KR20220081301A - Pharmaceutical composition for preventing or treating infalmmatory disease comprising glycyrrhiza glabra l. and orus alba l. - Google Patents

Abstract

The present invention relates to a pharmaceutical composition, a cosmetic composition and a food composition for preventing or treating bronchial inflammatory diseases, including licorice and epiphyllum, which contain licorice and epiphyllum extract complex, thereby reducing inflammation through improvement of inflammatory factors.

Description

Pharmaceutical composition for preventing or treating inflammatory diseases comprising licorice and epiphyllum {PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING INFALMMATORY DISEASE COMPRISING GLYCYRRHIZA GLABRA L. AND ORUS ALBA L.}

This specification claims the benefit of the filing date of Korean Patent Application No. 10-2020-0170811 filed with the Korean Intellectual Property Office on December 8, 2020, the entire contents of which are included in the present invention.

The present invention provides a pharmaceutical composition, a cosmetic composition, and a food composition for preventing or treating bronchial inflammatory diseases, including licorice and sangbaekpi.

Inflammation refers to the pathological condition of an abscess formed by the invasion of external infectious agents (bacteria, fungi, viruses, and various types of allergens). Specifically, when external bacteria invade and proliferate in a specific tissue, the white blood cells of the living body recognize this and actively attack the proliferated external bacteria. At the same time, the cell debris of the invading bacteria killed by the white blood cells melts into the invaded tissue to form an abscess. The treatment of abscesses caused by inflammation can be promoted through anti-inflammatory action, which means using an anti-inflammatory agent to suppress the growth of invading bacteria or to activate macrophages that phagocytose the foreign substances accumulated during the abscess to digest the foreign substances. and promoting the treatment of inflammation, such as enhancing the function of excreted macrophages.

In general, the inflammatory reaction is a biological defense reaction process for repairing and regenerating damage caused by invasion that causes organic changes in cells or tissues of the living body. This works. While the inflammatory response normally induced by external invading bacteria is a defense system to protect the living body, when an abnormally excessive inflammatory response is induced, various diseases appear. These diseases are called inflammatory diseases. The inflammatory disease is a disease in which various inflammatory mediators secreted from target cells activated by external stimuli amplify and sustain inflammation, thereby threatening the life of the human body. It includes skin diseases and allergic inflammatory diseases such as bronchial asthma.

The onset of many inflammation-related diseases is associated with the activation of macrophages and the excessive production of inflammation-related factors. Representative inflammation-related factors include IL-1β, TNF-α, and nitric oxide (NO). The production of nitric oxide (NO) by activated white blood cells (monocytes) and macrophages during the inflammatory response initiates a strong inflammatory response and is the most important part of the initial immune response to bacterial pathogens (Bogdan C, Nat. Immunol. , 2:907-916, 2001). In addition, as a result of the inflammatory response, the supraphysiological concentrations of nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) were observed in the pathology of various inflammatory diseases. It is believed to play a role (Clancy RM et al., Arthritis. Rheum., 41:1141-1151, 1998).

Current anti-inflammatory drugs include dexamethasone and cortisone using adrenocortical hormone components. However, although they act as anti-inflammatory drugs, they are highly toxic and may cause symptoms such as edema as a side effect. In addition, there are cases in which a problem of inducing severe immunosuppression occurs because it cannot selectively act on the cause of inflammation (Check WA & Kaliner MA, Am. Rev. Respir. Dis., 141:44-51. 1990). The existing anti-inflammatory agents are steroid drugs using adrenocortical hormone components, and since they exhibit severe side effects such as edema, there is an urgent need for materials that can prevent or treat new inflammatory diseases.

Accordingly, the present inventors completed the present invention by confirming and analyzing the cytotoxicity and inflammatory indicators of various drugs.

One aspect of the present invention aims to provide a pharmaceutical composition for preventing or treating inflammatory diseases comprising a licorice and epiphyllum extract complex.

Another aspect of the present invention aims to provide a cosmetic composition for alleviating skin inflammation comprising licorice and sangbaekpi extract complex.

Another aspect of the present invention aims to provide a food composition comprising a licorice and sangbaekpi extract complex.

It provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising a licorice and epiphyllum extract complex.

The "licorice" is a plant of the legume family, and the scientific name is Glycyrrhiza glabra L. As components, terpene compounds such as Glycyrrhetinic acid, Glycyrrhizin and Liquiritigenin, Liquiritin ( Liquiritin) and other flavonoids are known, and it has been reported that the difference in components appears depending on the collected species and cultivation method. When the active ingredient, Glycyrrhizin, induced toxicity in the nerve cell line PC12 cell, research results were reported on its anti-toxic activity, and Liquiritigenin was used in animal experiments with cadmium, a heavy metal contained in yellow dust. Studies have shown that it reduces toxicity.

The "sangbaekpi" is the bark of Morus alba L., a deciduous tree belonging to the family Moraceae, and the bark of animals and plants, and its components include umbelliferone, scopoletin, morusin. ), mulberrin, and the like, and are known to have diuretic and blood pressure lowering effects.

The "extraction complex" in the present invention includes not only mixing extracts extracted from each of a plurality of raw materials, but also extracting a plurality of raw materials together, extracting extracts extracted from some of the plurality of raw materials and extracting by mixing other raw materials.

The "extract" refers to a liquid component obtained by immersing a target substance in various solvents and then extracting it for a certain period of time at room temperature, low temperature, or heating condition, a solid obtained by removing the solvent from the liquid component, etc. means the result. In addition, in addition to the result, it can be comprehensively interpreted as including all of the dilutions of the results, their concentrates, their preparations, and their purified products.

In the present invention, "inflammatory disease" means acute or chronic inflammatory disease, chronic bronchitis and related obstructive airway disease, upper respiratory tract infection and rhinitis, arthritis or inflammatory bowel disease, pain, arteriosclerosis, heart disease, multiple sclerosis, Parkinson's disease, Diseases including Alzheimer's disease, colon cancer, etc., specifically chronic bronchitis related to bronchial tubes and related obstructive airway diseases, may be interpreted as including upper respiratory tract infection, and may also be interpreted as including bronchial inflammation in animals, but in particular Not limited. When an inflammatory disease occurs in an individual, the expression of various inflammatory factors, such as cytokines, or genes encoding them increases.

"Relief" in the present invention means any action of reducing or alleviating the symptoms of inflammatory disease by administration of the composition of the present invention.

"Prevention" in the present invention refers to any action that suppresses the symptoms of inflammatory disease or delays the progression by administration of the composition of the present invention.

"Treatment" in the present invention means any action that improves or beneficially changes the symptoms of inflammatory disease by administration of the composition of the present invention.

The pharmaceutical composition refers to a drug used for the purpose of treating, alleviating, treating or preventing inflammatory diseases in animals including humans. Specifically, it refers to a drug used for the purpose of treatment, alleviation, treatment or prevention of diseases of humans or companion animals.

In one embodiment of the present invention, the extract complex may be a mixture of a licorice extract and an epiphyllum extract, that is, a mixture of a licorice extract and an extract of epiphyllum.

In one embodiment of the present invention, the licorice extract and epiphyllum extract may be extracted with any one or more solvents of purified water and C1 to C4 alcohol, and specifically, the licorice extract and epiphyllum extract may be 50 to 70% ethanol solvent extract. . More specifically, the licorice extract and sangbaekpi extract may be extracted with 70% ethanol. The licorice extract and sangbaekpi extract may be prepared by adding 70% ethanol as a 16-fold solvent of licorice or sangbaekpi, respectively, extracting at 70 to 80° C. for 6 hours, concentrating using a rotary evaporator, and then freeze-drying.

In one embodiment of the present invention, the extract complex may include a licorice extract and a sangbaekpi extract in a ratio of 1:2 to 2:1. Specifically, the licorice extract and sangbaekpi extract may be included in a ratio of 1:2, 1:1, or 2:1, and more specifically, may be included in a ratio of 1:2 or 2:1. If it is outside the above ratio range, the inflammatory disease improvement effect through the improvement of the inflammatory factor targeted by the present invention cannot be obtained.

In another embodiment of the present invention, the extraction complex may have a concentration of 50 to 100 μg/ml, and more specifically, the extraction complex may have a concentration of 100 μg/ml. When the concentration of the extract complex of the present invention is out of the above range, the inflammatory disease improvement effect through improvement of the inflammatory factor targeted by the present invention cannot be obtained.

In one embodiment of the present invention, the composition may be for alleviating, preventing or treating inflammatory diseases. The composition for alleviating, preventing or treating inflammatory diseases refers to drugs used for the purpose of treating, alleviating, treating or preventing inflammatory diseases of animals including humans. Specifically, it refers to a drug used for the purpose of treatment, alleviation, treatment or prevention of diseases of humans or animals other than humans.

In one embodiment of the present invention, the composition may be for alleviating, preventing or treating respiratory diseases. The composition for alleviating, preventing or treating respiratory diseases refers to drugs used for the purpose of treating, alleviating, treating or preventing respiratory diseases in animals including humans. Specifically, it refers to a drug used for the purpose of treatment, alleviation, treatment or prevention of diseases of humans or animals other than humans. More specifically, the respiratory disease may be an inflammatory respiratory disease, and more specifically, airway hyperresponsiveness, asthma, chronic obstructive pulmonary disease (COPD), acute lung injury, empyema, lung abscess, pneumonia, pulmonary tuberculosis , cough, sputum, bronchitis, sore throat, tonsillitis, sinusitis, rhinitis, bronchiolitis obliterans and laryngitis may be any one selected from the group consisting of, but is not limited thereto.

In one embodiment of the present invention, "animal" includes companion animals, and the companion animals specifically include dogs, cats, hamsters, rabbits, chickens, iguanas, guinea pigs, gerbils, snails, raccoons, parrots, lizards, and tropical fish. It can include all kinds of animals that coexist with humans, such as fish, including fish.

As confirmed in the experimental examples to be described later, the extract complex of the present invention showed an even improvement ability in biomarkers related to inflammatory disease improvement compared to the single extract. In particular, the extract complex showed excellent overall efficacy, but among them, the most stable and excellent efficacy was confirmed when the concentration of 100 μg/ml of the licorice extract and the sangbaekpi extract in a 2: 1 ratio.

In one embodiment of the present invention, the content of the extract complex in the composition may be appropriately selected depending on the species, age, weight, and breeding conditions of the animal.

The pharmaceutical composition may include 0.001 wt% to 99.9 wt%, 0.1 wt% to 99 wt%, or 1 wt% to 20 wt% of the extract complex with respect to the total weight of the composition, but is not limited thereto. Depending on the mode of use and the method of use, the content of the extract may be appropriately adjusted to a pharmaceutically effective amount or a desired amount. The "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit or risk ratio applicable to medical treatment, which is the type, severity, drug activity, sensitivity to, administration of the subject's disease. Time, route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field may be determined.

The preferred dosage of the composition of the present invention varies depending on the type of administration subject, age, sex, weight, symptoms, disease severity, drug form, administration route and period, but may be appropriately selected by those skilled in the art. For a desirable effect, the composition of the present invention is preferably administered at 0.01 to 1000 mg/day. Administration may be done once a day, or may be divided into several times. Specifically, the dosage of the composition of the present invention may be 10-2000 mg/kg/day, and more specifically, 10 mg/kg/day or more, 20 mg/kg/day or more, 30 mg/kg/day or more, 40 mg/kg/day or more, 50 mg/kg/day or more, 60 mg/kg/day or more, 70 mg/kg/day or more, 80 mg/kg/day or more, 90 mg/kg/day or more, 100 mg/ kg/day or more, 110 mg/kg/day or more, 120 mg/kg/day or more, 130 mg/kg/day or more, 140 mg/kg/day or more, 150 mg/kg/day or more, 160 mg/kg/day or more ≥ 170 mg/kg/day, ≥ 180 mg/kg/day, ≥ 190 mg/kg/day, ≥ 200 mg/kg/day, ≥ 250 mg/kg/day, ≥ 300 mg/kg/day , 400 mg/kg/day or more, 500 mg/kg/day or more, 600 mg/kg/day or more, 700 mg/kg/day or more, 800 mg/kg/day or more, or 900 mg/kg/day or more; , 2000 mg/kg/day or less, 1900 mg/kg/day or less, 1800 mg/kg/day or less, 1700 mg/kg/day or less, 1600 mg/kg/day or less, 1500 mg/kg/day or less, 1400 mg/kg/day or less, 1300 mg/kg/day or less, 1200 mg/kg/day or less, 1100 mg/kg/day or less, 1000 mg/kg/day or less, 900 mg/kg/day or less, 800 mg/day kg/day or less, 700 mg/kg/day or less, 600 mg/kg/day or less, 500 mg/kg/day or less, 400 mg/kg/day or less, 300 mg/kg/day or less, 290 mg/kg/day Days or less, 280 mg/kg/day or less, 270 mg/kg/day or less, 260 mg/kg/day or less, 250 mg/kg/day or less, 240 mg/kg/day or less, 230 mg/kg/day or less , 220 mg/kg/day or less, 210 mg/kg/day or less, 200 mg/kg/day or less, 190 mg/kg/day or less, 180 mg/kg/day or less, 170 m g/kg/day or less, 160 mg/kg/day or less, 150 mg/kg/day or less, 140 mg/kg/day or less, 130 mg/kg/day or less, 120 mg/kg/day or less, 110 mg/day kg/day or less, 100 mg/kg/day or less, 90 mg/kg/day or less, 80 mg/kg/day or less, 70 mg/kg/day or less, 60 mg/kg/day or less, 50 mg/kg/day Days or less, 40 mg/kg/day or less, or 20 mg/kg/day or less, but is not limited thereto.

In addition, the dosage may be increased or decreased according to age, sex, weight, degree of disease, administration route, and the like. Therefore, the above dosage does not limit the scope of the present invention in any way. By "administration" is meant providing a given composition of the present invention to a subject by any suitable method. In this case, the subject refers to all animals, such as humans, monkeys, dogs, goats, pigs, or mice, having a disease in which the symptoms of skin inflammation can be improved by administering the composition of the present invention.

The pharmaceutical composition of the present invention may include the extract complex of the present invention alone, and may further include pharmaceutically acceptable carriers, excipients, diluents and/or sub-components according to other formulations, methods of use, and purposes of use. .

More specifically, in addition to the extract complex, nutrients, vitamins, electrolytes, flavoring agents, colorants, thickeners, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, It may further contain glycerin, alcohol, a carbonation agent used in carbonated beverages, and the like.

The 'pharmaceutically acceptable' means that it is physiologically acceptable and does not normally cause gastrointestinal disorders, allergic reactions such as dizziness or similar reactions when administered to animals, preferably humans. The pharmaceutically effective amount may be appropriately changed depending on the disease and its severity, the age, weight, health status, sex, administration route, and treatment period of the patient.

Examples of the pharmaceutically acceptable carrier, excipient or diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, propylhydroxybenzoate, talc, magnesium stearate and mineral oil , dextrin, calcium carbonate, dextrin, calcium carbonate, propylene glycol, liquid paraffin and at least one selected from the group consisting of saline, but is not limited thereto, and all common carriers, excipients or diluents can be used.

The dosage form of the pharmaceutical composition may vary depending on the method of use, and it is formulated using a method well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. can be Examples of the formulation include ointments, creams, tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, aqueous solutions, non-aqueous solutions, suspensions and emulsions. It may be a formulation selected from the group consisting of .

For the formulation, excipients, for example, conventional fillers, extenders, binders, disintegrants, surfactants, anti-aggregants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, sweeteners, fragrances or preservatives may be further included.

In general, solid preparations for oral administration include tablets (TABLETS), pills, soft or hard capsules (CAPSULES), pills (PILLS), powders (POWDERS), and granules (GRANULES), and such preparations include one or more An excipient, for example, may be prepared by mixing starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

In addition, liquid formulations for oral administration include suspensions (SUSTESIONS), internal solutions, emulsions (EMULSIONS), and syrups (SYRUPS). Wetting agents, sweetening agents, flavoring agents, preservatives, and the like may be included.

Examples that may be mentioned for dermal administration include dusting powders, emulsions, suspensions, oils, sprays, ointments, greasy ointments, cream pastes, gels, foams, or suitable for producing solutions, Transdermal therapeutic system (TTS) ) suitable carriers and/or excipients. The topical pharmaceutical preparation of the present invention may be in a semi-solid dosage form, and in particular, there are ointments (solution ointments, suspension ointments), creams, gels or pastes. Mainly used in the oil phase are fatty alcohols such as lauryl alcohol, cetyl alcohol, stearyl alcohol, fatty acids such as palmitic or stearic acid, liquid or solid paraffin or ozokerite, liquid to solid waxes, such as for example isopropyl myristate, natural fats or some synthetic fats such as coconut fatty acid triglycerides, hydrogenated oils such as hydrogenated peanut or castor oil, or fatty acid partial esters of glycerol such as glycerol monostearate or glycerol There is distearate. Suitable emulsifiers include surfactants, for example nonionic surfactants, for example fatty acid esters of polyalcohols or ethylene oxide adducts thereof, such as polyglycerol fatty acid esters or polyoxyethylene sorbitan fatty acid esters, sorbitan fatty acid esters such as sorbitan oleate and/or sorbitan isostearate and the like, isostearates, sterols, or polyoxyethylene fatty alcohol ethers or fatty acid esters such as anionic surfactants such as alkali metal salts of fatty alcohol sulfonates, Examples are sodium lauryl sulfate, sodium cetyl sulfate or sodium stearyl sulfate, which are usually used in the presence of the fatty alcohol, for example cetyl alcohol or stearyl alcohol. Among these, it is possible to add an agent, for example, polyalcohol, such as glycerol, sorbitol, propylene glycol and/or polyethylene glycol, to the aqueous phase, or a preservative, flavoring, etc., to the aqueous phase, especially for preventing cream from drying out.

The pharmaceutical composition of the present invention may be an anhydrous ointment, suitable for topical use and containing paraffin, particularly low-viscosity paraffin, which is liquid at body temperature, or the natural or partially synthetic fat, such as coconut fatty acid. Triglycerides, hydrogenated oils such as hydrogenated peanut or castor oil, fatty acid partial esters of glycerol such as glycerol monostearate and distearate, silicones such as polymethylsiloxanes such as hexamethyldisiloxane or octamethyl It may contain trisiloxanes, for example fatty alcohols associated with water-based creams and increasing the water absorption capacity, and sterols, wool waxes, other emulsifiers and/or other additives.

In the present invention, when the pharmaceutical composition is formulated as a pharmaceutical, reference may be made to the contents disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, and the documents are incorporated as a part of this specification.

Another aspect of the present invention provides a cosmetic composition for alleviating skin inflammation comprising a composition for improving skin.

The cosmetic composition for alleviating skin inflammation includes the above-described extract complex, and the extract complex has an anti-inflammatory effect through improvement of inflammatory factors.

The components included in the cosmetic composition of the present invention include components commonly used in cosmetic compositions in addition to the extract complex, for example, conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers. include

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , oil, powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto. More specifically, it may be prepared in the form of a softening lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. can

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additional chlorofluorohydrocarbon, propane /may contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Adult cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester may be used.

Another aspect of the present invention provides a food composition comprising a licorice and sangbaekpi extract complex.

The food composition includes the above-mentioned licorice and sangbaekhui extract complex, and the licorice and sangbaekhui extract complex has an anti-inflammatory effect through improvement of inflammatory factors.

"Food composition" in the present invention means a natural product or processed product containing one or more nutrients, and preferably means a state that can be directly eaten through a certain amount of processing, As a meaning, it includes all health functional foods, functional foods, beverages, food additives and beverage additives.

The food composition may be, for example, various foods, beverages, gum, tea, vitamin complex, health functional food, and the like. In addition, in the present invention, food includes special nutritional food (eg, formula milk, infant food, etc.), processed meat products, fish meat products, tofu, jelly, noodles (eg, ramen, noodles, etc.), health supplements, seasoned foods ( Ex, soy sauce, soybean paste, red pepper paste, mixed soy sauce, etc.), sauces, sweets (eg snacks), dairy products (eg fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi, pickles, etc.), beverages ( Examples include, but are not limited to, fruit and vegetable beverages, soy milk, fermented beverages, etc.) and natural seasonings (eg, ramen soup, etc.).

The food, functional food, health functional food, beverage, food additive and beverage additive may be prepared by a conventional manufacturing method.

The pharmaceutical composition, cosmetic composition, and food composition for preventing or treating bronchial inflammatory diseases, including licorice and epidermis of the present invention, contain the licorice and epiphyllum extract complex, thereby reducing inflammation through improvement of inflammatory factors.

1 is a graph showing the cytotoxic effect of distilled water or alcohol (70% ethanol) sample extract on RAW 264.7. Specifically, RAW 264.7 cells were treated at concentrations of 100 and 200 (㎍ / ㎖) of the sample for 24 hours. Cytotoxicity was measured by MTT assay, and measurements were expressed as a percentage of control (mean±standard deviation, n=3).
2 is a graph showing the HPLC chromatogram and UV spectrum of a glycyrrhizin reference solution (500 μg/ml) (A) and a licorice extract (B).
3 is a graph showing the effect of distilled water (A), 70% ethanol (B) sample extracts on nitric oxide (NO) levels of LPS-induced RAW 264.7 cells. Specifically, RAW 264.7 cells were treated with 100 μg/ml of ethanol/distilled water sample extract and LPS (1 μg/ml) for 24 hours, and nitric oxide was analyzed by ELISA. Measured values were expressed as a percentage of the control group (mean±standard deviation, n=3) (indicated when significant compared to the control group was indicated ^*** p<0 . 001, ^** p<0 . 01, ^* p<0 05) .
Figure 4 is a graph showing the effect of the 70% ethanol complex extract of licorice and sangbaekpi on the cell viability of RAW 264.7 cells. Specifically, RAW 264.7 cells were treated with sample extracts of 50 and 100 μg/ml for 24 hours, and cytotoxicity was analyzed by MTT. Measurements were expressed as a percentage of the control group (mean±standard deviation, n=3).
5 is a graph showing the effect of LPS-induced-RAW 264.7 cells on nitric oxide (NO) of 70% ethanol complex extract of licorice and sangbaekpi. Specifically, RAW 264.7 cells were treated with sample extracts of 50 and 100 μg/ml and LPS (1 μg/ml) for 24 hours, and NO levels were analyzed by ELISA. Measured values were expressed as a percentage of the control group (mean±standard deviation, n=3) (indicated if significant compared to the control group ^*** p<0 . 001).
Figure 6 is a graph showing the effect of LPS-induced-RAW 264.7 cells PGE ₂ production amount of the 70% ethanol complex extract of licorice and sangbaekpi. Specifically, RAW 264.7 cells were treated with sample extracts of 50 and 100 μg/ml and LPS (1 μg/ml) for 24 hours, and the level of PGE ₂ production was analyzed by ELISA. Measured values were expressed as a percentage of the control group (mean±standard deviation, n=3) (indicated when significant compared to the control group, ^** p<0 . 001, ^* p<0.05) .
7 is a graph showing the effect of LPS-induced-RAW 264.7 cells IL-1β production of licorice and sangbaekpi 70% ethanol complex extract. Specifically, RAW 264.7 cells were treated with sample extracts of 50 and 100 μg/ml and LPS (1 μg/ml) for 24 hours, and IL-1β production levels were analyzed with Luminex. Measured values were expressed as a percentage of the control group (mean±standard deviation, n=3) (indicated when significant compared to the control group was indicated ^*** p<0.0001 , ^** p<0.001) .
8 is a graph showing the effect of LPS-induced-RAW 264.7 cells IL-6 production of licorice and sangbaekpi 70% ethanol complex extract. Specifically, RAW 264.7 cells were treated with sample extracts of 50 and 100 μg/ml and LPS (1 μg/ml) for 24 hours, and IL-6 production levels were analyzed with Luminex. Measured values were expressed as a percentage of the control group (mean±standard deviation, n=3) (indicated when significant compared to the control group was indicated ^*** p<0.0001 , ^** p<0.001) .
9 is a graph showing the effect of LPS-induced-RAW 264.7 cells TNF-α production of licorice and sangbaekpi 70% ethanol complex extract. Specifically, RAW 264.7 cells were treated with sample extracts of 50 and 100 μg/ml and LPS (1 μg/ml) for 24 hours, and the TNF-α production level was analyzed with Luminex. Measured values were expressed as a percentage of the control group (mean±standard deviation, n=3) (indicated when significant compared to the control group was indicated ^*** p<0 . 001, ^** p < 0 . 01).
10 is a graph showing the effect of LPS-induced-RAW 264.7 cell iNOS expression of licorice and sangbaekpi 70% ethanol complex extract. Specifically, RAW 264.7 cells were treated with sample extracts of 50 and 100 μg/ml and LPS (1 μg/ml) for 24 hours, and iNOS expression levels were analyzed with Luminex. Measured values were expressed as a percentage compared to control group (mean±standard deviation, n=3) (indicated when significant compared to control group was indicated ^*** p<0 .0001, ^** p<0 . 001 , ^* p<0 005) .
11 is a graph showing the effect of LPS-induced-RAW 264.7 cells of licorice and sangbaekpi 70% ethanol complex extract on COX-2 expression level. Specifically, RAW 264.7 cells were treated with sample extracts of 50 and 100 μg/ml and LPS (1 μg/ml) for 24 hours, and the COX-2 expression level was analyzed by western blot. Measured values were expressed as a percentage compared to control group (mean±standard deviation, n=3) (indicated when significant compared to control group was indicated ^*** p<0 .0001, ^** p<0 . 001 , ^* p<0 005) .
Figure 12 is a graph showing the effect of LPS-induced-RAW 264.7 cells IL-1β expression of the 70% ethanol complex extract of licorice and sangbaekpi. Specifically, RAW 264.7 cells were treated with sample extracts of 50 and 100 μg/ml and LPS (1 μg/ml) for 24 hours, and the IL-1β expression level was analyzed by western blot. Measured values were expressed as a percentage of the control group (mean±standard deviation, n=3) (indicated when significant compared to the control group was indicated ^*** p<0 . 001, ^** p<0 . 01, ^* p<0 05) .
13 is a graph showing the effect of LPS-induced-RAW 264.7 cell IL-6 expression of a 70% ethanol complex extract of licorice and sangbaekpi. Specifically, RAW 264.7 cells were treated with sample extracts of 50 and 100 μg/ml and LPS (1 μg/ml) for 24 hours, and the IL-6 expression level was analyzed by western blot. Measured values were expressed as a percentage compared to the control group (mean±standard deviation, n=3) (indicated when significant compared to the control group, ^** p<0 .01) .
14 is a graph showing the effect of LPS-induced-RAW 264.7 cell TNF-α expression of licorice and sangbaekpi 70% ethanol complex extract. Specifically, RAW 264.7 cells were treated with 50 and 100 μg/ml of sample extract and LPS (1 μg/ml) for 24 hours, and the TNF-α expression level was analyzed by Western blot. Measured values were expressed as a percentage compared to the control group (mean±standard deviation, n=3) (indicated when significant compared to the control group, ^** p<0 .01) .
15 is a diagram showing an experimental protocol of sensitization, OVA (ovalbumin) challenge and treatment in a model of bronchial inflammatory asthma.
16 is a graph showing the effect of licorice complex on specific-IgE levels in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is a group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), using student's t-test, ^* p<0.05 with the control group Significant differences were compared.
17 is a graph showing the effect of the licorice complex on the PGE ₂ level in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); The bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is the group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), and ^*** p<0.001 using student's t-test. Significant differences were compared with the control group.
18 is a graph showing the effect of licorice complex on histamine levels in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); The bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is the group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), using student's t-test ^* p<0.05, ^** Significant differences were compared with the control group at p<0.01.
19 is a graph showing the effect of licorice complex on WBC (leukocyte) count in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); The bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is a group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), using student's t-test, ^* p<0.05 with the control group Significant differences were compared.
20 is a graph showing the effect of licorice complex on the number of eosinophils in leukocytes in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is the group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), ^** p<0.01, ^* using student's t-test. ^** Significant differences were compared with the control group at p<0.001.
21 is a graph showing the effect of licorice complex on the number of neutrophils in leukocytes in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is the group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), ^** p<0.01, ^* using student's t-test. ^** Significant differences were compared with the control group at p<0.001.
22 is a graph showing the effect of licorice complex on the number of monocytes in white blood cells in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is a group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), using student's t-test, ^* p<0.05 with the control group Significant differences were compared.
23 is a graph showing the effect of licorice complex on the number of lymphocytes in leukocytes in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); The bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is a group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), and student's t-test was used.
24 is a graph showing the effect of the licorice complex on the number of PLTs in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is the group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), using student's t-test, ^** p<0.01 in the control group. and significant differences were compared.
25 is a graph showing the effect of licorice complex on IL-1β expression in the spleen in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); The bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is a group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), using student's t-test, ^* p<0.05 with the control group Significant differences were compared.
26 is a graph showing the effect of licorice complex on IL-4 expression in the spleen in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is a group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), using student's t-test, ^* p<0.05 with the control group Significant differences were compared.
27 is a graph showing the effect of licorice complex on IL-5 expression in the spleen in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is a group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), using student's t-test, ^* p<0.05 with the control group Significant differences were compared.
28 is a graph showing the effect of licorice complex on IL-6 expression in the spleen in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); The bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is a group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), using student's t-test, ^* p<0.05 with the control group Significant differences were compared.
29 is a graph showing the effect of licorice complex on IL-10 expression in the spleen in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); The bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is a group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), using student's t-test, ^* p<0.05 with the control group Significant differences were compared.
30 is a graph showing the effect of licorice complex on IL-13 expression in the spleen in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); The bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is a group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), using student's t-test, ^* p<0.05 with the control group Significant differences were compared.
31 is a graph showing the effect of licorice complex on IL-17 expression in the spleen in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is a group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), using student's t-test, ^* p<0.05 with the control group Significant differences were compared.
32 is a graph showing the effect of licorice complex on IFN-γ expression in the spleen in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is a group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), using student's t-test, ^* p<0.05 with the control group Significant differences were compared.
33 is a graph showing the effect of licorice complex on splenic TNF-α expression in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is a group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), using student's t-test, ^* p<0.05 with the control group Significant differences were compared.
34 is a graph showing the effect of licorice complex on NFK-κB expression in the spleen in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is a group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), using student's t-test, ^* p<0.05 with the control group Significant differences were compared.
35 is a graph showing the effect of licorice complex on iNOS expression in the spleen in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); The bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is a group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), using student's t-test, ^* p<0.05 with the control group Significant differences were compared.
36 is a graph showing the effect of licorice complex on COX-2 expression in the spleen in OVA-induced bronchial immunocompromised mice. Specifically, the untreated group, the normal group (Nor); bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); A group administered orally with a licorice complex at a dose of 100 mg/kg/day (100); This is a group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day, and the data values were expressed as mean±SD (n=6), using student's t-test, ^* p<0.05 with the control group Significant differences were compared.
37 and 38 are photographs showing the effect of the licorice complex on histopathological changes in the lungs in OVA-induced bronchial immunocompromised mice. 38 is a diagram showing the results of staining lung tissue with PAS (x200). Specifically, the untreated group, the normal group (Nor); The bronchial immunosuppressed control group (Con); a positive control group (PC) treated with Yonggaksan; A group administered orally with a licorice complex at a dose of 50 mg/kg/day (50); The group (100), in which the licorice complex was orally administered at a dose of 100 mg/kg/day; The group (200) in which the licorice complex was orally administered at a dose of 200 mg/kg/day.

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

test material

1) sample

The 10 powder samples used in this experiment were prepared by Ruach, the leading institution, and stored in a cryogenic freezer (-80 ℃), and the required concentration was prepared with distilled water. Information on the solvent used for extraction is shown in Table 1.

Samples
(botanical name) Solvent DW 70% EtOH licorice
Glycyrrhiza glabra L. O O Gilkyung
Platycodon grandiflorum (Jacq.) A. DC. O O Control Zizyphus jujuba Mill. O O quince Chaenomeles japonica (Thunb.) Lindl. O O Packing Ecklonia kurome Okam. O O Acorus gramineus var. pusill (Siebold) Engl. O O Epiphyllum Morus alba L. O O Schisandra Schizandra sphenanthera Rehd . et Wils. O O Ulmus pumila L. O O Astragalus membranaceus var. mongholicus Bunge O O

Specifically, the powder samples were extracted with distilled water or alcohol (70% ethanol) using distilled water or alcohol (70% ethanol) and without pressure for 6 hours or more at 70 to 80° C. with 16-fold solvent (Gyeongseo E&P COSMO660). After that, it was concentrated using a rotary concentrator (rotary concentrator HS-2005S), and a freeze dryer. (Lyophilizer MLB-9003) was used as a powder sample after freeze-drying.

2) reagent

The reagents used in this experiment were dulbecco's phosphate buffered saline (D-PBS: Welgene, Korea), Dulbecco's Modified Eagle's Medium (DMEM: Gibco BRL Co., U.S.A.), fetal bovine serum (FBS: Invitrogen Co., U.S.A.), penicillin/streptomycin (Gibco BRL Co., U.S.A.), cell viability assay kit (Daeillab sevice, Korea), lipopolysaccharide (LPS:Sigma Co., U.S.A.), nitric oxide detection kit (Intron Biotechnology, Korea), As, Pb, Hg, Cd standard solution (SCP Science, Canada), water (Duksan, Korea), acetonitrile (Duksan, Korea), mouse cytokine milliplex map immunoassay kit (Millipore Co., U.S.A.), prostaglandin E2 parameter assay kit (R&D system) , U.S.A.), protease inhibitor cocktail (Sigma, U.S.A.), phosphatase inhibitor cocktail 2 (Sigma, U.S.A.), phosphatase inhibitor cocktail 3 (Sigma, U.S.A.), bovine serum albumin (BSA: Gendepot, U.S.A.), miracle-star™ detection system (Intron Biotechnology, Korea), IL-1β Antibody (Cell signaling, U.S.A.), IL-6 Antibody (Cell signaling, U.S.A.), TNF-α Antibod y (Cell signaling, U.S.A.), COX-2 Antibody (Cell signaling, U.S.A.), iNOS Antibody (Cell signaling, U.S.A.), β-actin Antibody (Cell signaling, U.S.A.), peroxidase-conjugated affinipure rabbit anti-goat IgG (Jackson immunoresearch,U.S.A.), peroxidase-conjugated affinipure goat anti-mouse IgG (H+L) (Jackson immunoresearch,U.S.A.), OVA albumin (Sigma, U.S.A.), Aluminum hydroxide gel (invivogen, U.S.A.), Mouse OVA specific IgE ELISA kit ( Biolegend, U.S.A.), Histamine assay kit (Biomax, Korea), Mouse PGE2 ELISA kit (Mybiosource, U.S.A.), total RNA prep kit (Intronbio. Co., U.S.A.), Accu Power RT Premix (Bioneer Co., Korea), Primer (Bioneer Co., Korea), Accu Power HotStart PCR Premix (Bioneer Co., Korea), Agarose A (Cosmo Co., U.S.A.), 50ХTAE (Bioneer Co., Korea) and the like were used.

3) device

The equipment used in this experiment was a CO ₂ incubator (Forma scientific Co., USA), a clean bench (Vision scientific Co., Korea), an autoclave (Sanyo Co., Japan), a vortex mixer (Vision scientific Co., Korea), centrifuge (Sigma Co., USA), ELISA reader (MolecularDevices Co., USA), ICP (Shimadzu, Co., Japan), mercury analyzer (TELEDYNE Leeman Labs, USA), HPLC (Shimadzu, Co., Japan), Luminex (Millipore, USA), mini trans-Blot® (Bio-RAD, USA), funsion FX (Vilber, USA), plate shaker (Lab-Line Co., USA), Submarine electrophoresis system (Mupid-exu Co., Japan) ), Fusion Quick Guide (Vilber Lourmant Co., France), Thermal Cycler D-360 (Thermal Dynamic, Co., USA), etc. were used.

4) Specification and management of laboratory animals

The experimental animals used in this experiment were 6-week-old BALB/c male mice (20 ~ 22 g) supplied by Raon Bio Co., Ltd., and sufficient solid feed and water were supplied until the day of the experiment. After 2 weeks of adaptation in an environment of 15%, 12 hours to 12 hours (light-dark cycle), it was used in the experiment.

Experimental method

1) RAW 264.7 cell culture

Frozen RAW 264.7 cells were transferred to a 50 ml tube, and 9 ml of PBS was added to float the cells, followed by centrifugation at 1,200 rpm for 5 minutes to remove the supernatant. Cells were suspended in 1 ml of DMEM medium composed of 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and cultured in a cell incubator (37° C., 5% CO ₂ ). The number of passages was 5 or more, and the samples were acclimatized for 24 hours before processing.

2) Measurement of cytotoxicity

In order to select the concentration at which the experiment is possible, the cytotoxicity measurement was performed as follows. RAW 264.7 cells were seeded in a 96-well plate at 1.5×10 ⁵ cells/well and cultured in a cell incubator (37° C., 5% CO ₂ ) for 24 hours. After culturing, it was replaced with a new culture medium, and 20 samples were treated with concentrations of 100 and 200 (㎍/㎖), and then cultured in a cell incubator for 24 hours again. After incubation, 10 μl of WST solution was added and reacted for 30 minutes in a cell incubator (37° C., 5% CO ₂ ). After the reaction, the absorbance was measured at 450 nm to express the cell viability as a percentage relative to the control.

Afterwards, based on the confirmed cytotoxicity results, the highest concentration in which no toxicity was detected in common among 20 samples was selected for the study. Toxicity was confirmed in the same manner as above at a concentration of μg/ml.

3) Heavy metal inspection

For lead, arsenic, and cadmium analysis, 0.5 g of the sample was placed in a microwave sample pre-processing device dedicated container, 10 ml of nitric acid was added, and then the container was placed in a hood to remove the generated gas and decomposed using the microwave sample pre-processing device. After decomposition, the decomposition solution was filtered with filter paper, put in a volumetric flask, and diluted with water to the concentration range of the standard solution appropriately to be used as the sample solution. Separately, 10 ml of nitric acid was placed in a special container for microwave sample pre-processing equipment and operated in the same manner as for preparing the sample solution, and used as a blank test solution. A calibration curve was prepared using an inductively coupled plasma spectrometer (ICP) with the prepared sample solution, standard solution and blank test solution, and the sample solution was measured by correcting it with the blank test solution.

For mercury analysis, 50 mg of the sample was measured using a mercury analyzer without a special pretreatment process.

4) Glycyrrhizic acid content analysis

In order to analyze the content of glycyrrhizic acid, a representative component of licorice, 0.5 g of dry licorice powder was extracted with an ultrasonic extractor (WUC-A22H, Daihan Scientific, Wonju, Korea) under the specified conditions, filtered and purified, and used as an analytical test solution. After filtering the assay solution into a vial with a 0.45 μm filter, the concentration was calculated by analysis with high-performance liquid chromatography (HPLC)/diode-array detector (DAD), and HPLC analysis conditions are shown in Table 2.

Item Condition instrument SHIMADZU LC20A series Column Agilent eclipse plus C18 (250×4.6) mm with Guard column Eluent A (DW), B (Acetonitrile) gradient 5%B (0~15 min), 5%B~10%B (15~100 min) Wave length UV 251 nm Column Temp. 30℃ Injection 10 μl

5) Nitric oxide (NO) production amount measurement

RAW 264.7 cells were seeded in a 96-well plate at 1.5×10 ⁵ cells/well and cultured in a cell incubator (37° C., 5% CO ₂ ) for 24 hours. After culturing, it was replaced with a new culture solution, and LPS was treated at a concentration of 1 μg/ml to a concentration of 100 μg/ml of 20 samples, and then cultured in a cell incubator for 24 hours again. Then, 50 μl of N1 buffer included in the kit was treated in each well and reacted at room temperature for 10 minutes, then 50 μl of N ₂ buffer was treated in each well and reacted for 10 minutes. After the reaction, absorbance was measured at 540 nm. The NO concentration of the culture medium was determined using a standard curve for each concentration of nitrite standard, and the NO production amount was confirmed by converting it into a percentage compared to the control.

After that, licorice and epiphyllum extracts were divided into complex ratios and treated with concentrations of 50 and 100 μg/ml, and NO production was performed in RAW 264.7 cells in the same manner as above.

6) Prostaglandin E2 (PGE ₂ ) measurement of production

RAW 264.7 cells were seeded in a 96-well plate at 1.5×10 ⁵ cells/well and cultured in a cell incubator (37° C., 5% CO ₂ ) for 24 hours. After culturing, the culture medium was replaced with a new culture medium, and the licorice and epiphysis extracts were divided into complex ratios and LPS was treated at a concentration of 50 and 100 μg/ml at a concentration of 1 μg/ml, and then cultured in a cell culture medium for 24 hours again. Thereafter, centrifugation was performed at 1,200 rpm for 5 minutes and the obtained supernatant was measured as follows.

150 μl of standard, control, and measurement sample was added to a 96-well plate, and 50 μl of primary antibody was added and mixed for 1 hour using a plate shaker. After that, 50 μl of PGE ₂ conjugate was added, mixed using a plate shaker for 2 hours, and washed with 400 μl of wash buffer. Then, 200 μl of substrate solution was added and reacted at room temperature with light blocking for 30 minutes. made it Finally, 100 μl of stop solution was added and the measurement was performed at a wavelength of 450 nm.

7) Measurement of cytokine (IL-1β, IL-6, TNF-α) production

RAW 264.7 cells were seeded in a 96-well plate at 1.5×10 ⁵ cells/well and cultured in a cell incubator (37° C., 5% CO ₂ ) for 24 hours. After culturing, the culture medium was replaced with a new culture medium, and the licorice and epiphysis extracts were divided into complex ratios and LPS was treated at a concentration of 50 and 100 μg/ml at a concentration of 1 μg/ml, and then cultured in a cell culture medium for 24 hours again. Thereafter, centrifugation was performed at 1,200 rpm for 5 minutes and the obtained supernatant was measured as follows.

Put 25 µl of standard, control, and measurement sample in a 96 well plate, add 25 µl each of assay buffer, matrix buffer, and antibody-immobilized beads, mix, react at room temperature for 2 hours, and wash twice using washing buffer did 25 μl of detection antibody was added thereto, and reacted with blocking light at room temperature for 1 hour. Then, 25 μl of Streptavidin-Phycoerythrin was added to react at room temperature for 30 minutes, and washed twice using washing buffer solution. After washing, 150 μl of PBS was added and measurement was performed using Luminex.

8) Protein (iNOS, COX-2, IL-1β, IL-6, TNF-α) expression level measurement

RAW 264.7 cells were seeded in a 96-well plate at 1.5×10 ⁵ cells/well and cultured in a cell incubator (37° C., 5% CO ₂ ) for 24 hours. After culturing, the culture medium was replaced with a new culture medium, and the licorice and epiphysis extracts were divided into complex ratios and LPS was treated at a concentration of 50 and 100 μg/ml at a concentration of 1 μg/ml, and then cultured in a cell culture medium for 24 hours again. Thereafter, centrifugation was performed at 1,200 rpm for 5 minutes and the obtained cells were measured as follows.

Proteins were extracted by adding RIPA buffer containing protease inhibitor cocktail I and phosphatase inhibitor II and III to the measurement sample cells. The extracted protein was quantified using a pierce BCA protein assay kit, and it was prepared by mixing with sample loading buffer and reacting at 95°C for 5 minutes. The prepared proteins were separated by size by SDS-PAGE through 10% acrylamide gel, and transferred to a PVDF membrane. The protein-transferred membrane was immersed in 3% BSA and reacted at room temperature for 2 hours. Wash 3 times using TBS-T buffer and IL-1β (1:1000), IL-6 (1:1000), TNF-α (1:1000), COX-2 (1:1000), iNOS (1:1000), β-actin (1:1000) Put the first antibody and react at 4℃ for 16 hours made it After washing 3 times again, secondary antibody (1:10000) was added and reacted at room temperature for 1 hour, washed again 10 times, and protein was developed through ECL solution. Then, the protein expression level was analyzed through chemidoc fusion FX. As for the analysis result, the results of the normal group not treated with LPS and the sample were calculated based on the expression level of β-actin, and the results of the control group and the experimental group were expressed as a percentage based on the normal group.

9) Production of egg albumin-induced bronchial immunity model and classification of experimental groups

To prepare an animal model for allergic asthma, first, 0.3 ml of a solution of 1 mg of ovalbumin (chicken egg albumin; OVA) mixed with PBS and aluminum hydroxide gel [Al(OH) ₃ gel] in a 1:1 ratio The mice were intraperitoneally injected on days 0, 7, and 14 once a day at intervals of 7 days from the start of the experiment. After that, from day 21, 7 days after the last intraperitoneal injection, the mice were placed in a 50x15x50 cm acrylic pie cage, connected to a nebulizer device, and 1% OVA solution diluted with PBS was administered once daily (30 minutes, driven). Asthma was induced through breathing by injection at a flow rate of 3 ml/min).

The experimental group consisted of a normal group that did not induce asthma (hereinafter, denoted as Nor), a negative control group that orally administered only distilled water immediately after OVA solution injection (hereinafter denoted as Con), and a daily dose of ryukaksan immediately after OVA solution injection was 1.8, the maximum daily dose for adults. A positive control group (hereinafter referred to as PC) administered by converting g to mouse body weight, a licorice complex (composite in which licorice and sagebrush were mixed in a 2: 1 ratio) was administered at 50 mg/kg/day (hereinafter, denoted as 50), The experiment was carried out by dividing it into a total of 6 experimental groups, such as 100 mg/kg/day (hereinafter, denoted as 100) and 200 mg/kg/day (hereinafter, denoted as 200).

Animals for analysis were sacrificed 2 days after the OVA solution injection and oral drug administration period (9 days in total) of the experimental group except for the normal group (9 days in total) and 2 days later (day 31), and blood collection and tissues (spleen, lung) Extraction and euthanasia were performed in accordance with the method approved by the Animal Experimental Ethics Committee.

10) blood analysis

For blood analysis, blood was collected using cardiac puncture at the time of animal sacrifice, and 200 μl of whole blood was transferred to an EDTA tube and the remaining blood was stored at room temperature for 30 minutes and centrifuged at 3,000 rpm for 15 minutes to separate the serum. .

(1) Specific-IgE production analysis (serum)

50 μl of standard and serum sample was added to a 96-well plate, and 50 μl of Matrix A solution (standard) or assay buffer (serum sample) was added, followed by reaction (plate shaker, 200 rpm) for 2 hours. After washing, 100 μl of IgE detection antibody was added, reacted for 1 hour, and washing was performed. Again, 100 μl of Avidin-HRP D solution was added and reacted for 30 minutes, followed by washing, and 100 μl of Substrate solution F was added and reacted for 15 minutes. Finally, 100 μl of Stop solution was added, and measurement was performed at a wavelength of 450 nm in an ELISA reader, and the results were derived based on a standard curve.

(2) Histamine content analysis (serum)

50 μl of standard and serum sample was added to a 96 well plate, and 50 μl of background solution (0 ppm) or reaction mix solution (standard and serum sample) was added, followed by reaction at 30° C. for 10 minutes. Then, the ELISA reader was measured at a wavelength of 570 nm, and the results were derived based on the standard curve.

(3) PGE ₂ Production analysis (serum)

100 μl of standard and serum samples were put in a 96 well plate, and reacted (37° C. incubator) for 1 hour and 30 minutes, washed, and then 100 μl of Biotinylated antibody solution was added and reacted for 1 hour. After that, washing was performed, 100 μl of Enzyme conjugate solution was added and reacted for 30 minutes, and then washing was performed again. Finally, 100 μl of Color reagent solution was added, and after 30 minutes of reaction, 100 μl of Color reagent C solution was added, and measurement was performed at a wavelength of 450 nm in an ELISA reader, and the results were derived based on a standard curve.

(4) Immune cell count and platelet count

For the number of immune cells (leukocytes, neutrophils in white blood cells, eosinophils, lymphocytes, monocytes) and platelets, whole blood from which serum was not separated was requested to be analyzed by KP&T (Korea) and the results were derived.

11) Gene expression measurement

For gene expression measurement, the spleen tissue obtained after laparotomy at the time of animal sacrifice was washed in PBS to remove impurities and the experiment was carried out.

(1) RNA extraction

The spleen tissue was homogenized, mixed with 400 μl of binding buffer, shaken slowly, and the solution was injected into the column. This was again centrifuged at 13,000 rpm with a 4°C centrifuge, washing buffer was added, and centrifuged again. After washing with washing buffer again, 50 μl of elution buffer was added, followed by centrifugation to extract total RNA.

(2) reverse transcription reaction

The mixture of the RT premix kit (reaction buffer, dNTPs mixture, RNase inhibitor, stabilizer, oligo dT15 primer) was added to diethyl pyrocarbonate (DEPC)-treated distilled water so that the final volume was 20 μl to 1 μg of total RNA. After mixing 20 μl of the reaction mixture well, react at 45°C for 60 minutes to synthesize first-strand cDNA, react at 95°C for 5 minutes, and inactivate M-MLV RT to use the synthesized cDNA for gene expression measurement. was used.

(3) Real-time PCR

Real-time PCR was performed to amplify the synthesized cDNA, and put 1 µl of cDNA, 2 µl of each primer, 10 µl of SYBR Green, and 5 µl of DEPC-DW into a real-time dedicated tube and react at 95°C for 2 minutes. After repeating 40 times for 5 seconds at 95°C and 30 seconds at 62.5°C, the gene expression level was then calculated compared to the control group, and the sequences of the primers used are shown in Table 3.

primer F/R sequence (5`->3`) size (bp) IL-1β F GCCACCTTTTGACAGTGATGAG 135 R ATGTGCTGCTGCGAGATTTG IL-2 F CTGAAACTCCCCAGGATGCTC 102 R CCGCAGAGGTCCAAGTTCATC IL-4 F CCATATCCACGGATGCGACA 118 R CGTTGCTGTGAGGACGTTTG IL-5 F TGAGACGATGAGGCTTCCTG 105 R CCCCCACGGACAGTTTGATT IL-6 F CCCCAATTTCCAATGCTCTCC 141 R CGCACTAGGTTTGCCGAGTA IL-10 F CGCTGTCATCGATTTCTCCC 106 R CATTCATGGCCTTGTAGACACC IL-13 F GGCAGCATGGTATGGAGTGT 132 R CTTGCGGTTACAGAGGCCAT IL-17A F TCCAAACACTGAGGCCAAGG 120 R GACGTGGAACGGTTGAGGTA TNF-α F GATCGGTCCCCAAAGGGATG 129 R TTTGCTACGACGTGGGCTAC IFN-γ F CACGGCCAGTCATTGAAAGC 115 R CATCCTTTTGCCAGTTCCTCC COX-2 F CCGTGGGGAATGTATGAGCA 128 R GGGTGGGCTTCAGCAGTAAT NF-KB1 F CTGCCAAAGAAGGACACGACA 130 R GGCAGGCTATTGCTCATCACA iNOS F GCTCCAGCATGTACCCTCAG 127 R AAGGCATCCTCCTGCCCACT β-actin F AGGGAAATCGTGCGTGACAT 95 R TCCAGGGAGGAAGAGGATGC

^* F:forward, R:reverse

12) Histopathological analysis

For histopathological analysis, the lung tissue obtained after laparotomy was fixed in 10% neutral formalin for 48 hours at the time of animal sacrifice, and then hematoxyline and eosin (H&E) staining and Periodic acid-Schiff (PAS) staining were performed in KP&T (Korea). were analyzed through the Motic DSAssistant (China) program.

3. Statistical processing

The experimental results of this study were expressed as mean ± standard deviation (mean ± SD) for each group. For comparison between groups, one-way analysis of variance (ANOVA) method was used, and statistical significance was verified using student's t-test ( ^*** p<0 . 001, ^** p < 0 . 01, ^* p <0 .05 marked if significant compared to control) .

Experiment result

1. Selection of extracts

1-1 Confirmation of cytotoxicity

The results of measuring the cytotoxicity of 20 samples through RAW 264.7 cells are shown in Table 4 and FIG. 1 . When the control group that was not treated with the sample was 100.0±1.0%, the cytotoxicity of the hot-water extract, Schisandra Schisandra, seokchangpo, Sangbaekpi, and Omija, etc. was confirmed at a concentration of 200 μg/ml out of 20 samples compared to the control group by 15% or more. (Table 4, Figures 1A and B). All extracts and concentrations except this showed a survival rate of 15% or less compared to the control group, confirming that it was safe. Based on these results, all subsequent experiments were conducted at a concentration of 100 μg/ml in which toxicity was not confirmed in all samples.

Sample Name Control DW extracts 70% EtOH extracts 100 μg/ml 200 μg/ml 100 μg/ml 200 μg/ml licorice 100.0±1.0 104.1±0.2 97.2±0.7 98.3±0.5 86.1±0.2 Gilkyung 107.3±2.2 105.3±1.7 94.4±1.2 90.6±0.0 contrast 93.0±0.8 90.0±0.3 92.6±0.1 88.0±0.6 Quince 96.2±0.7 90.4±1.1 94.5±0.3 90.6±0.1 bale 94.0±0.5 85.7±0.5 94.5±0.0 88.9±0.1 seokchangpo 97.2±0.2 86.7±0.2 94.7±1.0 84.8±0.7 sangbaekpi 97.6±0.5 93.1±0.4 86.0±0.1 80.1±0.3 Schisandra 88.2±0.3 81.2±1.1 86.6±0.6 79.4±0.9 You Geun-Pi 92.6±0.2 90.6±1.2 89.0±0.5 86.9±0.4 Astragalus 97.1±0.1 96.5±0.6 98.6±0.5 94.5±0.6

1-2. Check the heavy metal content

As a result of measuring the content of heavy metals in 20 samples, cadmium, lead, arsenic, and mercury were all or not detected below the standard value (Table 5).

Pb As CD Hg permissive density (mg/kg) 5 3 0.3 0.2 DW
extracts licorice ND ¹⁾ ND ¹⁾ 0.002 ND ¹⁾ Gilkyung ND ¹⁾ ND ¹⁾ 0.001 ND ¹⁾ contrast ND ¹⁾ ND ¹⁾ ND ¹⁾ ND ¹⁾ Quince ND ¹⁾ ND ¹⁾ ND ¹⁾ ND ¹⁾ bale ND ¹⁾ 0.178 0.003 ND ¹⁾ seokchangpo ND ¹⁾ ND ¹⁾ ND ¹⁾ ND ¹⁾ sangbaekpi ND ¹⁾ ND ¹⁾ 0.001 ND ¹⁾ Schisandra ND ¹⁾ ND ¹⁾ ND ¹⁾ ND ¹⁾ You Geun-Pi ND ¹⁾ ND ¹⁾ ND ¹⁾ ND ¹⁾ Astragalus ND ¹⁾ ND ¹⁾ ND ¹⁾ ND ¹⁾ 80% EtOH
extracts licorice ND ¹⁾ ND ¹⁾ 0.002 ND ¹⁾ Gilkyung ND ¹⁾ ND ¹⁾ 0.001 ND ¹⁾ contrast ND ¹⁾ ND ¹⁾ ND ¹⁾ ND ¹⁾ Quince ND ¹⁾ ND ¹⁾ ND ¹⁾ ND ¹⁾ bale ND ¹⁾ 2.115 0.015 ND ¹⁾ seokchangpo ND ¹⁾ ND ¹⁾ ND ¹⁾ ND ¹⁾ sangbaekpi 0.116 ND ¹⁾ ND ¹⁾ ND ¹⁾ Schisandra ND ¹⁾ ND ¹⁾ ND ¹⁾ ND ¹⁾ You Geun-Pi ND ¹⁾ ND ¹⁾ ND ¹⁾ 0.005 Astragalus ND ¹⁾ ND ¹⁾ ND ¹⁾ ND ¹⁾

¹⁾ ND : Not detected.

1-3. Check the content of glycyrrhizic acid

As a result of measuring the glycyrrhizic acid content of the licorice extract, it was confirmed that DW and alcohol extract were 16.98 and 15.44 mg/g, respectively (FIG. 2).

1-4. NO production check

The results of measuring the amount of NO production of 20 samples through RAW 264.7 cells are shown in Table 6 and FIG. 3 . When the control group treated only with LPS was 100.0±4.8%, compared to the control group of 20 samples, the hot water extract was packed, and the alcohol extract showed a significant decrease in production by about 15% or more, except for S. (Table 6, Figure 3).

Sample Name Normal Control 100 μg/ml DW 70% EtOH licorice 10.8±1.6 100.0±4.8 28.2±0.9 84.2±0.3 Gilkyung 78.6±0.9 78.7±0.3 contrast 85.0±0.4 82.4±0.3 Quince 76.7±0.4 74.4±0.3 bale 91.3±0.6 84.2±0.9 seokchangpo 74.0±0.7 89.4±0.9 sangbaekpi 61.1±0.8 71.4±0.5 Schisandra 76.1±0.4 92.1±0.5 You Geun-Pi 76.3±1.2 88.3±3.6 Astragalus 78.7±1.2 97.1±0.5

Summarizing the above experimental results, as a result of measuring the cytotoxicity, the hot-water extract of Schisandra Schisandra, alcohol extract Seokchangpo, Sangbaekpi, and Schisandra omija, etc. at a concentration of 200 μg/ml out of 20 samples, caused more than 15% apoptosis compared to the control group. Toxicity was confirmed, and the concentration of 100 μg/ml showed a survival rate of 85% or more for both hot water and alcoholic extracts. As a result of checking the content of heavy metals, cadmium, lead, arsenic, and mercury were all not detected or detected below the standard in 20 samples. As a result of checking the content of glycyrrhizic acid, an indicator component of the licorice extract, it was confirmed that the hot water extract and the alcohol extract were 16.98 and 15.44 mg/g, respectively. As a result of measuring NO production, compared to the control group, for 20 samples, the hot water extract showed a significant decrease in production by about 15% or more, except for seokchangpo, omija, Yugeunpi, and astragalus extract. Therefore, in the following, licorice extract and Sangbaekpi extract were selected and tested.

2. Characterization of the extract complex

2-1. Cytotoxicity check

The results of measuring the cytotoxicity of the licorice and epithelial complex through RAW 264.7 cells are shown in Table 7 and FIG. 4 . When the control group without sample treatment was expressed as 100.0±0.3%, the complex mixed with licorice root and licorice root in a ratio of 1:1, 1:2, and 2: 1 under the conditions of the control was 50 and 100 μg/ml concentration, respectively. After treatment, there was no difference from the control group, resulting in no toxicity (Table 7, FIG. 4 ).

Sample Name Control RAW 264.7 cells 50 μg/ml 100 μg/ml Licorice: Sangbaekpi
Complex (1:1) 100.0±0.3 100.6±3.5 99.1±0.7 Licorice: Sangbaekpi
Complex (1:2) 100.4±0.4 100.1±0.6 Licorice: Sangbaek Cover Complex (2:1) 100.8±2.0 100.3±1.3

2-2. NO production check

Table 8 and FIG. 5 show the results of measuring the NO production of the licorice and sangbaekpi complex through RAW 264.7 cells. The control group treated only with LPS was 100.0±5.5%, and the normal group not treated with LPS and the sample was 100.0±5.5%. As a result of treatment with 50 and 100 μg/ml concentrations of the complexes mixed with licorice and Sangbaekpi alcohol in 1:1, 1:2, and 2:1 ratios under the conditions of the control group, the licorice and Sangbaekpi alcohol complex was treated with all complex ratios There was a significant ( ^*** p <0.001) decrease in concentration compared to the control group (Table 8, FIG. 5 ).

Sample Name Normal Control RAW 264.7 cells 50 μg/ml 100 μg/ml Licorice: Sangbaekpi
Complex (1:1) 15.6±0.3 100.0±5.5 67.4±5.7 ^*** 49.5±3.1 ^*** Licorice: Sangbaekpi
Complex (1:2) 73.5±5.4 ^*** 53.9±1.8 ^*** Licorice: Sangbaek Cover Complex (2:1) 59.7±3.0 ^*** 39.1±1.9 ^***

^*** p<0 . 001 marked if significant compared to control

2-3. PGE ₂ Check production

The results of measuring the amount of PGE ₂ production of the licorice and epiphyllum complex through RAW 264.7 cells are shown in Table 9 and FIG. 6 . The control group treated only with LPS showed 584.8±33.0 pg/ml, and the normal group not treated with LPS and the sample showed 87.7±18.8 pg/ml. As a result of treatment with 50 and 100 μg/ml concentrations of a mixture of licorice and sangbaekpi alcohol in 1:1, 1:2, and 2:1 ratios, respectively, the licorice and sangbaekpi alcohol complex were 1:1 and 1 A significant ( ^** p<0.01, ^* p<0.05) decrease was observed compared to the control group at all complex ratios and treatment concentrations except for the 50 μg/ml concentration of :2 ratio (Table 9, FIG. 6).

Sample Name Normal Control RAW 264.7 cells 50 μg/ml 100 μg/ml Licorice: Sangbaekpi
Complex (1:1) 87.7±18.8 536.6±23.4 536.6±23.4 422.0±27.8 ^** Licorice: Sangbaekpi
Complex (1:2) 560.5±28.2 479.6±18.9 ^** Licorice: Sangbaek Cover Complex (2:1) 505.5±25.4 ^* 399.0±6.1 ^**

2-4. Cytokine production check

2-4-1. IL-1β production check

The results of measuring the amount of IL-1β production of the licorice and epiphyllum complex through RAW 264.7 cells are shown in Table 10 and FIG. 7 . The control group treated only with LPS showed 150.6±1.6 pg/ml, and the normal group not treated with LPS and the sample showed 13.8±2.6 pg/ml. As a result of treatment with 50 and 100 μg/ml concentrations of a mixture of licorice and Sangbaekpi alcohol in 1:1, 1:2, and 2:1 ratios, respectively, the licorice and Sangbaekpi alcohol complex was obtained in a 1:2 ratio under the conditions of the control group. Significant ( ^*** p<0.001, ^** p<0.01) reduction was observed compared to the control at all complex ratios and treatment concentrations except for the 50 μg/mL concentration (Table 10, FIG. 7).

Sample Name Normal Control RAW 264.7 cells 50 μg/ml 100 μg/ml Licorice: Sangbaekpi
Complex (1:1) 13.8±2.6 150.6±1.6 124.0±2.8 ^** 104.6±3.4 ^*** Licorice: Sangbaekpi
Complex (1:2) 133.6±2.8 103.7±2.5 ^*** Licorice: Sangbaek Cover Complex (2:1) 123.5±2.9 ^** 93.5±3.1 ^***

^*** p<0 . 001, ^** p<0 . 01 Marked when it was significant compared to the control group

2-4-2. IL-6 production check

The results of measuring the amount of IL-6 production of the licorice and epiphyllum complex through RAW 264.7 cells are shown in Table 11 and FIG. 8 . The control group treated only with LPS showed 235.8±8.8 pg/ml, and the normal group not treated with LPS and the sample showed 57.2±11.0 pg/ml. As a result of treatment with 50 and 100 μg/ml concentrations of a mixture of licorice and Sangbaekpi alcohol in 1:1, 1:2, and 2:1 ratios, respectively, the licorice and Sangbaekpi alcohol complex was obtained in a 1:2 ratio under the conditions of the control group. Significant ( ^*** p<0.001, ^** p<0.01) reduction was observed compared to the control at all compound ratios and treatment concentrations except for the 50 μg/mL concentration (Table 11, FIG. 8).

Sample Name Normal Control RAW 264.7 cells 50 μg/ml 100 μg/ml Licorice: Sangbaekpi
Complex (1:1) 57.2±11.0 235.8±8.8 176.7±8.2 ^** 139.8±9.3 ^*** Licorice: Sangbaekpi
Complex (1:2) 210.7±10.4 147.8±7.9 ^*** Licorice: Sangbaek Cover Complex (2:1) 178.9±9.8 ^** 129.8±11.4 ^***

^*** p<0 . 001, ^** p<0 . 01 Marked when it was significant compared to the control group

2-4-3. Confirmation of TNF-α production

The results of measuring the amount of TNF-α production of the licorice and sangbaekpi complex through RAW 264.7 cells are shown in Table 12 and FIG. 9 . The control group treated only with LPS was 953.7±53.4 pg/ml, and the normal group not treated with LPS and the sample showed 253.4±53.9 pg/ml. As a result of treatment with 50 and 100 μg/ml concentrations of a mixture of licorice and Sangbaekpi alcohol in 1:1, 1:2, and 2:1 ratios, respectively, the licorice and Sangbaekpi alcohol complex was obtained in a 1:2 ratio under the conditions of the control group. Significant ( ^*** p<0.001, ^** p<0.01) reduction compared to the control was shown at all compound ratios and treatment concentrations except for the 50 μg/mL concentration (Table 12, FIG. 9).

Sample Name Normal Control RAW 264.7 cells 50 μg/ml 100 μg/ml Licorice: Sangbaekpi
Complex (1:1) 253.4±53.9 953.7±53.4 735.5±34.3 ^** 553.5±55.2 ^*** Licorice: Sangbaekpi
Complex (1:2) 854.2±55.2 652.7±55.1 ^*** Licorice: Sangbaek Cover Complex (2:1) 654.1±56.1 ^*** 453.1±55.7 ^***

^*** p<0 . 001, ^** p<0 . 01 Marked when it was significant compared to the control group

2-5. Gene expression level check

2-5-1. Confirmation of iNOS gene expression level

Table 13 and FIG. 10 show the results of measuring the iNOS expression level of the licorice and epithelial complex through RAW 264.7 cells. The control group treated only with LPS was 1.00±0.06%, and the normal group not treated with LPS and the sample was 0.09±0.01%. As a result of treatment with 50 and 100 μg/ml concentrations of the complexes mixed with licorice and Sangbaekpi alcohol in 1:1, 1:2, and 2:1 ratios under the conditions of the control group, the licorice and Sangbaekpi alcohol complex was treated with all complex ratios A significant ( ^*** p<0.001, ^** p <0.01, ^* p <0.05) decrease in concentration compared to the control group was observed (Table 13, FIG. 10 ).

Sample Name Normal Control RAW 264.7 cells 50 μg/ml 100 μg/ml Licorice: Sangbaekpi
Complex (1:1) 0.09±0.01 1.00±0.06 0.68±0.02 ^** 0.38±0.02 ^*** Licorice: Sangbaekpi
Complex (1:2) 0.85±0.03 ^* 0.48±0.04 ^*** Licorice: Sangbaek Cover Complex (2:1) 0.67±0.06 ^** 0.29±0.02 ^***

^*** p<0 . 001, ^** p<0 . 01, ^* p<0 . 05 Marked when it was significant compared to the control group

2-5-2. Confirmation of COX-2 gene expression level

Table 14 and FIG. 11 show the results of measuring the COX-2 expression level of the licorice and sangbaekpi complex through RAW 264.7 cells. The control group treated with only LPS was 1.00±0.04%, and the normal group not treated with LPS and the sample was 0.03±0.01%. As a result of treatment with 50 and 100 μg/ml concentrations of a mixture of licorice and Sangbaekpi alcohol in 1:1, 1:2, and 2:1 ratios, respectively, the licorice and Sangbaekpi alcohol complex was obtained in a 1:2 ratio under the conditions of the control group. Significant ( ^*** p<0.001, ^** p<0.01, ^* p<0.05) reduction compared to the control was shown at all compound ratios and treatment concentrations except for the 50 μg/mL concentration (Table 14, FIG. 11).

Sample Name Normal Control RAW 264.7 cells 50 μg/ml 100 μg/ml Licorice: Sangbaekpi
Complex (1:1) 0.03±0.01 1.00±0.04 0.76±0.10 ^* 0.68±0.05 ^** Licorice: Sangbaekpi
Complex (1:2) 1.01±0.02 0.82±0.01 ^* Licorice: Sangbaek Cover Complex (2:1) 0.76±0.02 ^** 0.62±0.05 ^***

^*** p<0 . 001, ^** p<0 . 01, ^* p<0 . 05 Marked when it was significant compared to the control group

2-5-3. IL-1β gene expression confirmation

The results of measuring the IL-1β expression level of the licorice and epiphyllum complex through RAW 264.7 cells are shown in Table 15 and FIG. 12 . The control group treated with only LPS was 1.00±0.04%, and the normal group not treated with LPS and the sample was 0.10±0.05%. As a result of treatment with 50 and 100 μg/ml concentrations of a mixture of licorice and Sangbaekpi alcohol in 1:1, 1:2, and 2:1 ratios, respectively, the licorice and Sangbaekpi alcohol complex was obtained in a 1:2 ratio under the conditions of the control group. Significant ( ^*** p<0.001, ^** p<0.01, ^* p<0.05) reduction compared to the control was observed at all compound ratios and treatment concentrations except for the 50 μg/mL concentration (Table 15, FIG. 12).

Sample Name Normal Control RAW 264.7 cells 50 μg/ml 100 μg/ml Licorice: Sangbaekpi
Complex (1:1) 0.10±0.05 1.00±0.04 0.82±0.07 ^* 0.52±0.03 ^*** Licorice: Sangbaekpi
Complex (1:2) 0.95±0.01 0.63±0.08 ^** Licorice: Sangbaek skin complex (2:1) 0.76±0.07 ^* 0.46±0.02 ^***

^*** p<0 . 001, ^** p<0 . 01, ^* p<0 . 05 Marked when it was significant compared to the control group

2-5-4. IL-6 gene expression level check

Table 16 and FIG. 13 show the results of measuring the IL-6 expression level of the licorice and epidermal complex through RAW 264.7 cells. The control group treated only with LPS was 1.00±0.09%, and the normal group not treated with LPS and the sample was 0.03±0.00%. As a result of treatment with 50 and 100 μg/ml concentrations of the complexes mixed with licorice and Sangbaekpi alcohol in 1:1, 1:2, and 2:1 ratios under the conditions of the control group, the licorice and Sangbaekpi alcohol complex was treated with all complex ratios There was a significant ( ^** p <0.01) decrease in concentration compared to the control group (Table 16, FIG. 13 ).

Sample Name Normal Control RAW 264.7 cells 50 μg/ml 100 μg/ml Licorice: Sangbaekpi
Complex (1:1) 0.03±0.00 1.00±0.09 0.55±0.04 ^** 0.48±0.05 ^** Licorice: Sangbaekpi
Complex (1:2) 0.64±0.06 ^** 0.47±0.06 ^** Licorice: Sangbaek Cover Complex (2:1) 0.55±0.06 ^** 0.47±0.01 ^**

^** p<0 . 01 Marked when it was significant compared to the control group

2-5-5. Confirmation of TNF-α gene expression level

The results of measuring the TNF-α expression level of the licorice and sangbaekpi complex through RAW 264.7 cells are shown in Table 17 and FIG. 14 . The control group treated with only LPS was 1.00±0.06%, and the normal group not treated with LPS and the sample was 0.18±0.02%. As a result of treatment with 50 and 100 μg/ml concentrations of a mixture of licorice and Sangbaekpi alcohol in 1:1, 1:2, and 2:1 ratios, respectively, the licorice and Sangbaekpi alcohol complex was obtained in a 1:2 ratio under the conditions of the control group. Significant ( ^*** p<0.001, ^* p<0.05) reduction was observed compared to the control group at all compound ratios and treatment concentrations except for the 50 μg/ml concentration (Table 17, FIG. 14).

Sample Name Normal Control RAW 264.7 cells 50 μg/ml 100 μg/ml Licorice: Sangbaekpi
Complex (1:1) 0.18±0.02 1.00±0.06 0.83±0.02 ^* 0.55±0.02 ^*** Licorice: Sangbaekpi
Complex (1:2) 1.03±0.07 0.85±0.05 ^* Licorice: Sangbaek Cover Complex (2:1) 0.80±0.07 ^* 0.49±0.05 ^***

^*** p<0 . 001, ^* p<0 . 05 Marked when it was significant compared to the control group

3-1. Specific-IgE production amount

As a result of measuring the amount of Specific-IgE in the serum, the normal group was 2.4±0.5 ng/ml, the negative control group was 7.2±1.4 ng/ml, the positive control group was 7.3±2.7 ng/ml, the licorice complex 50 mg/kg/day administered group Silver showed 7.2±1.6 ng/ml, 100 mg/kg/day administration group, 5.9±1.2 ng/ml, 200 mg/kg/day administration group, 5.8±1.4 ng/ml. A significant ( ^* p<0.05) decrease was observed in the experimental group administered at kg/day ( FIG. 16 ).

3-2. PGE ₂ amount of production

As a result of measuring the amount of PGE ₂ in the serum, the normal group was 32.2±2.0 pg/ml, the negative control group was 71.5±5.1 pg/ml, the positive control group was 15.6±3.8 pg/ml, and the licorice complex 50 mg/kg/day administration group was The 33.2±8.8 pg/ml, 100 mg/kg/day administration group showed 32.3±3.1 pg/ml, and the 200 mg/kg/day administration group 31.1±3.9 pg/ml. , a significant ( ^*** p <0.001) decrease was observed in all experimental groups administered at 200 mg/kg/day ( FIG. 17 ).

3-3. Histamine production

As a result of measuring the amount of histamine in the serum, the normal group was 21.3±4.1 ppm, the negative control group was 39.2±3.0 ppm, the positive control group was 33.4±7.1 ppm, and the licorice complex 50 mg/kg/day administration group was 34.7±0.7 ppm, 100 mg The /kg/day administration group showed 32.1±4.5 ppm, and the 200 mg/kg/day administration group showed 29.3±0.5 ppm, which was significant ( ^** p<0.01, ^* p<0.05) decreased ( FIG. 18 ).

3-4. number of immune cells

3-4-1. White blood cells

As a result of measuring the number of white blood cells in the blood, the normal group was 2.1±0.2 (x10 ³ cells/μL), the negative control group was 3.0±0.3 (x10 ³ cells/μL), and the positive control group was 2.5±0.4 (x10 ³ cells/μL) , Licorice complex 50 mg/kg/day administration group 2.0±0.4 (x10 ³ cells/μl), 100 mg/kg/day administration group 1.8±0.4 (x10 ³ cells/μl), 200 mg/kg/day administration group 1.5 It appeared as ±0.5 (x10 ³ cells/μl), and there was a significant ( ^* p<0.05) decrease in the positive control group and all the experimental groups administered at 50, 100, 200 mg/kg/day of the licorice complex compared to the negative control group (FIG. 19). ).

3-4-2. Eosinophils in white blood cells

As a result of measuring the number of eosinophils in leukocytes in the blood, the normal group was 1.5±0.2%, the negative control group was 2.4±0.2%, the positive control group was 1.5±0.2%, the licorice complex 50 mg/kg/day administered group was 2.4±0.2%, The 100 mg/kg/day administration group showed 2.0±0.1%, and the 200 mg/kg/day administration group showed 1.8±0.1%, which was significant compared to the negative control group in the positive control group and the experimental group administered at 100 and 200 mg/kg/day. ( ^*** p<0.001, ^** p<0.01) decreased (Fig. 20).

3-4-3. Neutrophils in white blood cells

As a result of measuring the number of neutrophils in the blood leukocytes, 20.8±5.8% in the normal group, 26.9±5.2% in the negative control group, 25.4±9.5% in the positive control group, 25.7±5.8% in the group administered with licorice complex 50 mg/kg/day, The 100 mg/kg/day administration group showed 25.1±6.7%, and the 200 mg/kg/day administration group showed 25.4±4.1%, compared to the negative control group. was decreased, but did not show significance (FIG. 21).

3-4-4. Monocytes in white blood cells

As a result of measuring the number of monocytes in leukocytes in the blood, the normal group was 2.9±1.2%, the negative control group was 5.0±1.5%, the positive control group was 4.5±0.9%, the licorice complex 50 mg/kg/day administered group was 5.2±0.7%, The 100 mg/kg/day administration group showed 4.7±0.9%, and the 200 mg/kg/day administration group showed 4.2±0.3%, which was significant ( ^* p< 0.05) decreased (Fig. 22).

3-5-5. Lymphocytes in white blood cells

As a result of measuring the number of lymphocytes in leukocytes in the blood, 74.9±8.5% in the normal group, 65.7±5.6% in the negative control group, 68.6±10.2% in the positive control group, 67.0±11.6% in the group administered with licorice complex 50 mg/kg/day, The 100 mg/kg/day administration group showed 68.4±9.0%, and the 200 mg/kg/day administration group showed 68.0±7.2%, compared to the negative control group. was increased, but did not show significance (FIG. 23).

3-5. Platelet count

As a result of measuring the number of platelets in the blood, the normal group was 679.2±304.9 (x10 ³ cells/μL), the negative control group was 1637.4±312.0 (x10 ³ cells/μL), and the positive control group was 1378.0±229.8 (x10 ³ cells/μL) , Licorice complex 50 mg/kg/day administration group 1374.9±206.6 (x10 ³ cells/μL), 100 mg/kg/day administration group 1277.3±303.4 (x10 ³ cells/μL), 200 mg/kg/day administration group 1209.5 It appeared as ±95.6 (x10 ³ cells/μl), and there was a significant ( ^* p<0.05) decrease in the positive control group and all the experimental groups administered at 50, 100, 200 mg/kg/day of the licorice complex compared to the negative control group (Fig. 24). ).

3-6. gene expression level

3-6-1. IL-1β

As a result of measuring IL-1β gene expression with spleen tissue, the normal group was 0.1±0.1, the negative control group was 1.0±0.0, the positive control group was 0.4±0.0, and the licorice complex 50 mg/kg/day administered group was 0.7±0.1, 100 The mg/kg/day administration group showed 0.5±0.0 and the 200 mg/kg/day administration group 0.4±0.1, which was significant in all experimental groups administered at 50, 100, or 200 mg/kg/day of the positive control group and licorice complex compared to the negative control group. ( ^* p<0.05) decreased (Fig. 25).

3-6-2. IL-4

As a result of measuring the IL-4 gene expression level with spleen tissue, the normal group was 0.1±0.1, the negative control group was 1.0±0.0, the positive control group was 0.6±0.2, and the licorice complex 50 mg/kg/day administered group was 0.4±0.1, 100 The mg/kg/day administration group showed 0.3±0.0 and the 200 mg/kg/day administration group showed 0.3±0.1, which was significant compared to the negative control group in all experimental groups administered at 50, 100, or 200 mg/kg/day of the positive control and licorice complex. ( ^* p<0.05) decreased (Fig. 26).

3-6-3. IL-5

As a result of measuring the IL-5 gene expression level with spleen tissue, the normal group was 0.1±0.1, the negative control group was 1.0±0.0, the positive control group was 0.8±0.0, and the licorice complex 50 mg/kg/day administered group was 0.6±0.2, 100 The mg/kg/day administration group showed 0.6±0.2 and the 200 mg/kg/day administration group 0.4±0.2, which was significant ( ^* p<0.05) decreased ( FIG. 27 ).

3-6-4. IL-6

As a result of measuring the IL-6 gene expression level with spleen tissue, the normal group was 0.1±0.1, the negative control group was 1.0±0.0, the positive control group was 0.8±0.0, and the licorice complex 50 mg/kg/day administered group was 0.7±0.1, 100 The mg/kg/day administration group showed 0.6±0.0, and the 200 mg/kg/day administration group showed 0.6±0.1, which was significant ( ^* p<0.05) decreased ( FIG. 28 ).

3-6-5. IL-10

As a result of measuring IL-10 gene expression with spleen tissue, the normal group was 0.1±0.1, the negative control group was 1.0±0.0, the positive control group was 1.8±0.1, and the licorice complex 50 mg/kg/day administered group was 1.3±0.1, 100 The mg/kg/day administration group showed 1.5±0.1 and the 200 mg/kg/day administration group 1.6±0.1, which was significant compared to the negative control group in all experimental groups administered at 50, 100, or 200 mg/kg/day of the positive control and licorice complex. ( ^* p<0.05) increased (Fig. 29).

3-6-6. IL-13

As a result of measuring the IL-13 gene expression level with spleen tissue, the normal group was 0.1±0.1, the negative control group was 1.0±0.0, the positive control group was 0.2±0.1, and the licorice complex 50 mg/kg/day administered group was 0.6±0.0, 100 The mg/kg/day administration group showed 0.4±0.0, and the 200 mg/kg/day administration group showed 0.3±0.1, which was significant compared to the negative control group in all experimental groups administered at 50, 100, or 200 mg/kg/day of the positive control and licorice complex. ( ^* p<0.05) decreased (Fig. 30).

3-6-7. IL-17

As a result of measuring the IL-17 gene expression level with spleen tissue, the normal group was 0.1±0.1, the negative control group was 1.0±0.0, the positive control group was 0.6±0.1, and the licorice complex 50 mg/kg/day administered group was 0.6±0.2, 100 The mg/kg/day administration group showed 0.6±0.2, and the 200 mg/kg/day administration group showed 0.6±0.1, which was significant in all experimental groups administered at 50, 100, or 200 mg/kg/day of the positive control group and the licorice complex compared to the negative control group. ( ^* p<0.05) decreased (Fig. 31).

3-6-8. IFN-γ

As a result of measuring the IFN-γ gene expression level with spleen tissue, the normal group was 0.1±0.1, the negative control group was 1.0±0.0, the positive control group was 1.6±0.1, and the licorice complex 50 mg/kg/day administered group was 1.4±0.1, 100 The mg/kg/day administration group showed 1.3±0.1, and the 200 mg/kg/day administration group showed 1.4±0.2, which was significant in all experimental groups administered at 50, 100, or 200 mg/kg/day of the positive control group and the licorice complex compared to the negative control group. ( ^* p<0.05) increased (Fig. 32).

3-6-9. TNF-α

As a result of measuring TNF-α gene expression level with spleen tissue, the normal group was 0.1±0.1, the negative control group was 1.0±0.0, the positive control group was 0.5±0.2, and the licorice complex 50 mg/kg/day administered group was 0.3±0.2, 100 The mg/kg/day administration group showed 0.4±0.2, and the 200 mg/kg/day administration group showed 0.3±0.1, which was significant in all experimental groups administered at 50, 100, or 200 mg/kg/day of the positive control group and the licorice complex compared to the negative control group. ( ^* p<0.05) decreased (Fig. 33).

3-6-10. NF-κB

As a result of measuring NF-κB gene expression level with spleen tissue, the normal group was 0.1±0.1, the negative control group was 1.0±0.0, the positive control group was 0.5±0.2, and the licorice complex 50 mg/kg/day administered group was 0.3±0.2, 100 The mg/kg/day administration group showed 0.4±0.2, and the 200 mg/kg/day administration group showed 0.3±0.1, which was significant in all experimental groups administered at 50, 100, or 200 mg/kg/day of the positive control group and the licorice complex compared to the negative control group. ( ^* p<0.05) decreased (Fig. 34).

3-6-11. iNOS

As a result of measuring the iNOS gene expression level with spleen tissue, the normal group was 0.1±0.1, the negative control group was 1.0±0.0, the positive control group was 0.7±0.0, and the licorice complex 50 mg/kg/day administered group was 0.5±0.1, 100 mg/day. The kg/day administration group showed 0.5±0.1, and the 200 mg/kg/day administration group showed 0.5±0.1. ^* p<0.05) decreased ( FIG. 35 ).

3-6-12. COX-2

As a result of measuring COX-2 gene expression with spleen tissue, the normal group was 0.1±0.1, the negative control group was 1.0±0.0, the positive control group was 0.4±0.2, and the licorice complex 50 mg/kg/day administered group was 0.8±0.1, 100 The mg/kg/day administration group showed 0.5±0.2, and the 200 mg/kg/day administration group showed 0.5±0.1, which was significant ( ^* p<0.05) decreased ( FIG. 36 ).

3-7. histopathology analysis

3-7-1. H&E dyeing

As a result of analyzing the histopathology of lung tissue after H&E staining, infiltration of inflammatory cells around the bronchioles and increase in number were not observed in the normal group. No pathostructural changes were observed. On the other hand, in the bronchial immunosuppression test group, inflammatory cell infiltration and increase in number were observed around the bronchioles, damage to the epithelial cell layer inside the bronchioles was observed, and the airway area was narrowed due to thickening. In comparison of the negative control group, the positive control group, and the licorice complex administered group, the number and infiltration of inflammatory cells around the bronchioles were relatively low in the positive control group and the licorice complex administered group compared to the negative control group, and the epithelial cell layer damage and internal thickening were decreased. was observed (FIG. 37).

3-7-2. PAS staining

As a result of analyzing the histopathology of lung tissue after H&E staining, mucus secretion by goblet cell proliferation was not observed in the normal group with damage to the epithelial cell layer inside the bronchioles. On the other hand, in the bronchial immunosuppression test group, it was confirmed that the portion was deeply stained by PAS staining due to the damage to the epithelial cell layer inside the bronchioles and increased mucus secretion due to goblet cell proliferation. In the comparison between the negative control group, the positive control group, and the licorice complex administered group, it was observed that the PAS stained portion in the epithelial cell layer inside the bronchioles was significantly reduced in the positive control group and the licorice complex administered group compared to the negative control group (FIG. 38) ).

Combining the characteristics of the above-mentioned licorice and sangbaekpi complex, as a result of measuring cytotoxicity, the mixture of 1:1, 1:2, 2: 1 mixing ratio of licorice and sangbaekpi was compared with the control at 50 and 100 μg/ml concentrations. Safety was confirmed as there was no significant difference. As a result of measuring NO production, the compound of 1:1, 1:2, 2: 1 mixing ratio of licorice and sangbaekpi showed a significant reduction in production by about 27 to 61% or more compared to the control at 50 and 100 μg/ml concentrations. As a result of measuring the amount of PGE ₂ production, the mixture of 1:1, 1:2, and 2:1 mixing ratios of licorice and sangbaekpi were all ratios and treatment concentrations except for the 50 μg/ml concentration of 1:1 and 1:2 ratios. showed a significant decrease in production by about 14 to 32% or more compared to the control group. As a result of measuring the amount of cytokine production, the mixture of 1:1, 1:2, and 2:1 mixing ratios of licorice and Sangbaekpi was IL- 1β (about 18-38%), IL-6 (about 24-45%), and TNF-α (about 23-53%) showed a significant decrease in production. As a result of measuring the gene expression level, the complex of licorice and sangbaekpi 1:1, 1:2, and 2:1 mixing ratios showed iNOS and IL-6 expression at all mixing ratios and concentrations, COX-2, IL-1β, TNF -α expression showed iNOS (about 15-71%), COX-2 (about 17-38%), IL-1β (about 18-54%), IL-6 (about 36-53%), and TNF-α (about 15-51%) showed a significant decrease in production.

In addition, as a result of blood analysis, the licorice complex administration group (50, 100, or 200 mg/kg/day) showed the specific-IgE production amount, PGE ₂ production amount, Histamine production amount, white blood cell count, eosinophil count in white blood cell, monocyte count in white blood cell, and platelet count. significantly decreased. According to the results of gene expression analysis in the spleen, the licorice complex administered group (50, 100, or 200 mg/kg/day) had IL-1β, IL-4, IL-5, IL-6, IL-13, IL-17 , IFN-γ, TNF-α, NF-κB, iNOS, COX-2 expression was significantly reduced, and IL-10 expression was significantly increased. Furthermore, as a result of lung tissue pathology analysis, the number and infiltration of inflammatory cells around the bronchioles were less in the licorice complex administered group (50, 100, or 200 mg/kg/day) compared to the negative control group, and damage to the epithelial cell layer and internal thickening were reduced. In contrast to the negative control group, it was observed that the PAS-stained portion in the epithelial cell layer inside the bronchioles was significantly reduced, and thus the secretion of mucus was decreased.

Therefore, in immune-related biomarkers, it was concluded that the 100 μg/ml concentration of the complex mixed with licorice and sagebrush in a 2:1 ratio was comprehensively useful in transcription factors and inflammatory mediators. In particular, the significant efficacy of inflammatory mediators acting on the innate and acquired immune stages, respectively, as well as the result of consistent suppression of the gene expression and the production amount of IL-6, which is used as a major tracer in bronchial diseases, is associated with asthma, rhinitis, allergy and The possibility of using it for the same disease can be confirmed.

In addition, the licorice complex has the effect of reducing the regulation of Th1/Th2 cytokines, inflammatory transcription factors and related mediators, as well as the hematologic effects of immunoglobulins, histamine, prostaglandins, immune cells in the body, and platelet counts related to bronchial immunity and allergy. It can be confirmed that there is The efficacy of bronchial immunity-related biomarkers supported the results through histopathological analysis performed through lung tissue, and it was confirmed that the licorice complex is a possible raw material for improving bronchial immunity. These results indicate that it is possible to develop a functional feed for enhancing bronchial immunity for companion animals using the licorice complex extract, and furthermore, the results of this study show that it can be used not only for bronchial immunity but also for respiratory diseases including airway hypersensitivity and asthma.

The above detailed description illustrates and describes the present invention. In addition, the foregoing is merely to show and describe preferred embodiments of the present invention, and as described above, the present invention can be used in various other combinations, modifications and environments, the scope of the concept of the invention disclosed herein, and the writing Changes and modifications are possible within the scope equivalent to one disclosure and/or within the skill or knowledge in the art. Accordingly, the detailed description of the present invention is not intended to limit the present invention to the disclosed embodiments. In addition, the appended claims should be construed to include other embodiments as well.

Claims (8)

A pharmaceutical composition for preventing or treating inflammatory diseases comprising a licorice and epiphyllum extract complex.
The method of claim 1,
The extract complex is a pharmaceutical composition for preventing or treating inflammatory diseases that is a mixture of licorice extract and sangbaekpi extract.
3. The method of claim 2,
The licorice extract and sangbaekpi extract are purified water and a pharmaceutical composition for preventing or treating inflammatory diseases that is extracted with any one or more solvents of C1 to C4 alcohol.
3. The method of claim 2,
The licorice extract and sangbaekpi extract is a pharmaceutical composition for preventing or treating inflammatory diseases that is extracted with 50 to 70% ethanol solvent.
The method of claim 1,
The extract complex is a pharmaceutical composition for preventing or treating inflammatory diseases comprising a licorice extract and a sangbaekpi extract in a ratio of 1:2 to 2: 1.
The method of claim 1,
The extract complex is a pharmaceutical composition for preventing or treating an inflammatory disease having a concentration of 50 to 100 μg/ml.
A cosmetic composition for alleviating skin inflammation comprising a licorice and sagebrush extract complex.
A food composition comprising a licorice and sangbaekhu extract complex.

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