KR20200136729A - Pharmaceutical composition for preventing or treating atopic dermatitis comprising bio-fermented papaya - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating atopic dermatitis, and a quasi-drug composition, a food composition or a cosmetic composition for preventing or alleviating atopic dermatitis, all of which contain a papaya fermentation product. The papaya fermentation product according to the present invention not only has almost no side effects by using papaya, a natural product, but also effectively reduces skin lesions and the number of mast cells, and thus significantly reduces levels of NF-κB and IκBα in the cytoplasm involved in the expression of inflammatory genes, thereby being expected to be usefully used in the development of pharmaceuticals, health functional foods, and cosmetics for preventing, alleviating or treating atopic dermatitis.

Description

Pharmaceutical composition for preventing or treating atopic dermatitis comprising a papaya fermentation product {Pharmaceutical composition for preventing or treating atopic dermatitis comprising bio-fermented papaya}

The present invention relates to a pharmaceutical composition for the prevention or treatment of atopic dermatitis comprising a papaya fermented product, a quasi-drug composition for the prevention or improvement of atopic dermatitis, a food composition or a cosmetic composition.

Atopic dermatitis (AD) is a common chronic inflammatory skin disease.It increases the number of eosinophils, increases serum IgE levels, and is a proinflammatory cytokine including TNF-α and chemokine. cytokine). The IgE induces mast cells activation, and accordingly, inflammatory mediators including histamine, Th2 cytokines such as IL-4, IL-5, and IL-13 It is released from mast cells. The secretion of IgE is the most important and immediate hypersensitivity reaction of atopic dermatitis.

Currently, the number of atopic dermatitis patients in Korea is estimated at about 1 million as of 2017, and the global atopic dermatitis treatment market is expected to increase from $4.71 billion in 2017 to an average annual growth rate of 4.20%, reaching $5.56 billion by 2023. Expected (TechNavio, Global Atopic Dermatitis Drugs Market, 2017). In addition, the number of companion animals such as dogs and cats with hypersensitivity dermatitis corresponding to human atopic dermatitis is also increasing. Atopic dermatitis in dogs and cats begins with scratching at the age of 1 year old, and develops into typical symptoms such as skin redness, pustules and crust formation, along with scratching at the age of 3, and it is known that ear disease accompanies most of this.

On the other hand, in the case of steroid-based atopic treatments, which are widely used in general, the market size is shrinking due to side effects that are higher than their effects. The U.S. FDA has placed warnings about carcinogenicity for Elidel and Protopic, which are treatments for atopy, and the Korean Food and Drug Administration is also implementing restrictions on use. Accordingly, the necessity of developing a natural atopic dermatitis therapeutic agent that does not use steroids and antihistamines is on the rise.

Papaya ( Carica papaya Linn . ) is one of the most widely distributed and widely grown tropical plants, and has traditionally been used for nutritional and medicinal purposes. According to the literature, papaya has been reported to be effective in cardiovascular disease, dengue fever, digestive disorders, inflammatory diseases, wound healing, malaria, hypoglycaemia, and hyperlipidaemia. In addition, papaya contains active compounds having anticancer activity such as α-tocopherol, flavonoid, benzylisothiocyanate, and lycopene.

Under this background, the present inventors have researched to develop a therapeutic agent for atopic dermatitis using natural products, as a result of confirming that the papaya fermented product obtained by fermenting papaya with lactic acid bacteria can cure atopic dermatitis by suppressing the immune response. Was completed.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of atopic dermatitis comprising a papaya fermented product, a quasi-drug composition for preventing or improving atopic dermatitis, a food composition, and a cosmetic composition.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of atopic dermatitis comprising a papaya fermented product.

In addition, the present invention provides a quasi-drug composition for preventing or improving atopic dermatitis comprising a papaya fermented product.

In addition, the present invention provides a food composition for preventing or improving atopic dermatitis comprising a papaya fermented product.

In addition, the present invention provides a cosmetic composition for preventing or improving atopic dermatitis comprising a papaya fermented product.

The papaya fermentation product according to the present invention not only has almost no side effects using the natural papaya, but also effectively reduces the number of skin lesions and mast cells, and pays attention to the levels of NF-κB and IκBα in the cytoplasm involved in the expression of inflammatory genes. It is expected to be useful in the development of pharmaceuticals, health functional foods and cosmetics for the prevention, improvement or treatment of atopic dermatitis.

1 is a diagram showing the overall experimental protocol of a mouse experiment using the papaya fermentation product of the present invention.
Figure 2 is a diagram showing the observation results of the degree of improvement of atopic dermatitis for each group as a photograph.
3 is a graph showing the observation results of the degree of improvement of atopic dermatitis for each group by quantifying clinical skin severity scores (*P<0.05, **P<0.01, ***P<0.001 vs. control group; ###P) <0.001 vs. DNCB group; $$P <0.01 vs. papaya fermentation and DNCB+ papaya fermentation group).
4 is a view showing the result of observing the degree of atopic dermatitis improvement according to the papaya fermentation treatment of the present invention through histological analysis. 4A, B, and C are diagrams showing the results of H&E, toluidine blue, and Giemsa staining, respectively, and FIG. 4D is a diagram showing the result of measuring the thickness of the epidermis, and E and F of FIG. 4 are in the mouse dermis. Is a diagram showing the results of counting the number of mast cells and eosinophils (*P<0.05, **P<0.01, ***P<0.001 vs. control group; #P<005, ##P<0.01, ### P<0.001 vs. DNCB group).
5 is a diagram showing the results of observing changes in IgE and IgG levels of mice according to the papaya fermentation treatment of the present invention. 5A and B are diagrams showing changes in serum IgE and IgG levels, and FIG. 5C and E are diagrams showing changes in IgE and IgG secretion levels of spleen cells (*P<0.05, **P<0.01, * **P<0.001 vs. control group; #P<005, ###P<0.001 vs. DNCB group; $P<0.05, $$$P<0.001 vs. papaya fermented product and DNCB+ papaya fermented product group).
6 is a diagram showing the results of observing changes in the expression levels of IL-10, IFN-γ, and inflammatory proteins according to the papaya fermentation treatment of the present invention. 6A, B, D, and E are diagrams showing changes in the expression levels of IL-10, IFN-γ, NF-κB and IκBα, respectively, and C of FIG. 6 shows Western blot results for NF-κB and IκBα. Fig. (*P<0.05, **P<0.01 vs. control group; #P<005, ##P<0.01 vs. DNCB group)

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention or treatment of atopic dermatitis comprising a papaya fermented product.

In the present invention, "atopic dermatitis" or "atopic skin disease" refers to a skin disease accompanied by pruritus (itch), dry skin, and characteristic eczema as a chronic and recurrent inflammatory skin disease that begins mainly in infancy or childhood. In infancy, it starts with eczema on the spread side of the face and limbs, but as it grows, it appears in the form of eczema on the bending part of the arm and the bend behind the knee. In many cases, it tends to improve naturally as it grows. . In adults, lichenification occurs in which the skin at the folds becomes thick, and eczema on the face is more common than in childhood.

In the present invention, the term "treatment" refers to any act of improving or beneficially altering the symptoms of atopic dermatitis by administering or applying the composition. Those of ordinary skill in the technical field to which the present application pertains will know the exact criteria of atopic dermatitis or disease in which the composition of the present application is effective by referring to the data presented by the Korean Medical Association, and determine the degree of improvement, improvement and treatment. I will be able to.

In the present invention, the term "prevention" refers to any action that inhibits or delays the onset of diseases in which the present composition is effective by administration of the composition according to the present invention. It will be apparent to those skilled in the art that the composition of the present application, which is effective for the treatment of atopic dermatitis, can prevent such a disease if taken before the initial symptoms of the related disease or appear.

Atopic dermatitis is not only spreading to adults due to environmental pollution, stress, and changes in residential environment, but also due to the increase in the elderly population complaining of dry skin, the atopic market is rapidly growing. In addition, various kinds of skin disease treatments are currently on the market, but most of these drugs are artificially synthesized, and when used for a long time, they cause serious side effects or cause resistance of pathogens, so that the expected therapeutic effect cannot be obtained. Therefore, it would be said that the economic and industrial importance of the technology for manufacturing functional products for skin disease improvement from natural materials is very significant. Accordingly, the present inventors prepared a papaya fermented product by fermenting natural products such as papaya and pomegranate with lactic acid bacteria, and confirmed the excellent therapeutic effect on atopic dermatitis, thereby completing the present invention.

In the present invention, the papaya fermented product contains 1 to 10 parts by weight, preferably 1 to 5 parts by weight, more preferably 1 to 3 parts by weight, most preferably lactic acid bacteria based on 100 parts by weight of the papaya extract and pomegranate mixture. It may be characterized by inoculation in 2 parts by weight and prepared by fermentation.

In addition, the papaya extract and pomegranate mixture may be prepared by mixing papaya extract and pomegranate fruit, then adding fructooligosaccharide at the same weight as the mixed papaya extract and pomegranate fruit, and mixing evenly. That is, the papaya extract and pomegranate mixture may be prepared by mixing papaya extract, pomegranate and fructooligosaccharide in a ratio of 1:1:2 (w/w).

The fermentation may be performed at 25 to 40° C. for 1 to 5 days, preferably at 30 to 40° C. for 2 to 4 days, and in one embodiment of the present invention, at 37° C. for 3 days By fermentation, a papaya fermentation product according to the present invention was prepared.

In the present invention, the term "papaya extract" means an extract obtained by extracting papaya. For the purposes of the present invention, the papaya extract is characterized in that it is extracted with water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. . In this case, the water may be hot water of 90°C to 130°C, preferably 100°C to 120°C, more preferably 105°C to 115°C. The hot water extraction method can avoid the extraction of steroids, fat-soluble vitamins, and protein-based substances, and the extraction process is also simple compared to organic solvents and the time required is also short, so there are many advantages over the organic solvent extraction method. In addition, in the present invention, the extract may include an extract obtained by an extraction treatment, a dilution or concentrate of the extract, a dried product obtained by drying the extract, or any of these preparations or purified products.

In the present invention, the papaya extract may be extracted from at least one selected from the group consisting of papaya leaves, stems, roots, and fruits, and preferably extracted from papaya leaves, fruits, or a mixture of leaves and fruits. Can be used.

In the present invention, the lactic acid bacteria inoculated in the papaya extract and pomegranate are Lactobacillus acidophilus , Lactobacillus casei , Lactobacillus plantarum , Lactobacillus Bvlgari Lactobacillus bulgaricus , Lactobacillus lactis , Lactobacillus fermentum , Bifidobacterium Bifidobacterium bifidum ), Saccharomyces cerevisidae ( Saccharomyces cerevisidae ) and Saccharomyces boulardii ( Saccharomyces boulardii ) may be characterized in that it is one or more selected from the group consisting of, and preferably by inoculating all of the lactic acid bacteria. A papaya fermentation product according to the invention can be obtained.

The composition comprising the papaya fermented product according to the present invention may be characterized in that it further comprises vegetable glycerin. The vegetable glycerin may be added in an appropriate amount by a person skilled in the art according to the purpose of the invention. In addition to vegetable glycerin, the composition comprising the papaya fermented product according to the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stable It may contain additives such as an agent, a preservative, an alcohol, and a carbonation agent used in carbonated beverages. These ingredients may be used independently or in combination, and the ratio of these additives is not very important, but it is generally selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the papaya fermentation.

In addition, the pharmaceutical composition may further include an appropriate carrier, excipient, or diluent commonly used in the preparation of a pharmaceutical composition. Preferably, the composition is a tablet, pill, powder, granule, capsule, suspension, solution, emulsion, syrup, sterilized aqueous solution, non-aqueous solvent, suspension, emulsion, freeze-dried preparation, transdermal absorbent, gel, lotion, Ointments, creams, patches, cataplasmas, pastes, sprays, skin emulsions, skin suspensions, transdermal delivery patches, drug-containing bandages and suppositories may have any one formulation selected from the group consisting of, oral or It may be a variety of parenteral formulations, but is not limited thereto.

In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient, such as starch, calcium carbonate, sucrose, or lactose ( lactose), gelatin, etc. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives may be included. have. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.

The pharmaceutical composition of the present invention can be administered to mammals such as dogs, cats, mice, livestock, humans, etc. by various routes such as oral or parenteral, such as oral, rectal, intravenous, muscle, subcutaneous, intrauterine, dural It can be administered by intracranial or intraventricular injection. The preferred dosage of the pharmaceutical composition varies depending on the condition and weight of the patient, the degree of disease, the form of the drug, the route and duration of administration, but may be appropriately selected by those skilled in the art. For more effective prevention or treatment, the papaya fermented product of the present invention is preferably administered at 0.0001 to 100 mg/kg per day, preferably 0.001 to 100 mg/kg, and the administration may be administered once a day. Alternatively, it may be administered several times.

On the other hand, the pharmaceutical composition according to the present invention is characterized in that it can be applied to companion animals, such as dogs and cats, particularly having atopic dermatitis (or irritable dermatitis).

In addition, the present invention provides a quasi-drug composition for preventing or improving atopic dermatitis comprising a papaya fermented product.

In the present invention, "quasi-drug" refers to non-instrument, machine or device among articles used for the purpose of diagnosing, treating, alleviating, treating or preventing diseases of humans or animals, and pharmacological effects on the structure and function of humans or animals It refers to any item that is not an apparatus, machine, or device among the items used for the purpose of giving a message.

The quasi-drug composition is not particularly limited thereto, but preferably may be a disinfectant cleaner, a shower foam, a gagrin, a wet tissue, a detergent soap, a hand wash, a humidifier filler, a mask, an ointment or a filter filler. In addition, the quasi-drug composition may include skin external preparations and personal hygiene products. The external preparation for skin is not particularly limited thereto, but preferably may be prepared and used in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel or gel, and the personal hygiene product is specially used thereto. Without limitation, preferably soap, cosmetics, wet wipes, tissue paper, shampoo, skin cream, face cream, toothpaste, lipstick, perfume, make-up, foundation, cheek touch, mascara, eye shadow, sunscreen lotion, hair care products , Air freshener gel, cleaning gel, etc.

In addition, the present invention provides a food composition for preventing or improving atopic dermatitis comprising a papaya fermented product.

In the present invention, the "food composition" may include all foods in a conventional sense, and may be mixed with terms known in the art such as functional foods and health functional foods.

When the papaya fermented product of the present invention is used as a food additive, the papaya fermented product may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment), and may further include food additives acceptable as food. Since the composition of the present invention uses a substance derived from a natural substance as an active ingredient, there is no problem in terms of stability, and there is no significant limitation on the amount of mixing.

In the present invention, "functional food" means a food manufactured and processed using raw materials or ingredients having useful functions for the human body according to the Health Functional Food Act No. 6727, and "functional" It means ingestion for the purpose of obtaining useful effects for health purposes such as controlling nutrients for structure and function or for physiological effects.

In addition, in the present invention, "health functional food" refers to a food manufactured and processed by extracting, concentrating, refining, mixing, or extracting, concentrating, refining, or mixing a specific ingredient as a raw material for the purpose of health assistance. .

There is no limitation on the types of foods in which the papaya fermented product of the present invention can be used. In addition, the food composition comprising the fermented papaya product of the present invention as an active ingredient may be prepared by mixing appropriate other auxiliary ingredients and known additives that may be contained in food according to the choice of a person skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and There are vitamin complexes and the like, and can be prepared by adding the papaya fermented product according to the present invention as a main component to juice, tea, jelly, and juice.

In addition, foods that can be applied to the present invention include, for example, special nutritional foods (e.g., formula, infants, baby food, etc.), processed meat products, fish meat products, tofu, rice cakes, noodles (e.g., ramen, noodles, etc.), health supplements. , Seasoned foods (e.g. soy sauce, miso, red pepper paste, mixed sauce, etc.), sauces, sweets (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.) ), beverages (eg fruit, vegetable beverages, soy milk, fermented beverages, etc.), natural seasonings (eg, ramen soup, etc.).

When the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweetening agents, flavoring agents, natural carbohydrates, and the like as an additional component, like a normal beverage. In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin , Alcohol, carbonated beverages, etc. may contain. In addition, it may contain flesh for the manufacture of natural fruit juice, fruit juice beverage and vegetable beverage.

In addition, the present invention provides a cosmetic composition for preventing or improving atopic dermatitis comprising a papaya fermented product.

The papaya fermented product according to the present invention is a safe natural substance in the body, softening lotion, astringent lotion, nutrient lotion, nutrition cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder , Body lotion, body cream, body oil, body essence, makeup base, foundation, hair-dye, shampoo, conditioner or body cleaner, etc., but is not limited thereto.

When used as a cosmetic composition, additional substances may be added according to the formulation of external preparations for skin or cosmetics. For example, but not limited thereto, if the formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or Zinc oxide or the like may be used, and when the formulation is a powder or 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, additionally Propellants such as chlorofluorohydrocarbon, propane/butane or dimethyl ether. In addition, when the formulation is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, preferably water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan, but is not limited thereto. When the formulation is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, Aluminum metahydroxide, bentonite, agar, or tracant may be used, and when the formulation is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, Imidazolinium derivatives, methyltaurates, sarcosinates, fatty acid amide ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters are used. May be, but is not limited thereto.

The contents of the present invention described above are applied identically to each other, unless contradictory to each other, and it is also included in the scope of the present invention that those skilled in the art make appropriate changes.

Hereinafter, the present invention will be described in detail through experimental examples and examples, but the scope of the present invention is not limited to the following experimental examples and examples. All data are expressed as mean±standard error (SEM).

Example 1. Experiment preparation and method

1-1. papaya Fermented Produce

The papaya extract and pomegranate were mixed at a ratio of 1:1 (w/w), respectively, and fructooligosaccharide was added at the same weight as the mixed papaya extract and pomegranate, and mixed evenly. The mixture was finely pulverized with a grinder to prepare papaya blue. Thereafter, 2 parts by weight of lactic acid bacteria are inoculated with respect to 100 parts by weight of the prepared papaya green, and the lactic acid bacteria are mixed in an equal amount of 9 types of lactic acid bacteria and cultured at 37°C for 3 days to obtain 6 x 10 ⁸ cfu/mL to obtain a papaya fermented product. Was prepared. The papaya fermentation product was freeze-dried and stored at 4°C until use. The lactic acid bacteria used in the preparation of the papaya fermentation product were as follows: Lactobacillus acidophilus (KCCM 32820), Lactobacillus casei ; KCCM 12452), Lactobacillus plantarum ( KCCM 11322), Lactobacillus bulgaricus ( KCCM 35463), Lactobacillus lactis ( KCCM 34717), Lactobacillus fermentum ( Lactobacillus fermentum ; KCCM 40400), Bifidobacterium bifidum ( KCCM12096), Saccharomyces cerevisidae ( KCCM 11215) and Saccharomyces boulardii ( ATCC MYA-796 ).

Papaya extract is an extract of papaya leaf and fruit, purchased from STRAYA NATURAL Pharm (Sydney, Australia). Pomegranate fruit was purchased and used on the market. Meanwhile, the prepared papaya fermented product may be used in a form further including vegetable glycerin. For example, the papaya fermented product may be commercialized by mixing 94 to 96 parts by weight of the papaya fermentation product and 4 to 6 parts by weight of vegetable glycerin.

1-2. Set up fauna and induce atopic dermatitis

Animal experiments were conducted with the approval of the Seoul National University institutional animal care of use committee (SNU-160322-6). Six-week-old female BALB/c mice were purchased from Jungang Lab animal, Inc. and reared at 22 ± 20°C for 12 hours in light and dark cycles, and water and food were freely fed. Mice were acclimated to the above conditions for 1 week before starting the experiment. DNCB (2,4-Dinitrochlorobenzene, 2,4-dinitrochlorobenzene) is manufactured by Sigma-Aldrich Co. (St. Louis, Mo, USA) was purchased and dissolved in PBS to be used.

Mice were divided into 4 groups: normal (group 1), DNCB (group 2), papaya ferment (group 3), and DNCB + papaya ferment 100 μl (group 4). In order to prepare an atopic dermatitis (AD) animal model, 100 μl of 2% DNCB was repeatedly treated on the dorsal skin of a shaved mouse for 2 weeks using a 2 x 2 cm patch. After 3 weeks of AD induction, 100 μl of 2% DNCB was treated once more. Finally, the papaya fermented product was fed for 2 weeks with DNCB treatment. The papaya ferment was dissolved in drinking water and fed. The overall experimental protocol is shown in FIG. 1.

The severity of clinical skin was defined as the sum of the scores given for each of the four symptoms, erythema/hemorrhage, edema, erosion, and dryness (0-3 points). , None; 1, weak; 2, moderate; 3, severe). The observation results and clinical skin severity scores of the degree of atopic dermatitis improvement for each group are shown in FIGS. 2 and 3.

1-3. Histology and Immunohistochemistry ( immunihistochemistry , IHC ) Observation

For histopathological observation, dorsal skin samples of each group were immersed in a Tissue-Tek optimal cutting temperature (OCT) solution (Leica, USA) and stored in a refrigerator. Hematoxylin & Eosin (H&E) or toluidine blue (TB) and Kimja's dye (H&E) or toluidine blue (TB) and Kimja's dye to detect inflammatory cells or mast cells, respectively. Giemsa stain). Inflammatory cells and mast cells were counted in high-power fields (HPF) (250 μm×250 μm) of 40×, 400× and 1000× total 10 regions and expressed as cells/HPFs. In addition, the thickness of the epidermal layer was measured using an image analyzer. The results are shown in FIG. 4.

1-4. Enzyme-linked Immunosorbent Assay (ELISA)

Blood samples collected from the sacrificed mice were centrifuged at 5000 rpm for 30 minutes to separate the supernatant and stored at -800°C until use. The amount of serum IgE and IgG was measured using a sandwich ELISA (eBioscience, San Diego, CA, USA). Specifically, a monoclonal anti-mouse IgE, IgG primary antibody was dispensed on a microtiter plate, and then gradually diluted mouse IgE, IgG, HRP (horseradish peroxidase)-conjugated monoclonal anti-mouse IgE, IgG secondary antibodies, and serum collected from mice were sequentially added and cultured. OD was measured at 450 nm using a Bio-Rad microplate reader. The results are shown in FIG. 5.

1-5. Real-time Polymerase Chain Reaction (qPCR)

Total RNA was collected using TRIzol reagent (Invitrogen Life Technologies, Grand Island, NY, USA) according to the manufacturer's instructions. The amount and purification of total RNA was performed using a Nanodrop reader. 1 μg of total cellular RNA of each sample was reverse transcribed using a cDNA synthesis kit (TaKaRa, Japan), and quantitative PCR was performed using SYBR green iMaster and CFX 96 Real-Time System. The results are shown in FIG. 6.

1-6. Western Blot Western blot analysis

The spleen tissue was homogenized and dissolved. Protein concentration was measured using a Pierce® BCA protein assay kit (Thermo scientific, Rockford, IL 61101 USA). After loading 25 μg of protein into each well, and separating the protein using 10-15% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel under 100-250V conditions for 1-2 hours, i-Blot (Life Technology , Carlsbad, CA) to the membrane for 7 minutes. Thereafter, after blocking in 5% skim milk for 1 hour, the membrane was incubated with primary antibodies of IκBα and p65 NF-κB (Santa Cruz Biotech., Santa Cruz, CA) and then incubated with HRP ( horseradish peroxidase) conjugated secondary antibody (diluted 1:1000 in 5% skimmed milk solution) and incubated. After washing the membrane, the intensity of the protein band was quantified using ATTO CS image analyzer 3.0 (ATTO, Tokyo, Japan). As a control, GAPDH (Glyceraldehyde-3-phosphate Dehydrogenase) was used. The results are shown in FIG. 6.

Example 2. Results

2-1. On DNCB Caused by BALB /c Papaya for symptoms of atopic dermatitis in mice Fermented effect

As a result of repeatedly applying DNCB to the dorsal skin of the mouse for 2 weeks, it was confirmed that similar skin lesions occurred (Atopy dermatitis, AD) (FIG. 2). Dermatitis scores were calculated to evaluate the effect of papaya fermentation on AD symptoms in BALB/c mice induced by DNCB. Compared with the control group, it was confirmed that clinical symptoms such as erythema/bleeding, swelling, sores, and dryness became evident as the dermatitis score in the DNCB-treated group approached an average of 5 points. On the other hand, in the case of the DNCB + papaya fermented product treatment group, it was confirmed that the occurrence of AD-like skin lesions was significantly improved compared to the AD control group, and dermatitis scores for erythema/bleeding, edema, sores and dryness were also significantly reduced (Fig. 3 ).

As a result of histological analysis, it was confirmed that the skin of the DNCB-treated group showed remarkable epidermal hyperplasia that appears in atopic dermatitis compared to the control group. In the group treated with the DNCB + papaya fermentation, it was confirmed that the epidermal layer thickness decreased, and the papaya fermented product according to the present invention can effectively improve epidermal hyperkeratosis (Fig. 4D). In addition, in the results of H&E, toluidine blue and Giemsa staining, the number of mast cells and eosinophils in the dermis of DNCB-treated mice significantly increased compared to the control, and such an increase in inflammatory cells was observed. It was confirmed that it was significantly inhibited according to the papaya fermentation treatment according to the invention (E, F of Fig. 4). Histamine secreted by the degranulation of mast cells is known to play a major role in the progression of atopic dermatitis by damaging the skin barrier and causing skin inflammation by acting as a major cause of itching. Therefore, it was confirmed through the above results that the papaya fermented product according to the present invention has a remarkable effect in reducing the itching by reducing the number of mast cells.

2-2. On DNCB Caused by BALB /c of mouse IgE , IgG Papaya for shame Fermented effect

Atopic dermatitis increases the IgE antibody response, which increases the activity of the IgE-dependent histamine vitreous body to promote histamine secretion, and histamine induces eosinophil infiltration and causes acute hypersensitivity reaction and itching. Accordingly, serum IgE levels are known as an indicator of atopic dermatitis, and in particular, IgE is known to have a close correlation with clinical severity. Furthermore, it is known that serum IgE and IgG levels are related to Th1 or Th2 immunity.

In order to evaluate the effect of the papaya fermentation product according to the present invention on atopic dermatitis, IgE and IgG levels in serum and spleen cells were measured. Serum IgE and IgG levels were significantly increased in the DNCB-treated group compared to the control group, whereas the papaya fermented product-treated group in spleen cells significantly decreased IgE and IgG levels compared to the DNCB-treated group (FIG. 5). Through the above results, it was confirmed that the papaya fermented product according to the present invention can significantly reduce the amount of IgE and IgG secretion in the spleen cells, and thereby has an effect on inhibiting atopic dermatitis.

2-3. IL-10, IFN - γ and Papaya on inflammatory protein expression Fermented effect

In order to evaluate the effect of the papaya fermentation product according to the present invention on Th1 and Th2 immunity, the expression levels of IL-10 and IFN-γ mRNA in the spleen were observed. As a result, it was confirmed that the expression of IL-10 mRNA was significantly increased by the DNCB treatment, and IFN-γ mRNA was significantly decreased in the DNCB treated group compared to the control group (Fig. 6A, B). In the case of NF-κB and IκBα, it was confirmed that the expression level was significantly increased by the DNCB treatment, and then the expression level was significantly decreased by the papaya fermentation treatment (FIG. 6D, E). In addition, as a result of Western blot, the DNCB-treated group had higher NF-κB and IκBα levels than the control group (p<0.01), and the DNCB + papaya fermented product group significantly decreased NF-κB and IκBα levels compared to the AD control group. It was confirmed (Fig. 6C). Activation of NF-κB and IκBα induces the expression of proinflammatory chemokine, and the results show that the papaya fermentation product according to the present invention can improve or treat atopic dermatitis by inhibiting the expression of proinflammatory chemokine. Confirmed.

As described above, the present inventors experimented that the papaya fermented product according to the present invention effectively reduces the number of skin lesions and mast cells, and significantly reduces the levels of NF-κB and IκBα in the cytoplasm involved in the expression of inflammatory genes. It was confirmed through. In particular, it was confirmed that the papaya fermentation product can effectively improve atopic dermatitis without cytotoxicity in vivo using a mouse model of atopic dermatitis. Therefore, the papaya fermented product according to the present invention can be usefully used in the development of pharmaceuticals, health functional foods and cosmetics for the prevention, improvement or treatment of atopic dermatitis.

Hereinafter, examples of the preparation of a pharmaceutical composition for preventing or treating atopic dermatitis or a food composition for improving atopic dermatitis comprising the fermented papaya of the present invention as an active ingredient will be described, but not intended to limit the present invention, but to be described in detail. to be.

Formulation example 1. Preparation of pharmaceutical preparation

1-1. Powdery Produce

200 mg papaya fermented product

100 mg lactose

10 mg of talc

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

1-2. Manufacture of tablets

100 mg papaya fermented product

100 mg corn starch

100 mg lactose

2 mg of magnesium stearate

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet preparation method.

1-3. Preparation of capsules

100 mg papaya fermented product

3 mg of crystalline cellulose

14.8 mg lactose

Magnesium stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare a capsule.

1-4. Preparation of injections

100 mg papaya fermented product

Mannitol 180 mg

2974 mg of sterile distilled water for injection

Na ₂ HPO ₄ 2H ₂ O 26 mg

It is prepared with the above ingredients per ampoule (2 ml) according to a conventional injection preparation method.

1-5. Liquid Produce

200 mg papaya fermented product

10 g of isomerized sugar

5 g of mannitol

Purified water appropriate amount

According to the usual preparation method of liquid preparation, add and dissolve each ingredient in purified water, add lemon zest, and mix the above ingredients, add purified water to adjust the total to 100 ml, fill in a brown bottle, and sterilize the solution. To manufacture.

Formulation example 2. Preparation of food preparation

2-1. Manufacture of health food

100 mg papaya fermented product

Vitamin mixture right amount

70 g of vitamin A acetate

1.0 mg of vitamin E

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

0.5 mg of vitamin B6

0.2 g of vitamin B12

Vitamin C 10 mg

10 g biotin

1.7 mg of nicotinic acid amide

50 g folic acid

0.5 mg of calcium pantothenate

Suitable amount of inorganic mixture

1.75 mg ferrous sulfate

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dicalcium phosphate 55 mg

90 mg of potassium citrate

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixture is relatively suitable for health food, but it may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. , To prepare granules, and can be used to prepare a health food composition according to a conventional method.

2-2. Manufacturing of health drinks

100 mg papaya fermented product

15 g of vitamin C

100 g of vitamin E (powder)

19.75 g of iron lactate

Zinc oxide 3.5 g

3.5 g of nicotinic acid amide

0.2 g of vitamin A

0.25 g of vitamin B1

0.3 g of vitamin B2

Water quantity

After mixing the above ingredients according to a general health drink manufacturing method, stirring and heating at 85° C. for about 1 hour, the resulting solution is filtered and obtained in a sterilized container, sealed and sterilized, and then stored in a refrigerator. It is used to manufacture health beverage compositions.

The composition ratio is a mixture of ingredients suitable for a relatively preferred beverage in a preferred embodiment, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the demand class, the country of demand, and the purpose of use.

Claims (9)

A pharmaceutical composition for the prevention or treatment of atopic dermatitis comprising a papaya fermented product.
The pharmaceutical according to claim 1, wherein the papaya fermentation product is prepared by inoculating 1 to 10 parts by weight of lactic acid bacteria based on 100 parts by weight of a papaya extract and pomegranate mixture, and fermenting it. Ever composition.
The method of claim 2, wherein the papaya extract and pomegranate mixture is a mixture of papaya extract, pomegranate and fructooligosaccharide in a ratio of 1:1:2 (w/w), preventing or treating atopic dermatitis. Pharmaceutical composition for use.
The pharmaceutical composition for preventing or treating atopic dermatitis according to claim 2, wherein the fermentation is performed at 25 to 40°C for 1 to 5 days.
The method of claim 2, wherein the lactic acid bacteria are Lactobacillus acidophilus , Lactobacillus casei , Lactobacillus plantarum , Lactobacillus bulgaricus , Lactobacillus bulgaricus , Lactobacillus lactis , Lactobacillus fermentum , Bifidobacterium Bifidobacterium bifidum ), Saccharomyces cerevisidae ) and Saccharomyces bouladi ( Saccharomyces boulardii ), characterized in that at least one selected from the group consisting of, a pharmaceutical composition for the prevention or treatment of atopic dermatitis.
According to claim 1, wherein the composition is characterized in that it further comprises vegetable glycerin, a pharmaceutical composition for the prevention or treatment of atopic dermatitis.
A quasi-drug composition for preventing or improving atopic dermatitis comprising a papaya fermented product.
A food composition for preventing or improving atopic dermatitis comprising a papaya fermented product.
A cosmetic composition for preventing or improving atopic dermatitis comprising a papaya fermented product.

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