KR102159577B1 - A composition for improving skin wrinkle comprising Vaccinium ashei leaves extract - Google Patents

Abstract

The present invention relates to a composition for improving skin wrinkles comprising a blueberry ( Vaccinium ashei ) leaf extract as an active ingredient. The blueberry leaf extract contained as an active ingredient in the composition of the present invention promotes the synthesis of elastin and collagen, inhibits or promotes the expression of wrinkle-related proteins MMP-1, MMP-3, MMP-9, and procollagen proteins. It exhibits excellent anti-wrinkle effect through the molecular mechanism of, and has no cytotoxicity and excellent skin safety, so it can 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 a composition for improving wrinkles comprising a blueberry ( Vaccinium ashei ) leaf extract, and more specifically, a blueberry leaf extract having an effect of enhancing collagen synthesis, inhibiting collagen degradation, and inhibiting the expression of wrinkle-related proteins as an active ingredient. It relates to a cosmetic composition, a pharmaceutical composition, and a food composition for improving wrinkles, including as.

In the human body, aging inevitably occurs over time and is a phenomenon in which the structure and function of the body gradually deteriorate. Although this cannot be completely stopped, the progress of the process can be slowed through various modern technologies and applications, thereby improving the quality of life of members of the modern society.

Skin aging can be classified into extrinsic aging and intrinsic aging. Endogenous aging is aging that progresses due to the internal action of the human body, and its factor is the loss of telomere, a specific part 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, chronic wasting diseases, etc. . Exogenous aging is aging that is progressed by the surrounding environment, and the factors include climate, dust, chemicals, and ultraviolet rays (Kim Hyo-cheol, & Oh Kyung-dong. (2011). Research on causes, prevention, and treatment affecting skin aging) Jinjeon. Journal of Korean Society for Skin Beauty, 9(1), 1-9.). According to these factors, phenomena such as pigmentation, wrinkles, and inflammation appear on the skin and aging progresses.

On the other hand, modern people are increasingly interested in physical beauty and health, and recently, studies to prevent skin aging by using natural products that are more friendly to the skin have been actively conducted. Its contents include the elimination of oxidative stress in cells by free radicals that occur mainly in the metabolic process of the human body (Songhwa Hyun, Gayun Kim, Jiyeon Yoo, Jiwon Kim, Yerim Yang, Younghee Jeon, Sunam Park. (2016) ).Cytoprotective effects of calendula flower extract on antioxidant and oxidative stress in human skin cells Applied Chemistry for Engineering, 27(6), 620-626.), tyrosine is DOPA and DOPA quinone, indole- Inhibitory effect in the process of synthesizing melanin via 5,6-quinone (Choi, SW, & Kim, HJ (1998). Inhibitioin of tyrosinase activity by polyphenols of chestnut leaf. HSJAS, 6, 321-327.), It has the inhibitory effect of collagenase and elastase, substances that are involved in the decomposition of collagen and elastin, which are the major constituent proteins of the skin dermal layer produced from fibroblasts. , Do-Yeon Jang, Seok-Woo Nam, Yong Eo, & Kyung-Joo Lee. (2008) Inhibitory activity of Coenzyme Q10 derivatives against intracellular melanin production and Collagenase and Elastase. -170.).

Blueberry is a shrub plant belonging to the genus Vaccinium of the Ericaceae family, and is a low bush blueberry ( Vaccinium myritillus ) highbush blueberry ( Vaccinium corymbosum ), and rabbit eye blueberry ( Vaccinium ashei ) three cultivars dominate and are cultivated as commercially important fruits (Westwood, MN (1993). Hormones and growth regulators. Temparate Zone) Pomology: Physiology and Culture.Timber Press, Inc, 9999.). In recent years, interest and use of blueberries has rapidly increased 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 Korea Conference. Korea.), and thus, studies on the efficacy of blueberries have been conducted considerably (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, so related research is also relatively incomplete.In Korean Patent No. 1361785 and Korean Patent Publication No. 2011-0051547, blueberry leaf extract prevents degenerative neurological diseases or gout. It just discloses that it is useful.

Accordingly, the inventors of the present invention have been researching various methods for utilizing industrial by-products such as blueberry leaves, and found that blueberry leaves are very useful as food and cosmetic materials, and accordingly completed the present invention.

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

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

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

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

In the present invention, blueberries ( Vaccinium ashei ) is a fruit tree of the Ericaceae genus Vaccinium, and contains a large amount of vitamins K and C, and also contains a lot of manganese. Other nutrients such as vitamin E, thiamine, riboflavin, vitamin B6, and copper are evenly balanced, and anthocyanins, which exhibit the vivid color of blueberries, are also known to have the effect of neutralizing free radicals. In addition, blueberries are known as foods rich in nutrients because they are effective in protecting the retina of the eye as well as antioxidants.

In the present invention, "improving skin wrinkles" alleviates and removes wrinkles formed on the skin of animals, including humans, due to endogenous skin aging due to decreased hormone secretion or decreased physiological function, or exogenous skin aging caused by ultraviolet rays or pollution. Or means to prevent.

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

In addition, the blueberry leaf extract refers to all extracts, fractions, purified products obtained in each step of extraction, fractionation, or purification (separation, fractionation), their dilutions, concentrates, dried products, or any form formulated using.

As the extraction method of the blueberry leaf extract, cold needle extraction, ultrasonic extraction, reflux cooling extraction method, or supercritical extraction method are used.

In addition, the blueberry leaf extract may be extracted using a conventional solvent known in the art, and the extracted liquid may be used in a liquid form or concentrated and/or dried. At this time, as the solvent, water, anhydrous or aqueous 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 Any one of glycol, propylene glycol, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), or a mixed solvent thereof may be used. Preferably, water, an anhydrous or hydrated lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), and acetone are used. More preferably, water, ethanol, and acetone are used. However, it is not particularly limited thereto, and since the degree of extraction and loss of the active ingredient of the extract may vary depending on the organic solvent to be extracted, it is preferable to select and use an appropriate organic solvent.

As a specific example, the blueberry leaf extract may be obtained by pulverizing blueberry leaves, immersion extraction with a solvent, and then filtration after recovering the extract. The filtration is a process of removing suspended solid particles from the extract, and the particles may be filtered using cotton, nylon, or the like, or ultrafiltration, freezing filtration, centrifugation, etc. may be used, but is not limited thereto. In addition, a separation process by various chromatography (chromatography according to size, charge, hydrophobicity or affinity) may be additionally included.

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

In one specific embodiment, a 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 contains a blueberry leaf extract as an active ingredient in 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. I can. When the content is less than 0.00001% by weight, the effect of improving skin wrinkles cannot be expected, and when it exceeds 15.0% by weight, the difference in the effect of improving wrinkles is insignificant or there is difficulty in manufacturing the formulation.

The cosmetic composition may additionally include components commonly used in cosmetic compositions in addition to blueberry leaf extract. For example, it may contain conventional adjuvants and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments and perfumes.

The cosmetic composition of the present invention can be prepared in any formulation conventionally prepared in the art. For example, it can 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. More specifically, it may be prepared in a formulation such as a nourishing cream, an astringent lotion, a soft lotion, a lotion, an essence, a nourishing gel or a massage cream. However, it is not particularly limited thereto.

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

When the formulation of the cosmetic composition is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additional chlorofluorohydrocarbon, Propellants such as propane/butane or dimethyl ether.

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

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

When the formulation of the cosmetic composition is a surfactant containing cleansing, as a carrier component, 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 diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters, and the like may be used.

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

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

The pharmaceutical composition of the present invention comprises 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, as an active ingredient blueberry leaf extract. Can include. When the content is less than 0.00001% by weight, the effect of improving skin wrinkles cannot be expected, and when it exceeds 15.0% by weight, the difference in the effect of improving wrinkles is insignificant or there is difficulty in manufacturing the formulation.

The pharmaceutical composition of the present invention is prepared in unit dosage form or in a multi-dose form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person skilled in the art. It can be manufactured by incorporating into a container. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be in the form of an excir, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in the preparation of pharmaceutical compositions, and carbohydrate compounds (e.g., lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch) , Cellulose, etc.), gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, salt solution, alcohol, gum arabic, vegetable oil (e.g. corn Oil, cotton seed oil, soy milk, olive oil, coconut oil), polyethylene glycol, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto.

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

The pharmaceutical composition of the present invention can be formulated and used in the form of an injection or external preparation according to a conventional method.

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

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

The dosage of the pharmaceutical composition may vary depending on factors such as formulation method, administration method, age, weight, sex, and pathological condition of the patient, and the dosage is in the range of 0.001 to 300 mg/kg based on an adult. However, the dosage is not intended to limit the scope of the present invention.

The pharmaceutical composition of the present invention exhibits excellent skin wrinkle improvement effect through molecular mechanisms such as a fibroblast protective action and promotion of collagen synthesis, while also having low cytotoxicity.

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 skin wrinkles of an individual by intra-individual administration, subcutaneous administration, or topical administration by application.

In another specific embodiment, a 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, in the food composition according to another embodiment of the present invention, not only blueberry extract as an active ingredient, but also ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients, flavoring agents and flavoring agents It may further include.

Examples of the carbohydrate include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatin, stevia extract (eg, 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 as a drink, it may further include citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, cephalic extract, jujube extract, licorice extract, etc. in addition to the blueberry extract of the present invention. I can.

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

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

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 in an individual by intra-individual administration, subcutaneous administration, or topical administration by application.

In the present invention, the term "subject" refers to monkeys, cattle, horses, pigs, sheep, dogs, cats, rats, mice, including humans with conditions or diseases in which symptoms can be improved by administering the composition of the present invention, It means a mammal such as a chimpanzee.

In the present invention, the terms "treatment" and "improvement" mean destruction of inflammatory cells in an individual or inhibition of the development of inflammation (a), (b) alleviation, and (c) elimination.

In the present invention, the term "prevention" refers to any action that suppresses 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 a plant and is a safe material that is 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 contains blueberry leaf extract as an active ingredient, inhibits the decomposition and promotes synthesis of elastin and collagen in skin cells, inhibits MMP-1, inhibits MMP-3, and inhibits MMP-9. It exhibits excellent skin wrinkle improvement effects through detailed mechanisms such as inhibition and promotion of procollagen synthesis. In addition, the extract of the present invention is derived from natural materials and is a safe material that is harmless to the human body without toxicity and side effects as a result of skin irritation tests on the human body, and can be safely applied to cosmetics, pharmaceuticals, and food compositions.

1 is a diagram showing a manufacturing process of a blueberry leaf extract according to an embodiment of the present invention.
Figure 2 is a picture 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 human-derived fibroblasts CCD986sk by the blueberry leaf extract of the present invention.
5 is a diagram illustrating the effect of inhibiting the expression of MMP-1 protein by the hot water extract of blueberry leaves of the present invention in CCD986sk cells stimulated with UV-B.
Figure 6 is a picture analyzing the effect of inhibiting the expression of MMP-3 protein by the 70% ethanol extract of blueberry leaves of the present invention in CCD986sk cells stimulated with UV-B.
7 is a diagram illustrating the effect of inhibiting the expression of MMP-9 protein by 70% acetone extract of blueberry leaves of the present invention in CCD986sk cells stimulated with UV-B.

Hereinafter, examples, etc. will be described in detail to aid understanding of the present invention. However, the embodiments according to the present invention may be modified in various forms, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

All experiments were measured by repeating 3 times, and the results were expressed as mean value ± standard deviation. Statistical analysis was performed using SPSS program to analyze the variance to verify the significance (*p<0.05,**p<0.01) of each treatment section. After (analysis of variance, ANOVA), multiple comparisons were performed using the Turkish test.

Example 1: Preparation of blueberry leaf extract

The blueberry ( Vaccinium ashei ) leaves used in this experiment were supplied from the blueberry farm in Ulsan and used, and the extraction process of the blueberry leaves was performed as shown in FIG. 1. The hot water extract of blueberry leaves was 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 blueberry leaf and the acetone extract of blueberry leaf were extracted by adding 70% ethanol and 70% acetone at 20 times the weight of the dried sample, immersing for 24 hours at room temperature, and repeated 3 times. The yield of the hot water extract was 38.31%, the yield of the 70% ethanol extract was 21.78%, and the yield of the 70% acetone extract was 21.68%. The extracts were filtered with filter paper (No. 20 filter paper, Hyundai micro Co., Ltd., Korea), concentrated, freeze-dried, powdered, and stored in a refrigerator at 4°C, and used as samples for the following experiments.

Example 2: Reagent preparation

2-1: elastase and collagenase inhibitory effect test reagent

The reagents used in the experiment for measuring the wrinkle improvement effect are as follows. N-Succinyl-Ala-Ala-Ala-p-nitroanilide, Elastase from porcine pancreas, Collagenase from clostridium histdyticum, etc. are Sigma aldrich. Co., Ltd.; St. Louis, MO, USA) was used. 2-Amino-2-(hydroxymethyl)propane-1,3-diol, Hydrogen chloride, CaCl ₂ , Citric acid, Ethyl acetate, etc. are Duksan pure chemicals Co., Ltd. .; Korea) products were used. Pz-Pro-Leu-Gly-Pro-D-Arg-OH Trifluoroacetate was manufactured by 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, Fetal bovine serum (FBS), etc., were used by GE Healthcare (GE Healthcare life sciences hyclone laboratories; Logan, Utah, USA). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide), LPS (Lipopolysaccharides), Phosphatase inhibitor cocktail 3, Ripa buffer, Greece Reagents (Griess reagent (modified)), Protease inhibitor cocktail for use with mammalian cell and tissue extract, etc. were manufactured by Sigma (Sigma aldrich Co., Ltd.; St. Louis, MO, USA). . Prostaglandin E ₂ ELISA Kit was manufactured by R&D Systems (USA). Antibodies (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 others used a product of Santa cruz Co., Ltd.; USA. Acryl-bisacrylamide 30% stock solution (Acryl-bisacrylamide (29:1) 30% stock solution) was manufactured by m.biotech Co., Ltd.; Korea. Tris-base(2-Amino-2-(hydroxymethyl)propane-1,3-diol), glycine, 20X PBS buffer, sodium dodecyl sulfate (SDS), and TEMED are Biosesang Co., Ltd.; Korea) was used. Protein assay dye reagent concentrate was a product of Bio-rad laboratories Co., Ltd. (USA). pH 7 albumin-bovine was used by USB Co., Ltd. (Cleveland, OH, USA). PageRular™ prestained protein ladder, Western blot stripping buffer, Power SYBR green PCR master mix, NE-PER nuclear and cytoplasmic extraction reagents kit were manufactured by Thermo scientific Co., Ltd. (Lithuania). Tween 20 was manufactured by Daejung Chemicals & Metals Co., Ltd. (Korea). Nuclease-free water was used by Integrated DNA technologies Co., Ltd. (USA). Total RNA isolation regent-solution was a product of Bio science technology. The PCR primer was manufactured by Bionics Co., Ltd. (Korea). The transfer membrane was manufactured by Millipore Co., Ltd. (billerica, MA, USA). The 3mm CHR was manufactured by GE healthcare life sciences Co., Ltd. (China).

2-3. Instruments used in the experiment

The equipment used in this experiment was an ultraviolet/visible spectrophotometer (Hitachi, Tokyo, Japan), a polarization microscope (Olympus BX 51, Tokyo, Japan), and a 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 (Metrohm, Herisau Switzerland), BOD Incubator, Hanbaek Scientific Co., Bucheon, Korea ), an autoclave (Hanbaek Scientific Co., Bucheon, Korea), ELISA reader (Molecular Devices, California, USA) was used.

Example 3: Measurement of the elastase inhibitory effect of blueberry leaf extract

Elastase inhibitory effect was measured by 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.). 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 Tris(2-Amino-2-(hydroxymethyl)propane-1,3-diol) to a pH of 8.6. N-Succinyl-Ala-Ala-Ala-p-nitroanilide used as a substrate was diluted to a concentration of 3 mM using a 50 mM buffer solution as a solvent. 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 each buffer, enzyme, and sample solution to a 96 well plate, react at 37°C for 5 min, add 80 μL of the substrate solution, react for 15 min at 37°C, and measure the absorbance at 445 nm. The result was calculated using Equation 1.

[Equation 1]

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

Elastin is an important protein in maintaining skin elasticity in the dermis, and exists in the connective tissue of the skin and is largely involved in the elasticity and flexibility of the tissue. Elastase is an enzyme that decomposes elastin and is one of the causes of skin aging such as sagging and wrinkles in the skin (Bongjeon Ahn, Jintae Lee, & Chang-Eon Lee. (2009). New material for cosmetics, Paju, Gwangmungak, etc.) .p, 82.). Therefore, the elastase inhibitor acts to prevent aging through the improvement of skin wrinkles, and the results of measuring the elastase inhibitory activity effect 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 all extracts in a concentration-dependent manner. At 1000 μg/mL concentration, the effect of 70% acetone extract was 75.90%, which was higher than hot water extract (73.61%) and 70% ethanol extract (55.53%), showing that 70% acetone extract was the most effective among the three extracts. This is particularly higher than Epigallocatechin gallate (44.87%), which was used as a positive control, and blueberry leaf extract was considered to be a natural product and could provide excellent help in wrinkle improvement.

Example 4: Measurement of collagenase inhibitory effect of blueberry leaf extract

Collagenase inhibition effect was measured by Wunsch (Wnsch, E., & Heidrich, HG (1963). Zur quantitativen bestimmung der kollagenase. It was measured in accordance with the method of such as. Each sample was prepared by diluting at concentrations of 10, 100, and 1000 μg/mL, and EGCG (Epigallocatechin gallate) was used as a positive control. The buffer solution was used after adding hydrogen chloride to a 0.1 M Tris, 4 mM CaCl ₂ solution to a pH of 7.5. The substrate solution was diluted to a concentration of 0.3mg/ml using a buffer solution as a solvent in Pz-Pro-Leu-Gly-Pro-D-Arg-OH Trifluoroacetate. As for the enzyme solution, a buffer solution was used as a solvent, and collagenase from clostridium histdyticum was diluted to a concentration of 0.5 mg/ml. Put 500 μL of buffer, 250 μL of substrate, 150 μL of enzyme, and 100 μL of sample into a test tube, react at 37°C for 30 min, add 500 μL of 6% citric acid, add 3 ml of Ethyl acetate, shake vigorously, and adjust the absorbance of the supernatant to 320 nm. It was measured at and the result was calculated using Equation 2 below.

[Equation 2]

Collagenase inhibitory effect (%) = (1-absorbance in the sample added group/absorbance in the non-added group) × 00

Collagen is one of the major constituent proteins of the dermal layer of the skin. It is produced from fibroblasts and plays an important role in maintaining skin structure and elasticity. In the skin, collagen production decreases and decomposition increases due to various factors including natural aging. Collagen decomposition by collagenase is an intrinsic cause of skin sagging and wrinkles. . Therefore, substances that inhibit collagenase have a large protective effect against aging, such as preventing wrinkles, and can improve skin elasticity.

The results of measuring the collagenase inhibitory activity effect of blueberry leaf extract are shown in FIG. 3. As can be seen in Figure 3, the blueberry leaf extract showed a collagenase inhibitory effect in a concentration-dependent manner. At 1000 μg/mL concentration, the effect of 70% acetone extract was 94.62%, which was higher than that of hot water extract (93.88%) and 70% ethanol extract (92.78%), showing that 70% acetone extract was the most effective among the three extracts. This is particularly higher than Epigallocatechin gallate (86.48%) used as a positive control. Blueberry leaf extract is a natural product and is considered to be very helpful in improving wrinkles.

Example 5: Measurement of cell viability by MTT analysis of blueberry leaf extract

The CCD-986sk cell used in this experiment was subcultured in a DMEM medium supplemented with 10% FBS and 1% peniillin/streptomycin (100 unit/mL) at 37° C. and 5% CO ₂ incubator.

The cell line CCD-986sk was seeded into a 96 well plate so as to be 1 x 10 ⁴ cells/well and incubated in a 37°C, 5% CO ₂ incubator for 24 h. After that, the culture solution was exchanged with serum-free DMEM medium, and each sample solution was treated by concentration, followed by incubation in a 37°C, 5% CO ₂ incubator for 24 h. After that, the MTT solution prepared at a concentration of 5 mg/mL was added at 0.02 mL/well, incubated for 4 h, and then the culture medium was removed, and DMSO (dimethyl sulfoxide) was added at 0.1 mL/well to react at room temperature for 10 min. The absorbance was measured at 540 nm and the result was calculated using Equation 3 below.

[Equation 3]

Cell viability (%) = (absorbance in the sample added group/absorbance in the non-added group) × 100

The results for this are shown in FIG. 4. CCD986sk cells were treated with UV-B for 40 seconds and at the same time hot water, 70% ethanol, and 70% acetone extracts were treated at concentrations of 10, 25, 50, 100, 200, 400, 800 μg/mL, respectively, for 48 h. It has been shown to be usable up to a concentration of 50 μg/mL. In the subsequent anti-wrinkle cell experiment, the highest concentration of blueberry leaf extract that can be safely used for cells was set to 50 μg/mL of hot water extract.

Example 6: Measurement of the inhibitory effect of blueberry leaf extract on MMP-1, MMP-3, MMP-9, and Procollagen expression

To confirm the expression of MMP-1, MMP-3, MMP-9, and procollagen proteins, CCD-986sk cells were dispensed into 6 well plates at 1×10 ⁵ cells/well and incubated for 24 hours. 20 mJ/cm ² ) After irradiation, the blueberry leaf extract was treated by concentration and cultured for 48 hours. The cells were lysed in RIPA buffer, and the supernatant obtained after centrifugation was quantified by Brad-ford assay. The prepared protein sample was electrophoresed using 10% SDS-PAGE and transferred to the PVDF membrane. After blocking with a tris buffer solution containing 5% skim milk for 1 hour, pro-collagen, MMP-1, MMP-3, MMP-9 and β-actin were reacted with each of the primary and secondary antibodies. After the reaction, it was reacted for 5 minutes using an Immobilon Western Chemiluminescent HRP substrate (Millipore, Billerica, MA, USA), and then developed using Chemi-Doc. The results are shown in Figs. 5 to 7. As can be seen from FIGS. 5 to 7, it was confirmed that the blueberry leaf extract significantly reduced the expression of MMP-1, MMP-3, MMP-9, and Procollagen proteins.

Claims (3)

Blueberry ( Vaccinium ashei ) Cosmetic composition for improving skin wrinkles containing acetone extract as an active ingredient. The method of claim 1,
The skin wrinkle improvement cosmetic composition for improving skin wrinkles, characterized in that the relief, removal or prevention of wrinkles formed on the skin. delete

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