-specific IgE for 2 hours. The cells were washed twice with Tyrode's buffer. A water-soluble extract sample of cumin diluted with Tyrode's buffer was added at 200 μL/well and incubated for 10 minutes. After 10 minutes, the water-soluble extract sample solution of cumin diluted with Tyrode's buffer was aspirated and discarded, and Tyrode's buffer was added at 200 μL/well. The antigen DNP was then added and incubated for 30 minutes to induce degranulation, after which the supernatant was collected.

The degranulation inhibitory effect of the cumin water-soluble extract was evaluated using the amount of β-hexosaminidase released from the granules released by antigen stimulation as an index. Specifically, after collecting the supernatant, the cells were sonicated for 5 seconds on ice in 130 μL of improved Tyrode buffer containing 0.1% Triton X-100 to lyse the cells. Both the collected supernatant and the cell lysate obtained above were transferred to each well of a new 96-well (50 μL/well) microplate and incubated at 37° C. for 5 minutes. Then, 100 μL of 3.3 mM 4-nitrophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside (substrate solution) dissolved in 0.1 M citrate buffer (pH 4.5) was added to each well, and the wells were further incubated at 37° C. for 25 minutes. The enzyme reaction was stopped by adding 100 μL of 2 M glycine buffer (pH 10.4). The absorbance of the reaction solution was measured at a wavelength of 405 nm using a microplate reader (SH-8000Lab, Corona Electric Co., Ltd.) The β-hexosaminidase release rate (%), which is an index of degranulation, was calculated according to the following formula.

β-hexosaminidase release rate (%)=[(A _supernatant −A _{supernatant blank} )/(A _supernatant −A _{supernatant blank} )+(A _{cell lysate} −A _{cell lysate blank} )}]×100

A: absorbance of each well at a wavelength of 405 nm

Protein concentration was measured according to the Lowry method, specifically, using a DC protein assay kit from Bio-Rad.

The cumin water-soluble extract contains a substance whose color changes depending on the pH, which affects the activity evaluation in the degranulation test. Furthermore, this substance could not be removed by dialysis or ultrafiltration. Therefore, in the degranulation test, the cumin water-soluble extract diluted with Tyrode's buffer was removed by suction 10 minutes after the addition of the cumin water-soluble extract diluted with Tyrode's buffer, and the same amount of Tyrode's buffer in which the cumin water-soluble extract had been replaced with 10 mM NaPB was added before stimulation with the antigen. The degranulation tests for A23187 and thapsigargin described below were also performed using this method. This eliminated the variability in the data for the cumin water-soluble extract when a high protein concentration was added.

Because the cumin water-soluble extract itself contains β-hexosaminidase, endogenous β-hexosaminidase was inactivated by heating at 100°C for 5 minutes, and then a degranulation test was performed to evaluate activity. As a result, as shown in Figure 1, it was found that the cumin water-soluble extract inhibited β-hexosaminidase release in a concentration-dependent manner. When cytotoxicity was then evaluated using the WST-8 method, no cytotoxicity was observed at any concentration (not shown). This suggests that the cumin water-soluble extract inhibits degranulation without cytotoxicity.

Estimation of the molecular size of active molecules
The cumin extract sample was ultrafiltered using a membrane with a molecular weight cutoff of 3000, and the degranulation activity of the ultrafiltrate was evaluated. As a result, as shown in Figure 2, the ultrafiltrate sample inhibited β-hexosaminidase release in a concentration-dependent manner (UCEA in Figure 2). The β-hexosaminidase release rate of the control to which no cumin extract sample was added was 34.3 ± 2.6%. In this case, as shown in Figure 3, the ultrafiltrate sample did not show cytotoxicity even at high concentrations.

Furthermore, a degranulation test was performed on a filtrate sample ("500-3000 Da" in Figure 4) that was ultrafiltered using a membrane with a molecular weight cutoff of 3000, and then further dialyzed using a dialysis membrane with a molecular weight cutoff of 100-500. As shown in Figure 4, a decrease in maximum activity was observed, but activity was retained. The β-hexosaminidase release rate of the control without the addition of the cumin extract sample was 30.6±2.5%.

These findings suggest that the active substances in the water-soluble cumin extract that inhibit degranulation may include substances with molecular weights of 500 to 3000 as well as substances with molecular weights of 500 or less that have activity. In the following examples, the water-soluble cumin extract was ultrafiltered using a membrane with a molecular weight cutoff of 3000 and used as ultrafiltered water-soluble cumin extract (UCAE).

Estimation of active substances
In order to estimate the degranulation inhibitor contained in UCAE, the release rate of β-hexosaminidase was measured after heat treatment at 100°C for 30 minutes. As a result, as shown in Figure 5, no change in activity was observed after heat treatment at 100°C for 30 minutes (5 minutes treatment was the control: to inactivate endogenous β-hexosaminidase). The release rate of β-hexosaminidase in the control without addition of the cumin extract sample was 25.9 ± 1.9%. Therefore, it was inferred that the active substance is a heat-stable substance.
The active substance of the antiallergic agent of the present invention is not specified and may be a protein or a substance other than a protein, but for the sake of convenience, the concentration of the extract is shown in this specification using the concentration of protein in the extract.

《Effect of cumin water-soluble extract on degranulation signaling》
After culturing RBL-2H3 cells with the addition of the UCAE sample, a cell lysate was prepared and subjected to SDS-PAGE gel electrophoresis. After electrophoresis, the electrophoresed proteins were transferred to a PVDF membrane by Western blotting. The transferred PVDF membrane was blocked with skim milk and reacted with each signal factor-specific antibody (Cell Signaling Technology). After reacting with a peroxidase-labeled anti-IgG antibody, a substrate solution was added to detect bands.

As a result, as shown in Figures 6 and 7, it was found that UCAE inhibits the phosphorylation-induced activation of PI3K and Btk proteins. Similar results were obtained for PLCγ1 and PLCγ2 proteins, indicating that UCAE inhibits the phosphorylation-induced activation of PLCγ1 and PLCγ2 proteins.

Example 2: Antigen-antibody reaction inhibition test
In the degranulation test, the initial stage of degranulation induction by IgE antibody and antigen is an antigen-antibody reaction between IgE bound to the high affinity IgE receptor (FcεRI) on the cell surface and allergen. If the antigen-antibody reaction is inhibited, the subsequent degranulation reaction will also be suppressed. Therefore, the effect of cumin extract samples on the antigen-antibody reaction was examined by enzyme antibody method.

After coating a 96-well plate with DNP-HSA, a mixture of samples at various concentrations and anti-DNP-IgE was added. After incubation at 37°C for 1 hour, the plate was washed three times with 0.05% tween 20-PBS. Peroxidase-labeled anti-IgE antibody was added and incubated at 37°C for 1 hour. After washing three times with 0.05% tween 20-PBS, a peroxidase substrate was added to develop color.

As a result, as shown in Figure 8, it was confirmed that the cumin extract sample, UCAE (3000 Da↓), and the cumin extract sample containing a fraction with a molecular weight greater than 3000 (3000 Da↑) all inhibited the antigen-antibody reaction at high concentrations. However, the degree of inhibition is thought to be slight compared to the degranulation inhibitory effect shown in Figure 2, etc., and the degranulation inhibitory effect of the cumin extract sample is thought not to be due to inhibition of the antigen-antibody reaction.

Example 3: Analysis of degranulation inhibition mechanism
Calcium ions are involved in degranulation, and degranulation occurs when the intracellular calcium ion concentration increases (Figure 9). In this experiment, Fluo3-AM (Dojindo Chemical Industries, Ltd.) was used to measure the intracellular calcium ion concentration over time before and after stimulation with UCAE at a protein concentration of 400 μg/mL. As a result, as shown in Figure 10, the addition of UCAE suppressed the increase in intracellular calcium ion concentration. However, since the degree of suppression was not as great as the effect shown in Figure 2, etc., it is possible that UCAE also exerts an inhibitory effect on the stage after calcium ion influx during antigen stimulation.

<Study of the degranulation inhibitory effect using degranulation-inducing drugs>
Using rat basophil cell line RBL-2H3 cells, the effect of UCAE at a protein concentration of 400 μg/mL on degranulation stimulated by A23187 and thapsigargin was examined using procedures similar to those used in the antigen experiments. Thapsigargin is a calcium ion-ATPase inhibitor on the endoplasmic reticulum membrane, which causes calcium leakage from the endoplasmic reticulum. When calcium ions in the endoplasmic reticulum are depleted, calcium ions flow in from outside the cell. A23187 also induces calcium ion influx from outside the cell by increasing the permeability of Ca ion channels present in the cell membrane. As a result, intracellular calcium ion concentration increases, causing degranulation.

The results of a degranulation test on A23187- and thapsigargin-induced degranulation are shown in Figure 11. The control shows the results for RBL-2H3 cells to which UCAE was not added. It was found that UCAE concentration-dependently inhibited degranulation induced by both stimuli.

<Examination of the effect of degranulation-inducing drugs on the increase in intracellular calcium ion concentration>
Using rat basophil cell line RBL-2H3 cells, the effect of UCAE at a protein concentration of 400 μg/mL on the increase in intracellular calcium ion concentration induced by A23187 stimulation and thapsigargin stimulation was examined in a similar manner to the antigen-based experiments.

As a result, as shown in Figure 12, both stimuli had an inhibitory effect on intracellular calcium ion concentration. This suggests that the cumin water-soluble extract inhibits A23187- and thapsigargin-induced degranulation by inhibiting calcium ion influx.

Example 4: Oral administration experiment using mice
A passive cutaneous anaphylaxis (PCA) response test by IgE was performed using mice. 7-week-old female BALB/c mice were orally fed 10 mM NaPB, low-dose cumin water-soluble extract (25 mg/kg body weight/day), or high-dose cumin water-soluble extract (125 mg/kg body weight/day) for 7 days. Then, anti-DNP-IgE antibody (50 ng) diluted with PBS or PBS was intradermally injected at 10 μL into the left auricle using an airtight syringe. 23 hours later, 10 mM NaPB, low-dose cumin water-soluble extract (25 mg/kg body weight/day), or high-dose cumin water-soluble extract (125 mg/kg body weight/day) was orally administered again. One hour later, 200 μg of antigen (DNP-HSA) diluted with 200 μL of PBS containing 0.5% Evans blue was injected into the tail vein using an airtight syringe. After treatment with DNP-HSA, mice were euthanized and the ears were excised and collected. Evans blue dye was then extracted from each collected ear with 500 μL of formaldehyde overnight at 70° C. The absorbance of Evans blue dye was then measured at 620 nm using an Ultrospec 3000 spectrophotometer (Amersham Pharmacia Biotech).

As a result, a significant inhibitory effect was confirmed in the low-concentration group. A tendency for inhibition was also observed in the high-concentration group. The more the individual preferred to drink the sample, the greater the inhibitory effect. This suggests that cumin intake is effective in inhibiting the PCA reaction.

The antiallergic agent of the present invention can prevent the onset of allergic diseases or alleviate or treat their symptoms. The antiallergic agent of the present invention can provide foods and beverages that have an antiallergic effect and are highly safe.

Claims (2)

1. A therapeutic or preventive agent for hay fever, comprising ground cumin seeds or an extract thereof, the extract being an extract obtained from an aqueous solvent or a mixture of an aqueous solvent and an organic solvent . A food composition for preventing or treating hay fever, comprising the agent for treating or preventing hay fever according to claim 1 and a food or drink.