KR102169159B1 - Composition for preventing or treating of allergic comprising extract of Cyperus flavidus - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating allergies containing an extract of Cyperus globosus. More specifically, the present invention relates to a pharmaceutical composition for preventing and treating allergies which contains an extract of Cyperus globosus, which has an excellent effect of inhibiting interleukin-4 (IL-4) secretion, and histamine secretion, as an active component.

Description

Composition for preventing or treating of allergic comprising extract of Cyperus flavidus}

The present invention relates to a pharmaceutical composition for the prevention and treatment of allergies comprising an extract of Cyperus flavidus , and more particularly, an allergy comprising an extract of Drengbangdongsani having excellent inhibitory effect on IL-4 and histamine as an active ingredient. It relates to a pharmaceutical composition for prevention and treatment.

Cyperus flavidus Retz. ( Cyperus flavidus Retz.) grows in fields, reservoirs or rivers nationwide, and is also known as rice field. It is a perennial plant, and the stems are 10~50cm high and the stems under the ground are short. Leaves are 1~2mm wide, flat and linear, and the length of the stem is similar or short. Flowers bloom from August to October. The inflorescence is umbrella-shaped or head-shaped, reaches 8cm in length, and has 1-5 spikelets. A few are yellowish brown, 3~18mm long, flat, linear, and 20-50 small flowers are arranged in 2 rows. The scale piece is about 1.5mm long and has a dull end. Fruits are achene, obovate about 1mm in length, ripen brown, and tallow lenticular. The pistil head is split into two.

In general, allergy refers to a phenomenon or hypersensitivity reaction in which a living body in contact with a foreign substance exhibits a reaction different from normal to the substance, and is also called allergy. In addition, it also means that when an organism comes into contact with a foreign substance, a rapid change in the reaction capacity occurs in the living body by the antigen-antibody reaction.

The living body produces antibodies and lymphocytes that specifically respond to the antigen for foreign substances, and when it comes into contact with the antigen again, various immune reactions occur. The immune response is one of the important defense mechanisms for the self-preservation of the living body, and usually acts protectively against the living body, but sometimes this mechanism acts unfavorably to the living body and causes disorder.

The antigen that causes the allergic reaction is called an allergen, and typical allergens include pollen, drugs, vegetable fibers, bacteria, foods, hair dyes, and chemicals. The immune system has several defense mechanisms to protect the body against antigens. The most common types of these are lymphocytes, which are specialized to respond to specific antigens, and B-cells and T-cells correspond to this. Allergic reactions appear as immediate-type or delayed-type allergies, and depend on whether the antigen reacts with B cells or T cells.

The disease caused by allergies, that is, allergic diseases, is a generalization of extensive and complex pathological phenomena and is a systemic or local disorder of a living body based on an immune response. For example, diseases caused by allergies include autoimmune diseases, collagen diseases, atopic diseases, allergic rhinitis, hay fever, bronchial asthma, urticaria, eczema, or allergic contact dermatitis.

The various allergic diseases are largely classified into four types, that is, type I, type II, type III and type IV according to the immune mechanism. The most representative disease among the allergic diseases is type I allergy or type 1 allergy.

The type 1 allergy is an immediate reaction that occurs within minutes after the antigen binds to an antibody attached to an individual that is already sensitized to a certain antigen. The type 1 allergic disease is allergic rhinitis, atopic dermatitis, and bronchial asthma. And the like. The type 1 allergy is caused by activation of mast cells, and when histamine, a mediator of inflammation contained in large amounts in the granules of mast cells, is released, immediate type allergy including type 1 allergic diseases It is known that a reaction is induced.

More specifically, FceRI, a high-affinity IgE receptor expressed on the surface of mast cells, binds to IgE and specific allergens, thereby increasing the concentration of calcium in the cytoplasm and causing degranulation to cause inflammation mediators, specifically histamine, heparin, and hydrolase. In addition, various cytokines such as interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-13 (IL-13) and TNF-α, which are known as modulators of the immune response, are secreted. The secreted inflammatory mediators relax blood vessels, increase vascular permeability, and cause bronchi to contract.

In addition to immunological stimulation through these allergens and IgE, the activation of mast cells is compound 48/80, a non-immune stimulating substance, phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, and calcium ionophore (A23187). It is reported to be caused by such as.

Based on the above theory, rat-derived RBL-2H3 cells that contain hundreds of thousands of IgE receptors on the surface of the membrane and release histamine by reacting to antibodies after being sensitized with IgE are widely used as model cell lines for anti-allergic efficacy screening. It is being used.

On the other hand, it is rapidly increasing due to complex effects such as modern environmental pollution, changes in dietary life, and genetic influences, and the number of allergic disease patients worldwide including Korea is steadily increasing. For example, allergic asthma patients are known to make up about 10% of the world's population (about 27.5 million as of 1995), and 17 million suffer from asthma in the United States alone, of which about 5 million are reported to be adolescents. Has been. In addition, in 2000, the US market for allergic asthma drugs was about 6.4 billion dollars, and the Korean market was also reported to be about 1 billion dollars, accounting for about 20% of the total Korean pharmaceutical market.

In order to treat the allergic disease, anti-allergic agents such as antihistamine drugs are being administered. However, immunological or non-immune hypersensitivity reactions are temporarily relieved by antihistamine drugs, but are not completely cured. Therefore, there is a demand for the development of a material that prevents the release of histamine, an allergy mediator.

The present inventors have made research efforts result to develop allergic disease drugs of natural origin with no toxicity and side effects, the present inventors have deureong as a pharmaceutical composition for the prevention and treatment of allergic diseases bangdongsani (Cyperus flavidus Retz.) Extract of IL -4 It was confirmed the effect of inhibiting secretion and suppressing histamine, thereby completing the present invention.

Korean Patent Registration No. 10-1908221 (Name of invention: anti-obesity composition containing extract of Geumbangdongsani, Applicant: Dongeui University Industry-Academic Cooperation Foundation, Registration Date: 2018.10.08) Korean Patent Laid-Open Patent No. 10-2015-0026579 (Name of invention: Pharmaceutical composition and health functional food for the prevention and treatment of allergic or non-allergic skin diseases containing herbal extracts as an active ingredient, Applicant: YD Life Science Co., Ltd. , Publication date: 2015.03.11.)

It is an object of the present invention to provide a pharmaceutical composition for the prevention and treatment of allergies comprising an extract of Cyperus flavidus Retz. More specifically, an extract of Drengbangdongsani excellent in inhibiting IL-4 secretion and histamine secretion is an active ingredient. It is to provide a pharmaceutical composition for preventing and treating allergies, including as.

The present invention relates to a pharmaceutical composition for preventing or treating allergies comprising an extract of Cyperus flavidus Retz.

The extract may be extracted with water, a C1 to C6 lower alcohol, or a mixed solvent thereof.

The extract may have a function of inhibiting the secretion of interleukin-4 (IL-4).

The extract may have a function of inhibiting histamine secretion.

Another aspect of the present invention relates to a health functional food for preventing or improving allergies comprising the extract of Drengbangdongsani as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition for preventing or treating allergies comprising an extract of Cyperus flavidus Retz.

The drengbangdongsani extract may be extracted using water, C1 ~ C4 alcohol, or a mixed solution thereof as a solvent. Any water without impurities, such as distilled water or DI water, may be used as the water. The C1 ~ C4 alcohol may be selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol and isobutanol. At this time, Drengbangdongsani extract has a better anti-allergic effect than a water extract, but a C1~C4 alcohol extract or an aqueous solution extract of C1~C4 alcohol, and the antiallergic effect of the aqueous solution extract of C1~C4 alcohol is the best. As an aqueous solution of C1~C4 alcohol, 50 to 90% (v/v) ethanol aqueous solution is suitable for preparing an extract having good anti-allergic effect, and the most suitable is 60 to 70% (v/v) ethanol aqueous solution.

When preparing the drengbangdongsani extract, it may be extracted using a solvent having a weight of 5 to 40 times, preferably 10 to 20 times the dry weight of the drengbangdongsani. If the solvent is too small than the above range, when the extract is prepared by applying heat such as a hydrothermal reaction, extraction may be difficult due to evaporation of the solvent, and if there is too much solvent, there is a problem that the extraction time is too long.

Further, the extraction process may be performed while stirring at 10 to 40° C. for 24 to 48 hours, and additional ultrasonic waves may be applied. This may be repeated until 1 to 4 times and extracted to obtain a liquid extract.

The liquid drengbangdongsani extract may be filtered out using sterile gauze or filter paper. The filtrate through this filtration process may be concentrated under reduced pressure at 10 to 40° C. using a rotary vacuum concentrator, and an extract powder may be obtained after removing the remaining solvent by lyophilization. In addition, the extract powder may be dissolved in DMSO (dimethyl sulfoxide) to prepare a sample solution.

In addition, the present invention provides a pharmaceutical composition for preventing or treating allergies comprising an extract of Drengbangdongsani. The drengbangdongsani extract may be added to the pharmaceutical composition of the present invention in an amount of 0.01 to 100% by weight.

Examples of allergic diseases that have an effect on the Drengbangdongsani extract include hypersensitivity, allergic rhinitis, asthma, allergic conjunctivitis, allergic dermatitis, atopic dermatitis, allergic contact dermatitis, urticaria, insect allergy, food allergy or drug allergy. Include.

The pharmaceutical compositions may be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, and suppositories according to a conventional method. Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , Methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, such as starch, calcium carbonate, sucrose or lactose, in the extract of the present invention, It is prepared by mixing gelatin or the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like may be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration, and the judgment of the prescriber. Dosage determination based on these factors is within the level of one of ordinary skill in the art, and dosages generally range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is from 1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be divided several times. The above dosage does not in any way limit the scope of the present invention.

The pharmaceutical composition of the present invention can be administered to mammals such as mice, livestock, and humans by various routes. All modes of administration can be expected and can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injection. Since the extract of the present invention has almost no toxicity and side effects, it is a drug that can be safely used even when taken for a long time for prophylactic purposes.

The present invention can provide a health functional food for preventing or improving allergic diseases comprising the extract of Drengbangdongsani as an active ingredient.

The health functional food may contain food supplementary additives that are food wise acceptable. The health functional food of the present invention includes forms such as tablets, capsules, pills or liquids, and foods to which the extract of the present invention can be added include, for example, various drinks, meat, sausages, bread, candy, There are snacks, noodles, ice cream, dairy products, soups, ionized drinks, beverages, alcoholic beverages, gum, tea and vitamin complexes.

The present invention relates to a pharmaceutical composition for the prevention and treatment of allergies comprising an extract of Drengbangdongsani, and has excellent effects of inhibiting the secretion of interleukin-4 (IL-4) and secretion of histamine, thereby exhibiting an allergy prevention and treatment effect.

1 is a graph showing the IL-4 secretion inhibitory activity of the extract of Drengbangdongsani according to the present invention.
Figure 2 is a graph showing the histamine secretion inhibitory activity according to the concentration of Drungbangdongsani extract according to the present invention.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided to sufficiently convey the spirit of the present invention to those skilled in the art so that the contents introduced herein are thorough and complete.

Example 1. Preparation of Drengbangdongsani extract

First, the drengbangdongsani ( Cyperus flavidus Retz.) was washed with 3rd distilled water, and then finely crushed using a grain bean . The finely pulverized drengbangdongsani was extracted using 70% (v/v) ethanol aqueous solution. After making 100 g of finely pulverized drengbangdongsani sufficiently immersed in 1 kg of the ethanol aqueous solution corresponding to 10 times the weight, the extraction was performed by applying ultrasonic waves while stirring at 25° C. for 36 hours. Was prepared.

After the extraction solution was first filtered using sterile gauze, the filtrate was filtered again using a filter paper (Whatman No. 2: Watmam Ltd., Maidstone, Kent, UK) to filter out pulverized samples. Thereafter, it was concentrated under reduced pressure at 20° C. using a rotary vacuum concentrator, and the remaining solvent was removed by lyophilization to obtain an extract sample. The obtained lyophilized sample was stored at -20°C, and the extract sample obtained by this method was dissolved in DMSO (dimethyl sulfoxide) at the concentration required for the experiment and used.

Comparative Example 1. Preparation of plant extract of the genus Bangdongsani

Prepared in the same manner as in Example 1, but extracts of Geumbangdongsani ( Cyperus microiria Steud.), Soobangdongsani ( Cyperus orthostachyus Franch. & Sav.) or Bangdongsanidae ( Cyperus sanguinolentus Vahl) were obtained, respectively, and obtained. One lyophilized sample was stored at -20°C. The extract sample obtained by this method was dissolved in DMSO (dimethyl sulfoxide) at the concentration required for the experiment and used.

division Contents Comparative Example 1-1 Geumbangdongsani extract Comparative Example 1-2 Soobangdongsani extract Comparative Example 1-3 Bangdong Sanihead Extract

Experimental Example 1. Cytotoxicity confirmation experiment

Before performing the anti-allergic activity assay, a cytotoxicity experiment was performed using the MTT assay to determine whether it does not induce cytotoxicity.

In this experimental example, the effect of the extracts of Example 1 and Comparative Example 1 on the cell viability of RBL-2H3 (rat basophilic leukemia cell line) was examined.

Specifically, RBL-2H3 was distributed in Korea Cell Line Bank (KCLB) and cultured in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% heat-inactivated FBS (fetal bovine serum) and 1% streptomycin/penicillin. I did.

After suspending RBL-2H3 in DMEM medium, it was dispensed so that the number of cells per well of 48 well plate was 1.5x10 ⁵ cells/well, and cultured in a 37°C, 5% CO ₂ incubator for 4 hours. Thereafter, the culture solution was removed, and 100 µl of a 0.2 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution was added. After incubation for 4 hours, the MTT solution was removed, and the extract of Example 1 or Comparative Example 1 dissolved in DMSO (dimethyl sulfoxide) at a concentration of 20 and 100 μg/ml was treated. After reacting for 30 minutes, absorbance was measured at a wavelength of 545 nm using a microplate reader (Tecan, Switzerland). At this time, the cell viability was calculated as follows, and it is shown in Table 2.

* Cell viability (%)

= 100-{(absorbance of control group-absorbance of sample treatment group)/absorbance of control group) ㅧ100}

division Cell viability (%) 20㎍/㎖ 100㎍/㎖ Drengbangdongsani extract of Example 1 118 95 Geumbangdongsani extract of Comparative Example 1-1 101 94 Soebangdongsani extract of Comparative Example 1-2 99 97 Bangdong Sanidae extract of Comparative Example 1-3 96 82

Referring to Table 2, it was confirmed that when the extracts of Example 1 or Comparative Examples 1-1 to 1-3 were treated with RBL-2H3 cells, cytotoxicity was hardly induced at all treatment concentrations of 20 and 100 µg/ml. I can.

Experimental Example 2. Measurement of IL-4 secretion inhibitory activity

In this experimental example, an experiment was conducted to measure the IL-4 secretion inhibitory activity that can confirm the effect of the extract on anti-allergic activity.

When mast cells are activated, mast cells secrete arachidonic acid metabolites and various cytokines that induce inflammatory reactions through degranulation. Among them, IL-4 (interleukin-4) activates B-cells and thus Immunoglobulin E (IgE) produces antibodies and promotes differentiation of Th2 cells, so it has been reported to play an important role in allergic diseases such as asthma [J. Allergy Clin. Immunol. 99, 155-160]. TNF-α is a cytokine known to play an important role in allergic inflammatory reactions as a factor that is produced in mast cells and macrophages and induces many inflammatory reactions [Immunol Today 11, 458-464].

Therefore, in this experiment, IL-4 production assay was used to measure the amount of inflammatory cytokine IL-4 (interleukin-4) secreted from mast cells, and through this, it was confirmed how much the extract inhibited the production of IL-4. I did. In detail, when the cell culture solution was added to a plate coated with a primary cytokine antibody and an antibody for assay was added, the concentration of IL-4 could be confirmed through absorbance measurement that causes a color reaction, which is shown in FIG. 1 below.

First, when RBL-2H3 cells were treated with 12.5µg/ml of PMA (phorbol 12-myristate 13-acetate) and 125nM ionomycin (Io) at the same time, the IL-4 produced significantly increased to more than 200pg/ml. I did. Here, 12.5, 25, 50, 100, 200 ㎍ / ㎖ of Example 1 or Comparative Examples 1-1 to 1-3 were treated by concentration, and the results of the concentration change of IL-4 are shown in Table 3 and Fig. 1 Shown in.

division IL-4 concentration change (pg/ml) Control(+PMA/Io) 12.5㎍/㎖ 25㎍/㎖ 50㎍/㎖ 100㎍/㎖ 200㎍/㎖ Example 1 208.5 206.3 193.7 190.8 128.1 72.5 Comparative Example 1-1 208.5 203.7 198.1 193.3 189.0 172.4 Comparative Example 1-2 208.5 207.1 205.4 203.1 199.2 179.8 Comparative Example 1-3 208.5 198.6 194.8 191.6 184.7 174.0

Referring to Table 3 and FIG. 1, it was confirmed that the extract of Drengbangdongsani of Example 1 inhibited the secretion of IL-4 in a concentration-dependent manner, which indicates that the extract of Drengbangdongsani had an anti-allergic effect. In addition, it can be confirmed that the anti-allergic effect is more excellent through the fact that the value for inhibiting the secretion of IL-4 is higher than that of the extracts of Comparative Examples 1-1 to 1-3.

Experimental Example 3. Histamine release measurement experiment

Histamine release assay is a method of measuring the amount of histamine secreted by mast cells, and the ability to inhibit histamine production can be confirmed. Histamine secretion was measured to confirm the inhibitory effect on degranulation, which is an indicator of an immediate reaction to allergies. In more detail, the Histamine release assay is an experiment that is measured using the principle that the colorimetric probe mix containing WST-1, which is an electric donor, and the histamine reacts into a formazan form, and the measured absorbance reflects the concentration of histamine.

Using this principle, the ability of rat basophilic leukemia cells to inhibit histamine release was measured. When RBL-2H3 cells were treated with 12.5µg/ml of PMA (phorbol 12-myristate 13-acetate) and 125nM of ionomycin, the amount of histamine secretion increased to more than 240µg/ml. It was confirmed that histamine secretion was increased. Thereafter, 12.5, 25, 50, 100, and 200 µg/ml of Example 1 or Comparative Examples 1-1 to 1-3 were treated by concentration, and the results of histamine concentration change are shown in Table 4 and FIG. 2.

division Histamine concentration change (㎍/㎖) Control(+PMA/Io) 12.5㎍/㎖ 25㎍/㎖ 50㎍/㎖ 100㎍/㎖ 200㎍/㎖ Example 1 248.7 172.4 157.0 126.3 74.1 71.6 Comparative Example 1-1 248.7 226.3 210.6 199.4 184.2 182.3 Comparative Example 1-2 248.7 219.8 198.7 192.0 181.8 179.7 Comparative Example 1-3 248.7 223.4 212.4 208.1 195.9 191.8

Referring to Table 4, it can be seen that the extract of Drengbangdongsani of Example 1 lowers the concentration of histamine in a concentration-dependent manner, and through this, it can be confirmed that the extract of Drengbangdongsani of Example 1 has an effect of inhibiting the secretion of histamine. On the other hand, it can be seen that the histamine secretion inhibitory effect is superior to the extract of Comparative Examples 1-1 to 1-3 due to the higher histamine concentration reduction by the extract of Example 1.

In addition, referring to FIG. 2, it can be seen that the secretion of histamine is suppressed even when the extract of Example 1 is treated with the RBL-2H3 cells not treated with PMA/Io. You can see that the effect is excellent.

<Formulation Example 1. Preparation of pharmaceutical formulation>

Formulation Example 1-1. Preparation of powder

Drengbangdongsani extract ------------------------------------ 20 mg

Lactose ------------------------------------------------- -100 mg

Talc ------------------------------------------------- - 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 1-2. Manufacture of tablets

Drengbangdongsani extract ------------------------------------ 10 mg

Corn Starch -------------------------------------------- 100 mg

Lactose ------------------------------------------------- -100 mg

Magnesium stearate ------------------------------------- 2 mg

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet preparation method.

Formulation Example 1-3. Preparation of capsules

Drengbangdongsani extract ------------------------------------- 10 mg

Crystalline cellulose ---------------------------------------- 3 mg

Lactose ---------------------------------------------- 14.8 mg

Magnesium stearate ---------------------------------- 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare a capsule.

Formulation Example 1-4. Preparation of liquid

Drengbangdongsani extract ------------------------------------- 20 mg

Seonghwadang Lee ------------------------------------------------ 10 g

Mannitol ------------------------------------------------- - 5 g

Purified water ------------------------------------------------- Appropriate amount

According to the usual preparation method of liquid preparation, add each ingredient to purified water to dissolve it, add lemon zest, mix the above ingredients, add purified water, add purified water, adjust the total to 100 ml, and fill in a brown bottle. It is sterilized to prepare a liquid formulation.

Claims (7)

A pharmaceutical composition for the prevention or treatment of allergies containing the extract of Cyperus flavidus Retz. The method of claim 1,
The extract is a pharmaceutical composition for preventing or treating allergies, characterized in that the extract is extracted with water, C1 to C6 lower alcohol or a mixed solvent thereof. The method of claim 1,
The extract is a pharmaceutical composition for preventing or treating allergies, characterized in that it has a function of inhibiting the secretion of IL-4 (interleukin-4). The method of claim 1,
The extract is a pharmaceutical composition for preventing or treating allergies, characterized in that it has a function of inhibiting histamine secretion. A health functional food for preventing or improving allergies containing extracts of Cyperus flavidus Retz. The method of claim 5,
The extract is a health functional food for preventing or improving allergy, characterized in that it has a function of inhibiting the secretion of IL-4 (interleukin-4). The method of claim 6,
The extract is a health functional food for preventing or improving allergy, characterized in that it has a function of inhibiting histamine secretion.

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