KR20200099357A - Pharmaceutical Composition for Preventing or Treating Allergic Disease Containing Extract from Bark of Prunus serrulata - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of allergic diseases, comprising a Prunus serrulata bark extract as an active ingredient. According to an embodiment of the present invention, the Prunus serrulata bark extract excellent effect of inhibiting the activity of transcription factor NF-κB as a transcription factor, expression of TNF-α and IL-4 as inflammation-inducing cytokines, and the release of histamines from mast cells involved in allergic reactions. In addition, the Prunus serrulata bark extract shows effectiveness in treating passive local skin allergic reactions and systemic allergic reactions in animal models, thereby being usefully used in the prevention and treatment of allergic diseases.

Description

Pharmaceutical Composition for Preventing or Treating Allergic Disease Containing Extract from Bark of Prunus serrulata}

The present invention relates to a composition for preventing or treating allergic diseases comprising a cherry bark extract as an active ingredient.

Allergy is a systemic or local disorder of a living body based on an immune response, and allergies appearing in the human body are classified into types Ⅰ, Ⅱ, Ⅲ and Ⅳ according to the immune mechanism. Type I-III allergy is a humoral immune response (immediate type) in which antibodies are involved, and type IV is a cellular immune response (delayed type) in which cells are directly involved.

Most allergic reactions are known as type I. The antigen (allergen), a substance that causes allergies, enters the body through the respiratory tract, digestive tract, and skin, and binds to the IgE antibody attached to the receptor (FcεRI) on the surface of the mast cell in the tissue, and thus the mast cell Histamine is secreted through degranulation and type I allergy is immediately induced. Subsequently, leukotriene and various inflammatory cytokines are secreted from mast cells to induce late allergic reactions such as tissue damage and inflammatory reactions.

As allergy treatment drugs currently used clinically, there are degranulation inhibitors, chemical transporter inhibitors, and chemical transporter synthesis inhibitors depending on the mechanism of action. In the case of degranulation inhibitors, their mechanism of action is unclear, and because they show tolerance within a short time after administration, they often fail to improve the symptoms of patients after a certain period of time or when repeated administration. Most allergy treatments are being studied in the direction of relieving symptoms rather than eliminating the cause, and drugs with excellent therapeutic effects are widely used as steroids for allergic rhinitis and asthma, but long-term use of steroids is a serious side effect. Can lead to In addition, long-term use of antihistamines causes side effects such as lethargy, depression, and concentration disorders, and there is a problem that allergic diseases recur when the use is stopped. Accordingly, there is a need to develop a safe drug that has excellent anti-allergic effect and has no side effects even in long-term treatment.

Accordingly, in order to overcome the low recovery rate and side effects of the existing allergy treatment drugs, the present inventors completed the present invention by conducting research on drugs for the prevention, improvement or treatment of allergic diseases based on herbal materials.

Republic of Korea Patent Publication No. 10-2018-0122983

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating allergic diseases, comprising a cherry bark extract as an active ingredient.

Another object of the present invention is to provide a health functional food for preventing or improving allergic diseases, comprising a cherry tree bark extract as an active ingredient.

One aspect of the present invention provides a pharmaceutical composition for preventing or treating allergic diseases, comprising a bark extract of cherry tree ( Prunus serrulata ) as an active ingredient.

The cherry tree ( Prunus serrulata L. var. spontanea) is a deciduous arboreous tree that grows wildly near villages with an altitude of 100 to 1,560 m in the southern, central and northern regions of Korea. It is around 20 m in height and has more flowers than leaves in April-May. It blooms first, and white or red flowers cover the entire tree to form a flower mass. The varieties of cherry trees are very diverse, and it is known that cherry trees obtained by naturally or artificially crossing different types of cherry trees may become new species. The bark of a cherry tree ( Prunus serrulata ) is known to be effective in easing, jinhae, and detoxification.

The bark of a cherry tree used in an embodiment of the present invention is distinguished from the bark of a wild cherry tree ( Prunus sargentii ) or a bark of a Yoshino cherry tree ( Prunus yedoensis ), which is used as an epidermis in oriental medicine.

The cherry bark extract according to an embodiment of the present invention can (1) inhibit the release of histamine from activated mast cells, (2) reduce the amount of calcium in the cells to inhibit degranulation of mast cells, and , (3) can inhibit the expression of inflammatory cytokine genes, TNF-α and IL-4, and the activity of transcription factor NF-κB, and (4) in passive cutaneous anaphylaxis (PCA). It can suppress local skin allergic reactions in a concentration-dependent manner, and (5) it can suppress body temperature reduction and histamine release in an animal model that caused an active systemic anaphylaxis, showing excellent anti-allergic effect. It can be usefully used in the prevention, improvement and treatment of diseases or inflammatory allergic diseases.

The cherry tree bark extract according to an embodiment of the present invention is capable of fundamental treatment of allergic diseases by inhibiting or blocking the secretion of allergens from mast cells, unlike conventional treatments focused on relieving symptoms.

It was confirmed that the composition according to an embodiment of the present invention inhibits a local skin allergic reaction in a concentration-dependent manner when administered orally in a passive cutaneous anaphylaxis (PCA).

It was confirmed that the composition according to an embodiment of the present invention inhibits body temperature reduction and histamine release when administered orally in an animal model that caused an active systemic anaphylaxis.

According to one embodiment of the present invention, the cherry bark extract is water, alcohol having 1 to 4 carbon atoms, propylene glycol, butylene glycol, glycerin, acetone, ethyl acetate, butyl acetate, chloroform, diethyl ether, dichloromethane, hexane, And it may be extracted with a solvent selected from the group consisting of a mixture thereof.

According to an embodiment of the present invention, the alcohol having 1 to 4 carbon atoms may be methanol or ethanol.

According to one embodiment of the present invention, the cherry bark extract may be extracted with a solvent mixed with water and ethanol in a volume ratio of 3:7.

In the present invention, the "extract" refers to an extract obtained by extraction treatment of herbal substances, a dilution or concentrate of the extract, a dried product obtained by drying the extract, a preparation or purified product of the extract, or a mixture thereof, the extract itself and the extract. It includes extracts of all formulations that can be formed.

According to one embodiment of the present invention, the cherry tree bark extract may be prepared in a powder state by additional processes such as distillation under reduced pressure and freeze drying or spray drying.

According to one embodiment of the present invention, the cherry bark extract includes not only the extract by the above-described extraction method, but also an extract that has undergone a conventional purification process. For example, separation by using an ultrafiltration membrane having a certain molecular weight cut-off value, separation by various chromatography (made for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through various purification methods may also be included in the extract of the present invention.

The composition according to an embodiment of the present invention may contain a cherry tree bark extract in an amount of 0.001% to 99.99% by weight, preferably 0.1% to 99% by weight, based on the total weight of the composition, which prevents or treats allergic diseases. Depending on the method of use and the purpose of use of the composition, it may be appropriately selected by those of ordinary skill in the art to which the present invention belongs.

According to one embodiment of the present invention, the cherry bark extract is 1 to 1000 mg/ml, 10 to 500 mg/ml, 10 to 300 mg/ml, 10 to 200 mg/ml, 50 to 150 mg/ml, or 100 It may be included in a concentration of mg/ml.

Composition of the present invention For administration, the composition of the present invention may contain a pharmaceutically acceptable carrier, excipient, or diluent in addition to the above-described active ingredients. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.

The composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injectable solutions according to a conventional method. Specifically, when formulated, it may be prepared using diluents or excipients such as fillers, weight agents, binders, wetting agents, disintegrants, and surfactants that are commonly used.

According to one embodiment of the present invention, solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like with the cherry bark extract. Further, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. It can be prepared by adding various excipients, such as wetting agents, sweetening agents, fragrances, preservatives, and the like, in addition to oral liquids and liquid paraffin.

According to one embodiment of the present invention, formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyloleate, and the like can be used. As a base for suppositories, Witepsol, Macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.

According to one embodiment of the present invention, the external preparation may be formulated in a formulation selected from the group consisting of an external powder, an external tablet, an external liquid, an ointment, a cream, a gel, a warning agent, a dressing agent, a patch, a spray, and a suppository. I can.

According to an embodiment of the present invention, the composition may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) according to a desired method. According to an embodiment of the present invention, the composition may be for oral administration.

According to one embodiment of the present invention, the dosage of the composition varies depending on the condition and weight of the patient, the severity of the disease, the form of the drug, the route of administration and the time, but may be appropriately selected by those skilled in the art.

The composition according to an embodiment of the present invention may be administered in a pharmaceutically acceptable amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the individual, age, sex, drug activity, Sensitivity to drugs, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field.

However, for a desirable effect, according to an embodiment of the present invention, the cherry bark extract may be administered at 0.0001 to 500 mg/kg/day, 0.001 to 300 mg/kg/day, or 10 to 100 mg/kg/day.

The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with another therapeutic agent exhibiting an anti-allergic effect, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and can be easily determined by a person skilled in the art.

In the present invention, "allergic disease" refers to a disease caused by immune hypersensitivity reaction, and 　allergic 　 disease is a complex reaction in which various cells and mediators participate, and acts as a cause of various diseases.

According to one embodiment of the present invention, the allergic disease is asthma, anaphylaxyis, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, contact dermatitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, Colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatoid fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, peri-shoulderitis, tendinitis, tendonitis, peritonitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome ), multiple sclerosis, acute inflammation, chronic inflammatory disease, hay fever, digestive tract allergy, hay fever, urticaria, insect allergy, food allergy and drug allergy, may be one or more selected from the group consisting of.

The cherry tree bark extract according to an embodiment of the present invention has no cytotoxicity and has few side effects, so it can be usefully used as a safe medicine or health functional food.

Since the cherry tree bark extract according to an embodiment of the present invention has few side effects and can be used for a long time, from a long-term perspective, it may have superior allergy prevention, improvement, or treatment effects than drugs having an allergy treatment effect known in the art.

Another aspect of the present invention is to provide a health functional food for preventing or improving allergic diseases comprising a cherry tree bark extract as an active ingredient.

According to one embodiment of the present invention, the cherry bark extract is water, alcohol having 1 to 4 carbon atoms, propylene glycol, butylene glycol, glycerin, acetone, ethyl acetate, butyl acetate, chloroform, diethyl ether, dichloromethane, hexane, And it may be extracted with a solvent selected from the group consisting of a mixture thereof.

According to an embodiment of the present invention, the alcohol having 1 to 4 carbon atoms may be methanol or ethanol.

According to one embodiment of the present invention, the cherry bark extract may be extracted with a solvent mixed with water and ethanol in a volume ratio of 3:7.

According to one embodiment of the present invention, the allergic disease is asthma, anaphylaxyis, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, contact dermatitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, Colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatoid fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, peri-shoulderitis, tendinitis, tendonitis, peritonitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome ), multiple sclerosis, acute inflammation, chronic inflammatory disease, hay fever, digestive tract allergy, hay fever, urticaria, insect allergy, food allergy and drug allergy, may be one or more selected from the group consisting of.

The term "improvement" as used in the present invention means all actions in which symptoms of an allergic disease are improved by administration of a composition containing a cherry tree bark extract.

According to one embodiment of the present invention, the health functional food includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid, and Salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like. In addition, it may contain flesh for the manufacture of natural fruit juice and fruit juice beverages and vegetable beverages. These components may be used independently or in combination. In addition, health functional foods are in the form of any one of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, drink, alcoholic beverage and vitamin complex. Can be

According to one embodiment of the present invention, the health functional food may additionally contain a food additive, and whether or not it is suitable as a "food additive" is a general rule and general test of the Food Additive Code approved by the Food and Drug Administration unless otherwise specified. It is judged according to the standards and standards for the relevant item in accordance with the law.

According to one embodiment of the present invention, as the items described in the "Food Additives Code", for example, chemical synthetic products such as ketones, glycine, potassium citrate, nicotinic acid, cinnamic acid, reduced pigment, licorice extract, crystalline cellulose, high cooling pigment , Natural additives such as guar gum, sodium L-glutamate, alkali additives for noodles, preservatives, and mixed preparations such as tar colorants.

Cherry bark extract according to an embodiment of the present invention inhibits the release of histamine from mast cells involved in allergic reactions, the expression of inflammatory cytokines TNF-α and IL-4, and the activity of transcription factor NF-ĸB It has an excellent effect of doing so and has a therapeutic effect on passive local skin allergic reactions and systemic allergic reactions, which are animal models, so that it can be used not only as a pharmaceutical composition for preventing, treating or improving allergic diseases, but also as a health functional food. I can.

1A to 1C show the effect of inhibiting histamine release by the addition of a cherry bark extract according to an embodiment of the present invention in mast cells.
Specifically, Figure 1a shows the effect of hot water extract (AEBPS) and ethanol extract (EEBPS) of cherry tree bark on the inhibition of histamine release in RBL-2H3 (rat basophilic leukemia cell), which is a type of mast cells, Figure 1b is RBL-2H3 Shows the inhibitory effect of histamine release according to the addition of ethanol extract of cherry bark at various concentrations (1, 10, 100㎍/㎖), and FIG. 1C is a cherry tree in RPMC (rat peritoneal mast cells), which is a type of mast cells. It shows the inhibitory effect of histamine release according to various concentrations (1, 10, 100㎍/㎖) of the ethanol extract of bark. In Figures 1a to 1c, the data are expressed as mean±standard deviation values for two replicates.
In Figures 1a to 1c, AEBPS represents a cherry bark hot water extract (aqua extract of bark of Prunus serrulata ), EEBPS represents a cherry bark ethanol extract (ethanol extract of bark of Prunus serrulata ), and Dexa represents dexamethasone as a positive control. And * means that a statistically significant difference is indicated (p<0.05). It is the same in the following drawings.
2 shows the effect of reducing intracellular calcium by adding a cherry tree bark extract according to an embodiment of the present invention at various concentrations (1, 10, 100㎍/㎖) in RBL-2H3. BAPTA-AM, an intracellular calcium inhibitor, was used as a positive control. In FIG. 2, data are expressed as mean±standard deviation values for two replicate experiments.
3 shows the effect of reducing the secretion of TNF-α and IL-4 cytokines by addition of a cherry bark extract according to an embodiment of the present invention at various concentrations (1, 10, 100㎍/㎖) in RBL-2H3. In FIG. 3, the data are expressed as mean±standard deviation values for two replicate experiments.
Figure 4 shows the results of confirming the effect of inhibiting IκBα decomposition and NF-ĸB activity by the addition of a cherry bark extract according to an embodiment of the present invention by Western blot.
5A and 5B show the effect of cherry bark extract through passive cutaneous anaphylaxis (PCA). Specifically, FIG. 5A is a photograph showing the effect of a cherry tree bark extract on skin allergy through a passive local skin allergic reaction. Figure 5b shows the absorbance of the amount of Evans blue exuded when a cherry tree bark extract according to an embodiment of the present invention is orally administered at various concentrations. In Fig. 5b, the data are expressed as mean±standard deviation values for two replicate experiments.
6A and 6B show the anti-allergic effect of a cherry tree bark extract according to an embodiment of the present invention in an active systemic anaphylaxis. Specifically, FIG. 6A shows the inhibitory effect of reducing rectal temperature when treated with an ethanol extract of cherry bark according to an embodiment of the present invention in an active systemic allergy model induced by ovalbumin, and FIG. 6B is an active systemic allergy induced by ovalbumin. In the model, it shows the effect of reducing histamine concentration in serum when the ethanol extract of cherry tree bark according to an embodiment of the present invention is treated. Data in FIGS. 6A and 6B are expressed as mean±standard deviation values for two replicates, and OVA refers to ovalbumin used to induce an active systemic allergic reaction.
7 shows the cell viability of RBL-2H3 by the addition of ethanol extract of cherry tree bark according to an embodiment of the present invention at various concentrations (0.1 to 1000 μg/ml). Data are expressed as a percentage of the untreated control group and are mean±standard deviation values for two replicates.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1. Preparation of cherry bark extract

To prepare the bark extract of Prunus serrulata , the bark of cherry trees was purchased from Bohwadang (Jeonju) and used. The varieties of cherry trees are very diverse, and it is known that cherry trees obtained by naturally or artificially crossing different types of cherry trees may become new species. The cherry tree used in this example is different from the Yoshino cherry tree ( Prunus yedoensis ) and is distinguished from each other.

Example 1-1. Preparation of hot water extract

20 g of cherry bark was extracted in hot water (90 to 100° C.) for 3 hours to obtain a hot water extract. After extraction, the solids were removed by filtering with a decompression device using a filter paper (Whatman™ No.1 qualitative filter paper; GE Healthcare Life Sciences). The filtered extract was centrifuged at 13,000 rpm for 10 minutes, and then the supernatant was taken, excluding insoluble substances, and the obtained hot water extract of cherry tree bark was freeze-dried to obtain 1.96 g of a dried product. In the case of hot water extract, the extraction yield was 9.8%. The dried product was diluted in a PBS solution, filtered through a 0.22 μm syringe filter, and used for later experiments.

Example 1-2. Preparation of ethanol extract

20 g of cherry tree bark was added to 1 L of 70% ethanol, followed by reflux extraction at 80 to 100° C. for 2 hours to obtain an ethanol extract. After extraction, the solids were filtered off with a pressure reducing device using a filter paper (Whatman™ No.1 qualitative filter paper; GE Healthcare Life Sciences). The filtered cherry tree bark extract was concentrated in vacuo on a water bath and lyophilized to obtain 2.02 g of a dried product. In the case of the ethanol extract, the extraction yield was 10.1%. The dried product was diluted in saline, filtered through a 0.22 μm syringe filter, and used for later experiments.

Example 2. Histamine release inhibitory effect of cherry bark extract in mast cells

Example 2-1. RBL-2H3 cell culture

RBL-2H3 (rat basophilic leukemia cell, Korea Cell Line Bank), a type of rat mast cell line, was heat-inactivated under conditions of 37°C and 5% CO ₂ and added 10% FBS (Dulbecco's modified eagle medium; Cat No. 12800-017, Gibco, USA). RBL-2H3 has an IgE antibody receptor (FcεRI) on its surface, and is known to secrete mediators of allergic reactions such as histamine by degranulation when activated by IgE and antigen.

Example 2-2. Isolation and culture of rat peritoneal mast cells (RPMC)

To isolate rat peritoneal mast cells (RPMC), rats (Korea Laboratory Animal Center, Daejeon) (6 weeks old, 150 g) were euthanized with carbon dioxide and 20 ml of Tyrode's buffer (137 mM). NaCl, 5.6mM glucose, 12mM NaHCO ₃ , 2.7mM KCl, 0.3mM NaH ₂ PO ₄ , 0.1% gelatin) was injected into the abdominal cavity, and the left and right sides of the abdomen were gently massaged for about 90 seconds. After incision in the abdominal cavity of the rat, the Tyrode buffer solution in the abdominal cavity was recovered using a Pasteur pipette and then centrifuged at room temperature (150 g, 10 minutes) to precipitate. The precipitate was mixed with 1 ml of Tyrode buffer solution, and then carefully suspended on 2 ml of Histodenz solution (0.235 g/ml) and centrifuged at room temperature (400 g, 10 minutes). After removing the supernatant, the remaining precipitate was washed with 1 ml of Tyrode buffer solution, and mast cells were identified by staining with toluidine blue, and the survival of cells was confirmed by staining with trypan blue. I did. RPMC was cultured in α-MEM (α-Minimal Essential Media; Cat No. 11900-024, Gibco, USA) to which 10% FBS inactivated by heat was added under conditions of 37° C. and 5% CO ₂ .

Example 2-3. Histamine Release Inhibitory Effect of Cherry Bark Extract

In order to confirm the inhibitory effect on the release of histamine from mast cells, the mast cells (RBL-2H3 and RPMC) obtained in Examples 2-1 and 2-2 were treated with cherry bark extract, and the main substance and allergy of intracellular granules The degree of release of histamine, an inducer, was measured.

RBL-2H3 and RPMC in various concentrations (1, 10 or 100 ㎍ / ㎖) of the ethanol extract obtained in Example 1-2 or the cherry bark hot water extract obtained in Example 1-1 (100 ㎍ / ㎖) It was pretreated with. RBL-2H3 and RPMC were sensitized with anti-DNP-IgE (anti-dinitrophenyl-IgE; Cat No. D8406, Sigma, USA), and then DNP-HSA (DNP-human serum albumin; Cat No. A6661, Sigma, USA). ) To activate mast cells to release histamine. The culture medium of the activated mast cells was recovered and used in the experiment. 200 µl of a sample was taken into an Eppendorf tube, 80 µl of 0.1N hydrochloric acid and 20 µl of a 60% perchloric acid solution were mixed, followed by centrifugation (5415R; Eppendorf) at 13,000 g for 20 minutes. Then, the supernatant was taken 200㎕ 5N NaOH solution 100㎕, n - butanol (n -butanol) 800㎕, into mixing 5M NaCl 200㎕ in a test tube, and performing centrifugation for 20 min at 13,000g and then shaking it I did. Take 500 µl of butanol layer from the test tube, add 200 µl of 0.1N hydrochloric acid and 500 µl of n-heptane, shake, and centrifuge at 13,000 g for 20 minutes to add 1 N to 150 µl of the obtained supernatant. 40㎕ NaOH, 1% o - au taldi aldehyde (o -phthaldialdehyde) solution (Sigma Co., Cat No. P1378) was added to a mixed 10㎕, five minutes, and then allowed to stand, an emission wavelength of 440nm by adding 3N hydrochloric acid while 20㎕ The fluorescence intensity at (emission wavelength) and an excitation wavelength of 360 nm was measured using a fluorescence analyzer (GEMINIEM, Molecular Devices) to calculate the degree of histamine release.

As a positive control, dexamethasone (10 μM) exhibiting anti-allergic action was used as an adrenal cortical hormone.

Figure 1a shows the histamine release inhibitory activity of a cherry tree hot water extract or ethanol extract in RBL-2H3. 1B shows the histamine release inhibitory activity of ethanol extracts of cherry trees at various concentrations (1, 10 or 100 µg/ml) in RBL-2H3. Figure 1c shows the histamine release inhibitory activity of the ethanol extract of cherry trees at various concentrations (1, 10 or 100 ㎍ / ㎖) in RPMC. In FIGS. 1A to 1C, AEBPS represents an aqua extract of bark of Prunus serrulata, EEBPS represents an ethanol extract of bark of Prunus serrulata, and Dexa represents dexamethasone as a positive control. And * means that a statistically significant difference is indicated (p<0.05). It is the same in the following drawings.

As shown in FIGS. 1A to 1C, histamine release was increased in mast cells activated with DNP-HSA, but histamine release was decreased upon treatment with cherry bark extract. As shown in Figure 1a, when treated with the same concentration of the extract, when treated with ethanol extract (EEBPS) than hot water extract (AEBPS) of cherry tree bark, histamine release inhibitory activity was superior, and the release of histamine was significant when treated with ethanol extract. It could be confirmed that it was reduced to a degree. 1B and 1C, it was confirmed that the release of histamine was inhibited in RBL-2H3 and RPMC by the addition of the ethanol extract of cherry bark. In particular, it was found that the ethanol extract of cherry tree bark of the present invention has a similar level of histamine release inhibitory effect to dexamethasone used as a positive control.

Example 2-4. Effect of cherry bark extract on intracellular calcium in mast cells

Calcium plays an important role in the regulation of physiological activity as a secondary transport material in cells. Calcium is a signaling substance that regulates the degranulation of mast cells. As the amount of calcium in the cells increases, the mast cells are degranulated. Conversely, when the amount of calcium decreases, the degranulation of mast cells is inhibited. Therefore, by observing the effect of the cherry bark extract on intracellular calcium, the mechanism of histamine release of the ethanol extract of cherry bark in mast cells was confirmed.

RBL-2H3 was treated with anti-DNP-IgE and DNP-HSA, respectively, and activated mast cells were treated with ethanol extract of cherry bark at a concentration of 1, 10 or 100 µg/ml. The amount of calcium in the cells was Fluo-3 AM ( Cat No. F1242, Invitrogen, USA) was used for fluorescence staining and then measured with a fluorescence analyzer, and relative fluorescence intensity was calculated based on the control group not treated with anti-DNP-IgE, DNP-HSA and cherry tree extract. This was expressed as the amount of calcium in the cell.

2 shows the amount of calcium according to the addition of ethanol extracts of cherry trees at various concentrations (1, 10 or 100 μg/ml) in RPMC. As a positive control, intracellular calcium inhibitor BAPTA-AM (1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester)) was used.

As shown in FIG. 2, the amount of calcium was increased in the mast cells (RBL-2H3) activated with DNP-HSA, but the amount of calcium was decreased in a concentration-dependent manner when the cherry bark extract was treated. Since the amount of calcium was reduced by the addition of the cherry bark extract according to an embodiment of the present invention, it can be seen from this that the cherry bark extract is effective in treating allergies by reducing the amount of calcium to inhibit degranulation of mast cells. have. In particular, it was found that the ethanol extract of cherry tree bark of the present invention inhibited degranulation of mast cells at a level similar to that of BAPTA-AM, an intracellular calcium inhibitor used as a comparative example.

Example 3. Inhibitory effect of cherry bark extract

Example 3-1. Effect of Cherry Bark Extract on the Expression of Inflammatory Cytokines in Mast Cells

To confirm the effect of cherry tree bark extract on the expression of inflammatory cytokines such as TNF-α and IL-4, which are physiologically active substances that cause inflammatory allergy by being released from mast cells or newly synthesized and secreted, After sensitization with -DNP-IgE, it was activated with DNP-HSA to promote the secretion of inflammation-inducing cytokines, and then the amount of cytokine secretion was measured using an enzyme-linked immunosorbent assay (ELISA).

Rat TNF-a, IL-4 ELISA set (BD biosciences, San Diego, Cat No. 558535, 555198) was used to calculate the cytokine concentration in the culture medium by enzyme immunoassay.

100 µl of RBL-2H3 culture solution and 200 µl of a standard sample diluted by each concentration of cytokines for which expression levels are to be checked were added to the wells to which the antigen was attached, and left to stand. After 120 minutes, the plate was washed three times with a washing solution, and then 100 μl of the primary antibody was added. After mixing well and leaving for 60 minutes, after washing three times with a washing solution, 100 µl of a mixed reagent in which the secondary antibody and enzyme reagent were mixed was added. After the reaction was over, the mixture was discarded, and the plate was washed 4 times with a washing solution, and then 100 μl of the substrate solution was added each, and the mixture was left for 20 minutes at room temperature to block light. After the reaction, a stop reagent (2N H ₂ SO ₄ ) was added each 50 μl to stop the reaction, and the absorbance at 450 nm was measured with an absorbance analyzer (Sunnyvale, Molecular Devices), and the result of the secretion amount of each cytokine was shown in FIG. 3 Shown in.

As shown in FIG. 3, the secretion of TNF-α and IL-4 cytokines increased in RBL-2H3 activated with anti-DNP-IgE and DNP-HSA, and as a result of pretreatment of cherry bark ethanol extract by concentration, concentration It was confirmed that the secretion of TNF-α and IL-4 cytokine was dependently reduced. As a positive control, dexamethasone (10 μM) having anti-inflammatory activity was used.

Example 3-2. Effect of cherry tree bark extract on NF-ĸB

In order to reveal the signaling mechanisms involved in the process of suppressing the expression of TNF-α and IL-4, which are inflammation-inducing cytokines, cherry bark extract is a factor that regulates the transcription of inflammation-inducing cytokines. It was measured for the activation of NF-ĸB, an important signal transducer that mediates. In addition, NF-ĸB in an inactive state binds to IĸBα, an endogenous inhibitory protein, and maintains its inactive state.When NF-ĸB is activated, IĸBα is phosphorylated and decomposed, freed from these and moved to the nucleus, acting as a transcription factor, so IĸBα is decomposed. The degree to which it becomes is also measured.

After sensitizing mast cells (RBL-2H3) with anti-DNP-IgE, activating NF-ĸB by activating with DNP-HSA, and then having anti-inflammatory activity as a cherry bark ethanol extract (100 μg/ml) or positive control After dexamethasone (10 μM) was treated, the amount of NF-ĸB protein in the nucleus and the amount of IĸBα protein in the cytoplasm were measured using a Western blot.

RBL-2H3 is an extraction buffer containing 0.5 mM phenylmethanesulfonyl fluoride (Sigma, Cat No. P7626) and 5 µg/ml leupeptin (Sigma, Cat No. L2884). After dissolving with 0.5% triton X-100, PBS solution containing 1mM Na ₃ VO ₄ ), the DNA was fragmented by sonication. The amount of protein was measured using a protein assay kit (Cat No. 500-0002, Bio-Rad) using bovine serum albumin as a standard. After electrophoresis of 10 to 50 μg of total cell protein on an 8 to 12% (w/v) polyacrylamide gel containing 0.1% SDS, the protein present in the gel was electroblotting. After transferring to a nitrocellulose (NC) filter, in order to block non-specific binding, put the nitrocellulose filter in a TBS-tween (tris-buffered saline-tween; Sigma) solution containing 5% skim milk and 1 at room temperature. Reacted for hours. Thereafter, a nitrocellulose filter was put in a TBS-tween solution containing an antibody against the target protein and left at 4° C. for 12 hours, and then labeled with a secondary antibody labeled with HRP (horseradish peroxidase, Sigma, Cat No. P0889). And, the band was detected using ECL (Enhanced chemiluminescence, Thermo scientific company, Cat No. 34080).

4 shows the results of confirming the effect of inhibiting IκBα decomposition and NF-ĸB activity according to the addition of cherry bark extract by Western blot. As shown in FIG. 4, when RBL-2H3 is sensitized with anti-DNP-IgE and then activated with DNP-HSA, IĸBα is decomposed, and NF-κB (N-NF-κB) in the nucleus is increased. It was confirmed that the treatment of the ethanol extract of cherry bark on mast cells activated with DNP-HSA inhibited the degradation of IκBα due to the binding of anti-DNP-IgE and DNP-HSA, and the expression of NF-ĸB in the nucleus was reduced. From this, since the cherry bark extract has an inhibitory effect on the expression and activity of NF-ĸB, it can be seen that the cherry bark extract is involved in the gene expression of inflammation-inducing cytokines in the nucleus.

Example 4. Effect of cherry tree bark extract on local skin allergic reaction

Using a passive local skin allergic reaction (passive cutaneous anaphylaxis; PCA), the allergy inhibitory effect of the cherry tree bark extract according to an embodiment of the present invention was tested. The passive topical skin allergic reaction is an animal model that is widely used when developing drugs with anti-allergic effects. PCA is an Evans Blue solution containing IgE-specific antigens (Sigma, USA, Cat No. 48 hours after intradermal injection into the ears of 6-week-old ICR mice (Daehan Experimental Animal Center, Daejeon, 30g)). E2129) is an experimental method using the principle that the antigen induces degranulation by stimulating the ear mast cells sensitized to IgE, and increases the vascular permeability of substances in the granules, thereby exuding Evans blue into the ear. In this case, an antigen-antibody reaction occurs at the injection site of the antibody, and the mast cells present around it are activated, secreting histamine, and a local allergic reaction occurs.

In order to induce PCA, 0.5 μg of IgE was injected into one ear of the mouse. After about 12 hours, 1, 10 or 100 mg/kg concentration of cherry bark extract or 10 μM of dexamethasone as a positive control was orally administered to mice.

After 1 hour, DNP-HSA was diluted to a concentration of 5 mg/ml in 10 mg/ml Evans Blue staining solution, and 200 μl was administered to the tail vein. After an hour, the mouse was euthanized, the ear was put in 1 ml of potassium hydroxide, dissolved for 2 days, and then added to a mixture of acetone-phosphoric acid to stop the reaction. Then, after centrifugation to obtain a supernatant, the absorbance was measured at a wavelength of 620 nm.

Fig. 5a shows the ears of mice stained with Evans Blue after oral administration of cherry bark extract in PCA. 5B shows absorbance measured according to the concentration of various cherry bark extracts. The lower the absorbance, the less the amount of Evans blue exuded into the mouse ear, and it means that the antigen inhibits degranulation of the ear mast cells sensitized to IgE. As a result of treating the cherry bark extract as shown in FIGS. 5A and 5B, it was confirmed that the higher the concentration, the less the exudate part of Evans Blue, and the amount of exuded Evans Blue decreased. Therefore, it was found that the cherry bark extract according to an embodiment of the present invention suppresses degranulation of mast cells in a concentration-dependent manner, thereby suppressing allergic reactions.

Example 5. Effect of cherry tree bark extract on systemic allergic reactions

Sensitivity to OVA increases the production of IgE in serum, and when re-exposed to OVA afterwards, the antigen-IgE-receptor complex binds to the surface of mast cells, causing an allergic reaction (Lim et al., 2014; 30: 13- 18.). In particular, hypothermia, i.e., an allergic reaction to the OVA challenge, is triggered by an increase in serum histamine levels (Kim et al., 2017; 38: 90-99.), so the OVA-induced active systemic allergy model is based on mast cell mediated type I hypersensitivity. Is an appropriate animal model for (Finkelman et al., 2005; 115: 449-457.).

Using an active systemic allergy model (active systemic anaphylaxis) was confirmed the effect of the cherry bark extract according to an embodiment of the present invention on the systemic allergic reaction.

For the induction of a systemic allergic reaction, a 6-week-old male ICR of around 30 g after dissolving 100 μg of ovalbumin (OVA) and 2 mg of alum adjuvant in phosphate buffered saline (PBS) The mice (Daehan Experimental Animal Center, Daejeon) were first sensitized by intraperitoneal injection, and one week later, the same amount of ovalbumin and alum adjuvant were injected intraperitoneally to sensitize the mice. From the 4th day to the 6th day after the second sensitization, the ethanol extract of cherry bark obtained in Example 1-2 was orally administered 3 times a day at a dose of 1, 10, or 100 mg/kg. As a positive control, dexamethasone was orally administered 3 times a day at a dose of 10 mg/kg. On the 7th day after the second sensitization, ovalbumin was additionally administered intraperitoneally to induce anaphylaxis, and a change in rectal temperature was observed for 80 minutes, and this is shown in FIG. 6A.

6A shows the change in rectal temperature according to treatment with an ethanol extract of cherry bark in an active systemic allergy model induced with ovalbumin. As shown in FIG. 6A, a decrease in temperature was not observed in the mice administered with only PBS, and the control group administered with only ovalbumin showed a decrease in temperature of up to 2.95°C. When the ethanol extract of cherry tree bark was administered at a concentration of 100 mg/kg, a decrease in body temperature of 1.32°C was observed, and from this, it was found that the tree bark extract suppressed the decrease in body temperature induced by ovalbumin. In addition, it was confirmed that the decrease in body temperature was better than the positive control group administered with dexamethasone in the experimental group to which the cherry bark extract was administered.

After observing the change in body temperature of the mice for 80 minutes, blood was collected from the abdominal artery of each mouse and the histamine concentration in the serum was measured. Histamine concentration methods known in the prior (Kim et al, Food Chem Toxicol 2013; 57:.. 209-216.) O steps were analyzed using the fluorescence spectroscopy phthalic aldehyde (o -phthaldialdehyde spectrofluorometric procedure). Fluorescence intensity was detected using a fluorescence analyzer at an excitation wavelength of 360 nm and an emission wavelength of 440 nm.

6B shows the concentration of histamine in serum according to treatment with an ethanol extract of cherry bark in an active systemic allergy model induced with ovalbumin. As shown in FIG. 6B, the control group administered only ovalbumin showed high serum histamine secretion at a concentration of 134.97 ng/ml, but when the ethanol extract of cherry tree bark (1-100 mg/kg) was administered, the average histamine in serum was 103.34 ng/ml. It was confirmed that the concentration was decreased, and it was found that the cherry bark extract inhibited histamine secretion in a concentration-dependent manner.

Therefore, from these results, it was found that the ethanol extract of cherry bark according to an embodiment of the present invention has a therapeutic effect on systemic allergic reactions.

Example 6. Cytotoxicity test of cherry bark extract

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) experiment was performed to confirm the cytotoxicity of the cherry bark extract. The MTT solution forms formazan by mitochondrial dehydrogenases in living cells to confirm the survival of cells.

RBL-2H3 was dispensed into a 96-well plate at a concentration of 6×10 ⁴ cells/well, incubated at 37° C. for 6 hours, and the ethanol extracts of cherry bark obtained in Example 1-2 were respectively 0.1, 1, 10, 100 Alternatively, it was added at a concentration of 1000 μg/ml and incubated at 37° C. for 24 hours. After 24 hours, 20 µl of MTT solution (5 mg/ml) was added to each well, and after incubating for 2 hours, the culture solution was removed. In order to dissolve the formazan crystal, 100 µl of dimethylsulfoxide (DMSO) was added, the plate was wrapped with aluminum foil, and incubated for 30 minutes. The absorbance of each well was measured at 570 nm using a spectrophotometer (Molecular Devices, Sunnyvale, CA), and the cell viability (%) was calculated as shown in Equation 1 below as a percentage of the untreated control, and the results are shown in FIG. Done.

As shown in FIG. 7, it was confirmed that the ethanol extract of cherry tree bark did not show cytotoxicity up to a concentration of 1000 μg/ml, and was harmless.

Claims (4)

Cherry tree ( Prunus serrulata ) comprising a bark extract as an active ingredient, a pharmaceutical composition for the prevention or treatment of allergic diseases.
The method of claim 1, wherein the cherry tree bark extract is water, alcohol having 1 to 4 carbon atoms, propylene glycol, butylene glycol, glycerin, acetone, ethyl acetate, butyl acetate, chloroform, diethyl ether, dichloromethane, hexane, and A pharmaceutical composition that is extracted with a solvent selected from the group consisting of mixtures.
The method of claim 1, wherein the allergic disease is asthma, anaphylaxyis, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, contact dermatitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids , Gout, ankylosing spondylitis, rheumatoid fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, peri-shoulderitis, tendinitis, tendonitis, peritonitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple Sclerosis, acute inflammation, chronic inflammatory disease, hay fever, digestive tract allergy, hay fever, urticaria, insect allergy, food allergy and pharmaceutical composition that is one or more selected from the group consisting of allergy to drugs.
Health functional food for the prevention or improvement of allergic diseases, including cherry bark extract as an active ingredient.

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