JP6639162B2 - Antiallergic agent containing lotus root extract - Google Patents

Description

  The present invention relates to an interleukin-9 (IL-9) production inhibitor, an antiallergic agent, and an antiallergic composition.

  In recent years, the number of allergic patients has been increasing year by year due to changes in the natural environment and eating habits, increase in stress, and the like. The mechanism of onset of allergy is usually classified into four types, type I to type IV, and there are mixed types of these. Type I allergies such as hay fever, atopic dermatitis, bronchial asthma, allergic rhinitis, and urticaria are called immediate allergies because the symptoms appear in a short time after the action of the antigen.

  The mechanism of action of type I allergy is characterized by induction of IgE antibodies and release (degranulation) of chemical mediators such as histamine and leukotriene from mast cells and basophils. First, allergens (pollen, house dust, mites, mold, etc.) that invaded from the outside world are presented to T cells with a part of them bound to MHC class II molecules by antigen presenting cells such as dendritic cells and macrophages. As a result, Th2 cells are activated and differentiated. Th2 cytokines (IL-4, IL-5, IL-9, IL-13, etc.) released by Th2 cells induce class switching of B cells, and IgE antibodies are produced. Binds to FcεRI on the surface of basophils in blood. The allergen is recognized by the IgE antibody bound to the surface of mast cells and basophils at the next invasion, and a cross-link is formed between the IgE antibodies. This stimulation triggers mast cells and basophils to release chemical mediators ( Degranulation) causes allergic symptoms. For example, in allergic rhinitis (including hay fever), histamine released from mast cells and basophils of the nasal mucosa causes vasodilation and vascular hyperpermeability, resulting in sneezing, aqueous rhinorrhea, nasal congestion ( Unpleasant rhinitis symptoms such as stuffy nose appear. Heretofore, as an agent for suppressing and improving allergic symptoms mediated by IgE or a chemical mediator, for example, a chemical chemical mediator release inhibitor, which suppresses the release of a chemical mediator by weakening mast cell activity, and a histamine H1 receptor Histamine H1 antagonist that inhibits its function by competitively binding to histamine, leukotriene antagonist that inhibits its function by competitively binding to leukotriene to leukotriene receptor, and suppresses production and action of thromboxane In addition to antiallergic agents such as thromboxane inhibitors / antagonists, Th2 cytokine inhibitors that suppress the production of IL-4 and IL-5, steroid agents that reduce inflammation by weakening immune responsiveness, and the like. Among antihistamines, second- or third-generation antihistamines having both a histamine antagonistic action and an inhibitory action on release of chemical mediators from mast cells are sometimes referred to as antiallergic drugs in Japan. However, such drugs have not been sufficiently effective in treating allergic symptoms, and have serious side effects and are contraindicated in patients with other diseases. There is a problem. Furthermore, the long-term use of the above-mentioned drugs also imposes a heavy burden on medical economy. Therefore, there is a demand for the development of a safe and inexpensive next-generation antiallergic agent having a high therapeutic effect.

  The present inventors have been working on identification of an allergic disease susceptibility gene and elucidation of its expression mechanism as a novel therapeutic strategy for allergic disease. As a result, the histamine H1 receptor gene is an allergic disease susceptibility gene, and activation of protein kinase C-δ (PKCδ) is required for enhancing the expression of the histamine H1 receptor (H1R) gene. It has been clarified that increased gene expression is highly correlated with the severity of allergic rhinitis symptoms and IL-5 gene expression. Further, merchiain, which is a histamine H1 receptor gene / IL-4 gene expression inhibitor, was identified from an extract of the antiallergic Japanese medicine "Bushin" (Patent Document 1). Further, as a signal involved in the onset of allergy different from the PKCδ signal transduction system as described above, suplatast suppresses the enhanced expression of the IL-9 gene induced by activation of the calcineurin / NFAT signal transduction system, It has been reported that the concurrent oral administration of suplatast, a calcineurin / NFAT inhibitor, and epinastine, an antihistamine, which suppresses PKCδ, significantly improves the symptoms of nasal hypersensitivity (Patent Document 2).

JP 2011-126791 A JP 2013-173722 A

As described above, antihistamines are typical antiallergic agents, but cannot completely suppress allergic symptoms by themselves, and increasing the dosage to increase the effect may cause side effects or costs. Problem.
Accordingly, an object of the present invention is to provide a safe and inexpensive drug that enhances the therapeutic effect of an antihistamine on allergic diseases.

  The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, it has been found that an extract of a lotus root node has an action of suppressing the enhanced expression of the IL-9 gene, and that the extract and an antihistamine drug The present inventors have found that the combined use of the present invention markedly suppresses the nasal inflammation state in nasal hypersensitivity model rats, and has completed the present invention.

That is, the present invention includes the following inventions.
(1) An IL-9 production inhibitor containing an extract of lotus root as an active ingredient.
(2) An agent for enhancing the antiallergic activity of an antihistamine substance, comprising an extract of lotus root node as an active ingredient.
(3) An antiallergic agent containing an extract of lotus root and an antihistamine substance.
(4) An antiallergic composition comprising the antiallergic agent according to (3).
(5) The antiallergic composition according to (4), which is a pharmaceutical or a food or beverage.
(6) A method for producing an antiallergic agent, comprising a step of extracting an active ingredient for inhibiting IL-9 production from a lotus root node.

  ADVANTAGE OF THE INVENTION According to this invention, the IL-9 production inhibitor containing the extract of lotus root node as an active ingredient or the enhancer of the antiallergic activity of an antihistamine substance is provided. In addition, the extract of lotus root node can remarkably improve allergic symptoms such as allergic rhinitis by using an antihistamine substance in combination, as compared with the case of using the antihistamine substance alone. Therefore, according to the present invention, there is also provided an antiallergic agent comprising an extract of lotus root node and an antihistamine substance. Since the antiallergic agent of the present invention contains an extract of lotus root, which is a natural product, as an active ingredient, it has high safety without side effects even when used daily. Therefore, it can be used with confidence in pharmaceuticals and health foods. In addition, since lotus root is a material that is inexpensive and easily available, the burden of treating allergies is reduced.

FIG. 1 shows the IL-9 expression inhibitory activity of extracts (concentrations: 10, 50, 100 μg / ml) of lotus roots (nodes, edible parts, leaves, seeds) (negative control: lotus root extract) (Ionomycin (positive control): a test plot in which ionomycin was added without adding a lotus root extract). FIG. 2A shows extracts of lotus root nodes (nodes, nodes to 1 cm) and edible portions of lotus roots (1-2 cm, 2-3 cm, 3-4 cm, 4-5 cm, 5-6 cm, 6-7 cm). (Concentration: 50 μg / ml). FIG. 2B shows the IL-9 expression inhibitory activity of extracts (concentration: 1 to 150 μg / ml) of lotus root nodes (nodes, nodes to 1 cm) and edible portions of lotus roots (1 to 2 cm from nodes) (negative control) : A test group in which neither lotus root extract nor ionomycin was added, and ionomycin (positive control): a test group in which ionomycin was added without adding a lotus root extract). FIG. 3 shows a schedule for preparing a TDI-sensitized nasal hypersensitivity model rat. FIG. 4 shows the IL-9 expression inhibitory activity of the lotus root extract (concentration: 7.5, 25, 75, 753 μg / ml) (negative control: a test group in which neither the lotus root extract nor ionomycin was added; (Positive control): a test plot in which ionomycin is added without adding the lotus root extract. Shows nasal symptom scores of TDI-sensitized nasal hypersensitivity model rats by administration of lotus root node extract alone, epinastine alone, and combination administration of lotus root node extract and epinastine (negative control: same as sensitization and seizure induction by TDI) In a schedule, a test group in which an ethyl acetate solution was applied instead of TDI, TDI (positive control): a test group in which no drug was administered to a TDI-sensitized nasal hypersensitivity model rat, and epinastine: an epinastine in a TDI-sensitized nasal hypersensitivity model rat , A test group to which TDI-sensitized nasal hypersensitivity model rats were administered only a lotus root node extract, and a combination group: a test group to which TDI-sensitized nasal hypersensitivity model rats were administered epinastine and a lotus root extract Test group administered simultaneously).

Hereinafter, the present invention will be described in detail.
1. Interleukin-9 (IL-9) production inhibitor, enhancer of anti-allergic activity of antihistamine substance Interleukin-9 (hereinafter, referred to as IL-9) production inhibitor of the present invention or anti-allergy of antihistamine substance The activity enhancer contains an extract of lotus root node as an active ingredient. The lotus root (Lotus root) in the present invention refers to an enlarged rhizome of a lotus (Nelumbo nucifera). In the present invention, the term "lotus root" refers to a node or a portion near a node within 1 cm from the end of the node. Say. Examples of lotus root varieties (where the parentheses are produced) include, for example, Bicchu (from Naruto and Ishii, Tokushima Prefecture), Giant White (from Kawauchi in Tokushima Prefecture), and Lotus (from Kawauchi in Tokushima Prefecture). It is not particularly limited to these.

  The extraction method is not particularly limited, but can be performed by stirring or column extraction using water or hot water, or a mixed solvent of water and an organic solvent. As the organic solvent, water-soluble organic solvents, for example, lower alcohols such as methanol, ethanol, propyl alcohol, isopropyl alcohol, butanol, isobutanol or water-containing lower alcohols, polyhydric alcohols such as propylene glycol, 1,3-butylene glycol, glycerin, etc. Examples thereof include alcohol or water-containing polyhydric alcohol, acetone, ethyl acetate, and a mixed solvent of two or more thereof. Among these solvents, the solvent used in the present invention is preferably water, hot water or ethanol. The amount of the solvent used is not particularly limited, and may be, for example, 10 times or more, preferably 20 times or more, relative to the lotus root (dry weight). It is preferably 100 times or less for convenience of operation in that case. The extraction temperature and time depend on the type of the solvent used. For example, when water is used as the solvent, the temperature is 10 to 120 ° C., preferably 20 to 100 ° C., 10 minutes to 24 hours, and preferably 30 minutes to 5 hours. Can be exemplified.

  The extract may be centrifuged to remove insolubles and may be used as it is as a concentrated solution, or may be used in the form of a powder such as concentrated to dryness, spray drying, freeze drying or the like. If necessary, the extract may be purified by a suitable means after centrifugation and used. As means for purification, means known in the art, such as an ion exchange column, activated carbon, and gel filtration, can be used.

  As described in Examples below, the extract of the lotus root node obtained as described above was used to induce the IL-9 gene induced by influencing calcium ions by ionomycin stimulation in rat basophil leukemia cells RBL-2H3. It has the activity of suppressing the enhancement of expression. In addition, the extract of lotus root node, as shown in the examples below, significantly suppresses allergic symptoms of TDI-sensitized nasal hypersensitivity model rats by co-administration with an antihistamine substance than the antihistamine substance alone. Can be. Therefore, the extract of the lotus root node can be used as it is or by adding a suitable carrier to a desired form and formulated as an IL-9 production inhibitor or an anti-allergic activity enhancer of an antihistamine substance.

  The enhancement of IL-9 gene expression in RBL-2H3 stimulated by ionomycin is induced by the translocation of NFAT into the nucleus and binding to the NFAT binding site of the IL-9 gene. In addition, as one of the onset mechanisms of allergic nasal hypersensitivity, enhanced expression of IL-9 via a calcineurin / NFAT signal transduction system has been confirmed (Patent Document 2). The calcineurin / NFAT signal transduction system is formed by binding of activated T cell nuclear factor (NFAT) dephosphorylated by serine / threonine phosphatase calcineurin to the NFAT binding site on DNA. Refers to an intracellular signal transduction system in which downstream gene transcription is promoted. An agent that inhibits T cell activation by inhibiting this calcineurin / NFAT signal has been used as an immunosuppressant. In addition, IL-9 is one of the cytokines produced by Th2 cells along with IL-4, IL-5, and IL-13, and these Th2 cytokines are used in allergic reactions in adipocytes, basophils, and basophils. It is involved in the production of various chemical mediators including histamine from eosinophils, and the subsequent inflammation and tissue damage. Unlike IL-4, it is not known whether IL-9 is essential for the induction of IgE production, but IL-9 proliferates mast cells and is also produced from activated mast cells and basophils. I know that Therefore, based on these findings, the extract of the lotus root node can be used to alleviate the suppression of excessive allergic reactions based on the inhibitory action of the calcineurin / NFAT signaling system, which is different from the PKCδ signaling system which is inhibited by the antihistamine substance. Probably involved.

2. Antiallergic agent The antiallergic agent of the present invention contains an antihistamine substance in combination with the above extract of lotus root. The antihistamine substance may be a synthetic or natural substance as long as it has an antihistamine action, and is not particularly limited. Therefore, in the present invention, as the antihistamine substance, an antihistamine drug, a plant extract (herbal extract) having an antihistamine action, or the like can be used. The antihistamine may be any histamine H1 receptor antagonist, for example, epinastine or a salt thereof, chlorpheniramine, calevastatin, mepyramine, oxatomide, fexofenadine, cetirizine, loratadine, diphenhydramine, olopatadine and the like. However, epinastine or a salt thereof is preferable. Further, as epinastine or a salt thereof, epinastine hydrochloride, epinastine bromate, epinastine oxalate, epinastine nitrate, epinastine sulfonate, epinastine fumarate, epinastine maleate, epinastine sulfate, epinastine phosphate, etc. And epinastine hydrochloride, and epinastine hydrochloride is preferred. In addition, as plant extracts (herbal extract) having an antihistamine action, there are: Bitter ginseng (Kujin), Gyaku (Muresu sparrow), Natsuto (Murasaki Natsufuji), Western nettle, Tengcha (Tengcha), Sophora (Enju), Chamomile, life-prolonging grass (Emmeiso), hammerellis, hawthorn, red clover, mulberry branch (Soji), pine nodes (Shousettsu), agarwood (Jinko), cloves (Chouzi), fragrance (Koukou), and ginger (Hokyo) Extract.

  The antiallergic agent of the present invention may be composed of separate preparations of the extract of lotus root and an antihistamine substance, and may be composed of a single preparation containing a mixture of the extract of lotus root and an antihistamine substance. It may be configured.

  The method of administering the antiallergic agent of the present invention may be any method in which the extract of the lotus root node and the antihistamine substance are administered substantially simultaneously.For example, the extract of the lotus root node and the antihistamine substance are administered to the subject to be administered. May be administered completely simultaneously, or may be administered continuously in a short time (preferably within several minutes). In order to further maximize the effect, it is preferable to administer a preparation in which a lotus root node extract and an antihistamine substance are well mixed in advance. Therefore, the dosage form of the antiallergic agent of the present invention includes, for example, (a) administration of a single preparation obtained by simultaneously formulating an extract of a lotus root node and an antihistamine substance; Simultaneous administration by the same administration route of two preparations obtained by separately formulating an extract of the present invention and an antihistamine substance, and (c) obtaining an extract of the lotus root node and an antihistamine substance separately. (D) administration of the two preparations at different times by the same administration route (for example, administration of the extract of the lotus root node and the antihistamine substance in the order or reverse order); Co-administration of the two preparations obtained by separately formulating the extract of the head portion and the antihistamine substance by different administration routes, (e) formulating the extract of the lotus root node and the antihistamine substance separately Different administration of the two resulting formulations It includes administration mode at an interval via the road.

  The content of the extract of the lotus root node and the antihistamine substance in the above-mentioned preparations differs depending on the purpose and the dosage form, and is not particularly limited. The content is preferably 0.001 to 30% by weight, more preferably 0.01 to 10% by weight, and the antihistamine substance is preferably 0.01 to 30% by weight, more preferably 0.1 to 10% by weight. The method of adding the active ingredient in the formulation may be added in advance or during the production, and may be appropriately selected in consideration of workability.

3. Anti-allergic composition The anti-allergic agent of the present invention can be incorporated into the anti-allergic composition together with appropriate additives as long as the effects of the present invention are not impaired. Examples of the antiallergic composition include antiallergic drugs or antiallergic foods and drinks. The drug of the present invention includes drugs used for animals, that is, veterinary drugs.

  The medicament of the present invention is a preventive agent for suppressing the onset of diseases and conditions associated with allergic symptoms, based on the IL-9 production inhibitory effect of an extract of lotus root node as an active ingredient and the antiallergic activity of an antihistamine substance. It functions as a drug and / or as a therapeutic to improve normal conditions. That is, the medicament of the present invention can be used for preventing the occurrence of allergic symptoms and for alleviating or improving allergic symptoms after the onset.

  Examples of diseases and conditions accompanied by allergic symptoms include, for example, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, bronchial asthma, urticaria, food allergy, hay fever, runny nose, stuffy nose, sneezing, itching of nose or eyes, Examples include, but are not limited to, itching in the throat. Among them, nasal hypersensitivity, particularly allergic nasal hypersensitivity caused by increased expression of IL-9 via the calcineurin / NFAT signaling system is preferred. Here, “allergic nasal hypersensitivity” means an allergic disease characterized by exhibiting some symptoms of recurrent sneezing, aqueous rhinorrhea, and nasal congestion (congestion of the nose).

  Since the active ingredient of the drug of the present invention is derived from a natural product, it has high safety and no side effects, and when used as a drug for treating and preventing the above-mentioned diseases or conditions, humans, mice, rats, rabbits, dogs, It can be administered orally or parenterally to mammals such as cats in a wide range of dosages.

  The medicament of the present invention can be prepared by mixing the above-mentioned antiallergic agent with pharmacologically and pharmaceutically acceptable additives, and formulating various preparations in a form suitable for application to the affected area. Pharmaceutically and pharmaceutically acceptable additives include formulation base materials and carriers, excipients, diluents, binders, lubricants, and coatings that are appropriately selected depending on the dosage form and application. Agents, disintegrants or disintegration aids, stabilizers, preservatives, preservatives, extenders, dispersants, wetting agents, buffers, solubilizers or dissolution aids, isotonic agents, pH regulators, propellants , A coloring agent, a sweetening agent, a flavoring agent, a flavor, etc. may be appropriately added to prepare various preparations which can be orally or parenterally administered systemically or locally by various known methods. When the drug of the present invention is provided in each of the above-mentioned forms, it can be manufactured by a manufacturing method usually used by those skilled in the art, for example, a manufacturing method shown in each section of the formulation of the Japanese Pharmacopoeia [2].

  Formulations for oral administration include, for example, excipients such as starch, glucose, sucrose, fructose, lactose, sorbitol, mannitol, crystalline cellulose, magnesium carbonate, magnesium oxide, calcium phosphate, or dextrin; carboxymethylcellulose, calcium carboxymethylcellulose, Disintegrant or disintegration aid such as starch or hydroxypropylcellulose; binder such as hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, gum arabic, or gelatin; lubricant such as magnesium stearate, calcium stearate, or talc Coating agents such as hydroxypropylmethylcellulose, sucrose, polyethylene glycol, or titanium oxide; petrolatum, liquid paraffin, polyethylene glycol Call, gelatin, kaolin, glycerin, purified water, or the like can be used base hard fat, but are not limited to.

  The form of the medicament of the present invention is not particularly limited. For example, tablets, sugar-coated tablets, capsules, troches, granules, powders, solutions, pills, emulsions, syrups, suspensions, elixirs Preparations such as oral preparations, injections (eg, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drops, nasal drops, eye drops, suppositories, ointments, lotions, Parenteral preparations such as sprays, transdermal absorbents, transmucosal absorbents, patches and the like can be mentioned. It may also be a dry product to be re-dissolved when used, and in the case of an injection preparation, it is provided in the form of a unit dose ampoule or a multi-dose container. These preparations can be prepared by conventional methods for preparation.

  The dose of the medicament of the present invention can be appropriately determined according to the type of disease, age, sex, body weight, symptom, administration route, administration schedule, formulation form, etc. of the administration subject. For example, in the case of oral administration to an adult, as a daily dose, the extract of lotus root node is 1 to 1000 mg, preferably 5 to 500 mg, more preferably 10 to 100 mg, in terms of solid content, an antihistamine substance. Is 0.01 to 20 mg, preferably 0.1 to 10 mg, more preferably 1 to 5 mg.

  In addition, the antiallergic agent of the present invention can be blended in foods and drinks. The food or drink of the present invention can be used as a functional food used for prevention or treatment of patients with the above-mentioned diseases and conditions accompanied by allergic symptoms. In the present invention, foods and drinks include, in addition to general foods and drinks, foods that can be taken for the purpose of maintaining and promoting health other than pharmaceuticals, for example, health foods, functional foods, health functional foods, or special use foods. It is used in the sense to include. Health foods include foods provided under names such as dietary supplements, health supplements, and supplements. Health functional foods are defined by the Food Sanitation Act or the Food Promotion Act, and are based on foods for specified health use and nutritionally functional foods and scientific evidence that can indicate the effects of specific health, nutritional function, and reduction of disease risk. Functional foods that can indicate the content of the notified functionality to the Commissioner of Consumer Affairs Agency are included. The special use foods include foods for the sick, foods for the elderly, foods for infants, foods for pregnant women, etc., which indicate that the foods are suitable for a specific subject or a patient having a specific disease. The form of the food or drink may be a form suitable for food, for example, any of solid, liquid, granular, granular, powder, capsule, cream, and paste.

  The types of foods and drinks include breads, noodles, confectionery, dairy products, processed marine and livestock products, oils and fats and oils and processed foods, seasonings, and various beverages (soft drinks, carbonated drinks, nutritional drinks, fruit drinks, dairy drinks) And the like, a concentrated stock solution of the beverage, a powder for adjustment, and the like, but are not limited thereto.

  The food or drink of the present invention may be appropriately blended with additives usually used depending on the type. As the additive, any additives can be used as long as they are acceptable in food hygiene. For example, sweeteners such as glucose, sucrose, fructose, isomerized liquid sugar, aspartame, and stevia; citric acid, malic acid, Acidulants such as tartaric acid; excipients such as dextrin and starch; binders, diluents, flavors, coloring agents, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, and suspensions Agents, preservatives and the like. In addition to the essential components described above, foods may contain other materials and additives (eg, excipients, disintegrants, emulsifiers, stabilizers, lubricants, and buffering agents) that are commonly used in foods as long as the effects of the present invention are not impaired. , Fragrances, etc.) can be appropriately compounded as necessary.

  The content of the antiallergic agent in the food and drink of the present invention may be an amount capable of exerting an antiallergic effect, and the general intake amount of the food and drink, the form of the food and drink, taste, taste, cost, and the like are considered. It may be appropriately determined in consideration of the above, but for example, it can be blended so as to be 0.1 to 99.0% by weight with respect to the whole food and drink.

  When taken for the purpose of preventing or ameliorating the above-mentioned diseases or conditions, the amount of the food or drink of the present invention varies depending on the condition of the subject to be taken, the form of intake, the amount of food consumed, etc. The extract is 1 to 1000 mg, preferably 5 to 500 mg, more preferably 10 to 100 mg, and the antihistamine substance is 0.01 to 20 mg, preferably 0.1 to 10 mg, more preferably 1 to 5 mg in terms of solid content. It is. The above amount may be taken at one time, or may be taken in several times (2 to 4 times). In the food and drink of the present invention, it is preferable that the amount of food and drink to be taken at one time is packaged or filled in one container such as a bag or a bottle in order to measure the intake amount.

  Hereinafter, the present invention will be described more specifically with reference to Examples and Comparative Examples, but these contents do not limit the present invention.

(Example 1) Evaluation of IL-9 mRNA expression inhibitory action of lotus root extract (comparison by site)
1. Test method
(1) Preparation of powder of lotus root extract 500 ml of distilled water was added to 40 g of each of the edible part, knot part, leaf, and seed of the Bichchu seed lotus root collected in Tokushima Prefecture and boiled at 100 ° C. for 30 minutes. This broth was dispensed into 50 ml centrifuge tubes and centrifuged at 3000 g for 10 minutes. The supernatant after the centrifugation was filtered, and the filtrate was powdered by freeze-drying to prepare a powder of a lotus root extract, which was used in an IL-9 mRNA expression inhibitory activity test.

(2) IL-9 mRNA expression inhibitory effect test (real-time PCR)
(2-1) Culture of RBL-2H3 cells Culture of rat basophil leukemia cells RBL-2H3 cells (purchased from Human Science Research Resource Bank, Human Science Promotion Foundation) requires MEM (containing Earl salt, L -Glutamine-containing, non-essential amino acid-containing, sodium bicarbonate-free) were used. Inactivated fetal bovine serum (FBS) by incubating with 500 ml of MEM and 6 ml of antibiotic (0.9% saline containing 10,000 Units / ml sodium penicillin G and 10 mg / ml streptomycin) at 56 ° C for 30 minutes It was added to a concentration of 10%. RBL-2H3 cells were cultured in a 6-well plate, added with the lotus root extract (10, 50, 100 μg / ml) prepared in (1), incubated for 22 hours, and added with 1 μM ionomycin (ionomycin) for 2 hours. Stimulation was performed and this was used as a test sample. The test group to which ionomycin was added without adding the lotus root extract was used as a positive control, and the test group to which neither lotus extract nor ionomycin was added was used as a negative control.

(2-2) Extraction of total RNA from RBL-2H3 cells After washing the test sample twice with PBS (-), 700 μl of RNAiso Plus (Takara) was added and scraped. After adding 210 μl of chloroform and shaking vigorously for 15 seconds to separate into two layers, the mixture was centrifuged at 15,000 rpm for 15 minutes at 4 ° C. The upper layer containing the RNA was collected, the same amount of isopropanol as the upper layer was added, the mixture was vigorously shaken for 15 seconds, and centrifuged at 15,000 rpm for 15 minutes at 4 ° C. to obtain pelleted RNA. The pellet was washed by adding 0.5 ml of 75% ethanol (-20 ° C). Furthermore, after centrifugation at 15,000 rpm for 15 minutes at 4 ° C., ethanol was removed, and diethylpyrocarbonate (DEPC) water was added to the obtained pellet to prepare an RNA solution. The absorbance was measured at 260 nm and 280 nm using a spectrophotometer (Thermo: Nanodorop ND-1000), and the total RNA concentration and purity of each test sample were measured by an assay based on the absorbance at 260 nm and the ratio of the two wavelengths.

(2-3) Synthesis of cDNA DEPC water was added to a sample tube so as to obtain an RNA solution equivalent to 1.0 μg of total RNA to make a total volume of 5 μl. A reverse transcription reaction solution having the composition shown in Table 1 was prepared, and a reverse transcription reaction was performed using a PrimeScript ^™ RT reagent Kit (Takara) with a thermal cycler (Biometra: T3000 Thermocycler) according to the program shown in Table 2.

(2-4) Real-time PCR
A PCR reaction solution having the composition shown in Table 3 was prepared, and 18 μl of a reagent was prepared per well of a Micro Amp Optical 96-well Reaction Plate.

  The PCR reaction was performed using the Sequence Detector (Gene Amp 7300 Sequence Detection System; PE Applied Biosystems) .Then, hold at 95 ° C for 30 minutes, melt at 95 ° C for 5 minutes, and anneal / extend at 60 ° C for 31 minutes. 40 cycles), PCR product amplification curves were detected in real time, analyzed and quantified using Sequence Detection software. The rat IL-9 primers and probes used are shown below.

[Rat IL-9 primer and probe set]
Forward: 5'-GAC GAC CCA TCA TCA AAA TGC-3 '(SEQ ID NO: 1)
Reverse: 5'-CTG TGA CAT TCC CTC CTG GAA-3 '(SEQ ID NO: 2)
Probe: FAM-TTG TGC CTC CCC ATC CCA TCT GAT-TAMRA (SEQ ID NO: 3)

  As an internal control that compensates for differences in RNA purity and reverse transcription efficiency, which are the main causes of quantitative RT-PCR fluctuations, expression fluctuations due to environmental conditions such as cell activation or proliferation are theoretically Instead, GAPDH (Glycelaldehyde-3-phosphate dehydrogenase), a housekeeping gene considered to be always expressed at a constant level, was used. In addition, commercially available products (Applied Biosystems: TaqMan Rodent GAPDH control reagents) were used as primers and probes for rat GAPDH. Also. As a quantification method, a Relative Standard Curve Method (Separate Tubes) for obtaining a relative value of mRNA expression level was used.

(2-5) Statistical processing The experimental data was shown as mean ± SEM. In addition, statistical processing was performed by One-way ANOVA and Dunnet's multiple comparison test using GraphPad Prism software (GraphPad Software INC., La Jolla, CA, USA). P <0.05 was considered significant.

2. Test Results FIG. 1 shows the results of the IL-9 mRNA expression inhibitory effect test of the extracts of each lotus root. As shown in FIG. 1, the nodal extract clearly had an inhibitory effect on IL-9 mRNA expression, but could not be confirmed in leaves, seeds, and edible parts.

Example 2 Evaluation of IL-9 mRNA Expression Inhibitory Effect of Lotus root Extract (Comparison between Nodal and Edible Parts)
The nodes and edible parts of the Bichchu lotus root collected in Tokushima Prefecture were extracted in the same manner as in Example 1, and the IL-9 mRNA expression inhibitory activity of the extracts was measured. As shown in FIG. 2A, IL-9 mRNA expression inhibitory activity was significantly observed at the nodes and near the nodes (1 cm from the ends of the nodes). In addition, the same test was carried out at different concentrations for the extract in which the IL-9 mRNA expression inhibitory activity was observed and the extract near the node (part from the end of the node to 1 cm). Although the inhibitory activity was increased, the inhibitory activity was not observed in the edible portion 1 to 2 cm from the node where the inhibitory activity was not observed even if the concentration was increased (FIG. 2B).

Example 3 Evaluation of Suppressive Effect of Lotus Root Extract on Nasal Hypersensitivity 1. Test Method
(1) Preparation of powder of lotus root extract 500 ml of distilled water was added to 40 g of a lotus root lotus node (used in this example) collected in Tokushima Prefecture and boiled for 30 minutes. This broth was dispensed into 50 ml centrifuge tubes and centrifuged at 3000 g for 10 minutes. The supernatant after this centrifugation treatment was filtered, and the filtrate was powdered by freeze-drying to prepare a lotus root extract powder. A part of this powder was tested for its IL-9 mRNA expression inhibitory activity test (50% (Confirmation test of expression inhibition concentration).

(2) TDI-sensitized nasal hypersensitivity model rat test
(2-1) Rat sensitization by TDI
The antiallergic activity of the lotus root node extract prepared in (1) was evaluated using TDI-sensitized nasal hypersensitivity model rats.
Six-week-old male Brown-Norway rats (200 g, SLC, Hamamatsu, Japan) were used. Animals were housed at a room temperature of 22 ± 1 ° C. with a 12-hour day-night cycle. For sensitization of rats with TDI (toluene 2,4-diisocyanate), a modification of the method of Kitamura et al. (Acta Otolaryngol. 2004, 124, 1053-1058) was used. That is, 10 μl of a 10% TDI-ethyl acetate solution was applied twice a day for 5 days every day using a very fine nose swab in both nose vestibules of a Brown-Norway male rat (TDI sensitization). After 1 week of treatment-free period, seizures were induced by nasal vestibular application of 10% TDI solution. FIG. 3 shows a schedule for preparing TDI-sensitized nasal hypersensitivity model rats.

  For the observation of symptoms and comparison of test results, a test group coated with an ethyl acetate solution instead of TDI was used as a negative control on the same schedule as the above-described sensitization and seizure induction by TDI. Further, sensitization and seizure induction by the above-mentioned TDI were performed, but a test group to which no drug was administered was used as a positive control. During the preparation process, TDI-sensitized nasal hypersensitivity model rats were treated with drugs such as epinastine (epinastine hydrochloride, Sigma) 24 mg / kg, lotus root node extract 40 mg / kg, or epinastine 24 mg / kg and lotus root node extract 40 mg / kg were orally administered once a day for 3 consecutive weeks.

(2-2) Method for evaluating allergic symptoms of nasal hypersensitivity As for allergic symptoms of nasal hypersensitivity, “water-soluble rhinorrhea” and “nose swelling and redness” were evaluated and scored using the indexes shown in Table 4 below.

2. Test Results FIG. 4 shows the results of the IL-9 mRNA expression inhibitory effect test of the lotus root node extract. The 50% expression inhibitory concentration of the Lotus root node extract for inhibiting IL-9 mRNA expression was 27.4 μg / ml. FIG. 5 shows the results of evaluating the allergic symptoms of nasal hypersensitivity of the same lotus root extract. As shown in FIG. 5, when the lotus root node extract was mixed and administered with epinastine, which is used as an anti-allergic drug, the nasal symptoms (water-soluble rhinorrhea, nasal discharge, Swelling and redness) and a very strong synergistic effect was confirmed.

  INDUSTRIAL APPLICABILITY The present invention can be used in the field of manufacturing foods and drinks such as pharmaceuticals, health foods and functional foods for treating, improving and preventing diseases and conditions associated with allergic symptoms.

Claims (3)

An antiallergic agent comprising a hot water extract of lotus root and epinastine or a salt thereof . An antiallergic composition comprising the antiallergic agent according to claim 1 . The antiallergic composition according to claim 2 , which is a pharmaceutical or a food or drink.

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