KR102644047B1 - A composition for preventing or improving skin wrinkles or skin thermal aging comprising rosa multiflora extracts - Google Patents

Abstract

The present invention relates to a composition for preventing or improving skin wrinkles or thermal aging of the skin containing a wild rose extract ( Rosa multiflora ), wherein the composition does not exhibit cytotoxicity and has an activity inhibitory effect on elastase and collagenase. In addition to being excellent, it reduces the expression of TRPV-1 and MMP-1, which are indicators of heat stimulation inhibition proteins, and increases the expression of procollagen, ultimately inhibiting thermal aging of the skin, making the wild rose extract effective. The composition containing it as an ingredient can be usefully used as a cosmetic composition for preventing or improving skin wrinkles or heat aging, an external skin agent, or a health functional food.

Description

Composition for preventing or improving skin wrinkles or thermal aging of the skin containing wild rose extract

The present invention relates to a composition for preventing or improving skin wrinkles or heat aging of the skin, comprising an extract of Rosa multiflora . In detail, it relates to a cosmetic composition, food composition, or quasi-drug composition for preventing or improving skin wrinkles or heat aging, containing wild rose extract as an active ingredient.

Skin aging is largely divided into two types: natural aging, which causes wrinkles as people age, i.e., intrinsic aging, and extrinsic aging, caused by heat from the sun, ultraviolet rays, stress, etc. . Recently, as the ozone layer has been destroyed due to environmental pollution, ultraviolet rays have become stronger, and temperatures are gradually rising due to global warming. As a result, people's interest in heat aging is increasing.

Since ultraviolet rays and heat exist all year round and are common causes of aging in daily life, it is most important to protect and improve aged skin from ultraviolet rays and heat. Although a variety of product lines for UV protection are on the market, cosmetics containing heat-aging protection are not common.

Wild rose ( Rosa multiflora Thunb. ) is a plant belonging to the Rosaceae family. It can be easily found in mountains, the foothills of fields, and valleys. It has good medicinal efficacy and has been used in oriental medicine for a long time so that none of it is discarded. In addition, each part of the wild rose, including flowers, fruits, roots, and seeds, is known to be a material with excellent antioxidant, anti-inflammatory, antibacterial, and hair growth effects. The active ingredients of these wild roses include quercetin glycosides (multinoside A, multinoside B, quercitrin, etc.), which are part of the flavonoid family, and kaempferol glycosides (multiflorin A, multiflorin B, aphrodisiacs). Jelly, astragalin, etc.), tannin-based methyl gallate, and lycopene, a red pigment in fruit, have been reported (Hyun Woo Kim et.al. , Korean J. Medicinal Crop Sci , 26(4) ):308-316, 2018).

Thermal aging is a new concept of skin aging that accelerates aging by increasing the skin temperature when the skin is exposed to heat. The human skin temperature is maintained at an average of 31-32℃, but if the skin temperature rises above that, a skin inflammatory reaction occurs and heat aging begins. Meanwhile, human skin is frequently exposed to heat generated by using electronic devices such as smartphones, hair dryers, and stoves, which increases skin temperature and causes skin aging. As such, human skin is exposed to ultraviolet and infrared rays every day, and research results have reported that human skin temperature rises to 40-43℃ in direct sunlight in the middle of the summer within 15-20 minutes.

It can be confirmed that the cause of aging in the skin is not only ultraviolet rays in the sun, but also heat (infrared rays). Repeated exposure to infrared rays increases the synthesis of MMP (metallo matrix protease) present in the dermis, causing wrinkles and skin aging. accelerates.

Meanwhile, TRPV-1 (transient receptor potential vanilloid-1) ion channel, a heat receptor, is known to have the characteristic of responding to thermal stimulation above 43°C and acidic stimulation below pH 5.4. In addition, it is known to be involved in not only transmitting information about thermal and spicy stimuli, but also causing an increase in pain due to environments such as inflammation (Seung Jun Jung, Hanyang Med Rev. , 31(2):116-122, 2011 ).

Against this background, the present inventors made diligent efforts to find a natural product that improves skin wrinkles, prevents skin heat aging, and has fewer side effects. As a result, Rosa multiflora extract was found to be effective in elastase and collagenase. Not only does it have an excellent activity inhibition effect, but it also reduces the expression of TRPV-1 (transient receptor potential vanilloid-1) and MMP-1, which are indicators of heat stimulation inhibitory proteins, and increases the expression of procollagen, ultimately By confirming that skin thermal aging can be excellently inhibited, the present invention has been completed.

Hyun Woo Kim et.al., Korean J. Medicinal Crop Sci, 26(4):308-316, 2018 Seung Jun Jung, Hanyang Med Rev., 31(2):116-122, 2011

The purpose of the present invention is to provide a cosmetic composition for preventing or improving skin wrinkles or heat aging, containing Rosa multiflora extract as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving skin wrinkles or heat aging of the skin, containing a wild rose extract as an active ingredient.

Another object of the present invention is to provide a quasi-drug composition for preventing or improving skin wrinkles or heat aging of the skin, containing wild rose extract as an active ingredient.

To achieve the above object, the present invention provides a cosmetic composition for preventing or improving skin wrinkles or heat aging of the skin containing Rosa multiflora extract as an active ingredient.

In addition, the present invention provides a food composition for preventing or improving skin wrinkles or heat aging of the skin, comprising a wild rose extract as an active ingredient.

In addition, the present invention provides a quasi-drug composition for preventing or improving skin wrinkles or heat aging of the skin, containing a wild rose extract as an active ingredient.

The composition containing the wild rose extract of the present invention as an active ingredient not only exhibits no cytotoxicity but has an excellent inhibitory effect on the activity of elastase and collagenase, as well as TRPV-1 and MMP, which are indicators of heat stimulation inhibitory proteins. Because it can reduce the expression of -1, increase the expression of procollagen, and lower skin temperature, ultimately inhibiting heat aging of the skin, a composition containing wild rose extract as an active ingredient prevents skin wrinkles or heat aging. It can be usefully used as a preventive or improving cosmetic composition, external skin preparation, or health functional food.

Figure 1 is a diagram schematically showing a method for producing the Rosa multiflora extract of the present invention.
Figure 2 is a diagram showing the results of measuring the elastase inhibitory activity effect of the hot water extract (RMW), ethanol extract (RME), and acetone extract (RMA) of the present invention.
Figure 3 is a diagram showing the results of measuring the collagenase inhibitory activity effect of the hot water extract (RMW), ethanol extract (RME), and acetone extract (RMA) of the present invention.
Figure 4 shows the results of measuring the cell survival rate of Hs68 cells treated with the hot water extract (RMW), ethanol extract (RME), and acetone extract (RMA) of the present invention upon thermal stimulation at a temperature of 44°C. Here, N is a control group in which no sample treatment or thermal stimulation was applied, and C is a control group in which only thermal stimulation was applied at a temperature of 44°C without sample treatment.
Figure 5 is a diagram showing the results of measuring the cell survival rate of HaCaT cells treated with the hot water extract (RMW), ethanol extract (RME), and acetone extract (RMA) of the present invention upon thermal stimulation at a temperature of 44°C. Here, N is a control group in which no sample treatment or thermal stimulation was applied, and C is a control group in which only thermal stimulation was applied at a temperature of 44°C without sample treatment.
Figure 6 is a diagram showing Western blot results confirming changes in MMP-1, procollagen, and TRPV-1 protein expression levels in Hs68 dermal fibroblasts treated with the acetone extract (RMA) of the present invention. Here, N is a control group in which no sample treatment or thermal stimulation was applied, and C is a control group in which only thermal stimulation was applied at a temperature of 44°C without sample treatment.
Figure 7 is a diagram showing Western blot results confirming changes in MMP-1, procollagen, and TRPV-1 protein expression levels in human keratinocyte cells (HaCaT) treated with the acetone extract (RMA) of the present invention. Here, N is a control group in which no sample treatment or thermal stimulation was applied, and C is a control group in which only thermal stimulation was applied at a temperature of 44°C without sample treatment.

In one embodiment, the present invention provides a cosmetic composition for preventing or improving skin wrinkles or thermal aging, containing Rosa multiflora extract as an active ingredient.

In the present invention, the term " Rosa multiflora " is a deciduous shrub of the Rosaceae family, which is a dicotyledonous plant. Wild roses grow in abundance at the foot of mountains or in sunny streams and valleys in Korea and Japan. The flowers bloom in white or light red in May and grow in panicles at the ends of branches. In the private sector, it is known to be effective in treating edema, neuralgia, and urinary incontinence caused by poor blood circulation when made into tea.

The wild rose extract is preferably prepared by a production method comprising the following steps, but is not necessarily limited thereto:

1) Extracting the wild rose by adding an extraction solvent;

2) filtering the extract of step 1); and

3) Preparing a wild rose extract by concentrating the filtered extract of step 2) under reduced pressure and drying it.

In the above method, the wild roses of step 1) can be used without limitation, whether cultivated or commercially available. Additionally, in step 1), the extract may include, but is not limited to, leaves, fruits, seeds, roots, or two or more of these in addition to the wild rose.

In the above method, the extraction solvent in step 1) is preferably water, acetone, alcohol, for example, C ₁ -C ₆ alcohol, or a mixture thereof. The C ₁ -C ₆ alcohol may be ethanol, methanol, propanol, isopropanol, 1,3-propanediol, butanol, pentanol, hexanol, etc. The solvent may be, for example, a mixture of water and alcohol, that is, an aqueous alcohol solution. The alcohol concentration of the aqueous alcohol solution is 1 to 100 (v/v)%, for example, 1 to 99.5 (v/v)%, 10 to 100 (v/v)%, 20 to 100 (v/v)%, 30 to 100 (v/v)%, 40 to 100 (v/v)%, 50 to 100 (v/v)%, 60 to 100 (v/v)%, 65 to 100 (v/v)%, Or it may be 70 to 100 (v/v)%. The alcohol aqueous solution may be an ethanol aqueous solution. The concentration of acetone is 1 to 100 (v/v)%, for example, 1 to 99.5 (v/v)%, 10 to 100 (v/v)%, 20 to 100 (v/v)%, 30 to 100 (v/v)%, 40 to 100 (v/v)%, 50 to 100 (v/v)%, 60 to 100 (v/v)%, 65 to 100 (v/v)%, or It may be 70 to 100 (v/v)%. The extraction method is preferably shaken extraction, Soxhlet extraction, or reflux extraction, but is not limited thereto. It is preferable to extract by adding 1 to 30 times the amount of the extraction solvent to the amount of dried wild rose. The extraction temperature is preferably 20°C to 100°C, more preferably 20°C to 60°C, and most preferably room temperature, but is not limited thereto. In addition, the extraction time is preferably 10 to 48 hours, more preferably 15 to 30 hours, and most preferably 24 hours, but is not limited thereto. In addition, the number of extractions is preferably 1 to 5 times, more preferably 2 to 4 times, and most preferably 2 times, but is not limited thereto.

In the above method, filtration in step 2) can be performed by known methods such as filtering the extract using filter paper.

In the above method, the vacuum concentration in step 3) is preferably performed using a vacuum vacuum concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, drying is preferably performed by reduced pressure drying, vacuum drying, boiling drying, spray drying, or freeze drying, but is not limited thereto.

In the present invention, the term "wrinkle" refers to a state in which skin loses its elasticity and becomes loose. Wrinkles in the skin occur for various reasons, but are mainly caused by degeneration and atrophy of elastic fibers, connective fibers, and muscle fibers in the dermis. Wrinkles occur due to aging. Wrinkles occur when collagen or hyaluronic acid in the dermal layer decreases. The skin is composed of the epidermis, dermis, and subcutaneous tissue in order from the surface. Collagen in the dermis layer and elastic protein (hyaluronic acid) that maintains elasticity play a role in maintaining skin elasticity. However, as aging progresses, enzymes such as collagenase act that inhibit the connection between collagen and elastic proteins, and the sun's ultraviolet rays, which affect the dermal layer of the skin, also produce active oxygen and reduce skin elasticity. .

In a specific example of the present invention, in order to confirm the effect of the wild rose extract on improving skin wrinkles, the elastase activity inhibition effect and collagenase activity inhibition effect were respectively measured, and as a result, the wild rose extract, specifically the wild rose flower extract, and ethanol extract , and acetone extracts were confirmed to exhibit an elastase activity inhibitory effect and a collagenase activity inhibitory effect in a concentration-dependent manner (Figures 2 and 3). Therefore, the effect of preventing or improving skin wrinkles of the present invention may be achieved by inhibiting the activity of elastase or collagenase.

In the present invention, the term “thermal aging” refers to aging that occurs at a temperature higher than 37°C, which is the normal skin temperature, and preferably at a temperature between 38°C and 60°C. When the skin temperature exceeds 37°C, the area of blood vessels increases due to heat and collagen fibers and elastic fibers are destroyed, thereby accelerating skin aging. The heat aging can be caused by factors such as ultraviolet rays, infrared rays, visible light, smoking, drinking, burns, thermal erythema, and prolonged exercise, and is a concept that includes all skin aging that occurs due to an increase in skin temperature due to exposure of the skin to heat. am.

In a specific embodiment of the present invention, in order to confirm the effect of the wild rose extract on improving skin thermal aging, TRPV-1 (transient receptor potential vanilloid-1), MMP-1, and procollagen, which are indicators of thermal stimulation inhibitory proteins, were used. The expression level of was measured by Western blotting, and the results showed that the wild rose extract, specifically the wild rose hot water extract, ethanol extract, and acetone extract, concentration-dependently reduced the expression levels of TRPV-1 and MMP-1, and the expression of procollagen. It was confirmed that the amount increased (Figures 6 and 7). Therefore, the effect of preventing or improving skin thermal aging of the present invention may be achieved by reducing the expression of MMP-1 and TRPV-1 and increasing the expression of procollagen.

In the present invention, the term "prevention" includes suppressing the degree of increase in skin wrinkles or avoiding or minimizing skin roughness even when exposed to ultraviolet rays or infrared rays.

In the present invention, the term "improvement" includes suppressing the degree of increase in skin wrinkles or alleviating skin roughness even when exposed to ultraviolet rays or infrared rays.

The composition containing the wild rose extract of the present invention as an active ingredient not only has an excellent inhibitory effect on the activity of elastase and collagenase, but also reduces the expression of TRPV-1 and MMP-1, indicators of heat stimulation inhibitory proteins, and , it increases the expression of procollagen and lowers skin temperature, ultimately excellently inhibiting skin thermal aging, and thus can be usefully used as a cosmetic composition.

The cosmetic composition may preferably be in the form of a functional cosmetic.

In the present invention, "functional cosmetics (cosmedical, cosmeceutical)" refers to a product that has the specialized therapeutic function of a pharmaceutical introduced into cosmetics and has specialized functionality that emphasizes bioactive effects and effects, unlike general cosmetics. For the purpose of the present invention, the functional cosmetics refer to products that help improve skin wrinkles and prevent skin heat aging.

The cosmetic composition according to the present invention includes solution, external ointment, cream, foam, nourishing lotion, softening lotion, pack, softening water, emulsion, makeup base, essence, soap, liquid cleanser, bath agent, sunscreen cream, sun oil, suspension, Can be prepared in a formulation selected from the group consisting of emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations, patches and sprays. That is not the case.

In addition, the cosmetic composition of the present invention may additionally include one or more cosmetically acceptable carriers that are blended with general skin cosmetics, and common ingredients include, for example, oil, water, surfactant, moisturizer, lower alcohol, Thickeners, chelating agents, pigments, preservatives, fragrances, etc. may be appropriately mixed, but are not limited thereto.

Cosmetically acceptable carriers included in the cosmetic composition of the present invention vary depending on the formulation.

When the formulation of the present invention is an ointment, paste, cream or gel, the carrier ingredient may be animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or Mixtures of these can be used.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silcate, polyamide powder, or mixtures thereof may be used as the carrier ingredient, and especially in the case of a spray, chloro May contain propellants such as fluorohydrocarbons, propane/butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-Butylglycol oil may be used, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan. there is.

When the formulation of the present invention is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, and miso. Crystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.

When the formulation of the present invention is soap, alkali metal salts of fatty acids, hemiester salts of fatty acids, fatty acid protein hydrolyzates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol, sugars, etc. may be used as carrier ingredients. You can.

In another aspect, the present invention provides a food composition for preventing or improving skin wrinkles or heat aging, containing Rosa multiflora extract as an active ingredient.

The above-mentioned wildflower extract is as described above, and when using the composition of the present invention as a food additive, the wildflower extract can be added as is or used with other foods or food ingredients, and can be appropriately used according to conventional methods. there is. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use, and foodologically acceptable food auxiliary additives may be additionally included. Since the composition of the present invention uses extracts derived from natural products as an active ingredient, there is no problem in terms of stability, so there is no significant limitation on the mixing amount.

The food composition may preferably be in the form of a health functional food or food additive.

In the present invention, "health functional food" refers to a food group or food composition in which added value is added to a food to function and express the function of the food for a specific purpose using physical, biochemical, biotechnological methods, etc., biological defense rhythm regulation, It refers to food that has been designed and processed to fully express the body's regulatory functions related to disease prevention and recovery. For the purpose of the present invention, the health functional food may be used to prevent skin wrinkles and thermal aging of the skin, or to improve symptoms of skin wrinkles and thermal aging of the skin.

In the present invention, the food composition can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and components commonly added in the art. Additionally, the formulation of the food composition can be manufactured without limitation as long as it is a formulation recognized as a food composition.

Foods according to the present invention include, for example, various foods, beverages, gum, tea, vitamin complexes, functional foods, etc. In addition, foods include special nutritional foods (e.g., infant formula, infant and baby food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g., ramen, noodles, etc.), breads, health supplements, and seasoned foods (e.g., Soy sauce, soybean paste, red pepper paste, mixed sauce, etc.), sauces, confectionery (e.g., snacks), candy, chocolate, gum, ice cream, dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various) This includes, but is not limited to, kimchi, pickled vegetables, etc.), beverages (e.g. fruit drinks, vegetable drinks, soy milk, fermented drinks, etc.), natural seasonings (e.g. ramen soup, etc.), food additives, etc. The food, beverage or food additive can be manufactured by conventional manufacturing methods.

In addition to containing the active ingredient, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like a normal food composition. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavoring agents include natural flavoring agents (thaumatin), stevia extracts (e.g. rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.).

In addition, the food composition contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts. , organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice and fruit juice drinks and vegetable drinks.

In another aspect, the present invention provides a quasi-drug composition for preventing or treating skin wrinkles or heat aging, containing Rosa multiflora extract as an active ingredient.

As described above, the wild rose extract has the effect of improving skin wrinkles and inhibiting skin thermal aging, so it can be usefully used as a quasi-drug composition for the purpose of improving skin wrinkles and inhibiting skin thermal aging.

In addition to the above ingredients, the quasi-drug composition of the present invention may further include pharmaceutically acceptable carriers, excipients, or diluents as needed. The pharmaceutically acceptable carrier, excipient, or diluent is not limited as long as it does not impair the effect of the present invention, and includes, for example, fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, fragrances, preservatives, etc. It can be included.

The “pharmaceutically acceptable carrier” may refer to a carrier, excipient, or diluent that does not irritate living organisms and does not inhibit the biological activity and properties of the injected active ingredient. Specifically, it may refer to a non-natural carrier (non-natural carrier). It may be a naturally occurring carrier). The type of carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable can be used. Non-limiting examples of the carrier include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, etc. These may be used alone or in combination of two or more types.

The composition containing a pharmaceutically acceptable carrier may be a quasi-drug and may be in various parenteral formulations. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Specifically, solid preparations for parenteral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain the active ingredients plus at least one excipient, such as starch, calcium carbonate, sucrose, It can be prepared by mixing lactose, gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Additionally, preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

The quasi-drug composition of the present invention may include, but is not limited to, disinfectant cleaners, shower foams, ointments, wet tissues, coating agents, etc., and the formulation method, dosage, usage method, components, etc. of the quasi-drugs are commonly known in the technical field. It can be appropriately selected from the techniques.

In addition, it can be used in a method for improving skin wrinkles and thermal aging of the skin, which includes administering a quasi-drug composition containing the wild rose extract of the present invention as an active ingredient to an entity other than a human. The subject may include mammals including rats, livestock, humans, etc. without limitation.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Preparation Example 1: Cell preparation and reagent preparation

As an experiment measuring the effect of inhibiting heat aging, cells and reagents necessary for measuring cell viability and performing Western blot according to heat stimulation were prepared.

Preparation Example 1-1: Cell and reagent preparation

Hs68 dermal fibrobast cell (Hs68 cell), the cell line used for cell viability measurement and Western blot, was purchased from ATCC (Manassas, VA, USA), and keratinocyte cell (HaCaT) was purchased from the Oriental Medicine Promotion Foundation ( It was purchased and used from Gyungsan, Korea.

Fetal bovine serum (FBS), dulbecco's modified eagle medium (DMEM), trypan blue stain, trypsin-EDTA, and MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-dipheyl-tetrazolium bromide) reagent was purchased from Sigma aldrich Co., Ltd. It was purchased and used from (St. Louis, MO, USA).

In addition, TRPV-1 used in the experiment measuring the effect of inhibiting thermal aging was purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA), and MMP-1 and procollagen secondary antibodies were purchased from Santa Cruz (Biotech. CA, USA). It was used.

Preparation Example 1-2: Cell Culture

DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin (100 U/mL) was used to culture Hs68 dermal fibroblasts and HaCaT keratinocytes, and they were subcultured in an incubator at 37°C and 5% CO ₂ . HaCaT cells and Hs68 cells were washed with PBS (phosphate buffer saline), harvested using trypsin-EDTA, and then subcultured.

Preparation example 1-3: Preparation of equipment for use

ELISA reader (Spectra max 190, Moleculer devices, Sunnyvate, USA), UV/VIS spectrophotometer (Hitachi, Japan), freeze dryer (Ilshin, Korea), microscope (Olympus Co., Ltd, Japan), rotary vacuum evaporator ( Rikakikai Co., Ltd., Japan), digital reciprocating shaker (Daihan scientific Co., Ltd., Korea), Western imaging system (CAS-400SM, Davinch-K Co., Ltd., Korea), _CO2 incubator ( Equipment such as VS-9160GC, Hanbaek Scientific Co., Deajeon, Korea) was used to perform the examples described below.

Example 1: Preparation of wild rose extract

Wild rose ( Rosa multiflora ) was collected in Yeri 2-ri, Gaknam-myeon, Cheongdo, and water, 70% ethanol, and 70% acetone were used as solvents, respectively, to obtain wild rose hot water extract, 70% ethanol, and 70% acetone extract.

Specifically, as in Examples 1-1 to 1-3 below, a hot water extract of Wildflowers, 70% ethanol, and 70% acetone extract were obtained. At this time, the yield of the wild rose extract (RMW) was 6.93%, the yield of the 70% ethanol extract (RME) was 23.66%, and the yield of the 70% acetone extract (RMA) was 20.08%. Each of the powdered extracts was stored in a refrigerator at 4°C and used as samples to measure the wrinkle improvement effect and heat aging inhibition effect in Examples 2 and 3 below, respectively.

Example 1-1: Preparation of wildflower hydrothermal extract

The hot water extract of wild roses was extracted twice for 24 hours at 50°C using ultrapure water 20 times the weight of the dried sample as a solvent, and the extract was filtered on filter paper (No. 20 filter paper, Hyundai micro Co., Ltd., Korea), filtered, concentrated, freeze-dried, and powdered (Figure 1).

Example 1-2: Preparation of ethanol extract of Wildflowers

The ethanol extract of wild roses was extracted twice for 24 hours at room temperature using 70% ethanol (20 times the weight of the dried sample) as a solvent, and then the extract was filtered in the same manner as in Example 1-1. It was concentrated, freeze-dried, and powdered (Figure 1).

Example 1-3: Preparation of wild rose acetone extract

The acetone extract of wild roses was extracted twice for 24 hours at room temperature using 70% acetone (20 times the weight of the dried sample) as a solvent, and the extract was filtered in the same manner as in Example 1-1. It was concentrated, freeze-dried, and powdered (Figure 1).

Example 2: Measurement of wrinkle improvement effect of wild rose extract

As an experiment to measure the wrinkle improvement effect, an elastase inhibition effect measurement and a collagenase inhibition activity measurement experiment were conducted. The experiment included N-Succinyl-Ala-Ala-Ala-p-nitroanilide (N-Succinyl-Ala-Ala-Ala-Ala-p-nitroanilide) from Sigma aldrich Co., Ltd. (St. Louis, MO, USA). ), Elastase derived from porcine pancreas, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg), collagenase Reagent products such as (derived from Clostridium histohyticum, type 1) were purchased and used.

Example 2-1: Measurement of elastase inhibition effect

Elastase inhibitory activity was tested by referring to the method of Cannell (Cannell et al., 1988). Using N-syccinyl-(L-Ala) ₃ -p-nitroanilide, the amount of p-nitroanilide generated from the substrate at 37°C for 25 minutes was measured by ELISA at 405 nm.

Specifically, the sample solution diluted by concentration (5, 10, 50, 100, 500, 1000 μg/mL), that is, the wild water extract (RMW), ethanol extract (RME), and acetone extract (RMA) prepared in Example 1. ) was taken, and 40 μl of Porcine pancreas elastase (0.5 unit/mL) solution dissolved in 50 mM Tris-HCl buffer (pH 8.6) was added. Afterwards, N-succinyl-(L-Ala) ₃ -p-nitroanailide dissolved in 50 mM Tris buffer (pH 8.6) was added as a substrate, reacted for 25 minutes, and the results were measured. At this time, epigallocatechin gallate (EGCG) was used as a positive control.

As a result of measuring the elastase inhibitory effect, the wild rose extract (RMW), ethanol extract (RME), and acetone extract (RMA) all showed concentration-dependent effects, and had higher inhibitory activity than the positive control EGCG at a concentration of 50 ㎍/mL. showed. At a concentration of 50 μg/mL, which showed higher inhibitory activity than the positive control group, RMW showed an inhibitory effect of 1.14%, RME showed an inhibitory effect of 21.83%, and RMA showed an inhibitory effect of 28.11% (Figure 2). Through this, it was confirmed that the elastase inhibitory effect was higher than that of EGCG, which was a positive control, and that the wild rose extract according to the present invention could be usefully used as a wrinkle-improving functional material.

Example 2-2: Measurement of collagenase inhibitory activity

Collagenase inhibitory activity was measured according to the method of Cannell (Cannell et al., 1988). Specifically, a buffer was prepared by adding 4 mM CaCl ₂ (Junsei chemical Co., Ltd., Tokyo, Japan) to 0.1 M tris-HCl buffer (pH 7.5). 4-phenylazobenyloxycarbonly-Pro-Leu-Gly-Pro-D-Arg (0.3 mg/mL) was dissolved in buffer and mixed with 0.25 mL of substrate and samples of various concentrations (31, 62, 125, 250, 500 ㎍/mL). After mixing 0.1 mL of hot water extract (RMW), ethanol extract (RME), and acetone extract (RMA), 0.15 mL of collagenase (0.2 mg/mL) was added and reacted at room temperature for 20 minutes. At the end of the reaction time, 0.5 mL of 6% citric acid was added to stop the reaction, and then 1.5 mL of ethyl acetate was added and the absorbance was measured at 320 nm.

As a result of measuring collagenase inhibitory activity, the wild water extract (RMW), ethanol extract (RME), and acetone extract (RMA) all showed concentration-dependent effects. At the highest concentration of 500 μg/mL, RMW showed inhibitory activity of 40.95%, RME of 43.12%, and RMA of 85.48% (Figure 3). Through this, it was confirmed that the wild rose acetone extract (RMA) showed similar activity to EGCG, a positive control, and it was confirmed that RMA according to the present invention can be usefully used as a wrinkle-improving functional material.

Example 3: Measurement of heat aging inhibition effect

As an experiment to measure the effect of inhibiting thermal aging, cell survival rate according to heat stimulation and protein expression level were measured through Western blot.

Example 3-1: Measurement of cell viability according to thermal stimulation

To measure the effect of inhibiting heat aging, an experiment was conducted referring to the method of Dahee Son (Dahee Son et al., 2018). Specifically, Hs68 cells were distributed in a 96-well plate at 2×10 ⁴ cells/well and HaCaT cells were distributed at 5×10 ⁴ cells/well, and then cultured in an incubator at 37°C and 5% CO ₂ for 24 hours. . Afterwards, the temperature was placed in an incubator set at 44°C for 30 minutes to provide thermal stimulation, and then the samples (red water extract (RMW), ethanol extract (RME), and acetone extract (RMA)) were added at 25, 50, 100, 250, and 500 μg. /mL It was prepared according to concentration and 0.02 mL was added. After addition, both HaCaT and Hs68 cells were cultured in an incubator at 37°C and 5% CO ₂ for 6 hours. Afterwards, 0.02 mL of MTT solution prepared at a concentration of 2.5 mg/mL was added and cultured for 4 hours. Afterwards, the culture medium was removed as much as possible, 0.1 mL of DMSO (Dimethyl sulfoxide, DUSAN) was added to each well, and the mixture was sufficiently reacted at room temperature for 30 minutes, and the absorbance was measured at 540 nm with an ELISA reader.

As described above, the survival rate of heat stimulation in Hs68 and HaCaT cells was confirmed through absorbance measurement. Specifically, the survival rate of Hs68 cells when thermal stimulation was applied and samples (RMW, RME, RMA) were treated at different concentrations of 25, 50, 100, 250, and 500 μg/mL are shown in Figure 4, and the survival rate of HaCaT cells was It is shown in Figure 5. When only a heat stimulus of 44°C was applied to Hs68 cells, approximately 60% of cell death was observed, and when only a heat stimulus of 44°C was applied to HaCaT cells, approximately 50% of cell death was observed. On the other hand, when the samples (RMW, RME, RMA) were treated at the highest concentration of 500 ㎍/mL, Hs68 cells showed 70.71% cell viability for RMW, 89.01% for RME, and 99.76% for RMA (Figure 4). , HaCaT cells showed a cell survival rate of over 80% when samples (RMW, RME, RMA) were treated at a concentration of 250 μg/mL (Figure 5). Through this, it was confirmed that the wild water extract (RMW), ethanol extract (RME), and acetone extract (RMA) showed an excellent inhibitory effect on cell death caused by heat stimulation, and it was confirmed that the wild rose extract according to the present invention has thermal aging improvement functionality. It was found that it could be usefully used as a material.

Example 3-2: heat aging Measurement of expression level of related proteins

The expression levels of heat aging-related proteins, specifically MMP-1, Procollagen, and TRPV-1, were measured through Western blot. Specifically, HaCaT cells were distributed in a 6-well plate at 5×10 ⁵ cells/well and Hs68 cells were distributed at 2×10 ⁵ cells/well, and then cultured in an incubator at 37°C and 5% CO ₂ for 24 hours. , the culture medium was exchanged for serum-free DMEM medium. Then, in order to measure the wrinkle improvement effect and heat aging inhibition effect, Hs68 cells and HaCaT cells were stimulated with heat at 44°C and each sample (Wildflower hot water extract (RMW), ethanol extract (RME), acetone extract (RMA)) solution was added. After treatment at different concentrations (50, 100, and 250 ㎍/mL), the cells were cultured in a 5% CO ₂ incubator at 37°C for 6 hours to measure the protein expression levels of MMP-1, procollagen, and TRPV-1. The experiment was conducted with the treatment concentration of each sample set to 250 ㎍/mL or less based on the results of Example 3-1.

After culturing for 6 hours, the medium was removed, washed once with PBS, and all cells were treated with RIPA buffer to lyse the cells. After dissolution, the solution was centrifuged at 4°C and 12,000 rpm for 15 minutes using a centrifuge, and the obtained supernatant was transferred to a new tube. The membrane was blocked with 5% skim milk for 2 hours using a digital reciprocating shaker device. Afterwards, it was reacted with primary antibody for 3 hours and then washed three times for 10 minutes with TBST solution. Afterwards, the membrane with the primary antibody bound to a specific protein was reacted with the secondary antibody corresponding to the host of each antibody for 1 hour. After the reaction, washing was repeated three times for 10 minutes with TBST solution, and then the protein expression level was measured using a da Vinci Western imaging system.

Result (1): Confirmation of MMP-1, procollagen, and TRPV-1 expression through Western blot in Hs68 cells

After heat stimulation was applied for 30 minutes in an incubator at 44°C, protein expression for heat aging-related proteins MMP-1, procollagen, and TRPV-1 was confirmed by treatment with rosehip acetone extract (RMA). As a result, when treated at the highest concentration of 250 ㎍/mL, MMP-1 decreased by about 60.94% compared to the control, and procollagen increased by 20.93% compared to the control. Lastly, TRPV-1, an indicator of heat aging, was confirmed to decrease by 53.15% compared to the control group (Figure 6). Through this, it was confirmed that the wild rose acetone extract (RMA) decreased the expression level of MMP-1 and TRPV-1 and increased the expression level of procollagen compared to the control group to which only heat stimulation was applied. RMA according to the present invention was treated with heat. It was found that it can be usefully used as an aging-improving functional material.

Result (2): Confirmation of MMP-1, procollagen, and TRPV-1 expression through Western blot in HaCaT cells

After thermal stimulation was applied for 30 minutes in an incubator at 44°C, protein expression for heat aging-related proteins MMP-1, procollagen, and TRPV-1 was confirmed by treatment with acetone extract of wild rose (RMA). As a result, when treated at the highest concentration of 250 ㎍/mL, MMP-1 decreased by 70.14% compared to the control group, and procollagen increased by 16.29% compared to the control group. Lastly, TRPV-1, an indicator of heat aging, was confirmed to decrease by 58.18% compared to the control group (Figure 7). Through this, it was confirmed that the wild rose acetone extract (RMA) decreased the expression level of MMP-1 and TRPV-1 and increased the expression level of procollagen compared to the control group to which only heat stimulation was applied. RMA according to the present invention was treated with heat. It was found that it can be usefully used as a functional material to improve aging.

Example 4: Evaluation of skin temperature reduction and skin stability by wearing a mask containing wild rose extract

A mask containing 0.2% of the wild rose acetone extract of Example 1-3 was prepared according to a conventional method. After having a tester (female) in her 20s to 50s wear the manufactured mask for 1 hour, the test area (alare) was taken before and after wearing the mask using a thermal imaging camera (FTIR T0420, FLIR system, Sweden). We spot analyzed the temperature of the temperature measurement point in the thermal image of the horizontal line and the ipsilateral mid-pupillary line (intersection point of the ipsilateral mid-pupillary line) and confirmed a decrease in skin temperature, as well as side effects (erythema, edema, scaling, itching, and stinging) caused by wearing the mask. , burning sensation, stiffness, tingling, and other abnormalities) were checked. This experiment was conducted at the request of the Korea Dermatological Research Institute, and the results are shown in Table 1 below.

division Skin temperature (℃) p-value test site Before application 34.48 <0.001 After application 27.25 ^***

Significant probability: ^* p<0.05, ^** p<0.01, ^*** p<0.001

As can be seen in Table 1, it showed an average skin temperature reduction (cooling) effect of 7.23°C before and after the test, and no special skin abnormalities occurred in the tester during the test period, confirming that it was safe for the skin.

Through the above series of results, it can be seen that the wild rose extract of the present invention does not exhibit cytotoxicity, but has an excellent inhibitory effect on the activity of elastase and collagenase, and TRPV-1 and MMP-1, which are indicators of heat stimulation inhibitory proteins. It was confirmed that it reduces the expression of It was found that it could be usefully used as a material.

Claims (6)

A cosmetic composition for preventing or improving heat aging of the skin containing Rosa multiflora acetone extract as an active ingredient. delete delete According to paragraph 1,
A cosmetic composition in which the prevention or improvement of skin thermal aging is achieved by decreasing the expression of MMP-1 and TRPV-1 and increasing the expression of procollagen. A food composition for preventing or improving heat aging of the skin containing acetone extract of wild roses as an active ingredient. A quasi-drug composition for preventing or improving skin heat aging containing acetone extract of wild roses as an active ingredient.