KR102393324B1 - Composition for preventing or treating inflammatory skin diseases comprising Japanese pepper hydrozol and Cordyceps mycelium extract as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or mitigating inflammatory skin diseases containing a mixture comprising Zanthoxylum schinifolium hydrosol and Cordyceps mycelium extract as active ingredients. More specifically, Zanthoxylum schinifolium hydrosol extracted from Zanthoxylum schinifolium fruits reduces expression of SNARE protein which is inflammatory mediating protein, and inhibits activity of reactive oxygen species, NO and β-hexasosaminidase which induce inflammation. Also, a mixed composition of Zanthoxylum schinifolium hydrosol and Cordyceps mycelium extract improves stability of Zanthoxylum schinifolium hydrosol and exhibits an excellent β-hexasosaminidase activity inhibitory effect. As a result, the mixture may be provided as a cosmetic composition, health food and pharmaceutical composition for preventing or mitigating inflammatory skin diseases including atopic dermatitis.

Description

Composition for preventing or treating inflammatory skin diseases comprising Japanese pepper hydrozol and Cordyceps mycelium extract as an active ingredient

The present invention relates to a composition for preventing or improving inflammatory skin diseases, comprising Sancho hydrosol and Cordyceps mycelium extract as active ingredients.

Atopic dermatitis is a common skin disease with a high incidence mainly in infancy and childhood. The prevalence of atopic dermatitis has been reported from 3% to 15%, and since World War II, extensive epidemiological investigations have been conducted and many studies have been conducted in Korea. Atopic dermatitis is a chronic recurrent dermatitis, and it appears to have a genetic predisposition with severe pruritus, characteristic shape and distribution of skin lesions, and a personal or familial history of atopic dermatitis. Recently, atopic dermatitis is on the rise due to the influence of food additives or environmental pollution, and although the exact etiology is not known, it is assumed that genetic, environmental and immunological factors are complicatedly related to the onset.

In atopic dermatitis, a decrease in peripheral blood CD8 ⁺ T lymphocytes is observed, resulting in an increase in the CD4:CD8 ratio. Although this phenomenon does not induce a decrease in systemic cell-mediated immunity, it causes a decrease in skin immunity, which causes frequent viral or bacterial infections in patients with atopic dermatitis, and a decrease in response to contact allergens. In addition, the number of Th1 involved in normal cell-mediated immunity by secreting INF-γ and TNF-β among CD4 ⁺ T lymphocytes is decreased, and the production of IgE that induces an allergic response by secreting IL-4 is increased and allergic reaction The number of Th2 directly involved in Th2 increases.

On the other hand, 80% of patients with atopic dermatitis have higher serum IgE than normal people, and the more severe the symptoms, the higher the level tends to be. It is still unclear what causes it. In addition, damage to the epidermal barrier is observed in atopic patients. It is presumed that the lack of ceramide, a lipid component between keratinocytes in the epidermal stratum corneum, increases percutaneous moisture loss in atopic dermatitis and causes dryness.

Currently, atopy-related products on the market for the purpose of preventing and/or treating atopy are made from steroids or plant extracts. However, the steroid system is a situation that consumers avoid due to side effects and tolerance, and most atopy-related products use plant extracts as raw materials, but their efficacy does not satisfy atopic patients. Therefore, despite the fact that there are many known literatures and prototypes for the prevention, improvement, and treatment of atopic dermatitis, the development of new substances and formulations that are highly effective against atopic dermatitis and have no toxicity is required.

Republic of Korea Patent Publication No. 10-2014-0017761 (published on Feb. 12, 2014)

An object of the present invention is to provide a cosmetic composition, a health food and a pharmaceutical composition for improving or treating inflammatory skin diseases such as atopic dermatitis by containing a mixture consisting of Sancho hydrosol and Cordyceps mycelium extract as an active ingredient.

The present invention provides a cosmetic composition for preventing or improving inflammatory skin diseases, comprising Sancho hydrosol and Cordyceps mycelium extract as active ingredients.

The present invention provides a health food for preventing or improving inflammatory skin disease, comprising Sancho hydrosol and Cordyceps mycelium extract as active ingredients.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory skin diseases containing the extract of Sancho hydrosol and Cordyceps mycelium as active ingredients.

According to the present invention, it was confirmed that sancho hydrosol extracted from sancho fruit reduced the expression of SNARE protein, which is an inflammation mediating protein, and inhibited the activity of reactive oxygen species, NO and β-hexasosaminidase that cause inflammation. , As it was confirmed that the composition in which Sancho hydrosol and Cordyceps mycelium extract are mixed improves the stability of Sancho hydrosol and exhibits an excellent inhibitory effect on β-hexasosaminidase activity, the mixture prevents inflammatory skin diseases including atopic dermatitis It may be provided as a cosmetic composition, health food, and pharmaceutical composition for improving or improving.

1 is a schematic diagram showing a manufacturing step of a cosmetic composition for inhibiting inflammation.
FIG. 2 is a schematic diagram showing a process of extracting hydrosol from sancho fruit using a steam distillation apparatus.
3 is a result of confirming the GC-MS chromatogram and main compounds of Sancho hydrosol.
4 is a result of confirming the SNARE protein expression level according to the concentration of Sancho hydrosol (25, 37.5, 50, 75 and 100 ppm).
5 is a result confirming the inhibitory activity of β -hexosaminidase according to Sancho hydrosol treatment concentrations (25, 50 and 100 ppm). +++: p<0.001 compared DNP-IgE; *: p<0.05 compared DNP-BSA; **: p<0.01 compared DNP-BSA; ***: p<0.001 compared DNP-BSA.
6 is a result confirming the active oxygen species (ROS) inhibitory effect of Sancho hydrosol (25, 50 and 100 ppm).
7 is a result confirming the NO production inhibitory effect induced by LPS of Sancho hydrosol (25, 50 and 100 ppm).
8 is a result confirming the inhibitory activity of β-hexasosaminidase of a mixture consisting of an ethanol extract of Cordyceps hydrosol and Cordyceps mycelium. +++: p<0.001 compared DNP-IgE; *: p<0.05 compared DNP-BSA; **: p<0.01 compared DNP-BSA; ***: p<0.001 compared DNP-BSA.
9 is a result confirming the effect of sancho oil and sancho hydrosol on the SNARE protein expression level.
10 is a result confirming the inhibitory activity of β-hexosaminidase ( β -hexosaminidase) of sancho oil and sancho hydrosol. +++: p<0.001 compared DNP-IgE; *: p<0.05 compared DNP-BSA; **: p<0.01 compared DNP-BSA; ***: p<0.001 compared DNP-BSA.

Hereinafter, the present invention will be described in more detail.

The present invention can provide a cosmetic composition for preventing or improving inflammatory skin disease containing a mixture consisting of Sancho hydrosol and Cordyceps mycelium extract as an active ingredient.

The Sancho hydrosol may be a water-soluble hydrosol obtained by condensing the steam produced by steam distillation of the Sancho fruit.

In the present invention, Sancho hydrosol is obtained by steam distillation, which is a method of extracting hydrophobic oil and hydrophilic hydrosol from a raw material using steam of water. When water vapor is applied from the part, the cell wall of the raw material is destroyed, and oil and water vapor are collected between the cell walls.

In more detail, the step of generating steam from 750 mL of boiling water at the bottom of the container by putting 250 g of Sancho fruit in a distillation container using a distillation apparatus (see FIG. 2) for the hydrosol of Sancho; It may be extracted by condensing the vapor to obtain a water-soluble hydrosol, but is not limited thereto.

The cordyceps mycelium extract may be extracted with water, C1 to C4 alcohol or a mixed solvent thereof, and more preferably, an aqueous ethanol solution, but is not limited thereto.

In more detail, the Cordyceps mycelium extract comprises the steps of culturing the Cordyceps mycelium in a liquid medium; obtaining mycelium after centrifuging the cultured Cordyceps mycelium culture solution; and extracting the obtained Cordyceps mycelium by mixing with 98% ethanol and then sonicating at 60° C. for 1 hour; may be extracted, but this is not the case.

The inflammatory skin disease may be selected from the group consisting of atopic dermatitis, psoriasis, contact dermatitis and seborrheic dermatitis.

The Sancho hydrosol may inhibit the expression of inflammation-mediated proteins SNAP23, Syntaxin4, VAMP7 and VAMP8.

The cosmetic composition may be mixed with 80 to 90% by weight of Sancho hydrosol and 10 to 20% by weight of the Cordyceps mycelium extract.

The cosmetic composition may be comprised of 5 to 10 parts by weight of Sancho hydrosol and Cordyceps mycelium extract based on 100 parts by weight of the total composition.

The cosmetic composition may be in any one formulation selected from the group consisting of a solution, a softening lotion, a nutritional lotion, a nourishing cream, a moisturizing cream, a nourishing essence, a gel, and a lotion.

The cosmetic composition may include conventional adjuvants such as stabilizers, solubilizers, vitamins, pigments and fragrances, and a carrier in addition to the active ingredient, stone maple extract.

The cosmetic composition may be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, oil, powder foundation, emulsion foundation, It may be formulated as a wax foundation or a spray, but is not limited thereto. More specifically, it may be prepared in the form of a sun cream, a softening lotion, astringent lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a pack, a spray, or a powder.

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

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

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

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

The present invention can provide a health food for preventing or improving inflammatory skin disease containing Sancho hydrosol and Cordyceps mycelium extract as active ingredients.

The health food is used together with other foods or food additives in addition to the Sancho hydrosol and Cordyceps mycelium extract, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined according to the purpose of its use, for example, prophylactic, health or therapeutic treatment.

The effective dose of the compound contained in the health food may be used according to the effective dose of the therapeutic agent, but in the case of long-term intake for health and hygiene or health control, it may be less than or equal to the above range, It is clear that the ingredient can be used in an amount beyond the above range because there is no problem in terms of safety.

The type of health food is not particularly limited, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes.

In addition, the present invention can provide a pharmaceutical composition for preventing or treating inflammatory skin disease containing Sancho hydrosol and Cordyceps mycelium extract as active ingredients.

The pharmaceutical composition may be in any one formulation selected from the group consisting of oral administration agents, sprays, gels, ointments, creams, and external preparations.

In another embodiment of the present invention, the pharmaceutical composition for preventing or treating inflammatory skin disease containing a mixture consisting of Sancho hydrosol and Cordyceps mycelium extract as an active ingredient is an appropriate carrier, excipient, disintegrant commonly used in the manufacture of a pharmaceutical composition. , a sweetener, a coating agent, a swelling agent, a lubricant, a flavoring agent, an antioxidant, a buffer, a bacteriostatic agent, a diluent, a dispersant, a surfactant, one or more additives selected from the group consisting of a binder and a lubricant may be further included.

Specifically, carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, and capsules. agent, and the like, and the solid preparation may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like in the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, solutions, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included in addition to commonly used simple diluents such as water and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base material for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

According to an embodiment of the present invention, the pharmaceutical composition is administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered to the subject.

A preferred dosage of the mixture consisting of the Sancho hydrosol and Cordyceps mycelium extract may vary depending on the condition and weight of the subject, the type and extent of the disease, the drug form, the route and duration of administration, and may be appropriately selected by those skilled in the art. According to an embodiment of the present invention, although not limited thereto, the daily dose may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or may be administered in several divided doses, thereby not limiting the scope of the present invention.

In the present invention, the 'subject' may be a mammal including a human, but is not limited to these examples.

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Example 1> Sancho hydrosol extract preparation and activity confirmation

1-1. Sancho Hydrosol Extraction

The hydrosol was extracted from the sancho fruit in the same manner as in FIG. 2 by steam distillation using a steam distillation apparatus.

First, 250 g of ginseng fruit was put into a distillation container, and 750 mL of distilled water was added to generate steam. Then, the water vapor containing essential oil was condensed as shown in FIG. 2 through a water-cooled condenser, and the obtained hydrosol was stored at -4 ℃. It was refrigerated and used in the study.

Steam distillation is a method for obtaining oil or hydrosol. It is a method of extracting hydrophobic oil and hydrophilic hydrosol from a raw material using water vapor. Oil and water vapor collected from steam distillation are passed through a pipe into a cooling tank. During the passage, the essence is classified as an oil, and the water vapor is classified as a hydrosol.

By the steam distillation method, sancho hydrosol was produced in an amount of 16 to 23% compared to the raw material, and a small amount of oil was produced in an amount of 0.3 to 1.3%.

1-2. Identification of volatile components of hydrosol

The components of the Sancho hydrosol obtained in the previous process were analyzed under the analysis conditions shown in Table 1 using Gas Chromatography and Mass Spectrometry (GC-MS) equipped with a head space.

Apparatus Item Condition Headspace Instrument Agilent 7694 Vial oven temp. 95 ℃ Loop Temp. 98 ℃ transfer line temp. 100 ℃ GC Instrument Agilent 7890A Column HP-5MS (Length 30, diam 0.250, film 0.25) carrier gas He 1.57 mL/min Split splitless MS Ion source EI, 70 eV Ms source 230 ℃ Scan range 50-550 m/s Library w9N11.L SPME SUPELCO SPME Fiber GRAY (DVB/CAR/PDMS)

As a result of hydrosol GC-MS analysis as shown in Table 2, a total of 77 volatile components were contained, and as the main components, linalool, camphor, estragole, R (+ )-limonene and elemol were identified. Among them, estragol showed the highest content with about 33%, and camphor showed the second highest content with about 21%.

Peak RT Area Library/ID Peak RT Area Library/ID One 2.55 0.01 Octyl acetate 52 14.42 0.09 Carvenone 2 2.69 0.02 2-Propen-1-amine 53 14.55 0.03 1,2-Di(ethylidene)cyclopentane 3 2.81 0.03 1-Butanol 54 14.75 0.04 Anethole 4 2.87 0.02 1-Pentene 55 14.82 0.03 Methyl 6-oxabicyclo 5 2.90 0.02 2-Butanone 56 14.89 0.03 (+)-Isopiperitenone 6 2.95 0.01 3-Methyl acetate 57 15.35 0.92 Bicyclo[2.2.1]heptan-2-ol 7 4.27 0.01 3,4-Dihydropyran 58 15.72 0.13 2-Carene 8 4.29 0.01 3-Methyl-2-butenal 59 15.79 0.02 3-Carene 9 4.32 0.01 3,4-Dihydro-2H-pyran 60 15.97 0.06 p-Diisopropylbenzene 10 4.48 0.27 Hexanal 61 16.03 0.04 3,5-Xylidine 11 5.19 0.11 2-Hexenal 62 16.68 0.10 Cyclohexasiloxane 12 5.76 0.01 p-Toluic acid 63 17.31 0.01 1H-Naphthalen-2-one 13 5.80 0.02 Heptanal 64 17.37 0.01 Mentha-1,4,8-triene 14 6.21 0.01 Cyclohexanol 65 17.59 3.75 R(+)-Limonen 15 6.30 0.01 Pyridine 66 18.40 0.01 7-Amino-5,6,7,8-tetrahydronaphthalen 16 6.69 0.03 2-Heptenal 67 18.55 0.01 α-ylangene 17 6.84 0.03 Benzaldehyde 68 18.90 0.01 Salvial-4(14)-en-1-one 18 7.05 0.03 Amyl vinyl carbinol 69 19.11 0.05 β -elemene 19 7.19 0.04 6-Methyl-5-hepten-2-one 70 19.37 0.01 α-Longipinene 20 7.29 0.95 Cyclotetrasiloxane 71 19.46 0.01 Methyleugenol 21 7.48 0.07 2-Cyclohepten-1-ol 72 19.56 0.01 1,6-Dimethyltricyclo[3.2.1]octane 22 7.64 0.26 3-Fluoro-o-xylene 73 19.91 0.03 Cedrene 23 8.00 0.06 1-Hexanol 74 20.01 0.01 1-Cyclobutylethanol 24 8.13 0.07 DL-Limonene 75 20.15 0.10 Caryophyllene 25 8.22 0.04 2,5-Dihydroanisole 76 20.36 0.01 α-Ionone 26 8.30 0.09 3,4,5-Trimethylpyrazole 77 20.46 0.01 5,6-Decadien-3-yne 27 8.66 0.03 2-Dodecenal 78 20.57 0.14 Thujopsene 28 8.79 0.02 1H-pyrazole 79 20.75 0.01 Isoterpinolene 29 8.93 0.15 Cyclooctane 80 21.09 0.02 (+)-epi-Bicyclosesquiphellandrene 30 9.07 0.08 Pinol 81 21.37 0.05 1,4,7,-Cycloundecatriene 31 9.15 0.02 3,5-Hexadien-2-ol 82 21.72 0.09 β -Cubebene 32 9.28 0.02 1-Vinyl-2-cyclohexen-1-ol 83 21.83 0.01 4,7-Methanoazulene 33 9.51 0.34 4-Ethyl-resorcinol 84 22.17 0.01 γ-Muurolene 34 9.70 1.10 Linalool 85 22.25 0.01 Spiro[5.5]undec-2-ene 35 9.92 0.02 9-Dodecyn-1-ol 86 22.37 0.02 Germacrene 36 9.94 0.01 2,3-Dimethyl-2-cyclopenten-1-one 87 22.47 0.01 2-Cyclohexene-1-butanal 37 9.96 0.01 Cyclohept-4-enone 88 22.56 0.01 α-Guaiene 38 10.07 0.71 succinic acid 89 22.72 0.01 2H-Isoindole 39 10.20 0.04 D-fenchyl alcohol 90 22.87 0.03 1,4-Dimethyladamantane 40 10.32 0.06 2-Butanone 91 22.98 0.12 1H-Cyclopropazulene 41 10.59 0.11 2-Methylenebornane 92 23.06 0.03 β -Guaiene 42 10.69 0.01 α-Terpinene 93 23.27 0.10 methyl benzene 43 10.71 0.07 Cyclopentane 94 23.38 0.04 2,4-Di-tert-butylphenol 44 11.19 30.68 Camphor 95 23.55 0.04 α-amorphene 45 11.37 0.33 Isoborneol 96 23.86 0.24 4-Isopropyl-1,6-dimethyldecahydronaphthalene 46 11.42 0.22 5-Tert-butyl-3-hydroxy-3H-furan-2-one 97 24.39 0.01 γ-cadinene 47 11.55 0.69 1,3-Cyclopentadiene 98 24.46 0.01 Isolongifolene 48 11.62 0.76 endo-(-)-Borneol 99 24.60 0.02 α-calacorene 49 11.77 0.06 Bicyclo[2.2.1]heptan-2-one 100 24.85 1.89 Elemol 50 11.89 0.12 Bicyclo[3.1.1]heptan-3-one 101 25.12 0.01 Germacrene B 51 12.76 50.86 Estragole 102 25.20 0.01 Camphene

1-3. Confirmation of SNARE protein (SNAP23, Syntaxin4, VAMP7 and VAMP8) expression change in RBL-2H3 mast cells by hydrosol

RBL-2H3 mast cells were purchased from ATCC (Washington, DC; ATCC number: CRL-2256; Fig. 3), and DMEM (Dulbecco's modified Eagle's) supplemented with glucose, 10% thermally inactivated fetal bovine serum and antibiotics (Dulbecco's modified Eagle's) medium; Merck KGaA, Darmstadt, Germany) at 37 ° C., 5% CO ₂ condition, and incubated in a CO ₂ incubator (BB15, Thermo Scientific HERAEUS® BB 15).

Hydrosol was dissolved in DMSO (Dimethyl sulfoxide; Merck KGaA, Darmstadt, Germany) and treated at 25, 37.5, 50, 75 and 100 ppm at 0.5 ^-1 × 10 ⁶ mast cells cultured 6-well.

Hydrosol-treated total 0.5 ^-1 × 10 ⁶ cells were treated with 1 mL of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1 mL of a protease inhibitor (Calbiochem, Germany)) mM EDTA, pH 7.5), and the lysate was centrifuged at 13,000 rpm, 4° C. to remove cell debris. Thereafter, 100 μL of protein G Sepharose (GE healthcare) was treated at 4° C. for 2 hours.

After that, the antibody against the SNARE protein (SNAP-23, Syntaxin 4, VAMP2, and VAMP8) was added to the lysate and bound at 4° C. for 2 hours, and the antibody-antigen complex was incubated overnight after the protein G suspension was added. , after washing with lysis buffer, immunoblot analysis of the precipitate was performed.

Immunoprecipitated proteins and cell lysates were separated on a 12% SDS-PAGE gel, transferred to a nitrocellulose membrane (Whatman, Germany), and TBST (10 mM Tris-HCl, 10 mM Tris-HCl; The blot was probed with the primary antibody of SNAP-23 overnight by blocking with 150 mM NaCl, 0.05% Tween-20, pH 8.0) at room temperature for 1 hour.

After washing with TBST, the membrane was treated with a secondary anti-mouse IgG antibody (A4416, Sigma) mixed with horseradish peroxidase, and the band was detected with an enhanced chemiluminescence (ECL) solution to detect the hydrosol SNARE protein (SNAP23, Syntaxin4, VAMP7 and VAMP8) expression changes in RBL-2H3 mast cells by

As a result, it was confirmed that the expression of SNAP23, Syntaxin4, VAMP7 and VAMP8 is concentration-dependently suppressed as shown in FIG. In particular, the expression of VAMP7 of the protein was inhibited in the hydrosol treatment of 37.5 ppm or more, and VAMP8 was confirmed to be suppressed in protein expression when the hydrosol treatment of 75 ppm or more.

From the above results, it was confirmed that Sancho hydrosol was effective in suppressing the expression of VAMP7 and VAMP8, which are inflammatory mediating proteins, and hydrosol capable of inhibiting all of NAP23, Syntaxin4, VAMP7 and VAMP8 was suitable as a raw material for a cosmetic composition for improving inflammation. Confirmed.

1-4. Inhibitory efficacy of β-hexosaminidase according to hydrosol treatment concentration

To determine the effect of hydrosol-induced mast cell SNARE protein expression inhibition on mast cell degranulation, β-induced by anti-DNP IgE (200 ng/mL) and DNP-BSA (10 ng/mL) Degranulation of RBL-2H3 mast cells was measured by confirming the secretion of hexosaminidase. In addition, the concentration of hydrosol inhibiting the secretion of β-hexosaminidase in mast cells induced by stimulants (anti-DNP IgE + DNP-BSA) was confirmed.

β-hexosaminidase is an enzyme known as an indicator for mast cell degranulation and histamine release. Inhibition of β-hexosaminidase reduces mast cell degranulation and inflammation caused by histamine release.

First, RBL-2H3 cells were cultured by dispensing 2 × 10 ⁵ cells/well in 500 μL of culture solution in a 24-well plate. Cells were washed with HEPES-buffered saline (140 mM NaCl, 5 mM KCl, 1 mM CaCl ₂ , 0.6 mM MgCl ₂ , 0.1% glucose, 0.1% BSA and 10 mM HEPES) and anti-dinitrophenyl (DNP) )-IgE (100 ng/mL, Sigma) was incubated for 3 hours, followed by incubation with or without hydrosol (25 ppm, 50 ppm and 100 ppm) for 30 minutes.

After incubation, cells were stimulated with DNP-group (200 ng/mL, DNP-BSA) conjugated with bovine serum albumin (BSA) for antigen-induced degranulation. Degranulation was quantified by measuring β-hexosaminidase in the supernatant through a colorimetric biochemical assay in HEPES-buffered saline at 37° C. for 30 minutes.

The supernatant was transferred to a 96-well plate (50 μL/well) and incubated in 50 μL substrate solution at 37° C. for 3 hours. After stopping the reaction with 100 μL of NaCO ₃ -NaHCO ₃ buffer (1:1 mixture, 100 mM NaCO ₃ -NaHCO ₃ , pH 10.0), absorbance was measured at 405 nm using a micro plate reader. Secretory activity was calculated as the relative total β-hexosaminidase content in the cells. The total β-hexosaminidase content was determined by cells lysed with 0.1% Triton X-100, and the percentage of β-hexosaminidase in the supernatant was calculated using the following formula.

[formula]

β-hexosaminidase secretion (%) = ((T - B - N)/(C - N)) × 100

T (Test): DNP-BSA (+), sample (+)

B (Blank): DNP-BSA (-), sample (+)

C (Control): DNP-BSA (+), sample (-)

N (Normal): DNP-BSA (-), sample (-)

As a result, as shown in FIG. 5 , when DNP-BSA was added to DNP-IgE of mast cells, β-hexosaminidase was rapidly increased, and hydrosol decreased β-hexosaminidase in a concentration-dependent manner. In particular, it was confirmed that the significance level of p <0.001 when treated with 100 ppm.

1-5. Confirmation of the effect of hydrosol on the generation of reactive oxygen species (ROS) in skin keratinocytes

When ROS (reactive _oxigen species) is abnormally and excessively produced, skin diseases such as inflammation and aging are known to be induced by oxidative stress _. The effect of hydrosol on ROS was confirmed by exposure.

DCF-DA (2′7′-dichloro-fluorescein diacetate) that emits fluorescence when reacted with active oxygen for the effect of hydrosol on NO (nitrite scavenging) induced by lipopolysaccharide (LPS) in RAW 264.7 (Sigma, St. Louis, MO, USA) was used.

First, after culturing RAW 264.7 cells in a 96-well plate at a concentration of 5×10 ⁴ cells/well, hydrosol was treated at 25 to 100 ppm for 4 hours. Thereafter, LPS was treated at a concentration of 1 μg/mL and cultured for 24 hours, then the medium was removed and DCF-DA was added to PBS. After reacting at 37°C for 30 minutes, active oxygen was identified using a fluorescence microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

As a result, as shown in FIG. 6 , ROS in RAW 264.7 cells treated with LPS increased by 150% or more, whereas in RAW 264.7 cells treated with 25 ppm, 50 ppm and 100 ppm hydrosol, ROS was 1.9 times, 2.2 times and 2.6 times, respectively. It was confirmed that the fold was reduced, and it was confirmed that Bayll-7080 (Sigma-Aldrich, Germany) used as a positive control appeared at a level similar to the ROS reduction effect of RAW 264.7 cells treated.

ROS induces oxidative stress when present in high concentrations in the body, promotes DNA mutation and aging, and secretes various proinflammatory cytokines, chemokines, and eicosanoid metabolites promotes the continuation and exacerbation of the inflammatory response. This increase in ROS means that the skin is subjected to oxidative stress, and oxidation can cause damage to the epidermal barrier and loss of moisture, which can exacerbate skin inflammation such as atopic dermatitis.

From the above results, it was confirmed that hydrosol, which showed a reducing effect on ROS, could be used as a therapeutic agent for improving inflammation.

1-6. Confirmation of the effect of hydrosol on the inhibitory effect of NO production induced by LPS

RAW 264.7 cells, a mouse macrophage cell line, were received from the Korea Cell Line Bank (KCLB) and cultured. For cell culture, DMEM (Dulbecco's Modified Eagle Medium) medium containing 10% FBS and 1% penicillin-streptomycin was used, _{and a CO 2 incubator} (BB15, Thermo Scientific HERAEUS® BB 15).

RAW 264.7 cells were treated with hydrosol (25 ppm, 50 ppm and 100 ppm) and after 1 hour, treated with 100 ng/mL LPS and cultured for 18 hours, and the same amount of Griess Reagent (Merck KGaA, Darmstadt) as 50 μl of the culture solution. , Germany) was added, reacted at room temperature for 10 minutes, absorbance was measured at 540 nm with an ELISA reader, and the NO concentration in the culture was confirmed using a standard curve for each concentration of sodium nitrite.

As a result, as shown in Figure 7, NO increased by LPS in RAW 264.7 cells significantly reduced NO in all concentrations of 25 to 100 ppm hydrosol treatment, in particular, cells treated with 100 ppm hydrosol were treated as a positive control. It was confirmed that the used Bayll-7080 (place of purchase) had a similar level of NO inhibitory effect to the treated cells (1.6-fold inhibition), but no significance was confirmed.

Therefore, it was confirmed that the hydrosol is most suitable to be treated at 50 ppm for ROS and NO reduction.

1-7. Confirmation of anti-inflammatory effect of sancho oil and sancho hydrosol

In order to compare the anti-inflammatory effects of sancho oil and sancho hydrosol, the effects of VAMP7 and VAMP8 expression, which are representative R-SNARE proteins, which exist in cell membranes and are involved in mediators that transport inflammatory substances, were evaluated in the experiments of 1-3. method was confirmed.

As a result, as shown in FIG. 9 , it was confirmed that sancho hydrosol showed a definite inhibition of VAMP7 and VAMP8, whereas it was confirmed that sancho oil was only involved in the inhibition of VAMP7.

In addition, as a result of confirming the effect of Sancho oil and Sancho hydrosol on β-hexosaminidase in the same manner as in 1-4, β rapidly increased in mast cells by DNP-IgE and DNP-BSA treatment as shown in FIG. 10 . -Hexosaminidase was decreased in a concentration-dependent manner in Sancho hydrosol-treated cells, particularly when it was treated with 100 ppm, it was confirmed that it showed a significance level of p<0.001, whereas in Sancho oil, very low β-hexosaminidase The effect of reducing the enzyme was confirmed.

From the above results, it was confirmed that the anti-inflammatory effect of sancho oil and sancho hydrosol was different, and it was confirmed that the anti-inflammatory effect of sancho hydrosol was superior to that of sancho oil.

<Example 2> Confirmation of hydrosol stability

1. Identification of mixtures to improve stability

In order to increase the stability of the hydrosol, tocopherol (tocopherol), ascorbic acid (ascorbic acid), heat-treated wood flour hot water / ethanol extract or mushroom mycelium hot water / ethanol extract was mixed at 5 to 20% to confirm the stability.

First, heat-treated wood flour was prepared by manufacturing oak ( Quercus variabilis Blume ) into chips, heat-treating it at 220 ° C. for 1 minute, and classifying it with 20 mesh pass 80 mesh on, and Cordyceps militaris ) was prepared by PDB (Potato Dextrose Broth). ) cultured for 7 days at 100 rpm at 24 ° C. using a medium, and then centrifuged to obtain mycelium.

The hot water extraction of the heat-treated oak wood flour and cordyceps mycelium was obtained by weighing 1 g of the sample on a dry weight basis, mixing it with 20 mL of distilled water, and performing ultrasonication at 60° C. for 30 minutes.

In addition, the ethanol extraction of heat-treated oak wood flour and Cordyceps mycelium was extracted using 98% ethanol, and the sample was weighed 1 g on a dry weight basis, mixed with 20 mL of ethanol, and sonicated at 60 ° C. for 30 minutes. As a control, it was obtained. Tocopherol (Merck KGaA, Darmstadt, Germany) and ascorbic acid (Merck KGaA, Darmstadt, Germany) used were dissolved in ethanol and used.

The stability measurement was compared with the oil stability index (OSI) using an electrical conductivity meter according to the AOCS analysis method (Cd 12b-92). After sampling a certain amount of sample (2.5±0.2 g), the temperature was maintained at 100 °C to check the OSI values as shown in Table 3.

The OSI measurement principle is that when polar volatile substances are volatilized by oxidation of fat and collected in distilled water, the conductivity increases. This is detected through the electrode connected to the distilled water bottle. The time up to this point is expressed as OSI (time), and the longer the time derived from the result, the higher the stability.

As a result, as shown in Table 3, when the ethanol extract of the Cordyceps mycelium and the hydrosol were mixed, it was confirmed that the stability was maintained for the longest time. In particular, it was confirmed that the ethanol extract of Cordyceps mycelium exhibited higher stability than tocopherol and ascorbic acid, which are generally used to maintain stability.

Samples ppm Electrical conductivity (μs/cm)/hour result
(hours) 0 12 24 36 48 60 72 84 Tocopherol (Control 1)

200 363 355 312 50 51 55 49 50 36 400 363 357 351 333 50 51 55 49 48 600 363 394 344 315 49 59 55 44 48 Ascorbic acid (control 2)

200 363 355 312 50 51 55 49 50 36 400 363 357 351 362 50 51 55 41 48 600 363 357 346 329 45 54 55 50 48 Steam exploded wood chip hot water extract (Control 3)

200 363 345 50 51 50 52 57 57 24 400 363 357 48 44 61 62 50 59 24 600 363 351 61 52 49 50 50 50 24 Steam exploded wood chip ethanol extract (control 4)

200 363 345 50 51 50 52 57 57 24 400 363 388 59 44 61 52 53 59 24 600 363 372 65 50 49 51 51 48 24 Cordyceps hot water extract (Control 5)

200 363 357 351 333 50 51 51 49 48 400 363 394 344 315 49 59 44 41 48 600 363 394 344 300 312 59 55 49 60 Cordyceps ethanol extract 200 363 394 344 315 332 59 55 44 60 400 363 313 319 335 329 319 49 52 72 600 363 313 319 335 329 319 49 52 72

2. Check the mixing ratio to improve stability

To improve the stability of the hydrosol, the optimal mixing ratio of the ethanol extract of Cordyceps mycelium and the hydrosol confirmed from the above results was confirmed.

The hydrosol and the ethanol extract of the Cordyceps mycelium were mixed at 60:40, 70:30, 80:20 and 90:10 by mass to confirm stability (OSI, time). The stability was measured using an electrical conductivity meter according to the AOCS analysis method (Cd 12b-92), and the electrical conductivity was measured at 12 hour intervals for a total of 84 hours, and the oil stability index (OSI) was compared.

As a result, as shown in Table 4, it was confirmed that mixing the hydrosol and the Cordyceps mycelium ethanol extract at 80:20 (w:w) and 90:10 (w:w) showed the highest stability, especially hydrosol and Cordyceps cordyceps. The condition of mixing the mycelium ethanol extract at 90:10 (w:w) showed high stability for 72 hours, and since a relatively high amount of sancho oil was mixed, it would be suggested to be the most suitable raw material for product development for alleviating atopic dermatitis. can

Samples
Electrical conductivity (μs/cm)/hour result
(hours) 0 12 24 36 48 60 72 84 80:20 ^One) 363 313 325 342 312 319 48 52 72 90:10 361 352 311 346 322 315 49 52 72 95:5 355 364 354 344 322 49 51 42 48 100:0 349 342 361 352 312 59 44 40 48

¹⁾ Hydrosol: Cordyceps mycelium ethanol extract (w:w)

3. Confirmation of β-hexosaminidase inhibitory effect of hydrosol and ethanol extract of Cordyceps mycelium

By the method described in Example 1, the β-hexosaminidase inhibitory effect of the mixture using hydrosol and ethanol extract of Cordyceps mycelium as raw materials was confirmed.

As a result, as shown in FIG. 8, the mixed formulation showed an inhibitory effect on β-hexosaminidase with a significance of p<0.01. In particular, it was confirmed that the mixed formulation using hydrosol and ethanol extract of Cordyceps mycelium as raw materials reduced β-hexosaminidase expressed by DNP-IgE and DNP-BSA by more than 40%.

From the above results, it was confirmed that a formulation in which hydrosol and an ethanol extract of Cordyceps mycelium were mixed was suitable as a raw material for atopic dermatitis alleviation formulation.

<Example 3> Cosmetic composition using a mixture of hydrosol and ethanol extract of Cordyceps mycelium as a raw material

As described above, a cosmetic composition was prepared using a mixed formulation of hydrosol, which was confirmed to have stability and anti-inflammatory effect, and an ethanol extract of Cordyceps mycelium.

As shown in Table 5, green tea water, yeast fermentation filtrate, butylene glycol, niacinamide, glycerin, PEG-60 hydrogenated castor oil, panthenol, tocopheryl acetate, disodium EDTA, 1, It was prepared by mixing 2-hexanediol, and the mixture of Sancho hydrosol and Cordyceps mycelium ethanol extract was added in an amount of 5 to 10 parts by weight based on 100 parts by weight of the base formulation and homogenized.

No. composition Content (parts by weight) One green tea water 53-55 2 Yeast fermentation filtrate 27-30 3 Butylene Glycol 2 4 niacinamide One 5 glycerin 2 6 PEG-60 Hydrogenated Castor Oil One 7 panthenol One 8 tocopheryl acetate One 9 Disodium EDIT One 10 1,2-Hexanediol One 11 Sancho Hydrosol 4.5-9 12 Cordyceps mycelium ethanol extract 0.5-1

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (11)

A cosmetic composition for preventing or improving inflammatory skin disease, comprising 80 to 90 parts by weight of Sancho hydrosol and 10 to 20 parts by weight of Cordyceps mycelium extract as active ingredients. The cosmetic composition for preventing or improving inflammatory skin disease according to claim 1, wherein the Sancho hydrosol is a water-soluble hydrosol obtained by condensing the steam produced by steam distillation of Sancho fruit. The cosmetic composition for preventing or improving inflammatory skin disease according to claim 1, wherein the Cordyceps mycelium extract is extracted with water, C1 to C4 alcohol, or a mixed solvent thereof. The cosmetic composition for preventing or improving inflammatory skin disease according to claim 1, wherein the inflammatory skin disease is selected from the group consisting of atopic dermatitis, psoriasis, contact dermatitis and seborrheic dermatitis. The cosmetic composition for preventing or improving inflammatory skin disease according to claim 1, wherein the Sancho hydrosol inhibits the expression of SNAP23, Syntaxin4, VAMP7 and VAMP8, which are inflammatory mediating proteins. delete The cosmetic composition for preventing or improving inflammatory skin disease according to claim 1, wherein the cosmetic composition contains 5 to 10 parts by weight of Sancho hydrosol and Cordyceps mycelium extract based on 100 parts by weight of the total composition. The method according to claim 1, wherein the cosmetic composition is for preventing or improving inflammatory skin disease, characterized in that any one formulation selected from the group consisting of a solution, a flexible lotion, a nutritional lotion, a nutritional cream, a moisturizing cream, a nutritional essence, a gel, and a lotion cosmetic composition. A health food for preventing or improving inflammatory skin disease containing 80 to 90 parts by weight of Sancho hydrosol and 10 to 20 parts by weight of Cordyceps mycelium extract as active ingredients. A pharmaceutical composition for preventing or treating inflammatory skin diseases, comprising 80 to 90 parts by weight of Sancho hydrosol and 10 to 20 parts by weight of Cordyceps mycelium extract as active ingredients. The pharmaceutical composition for preventing or treating inflammatory skin diseases according to claim 10, wherein the pharmaceutical composition is any one selected from the group consisting of oral administration agents, sprays, gels, ointments, creams, and external preparations.

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