KR102416653B1 - Cosmetic composition for improving skin conditions comprising protein hydrolysate of Agastache rugosa - Google Patents

Abstract

It relates to a composition comprising a protein hydrolyzate of baecho flavor and its use for improving skin condition.

Description

Cosmetic composition for improving skin conditions comprising protein hydrolysate of Agastache rugosa

It relates to the use of baechohyang to improve skin condition.

The skin is a primary barrier of the human body from the external environment and is one of the important organs of the human body that regulates body temperature, barrier function, and various physiological functions. Human skin undergoes an aging process as it ages, and the causes can be largely divided into intrinsic aging and extrinsic aging. Intrinsic aging is a process that cannot be avoided by anyone with aging, and wrinkles occur, the skin loses elasticity, moisture in the skin cells is lost, and the structure of the stratum corneum changes. On the other hand, exogenous aging is caused by external stress such as environmental factors, and among them, ultraviolet rays are the main cause, also called photoaging.

In aging skin, the thickness of the epidermis, dermis, and subcutaneous fat decreases. Also, there is a change in the extracellular matrix (ECM) component of the dermis, which is responsible for skin elasticity and tension. Collagen and elastin fibers, which occupy most of the extracellular matrix, give skin tension and elasticity, and fibrillin 1 (FBN1; fibrilin 1) is also produced by fibroblasts in the dermis, mainly forming elastic fibers such as elastin. It is responsible for the function of bone formation when As we age, the production of collagen and elastin decreases, while the amount of proteolytic enzyme (MMP; matrix metalloproteinase, collagenase 1) increases. As a result, proteolytic enzymes break down collagen and elastin, and aging skin becomes thin and tension is reduced, making it weak and brittle.

On the other hand, another phenomenon of skin aging is that the ability to retain moisture is reduced and the skin becomes dry and rough. Water constitutes about 70% of the human body and helps to keep the skin moist and shiny. The stratum corneum of a healthy epidermis contains 15-20% of moisture, and when the level falls below 10%, an abnormality occurs in the skin barrier and the skin becomes dry and wrinkles increase. The water content of the stratum corneum is determined by the sebum membrane, a lipid mixture produced and secreted by the epidermis, and natural moisturizing factor (NMF), a water-soluble component present in the stratum corneum. Hyaluronic acid, one of the natural moisturizing factors, is formed in fibroblasts and keratinocytes, and as a component of point polysaccharide, it contains a lot of polysaccharides, so it has the characteristic that it can contain a considerable amount of moisture. The cycle is 2 to 4.5 days, and by combining with water that is 1,000 times its own weight, it regulates the skin barrier function and hydrates the extracellular matrix to maintain the homeostasis of water in the tissue. Therefore, when the moisture content in the skin decreases and the amount of hyaluronic acid decreases, it is directly related to skin aging. Hyaluronic acid synthase (HAS3; Hyaluronan synthase 3) has HAS-1, 2, and 3, and glucuronic acid and N-acetylglucosamine are repeatedly added to polysaccharides in the initial stage of synthetase to produce hyaluronic acid.

Among proteins that control the movement of water, aquaporin (AQP; aquaporin) and claudin (CLD; claudin) exist among proteins that perform a tight junction function. Aquaporin-3 (AQP3; aquaporin3), which is mainly present in keratinocytes of the epidermis and is a water channel through which water, glycerol, urea, etc. move, provides smooth moisture between cells. Claudine, a tight junction protein, has various functions, such as controlling the movement of electrolytes and water by connecting and bonding between adjacent epidermal cells, as well as transmitting intracellular signals.

Accordingly, there is a need for the development of ingredients derived from natural products that can be usefully used for skin-related conditions.

KR 10-1686399 B1

It provides a composition comprising a protein hydrolyzate of baechohyang.

A method for preparing a protein hydrolyzate of baechohyang is provided.

Provided is a method for improving a skin condition in a subject comprising administering to the subject in need thereof an effective amount of the composition described above.

Provided is the use of a protein hydrolyzate of baechohyang for preparing a composition for improving skin condition.

One aspect provides a composition comprising a protein hydrolyzate of baechohyang ( Agastache rugosa ).

The "baechohyang ( Agastache rugosa )" is also called "banga" or "bangae", and dried baechohyang collected in summer and early autumn is also called "gwakhyang". Baecho-hyang is a perennial plant of the Lamiaceae family, with green stems and many leaves, and has a distinctive strong scent. It is distributed from temperate to temperate zones, especially in the southern regions of Northeast Asia and Korea. The stems of baechohyang are dark brown, have vertical patterns and nodes, and grow well in poor environments.

The composition may be a composition for improving skin condition, improving skin beauty, preventing, improving or treating skin diseases.

The composition may be for improving skin condition.

The skin condition improvement may be anti-aging, skin wrinkle improvement, skin elasticity enhancement, skin moisturizing, skin barrier strengthening, or a combination of two or more thereof.

The term "skin aging" refers to the tangible and intangible changes that appear on the skin with age, such as a thinning of the epidermis, the number of cells or blood vessels in the dermis, DNA damage repair ability, cell replacement cycle, wound healing, It refers to a decrease in skin barrier function, epidermal moisture retention, sweat secretion, sebum secretion, vitamin D production, physical damage protection, chemical removal ability, immune response, sensory function, and thermoregulation.

The baechohyang protein hydrolyzate may be for improving skin aging or anti-aging caused by extrinsic factors or intrinsic factors. The extrinsic factor refers to various external factors, such as ultraviolet rays (light), and the intrinsic factor is also referred to as a chronological factor, and refers to a factor mainly caused by the passage of time. That is, the skin aging specifically refers to not only the premature aging symptoms induced by external stimuli such as ultraviolet rays, pollution, cigarette smoke, chemical substances, etc., but also a natural aging phenomenon that occurs as the proliferation of skin cells decreases with aging. It is a concept that includes all of wrinkles, loss of elasticity, skin sagging and dryness. In addition, wrinkles include those that cause wrinkles by changing the components constituting the skin tissue by stimulation by changes in internal and external factors.

The aging may be photoaging. The term “photoaging” is a phenomenon induced by external environmental factors, and the most representative factor is ultraviolet rays. Ultraviolet rays cause damage to biological components such as activation of proteolytic enzymes, chain cleavage of matrix proteins, and abnormal cross-linking, and repetition of these mechanisms leads to apparent skin aging. Accordingly, the composition may be for preventing or improving photoaging by ultraviolet B (UVB).

The term "skin wrinkle improvement" is interpreted to include not only an action of reducing the number or depth of skin wrinkles, but also inhibiting skin aging through wrinkle improvement.

The term “enhancement of skin elasticity” may refer to any action that increases skin elasticity, which is reduced due to aging, or prevents deterioration of skin elasticity.

The term “moisturizing the skin” may refer to any action that retains moisture in the skin or prevents moisture loss. The skin 　 moisturizing effect can help improve skin wrinkles and increase elasticity.

The term “strengthening the skin barrier” may refer to any action that enhances the function of the skin barrier, which is located at the outermost part of the skin and prevents loss of moisture and nutrients.

The term “skin disease” may be a disease caused by skin aging, or damage to the skin barrier function. The term “prevention” includes inhibiting the development of a disease. The term “treatment” includes inhibiting, alleviating, or eliminating the development of a disease.

The damage to the skin barrier function may refer to any change that appears in the skin as the function of the skin barrier is reduced or damaged. For example, it may include increased skin wrinkles, dryness, dermatitis, atopic dermatitis, allergic dermatitis, acne, and the like.

The protein hydrolyzate of baechohyang may be prepared by enzymatically reacting baechohyang with a proteolytic enzyme.

The enzymatic reaction may be performed by incubating a mixture of baechohyang, protease, and purified water. The baechohyang may use an outpost or a part of baechohyang, for example, leaves, stems, flowers, etc. may be used, but is not limited to a specific part thereof. The baecho flavor may be used, for example, in a dry powder form, but is not limited thereto.

The enzymatic reaction may be performed at an appropriate temperature depending on the type of enzyme. For example, the enzymatic reaction may be carried out at 25 to 70 ℃, 30 to 70 ℃, 35 to 70 ℃, 40 to 70 ℃, 45 to 70 ℃, 50 to 70 ℃, or 55 to 65 ℃, It is not limited thereto.

The enzymatic reaction may be performed for an appropriate time so that the enzymatic reaction can be sufficiently performed. For example, the enzymatic reaction is 1 minute to 24 hours, 1 minute to 20 hours, 1 minute to 16 hours, 1 minute to 12 hours, 1 minute to 8 hours, 30 minutes to 24 hours, 30 minutes to 20 hours, 30 minutes to 16 hours, 30 minutes to 12 hours, 30 minutes to 8 hours, 1 hour to 24 hours, 1 hour to 20 hours, 1 hour to 16 hours, 1 hour to 12 hours, or 1 hour to 8 hours may be, but is not limited thereto.

The term "protease (protease, peptidase, proteinase)" refers to an enzyme that hydrolyzes peptide bonds between amino acids constituting a protein.

The proteolytic enzyme may be an endopeptidase. The term "endopeptidase", also referred to as "peptide endohydrolase", is a type of proteolytic enzyme that cuts the middle of a protein.

The protease may be EC 3.4.21 or EC 3.4.16.

The protease may be a serine protease. The serine protease may be a microorganism-derived serine protease. The serine protease may be a microorganism-derived alkaline serine protease.

The proteolytic enzyme may be an alcalase. The "Alcalase" is also referred to as "Subtilisin", and is a serine proteolytic enzyme derived from Bacillus sp. microorganisms.

The protein hydrolyzate is a protein hydrolyzate obtained by enzymatically reacting baechohyang with a proteolytic enzyme, a diluted or concentrated solution of a protein hydrolyzate, a dried product obtained by drying the protein hydrolyzate, or these prepared or purified products, It may include a fractionated fraction.

The protein hydrolyzate may include a glycoprotein. Plant glycoproteins are present in the primary cell wall in plant cells and protect the cell wall when the plant is growing or the cell wall is damaged. Plant glycoproteins contain a greater amount of sugar than animal collagen, so they are convenient to use, and because of their low molecular weight, they can be absorbed well into the skin. In addition, since it is composed of amino acids, which are biological substances, it can be safely absorbed into the skin and prevent skin aging by providing amino acids directly to the skin.

The protein hydrolyzate may include flavonoids, polyphenols, or a combination thereof. The protein hydrolyzate of baechohyang may include flavonoid components such as agastachoside, acacetin, and tyrianin. The protein hydrolyzate of baechohyang may include essential oil components such as monoterpene, sesquiterpene, methylchavicol, and limonene.

The composition may inhibit the expression of collagenase (matrix metalloproteinase, collagenase 1, MMP-1). The collagen-degrading enzyme decomposes collagen and elastin to make the skin thinner and the tension to decrease, thereby reducing elasticity. Therefore, the composition may exhibit the effect of improving skin wrinkles and enhancing skin elasticity by inhibiting the expression of MMP-1.

The composition may increase the expression of collagen synthesis factor (Collagen, type 1, alpha 1, COL1A1). The composition induces collagen synthesis in the skin by increasing the expression of COL1A1, thereby improving skin wrinkles and enhancing skin elasticity.

The composition may increase the expression of fibrillin 1 (FBN1). By increasing the expression of FBN1, the composition forms a skeleton when elastic fibers such as elastin are formed, thereby improving skin wrinkles and enhancing skin elasticity.

Accordingly, the composition may improve skin aging by inhibiting the expression of MMP-1, increasing the expression of COL1A1, increasing the expression of FBN1, or a combination thereof.

The composition may increase the expression of aquaporin 3 (AQP3). The composition may exhibit a skin moisturizing effect by smoothly supplying moisture to skin cells by increasing the expression of AQP3.

The composition may increase the expression of hyaluronic acid synthase 3 (HAS3). The composition induces hyaluronic acid synthesis by increasing the expression of HAS3, thereby exhibiting skin moisturizing and skin barrier strengthening effects.

The composition may increase the expression of claudin 1 (CLD1). The composition may exhibit skin moisturizing and skin barrier strengthening effects by connecting and bonding adjacent epidermal cells to each other by increasing the expression of CLD1.

Accordingly, the composition may exhibit an effect of moisturizing the skin or strengthening the skin barrier by increasing the expression of AQP3, increasing the expression of HAS3, increasing the expression of CLD1, or a combination thereof.

Therefore, the composition inhibits the expression of collagenase (MMP-1), collagen synthesis factor (COL1A1), fibrillin (FBN1), aquaporin 3 (AQP3), hyaluronic acid synthase 3 (HAS3) and claudin 1 (CLD1) may increase the expression of any one or more.

In one embodiment, the protein hydrolyzate of baechohyang is compared to the hydrolyzate of baechohyang by hot water extract of baechohyang, ethanol extract of baechohyang, or sugar hydrolyzate of baechohyang by complex enzyme treatment that breaks down sugar bonds, skin wrinkle improvement, skin elasticity enhancement, etc. It was confirmed that the anti-aging effect, skin moisturizing and skin barrier strengthening effect were excellent.

The composition is 0.1 ppm to 1000 ppm, 0.1 ppm to 500 ppm, 0.1 ppm to 100 ppm, 1 ppm to 1000 ppm, 1 ppm to 500 ppm, 1 ppm to 100 ppm, It may be included in a concentration of 10 ppm to 1000 ppm, 10 to 500 ppm, or 10 to 100 ppm. In one embodiment, it was confirmed that the anti-aging effect and skin moisturizing and skin barrier strengthening effect by treating the protein hydrolyzate of baecho-hyang directly into the cells at a concentration of 1 ppm or 10 ppm, improving skin wrinkles, enhancing skin elasticity, etc. were confirmed. When the protein hydrolyzate of baechohyang is applied to the cosmetic composition formulation, a person skilled in the art can appropriately select a concentration of the protein hydrolyzate of baechohyang to exhibit the above-mentioned effects. For example, when the protein hydrolyzate of baechohyang is applied to the cosmetic composition formulation, it can be used at a concentration of 10 times or 100 times compared to the concentration used for cell experiments.

The composition contains 0.0001 to 10 wt%, 0.0001 to 5 wt%, 0.0001 to 1 wt%, 0.0001 to 0.5 wt%, 0.0001 to 0.1 wt%, 0.001 to 10 wt%, It may be included in an amount of 0.001 to 5% by weight, 0.001 to 1% by weight, 0.001 to 0.5% by weight, or 0.001 to 0.1% by weight.

The term, “included as an active ingredient” means that the protein hydrolyzate of the present specification is added to an extent capable of exhibiting the above-mentioned effects. In addition, this may include being formulated in various forms by adding various components as sub-components for drug delivery and stabilization.

The composition may be a cosmetic composition.

The cosmetic composition may be prepared in any conventionally prepared formulation. For example, the cosmetic composition may be a lotion, cream, essence, cleansing foam, cleansing water, pack, ampoule, body lotion, body oil, body gel, shampoo, conditioner, hair conditioner, hair gel, foundation, lipstick, mascara, makeup It may have a cosmetic formulation of a base or skin adhesion type.

Components included in the cosmetic composition may include components commonly used in cosmetic compositions in addition to the composition as an active ingredient, for example, conventional adjuvants and carriers such as stabilizers, solubilizers, vitamins, pigments and fragrances. may include.

In addition, the composition may be a composition for external application to the skin.

The external preparation for skin may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, drug-containing bandage, lotion, or a combination thereof. The external preparation for skin is a component usually used in external preparations for skin such as cosmetics or pharmaceuticals, for example, an aqueous component, an oily component, a powder component, alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, a preservative, an antioxidant, a surfactant, a fragrance , colorant, various skin nutrients, or a combination thereof may be appropriately formulated as needed. The external preparation for skin includes metal-blocking agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belapamil, licorice extract, glablidine, and kaline. Fruit hot water extracts, various herbal medicines, tocopherol acetate, glitylittic acid, tranexamic acid and derivatives or salts thereof, vitamin C, magnesium ascorbate phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, Sugars, such as trehalose, etc. can be mix|blended suitably.

The composition may be a food composition. The composition may be a health functional food composition.

The food composition may be used alone or in combination with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prophylactic, health or therapeutic treatment). In general, in the production of food or beverage, the composition of the present specification may be added in an amount of 15 parts by weight or less based on the raw material. There is no particular limitation on the type of the health functional food. Among the types of health functional food, the beverage composition may contain various flavoring agents or natural carbohydrates as an additional component like a conventional beverage. The natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumartin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used. The health food composition can also be added to nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonated beverages carbonation agent used, or a combination thereof. The health functional food composition may also contain natural fruit juice, fruit juice beverage, fruit flesh for the production of vegetable beverage, or a combination thereof.

The composition may be a pharmaceutical composition.

The pharmaceutical composition may further include a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and the lubricant may be magnesium stearate, talc, or a combination thereof. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be carboxymethyl cellulose calcium, sodium starch glycolate, anhydrous calcium monohydrogen phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The pharmaceutical composition may be formulated as an oral or parenteral dosage form. Oral dosage forms may be granules, powders, solutions, tablets, capsules, dry syrups, or a combination thereof. The parenteral dosage form may be an injection.

Another aspect provides a method for preparing a protein hydrolyzate of baecho flavor.

The method includes the step of incubating a mixture of baechohyang, protease, and purified water to obtain a baechohyang protein hydrolyzate.

Details regarding the baechohyang, proteolytic enzyme, and baechohyang protein hydrolyzate are as described above.

The baechohyang may use an outpost or a part of baechohyang, for example, leaves, stems, flowers, etc. may be used, but is not limited to a specific part thereof. The baecho flavor may be used, for example, in a dry powder form, but is not limited thereto.

The protease may be used in an amount sufficient to allow the enzymatic reaction to occur. Specifically, the protease is 0.1 to 10.0 parts by weight, 0.1 to 5.0 parts by weight, 0.1 to 3.0 parts by weight, 1.0 to 10.0 parts by weight, 1.0 to 5.0 parts by weight, 1.0 to 3.0 parts by weight, based on 100 parts by weight of baechohyang, Or about 2.0 parts by weight may be used, but is not limited thereto.

The purified water may be used in an amount sufficient to allow the enzymatic reaction to occur. Specifically, the purified water is 100 to 100000 parts by weight, 100 to 10000 parts by weight, 100 to 5000 parts by weight, 100 to 3000 parts by weight, 100 to 2000 parts by weight, 1000 to 100000 parts by weight, 1000 to 10000 parts by weight, based on 100 parts by weight of baecho flavor. 10000 parts by weight, 1000 to 5000 parts by weight, 1000 to 3000 parts by weight, or about 2000 parts by weight may be used, but is not limited thereto.

The enzymatic reaction may be performed at an appropriate temperature depending on the type of enzyme. For example, the enzymatic reaction may be carried out at 25 to 70 ℃, 30 to 70 ℃, 35 to 70 ℃, 40 to 70 ℃, 45 to 70 ℃, 50 to 70 ℃, or 55 to 65 ℃, It is not limited thereto.

The enzymatic reaction may be performed for an appropriate time so that the enzymatic reaction can be sufficiently performed. For example, the enzymatic reaction is 1 minute to 24 hours, 1 minute to 20 hours, 1 minute to 16 hours, 1 minute to 12 hours, 1 minute to 8 hours, 30 minutes to 24 hours, 30 minutes to 20 hours, 30 minutes to 16 hours, 30 minutes to 12 hours, 30 minutes to 8 hours, 1 hour to 24 hours, 1 hour to 20 hours, 1 hour to 16 hours, 1 hour to 12 hours, or 1 hour to 8 hours may be, but is not limited thereto.

The method may further include concentrating the protein hydrolyzate after filtration. The filtration and concentration method may use a known conventional method.

The method may further include the step of freeze-drying the concentrate obtained by the step of concentrating. For the freeze-drying method, a known conventional method may be used. By the freeze-drying, it is possible to obtain a powdery baecho-hyang protein hydrolyzate.

Another aspect provides a method of improving a skin condition in a subject comprising administering to the subject in need thereof an effective amount of the composition described above.

The skin condition improvement may be anti-aging, skin wrinkle improvement, skin elasticity enhancement, skin moisturizing, skin barrier strengthening, or a combination of two or more thereof.

The term "effective amount" means an amount effective enough to produce the above-mentioned effects.

The terms "administering", "introducing", and "implanting" are used interchangeably and in one embodiment into a subject by a method or route that results in at least partial localization of the composition according to one embodiment to a desired site. It may mean the arrangement of the composition according to

Administration may be administered by methods known in the art. Administration can be administered directly to a subject by any means, for example, intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. can The administration may be systemically or locally. The administration may be applied to the skin.

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be an individual in need of skin beauty improvement, for example, skin wrinkle improvement, skin elasticity enhancement, anti-aging, skin moisturizing, and skin barrier strengthening effect.

The administration is 0.1 mg to 1,000 mg, for example, 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1 mg of the composition according to one embodiment per day 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg may be administered. However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity, and those skilled in the art The dosage may be appropriately adjusted in consideration of these factors. The number of administration may be once a day or twice or more within the range of clinically acceptable side effects, and may be administered to one or two or more sites for the administration site, and total daily or at intervals of 2 to 5 days The number of days of administration may range from 1 to 30 days per treatment. If necessary, the same treatment can be repeated after a titration period. For animals other than humans, the dose is the same as that of a human per kg, or the above dose is converted, for example, by the volume ratio (for example, average value) of the target animal and the organ (heart, etc.) of the human One dose can be administered.

Another aspect provides the use of a protein hydrolyzate of baechohyang for preparing a composition for improving skin conditions.

Details regarding the improvement of the skin condition and the protein hydrolyzate of baechohyang are as described above.

A composition comprising a baecho-hyang protein hydrolyzate according to an aspect may be usefully used for preventing, improving, or treating skin-related conditions such as skin wrinkle improvement, skin elasticity enhancement, anti-aging, skin moisturizing, skin barrier strengthening, and the like.

1 is a graph showing the relative expression level of collagen degrading enzyme (Matrix metalloproteinase-1, MMP-1). None, untreated group; UV, UVB irradiation group; EGCG, Epigallocatechin gallate treated group as a positive control; Baechohyang DW_1, 1 ppm treatment group of baechohyang hot water extract of Comparative Example 1; Baechohyang DW_10, 10 ppm treatment group of baechohyang hot water extract of Comparative Example 1; Baechohyang EtOH_1, Comparative Example 2 baechohyang 70% ethanol extract 1 ppm treatment group; Baechohyang EtOH_10, 10 ppm treatment group of baechohyang 70% ethanol extract of Comparative Example 2; Baechohyang GP_1, 1 ppm treatment group of baechohyang protein hydrolyzate of Example 1; Baechohyang GP_10, 10 ppm treatment group of baechohyang protein hydrolyzate of Example 1; Baechohyang Complex Enzyme_1, Comparative Example 3 baechohyang sugar hydrolyzate 1 ppm treatment group; Baechohyang complex enzyme_10, 10 ppm treatment group of baechohyang sugar hydrolyzate of Comparative Example 3.
2 is a graph showing the relative expression level of collagen synthesis factors (Collagen, type 1, alpha 1, COL1A1). None, untreated group; UV, UVB irradiation group; EGCG, Epigallocatechin gallate treated group as a positive control; Baechohyang DW_1, 1 ppm treatment group of baechohyang hot water extract of Comparative Example 1; Baechohyang DW_10, 10 ppm treatment group of baechohyang hot water extract of Comparative Example 1; Baechohyang EtOH_1, Comparative Example 2 baechohyang 70% ethanol extract 1 ppm treatment group; Baechohyang EtOH_10, 10 ppm treatment group of baechohyang 70% ethanol extract of Comparative Example 2; Baechohyang GP_1, 1 ppm treatment group of baechohyang protein hydrolyzate of Example 1; Baechohyang GP_10, 10 ppm treatment group of baechohyang protein hydrolyzate of Example 1; Baechohyang Complex Enzyme_1, Comparative Example 3 baechohyang sugar hydrolyzate 1 ppm treatment group; Baechohyang complex enzyme_10, 10 ppm treatment group of baechohyang sugar hydrolyzate of Comparative Example 3.
3 is a graph showing the relative expression level of fibrillin (Fibrillin 1, FBN1). None, untreated group; UV, UVB irradiation group; EGCG, Epigallocatechin gallate treated group as a positive control; Baechohyang DW_1, 1 ppm treatment group of baechohyang hot water extract of Comparative Example 1; Baechohyang DW_10, 10 ppm treatment group of baechohyang hot water extract of Comparative Example 1; Baechohyang EtOH_1, Comparative Example 2 baechohyang 70% ethanol extract 1 ppm treatment group; Baechohyang EtOH_10, 10 ppm treatment group of baechohyang 70% ethanol extract of Comparative Example 2; Baechohyang GP_1, 1 ppm treatment group of baechohyang protein hydrolyzate of Example 1; Baechohyang GP_10, 10 ppm treatment group of baechohyang protein hydrolyzate of Example 1; Baechohyang Complex Enzyme_1, Comparative Example 3 baechohyang sugar hydrolyzate 1 ppm treatment group; Baechohyang complex enzyme_10, 10 ppm treatment group of baechohyang sugar hydrolyzate of Comparative Example 3.
4 is a graph showing the relative expression level of aquaporin 3 (Aquaporin 3, AQP3). None, untreated group; RA, retinoic acid treated group as a positive control; Baechohyang DW_1, 1 ppm treatment group of baechohyang hot water extract of Comparative Example 1; Baechohyang DW_10, 10 ppm treatment group of baechohyang hot water extract of Comparative Example 1; Baechohyang EtOH_1, Comparative Example 2 baechohyang 70% ethanol extract 1 ppm treatment group; Baechohyang EtOH_10, 10 ppm treatment group of baechohyang 70% ethanol extract of Comparative Example 2; Baechohyang GP_1, 1 ppm treatment group of baechohyang protein hydrolyzate of Example 1; Baechohyang GP_10, 10 ppm treatment group of baechohyang protein hydrolyzate of Example 1; Baechohyang Complex Enzyme_1, Comparative Example 3 baechohyang sugar hydrolyzate 1 ppm treatment group; Baechohyang complex enzyme_10, 10 ppm treatment group of baechohyang sugar hydrolyzate of Comparative Example 3.
5 is a graph showing the relative expression level of hyaluronic acid synthase 3 (Hyaluronan synthase 3, HAS3). None, untreated group; RA, retinoic acid treated group as a positive control; Baechohyang DW_1, 1 ppm treatment group of baechohyang hot water extract of Comparative Example 1; Baechohyang DW_10, 10 ppm treatment group of baechohyang hot water extract of Comparative Example 1; Baechohyang EtOH_1, Comparative Example 2 baechohyang 70% ethanol extract 1 ppm treatment group; Baechohyang EtOH_10, 10 ppm treatment group of baechohyang 70% ethanol extract of Comparative Example 2; Baechohyang GP_1, 1 ppm treatment group of baechohyang protein hydrolyzate of Example 1; Baechohyang GP_10, 10 ppm treatment group of baechohyang protein hydrolyzate of Example 1; Baechohyang Complex Enzyme_1, Comparative Example 3 baechohyang sugar hydrolyzate 1 ppm treatment group; Baechohyang complex enzyme_10, 10 ppm treatment group of baechohyang sugar hydrolyzate of Comparative Example 3.
6 is a graph showing the relative expression level of claudin-1 (CLD1). None, untreated group; RA, retinoic acid treated group as a positive control; Baechohyang DW_1, 1 ppm treatment group of baechohyang hot water extract of Comparative Example 1; Baechohyang DW_10, 10 ppm treatment group of baechohyang hot water extract of Comparative Example 1; Baechohyang EtOH_1, Comparative Example 2 baechohyang 70% ethanol extract 1 ppm treatment group; Baechohyang EtOH_10, 10 ppm treatment group of baechohyang 70% ethanol extract of Comparative Example 2; Baechohyang GP_1, 1 ppm treatment group of baechohyang protein hydrolyzate of Example 1; Baechohyang GP_10, 10 ppm treatment group of baechohyang protein hydrolyzate of Example 1; Baechohyang Complex Enzyme_1, Comparative Example 3 baechohyang sugar hydrolyzate 1 ppm treatment group; Baechohyang complex enzyme_10, 10 ppm treatment group of baechohyang sugar hydrolyzate of Comparative Example 3.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of baechohyang protein hydrolyzate

In order to prepare a protein hydrolyzate of baechohyang by proteolytic enzyme treatment, the baechohyang was naturally dried and then pulverized to prepare the raw material in powder form. 2.0 kg of purified water corresponding to 20 times the weight of 100 g of pulverized baechohyang raw material was added, and NaOH was added to adjust the pH to 7.0. 2 parts by weight (2 g) of Alcalase enzyme was added based on 100 parts by weight (100 g) of baechohyang raw material, followed by enzyme extraction at 60° C. for 5 hours. The alkalase enzyme corresponds to an endopeptidase among proteolytic enzymes. Upon completion of the enzyme extraction, inactivation treatment was performed at 90° C. for 1 hour, filtered through filter paper (0.5 μm porosity), and the filtrate was concentrated under reduced pressure. In order to remove the solvent remaining in the filtrate concentrated under reduced pressure, it was freeze-dried to obtain a powdery baecho-hyang protein hydrolyzate.

Comparative Example 1. Preparation of baechohyang hot water extract

In order to prepare the hot water extract of baechohyang, after natural drying of baechohyang, the raw material was prepared in powder form by pulverization. After adding distilled water corresponding to 20 times the weight of 100 g of pulverized baechohyang raw material and mixing, it was extracted at a temperature of 90° C. for 4 hours and 30 minutes. The obtained extract was filtered using filter paper, and then the filtrate was concentrated under reduced pressure using a rotary evaporator. In order to remove the solvent remaining in the filtrate concentrated under reduced pressure, it was freeze-dried to obtain a powdery hot water extract of baechohyang.

Comparative Example 2. Preparation of ethanol extract of baechohyang

In order to prepare an ethanol extract of baechohyang, after natural drying of baechohyang, the raw material was prepared in powder form by pulverization. 100 g of pulverized baechohyang raw material was immersed in 70% ethanol aqueous solution corresponding to 20 times the weight and left at room temperature for 48 hours to obtain an extract. The obtained extract was filtered using filter paper, and then the filtrate was concentrated under reduced pressure using a rotary evaporator. In order to remove the solvent remaining in the filtrate concentrated under reduced pressure, it was freeze-dried to obtain a powdery 70% ethanol extract of baechohyang.

Comparative Example 3. Preparation of hydrolyzate of baechohyang sugar

Baechohyang through an enzymatic digestion method using a complex enzyme containing β-glucosidase, α,β-arabinosidase and α,β-ramosidase that breaks down sugar bonds A sugar hydrolyzate was prepared.

experimental method

(1) Protein quantification method

Lowry assay, one of protein quantification methods, was performed, and Bio-Rad's DC Protein Assay (500-0116) product was used. After reacting with Reagent A: Reagent S = 50:1, Reagent B was treated and reacted at room temperature for 20 minutes. Absorbance was measured at 750 nm with an ELISA reader (Thermo, 51119300), and the amount of total protein was determined using a standard curve obtained from bovine serum albumin.

(2) Total flavonoid quantification method

For flavonoid quantification, the method of Moreno et al. (Moreno et al., J Ethnopharmacol. 2000; 71:109-114) was used. To the 0.5 mL test solution, 0.1 mL of 10% aluminum nitrate, 0.1 mL of 1 M potassium acetate, and ethanol were sequentially added, mixed and left at room temperature for 40 minutes, using an ELISA reader (Thermo, 51119300). to measure the absorbance at 415 nm. Quercetin was accurately taken, operated in the same manner as the sample solution, and a calibration curve was prepared, and the total flavonoid content in the raw material was quantified from this calibration curve.

(3) Total polyphenol quantification method

Total polyphenol content was measured using the Folin-Denis method (Folin et al., 1912). Folin-Ciocalteu (F-C) reagent is prepared by reacting sodium molybdate and sodium tungstate with phosphoric acid solution. By donating electrons, a blue-violet reduced product is produced.

The sample solution to be measured was reacted with 2N Folin-Ciocalteu reagent. After adding distilled water immediately, the reaction was carried out at room temperature for 3 minutes. 20% Na ₂ CO ₃ was added, and the mixture was stirred and allowed to develop color at room temperature for 1 hour. Absorbance was measured at 765 nm using an ELISA reader (Thermo, 51119300). Gallic acid was accurately taken and operated in the same manner as the sample solution to prepare a calibration curve, and the total polyphenol content in the raw material was quantified from this calibration curve.

(4) Total sugar quantification method

To measure the total sugar content, a phenol-sulfuric acid method was performed. In the phenol-sulfuric acid method, when reducing sugar is treated with concentrated sulfuric acid, it is dehydrated to form furfural or a derivative thereof. The absorbance of this derivative is measured and quantified at 485 nm. The experimental method is as follows.

To 1 ml of the sample solution, add 1 ml of 5% phenol, and then add 5 ml of sulfuric acid. Then, after vortexing for 30 seconds using a vortex mixer, it was left at 30° C. for 30 minutes, and absorbance was measured at 485 nm using an ELISA reader (Thermo, 51119300). Glucose was accurately taken and operated in the same manner as in the sample solution to prepare a calibration curve, and the total sugar content in the raw material was quantified from this calibration curve.

(5) UV irradiation

The fibroblast line Hs68 was aliquoted in the number of 3x10 ⁵ in a 6-well plate, and then cultured in an incubator at 37° C., 5% CO ₂ condition for 24 hours. After removing the medium and adding DPBS, the remaining cell groups except for the UVB non-irradiated group were irradiated with UVB at 12 mJ/cm ² .

(6) RNA isolation and real-time RT-PCT analysis

The expression levels of genes related to anti-aging, skin moisture and skin barrier enhancement were measured using the human fibroblast line Hs68 or human Keratinocyte Hacat. RNA was isolated from the cells treated with each sample using Trizol (RNA iso, DAKARA, Japan), and then RNA was quantified at 260 nm using a nanodrop, and 2 μg of each RNA was used. cDNA was synthesized in an amplifier (C1000 Thermal Cycler, Bio-Rad, USA). Finally, a real-time polymerase chain reaction is performed in a real-time PCR machine using a mixture of a target gene-specific primer and a cyanine dye, SYBR Green supermis, Applied Biosystems, USA) added to the synthesized cDNA. to evaluate the expression level of the target gene. The sequence and reaction conditions of the primers are shown in Table 1 below, and the expression level of the gene was finally analyzed through correction for the β-actin gene.

gene Primer direction Sequence (5'→3') SEQ ID NO: reaction conditions MMP-1 F 5'-CGAATTGCCGACAGAGATGA-3' One Polymerase activation at 94°C for 5 minutes, polymerization reaction in 40 cycles at 95°C 30 seconds, 55°C 30 seconds, 72°C 30 seconds R 5'-GTCCCTGAACAGCCCAGTACTT-3' 2 COL1A1 F 5'-TGACGTTCCCATTAGACAACTG-3' 3 R 5'-CCGTCTTTCATTACACAGGACA-3' 4 CLD1 F GCTCTAGAATTCCGAGCGAGTCATGGCCAACGC 5 R GCTCTAGAATTCTCACACGTAGTCTTTCCCGCT 6 FBN1 F AATGTCAGACGAAGCCAGGG 7 R GATTTGGTGACGGGGTTCCT 8 HAS-3 F CTTAAGGGTTGCTTGCTTGC 9 R GTTCGTGGGAGATGAAGGAA 10 AQP3 F GTCACTCTGGGCATCCTCAT 11 R CTATTCCAGCACCCAAGAAGG 12 β-actin F 5'-GGCCATCTCTTGCTCGAAGT-3' 13 R 5'-GAGACCTTCAACACCCCAGC-3' 14

Experimental Example 1. Protein, sugar, flavonoid, polyphenol content analysis

Protein, sugar, flavonoid, and polyphenol content were analyzed for the hydrolyzate of baechohyang protein of Example 1 and the hot water extract of baechohyangyi of Comparative Example 1, and the results are shown in Table 2 below. As shown in Table 2, compared to Comparative Example 1, Example 1 significantly increased the total protein, sugar, flavonoid, and polyphenol content.

Comparative Example 1 Example 1 protein content 14% 20% sugar content 16% 21% Total flavonoid content 1.8% 2.7% Total polyphenol content 4.1% 5%

Experimental Example 2. Confirmation of anti-aging efficacy

The hydrolyzate of baechohyang protein of Example 1, the hot water extract of baechohyang of Comparative Example 1, 70% ethanol extract of baechohyang of Comparative Example 2, and the hydrolyzate of baechohyang of Comparative Example 3 were added to the Hs68 fibroblast line, respectively, and anti-aging genes The expression level was analyzed.

1 is a graph showing the relative expression level of collagen degrading enzyme (Matrix metalloproteinase-1, MMP-1).

2 is a graph showing the relative expression levels of collagen synthesis factors (Collagen, type 1, alpha 1, COL1A1).

3 is a graph showing the relative expression level of fibrillin (Fibrillin 1, FBN1).

As shown in FIG. 1 , while the expression level of MMP-1 rapidly increased in the UVB irradiation group, the MMP-1 expression inhibition ability was the best in the 1 ppm treatment group of the baechohyang protein hydrolyzate of Example 1.

As shown in FIG. 2 , while the expression level of COL1A1 was rapidly decreased in the UVB irradiation group, the COL1A1 expression increase ability was the best in the 10 ppm treatment group of the baechohyang protein hydrolyzate of Example 1.

As shown in FIG. 3 , while the FBN1 expression level in the UVB-irradiated group decreased rapidly, the FBN1 expression increase ability was excellent in the 1 ppm and 10 ppm treatment groups of the baechohyang protein hydrolyzate of Example 1.

Through these results, it was confirmed that the hydrolyzate of baechohyang protein of Example 1 has excellent anti-aging activity on skin cells by improving skin wrinkles and enhancing skin elasticity.

Experimental Example 3. Confirmation of skin moisturizing and skin barrier strengthening effect

The hydrolyzate of baechohyang protein of Example 1, the hot water extract of baechohyang of Comparative Example 1, the 70% ethanol extract of baechohyang of Comparative Example 2, and the hydrolyzate of baechohyang of Comparative Example 3 were added to the HaCaT cell line, respectively, and related to skin moisturizing and skin barrier The gene expression level was analyzed.

4 is a graph showing the relative expression level of aquaporin 3 (Aquaporin 3, AQP3).

5 is a graph showing the relative expression level of hyaluronic acid synthase 3 (Hyaluronan synthase 3, HAS3).

6 is a graph showing the relative expression level of claudin-1 (CLD1).

As shown in FIG. 4 , the baechohyang protein hydrolyzate treatment group of Example 1 had the best ability to increase the expression of AQP3, a water channel.

As shown in FIG. 5 , the baechohyang protein hydrolyzate treatment group of Example 1 had the best ability to increase the expression of HAS3.

As shown in FIG. 6 , the baechohyang protein hydrolyzate treatment group of Example 1 was overall excellent in the ability to increase CLD1 expression.

Through these results, it was confirmed that the hydrolyzate of baecho-hyang protein of Example 1 was excellent in skin moisturizing or skin barrier strengthening effect.

SEQUENCE LISTING <110> Cosmax, INC. <120> Cosmetic composition for improving skin conditions comprising protein hydrolysate of Agastache rugosa <130> PN135837 <160> 14 <170> PatentIn version 3.2 <210> 1 <211> 20 <212> DNA <213> <220> <223> MMP-1 forward primer <400> 1 cgaattgccg acagagatga 20 <210> 2 <211> 22 <212> DNA <213> <220> <223> MMP-1 reverse primer <400> 2 gtccctgaac agcccagtac tt 22 <210> 3 <211> 22 <212> DNA <213> <220> <223> COL1A1 forward primer <400> 3 tgacgttccc attagacaac tg 22 <210> 4 <211> 22 <212> DNA <213> <220> <223> COL1A1 reverse primer <400> 4 ccgtctttca ttacacagga ca 22 <210> 5 <211> 33 <212> DNA <213> <220> <223> CLD1 forward primer <400> 5 gctctagaat tccgagcgag tcatggccaa cgc 33 <210> 6 <211> 33 <212> DNA <213> <220> <223> CLD1 reverse primer <400> 6 gctctagaat tctcacacgt agtctttccc gct 33 <210> 7 <211> 20 <212> DNA <213> <220> <223> FBN1 forward primer <400> 7 aatgtcagac gaagccaggg 20 <210> 8 <211> 20 <212> DNA <213> <220> <223> FBN1 reverse primer <400> 8 gatttggtga cggggttcct 20 <210> 9 <211> 20 <212> DNA <213> <220> <223> HAS-3 forward primer <400> 9 cttaagggtt gcttgcttgc 20 <210> 10 <211> 20 <212> DNA <213> <220> <223> HAS-3 reverse primer <400> 10 gttcgtggga gatgaaggaa 20 <210> 11 <211> 20 <212> DNA <213> <220> <223> AQP3 forward primer <400> 11 gtcactctgg gcatcctcat 20 <210> 12 <211> 21 <212> DNA <213> <220> <223> AQP3 reverse primer <400> 12 ctattccagc acccaagaag g 21 <210> 13 <211> 20 <212> DNA <213> <220> <223> beta-actin forward primer <400> 13 ggccatctct tgctcgaagt 20 <210> 14 <211> 20 <212> DNA <213> <220> <223> beta-actin reverse primer <400> 14 gagaccttca acaccccagc 20

Claims (10)

Contains a protein hydrolyzate of baechohyang ( Agastache rugosa ),
The protein hydrolyzate is prepared by enzymatically reacting baechohyang with a proteolytic enzyme,
The proteolytic enzyme is an endopeptidase (endopeptidase), the cosmetic composition for improving skin condition. The cosmetic composition of claim 1, wherein the skin condition improvement is skin wrinkle improvement, skin elasticity enhancement, skin moisturizing, skin barrier strengthening, or a combination of two or more thereof. delete delete The cosmetic composition of claim 1, wherein the proteolytic enzyme is a serine protease. The cosmetic composition according to claim 1, wherein the proteolytic enzyme is Alcalase. The method according to claim 1, which inhibits the expression of collagenase (MMP-1) or collagen synthesis factor (COL1A1), fibrillin (FBN1), aquaporin 3 (AQP3), hyaluronic acid synthase 3 (HAS3) and claudin 1 (CLD1) to increase the expression of any one or more of the cosmetic composition. The cosmetic composition of claim 1, wherein the protein hydrolyzate of baechohyang is included in a concentration of 0.1 ppm to 1000 ppm based on the total weight of the composition. Contains a protein hydrolyzate of baechohyang ( Agastache rugosa ),
The protein hydrolyzate is prepared by enzymatically reacting baechohyang with a proteolytic enzyme,
The proteolytic enzyme is an endopeptidase (endopeptidase), the skin condition improvement food composition. Incubating a mixture of baechohyang ( Agastache rugosa ), a proteolytic enzyme, and purified water to obtain a baechohyang protein hydrolyzate,
The protease is an endopeptidase (endopeptidase), the method for producing a baechohyang protein hydrolyzate.

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