Composition for improving allergic rhinitis and / or sinusitis symptoms

Abstract

To provide a composition for improving allergic rhinitis and / or sinusitis and their symptoms due to air pollutants.SOLUTION: The present disclosure provides a composition for improving allergic rhinitis and / or sinusitis and their symptoms due to air pollutants, the composition containing Magnolia flower or extract thereof as an active ingredient.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a composition for improving allergic rhinitis and / or sinusitis symptoms. More specifically it relates to a composition for improving allergic rhinitis and / or sinusitis symptoms caused by air pollutants .

Background Art

[0002] With the development of industry and the increase in population, air pollution has become a serious problem worldwide. Among various air pollutants such as PM2 .5, exhaust gas, and yellow sand, it has been reported that they can cause respiratory diseases such as asthma and bronchitis and circulatory diseases such as heart disease.

[0003] The involvement of air pollutants in allergic rhinitis and sinusitis has been attracting attention for many years. In Non-Patent Document 1, it has been clarified by an epidemiological survey that the prevalence of allergic rhinitis and sinusitis is higher in polluted areas than in non-polluted areas. In Non-Patent Document 2, in the nasal secretions of men and women who inhaled urban air dust, the inflammatory cytokine IL-1β was significantly increased by 72. 3%, IL-6 was 42.2%, and IL-8 was 19.7%, indicating that these cytokines are closely related to nasal inflammation. In addition, in Non-Patent Document 3, IL-1β was significantly increased in the nasal secretions of patients with sinusitis, indicating that it is the cause of sinusitis. In Non-Patent Document 4, it has been shown that the water-soluble components of mast cell lines in PM2.5 cause degranulation, and diesel exhaust particles, etc. act as adjuvants for the antigen-antibody reaction of allergic rhinitis (Non-Patent Document 5). 3%, IL-6 was 42.2%, and IL-8 was 19.7%, indicating that these cytokines are closely related to nasal inflammation. In addition, in Non-Patent Document 3, IL-1β was significantly increased in the nasal secretions of patients with sinusitis, indicating that it is the cause of sinusitis. In Non-Patent Document 4, it has been shown that the water-soluble components of mast cell lines in PM2.5 cause degranulation, and diesel exhaust particles, etc. act as adjuvants for the antigen-antibody reaction of allergic rhinitis (Non-Patent Document 5). In Non-Patent Document 4, it has been shown that the water-soluble components of mast cell lines in PM2.5 cause degranulation, and diesel exhaust particles, etc. act as adjuvants for the antigen-antibody reaction of allergic rhinitis (Non-Patent Document 5). In Non-Patent Document 4, it has been shown that the water-soluble components of mast cell lines in PM2.5 cause degranulation, and diesel exhaust particles, etc. act as adjuvants for the antigen-antibody reaction of allergic rhinitis (Non-Patent Document 5). In Non-Patent Document 4, it has been shown that the water-soluble components of mast cell lines in PM2.5 cause degranulation, and diesel exhaust particles, etc. act as adjuvants for the antigen-antibody reaction of allergic rhinitis (Non-Patent Document 5). In Non-Patent Document 4, it has been shown that the water-soluble components of mast cell lines in PM2.5 cause degranulation, and diesel exhaust particles, etc. act as adjuvants for the antigen-antibody reaction of allergic rhinitis (Non-Patent Document 5).

Prior Art Documents

Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

[0005] However, there is little information on substances that are effective against allergic rhinitis and sinusitis caused by air pollutants. There have been few reports on this phenomenon, and it is necessary to propose countermeasures based on molecular mechanisms. There are.

[0006] Therefore, the present invention provides a method for effectively improving allergic rhinitis and sinusitis caused by air pollutants. The object of the present invention is to provide a composition which [Means for solving the problem]

[0007] In order to solve the above problems, the present inventors have conducted extensive research and have found that Xin-i, a type of herbal medicine, (Mahiura japonica) and its extracts suppress the expression of IL-1β, an inflammatory cytokine. They found that this inhibits the release of β-hexosaminidase, and thus completed the present invention.

[0008] That is, the present invention provides the compositions described below. [1] A drug for treating allergies caused by air pollutants, containing scutellaria or its extract as an active ingredient. A composition for improving chronic rhinitis and / or sinusitis and their symptoms. [2] The symptoms are selected from one or more of sneezing, runny nose, nasal congestion, watering eyes, sore throat, and feeling of heaviness in the head The composition according to [1], wherein the symptoms are one or more selected from the group consisting of [3] A composition for inhibiting the release of β - hexosaminidase or for inhibiting the expression of IL - 1β, containing Shin - i or an extract thereof as an active ingredient [4] The composition according to [3], which improves allergic rhinitis, sinusitis caused by air pollutants, and their symptoms [5] The composition according to [4], wherein the air pollutants are urban air dust and / or diesel exhaust particles [6] A composition for improving the ciliary function of mucosal epithelial cells, containing Shin - i or an extract thereof as an active ingredient [7] The composition according to [6], which improves cough due to nasal congestion (or nasal congestion accompanied by cough), ease of nasal discharge, or sputum congestion

Advantages of the Invention

[0009] According to the present invention, when allergic rhinitis and / or sinusitis caused by air pollutants occur, it is possible to effectively improve their symptoms.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0011] [Composition for Improving Allergic Rhinitis and / or Sinusitis Caused by Air Pollutants and Their Symptoms [Composition for Use] In one embodiment, the composition of the present invention contains Shin-yi, a crude drug, or an extract thereof . Further, the present invention is particularly suitable for improving allergic rhinitis and / or sinusitis caused by air pollutants and their symptoms. Further, the present invention is applicable, although not limited thereto, as a daily anti-pollution measure.

[0012] In addition, the present invention is suitable for those who are concerned about the air (atmosphere) in the city, those who are concerned about the exhaust gas of automobiles, those who are concerned about PM10 or PM2.5, those who are concerned about yellow sand, those who want to take anti-pollution measures , and those who are concerned about tobacco smoke, etc.

[0013] In the present specification, Shin-yi is, although not limited thereto, the flower buds of Magnoliaceae plants such as Magnolia denudata, Magnolia biondii, Magnolia sieboldii , Kobus magnolia, Michelia compressa, etc., or other related plants, dried and contains magnoflorine, etc.

[0014] When using Shin-yi as an extract, as the extraction solvent, water (including hot water), alcohols such as methanol, ethanol, isopropanol, ethylene glycol, glycerin, etc., esters such as ethyl acetate, ketones such as acetone and methyl ethyl ketone, nitriles such as acetonitrile, ethers such as diethyl ether and tetrahydrofuran , saturated hydrocarbons such as pentane, hexane, cyclopentane, cyclohexane, toluene Aromatic hydrocarbons such as benzene, halogenated hydrocarbons such as dichloromethane and chloroform , and other organic solvents such as dimethylformamide and dimethyl sulfoxide (all of which may contain water) can be appropriately used, and it may be a single kind or an arbitrary mixture of two kinds . Among these solvents, water, ethanol, or a mixed solution thereof is preferable from the viewpoint of safety .

[0015] In addition, when using the extract of Shin-yi as an extract, those produced according to the method for producing the crude drug-related preparation "extract preparation" described in the General Rules of Preparations of the 17th Revised Japanese Pharmacopoeia can also be used . As the above method, for example, an appropriate extractant is added to the crude drug of appropriate size , and the extract is obtained by cold extraction or warm extraction for a certain period of time, or the crude drug of appropriate size is weighed out in accordance with the prescription in a certain amount, about 10 to 20 times the amount of water is added to the total amount and heated for a certain period of time to obtain an extract . The obtained extract is usually subjected to solid-liquid separation such as centrifugation and filtration , and the solid content is removed before use .

[0016] As the extraction method with hot water, for example, the method of immersing Shin-yi as it is or after crushing in hot water can be mentioned . The extraction temperature with hot water is preferably 40 to 100°C, more preferably 70 to 100°C . The extraction time with hot water is preferably 10 to 120 minutes, more preferably 60 to 100 minutes . As the extraction method with alcohols, it is preferable to use ethanol , the extraction temperature is preferably 20 to 50°C, the extraction time is preferably 10 to 120 minutes, and 60 to 100 minutes is more preferable

[0017] The extract of Shin-yi may be in a liquid state, but if necessary, it can be dried under reduced pressure or freeze-dried By performing a drying treatment such as air drying, spray drying, etc. to reduce or remove the liquid content, a concentrated liquid, semi- solid, solid, or powdered product may be used.

[0018] The extract of Xin Yi can also use commercially available products. For example, it is possible to use Xin Yi extracts from companies such as Delta International Co., Ltd. and Matsuura Pharmaceutical Co., Ltd.

[0019] Xin Yi or its extract in the present invention also includes those that are constituent crude drugs in traditional Chinese medicine prescriptions. Specific examples of traditional Chinese medicine prescriptions containing Xin Yi as a constituent crude drug include Chai Ge Tang plus Chuanxiong and Xin Yi, Xin Yi Qing Fei Tang, Li Ze Tong Qi Tang plus Xin Yi, etc., but are not limited thereto.

[0020] The daily dosage (administered amount) of Xin Yi in the composition of the present invention can be appropriately set according to the type and amount of other components, the condition of the user (weight, age, gender, symptoms, physical condition, etc.), and the dosage form, etc., and is not limited. However, from the perspective of more significantly exerting the effects of the present invention, expressed in terms of the equivalent amount of crude drug, for example, it can be about 1 to about 5000 mg, more preferably about 50 to about 4 000 mg, even more preferably about 100 to about 3500 mg, particularly preferably about 150 to about 3500 mg, and most preferably about 200 to about 3000 mg. Also, in another embodiment, the daily dosage (administered amount ) of Xin Yi in the composition of the present invention, expressed in terms of the equivalent amount of crude drug, can be, for example, about 200 to about 2500 mg, about 200 to about 220 0 mg, about 200 to about 1800 mg, about 200 to about 1500 mg. Moreover, the daily oral dosage of the Xin Yi extract, expressed in terms of the dry solid content of the extract,

[0021] can be, for example, about 1 to about 5000 mg, more preferably about 50 to about 4 For example, it can be about 0.01 to about 2700 mg, more preferably about 0.1 to about 1800 mg, even more preferably about 0.5 to about 1500 mg, particularly preferably about 1 to about 1200 mg, and most preferably about 3 to about 900 mg. In another embodiment, the oral dosage of the Shin'ei extract per day, in terms of the dry solid content of the extract, for example, can be about 3 to about 900 mg, about 3 to about 800 mg, about 3 to about 700 mg, about 3 to about 600 mg, about 3 to about 500 mg, about 3 to about 400 mg, about 3 to about 300 mg, about 3 to about 250 mg.

[0022] As used herein, "in terms of crude drug" means the weight (dry weight) of the crude drug (crude drug mixture) required to obtain the amount of its components. When using an extract as the crude drug, the dry weight of the crude drug required to obtain the amount of the extract is the amount in terms of crude drug. The dosage per day can be taken in 1 to 6 times, preferably 1 to 3 times. The content of Shin'ei may vary depending on the type and amount of other components, the condition of the user (weight, age, symptoms, physical condition, etc.), and the dosage form, etc. When the composition is a solid preparation, based on the total amount of the composition,

[0023] it is usually 0.01 to 99% by weight, preferably 0.05 to 95% by weight, more preferably 0.1 to 90% by weight, and particularly preferably 1 to 85% by weight. Also, when the total content of the crude drugs for rhinitis is considered, when the composition is a liquid preparation, it is usually 0.01 to 80% by weight with respect to the total amount of the composition, preferably 0.05 to 70% by weight, more preferably 0.1 to 60% by weight, and particularly preferably 1 to 55% by weight. When the solid preparation includes a liquid preparation, the liquid preparation is considered in the same way. Note that when the solid preparation includes a liquid preparation, the liquid preparation Based on the total amount, it is usually 0.01 to 70% by weight, preferably 0.05 to 60% by weight, more preferably 0.1 to 50% by weight, and particularly preferably 1 to 50% by weight.

[0024] If desired, the composition of the present invention may contain other physiologically active components in addition to Shin-i. That's fine.

[0025] Examples of such physiologically active components include (1) antihistamine components (e.g., mequitazine, loratadine, ketotifen fumarate , cetirizine hydrochloride, epinastine hydrochloride, fexofenadine hydrochloride, olopatadine hydrochloride , isothipendyl hydrochloride, iproheptine hydrochloride, dipheteol hydrochloride, diphenyl pyrraline hydrochloride, diphenhydramine hydrochloride, triprolidine hydrochloride hydrate, tripelennamine hydrochloride, tonzylamine hydrochloride, promethazine hydrochloride, methdilazine hydrochloride, diphen hydramine salicylate, diphenyl disulfonic acid carbinoxamine, alimemazine tartrate, diphenhydramine tannate, diphenylpyrraline theophyllinate, carbinoxamine maleate, chlorpheniramine maleate, promethazine methylenedisalicylate ), (2) parasympathetic nerve blocking components (e.g., belladonna total alkaloids, isopropamide iodide , datura extract, rhubarb extract, etc.), (3) sympathetic nerve stimulating components (e.g., methyl ephedrine, pseudoephedrine, phenyl ephrine, methoxyphenamine or their salts, etc.), (4) anti-inflammatory enzymes (e.g., lysozyme, bromelain, etc.), (5) glycyrrhizic acids (e.g., glycyrrhizic acid or its salts, etc.), (6) xanthine derivatives (e.g., sodium benzoate caffeine, caffeine hydrate , caffeine such as anhydrous caffeine, and the like.

[0026] These bioactive components may be in the free form or in the form of salts.

[0027] From the viewpoint of stably exerting the effects of the present invention, as these bioactive components, mexitil , methyl ephedrine and its salts (such as methyl ephedrine hydrochloride), pseudoephedrine and its salts (such as pseudoephedrine hydrochloride), belladonna total alkaloids, glycyrrhizin acid or its salts (such as dipotassium glycyrrhizinate), and at least one selected from the group consisting of anhydrous caffeine are preferable.

[0028] The composition of the present invention can be, for example, a pharmaceutical product, a quasi-drug, or a raw material thereof.

[0029] The composition of the present invention can be prepared into various dosage forms as solid preparations according to methods known to those skilled in the art. There are no particular limitations on the shape and size of the solid preparations. For example, as oral preparations, tablets [including orally disintegrating tablets, chewable tablets, effervescent tablets, dispersible tablets, soluble tablets, film-coated tablets, plain tablets, sugar-coated tablets, etc.], capsules [including hard capsules and soft capsules, etc.], granules [including effervescent granules], powders, fine powders, fine granules, pills, oral tablets [including lozenges, sublingual tablets, buccal tablets, adhesive tablets, and gums, etc.], film agents, dry syrups, jelly agents, oral semi-solid agents, confectionery products [including candies (sweets), gummies, and nougat agents, etc.], etc. can be mentioned. Further, the composition of the present invention can be prepared into various dosage forms as liquid preparations. For example, as oral preparations, syrups, solutions, suspensions, etc. [including troches, sublingual tablets, buccal tablets, adhesive tablets, and gums, etc.], film agents, dry syrups, jelly agents, oral semi-solid agents, confectionery products [including candies (sweets), gummies, and nougat agents, etc.], etc. can be mentioned. Further, the composition of the present invention can be prepared into various dosage forms as liquid preparations. For example, as oral preparations, syrups, solutions, suspensions, etc. etc. Among these, liquid preparations that can more significantly exhibit the effects of the present invention are From this viewpoint, the composition of the present invention is preferably used in a solid preparation, such as a tablet, a capsule, or a granule. It is more preferable to use it in the form of a tablet, a powder, or a film, and more preferably in the form of a tablet or a capsule. More preferred.

[0030] [Application] By using the present invention, it is possible to suppress the expression of IL-1β, an inflammatory cytokine, and This makes it possible to effectively suppress the release of β-hexosaminidase. , prevention and treatment of symptoms and conditions involving the expression of IL-1β and the release of β-hexosaminidase. It is effective for improvements, etc.

[0031] The composition of the present invention is effective in treating conditions accompanied by rhinitis as a symptom and symptoms associated with rhinitis (nasal discharge (rhinorrhea, runny nose, excessive nasal discharge), nasal congestion (stuffy nose), sneezing, watery eyes, itching, sore throat, heavy head (headache) severe), swelling of the nasal mucosa, congestion of the nasal cavity, nosebleeds, and atrophy or hardening of the nasal mucosa. It is believed to be applicable to a wide range of conditions, and is highly effective in treating acute rhinitis, sinusitis, and allergic rhinitis. The composition of the present invention is useful for treating nasal discharge, nasal congestion, or sneezing, or acute rhinitis caused by allergens. The present invention can be suitably used for, but is not limited to, rhinitis, sinusitis, or allergic rhinitis. The composition of Ming is sticky nasal mucus, mucus dripping down the throat, mucus coming out of the nostrils, shortness of breath, headaches, etc. The composition of the present invention can be preferably used for treating symptoms such as drowsiness. (for oral administration).

[0032] In particular, by suppressing inflammation, it activates the cilia movement of the nasal mucosa and reduces coughing (or is expected to be able to improve nasal congestion accompanied by cough, nasal discharge, or phlegm congestion (Non-Patent Documents 6 and 7). It is measured (Non-Patent Documents 6 and 7).

[0033] When the above allergen is an air pollutant, the composition of the present invention is also useful as an anti-pollution measure. Although anti-pollution measures for the body have been emphasized in recent years, many products are external skin preparations. In the composition of the present invention, for example, but not limited to, by using it for internal use (for oral administration), anti-pollution measures can be taken from within the body, which is useful. As described in Non-Patent Document 7, for anti-pollution measures, biological defense mechanisms such as mucus secretion and mucociliary movement are developed, but when inflammation occurs in mucosal epithelial cells and the like, these biological defense mechanisms break down, making it difficult to appropriately discharge air pollutants. By using the composition of the present invention, by suppressing inflammation in mucosal epithelial cells and the like, the ciliary movement of the nasal mucosa can be activated, and it becomes possible to improve and enhance the biological defense mechanism against air pollutants. Although anti-pollution measures for the body have been emphasized in recent years many products are external skin preparations. In the composition of the present invention, for example, but not limited to, by using it for internal use (for oral administration), anti-pollution measures can be taken from within the body which is useful. As described in Non-Patent Document 7, for anti-pollution measures biological defense mechanisms such as mucus secretion and mucociliary movement are developed, but when inflammation occurs in mucosal epithelial cells and the like, these biological defense mechanisms break down, making it difficult to appropriately discharge air pollutants By using the composition of the present invention, by suppressing inflammation in mucosal epithelial cells and the like the ciliary movement of the nasal mucosa can be activated, and the biological defense mechanism against air pollutants can be improved and enhanced.

[0034] The dosage of the composition of the present invention is appropriately set according to its form, administration method, administration purpose, and the age, weight, symptoms, and physical condition of the subject to whom the composition is administered, and is not constant. In addition, the administration of the composition of the present invention may be carried out once a day or divided into several times within the desired dosage range, and may be administered before meals, between meals, after meals, or simultaneously with meals. Note that the term "administration" in this specification is used with the intention of including "taking". The dosage of the composition of the present invention is appropriately set according to its form, administration method, administration purpose, and the age, weight, symptoms, and physical condition of the subject to whom the composition is administered, and is not constant. Also, the administration of the composition of the present invention may be carried out once a day or divided into several times within the desired dosage range, and may be administered before meals, between meals, after meals, or simultaneously with meals Note that the term "administration" in this specification is used with the intention of including "taking". The composition of the present invention can usually be administered 1 to 6 times a day, preferably 1 to 3 times a day

[0035] It can be administered Therefore, the composition of the present invention for a single administration preferably contains an amount obtained by dividing the above-mentioned daily dosage by the number of administrations per day. In addition, since the composition of the present invention has improved solubility in water and is excellent in the rapid solubility and / or rapid release property of the drug, administration 1 to 3 times a day is preferable, and it can be used by administration once a day, twice a day, or three times a day. It is preferably divided by the number of administrations per day. Since the composition of the present invention has improved solubility in water and is excellent in the rapid solubility and / or rapid release property of the drug, administration 1 to 3 times a day is preferable, and it can be used by administration once a day, twice a day, or three times a day.

Examples

[0036] Next, the present invention will be specifically described by way of examples, but the present invention is not limited to the following examples. When there is no special description of the reagents used, special grade reagents from Wako Pure Chemical Industries were used. When there is no special description of the reagents used, special grade reagents from Wako Pure Chemical Industries were used.

[0037] [Test Example 1: IL-1β inhibition test of Shin-i in human nasal septum squamous epithelial cells] (1-1) Culture conditions Human nasal septum squamous epithelial cells (RPMI2650 cells) were cultured in EMEM (ATCC) supplemented with Fetal bovine serum (FBS) as serum, and Anti biotic-Antimycotic (hereinafter also referred to as A.A) (gibco), L-Glutamine (gibco). EMEM (10% FBS, +A.A ) was used. The culture was carried out using tissu Culture Dish 60 mm and 100 mm. The medium was changed every 3 days. In this test, a cell suspension prepared at 1.0 x 10 6 cells / ml was seeded at 1 ml per well, and the experiment was conducted with 1 .0 x 10 6 cells per well.

[0038] (1-2) Method for preparing the added sample ​​Shin-ie extract (meeting the requirements of the item on Shin-ie in the 17th Revised Japanese Pharmacopoeia; the same shall apply hereinafter) was adjusted to 10 and 50 mg / ml (in terms of crude drug equivalent) to 125 mg / ml and 625 mg / ml, respectively, with EMEM (10% FBS, +A.A), centrifuged at 12,000 rpm for 2 min, and the supernatant was used as the Shin-ie extract addition solution.

[0039] TNFα (Recombinant Human TNFα R&D systems ) was diluted 100-fold with EMEM (10% FBS, +A.A) from a 100 μg / ml stock to obtain the TNFα addition solution.

[0040] (1-3) Test conditions To RPMI2650 cells cultured in a 12-well plate for 48 h, the Shin-ie extract addition solution (10 and 50 mg / ml) was added at 10 μl / well, and the mixture was incubated at 37°C and 5% CO 2 for 1 h. Thereafter, the TNFα addition solution (1 μg / ml) was added at 10 μl / well, and the mixture was incubated at 37°C and 5% CO2 for 6 h. For Veh icle ((-)), the same volume of EMEM (10% FBS, +A .A) was added at the time of adding each sample.

[0041] (1-4) Gene analysis method RNA was extracted using the RNeasy (registered trademark) Mini kit (QIAGEN) according to the recommended protocol. The RNA sample was measured for RNA concentration using a NanoDr op ND-1000 (Thermo Fisher Scientific), treated with DNase, and converted to cDNA by reverse transcription reaction. The cDNA sample after the reverse transcription was diluted 5-fold with RNase free water It was stored at -30°C.

[0042] Gene expression analysis was performed by the Real-Time PCR method using TaqMan probes with a QuantStudio (registered trademark) 3 real-time PCR system (Thermo Fisher Scientific). ACTB was used as the internal standard and data analysis was performed using the ΔΔCT method.

[0043] (1-5) Results The results of verifying the anti-inflammatory effect of Shin-ie extract are shown in Figure 1. Shin-ie extract significantly decreased the mRNA expression level of IL-1β, which is increased by TNFα, in a concentration-dependent manner (*p < 0.05, ***p < 0.0005).

[0044] [Test Example 2: Inhibition test of β-hexosaminidase release of Shin-ie in rat basophilic leukemia cells To verify the effect of Shin-ie extract on degranulation, the inhibitory activity of the release of β-hexosaminidase induced by Fc R1-IgE-antigen crosslinking was evaluated.

[0045] (2-1) Culture conditions Rat basophilic leukemia cells (RBL-2H3 cells) were cultured in DMEM supplemented with fetal bovine serum (FBS) as serum, Antibiotic-Antimycotic (A.A) (gibco) as an antibiotic, and L-Glutamine (gibco). DMEM (10% FBS, +A.A) was used. The culture was carried out using Cell Culture Flasks 70ml (Falcon). The medium was changed every 3 days. In this test, it was prepared at 5.0x10 cells / ml in a 24-well plate 5 ​ 0.5 ml of the prepared cell suspension was seeded with 2.5 x 10 5 cells per well. Then, RBL-2 H3 cells cultured overnight at 37°C under 5% CO2 were used for the assay.

[0046] (2-2) Method for preparing the added sample The Shin-ye extract was adjusted to 0.1, 1, and 10 mg / ml (equivalent amounts of crude drugs, 1.25 mg / ml, 12.5 mg / ml, and 125 mg / ml, respectively) with PIPES buffer, centrifuged at 12,000 rpm for 2 min, and the supernatant was used as the Shin-ye extract-added solution.

[0047] (2-3) Test conditions The medium of RBL-2H3 cells was removed and the cells were washed twice with PBS. An anti-DNP-IgE antibody mixed medium adjusted to 100 ng / ml with DMEM (10% FBS, +A.A) was added, and the cells were cultured at 37°C under 5% CO2 for 2 hours.

[0048] The mixed medium was removed and the cells were washed twice with PIPE buffer. 490 μl of the Shin-ye extract, tranilast, or PIPES buffer alone dissolved in PIPES buffer was added, and the mixture was reacted at 37°C under 5% CO2 for 10 minutes. 10 μl of DNP-BSA at 150 ng / ml dissolved in PIPES buffer was added, and the cells were cultured at 37°C under 5% CO2 for 30 minutes. After collecting the culture supernatant, the cells were lysed with 0.1% Triton X-100 / PIPES buffer, and the cell lysate was collected. 50 μl of the supernatant and the lysate were added to a 96-well plate and heated at 37°C for 5 minutes. 50 μl of Substrate and the lysate were added to a 96-well plate and heated at 37°C for 5 minutes. 50 μl of Substrate Buffer (prepared according to the formulations shown in Tables 1 and 2 below) was added, and the reaction was allowed to proceed at 37 °C for 25 minutes. To the reaction solution, 100 μl of Stop Buffer (prepared according to the formulation shown in Table 3 below) was added to stop the reaction, and then the absorbance at 405 nm was measured. The release rate of β-hexosaminidase was determined by the following Equation 1.

[0049] [Table 1]

[0050] [Table 2]

[0051] [Table 3]

[0052] [Equation 1] β-hexosaminidase release rate = (Absorbance of culture supernatant / Absorbance of culture supernatant + Absorbance of cell lysate) × 100

[0053] (2 - 4) Results The results of verifying the effect of Shin-ye extract on the release of β-hexosaminidase are shown in Figure 2. Pretreatment with Shin-ye extract significantly inhibited the release of β-hexosaminidase in a concentration-dependent manner. (*p < 0.00001, **p < 0.0005 (vs. control))

[0054] [Test Example 3: Inhibitory effect of Shin-ye extract on the expression of adhesion factor VCAM-1 in human vascular endothelial cells Test] (3 - 1) Culture conditions Human umbilical vein endothelial cells (HUVEC) were cultured using HuMedia EG2 (KURABO). ​​​​It was cultured in Corning (registered trademark) CellBIND (registered trademark) Surface cell culture flasks CellBIND 75cm 2 (Cornin g) was used. The medium was changed every three days. In this experiment, 2.0x10 5 cells / ml of the prepared cell suspension was seeded at 0.5 ml per well, and ell was performed at a cell count of 1.0x10 5 cells per well.

[0055] (3-2) Preparation method of the addition sample The Shin-ie extract was adjusted to 1 mg / ml in HuMedia EG2 (in terms of the equivalent amount of crude drug, 12. 5 mg / ml), centrifuged at 12,000 rpm for 2 min, and the supernatant was used as the Shin-ie extract-added medium.

[0056] TNFα (Recombinant Human TNFα R&D systems ) was diluted 100-fold with HuMedia EG2 at 100 μg / ml to 1 μg / ml as the TN Fα addition solution.

[0057] (3-3) Test conditions HUVEC cultured in a 24-well plate for 24 hours was changed to a medium of 0.5 ml of Shin-ie extract-added medium (1 mg / ml), and incubated at 37°C under 5% CO2 for 1 hour in an incubator. Then, the TNFα addition solution (1 μg / ml) was added at 5 μl / well, and 37 °C, incubated for 4 hours under 5% CO2. For Vehicle ((-)), the same amount of HuMedia EG2 was added at the time of adding each sample.

[0058] (3-4) Gene analysis method Gene analysis was performed in the same manner as in Methods 1-4. In addition, the internal standard was GAPDH.

[0059] (3-5) Results The results of verifying the effect of Shin-ye extract on the adhesion factor VCAM-1 are shown in Figure 3. Shin extract significantly decreased the mRNA expression level of VCAM-1, which is increased by TNFα. (*p < 0.001, **p < 0.01) (*p < 0.001, **p < 0.01)

Claims

[Claim 1] A composition for inhibiting the release of β-hexosaminidase or for inhibiting the expression of IL-1β, comprising Aspergillus or an extract thereof as an active ingredient.

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