KR20200014570A - Cosmetic Composition for Improving Skin Condition Comprising Passiflora alata to improve skin radiance and vitality - Google Patents

Abstract

The present invention relates to a cosmetic composition for improving skin conditions, comprising a Passiflora alata callus culture or an extract thereof. More particularly, the present invention relates to a cosmetic composition for improving skin radiance, enhancing skin vitality, preventing or reducing skin wrinkles, enhancing skin elasticity, providing skin astringency, protecting a skin barrier function, protecting the skin from UV rays, and having anti-atopic dermatitis, anti-inflammation, anti-oxidation and anti-aging functions, etc., by including a Passiflora alata callus culture or an extract thereof as an active ingredient.

Description

Composition for Improving Skin Condition Comprising Passiflora alata to improve skin radiance and vitality

The present invention relates to a cosmetic composition for skin improvement comprising the wing stem fashion flower callus culture or its extract, and more particularly, by providing the wing stem fashion flower callus culture or its extract as an active ingredient, giving skin vitality The present invention relates to a cosmetic composition having functions of increasing skin vitality, preventing or improving skin wrinkles, enhancing skin elasticity, improving skin convergence, protecting skin barrier function, protecting skin from ultraviolet rays, antioxidants, and anti-aging.

Recent research has identified a biological clock molecular network that governs circadian rhythms. Since the 1970s, the biological clock that regulates the expression of genes related to physiological metabolism and body rhythms of organisms according to the day and night cycles of the earth's rotation is becoming more detailed since the 1970s, and the study of diseases related to the cycle is being actively conducted. Jeffrey C. Hall, Professor, Maine University, Michael Rosbash, Brandeis University, and Michael W. Young, Professor Rockefeller University, have a model animal, Drosophila, in a 24-hour day and night cycle. He was honored with the 2017 Nobel Prize for his work on identifying the mechanisms by which circadian genes appear. The winners followed up the genes in the 1980s, after the earliest existence of a gene associated with the clock, revealed the basic mechanism of the clock. The molecular network of these biological clock genes contains clock genes that develop as they adapt to day and night changes. Clock genes called CLOCK (Clock circadian regulator) and PER1 (Period circadian protein) are present in organs such as the heart, lungs, fat, blood vessels and kidneys, as well as brain tissues such as the cerebellum, midbrain and hypothalamus. .

Their research has shown that the biological cycle is a biological phenomenon that occurs when the concentration of a protein (PER) expressed by a gene called pyriod changes every 24 hours. As the PER protein produced by the pyriod gene increases and continues to accumulate in the cytoplasm outside the nucleus, the PER protein now binds to other enzymes and enters the nucleus to inhibit the expression of the pyriod gene. The cycle of the livelihood clock appears as a molecular oscillation that increases and decreases the concentration of PER protein. Later, as other genes (proteins) were discovered that regulate the stability and functionality of these basic mechanisms, the mechanism of the 'bio clock', which is precisely and finely regulated, became known. At this stage, the dimer of CLOCK and BMAL1 protein triggers the expression of Period (PER) and Cryptochrome (CRY) family of feedback genes located in the lower stages as transcription factors, and the gene products burn higher level transcription factors. It forms a series of molecular circulatory structures that activate. These molecular networks are known to affect a variety of physiological processes by triggering the circadianity of genes regulated downstream (Stratmann and Schibler, 2006, J Biol Rhythms 21: 494). In other words, the circadian rhythm is controlled to be repeated daily through the associated feedback of the CLOCK / PER protein to activate the biological clock.

Skin cells, keratinocytes, melanocytes, and fibroblasts also have independent biological clock systems. The skin biological clock regulates skin physiological activity, which is the circadian rhythm of skin cell proliferation and differentiation, hydration and transepidermal water loss (TEWL), capillary blood flow, sebum production, temperature, surface pH, and wrinkle formation. Seems. If the rhythm of this process is broken, it will not produce the necessary physiological response at the right time, break the homeostasis of the skin, promote skin aging, and seriously cause skin cancer.

As human skin ages, it changes with a variety of internal and external factors. Internally, the secretion of various hormones that regulate metabolism is reduced, and the function and activity of cells of immune cells is reduced, thereby reducing the biosynthesis of immune proteins and constituent proteins necessary for the living body. Externally, due to the destruction of the ozone layer, the amount of ultraviolet rays reaching the surface of the sunlight increases and environmental pollution is intensified, resulting in the increase of free radicals and active harmful oxygen, which reduces the thickness of the skin and increases wrinkles. Not only does it reduce elasticity, but it also causes various changes such as frequent skin problems.

As skin aging progresses, keratinocytes and fibroblasts may divide, reduce collagen synthesis, increase MMP production, and increase melanin signaling, resulting in increased wrinkles and reduced skin elasticity. And blemishes, freckles and blotch are to be increased. In particular, the hormone balance is broken around 40s, and the moisture content of the stratum corneum, which is important for cell regeneration, collagen synthesis, and skin moisturization, is significantly lowered. It is suggested that this is due to the decrease in the stratum corneum lipid composition, the decrease in the natural moisturizing factor, the decrease in the amount of glycerol in dry skin. As a result, in aging skin, the amount of water supplied from the lower epidermis to the stratum corneum is insufficient, resulting in severe skin dryness, which causes inflammation and pruritus. On the other hand, the wrinkles of the skin are due to the imbalance of collagen synthesis and degradation, which is usually balanced by the synthesis of type I collagen and MMP-1, its degradation enzyme. However, in photo-aged skin, the synthesis of type I and III collagen is decreased, and the activity of MMP-1 is increased. These MMPs (matrix metalloproteinases) are proteolytic enzymes that degrade the extracellular matrix and are known to be involved in wound healing or tissue regeneration during normal conditions.

The present inventors have made efforts to discover materials having various functionalities such as skin vitality, skin vitality improvement, skin wrinkle improvement, skin regeneration, skin barrier function enhancement, wrinkle improvement, etc., Wingstem fashion flower callus culture or extract thereof It revealed that it has a biological clock control effect, confirmed that it can be a variety of functional cosmetic material, and completed the present invention.

Therefore, an object of the present invention to provide a cosmetic composition for skin improvement Wingstem fashion flower callus culture or its extract as an active ingredient.

Another object of the present invention is to provide a method for preparing a cosmetic composition for skin improvement comprising the wing stem fashion flower callus culture or its extract as an active ingredient.

In order to solve the above problems, the present invention provides a Winstem fashion flower callus culture or its extract and its preparation method, a cosmetic composition containing the extract.

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

The present invention relates to a cosmetic composition for skin improvement comprising a wing stem fashion flower callus culture or its extract as an active ingredient in one aspect. Specifically, the wing stem fashion flower callus culture or its extract is not limited to the content, it may contain 0.1 to 10% by weight relative to the total weight of the composition.

In the present invention, there are several kinds of wing stem fashion flowers, but a kind of Alata passionflower (Passiflora alata) grows about 4m in stem, and the leaves are deeply divided into five branches like palms. A flower about 8 cm in diameter has five petals and calyx leaves, each with light red or blue inside, and the calyx leaves are white, light pink, or light blue inside. The fruit is egg-shaped and has a sweet and sour taste that is also used for food.

 In the present invention, 'callus' is an oily tissue in which a cell recovers division ability and prevents and enlarges the wound when the plant is injured. The tissue cut out of the plant is cultured in an auxin-containing medium, It refers to undifferentiated tissue or cell mass generated when wounding certain kinds of plants or treating wound sites with auxin, and can be permanently divided and proliferated by passage in fresh medium. These callus are amorphous cells or masses of cells that have lost their ability to cause normal organogenesis or tissue differentiation, and are mostly flow cells, and broadly include plant tumor tissues caused by infections such as agrobacterium. In other words, the cells in the plant are immediately oriented as they proliferate and are assembled regularly into organs as well as specific tissues. However, callus, an undifferentiated cell mass formed through tissue culture, can be interpreted to proliferate arbitrarily by releasing control that has been maintained until then when various plant growth hormones are stimulated from the outside.

When the callus obtained by dedifferentiation is put into a liquid medium having the same composition and shaken, the cells are separated and continue to proliferate as a suspended state. The callus or cultured cells are essentially different from the differentiated cells in which the whole plant ages and dies by being able to divide and proliferate permanently by passage in fresh medium. Callus or cultured cells, which have been passaged for several generations, are applied to a solid medium from which various plant growth hormones have been removed. This regenerated plant is derived from the division and proliferation of a single cultured cell, and the source of the cultured cell is derived from axons or aquatic tissues.

In the present invention, "wingstem fashion flower callus" can be any one derived from any part of the wing stem fashion flowers, in one embodiment, wing petals, calyx, fruit, ovary, placenta tissue of the wing stem fashion flower Or callus derived by cutting cotyledon from germinated seedlings of seeds.

In the present invention, the "wingstem fashion flower callus culture" is a culture of the wingstem fashion flower callus in the medium. The medium can be used without limitation as long as there is a medium generally used for callus culture in the art.

In the present invention, the skin improvement for skin vitality, skin vitality, skin convergence, wrinkle improvement, skin barrier strengthening or improvement, skin regeneration, moisturizing, maintaining elasticity, skin barrier function protection, improvement, anti-inflammatory, It contains various skin-activating functions such as atopy, antioxidant, and anti-aging.

The present invention provides a method for preparing a cosmetic composition for skin improvement comprising a callus culture of Wingstem fashion flowers or an extract thereof:

(a) separating the cells from the wing stem fashion flower tissues and incubating by inducing callus; And (b) preparing a composition containing the culture obtained from step (a) or an extract thereof.

In the present invention, the step (b) may include the step of preparing a composition by drying, powdering and mixing the Winstem fashion flower callus culture obtained in step (a).

In the present invention, step (b), the Winstem fashion flower callus culture obtained in step (a) is dried, powdered and mixed in purified water, and then ultrasonic extraction or hot water extraction to prepare a composition It may include.

In the present invention, the Winstem fashion flower callus culture (culture) itself, the culture is used by filtration, the culture is used by crushing, or the culture is dried and powdered, and then dissolved in purified water Can be used

In the present invention, "extract" refers to an extract obtained by various extracting methods known in the art such as powdered extract of the Winstem fashion flower callus culture, cold water extraction method, hot water extraction method, ethanol extraction method.

The extraction method in the present invention is not particularly limited, and for example, cold extraction, ultrasonic extraction, reflux extraction, hot water extraction and the like. Here, in the case of hot water extraction, heated to 80 ~ 100 ℃ for 8 to 48 hours in a boiling water distillation to obtain a hot water extract.

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

Or by extraction using water, an organic solvent, or a mixed solvent thereof. The extracted solution can be used directly or can be concentrated and / or dried. When extracted with an organic solvent, methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof may be used and extracted by room temperature or warming under conditions where the active ingredient of the crude drug is not destroyed or minimized. Depending on the organic solvent to be extracted, the degree of extraction and loss of the active ingredient of the drug may vary, so select an appropriate organic solvent. In particular, in the present invention, an ethanol extract, preferably 20-50% ethanol extract, in one embodiment 50% ethanol extract is preferred, and the extraction method is 3 days after mixing in 50% ethanol, as described in the Examples Obtained by stirring.

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

In the present invention, the meaning of "contained as an active ingredient", as a cosmetic composition can exhibit the skin improvement effect of anti-aging, including skin vitality, skin vitality, skin convergence, skin barrier strengthening, improvement, wrinkle improvement For example, it means that the cosmetic composition contains an effective amount that can exhibit functionalities such as collagen synthesis related to the anti-wrinkle effect, increased elastin invention, skin convergence, wrinkle improvement, skin barrier enhancement, improvement and the like.

In the present invention, the principle of skin astringent action refers to a phenomenon in which the skin contracts by forming crosslinks by binding to polymer flavonoids of the skin protein. Convergence basically means wrinkles or shrinks, and astringents constrict the skin and mucous vessels, and block the cell and lymph gaps to inhibit the secretion of mucus. In addition, since astringents have a property of binding to proteins, the degree of convergence can be determined according to the degree of binding of hemoglobin proteins to extracts. This astringent action can be said to form an insoluble film on the surface of the skin and mucous membranes externally or internally, thereby protecting the topical or densifying tissues, thereby reducing the permeability of the cell membrane.

There are two types of astringent action on the skin: chemical agglomeration and physical abortion. Chemical convergence refers to the temporary contraction of sweat glands and pores by substances that bind protein, such as tannins. Physical astringent, on the other hand, refers to the use of cold water on the skin or to the temporary contraction of sweat glands and pores by lowering the temperature of the skin through the volatilization of ethanol.

In addition, such a skin convergence is caused in the summer, or due to stress, when more sebum occurs, the skin trouble occurs, and the area where the skin trouble occurs becomes more and more reddened, and skin soothing means to alleviate such skin troubles. This is a distinct action from skin convergence.

In the skin barrier protection function according to the present invention, the skin barrier (stratum corneum), which is the outermost layer of the epidermis, is mainly composed of non-nuclear flat corneocytes. Multi lamella lipid layer formed by intercellular lipids such as ceramide, cholesterol, and fatty acid synthesized by keratinocytes of skin barrier maintained through normal epidermal cell division and differentiation process prevents moisture from evaporating inside skin Play a role. On the other hand, of these intercellular lipids, omega hydroxy ceramides are chemically covalently linked to involucrin, a protein in the outer layer of corneocytes, to form a corneocyte lipid envelope (CLE). It acts to physically stabilize the intercellular lipids of the form to strengthen the barrier function.

The composition of the present invention is delivered to the stratum corneum through the skin coating to promote the differentiation of keratinocytes, not only has the effect of thickening the thickness of the epidermal layer, but also has an excellent effect of restoring damage to the skin barrier by damage to the skin barrier. It can be usefully used for the treatment and prevention of induced skin diseases. Skin diseases caused by damage to the skin barrier include atopic dermatitis (atopic dermatitis), dry skin (xeroderma), psoriasis, psoriasis, ichthyosis, acne and the like, but are not limited thereto.

In addition, the skin barrier protection mechanism was confirmed by increasing the amount of protein of occludin and claudin-1, which is a close junction protein. Filaggrin is one of several structural proteins expressed by keratinocytes in the differentiation stage and is involved in the differentiation of the epidermis from the basal layer of the epidermis to the stratum corneum. It is the main agent of the natural moisture factor (NMF), which is essential for the maintenance of skin tissue moisture. It is used as a major indicator of skin moisture retention and skin membrane function. The wing stem fashion flower callus culture according to the present invention has excellent skin barrier protection, strengthening and improving function as the gene expression of filaggrin is greatly increased. May have

In addition, the wing stem fashion flower callus culture or its extract according to the present invention can be utilized for protecting the skin by ultraviolet irradiation.

In addition, the wing stem fashion flower callus culture according to the present invention has an ability to increase elastin expression. Elastin (Elastin) is a structural material present in the extracellular matrix of connective tissue and is present in the skin to impart elasticity. Elastin is synthesized in cells with a polypeptide called Tropo elastin and then produced by cross-linking between tropoelastin extracellularly. Therefore, elastin has a second or three-dimensional elasticity by crosslinking of tropoelastin. Therefore, the wing stem fashion flower callus culture according to the present invention can be utilized for skin elasticity enhancement and improvement.

Therefore, the cosmetic composition according to the present invention, in particular, has the ability to increase the procollagen, increase collagen biosynthesis, can be applied for preventing skin aging, skin wrinkle improvement / prevention, in addition to enhancing skin elasticity, skin barrier according to the enhancement of elastin expression It can be applied for reinforcement and improvement.

In another aspect, the present invention comprises the steps of: (a) inducing and culturing callus from the wing stem fashion flower tissue; And (b) relates to a method for producing a cosmetic composition for skin improvement Wingstem fashion flower callus culture or extract comprising the step of preparing a composition containing the Winstem fashion flower callus culture or its extract. .

In the step (a), specifically, a suitable medium may be selected for induction and cultivation of Winstem fashion flower callus. If there is a medium generally used in the art, it can be used without limitation. In plants, MS medium and B5 medium are generally used.For example, the composition of MS medium (1 L standard) is NH ₄ NO ₃ 1650 mg, KNO ₃ 1900 mg, CaCl ₂ H ₂ O 440 mg, MgSO _{4 .7H 2 O 370 mg, KH} 2 PO 4 170 mg, KI 0.83 mg, H 3 BO 3 6.2 mg, MnSO 4 .4H 2 O 22.3 mg, ZnSO 4 .7H2O 8.6 mg, Na 2 MoO 4 .2H 2 O _{0.25 mg, 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, Nicotinic acid 0.5 mg, Pyridoxine -HCl 0.5 mg, Thiamine-HCl 0.5 mg, Glycine 2 mg, Sucrose 30000 mg.

In the present invention, in step (b), the composition comprising the Winstem fashion flower callus culture as obtained through the culture of step (a) as prepared in a cosmetic composition of the appropriate form, or the culture is known It can be obtained in the form of extract through the extraction method can be prepared into a cosmetic composition of the appropriate form.

For example, in step (b), the Winstem fashion flower callus culture obtained in step (a) is dried, powdered and mixed in purified water to prepare a composition,

The Winstem fashion flower callus culture obtained in step (a) may be dried, powdered and mixed with purified water, followed by cold water extraction, hot water extraction or ethanol extraction to prepare a composition.

As another example, the culture obtained in step (a) may be prepared by drying the powder after hot air drying and evaporating moisture.

In the cosmetic composition, an acceptable carrier may be included in the cosmetic formulation. Herein, "acceptable carrier in cosmetic preparation" means a compound or composition already known and used that can be included in cosmetic preparations, or a compound or composition which will be developed in the future, which has no toxicity, instability or irritation that can be adapted to the human body upon contact with skin. Say nothing.

The carrier may be included in the cosmetic composition of the present invention in an amount from about 1% to about 99.99% by weight, preferably from about 90% to about 99.99% by weight of the composition. However, since the ratio depends on the formulation as described below in which the cosmetic composition of the present invention is prepared and its specific application site (face, neck, etc.), its preferred application amount, etc., the ratio is in any aspect to the present invention. It should not be understood as limiting the scope of.

Examples of the carrier include alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizers, viscosity modifiers, emulsions, stabilizers, ultraviolet scatterers, ultraviolet absorbers, colorants, fragrances, and the like. The alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizers, viscosity modifiers, emulsions, stabilizers, UV scattering agents, UV absorbers, colorants, compounds / compositions that can be used as fragrances are already known in the art. As such, those skilled in the art can select and use appropriate materials / compositions.

As an embodiment of the present invention, the cosmetic composition according to the present invention includes glycerin, butylene glycol, propylene glycol, rolled polyoxyethylene hydrogenated castor oil, ethanol, triethanolamine, etc., in addition to the Wingstem fashion flower callus extract, which is the active ingredient. It may be, and may contain a small amount of preservatives, paints, coloring, purified water and the like as necessary.

Cosmetic compositions according to the invention can be prepared in various forms, for example, lotions, essences, gels, emulsions, lotions, creams (oil-in-water, water-in-oil, multiphase), solutions, suspensions (anhydrous and aqueous) , Anhydrous products (oil and glycol based), gels, masks, packs, powders, or gelled capsules (soft capsules, hard capsules) formulations and the like.

The skin in the present invention is a concept that includes not only a face but also a scalp and a whole body, and a cosmetic composition that can be applied to such a scalp, such as a shampoo, a rinse, a treatment, a hair regrowth, and a body cleanser that can be applied to the whole body. It can be prepared in various forms as the use of.

The manufacturing method of the cosmetic composition according to the present invention is not limited to the above-described manufacturing method, and those skilled in the art can also be prepared by a method of partially modifying the manufacturing method.

When the cosmetic composition is prepared, cosmetics of the emulsion formulations include nutrient cosmetics, creams, essences, etc., and cosmetics of the solubilized formulations include soft cosmetics. In addition, by containing a dermatologically acceptable medium or base, it can be prepared in the form of topical or systemically applicable adjuvants commonly used in the field of dermatology.

In addition, suitable formulations of cosmetics include, for example, emulsions, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes), nonionics obtained by dispersing an oil phase in a solution, gel, solid or pasty anhydrous product, aqueous phase, for example. It may be provided in the form of a vesicle dispersant in the form of a cream, skin, lotion, powder, ointment, spray or concealed stick. It may also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

In addition, the cosmetic composition of the present invention may further comprise fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic Or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other commonly used in cosmetics It may contain adjuvants conventionally used in the cosmetic or dermatological fields, such as ingredients. In addition, the above components may be introduced in an amount generally used in the dermatology field.

Wingstem fashion flower callus culture according to the invention or a cosmetic composition containing the extract is a skin vitality, skin vitality enhancement, skin wrinkle improvement, skin regeneration, skin barrier function protection, skin protection from ultraviolet rays, atopy, anti-inflammatory, As a cosmetic composition having functions such as antioxidant and anti-aging, it can be used as a material of various functional cosmetics.

Figure 1 is a result graph showing the cell survival rate according to the concentration treatment of Wingstem fashion flower callus extract.
Figure 2 is a result graph showing the expression of genes related to biological control according to the treatment of Wingstem fashion flower callus extract.

Unless defined otherwise, 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: Wingstem fashion flower callus production and extract preparation according to the present invention

1-1: Surface disinfection of wing stem fashion flower petals

In order to sterilize the surface of the wing stem fashion flower petals, it was immersed in 70% ethanol for 30 seconds, shaken with 2% sodium hypochlorite for 5 minutes, and then washed with sterile water.

1-2: Callus derived from wing stem flower petals

Sterilized Wingstem fashion flower petals were placed in 1/2 MS (Murashinge and Skoog) medium, and then germinated by incubating for 7 days in 25 ° C. and 70% humidity. Germinated hawthorn leaves were cut at once with a sharp knife to remove 0.5 mg / L of IAA (indole-3-acetic acid), 0.2 mg / L of Zeatin, 30 g / L of Sucrose, and Gelite (Gelite). ) After inoculation into the MS basic medium containing 2.0 g / L and incubated for 8 weeks in a culture room of 23 ~ 27 ℃ to induce callus. Induced callus was proliferated by subculture at 4 week intervals.

1-3: Callus culture and mass production according to the present invention

Aseptically harvested Winstem fashion flower callus was passaged at regular intervals of 2 to 3 weeks in MS basal medium containing 3% Sucrose and agar 8 g / L. Thereafter, in a liquid medium having the same composition except for agar, the bioreactor was incubated at a temperature of 25 ° C., a humidity of 70%, and an air supply amount of 0.1 vvm at 14 days intervals.

1-4: Preparation of wing stem fashion flower callus extract according to the present invention

The propagated Winstem fashion flower callus culture was harvested and sufficiently watered with a clean tissue, and then dried in a drier at 60 ° C. for 2 days. 10 g of dried callus culture powder was placed in a container, and 1 L of purified water was added thereto, followed by hot water extraction. Hot water extraction was carried out by heating to 80 ~ 100 ℃, about 8 ~ 48 hours. After extraction, the solids were removed by filtration through a mesh.

Experimental Example 1: Influence test on cell viability

In this experiment, the proliferation activity of the cells for the Winstem fashion flower callus extract prepared in Example 1 was examined.

To this end, first, HaCaT (human keratinocyte) and Detroit551 (human fibroblast, fibroblast) were dispensed in 96 well plate with DMEM medium containing 10% FBS and in a thermostat at 5% CO ₂ , 37 ℃. Incubated for 24 hours. After 24 hours, the cells were treated with a concentration of 0.5, 1, and 2% of the Winstem fashion flower callus extract, and further incubated for 24 hours. Then 5 mg / mL MTT solution was added to each well 4 μL and incubated for 4 hours. After removing the culture medium and lysing the cells by adding 100 μL of DMSO (Dimethyl sulfoxide) solution, the absorbance was measured at 540 nm using a spectrophotometer.

The survival rate of the skin cells was calculated according to the following equation based on the absorbance intensity of the control group using purified water and expressed as a percentage, and the results were shown in FIG. 1.

Equation 1] cell survival rate (%) = [(experimental absorbance-control absorbance) / control absorbance] × 100

As shown in Figure 1, the callus extract of the present invention did not affect the cell viability in the treatment concentration. That is, the Winstem fashion flower callus extract of the present invention prepared in Example 1 was confirmed that there is no toxicity in HaCaT cells and Detroit551 cells at 0.5-2%.

Experimental Example 2: Gene Expression Test Related to Biological Clock Control

In this experiment, in order to examine the biological clock control effect of the Wingstem fashion flower callus extract prepared in Example 1, the expression changes of the related genes CLOCK and PER1 were investigated.

HaCaT cells were dispensed into 96 well plates, and then cultured for 24 hours under cell culture conditions. After 24 hours of induction of UVB, the test substance Wingstem fashion flower callus extract was treated with cells to a final concentration of 1% with DMEM medium containing no serum and further incubated for 8, 16, 24 and 32 hours. It was. Real-time PCR was performed to check gene levels of CLOCK and PER1. The test sequence is as follows.

SuperPrep ^™ cell lysis & RT Kit for qPCR (TOYOBO, Cat. # SCQ-101) was used for RNA isolation and cDNA synthesis. The cells from which the medium was removed were washed once with PBS, 50 μL cell lysis mixture (including a gDNA remover) was added and reacted for 5 minutes, and then a stop solution was added. 8 μL of the extracted mRNA was added to 32 μL of the RT reaction mixture, and cDNA was synthesized using PCR at 37 ° C. 15 min, 50 ° C. 5 min, and 95 ° C. 5 min. To compare the expression of genes, cDNA synthesized above was used as a template and real-time PCR analysis was performed using Thunderbird ^TM SYBR qPCR Mix (TOYOBO, Cat. # QPS-201). The primers used in the experiment were QuantiTect primer assays (GAPDH; Cat. # QT01192646, PER; Cat. # QT00069265, CLOCK; Cat. # QT00054481) from Qiagen. Real-time qPCR conditions were first conducted at 95 ° C. for 15 min, followed by 40 cycles of 94 ° C. 15 s, 60 ° C. 30 s, and 72 ° C. 30 s per cycle.

As a result, as shown in FIG. 2, when UVB stimulation was applied to HaCaT cells, the CLOCK / PER expression pattern in the cells was changed irregularly (UV treatment group). It can be seen that the / PER expression pattern is restored close to the expression pattern of the cells without UV stimulation.

Experimental Example 3: skin barrier improvement gene expression test

In this experiment, in order to examine the skin barrier improvement effect of Wingstem fashion flower callus extract prepared in Example 1, the expression changes of the water / glycerol transporter AQP3 and FLG known as a natural moisturizing factor was investigated.

HaCaT cells were dispensed into 96 well plates, and then cultured for 24 hours under cell culture conditions (5% CO ₂ , 37 ° C). After 24 hours, the Winstem Fashion Flower Callus extract, a test substance, was treated with cells to a final concentration of 1% with DMEM medium containing no serum, and further incubated for 24 hours. Real-time PCR was performed to confirm the gene levels of AQP3 and FLG, and the test sequence was as follows.

SuperPrep ^™ cell lysis & RT Kit for qPCR (TOYOBO, Cat. # SCQ-101) was used for RNA isolation and cDNA synthesis. The cells from which the medium was removed were washed once with PBS, 50 μL cell lysis mixture (including a gDNA remover) was added and reacted for 5 minutes, and then a stop solution was added. 8 μL of the extracted mRNA was added to 32 μL of the RT reaction mixture, and cDNA was synthesized using PCR at 37 ° C. 15 min, 50 ° C. 5 min, and 95 ° C. 5 min. To compare the expression of genes, cDNA synthesized above was used as a template and real-time PCR analysis was performed using Thunderbird ^TM SYBR qPCR Mix (TOYOBO, Cat. # QPS-201). The primers used in the experiment were QuantiTect primer assays (GAPDH; Cat. # QT01192646, AQP3; Cat. # QT00212996, FLG; Cat. # QT02448138) from Qiagen. Real-time qPCR conditions were first conducted at 95 ° C. for 15 min, followed by 40 cycles of 94 ° C. 15 s, 60 ° C. 30 s, and 72 ° C. 30 s per cycle.

Experimental Example 4: Skin regeneration and wrinkle improvement effect test

In this experiment, in order to examine the skin regeneration and wrinkle improvement effect of the Wingstem fashion flower callus extract prepared in Example 1, procollagen (Procollagen type-), which is a precursor of collagen decomposition-related enzyme MMP-1 and collagen synthesis 1, PIP) biosynthesis amount was measured.

<4-1> MMP-1 biosynthesis measurement

Detroit551 cells were dispensed into 48 well plates and incubated for 24 hours under cell culture conditions. After 24 hours, the test material Wingstem Fashion Flower Callus Extract with serum-free DMEM medium was treated to cells to a final concentration of 1% and further incubated for 48 hours.

100 μL of the diluted supernatant of the cultured medium was taken and placed in an antibody coated microtiter plate in Matrix Metalloproteinase-1, Biotrak Activity Assay Kit (Amersham, Cat. # RPN2629) and reacted at 25 ° C. for 3 hours. . After the medium was removed and washed four times with 200 μL of PBS, 100 μL of antiserum was added to each well and reacted at 25 ° C. for 3 hours. Then, the solution was removed and washed four times with 200 μL of PBS, 100 μL of peroxidase-labeled antibody solution was added to each well, and further reacted at 25 ° C. for 1 hour. After removing the solution, 100 μL of substrate solution was added to each well, and the reaction was performed at room temperature for 15 minutes in a shaded state. Thereafter, 100 μL stop solution (1 NH ₂ SO ₄ ) was added thereto and the absorbance at 450 nm was measured. After measuring the absorbance at 450 nm, a standard concentration curve of MMP-1 was prepared to determine the amount of MMP-1 in the sample.

<4-2> PIP biosynthesis measurement

Detroit551 cells were dispensed into 48 well plates and incubated for 24 hours under cell culture conditions. After 24 hours, the test material Wingstem Fashion Flower Callus Extract with serum-free DMEM medium was treated to cells to a final concentration of 1% and further incubated for 48 hours.

Take 20 μL of the diluted supernatant of the culture medium through the above procedure and prepare antibody coated microtiter plate in Procollagen Type I C-Peptide EIA Kit (Takara, Cat. # MK101) with 100 μL of peroxidase-labeled antibody solution. It was added to the well, and reacted at 37 ° C. for 3 hours. After the medium was removed and washed four 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 a shaded state. Then 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 to determine the amount of PIP in the sample.

Experimental Example 5: Skin Energy Activity Enhancement Effect Test

In this experiment, to examine the skin energy activity enhancing effect of Wingstem fashion flower callus extract, the activity of the energy of the cells (ATP) was measured.

Detroit551 cells were dispensed into 12 well plates and incubated for 24 hours under cell culture conditions. Thereafter, the Winstem Fashion Flower Callus extract, a test substance, was treated with the cells to a concentration of 0.5, 1, and 2% of the final test together with DMEM medium containing no serum, and further cultured for 24 hours.

50 μL of the diluted lysate of the cultured cells was taken, and 50 μL of ATP standard in ATP assay Kit (Abcam, Cat. Ab83355) was added to each 96 well plate and 50 μL of ATP reaction mix solution was added. In addition, it was added to each well and allowed to react at room temperature for 30 minutes in a shaded state. Then, after measuring the absorbance at 570 nm, the ATP standard concentration curve was prepared to calculate the amount of ATP in the sample. The degree of skin energy activity was calculated according to the following equation based on the absorbance intensity of the control group using purified water and expressed as a percentage, and the results are as follows.

[Equation 1] energy activity effect (%) = [(experimental absorbance-control absorbance) / control absorbance] × 100

Skin Energy Activity Effect by Wingstem Fashion Flower Callus Extract of the Present Invention
Test substance
Skin energy activation effect (%)
Control
0
Wingstem Fashion Flower Callus Extract 0.5%
33.53
Wingstem Fashion Flower Callus Extract 1%
44.77
Wingstem Fashion Flower Callus Extract 2%
51.07

As a result, as shown in Table 1, it can be seen that the energy activity effect of the skin cells in the Wingstem fashion flower callus extract treatment concentration of the present invention.

Experimental Example  6: skin vitality effectiveness test

In this study, to examine the skin-enhancing effect of Wingstem Fashion Flower Callus Extract, 20 test subjects with 20-45 year old females with BMI (Body Mass Index) 21-27 shower the test substance once a day for 12 weeks. After the use of the massage, and compared with before and after 12 weeks of use was determined whether the effect. The evaluation items are shown in Table 2 below the number of subjects who responded that they felt the effect on skin cell activation and skin vitality by performing a survey. The results are as follows.

Skin Vitality Enhancement Effect by Wingstem Fashion Flower Callus Extract of the Present Invention
Test substance
Skin cell activation
Skin vitality
Control
0
2
Wingstem Fashion Flower Callus Extract 0.5%
5
7
Wingstem Fashion Flower Callus Extract 1%
13
8
Wingstem Fashion Flower Callus Extract 2%
15
11

As a result, as shown in Table 2, Wingstem fashion flower callus extract of the present invention almost no effect on skin cell activation and skin vitality after 12 weeks in the case of the control group in the evaluation of feeling in the treatment concentration However, the response of Wingstem Fashion Flower Callus extract showed skin cell activation (5, 13, 15) and skin vitality enhancement (7, 8, 11).

Claims (10)

Wingstem fashion flower callus culture or cosmetic composition for skin improvement containing the extract as an active ingredient. The cosmetic composition according to claim 1, wherein the composition is for preventing or improving wrinkles. According to claim 1, wherein the composition is a cosmetic composition, characterized in that for protecting or enhancing the skin barrier function. According to claim 1, wherein the composition is a cosmetic composition, characterized in that for enhancing skin elasticity. According to claim 1, wherein the composition is a cosmetic composition, characterized in that for protecting the skin by ultraviolet radiation. The cosmetic composition according to claim 1, wherein the composition is for skin whitening. According to claim 1, wherein the composition is a cosmetic composition, characterized in that for anti-aging or antioxidant. A method for preparing a cosmetic composition according to any one of claims 1 to 7, containing an extract of a wing stem fashion flower callus culture or a wing stem fashion flower callus culture comprising the following steps:
(a) isolating cells from petals, seeds, or fetal tissue from the wing stem fashion flower to induce callus and incubate; And
(b) preparing a culture containing the culture obtained in step (a) or an extract of the culture. The method of claim 8, wherein step (b) comprises drying the Winstem fashion flower callus culture obtained in step (a), pulverizing and mixing it with purified water to prepare a composition. . The method of claim 8, wherein step (b) comprises drying the Winstem fashion flower callus culture obtained in step (a), pulverizing and mixing the mixture with purified water, and then preparing a composition by ultrasonic extraction or hot water extraction. Method comprising a.

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