KR20190138946A - A composition for improving skin wrinkle comprising Vaccinium ashei leaves extract - Google Patents

Abstract

The present invention relates to a composition for reducing skin wrinkles, comprising a Vaccinium ashei leaf extract as an active ingredient. The Vaccinium ashei leaf extract contained as an active ingredient in the composition of the present invention exhibits an excellent effect of reducing skin wrinkles through a molecular mechanism such as promoting synthesis of elastin and collagen, inhibiting or promoting the expression of wrinkle related proteins, MMP-1, MMP-3, MMP-9, and procollagen proteins, and the like, has no cytotoxicity and excellent skin safety, and thus may be usefully used in cosmetics, pharmaceuticals, and food compositions.

Description

A composition for improving skin wrinkle comprising Vaccinium ashei leaves extract}

The present invention relates to an anti-wrinkle composition comprising a blueberry ( Viccinium ashei ) leaf extract, and more specifically, an active ingredient of a blueberry leaf extract having an effect of enhancing collagen synthesis, inhibiting collagenase, and inhibiting expression of wrinkle-related proteins. It relates to a cosmetic composition for improving wrinkles, a pharmaceutical composition, and a food composition comprising a.

Aging in the human body inevitably occurs over time, and the structure and function of the body gradually degrades. Although this can not be stopped completely, it is possible to slow the progress through various modern technologies and their applications, thereby improving the quality of life of members of modern society.

Skin aging can be divided into extrinsic aging and intrinsic aging. Endogenous aging is the aging that occurs due to internal action of the body, including the loss of telomere, a specific region of the gene (Imbert, I., Botto, JM, Farra, CD, & Domloge, N. (2012) Modulation of telomere binding proteins: a future area of research for skin protection and anti-aging target.Journal of cosmetic dermatology, 11 (2), 162-166.), Gravity, decreased endocrine function in the body, and chronic wasting diseases. . Exogenous aging is the aging that is caused by the environment, and the factors include climate, dust, chemicals, and ultraviolet rays (Hyo-Chul Kim, & Kyung-Dong Oh. (2011). Research on the causes, prevention and treatment of skin aging Progress, Korean Society for Skin Beauty, 9 (1), 1-9.). According to these factors, the skin develops pigmentation, wrinkles, and inflammation, and ages.

On the other hand, modern people are increasing interest in physical beauty and health, and in recent years, research is being actively conducted to prevent skin aging by using natural products more friendly to the skin. Its contents are mainly the oxidative stress scavenging effect of the cells by free radicals occurring in the metabolic process of the human body (Song Hwa Song, Ga-Yun Kim, Ji-Yeon Ji, Kim Ji-won, Yang Ye-Lim, Jeon Young-Hee, Park Soo-Nam. (2016) Cytoprotective Effect of Calendula Flower Extracts on Human Skin Cells Against Antioxidant and Oxidative Stress Applied Chemistry for Engineering, 27 (6), 620-626.), Tyrosine is DOPA and DOPA quinone, indole- Inhibitory effect on melanin synthesis through 5,6-quinone (Choi, SW, & Kim, HJ (1998). Inhibitioin of tyrosinase activity by polyphenols of chestnut leaf. HSJAS, 6, 321-327.), Inhibitory effects of collagenase and elastase, which are substances involved in the degradation of collagen and elastin, which are the major constituent proteins of the dermal layer produced from fibroblasts (Cho, Won-Sik) , Jang Do Yeon, Nam Seok Woo, Jin Yong Yong, & Racing. (2008) in inhibiting melanogenesis and Biological Activity of Korea Application of Coenzyme Q10 derivatives for Collagenase and Elastase Journal cells (old Journal of Korea thickening), 51 (3), 164-170.).

Blueberry is a shrub plant that belongs to the Vaccinium (Vaccinium) of the Ericaceae (Ericaceae), low-bush blueberry (Vaccinium myritillus ) Three varieties of high bush blueberry ( Vaccinium corymbosum ) and rabbit eye blueberry ( Vaccinium ashei ) are dominant and are grown as commercially important fruit (Westwood, MN (1993) .Hormones and growth regulators.Temparate Zone Pomology: Physiology and Culture.Timber Press, Inc, 9999.). In recent years, interest and utilization of blueberries has increased rapidly in Korea (Song, GC (2012) .The cultural status and the industrial prospects on blueberry.In Proceedings of the Plant Resources Society of Korea Conference.The Plant Resources Society of (Woo, NRY, Chung, HK, Kim, JH, & Lee, TR (2010) .Development of rice noodles with lotus leaf.In Proceedings of the KAIS) Fall Conference.The Korea Academia-Industrial Cooperation Society.). On the other hand, blueberry leaves are treated as industrial by-products of blueberry cultivation and related studies are relatively inadequate. Thus, blueberry leaf extracts in Korean Patent No. 1381785 and Korean Patent Application Publication No. 2011-0051547 prevent degenerative neuropathy or gout. It is only disclosed as useful.

Accordingly, the inventors of the present invention have been researching various ways to utilize industrial by-products such as blueberry leaves, and found that blueberry leaves are very useful as food and cosmetic materials, thereby completing the present invention.

One object of the present invention is to provide a cosmetic composition for improving wrinkles comprising a blueberry ( Vacccinium ashei ) leaf extract as an active ingredient.

It is another object of the present invention to provide a pharmaceutical composition for improving wrinkles comprising blueberry ( Vaccinium ashei ) leaf extract as an active ingredient.

Yet another object of the present invention is to provide a food composition for improving wrinkles comprising blueberry ( Vacniumnium ashei ) leaf extract as an active ingredient.

As one embodiment, the present invention provides a composition for improving wrinkles comprising blueberry ( Vacccinium ashei ) leaf extract as an active ingredient.

In the present invention, the blueberry ( Viccinium ashei ) is a fruit tree of the genus Azalea (Vaccinium), which contains a large amount of vitamin K and C, and also contains a lot of manganese. Other nutrients, such as vitamin E, thiamine, riboflavin, vitamin B6, and copper, are balanced evenly, and anthocyanins, which are bright colors of blueberries, are known to neutralize free radicals. In addition, blueberries are known to be rich in nutrients because they are effective in protecting antioxidants and the retina of the eye.

In the present invention, "improvement of skin wrinkles" is to relieve and remove wrinkles formed on the skin of animals including humans by endogenous skin aging due to hormonal secretion reduction or physiological function degradation or exogenous skin aging due to ultraviolet rays or pollution. Or to prevent.

The blueberry leaf extract of the present invention refers to a substance having the activity of improving skin wrinkles extracted from or separated from the blueberry leaf which is treated as a by-product in the blueberry cultivation industry.

In addition, the blueberry leaf extract refers to all extracts, fractions, purified products, diluents thereof, concentrates, dried products or all forms formulated using each step of extraction, fraction, or purification (separation, fraction).

Extraction method of the blueberry leaf extract uses a cold needle extraction, ultrasonic extraction, reflux cooling extraction method or supercritical extraction method.

In addition, the blueberry leaf extract may be extracted using a conventional solvent known in the art, the extracted liquid may be used in liquid form or concentrated and / or dried. At this time, the solvent is water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), ethylene, acetone, hexane, ether, ethyl acetate, butyl acetate, chloroform, 1,3-butylene A solvent of any one of glycol, propylene glycol, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), or a mixed solvent thereof can be used. Preferably, water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), acetone are used. More preferably, water, ethanol and acetone are used. However, the present invention is not limited thereto, and the extraction degree and loss degree of the active ingredient of the extract may vary depending on the organic solvent to be extracted. Therefore, it is preferable to select and use an appropriate organic solvent.

As one specific example, the blueberry leaf extract may be obtained by crushing the blueberry leaf, dipping and extracting with a solvent, and then recovering the extract and then filtering. The filtration is a process of removing the suspended solid particles from the extract, it may be used to filter the particles using cotton, nylon, or the like, ultrafiltration, cryofiltration, centrifugal separation, etc., but is not limited thereto. It may also further comprise a separation process by various chromatography (chromatography according to size, charge, hydrophobicity or affinity).

As one specific example, the blueberry leaf extract may be obtained by drying the filtrate. Freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, foam drying, high frequency drying or infrared drying may be used as a method of drying the filtrate, but is not limited thereto.

In one specific embodiment, the composition for improving skin wrinkles comprising a blueberry leaf extract as an active ingredient is a cosmetic composition.

The cosmetic composition of the present invention comprises blueberry leaf extract as an active ingredient in an amount of 0.00001 to 15.0% by weight, preferably 0.001 to 10% by weight, more preferably 0.01 to 5% by weight, based on the total weight of the total cosmetic composition. Can be. If the content is less than 0.00001% by weight can not be expected to improve the skin wrinkles, if the content exceeds 15.0% by weight difference in the wrinkle improvement is insignificant or difficult to prepare a formulation.

The cosmetic composition may further include components commonly used in cosmetic compositions in addition to the blueberry leaf extract. For example, it may include conventional auxiliaries and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments and agents.

The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art. For example, it may be formulated as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation or spray, and the like. More specifically, it may be prepared in a formulation such as nourishing cream, astringent lotion, flexible lotion, lotion, essence, nourishing gel or massage cream. However, it is not particularly limited to this.

When the formulation of the cosmetic composition is a paste, cream or gel, the carrier component is animal oil, vegetable oil, wax, paraffin, starch, tragacanth gum, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide. This can be used.

When the formulation of the cosmetic composition is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, and in the case of a spray, additionally chlorofluorohydrocarbon, Propellant such as propane / butane or dimethyl ether.

When the formulation of the cosmetic composition is a solution or emulsion, a solvent, a solubilizer or an emulsifier may be used as a carrier component. For example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic esters, polyethylene glycols or sorbitan fatty acid esters can be used. have.

When the formulation of the cosmetic composition is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester as a carrier component Crystalline cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the cosmetic composition is a surfactant-containing cleansing agent, the carrier component is aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid. Amide ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The cosmetic composition of the present invention exhibits excellent skin wrinkle improvement effect through molecular mechanisms such as fibroblast protection and promotion of collagen synthesis while having low cytotoxicity.

In one specific embodiment, the composition for improving skin wrinkles comprising a blueberry leaf extract as an active ingredient is a pharmaceutical composition.

In the pharmaceutical composition of the present invention, the blueberry leaf extract as an active ingredient is 0.00001 to 15.0% by weight, preferably 0.001 to 10% by weight, more preferably 0.01 to 5% by weight, based on the total weight of the total pharmaceutical composition. It may include. If the content is less than 0.00001% by weight can not be expected to improve the skin wrinkles, if the content exceeds 15.0% by weight difference in the wrinkle improvement is insignificant or difficult to prepare a formulation.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form or in multidose form by formulating with a pharmaceutically acceptable carrier and / or excipient, according to methods which can be easily carried out by those skilled in the art. It may be prepared by incorporation into a container. In this case, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or an aqueous medium, or may be in the form of an exir, powder, powder, granule, tablet or capsule, and may further include a dispersing or stabilizing agent.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are commonly used in the preparation of pharmaceutical compositions, and include carbohydrate compounds (eg, lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch). , Cellulose, etc.), acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, salt solutions, alcohols, gum arabic, vegetable oils (e.g. corn Oils, cotton seed oil, soy milk, olive oil, coconut oil), polyethylene glycol, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil, and the like.

In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical compositions of the present invention can be used in the form of injections or external preparations according to conventional methods.

The pharmaceutical composition of the present invention has an effect of improving skin wrinkles, and the method of administration includes administering the pharmaceutical composition in a pharmaceutically effective amount to mammals such as rats, mice, livestock, humans, and the like by various routes.

The pharmaceutical composition may be administered orally or parenterally, and is preferably applied by topical application by parenteral administration, more preferably by application.

The dosage of the pharmaceutical composition may vary depending on factors such as the method of formulation, the mode of administration, the age, weight, sex, morbidity, etc. of the patient, and the dosage ranges from 0.001 to 300 mg / kg on an adult basis. However, the dosage does not limit the scope of the present invention.

Although the pharmaceutical composition of the present invention has low cytotoxicity, it has an excellent skin wrinkle improvement effect through molecular mechanisms such as fibroblast protection and promotion of collagen synthesis.

The pharmaceutical composition comprising the blueberry leaf extract of the present invention as an active ingredient has the advantage of achieving the purpose of improving the skin wrinkles of the subject by topical administration, such as administration in the subject, subcutaneous administration or application.

In another specific embodiment, the composition for improving skin wrinkles comprising a blueberry leaf extract as an active ingredient is a food composition.

It provides a food composition comprising a blueberry extract as an active ingredient.

In addition, the food composition according to another embodiment of the present invention contains not only blueberry extract as an active ingredient but also components commonly added in food preparation, such as proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. It may further include.

Examples of such carbohydrates are monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. As the flavoring agent, natural flavoring agents (tauumatin, stevia extract (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

For example, when the food composition of the present invention is prepared with a drink, in addition to the blueberry extract of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, tofu extract, jujube extract, licorice extract, etc. Can be.

Although the food composition of the present invention has low cytotoxicity, it has an excellent skin wrinkle improvement effect through molecular mechanisms such as fibroblast protection and promotion of collagen synthesis.

As another aspect, the present invention provides a method for treating, improving and preventing wrinkles of an individual by administering to the individual a composition comprising a blueberry leaf extract as an active ingredient.

The composition comprising the blueberry leaf extract of the present invention as an active ingredient has the advantage of achieving the purpose of improving and preventing wrinkles of the individual by topical administration, such as administration in the subject, subcutaneous administration or application.

In the present invention, the term "individual" refers to monkeys, cows, horses, pigs, sheep, dogs, cats, rats, mice, including humans with a condition or disease in which symptoms may be improved by administering the composition of the invention. It means a mammal such as a chimpanzee.

In the present invention, the terms “treatment” and “improvement” refer to the destruction of inflammatory cells or the inhibition of (a) development, (b) alleviation, and (c) removal of inflammatory cells in a subject.

In the present invention, the term "prevention" means any action that inhibits or delays inflammation by administration of the composition.

On the other hand, the blueberry leaf extract of the present invention is a natural extract derived from plants as a safe substance harmless to the human body without toxicity and side effects, and can be safely applied to cosmetics, pharmaceuticals and food compositions as an active ingredient for improving skin wrinkles.

The composition for improving skin wrinkles of the present invention includes blueberry leaf extract as an active ingredient, inhibits degradation of elastin and collagen in skin cells and promotes synthesis, inhibits MMP-1, inhibits MMP-3, and MMP-9 It exhibits excellent skin wrinkle improvement effect through detailed mechanisms such as inhibition and promotion of procollagen synthesis. In addition, the extract of the present invention is derived from a natural material can be safely applied to cosmetics, pharmaceuticals, and food compositions as a safe substance harmless to the human body without toxicity and side effects as a result of skin irritation experiments on the human body.

1 is a view showing a manufacturing process of a blueberry leaf extract according to an embodiment of the present invention.
Figure 2 is a diagram showing the results of measuring the elastase inhibitory activity by the blueberry leaf extract of the present invention.
Figure 3 is a diagram showing the results of measuring the collagenase inhibitory activity by the blueberry leaf extract of the present invention.
Figure 4 is a diagram showing the cell viability of the human-derived fibroblast CCD986sk by the blueberry leaf extract of the present invention.
5 is a picture analyzing the effect of inhibiting the expression of MMP-1 protein by hydrothermal extract of the blueberry leaf of the present invention in UV-B stimulated CCD986sk cells.
Figure 6 is a picture analyzing the effect of inhibiting the expression of MMP-3 protein by 70% ethanol extract of blueberry leaves of the present invention in UV-B stimulated CCD986sk cells.
Figure 7 is a picture analyzing the effect of inhibiting the expression of MMP-9 protein by 70% acetone extract of the blueberry leaf of the present invention in UV-B stimulated CCD986sk cells.

Hereinafter, examples and the like will be described in detail to help understand the present invention. However, embodiments according to the present invention can be modified in many different forms, the scope of the invention should not be construed as limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

All experiments were repeated three times, and the results were expressed as mean ± standard deviation. Statistical analysis was performed using the SPSS program to analyze the variance (* p <0.05, ** p <0.01) for the significance of each treatment. After the analysis of variance (ANOVA), multiple comparisons were carried out using the Turkish test.

Example 1: Preparation of Blueberry Leaf Extract

The blueberries ( Vacniumnium ashei ) leaves used in this experiment were supplied from a blueberry farm in Ulsan, and the extraction process of the blueberry leaves was carried out as shown in FIG. 1. The hot water extract of the blueberry leaf was repeatedly extracted three times at 90 ° C. for 4 hours using ultrapure water 20 times the weight of the dried sample as a solvent. The ethanol extract of the blueberry leaf and the acetone extract of the blueberry leaf were extracted by immersing at 70% ethanol and 70% acetone at 20 times the weight of the dried sample for 24 hours at room temperature, and repeated three times. The yield of hot water extract was 38.31%, the yield of 70% ethanol extract was 21.78%, the yield of 70% acetone extract was 21.68%. The extracts were filtered through a filter paper (No. 20 filter paper, Hyundai micro Co., Ltd., Korea), concentrated, lyophilized, powdered and stored in a 4 ° C. cold storage room.

Example 2: Reagent Preparation

2-1: Elastase and Collagenase Inhibitory Effect Experimental Reagent

The reagents used in the wrinkle improvement effect measurement experiment are as follows. N-Succinyl-Ala-Ala-Ala-p-nitroanilide, Elastase from porcine pancreas, and Collagenase from clostridium histdyticum from Sigma aldrich Co., Ltd .; St. Louis, Mo., USA). 2-Amino-2- (hydroxymethyl) propane-1,3-diol, Hydrogen chloride, CaCl ₂ , Citric acid, Ethyl acetate, etc. are made by Duksan pure chemicals Co., Ltd. .; Korea). Pz-Pro-Leu-Gly-Pro-D-Arg-OH Trifluoroacetate was a product of BACHEM Co., Ltd.

2-2: Cell Lines and Reagents for Cell Experiments

Cell lines Murine macrophage cells (RAW 264.7) and human fibroblast cells (CCD986sk) were manufactured by ATCC (American type culture collection; Manassas, VA, USA). DMEM (Dulbecco's modified eagle medium), penicillin (Penicillin), FBS (Fetal bovine serum) and the like were used by GE Healthcare life sciences hyclone laboratories (Logan, Utah, USA). 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl-tetrazolium bromide (MTT), lipopolysaccharides (LPS), phosphatase inhibitor cocktail 3, ripa buffer, grease Griess reagent (modified), Protease inhibitor cocktail for use with mammalian cell and tissue extract, and the like were used by the product of Sigma aldrich Co., Ltd .; St. Louis, MO, USA. . Prostaglandin E ₂ ELISA Kit was used from R & D Systems (USA). Antibodies (goat anti-rabbit IgG-HRP, donkey anti-mouse IgG-HRP, Bovine anti-goat IgG-HRP, COX-2, iNOS, NF-κB, MMP-1, MMP-3, MMP-9, Procollagen)) and the like used a product of Santa Cruz (Santa cruz Co., Ltd .; USA). Acryl-bisacrylamide (29: 1) 30% stock solution was used as the product of m. Biotech Co., Ltd .; Korea. Tris-base (2-Amino-2- (hydroxymethyl) propane-1,3-diol), glycine, Glycine, 20X PBS buffer, sodium dodecyl sulfate (SDS), and TEMED are available from Bioosesang Co., Ltd .; Korea). Protein assay dye reagent concentrate was used as a product of Bio-rad laboratories Co., Ltd. (USA). pH 7 albumin-bovine was used by USB Co., Ltd. (Cleveland, OH, USA). The PageRular ™ prestained protein ladder, Western blot stripping buffer, Power SYBR green PCR master mix, and NE-PER nuclear and cytoplasmic extraction reagents kit were manufactured from Thermo scientific Co., Ltd. (Lithuania). Tween 20 used a product of Daejung chemicals & metals Co., Ltd. (Korea). Nuclease-free water used a product of Integrated DNA technologies Co., Ltd. (USA). Total RNA isolation regent-solution uses a product of Bio science technology. PCR primers were used by Bionics Co., Ltd. (Korea). As the transfer membrane, a product of Millipore Co., Ltd. (billerica, MA, USA) was used. 3 mm CHR was used by GE healthcare life sciences Co., Ltd. (China).

2-3. Device used for experiment

Instruments used in this experiment were UV / Visible Spectrophotometer (Hitachi, Tokyo, Japan), Polarization Microscope (Olympus BX 51, Tokyo, Japan), Western Imaging System (CAS-400SM, Davinch-). K, Seoul, Korea), rotary vacuum evaporator (EYELA, Tokyo, Japan), centrifuge (Hitachi, Tokyo, Japan), freeze drier (Ilshin, Gimpo, Korea), microscope (microscope) , Olympus, Tokyo, Japan), CO2 incubator, Hanbaek Scientific Co., Bucheon, Korea, pH meter (pH meter, Metrohm, Herisau Switzerland), BOD incubator, Hanbaek Scientific Co., Bucheon, Korea ), Autoclave (Hanbaek Scientific Co., Bucheon, Korea) and ELISA reader (ELISA reader, Molecular Devices, California, USA) were used.

Example 3: Determination of Elastase Inhibitory Effect of Blueberry Leaf Extract

The measurement of elastase inhibitory effect is Cannell (Cannell, RJ, Kellam, SJ, Owsianka, AM, & Walker, JM (1988) .Results of a large scale screen of microalgae for the production of protease inhibitors.Planta medica, 54 (01 ), 10-14.) And the like. Each sample was prepared by diluting at concentrations of 5, 10, 50, 100, 500, and 1000 μg / mL, and EGCG (Epigallocatechin gallate) was used as a positive control. The buffer solution was used after adding hydrogen chloride to pH 8.6 in Tris (2-Amino-2- (hydroxymethyl) propane-1,3-diol). N-Succinyl-Ala-Ala-Ala-p-nitroanilide used as a substrate was used diluted to 3 mM concentration using a 50 mM buffer solution as a solvent. Porcine pancreas-derived elastase (Elastase from porcine pancreas) used as an enzyme was diluted to a concentration of 1 unit / ml using a 50 mM buffer solution as a solvent. Add 40 μL of buffer, enzyme and sample solution to each 96 well plate and react for 5 min at 37 ℃. Add 80 μL of substrate solution, further react for 15 min at 37 ℃ and measure absorbance at 445 nm. Equation 1 was used to calculate the result.

[Equation 1]

Elastase inhibitory effect (%) = (1-sample group absorbance / no group absorbance) × 00

Elastin is an important protein for maintaining skin elasticity in the dermis and is present in the connective tissue of the skin and is greatly involved in the elasticity and flexibility of the tissue. Elastase is an enzyme that breaks down elastin and is one of the causes of skin aging such as sagging skin and wrinkles (Ahn Bong Jeon, Jin Tae Lee, & Chang-Eon Lee. (2009). p, 82.). Therefore, the elastase inhibitor acts to prevent aging through skin wrinkle improvement. The results of measuring the elastase inhibitory activity of the blueberry leaf extract are shown in FIG. 2.

As can be seen in Figure 2, blueberry leaves showed an elastase inhibitory effect in a concentration-dependent manner in all extracts. At the concentration of 1000 μg / mL, the effect of 70% acetone extract was 75.90%, which was higher than that of hydrothermal extract (73.61%) and 70% ethanol extract (55.53%), showing that 70% acetone extract was the most effective among the three extracts. In particular, it is much higher than Epigallocatechin gallate (44.87%) used as a positive control. Blueberry leaf extract is a natural product, which can be very effective in improving wrinkles.

Example 4: Determination of Collagenase Inhibitory Effect of Blueberry Leaf Extract

Measurement of collagenase inhibitory effect is described by Wunsch (W ㆌ nsch, E., & Heidrich, HG (1963) .Zur quantitativen bestimmung der kollagenase. It measured according to methods, such as these. Each sample was prepared by diluting at a concentration of 10, 100, 1000 μg / mL, and EGCG (Epigallocatechin gallate) was used as a positive control. The buffer solution was used after adding hydrogen chloride to pH 7.5 in 0.1 M Tris, 4 mM CaCl ₂ solution. The substrate solution was diluted with 0.3 mg / ml concentration using Pz-Pro-Leu-Gly-Pro-D-Arg-OH Trifluoroacetate as a buffer solution as a solvent. The enzyme solution was used by diluting Clostridium histidicum-derived collagenase (Collagenase from clostridium histdyticum) to a concentration of 0.5 mg / ml using a buffer solution as a solvent. Add 500 μL of buffer, 250 μL of substrate, 150 μL of enzyme, 100 μL of sample, react for 30 min at 37 ℃, add 500 μL of 6% citric acid, shake 3 ml of Ethyl acetate violently, and absorb the supernatant at 320 nm. Measured at and calculated the result using the following equation (2).

[Equation 2]

Collagenase inhibitory effect (%) = (1-sample group absorbance / no group absorbance) × 00

Collagen (Collagen) is one of the major constituent proteins of the dermal dermal layer and is produced from fibroblasts and plays an important role in maintaining skin structure and elasticity. The skin causes a decrease in the production and degradation of collagen due to various factors including natural aging. Collagen breakdown by collagenase is an endogenous cause of skin sagging and wrinkles. . Therefore, the substances that inhibit collagenase have a large protective effect against aging, such as preventing wrinkles, and may improve skin elasticity.

The results of measuring the collagenase inhibitory activity of the blueberry leaf extract is shown in FIG. As can be seen in Figure 3, blueberry leaf extract showed a collagenase inhibitory effect in a concentration-dependent manner. At the concentration of 1000 μg / mL, the effect of 70% acetone extract was 94.62%, which was higher than that of hydrothermal extract (93.88%) and 70% ethanol extract (92.78%), indicating that 70% acetone extract was the most effective among the three extracts. This is especially higher than Epigallocatechin gallate (86.48%) used as a positive control, and blueberry leaf extract is a natural product, which can be very excellent for wrinkle improvement.

Example 5: Measurement of Cell Viability by MTT Analysis of Blueberry Leaf Extract

The CCD-986sk cells used in this experiment were subcultured by adapting to 37 ° C and 5% CO ₂ incubator using DMEM medium containing 10% FBS and 1% peniillin / streptomycin (100 unit / mL).

Cell line CCD-986sk was seeded to 1 x 10 ⁴ cells / well in a 96 well plate and incubated for 24 h in a 37 ° C., 5% CO ₂ incubator. Thereafter, the culture solution was exchanged with serum-free DMEM medium, and each sample solution was treated by concentration, followed by incubation for 24 h in a 37 ° C., 5% CO ₂ incubator. After that, MTT solution prepared at 5 mg / mL concentration was added at 0.02 mL / well, followed by incubation for 4 h, and then the culture medium was removed. Absorbance was measured at 540 nm, and the result was calculated using Equation 3 below.

[Equation 3]

Cell survival rate (%) = (Sample absorbance / no absorbance) × 100

The results for this are shown in FIG. 4. UV-B was treated in CCD986sk cell for 40 seconds and hot water, 70% ethanol and 70% acetone extracts were treated for 10 hours at 10, 25, 50, 100, 200, 400 and 800 μg / mL concentrations. It has been shown that it can be used up to this 50 μg / mL concentration. In the subsequent anti-wrinkle cell experiment, the highest concentration of the blueberry leaf extract that can be safely used in the cells was set to 50 μg / mL of hydrothermal extract.

Example 6: Determination of MMP-1, MMP-3, MMP-9, and Procollagen Expression Inhibitory Effect of Blueberry Leaf Extract

In order to confirm the expression of MMP-1, MMP-3, MMP-9 and procollagen proteins, CCD-986sk cells were dispensed into 6 well plates at 1 ㅧ 10 ⁵ cells / well for 24 hours, followed by UVB ( 20 mJ / cm ² ) was investigated and the blueberry leaf extract was treated by concentration to incubate for 48 hours. Supernatants obtained after lysis and centrifugation of cells in RIPA buffer were quantified by Bradford assay. The prepared protein sample was electrophoresed using 10% SDS-PAGE, transferred to PVDF membrane, blocked with tris buffer solution containing 5% skim milk for 1 hour, and then pro-collagen, MMP-1, MMP-3, Reacted with primary and secondary antibodies of MMP-9, β-actin, respectively. After the reaction, the reaction was performed using an Immobilon Western Chemiluminescent HRP substrate (Millipore, Billerica, MA, USA) for 5 minutes and then developed using Chemi-Doc. The results are shown in FIGS. 5 to 7. As can be seen from Figures 5 to 7, blueberry leaf extract was confirmed that significantly reduced the expression of MMP-1, MMP-3, MMP-9, Procollagen protein.

Claims (3)

A cosmetic composition for improving skin wrinkles comprising blueberry ( Vacniumnium ashei ) leaf extract as an active ingredient. The method of claim 1,
The skin wrinkle improvement is a cosmetic composition for improving skin wrinkles, characterized in that the relief, removal or prevention of wrinkles formed on the skin. The method of claim 1,
The blueberry leaf extract is a cosmetic composition for improving skin wrinkles, characterized in that 70% acetone extract of the blueberry leaf.

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