KR20190055030A - Composition for Improving Atopy Dermatitis Using an Extract of Ecklonia cava and an Extract of Sargassum horneri - Google Patents

Abstract

The present invention discloses a composition for alleviating atopic dermatitis using an extract of Ecklonia cava and an extract of Sargassum horneri, wherein the composition is developed by confirming that the extract of Ecklonia cava and the extract of Sargassum horneri exhibit anti-inflammatory activity and anti-allergic activity in cell experiments and atopic dermatitis-alleviating activity in animal experiments.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for improving atopic dermatitis using an extract of Ganoderma lucidum and a hornblende extract,

The present invention relates to a composition for improving atopic dermatitis using an extract of Ecklonia cava and a extract of Sargassum horneri .

Atopic dermatitis is a recurrent chronic skin disease that occurs frequently in infancy, but it may persist in adults or develop into an adult, and its prevalence is increasing recently (Kor J Pharmacogn., 43: 59-65, 2012 ). Atopic dermatitis is a type of allergic disease characterized by symptoms such as dry skin, itching, irritation, pruritus, swelling around the hair follicle, hypophytosis, ecchymosis, and eczema lesions (Korean J Invest Dermatol., 14: 67-72 , 2007).

The cause of atopic dermatitis has not yet been clarified yet. However, it has been suggested that the abnormal increase of IgE, which is the causative factor of allergic reaction, the decrease in the number of T cells and the decline of T cells which play a pivotal role in cell immunity, the infiltration of monocytes and macrophages, (J Invest Dermatol., 96: 523-526, 1991; J Invest Dermatol., 97: 389-394, 1991; Immunol , 11: 81-88, 1999, Curr Drug Targets Inflamm Allergy., 2: 199-120, 2003; J Allergy Clin Immunol., 107: 871-877, 2001; Adv Immunol., 78:57, 2001; International Th1 / Th2 imbalance due to the increase in the number of Th2 cells is known to be an important factor, especially in Th1 cells (Pediatr Allergy Immunol., 14: 775-773, 2002; Pediatr Allergy Immunol., 19: 605-613, 2008) Kor J Pharmacogn, 43: 59-65, 2012).

The immune system, a defense mechanism against external invasion of the human body, is centered on the activation of T cells. The cytokines IL-2, IFN-γ and TNF produced by Th1 cells induce the differentiation of Th1 cells and suppress the proliferation and differentiation of Th2 cells, whereas IL-4, IL-5, IL Cytokines such as IL-6, IL-10, IL-13 and TSLP (thymic stromal lymphopoietin) induce the proliferation and differentiation of Th2 cells and inhibit the differentiation of Th1 cells (J Am Acad Dermatol., 44, S1- 2001). Under normal conditions, the interaction between Th1 and Th2 cells is balanced by maintaining the immune response. Atopic dermatitis promotes the conversion of T cells into Th2 cells, and increased Th2 cells secrete a large amount of cytokines to produce IgE (J Clin Invest. 113 (5): 651-7, 2004; J Allergy Clin Immunol. 2003; 112 (2)), which induces hypersensitive responses by inducing mast cell and eosinophil differentiation : 252-62; Kor J Pharmacogn, 43: 59-65, 2012). In particular, TSLP is overexpressed in skin lesions in patients with atopic dermatitis and overexpression of TSLP by keratinocyte in mice has been reported to cause atopic dermatitis (Nature immunology. 2002; 3 (7): 673-80 ; Methods in enzymology. 2014; 535: 371-87; The Journal of experimental medicine. 2005; 202 (4): 541-9).

Chemokine is a low-molecular protein that displays chemoattractants and is divided into four types according to its structure and function: C, CC, CXC, and CX3C. Recently, these chemokines have been implicated in the development and maturation of immune cells, And migration, Th1 / Th2 balance, and so on.

Among the various chemokines, TARC (Thymus & activation-regulated chemokine) is a typical CC chemokine that acts on Th2 cells and acts on CCR4 (CC chemokine receptor 4) of Th2 cells to induce migration and invasion of Th2 cells as inflammatory lesions J Clin Invest. 113 (5): 651-7, 2004, Curr Allergy Asthma Rep. 5 (4): 284-90, 2005). Recently, there has been a report that the serum concentration of TARC is significantly increased in patients with atopic dermatitis (J Allergy Clin Immunol., 113 (2): 334-340), and the skin expression of TARC is increased in an atopic dermatitis animal model, It has been reported that the serum concentration of TARC is decreased when ciclosporin A or corticosteroid used as a treatment for dermatitis is administered to patients with atopic dermatitis (J Dermatol Sci., 34: 201-208, 2004). In addition, in vitro experiments, when HaCaT cells, which are human keratinocyte cell lines, were treated with INF-γ or TNF-α, a large amount of TARC was expressed, and a substance capable of inhibiting such expression could be used as a therapeutic agent for atopic dermatitis Int Immunol., 14 (7): 767-773, 2002).

In recent years, not only the immunological mechanism but also the damage of the skin barrier has become a major factor in the aggravation of atopic dermatitis. The outer most stratum corneum is formed from keratinocytes and consists of differentiated keratinocytes and surrounding lipid layers. These skin barriers are easily damaged due to intrinsic diseases such as lifestyle such as excessive cleansing and bathing, environmental factors such as dry atmosphere, pollutants, and degradation of lipid synthesis ability of keratinocytes, and thereby cause atopic dermatitis. Thus, atopic dermatitis presents symptoms such as folliculitis, scurvy and dryness. In addition, a feeling of pruritus occurs throughout the acute phase and the chronic phase, which is important in the treatment of atopic dermatitis because it causes secondary skin symptoms such as wound infection and secondary infection due to scratching.

Accordingly, at the time of the atopic dermatitis test, not only the production of the blood immunity substance is confirmed but also the direct skin symptom is confirmed using the animal model such as the skin irritation model, the dietary induction model, the environmental induction model, and the ovalbumin skin sensitization model . In general, ovalbumin skin sensitization model and skin irritation model are used frequently, but ovalbumin skin sensitization model has relatively long time to induce relative to chemical skin irritation model, and the actual dermatitis symptom is difficult to confirm with eyes Therefore, the skin stimulation model tends to be preferred. The skin irritation model is a method of inducing atopic dermatitis using chemical (DNCB: dinitrofluorobenzene, DNFB: dinitrofluorobenzene, PiCl: picryl chloride), which can induce dermatitis in a relatively short period of time with acute atopy induction model, It is useful for research because it has the merit of being able to visually confirm the wind and wind blasts.

Currently, steroid drugs are used to treat inflammation and cytokine production. However, long-term administration of steroids is associated with various side effects such as skin atrophy and growth retardation, and the use of non-steroids is increasing in recent years . However, non-steroids also have a variety of side effects such as erythema, itching, edema, erythematosus, and hypoglycemia and weakness of immunity, making it difficult to treat essential atopic dermatitis (Arellano FM et al., J Invest Dermatol 2007 Apr ; 127 (4): 808-16.). Therefore, research is needed to find a substance with superior therapeutic effects from relatively safe natural products.

On the other hand, algae have historically been used extensively by mankind for edible and industrial purposes. In Asia, they have long been used only as food materials, while in the West they have been used to produce valuable chemical materials, and research has been conducted on them. Aquatic organisms are abundant in seaweeds containing physiologically active substances slightly different from those of terrestrial organisms. Seaweeds are known to contain various components such as polyphenols, polysaccharides, minerals and alginic acid.

The present invention relates to a method for inhibiting the growth and the proliferation of hornblende and hornblende extracts, which are a kind of seaweeds, have antiinflammatory and antiallergic activity in cell experiments, It was confirmed that it has atopic dermatitis improving activity.

It is an object of the present invention to provide a composition for improving atopic dermatitis using the extracts of Ganoderma lucidum and Horns mellifera.

Other and further objects of the present invention will be described below.

As shown in the following Examples and Experimental Examples, the present invention inhibits NO production in a concentration-dependent manner in a mouse macrophage cell line (RAW 264.7 cells) stimulated with LPS (lipopolysaccharide) In addition, it inhibits the production of inflammatory cytokines (IL-1β, IL-6 and TNF-α) and exhibits anti-inflammatory activity, as well as mouse bone marrow activated with IgE (anti-DNP-IgE) and antigen (DNP-BSA) Derived dendritic cell line BMCMCs, and exhibits antiallergic activity such as inhibition of degranulation. When administered orally in a mouse animal model in which atopic dermatitis is induced by DNCB (dintrochlorobenzene), it can be used to alleviate ear edema, water loss and skin itching symptoms, It was completed by confirming that the clinical symptoms improved significantly.

In view of the foregoing, the present invention can be understood as a composition for improving atopic dermatitis, which comprises a mixture of a gentian extract and a hornblende extract as an active ingredient.

In the present specification, the term " Ganoderma lucidum or P. vaginalis extract " means water, a lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.) Methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N, N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), 1,3-butylene glycol, Extracts obtained by leaching using a mixed solvent of carbon dioxide and pentane, or fractions obtained by fractionating the extracts obtained by using a supercritical extraction solvent such as pentane, Any method such as cold rolling, refluxing, heating, ultrasonic irradiation, supercritical extraction, etc. can be applied. In the case of the fractionated extract, a fraction obtained by suspending the extract in a specific solvent and mixing and leaving with a solvent having a different polarity, the crude extract is adsorbed on a column packed with silica gel, and then a hydrophobic solvent, a hydrophilic solvent, Quot; means fractions obtained as a mobile phase. Also, the meaning of the extract includes a concentrated liquid extract or a solid extract in which the extraction solvent is removed by a method such as freeze drying, vacuum drying, hot air drying, spray drying and the like. Preferably an extract obtained by using water, ethanol or a mixed solvent thereof as an extraction solvent, and more preferably an extract obtained by using a mixed solvent of water and ethanol as an extraction solvent.

As used herein, the term " active ingredient " alone means an ingredient which exhibits the desired activity or which can exhibit activity together with a carrier which itself is not active.

As used herein, " atopic dermatitis " is defined to include all diseases classified as atopic dermatitis in the art accompanied by a skin irritable immune response, regardless of the cause, either directly or indirectly. In general, atopic dermatitis is classified into infantile atopic dermatitis, infantile atopic dermatitis, adult atopic dermatitis and maternal atopic dermatitis according to the onset period or the object of the invention. In this specification, atopic dermatitis includes all these types of atopic dermatitis .

In the present specification, the term " improvement " is meant to include atopic dermatitis treatment, prevention and improvement (alleviation of symptoms).

The composition of the present invention may contain any amount (effective amount) of the active ingredient as long as it can exhibit the atopic dermatitis improving activity that is intended to be treated according to the purpose of use, formulation, blending purpose and the like. And will be determined within the range of from 0.001% by weight to 15% by weight, based on the total weight of the composition. The term "effective amount" as used herein means a medical or pharmacological effect that is intended to be applied to a mammal, preferably a human, when the composition of the present invention is administered during a period of administration by a medical professional or the like, Refers to the amount of active ingredient contained in the composition of the present invention. Such effective amounts can be determined experimentally within the ordinary skill of those skilled in the art.

The composition of the present invention can be used not only for the effective ingredient but also for the purpose of increasing or enhancing the effect of improving atopic dermatitis or providing convenience or convenience through addition of similar activities such as skin protecting activity (skin damaging by ultraviolet rays, skin moisturizing etc.) In order to enhance, it may further comprise any compound or natural extract which is already known in the art to be safe and known to have that activity.

Such compounds or extracts include compounds or extracts listed in the official pamphlet of each country's pharmacopeia ("Korea Pharmacopoeia" in Korea), each country's health functional foods (in Korea, "Health Functional Food Standards and Specifications" (The "Act on Health Functional Foods" in Korea), which regulates the manufacture and sale of compounds or extracts and health functional foods approved by the respective countries in accordance with the laws of the respective countries ("Pharmaceutical Affairs Law" in Korea) &Quot;).≪ / RTI > For example, the Enterococcus faecalis heat-treated dry powder and the complex of Guava leaf extract, which are recognized individually as "functional immunity to the irritable immune response" in accordance with the Korean Health Functional Food Act, extracts of Quercus fruticosa, extracts of lobules, (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol), and L. sakei Probio 65, which has been individually recognized for its 'improved skin condition'. Lactic acid bacteria L. plantarum CJLP133, probiotic ATP, and the like will correspond to these compounds or extracts.

Such compounds or natural extracts may be included in the compositions of the present invention in combination with one or more of their active ingredients.

In a specific embodiment, the composition of the present invention can be identified as a food composition. When the composition of the present invention is identified as a food composition, its use can be understood as suppression of skin hypersensitivity.

The food composition of the present invention can be produced in any form and can be used in various forms such as beverages such as tea, juice, carbonated drink, ionic drink, processed oil such as milk and yogurt, gum, rice cake, Korean confectionery, A food, a health food, a food, a tablet, a capsule, a ring, a granule, a liquid, a powder, a slice, a paste, a syrup, a gel, a jelly and a bar.

In addition, the food composition of the present invention may be classified into any product category as long as it meets the laws and regulations on the time of manufacture and distribution in the legal and functional category. For example, it is a health functional food according to the 「Health Functional Food Act」 in Korea, or a food functional food according to the Korean Food Sanitation Law (Food Standards and Specifications) , Special-purpose food, and the like.

The food composition of the present invention may contain food additives in addition to the active ingredients thereof. Food additives are generally understood to be substances that are added to foods and mixed or infiltrated into food in the manufacture, processing or preservation of food, and their safety must be ensured since they are ingested daily with food and for long periods of time. In food additives according to the laws of the respective countries ("Food Sanitation Act" in Korea) regulating the manufacture and distribution of food, food additives with safety are specified in terms of ingredient or function. In the Food Additives Code of Korea (Food Additives Standards and Standards), the food additives are classified into chemical compounds, natural additives and mixed preparations in terms of ingredients. Such food additives are classified into sweeteners, flavors Preservatives, emulsifiers, acidulants, and thickeners.

The sweetener is used for imparting a sweet taste suitable for foods, and both natural and synthetic sweeteners can be used in the composition of the present invention. Preferably, natural sweeteners are used. Examples of natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.

Flavors may be used to enhance taste or flavor, both natural and synthetic. Preferably, a natural one is used. When using natural ones, the purpose of nutritional fortification can be performed in addition to the flavor. Examples of natural flavoring agents include those obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or those obtained from green tea leaves, Asiatica, Daegu, Cinnamon, Chrysanthemum leaves and Jasmine. Also, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, banks and the like can be used. The natural flavoring agent may be a liquid concentrate or a solid form of extract. Synthetic flavors may be used depending on the case, and synthetic flavors such as esters, alcohols, aldehydes, terpenes and the like may be used.

As the preservative, calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate and EDTA (ethylenediaminetetraacetic acid) can be used. As the emulsifier, acacia gum, carboxymethyl cellulose, Pectin and the like. As the acidulant, math, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid and the like can be used. The acidulant may be added so that the food composition has a proper acidity for the purpose of inhibiting the growth of microorganisms other than the purpose of enhancing the taste.

Examples of the thickening agent include suspending agents, sedimentation agents, gel-forming agents, bulking agents and the like.

The food composition of the present invention may contain physiologically active substances or minerals which are known in the art and which are stable as a food additive in addition to the above-mentioned food additives in order to supplement and supplement functional and nutritional properties.

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

The food composition of the present invention may contain an appropriate amount of the above-mentioned food additives according to the product type so as to achieve the purpose of addition thereof.

With regard to other food additives that may be included in the food composition of the present invention, reference may be made to the Food Code of the respective countries or the Food Additives Code.

In another specific embodiment, the composition of the present invention can be identified as a pharmaceutical composition.

The pharmaceutical composition of the present invention may be prepared into oral formulations or parenteral formulations according to the route of administration by conventional methods known in the art, including pharmaceutically acceptable carriers in addition to the active ingredient. Where the route of administration may be any suitable route including local routes, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucosal tissues, and combinations of two or more routes may be used. An example of a combination of two or more routes is a combination of two or more formulations of the drug according to the route of administration, for example, one drug is administered intravenously and another drug is administered via a local route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration and formulation, and specific reference may be made to the pharmacopoeia of each country, including the " Korean Pharmacopoeia ".

When the pharmaceutical composition of the present invention is prepared into an oral formulation, it may be formulated into powder, granules, tablets, pills, sugar tablets, capsules, solutions, gels, syrups, suspensions, wafers And the like. Examples of suitable carriers include starches such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol and xylitol, corn starch, potato starch and wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Hydroxypropylmethylcellulose and the like; polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol Serol, and the like. In case of formulation, suitable binders, lubricants, disintegrants, coloring agents, diluents and the like may be included as needed. Examples of suitable binders include starch, magnesium aluminum silicate, starch pellets, gelatin, methyl cellulose, sodium carboxymethyl cellulose, polyvinyl pyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax and the like. Examples of the disintegrating agent include starch, methylcellulose, magnesium stearate, magnesium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, Agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt, and the like. Examples of the diluent include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine and the like.

When the pharmaceutical composition of the present invention is prepared into a parenteral dosage form, it may be formulated in the form of an injection, transdermal drug delivery, nasal aspirate and suppository together with a suitable carrier according to methods known in the art. As the carrier suitable for injection preparation, aqueous isotonic solutions or suspensions may be used. Specifically, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, and isotonic solution such as 5% dextrose may be used . When formulated with a transdermal preparation, it can be formulated in the form of ointments, creams, lotions, gels, external liquids, pastes, liniments, and air-lozenges. Nasal inhalers may be formulated in the form of aerosol sprays using suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc., and when formulated as a suppository, witepsol, tween 61, polyethylene glycols, cacao butter, laurin, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, and sorbitan fatty acid esters.

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

The preferred dosage of the pharmaceutical composition of the present invention is 0.001 mg / kg to 10 g / kg per day, preferably 0.001 mg / kg to 1 g / day, depending on the patient's condition, body weight, sex, age, / kg < / RTI > The administration can be carried out once or several times a day. Such dosages should in no way be construed as limiting the scope of the invention.

In another specific embodiment, the composition of the present invention can be identified as a cosmetic composition. When the composition of the present invention is identified as a cosmetic composition, its use can be understood as application to skin irritation or suppression of skin troubles such as erythema, dryness, burning, erythema, drying and bleeding caused by abnormal immunity.

Even when the composition of the present invention is identified as a cosmetic composition, the cosmetic composition may be classified into any product category according to the use of the cosmetic composition. Specifically, the cosmetic composition may contain functional cosmetic products having applications such as improvement of skin troubles and improvement of atopic dermatitis, General cosmetics, and the like. Specific examples of the product form include a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing oil, powder foundation, emulsion foundation, wax Foundation, spray, and the like. In a specific product form, it may be a form of flexible lotion, nutritional lotion, nutritional cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

The cosmetic composition of the present invention may contain, in addition to its active ingredient, conventional additives such as stabilizers, solubilizing agents, surfactants, vitamins, colorants and antioxidants, and carriers commonly used in cosmetic compositions.

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

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 the case of a spray, in particular, / Propane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component. Specifically, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid esters of sorbitan, and the like.

When the formulation of the present invention is a suspension, a carrier, such as water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component is selected from aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

The cosmetic composition of the present invention can be produced according to a method for producing a cosmetic composition which is usually carried out in the art, except that it contains an active ingredient showing atopic dermatitis improving activity or skin immunity hypersensitivity inhibitory activity.

As described above, according to the present invention, it is possible to provide a composition for improving atopic dermatitis using a Gentian extract and a Houttuynia cordata extract.

The composition for improving atopic dermatitis of the present invention can be commercialized into foods, cosmetics, medicines, etc. for the purpose of improving atopic dermatitis or suppressing skin irritation of immune system.

Fig. 1 shows the results of cytotoxicity evaluation of mouse macrophage cell line of hoe saengmyung mackerel, ganoderma lucidum extract and mixture thereof.
FIG. 2 shows the results of evaluating the effect of hoe saengmyung mackerel, ganoderma lucidum extract, and mixtures thereof on cell viability of mouse macrophage cell line by LPS stimulation.
FIG. 3 shows the results of evaluating the effects of Hornsby moth, Liliaceae extract, and mixtures thereof on NO production of mouse macrophage cell line by LPS stimulation.
FIGS. 4 to 6 are the results of evaluating the effects of the hornbill venom, phlegmon extract and mixtures thereof on the secretion of inflammatory cytokines (IL-1β, IL-6 and TNF-α) of mouse macrophages by LPS stimulation.
FIG. 7 shows the β-hexosaminidase assay results of inhibiting the degranulation inhibition of the hoe saengmyung mackerel, ganoderma lucidum extract and mixtures thereof.
FIG. 8 shows RT-PCR results on the effect of the 5: 5 mixture of hornblende extract and gentian extract on the expression of inflammatory cytokines.
FIG. 9 is a western blot result of the effect on the expression of NF-.kappa.B, a transcription factor of inflammatory cytokine, of a mixture of Horns Cyprinus mellifera and a 5: 5 mixture of moxibustion extracts.
FIG. 10 shows the results of evaluating the effect on the clinical symptoms of the skin of a mixture of hoe saengmyung mackerel extract and phytotoxic extract 5: 5 in an atopic dermatitis animal model.
FIG. 11 shows the results of evaluating the effect of a mixture of hornblende extract and a 5: 5 mixture of hornblende extract on ear edema in an animal model of atopic dermatitis.
FIG. 12 shows the results of evaluating the effect of the mixture of hornblende extract and a 5: 5 mixture of hornblende extracts on moisture change in an atopic dermatitis animal model.
FIG. 13 shows the results of evaluating the effects of the mixture of hornblende extract and moxibustion extract 5: 5 on skin itch symptoms in an atopic dermatitis animal model.
FIG. 14 shows the results of evaluating the effect of the mixture of hoe saengmyung mackerel extract and gentle extract 5: 5 on the spleen size in an animal model of atopic dermatitis.
FIG. 15 shows the results of evaluating the effect of the mixture of hornblende extract and moxibustion extract 5: 5 on lymph node size in an atopic dermatitis animal model.
FIG. 16 shows the results of evaluating the effect of the mixture of hornblende extract and moxibustion extract 5: 5 on the blood levels of IgE, IgG1, and IgG2a in an animal model of atopic dermatitis.

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

< Example > Preparation of Ganoderma lucidum extract, Hornbill extracts and mixtures thereof

&Lt; Example 1 > Preparation of gut extract

Ecklonia cava (outpost) was collected from July to August 2016 in the coast of Jeju Island, Korea and washed with water to remove salt and other impurities. After washing with water, 20 g of ethanol was added to 70% ethanol for 24 hours, and filtered. The filtrate was concentrated under reduced pressure and lyophilized to prepare a powdery extract (ECE, Ecklonia cava ethanol extract).

&Lt; Example 2 > Manufacture of extracts of hornbill

In the same manner as in <Example 1>, powdery Horseshoe horneri ethanol extract (SHE) was prepared by extracting 70% ethanol from a horseshoe crab (outpost).

&Lt; Example 3 > Preparation of mixtures

The ECE of Example 1 and the SHE of Example 2 were changed to 0:10 (SHE alone), 2: 8, 4: 6, 5: 5, 6: 4, 8: ECE alone) (ECE: SHE) (mixture: MES).

<Experimental Example> Experiment to improve atopic dermatitis

< Experimental Example 1 > Anti-inflammatory activity experiment

<Experimental Example 1-1> Assessment of cytotoxicity against macrophages

The macrophage RAW264.7 cells were suspended in DMEM medium containing 10% FBS and 1% antibiotics and cultured at 37 ° C in a 5% CO ₂ incubator. 1 × 10 ⁵ cells per well were dispensed in 96-well plates and cultured for 16 hours. After incubation for 24 h at 37 ° C in a 5% CO ₂ incubator, 50 μl of MTT (2 mg / ml) was added to each well. After incubation at 37 ° C in a 5% CO ₂ incubator for 2 hours, the solution was removed and treated with 150 L of DMSO to dissolve the Formazan precipitate formed and the absorbance was measured at 540 nm using ELISA Was measured.

The results are shown in Fig. Figure 1 shows that ECE, SHE and mixtures thereof show no cytotoxicity in particular.

<Experimental Example 1-2> Evaluation of cell survival rate for LPS stimulation

The macrophage RAW264.7 cells were suspended in DMEM medium containing 10% FBS and 1% antibiotics and cultured at 37 ° C in a 5% CO ₂ incubator. 1 × 10 ⁵ cells per well were dispensed in 96-well plates and cultured for 16 hours. After 1 hour, LPS (lipopolysaccharide, 1 ㎍ / mL) inducing inflammation was treated and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. 50 μl of MTT (2 mg / ml) Respectively. After incubation at 37 ° C in a 5% CO ₂ incubator for 2 hours, the solution was removed and treated with 150 μl of DMSO to dissolve the Formazan precipitate and the absorbance was measured at 540 nm using ELISA Was measured.

The results are shown in FIG. 2, where ECE, SHE, and mixtures thereof all suppressed some cytotoxicity by LPS in proportion to the treatment concentration.

<Experimental Example 1-3> Assessment of NO production on LPS stimulation

The macrophage RAW264.7 cells were suspended in DMEM medium containing 10% FBS and 1% antibiotics and cultured at 37 ° C in a 5% CO ₂ incubator. 1 × 10 ⁵ cells per well were dispensed in 96-well plates and cultured for 16 hours. After 1 hour, LPS (lipopolysaccharide, 1 ㎍ / mL) inducing inflammation was treated and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. Then, 100 μl of the cell culture supernatant and 1 μl of Griess reagent [1% ) sulfanilamide in 100 μl of 5% (v / v) phosphoric acid and 0.1% (w / v) aphtylethylenediamine-HCl) were reacted for 10 min on 96-well plates and the absorbance was measured at 540 nm using ELISA NO production amount was measured.

The results are shown in FIG. 3. As shown in FIG. 3, ECE, SHE, and mixtures thereof inhibited NO production significantly in the sample-untreated group (LPS only treatment group) : 4 The mixture was excellent.

<Experimental Example 1-4> Evaluation of Inflammatory Cytokine Secretion to LPS Stimulation

The macrophage RAW264.7 cells were suspended in DMEM medium containing 10% FBS and 1% antibiotics and cultured at 37 ° C in a 5% CO ₂ incubator. 1 × 10 ⁵ cells per well were dispensed in 96-well plates and cultured for 16 hours. After 1 hour, the cells were treated with LPS (lipopolysaccharide, 1 / / mL) inducing inflammation and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. IL-1β, IL-6 And TNF- [alpha] cytokines were analyzed using real-time PCR. The samples used here were ECE, SHE and mixtures of 5: 5 and 8: 2.

primer TNF- F: 5 'CAA GGG ACT AGC CAG GAG 3' (SEQ ID NO: 1) R: 5 'TGC CTC TTC TGC CAG TTC 3' (SEQ ID NO: 2) IL-6 F: 5 'TGT GCA ATG GCA ATT CTG ATT GTA 3' (SEQ ID NO: 3) R: 5 'ATG GTC TTG GTC CTT AGC CAC TCC 3' (SEQ ID NO: 4) IL-1 F: 5 'GCT ACC TGT GTC TTT CCC GTC G 3' (SEQ ID NO: 5) R: 5 'TTG TCG TTG CTT GGT TCT CCT TG 3' (SEQ ID NO: 6) GAPDH F: 5 'CCC AAT GTG TCC GTC GTG 3' (SEQ ID NO: 7) R: 5 'CCT GCT TCA CCA CCT TCT TG 3' (SEQ ID NO: 8)

The results are shown in Fig. 4 to Fig. Figures 4 to 6 show that all samples significantly inhibit the secretion of inflammatory cytokines (IL-1 [beta], IL-6 and TNF-a). In particular, the 5: 5 mixture and the 8: 2 mixture of the samples were excellent in the suppression of secretion.

Statistical analysis

All of the above data were expressed as mean ± standard deviation based on the results of three repeated experiments. Statistical comparisons of mean values were performed by ANOVA analysis followed by Duncan's multi-range test using SPSS software. P-values were compared with the LPS-stimulating factor group †; p < 0.05, *; p < 0.005, and compared to a single extract of Horns mellifera (mixture 0:10); p < 0.05, b; p <p.005, and compared with the sensory extract alone (mixing ratio 10: 0), c; p < 0.05, d; p < 0.005.

<Experimental Example 2> Antiallergic activity experiment

<Experimental Example 2-1> Verification of β-hexosaminidase secretion inhibition

The secretion of β-hexosaminidase was measured in order to determine inhibitory effects of BMCMC on detachment, hornbill, hornblende and hornblende. Cells (5 × 10 ⁵ cells) were sensitized with anti-DNP-IgE (1 μg / ml) for 1 hour after preincubation at 37 ° C. for 2 hours with various concentrations of ECE, SHE and MES. Cells were washed with tyrode buffer (10 mM HEPES, 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl ₂ , 1 mM MgCl ₂ , 5.6 mM glucose and 0.1% BSA, pH 7.4) and incubated with DNP-BSA ) At 37 &lt; 0 &gt; C for 1 hour. After obtaining 30 μl of supernatant, the cells were lysed by tyroide buffer containing 1% Triton X-100. The supernatant (5 μl) and the dissolved cells (5 μl) were incubated with 50 μl of substrate solution (2 mM p-nitrophenyl-N-acetyl-D-glucosaminide, pH 4.5) in a 96- And reacted at 37 ° C. The reaction was terminated by treating 150 μl of 0.2 M Glycine-NaOH buffer (pH 10.7), and the absorbance was measured at 405 nm using a microplate reader. The percentage of β-hexosaminidase secreted from the supernatant was calculated by the following formula.

β-hexosaminidase% = [absorbance of supernatant / absorbance of supernatant + absorbance of cell] × 100

The results are shown in Fig. Figure 7A shows that the treated samples significantly inhibited the secretion of β-hexosaminidase, an indicator factor of degranulation, and showed the best activity of the 5: 5 mixture in particular. 7B shows that the 5: 5 mixture inhibits the secretion of? -Hexosaminidase in a concentration-dependent manner. A 5: 5 mixture with the best activity was used as a sample.

<Experimental Example 2-2> Inhibitory effect of inflammatory cytokine and chemokine expression

Evaluation of expression of inflammatory factor was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). BMCMC cells were treated with various concentrations of BMCMC for 2 hours and sensitized with anti-DNP-IgE (1 μg / ml) for 4 hours. Cells were washed with tyrode buffer and stimulated with DNP-BSA (100 ng / ml) for 24 h at 37 ° C. Twenty-four hours later, the supernatant was removed, and triazol reagent was added to the cells. After adding chloroform, the cells were vortexed for 10 seconds and centrifuged at 12,000 rpm for 15 minutes. The supernatant was removed and mixed with isopropanol. After centrifugation at 12000 rpm for 15 minutes, the supernatant was removed and the pellet was mixed and used for RT-PCR. CDNA was synthesized using a cDNA synthesis kit, and the cDNAs were denatured at 45 ° C for 30 minutes and at 94 ° C for 30 seconds using the cytokine and chemokine primer and RT-PCR kit shown in Table 2, After 30 seconds of annealing, the cycle of extraction at 72 ° C for 30 seconds was repeated 32 times and the final extension was performed at 72 ° C for 5 minutes. Each PCR product was loaded on 1.5% agarose gel and analyzed by electrophoresis for 30 minutes at 100V. The results are shown in Fig.

FIG. 8 shows that the 5: 5 mixture was increased in concentration-dependent manner by treatment with anti-DNP-IgE and DNP-BSA, and IL-1β, IL-4, IL-5, IL-6, TNF- Inhibit the expression of cytokines and suppress the expression of TSLP and TARC, which are known to increase in atopic dermatitis.

<Experimental Example 2-3> Verification of Expression of Inflammatory Cytokine Transcription Factor

Expression of inflammation-related proteins in BMCMC cells was confirmed by western blot. That is, BMCMC cells were cultured for 2 hours at various concentrations and sensitized with anti-DNP-IgE (1 μg / ml) for 4 hours. Cells were washed with tyrode buffer and stimulated with DNP-BSA (100 ng / ml) for 30 min at 37 ° C. After 30 minutes, the supernatant was removed, and lysis buffer was added to the cells. The vortexing was carried out for 6 times for 10 minutes. The supernatant was centrifuged at 14,000 rpm for 10 minutes at 4 ° C and proteins were quantified using BCA kit. The same concentration of protein and sample buffer was mixed and boiled at 100 ° C for 3 minutes to induce protein denaturation. Proteins were separated by 10% SDS-PAGE and transferred to PVDF membrane. Non-specific protein binding sites were blocked by reaction with Tris-buffered saline (TBS) containing 0.1% Tween 20 and bloking buffer for 2 h. Then, the solution containing the primary antibodies IκBα, phospho-IκBα, p65 (NF-κB) and phospho-p65 (p-NF-κB) was mixed for 12 hours and washed three times with TBST for 10 minutes. Then, the membrane was incubated with the secondary antibody goat anti-rabbit IgG at room temperature for 1 hour and 30 minutes, and then reacted with TBST for 10 minutes three times. ECL was used to detect the target The expression of the protein was measured.

The results are shown in Fig. 9, when the 5: 5 mixture was treated, NF-κB (p-NF-κB), which was increased by treatment with anti-DNP-IgE and DNP-BSA in cytoplasm and nucleus, Expression is decreased. The expression of p-IκBα was similarly decreased.

Statistical processing

Statistical significance was assessed using the PASW Statistics 19.0 software (SPSS, Chicago, IL, USA). The significance of the mean values between the two groups was compared using the Duncan's test at P <0.05.

<Experimental Example 3> Atopic dermatitis animal model experiment

<Experimental Example 3-1> Induction of atopic dermatitis and sample administration

In an animal model test of atopic dermatitis, a mixture of 5: 5 of a moxibustion extract and a hornblende extract, which showed the highest activity in the anti-inflammatory test and anti-allergy test, was used as a sample, After oral administration, clinical symptoms and degree of skin itching were evaluated.

Specifically, 1% DNCB (Dinitrochlorobenzene) was applied to Nc / Nga mice deprived of the back region to induce atopic dermatitis first. One week later, 1% DNCB and AD ointment 100 mg were applied to the depilated area, Induced atopic dermatitis. After this, oral administration was performed daily for 2 weeks and clinical analysis and scratching analysis were performed weekly. After the end of the experiment, the animals were sacrificed and the ear thickness, transepidermal water loss (TEWL), water and pH were measured and the blood, spleen, and lymph nodes were separated and analyzed.

<Experimental Example 3-2> Evaluation of the effect on the clinical symptom change of skin

In order to evaluate the effect of 5: 5 mixture of moxa and hornblende extracts on atopic dermatitis in animal models, changes in skin clinical symptoms were observed weekly during an animal experiment. It was observed that itching, erythema and bleeding, edema, And drying were evaluated as follows, as shown in [Table 3] below.

The results are shown in Fig. 10, a mixture of 5: 5 (Mix-ECE: SHE 5: 5) of the extracts of Ganoderma lucidum and Hornfish moth, Mycobacterium tuberculosis extracts was significantly different from that of atopic dermatitis causing vehicle (CJLP-133) (Novarax, Inc., Korea) in terms of the dose.

<Experimental Example 3-3> Evaluation of effects on ear edema

To evaluate ear swelling, a clinical symptom of atopic dermatitis, ear thickness of each individual was measured at 4th week. After animals were anesthetized, the right ear and left ear were divided into three parts, respectively, and caliper was used to measure the thickness.

The results are shown in Fig. 11. In the evaluation of the effect on edema of the ear, the 5: 5 mixture of the extracts of Ganoderma lucidum and Horns mellifera almond extract significantly alleviated ear edema.

<Experimental Example 3-4> Evaluation of effect on skin moisture change

① Transepidermal water loss (TEWL): Loss of water through the skin is caused by evaporation of skin moisture and perspiration. Among these, transdermal water loss is caused by passive diffusion of moisture from the inside of the body to the skin, It is called water loss. To evaluate skin dryness, which is a typical clinical symptom of atopic dermatitis, transdermal water loss was measured at the 4th week of the animal experiment. After the animals were anesthetized, the atrophy-induced back area was measured for 30 seconds using a transcutaneous water loss measuring device (AquaFlux, Biox).

② Moisture: Moisture was measured at 4th week after the end of the animal experiment. Skin-o-mat (Cosmomed Medical Beauty) was used as an index to evaluate the degree of skin dryness, which is a symptom of atopic dermatitis. To measure the skin skin moisture after depilating the area to be measured.

The results are shown in FIG. 12. The 5: 5 mixture of the extracts of Ganoderma lucidum and Rhododendron japonica significantly inhibited the transdermal water loss and the water content was markedly increased.

<Experimental Example 3-5> Evaluation of effects on skin itching symptom (Scratching) change

We evaluated the change of skin itching symptom which is a typical clinical symptom of atopic dermatitis. In order to observe the itching symptoms before and after the induction of atopic dermatitis, the behavior of the animal was photographed for 1 hour in the dark condition once every two weeks, and the number of scratches was measured by observing the behavior.

The results are shown in FIG. 13, wherein a 5: 5 mixture of the extracts of Ganoderma lucidum and P. vaginalis extracts markedly alleviated the symptoms of skin itch in the 4th week.

<Experimental Example 3-6> Evaluation of effects on immune-related tissue size changes

Atopic dermatitis reactions cause a variety of responses in the immune system, primarily causing hypertrophy of the immune system. Therefore, the spleen and lymph node weights were measured in order to observe the effect of the 5:

The results for the change in spleen size are shown in Fig. 14, and the sample clearly alleviated the spleen enlargement.

The results for the lymph node size are shown in Fig. 15, which alleviated the hypertrophy of both superficial cervical lymph nodes (cLN), axillary lymph nodes (aLN), brachial lymph nodes (bLN) and inguinal lymph nodes (dLN).

<Experimental Example 3-8> Evaluation of the effect on the production of immune related antibodies

Overexpression of IgE in serum is known to be the most representative marker of atopic disease. Therefore, in order to evaluate the effect of the 5: 5 mixture of the extracts of Ganoderma lucidum and Horns mellifera extract on the production of IgE in the animals with atopic dermatitis, the amounts of IgE, IgG1 and IgG2a were determined by ELISA kit (Bethyl Laboratories, Inc.).

The results are shown in FIG. 16, which shows that the sample lowers all of the blood concentrations of IgE, IgG1, and IgG2a.

Statistical processing

Statistical significance was assessed using the PASW Statistics 19.0 software (SPSS, Chicago, IL, USA). The significance of the mean values between the two groups was compared using the Duncan's test at P <0.05.

Claims (6)

A composition for improving atopic dermatitis comprising as an active ingredient, a mixture of an extract of Ganoderma lucidum and a extract of Horns mosaic virus.
The method according to claim 1,
The extract is a mixed solvent extract of water and ethanol,
Wherein the mixture is a mixture of a ghatti extract and a hornblende extract at a weight ratio of 5: 5 to 8: 2.
The method according to claim 1,
Wherein the mixture is a 5: 5 weight ratio mixture of a Gentian extract and a Horns mellifera extract.
4. The method according to any one of claims 1 to 3,
Wherein the composition is a pharmaceutical composition.
4. The method according to any one of claims 1 to 3,
Wherein the composition is a food composition.
4. The method according to any one of claims 1 to 3,
Wherein the composition is a cosmetic composition.

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