KR102052001B1 - Cosmetic composition for skin anti-aging or improving skin wrinkle comprising Kudzu root vinegar and adenosine - Google Patents

Abstract

The present invention relates to a cosmetic composition for preventing skin aging or for improving wrinkles, including gallium herb and adenosine. The present invention is safe to the skin, but also excellent in the effect of inhibiting skin aging, wrinkle improvement or skin elasticity by simultaneous treatment of the reed root and adenosine.

Description

Cosmetic composition for skin anti-aging or wrinkle improvement comprising brown root vinegar and adenosine {Kdzu root vinegar and adenosine}

The present invention relates to a cosmetic composition for preventing skin aging or for improving wrinkles, including gallium herb and adenosine.

Skin aging can be divided into endogenous skin aging and exogenous skin aging. Endogenous skin aging is a natural aging phenomenon that occurs over time, resulting in fine lines, dry skin, and reduced elasticity. Exogenous skin aging is an aging phenomenon caused by external factors such as ultraviolet rays, pollution, cold weather, and cigarette smoke. Photoaging occurs in areas exposed mainly to the sun, such as the face, nape, and back of the hand, and causes symptoms including thick and deep wrinkles, decreased skin elasticity, and pigmentation. Compared with endogenous aging, exogenous aging (photoaging) is characterized by severe phenomena and promoting endogenous effects.

Natural fermented brown root vinegar is produced through fermentation in the root root concentrate, and the root root root concentrate contains isoflavones 300 times more concentrated than soybeans. Fermenting the root extract concentrate produces a variety of organic acids. Kudzu vinegar is a natural acetic acid that exfoliates the skin and safely fills the skin and quickly replaces it with a new skin layer. The company has launched a natural lifting peeling agent under the name of Pink Peel to help improve wrinkles, and it uses kudzu-THERA, a root extract. Wrinkle improvement cosmetic composition using natural fermented brown root vinegar is the only product of its own.

At the present time when there is an explosive interest in skin care, especially anti-aging, there is a need to develop cosmetic raw materials from natural materials that can be used for a long time and have less damage to normal skin, which are safe for the skin and excellent in improving wrinkles. .

Thus, the inventors of the present invention while researching a cosmetic composition for improving skin anti-aging or wrinkles improved its pink peel, it was confirmed that the cosmetic composition containing adenosine with natural fermented brown vinegar has an excellent anti-aging or wrinkle improvement effect This invention was completed.

Accordingly, an object of the present invention is to provide a cosmetic composition for preventing skin aging or improving wrinkles, including gallium herb and adenosine.

In order to achieve the above object, the present invention provides a cosmetic composition for preventing skin aging or improving wrinkles, including gallium herb and adenosine.

In addition, the present invention provides a pharmaceutical composition for preventing skin aging or improving wrinkles, including gallium herb and adenosine.

In addition, the present invention provides a quasi-drug for preventing skin aging or improving wrinkles, including gallium herb and adenosine.

The present invention is safe to the skin, but also excellent in the effect of inhibiting skin aging, wrinkle improvement or skin elasticity by simultaneous treatment of the reed root and adenosine.

BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the schematic diagram of the manufacturing method of a cut root.
Figure 2 is a diagram showing the results of cell viability analysis according to the treatment of reed root.
Figure 3 is a diagram showing the results of analysis of cell viability before and after ultraviolet irradiation by adenosine alone treatment.
Figure 4 is a diagram showing the results of analysis of cell viability before and after ultraviolet irradiation according to the treatment of the mixture of brown root and adenosine.
5 is a diagram showing the results of confirming the protective effect of the cell according to the treatment of the mixture of brown root and adenosine before UV irradiation.
6 is a diagram showing the results of confirming the cell protective effect of the treatment of the mixture of brown root and adenosine after ultraviolet irradiation.
Figure 7 is a diagram showing the results of evaluation of apoptosis inhibitory effect according to the treatment of the reed root and adenosine (A: FACS results, B: apoptosis effect).
8 is a diagram showing the results of evaluation of the ability to remove the reactive oxygen species (ROS) in the cells according to the treatment of the mixture of the reed root and adenosine (A: fluorescence intensity measurement results according to DCF-DA staining, B: RFU (Relative fluorescence unit) One fluorescence intensity).
Figure 9 is a diagram showing the results of the evaluation of cell aging inhibitory ability according to the treatment of the root and the adenosine mixture (A: SA-β staining results, B: quantified β-galactosidase fluorescence intensity for the control group).
10 is a diagram showing the results of evaluating the inhibitory ability of the MAPKs signaling pathway in accordance with the treatment of the mixture of the reed root and adenosine (A: Western blot results, B: the result of quantifying the degree of p-JNK phosphorylation).
11 is a diagram showing the results of evaluating the inhibition of MMP-9 expression according to the treatment of brown root and adenosine mixture.
12 is a view showing the results of a clinical test on the skin wrinkles with a cosmetic composition comprising the gallon herb and adenosine of the present invention.
Figure 13 is a diagram showing the results of a clinical test on the skin wrinkles cosmetic composition comprising the root knotweed and adenosine of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a cosmetic composition for preventing skin aging or improving wrinkles, including gallium herb and adenosine.

The anti-aging or anti-wrinkle cosmetic composition comprising the reed root and adenosine according to the present invention by inhibiting skin damage, cell aging and cell death caused by ultraviolet rays and increase skin elasticity by co-treatment with the reed root and adenosine It is characterized by an excellent wrinkle improvement effect.

In the present invention, the term "skin aging" refers to the aging of the skin caused by intrinsic and external factors, preferably may be skin photoaging accompanied with the formation of skin wrinkles, more preferably May be skin photoaging due to ultraviolet stimulation accompanied with skin wrinkle formation, but is not limited thereto.

The term "skin wrinkle" also refers to wrinkle formation on skin.

In the present invention, the "brown root vinegar" may be fermented vinegar or synthetic vinegar, and preferably fermented vinegar, depending on the preparation method. The fermented vinegar is divided into natural fermented vinegar or alcohol fermented vinegar according to the method of making, which means that the natural fermented vinegar is made by fermenting sugar and acetic acid fermentation again, alcohol fermented vinegar is 99% pure alcohol alcohol It means that the acetic acid is fermented as a raw material, and the brown root vinegar of the present invention is preferably a natural fermented vinegar.

More specifically, the reed root according to the present invention comprises the steps of (a) washing and grinding the root; (b) extracting the juice by mixing 1.8 to 2.2 parts by weight of water with respect to 1 part by weight of the pulverized root; (c) first filtering the extracted juice; (d) adding and mixing 0.12 to 0.18 parts by weight of starch and 0.08 to 0.12 parts by weight of yeast with respect to 1 part by weight of the first filtered brown root juice and fermenting at a temperature of 20 to 40 ° C .; And (e) aging the fermented root juice after the second filtration and adding the yeast to a temperature of 20 to 40 ° C.

In one embodiment of the present invention (a) washing and grinding the roots; (b) extracting juice by mixing and compressing 2 parts by weight of water with respect to 1 part by weight of pulverized root; (c) first filtering the extracted juice; (d) adding 0.15 parts by weight of starch and 0.10 parts by weight of koji with respect to 1 part by weight of the primary filtrate, and fermenting at a temperature of 30 ° C. for 1 month; And (e) fermenting the rooted black root juice after filtration and aging at a temperature of 30 ° C. by adding yeast; to prepare the rooted root, and the manufacturing process is described in FIG. 1.

In the present invention, the root may be 10 to 50 parts by weight, preferably 20 to 40 parts by weight, more preferably 25 to 35 parts by weight with respect to 100 parts by weight of the total cosmetic composition, the adenosine is a total cosmetic 0.01 to 0.10 parts by weight, preferably 0.02 to 0.08 parts by weight, and more preferably 0.03 to 0.05 parts by weight, based on 100 parts by weight of the composition. When the content of the root knotweed and adenosine falls within the aforementioned range, it is possible to significantly exert an anti-aging and anti-wrinkle effect without skin toxicity.

In the present invention, the cosmetic composition for preventing skin aging or improving wrinkles may further comprise an organic solvent. The organic solvent may be at least one selected from the group consisting of ethanol, methanol, isopropanol, propylene glycol, butylene glycol, 1,2-hexanediol, glycerin and acetic acid ethyl ester, preferably ethanol or 1,2- Hexanediol.

In the present invention, the organic solvent may be ethanol and 1,2-hexanediol, 1 to 5 parts by weight of ethanol, and 1 to 5 parts by weight of 1,2-hexanediol, respectively, based on 100 parts by weight of the total cosmetic composition And preferably 1.5 to 2.5 parts by weight of ethanol, and 1.5 to 2.5 parts by weight of 1,2-hexanediol, respectively.

In addition, the cosmetic composition according to the present invention may further include auxiliary ingredients such as pigments, fragrances, preservatives and other additives commonly used in the manufacture of cosmetic compositions for preventing skin aging or improving wrinkles, in addition to the above components. The type and content of ingredients are well known to those skilled in the art, and those skilled in the art can select and combine them without difficulty. In addition, their content may be appropriately adjusted according to the use of the final product and the purpose of use thereof.

According to an embodiment of the present invention, the cosmetic composition is 30 parts by weight of brown root, 0.04 parts by weight of adenosine, 8.30 parts by weight of pH adjusting agent, 2 parts by weight of ethanol, 1,2-hexanediol 2 based on 100 parts by weight of the total cosmetic composition Parts by weight, 0.70 parts by weight of panthenol, 0.54 parts by weight of viscosity modifier, 0.15 parts by weight of emulsifier, 0.10 parts by weight of pigment and 0.02 parts by weight of disodium ethane, and the remaining amount of solvent to adjust the total amount of the mixture to 100 parts by weight. It may be a composition. At this time, the solvent may be used without limitation conventional solvents known in the art, preferably can be used in the root.

The cosmetic composition according to the present invention may be prepared in any formulation commonly prepared in the art, such as lotion, latex, gel, cream, essence, pack, ampoule, lotion, cleaning agent, soap, body products, soap, It may be any one of the formulations selected from oils, lipsticks, sprays, powders and foundations. More specifically, it may have a formulation of a flexible lotion (skin), astringent lotion, nourishing lotion (milk lotion), nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder Can be.

When the formulation of the present invention is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. Can be.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, in particular in the case of a spray, additionally chlorofluorohydrocarbon, propane Propellant such as butane or dimethyl ether.

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

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, and microcrystals are used as carrier components. Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The cosmetic composition of the present invention may be used daily and may also be used for an indefinite period of time. Preferably, the amount of use, the number of times and the period can be appropriately adjusted according to the age, skin condition or skin type of the user.

In addition, the present invention provides a pharmaceutical composition for preventing skin aging or improving wrinkles, including gallium herb and adenosine.

The composition may further comprise a pharmaceutically acceptable carrier, excipient and diluent. Pharmaceutically acceptable carriers, excipients and diluents included in the composition are those commonly used in the formulation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. It doesn't happen. The pharmaceutical composition may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, in addition to the above components.

When the pharmaceutical composition of the present invention is formulated as a solid preparation for oral administration, the solid preparation for oral administration includes tablets, pills, powders, granules, capsules, and the like, which are mixtures of the above-mentioned root herb and adenosine. Formulated by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. In addition, the pharmaceutical compositions of the present invention may be formulated into liquid preparations for oral administration. Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups. These liquid preparations include various excipients in addition to commonly used inert diluents (e.g., purified water, GaIN / LPS, liquid paraffin), For example, wetting agents, sweetening agents, fragrances, preservatives and the like can be included.

In addition, the pharmaceutical compositions of the present invention may be formulated into preparations for parenteral, preferably intraperitoneal administration. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As a sterile aqueous solution, suitable buffer solutions such as Hanks' solution, Ringer's solution, or physically buffered saline can be used.For non-aqueous solvents, suspensions include propylene glycol, polyethylene glycol, olive oil, Same vegetable oils, injectable esters such as ethyloleate, and the like can be used. If necessary, preservatives, stabilizers, wetting or emulsifying agents, salts for controlling osmotic pressure and / or buffers may also be used. Meanwhile, in the case of suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, and the like, which are conventional bases thereof, may be used.

As used herein, the term "administration" means providing a subject with a composition of the present invention in any suitable manner. Compositions formulated in such a manner can be administered in various amounts, including parenteral or oral (dermal, subcutaneous, intravenous, intramuscular, intraperitoneal) in an effective amount. As used herein, an "effective amount" refers to an amount exhibiting a prophylactic or therapeutic effect when administered to a patient. The dosage of the composition according to the present invention may be appropriately selected depending on the route of administration, subject of administration, age, sex, weight, individual difference and disease state. Preferably, the composition of the present invention may vary the content of the active ingredient depending on the degree of disease, preferably 0.1 to 10000 mg / kg / day, more preferably 10 ~ 1000 mg / kg / day of weight It may be administered in an effective amount.

In addition, the present invention provides a quasi-drug for preventing skin aging or improving wrinkles, including gallium herb and adenosine.

The quasi-drugs refer to drugs based on classification criteria set by the Ministry of Health and Welfare for articles having a slight effect on the human body rather than drugs used to treat or prevent diseases. According to the Pharmaceutical Affairs Law, except for articles used for medicines, textiles and rubber products used for the treatment or prevention of diseases of humans and animals, and those with slight or no direct action on the human body, and similar to those that are not instruments or machines. And antiseptic or insecticides to prevent infectious diseases.

Hereinafter, the present invention will be described in more detail with reference to the following examples and experimental examples. The following examples and experimental examples are provided only for the purpose of illustration to help understanding of the present invention, but the scope and scope of the present invention is not limited thereto.

Example  One. Reed  Produce

The schematic diagram of the manufacturing method of the brown root herb used by this invention is shown in FIG.

First, the roots were washed and pulverized, and then a pretreatment process was performed to make the liquefaction process easier and faster. 2 parts by weight of water was mixed with 1 part by weight of the pulverized root, and the root was extracted. Thereafter, the extracted root juice was first filtered. 갈 starch and natural yeast powder mixed at 4: 1 to the root extract obtained through primary filtration were mixed and fermented at 30 ° C. for 1 month. The fermented rooted root juice was filtered second, and then aged at 30 ° C. by adding yeast. Brown root herb prepared through the above process (hereinafter, in the Examples or Experimental Examples, the term “brown root herb” was used in combination with “brown root”) in the following examples.

Example  2. Cell Culture

HaCaT cells, a human keratinocyte cell line, were distributed from ATCC and used in subsequent experiments. The cell line was prepared at 37 ° C. with 5% CO ₂ using DMEM (Dulbecco's Modified Eagle Medium) medium containing 10% FBS (fetal bovine serum) and 1% antibiotic (penicillin / streptomycin). Maintained in the incubator and passaged 2-3 times a week.

Example  3. Cell viability measurement

3-1. Brown root  Sole treatment

The cultured subcutaneous herbaceous root prepared in Example 1 was treated for each concentration in the culture medium passaged in Example 2.

Cell viability was measured by MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt) assay. Specifically, the treated group of the reed knotweed in a concentration of 10, 50, 100, 200, 500μg / ml incubated for 24 hours and treated with MTS dye. At this time, the living cells produce purple formazan by the treated MTS dye by reductase in the mitochondria, but the dead cells do not produce formazan because the mitochondria do not function. The formed formazan was measured by absorbance at 490/590 nm with a microplate reader to determine the effect of the reed treatment on cell viability. The results are shown in FIG.

As shown in Figure 2, when treated with the concentration of 10 ~ 100μg / ml of the cutlery did not have a significant effect on the cell viability, the cell survival rate is about 20%, 500μg / ml when treated at a concentration of 200μg / ml When treated with the concentration of the cell survival rate was reduced by about 30%. Thus, it was determined that there is cytotoxicity when treated with more than 200μg / ml of the cutlery, and in the subsequent experiments were limited to the concentration of the cuttleweed to 100μg / ml appearing to be less than 10% cytotoxicity.

3-2. Adenosine alone treatment

Adenosine was cultured by concentration in the passage cultured in Example 2, and cell viability was analyzed before and after UV irradiation according to adenosine alone treatment. Specifically, the cells were treated with 1, 10, 20, 50, 100 μM adenosine, and then cultured for 24 hours to measure cell viability, and treated with the same concentration of adenosine after UV irradiation in the same medium, followed by culture for 24 hours. Cell viability was measured. The results are shown in FIG.

As shown in Figure 3, adenosine alone treatment did not show a significant difference in cell viability compared to the control (con) not treated with adenosine. In addition, the results of treatment with adenosine after irradiation with ultraviolet rays did not show a significant difference in cell activity compared to the untreated group. The results confirmed that there was no change in the cells before and after ultraviolet irradiation when adenosine alone treatment.

3-3. Brown root  And adenosine mixture treatment

In the medium passaged in Example 2, the mixture of brown root and adenosine prepared in Example 1 was treated for each concentration to further incubate. Specifically, the HaCaT cells were treated with 10, 50 or 100 μg / ml of the root herb, and the mixture of the root of the root and 50 μM or 100 μM adenosine (the group of the root and the adenosine mixture), respectively, and cultured for 24 hours. Post cell viability was measured. Subsequently, ultraviolet rays were irradiated to the same medium, and treated with gallium herb alone or with the mixture of gallium herb and adenosine under the same concentration conditions, and cell viability after culture for 24 hours was measured. As a control group, the untreated group or the root treated with only black root grass was analyzed as a control group and the cell viability (Cell viability) treated with the mixture of the root and the adenosine, and the results are shown in FIG.

As shown in FIG. 4, it was confirmed that the cell survival rate after the UV irradiation was increased in a concentration-dependent manner compared to the control group in the case of the group of the brown root and the adenosine. In particular, it was confirmed that the cell survival rate increased by about 21% compared to the control group when treated with a mixture of 100 μg / ml and 100 μM of adenosine in a medium having reduced cell viability by about 35% by irradiation with ultraviolet rays. Through the above results, it was confirmed that the brown root and adenosine mixture of the present invention does not exhibit cytotoxicity, excellent protection against ultraviolet rays, and activate keratinocytes. Thus, in the subsequent experiments, a mixture of brown root herb and adenosine mixed with adenosine concentration of 100 μM was used.

Example  4. Observation of Cell Morphology by UV Irradiation

UVB irradiation damages proteins, DNA, lipid membranes, etc., and induces apoptosis. The cell protective effect of brown root and adenosine against such cell damage was confirmed through morphological observation. The change of cells was observed with a phase contrast microscope with respect to the group of the group which treated the root of the root of the root of the root of the root of the root of the root of the root, and the combination of the root of the root with the same concentration and 100 µM of adenosine 100µM and 20 µg / ml and 100 µg / ml, respectively. 5 shows the results of treatment of the above-mentioned roots and adenosine on the cells not irradiated with UVB, and the results of the treatment of the roots and adenosine on the cells irradiated with UVB (30mJ / cm 2) are shown in FIG. 6.

As shown in FIG. 5, in the case of the cells not irradiated with UVB, both the group of the group treated with the root of black root and the group of the group of the group of the group of root of root and adenosine did not show any significant difference. On the other hand, as shown in Figure 6, when irradiated with UVB, unlike the normal cells, the morphological changes of cells due to UVB-induced damage was confirmed, UVB both in the group treated with karrowweed alone and the group treated with karrowweed and adenosine It was confirmed that cell suicide caused by. Among them, 100 μg / ml of rootwort and 100 μM of adenosine were the least byproducts due to cell death, and the number of cells was significantly higher and the cell morphology was not broken much compared to UVB-only control. It was confirmed that the percent increase. Through the above results, it was confirmed that the brown root and adenosine mixture of the present invention shows an excellent cell protection effect.

Example  5. Apoptosis Inhibitory ability  evaluation

UVB-irradiated HaCaT cells and FITC-conjugated annexin-V detection kits in order to confirm a direct association with apoptosis and cell death due to ultraviolet (UVB) irradiation of the root herb and adenosine mixtures according to the present invention. Fluorescence-activated cell sorting (FACS) analysis was performed. First, HaCaT cells were incubated for 24 hours in medium with 10% FBS. Thereafter, the cells were diluted with DMEM medium to 2 × 10 ⁵ cells / well, and the cell suspension was placed in a 6-well culture vessel with 2 ml of each well and incubated for 24 hours to attach the cells to the culture vessel. After 24 hours, a mixture of 100 μg / ml of brown root sheath and 100 μM of adenosine used in Example 3 was treated, and further incubated for 24 hours. After 24 hours, the cells were washed with PBS, suspended again with 100 μl of binding buffer, and then 2 μl of annexin V-FITC was added and reacted in the dark for 15 minutes. 500 μL FACS buffer and 5 μL propium iodide (PI) were then added and analyzed using flow cytometry (BD Biosciences, Calif.). The results are shown in FIG.

As shown in FIG. 7, the apoptosis was increased to about 30% when UVC was treated to HaCaT cells, but the apoptotic and adenosine mixture treatment groups showed a decrease in cell death to 15%. Through this, brown root and adenosine mixture was confirmed to exhibit an excellent effect in preventing skin aging by inhibiting cell death by ultraviolet rays in human skin cells.

Example  6. Within Cells Reactive oxygen species ( ROS ) Removability  evaluation

ROS production using DCF-DA (2'-7'dichlorofluorescin diacetate) to determine the effect of the mixture of the root and the adenosine according to the present invention on the quantitative change of reactive oxygen species (ROS) caused by UVB irradiation It was confirmed. Specifically, the HaCaT cells prepared by attaching to the culture vessel in Example 5 were treated with a mixture of 100 μg / ml of the root herb used in Example 3 and 100 μM of adenosine, and washed twice with PBS after 24 hours of incubation. Then, the medium was changed to medium to which 25 μM of DCF-DA was added and incubated at 37 ° C. for 30 minutes. This was again washed twice with PBS and analyzed using flow cytometry (BD Biosciences, CA). The results are shown in FIG.

As shown in FIG. 8, reactive oxygen species increased by 60% in the UVB-only control group (UVB) compared to the control group without UVB treatment. On the other hand, it was confirmed that the accumulation of reactive oxygen species in the group treated with the mixture of brown root and adenosine (UVB + brown root + Adenosine) by 27% compared to the control group. Through this, it was confirmed that the ROS removal effect of the brown root and adenosine mixture of the present invention.

Example  7. Cell Aging Inhibition  evaluation

In order to confirm the cellular aging inhibitory ability of UV-irradiated (UVB) irradiation of the mixture of the reed root and adenosine according to the present invention, SA-βSenescence Associated-ß-galatoctosidase, an indicator of aging marker, was analyzed (senescence detection kit; Biovision, USA ).

Specifically, the HaCaT cells prepared by attaching to the culture vessel in Example 5 were treated with a mixture of 100 μg / ml of the root herb used in Example 3 and 100 μM of adenosine, and irradiated with UVB 30 mJ / cm 2 and incubated for 24 hours. Thereafter, the medium was removed, washed with 1 ml PBS, and 0.5 ml of a fixing solution was added thereto, and the mixture was left at room temperature for 15 minutes to fix. To the immobilized HaCaT, 0.5 ml of a staining solution mixture (staining solution 470 μl, staining supplement 5 μl, 20 mg / ml X-gal in DMF (dimethylformamide) 25 μl) was added and cultured at 37 ° C. for 24 hours for cell staining. After washing with PBS, the number of stained cells was measured by a fluorescence microscope (Olympus Co, Japan). In addition, absorbance was measured using a microplate reader and fluorescence intensity was analyzed for the control group. The results are shown in FIG.

Aged cells have higher expression of β-galactosidase, and thus, the progress of aging can be confirmed by comparing the number of stained cells. As shown in FIG. 9, while aging was progressed due to the large number of stained cells during UVB treatment, it was confirmed that cell aging was markedly inhibited in the group treated with the root herb and adenosine mixture (UVB + brown root + Adenosine). Through this, it was confirmed that the effect of preventing cell aging when the mixture of brown root and adenosine after UV irradiation.

Example  8. MAPKs  Of signaling path Inhibitory ability  evaluation

HaCaT cells were treated with the mixture and Western blots were performed to confirm the effect of the mixture of brown root and adenosine according to the present invention on the phosphorylation of JNK MAPKs induced by UVB.

First, HaCaT cells were incubated for 24 hours in a medium containing 10% FBS, and then the cells were diluted with DMEM medium so as to be 1.5 × 10 ⁶ cells / well, and the cell suspension was placed in a 100 mm culture vessel in 10 ml portions and cultured for 24 hours. Cells were attached to the culture vessel. After 24 hours, a mixture of 100 μg / ml of brown root sheath used in Example 3 and 100 μM of adenosine was treated, irradiated with UVB 30 mJ / cm 2, and then incubated for another 24 hours. After 24 hours, the cells were washed 2-3 times with PBS, and then lysed for 30 minutes by addition of lysis buffer. Centrifugation at 13,000 xg removed cell membrane components, and only the supernatant was collected for bicinchoninic acid protein assay kit; Proteins were quantified using Pierce Biotechnology, USA. Thereafter, SDS-PAGE electrophoresis was performed on 20-50 μg of the separated protein using a 10% running gel and a 4.5% stacking gel (0.02 mA). Proteins isolated by electrophoresis were transferred to membranes at 100 V for 1 hour using polyvinylidene difluoride (PVDF) membrane and transfer buffer (20% methanol, 25 mM Tris, 192 mM glycine). The protein-transferred membrane was blocked for 1 hour with 5% skim milk solution.

Primary antibodies against JNK, p-JNK (Cell Signaling, USA) and GAPDH (Sigma, USA) at 0.1% TB at 0.1% TBST (10 mM Tris, 150 mM NaCl, pH 7.5 and 10% Tween 20) Diluted in the buffer solution and reacted overnight at 4 ℃ and washed three times with 0.1% TBST. After washing, the secondary antibody (Santa Cruz Biotechnology, USA) was diluted 1: 5000, reacted at room temperature for 1 hour, washed three times with 0.1% TBST, and then ECL Western blot detection system (ECL detection kit; Thermo Scientific, USA). And protein expression was analyzed using imagequantTM LAS 4000 (Fujifilm Life Science, Japan). The results are shown in FIG.

As shown in FIG. 10, it was confirmed that phosphorylation (p-JNK) of JNK was significantly reduced in the treatment group of brown root and adenosine mixtures, so that the brown root and adenosine mixture of the present invention can effectively regulate the MAPK signaling pathway. Through this, it was confirmed that the mixture according to the present invention can be usefully used as a skin anti-aging, anti-wrinkle, anti-inflammatory cosmetic composition through the improvement on skin aging, skin inflammation and skin wrinkles.

Example  9. MMP -9 expression Inhibitory ability  evaluation

Matrix metalloproteinases (MMPs) are matrix proteinases that increase the expression of certain MMP families such as MMP-1, MMP-2, and MMP-9 upon UV irradiation, which inhibits the synthesis of protocols and reduces collagen content. It promotes wrinkles of the skin and causes pigmentation, making the skin dull. Gelatin zymography was performed to confirm the effect of the mixture of the Korean herb and adenosine according to the present invention on the amount of MMP-9 expression.

Specifically, the HaCaT cells prepared by attaching to the culture vessel in Example 5 were treated with a mixture of 100 μg / ml of the root herb used in Example 3 and 100 μM of adenosine, and incubated for 24 hours after irradiating UVB 30 mJ / cm 2. After incubation, the supernatant was collected and mixed with 5X sample buffer solution, followed by electrophoresis using acrylamide gel containing 0.1% gelatin. The gel was then washed twice with 2.5% Tripton X-100 solution at room temperature for 10 minutes and then incubated with culture buffer for 48 hours at 37 ° C. Thereafter, the gel was stained with Coomassie blue solution for 1 hour and fixed with a destaining solution so that the gelatine degradation activity (destained white band) by MMP-9 was imagequantTM LAS 4000 (Fujifilm Life Science, Japan). ). The results are shown in FIG.

As shown in FIG. 11, MMP-9 protein activity was found to be significantly lower in the group treated with brown root and adenosine. Through the above results, it was confirmed that the knotweed and adenosine mixture according to the present invention can be usefully used as an anti-aging, anti-wrinkle cosmetic composition by inhibiting the expression of MMP-9 induced by UVB.

Example  10. Brown root  And adenosine mixtures Cosmetics  Composition preparation

In Example 3 to 4 was prepared in the composition of Table 1 containing a mixture of brown root and adenosine mixture confirmed to be excellent in cell viability and protective effect. In addition, the composition of the commercially available pink peel of the company was shown together as the comparative example 1.

No. Raw material The present invention
( weight% ) Comparative example  One
( weight% ) One Roots QS QS 2 Brown root 30.00 30.00 3 pH regulator 8.30 8.30 4 ethanol 2.00 2.00 5 1,2-hexanediol 2.00 2.00 6 Panthenol 0.70 0.70 7 Viscosity Modifier 0.54 0.54 8 Emulsifier 0.15 0.15 9 Pigment 0.10 0.10 10 Adenosine 0.04 - 11 Disodium ID 0.02 0.02 T O T A L 100.00 100.00

Experimental Example  1.of the present invention Cosmetics  Evaluation of Stability of the Composition Over Time

Comparative evaluation of stability over time at temperatures of 4 ° C., 25 ° C. and 40 ° C. was carried out using the cosmetic composition comprising the brown root herb and adenosine of the present invention prepared in Example 10. As a method of comparing stability over time, changes in appearance characteristics at refrigeration (4 ° C.), room temperature (25 ° C.) and high temperature (40 ° C.) were observed, and the results observed for a period of 3 months are shown in Table 2 0: good stability over time, ±: slight stability over time, +: stability over time).

Test substance Storage temperature (℃) 4 ℃ 25 ℃ 40 ℃ Example 10 0 0 0

As shown in Table 2, it was confirmed that the cosmetic composition of the present invention exhibits excellent temperature stability in all of the range of 4 to 40 ℃ can maintain the formulation stability of the cosmetic composition at refrigeration, room temperature and high temperature.

Experimental Example  2. of the present invention Cosmetics  Wrinkle improvement effect of the composition

Using a cosmetic composition comprising the root knotweed and adenosine of the present invention prepared in Example 10 was carried out a questionnaire about the skin improvement satisfaction. The cosmetic composition was used twice at intervals of two weeks after one application, with the application amount being 2 cc. A total of 20 evaluators were included and the results are shown in Table 3.

Evaluation item Score (persons) The complexion became clear. 17 Fine wrinkles are reduced 18 Increased elasticity 18 Skin became smooth 18 Moist 17

As shown in Table 3, the skin improvement satisfaction evaluation results of the cosmetic composition of the present invention was confirmed that the skin improvement satisfaction for clearing the complexion, decrease the fine wrinkles, increase the elasticity, increase the smoothness.

Experimental Example  3. of the present invention Cosmetics  Evaluation of skin response of the composition

The skin reaction evaluation was performed using the cosmetic composition containing the myofibril and adenosine of the present invention prepared in Example 10. The evaluator measured the average skin reactivity of the cosmetic composition of the present invention according to the evaluation criteria of Table 4, targeting a total of 20 people. The evaluation results are shown in Table 5.

sign score Evaluation standard - 0 no response ± One Faint erythema + 2 Weak erythema, edema ++ 3 Pronounced erythema. Edema, vesicles +++ 4 Severe erythema, edema, alveoli

Test substance 24 hours later Reaction Rate (%) ± + ++ +++ Example 5 - - - - 0

As shown in Table 5, it was confirmed that the cosmetic composition of the present invention is a cosmetic composition without irritation to the skin.

Experimental Example  4. Clinical Trial Example

The results of clinical trials of the cosmetic composition of the present invention are shown in FIGS. 12 and 13. Specifically, using the cosmetic composition of the present invention was applied once the amount of coating 2cc and once applied four times at intervals of two weeks after the application was taken pictures. In addition, the skin diagnostic device Derma Vision was used to analyze the wrinkles and elasticity of the patient's skin using UV.

As shown in Figure 12 and 13, when the cosmetic composition of the present invention was applied to the wrinkles, it was confirmed that the fine wrinkles and rough skin is improved to have an excellent effect in improving wrinkles.

As confirmed in the Examples and Experimental Examples it could be confirmed that the cosmetic composition for improving wrinkles of the present invention comprising gallium herb and adenosine has excellent properties in inhibiting skin aging and wrinkle improvement by ultraviolet rays.

Claims (8)

As a cosmetic composition for preventing skin aging or for improving wrinkles comprising gallium herb and adenosine,
The skin aging is characterized in that the skin photoaging (photoaging), the root knotweed is included in 25 to 35 parts by weight based on 100 parts by weight of the total composition, the adenosine is contained in 0.03 to 0.05 parts by weight based on 100 parts by weight of the total composition,
The knotweed is
(a) washing and grinding the roots;
(b) extracting the juice by mixing 1.8 to 2.2 parts by weight of water with respect to 1 part by weight of the pulverized root;
(c) first filtering the extracted juice;
(d) adding and mixing 0.12 to 0.18 parts by weight of starch and 0.08 to 0.12 parts by weight of yeast with respect to 1 part by weight of the first filtered brown root juice and fermenting at a temperature of 20 to 40 ° C; And
(e) aging the fermented root juice after the second filtration to add a yeast at a temperature of 20 to 40 ℃; A cosmetic composition for preventing skin aging or improving wrinkles, which is prepared by the method.
delete delete The method of claim 1, wherein the composition further comprises at least one organic solvent selected from the group consisting of ethanol, methanol, isopropanol, propylene glycol, butylene glycol, 1,2-hexanediol, glycerin and acetic acid ethyl ester Characterized in that the anti-aging or wrinkle cosmetic composition.
The organic solvent of claim 4, wherein the organic solvent is ethanol and 1,2-hexanediol, and 1 to 5 parts by weight of ethanol and 1 to 5 parts by weight of 1,2-hexanediol, respectively, based on 100 parts by weight of the total cosmetic composition. A cosmetic composition for preventing skin aging or improving wrinkles, characterized in that.
The composition of claim 1, wherein the composition is selected from the group consisting of lotion, emulsion, gel, cream, essence, pack, ampoule, lotion, cleaning agent, soap, body products, soap, oil, lipstick, spray, powder and foundation. A cosmetic composition for preventing skin aging or improving wrinkles, which is formulated as a species.
delete As quasi-drug composition for preventing skin aging or improving wrinkles, comprising gallium herb and adenosine,
The skin aging is characterized in that the skin photoaging (photoaging), the root knotweed is included in 25 to 35 parts by weight based on 100 parts by weight of the total composition, the adenosine is contained in 0.03 to 0.05 parts by weight based on 100 parts by weight of the total composition,
The knotweed is
(a) washing and grinding the roots;
(b) extracting the juice by mixing 1.8 to 2.2 parts by weight of water with respect to 1 part by weight of the pulverized root;
(c) first filtering the extracted juice;
(d) adding and mixing 0.12 to 0.18 parts by weight of starch and 0.08 to 0.12 parts by weight of yeast with respect to 1 part by weight of the first filtered brown root juice and fermenting at a temperature of 20 to 40 ° C; And
(e) aging the fermented root juice after the second filtration to add a yeast at a temperature of 20 to 40 ℃; A quasi-drug composition for preventing skin aging or improving wrinkles, which is prepared by the method.

Images