Composition for preventing, alleviating, or treating respiratory diseases caused by fine dust, comprising extract of phlomis umbrosa

Abstract

The present invention relates to a composition for preventing, alleviating, or treating respiratory diseases caused by fine dust, the composition comprising an extract of Phlomis umbrosa. The extract of Phlomis umbrosa according to the present invention enhances antioxidant activity in the bronchi and lungs, reduces inflammatory factors caused by fine dust, and exhibits expectorant activity, and can thus be used as a food composition for preventing or alleviating respiratory diseases caused by fine dust, or as a pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust.

Description

Composition for preventing, improving or treating respiratory diseases caused by fine dust, containing extract of Hansokdan as an active ingredient

[0001] The present invention was made under the support of the Rural Development Administration under the project number 1395070339, and the research management specialized institution of the project is the Rural Development Administration, the research project name is "Development of core technology for increasing useful components of crops", the research project name is "Development of technology for producing Hansokdan and processing / materialization technology of black ginseng for increasing useful components", the main institution is Natural Endotech Co., Ltd., and the research period is from January 1, 2019 to December 31, 2023.

[0002] The present invention relates to a composition for preventing, improving or treating respiratory diseases caused by fine dust, comprising an extract of Hansokdan as an effective ingredient.

[0003] The respiratory system governs the body's ability to inhale and exhale air. The trachea, which serves as a passage connecting the throat to the bronchi, has a branched structure. This structure slows the airflow, preventing foreign substances like bacteria and dust from reaching the terminal alveoli and allowing them to settle on the bronchial walls. These deposited foreign substances are then transported out of the lungs and eliminated by the mucus secreted within the bronchi and the movement of cilia.

[0004] Respiratory diseases, which occur when the respiratory system does not function normally, are diseases that affect the organs related to breathing. The main symptoms include coughing, hemoptysis, chest pain, sputum, and shortness of breath, and are caused by chronic inflammatory reactions in the respiratory tract and damage to the respiratory organs.

[0005] In particular, the incidence of respiratory diseases has been increasing recently due to inflammation and damage to the respiratory organs caused by external factors such as air pollution. According to data from the World Health Organization (WHO) published in 2018, as of 2016, more than 91% of the world's population lived in areas where air quality did not meet the WHO air quality guidelines, and premature deaths due to air pollution reached 4.2 million people annually. Exposure to air pollution is reported to have a higher risk of disease than generally known modifiable disease risk factors such as lack of exercise, a high-salt diet, hyperlipidemia, and drug abuse. According to the Organization for Economic Co-operation and Development (OECD), the number of premature deaths worldwide due to air pollution is projected to increase from 3 million in 2010 to 6-9 million in 2060. In Korea, the number of premature deaths is projected to surge from 359 in 2010 to 1,109 in 2060, making additional responses essential. (Yu, I-ran, Kim, Jin-hoo, Yang, Won-kyung, Kim, Seung-hyung, and Park, Yang-chun. (2019). Clinical research trends in fine dust-related respiratory diseases. Journal of Korean Oriental Internal Medicine, 40(3), 443-457.)

[0006] Fine dust is defined as particulate matter (PM) with a diameter of 10 μm or less among total suspended particles (TSP) in the air, and is divided into fine dust (PM10) with a diameter of less than 10 μm and ultrafine dust (PM2.5) with a diameter of less than 2.5 μm.

[0007] Fine dust is classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC), an affiliate of the World Health Organization (WHO). Ultrafine dust (PM2.5) is known to be harmful to the human body, causing respiratory diseases and other health problems by passing through the respiratory system and reaching the alveoli directly. Fine dust occurs in high concentrations from winter to spring, causing significant tangible and intangible damage and adverse effects on daily life. South Korea, however, faces significant challenges in managing fine dust due to the geographical influence of westerly winds blowing from China and seasonal characteristics such as heavy rain concentrated in the summer.

[0008] Phlomite (Phlomoides umbrosa Turczaninow.) is the root of a perennial plant in the Labiatae family. It is commonly found on mountain slopes throughout Korea, except in the northern mountainous regions. Phlomite has multiple long, cylindrical or spindle-shaped roots with longitudinal wrinkles on its outer surface. Phlomite is known to be effective in relieving pain, treating inflammation, and allergies, and its roots and leaves are known to be useful as food ingredients.

[0009] However, the use of Hansokdan extract for preventing, improving, or treating respiratory diseases caused by fine dust is unknown, and no research on its mechanism has been conducted.

[0010] Accordingly, the present inventors conducted a direct efficacy study on the prevention, improvement, or treatment of respiratory diseases induced by fine dust with the extract of Hansokdan. As a result, it was confirmed that the extract of Hansokdan increased antioxidant enzyme activity and decreased the levels of Th2 (T helper type 2) cytokines such as interleukin 4 (IL-4), interleukin 5 (IL-5), and interleukin 13 (IL-13) and the concentrations of leukotrienes and prostaglandin E2, which are respiratory disease factors. In addition, it was confirmed that the thickness and degree of contraction of the alveolar wall were improved, and it was confirmed that the expression of inflammation-related factors in lung tissue was suppressed in a concentration-dependent manner. In addition, it was confirmed that the expectoration ability in the bronchial tubes was improved through the secretion of phenol red. As a result, it was confirmed that the prevention, improvement, or treatment of respiratory diseases caused by fine dust was possible, and the present invention was completed.

[0011] The purpose of the present invention is to provide a food composition for preventing or improving respiratory diseases caused by fine dust, including an extract of Hansokdan.

[0012] In addition, the present invention aims to provide a pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust, including an extract of Hansokdan.

[0013] To achieve the above purpose, the present invention provides a food composition for preventing or improving respiratory diseases caused by fine dust, including an extract of Hansokdan.

[0014] In addition, the present invention provides a pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust, including an extract of Hansokdan.

[0015] The extract of the present invention increases antioxidant activity in the bronchi and lungs, reduces inflammatory factors caused by fine dust, and has expectorant activity, so it has excellent effects in preventing, improving, and treating respiratory diseases caused by fine dust, and thus can be used as a food composition for preventing or improving respiratory diseases caused by fine dust or a pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust.

[0016] Figure 1 is a graph showing the results of measuring cell viability of Hansokdan extract.

[0017] Figure 2 is a graph showing the results of an analysis of the expression of inflammatory cytokines in a cell model of the extract of Hansokdan.

[0018] Figure 3 is a graph showing the results of analysis of the expression regulation of proteins that regulate inflammation-inducing signaling pathways in a cell model of the extract of Hansokdan.

[0019] Figure 4 is a graph measuring the antioxidant activity of the extract of Hansokdan in an animal model.

[0020] Figure 5 is a graph showing the results of an analysis of the expression of inflammatory cytokines in an animal model of the extract of Hansokdan.

[0021] Figure 6 is a photograph showing the results of an analysis of the regulation of alveolar wall thickness in an animal model of a Korean herb extract.

[0022] Figure 7 is a graph showing the results of protein expression regulation analysis of the inflammatory signaling pathway of bronchial and lung tissue in an animal model of Hansokdan extract.

[0023] Figure 8 is a graph showing the results of analysis of the regulation of expression of leukotrienes and prostaglandin E2 related to the inflammatory mechanism in an animal model of the extract of Hansokdan.

[0024] Figure 9 is a graph analyzing the expectorant activity of the extract of Hansokdan in an animal model.

[0025] A food composition for preventing or improving respiratory diseases caused by fine dust, comprising an extract of Hansokdan.

[0026] Hereinafter, the present invention will be described in detail with reference to the contents described in the attached drawings.

[0027] Throughout this specification, '%' used to indicate the concentration of a particular substance is (w / w) % for solid / solid, (w / v) % for solid / liquid, and (v / v) % for liquid / liquid, unless otherwise stated.

[0028] The term "prevention" used in the present invention means any act of inhibiting or delaying the occurrence, development, and recurrence of respiratory diseases caused by fine dust by administering a composition according to the present invention.

[0029] The term "improvement" used in the present invention means any action that reduces a parameter related to a respiratory disease caused by fine dust, for example, the severity of symptoms, by administering a composition according to the present invention.

[0030] The term "treatment" as used herein refers to any action that improves or beneficially alters the symptoms of respiratory diseases and complications caused by fine dust by administering a composition according to the present invention. Those skilled in the art to which the present invention pertains will be able to reference materials provided by the Korean Medical Association and other sources to determine the precise criteria for diseases for which the composition of the present invention is effective, and to determine the degree of improvement, enhancement, and treatment.

[0031] One aspect of the present invention is a food composition for preventing or improving respiratory diseases caused by fine dust, including an extract of Hansokdan.

[0032] In the present invention, "Phlomoides umbrosa Turczaninow." is a perennial herbaceous plant belonging to the Labiatae family, and its main physiologically active components include flavonol glycoside, phenylethanoid glycoside, amino acid, umbroside, etc., and these components are known to exhibit anti-inflammatory, antioxidant, and immunomodulatory activities.

[0033] The extract of Hansokdan used in the present invention can be obtained using various organs and tissues of Hansokdan (e.g., roots, leaves, flowers, stems, fruits, seeds, etc.), and most preferably, it is an extract obtained from the roots of Hansokdan.

[0034] The extract of Hansokdan according to the present invention can be obtained by extracting and separating from nature using extraction and separation methods known in the art, and the "extract" defined in the present invention is extracted from Hansokdan using an appropriate solvent, and includes, for example, a crude extract, a polar solvent-soluble extract, or a non-polar solvent-soluble extract. Any organic solvent acceptable in the fields of food science / pharmaceutics / cosmetics may be used as an appropriate extraction solvent for extracting the extract from Hansokdan, and water or an organic solvent may be used, but is not limited thereto, for example, water; straight-chain or branched alcohols having 1 to 4 carbon atoms, including methanol, ethanol, propanol, isopropanol, and butanol; acetone; ether; benzene; chloroform; Various solvents such as ethyl acetate; methylene chloride; hexane; and cyclohexane may be used alone or in combination. Preferably, the extract may be extracted with one or more solvents selected from the group consisting of water and straight-chain or branched alcohols having 1 to 4 carbon atoms. More preferably, the extract may be extracted with water as a solvent. Any one of the following methods may be selected for the extraction method: hot water extraction, cold immersion extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, dissolution, and compression. In addition, the desired extract may additionally undergo a conventional fractionation process and may be purified using a conventional purification method.

[0035] There is no limitation on the method for preparing the extract of the present invention, and any known method can be used. For example, the extract included in the composition of the present invention can be prepared in a powder form by additional processes such as reduced pressure distillation and freeze drying or spray drying of the primary extract extracted by the above-mentioned hot water extraction or solvent extraction method. In addition, the primary extract can be obtained by further purifying fractions using various chromatography methods such as silica gel column chromatography, thin layer chromatography, and high performance liquid chromatography. Therefore, in the present invention, the extract is a concept that includes all extracts, fractions, and purified products obtained at each stage of extraction, fractionation, or purification, as well as their dilutions, concentrates, or dried products.

[0036] Additionally, the extract of the present invention includes an extract obtained by the above-described extraction solvent. The extract of the present invention can be prepared into a powder form by additional processes such as reduced pressure distillation and freeze drying or spray drying.

[0037] According to a preferred embodiment of the present invention, the amount of the extract of Hansokdan as an active ingredient in the composition of the present invention is 0.10-90 wt% with respect to the total weight of the composition.

[0038] In addition, the composition suppresses an inflammation-related signal pathway caused by fine dust, and the inflammation-related signal may be at least one selected from the group consisting of ERK (Extracellular signal-regulated kinases), JNK (c-jun N-terminal kinase), NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) p65, PI3K / AKT, and STAT6, and for example, the signal may be ERK (Extracellular signal-regulated kinases), JNK (c-jun N-terminal kinase), NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) p65, PI3K / AKT, and STAT6, but is not limited thereto.

[0039] In one embodiment of the present invention, the composition suppresses an inflammation-related signal pathway caused by fine dust, and the inflammation-related signal may be STAT6.

[0040] In addition, the composition increases the activity of antioxidant enzymes, and the antioxidant enzymes may be, but are not limited to, superoxide dismutase, catalase, and glutathione peroxidase.

[0041] Additionally, the composition may reduce the secretion of inflammatory factors caused by fine dust or inflammatory cytokines caused by fine dust.

[0042] In addition, the inflammatory factor may be at least one selected from the group consisting of leukotrienes, prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS), and may be, for example, leukotrienes, prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), and iNOS, but is not limited thereto.

[0043] In addition, the inflammatory cytokine may be at least one selected from the group consisting of interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 13 (IL-13), and interferon gamma (IFN-γ), and may be, for example, interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 13 (IL-13), and interferon gamma (IFN-γ), but is not limited thereto.

[0044] In addition, the respiratory disease may be at least one disease selected from the group consisting of respiratory inflammatory lung disease, chronic obstructive pulmonary disease, allergic rhinitis, sinusitis, upper respiratory tract infection, lower respiratory tract infection, chronic bronchitis, bronchiectasis, pneumonia, sequelae of pulmonary tuberculosis, acute respiratory distress syndrome, cystic fibrosis, otitis media, pulmonary fibrosis, asthma, emphysema, pharyngitis, laryngitis, and tonsillitis, but is not limited thereto.

[0045] The extract of the present invention can be added to food for the purpose of preventing or improving respiratory diseases caused by fine dust. When the extract of the present invention is used as a food additive, the extract of the present invention can be added as is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method. The amount of the active ingredient mixed can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing a food or beverage, the composition of the present invention is added in an amount of 15 wt% or less, preferably 10 wt% or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount can be below the above range, and since there is no problem in terms of safety, the active ingredient can also be used in an amount above the above range.

[0046] There are no specific restrictions on the types of the above foods. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and all foods in the conventional sense are included.

[0047] The health beverage composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, similar to conventional beverages. The natural carbohydrates mentioned above include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the natural carbohydrate is generally about 0.01-0.20 g, preferably about 0.04-0.10 g, per 100 mL of the composition of the present invention.

[0048] In addition to the above, the composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in combination. The proportion of these additives is not particularly critical, but is typically selected within the range of 0.01 to 0.20 parts by weight per 100 parts by weight of the composition of the present invention.

[0049] Another aspect of the present invention is a pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust, comprising an extract of Hansokdan.

[0050] The composition according to the present invention may contain a pharmaceutically effective amount of the extract of Hansokdan alone, or may contain one or more pharmaceutically acceptable carriers, excipients, or diluents. The "pharmaceutically effective amount" herein refers to an amount sufficient to prevent, improve, and treat symptoms of respiratory diseases. The term "pharmaceutically acceptable" herein refers to a composition that is physiologically tolerable and, when administered to humans, typically does not cause allergic reactions such as gastrointestinal upset, dizziness, or similar reactions.

[0051] In addition, the composition of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of a general pharmaceutical preparation. The dosage form can be oral, mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g., subcutaneous, intravenous, bolus injection, intramuscular, or intraarterial), topical (e.g., ophthalmic), transdermal, or transcutaneous, but is not limited thereto. Examples of dosage forms include, but are not limited to, tablets; caplets; capsules such as soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; powders; aerosols (e.g., nasal sprays or inhalers); gels; Liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions and elixirs; liquid dosage forms suitable for injectable administration to a patient; eye drops or other ophthalmic preparations suitable for topical administration; and sterile solid preparations (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for injectable administration to a patient. The type, shape, and form of the dosage forms of the present invention will generally vary greatly depending on their use. For example, a dosage form used for the acute treatment of a disease may contain a greater amount of active ingredient than a dosage form used for the chronic treatment of the same disease. Furthermore, a parenteral dosage form may contain a lesser amount of active ingredient than an oral dosage form used to treat the same disease. The dosage forms and methods encompassed by the present invention are very diverse, and this will be apparent to those skilled in the art to which the present invention pertains.

[0052] Additionally, compositions containing pharmaceutically acceptable carriers may be administered orally or parenterally in various dosage forms. When formulated, they may be prepared using commonly used diluents or excipients, such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. The above carrier, excipient and diluent may be at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, physiological saline, methyl hydroxybenzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol and liquid paraffin, but is not limited thereto, and all conventional carriers, excipients or diluents may be used. The above components may be added independently or in combination to the active ingredient, Hansokdan extract.

[0053] The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.

[0054] In addition, solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and these solid preparations may be prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.

[0055] Additionally, preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, etc. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cocoa butter, laurin butter, glycerol, gelatin, etc.

[0056] In addition, the pharmaceutical composition of the present invention can be used parenterally in the form of a sterile solution or suspension injection with water or other pharmaceutically acceptable liquids, as needed. For example, it can be formulated by mixing it appropriately with a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, excipient, vehicle, preservative, binder, etc., and mixing it in a unit dosage form required for generally recognized pharmaceutical practice. In the above formulation, the amount of the active ingredient can be such that an appropriate dosage within the indicated range can be obtained.

[0057] Additionally, a sterile composition for injection can be prescribed according to the usual formulation practice using a vehicle such as distilled water for injection.

[0058] In addition, as an aqueous solution for injection, examples thereof include physiological saline, isotonic solutions containing glucose or other auxiliary agents, for example, D-sorbitol, D-mannose, D-mannitol, sodium chloride, and may be used in combination with appropriate solubilizing agents, for example, alcohol, specifically ethanol, polyalcohols, for example, propylene glycol, polyethylene glycol, and nonionic surfactants, for example, polysorbate 80(TM), HCO-50.

[0059] In addition, the above-mentioned oily liquid may include sesame oil and soybean oil, and may be used in combination with benzyl benzoate and benzyl alcohol as solubilizing agents. In addition, it may be combined with buffers such as phosphate buffer and sodium acetate buffer, analgesics such as procaine hydrochloride, stabilizers such as benzyl alcohol and phenol, and antioxidants. The prepared injection solution can usually be filled into an appropriate ampoule.

[0060] In addition, the pharmaceutical composition of the present invention may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As a base for the suppository, witepsol, macrogol, tween 61, cocoa butter, laurin butter, glycerol, gelatin, etc. can be used.

[0061] In addition, the effective dosage for the human body of the pharmaceutical composition containing the extract of the present invention may vary depending on the patient's age, body weight, sex, dosage form, health condition, and disease severity.

[0062] The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, hormone therapy, drug therapy, and biological response modifiers to prevent or treat respiratory diseases caused by fine dust.

[0063] Since the pharmaceutical composition of the present invention includes the above-described Hansokdan extract, descriptions of the overlapping contents with the above-described Hansokdan extract of the present invention are omitted to avoid excessive complexity of the present specification due to descriptions of overlapping contents.

[0064] The present invention will be described in more detail through the following examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited by these examples.

[0065]

[0066] <Manufacturing Example 1> Manufacturing of Hansokdan extract

[0067] The root of Phlomium umbrosa Turczaninow (Naturalendo Tech Co., Ltd., Republic of Korea), a natural herbal medicine, was cut into pieces of 2 to 10 cm in size, distilled water was added as a solvent in an amount 10 times (w / v) relative to the weight of the raw material, extracted at 100°C for 8 hours, filtered, and then concentrated and dried using a vacuum concentrator to prepare an extract.

[0068]

[0069] <Example 1> Materials and methods

[0070] 1-1. Cell culture

[0071] RAW264.7 cells used in this experiment were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). The medium used was Roswell Park Memorial Institute (RPMI-1640) medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (100 units / mL), and the cells were cultured in an incubator (Thermo Fisher Scientific Inc., Pittsburgh, PA, USA) controlled at 37°C, 5% CO2, and 95% humidity.

[0072] 1-2. Cell viability test (MTT assay)

[0073] Cell viability tests were performed with reference to the method of Berridge et al. First, RAW264.7 cells were seeded in 96-well plates at a density of 5X10 5 After dispensing cells / well and stabilizing, each well was treated with samples at different concentrations. After 24 hours, 20 μL of 5 mg / mL MTT was added and incubated for 4 hours. After removing the medium, 100 μL / well of DMSO was added, and the absorbance was measured at 560 nm.

[0074] 1-3. Positive control group

[0075] Dexamethasone (Sigma-Aldrich, Korea) was used as a positive control (PC).

[0076] 1-4. Preparation of animal models

[0077] In this experiment, 6-week-old male Balb / c mice weighing approximately 20 g were supplied by Saeron Bio Co., Ltd. (Uiwang-si, Korea). After a 1-week adaptation period under conditions of a 12-h light / dark cycle, temperature of 23±2°C, and relative humidity of 50±5%, they were used in the experiment. During the adaptation period, food and drinking water were freely available, and the mice were randomly divided into 5 groups of 8 mice each to ensure similar average body weights. The experiments were conducted in each experimental group under the conditions shown in Table 1 below.

[0078] GroupsIPDietary administrationINTNormal ControlPBSWaterPBSControl25 μg OVA + 1mg AlumWater200 μg OVAPC25 μg OVA + 1mg AlumDexamethasone 3 mg / kg bw200 μg OVAPU-L25 μg OVA + 1mg AlumPhlomoides umbrosaTurczaninow.100 mg / kg bw200 μg OVAPU-H25 μg OVA + 1mg AlumPhlomoides umbrosaTurczaninow.200 mg / kg bw200 μg OVA

[0079] The intake and body weight of each experimental group were measured weekly. Low-dose ovarian cyst nematode (OVA) and alum were injected intraperitoneally on days 1, 8, and 15 from the start of the experiment. Hansokdan extract (PU) was administered orally at various concentrations daily for 7 days, starting one week before the end of the experiment. For 3 days prior to the end of the experiment, high-dose OVA was administered intranasally (INT) to induce airway and lung damage and inflammation. INT was administered by securing the airway of the mice, allowing them to breathe through the nose and enter the lungs through the trachea. After fasting for 12 hours, the experimental animals were anesthetized, opened, and blood samples were collected through the abdominal aorta. 1-5. Histopathological observation (H&E staining)

[0080] Lung tissues obtained by sacrificing animals were fixed in 10% neutral formalin for 24 hours, then embedded in paraffin after general tissue processing, sectioned into 4 μm thick sections using a tissue sectioner (RM2125, Leica, Wützlar, Germany), and then mounted on polylysine-coated slides. Paraffin was removed from the tissue sections using xylene, and the sections were soaked in alcohol and distilled water for 10 minutes, washed with distilled water, and then stained with H&E (Hematoxylin & Eosin) to observe the size of fat globules.

[0081] 1-6. Measurement of antioxidant enzymes

[0082] The effect of the extract of the present invention on antioxidant enzymes in the bronchoalveolar lavage fluid (BALF) of mice induced with OVA after administration was measured. The animals were sacrificed and the absorbance was measured at 450 nm in the BALF using SOD, GPx kits (BioVison Inc., Mountain View, CA, USA), and CAT (Biomax Inc., Seoul, Korea) with an ELIAS plate reader (Bio-Rad Laboratories Headquarters, Hercules, CA, USA).

[0083] 1-7. Measurement of inflammatory cytokines

[0084] 1-7-1. Cell experiment

[0085] To determine the effect of the extract of Hansokdan on the inflammatory response in LPS-induced RAW264.7 cells, cytokine secretion was measured. RAW264.7 cells were seeded in 96-well plates (5x10) 5After dispensing cells / well and stabilizing, each well was treated with samples at different concentrations, and LPS was treated together at a concentration of 1 μg / mL. After 24 hours, the secretion levels of IL-4, IL-5, IL-13, and TNF-α were measured using a Duoset ELISA kit (R&D system, Minneapolis, MN, USA) at 655 nm with an ELIAS plate reader (Bio-Rad Laboratories Headquarters, Hercules, CA, USA) using the supernatant.

[0086] 1-7-2. Animal testing

[0087] The effects of Hansokdan extract on cytokine secretion in an animal model induced by OVA-induced immune hypersensitivity were measured in the same manner as in the cell experiment. Specifically, the secretion levels of IL-4, IL-5, IL-13, and IFN-γ were measured using a Duoset ELISA kit (R&D system, Minneapolis, MN, USA) and an ELIAS plate reader (Bio-Rad Laboratories Headquarters, Hercules, CA, USA) at 655 nm in bronchoalveolar lavage fluid (BALF) obtained by sacrificing the animals.

[0088] 1-8. Measurement of NF-κB pathway, MAPK pathway, and PTEN protein expression

[0089] 1-8-1. Cell experiment

[0090] To confirm the effect of Hansokdan extract on inducing respiratory diseases caused by fine dust in RAW264.7 cells induced by LPS-induced immune hypersensitivity, the expression of NF-κ pathway, MAPK pathway, and MMP-9 protein, which are signal transduction pathways that induce inflammation caused by fine dust, was measured through western blot. RAW264.7 cells were seeded in 6-well plates at a density of 1 × 10 6After dispensing cells / well and stabilizing, the extract of Hansokdan was treated at concentrations of 100 and 500 μg / mL and LPS was treated together at a concentration of 1 μg / mL. After 24 hours, the medium was removed, and lysis buffer containing protease inhibitor was added, homogenized, kept on ice for a certain period of time, and centrifuged (12,000 rpm, 20 min, 4°C) to isolate proteins. Protein quantification was performed using BSA and bio-rad protein assay dye reagent concentrate (Bio-Rad, Hercules, CA USA) using the protein quantification method (Bradford). Each sample was loaded with 20 μL using 10% SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide) gel, separated by electrophoresis, and the proteins were transferred to a nitrocellulose membrane. The membrane was blocked for 1 hour with 5% skim milk (TBS containing 0.5% Tween 20) dissolved in TBST buffer, and the membrane was reacted with the primary antibody p-p65, p-ERK, p-JNK, PTEN, iNOS, MMP-9, beta-actin (Cell signaling Technology, Beverly, MA, USA) for 3 hours at room temperature. The membrane was then reacted with the secondary antibody polymerized with HRP (Cell signaling Technology, Beverly, MA, USA) for 60 minutes, and then enhanced chemiluminescent (ECL, Amershampharmacia) was used. After color development using a fluorescence microscope (Biotech, UK), the images were photographed using Easy Photo. The captured Western blot band images were measured for band density using Image J software (NIH, Bethesda, MD).

[0091] 1-8-2. Animal model experiments

[0092] To determine the effects of the extract of Hansokdan on the lung tissue of an animal model induced by fine dust-induced immune hypersensitivity reaction with OVA, the expression of NF-κB pathway and MAPK pathway proteins was measured using Western blotting, in the same manner as in the cell experiment. Lung tissue was homogenized with lysis buffer containing protease inhibitors, kept on ice for a certain period of time, and centrifuged (12,000 rpm, 20 min, 4°C) to isolate proteins. Protein quantification was performed using the protein quantification method (Bradford) using BSA and Bio-Rad protein assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA USA). Using sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE, 10%), 20 μL of each sample was loaded and separated by electrophoresis, and then the proteins were transferred to a nitrocellulose membrane, blocked for 1 hour with 5% skim milk (TBS containing 0.5% Tween 20) dissolved in TBST buffer, and reacted with primary antibodies p-p65, p-ERK, p-JNK, p-AKT, p-STAT6, p-PTEN, COX2, iNOS, beta-actin (Cell signaling Technology, Beverly, MA, USA) for 3 hours at room temperature, and then the membrane was reacted with HRP-conjugated secondary antibody (Cell signaling Technology, Beverly, MA, USA) for 60 minutes, and enhanced After color development using chemiluminescent (ECL, Amershampharmacia Biotech, UK), the image was taken using Easy Photo.The captured Western blot band images were analyzed for band density using Image J software (NIH, Bethesda, MD).

[0093] 1-9. Leukotriene and prostaglandin E2 analysis

[0094] Analysis of bronchial constrictors, bronchitis- and asthma-inducing factors (leukotrienes) and prostaglandin E2) present in bronchoalveolar lavage fluid (BALF) using an animal model was performed using a competitive ELISA assay kit (Invitrogen USA).

[0095] 1-10. Measurement of expectorant activity

[0096] The expectorant effect was measured using the phenol red excretion method using an animal model. Ambroxol 250 mg / kg, known as an expectorant drug, was used as a positive control (Amb). First, Hansokdan extract (PU) was administered orally, and 30 minutes later, 500 mg / kg phenol red was injected intraperitoneally. 30 minutes later, the animals were anesthetized, bled through the abdominal aorta, and the entire trachea was cut. The isolated trachea was washed in 1 mL of saline for 30 minutes, centrifuged at 10,000 rpm for 5 minutes at room temperature, and 1 N caustic soda (NaOH) was added to the supernatant (0.1 mL of 1 N NaOH per 1 mL of supernatant). The absorbance was measured at 546 nm to determine the expectorant activity as the phenol red concentration.

[0097] 1-11. Statistical processing

[0098] The experiments were analyzed using SPSS (Statistical Package for the Social Science) version 22, and the results of all experiments were expressed as the mean ± standard deviation (SD). Statistical significance between groups was assessed using Duncan's multiple range test, and significance was verified at the p<0.05 level.

[0099]

[0100] <Example 2> Cell model experiment results (in vitro)

[0101] 2-1. Cell viability measurement results

[0102] As a result of examining the cell survival rate treated with the extract of the present invention at different concentrations, it was confirmed that the higher the concentration of the extract of the present invention, the lower the cell survival rate compared to cells not treated with the sample (Fig. 1).

[0103] 2-2. Efficacy of Hansokdan extract on inflammatory cytokines (in vitro)

[0104] When macrophages are activated to primarily remove antigens that have entered the lungs, the macrophages stimulate B cells to produce antigen-specific antibodies, which in turn secrete antigen-specific IgE. The produced IgE activates mast cells, and when it degranulates from mast cells through binding to the antigen, it causes symptoms of respiratory diseases such as bronchoconstriction. In addition, when airway inflammation generally occurs, the absolute total cell count of granulocytes and lymphocytes in bronchoalveolar lavage fluid (BALF) increases and they infiltrate the airways and alveoli. During this process, inflammatory cytokines such as TNF-α, IL-4, IL-5, and IL-13 secreted by inflammatory cells such as eosinophils, Th2 cells, and mast cells induce an inflammatory response by increasing the size of mucus cells and the amount of mucus.

[0105] Therefore, an experiment was conducted to determine the anti-inflammatory effect of the extract of Hansokdan through the cytokine suppression ability, which is a factor causing respiratory disease due to fine dust, and the results are shown in Fig. 2 of the present invention.

[0106] As a result of the experiment, it was confirmed that when the extract of Hansokdan was treated, the secretion of TNF-α, IL-4, IL-5, IL-13, and NO (Nitric oxide) were all significantly reduced compared to the control group (C) in which inflammation was induced by LPS, and it was confirmed that the secretion of each cytokine decreased in a concentration-dependent manner as the treatment concentration of Hansokdan extract increased (100, 250, 500 μg / mL) (Fig. 2).

[0107] 2-3. Effect of Hansokdan extract on the expression of signaling pathway regulatory protein factors associated with inflammation in bronchial and lung tissue (in vitro)

[0108] Among the systems involved in intracellular signal transduction, mitogen-activated protein kinases (MAPKs) include, in addition to ERK1 / 2, p38 MAPK and jun N-terminal kinase (JNK). Most of their target genes play an important role in producing hormones that promote cell division or growth, which are necessary for cell growth, and in regulating inflammatory substances. NF-κB, like MAPKs, regulates various biological functions, including cell proliferation and differentiation, while PTEN, a tumor suppressor gene, induces cell cycle arrest and apoptosis through the PI3K / AkT signaling pathway. In addition, MMPs are proteolytic enzymes secreted by neutrophils, and in the case of MMP-9 (Matrix metalloproteinase-9), it promotes the decomposition of lung tissue and fibrosis around the small airways by activating TGF-β, and macrophages stimulated by allergens express iNOS (inducible nitric oxide synthase) within the cells, promoting the production of a large amount of NO (nitric oxide) from L-arginine and oxygen molecules.

[0109] Accordingly, the effect of the extract of Hansokdan on protein factors related to inflammation response was analyzed, and the results are shown in Fig. 3 of the present invention.

[0110] As a result of the experiment, it was confirmed that the expression levels of all proteins (p-ERK, p-JNK, p-p65, MMP-9, iNOS, and p-PTEN) were significantly reduced when treated with the extract of Hansokdan compared to the control group (C) in which inflammation was induced by LPS, and it was confirmed that the protein expression levels decreased in a concentration-dependent manner at a high concentration (500 μg / mL) of the extract of Hansokdan (Fig. 3).

[0111] <Example 3> Animal model experiment results (in vivo)

[0112] 3-1. Efficacy of Hansokdan extract on the antioxidant activity of bronchoalveolar lavage fluid (BALF) (in vivo)

[0113] Inflammatory responses in the bronchi and lungs caused by fine dust, etc., are inevitably accompanied by oxidative stress, which acts as a mediator that worsens symptoms. Therefore, to examine the effect of the extract of Hansokdan on the antioxidant enzyme system that removes oxidative stress, the treated bronchoalveolar lavage fluid (BALF) was collected and the activities of the antioxidant enzymes SOD (superoxide dismutase), CAT (catalase), and GPx (glutathione peroxidase) were measured. The results are shown in Figure 4 of the present invention.

[0114] As a result of measuring antioxidant activity, the extract of Hansokdan showed consistent and significant differences in antioxidant activity compared to the control group (C) at 200 mg / kg of high concentration of Hansokdan extract (PU-H) for all antioxidant enzymes SOD, CAT, and GPx (Fig. 4).

[0115] 3-2. Efficacy of Hansokdan extract on inflammatory cytokines (in vivo)

[0116] As in the cell experiment, the anti-inflammatory effect of the extract of Hansokdan was cross-validated with the type 2 cytokines IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluid (BALF) and IFN-γ, which cross-inhibits type 2 cytokines among type 1 cytokines, to determine whether similar results could be obtained in animal experiments.

[0117] As a result of the experiment, for IL-4, IL-5, and IL-13, the secretion of inflammatory cytokines was significantly reduced by 17.6%, 14.2%, and 17.7%, respectively, at 200 mg / kg of high-concentration Hansokdan extract (PU-H) compared to the control group (C). However, no statistically significant difference was observed in the Hansokdan extract for IFN-γ (Fig. 5).

[0118] 3-3. Efficacy of Hansokdan extract on reducing alveolar wall thickness (in vivo)

[0119] In the control group (C) treated with only OVA, it was confirmed that the thickness and degree of contraction of the alveolar wall were significantly reduced compared to the normal control group (NC), and in the case of 200 mg / kg of high-concentration PU-H extract, it was confirmed that the thickness and degree of contraction of the alveolar wall were restored compared to the control group (C) (Fig. 6).

[0120] 3-4. Efficacy of Hansokdan extract on the expression of signaling pathway regulatory protein factors associated with inflammation in bronchial and lung tissue (in vivo)

[0121] As in the cell experiment, the expression levels of p-ERK, p-JNK, p-p65, p-PTEN and iNOS, which are inflammation-inducing factors, as well as p-AKT and p-STAT6 and COX-2, which are inflammation-inducing mechanism-related factors, were additionally evaluated using lung tissue, and the results are shown in Fig. 7 of the present invention.

[0122] As a result of the experiment, the expression of p-JNK, p-STAT6, p-AKT, and COX-2 was significantly reduced compared to the control group (C) treated with only OVA at low concentrations (PU-L) of 100 mg / kg and high concentrations (PU-H) of 200 mg / kg of Hansokdan extract, and the high effect of Hansokdan extract was confirmed to be concentration-dependent (PU-L; 19.2%, 17.9%, 17.6%, and 37.5%, respectively; PU-H; 74.2%, 33.0%, 28.2%, and 51.0%, respectively), and it was confirmed that the expression levels of p-ERK, p-p65, p-PTEN, and iNOS were significantly reduced at high concentrations (PU-H) of 200 mg / kg of Hansokdan extract (Fig. 7).

[0123] These results indicate that the extract of Hansokdan is effective in improving inflammation of the bronchial and lung tissues caused by fine dust.

[0124] 3-5. Effect of Hansokdan extract on leukotrienes and prostaglandin E2 (PGE2) (in vivo)

[0125] Leukotrienes are factors related to bronchial constriction, and when asthma and bronchitis occur, their excessive production acts as a factor that worsens the disease, and prostaglandin E2 (PGE2) is known to be an important substance that cyclically induces inflammation in the inflammatory mechanism. Therefore, by measuring the two factors in an animal model, the improvement effect of Hansokdan extract and fine dust-induced respiratory inflammation was confirmed, and the results are shown in Fig. 8 of the present invention.

[0126] As a result of the experiment, the high concentration of Hansokdan extract (PU-H) at 200 mg / kg significantly decreased (23.0% and 29.6%, respectively) compared to the control group (C) treated with only OVA, confirming that Hansokdan extract can reduce inflammation-inducing substances caused by fine dust (Fig. 8).

[0127] 3-6. Efficacy of Hansokdan extract on expectorant activity (in vivo)

[0128] In order to confirm the expectorant effect by phenol red secretion, an animal model was used to confirm the expectorant effect, and the results are shown in Fig. 9 of the present invention.

[0129] As a result of the experiment, the amount of phenol red released tended to increase in the low concentration (PU-L) of Hansokdan extract compared to the control group (C) treated with only OVA, but there was no statistically significant difference. In the high concentration (PU-H) of Hansokdan extract at 200 mg / kg, the amount of phenol red released increased by 31.2% compared to the control group (C), confirming a significant result (Fig. 9).

[0130] Therefore, it was confirmed that the extract of Hansokdan can be used to prevent and treat respiratory diseases caused by fine dust through the expectorant activity of the extract of Hansokdan.

[0131] While the present invention has been described in detail through representative examples above, those skilled in the art will understand that various modifications to the above-described embodiments are possible without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the described embodiments, but should be defined not only by the claims described below but also by all changes or modifications derived from the claims and equivalent concepts.

[0132] The purpose of the present invention is to provide a food composition for preventing or improving respiratory diseases caused by fine dust, including an extract of Hansokdan.

[0133] In addition, the present invention aims to provide a pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust, including an extract of Hansokdan.

Claims

1. A food composition for preventing or improving respiratory disease caused by fine dust, containing an extract of Hansokdan.

2. In paragraph 1, A food composition for preventing or improving respiratory diseases caused by fine dust, wherein the extract is extracted with water and at least one solvent selected from the group consisting of straight-chain or branched alcohols having 1 to 4 carbon atoms.

3. In paragraph 1, The composition above suppresses a signal pathway related to inflammation caused by fine dust, and the inflammation-related signal is at least one selected from the group consisting of ERK (Extracellular signal-regulated kinases), JNK (c-jun N-terminal kinase), NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) p65, PI3K / AKT, and STAT6, a food composition for preventing or improving respiratory disease caused by fine dust.

4. In paragraph 1, The above composition is a food composition for preventing or improving respiratory disease caused by fine dust, which reduces the secretion of inflammatory factors caused by fine dust or inflammatory cytokines caused by fine dust.

5. In paragraph 4, A food composition for preventing or improving respiratory disease caused by fine dust, wherein the inflammatory factor is at least one selected from the group consisting of leukotrienes, prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), and iNOS.

6. In paragraph 4, A food composition for preventing or improving respiratory disease caused by fine dust, wherein the inflammatory cytokine is at least one selected from the group consisting of interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 13 (IL-13), and interferon gamma (IFN-γ).

7. In paragraph 1, A food composition for preventing or improving respiratory diseases caused by fine dust, wherein the respiratory diseases are at least one disease selected from the group consisting of respiratory inflammatory lung disease, chronic obstructive pulmonary disease, allergic rhinitis, sinusitis, upper respiratory tract infection, lower respiratory tract infection, chronic bronchitis, bronchiectasis, pneumonia, sequelae of pulmonary tuberculosis, acute respiratory distress syndrome, cystic fibrosis, otitis media, pulmonary fibrosis, asthma, emphysema, pharyngitis, laryngitis, and tonsillitis.

8. A pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust, containing an extract of Hansokdan.

9. In paragraph 8, A pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust, wherein the extract is extracted with water and at least one solvent selected from the group consisting of straight-chain or branched alcohols having 1 to 4 carbon atoms.

10. In paragraph 8, A pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust, wherein the respiratory disease is at least one disease selected from the group consisting of respiratory inflammatory lung disease, chronic obstructive pulmonary disease, allergic rhinitis, sinusitis, upper respiratory tract infection, lower respiratory tract infection, chronic bronchitis, bronchiectasis, pneumonia, sequelae of pulmonary tuberculosis, acute respiratory distress syndrome, cystic fibrosis, otitis media, pulmonary fibrosis, asthma, emphysema, pharyngitis, laryngitis, and tonsillitis.

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