KR20220157217A - 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 containing a Rosa multiflora extract for preventing or alleviating skin wrinkles or skin heat aging, wherein the composition exhibits excellent activity inhibitory effects on elastase and collagenase without exhibiting cytotoxicity and reduces the expression of TRPV-1 and MMP-1, which are indicators of heat stimulation inhibitory proteins. The composition also increases the expression of procollagen, and ultimately inhibits skin heat aging to a great extent. Thus, the composition containing a Rosa multiflora extract as an active ingredient can be usefully used as a cosmetic composition for preventing or alleviating skin wrinkles or heat aging, an external skin preparation, or a healthy functional food.

Description

Composition for preventing or improving skin wrinkles or skin thermal aging containing a rose flower extract

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

Skin aging is largely divided into two types of aging: natural aging in which wrinkles form as a person ages, that is, intrinsic aging, and extrinsic aging caused by heat, ultraviolet rays, or stress caused by the sun. . Recently, as the ozone layer is destroyed due to environmental pollution, ultraviolet rays become stronger and temperatures are gradually rising due to global warming, so people's interest in heat aging is gradually increasing.

Because ultraviolet rays and heat exist all year round and are common causes of aging in everyday life, it is most important to protect and improve aging skin from ultraviolet rays and heat. Various product groups for sun protection are released, but cosmetics containing heat aging protection are not common.

Brier flower ( Rosa multiflora Thunb. ) is a plant belonging to the Rosaceae family and can be easily seen at the foot of mountains or fields, valleys, etc. In addition, it is known as a material with excellent effects such as antioxidant, anti-inflammatory, antibacterial, and hair growth by parts such as flowers, fruits, roots, and seeds of wild rose flower. As active ingredients of these wild rose flowers, quercetin glycosides (multinoside A, multinoside B, quercitrin, etc.), which are a family of flavonoids, kaempferol glycosides (multiflorin A, multiflorin B, gelatin, astragalin, etc.), tannin-based methyl gallate, and lycopene, a red pigment of 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 temperature of the skin when the skin is heated. Human skin temperature is maintained at an average of 31 to 32 ° C. When the skin temperature rises above that level, a skin inflammatory reaction appears and thermal aging begins. On the other hand, human skin is often exposed to heat generated by using electronic devices such as smart phones, hair dryers, stoves, etc., 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 also been reported that the human skin temperature rises to 40 to 43 ° C in direct sunlight in the middle of summer within 15 to 20 minutes.

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

On the other hand, it is known that the thermoreceptor TRPV-1 (transient receptor potential vanilloid-1) ion channel responds even to thermal stimulation of 43° C. or higher and acidic stimulation of pH 5.4 or less. In addition, it is known to be involved in inducing an increase in pain by an environment such as inflammation as well as transmitting information of heat stimulation and spicy stimulation (Seung Jun Jung, Hanyang Med Rev. , 31(2):116-122, 2011 ).

Under this background, the present inventors have made diligent efforts to find a natural product with fewer side effects while improving skin wrinkles and preventing skin thermal aging, and as a result, rose flower ( Rosa multiflora ) extract has been shown to be effective against elastase and collagenase. It not only has excellent activity inhibition effect, but also reduces the expression of TRPV-1 (transient receptor potential vanilloid-1) and MMP-1, which are indicators of heat stimulation inhibitory protein, and increases the expression of procollagen, ultimately leading to By confirming that the skin heat aging can be inhibited excellently, the present invention was 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

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

In order to achieve the above object, the present invention provides a cosmetic composition for preventing or improving skin wrinkles or skin heat aging comprising a Rosa multiflora extract as an active ingredient.

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

The composition containing the wild rose extract of the present invention as an active ingredient has excellent activity inhibitory effects on elastase and collagenase without exhibiting cytotoxicity, as well as TRPV-1 and MMP, which are indicators of heat stimulation inhibitory proteins Since it can reduce the expression of -1, increase the expression of procollagen, lower the skin temperature, and ultimately inhibit skin heat aging excellently, a composition containing a wild rose extract as an active ingredient can be used to treat skin wrinkles or heat aging It can be usefully used as a cosmetic composition for prevention or improvement, an external skin preparation, or a health functional food.

1 is a view schematically showing a method for preparing a rose flower ( Rosa multiflora ) extract of the present invention.
Figure 2 is a view showing the results of measuring the elastase inhibitory activity of the wild rose extract (RMW), ethanol extract (RME), and acetone extract (RMA) of the present invention.
Figure 3 is a view showing the results of measuring the collagenase inhibitory activity of the wild rose flower extract (RMW), ethanol extract (RME), and acetone extract (RMA) of the present invention.
4 is a view showing the results of measuring the cell viability of Hs68 cells treated with hot water extract (RMW), ethanol extract (RME), and acetone extract (RMA) of the present invention according to thermal stimulation at 44 ° C. Here, N is a control group in which no sample treatment and thermal stimulation are applied, and C is a control group in which only thermal stimulation is applied at a temperature of 44° C. without sample treatment.
5 is a view showing the results of measuring the cell viability of HaCaT cells treated with hot water extract (RMW), ethanol extract (RME), and acetone extract (RMA) of the present invention according to thermal stimulation at 44°C. Here, N is a control group in which no sample treatment and thermal stimulation are applied, and C is a control group in which only thermal stimulation is applied at a temperature of 44° C. without sample treatment.
Figure 6 is a diagram showing the results of Western blotting to confirm 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 and thermal stimulation are applied, and C is a control group in which only thermal stimulation is applied at a temperature of 44° C. without sample treatment.
Figure 7 is a diagram showing the results of Western blot 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 and thermal stimulation are applied, and C is a control group in which only thermal stimulation is applied at a temperature of 44° C. without sample treatment.

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

In the present invention, the term "briar flower ( Rosa multiflora )" is a dicotyledonous plant, a deciduous shrub plant of the Rosaceae family. Wildflowers grow a lot at the foot of mountains or in sunny streams and valleys in Korea and Japan, and the flowers bloom in May in white or light red, and run in panicles at the ends of branches. In the private sector, it is known to be effective in edema caused by poor blood circulation, neuralgia, and urinary incontinence.

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

1) extracting by adding an extraction solvent to wild rose;

2) filtering the extract of step 1); and

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

In the above method, the wild rose flower of step 1) can be used without limitation, such as cultivated or commercially available. In addition, in step 1), but not limited thereto, it may be extracted by including leaves, fruits, seeds, roots, or two or more of them in addition to wild rose flowers.

In the 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, and the like. The solvent may be, for example, a mixture of water and alcohol, i.e., 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 the 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)%. As an extraction method, shaking extraction, Soxhlet extraction, or reflux extraction is preferably used, but is not limited thereto. It is preferable to perform extraction by adding 1 to 30 times the amount of the extraction solvent to the amount of dried wild rose flowers. 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 repeated extractions, and most preferably 2 times, but is not limited thereto.

In the above method, the filtration of step 2) may be performed by a known method such as filtering the extract using filter paper.

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

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

In a specific embodiment of the present invention, in order to confirm the skin wrinkle improvement effect of the rose flower extract, as a result of measuring the elastase activity inhibitory effect and the collagenase activity inhibitory effect, respectively, the rose flower extract, specifically the rose flower hot water extract, ethanol extract , And it was confirmed that the acetone extract exhibited an elastase activity inhibitory effect and a collagenase activity inhibitory effect in a concentration-dependent manner (Figs. 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 normal skin temperature of 37° C., preferably at a temperature of 38° C. to 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 thermal aging may be caused by factors such as ultraviolet rays, infrared rays, visible rays, smoking, drinking, burns, thermal erythema, and long-term exercise, and includes all skin aging caused by an increase in skin temperature due to exposure to heat. to be.

In a specific embodiment of the present invention, in order to confirm the effect of improving skin heat aging of wild rose flower extract, TRPV-1 (transient receptor potential vanilloid-1), MMP-1, and procollagen, indicators of heat stimulation inhibitory proteins As a result of measuring the expression level of by Western blot, respectively, the wild rose extract, specifically, the wild rose flower extract, the ethanol extract, and the acetone extract decreased the expression levels of TRPV-1 and MMP-1 in a concentration-dependent manner, and the expression of procollagen It was confirmed that the amount increased (Figs. 6 and 7). Therefore, the effect of preventing or improving skin aging by reducing the expression of MMP-1 and TRPV-1 and increasing the expression of procollagen may be achieved.

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

In the present invention, the term “improvement” includes those capable of suppressing the increase in 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 activity inhibitory effect on elastase and collagenase, but also reduces the expression of TRPV-1 and MMP-1, which are indicators of heat stimulation inhibitory proteins, , Since it increases the expression of procollagen and lowers the skin temperature, ultimately it can excellently inhibit skin heat aging, so it 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 having professional functionality in which the professional therapeutic function of a drug is introduced into cosmetics, and physiologically active efficacy and effects are emphasized, unlike general cosmetics. For the purpose of the present invention, the functional cosmetics refer to products that help improve skin wrinkles and prevent skin aging due to heat.

The cosmetic composition according to the present invention is a solution, external ointment, cream, foam, nutrient lotion, softening lotion, pack, softening water, emulsion, makeup base, essence, soap, liquid cleanser, bath additive, sunscreen cream, sun oil, suspension, Emulsion, paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, may be prepared in a formulation selected from the group consisting of patches and sprays, but is limited thereto It is not.

In addition, the cosmetic composition of the present invention may further include one or more cosmetically acceptable carriers formulated in general skin cosmetics, and as typical ingredients, for example, oil, water, surfactants, moisturizers, lower alcohols, A thickener, a chelating agent, a colorant, a preservative, a flavoring agent, and the like may be suitably blended, but are not limited thereto.

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

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

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or a mixture thereof may be used as a carrier component, and in particular, in the case of a spray, chloro 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-butyl glycol oil may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, fatty acid esters of glycerol, polyethylene glycol or sorbitan may be used. have.

In the case where the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohols, suspending agents such as polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like may be used.

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

As another aspect, the present invention provides a food composition for preventing or improving skin wrinkles or skin heat aging comprising a rose flower ( Rosa multiflora ) extract as an active ingredient.

The wild rose extract is as described above, and when the composition of the present invention is used as a food additive, the wild rose extract can be added as it is or used together with other foods or food ingredients, and can be appropriately used according to a conventional method have. The mixing amount of the active ingredient may be suitably determined depending on the purpose of use, and may further include food additives acceptable in food science. Since the composition of the present invention uses an extract derived from a natural product as an active ingredient, there is no problem in terms of stability, so there is no great 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 that has added added value so that the function of the food acts for a specific purpose by using physical, biochemical, or bioengineering methods, etc. It refers to food 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 heat aging, or to improve skin wrinkles and symptoms of skin heat aging.

In the present invention, the food composition can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the preparation. In addition, the formulation of the food composition may also be prepared without limitation as long as the formulation is recognized as a food composition.

Foods according to the present invention include, for example, various foods, beverages, chewing gum, tea, vitamin complexes, and functional foods. In addition, foods include special nutritional foods (eg, formula milk, infant food, baby food, etc.), processed meat products, fish meat products, tofu, jelly, noodles (eg, ramen, noodles, etc.), breads, health supplements, seasoning foods (eg, Soy sauce, soybean paste, gochujang, mixed paste, etc.), sauces, confectionery (eg, snacks), candies, chocolates, chewing gum, ice cream, dairy products (eg, fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various types Kimchi, pickles, etc.), beverages (eg, fruit drinks, vegetable drinks, soy milk, fermented beverages, etc.), natural seasonings (eg, ramen soup, etc.), food additives, etc. are included, but are not limited thereto. The food, beverage or food additive may be prepared by a conventional manufacturing method.

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 conventional food compositions. Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as common sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agents described above, natural flavoring agents (thaumatin), stevia extracts (eg rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used.

In addition, the food composition includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts , organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain natural fruit juice and fruit flesh for preparing fruit juice beverages and vegetable beverages.

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

The wild rose extract is as described above, and since the wild rose flower extract has an effect of improving skin wrinkles and inhibiting skin heat aging, it can be usefully used as a quasi-drug composition for the purpose of improving skin wrinkles and inhibiting skin heat aging.

The quasi-drug composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent as needed in addition to the above components. The pharmaceutically acceptable carrier, excipient or diluent is not limited as long as the effect of the present invention is not impaired, and examples thereof include fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, aromatics, preservatives, and the like. can include

The "pharmaceutically acceptable carrier" may refer to a carrier, excipient, or diluent that does not inhibit the biological activity and properties of the injected active ingredient without irritating the living organism, and specifically, a non-natural carrier (non-natural carrier). naturally occurring carrier). The type of the 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, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or in combination of two or more.

The composition containing a pharmaceutically acceptable carrier may be in various parenteral formulations as a quasi-drug. 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 at least one excipient in addition to the active ingredient, for example, starch, calcium carbonate, sucrose, It may be prepared by mixing lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. In addition, preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.

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. It can be appropriately selected from the description of.

In addition, it can be used in a method for improving skin wrinkles and skin aging by administering a quasi-drug composition containing the wild rose flower extract as an active ingredient to a non-human subject. The subject may include without limitation mammals including mice, livestock, humans, and the like.

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

Preparation Example 1-1: Preparation of cells and reagents

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

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

In addition, TRPV-1 used in the experiment to measure the effect of inhibiting heat 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). and used it.

Preparation Example 1-2: Cell Culture

For the culture of Hs68 dermal fibroblasts and HaCaT keratinocytes, DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin (100 U/mL) was used, and subcultures were adapted to a 37°C, 5% CO ₂ incubator. HaCaT cells and Hs68 cells were subcultured after washing with PBS (phosphate buffer saline), harvesting the cells using trypsin-EDTA.

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), CO ₂ incubator ( An instrument such as VS-9160GC, Hanbaek Scientific Co., Deajeon, Korea) was used for carrying out the examples described below.

Example 1: Preparation of Wildflower Extract

Brier flower ( Rosa multiflora ) was collected in Yeri 2-ri, Gaknam-myeon, Qingdao, and water, 70% ethanol, and 70% acetone were used as solvents to obtain hot-water extracts of wild rose flowers, 70% ethanol, and 70% acetone extracts.

Specifically, as in Examples 1-1 to 1-3, wild-water extracts of wild rose flowers, 70% ethanol, and 70% acetone extracts 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 4° C. refrigerator and used as a sample when measuring the effect of improving wrinkles and inhibiting heat aging in Examples 2 and 3, respectively.

Example 1-1: Preparation of wild rose flower extract

The hot-water extract of wild rose flowers was extracted twice at 50 ° C for 24 hours using ultrapure water 20 times the weight of the dried sample as a solvent, and then the extract was placed on filter paper (No. 20 filter paper, Hyundai micro Co., Ltd., Korea), and powdered by filtration, concentration, and lyophilization (FIG. 1).

Example 1-2: Preparation of Wildflower Ethanol Extract

The wild rose flower ethanol extract was extracted twice at room temperature for 24 hours 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, lyophilized and powdered (FIG. 1).

Example 1-3: Preparation of acetone extract of wild rose flower

Wildflower acetone extract was extracted twice at room temperature for 24 hours using 70% acetone 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, lyophilized and powdered (FIG. 1).

Example 2: Measurement of anti-wrinkle effect of wild rose extract

As an experiment for measuring the wrinkle improvement effect, an experiment for measuring the elastase inhibitory effect and measuring the collagenase inhibitory activity was conducted. For the experiments, N-succinyl-Ala-Ala-Ala-p-nitroanilide (N-Succinyl-Ala-Ala-Ala-p-nitroanilide) from Sigma aldrich Co., Ltd. (St. Louis, MO, USA) was used. ), porcine pancreatic elastase, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg), collagenase A reagent product such as (derived from Clostridium histohyticum, type-1) was purchased and used.

Example 2-1: Measurement of elastase inhibitory effect

Elastase inhibitory activity was tested by referring to the method of Cannell (Cannell et al., 1988) and the like. Using N-syccinyl-(L-Ala) ₃ -p-nitroanailide, 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 rose 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. Thereafter, as a substrate, N-succinyl-(L-Ala) ₃ -p-nitroanailide dissolved in 50 mM Tris buffer (pH 8.6) was added and reacted for 25 minutes, and the result was measured. At this time, as a positive control, epigallocatechin gallate (EGCG) was used.

As a result of measuring the elastase inhibitory effect, all of the wild rose extract (RMW), ethanol extract (RME), and acetone extract (RMA) showed concentration-dependent effects, and from a concentration of 50 μg/mL, higher inhibitory activity than EGCG, a positive control showed At a concentration of 50 μg/mL, which showed higher inhibitory activity than the positive control group, RMW showed 1.14%, RME 21.83%, and RMA 28.11% inhibitory effects (FIG. 2). Through this, it was confirmed that the elastase inhibitory effect was higher than that of the positive control EGCG, and it was found that the wild rose extract according to the present invention can 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). Dissolve 4-phenylazobenyloxycarbonly-Pro-Leu-Gly-Pro-D-Arg (0.3 mg/mL) in buffer and prepare 0.25 mL of substrate and samples of various concentrations (31, 62, 125, 250, 500 μg/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. After the reaction time was over, 0.5 mL of 6% citric acid was added to stop the reaction, and 1.5 mL of ethyl acetate was added to measure the absorbance at 320 nm.

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

Example 3: Measurement of thermal aging inhibitory effect

As an experiment to measure the effect of inhibiting heat aging, an experiment was conducted to measure cell viability according to heat stimulation and to measure protein expression level through Western blot.

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

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

Viability of heat stimulation in Hs68 and HaCaT cells was confirmed by measuring absorbance as described above. Specifically, the survival rate of Hs68 cells when samples (RMW, RME, RMA) were treated with concentrations of 25, 50, 100, 250, and 500 μg/mL with thermal stimulation is shown in FIG. 4, and the survival rate of HaCaT cells was shown in Figure 5. When only 44°C heat stimulation was applied to Hs68 cells, about 60% cell death was shown, and when only 44°C heat stimulation was applied to HaCaT cells, about 50% cell death was shown. In contrast, when samples (RMW, RME, RMA) were treated at the highest concentration of 500 μg/mL, the cell viability was 70.71% for RMW, 89.01% for RME, and 99.76% for RMA in Hs68 cells (FIG. 4). , In HaCaT cells, when the samples (RMW, RME, RMA) were treated at a concentration of 250 μg/mL, all showed cell viability of 80% or more (FIG. 5). Through this, it was confirmed that the wild rose flower extract (RMW), ethanol extract (RME), and acetone extract (RMA) exhibited excellent inhibitory effects on cell death caused by heat stimulation, and the wild rose flower extract according to the present invention had a thermal aging improvement function It was found that it can be usefully used as a material.

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

Expression levels of heat aging-related proteins, specifically MMP-1, procollagen, and TRPV-1, were measured through Western blotting. Specifically, HaCaT cells are 5×10 ⁵ cells/well and Hs68 cells are 2×10 ⁵ cells/well, and then seeded in a 6-well plate, 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 anti-wrinkle effect and the effect of inhibiting heat aging, Hs68 cells and HaCaT cells were stimulated with heat at 44° C., and each sample (Hot Water Extract (RMW), Ethanol Extract (RME), Acetone Extract (RMA)) was added to each sample. After treatment with different concentrations (50, 100, and 250 μg/mL), the cells were cultured in an incubator at 37° C. and 5% CO ₂ for 6 hours to measure the expression levels of MMP-1, procollagen, and TRPV-1 proteins. The experiment was conducted by setting the concentration of each sample to 250 μg/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, centrifugation was performed for 15 minutes at 4° C. and 12,000 rpm using a centrifuge, and the obtained supernatant was transferred to a new tube. The membrane was blocked for 2 hours with 5% skim milk using a digital reciprocating shaker machine. Thereafter, the reaction was performed with the primary antibody for 3 hours and then washed three times for 10 minutes with TBST solution. Thereafter, the membrane in which the primary antibody was 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 a 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 in Hs68 cells through Western blotting

After applying thermal stimulation for 30 minutes in an incubator at 44° C., acetone extract (RMA) was applied to confirm protein expression of MMP-1, procollagen, and TRPV-1, which are related to heat aging. As a result, when treated with the highest concentration of 250 μg/mL, it was confirmed that MMP-1 decreased by 60.94% compared to the control group, and procollagen increased by 20.93% compared to the control group. Finally, it was confirmed that TRPV-1, a heat aging index, decreased by 53.15% compared to the control group (FIG. 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. 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 in HaCaT cells by Western blotting

After applying thermal stimulation for 30 minutes in an incubator at 44° C., acetone extract (RMA) was applied to confirm protein expression of MMP-1, procollagen, and TRPV-1, which are related to heat aging. As a result, when treated with the highest concentration of 250 μg/mL, it was confirmed that MMP-1 decreased by 70.14% compared to the control group, and procollagen increased by 16.29% compared to the control group. Finally, it was confirmed that TRPV-1, a heat aging index, decreased by 58.18% compared to the control group (FIG. 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. It was found that it can be usefully used as an aging-improving functional material.

Example 4: Evaluation of Skin Temperature Reduction and Skin Stability by Wearing a Mask Containing Wildflower Extract

A mask containing 0.2% of the acetone extract of the wild rose flower of Example 1-3 was prepared according to a conventional method. After having the manufactured mask worn by a tester (female) in her 20s to 50s for 1 hour, a thermal imaging camera (FTIR T0420, FLIR system, Sweden) was used to examine the test area (alare) before and after wearing the mask. The temperature of the temperature measurement point in the thermal image of the horizontal line and the ipsilateral mid-pupillary line) was spot-analyzed, and the skin temperature drop was confirmed. , burning sensation, stiffness, tingling sensation, and other abnormal symptoms) were checked. These experiments were conducted by requesting 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 lowering (cooling) effect of 7.23 ° C. before and after the test, and it was confirmed that it was safe for the skin as no special skin abnormalities occurred to the tester during the test period.

Through the above series of results, the wild rose extract of the present invention has excellent activity inhibitory effects on elastase and collagenase without exhibiting cytotoxicity, and TRPV-1 and MMP-1, which are indicators of heat stimulation inhibitory proteins, As it was confirmed that it reduces the expression of procollagen, increases the expression of procollagen, and lowers the skin temperature, it is confirmed that cosmetics and health functional foods that can prevent or alleviate heat aging as well as ultimately improve skin wrinkles. It was found that it could be usefully used as a material.

Claims (6)

Brier flower ( Rosa multiflora ) A cosmetic composition for preventing or improving skin wrinkles or skin heat aging comprising an extract as an active ingredient. According to claim 1, wherein the extract is water, acetone, C ₁ -C ₆ alcohol, or extracted in a combination thereof, the cosmetic composition. According to claim 1,
The skin wrinkle prevention or improvement is achieved by inhibiting the activity of elastase (elastase) or collagenase (collagenase), a cosmetic composition. According to claim 1,
The prevention or improvement of skin aging is a cosmetic composition that is achieved by reducing the expression of MMP-1 and TRPV-1 and increasing the expression of procollagen. A food composition for preventing or improving skin wrinkles or skin heat aging comprising a wild rose extract as an active ingredient. A quasi-drug composition for preventing or improving skin wrinkles or skin heat aging comprising a wild rose flower extract as an active ingredient.

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