JP7801145B2 - Pharmaceutical and food compositions for treating allergies - Google Patents

Description

Article 30, Paragraph 2 of the Patent Act will be applied. December 19, 2021, Pacifichem 2021, held online (https://pacifichem.org/)

The present invention relates to pharmaceutical compositions and food compositions for treating allergies.

For example, type I allergies are caused by IgE produced by B cells, a type of lymphocyte. In the case of hay fever, the body recognizes pollen as an allergen (antigen) and produces IgE that specifically binds to it. The IgE then binds to the surface of basophils and mast cells via the IgE receptor (FcεRI). When pollen, which is an allergen, enters the body, it binds to IgE, stimulating a signal that is transmitted within the basophils and mast cells, causing the release of granules containing substances that cause allergic symptoms, such as histamine. This phenomenon is called degranulation. The histamine released outside the cells by degranulation irritates the mucous membranes, resulting in the onset of allergic symptoms. Therefore, in order to prevent or treat allergic symptoms, it is important to inhibit the release of granules by basophils and mast cells.

Japanese Patent Application Laid-Open No. 2020-26395 Japanese Patent Application Laid-Open No. 2019-127455

For example, Patent Document 1 describes an anti-type I allergy agent containing phlestoic acid. Patent Document 2 describes an anti-allergy agent containing p-coumaric acid. However, the development of new anti-allergy agents is desired.
An object of the present invention is to provide a novel antiallergic agent.

The present inventors have conducted extensive research into novel antiallergic agents and have surprisingly found that certain novel compounds exhibit antiallergic activity.
The present invention is based on this finding.
Therefore, the present invention provides
[1] The following formula (1):
3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucoside represented by the formula:
[2] The following formula (2):
7,8-dihydro-8-β-D-glucopyranosyloxy-4-methoxy-7-methyl-5H-furo[2,3-g][2]benzopyran-5-one represented by the formula
[3] The following formula (3):
(wherein R ¹ is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R ² is a group having 1 to 5 carbon atoms and each independently represents a hydroxy group, a carboxy group, or an alkyl group having 1 to 6 carbon atoms; and R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ are each independently a hydrogen atom, a hydroxy group, a carboxy group, or an alkyl group having 1 to 6 carbon atoms),
[4] Formula (5):
2-O-trans-caffeoylhydroxycitric acid represented by
[5] The following formula (6):
2-O-trans-caffeoylhydroxycitric acid 6'-O-methyl ester represented by the formula:
[6] A pharmaceutical composition comprising, as an active ingredient, a compound selected from the group consisting of the compounds according to [1] to [5] or a salt thereof.
[7] The pharmaceutical composition according to [6], which is for preventing or treating allergies.
[8] The pharmaceutical composition according to [7], wherein the allergy is type I allergy.
[9] The pharmaceutical composition according to any one of [6] to [8], which suppresses degranulation.
[10] A food composition for suppressing allergic symptoms, comprising, as an active ingredient, a compound selected from the group consisting of the compounds according to [1] to [5] or a salt thereof.
[11] The food composition according to [10], wherein the allergic symptoms are type I allergic symptoms; and [12] the food composition according to [10] or [11], wherein the suppression of the allergic symptoms is suppression of degranulation.
Regarding.

The present invention provides a novel pharmaceutical composition, particularly a pharmaceutical composition for preventing or treating allergies. The present invention also provides a food composition for suppressing allergic symptoms.

1 is a graph showing the inhibitory effect of (3S)-3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucoside on degranulation of RBL-2H3 cells due to antigen stimulation. 1 is a graph showing the inhibitory effect of 7,8-dihydro-8-β-D-glucopyranosyloxy-4-methoxy-7-methyl-5H-furo[2,3-g][2]benzopyran-5-one on antigen-stimulated degranulation of RBL-2H3 cells. Graph (A) shows the inhibitory effect of 2-O-trans-caffeoylhydroxycitric acid or 2-O-trans-caffeoylhydroxycitric acid 6'-O-methyl ester on the degranulation of RBL-2H3 cells, and graph (B) shows the results of examining their cytotoxicity. 1 is a graph showing the inhibitory effect of adenosine on degranulation caused by antigen stimulation. 1 is a graph showing the inhibitory effect of maltotriose on degranulation caused by antigen stimulation. 1 is a graph showing the inhibitory effect of DL-malic acid on degranulation caused by antigen stimulation. 1 is a graph showing the inhibitory effect of L-malic acid on degranulation caused by antigen stimulation. 1 is a graph showing the inhibitory effect of D-malic acid on degranulation caused by antigen stimulation.

[1] Pharmaceutical Composition The 3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucoside of the present invention is represented by the following formula (1):
The compound is a novel compound represented by the formula (hereinafter, sometimes referred to as Compound A), and is preferably (3S)-3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucoside.

The 7,8-dihydro-8-β-D-glucopyranosyloxy-4-methoxy-7-methyl-5H-fro[2,3-g][2]benzopyran-5-one of the present invention is a compound represented by the following formula (2):
The compound is a novel compound represented by the formula (hereinafter, may be referred to as Compound B), and is preferably (7S,8R)-7,8-dihydro-8-β-D-glucopyranosyloxy-4-methoxy-7-methyl-5H-fro[2,3-g][2]benzopyran-5-one.

The compound of the present invention is represented by the following formula (3):
(wherein R ¹ is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R ² is a group having 1 to 5 carbon atoms and each independently represents a hydroxy group, a carboxy group, or an alkyl group having 1 to 6 carbon atoms; and R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ are each independently a hydrogen atom, a hydroxy group, a carboxy group, or an alkyl group having 1 to 6 carbon atoms) (hereinafter, this may be referred to as Compound C). Compound C is water-soluble and can be easily dissolved in water.
The compound C is preferably represented by the following formula (4):
(wherein R ¹ is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).
The compound C is more preferably 2-O-trans-caffeoyl hydroxycitric acid, which is represented by the following formula (5):
The compound is a novel compound represented by the formula (hereinafter, may be referred to as compound C1).

The compound C is more preferably 2-O-trans-caffeoyl hydroxycitric acid 6'-O-methylester, which is represented by the following formula (6):
The compound is a novel compound represented by the formula (hereinafter, may be referred to as compound C2).

The salt of the compound is not limited to a salt with an inorganic base or an organic base, or a salt with an acid, as long as it is acceptable as a pharmaceutical or food product. Specific examples of salts with inorganic bases or organic bases include salts with inorganic bases, organic bases, or metal alkoxides. The salts can be produced by mixing the compound with an inorganic base, organic base, or metal alkoxide.
Inorganic bases capable of forming salts include hydroxides, carbonates, bicarbonates, acetates, or hydrides of alkali metals (e.g., lithium, sodium, or potassium); and hydroxides or hydrides of alkaline earth metals (e.g., magnesium, calcium, or barium). Organic bases capable of forming salts include dimethylamine, triethylamine, piperazine, pyrrolidine, piperidine, 2-phenylethylamine, benzylamine, ethanolamine, diethanolamine, pyridine, or collidine. Metal alkoxides include sodium methoxide, potassium tert-butoxide, or magnesium methoxide. Preferred salts of β-alanine are sodium salts, potassium salts, calcium salts, or combinations thereof.
Specific examples of salts with acids include salts with inorganic acids and organic acids. Examples of inorganic acids that can form salts include hydrochloric acid.

The pharmaceutical composition of the present invention comprises, as an active ingredient, a compound selected from the group consisting of compounds represented by formulas (1) to (5) or a salt thereof. The pharmaceutical composition of the present invention is preferably a pharmaceutical composition for type I allergy and may be an agent having the effect of suppressing type I allergic symptoms. That is, it can alleviate, treat, or prevent symptoms caused by type I allergy, such as hay fever, urticaria, allergic rhinitis, or bronchial asthma. Furthermore, the effect of suppressing type I allergic symptoms may be the effect of suppressing degranulation of mast cells or basophils in the mechanism of type I allergic reactions. Therefore, the pharmaceutical composition of the present invention may be an agent for suppressing degranulation of mast cells or basophils.
That is, the pharmaceutical composition of the present invention can inhibit degranulation of at least mast cells or basophils, and therefore can effectively suppress the symptoms of type I allergy.
The pharmaceutical composition of the present invention may contain the above-mentioned maltotriose, adenosine, malic acid, or a salt thereof as an active ingredient.
In one embodiment, the pharmaceutical composition of the present invention contains one of the above compounds. In another embodiment, the pharmaceutical composition of the present invention contains a combination of two or more of the above compounds. Preferably, the pharmaceutical composition of the present invention contains a combination of two or more of the above compounds.

Maltotriose has the following formula (7):
It is a trisaccharide formed by the α1-4 glycosidic bond of three glucose molecules, represented by the following formula: For example, it is produced by the degradation of amylose or starch by amylase.

Adenosine has the following formula (8):
It is a nucleoside formed by the β-N ₉ -glycosidic bond between adenine (a type of purine base) and ribose, and is used in the body as a component of DNA and RNA.

Malic acid is represented by the following formula (9):
L-Malic acid is a type of hydroxy acid represented by the following formula (10):
D-malic acid is a compound represented by the following formula (11):
Both L-malic acid and D-malic acid are effective as active ingredients in the antiallergic agent of the present invention. DL-malic acid, which is a mixture of L-malic acid and D-malic acid, may also be used as the active ingredient.

"allergy"
The allergies targeted by the pharmaceutical composition of the present invention include, but are not limited to, type I allergies. Examples of type I allergies include atopic dermatitis, bronchial asthma, allergic rhinitis, hay fever, hives, food allergies, animal allergies, allergic conjunctivitis, anaphylactic shock, and allergic gastroenteritis. The pharmaceutical composition of the present invention has the effect of preventing the onset of allergic diseases or alleviating or treating allergic diseases that have developed in subjects who have developed or are at risk of developing allergic diseases.

Type I allergic reactions involve antigen-specific IgE antibodies and mast cells and basophils that possess 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, resulting in the release of intracellular granule contents such as histamine, leukotriene C4, PAF, or eosinophil chemotactic factor. This phenomenon is called degranulation. In tissues 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 pharmaceutical composition of the present invention can inhibit antigen-stimulated degranulation of mast cells and/or basophils. In particular, the pharmaceutical composition of the present invention has the effect of inhibiting the extracellular release (degranulation) of granules containing chemical mediators such as histamine and leukotrienes from mast cells or basophils (degranulation inhibitory effect). Therefore, the pharmaceutical composition of this embodiment can effectively suppress, treat, or prevent symptoms caused by type I allergic reactions.

The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and examples include oral and parenteral preparations, with oral preparations being preferred. Examples of oral preparations 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 pharmaceutical composition of the present invention may consist solely of the compound, or may contain the compound. When the pharmaceutical composition of the present invention contains the compound, it may contain other additives.

When the pharmaceutical composition of the present invention is an oral dosage form, other additives may include excipients, binders, disintegrants, emulsifiers, lubricants, flow enhancers, diluents, preservatives, colorants, flavorings, taste-correcting agents, stabilizers, humectants, antiseptics, antioxidants, or suspending agents. Specific examples include gelatin, sodium alginate, starch, corn starch, sucrose, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soybean lecithin, sucrose, fatty acid esters, talc, magnesium stearate, polyethylene glycol, magnesium silicate, anhydrous silicic acid, and synthetic aluminum silicate.

When the pharmaceutical composition of the present invention is a parenteral preparation, other additives may include water-soluble solvents such as saline or Ringer's solution, water-insoluble solvents such as vegetable oils or fatty acid esters, isotonic agents such as glucose or sodium chloride, solubilizers, stabilizers, preservatives, suspending agents, or emulsifiers.

The pharmaceutical composition of the present invention may contain the above-mentioned compound 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 pharmaceutical composition of the present invention can be adjusted as appropriate depending on the formulation, as well as the age, sex, weight, and severity of allergic symptoms of the subject. However, it is preferable that the administration or intake of the pharmaceutical composition be an amount that can prevent the onset of allergic diseases or alleviate or treat existing allergic diseases. Specifically, the amount of the compound added can be 0.01 to 1,000 mg/kg body weight/day, preferably 0.1 to 750 mg/kg body weight/day, more preferably 1 to 500 mg/kg body weight/day, even more preferably 5 to 400 mg/kg body weight/day, even more preferably 10 to 300 mg/kg body weight/day, even more preferably 15 to 200 mg/kg body weight/day, or most preferably 20 to 150 mg/kg body weight/day. Of course, the above administration method is only one example, and other administration methods are also possible. The administration method, dosage, administration period, administration interval, etc. of the pharmaceutical composition to humans are preferably determined through controlled clinical trials.

The pharmaceutical composition of the present invention can be administered to humans, but it 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 pharmaceutical composition of the present invention may be a pharmaceutical composition for preventing or treating allergies. The pharmaceutical composition includes pharmaceuticals and quasi-drugs. Pharmaceuticals include, for example, herbal medicine preparations and herbal medicine preparations. Quasi-drugs include, for example, nutritional drinks and health supplements containing herbal medicines.

Whether or not the pharmaceutical composition of the present invention has a degranulation inhibitory effect can be tested using cells, such as rat basophilic leukemia cells (RBL-2H3 cells), which release granulocytes containing histamine and other substances extracellularly when IgE bound to the cell surface is crosslinked by an antigen. This can be confirmed by stimulating such cells with an antigen and calculating the degree to which degranulation is inhibited in a sample to which the pharmaceutical composition has been added compared to a sample to which the pharmaceutical composition has not been added.

The pharmaceutical composition of the present invention may contain an ingredient other than maltotriose or adenosine that is effective against allergies, such as maltotriose, adenosine, malic acid, (3S)-3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucoside, 7,8-dihydro-8-β-D-glucopyranosyloxy-4-methoxy-7-methyl-5H-furo[2,3-g][2]benzopyran-5-one, 2-O-trans-caffeoylhydroxycitric acid, or 2-O-trans-caffeoylhydroxycitric acid 6'-O-methyl ester.
The pharmaceutical composition of the present invention can contain two or more of these active ingredients, and by containing multiple active ingredients, allergic symptoms can be suppressed synergistically.

[2] Food composition for suppressing allergic symptoms As used herein, the term "food composition" refers to a food composition containing the compound or a salt thereof as an active ingredient. The food composition of the present invention can be used as a food composition for suppressing allergic symptoms. Examples of the food composition include foods and beverages.

Specific examples of foods include fresh prepared foods such as salads; cooked foods such as steak, pizza, and hamburger steak; stir-fried foods such as stir-fried vegetables; vegetables such as tomatoes, bell peppers, celery, bitter melon, carrots, potatoes, and asparagus, and processed foods made from these vegetables; meats such as beef, pork, and chicken, and processed foods made from these meats; seafood such as salmon, tuna, sea bream, flounder, trout, octopus, and squid, and processed foods made from these seafood; cookies, bread, biscuits, hardtack, cakes, rice crackers, yokan, pudding, jelly, Examples include confectioneries such as ice cream, chewing gum, crackers, chips, chocolate, and candy; 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; condiments such as miso, soy sauce, dressing, ketchup, mayonnaise, soup base, noodle soup, curry powder, mirin, and roux; soy foods such as tofu; processed agricultural and marine products such as furikake, tsukudani, and cereal; fruits such as apples or grapes; and konjac.

Examples of beverages include coffee drinks; cocoa drinks; vegetable juices obtained from vegetables; fruit juice drinks such as grapefruit juice, orange juice, grape juice, apple juice, and lemon juice; tea drinks 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; dairy drinks; soy milk drinks; liquid diets; and sports drinks.

Food or beverages include feed and beverages 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 determined appropriately within the range that does not impair the objectives of the present invention.

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

Foods and beverages include functional foods (drinks) and health foods (drinks). As used herein, "health foods (drinks)" refers to foods or beverages that have or are expected to have some effect on health, and "functional foods (drinks)" refers to foods or beverages among the "health foods (drinks)" that have been designed and processed to fully exhibit bioregulatory functions (i.e., the function of preventing the onset of allergic symptoms or alleviating or treating allergic symptoms). Functional foods and health foods can be in granular, solid, liquid, capsule, gel, or tablet form.
The food composition of the present invention can be prepared by adding one or more of the above compounds to the above food or beverage.

《Action》
The mechanism by which the pharmaceutical composition and food composition for suppressing allergic symptoms of the present invention can suppress the symptoms of type I allergy has not been analyzed in detail, but can be assumed as follows.
There are various antigens that cause type I allergies, including hay fever and food. However, the mechanism of type I allergy development is that 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, resulting in the release of intracellular granule contents such as histamine, leukotriene C4, PAF, or eosinophil chemotactic factor. That is, although the causative antigen is different, the symptoms are caused by degranulation of mast cells or basophils. The allergy agent of the present invention inhibits degranulation from mast cells and basophils, which is involved in the development of symptoms common to type I allergies. Therefore, even if the antigen causing type I allergy is different, it is presumed that inhibiting degranulation can suppress symptoms of type I allergies such as atopic dermatitis, bronchial asthma, allergic rhinitis, hay fever, urticaria, food allergy, animal allergy, allergic conjunctivitis, anaphylactic shock, or allergic gastroenteritis.

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

Example 1
In this example, a degranulation test was carried out using (3S)-3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucoside.
(3S)-3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucoside was dissolved in distilled water and adjusted to pH 7.0 for use as a sample. Rat basophilic cell line RBL-2H3 cells were seeded into a 96-well culture plate at a cell density of 4 x ¹⁰⁴ cells/well and sensitized for 20 hours in 5% FBS-DMEM medium containing dinitrophenyl phosphate (DNP)-specific IgE. After washing the cells twice with Tyrode's buffer, 120 μL/well of Tyrode's buffer containing various concentrations of (3S)-3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucoside was added and the cells were incubated at 37°C for 10 minutes. After 10 minutes, the antigen DNP-HSA was added and the cells were incubated at 37°C for 30 minutes to induce degranulation, after which the supernatant was collected.

The degranulation inhibitory effect of (3S)-3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucoside was evaluated using the amount of β-hexosaminidase released from granules upon antigen stimulation as an index. Specifically, after collecting the supernatant, cells were lysed in 130 μL of Tyrode's buffer containing 0.1% Triton X-100 by sonication on ice for 5 seconds. 50 μL of both the collected supernatant and the obtained cell lysate were transferred to each well of a new 96-well microplate and incubated at 37°C for 5 minutes. Subsequently, 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, followed by further incubation 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 415 nm using a microplate reader (iMark Microplate Reader, Bio-Rad). The β-hexosaminidase release rate (%), as 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 at 415 nm for each well

As shown in FIG. 1, (3S)-3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucoside inhibited degranulation of RBL-2H3 cells in a concentration-dependent manner.

Example 2
In this example, a degranulation test was performed using (7S,8R)-7,8-dihydro-8-β-D-glucopyranosyloxy-4-methoxy-7-methyl-5H-furo[2,3-g][2]benzopyran-5-one. The procedure of Example 1 was repeated, except that (7S,8R)-7,8-dihydro-8-β-D-glucopyranosyloxy-4-methoxy-7-methyl-5H-furo[2,3-g][2]benzopyran-5-one was used instead of (3S)-3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucoside. As shown in Figure 2, (7S,8R)-7,8-dihydro-8-β-D-glucopyranosyloxy-4-methoxy-7-methyl-5H-furo[2,3-g][2]benzopyran-5-one inhibited degranulation of RBL-2H3 cells in a concentration-dependent manner.

Example 3
In this example, degranulation tests and cytotoxicity tests were carried out using 2-O-trans-caffeoylhydroxycitric acid and 2-O-trans-caffeoylhydroxycitric acid 6'-O-methyl ester.
Rat basophilic cell line RBL-2H3 cells were seeded in 96-well culture plates at a cell density of 4 x ¹⁰ cells/mL. The cells were sensitized for 24 hours with 5% FBS-DMEM medium containing dinitrophenyl phosphate (DNP)-specific IgE. 2-O-trans-Caffeoylhydroxycitric acid and 2-O-trans-Caffeoylhydroxycitric acid 6'-O-methyl ester diluted with distilled water were added at 200 μL/well and incubated for 10 minutes. After 10 minutes, a solution of (3S)-3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucoside 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 inhibitory effect of (3S)-3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucoside on degranulation was evaluated using the amount of β-hexosaminidase released from granules upon antigen stimulation as an index. Specifically, after collecting the supernatant, cells were lysed by sonication for 5 seconds on ice in 130 μL of modified Tyrode's buffer containing 0.1% Triton X-100. Both the collected supernatant and the obtained cell lysate were transferred to each well of a new 96-well microplate (50 μL/well) and incubated at 37°C for 5 minutes. Subsequently, 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, followed by further incubation 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). The β-hexosaminidase release rate (%), as 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 at 405 nm for each well

As shown in FIG. 3(A), 2-O-trans-caffeoylhydroxycitric acid and 2-O-trans-caffeoylhydroxycitric acid 6'-O-methyl ester inhibited the degranulation of RBL-2H3 cells in a concentration-dependent manner.
Furthermore, when the cytotoxicity of the compound was measured by the WST-8 method, the compound did not exhibit cytotoxicity at a concentration that inhibited degranulation, as shown in FIG. 3(B).

Example 4
In this example, a degranulation test was performed using adenosine. The procedure of Example 1 was repeated except that maltotriose was used instead of (3S)-3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucoside. As shown in Figure 4, adenosine inhibited degranulation of RBL-2H3 cells in a concentration-dependent manner.

Example 5
In this example, a degranulation test was performed using maltotriose. The procedure of Example 1 was repeated, except that maltotriose was used instead of (3S)-3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucoside. As shown in Figure 5, maltotriose inhibited the degranulation of RBL-2H3 cells in a concentration-dependent manner.

Example 6
In this example, a degranulation test was performed using DL-malic acid. The procedure of Example 1 was repeated, except that DL-malic acid was used instead of (3S)-3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucoside. As shown in Figure 6, DL-malic acid inhibited degranulation of RBL-2H3 cells in a concentration-dependent manner.

Example 7
In this example, a degranulation test was performed using L-malic acid. The procedure of Example 1 was repeated, except that L-malic acid was used instead of (3S)-3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucoside. As shown in Figure 7, L-malic acid inhibited the degranulation of RBL-2H3 cells in a concentration-dependent manner.

In this example, a degranulation test was performed using D-malic acid. The procedure of Example 1 was repeated, except that D-malic acid was used instead of (3S)-3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucoside. As shown in Figure 8, D-malic acid inhibited degranulation of RBL-2H3 cells in a concentration-dependent manner.

The pharmaceutical and food compositions of the present invention can alleviate, treat, or prevent symptoms caused by type I allergies, such as hay fever, urticaria, allergic rhinitis, or bronchial asthma.

Claims (14)

The following formula (1):
3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucoside represented by the formula: The following formula (2):
7,8-Dihydro-8-β-D-glucopyranosyloxy-4-methoxy-7-methyl-5H-furo[2,3-g][2]benzopyran-5-one represented by the formula: A pharmaceutical composition comprising, as an active ingredient, a compound selected from the group consisting of the compounds according to claims 1 and 2 or a salt thereof. The pharmaceutical composition according to claim 3 , which is for preventing or treating allergies. The pharmaceutical composition according to claim 4 , wherein the allergy is type I allergy. The pharmaceutical composition according to claim 5, which is for preventing or treating hay fever. The pharmaceutical composition according to claim 5, which is for the prevention or treatment of allergic rhinitis. The pharmaceutical composition according to any one of claims 3 to 7 , which inhibits degranulation. A food composition for suppressing allergic symptoms, comprising a compound selected from the group consisting of the compounds according to claims 1 and 2 or a salt thereof as an active ingredient. The food composition according to claim 9 , wherein the allergic symptoms are type I allergic symptoms. The food composition according to claim 10, which is for preventing or treating hay fever. The food composition according to claim 10, which is for preventing or treating allergic rhinitis. The food composition according to any one of claims 9 to 12 , which inhibits degranulation . A degranulation inhibitor comprising, as an active ingredient, a compound selected from the group consisting of the compounds according to claims 1 and 2 or a salt thereof.