KR101698410B1 - Composition for improving skin conditions and preventing or treating proliferative skin diseases containing fermented extracts of sedum as active ingredient - Google Patents

Abstract

The present invention relates to a cosmetic composition for improving skin conditions comprising fermented extracts of Sedum kamtschaticum as active ingredients, a pharmaceutical composition for preventing or treating proliferative skin diseases, and a food additive or a health food for preventing proliferative skin diseases and improving skin conditions.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a composition for improving skin and a composition for preventing or treating a proliferative skin disease, which comprises a fermented extract of Kirinchos, as an active ingredient, and a composition for prevention or treatment of a proliferative skin disease.

The present invention relates to a cosmetic composition for skin improvement and a pharmaceutical composition for preventing or treating a proliferative skin disease, which comprises an extract of Kirinchosho fermented extract as an active ingredient.

In addition to rapid industrialization, the generation of strong oxidizing substances such as active oxygen species due to air pollution and the increase of UV amount due to ozone layer destruction cause skin aging and pigmentation in humans. Various antioxidants such as vitamin C, tocopherol and coenzyme Q10 have been used for the production of cosmetics for the purpose of protecting the skin from these various oxidizing substances. Recently, it has been found that various physiological active substances such as whitening, anti-aging, anti-wrinkle and antioxidant exist in various plants including herbal medicines, and thus their extracts are widely used in the production of natural cosmetics or herbal cosmetics.

An abnormality in the rate of cell proliferation within keratinocytes can sometimes lead to hyperproliferation, manifesting in many proliferative skin diseases, including various forms of psoriasis, coupled with an abnormal rate of inflammation and / or apoptosis. For psoriasis, which is a typical proliferative skin disease, there is currently no superior therapy, and only inhibition therapy is present.

Sedum kamtschaticum (Sedum kamtschaticum) is a perennial herbaceous plant belonging to the fusiforme family Rodeoblus chinensis and has five yellow petals of Bassocont. In folk medicine, it is used as a treatment for hemostasis, diuretic, sedation, swelling, bruising, pulmonary tuberculosis and heart disease, and is known to have radioprotective effect and vasodilation effect. The prior art discloses only antibacterial activity of Kirinchos extract, but does not disclose its skin improving effect.

Fermentation refers to the process by which microorganisms degrade organic matter using their own enzymes. It is known that raw materials after fermentation through microorganisms are at least two times to several tens of times more effective than before fermentation. Fermented metabolites contain various amino acids, organic acids and antioxidants that are good for the skin, promoting skin metabolism and making skin texture resilient and smooth. In addition, it has been reported that the fermentation process reduces the particle size, decomposes toxic substances such as heavy metals, so that it has good absorption rate and alleviates skin troubles and allergy side effects.

 There have been active researches on skin condition improvement and proliferative skin disease treatment, but there is still a need for a new method for these. Accordingly, the present invention is intended to apply a new natural fermented extract to the above-mentioned necessity.

Korean Patent Publication No. 10-2015-0107376 Korean Patent Registration No. 10-1330591

The present invention is to provide a cosmetic composition for skin improvement comprising a fermented Kirinchos extract as an active ingredient, a pharmaceutical composition for preventing or treating proliferative skin diseases, a prophylactic skin disease prevention, a food additive for skin improvement, or a health food.

One embodiment of the present invention provides a cosmetic composition for skin improvement comprising a fermented extract of Kirinchos as an active ingredient.

As used in the present invention, " girinchus " is a perennial herbaceous plant belonging to the fusarium waxy rhododendron family, and refers to a plant having five yellow petals of balsonic.

The " Kirinchose extract " used in the present invention can be prepared by mixing Kirinchase crushed by an extraction method known in the art into an extraction solvent and then extracting it. Wherein the extraction solvent is at least one aqueous solution selected from the group consisting of water, an anhydrous or a lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, butyl acetate, dichloromethane, chloroform, hexane and 1,3- Preferably, it may be sterilized water or 60% ethanol, and two or more different solvents may be mixed or used sequentially, but the present invention is not limited thereto.

The "Kirinchosu fermented extract" used in the present invention is prepared by drying the Kirinchos extract with the use of a device or, specifically, a rotary vacuum concentrator and a freeze drier, and then drying the Kirinchiso extract in a solvent, , Preferably 1 to 10% by weight, and then inoculating and fermenting the fermentation microorganism.

The fermentation microorganism refers to fermentation microorganisms commonly used in the fermentation related industry, and specifically includes, but is not limited to, lactic acid bacteria, enzymes, yeast, fungi, actinomycetes and the like. The fermentation microorganism is Bifidobacterium longum , Streptococcus thermophilus Or Lactobacillus fermentum is preferred.

According to one embodiment of the present invention, the fermentation extract may be contained in an amount of 2 to 10% by weight, preferably 5% by weight based on the total weight of the composition.

According to one embodiment of the present invention, the skin improvement is characterized in that the skin is selected from the group consisting of antioxidant, dermal cell growth promotion, collagen increase, collagenase reduction, immune cell activity inhibition, inflammation inhibition, skin hydration control, Improvement of skin barrier, improvement of skin texture, reduction of skin erythema and reduction of pigmentation.

Another embodiment of the present invention provides a cosmetic comprising the cosmetic composition for skin improvement.

The cosmetic may have formulations of ointments, solutions, suspensions, emulsions, pastes, gels, creams, lotions, serums, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays , Preferably a formulation of a skin toner, a water cream or a cream, but is not limited thereto.

One embodiment of the present invention provides a pharmaceutical composition for preventing or treating a proliferative skin disease containing an extract of Kirinchos fermented extract as an active ingredient.

According to one embodiment of the present invention, the fermented extract may be prepared by diluting the dried product of the extract obtained from Kirinchos by the Kirinchosu fermentation extract in a solvent, preferably purified water, inoculating the fermentation microorganism and fermenting the fermented extract.

According to one embodiment of the present invention, the fermentation extract may be contained in an amount of 2 to 10% by weight, preferably 5% by weight based on the total weight of the composition.

According to one embodiment of the present invention, the proliferative dermatosis is selected from the group consisting of actinic keratosis, basal cell carcinoma, squamous cell carcinoma, fibrous histiocytoma, elevated dermatofibrosis, angioma, flammei, A malignant melanoma, metastatic carcinoma, and psoriasis, in a mammal, including a human, a human,

The pharmaceutical composition of the present invention may be formulated into various oral or parenteral dosage forms, preferably parenteral dosage forms, more preferably ointments.

Examples of formulations for oral administration include, but are not limited to, tablets, pills, hard, soft capsules, liquids, suspensions, emulsions, syrups, granules and the like. These formulations may contain, in addition to the active ingredient, a diluent such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine, a lubricant such as silica, talc, stearic acid and its magnesium or calcium salt and / Or polyethylene glycol). The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally mixed with starch, agar, alginic acid Or a disintegrating or boiling mixture such as its sodium salt and / or an absorbent, a colorant, a flavoring agent and a sweetening agent. The formulations may be prepared by conventional mixing, granulating or coating methods.

Examples of the formulations for parenteral administration include injections, ointments, suppositories, and the like, but are not limited thereto. Depending on the characteristics of each formulation, appropriate additives such as solvents can be selected according to common knowledge of those skilled in the art.

Another embodiment of the present invention provides a pharmaceutical preparation containing the pharmaceutical composition for preventing or treating the proliferative skin disease.

Another embodiment of the present invention provides a food additive for prevention of proliferative skin disease and skin improvement comprising a fermented Kirinchos extract as an active ingredient.

Another embodiment of the present invention provides a health functional food for preventing proliferative skin diseases and improving skin comprising a fermented Kirinchos extract as an active ingredient.

According to an embodiment of the present invention, the health functional food may be, for example, but not limited to beverage, gum, tea, vitamin complex, and other health supplement foods. The health functional food may be in the form of a capsule, tablet, powder, granule, liquid, ring, flake, paste, syrup, gel, beverage, jelly or varnish.

According to an embodiment of the present invention, the Kirinchos fermented extract may contain 0.05 to 3% by weight based on the total weight of the health functional food.

According to an embodiment of the present invention, the health functional food may include a food supplementary additive suitable in view of common knowledge of those skilled in the art, for example, various nutrients, vitamins, electrolytes, flavors, colorants, , Alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like.

The present invention provides a novel use of the composition containing the fermented Kirinchos extract as an active ingredient for skin improvement or prevention and treatment of proliferative skin diseases. The fermented extract of the present invention is useful as an antioxidant, a dermis cell growth promoting agent, a collagen enhancing agent, a collagen degrading enzyme inhibitor, an immune cell activity inhibitor, an inflammation inhibitor, an inhibitor of skin irritation, Improvement of skin texture, reduction of skin erythema and reduction of pigment deposition. In addition, the fermented extract of the present invention can be used for the treatment and / or prophylaxis of diseases such as actinic keratosis, basal cell carcinoma, squamous cell carcinoma, fibrous histiocytoma, hypertrophic dermatofibroma, hemangioma, flamma, hematoma, Kaposi sarcoma, mastocytosis, And proliferative skin diseases such as dysplasia, nevus cellulata, malignant melanoma, malignant melanoma, metastatic carcinoma and psoriasis.

Fig. 1 shows the effect of the present invention on the active oxygen decomposition of the fermented Kirinchos extract, alpha -tocopherol and BHT.
FIG. 2 shows the cell activity of human dermal fibroblasts (nHDF) according to the concentration of the Kirinchos fermented extract of the present invention.
FIGS. 3 and 4 show changes in expression of COL1A1 and collagenase MMP1 in human dermal fibroblasts, respectively.
FIG. 5 shows the cell activity of mouse macrophages (RAW 264.7) according to the concentration of the Kirinchos fermented extract of the present invention.
FIG. 6 shows the activity of luciferase in immune cells according to the concentration of the fermented extract of Kirinchos.
FIG. 7 and FIG. 8 show changes in IL-1β and TNF-α gene expression according to the concentration of the fermented Kirinchos extract of the present invention, respectively.
9 to 17 are graphs showing skin moisture content, skin oil content, degree of skin wrinkle, dermal density, skin elasticity, percutaneous water loss, skin Degree of improvement of skin texture, degree of skin erythema and change of pigmented area.

Hereinafter, the present invention will be described in more detail with reference to one or more embodiments. However, these embodiments are illustrative of one or more embodiments, and the scope of the present invention is not limited to these embodiments.

1. Preparation of sample

1.1. Preparation of Kirinchosu fermented extract

The Kirinchos fermented extract was obtained using an ethanol extraction method commonly used in the art. Specifically, 100 g of crushed giraffe first was mixed with 1 L of 60% ethanol at a ratio of 10% by weight. The mixed raw materials were extracted by stirring at room temperature for 48 hours. The Kirinchos extract was filtered through 250 mesh and 0.5 mu m filter paper to obtain a liquid extract. Extraction dried material was obtained from the filtered liquid extract using a rotary vacuum concentrator and a freeze dryer. The extract was diluted to 5% by weight with purified water, and the microorganisms for fermentationBifidobacterium longumAnd fermented. The fermented extract was filtered through 500 mesh and 0.2 mu m filter paper to obtain a liquid fermentation extract. As a result of carrying out a related test example of the composition according to the present invention thus formed, it was confirmed that there is no abnormality in the skin stability.

1.2. Manufacture of skin toner

The composition of the ingredients for making a skin toner containing the composition of the present invention is shown in the following Table 1 and is prepared by the following procedure. The A phase (water phase) was completely dissolved at room temperature and the B phase (alcohol phase) was heated to 50 DEG C using a heater. The phase B was mixed with the phase A and solubilized at 1000 rpm for 10 minutes using a disperser. The mixture was cooled, filtered, and maintained at a pH of 5.2 to complete a skin toner. The Kirinchos fermented extract was prepared by adding 5% by weight of the extract.

Prize Raw material Content (% by weight) Control group Positive control group Experimental group A phase Purified water 5 - - Aloe - 5 - Kirinchu fermented extract - - 5 Disodium EDTA 0.01 0.01 0.01 1,3-butylene glycol 4 4 4 Citric acid 0.04 0.04 0.04 Sodium citrate 0.1 0.1 0.1 B phase Hyaluronic acid 2 2 2 Hydrogenated 0.4 0.4 0.4 Castor oil Suitable amount Suitable amount Suitable amount C phase Purified water Up to 100 Up to 100 Up to 100

1.3. Manufacture of moisture cream

The composition of the composition for making the water cream containing the composition of the present invention is shown in Table 2 below. Concretely, the A phase was heated to 80 DEG C and stirred, and the B phase was heated to 80 DEG C to dissolve, and then the mixture was put into the phase A and emulsified at 2000 rpm for 10 minutes. The C phase was evenly mixed, and the mixture was put on the A + B phase at 80 ° C and neutralized at 2000 rpm for about 5 minutes. The solution was cooled to 30 DEG C while stirring slowly, and adjusted to pH 6.15. The Kirinchos fermented extract was prepared by adding 5% by weight of the extract.

Phase Raw material Content (% by weight) Control group Positive control group Experimental group A phase Purified water 5 - - Aloe - 5 - Kirinchu fermented extract - - 5 Disodium EDTA 0.01 0.01 0.01 Paraoxybenzoic acid ester 0.2 0.2 0.2 Clofenine 0.2 0.2 0.2 Hyaluronic acid 5 5 5 Aloe gel 5 5 5 1% aqueous solution of xanthan gum 5 5 5 Carboxyvinyl polymer 15 15 15 B phase Squall 7 7 7 Dimethicone 3 3 3 Cyclomethicone 3 3 3 Polysorbate 60 2 2 2 Sorbitolate 0.5 0.5 0.5 Glyceryl stearate One One One Stearic acid One One One C phase Triethanolamine One One One Purified water Up to 100 Up to 100 Up to 100

1.4. Manufacture of cream

The constituents for preparing the cream containing the composition of the present invention are shown in Table 3 below. Concretely, the A phase was heated to 80 DEG C and stirred, and the B phase was heated to 80 DEG C to dissolve, and then the mixture was put into the phase A and emulsified at 2000 rpm for 10 minutes. The C phase was evenly mixed, and the mixture was put on the A + B phase at 80 ° C and neutralized at 2000 rpm for about 5 minutes. Subsequently, the mixture was gradually cooled to 30 DEG C while being mixed and stirred, and adjusted to pH 6.23. The Kirinchos fermented extract was prepared by adding 5% by weight of the extract.

Prize Raw material Content (% by weight) Control group Positive control group Experimental group A phase Purified water 5 - - Aloe - 5 - Kirinchu fermented extract - - 5 Disodium EDTA 0.01 0.01 0.01 Paraoxybenzoic acid ester 0.2 0.2 0.2 Clofenine 0.2 0.2 0.2 Hyaluronic acid 5 5 5 1% aqueous solution of xanthan gum 5 5 5 Carboxyvinyl polymer 15 15 15 B phase Squall 7 7 7 Dimethicone 3 3 3 Cyclomethicone 3 3 3 Polysorbate 60 2 2 2 Sorbitolate 0.5 0.5 0.5 Sheer butter 5 5 5 Glyceryl stearate One One One Cetostearyl alcohol One One One Stearic acid One One One C phase Purified water Up to 100 Up to 100 Up to 100

2. Experimental methods and results

Example 1. Confirmation of the effect of decomposition of active oxygen by the fermented extract of Kirinchosu

In order to investigate the effect of the extract of fermented Kirinchoshi on the skin, we investigated whether the active oxygen species, which plays a major role in skin aging, are eliminated by the Kirinchoshi fermentation extract.

A method for measuring free radical scavenging activity using 1,1-diphenyl-2-picryl hydrazyl (DPPH), a free radical, Was used. 4 mL of methanol was added to the test tube, and the Kirinchosol fermentation extract was added to each concentration. 1 mL of 0.15 mM DPPH solution was added thereto, followed by reaction at room temperature for 30 minutes, and the absorbance at 520 nm was measured. Initial (Ai) and blank (Ab) without substrate and DPPH were measured, and positive control group compared α-tocopherol and BHT.

As a result of the analysis, the Kirinchos fermented extract according to the present invention exhibited a higher activity of oxygen scavenging ability than the positive control groups of? -Tocopherol and BHT (see Table 4 and FIG. 1). Therefore, it was confirmed that the degradation of active oxygen was promoted by the Kirinchoso fermented extract, which was effective in improving skin beauty.

Concentration (mg / L) 0 One 10 100 extract 0 34.1 49.5 80.7 alpha -tocopherol 0 33.7 43.1 75.9 BHT 0 26.4 39.4 64.8

Example 2 Confirmation of Growth Promoting Effect of Human Dermal Fibroblast by Kirinchoso Fermented Extract

In order to confirm the improvement of skin beauty by Kirinchosu fermentation extract, we examined whether fibroblast growth promoting collagen, which is a constituent of dermis, is promoted by Kirinchoshi fermentation extract.

Human dermal fibroblast (nHDF) was purchased from Lonza. The cell culture medium was supplemented with 10% fetal bovine serum (FBS; Gibco), 10% penicillin (100 unit / ml) and streptomycin (100 g / ml) in Dulbecco's modified Eagle's sediment (DMEM; Gibco) / mL) was added and used. The cultivation was carried out under the conditions of 37 ° C, 95% humidity, 5% CO ₂ incubator, and cells between 3 and 10 generations were used for the experiment. To examine the degree of cytotoxicity of the human dendritic fibroblast nHDF cells treated with the Kirinchos fermented extract, the cells were cultured for 24 hours after treatment with the respective concentrations of the Kirinchos fermented extract, and then the cell MTT The viability was measured by using

As a result, the cell growth was increased in a concentration-dependent manner after the treatment of the Kirinchosho fermented extract (see Table 5 and FIG. 2), compared to the control group (untreated group).

Time (hr) Concentration (mg / L) 0 One 10 100 0 100 100 100 100 24 100.96 101.51 105.28 108.43 48 102.57 105.62 106.64 110.91 72 103.63 107.91 110.15 114.16

Example 3 Confirmation of Increased Expression of Col1A1 and Decreased Expression of MMP1 by Kirinchose Fermented Extract

In order to confirm the skin cosmetic improvement effect of the Kirinchos fermented extract, the expression of COL1A1, one of typical collagen expressed by human dermal fibroblasts, and MMP1, a collagenase, was measured. In order to confirm the degree of collagen expression change by the Kirinchos fermented extract, the human dermal fibroblasts were treated with the respective concentrations of the Kirinchos fermented extract and cultured for 24 hours.

Herein, quantitative real-time PCR (qRT-PCR), which is a method of continuously detecting a fluorescent substance by attaching a fluorescent substance to a DNA product amplified during a polymerase chain reaction (PCR) PCR). SYBR Green I (Invitrogen) was used to quantitatively determine the expression of COL1A1 and MMP1 gene expression by Kirinchos fermented extracts, using the fluorescence values emitted by the PCR products. The reaction solution was prepared by mixing 0.2 μM primer, 50 mM KCl, 20 mM Tris / HCl pH 8.4, 0.8 mM dNTP, 0.5 U Extaq DNA polymerase, 3 mM MgCl ₂ and 1 × green. The denaturation, annealing and polymerization were carried out at 94 ° C for 30 seconds, 58 ° C for 30 seconds, and 72 ° C for 30 seconds, respectively, after primary denaturation at 94 ° C for 3 minutes using Linegene K (BioER) 40 cycles and fluorescence intensity was measured after each cycle. PCR results were verified by melting curve for each result. The threshold cycle (Ct) value of each gene was normalized to the Ct value of β actin, and then the change amount of the Ct value was compared and analyzed. The Ct value was the number of cycles when the amount of fluorescence generated by the PCR product (the number of amplified PCR products) reached a constant reference value above the basal value, and the amount of gene expression was confirmed through the Ct value. The respective genes used in the experiment are shown in Table 6 below.

gene Forward primer Reverse primer beta -actin 5-GGATTCCTATGTGGGCGACGA-3 5-CGCTCGGTGAGGATCTTCATG-3 COL1A1 5-AGGGCCAAGACGAAGACATC-3 5-AGATCACGTCATCGCACAACA-3 MMP1 5-TCTGACGTTGATCCCAGAGAGCAG-3 5-CAGGGTGACACCAGTGACTGCAC-3

As a result, the expression of Col1A1 mRNA was increased in a concentration-dependent manner after treatment with the extract of the Kirinchosho fermentation extract (see Table 7 and FIG. 3). In addition, the expression of MMP1 mRNA was decreased in a concentration-dependent manner after the treatment of the Kirinchozo fermentation extract (see Table 8 and Fig. 4).

Concentration (mg / L) 0 One 10 100 Expression of Col1A1 (%) 100 107.64 155.65 214.73

Concentration (mg / L) 0 One 10 100 MMP1 expression (%) 100 97.65 85.65 60.41

Example 4. Confirmation of growth inhibition effect of immune cells by Kirinchoso fermented extract

In order to confirm the improvement of skin cosmetic effect by the Kirinchosu fermentation extract, we examined whether the growth of mouse macrophage (RAW 264.7), which acts on inflammation and erythema of the skin, is inhibited by the Kirinchose fermentation extract. Mouse immune cells were purchased from Korean Cell Line Bank (KCTC). Cell culture medium was prepared by adding 10% FBS (Gibco), 10% penicillin (100 units / mL) and streptomycin (100 g / mL) to DMEM (Gibco). The cultivation was carried out at 37 ° C, 95% humidity, 5% CO ₂ incubator conditions, and cells between 3 and 10 generations were used for the experiment. To investigate the extent of cytotoxicity of the RAW 264.7 cells treated with Kirinchos fermented extract, the cells were cultured for 24 hours after treatment with the respective concentrations of the Kirinchos fermented extract, and then the cell activity Were measured.

As a result of analysis, cell growth was reduced in a concentration-dependent manner after treatment with the extract of the Kirinchosho fermentation extract (see Table 9 and FIG. 5), as compared with the control (no treatment) group.

Time (hr) Concentration (mg / L) 0 One 10 100 0 100 100 100 100 24 99.78 98.68 99.41 97.18 48 99.54 96.86 94.81 94.03 72 98.96 95.41 92.96 88.94

Example 5 Confirmation of NF-kB Transcription Inhibitory Effect by the Kirinchos Fermented Extract

In order to confirm the improvement of skin cosmetic effect by the Kirinchosu fermentation extract, the transcriptional ability of NF-kB, which is a transcription factor of cytokine which plays an important role in the immune response, was inhibited by the Kirinchosan fermentation extract.

NF-kB reporter analysis was performed to determine the degree of suppression of NF-kB transcriptional activity when the Kirinchos fermented extract was treated with immune cells. The promoter region of the gene known to be transcribed by NF-kB was cloned into a vector containing the luciferase gene and inserted into the immune cells. The NF-kB transcription-inducing LPS was treated to activate the NF-kB transcriptional activity. Then, the Kirinchozo fermented extract was treated with each concentration and the luciferase activity was measured. The luciferase activity is determined by the presence of the IgG kappa NF- kappa B region (SEQ ID NO: 5-GGGGACTTTCC-3) oligonucleotides upstream of the minimal IL-8 promoter (positions 67 to +44), the NF- (Gibco-BRL), and the gene was inserted into the immune cells according to the reagent manual. The lipofectamine plus (Gibco-BRL) After transfection of NF-kB with LPS was performed on the immune cells with the gene inserted, the Kirinchos fermented extract was treated at 0, 1, 10, and 100 mg / L and cultured for 24 hours. The cultured immune cells were directly added with 0.1 mL dissolution buffer (0.1 M HEPES, pH 7.6, 1% Triton-X, 1 mM DTT, and 2 mM EDTA) at the time of collection to collect the cell lysate, The cells were centrifuged at rpm to obtain cell proteins. Protein concentration was measured using a Bradford assay (Bio-Rad Laboratories), and then 20 μg of protein was added to a luciferase assay mixture (25 mM glycylglycine, 15 mM MgSO ₄ , 1 mg / mL BSA, ATP and 1 mM D-luciferin (Analytical Luminescence Laboratory), and the luminescence was measured three times repeatedly for 20 seconds using Monolight 2010 (Analytical Luminescence Laboratory) to obtain an average value.

As a result, the transactivity of NF-κB increased by LPS after treatment with the Kirinchos fermented extract was decreased in a concentration-dependent manner (see Table 10 and FIG. 6).

Extract concentration (mg / L) 0 0 One 10 100 LPS - + + + + Luciferase activity (multiples) One 4.17 3.64 2.26 1.34

Example 6 Confirmation of Inflammatory Cytokine Synthesis Inhibitory Effect by Kirinchosu Fermentation Extract

In order to confirm the improvement of skin cosmetic effect by the Kirinchosu fermentation extract, it was confirmed that the synthesis of the inflammatory cytokine inducing the immune response was inhibited by the Kirinchosu fermentation extract.

In order to confirm the degree of expression of inflammatory cytokine by the Kirinchos fermented extract, the LPS-treated immune cells were treated with various concentrations of the Kirinchos fermented extract and cultured for 24 hours. The gene expression changes were then measured by qRT-PCR. SYBR Green I (Invitrogen) was used to quantitatively determine changes in IL-1β and TNF-α gene expression by Kirinchos fermented extracts, using fluorescence values emitted by PCR products. The reaction solution was prepared by mixing 0.2 μM primer, 50 mM KCl, 20 mM Tris / HCl pH 8.4, 0.8 mM dNTP, 0.5 U Extaq DNA polymerase, 3 mM MgCl ₂ and 1 × green. The denaturation, annealing and polymerization were carried out at 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds, respectively, after primary denaturation at 94 ° C for 3 minutes using Linegene K (BioER) 40 cycles and fluorescence intensity was measured after each cycle. PCR results were verified by melting curve for each result. The Ct value of each gene was normalized to the Ct value of β - actin, and the change of the Ct value was compared and analyzed. The Ct value was the number of cycles when the amount of fluorescence generated by the PCR product (the number of amplified PCR products) reached a constant reference value above the basal value, and the amount of gene expression was confirmed through the Ct value. Primers for each gene used in the experiment are shown in Table 11 below. L-NG-monomethyl arginine and acetate salt (L-NG-Monomethylarginine, Acetate Salt; L-NMMA) were used as the positive control.

gene Sequential primer Reverse primer beta -actin 5-CCAAGGCCAACCGCCGC-3 5-AGGGTACATGGTGCCGCC-3 IL-1? 5-CTCAATGGACAGAATATCAACCAACA-3 5-ACAGGACAGGTATAGATTCTTTCCTTTG-3 TNF-a 5-TGCCTATGTCTCAGCCTCTTC-3 5-GAGGCCATTTGGGAACTTCT-3

As a result, the expression of IL-1β mRNA increased by LPS in a concentration-dependent manner after the treatment of the Kirinchos fermented extract was decreased as compared with the LPS-treated group (see Table 12 and FIG. 7). In addition, the expression of TNF-α mRNA increased by LPS in a dose-dependent manner after treatment with the Kirinchos fermented extract was decreased as compared with the LPS-treated group (see Table 13 and FIG. 8).

Extract concentration (mg / L) 0 0 One 10 100 LPS - + + + + IL-1? MRNA expression
(Drainage) One 4.35 3.58 2.11 1.25

Extract concentration (mg / L) 0 0 One 10 100 LPS - + + + + Expression of TNF-α mRNA
(Drainage) One 4.08 3.34 2.44 1.43

Example 7. Confirmation of changes in skin oil content index by Kirinchoso fermented extract

In order to confirm the effect of improving the skin beauty by Kirinchoso fermented extract, the skin water content was analyzed using a corneometer and a sebumeter.

First, in order to examine the effect of the Kirinchos fermented extract on the improvement of skin oil, 30 adult women over 30 years old were selected and the cream and toner were used. Cream and toner were used for the control, the positive control and the experimental group for 2 weeks and 4 weeks, respectively, and skin moisture index was measured according to the instrument manual using a moisture analyzer (MPA5, Courage-Khazaka Electronic GmbH). Using a moisture analyzer, the same test person measured the right ball portion of the subject to be measured three times in succession. Skin oil index was measured according to the instrument manual using a sebum analyzer (MPA5, Courage-Khazaka Electronic GmbH). Using the sebum analyzer, the same person in charge of the test measured the area of the forehead which is the measurement site of the subject.

As a result of analysis, skin moisture increased in a time-dependent manner after treatment with the Kirinchos extract (see Table 14 and FIG. 9), as compared with the control (no treatment) group. Also, in the case of oil, skin moisture was increased in a concentration-dependent manner after treatment of the Kirinchotan fermented extract, as compared with the control (no treatment group) (see Table 15 and FIG. 10).

Before experiment after 2 weeks After 4 weeks Control group 251 252 256 Positive control group 247 259 264 Experimental group 254 284 297

Before experiment after 2 weeks After 4 weeks Control group 323 327 330 Positive control group 330 345 353 Experimental group 327 348 360

Example 8. Confirmation of Skin Wrinkle Reduction Effect by Fermented Extract of Kirinchosho

Skin wrinkles were analyzed by using a skin analyzer in order to confirm the effect of improving the skin wrinkles by the Kirinchos fermented extract.

First, in order to confirm the effect of the Kirinchoso fermentation extract on the improvement of skin wrinkles, 30 adult women aged 30 years or older were selected to use the cream and toner prepared above. After using the cream and toner for 2 weeks and 4 weeks for the control, the positive control and the experimental group respectively, the degree of facial wrinkles was measured according to the machine manual using PRIMOS Lite (field of view 18 flexible 3D measuring, GFMesstechnik GmbH) Respectively. Using the PRIMOS light, the same test person fixes the face of the subject on a specially prepared PRIMOS facial fixation kit and adjusts the tip of the eye, the measurement site, to match the focus pattern of the PRIMOS light Respectively.

As a result of the analysis, the wrinkles of the tail of the tail were decreased in a time-dependent manner after the treatment of the Kirinchotan fermentation extract (see Table 16 and Fig. 11 below), as compared with the control (no treatment group).

Before experiment after 2 weeks After 4 weeks Control group 32.51 32.13 31.67 Positive control group 32.05 31.28 29.13 Experimental group 33.10 31.15 28.91

Example 9. Confirmation of Skin Thickening Effect by Fermented Extract of Kirinchosho

The increase of dermal fibroblast proliferation and the increase of collagen expression by Kirinchoso fermented extract were confirmed. Thus, it was analyzed whether the thickness of the dermal layer was increased by the collagen produced, and the effect of improving the skin thickness by the Kirinchosu fermentation extract was confirmed.

First, to examine the effect of Kirinchoso fermented extract on skin thickness, 30 adult women over 30 years old were selected and cream and toner were used. The control, positive control and experimental groups were allowed to use cream and toner for 2 and 4 weeks, respectively, and then measured according to the instrument manual using a DUB-skin scanner (tpm taberna pro medicum). A DUB-skin scanner was applied with a gel for ultrasound examination and the probe was placed at right angles to the skin, and then the same test person was measured with the same pressure on the right side of the subject's right eye.

As a result of the analysis, skin thickness was increased in a time-dependent manner after treatment with the Kirinchotan fermented extract (see Table 17 and FIG. 12), as compared to the control group (no treatment group).

Before experiment after 2 weeks After 4 weeks Control group 56.02 56.21 56.30 Positive control group 56.44 56.96 57.24 Experimental group 56.30 56.91 57.67

Example 10. Confirmation of Skin Elasticity Improvement Effect by Fermented Extract of Kirinchosho

In order to confirm the improvement effect of skin beauty by the Kirinchosu fermentation extract, the skin elasticity improvement effect was confirmed. In order to confirm the effect of Kirinchoso fermented extract on skin thickness, 30 adult women over 30 years old were selected and cream and toner were used. After using cream and toner for 2 weeks and 4 weeks for control, positive control and experimental group respectively, the degree of change of skin elasticity was measured with DermaLab USB elasticity probe. The DermaLab USB elastic probe shows the skin change and resilience with time of inhalation and inhalation time. The same test person measured the probe three times after fixation to the left ball with tape.

As a result of the analysis, elasticity of the skin was increased in a time-dependent manner after treatment of the Kirinchotan fermented extract (see Table 18 and FIG. 13), as compared with the control (no treatment) group.

Before experiment after 2 weeks After 4 weeks Control group 11.42 11.44 11.54 Positive control group 11.10 11.95 12.24 Experimental group 11.38 12.15 13.59

Example 11 Confirmation of Skin Barrier Improvement Effect by Kirinchoso Fermented Extract

In order to confirm the improvement effect of the skin beauty by the Kirinchosu fermentation extract, the skin barrier improvement effect was confirmed.

First, in order to confirm the effect of the Kirinchos fermented extract on the improvement of the skin barrier, 30 adult women aged 30 years or older were selected and the cream and toner were used. Cream and toner were used for 2 weeks and 4 weeks for the control, the positive control and the experimental group, respectively. Then, the TEWL (TransEpidermal Water Loss) was measured using a DermaLab USB TEWL probe (Cortex Technology, Inc.) It was measured according to the manual of the apparatus.

 As a result of analysis, the amount of transdermal water loss was reduced in a time-dependent manner after the treatment of the Kirinchosho fermented extract, as compared with the control (no treatment group) (see Table 19 and FIG.

Before experiment after 2 weeks After 4 weeks Control group 32.17 31.53 31.39 Positive control group 31.79 31.02 30.27 Experimental group 33.09 31.28 28.74

Example 12. Confirmation of improvement of skin texture by Kirinchose fermented extract

In order to confirm the skin cosmetic improvement effect by the Kirinchosu fermentation extract, the skin texture improving effect was confirmed.

First, in order to examine the effect of the Kirinchos fermented extract on the improvement of skin texture, 30 adult women over 30 years old were selected and the cream and toner were used. After 2 and 4 weeks of cream and toner were applied to the control, positive control and experimental groups respectively, skin texture was measured according to the instrument manual using PRIMOS light (field of view 45, flexible 3D measuring, GFMesstechnik GmbH). Using the PRIMOS light, the same test person measured the area of the forehead of the subject three times in succession and measured the same area.

As a result of analysis, the depth of wrinkles generated on the skin to determine skin texture decreased in a time-dependent manner after treatment of the Kirinchotan fermented extract (see Table 20 and Fig. 15 below), as compared with the control group (untreated group).

Before experiment after 2 weeks After 4 weeks Control group 15.59 15.33 15.07 Positive control group 15.28 14.96 14.29 Experimental group 15.10 14.67 13.51

Example 13. Confirmation of erythema improvement effect by the fermented Kirinchos extract

Through the above Examples 5, 6 and 7, it was confirmed that the growth inhibition of the immune cells and the decrease of the inflammatory cytokine expression by the fermentation extract of Kirinchoshi were confirmed. Therefore, it was analyzed whether the erythema decreased by the reduced cytokine, and the erythema improvement effect of the Kirinchos fermented extract was confirmed.

First, in order to confirm the effect of the Kirinchos fermented extract on the erythema improvement, 30 adult women over 30 years old were selected and the cream and toner were used. The control, positive control, and experimental groups were allowed to use cream and toner for two and four weeks, respectively, followed by spectrophotometer (Spectrophotometer CR-2600D, Konica Minolta) according to the instrument manual. The same test person measured the right side of the subject's right side of the ball three times in succession, and the mean value was calculated and used for the analysis. The values measured in the spectrophotometer are L *, a *, and b *, and the red value indicates a lot of red as the value of a * increases to a value indicating erythema.

As a result of analysis, erythema decreased in a time-dependent manner after the treatment of the Kirinchosu fermented extract (see Table 21 and Fig. 16 below), as compared with the control group (untreated group).

Before experiment after 2 weeks After 4 weeks Control group 12.19 11.93 11.76 Positive control group 12.31 11.73 11.21 Experimental group 12.99 11.62 10.29

Example 14 Confirmation of Improvement of Pigmentation by Kirinchose Fermented Extract

In order to confirm the skin cosmetic improvement effect by the Kirinchosu fermentation extract, the improvement effect of the pigment deposition was confirmed.

First, in order to examine the effect of the Kirinchoso fermentation extract on the improvement of pigmentation, 30 adult women over 30 years old were selected and the cream and the toner were used. The control, positive control and experimental groups were allowed to use cream and toner for 2 and 4 weeks, respectively, and then measured according to the instrument manual using ANTERA 3D (Miravex). The same examiner used the left ball area three times in a row for analysis.

As a result of analysis, the pigmented area decreased in a time-dependent manner after the treatment of the Kirinchotan fermented extract, as compared to the control (no treatment group) (see Table 22 and Fig. 17 below).

Before experiment after 2 weeks After 4 weeks Control group 221.94 220.13 218.48 Positive control group 220.18 213.43 193.09 Experimental group 218.29 179.31 159.37

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than the foregoing description, and all changes or modifications derived from the meaning and scope of the claims and the equivalents thereof are included in the scope of the present invention. .

Claims (12)

A cosmetic composition for skin moisturizing and wrinkle improvement comprising an extract of Kirinchos fermented extract as an active ingredient.
The method according to claim 1,
Wherein the fermented extract is a fermented extract prepared by diluting a dried product of the extract obtained from the Kirinchoshi with a solvent, inoculating the microorganism for fermentation, and fermenting the fermented extract.
The method according to claim 1,
Wherein the fermentation extract is contained in an amount of 2 to 10% by weight based on the total weight of the composition.
The method according to claim 1,
The cosmetic composition for skin moisturizing and wrinkle improvement, wherein the improvement in skin wrinkles is caused by an increase in collagen due to promotion of dermal cell growth and reduction of collagenase.
A cosmetic comprising the cosmetic composition for skin moisturizing and wrinkle improvement according to any one of claims 1 to 4. delete delete delete delete delete delete delete

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