KR101998032B1 - Cosmetic Composition for Improving Skin Condition Comprising Rosa Damascena Callus Culture Extract or the fermented filtrates to improve periorbital wrinkles, skin brightness and calm troubled skin - Google Patents

Abstract

The present invention relates to a composition for improving skin condition comprising a damask rose callus culture extract or fermented filtrate thereof to improve skin elasticity, skin tone and calm troubled skin, and a manufacturing method thereof. More specifically, the present invention relates to cosmetic composition, by comprising a damask rose callus culture extract or fermented filtrate thereof as an active ingredient, helping skin whitening and skin tone improvement by inhibiting melanin synthesis, skin barrier improvement and skin elasticity by increasing FLG gene expression, and skin wrinkle reduction by increasing PIP production amount, and also helping to soothe and calm troubled skin caused by the reduction of COX-2, iNOS expression, and having an acne improvement effect, skin pore contraction effect and skin moisturizing effect due to increased AQP3 expression; and to a manufacturing method thereof.

Description

TECHNICAL FIELD [0001] The present invention relates to a cosmetic composition for enhancing skin elasticity, skin tone, and skin trouble, periorbital wrinkles, skin brightness and calm troubled skin}

The present invention relates to a composition for skin improvement comprising a multimask rose callus culture extract or a fermentation filtrate thereof for improving skin elasticity, improving skin tone and alleviating skin troubles, and a method for preparing the same, and more particularly, By containing the culture extract or the fermented filtrate thereof as an active ingredient, skin whitening and skin tone due to inhibition of melanin synthesis can be improved, skin barrier improvement and skin elasticity enhancement due to an increase in FLG gene expression, and improvement of skin wrinkles due to an increase in PIP production The present invention relates to a cosmetic composition having a skin moisturizing effect, a skin pore shrinking effect, and an acne improvement effect according to an increase in expression of AQP3, which is effective for relieving and relieving troubles associated with reduction of COX-2 and iNOS expression, and a preparation method thereof.

The human skin undergoes changes due to various internal and external factors as it gets older. Internally, secretion of various hormones that regulate metabolism is reduced, and immune cell function and cell activity are lowered, so that biosynthesis of immune proteins and biologic proteins necessary for the living body is reduced. As a result of the destruction of the ozone layer, the amount of ultraviolet rays reaching the surface of the sunlight increases and the environmental pollution is further increased. As a result, free radicals and active noxious oxygen are increased and the thickness of the skin decreases, Not only the elasticity is reduced but also skin troubles frequently occur. As skin aging progresses, there is a decrease in keratinocyte and fibroblast cleavage, a decrease in collagen synthesis, an increase in MMP production, and an increase in signal transmission of melanin production. As a result, wrinkles increase and skin elasticity decreases , And spots, freckles, and black spots are increased. Particularly, the balance of hormones is broken up in the forties, and cell regeneration ability, collagen synthesis ability, moisture content of the stratum corneum which is important for skin moisturization are significantly lowered. This is due to the reduction of stratum corneum lipid components, the decrease of natural moisturizing factors, and the decrease of clinical glycerol content of dry skin. As a result, in aging skin, the amount of water supplied from the lower epidermis to the stratum corneum is insufficient, so that the skin becomes more dry, resulting in inflammation and pruritus. On the other hand, the wrinkles of the skin are caused by the imbalance of synthesis and decomposition of collagen, and the synthesis of type I collagen in skin and the degradation enzyme MMP-1 are in balance. However, in photoaged skin, the synthesis of type I and III collagen is reduced and the activity of MMP-1 is increased.

Various methods for improving the skin aging phenomenon have been applied. Recently, a variety of plant resources have been studied for restoring skin damage as described above. In addition, researches are actively conducted to utilize various useful ingredients obtained from fermentation of various natural materials present in nature as cosmetic materials.

The present inventors have found that it is possible to improve skin whitening and skin tone by inhibiting melanin synthesis, improve skin barrier and skin elasticity by increasing FLG gene expression, improve skin wrinkles by increasing PIP production amount, and decrease COX-2 and iNOS expression As a result of efforts to discover materials having various functions such as skin moisturizing effect, skin pore shrinking effect and acne improvement effect according to the increase of expression of AQP3, It has been confirmed that water can be a cosmetic material having these various functionalities, and the present invention has been completed.

Another object of the present invention is to provide a method for producing a cosmetic composition for skin improvement comprising the above-described multimask rose callus extract or its fermented filtrate as an active ingredient.

 Accordingly, it is an object of the present invention to provide a cosmetic composition for improving skin comprising an extract of rose callus cultured in Da Mask or a fermented filtrate thereof as an active ingredient.

In order to solve the above problems, the present invention provides a multimask rose callus culture extract or a fermented filtrate thereof, a process for producing the same, a cosmetic composition containing the extract, and a process for producing the same.

Hereinafter, the present invention will be described more specifically.

The present invention relates to a method for producing a cosmetic composition for improving skin comprising a multimask rose culture or extract thereof, or a fermentation filtrate thereof, comprising the steps of:

Separating the cells from the plant tissue of Da Mask rose to induce callus induction; And a step of preparing a composition containing the obtained culture or an extract thereof.

In the present invention, the obtained multimask rose callus culture may be dried, pulverized and mixed with purified water to prepare a composition.

In the present invention, the obtained multimask rose callus culture may be dried, powdered, mixed with purified water, and then subjected to ultrasonic extraction or hot water extraction to prepare a composition.

In the present invention, the callus culture (culture) itself may be used, the culture may be used by filtration, the culture may be used after being crushed, or the culture may be dried and powdered and then dissolved in purified water.

In the present invention, "extract" means an extract obtained by powdering the extract of the callus culture, or by various known extraction methods such as a cold water extraction method, a hot water extraction method and an ethanol extraction method.

The extraction method in the present invention is not particularly limited, and examples thereof include cold extraction, ultrasonic extraction, reflux extraction, hot water extraction and the like. Here, in the case of hot water extraction, the hot water extract is obtained by heating at 80 to 100 ° C for 8 to 48 hours in a hot water distiller.

In the case of cold water extraction, cold water (15-25 ° C), for example, is mixed with the above-mentioned placental tissue culture or its dried powder and extracted for 3 days to obtain cold water extract.

Alternatively, it can be produced by a method of extracting using water, an organic solvent, or a mixed solvent thereof. The extracted liquid can be used directly or by concentrating and / or drying. In the case of extraction using an organic solvent, an organic solvent such as methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof. The active ingredient of the herbal medicine may be extracted at room temperature or warmed under the condition that the active ingredient is not destroyed or minimized. Depending on the organic solvent to be extracted, the degree of extraction and the degree of loss of the active ingredient of the medicament may differ. Therefore, an appropriate organic solvent should be selected and used. Particularly, in the present invention, an ethanol extract, preferably a 20-50% ethanol extract, specifically 50% ethanol extract, is preferable. As described in the examples, the extract is mixed with 50% ≪ / RTI >

In the present invention, the above extract may be used by concentration or dilution, and a distillate of the extract may be used.

(A) inducing and culturing callus from a multiparous rose tissue; And (b) a method for producing a cosmetic composition for skin improvement comprising a multimask rose callus culture or an extract thereof, comprising the step of producing a composition containing the callus culture or an extract thereof.

In one aspect, the present invention relates to a method for inducing and culturing callus from (a) damask rose tissue, treating the obtained damask rose callus culture with high frequency waves, And (b) preparing a composition containing the high-frequency treated callus culture or an extract thereof. The present invention also relates to a method for producing a skin-improving cosmetic composition containing the extract.

The present invention provides, in one aspect, (a) inducing and culturing a callus from a damask rose tissue and treating the resultant damask rose callus culture with high frequency; (b) preparing an extract of said high-frequency treated culture; (c) inoculating and fermenting the microorganism to the extract of the high-frequency treated culture; And (d) filtering the fermentation product;

And a fermentation filtrate of a multimask rose callus culture extract.

In the step (a), specifically, a medium suitable for inducing and culturing the calli can be selected. Any medium commonly used in the art can be used without limitation. For example, the composition of the MS medium (1 L standard) is 1650 mg of NH ₄ NO _3, 1900 mg of KNO ₃ , 440 mg of CaCl ₂ .2H ₂ O, MgSO ₄ .7H _{2 O370mg, KH 2 PO 4 170mg,} KI0.83mg, H 3 BO 3 6.2mg, MnSO 4 .4H 2 O22.3mg, ZnSO 4 .7H2O8.6mg, Na 2 MoO 4 .2H 2 O0.25mg, CuSO 4, 5H ₂ O 0.025 mg, CoCl ₂ .6H ₂ O 0.025 mg, FeSO ₄ .7H ₂ O 27.8 mg, Na ₂ EDTA.2H ₂ O 37.3 mg, Myoinositol 100 mg, Nicotinicacid 0.5 mg, Pyridoxine-HCl 0.5 mg, Thiamine- , Glycine 2mg, Sucrose 30000mg.

In the present invention, in the step (b), a composition containing each callus culture obtained as a result of culturing in step (a) may be prepared as a cosmetic composition of a proper form, or the culture may be prepared by a known extraction To obtain an extract in the form of a suitable cosmetic composition.

For example, in step (b), each of the callus cultures obtained in step (a) is dried, powdered and mixed with purified water to prepare a composition,

drying the callus culture obtained in step (a), pulverizing it, mixing it with purified water, and then preparing the composition by cold extraction, hot water extraction or ethanol extraction.

As another example, the culture obtained in the step (a) may be dried by hot air and powdered to evaporate water.

In the present invention, the fermentation filtrate means filtration of fermentation products obtained by inoculation with various microorganisms such as yeast and lactic acid bacteria.

There is no particular limitation to the types of microorganisms, but the products resulting from microbial fermentation can be used individually or in combination according to various species.

In the present invention, in the step (c), the microorganism may be yeast, lactic acid bacteria, or the like. For example, Galactomyces reesei, Saccharomyces cerevisiae, Lactobacillus plantarum, and Lactobacillus rhamnosus were used as yeast strains, and the yeast strains were YPD medium (BD, Cat. 242820) and the lactic acid bacteria were MRS medium (BD, Cat. Lt; / RTI >

In the present invention, the high-frequency processing may be performed four times at intervals of 15 minutes for one day at 100 or 400 kHz.

In one aspect, the present invention relates to a cosmetic composition for improving skin comprising, as an active ingredient, a multimask rose callus extract or a fermented filtrate thereof. Specifically, the content of the callus cultured extract or the fermentation filtrate thereof is not limited, but may be 0.1 to 10% by weight based on the total weight of the composition.

In the present invention, Rosa damascene which is an effective ingredient is Rosa damascene, a representative cultivar, which is grown in ancient times as Bulgaria, Turkey, France, and India, which are the largest producing areas, and the fragrance of flowers is silver and strong.

In the present invention, 'callus' is an oily tissue in which a cell restores the ability to divide and protects against scratches when the plant is injured, the tissue cut out from the plant is cultured in a medium containing auxin, An undifferentiated tissue or cell mass that occurs when a wound of a certain kind is injured or treated with auxin is permanently cleaved and proliferated by subculture in a new medium. These calluses are amorphous tissues or cell masses that lose their ability to induce normal organogenesis or tissue differentiation, and are mostly flow cells, and in a broad sense they also contain plant tumor tissues caused by infection such as agrobacterium. In other words, the cells in the plant body are immediately orientated when they multiply, and are assembled regularly as well as specific tissues. However, callus, which is a undifferentiated cell mass formed through tissue culture, is interpreted that when the stimulus such as various plant growth hormones is given from the outside, the control that has been maintained until then is released, and cells are spontaneously proliferated.

When the callus obtained by dedifferentiation is put into a liquid medium having the same composition and cultured under shaking, the cells are kept apart from each other and continue to multiply. This callus or cultured cell can be permanently cleaved and propagated by subculture in a new medium, which is essentially different from the differentiated cells in which the whole plant ages and dies. After several generations of subcultured callus or cultured cells are applied to a solid medium from which various plant growth hormones have been removed, eyes and roots come out and individual plants are restored. This regenerated plant is derived from the diagenesis of a single cultured cell, and the source of the cultured cell is derived from hypocotyledon or vesicular tissue.

In the present invention, "callus" may be derived from any part of the plant, but in one embodiment, it may be part of petal, calyx, fruit, ovary, ≪ / RTI >

In the present invention, "callus cultured" is a callus cultured in a medium. The medium can be used without limitation if there is a medium commonly used in callus culture in the technical field.

In the present invention, the skin improving agent is useful for improving skin whitening and skin tone by inhibiting melanin synthesis, improving skin barrier and skin elasticity by increasing FLG gene expression, improving skin wrinkles due to an increase in PIP production, 2, and iNOS expression, and also includes skin moisturizing effect, skin pore shrinking effect and acne improvement effect according to the increase of AQP3 expression, and various skin activity improvement functions.

In the present invention, the expression "comprising as an active ingredient" means that the cosmetic composition of the present invention can be used as a cosmetic composition for skin care, skin tone improvement, skin vitality enhancement, skin convergence, skin barrier enhancement, For example, collagen synthesis, elastin increase, skin convergence, wrinkle improvement, skin barrier enhancement, improvement, skin moisturizing, pore contraction, skin trouble relief, soothing, sebum suppression, acne improvement , Whitening, anti-inflammation, and the like.

Accordingly, the cosmetic composition according to the present invention is useful for improving skin, for example, for improving skin whitening and skin tone, for improving skin barrier and improving skin elasticity, for improving skin wrinkles, Skin pore contraction, and acne improvement.

In the skin barrier protection function according to the present invention, the skin barrier is composed of a stratum corneum, which is the outermost layer of the epidermis, and a corneocyte, which is mainly non-nucleated. The multilamellar lipid layer, composed of intercellular lipids such as ceramides, cholesterol, and fatty acids synthesized by keratinocytes of the skin barrier maintained through normal division and differentiation of epidermal cells, It plays a role. Meanwhile, omega hydroxyceramide in the intercellular lipids is chemically covalently bound to involucrin, which is a protein of the corneocyte outer layer, to form a corneocyte lipid envelope (CLE) Type interstitial lipids physically stabilizes the barrier function of the role is to play a role.

The composition of the present invention is delivered to the stratum corneum through skin application to promote the differentiation of keratinocytes, thereby improving the thickness of the skin layer. In addition, the composition has excellent effect of restoring skin barrier damage, And can be usefully used for the treatment and prevention of skin diseases caused. Skin diseases caused by skin barrier damage include, but are not limited to, atopic dermatitis, xeroderma, psoriasis, ichthyosis, acne and the like.

In addition, the mechanism of skin barrier protection was confirmed by increasing the amount of occludin and claudin-1 protein, which are closely related to each other. Filaggrin is one of several structural proteins that keratinocytes express at the differentiation stage. It is responsible for differentiation from the basal layer to the stratum corneum of the epidermis. It is responsible for the natural moisture factor (NMF) The present invention relates to the use of a multimask rose callus culture according to the present invention for the protection of skin barrier, enhancement and improvement of function as the gene expression of pilar green is significantly increased. .

In addition, the callus culture or the extract thereof or the fermentation filtrate thereof according to the present invention can also be used for skin protection by ultraviolet irradiation.

Also, the multimask rose callus culture according to the present invention or an extract thereof or a fermentation filtrate thereof has elastin expression ability. Elastin is a structural substance that exists in the extracellular matrix of connective tissue and is a substance that is present in the skin and imparts elasticity. Elastin is synthesized in cells with a polypeptide called Tropo elastin and then cross-linked between the tropoelastins outside the cell. Thus, elastin has a secondary or three-dimensional elasticity due to cross-linking of tropoelastin. Therefore, the callus culture or the extract thereof or the fermentation filtrate thereof according to the present invention can be utilized for enhancing and improving skin elasticity.

The cosmetic composition of the present invention may contain an acceptable carrier in cosmetic preparations. Herein, "an acceptable carrier for a cosmetic preparation" is a known or used compound or composition that can be contained in a cosmetic preparation, or a compound or composition to be developed in the future, which is toxic, instable or irritating It says nothing.

The carrier may be included in the cosmetic composition of the present invention in an amount of about 1% by weight to about 99.99% by weight, preferably about 90% by weight to about 99.99% by weight of the composition, based on the total weight thereof. However, since the ratio varies depending on the formulations described below in which the cosmetic composition of the present invention is prepared, and on the specific application site (face, neck, etc.) of the cosmetic composition and its preferable application amount, And should not be construed as limiting the scope of the invention.

Examples of the carrier include an alcohol, an oil, a surfactant, a fatty acid, a silicone oil, a wetting agent, a moisturizer, a viscous modifier, an emulsifier, a stabilizer, an ultraviolet scattering agent, an ultraviolet absorber, a coloring agent and a perfume. The compounds / compositions which can be used as the above-mentioned alcohol, oil, surfactant, fatty acid, silicone oil, humectant, humectant, viscous modifier, emulsifier, stabilizer, ultraviolet scattering agent, ultraviolet absorber, The person skilled in the art can select and use the appropriate substance / composition.

As an embodiment of the present invention, the cosmetic composition according to the present invention may contain glycerin, butylene glycol, propylene glycol, polyoxyethylene hydrogenated castor oil, ethanol, triethanolamine, etc., in addition to the above- Antiseptic, antiseptic, coloring agent, purified water and the like can be contained as needed.

The cosmetic composition according to the present invention can be prepared in various forms such as lotion, essence, gel, emulsion, lotion, cream (water-in-oil type, water-in-water type, multiphase), solution, suspension (Soft capsules, hard capsules) with a coating such as anhydrous products (oil and glycol), gel, mask, pack, powder, or gelatin.

The skin of the present invention includes not only a face but also a scalp and a whole body. The cosmetic composition applicable to such scalp is a shampoo, a rinse, a treatment, a hair removal agent, etc., and a body cleanser And can be manufactured in various forms as an application of the composition.

The method for manufacturing the cosmetic composition according to the present invention is not limited to the above-described manufacturing method, and may be manufactured by a method partially modified by a person having ordinary skill in the art.

When the cosmetic composition is manufactured from a cosmetic composition, the cosmetic composition of the emulsified formulation includes nutritional lotion, cream, essence and the like. It can also be prepared in the form of adjuvants which can be applied topically or systemically, which are conventionally used in the field of dermatology by containing a dermatologically acceptable medium or base.

In addition, suitable cosmetic formulations include, for example, solutions, gels, solid or kneaded anhydrous products, emulsions obtained by dispersing an oil phase in water, suspensions, microemulsions, microcapsules, microgranules or ionic forms (liposomes) May be provided in the form of a follicular dispersing agent of the type, cream, skin, lotion, powder, ointment, spray or conical stick. It can also be prepared in the form of a foam or an aerosol composition further containing a compressed propellant.

The cosmetic composition of the present invention may further contain at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softening agents, antioxidants, suspending agents, stabilizers, foaming agents, perfumes, Or any other conventionally used in cosmetics such as nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, barrier agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, ≪ RTI ID = 0.0 > cosmetics < / RTI > such as botanical ingredients, or adjuvants conventionally used in the field of dermatology. And, the above ingredients can be introduced in amounts commonly used in the field of dermatology.

The cosmetic composition containing the multimask rose callus extract or the fermentation filtrate thereof according to the present invention can be used for skin barrier improvement, skin wrinkle protection, skin whitening improvement, skin inflammation inhibition, skin moisturizing, skin pore contraction, sebum suppression, It can be used as a material for various functional cosmetics.

FIG. 1 shows the results of examining the degree of toxicity of skin cell to the multimask rose callus extract obtained according to the present invention and the fermented extract of the multimask rose callus cultured.
FIG. 2 shows the results of confirming the increase of FLG gene expression in the multi-mask rose callus extract obtained according to the present invention and the fermented extract of the multi-mask rosette callus.
FIG. 3 shows the results of confirming the effect of improving the skin wrinkles on the multi-mask rose callus extract and the multi-mask rose callus fermentation extract obtained according to the present invention.
FIG. 4 shows the result of skin whitening effect on the extract of the multi-mask rose callus obtained according to the present invention and the fermented extract of the multi-mask rose callus.
FIG. 5 shows the results of confirming the skin inflammation improving effect of the multi-mask rose callus extract obtained according to the present invention and the fermented extract of the multi-mask rose callus.
FIG. 6 shows the skin moisturizing effect of the multi-mask rose callus extract obtained according to the present invention and the fermented extract of the multi-mask rose callus.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

Example 1-1: induction of damask rose callus according to the present invention

In order to induce the callus from the damask rose petals, the petals were immersed in 70% ethanol for 30 seconds, sterilized by shaking in 2% sodium hypochlorite for 5 minutes, and then sterilized by washing with sterilized water. The disinfected rose petals were cut with a sharp knife so that a growth regulator of 0.5 mg / L of indole-3-acetic acid, 0.2 mg / L of Zeatin, 30 g / L of sucrose, After inoculating MS base medium containing 2.0 g / L, callus was induced by culturing in a culture room at 23 ~ 27 ° C for 8 weeks. The induced calli were propagated by subculture at intervals of 4 weeks.

Example 1-2: Culture and mass production of damask rose callus according to the present invention

The polysaccharide rosette collected through aseptic subculture was then uniformly adjusted to a temperature of 25 ° C, a humidity of 70% and an air supply of 0.1 vvm in a liquid medium of the same composition except agar using a bioreactor. And then mass-produced.

The mass-produced damask callus was mass-produced in the bioreactor and connected to a high-frequency wave generator at 100 or 400 kHz for four times at intervals of 15 minutes for one day, and repeated for one week or two weeks. The samples were incubated for one week in the medium and the high-frequency treated samples were collected and vacuum-dried at 60 DEG C for 2 to 5 days.

Example 1-3: Multicolor rose callus culturing extract according to the present invention Produce

10 g of the dried callus cultured powder was placed in a vessel, and 1 L of purified water was added thereto, followed by hot extraction. The hot water extraction was carried out by heating at 80 ~ 100 ℃ for 8 ~ 48 hours. After extraction, the solid matter was removed by filtration through a mesh.

Examples 1-4: Preparation of the multi-mask rose callus fermentation filtrate according to the present invention

The microorganisms used for grafting the previously prepared polysaccharide rosette were the two kinds of yeast Galactomyces reessii and Saccharomyces cerevisiae and the two lactic acid bacteria Lactobacillus plantarum and Lactobacillus rhamnosus. Yeast strains were cultured on YPD medium (BD, Cat. 242820) Were prepared by culturing in MRS medium (BD, Cat. 288130).

The fermentation was carried out at 30 ° C and 100 rpm shaking incubator conditions to inoculate the culture broth of each strain to 1% After fermentation, the supernatant was obtained by centrifugation for sterilization, and then sterilized at high temperature and high pressure (121 ° C, 1.5 atm) and filtered through 0.2 μm syringe filtration to produce a fermented product.

Experimental Example 1: Test for effect on skin cell survival rate

In this experiment, the degree of toxicity of the skin cancer cells to the extracts of the multimask rose callus cultured from Example 1 and the fermented extracts of the multiparous rose callus were examined.

To this end, HaCaT cells (human keratinocyte, keratinocyte), Detroit551 cells (human fibroblast) and B16F1 cells (mouse melanocyte, melanocyte) were cultured in 96 well plates with DMEM medium containing 10% FBS And cultured for 24 hours in a thermostat under the conditions of 5% CO ₂ and 37 ° C. Then, the extracts of the test substances were treated with cells to a final concentration of 0.5, 1, 2, 5%, and further cultured for 24 hours. Then, 4 μL of 5 ㎎ / mL MTT solution was added to each well and cultured for 4 hours. After removing the culture medium, 100 μL of DMSO (dimethyl sulfoxide) solution was added to dissolve the cells, and the absorbance was measured at 540 nm using a spectrophotometer. The survival rate of skin cells was calculated as a percentage based on the absorbance intensity of the control group using purified water according to the following formula.

Cell viability (%) = [(absorbance of test group - absorbance of control group) / absorbance of control group] × 100

As a result, as shown in Fig. 1, each of the multi-mask rose callus extract of the present invention, two kinds of yeast fungi and two kinds of lactic acid bacteria were inoculated, Survival rates were not affected. Therefore, it was confirmed that both the damask rose callus extract and the damask rose callus extract did not stimulate skin cells.

Experimental Example 2: Skin barrier improvement gene expression test

In order to examine the skin barrier improvement effect of the multi-mask rosette callus extract and the multi-masked rosette callus filtrate prepared in Example 1, the present inventors examined the expression level of FLG (filaggrin) known as a natural moisturizing factor .

HaCaT cells were seeded in 96-well plates and cultured for 24 hours under cell culture conditions. Thereafter, the test substance extracts were treated with cells to a final concentration of 0.5, 2%, and further cultured for 24 hours. Real-time PCR was performed to confirm the gene level of FLG, and the test procedure was as follows.

SuperPrep ^TM celllysis & RTKitforq PCR (TOYOBO, Cat. SCQ-101) was used for RNA isolation and cDNA synthesis. The cells were washed once with PBS (Phosphate Buffer Saline), and reacted for 5 minutes with 50 μL cell lysis mixture (containing gDNA remover). Then stop solution was added. 8 μL of extracted mRNA was added to 32 μL of RT reaction mixture, and cDNA was synthesized by PCR at 37 ° C for 15 minutes, 50 ° C for 5 minutes, and 95 ° C for 5 minutes. Real-time PCR analysis was carried out using Thunderbird ^TM SYBRqPCRMix (TOYOBO, Cat. QPS-201) as the template synthesized above to compare and analyze gene expression. The primers used in this study were QuantiTect ^® primerassays (GAPDH; Cat.QT01192646, FLG; Cat.QT02448138) from Qiagen, and the amount of FLG mRNA expression was quantified by GADPH. Real-time qPCR conditions were first performed at 95 ° C for 15 minutes, followed by 40 cycles of 94 ° C for 15 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds per cycle.

As a result, as shown in FIG. 2, both of the multimask rose callus extract and the multimask rose callus fermentation extract of the present invention showed the FLG mRNA expression level increased in a dose-dependent manner in comparison with the control group. Therefore, both the extracts of DAMAS rose callus and the extracts of DAMAS rose callus showed improvement of skin barrier and skin elasticity with increasing FLG gene expression.

Experimental Example 3: Test for improving skin wrinkles

In order to examine the wrinkle-improving effect of the multi-mask rosette callus extract and the multi-mask rosette callus extract prepared through Example 1, procollagen type I C-peptide (PIP), which is a precursor of collagen synthesis, ) Were measured.

Detroit551 cells were plated in 48 well plates and cultured for 24 hours under cell culture conditions. Then, the extracts of the test substances were treated with cells to a final concentration of 0.5, 2%, and further cultured for 48 hours.

A 20 μL aliquot of the supernatant from the culture medium was transferred to an antibody coated microtiter plate with 20 μL of the PIP standard in a PIP EIA Kit (Takara, Cat. MK101), and 100 μL of peroxidase labeled antibody solution Were added to each well and reacted at 37 캜 for 3 hours. After removing the medium and washing 4 times with 200 μL of PBS, 100 μL of substrate solution was added to each well and reacted at room temperature for 15 minutes in the shade condition. After that, 100 μL stop solution (1 NH ₂ SO ₄ ) was added and the absorbance at 450 nm was measured. After measuring the absorbance at 450 nm, the PIP standard concentration curve was prepared and the amount of PIP formation in the sample corrected to the protein amount was calculated.

As a result, as shown in FIG. 3, the PMA production was increased in the treatment-concentration-dependent manner in each of the multimask rose callus extract of the present invention, the two yeasts and two kinds of lactic acid bacteria inoculated with the multimask rose callus fermentation filtrate . Therefore, it was confirmed that both of the extracts of Daum mask rose callus and Daum mask rose callus helped to improve the wrinkles of the skin due to the increase of PIP production.

Experimental Example 4: Skin whitening improvement test

In order to examine skin whitening effect of the multi-mask rose callus extract and the multistage rose callus fermentation filtrate prepared in Example 1, the inhibition rate of the melanin pigment, which is a black pigment, was examined.

B16F1 cells were plated in 6 well plates and cultured for 24 hours under cell culture conditions. Subsequently, the extracts of the test substances were treated with cells to a final concentration of 0.5, 2%, and further cultured for 72 hours. At this time, α-MSH (Melanin stimulating hormone) was added to the final 10 nM to promote melanin production.

After the culture medium was removed, 400 μL of 1 N NaOH was added, and the cells were lysed by incubating at 70 ° C for 30 minutes in a thermostatic chamber. The dissolved solutions were measured for absorbance at 492 nm using a spectrophotometer for melanin content analysis. The inhibition rate of melanin production was calculated according to the following equation and corrected by the amount of protein and expressed as a percentage.

(%) = [1- (C-A) / (B-A)] x 100

A = amount of melanin synthesis per protein in the control group not treated with? -MSH

B = amount of melanin synthesis per protein in control group treated with? -MSH

C = amount of melanin per protein in the test group treated with? -MSH

As a result, as shown in Fig. 4, the extract of the multimask rose callus of the present invention, the two kinds of yeast fungi and two kinds of lactic acid bacteria were inoculated with the multimask rose callus fermentation filtrate, all of which were significantly inhibited by melanin synthesis Respectively. Therefore, it was confirmed that both the extracts of Daum mask rose callus and the extracts of Daum mask rose callus help to improve whitening and skin tone by inhibiting melanin synthesis.

Experimental Example 5: Skin inflammation inhibitory effect test

In order to investigate the skin inflammation inhibitory effect of the multi-mask rosette callus extract and the multi-masked rosette callus filtrate prepared in Example 1, COX-2 (Cyclooxygenase-2), which is known to be involved in the inflammatory reaction, , and iNOS (Inducible Nitric Oxide Synthase).

HaCaT cells were seeded in 96-well plates and cultured for 24 hours under cell culture conditions. Thereafter, the inflammatory reaction was induced by UVB irradiation, and the test substance extracts were treated with cells to a final concentration of 0.5, 2%, and further cultured for 24 hours. Real-time PCR was performed to confirm the gene level of COX-2 and iNOS, and the test procedure was as follows.

SuperPrep ^TM celllysis & RTKitforq PCR (TOYOBO, Cat. SCQ-101) was used for RNA isolation and cDNA synthesis. The cells were washed once with PBS, and reacted with 50 μL cell lysis mixture (containing gDNA remover) for 5 minutes. Then stop solution was added. 8 μL of extracted mRNA was added to 32 μL of RT reaction mixture, and cDNA was synthesized by PCR at 37 ° C for 15 minutes, 50 ° C for 5 minutes, and 95 ° C for 5 minutes. Real-time PCR analysis was carried out using Thunderbird ^TM SYBRqPCRMix (TOYOBO, Cat. QPS-201) as the template synthesized above to compare and analyze gene expression. The primers used in this study were QuantiTect ^® primerassays (GAPDH; Cat.QT01192646, COX-2; Cat. QT00040586, iNOS; Cat. QT01323189) from Qiagen and the amounts of mRNA expression of COX-2 and iNOS in the samples were quantified by GADPH . Real-time qPCR conditions were first performed at 95 ° C for 15 minutes, followed by 40 cycles of 94 ° C for 15 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds per cycle.

As a result, as shown in FIG. 5, both of the multi-mask rosette callus extract of the present invention, the two-species yeast fungus and the two kinds of lactic acid bacteria inoculated with the multimask rose callus fermentation filtrate were significantly more COX-2, iNOS expression. Therefore, it was confirmed that the inhibition of inflammation was mitigated by the reduction of expression of COX-2 and iNOS, which are inflammatory factors of both damask rose callus and fermented rosemary callus.

Experimental Example 6: Skin moisturizing effect test

In this experiment, changes in the expression level of AQP3 as water / glycerol transport were investigated in order to examine the skin moisturizing effect of the multi-mask rose callus extract prepared in Example 1 and the multi-masked rosette callus filtrate.

HaCaT cells were seeded in 96-well plates and cultured for 24 hours under cell culture conditions. Thereafter, the test substance extracts were treated with cells to a final concentration of 0.5, 2%, and further cultured for 24 hours. Real-time PCR was performed to confirm the gene level of AQP3, and the test procedure was as follows.

SuperPrep ^TM celllysis & RTKitforq PCR (TOYOBO, Cat. SCQ-101) was used for RNA isolation and cDNA synthesis. The cells were washed once with PBS, and reacted with 50 μL cell lysis mixture (containing gDNA remover) for 5 minutes. Then stop solution was added. 8 μL of extracted mRNA was added to 32 μL of RT reaction mixture, and cDNA was synthesized by PCR at 37 ° C for 15 minutes, 50 ° C for 5 minutes, and 95 ° C for 5 minutes. Real-time PCR analysis was carried out using Thunderbird ^TM SYBRqPCRMix (TOYOBO, Cat. QPS-201) as the template synthesized above to compare and analyze gene expression. The primers used in the experiments were QuantiTect ^® primerassays (GAPDH, Cat. QT01192646, AQP3, Cat. QT00212996) from Qiagen and the quantities of mRNA expression of AQP3 in the samples were quantified by GADPH. Real-time qPCR conditions were first performed at 95 ° C for 15 minutes, followed by 40 cycles of 94 ° C for 15 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds per cycle.

As a result, as shown in FIG. 6, both of the multimask rose callus extract and the multimask rose callus fermentation extract of the present invention showed the amount of mRNA expression of AQP3 which was increased depending on the treatment concentration. Therefore, both moisturizing effect of AQP3 gene expression was confirmed by both the extract of rose callus and the extract of rosemary callus.

Experimental Example 7: In Vitro Test for Skin Pore Shrinkage

In order to measure the pore-shrinking effect of the multimask rose callus extract of the present invention, the two-type yeast fungus and two lactic acid bacteria inoculated with the multimask rose callus filtration product, the pore shrinking effect was tested using hemoglobin as a protein replacing the skin The test procedure is as follows.

2 mg of 1 mg / mL hemoglobin solution was added to each of 2 ml of the callus cultured from Daimark rose callus and 2 ml of the callus fermented with rosemary rosette, followed by shaking for 30 seconds, followed by centrifugation at 3,500 rpm for 10 minutes. 2 mL of purified water was added to 1 mL of the supernatant, and the absorbance was measured at 407 nm using a spectrophotometer.

As a result, as shown in Table 1 below, the shrinkage effect was evaluated by measuring the amount of hemoglobin deposited in a composition containing a 0.5-2% concentration of a multimask rose callus extract and a multimask rose callus fermentation filtrate, and the hemoglobin precipitation (%) Was higher, the pore shrinkage effect was judged to be better. Therefore, it was confirmed that the pore shrinking effect was excellent in the concentration - dependent manner of the multi - masked callus culture extract and the multistage rosemary callus fermentation filtrate.

Concentration (%) Precipitation of hemoglobin (%) Purified water (negative control) 0 0.1 Tannic acid (positive control) One 40 Damask rose
extract 0.5 One 1.0 3 2.0 5 Damask rose
Callus culture extract 0.5 13 1.0 20 2.0 33 Damask rose
Callus fermentation filtrate 0.5 8 1.0 23 2.0 35

Experimental Example 8: Anti-sebum effect test

In order to confirm the sebum inhibitory effect of the callus extracts of Da masked rose callus and Da masked rose callus, sebum secretion amount of skin was measured using Sebum meter SM810 4 weeks after 10 subjects. Experiments were performed at 22 ± 2 ° C and 40 ± 5% humidity. When the oily and the measured value is more than 220 ㎍ / cm ^2, when the normal skin is 100~220 ㎍ / cm ^2.

As a result, as shown in Table 2, the 1% Da Mask rose callus culture extract and Da Mask rose callus fermentation filtrate showed a superior inhibitory effect on sebum secretion compared to the control group.

sample Sebum suppression effect 0 day (before use) 28 days (after use) Control group (purified water) 240 233 One 1% damask rose extract 241 193 2 1% Da Mask Rose Callus Culture Extract 240 173 3 1% Da Mask Rose callus fermentation filtrate 242 161

Experimental Example 9: Effect of improving acne

10 male and female acne sufferers were given lotion callus culture extracts of 1% concentration prepared in Example 1 throughout the face for 4 weeks in morning and evening. The evaluation of the acne improvement effect was made by evaluating the pre-test condition by 1-3 points according to the degree of the person who participated in the experiment, and then evaluating the acne healing effect. The results are shown in the table.

As a result, as shown in Table 3, the improvement effect of acne was confirmed in the majority of subjects after using the flexible lotion containing the 1% Damask rose callus extract for 4 weeks.

Subject Pre-test condition Effect after 2 weeks Effect after 4 weeks Subject 1 2 + + Subject 2 One + ++ Subject 3 One - + Subject 4 2 - - Subject 5 2 + ++ Subject 6 2 + + Subject 7 3 ++ +++ Subject 8 2 - ++ Subject 9 3 + + Subject 10 2 + ++

<Evaluation before the test> 1 = a little acne 2 = a little acne 3 = severe acne

<Acne Effect> -: Almost no effect, +: slight effect, ++: Much relief, +++: Completely healed

Claims (10)

(a) inducing and culturing a callus from a multi-masked rosette and treating the resulting multimask rose callus culture with high frequency of 100 to 400 kHz four times at intervals of 15 minutes;
(b) preparing an extract of said high-frequency treated culture;
(c) adding to the extract of the high-frequency treated culture a yeast strain of Galactomyces reesei, Saccharomyces cerevisiae, lactobacillus plant Lactobacillus plantarum or Lactobacillus lambosus (Lactobacillus rhamnosus) microorganism; And
(d) filtering the fermentation product;
Wherein the composition comprises a fermented filtrate of a multimask rose callus extract. A cosmetic composition for skin improvement comprising a fermented filtrate of a multimask rose callus extract according to claim 1. The cosmetic composition for improving skin according to claim 2, wherein the composition is used for skin whitening and skin tone improvement. [Claim 3] The cosmetic composition for improving skin according to claim 2, wherein the composition is for skin barrier improvement and skin elasticity enhancement. The cosmetic composition for improving skin according to claim 2, wherein the composition is for improving skin wrinkles. [Claim 3] The cosmetic composition for improving skin according to claim 2, wherein the composition is used for alleviating and relieving skin troubles. The cosmetic composition for improving skin according to claim 2, wherein the composition is for skin moisturizing. [Claim 3] The cosmetic composition for improving skin according to claim 2, wherein the composition is for skin pore shrinkage. The cosmetic composition for improving skin according to claim 2, wherein the composition is for improving acne. delete

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