KR20220058751A - Composition for preventing or treating ultraviolet induced skin damage containing extract of oenantae javanica - Google Patents

Abstract

The present invention relates to an external composition and a cosmetic composition for preventing or alleviating skin damage exposed to ultraviolet rays and, more specifically, to a cosmetic composition for protecting skin against ultraviolet rays, which includes an extract of Oenanthe javanica as an active ingredient. The Oenanthe javanica extract can suppress skin inflammation by effectively suppressing the generation of ROS and the expression of MMPs, which are inflammatory mediators, and reduce epidermal damage on the skin induced by ultraviolet irradiation, does not cause skin irritation, can suppress skin inflammation, and has an effect of suppressing wrinkles on the skin by increasing collagen synthesis. Accordingly, it is expected that the Oenanthe javanica extract can be used as an external composition for suppressing skin damage and for protecting skin by ultraviolet exposure, in particular as a cosmetic composition.

Description

Composition for preventing or improving skin damage caused by UV rays comprising shiitake extract {COMPOSITION FOR PREVENTING OR TREATING ULTRAVIOLET INDUCED SKIN DAMAGE CONTAINING EXTRACT OF OENANTAE JAVANICA}

The present invention relates to a cosmetic composition for preventing or improving skin damage comprising, as an active ingredient, a shiitake mushroom extract, which is harmless to the human body, which effectively reduces skin damage caused by ultraviolet rays.

The skin is located at the outermost part of the human organ and serves as a primary protective barrier against the external environment such as bacteria, viruses, physical damage, and UV rays. Among the environmental factors that affect the skin's defense function, the most important is ultraviolet rays, and ultraviolet rays irradiated to the skin cause a photochemical reaction and cause inflammatory lesions of the skin.

Ultraviolet rays are divided into ultraviolet C (UVC, 200 to 280 nm), ultraviolet B (UVB, 280 to 320 nm), and ultraviolet A (UVA, 320 to 400 nm) according to their wavelength. It is absorbed and does not reach the surface of the earth, and UVB and UVA rays have a direct effect on the skin mainly. In the case of the ultraviolet A, since it hardly absorbs the wavelength in the epidermis with a long wavelength, it can penetrate more deeply than the ultraviolet B, and mainly causes damage to blood vessels and damage to the dermis, whereas the ultraviolet B, which is the main cause of sunburn In this case, it mainly causes damage to the epidermis because proteins and DNA bases of the epidermis absorb most of the ultraviolet B wavelength region.

Recently, due to the destruction of the ozone layer, which absorbs ultraviolet rays due to air pollution, the amount of ultraviolet rays reaching the earth's surface increases, and skin problems caused by ultraviolet rays are rapidly increasing. The effect of ultraviolet rays, which is most sensitively increased due to the destruction of the ozone layer, is caused by the ultraviolet B. In acute exposure, inflammation such as erythema, edema, and pain occurs, the epidermis and keratin thicken, and chronic exposure occurs. It leads to serious skin lesions, causing changes in skin appearance such as wrinkles, and even leads to skin cell death and development of malignant tumors. On the other hand, although ultraviolet A has an energy intensity equivalent to 1/1,000 of that of ultraviolet B, the amount of light reaching the earth's surface is about 100 times greater than that of ultraviolet B, and it is reported that the skin toxicity is stronger than that of ultraviolet B.

Tumor necrosis factor (TNF-α), interleukin (IL)-1, IL-6, IL-8 and cyclooxygenase (COX)-2 are known as major inflammatory mediators involved in causing the inflammatory response of the skin by the ultraviolet rays. If their expression is continuously increased, the inflammatory response is prolonged. Therefore, suppressing the expression of inflammation-related mediators is very important for alleviating inflammation, and substances that inhibit the expression and function of these inflammatory mediators will be able to suppress the inflammatory response that occurs in the skin after UV irradiation. , it can be used as a useful ingredient in the development of various products for preventing and regenerating skin damage caused by UV rays.

In addition, due to UV exposure, intracellular reactive oxygen species (ROS) is generated, and the excessively generated reactive oxygen species oxidizes collagen and elastin proteins constituting the skin tissue to change the structure of the skin. Photoaging, which causes damage to skin, is one of the important problems in modern society where skin beauty is becoming more important. In this respect, the increase of reactive oxygen species by exposure to UV light promotes the secretion of various inflammatory cytokines, thereby activating various signaling systems.

In particular, matrix metalloproteinases (MMPs), which are activated through a signal transduction system according to UV exposure, degrade the extracellular matrix and cause structural transformation of the skin. The matrix metalloproteinase can be divided into collagenase, gelatinase, stromelysin, matrilysin, and membrane-type according to their function. Among them, MMP-1 is an interstitial collagenase that decomposes type I collagen and type III collagen, which are structural proteins most abundant in skin cells. The decomposed collagen and type IV collagen are broken down into smaller molecules by MMP-9, a gelatinase B. This decomposition of collagen causes structural changes in the skin, which leads to skin aging such as wrinkles and loss of elasticity of the skin.

However, there are still few studies on biologics that can effectively suppress skin damage caused by exposure to ultraviolet rays, and it has been confirmed that, in particular, studies related to natural extracts that do not have side effects or tolerance have hardly been conducted.

KR 10-2128168B KR 10-1479162B KR 10-0873998B KR 10-0848515B KR 10-0385335B KR 10-2011-0131866A

An object of the present invention is to prevent or improve skin damage caused by ultraviolet rays, which contains an extract of a natural plant that is excellent in preventing or improving skin damage caused by ultraviolet irradiation, which is a function related to skin protection, and is harmless to the human body as an active ingredient. An object of the present invention is to provide a cosmetic composition that can prevent or improve various skin diseases caused by ultraviolet rays.

In order to achieve the above object, the present invention provides an external composition for skin protection against ultraviolet rays comprising an extract of shiitake mushroom ( Lentinuls edodes ) as an active ingredient. Specifically, the shiitake mushroom may be a shiitake mushroom having a beta glucan content of 165 mg/g (beta glucan / shiitake) to 170 mg / g (beta glucan / shiitake) compared to shiitake, and in this regard, the shiitake The mushroom extract may be a shiitake extract having a beta-glucan content of 165 mg/g (beta-glucan/shiitake) to 170 mg/g (beta-glucan/shiitake) compared to shiitake. The composition for external use for skin protection against ultraviolet rays may have an effect of preventing or improving skin damage caused by ultraviolet rays.

The shiitake mushroom extract may inhibit skin damage and inflammation caused by exposure to ultraviolet rays.

In addition, in order to achieve the above object, the present invention provides a cosmetic composition for protecting the skin against ultraviolet rays comprising an extract of shiitake mushrooms ( Lentinuls edodes ) as an active ingredient. The cosmetic composition for skin protection against ultraviolet rays may have an effect of preventing or improving skin damage caused by ultraviolet rays.

The shiitake mushroom may be a shiitake mushroom having a beta glucan content of 165 mg/g (beta glucan / shiitake) to 170 mg / g (beta glucan / shiitake) compared to shiitake, in this regard, the shiitake extract is shiitake It may be a shiitake extract having a beta-glucan content of 165 mg/g (beta-glucan/shiitake) to 170 mg/g (beta-glucan/shiitake) compared to mushrooms.

The shiitake mushroom extract may be any one selected from the group consisting of water, an organic solvent, and a mixture thereof, preferably water extracted from shiitake mushrooms. In this regard, the shiitake mushroom extract may be a shiitake mushroom water extract.

The organic solvent is an alcohol having 1 to 5 carbon atoms including methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene ( It may be at least one selected from the group consisting of benzene), chloroform, ethyl acetate, methylene chloride, hexane, and cyclohexane.

In addition, the shiitake mushroom extract may inhibit skin damage and inflammation caused by exposure to ultraviolet rays.

In addition, the cosmetic composition for protecting the skin against ultraviolet rays may contain the shiitake mushroom extract as an active ingredient in an amount of 0.001 to 99.999% by weight based on the total weight of the composition.

The active ingredient of the present invention, the shiitake mushroom extract, has been used for food and medicine for a long time, and has not only been recognized for safety because it has no side effects or tolerance, but also confirmed by the present inventors, that the shiitake mushroom extract has Since it is possible to effectively prevent and improve the skin protection effect, specifically, the skin damage caused by UV rays, the shiitake mushroom extract can be usefully used to prevent or improve various skin diseases caused by excessive UV irradiation, so that an external composition or cosmetic It can be widely used as an active ingredient in the composition.

1 is a result of confirming the cytotoxicity of a shiitake mushroom water extract according to an embodiment of the present invention. Specifically, after UV treatment with a human keratinocyte line (HaCaT), the result of performing an MTT assay (MTT assay) This is the graph shown. In FIG. 1 , the horizontal axis indicates the amount of treatment of the shiitake mushroom water extract, and the vertical axis indicates the cytotoxicity compared to the control group (0).
2 is a result of confirming the skin regeneration effect of the shiitake mushroom water extract according to an embodiment of the present invention. This is a picture showing the result of performing In FIG. 2 , the horizontal axis means the amount of treatment of the shiitake mushroom water extract, and the vertical axis means the treatment time.
3 is a result of confirming the UV protection effect of the shiitake mushroom water extract according to an embodiment of the present invention, specifically, according to an embodiment of the present invention, the shiitake mushroom water extract according to the irradiation It is a graph confirming the anti-apoptosis effect. In detail, it shows the results of performing the MTT assay (MTT assay) after UV A or UV B treatment with a human keratinocyte line (HaCaT). The results are the irradiation results, and in FIG. 3 , the horizontal axis indicates the amount of treatment and whether the shiitake mushroom water extract is irradiated with UV light, and the vertical axis indicates the degree of cell proliferation compared to the control group (when neither the mushroom extract nor the UV light is treated).
4 is a result of confirming the effect of inhibiting reactive oxygen species (ROS) generation according to ultraviolet irradiation according to an embodiment of the present invention, specifically, according to an embodiment of the present invention, the shiitake mushroom water extract is ultraviolet A or ultraviolet It is a graph confirming the inhibitory effect on the generation of reactive oxygen species according to the irradiation of B. In detail, after UV A or UV B treatment with a human keratinocyte line (HaCaT), the result of confirming the degree of scavenging of reactive oxygen species through DCF-DA was compared with the result of hydrogen peroxide treatment, Figure 4a shows hydrogen peroxide treatment 4b is the result of irradiating UV B, FIG. 4c is the result of irradiating UV A, the horizontal axis in FIG. 4 means the amount of shiitake water extract and whether UV irradiation or hydrogen peroxide treatment, and the vertical axis is the control This is the result showing the degree of removal of reactive oxygen species, that is, the generation of reactive oxygen species, compared to (in the case of no mushroom extract treatment and UV irradiation or hydrogen peroxide treatment).
5 is a result of confirming the UV protection effect of the shiitake mushroom water extract according to an embodiment of the present invention, specifically, according to an embodiment of the present invention, the shiitake mushroom water extract according to the irradiation It is a graph confirming the effect of inhibiting MMPs activation. In detail, after UV-B treatment with a human keratinocyte line (HaCaT), MMPs (MMP-1 and MMP-9) and type I procollagen production levels according to whether or not the shiitake mushroom water extract was treated were evaluated by RT-PCR (FIG. 5a) and Photos and graphs confirmed through Western blotting (B). In the photo, the horizontal axis means the throughput and UV irradiation of the shiitake mushroom water extract, the vertical axis means the type of MMPs, and the order of each graph bar graph. matches the order of the pictures.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be embodied in several different forms and is not limited to the embodiments described herein.

The present invention relates to an external composition for skin protection against ultraviolet rays comprising shiitake mushroom extract ( Lentinuls edodes ) as an active ingredient. The composition for external use for skin protection against ultraviolet rays may have an effect of preventing or improving skin damage caused by ultraviolet rays.

The shiitake may be a shiitake mushroom having a beta-glucan content of 165 mg/g (beta-glucan/shiitake) to 170 mg/g (beta-glucan/shiitake) compared to the shiitake mushroom. The shiitake mushroom extract may be a shiitake mushroom extract.

The shiitake mushroom extract may inhibit skin damage and inflammation caused by exposure to ultraviolet rays.

In addition, in order to achieve the above object, the present invention relates to a cosmetic composition for protecting the skin against ultraviolet rays comprising shiitake mushroom extract ( Lentinuls edodes ) as an active ingredient. The cosmetic composition for skin protection against ultraviolet rays may have an effect of preventing or improving skin damage caused by ultraviolet rays.

The shiitake may be a shiitake mushroom having a beta glucan content of 165 mg/g (beta glucan / shiitake) to 170 mg / g (beta glucan / shiitake) compared to shiitake, in this regard, the shiitake extract It may be a shiitake mushroom extract having a beta-glucan content of 165 mg/g (beta-glucan/shiitake) to 170 mg/g (beta-glucan/shiitake) compared to shiitake.

The shiitake mushroom extract may be any one selected from the group consisting of water, an organic solvent, and a mixture thereof, preferably water extracted from shiitake mushrooms.

The organic solvent is an alcohol having 1 to 5 carbon atoms including methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene ( It may be at least one selected from the group consisting of benzene), chloroform, ethyl acetate, methylene chloride, hexane, and cyclohexane.

In addition, the shiitake mushroom extract may inhibit skin damage and inflammation caused by exposure to ultraviolet rays.

In addition, the cosmetic composition for protecting the skin against ultraviolet rays may contain the shiitake mushroom extract as an active ingredient in an amount of 0.001 to 99.999% by weight based on the total weight of the composition.

Unless otherwise stated herein, composition is meant to include products comprising the specified ingredients in the specified amounts, as well as products resulting directly or indirectly from combinations of the specified ingredients in the specified amounts.

Unless otherwise specified herein, an effective amount means an amount of the extract or composition of the present invention effective to produce a desired therapeutic, alleviating, inhibiting or prophylactic effect.

The inventors of the present invention are hypoallergenic and safe, unlike the active ingredients of cosmetic compositions for UV protection, which have been problematic in the past, have developed ingredients with excellent UV protection effects, especially from substances derived from natural plants that have been used as food for a long time. Shiitake mushrooms, especially shiitake mushrooms grown in smart farms, have a higher beta-glucan content than general shiitake mushrooms, from 165 mg/g (beta glucan / shiitake) to 170 mg/g (beta glucan / shiitake) Phosphorus shiitake mushroom water extract has the effect of preventing or improving skin damage caused by UV rays, specifically, has the effect of restoring and preventing skin damage despite UV irradiation, and effectively suppressing the expression of inflammatory mediators COX-2 and TNF-α. By suppressing the MMPs (MMP-1 and MMP-9), it was confirmed that the effect of inhibiting the skin inflammation caused by UV rays was excellent, and the skin regeneration effect was excellent by promoting the production of type I procollagen. Based on this, it was confirmed that the present invention was completed.

The shiitake mushroom extract, which is a natural plant-derived extract having an effect of protecting the skin against UV rays or preventing or improving skin damage caused by UV rays, preferably has a beta glucan content of 165 mg/g (beta glucan / shiitake mushroom) compared to shiitake mushrooms. 170 mg/g (beta glucan/shiitake) may be a shiitake extract.

The shiitake mushroom ( Lentinuls edodes ) is a mushroom of the basidiomycetes wrinkle mycobacteriaceae, distributed in Korea, Japan and China, and is also called an oak mushroom. The surface is dark brown, the epidermis is cracked and white flesh is visible, and the diameter of the mushroom cap is about 4 to 10 cm, and it mainly inhabits dry trees of broad-leaved trees such as oak, chestnut, or hornbeam. It is reported that the shiitake mushroom has a very high nutritional value because it is rich in various vitamins including vitamin D and minerals including calcium, phosphorus, iron and zinc, while having low calories and fat. As a function of the shiitake mushroom, eritadenine contained in the shiitake mushroom promotes cholesterol metabolism and discharges it out of the body to improve blood circulation, which is useful for the treatment of cardiovascular diseases such as hyperlipidemia and hypertension, and is rich in dietary fiber. It has been reported that it can prevent adult diseases. The shiitake mushroom has been widely used for food from a long time ago, and recently, dried shiitake mushroom powder has been used as a natural seasoning. The shiitake mushroom powder is widely used as a representative of a natural seasoning that gives a light taste and umami to soup dishes.

The shiitake mushroom may be cultivated in a smart farm, and specifically, the beta glucan content compared to shiitake mushrooms is 150 mg/g (beta glucan / shiitake) to 170 mg / g (beta glucan / shiitake), preferably 165 mg/g (beta glucan / shiitake) to 170 mg / g (beta glucan / shiitake) may be shiitake.

The shiitake mushroom includes any form commonly used to obtain an extract in the field of extract without any particular limitation on its form.

The extract may be prepared according to a conventional method for preparing a plant extract. More specifically, it may be extracted by performing a solvent extraction method, a supercritical extraction method, or an ultrasonic extraction method on the shiitake mushroom, the pulverized product of the shiitake mushroom, or the dried product of the pulverized shiitake mushroom from which impurities have been removed. In this sense, the extract is a solvent extract extracted with an extraction solvent, a supercritical extract, that is, an extract or ultrasonic extract through supercritical extraction with a supercritical fluid, a fraction fractionated by adding a fractionation solvent to the extract, or chromatography on the fraction It may be a purified product obtained by carrying out

The solvent extract may be prepared by using a conventional solvent, specifically an extraction solvent, under conditions of conventional temperature and pressure, according to a conventional method known in the art.

The extraction solvent may be any one selected from the group consisting of water, an organic solvent, or a mixture thereof, preferably water, an alcohol having 1 to 5 carbon atoms, and mixtures thereof, which can be used for natural product extraction, more preferably water. . The organic solvent is a linear or branched alcohol having 1 to 5 carbon atoms such as methanol and ethanol, a polar solvent such as ethyl acetate or acetone, a non-polar solvent such as hexane or dichloromethane, a neutral solvent such as a ketone having 3 to 5 carbon atoms, or these It may be a mixed solvent, preferably 30 to 90% of an aqueous solution of a linear or branched alcohol having 1 to 5 carbon atoms such as ethanol or 30 to 90% of a ketone having 3 to 5 carbon atoms, more preferably 50 to 80% ethanol It may be an aqueous solution or 50 to 80% acetone aqueous solution, more preferably 60 to 75% ethanol aqueous solution.

The fractionation solvent may be water, a C5 to C7 alkane, a C1 to C5 straight-chain or branched alcohol including butanol, ethyl acetate, methylene chloride, chloroform, hexane, or a mixture thereof.

On the other hand, the extraction solvent or the fractionation solvent may be replaced with other common solvents known to those skilled in the art that can exhibit substantially the same effect.

The supercritical extract may be extracted by a supercritical extraction method or supercritical fluid extraction in which carbon dioxide is used as a fluid under supercritical conditions by reduced pressure and high temperature. In general, supercritical fluids have liquid and gas properties when the gas reaches a critical point under high temperature and high pressure conditions, and have a polarity similar to that of a chemically non-polar solvent, so it is effective for extraction of fat-soluble substances. Specifically, the carbon dioxide becomes a supercritical fluid having liquid and gas properties at the same time as the pressure and temperature reach the critical point due to the operation of the supercritical fluid device, and as a result, the solubility in the fat-soluble solute increases. Therefore, when the supercritical carbon dioxide passes through the extraction vessel containing a certain amount of the sample, the fat-soluble substances contained in the sample are extracted into the supercritical carbon dioxide, and after extracting the fat-soluble substances, a small amount of cosolvent is added to the sample remaining in the extraction vessel. If the supercritical carbon dioxide contained therein is passed through, components that were not extracted with pure supercritical carbon dioxide alone can be extracted.

In this aspect, the supercritical fluid used in the supercritical extraction method may be supercritical carbon dioxide or a mixed fluid in which a cosolvent is additionally mixed with carbon dioxide. The cosolvent may be any one selected from the group consisting of chloroform, ethanol, methanol, water, ethyl acetate, hexane, diethyl ether, and mixtures thereof.

Since carbon dioxide used in the extraction process is volatilized into the air at room temperature, the extract obtained by the above method can be used as a cosmetic composition, and in the case of a co-solvent, it can be removed by a vacuum evaporator.

The ultrasonic extract means that it is extracted by the ultrasonic extraction method. The ultrasonic extraction method is an extraction method using energy generated by ultrasonic vibration. When the ultrasonic waves are delivered to the sample in the extraction solvent, the temperature rises by the ultrasonic waves, and the reactant particles located in the vicinity due to the high local temperature By increasing their kinetic energy, sufficient energy necessary for the reaction is obtained, and a high pressure is induced by the impact effect of ultrasonic energy, which increases the mixing effect of the material contained in the sample and the extraction solvent, thereby increasing the extraction efficiency.

The extraction solvent that can be used for the ultrasonic extraction method may be any one selected from the group consisting of chloroform, ethanol, methanol, water, ethyl acetate, hexane, diethyl ether, and mixtures thereof.

The solvent extraction method may be a cold extraction method, a hot extraction method, a pressure extraction method, or a reflux extraction method.

The preparation of the fraction of the shiitake mushroom extract can be performed by adding a fractionation solvent to the extract prepared by the above extraction method, that is, the crude extract or the crude extract, and then obtaining the fraction according to the polarity of the fractionation solvent. The method for obtaining the fraction may be performed by a separation method by layer separation or a fractionation method. For example, in the order of hexane, chloroform, methylene chloride, ethyl acetate, butanol and water, preferably hexane, methylene chloride, ethyl acetate, butanol and water, more preferably hexane, methylene chloride, ethyl acetate, butanol and water It can be carried out as a method of obtaining a hexane fraction, a chloroform fraction, an ethyl acetate fraction, a butanol fraction, and a water fraction, which are layer-separated after addition.

The fractionation method by layer separation may be a method of obtaining a fraction obtained for each application by applying the solvent in a non-polar order, for example, hexane obtained by fractionating the layer-separated hexane layer by adding hexane to an ethanol aqueous solution extract. fraction; a methylene chloride fraction obtained by fractionating the methylene chloride layer separated by adding methylene chloride to the aqueous layer remaining after separating the hexane fraction; The ethyl acetate fraction obtained by fractionating the layer-separated ethyl acetate layer by adding ethyl acetate to the aqueous layer remaining after separating the methylene chloride fraction and the water fraction remaining after separating the ethyl acetate fraction may be sequentially obtained. .

The extract also includes a purified product that is further subjected to a conventional purification process in addition to the extract or fraction.

Purification of the extract or fraction may be purification using an ultrafiltration membrane, purification by various chromatography methods, and the like.

Chromatography for purification of the extract or fraction is various chromatography methods such as silica gel column chromatography, thin layer chromatography, TLC, or high performance liquid chromatography (HPLC). It can be carried out using a chromatography prepared for separation according to size, charge, hydrophobicity or hydrophilicity, and can be variously performed.

In addition, the extract may be concentrated or the solvent may be removed by performing extraction, fractionation, or purification, followed by filtration under reduced pressure or additional concentration and/or freeze-drying. Therefore, the shiitake mushroom extract in the present invention is used in the sense of including a dry product of the extract dried by a conventional method, a concentrate of the extract concentrated by a conventional method, and a diluted solution of the extract, the dried product or the concentrate. The obtained shiitake mushroom extract may be stored in a deep freezer until use.

It was confirmed that the shiitake mushroom extract has excellent skin protection effect, specifically, the effect of preventing and improving skin damage caused by ultraviolet rays as a result of an animal test through an experiment confirming the effect of the present invention. Therefore, the shiitake mushroom extract may be used in an external composition or cosmetic composition for the purpose of protecting the skin.

In this aspect, the present invention provides an external composition for skin protection against ultraviolet rays comprising a shiitake mushroom extract as an active ingredient.

In addition, the present invention may be a cosmetic composition for protecting the skin against ultraviolet rays comprising a shiitake mushroom extract as an active ingredient.

The composition for external application for skin protection may be used as an external preparation for skin protection including a cosmetic composition for skin protection. All simple modifications or changes of the present invention can be easily carried out by those of ordinary skill in the art to which the present invention pertains, and all such modifications or changes can be considered to be included in the scope of the present invention.

In the present invention, the composition for external application is also called an external preparation, and the external composition is a concept that encompasses the overall composition used for external application. Specifically, it refers to a cosmetic composition including various cosmetics such as basic cosmetics, makeup cosmetics, and hair cosmetics, and various pharmaceuticals such as ointments or quasi-drugs, etc., preferably a cosmetic composition, more preferably a functional cosmetic composition or functional It may be cosmetics.

The composition for external use includes, in addition to active ingredients, ingredients commonly formulated in external compositions according to the intended use and properties of the external composition, for example, moisturizers, ultraviolet absorbers, vitamins, animal and plant extracts, digestive agents, whitening agents, vasodilators, astringents, refreshing agents, It may be one in which a hormonal agent or the like is additionally formulated.

The formulation of the composition for external use may take an appropriate form depending on the intended use and the nature of the composition for external use, for example, an aqueous solution-based, solubilizing-based, emulsified, emulsified, gel-based, paste-based, ointment-based, aerosol-based, water-oil-based formulation. It may be a two-layer system or a three-layer water-oil-powder system, and the dosage form and form of the external composition of the present invention are not limited by the dosage form.

In addition, the external preparation is an active ingredient, that is, a shiitake mushroom extract, preferably a shiitake mushroom extract having a beta glucan content of 165 mg/g (beta glucan / shiitake) to 170 mg/g (beta glucan / shiitake) compared to shiitake mushrooms It may further include a mechanism necessary to penetrate or migrate into the skin tissue.

The active ingredient may be 0.001 to 99.999 wt%, preferably 0.01 to 30 wt%, more preferably 0.05 to 20 wt%, based on the total weight of the composition for external use, but the content of the active ingredient contained in the formulation or composition for external use It can be appropriately adjusted according to the content of other ingredients.

It may be a cosmetic composition for protecting the skin against ultraviolet rays comprising the shiitake mushroom extract as an active ingredient.

In the cosmetic composition for skin protection, in addition to active ingredients, ingredients commonly formulated in cosmetic compositions depending on the intended use and the properties of the cosmetic composition, for example, moisturizers, ultraviolet absorbers, vitamins, animal and plant extracts, digestive agents, whitening agents, vasodilators, astringents, A refreshing agent, a hormone agent, etc. may be additionally formulated.

The formulation of the cosmetic composition may take an appropriate form depending on the intended use and the nature of the composition for external use, for example, an aqueous solution-based, solubilizing-based, emulsified, emulsified, gel-based, paste-based, ointment-based, aerosol-based, water-oil-based formulation It may be a two-layer system, water-oil-powder three-layer system, and the formulation and form of the cosmetic composition of the present invention are not limited by the formulation.

In addition, the cosmetic composition is an active ingredient, that is, a shiitake extract, preferably a shiitake mushroom having a beta glucan content of 165 mg/g (beta glucan / shiitake) to 170 mg / g (beta glucan / shiitake) compared to shiitake mushrooms. It may further include a base necessary to penetrate or migrate the extract into the skin tissue.

The active ingredient may be 0.001 to 99.999% by weight, preferably 0.01 to 30% by weight, more preferably 0.05 to 20% by weight, based on the total weight of the cosmetic composition, but the content is the active ingredient contained in the formulation or cosmetic composition It can be appropriately adjusted according to the content of other ingredients.

The cosmetic composition for skin care containing the shiitake mushroom extract as an active ingredient includes, in addition to the active ingredient, all kinds of ingredients that can be used in conventional cosmetics, such as fragrances, pigments, sterilizers, antioxidants, preservatives, moisturizers, thickeners, and excipients. , a diluent, inorganic salts, and synthetic polymer materials may be further included.

The cosmetic composition may be provided for use in basic cosmetics, makeup cosmetics, body cosmetics, hair cosmetics, scalp cosmetics, or shaving cosmetics.

Examples of the basic cosmetics include creams, lotions, packs, massage creams, and emulsions, and the makeup cosmetics include foundation, makeup base, lipstick, eye shadow, eyeliner, mascara, eyebrow pencil, etc., and the body As cosmetics, there are soap, liquid detergent, bath agent, sunscreen cream, sun oil, etc., and the cosmetics for hair include shampoo, conditioner, hair treatment, hair mousse, hair liquid, pomade, hair color agent, hair bleach. , color rinse, the scalp cosmetics include hair tonic and scalp treatment, and the shaving cosmetics include aftershave lotion, shaving cream, and the like.

The cosmetic composition for skin protection comprising the shiitake mushroom extract as an active ingredient may be a cosmetic composition for repairing skin damage caused by ultraviolet rays or a cosmetic composition for blocking ultraviolet rays.

Since the shiitake mushroom extract, which is an active ingredient of the cosmetic composition for repairing damage caused by ultraviolet rays or the sunscreen cosmetic composition, has an excellent effect of repairing damage caused by ultraviolet rays, it can be variously used in cosmetics having an effect of repairing damage caused by ultraviolet rays.

The cosmetic composition for skin protection against UV rays may further include at least one selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids, and seaweed extract.

The water-soluble vitamins can maintain the effects of the present invention, and can be included in any form as long as they can be formulated in cosmetics. Specifically, vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, nicotinic acid, nicotinic acid amide , folic acid or vitamin C, salts thereof (thiamine hydrochloride, sodium ascorbate, etc.) or derivatives thereof (sodium ascorbic acid-2-phosphate, magnesium ascorbic acid-2-phosphate, etc.) . The water-soluble vitamin may be obtained by a conventional method such as a microorganism transformation method, a purification method from a microbial culture, an enzymatic method or a chemical synthesis method.

The oil-soluble vitamins can maintain the effects of the present invention and may include any type of compound that can be formulated in cosmetics. Specifically, vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (D-alpha tocopherol, D-alpha tocopherol) , D-alpha tocopherol), and their derivatives (ascorbine palmitate, ascorbine stearate, ascorbine dipalmitate, acetate-alpha tocopherol, nicotinic acid-alpha tocopherol vitamin E, D-pantothenyl alcohol, D- pantothenyl alcohol, pantothenyl ethyl ether, etc.). The oil-soluble vitamin can be obtained by a conventional method such as a microorganism transformation method, a purification method from a microbial culture, an enzymatic method or a chemical synthesis method.

The polymer peptide can maintain the effects of the present invention, and may be included in any form as long as it can be formulated in cosmetics, and specifically, it may be collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin or keratin. The polymer peptide can be obtained through a purification process after being obtained by a conventional method such as a purification method from a microorganism culture solution, an enzyme method or a chemical synthesis method, and usually natural products such as the dermis of pigs or cattle, silk fibers of silkworms, etc. It can also be purified from and used.

The high molecular weight polysaccharide can maintain the effects of the present invention and may include any compound that can be formulated in cosmetics, specifically hydroxyethyl cellulose, xanthan gum, sodium hyaluronate or chondroitin sulfate, and their salts (sodium salt, etc.). The chondroitin sulfate or a salt thereof may be purified and used from mammals or fish.

The sphingolipid may maintain the effects of the present invention and may include any compound that can be formulated in cosmetics, and specifically may be ceramide, phytosphingosine, or sphingoglycolipid. The sphingo lipid may be purified by a conventional method from mammals, fish, shellfish, yeast or plants, or may be obtained by chemical synthesis.

The seaweed extract can maintain the effect of the present invention, and can include any that can be formulated in cosmetics, specifically brown algae extract, red algae extract, green algae extract, etc., and the seaweed extract includes a color purified from the seaweed extract. Also included are ginan, arginic acid, sodium alginate, potassium alginate, and the like. The seaweed extract may be obtained by purification from seaweed by a conventional method.

In addition, the cosmetic composition of the present invention may further include other ingredients commonly formulated in cosmetics. More specifically, the other ingredients commonly formulated in the cosmetic are oil and fat components, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, UV absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters , alcohol, color, fragrance, blood circulation promoter, cooling agent, limiting agent, or purified water.

Specifically, the fat and oil component may be an ester-based fat, hydrocarbon-based fat, silicone-based fat, fluorine-based fat, animal fat, or vegetable fat.

The ester-based oils and fats are, for example, tri-2-ethylhexanoate glyceryl, 2-ethylhexanoate cetyl, isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl stearate, octyl palmitate, isostearic acid Socetyl, butyl stearate, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl myristate, isostearyl myristate, isostearyl palmitate, octyldodecyl myristate, isocetyl isostearate, sebacic acid Diethyl, diisopropyl adipate, isoalkyl neopentanoate, tri(capryl, capric acid) glyceryl, tri-2-ethylhexanoate trimethylolpropane, triisostearate trimethylolpropane, tetra-2- Pentalyslitol ethyl hexanoate, cetyl caprylate, decyl laurate, hexyl laurate, decyl myristate, myristyl myristate, cetyl myristate, stearyl stearate, decyl oleate, ricinoleic acid Cetyl, isostearyl laurate, isotridecyl myristate, isocetyl palmitate, octyl stearate, isocetyl stearate, isodecyl oleate, octyldodecyl oleate, octyldodecyl linoleate, isostearate isopropyl, cetostearyl 2-ethylhexanoate, stearyl 2-ethylhexanoate, hexyl isostearate, ethylene glycol dioctanoate, ethylene glycol dioleate, propylene glycol dicapric acid, di(caprylic acid, capric acid) propylene glycol, Propylene glycol dicaprylate, neopentyl glycol dicapric acid, neopentyl glycol dioctanoate, glyceryl tricaprylate, glyceryl triundecyl acid, glyceryl triisopalmitate, glyceryl triisostearate, octyldode neopentanoate Thread, isostearyl octanoate, octyl isononanoate, hexyldecyl neodecanoate, octyldodecyl neodecanoate, isocetyl isostearate, isostearyl isostearisostearate, octyldecyl isostearate, polyglycerol oleic acid ester, Polyglycerol isostearate, triisocetyl citrate, triisoalkyl citrate, triisooctyl citrate, lauryl lactate, myristyl lactate, cetyl lactate, octyldecyl lactate, triethyl citrate, acetyl triethyl citrate, acetyl tributyl citrate , trioctyl citrate, diisostearyl malate, 2-ethylhexyl hydroxystearate, di2-ethylhexyl succinate, diisobutyl adipate, diisopropyl sebacate, Dioctyl sebacate, cholesteryl stearate, cholesteryl isostearate, cholesteryl hydroxystearate, cholesteryl oleate, dihydrocholesteryl oleate, phytsteryl isostearate, phytsteryl oleate, 1,2-stealoylhydroxystearate isocetyl, 1,2-stealoylhydroxystearate stearyl or 1,2-stealoylhydroxystearic acid is sostearyl and the like.

The hydrocarbon-based oil may be, for example, squalene, liquid paraffin, alpha-olefin oligomer, isoparaffin, ceresin, paraffin, liquid isoparaffin, polybudene, microcrystalline wax or petrolatum.

The silicone-based fat is, for example, polymethylsilicone, methylphenylsilicone, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane/methylcetyloxysiloxane copolymer, dimethylsiloxane methylstealloxysiloxane copolymer , alkyl-modified silicone oil or amino-modified silicone oil.

The fluorine-based oil may be perfluoropolyether or the like.

The animal oil or vegetable oil is avocado oil, almond oil, olive oil, sesame oil, rice bran oil, bird flower oil, soybean oil, corn oil, rapeseed oil, sesame oil, palm kernel oil, palm oil, castor oil, sunflower oil, grape seed oil, Cottonseed oil, palm oil, kukui nut oil, wheat germ oil, rice germ oil, shea butter, moonflower colostrum, marker damia nut oil, meadow home oil, egg yolk oil, tallow, horse oil, mink oil, orange rafi oil, jojoba oil, candelabra wax , carnauba wax, liquid lanolin, or hydrogenated castor oil.

The surfactant may be specifically an anionic surfactant, a cationic surfactant, or an amphoteric surfactant.

The anionic surfactant is, for example, fatty acid soap, alpha-acyl sulfonate, alkyl sulfonate, alkyl allyl sulfonate, alkyl naphthalene sulfonate, alkyl sulfate, POE alkyl ether sulfate, alkylamide sulfate, alkyl phosphate, POE alkyl ginseng Salt, alkylamide phosphate, alkyloyl alkyl taurine salt, N-acyl amino acid salt, POE alkyl ether carboxylate, alkyl sulfosuccinate, alkyl sulfoacetate sodium, acylated hydrolyzed collagen peptide salt or perfluoroalkyl phosphate ester etc.

The cationic surfactant is, for example, alkyltrimethylammonium chloride, stearyltrimethylammonium chloride, stearyltrimethylammonium bromide, cetostearyltrimethylammonium chloride, distearyldimethylammonium chloride, stearyldimethylbenzylammonium chloride, behenyl bromide trimethylammonium, benzalkonium chloride, diethylaminoethylamide stearate, dimethylaminopropylamide stearate, or a quaternary ammonium salt of a lanolin derivative.

The amphoteric surfactant is, for example, a carboxybetaine type, an amidebetaine type, a sulfobetaine type, a hydroxysulfobetaine type, an amidesulfobetaine type, a phosphobetaine type, an aminocarboxylate type, an imidazoline derivative type or an amide It may be an amine-type amphoteric surfactant or the like.

The pigment may be an organic pigment, an inorganic pigment, and a complex pigment thereof.

The inorganic pigment is, for example, silicic acid, silicic anhydride, magnesium silicate, talc, sericite, mica, kaolin, bengala, clay, bentonite, titanium coated mica, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, oxide aluminum, calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, ultramarine blue, chromium oxide, chromium hydroxide, calamine, and complexes thereof.

The organic pigment is, for example, polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenol resin, fluororesin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, divinyl It may be a benzene-styrene copolymer, silk powder, cellulose, CI pigment yellow, CI pigment orange, and complexes thereof.

The organic powder may include, for example, metal soap such as calcium stearate; alkyl phosphate metal salts such as sodium zinc cetylate, zinc laurylate, and calcium laurylate; acylamino acid polyvalent metal salts such as calcium N-lauroyl-beta-alanine, zinc N-lauroyl-beta-alanine, and calcium N-lauroyl glycine; amidesulfonic acid polyvalent metal salts such as calcium N-lauroyl-taurine and calcium N-palmitoyl-taurine; N such as N-epsilon-lauroyl-L-lysine, N-epsilon-palmitoylizine, N-alpha-paritoylolnithine, N-alpha-lauroylarginine, N-alpha-hydrogenated beef fatty acid acylarginine, etc. -acyl basic amino acids; N-acyl polypeptides such as N-lauroylglycylglycine; alpha-amino fatty acids such as alpha-aminocaprylic acid and alphaaminolauric acid; It may be polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene, ethylene tetrafluoride, or the like.

The cosmetic composition for protecting the skin against ultraviolet rays may further include a known sunscreen agent. The sunscreen may be an organic sunscreen and/or an inorganic sunscreen.

The cosmetic composition for preventing or recovering damage to UV rays may be prepared in any formulation conventionally prepared in the art, for example, in emulsion, cream, lotion, pack, foundation, lotion, cosmetic liquid, hair cosmetic, etc. can be

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be embodied in various different forms, and the examples described herein are only for illustrating the present invention, and the present invention is not limited to the following examples.

In this experiment, the same experiment was repeated three or more times, and the data obtained from each experiment result were average and standard deviation (Mean) using SPSS/WIN 12.0 version statistical program (Statistical Package for social Science, IBM, USA). ± SEM). To verify the significance between groups, analysis was performed through one-way ANOVA, and the post-Hoc test verified the significance between each experimental group at the level of P < 0.05 by Duncan's multiple range test.

Example 1. Preparation of Shiitake Mushroom Extract

Shiitake mushrooms were used with samples grown at Smart Farm (OLCHI, Korea) located in Yeonje-gu, Busan.

Shiitake mushroom extract was prepared as follows.

First, the provided raw shiitake mushrooms (50 kg) were sliced and dried in a hot air dryer (Cheil Machinery Co., Ltd., Korea) at 65 ° C. Extracted and recovered for 8 hours in an extraction tank of (Cheil Machinery Co., Ltd., Korea). The same amount of distilled water as the recovered shiitake mushroom extract was added and re-extracted under the same conditions. The obtained extract is concentrated in a concentrator (Thicking tank, Cheil Machinery Co., Ltd., Korea) at 60 ° C, and then subjected to a rapid freezing process at -70 ° C using a freeze dryer (PVTFD30R, IlShinBioBase, Korea) to extract powdered shiitake mushrooms A concentrated powder (3.10 kg) was obtained.

The beta-glucan (β-Glucan) content of the shiitake mushroom extract grown in the smart farm was measured. The beta glucan content was measured using the Yeast & Mushroom β-Glucan Assay Kit (Megazyme Ltd., Bray, Wicklow County, Ireland). Specifically, in a method to obtain the content of beta-glucan by subtracting alpha-glucan from total glucan, it was decomposed into glucan form through acid decomposition and enzyme treatment, followed by color development by measuring absorbance and quantification.

The total glucan content was measured as follows. First, 1.5 ml of 37% HCl was added to a 100 μl sample (100 mg/ml) and vortexed, followed by reaction in a thermostat at 40° C. for 45 minutes, followed by vortexing by adding 10 ml of distilled water, 100 After reacting for 2 hours in a constant temperature water bath at ℃, 10 ml of 2 M KOH was added after cooling, and 200 mM sodium acetate buffer (pH 5) was added to make 100 ml. 0.1 ml of the test solution prepared above was mixed with 0.1 ml of the beta-glucan enzyme mixture, reacted at 40° C. for 30 minutes, 3 ml of a color reagent (glucose oxidase/peroxidase reagent, GOPOD reagent) was added, and reacted at room temperature for 10 minutes, This was performed by measuring the absorbance at 510 nm to calculate the total glucan content.

항온수조에서 30분간 반응시키고, 1,500 x g의 조건에서 10분 동안 원심분리한 후, 상층액 0.1 ml와 발색시약(GOPOD) 3 ml을 혼합하여 실온에서 10분간 반응시킨 다음 증류수를 대조액으로 분광광도계 파장 510 nm에서 흡광도를 측정하여 알파 글루칸 함량을 계산하는 방법으로 수행하였다.The alpha glucan content was performed in the following way. First, 2 ml of 2 M KOH was added to a 100 μl sample (100 mg/ml) and stirred, then 8 ml of 1.2 M sodium acetate buffer (pH 3.8) was added, and then 0.2 ml of alpha-glucan enzyme mixture was added. 40

After 30 minutes of reaction in a constant temperature water bath, centrifugation at 1,500 x g for 10 minutes, 0.1 ml of the supernatant and 3 ml of the color developing reagent (GOPOD) were mixed and reacted for 10 minutes at room temperature, and then distilled water as a control solution for spectrophotometric wavelength This was performed by measuring the absorbance at 510 nm to calculate the alpha glucan content.

The beta glucan content was calculated by subtracting the alpha glucan content from the total glucan content.

As a result of the measurement by the above method, the beta-glucan content of the shiitake mushroom extract was confirmed to be 165.96 mg/g (166%), which was confirmed to be higher than the beta-glucan content of the normal shiitake mushroom.

Experimental Example 2: Protective Effect of Shiitake Mushroom Extract from UV-induced Skin Damage

Experimental Example 2-1. Cultivation of test target cells

HaCaT cells, which are human keratinocyte cells, which are test target cells for confirming the skin damage protection effect caused by UV rays, were purchased from CLS-Cell Line Service (200493, Eppelheim, Germany). The HaCaT cells were cultured in an incubator at 37° C. and 5% CO ₂ using Dulbecco's Modified Eagle Medium (DMEM, Welgene, Korea) containing 10% fetal bovine serum and 100 units/ml penicillin/streptomycin, and 2 to The culture was replaced with a new medium once every 3 days.

Example 2-2. Confirmation of Cytotoxicity of Shiitake Mushroom Extract

To confirm the cytotoxicity of the shiitake extract, MTT assay was performed using a HaCaT cell line.

Specifically, HaCaT cells were dispensed in a 96-well plate at 3X10 ⁴ cells/well and cultured for 24 hours, then the extract prepared in Example 1 was treated at a concentration of 0 to 5 mg/ml, and after 24 hours, 100 After adding μl of MTT solution (1 mg/ml) and incubating for 4 hours, the resulting formazan crystal was dissolved in DMSO and absorbance was measured at 540 nm with a GENios FL microplate reader (Tecan Austria GmbH, Grodig, Australia). was calculated as a percentage compared to the control group not treated and shown in FIG. 1 .

As shown in FIG. 1, when the shiitake mushroom water extract was treated at a concentration of 5 mg/ml, 83.6% of HaCaT cells survived, and at a concentration of 2 mg/ml or less, it was confirmed that more than 97.2% of the cells survived. , it was confirmed that there was no cytotoxicity at 2 mg/ml or less. Then, in order to measure the skin aging improvement effect, it was confirmed that the shiitake mushroom water extract was used at a concentration of 2 mg/ml or less.

Example 2-3. Measurement of skin damage protection effect

In order to confirm the skin damage protection effect of the shiitake mushroom extract, the regeneration effect of the shiitake mushroom extract by scratch was confirmed by a scratch wound healing assay.

Specifically, HaCaT cells were seeded in a 12 well plate at 3X10 ⁵ cells/well and cultured, and then terminated with a 200 μl yellow tip to induce cell wounding. Thereafter, the prepared shiitake mushroom water extract sample was treated with different concentrations (0.5, 1, 2 mg/ml) and cultured for 24 hours, and then the degree of cell migration was observed using a microscope to determine if the sample was not treated. The results observed in comparison with the control group are shown in FIG. 2 .

As shown in FIG. 2 , it was confirmed that the HaCaT cells moved to the wound site and the wound interval was narrowed as the treatment concentration of the shiitake mushroom water extract increased.

Example 2-4. Measurement of skin damage protection effect by UV rays1

Ultraviolet (UV) irradiation of HaCaT cells was performed in the following way to confirm the skin damage protection effect by ultraviolet rays. First, the culture medium of the HaCaT cell line was removed and washed with PBS. Then, in the presence of PBS, Bio-Sun UV irradiation system (Vilber Lourmat, France) was used at 10 J/cm ² for UVA and 20 for UVB. m J/cm ² It was carried out by irradiating each.

First, the cytoprotective effect of the shiitake mushroom extract according to ultraviolet (UVA/UVB) irradiation was confirmed by MTT assay. First, HaCaT cells were aliquoted at 3X10 ⁴ cells/well in a 24 well plate and cultured for 24 hours. After removing the medium and washing with PBS, 100 μl PBS was added and UVA (10 J/cm ² ) and UVB ( 20 mJ/cm ² ) was irradiated, washed with PBS, and replaced with a serum-free medium, and the prepared shiitake mushroom water extract samples were treated with different concentrations (0.5, 1, 2 mg/ml) and cultured for 24 hours.

After that, as in the above MTT assay, MTT solution (1 mg/ml) was added and incubated for 4 hours, the formazan crystal was dissolved in DMSO and absorbance was measured at 540 nm with a microplate reader (Tecan Austria GmbH, Australia). The degree of cell proliferation as a percentage is shown in FIG. 3 .

As shown in FIG. 3a, when HaCaT cells were irradiated with UVA (10 J/cm ² ), about 19% of cells were killed compared to a negative control that was not irradiated with UVA, and when treated with shiitake mushroom water extract, some cell proliferation The effect was confirmed. In addition, as shown in FIG. 3b, the control group irradiated with UVB (20 mJ/cm ² ) without any sample treatment resulted in more than 30% cell death compared to the negative control group not irradiated with UVB, and after UVB irradiation, shiitake mushroom water extract was treated at concentrations of 0.5, 1, and 2 mg/ml, the cells proliferated in a concentration-dependent manner at 14.4%, 18.7%, and 22.8%, respectively, and a significant effect was confirmed.

Example 2-5. Measurement of the protective effect of skin damage caused by UV rays2

In order to confirm the skin damage protection effect of the shiitake mushroom water extract by UV rays, the ROS scavenging effect was measured by the DCF-DA method.

Specifically, intracellular ROS generation was induced by H ₂ O ₂ treatment and UVA/UVB irradiation. HaCaT cells were seeded in a 96-well black plate at 3 x 10 ⁴ cells/well and cultured for 24 hours.

To measure the H ₂ O ₂ -induced ROS scavenging effect, HaCaT cells were cultured, washed twice with PBS, 20 μM DCF-DA (100 mM Hank’s buffered salt solution, HBSS) was added 100 μl, and incubated for 20 minutes. did After that, shiitake mushroom water extract samples were added by concentration and incubated for 1 hour, washed twice with PBS, 200 μM H ₂ O ₂ (in HBSS) was added, and excitation using a fluorometer after 120 minutes The intensity of fluorescence was measured at 485 nm and emission 530 nm, and it was expressed as a percentage compared to the control to which the sample was not added, and is shown in FIG. 4A .

In addition, the ROS scavenging effect in cells according to UVA and UVB irradiation was irradiated with UVA (10 J/cm ² ) and UVB (20 mJ/cm ² ) instead of H ₂ O ₂ treatment, excitation 485 nm, emission 530 nm after 120 minutes This was performed by measuring the intensity of fluorescence in the cell to confirm the ROS scavenging effect, and is shown in FIGS. 4b and 4c.

As shown in FIG. 4a, the ROS scavenging ability generated by hydrogen peroxide (H ₂ O ₂ ) was observed and the ROS generated in a concentration-dependent manner compared to the control group to which the sample was not added was eliminated, and each ROS scavenging rate was 0.5 , showed 45.3%, 54.5%, and 64.5% at concentrations of 1 and 2 mg/ml, respectively.

In addition, as shown in FIG. 4b, ROS generated by UVB irradiation was removed in a concentration-dependent manner, and the respective elimination rates were 16.9, 22.7, and 32.9% at the concentrations of 0.5, 1, and 2 mg/ml, respectively. In addition, as shown in Fig. 4c, the ROS scavenging effect of the shiitake mushroom water extract according to UVA irradiation increased in a concentration-dependent manner and was 17.0%, 21.1%, and 24.6%, respectively (Fig. 4C). From the above results, it was confirmed that the shiitake mushroom water extract effectively inhibited cell damage and excessive ROS generation due to UV irradiation.

Example 2-6. Measurement of skin damage protection effect by UV rays3

In order to confirm the protective effect of the shiitake mushroom water extract on skin damage caused by ultraviolet rays, MMPs, which are extracellular matrix proteolytic enzymes, were identified.

First, HaCaT cells were aliquoted in a 6 well plate at 3X10 ⁵ cells/well and cultured for 24 hours, then UVB was irradiated and samples were treated with serum-free medium for each concentration and cultured for 24 hours. The cultured cells used AccuPrep ^® Universal RNA Extraction Kit (Bioneer Corp. Korea) to extract total RNA. cDNA was synthesized according to the manufacturer's method using Cell ScriptTM cDNA master Mix (Cellsafe, Korea). For the target cDNA, the following primers were used. For MMP-1, forward 5'-TCT GAC GTT GAT CCC AGA GAG CAG-3' and reverse 5'-CAG GGT GAC ACC AGT GAC TGC AC-3' were used, and for MMP-9, forward 5'-CAC was used. TGT CCA CCC CTC AGA GC-3' and reverse 5'-CAC TTG TCG GCG ATA AGG-3' were used, and for Type 1α procollagen, forward 5'-CTC GAG GTG GAC ACC ACC CT-3' and reverse 5' -CAG CTG GAT GGC CAC ATC GG-3' was used, and for β-actin, forward 5'-AGA TCA AGA TCA TTG CTC CTC CTG-3' and reverse 5'-CAA AGG GTG TAA CGC AAC TAA G-3 ' was used. RT-PCR was performed using the primer and AccuPrep ^® PCR PreMix & Master Mix (Bioneer Corp. Korea), and the reaction product was observed using Davinch-Chemi imager ^TM (CAS-400SM, Korea), and then a control gene Rho was used as β-actin for relative quantification, and the results are shown in FIG. 5a.

In addition, the content of the produced protein was performed by Western blotting.

Specifically, HaCaT cells were seeded in a 6 well plate at 3X10 ⁵ cells/well and cultured for 24 hours, then UVB was irradiated, and samples were treated with serum-free medium for each concentration and cultured for 24 hours. Proteins were extracted from the cultured cells using RIPA lysis buffer (Sigma-Aldrich Corp., USA), and proteins were quantified using a BCA protein assay kit (Thermo Fisher Scientific, USA). Each protein was added to sodium dodecyl sulfate (SDS) sample buffer, boiled at 100°C for 5 minutes, loaded on 10% SDS-polyacrylamide gel, and electrophoresed. After electrophoresis, it was transferred to a PVDF membrane (Amersham GE Healthcare Life Sci., Germany). transferred. Membrane was blocked with 5% skim milk blocking solution (0.1% Tween-20, tris-buffered saline, pH 8.0), and primary antibody diluted 1:1,000 was added and incubated overnight at 4°C, Horseradish peroxidase (HRP) ) The enzyme-bound secondary antibody was diluted 1:5,000 and reacted at room temperature for 1 hour. Protein bands were analyzed using ECL solution with CAS-400SM Davinch-Chemi imager? (Davinch-K). The primary and secondary antibodies used were purchased from Cell Signaling Technology (Danvers, USA) and Santa Cruz Biotechnology (USA), and relative quantification was performed using β-actin as a control gene. The measurement results are shown in FIG. 5B.

As shown in FIG. 5a, in the case of the control group irradiated with UVB without treating the sample, the expression of MMP-1 and MMP-9 was increased, but when the shiitake mushroom water extract was treated, the expression of MMP-1 and MMP-9 was decreased. decreased was confirmed. In addition, in the case of Type I procollagen, it was confirmed that the expression was increased according to the treatment with the shiitake mushroom water extract, while almost no expression in the control group. In addition, as shown in FIG. 5b , it was confirmed that the shiitake mushroom water extract decreased the expression of MMP-1 and MMP-9 increased by UVB irradiation, and increased the synthesis of type I procollagen.

Claims (8)

An external composition for skin protection against UV rays comprising shiitake mushroom extract ( Lentinuls edodes ) as an active ingredient. The method of claim 1,
The shiitake mushroom has a beta glucan content of 165 mg/g (beta glucan / shiitake) to 170 mg / g (beta glucan / shiitake) compared to shiitake. The method of claim 1,
The shiitake mushroom extract is an external composition for skin protection against UV rays, characterized in that it suppresses skin damage and inflammation caused by exposure to UV rays. A cosmetic composition for protecting the skin against UV rays comprising shiitake mushroom extract ( Lentinuls edodes ) as an active ingredient. 5. The method of claim 4,
The shiitake mushroom has a beta glucan content of 165 mg/g (beta glucan / shiitake) to 170 mg / g (beta glucan / shiitake) compared to the shiitake mushroom. 5. The method of claim 4,
The shiitake mushroom extract is a cosmetic composition for skin protection against ultraviolet rays, characterized in that it suppresses skin damage and inflammation caused by exposure to ultraviolet rays. 5. The method of claim 4,
The shiitake mushroom extract is a cosmetic composition for skin protection against ultraviolet light, characterized in that it is a shiitake mushroom water extract. 5. The method of claim 4,
The shiitake mushroom extract is a cosmetic composition for skin protection against UV rays, characterized in that it is included in an amount of 0.01 to 30% by weight based on the total weight of the composition.

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