KR101840758B1 - Composition for preventing or treating nasal polyp comprising extract of Distromium decumbens - Google Patents

Abstract

The present invention relates to a composition for preventing or treating nosal polyp diseases containing an extract of Distromium decumbens as an active ingredient, wherein the extract of Distromium decumbens reduces an expression of IL-6 and IL-8 in nosal polyp fibroblast, and reduces phosphorylation of AKT, ERK and JNK. Moreover, the extract of Distromium decumbens reduces an expression of P65, and reduces a DNA coupling activity of NF-κB. As a result, the composition having the above described effects of the present invention, which contains the extract of Distromium decumbens as an active ingredient, can be utilized as a pharmaceutical composition for preventing or treating nosal polyp diseases, a functional food composition for preventing or alleviating nosal polyp diseases, a pharmaceutical composition for preventing or treating paranasal sinusitis, and a functional food composition for preventing or alleviating paranasal sinusitis.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating a nasal polyposis,

The present invention relates to a composition for preventing or treating a nontoxic disease containing an extract of Angelica keiskei koidz. As an active ingredient.

A nasal polyp or nasal polyp is a benign edematous mucosa with a protruding structure that secretes mucus secretion in the nose, and local edema of the nasal sinus membrane recurs and gradually develops. Most of the costal mucosa is composed of respiratory epithelium, and the submucosal layer shows severe edema and is known to infiltrate a large number of inflammatory cells, especially eosinophils, within the fibrotic tissue.

Until now, the exact cause of the costum is unknown, but it is chronic inflammatory disease. Cost species are formed and grow by the interaction of inflammatory cells with infectious or noninfectious inflammation. The eosinophil infiltration is promoted by various inflammatory mediators secreted from mast cells, and tissue damage is caused by various chemical mediators secreted from eosinophils, and it is exacerbated by edema.

Cost species are known to be more common in people with asthma or rhinitis with inflammation in the mucous membranes of the nose and neck. Symptoms of the costa species develop slowly over several months, and the severity of symptoms is determined by the number and size of the polyps. There are no special symptoms when the size of the cost species is small, but various symptoms may appear with increasing size. The main symptom of the nonspecific species is persistent mucus secretion, nasal obstruction by the polyp, and sensory slowing of the smell. In some cases, an anatomical change may occur and the nose may become wider.

The purpose of the treatment is to eliminate the symptoms caused by the cost species, to remove the cost species, to restore the function of the nasal cavity as a respiratory tract, and to induce drainage and ventilation of the sinus. In addition, the purpose of the treatment is to restore the olfactory sense and prevent the recurrence of costly diseases. Cost-type therapy can be divided into pharmacotherapy and surgery. Currently, the use of steroids as medication is known to be effective in the treatment of cost species. In order to alleviate rhinorrhea and nasal obstruction, there is a drug therapy in which a steroid agent is sprayed on the nose or a steroid agent is injected into a direct polyp to reduce the size of the polyp. In the case of a large polyp, this is called a polypectomy. Costs are often accompanied by chronic sinusitis rather than alone.

Dukyeo debt (Distromium decumbens ) are brown algae in the sea, and plants are attached to rocks. It grows at lunch and mainly appears from April to October. It has been collected from the coast of southern Australia and one species has been reported in Jeju Island, Ulleungdo and the southern coast of Korea. In addition, it lives in the intertidal and subterranean basins of the eastern and southern coast of Korea and is known to be distributed in Japan and southern Australia. The body is crawled, initially a single fan or kidney, and then spreads radially to form a wedge-shaped lobe. When expanded, it becomes fan-shaped and the edges overlap each other. The body is 2 to 4 cm in diameter, the lower part of the body becomes thicker and sometimes forms a stem. Its color is pale brown or dark brown, and consists of two cell layers with platelets. The effect of the swallow debt on the cost-benefit disorder has not been reported so far.

Korean Patent Publication No. 10-2012-0092742 (published on Aug.22, 2012)

It is an object of the present invention to provide a method and apparatus, decumbens extract as an active ingredient, or a health food composition for preventing or ameliorating a nontoxic disease.

Another object of the present invention is to provide a method and apparatus for detecting disturbance decumbens extract as an active ingredient, or a health food composition for preventing or ameliorating a sinusitis disease.

In order to achieve the above object, the present invention dukyeo fan (Distromium decumbens s) extract as an active ingredient, or a health food composition for preventing or ameliorating an idiopathic disease.

In addition, the present invention relates to the use of Distromium decumbens extract as an active ingredient, or a health food composition for preventing or ameliorating a sinusitis disease.

The present invention relates to the use of Distromium The present invention relates to a composition for the prevention or treatment of a nontoxic disease which comprises as an active ingredient an extract of dicotyledonous decubens, which reduces the expression of IL-6 and IL-8 in costodermal fibroblasts and inhibits AKT, ERK and JNK Phosphorylation can be reduced. In addition, Sukwi's fan extract reduces P65 expression and can reduce NF-κB DNA binding activity.

Therefore, the composition of the present invention comprising the extract of Chenopodium albumen having the above effect as an active ingredient is useful as a pharmaceutical composition for preventing or treating nontoxic diseases, a health food composition for preventing or improving nontoxic diseases, a pharmaceutical composition for the prevention or treatment of sinusitis A composition, and a health food composition for preventing or ameliorating sinusitis.

FIG. 1 shows the cytotoxicity of the extract of Chenopodium ambrosioides in the fibroblast cell line by MTT assay.
FIG. 2 is a graph showing the inhibitory effect of the extracts on the expression of IL-6 and IL-8 in the fibroblast cell line by the enzyme immunoassay.
FIG. 3 shows Western blot analysis of inhibition of MAPK and AKT phosphorylation by the extract of Chenopodium ambrosioides in costal fibroblast cells.
FIG. 4 shows that the expression of IL-6 and IL-8 in the fibroblast cell line is associated with JNK and ERK signal transduction by enzyme immunoassay.
FIG. 5 shows that the expression of IL-6 and IL-8 in the fibroblast cell line was associated with NF-κB transcriptional regulatory inhibitors by enzyme immunoassay.
FIG. 6 shows the inhibitory effect of P65 protein on NF-κB DNA binding inhibition by the scavenging agent extract and the electrophoretic mobility analysis of western blot.

In the present invention, the inventors of the present invention prepared an extract of Chenopodium ambrosioides using an aqueous solution of ethanol as an extraction solvent. The extract of Chenopodium acuminata decreased the expression of IL-6 and IL-8 in the fibroblast cell line and phosphorylated AKT, ERK and JNK And decreased the DNA binding activity of P65 and NF-kB, thus completing the present invention.

As used herein, the term " nasal polyp " refers to a protruding nasal passageway in the nasal cavity of a benign edematous mucosa in the shape of a grapevine derived from a middle degree (median nasal route). Most of the costal mucosa is composed of respiratory epithelium, and the submucosal layer is severely edematous, and there is a large number of inflammatory cells, especially eosinophil infiltration, within the fibrotic tissue. Cost is a chronic inflammatory disease whose cause is not clearly known.

The term " sinusitis, " as used herein, refers to inflammation of the sinus, an empty space in the facial bone around the nose. The sinus is connected to the nose through a small life span (natural ball), through which air in the sinuses is ventilated and secretions are excreted. Sinusitis is a condition in which the natural ball is blocked and the sinus is not properly ventilated and excreted, resulting in secondary inflammation and inflammation of the massive secretions. Sinusitis can be divided into two types, acute and chronic. Acute sinusitis usually occurs as a late complication of the cold. Chronic sinusitis is often accompanied by a nonspecific disease.

As used herein, the term " Distromium & decumbens "is a brown algae in the sea, and plants grow on rocks. It grows at lunch and mainly appears from April to October. It has been collected from the coast of southern Australia and one species has been reported in Jeju Island, Ulleungdo and the southern coast of Korea. In addition, it lives in the intertidal and subterranean basins of the eastern and southern coast of Korea and is known to be distributed in Japan and southern Australia.

The present invention provides a pharmaceutical composition for the prevention or treatment of nontoxic diseases containing an extract of Angelica keiskei koidz. As an active ingredient.

Preferably, the scalloped iron extract may be extracted using an aqueous 60 to 80% ethanol solution as an extraction solvent, but it is not limited thereto.

Preferably, the scalloped iron extract may inhibit the expression of IL-6 and IL-8.

Preferably, the scalloped iron extract may inhibit phosphorylation of AKT, ERK and JUK.

Preferably, the scalloped fan extract may reduce the expression of P65 and reduce NF-κB DNA binding activity.

In addition, the present invention provides a pharmaceutical composition for preventing or treating sinusitis, which comprises an extract of Sambrook, as an active ingredient.

When the composition of the present invention is a pharmaceutical composition, it may contain a pharmaceutically acceptable carrier in addition to the above-mentioned active ingredients. Such pharmaceutically acceptable carriers are those conventionally used in pharmaceutical preparations, and include lactose, dextrose, The present invention relates to a process for the preparation of a medicament for the treatment and / or prophylaxis of cancer, comprising administering a therapeutically effective amount of a compound selected from the group consisting of cross, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, Carboxymethylcellulose, carboxymethylcellulose, hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. The pharmaceutical composition may further contain a filler, a weight agent, a binder, a lubricant, a wetting agent, a disintegrant, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a fragrance, a preservative and the like as an additive and an auxiliary agent.

The pharmaceutical composition may be formulated into tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, nonaqueous solutions, emulsions, lyophilized preparations, suppositories and sterile injectable solutions.

The pharmaceutical composition may be administered by intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, subcutaneous, intradermal, intranasal, intrapulmonary, intraocular, intramuscular, Rectally, topically, orally, and by inhalation.

An effective amount of the active ingredient of the above pharmaceutical composition means an amount required for prevention or treatment of the disease. Accordingly, the present invention is not limited to the particular type of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, body weight, general health status, sex and diet, But are not limited to, various factors, including, for example, the rate of administration, the rate of administration, the duration of the treatment, the drugs used concurrently.

The present invention provides a health food composition for preventing or ameliorating a nontoxic disease containing an extract of Angelica keiskei koidz. As an active ingredient.

Further, the present invention provides a health food composition for preventing or ameliorating sinusitis disease, which comprises an extract of Angelica keiskei koidz. As an active ingredient.

The health food composition may be provided in the form of powder, granules, tablets, capsules, syrups or beverages. The health food composition may be used in combination with other food or food additives other than the active ingredient, . The amount of the active ingredient to be mixed can be suitably determined according to its use purpose, for example, prevention, health or therapeutic treatment.

The effective dose of the active ingredient contained in the health food composition may be used in accordance with the effective dose of the pharmaceutical composition. However, in the case of long-term consumption intended for health and hygiene purposes or health control purposes, And it is clear that the active ingredient can be used in an amount exceeding the above range since there is no problem in terms of safety.

There is no particular limitation on the type of the health food, and examples thereof include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Drinks, alcoholic beverages and vitamin complexes.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  One : Duck fan  Extract preparation

Algae that inhabit the coast of Jeju dukyeo debt (Distromium decumbens extracts were obtained from the National Marine Life Resource Center. The method of manufacturing the duck pan fan extract is as follows. First, the scoop fan was washed to remove impurities and then dried and crushed to prepare a powder. 1 L of 70% ethanol aqueous solution was added to 100 g of the pan powder, and the mixture was immersed at room temperature for 24 hours, and then centrifuged at 3,500 rpm at 4 ° C for 20 minutes using a centrifuge (Hanil Co., Seoul, Korea) The supernatant was obtained by centrifugation. Thereafter, a filtrate was obtained from the supernatant using a filter paper (Whatman), and a 70% ethanol aqueous solution was removed with a rotary evaporator (Eyela, Tokyo, Japan) at 40 ° C to obtain a dried sample . The dried samples were dissolved in 70% aqueous ethanol solution and used in the experiment.

Example  2: Cost Fibroblast cell line  culture

Nasal polyp fibroblast (NPF) was cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) at 37 ° C in a CO ₂ incubator.

Example  3: Cost In fibroblast cells Duck fan  Cytotoxicity analysis by extract

Cost Fibroblast cells were divided into 1 × 10 ⁵ cells / mL and cultured in DMEM medium without fetal bovine serum for 16 hours to produce serum starvation status. After that, ethanol extract of Distromium decumbens (EDD) was cultured for 24 hours and MTT analysis was performed. After incubation at 37 ° C for 1 hour, the absorbance was measured at 450 nm using a microplate reader (SpectraMax M2, Molecular Devices, CA) Survival and cytotoxicity were analyzed.

As a result, referring to FIG. 1, it was confirmed that the extract of Chenophelenchii extract in cost fibroblasts showed almost no toxicity to cells up to a concentration of 25 / / mL. Therefore, in the following experiment, Were used.

Example  4: Cost In fibroblast cells Duck fan  Inhibitory Effect of Extracts on Expression of IL-6 and IL-8

Cost Fibroblast cells were dosed at 1 × 10 ⁵ cells / mL and cultured in DMEM medium without fetal bovine serum for 16 hours to produce serum depletion. Subsequently, the extracts were incubated for 24 hours with 10 μg / mL of Pseudomonas aeruginosa LPS (P. LPS). In order to measure the concentration of IL-6 and IL-8 secreted out of the cells, enzyme immunoassay (BioLegend ELISA kit, San Diego, Calif.) Was performed using culture supernatant. Absorbance was measured and quantified.

As a result, referring to FIG. 2, it was confirmed that IL-6 and IL-8 increased by P.LPS treatment were suppressed by the panicle extract treatment, and as the treatment concentration of panicle extract increased, IL-6 < / RTI > and < RTI ID = 0.0 > IL-8. ≪ / RTI >

Example  5: Cost In fibroblast cells Duck fan  By extract MAPK  And AKT  Phosphorylation inhibitory effect

Cost Fibroblast cells were dosed at 1 × 10 ⁵ cells / mL and cultured in DMEM medium without fetal bovine serum for 16 hours to produce serum depletion. After that, the extracts were incubated for 1 hour and then treated with 10 μg / mL of P. LPS for 2 hours.

Cost Fibroblasts were washed three times with phosphate buffered saline (PBS), and the proteins were extracted using cell lysis buffer (Mammalian Cell-PE LB, G Biosciences, St. Louis, Mo.) Respectively. After protein determination, the same amount of protein was separated by 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Western blotting was performed. The proteins separated by electrophoresis were transferred to a nitrocellulose membrane (NC membrane) at 350 mA for 120 minutes using a transfer buffer (20% methanol, 25 mM Tris-HCl, 192 mM glycine) . The NC membrane was blocked with 3% non-fat dry milk (skim milk solution), reacted with primary antibody for 24 hours at 4 ° C, treated with tris-buffered saline (TBST) and tween 20). The HRP-conjugated secondary antibody was then reacted at room temperature for 1 hour and washed three times with TBST. After washing once more with distilled water, a mixed solution of ECL detection kit (Advansta) coloring reagent I and II in a ratio of 1: 1 was applied to the membrane, and an X-ray film (CP-G plus, Agfa Healthcare Ltd, New Orleans, LA, USA), and then the degree of protein expression on the film was observed.

As a result, referring to FIG. 3, the treatment of P. LPS with the fibroblast cell line induced phosphorylation of ERK, JNK and AKT, but did not induce phosphorylation of P38. It was confirmed that the treatment of duckweed fan extract inhibited the phosphorylation of ERK, JNK and AKT increased by P. LPS.

Example  6: Cost In fibroblast cells MAPK  And AKT  Expression of IL-6 and IL-8 by signaling inhibitors

Cost Fibroblast cells were dosed at 1 × 10 ⁵ cells / mL and cultured in DMEM medium without fetal bovine serum for 16 hours to produce serum depletion. Then, the ERK, JNK and AKT signal inhibitors were pretreated for 1 hour and then treated with 10 μg / mL of P. LPS and cultured for 24 hours.

To measure the concentration of IL-6 and IL-8 secreted out of the cell, enzyme immunoassay was performed using culture supernatant and the absorbance at 450 nm was measured using a microplate reader.

As a result, referring to FIG. 4, it was confirmed that the treatment of AKT signal inhibitor (LY294002) increased expression levels of IL-6 and IL-8 protein and showed higher expression levels than P. LPS treatment group. However, the expression of IL-6 and IL-8 protein was decreased in the treatment with JNK signaling inhibitor (SP600126) and ERK signaling inhibitor (U012) than in the LPS-treated group. Therefore, it was confirmed that IL-6 and IL-8 protein expression induced by P. LPS is involved in signal transduction of JNK and ERK.

Example  7: Cost In fibroblast cells Transcription regulator  Expression of IL-6 and IL-8 by inhibitor

Cost Fibroblast cells were dosed at 1 × 10 ⁵ cells / mL and cultured in DMEM medium without fetal bovine serum for 16 hours to produce serum depletion. Thereafter, the NF-κB transcription factor (bay and parthenolide (Par)) and the AP-1 transcription factor (SR11302 (SR)) were pretreated for 1 hour and then treated with 10 μg / Lt; / RTI >

To measure the concentration of IL-6 and IL-8 secreted out of the cell, enzyme immunoassay was performed using culture supernatant and the absorbance at 450 nm was measured using a microplate reader.

As a result, referring to FIG. 5, it can be seen that increased IL-6 and IL-8 protein expression by P. LPS treatment is inhibited by both the IκBα inhibitor Bay and the specific inhibitor of NF-κB, parthenolide Respectively. On the other hand, treatment with SR11302, an inhibitor of AP-1, did not affect IL-6 and IL-8 protein expression.

Example  8: Cost In fibroblast cells Duck fan  By extract NF - κB  Activity inhibitory effect

Cost Fibroblast cells were dosed at 1 × 10 ⁵ cells / mL and cultured in DMEM medium without fetal bovine serum for 16 hours to produce serum depletion. After that, the extracts were incubated for 1 hour and then treated with 10 μg / mL of P. LPS for 2 hours. Cost Fibroblasts were washed three times with phosphate-buffered saline, and proteins were extracted using protein extraction reagents (NE-PER nuclear and cytoplasmic extraction reagents, Thermo Fisher Scientific, Waltham, Mass.). After quantification of the proteins, 5 ㎍ of the same amount of protein was separated by 10% SDS-PAGE and Western blotting was performed. The proteins separated by electrophoresis were transferred to a nitrocellulose membrane at 350 mA for 120 minutes using a transfer buffer. The NC membrane with protein transfer was blocked with 3% skim milk, reacted with primary antibody at 4 ° C for 24 hours, and washed three times with TBST. The HRP-conjugated secondary antibody was then reacted at room temperature for 1 hour and washed three times with TBST. After washing once more with distilled water, a mixture of 1: 1 mixture of color development reagent I and II in an ECL detection kit was applied to the membrane, and X-ray film (CP-G plus, Agfa Healthcare Ltd, New Orleans, LA, USA), and then the degree of protein expression on the film was observed.

As a result, referring to FIG. 6, it was confirmed that the treatment of P. LPS increased the expression of P65, and the treatment of the panicle extracts depressed the expression of P65 protein in the nucleus induced by P. LPS.

Example  9: Cost In fibroblast cells Duck fan  The electrophoretic mobility shift assay, EMSA )

Proteins in the nucleus were extracted from the sample of Example 7 using a protein extraction reagent (NE-PER nuclear extraction reagent, Pierce, Rockford, Ill.) And the probe was immunglobulin k- binding site (kB, 5'-GATCTCAGAGGGGACTTTCCGAGAGA-3 ') oligonucleotide was synthesized and biotin (Pierce, Rockford, Ill.) was attached at the 3' end. 5 ng of nuclear protein, buffer (10 mM Tris, pH 7.5, 50 mM KCl, 5 mM MgCl ₂ , 1 mM dithiothreitol, 0.05% Nonidet P-40 and 2.5% glycerol), 50 ng poly ) And 20 [mu] M biotin labeled DNA was added to a final volume of 20 [mu] l, and reacted at room temperature for 20 minutes. As a negative control, a 100-fold dense oligonucleotide not labeled with biotin was added to the reaction solution. The reaction mixture was transferred to a 5% polyacrylamide gel with 0.5X TBE buffer, and the separated proteins were transferred to a nylon membrane and analyzed with an EMSA assay kit (Pierce, Rockford, Ill.) Using a LightShift chemiluminescent electrophoretic mobility shift assay kit ) Was used to identify the biotin-labeled DNA.

As a result, referring to FIG. 6, the treatment of P.LPS increased the DNA binding activity of NF-κB, and the treatment of panicle extracts showed that NF-κB DNA binding Lt; / RTI >

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims (8)

Dukyeo debt (Distromium decumbens extract of the present invention as an active ingredient. [Claim 7] The pharmaceutical composition according to claim 1, wherein the extract is extracted using an aqueous 60 to 80% ethanol solution as an extraction solvent. The pharmaceutical composition according to any one of claims 1 to 3, wherein the extract has a suppression of the expression of IL-6 and IL-8. [Claim 2] The pharmaceutical composition according to claim 1, wherein the extract of Angelica keiskei koidz. Inhibits phosphorylation of AKT, ERK and JUK. The pharmaceutical composition according to claim 1, wherein the extract has a reduced expression of P65 and a reduced NF-κB DNA binding activity. Dukyeo debt (Distromium decumbens extract of the present invention as an active ingredient. Dukyeo debt (Distromium decumbens extract as an active ingredient for the prevention or treatment of sinusitis. Dukyeo debt (Distromium decumbens extract as an active ingredient for preventing or ameliorating sinusitis diseases.

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