KR102245814B1 - Composition for Improving Respiratory Disease Using an Extract of Sargassum horneri - Google Patents

Abstract

The present invention reduces the invasion of inflammatory cells such as leukocytes and lymphocytes in trachea and lung tissue in an animal model experiment in which fine dust is inhaled, suppresses oxidative damage, and reduces the infiltration of mast cells, Furthermore, it discloses a composition for improving respiratory diseases using a hoeax saengimopura extract having an activity of inhibiting the proliferation of goblet cells, which are mucous (sputum) secreting cells, and thereby reducing the increase in mucus secretion.

Description

Composition for Improving Respiratory Disease Using an Extract of Sargassum horneri}

The present invention relates to a composition for improving respiratory diseases using an extract of Sargassum horneri , in particular, to a composition for improving respiratory diseases due to fine dust and the like.

Air pollution such as particulate matter (PM) is increasing due to advanced industrialization. In addition, due to the desertification of Asian continents such as China due to climate change, the occurrence of domestic yellow dust is increasing, and the public's interest in fine dust is growing (Kim HS, Chung YS, Yoon MB. An analysis on the impact of large-scale transports) of dust pollution on air quality in East Asia as observed in central Korea in 2014. Air Qual Atmos Health, 2015 Jan 15 [Epub].http://dx.doi.org/10.1007/s11869-014-0312-5). Fine dust contains various components such as carbon components such as soot and organic carbon of living organisms, ionic components such as chlorine, nitric acid, ammonium, sodium and calcium, metal components such as lead, arsenic, and mercury, and polycyclic aromatic hydrocarbons such as benzopyrene ( Jang An-Soo.Impact of particulate matter on health.J Korean Med Assoc, 2014;57:763-768). Secondary particles such as sulfate, nitrate, sulfur dioxide, nitrogen oxides, ammonia, and volatile organic compounds affect the generation of fine dust. In addition, fine dust is classified according to its size as a particulate matter, and the particle diameter is divided into 2.5-10 µm and 2.5 µm or less, and fine dust of 2.5 µm or less is called ultrafine dust. The dust gets caught in the nose or throat and does not affect the airways, but if it is smaller than 10 μm, it deposits in the upper respiratory tract, bronchi, small airways and alveoli, affecting the respiratory tract, causing allergic rhinitis, bronchitis, asthma, and alveolar damage (Allergy Asthma Respir Dis, 2015, 3:313-319). In addition, chronic obstructive pulmonary disease (COPD) that causes shortness of breath due to a decrease in lung function may occur if it progresses to chronic inflammation (J Int. Krean Med, 2017, 38:353-366). Fine dust can cause not only respiratory tract, but also allergic conjunctivitis, keratitis, cardiovascular disease, etc., and these human effects are known to be caused by inflammatory reactions due to the secretion of cytokines and chemokines, an increase in the number of white blood cells, and the production of free radicals. (Int J Environ Res Public Health 2018, 15(7)) There is a need for research on substances that can inhibit it.

Sargassum horneri is a perennial brown algae of the family of the family of the family, and is widely distributed along the coasts of Korea, Japan, and China, and is known as a major constituent of the floating hatch, which travels along the sea currents of the East Sea and Japan (Korean). J Fish Aquat Sci, 2016, 49:689-693).

As it contains a variety of ingredients such as minerals and polyphenols including fucoidan and alginic acid, Hoesengi Hatban is known to have various pharmacological and physiological functions that are beneficial to beauty and health. Phosphorus fucoidan has been reported to reduce oxidative stress in macrophages stimulated with LPS (Int J Biol Macromol, 2014, 68:98-106). In addition, the polyphenol component of the hoesaengyimojaban has a strong antioxidant activity (Journal of Medicinal Food, 2016, 19:615-628), and Fucoxanthin, a type of carotenoid pigment, has been reported to exhibit antioxidant, antibacterial, and antihypertensive effects. (Mar Drugs, 2015, 13:3422-3442).

The present invention discloses the improvement activity of respiratory diseases by fine dust and the like of the extract of hoesaengyimopura.

It is an object of the present invention to provide a composition for improving respiratory diseases due to fine dust, etc. using a hoesaengyimojaban extract.

Other or specific objects of the present invention will be presented below.

The present inventors, as confirmed in the following Examples and Experimental Examples, in an animal model experiment in which fine dust was inhaled, the extract of erythema chinensis was extracted from trachea, bronchus, and lung tissue, etc., neutrophils , Eosinophils, basophils, granulocytes, macrophages, and other white blood cells and helper T cells, cytotoxic T cells, and lymphocytes such as dendritic cells. It suppresses the invasion of mast cells, which are involved in inflammatory reactions and hypersensitivity reactions, causing bronchoconstriction and secretion of mucus, and further inhibits the proliferation of goblet cells, which are cells secreting mucus (sputum), and thereby inhibits the secretion of mucus. It was confirmed that the increase was reduced.

In consideration of the experimental results as described above, the present invention can be understood as a composition for improving respiratory diseases, in particular, a composition for improving respiratory diseases by fine dust, including the extract of chinensis chinensis as an active ingredient, in one aspect, In another aspect, it can be identified as a composition for inhibiting the secretion of sputum (mucus secreted from the bronchi or lungs) containing the extract as an active ingredient, and in another aspect, the bronchial tube containing the extract as an active ingredient It can be recognized as a composition for expansion.

In the present specification, the "e.g. hoesaeng imojaban extract" refers to a stem, leaf, root, outpost, or a mixture thereof, which is an extraction target, in water, a lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, Ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof It refers to an extract obtained by leaching by using (ie, an extract soluble in the extraction solvent), an extract obtained using a supercritical extraction solvent such as carbon dioxide, pentane, or a fraction obtained by fractionating the extract, and the extraction method includes the polarity of the active substance, In consideration of the degree of extraction and preservation, arbitrary methods such as cold sedimentation, reflux, warming, ultrasonic radiation, and supercritical extraction can be applied. In the case of a fractionated extract, a fraction obtained by suspending the extract in a specific solvent and then mixing and policing it with a solvent having a different polarity, the crude extract is adsorbed on a column filled with silica gel, etc., and then a hydrophobic solvent, a hydrophilic solvent, or a mixed solvent thereof is added. It means including the fraction obtained as a mobile phase. In addition, the meaning of the extract includes a concentrated liquid extract or a solid extract from which the extraction solvent has been removed by a method such as freeze drying, vacuum drying, hot air drying, spray drying, or the like. Preferably, it means an extract obtained by using water, ethanol or a mixed solvent thereof as an extraction solvent, more preferably an extract obtained by a mixed solvent of water and ethanol as an extraction solvent, and more preferably 70% ethanol is used. It refers to an extract (ie, extract soluble in 95% ethanol) that is the supernatant obtained by centrifugation after adding 95% ethanol to the concentrate after concentrating the obtained extract under reduced pressure and then allowing it to stand.

In addition, in the present specification, the term "active ingredient" refers to an ingredient capable of exhibiting a desired activity alone or exhibiting activity together with a carrier that is not itself active.

In addition, in the present specification, "respiratory disease" refers to asthma, chronic obstructive pulmonary disease (COPD, ie emphysema), tracheitis, bronchitis, as a respiratory disease caused by an inflammatory response or an irritable immune response (allergic reaction) or both mechanisms. (bronchitis) or rhinitis (rhinitis), preferably an inflammatory reaction due to fine dust, an irritable immune reaction, or a mechanism of both, coughing, excessive secretion of mucus (sputum), respiratory tract accompanied by difficulty breathing It refers to a disease such as asthma, chronic obstructive pulmonary disease or rhinitis. In particular, in the case of chronic obstructive pulmonary disease, smoking or fine dust is the cause of the disease. Chronic inflammation and oxidative stress are involved in the onset mechanism. (Biol Pharm Bull, 2012, 35:1752-1760; Am J Physiol Lung CellMol Physiol, 2010, 298:L262-L269), The following experimental example of the present invention is the inflammatory reaction due to fine dust in lung tissue, etc. In that it shows a clear relief of oxidative stress, the "respiratory disease" refers to chronic obstructive pulmonary disease.

In the composition of the present invention, the active ingredient may be included in an arbitrary amount (effective amount) depending on the use, formulation, etc., as long as it can exhibit the effect of improving respiratory diseases, etc., and the usual effective amount is 0.001 weight based on the total weight of the composition. % To 15% by weight. Here, the term "effective amount" refers to the intended medical and pharmacological effects, such as the effect of improving respiratory diseases, when the composition of the present invention is administered to a mammal, preferably a person, to which the composition of the present invention is administered during the administration period according to the advice of a medical expert, etc. Yes, it refers to the amount of the active ingredient contained in the composition of the present invention. Such effective amounts can be determined empirically within the range of ordinary skill in the art.

The composition of the present invention can be grasped as a food composition in a specific aspect.

The food composition of the present invention may be prepared in any form, such as beverages such as tea, juice, carbonated beverages, ionized beverages, processed oils such as milk, yogurt, gums, rice cakes, Korean confectionery, bread, Foods such as confectionery and noodles, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jelly, and health functional food preparations such as bars can be prepared. In addition, the food composition of the present invention can be classified as a product as long as it conforms to the enforcement regulations at the time of manufacture and distribution in terms of legal and functional classification. For example, it is a health functional food in accordance with the Korean "Health Functional Food Act", or confectionery, bean, tea, etc. according to each food type according to the food code of the Korean "Food Sanitation Act" (“Food Standards and Standards” notified by the Ministry of Food and Drug Safety). It may be beverages, special-purpose foods, and the like.

The food composition of the present invention may contain food additives in addition to the active ingredients. Food additives can generally be understood as substances that are added to food and mixed or infiltrated in manufacturing, processing, or preserving food. Since they are consumed daily and for a long time with food, their safety must be ensured. In the Food Additive Code (“Food Sanitation Act” in Korea) governing the manufacture and distribution of food, food additives with guaranteed safety are limited in terms of ingredients or functions. In the Korean Food Additives Code (KFDA notice "Food Additive Standards and Standards), food additives are classified into chemical synthetic products, natural additives, and mixed preparations in terms of ingredients. These food additives are sweetening agents and flavoring agents in terms of function. , Preservatives, emulsifiers, acidulants, thickeners, etc.

Sweeteners are used to impart a suitable sweetness to food, and either natural or synthetic can be used in the food composition of the present invention. Preferably, a natural sweetener is used, and examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents are used to improve taste or flavor, and can be used both natural and synthetic. Preferably, it is the case of using a natural one. In the case of using natural ones, the purpose of nutrient enhancement can be combined in addition to flavor. As a natural flavoring agent, it may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, or the like, or from green tea leaves, roundtails, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, you can use those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo. The natural flavoring agent may be a liquid concentrate or a solid extract. In some cases, synthetic flavoring agents may be used. As synthetic flavoring agents, esters, alcohols, aldehydes, terpenes, and the like may be used.

As a preservative, calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. can be used, and as an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, pectin And the like may be used, and as the acidulant, arithmetic, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like can be used. In addition to the purpose of enhancing taste, the acidulant may be added so that the food composition has an appropriate acidity for the purpose of inhibiting the growth of microorganisms. As the thickening agent, a suspending agent, a settling agent, a gel-forming agent, a swelling agent, and the like may be used.

In addition to the food additives described above, the food composition of the present invention may include a physiologically active substance or minerals known in the art for the purpose of supplementing and reinforcing functionality and nutritional properties and ensuring stability as a food additive.

Examples of such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoyl thiamine, etc., and minerals include calcium preparations such as calcium citrate, magnesium stearate. Magnesium preparations such as, iron preparations such as iron citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, and zinc.

In the food composition of the present invention, the food additive described above may be included in an appropriate amount to achieve the purpose of addition according to the product type.

With regard to other food additives that may be included in the food composition of the present invention, reference may be made to the food code or food additive code according to the laws of each country.

The composition of the present invention may be understood as a pharmaceutical composition in other specific embodiments.

The pharmaceutical composition of the present invention may be prepared in an oral dosage form or a parenteral dosage form according to an administration route by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, "pharmaceutically acceptable" means that the application (prescription) does not have toxicity beyond adaptable without inhibiting the activity of the active ingredient.

When the pharmaceutical composition of the present invention is prepared in an oral dosage form, powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, wafers according to a method known in the art together with a suitable carrier It can be prepared in a formulation such as. Examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, corn starch, potato starch, wheat starch, and other starches, cellulose, methylcellulose, ethylcellulose, Celluloses such as sodium carboxymethylcellulose and hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable Yu, etc. are mentioned. In the case of formulation activity, if necessary, it can be formulated including diluents and/or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

When the pharmaceutical composition of the present invention is prepared in a parenteral dosage form, it may be formulated in the form of an eye drop, an injection, a transdermal administration, a nasal inhalation, and a suppository according to a method known in the art together with a suitable carrier. When formulated as an eye drop, suitable carriers may include sterile water, saline, and isotonic solutions such as 5% dextrose, and benzalkonium chloride, mefilparaben, ethylparaben, etc. may be added as necessary for preservative purposes. When formulated as an injection, sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof may be used as suitable carriers, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanol amine, or sterilization for injection. Water, isotonic solutions such as 5% dextrose, etc. can be used. When formulated as a transdermal administration, it can be formulated in the form of an ointment, cream, lotion, gel, external solution, pasta, liniment, air roll, and the like. In the case of nasal inhalants, suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide can be used to form an aerosol spray.When formulated as a suppository, the base is Withepsol ( witepsol), tween 61, polyethylene glycols, cacao butter, laurin paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, and the like.

The specific formulation of pharmaceutical compositions is known in the art, and for example, Remington's Pharmaceutical Sciences (19th ed., 1995) may be referred to. This document is considered as part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, sex, age, patient severity, and route of administration. May be in the /kg range. Administration can be made once a day or divided into several times. Such dosage should not be construed as limiting the scope of the invention in any aspect.

As described above, according to the present invention, it is possible to provide a composition for improving respiratory diseases due to fine dust and the like, using a hoesaengyimojaban extract. The composition of the present invention may be commercialized as a health functional food or medicine.

1 is a diagram schematically showing the configuration of an experimental group and the timing and duration of sample administration in an experimental animal model.
Figures 2 and 3 are results of evaluating the effect of the extract on the leukocyte percentage change (differential cell count) of the blood in the animal model inhaled fine dust.
4 and 5 are the results of evaluating the effect of the extract on the white blood cell count (differential cell count) of the bronchialveolar lavage fluid (BALF) in the animal model inhaled with fine dust.
FIG. 6 is a result of evaluating the effect of the extract on the pathologic changes of organs and lungs in an animal model inhaled with fine dust (0, normal; 1, few cells observed; 2, a ring of inflammatory cells) one cell layer deep; 3, a ring of inflammatory cells 2-4 cells deep; and 4, a ring of inflammatory cells >4 cells deep).
FIG. 7 is a result of evaluating the effect of the extract of hoeosaengi hairworm on the expression of 8-OHdG in the lungs in an animal model inhaling fine dust.
FIG. 8 is a result of evaluating the effect of the extract of hoeosaengi hairworm on the change in the expression of Gr-1 in the organs and lungs in an animal model inhaled with a microbungee.
FIG. 9 is a result of evaluating the effect of the extract of hoeax saengi hatpidae on the eosinophilic leukocyte infiltration of organs and lung tissues in an animal model inhaled with fine dust.
FIG. 10 is a result of evaluating the effect of the extract of hoeosaengi hairpinae on the invasion of mast cells in organs in an animal model in which fine dust was inhaled.
11 and 12 are the results of evaluating the effect of the extract on the mucus secretion and gablet cell proliferation in the animal model inhaled fine dust.
FIG. 13 is a result of evaluating the effect of the extract on the cell population change in the lung cells of the animal model inhaled with fine dust.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these Examples and Experimental Examples.

<Example> Preparation of hoesaengyimojaban extract

After washing three times for 30 minutes with purified water, the domestic hoesaengi hatban (outpost) was dried with a hot air dryer at 50°C for 24 hours to a moisture content of 10% or less, and pulverized into a size of 40-50 mesh with a pin mill. Add 10-15% (w/v) of hoesaengi hatban powder to 70% alcohol and extract for 12 hours at 70℃ using a circulation extractor. Treated clay for food, stirred at 60rpm for 2 hours, and then centrifuged at 12,000rpm. Separated and recovered the supernatant. The recovered supernatant was concentrated under reduced pressure at 60° C. to 1/5 volume with a vacuum concentrator, 95% alcohol was added to 3 times the volume of the concentrated solution, allowed to stand for 12 hours, and then centrifuged and filtered to recover the supernatant. The recovered extract was concentrated to 1/5 volume with a vacuum concentrator, and then dried with a freeze dryer to recover Ethanol Extract Powder (SHE).

<Experimental Example> Respiratory disease improvement activity test of the extract of chinensis mosquito

<Experimental Example 1> Experimental Animal Model Composition of Experimental Group and Sample Administration

As for the experimental animals, 7-8 weeks old BALB/c mice were divided into 4 for each group and used. The experimental group was a normal control group (hereinafter referred to as Healthy control group), fine dust inhalation group (hereinafter referred to as Dust only group), ovalbumin (hereinafter referred to as OVA) sensitized group (hereinafter referred to as OVA only), and OVA sensitization + fine dust inhalation group (hereinafter referred to as OVA+Dust group) , OVA sensitization+fine dust inhalation and sample administration group (hereinafter, OVA+Dust+SHE 200 mg/kg, OVA+Dust+SHE 400 mg/kg group), OVA sensitization+fine dust inhalation and allergic respiratory inflammatory drug (Prednisone) The experiment was divided into administration groups (OVA+Dust+Prednisone 5mg/kg group). BALB/c mice were mixed well with 10 μg of OVA and 2 mg of Al(OH) _{3 and} 200 μl of physiological saline and administered intraperitoneally once (intraperitoneal, ip). After 15 days, the fine dust inhalation group was inhaled daily using a nebulizer at a concentration ^{of 5 mg/m 3 for 30 minutes for 7 days.} Along with inhalation of fine dust, SHE was administered orally at a concentration of SHE 200 and 400 mg/kg to the group administered with SHE, and prednisone was administered orally at a concentration of 5 mg/kg to the allergic respiratory inflammatory drug treatment group (Fig. 1 ).

<Experimental Example 2> The effect of white blood cell count (differential cell count) of the extract (SHE) in an animal model in which fine dust was inhaled

(1) After euthanizing BALB/c mice, blood was collected through cardiac blood collection using a heparin syringe to obtain mouse blood for each group. Thereafter, after thinly spreading on a slide glass, staining using Diff Quick solution, the number of neutrophils, lymphocytes, monocytes, eosinophils, and basophils were measured and compared under a microscope.

(2) Differential cell count results are shown in FIGS. 2 and 3. Compared to the healthy control group, infiltration of neutrophils, eosinophils, and basophils was significantly increased in the OVA+Dust group (Fig. 2A and Fig. 3A and B). In the OVA+Dust+SHE 200 mg/kg group administered with SHE, eosinophil infiltration decreased 6.6 times, and 10.0 times in the OVA+Dust+SHE 400 mg/kg group (Fig. 3A). In addition, in the OVA+Dust+SHE 400 mg/kg group, the infiltration of neutrophils was reduced by 1.5 times (Fig. 2A) and the infiltration of basophils by 3.0 times (B in Fig. 3). On the other hand, the degree of infiltration of lymphocytes and monocytes did not change due to OVA and fine dust (FIG. 2B and C).

<Experimental Example 3> Effect of white blood cell count (differential cell count) in bronchialveolar lavage fluid (BALF) in animal model inhaled with fine dust

(1) Because the cells contained in the bronchoalveolar lavage fluid reflect the inflammatory state of lung tissue and bronchi (J Toxicol Environ Health A 2017, 80(4), 197-207), the change in the percentage of leukocytes in the bronchoalveolar lavage fluid was observed. Thus, it was confirmed the effect of the extract (SHE) on the inflammation of the lungs and bronchi caused by inhalation of fine dust.

(2) After euthanizing BALB/c mice, DPBS was injected into the airway using an intratracheal catheter to collect bronchoalveolar lavage fluid. The collected bronchial alveolar lavage solution was centrifuged for 5 minutes at 3000 g at 4° C., fixed with methanol, and attached to the slide. The slides with attached cells were stained using Diff-Qick solution, and the number of neutrophils, lymphocytes, monocytes, eosinophils, and basophils were measured and compared under a microscope.

(3) Differential cell count results are shown in FIGS. 4 and 5. Compared to the healthy control group, the infiltration of lymphocytes and eosinophils was significantly increased in the OVA+Dust group (FIG. 4B and FIG. 5A), and OVA+Dust+SHE 400 administered SHE in parallel. In the group administered with the mg/kg group and the allergic respiratory inflammatory drug concurrently, the infiltration of lymphocytes and eosinophils was significantly reduced, and basophils were not significant, but showed a tendency to decrease (B in Fig. 4 and Fig. 5). A and B). On the other hand, the degree of infiltration of neutrophils and monocytes did not change due to OVA and fine dust (Fig. 4A and C).

<Experimental Example 4> Influence of the extract (SHE) on the pathological changes of the trachea and lung in the animal model inhaled fine dust

(1) BALB/c mice were euthanized, autopsied, trachea and lungs were collected, and paraffin blocks were prepared according to a conventional method. The paraffin block was cut into 3 μm and attached to the slide, followed by deparaffining and hydration, followed by staining with Mayer's hematoxylin solution and eosin solution, followed by dehydration and transparence and encapsulation.

(2) The results are shown in FIG. 6. Compared to the healthy control group, inflammatory cell infiltration was significantly increased in the organs of the OVA+Dust group (Fig. 6A), whereas the invasion of inflammatory cells in the organs in the OVA+Dust+SHE 200 mg/kg group administered concurrently with SHE. In particular, in the OVA+Dust+SHE 400 mg/kg group, it was confirmed that the invasion of inflammatory cells was effectively reduced, similar to the group administered with the allergic respiratory inflammatory drug concurrently (FIG. 6A). Similar to organs, inflammatory cell infiltration was significantly increased in the organs of the OVA+Dust group compared to the healthy control group (Fig. 6B), whereas the concurrent administration of SHE OVA+Dust+SHE 200, 400 mg/kg In the group, the infiltration of inflammatory cells in the lung tissue was significantly reduced depending on the SHE concentration (FIG. 6B). In the graph of Fig. 6, 0, normal; 1, few cells observed; 2, a ring of inflammatory cells one cell layer deep; 3, a ring of inflammatory cells 2-4 cells deep; and 4, a ring of inflammatory cells >4 cells deep.

<Experimental Example 5> Influence of Shoe Hatchery Spot Extract (SHE) on Changes in Expression of 8-OHdG in Lungs in an Animal Model Inhaled with Fine Dust

(1) 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is an indicator of DNA damage caused by oxidative stress. Is created. 8-OHdG is used as an index to evaluate the degree of DNA damage caused by reactive oxygen species (ROS) that induce oxidative stress (Particle and fiber toxicology, 2017, 14(38)). Therefore, the degree of oxidative stress caused by the inhalation of fine dust and the antioxidant effect of the extract (SHE) were confirmed through immunohistochemistry (IHC).

(2) BALB/c mice were euthanized and then autopsied to collect lung tissue, and then paraffin blocks were prepared according to a conventional method. The paraffin block was cut into 3 μm and attached to the slide, followed by immunohistochemistry (IHC). In order to confirm the expression of 8-OHdG, the 8-OHdG antibody (1:2000) was reacted at room temperature for 1 hour, and then reacted again at 4° C. overnight. After the reaction was over, biotinylated anti-goat IgG was reacted at room temperature, followed by color development with 3,3'-diaminobenxidine (DAB). Cells showing a positive reaction were counter-stained with hematoxylin solution and observed with an optical microscope.

(3) The results of confirming the expression of 8-OHdG are shown in FIG. 7. In the nuclei of neutrophils, eosinophils, and epithelial cells, 8-OHdG-positive cells were significantly increased in the Dust olny group, OVA only group, and OVA+Dust group compared to the Healthy control group, but OVA+Dust+SHE 8-OHdG positive cells were significantly decreased in the 200 mg/kg group and the OVA+Dust+SHE 400 mg/kg group, and in particular, the OVA+Dust+Prednisone group administered concurrently with the drug in the OVA+Dust+SHE 400 mg/kg group. The 8-OHdG-positive cells significantly decreased at a level similar to that of the 5 mg/kg group.

<Experimental Example 6> Influences of S. S. hairpinae extract (SHE) on the change of Gr-1 expression in organs and lungs in an animal model inhaled with microbundles

(1) Gr-1 is a granulocyte marker that is involved in the differentiation and maturation of granulocytes and is known to secrete cytokines and act on immune mechanisms when infection occurs (Immunity 2012, 36(3), 451-63). ). Therefore, it was confirmed that the effect of the extract (SHE) on granulocyte infiltration of trachea and lung tissue due to inhalation of fine dust.

(2) BALB/c mice were euthanized and then autopsied to collect trachea and lung tissue, and then paraffin blocks were prepared according to a conventional method. The paraffin block was cut into 3 μm and attached to the slide, followed by deparaffining and hydration, and immersed in a 0.3% hydrogen peroxide solution to inhibit endogenous peroxidase in the tissue. Afterwards, in order to suppress the non-specific immune response, blocking rat serum was reacted for 30 minutes, and then mouse Ly-6G/Ly-6C (1:200, R&D systems) was reacted overnight at 4°C. After the reaction was completed, biotinylated anti-rabbit serum was reacted at room temperature for 45 minutes, followed by color development using 3, 3'-diaminbenzidine (DAB, Vector), and counter-stained with hematoxylin solution. Between each step, it was sufficiently washed with phosphate buffered saline (PBS), 0.3% PBS-triton X100, and sealed after dehydration and clarification process.

(3) The results are shown in FIG. 8. Compared to the healthy control group, granulocyte infiltration was significantly increased 5.2 times in the organs of the OVA+Dust group, whereas in the OVA+Dust+SHE 200 and 400 mg/kg group administered concurrently with SHE, the infiltration of granulocytes decreased. When SHE was administered concurrently at 400 mg/kg, it was significantly decreased by 2.2 times, which was confirmed that the infiltration of granulocytes was reduced to a level similar to that of the group administered with the allergic respiratory inflammatory drug (FIG. 8A). Similar to organs in lung tissue, granulocyte infiltration was significantly increased by 5.6 times in the organs of the OVA+Dust group compared to the healthy control group, whereas in the OVA+Dust+SHE 200 and 400 mg/kg groups administered with SHE concurrently, granulocyte infiltration was observed. These were significantly reduced by 1.5 and 1.7 times, respectively (FIG. 8B). This is similar to that of the group administered with allergic respiratory inflammatory drugs, and the infiltration of granulocytes was reduced (2.0 times less granulocyte infiltration compared to the Healthy control group), so the administration of SHE reduced the infiltration of granulocytes in the organs and lung tissues due to fine dust. It was confirmed that it was effectively suppressed.

<Experimental Example 7> Influence of the extract (SHE) on eosinophilic leukocyte infiltration of organs and lung tissues in an animal model inhaled with fine dust

(1) The characteristic pathologic finding of chronic allergic respiratory inflammatory disease is an increase in eosinophilic leukocytes, and eosinophils containing inflammatory proteins induce airway epithelial cell damage, increase airway irritability, and induce degranulation of mast cells, leading to allergic reactions. It plays an important role in the onset of sexual respiratory inflammation (World Allergy Organ J 2016, 9, 7). Therefore, it was confirmed that the effect of the extract (SHE) on eosinophilic leukocyte infiltration of organs and lung tissues due to inhalation of fine dust.

(2) BALB/c mice were euthanized and then autopsied to collect trachea and lung tissue, and then paraffin blocks were prepared according to a conventional method. The paraffin block was cut into 3 μm and attached to the slide, and the tissue was stained and sealed using 0.05% Congo red in 50% EtOH solution.

(3) The results are shown in FIG. 9. Compared to the healthy control group, the infiltration of eosinophilic leukocytes was significantly increased by 4.4 times in the organs of the OVA+Dust group, whereas in the OVA+Dust+SHE 200 and 400 mg/kg group administered with SHE, the infiltration of eosinophilic leukocytes was significantly increased In particular, in the group administered with 400 mg/kg of SHE, the infiltration of eosinophilic leukocytes was significantly decreased by 1.9 times (Fig. 9A). Similar to organs in lung tissue, eosinophilic leukocyte infiltration was significantly increased in the organs of the OVA+Dust group compared to the healthy control group, and when SHE was administered concurrently, the infiltration of eosinophilic leukocytes tended to decrease in a concentration-dependent manner. In particular, in the OVA+Dust+SHE 400 mg/kg group, eosinophil infiltration was reduced by 1.7 times (FIG. 9B).

<Experimental Example 8> Influence of Shoe Hatchery Extract (SHE) on Invasion of Mast Cells in Organs in an Animal Model Inhaled with Fine Dust

(1) Mast cells are mainly present in connective tissues and mucous membranes, and are characterized by having granules in the cytoplasm.The granules include chemical mediators such as histamine, prostaglandin, and cytokines. They are released when mast cells are activated by external stimuli, and are involved in inflammatory reactions and hypersensitivity reactions, causing bronchoconstriction and increased mucus secretion (World Allergy Organ J 2016, 9, 7). Therefore, it was confirmed that the effect of the extract (SHE) on the infiltration of mast cells in the organs due to the inhalation of fine dust.

(2) BALB/c mice were euthanized, autopsied to collect organs, and paraffin blocks were prepared according to a conventional method. The paraffin block was cut into 3 μm and attached to the slide, and the tissue was stained and sealed with 0.05% Toluidine blue solution.

(3) The results are shown in FIG. 10. Compared to the healthy control group, the infiltration of mast cells into the trachea was significantly increased in the OVA+Dust group, and the infiltration of mast cells into the trachea was significantly increased compared to the OVA only group and the Dust only group (Fig. 10A). On the other hand, in the OVA+Dust+SHE 200, 400 mg/kg group administered with SHE in parallel, the invasion of mast cells in the trachea was significantly reduced (FIG. 10A). In addition, the degranulation rate of mast cells was increased in the OVA+Dust group compared to the healthy control group (FIG. 10B), and the degranulation rate of mast cells was significantly increased compared to the OVA only group and the Dust only group (FIG. 10B). However, in the OVA+Dust+SHE 200 and 400 mg/kg group administered with SHE, the degranulation rate of mast cells decreased. In particular, in the OVA+Dust+SHE 400 mg/kg group, OVA+Dust+Prednisone 5 It reduced the degranulation rate of mast cells more effectively than the mg/kg group (FIG. 10B).

<Experimental Example 9> Influence of the extract (SHE) on mucus secretion and proliferation of gablet cells in an animal model inhaled with fine dust

(1) When exposed to repetitive airway inflammation by foreign substances, airway remodeling occurs, and symptoms such as an increase in goblet cells and mucous gland hyperplasia appear (Chest 2018, 154(1). ), 169-176). Therefore, it was confirmed that the effect of the extract (SHE) on the secretion of mucus and proliferation of goblet cells due to inhalation of fine dust.

(2) BALB/c mice were euthanized and then autopsied to collect trachea and lung tissue, and then paraffin blocks were prepared according to a conventional method. The paraffin block was cut into 3 μm and attached to the slide, followed by periodic acid of Schiff (PAS) staining. In addition, for anti-mucin 5AC antibody (R&D abcam) staining, a paraffin block was cut to 3 μm and attached to the slide, followed by deparaffining and hydration, and immersed in a 0.3% hydrogen peroxide solution to inhibit endogenous peroxidase in the tissue. Afterwards, blocking horse serum was reacted for 30 minutes to suppress non-specific immune responses, and then anti-mucin 5AC antibody (1:500, R&D abcam) was reacted overnight at 4°C. After the reaction, biotinylated anti-mouse serum was reacted at room temperature for 45 minutes, followed by color development with 3, 3'-diaminbenzidine (DAB, Vector), and counterstained with hematoxylin solution. Between each step, it was sufficiently washed with phosphate buffered saline (PBS), 0.3% PBS-triton X100, and sealed after dehydration and clarification process.

(3) The results are shown in Figs. 11 and 12. In the results of immunohistochemical staining for organ PAS and mucin-5AC antibodies, compared to the healthy control group, mucus secretion was increased in the OVA+Dust group, and hyperproliferation of goblet cells was observed, whereas concentration-dependent in the group administered with SHE. The secretion of mucus and proliferation of goblet cells were decreased, and in particular, the OVA+Dust+SHE 400 mg/kg group decreased to a level similar to that of the OVA+Dust+Prednisone 5 mg/kg group (Fig. 11A and B). Meanwhile, in the OVA+Dust group, mucus secretion into the trachea was significantly increased compared to the OVA only group and the Dust only group (FIG. 11B).

(4) In addition, compared to the healthy control group, mucus secretion was increased in the lung tissue in the OVA+Dust group, and hyperproliferation of goblet cells was observed, whereas in the OVA+Dust+SHE 200, 400 mg/kg group administered with SHE. The secretion of mucus and proliferation of goblet cells decreased in a concentration-dependent manner, and decreased to a level similar to that of the OVA+Dust+Prednisone 5 mg/kg group administered with the drug (Fig. 12A and B).

<Experimental Example 10> Influence of the extract (SHE) on the change of cell population in the lung cells of the animal model inhaled with fine dust

(1) Allergic respiratory inflammatory disease is a chronic inflammatory disease involving the immune system and is characterized by T cells and eosinophil infiltration, mast cells and basophil activation (World Allergy Organ J 2016, 9, 7). Therefore, in order to investigate the effect of S. S. hairworm spot extract (SHE) on the change of immune cell population due to inhalation of fine dust, it was analyzed using flow cytometry.

(2) After euthanasia of BALB/c mice, the lungs were collected by autopsy, sectioned finely, added 0.4 mg/ml of collagenase, reacted at 37° C. for 30 minutes, and mixed well every 8-10 minutes. Then, PBS was added and mixed well at 1500 rpm for 5 minutes, and then ACK solution was added to dissolve red blood cells, reacted at room temperature for 10 minutes, and washed with DPBS. The nonspecific reaction was reduced by reacting the Fc blocker to the single cell suspension in the lung at 4°C for 15 minutes. Then FITC- or PE-, PerCp-CyTM5.5-, BV421-, AF700-labeled CD3e (145-2C11), CD4 (H129.19), CD8a (53-6.7), CD45 (30-F11), CD11b (M1/70) and CD11c (HL3) antibodies were reacted at 4°C for 15 minutes. Then, the cells were washed with PBS and analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Inc., Kraemer Blvd, CA, USA).

(3) The results are shown in FIG. 13. Compared to the healthy control group, the number of CD3e ⁺ CD4 ⁺ helper T cells increased by 1.3 times in the OVA + Dust group, and 1.2 times significantly in the OVA + Dust + SHE 400 mg/kg group administered with SHE. Decreased (Fig. 13A). In addition, the number of CD3e ⁺ CD8 ⁺ cytotoxic T cells was significantly increased in the OVA+Dust group compared to the Healthy control group, and the OVA+Dust+SHE 200, 400 mg/kg group administered with SHE concurrently. At, respectively, the concentration-dependently decreased 1.5 times and 2.0 times (FIG. 13B). The number of CD45 ⁺ CD11c ⁺ dendritic cells increased by 4.0 times in the OVA+Dust group compared to healthy control, and 1.3 times and 1.4 in the OVA+Dust+SHE 200 and 400 mg/kg groups, respectively, administered with SHE. The concentration-dependently decreased with double significance (FIG. 13C). The number of CD11c ⁺ CD11b ⁺ macrophages increased in the OVA+Dust group compared to the Healthy control group, and significantly decreased in the OVA+Dust+SHE 200, 400 mg/kg group administered with SHE. (Fig. 13D).

Statistical processing

Each experiment was repeated three or more times (n=3 or more per group). Each experimental result was expressed as mean value ± standard deviation, and using Microsoft Office Excelj's Student's t-test **; P <0.005, ***; P <0.0005, ††; P <0.005, †††; P <0.0005, #; Significance was tested at a level of P <0.05.

Claims (15)

Including the extract of hoesaengyimojaban as an active ingredient,
The extract is treated with water, ethanol, or a mixed solvent extract thereof from white clay for food, centrifuged, and the supernatant is recovered, and then a mixed solvent of water and ethanol is added to the supernatant, followed by centrifugation. A composition for improving respiratory diseases, characterized in that it is a supernatant obtained by separation and filtration or a concentrate thereof.
The method of claim 1,
The composition, characterized in that the respiratory disease is a respiratory disease caused by fine dust.
The method of claim 1,
The respiratory disease is a composition, characterized in that asthma, chronic obstructive pulmonary disease, tracheitis, bronchitis or rhinitis.
delete The method of claim 1,
The respiratory disease is a composition, characterized in that the chronic obstructive pulmonary disease.
The method according to any one of claims 1 to 3 and 5,
The composition is a composition, characterized in that the food composition.
The method according to any one of claims 1 to 3 and 5,
The composition is a pharmaceutical composition, characterized in that the composition.
Including the extract of hoesaengyimojaban as an active ingredient,
The extract is treated with water, ethanol, or a mixed solvent extract thereof from white clay for food, centrifuged, and the supernatant is recovered, and then a mixed solvent of water and ethanol is added to the supernatant, followed by centrifugation. A composition for inhibiting sputum secretion, characterized in that it is a supernatant obtained by separation and filtration or a concentrate thereof.
delete The method of claim 8,
The composition is a composition, characterized in that the food composition.
The method of claim 8,
The composition is a pharmaceutical composition, characterized in that the composition.
delete delete delete delete

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