Chrysanthemum extract with anti-allergic activity

Abstract

To provide an edible chrysanthemum extract having a high anti-type I allergic effect, an agent or a composition.SOLUTION: The present invention provides a hydrous ethanol extract of the flower part of Chrysanthemum morifolium Ramat., an edible chrysanthemum, or an anti-type I allergic agent or a composition each including the same.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a composition having anti-type I allergic activity. [Background technology]

[0002] Hay fever, allergic rhinitis, food allergies, asthma, urticaria, conjunctivitis, and anaphylactic shock are known as diseases classified as type I allergies. Among type I allergies, allergic rhinitis is particularly common, with some studies reporting that as many as one in two Japanese people suffer from it, highlighting the need for effective treatments and functional foods for type I allergies.

[0003] The mechanism of type I allergy is known to involve the production of antigen-specific immunoglobulin E (IgE). When an antigen enters the body, B lymphocytes begin to produce antigen-specific IgE through antigen presentation by macrophages and other cells that recognize the antigen. Sensitization occurs when IgE binds to high-affinity IgE receptors (FcεRI) expressed on the surface of cells such as mast cells and basophils. When an antigen enters the body again, it binds to IgE and cross-links the antibodies, causing degranulation from the cells and the release of inflammatory mediators such as histamine and leukotrienes. These inflammatory mediators trigger biological reactions such as increased vascular permeability, vasodilation, and itching.

[0004] On the other hand, the chrysanthemum flower (Chrysanthemum morifolium Ramat.), belonging to the genus Chrysanthemum in the family Asteraceae, is used as a food ingredient, but it is also known as a crude drug listed in the Japanese and Chinese pharmacopoeias and is known to have various effects. Patent Document 1 shows that a specific triterpene compound obtained from a methanol extract of dried edible chrysanthemum petals suppresses histamine release and leukotriene release by basophils, and describes an invention relating to an anti-allergic agent using a methanol extract of dried edible chrysanthemum petals. Non-Patent Document 1 reports that the production of Tumor Necrosis Factor-α (TNF-α) in microglial cells was suppressed by a hexane extract of edible chrysanthemum, suggesting that the hexane extract of edible chrysanthemum has neuroprotective effects. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2003-277267 [Non-patent literature]

[0006] [Non-Patent Document 1] Nippon Shokuhin Kogaku Kaishi, 66(4), 127-138, 2019 [Overview of the project] [Problems that the invention aims to solve]

[0007] However, Non-Patent Document 1 shows that while a hexane extract of the 'Enmeiraku' variety of Chrysanthemum morifolium Ramat. (Chrysanthemum genus, Asteraceae family) showed an inhibitory effect on TNF-α production, this effect was not obtained with a hexane extract of the 'Abokyu' variety. This suggests that the composition and effects of extracts vary greatly depending on the variety of Chrysanthemum genus, Asteraceae family. Furthermore, since the edible chrysanthemum variety is not specified in the invention described in Patent Document 1, it is presumed that the anti-allergic effect described in Patent Document 1 may not be achieved depending on the variety of edible chrysanthemum.

[0008] The object of this invention is to provide an extract, agent, or composition of edible chrysanthemum that has a high anti-type I allergy effect. [Means for solving the problem]

[0009] Therefore, the inventors conducted research with the objective of providing an extract of edible chrysanthemum that has effective anti-type I allergic activity. Using various parts of various Asteraceae plants such as buds, flowers, leaves, and stems, and various extraction solvents such as methanol, ethanol, water, and hot water, 280 types of extracts were prepared, and screening for anti-type I allergic activity was performed. As a result, it was found that an aqueous ethanol extract (AEtE) of the flower part of Chrysanthemum morifolium Ramat., a type of edible chrysanthemum, has a degranulation inhibitory effect. Furthermore, it was discovered that AEtE has the effect of suppressing TNF-α release from leukocytes and suppressing intracellular signal transduction by histamine, thus completing the present invention.

[0010] The present invention provides the following: [1] Aqueous ethanol extract of the flower of Chrysanthemum morifolium Ramat. [2] An anti-type I allergy agent containing an aqueous ethanol extract of the flower of the Epanguan flower. [3] The anti-type I allergic agent according to claim 2, which suppresses one or more of the histamine effects involved in (1) degranulation, (2) release of Tumor Necrosis Factor-α (TNF-α), and (3) an increase in intracellular calcium ion concentration. [4] A composition for the treatment of type I allergies, comprising an aqueous ethanol extract of the flower of the Epanguite. [5] The composition according to claim 4, which suppresses one or more of the histamine effects involved in (1) degranulation, (2) release of TNF-α, and (3) an increase in intracellular calcium ion concentration. [6] The composition according to claim 4 or 5, which is a food product. [7] The composition according to claim 4 or 5, which is a cosmetic. [8] The composition according to claim 4 or 5, which is a medicament.

[0011] [A] A method for suppressing type I allergy, comprising administering to the water-containing ethanol extract of the flower part of Chrysanthemum morifolium Ramat. [B] The water-containing ethanol extract of the flower part of Chrysanthemum morifolium Ramat. for use in the treatment of type I allergy. [C] Use of the water-containing ethanol extract of the flower part of Chrysanthemum morifolium Ramat. for the manufacture of an anti-type I allergy agent. [D] Use of the water-containing ethanol extract of the flower part of Chrysanthemum morifolium Ramat. for the manufacture of a composition for the treatment of type I allergy.

Advantages of the Invention

[0012] The water-containing ethanol extract of the flower part of Chrysanthemum morifolium Ramat. of the present invention can be used for the treatment of type I allergy.

Brief Description of the Drawings

[0013] [Figure 1] Figure 1 is a diagram showing the inhibitory effect of AEtE on the degranulation of RBL-2H3 cells. β-hex: β-hexosaminidase, CON: non-sensitized group, NC: no addition group, WM: wortmannin addition group p<0.001 (vs CON), *** p<0.001 (vs NC) [Figure 2] Figure 2 is a diagram showing the inhibitory effect of AEtE on passive cutaneous anaphylaxis. a in Figure 2 is a diagram showing the creation of a passive cutaneous anaphylaxis model of mouse auricles. b and c in Figure 2 are diagrams showing the results of quantifying the extravasation of mouse auricles due to the passive cutaneous anaphylaxis reaction. KET: ketotifen administration group [Figure 3] Figure 3 is a diagram showing the result that AEtE inhibited the increase in intracellular Ca2+ concentration in RBL-2H3 cells caused by the antigen-antibody reaction. [Figure 4] Figure 4 is a diagram showing the result that AEtE inhibited the release of TNF-α from RBL-2H3 cells induced by the antigen-antibody reaction. DEX: dexamethasone addition group [Figure 5] Figure 5 shows the results of AEtE suppressing the increase in intracellular Ca2+ concentration in HeLa cells caused by histamine addition. [Modes for carrying out the invention]

[0014] (The present invention extract) The extract of this invention is an aqueous ethanol extract of the flower part of the edible variety of Chrysanthemum morifolium Ramat., a member of the Asteraceae family. The 'Abokyu' chrysanthemum is mainly produced in Aomori Prefecture and other areas of Japan, and is used as an edible chrysanthemum. The flower part of the 'Abokyu' chrysanthemum is used as the raw material for extraction in this invention. In this invention, the term "flower portion" refers to the entire part generally known as a flower. In this invention, the tip portion of the flower stalk can be used, and it is also possible to use only a part of the flower portion, such as only the petals or ovary. The production method for the Epangong used in this invention is not particularly limited. Furthermore, there are no particular limitations on the harvesting time, growth period, cultivation method, or cultivation period of the Epangong used in this invention.

[0015] Extracts of the flowers of the Epangu plant are obtained by extracting the flowers of the Epangu plant fresh or dried, either directly or crushed using a crusher, with an extraction solvent. Drying may be carried out in the sun or using a commonly used drying machine, but is not limited to these methods. Drying can be carried out at a temperature of 20-80°C, preferably 30-70°C, more preferably 40-60°C, and most preferably 50°C. The drying time is 24-120 hours, preferably 36-108 hours, more preferably 48-96 hours, even more preferably 60-84 hours, and most preferably 72 hours.

[0016] The extraction process is not particularly limited as long as the active ingredients contained in the raw material are dissolved in aqueous ethanol, which is the extraction solvent, and can be carried out according to conventional methods. For example, the raw material is suspended in 1 to 100 times (by mass), preferably 5 to 80 times, more preferably 10 to 60 times, even more preferably 15 to 40 times, and most preferably 20 times, the extraction solvent. The active ingredients are extracted at room temperature or under reflux heating for 4 to 96 hours, preferably 8 to 72 hours, particularly preferably 12 to 60 hours, more preferably 16 to 48 hours, even more preferably 20 to 36 hours, and most preferably 24 hours. After extraction, the extraction residue can be removed by centrifugation or filtration to obtain the extract. The solvent is evaporated from the obtained extract to obtain the extract.

[0017] In the present invention, aqueous ethanol is used as the extraction solvent. Aqueous ethanol with an ethanol concentration of 1 to 99%, preferably 10 to 90%, particularly preferably 20 to 80%, more preferably 30 to 70%, even more preferably 40 to 60%, and most preferably 50% can be used.

[0018] The obtained extract may be subjected to dilution, concentration, drying, purification, and other treatments according to conventional methods in order to obtain a diluted or concentrated extract, a dried extract, or a crude or purified product thereof.

[0019] (Anti-type I allergy agent and composition of the present invention) The aqueous ethanol extract (AEtE) of the flower of the Aboganza plant according to the present invention has the effect of suppressing type I allergic reactions mediated by the production of antigen-specific IgE, and therefore can be used as an anti-type I allergy agent and a composition for the treatment of type I allergies. Here, "treatment" refers to prevention or treatment of a disease.

[0020] Here, the anti-type I allergic effect of AEtE is exerted based on its ability to suppress one or more of the following: degranulation, release of Tumor Necrosis Factor-α (TNF-α), and histamine activity related to the increase in intracellular calcium ion concentration. However, the anti-type I allergic effect of AEtE is not limited to the anti-type I allergic effect exerted based on the above-mentioned activity. Furthermore, since AEtE has degranulation inhibitory activity, TNF-α release inhibitory activity, and histamine-induced increase in intracellular calcium ion concentration inhibitory activity, it can be used as a degranulation inhibitor, TNF-α release inhibitor, and intracellular Ca 2+ It can also be used as an active ingredient in concentration-increasing inhibitors.

[0021] In this invention, degranulation refers to the release of intracellular granules from mast cells or basophils into the extracellular space. When an antigen binds to IgE antibodies bound to the surface of cells such as mast cells or basophils, and the antibodies cross-link, the mast cells or basophils become activated, causing degranulation. The granules released from mast cells or basophils contain inflammatory substances such as histamine, leukotrienes, and serotonin, and these inflammatory substances trigger type I allergic reactions such as increased vascular permeability, vasodilation, or itching.

[0022] The degranulation inhibitory effect can be evaluated according to methods known to those skilled in the art. One specific example is a method of measuring the enzymatic activity of β-hexosaminidase released from cells by degranulation. β-hexosaminidase is released simultaneously with inflammatory substances such as histamine upon degranulation. Evaluation of the inhibitory effect on β-hexosaminidase release from RBL-2H3 cells (rat basophilic leukemia cells) is widely used as one of the indicators of type I allergy suppression effects.

[0023] TNF-α is a type of inflammatory cytokine that is primarily released by activated macrophages, but is also produced by various cells, including mast cells, T cells, and adipocytes. While TNF-α released from these cells activates the immune system, excessive release can cause inflammation and tissue damage. Furthermore, TNF-α is known to have vasodilatory and vascular permeability-enhancing effects and is involved in various immune responses.

[0024] The inhibitory effect on TNF-α release can be evaluated by methods known to those skilled in the art. One specific example is the quantification of the concentration of TNF-α released extracellularly from RBL-2H3 cells activated by antigen-antibody reaction using ELISA.

[0025] Histamine released by degranulation is known to cause symptoms of type I allergies such as increased vascular permeability, vasodilation, and itching. These type I allergic reactions occur when released histamine acts on histamine receptors expressed in vascular endothelial cells, nerve cells, mast cells, etc., and triggers the release of Ca from the intracellular endoplasmic reticulum. 2+ Ca release and extracellular calcium 2+ Ca influx into cells 2+ It is caused by an increase in concentration.

[0026] Histamine-induced intracellular Ca 2+ The inhibitory effect on the increase in concentration can be evaluated by methods known to those skilled in the art. One specific example is intracellular Ca 2+ A detection reagent whose fluorescence intensity changes depending on the concentration is incubated with cells to allow it to be taken up into the cells, and the fluorescence intensity is measured to detect intracellular Ca 2+ One method is to quantify the change in concentration.

[0027] The present invention's anti-type I allergy agent or composition for treating type I allergy may contain 0.01 to 100% by mass of AEtE, preferably 0.1 to 90% by mass, particularly preferably 1.0 to 80% by mass, more preferably 2.0 to 70% by mass, even more preferably 3.0 to 60% by mass, and most preferably 3.0 to 50% by mass.

[0028] The form of the composition containing AEtE in the present invention is not particularly limited. The extract obtained by production may be used as is, but it may also be used in the form of a composition consisting of the extract and other components added as needed. Specific examples of such compositions include, for example, food (including beverages), food ingredients, cosmetics, quasi-drugs, pharmaceuticals, animal feed, etc.

[0029] The food of the present invention can be in any form that can be taken orally, such as a solution, suspension, emulsion, powder, or solid molded product. Specific examples of the food include soft drinks, juices, coffee, tea, liqueurs, dairy beverages, lactic acid bacteria beverages, candies, gum, chocolate, gummies, yogurt, ice cream, and the like. The foods of the present invention include supplements, functional foods, and foods for specified health uses. The form of supplements, functional foods, or foods for specified health uses is not particularly limited and can be any form such as tablets, capsules, granules, powders, syrups, or lozenges. The food product of the present invention may contain, as necessary, sweeteners, colorants, preservatives, thickeners, stabilizers, antioxidants, fungicides, flavorings, seasonings, vitamins, minerals, and the like. The food product of the present invention can be produced by combining the composition for treating type I allergies of the present invention with other food materials and additives, etc., using a food production method known to those skilled in the art.

[0030] The form of the cosmetic composition of the present invention is arbitrary and includes, for example, lotion, emulsion, cream, serum, mask, sunscreen, soap, facial cleanser, foundation, concealer, makeup base, lipstick, blush, eyeshadow, eyeliner, shampoo, conditioner, perfume, and the like. The cosmetic composition of the present invention may optionally contain vegetable oils, animal oils, higher alcohols, higher fatty acid esters, polyhydric alcohols, surfactants, humectants, water-soluble polymers, thickeners, preservatives, bactericides, UV absorbers, antioxidants, fragrances, pH adjusters, ethanol, water, whitening agents, wrinkle inhibitors, vitamins, and the like. The cosmetic composition of the present invention can be manufactured by combining the composition for treating type I allergies of the present invention with other raw materials and additives, etc., using methods for manufacturing cosmetic compositions known to those skilled in the art. In the cosmetic composition of the present invention, the AEtE content can be appropriately set according to the form of the cosmetic composition, the age of the person using it, etc.

[0031] The pharmaceutical product of the present invention is administered orally or parenterally, depending on the dosage form. Parenteral administration methods include, for example, transdermal absorption, topical application, subcutaneous injection, and intravenous injection. When the pharmaceutical product of the present invention is an oral preparation, AEtE is added as an active ingredient and it can be manufactured in any dosage form such as tablets, capsules, granules, powders, syrups, or oral liquids. The pharmaceutical product of the present invention may optionally contain excipients, disintegrants, binders, lubricants, coating agents, dispersants, emulsifiers, solubilizers, colorants, etc. Preferably, examples include starch, lactose, carboxymethylcellulose, distilled water, physiological saline, glucose solution, alcohol, propylene glycol, polyethylene glycol, animal and vegetable oils, white petrolatum, paraffin, wax, etc. The pharmaceutical product of the present invention can be formulated by combining the composition for the treatment of type I allergies of the present invention with other additives, using pharmaceutical manufacturing methods known to those skilled in the art.

[0032] When the composition of the present invention is taken in the form of food or medicine, the amount of AEtE taken by a human is preferably in the range of 0.01 to 1500 mg / kg body weight per day, and more preferably in the range of 0.1 to 300 mg / kg body weight per day, but is not limited to this range.

[0033] The anti-type I allergy agents, compositions, foods, cosmetics, and pharmaceuticals of the present invention described above are suitably applied to humans, but they can also be applied to animals other than humans, as long as their respective effects are achieved. [Examples]

[0034] The present invention will be described in more detail below with reference to examples, but the present invention is not to be limited thereto.

[0035] [Example 1. Preparation of aqueous ethanol extract (AEtE) from the flowers of the Abogyo Palace] The Abōkyū palace was constructed using materials donated by the Hachinohe City Agricultural Management Promotion Center. The petals of Chrysanthemum morifolium Ramat. were air-dried at 50°C for 3 days, and then crushed into a powder using a bead crusher (Multi-Bead Shocker: Yasui Kikai Co., Ltd.). The resulting powder was suspended in 20 times its volume of 50% ethanol and shaken for 24 hours. Then, centrifugation (4°C, 3500 rpm, 10 minutes) was performed, the supernatant was collected, and the extraction solvent was evaporated and removed using a centrifugal evaporator to obtain an aqueous ethanol extract (AEtE) of the Chrysanthemum morifolium flower.

[0036] [Example 2. Degranulation inhibition test in RBL-2H3 cells] RBL-2H3 cells (rat basophilic leukemia cells) were obtained from the JCRB cell bank (cell number JCRB0023). RBL-2H3 cells were suspended in DMEM (Dulbeccoo's Modified Eagle Medium) containing 10% fetal bovine serum and gentamicin at a concentration of 5 μg / mL, and then placed in a 96-well microplate in a 2.5 × 10⁶ container. 4Cells were seeded at a rate of 1 cell / well and simultaneously sensitized by adding an Anti-DNP-IgE (anti-dinitrophenyl IgE) solution to a final concentration of 100 ng / mL. The cells were then cultured at 37°C and 5% CO2 for 24 hours. Afterward, the culture medium was aspirated and the cells were washed twice with MT Buffer (137 mM sodium chloride, 2.7 mM potassium chloride, 1.8 mM calcium chloride, 1.0 mM magnesium chloride hexahydrate, 5.6 mM glucose, 20 mM hepes, 0.1% bovine serum albumin, pH 7.3). Finally, 100 μL of MT Buffer containing AEtE at concentrations of 25-400 μg / mL was added to each well, and the cells were cultured for 1 hour. As a positive control group, MT Buffer containing 5 μM woltmannin, a PI3K (phosphatidylinositol 3-kinase) inhibitor that suppresses signal transduction from IgE receptors on the surface of RBL-2H3 cells, was added simultaneously. To induce an antigen-antibody reaction, dinitrophenyl-conjugated human serum albumin (DNP-HSA) solution was added to a final concentration of 100 ng / mL, and the cells were cultured for 30 minutes. The reaction was then stopped by freezing the microplate on ice for 15 minutes. The supernatant was collected completely, and a portion of it was used to measure β-hexosaminidase, an enzyme contained in the granules released by the antigen-antibody reaction. 110 μL of 0.1% Triton® X-100 solution was added to each well of the remaining cells, and the mixture was stirred at 1000 rpm for 15 minutes to prepare a cell lysate. To 50 μL each of supernatant and cell lysate, 100 μL of β-hexosaminidase substrate solution (3.3 M p-nitrophenyl-2-acetamido-2-deoxy-β-D-glucopyranoside, 100 mM citrate, pH 4.5) was added, and the enzymatic reaction was carried out at 37°C for 1 hour with shaking at 60 rpm. After adding 100 μL of stop solution (2 M glycine, pH 10.4), the absorbance at 405 nm was measured. As a blank, the absorbance of the reaction solution to which the stop solution was added before the substrate solution was measured. The release rate of β-hexosaminidase was calculated using the following formula. β-hex release(%)=100×((S-Sb) / ((S-Sb)+(CL-CLb))) S: Absorbance of the supernatant. Sb: Absorbance of the supernatant blank. CL: Absorbance of the cell lysate. CLb: Absorbance of the cell lysate blank.

[0037] As shown in Figure 1, degranulation induced by the antigen-antibody reaction was inhibited in a concentration-dependent manner by the addition of AEtE.

[0038] [Example 3. Passive cutaneous anaphylaxis inhibition test] ICR mice (male, 5 weeks old) were used in the experiment after a 1-week acclimation period after arrival. Under isoflurane inhalation anesthesia, an Anti DNP-IgE solution (0.5 μg / 20 μL) was administered intradermally to the left ear of the mice. 23 hours later, AEtE suspended in physiological saline was administered orally at 125 - 500 mg / kg. As a positive control group, a ketotifen fumarate solution (25 mg / kg body weight) having an inhibitory effect on degranulation of mast cells and basophils was administered intraperitoneally. As a negative control group, physiological saline was administered orally. 1 hour later, under isoflurane inhalation anesthesia, a mixed solution of DNP-HSA (human serum albumin) (100 μg / mouse) and 2% Evans blue, 200 μL, was injected into the tail vein, and 30 minutes later, the mice were euthanized using carbon dioxide gas. The auricles of the mice were photographed with a digital camera and then cut off and collected. The Evans blue leaked into the auricles was extracted by immersing the auricles in 1 mL of formamide and incubating at 63 °C for 48 hours. The amount of leakage from the blood vessels was quantified by measuring the absorbance at 620 nm using the extracted Evans blue (Figure 2a).

[0039] As shown in Figures 2b and c, in the auricles of the mice administered with AEtE, extravasation induced by the antigen-antibody reaction was suppressed, and a significant inhibitory effect was observed in the group administered with 250 mg / kg of AEtE.

[0040] [Example 4. Intracellular Ca 2+ concentration increase inhibition test in RBL-2H3 cells] Intracellular Ca 2+The concentration was measured using Calcium Kit-Fura 2 (Dojin Chemical) according to the kit instructions. RBL-2H3 cells were sensitized in the same manner as in Example 2. After that, the culture medium was aspirated and removed, the cells were washed twice with MT Buffer, and then Loading Medium (5 μg / mL Fura 2-AM, 20 mM hepes, 115 mM sodium chloride, 5.4 mM potassium chloride, 0.8 mM magnesium chloride, 1.8 mM calcium chloride, 13.8 mM glucose, 1.25 mM probenecid, 0.04% Pluronic® F-127, pH 7.4) was added and the cells were incubated for 1 hour. After washing the cells twice with MT Buffer, 100 μL / well of Recording Medium containing AEtE (20 mM hepes, 115 mM sodium chloride, 5.4 mM potassium chloride, 0.8 mM magnesium chloride, 1.8 mM calcium chloride, 13.8 mM glucose, 1.25 mM probenecid, pH 7.4) was added to each well at concentrations ranging from 100 to 400 μg / mL, and the cells were incubated for 1 hour. As a positive control group, Recording Medium containing 5 μM woltmannin was added simultaneously. To induce an antigen-antibody reaction, DNP-HSA solution was added to each well to a final concentration of 100 ng / mL, and the fluorescence intensity (excitation wavelength: 340 / 380 nm, fluorescence wavelength: 510 nm) was immediately measured.

[0041] As shown in Figure 3, intracellular Ca2+ induced by antigen-antibody reaction 2+ The increase in concentration was suppressed in a concentration-dependent manner by the addition of AEtE.

[0042] [Example 5. TNF-α release inhibition test in RBL-2H3 cells] RBL-2H3 cells were sensitized in the same manner as in Example 2. After aspirating and removing the culture medium, the cells were washed twice with MT Buffer. Then, 100 μL of DMEM containing AEtE at concentrations of 100-400 μg / mL was added to each well, and the cells were cultured for 1 hour. As a positive control group, DMEM containing the steroid dexamethasone at a concentration of 10 μM was added simultaneously. To induce an antigen-antibody reaction, DNP-HSA solution was added to a final concentration of 100 ng / mL, and the cells were cultured for 6 hours before the cell supernatant was collected. The TNF-α contained in the supernatant was analyzed using Rat TNF-α ELISA MAX. TM The data was quantified using the Deluxe Sets (BioLegend) according to the kit's instructions.

[0043] As shown in Figure 4, the release of TNF-α from RBL-2H3 cells induced by the antigen-antibody reaction was suppressed in a concentration-dependent manner by the addition of AEtE.

[0044] [Example 6. Intracellular Ca in HeLa cells due to histamine 2+ [Concentration increase suppression test] HeLa cells were obtained from RIKEN BRC CELL BANK (cell number RCB0007). Intracellular Ca 2+ The concentration was measured using Calcium Kit-Fura 2 (Dojin Chemical) according to the kit instructions. HeLa cells were suspended in DMEM containing 10% fetal bovine serum and gentamicin at a concentration of 5 μg / mL, and 2.5 × 10⁶ cells were placed in a 96-well microplate. 4Cells were seeded at 1 cell / well and cultured for 24 hours at 37°C and 5% CO2. The culture medium was aspirated and removed, the cells were washed twice with MT Buffer, and then Loading Medium was added and cultured for 1 hour. After washing the cells twice with MT Buffer, 100 μL of Recording Medium containing AEtE at concentrations of 100-400 μg / mL was added to each well and cultured for 1 hour. As a positive control group, Recording Medium containing diphenhydramine hydrochloride, which has histamine H1 receptor antagonist activity, at a concentration of 1 μM was added simultaneously. To induce an antigen-antibody reaction, histamine solution was added to a final concentration of 100 μM, and the fluorescence intensity (excitation wavelength: 340 / 380 nm, fluorescence wavelength: 510 nm) was measured immediately afterward.

[0045] As shown in Figure 5, intracellular Ca iontophoresis caused by the addition of histamine. 2+ The increase in concentration was suppressed in a concentration-dependent manner by the addition of AEtE.

Claims

1. An anti-type I allergy agent containing an aqueous ethanol extract of the flower of the Epanguo Palace.

2. The anti-type I allergy agent according to claim 1, which suppresses one or more of the histamine effects involved in (1) degranulation, (2) release of Tumor Necrosis Factor-α (TNF-α), and (3) an increase in intracellular calcium ion concentration.

3. A composition for the treatment of type I allergies, comprising an aqueous ethanol extract of the flower of the Epangu flower.

4. The composition according to claim 3, which suppresses one or more of the following: (1) degranulation, (2) release of TNF-α, and (3) an increase in intracellular calcium ion concentration due to histamine.

5. The composition according to claim 3 or 4, which is a food product.

6. The composition according to claim 3 or 4, which is a cosmetic.

7. A composition according to claim 3 or 4, which is a pharmaceutical product.

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