Cosmetic composition for wrinkle improvement containing extract of quercus acuta thunb. branches as active ingredient

Abstract

To provide a cosmetic composition, more specifically a cosmetic composition having wrinkle-improving activity and containing an extract of Quercus acuta THUNB. branches as an active ingredient.SOLUTION: The extract of Quercus acuta THUNB. branches according to the present invention includes, as useful components, a large amount of catechin and tannic acid effective for suppressing wrinkle formation, together with a strong sulfated substance. The extract of Quercus acuta THUNB. branches according to the present invention effectively inhibits collagenase, elastase, and hyaluronidase, and suppresses the expression of MMP-3 and MMP-9 genes associated with wrinkle formation, thereby producing an excellent wrinkle-improving effect. Accordingly, the composition of the invention containing the extract as an active constituent is useful as a functional cosmetic composition for wrinkle improvement. Since the extract of the invention is a natural material derived from branches of Quercus acuta THUNB., the cosmetic composition containing the same has the advantage of safe long-term use.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a cosmetic composition, and more particularly to a cosmetic composition having wrinkle-reducing activity and containing an extract of red oak branches as an active ingredient. [Background technology]

[0002] The skin is in direct contact with the external environment and has the function of protecting itself from temperature, humidity, antigens, ultraviolet rays, etc. However, physical and chemical stimuli such as external pollutants, ultraviolet rays, and stress can cause skin function to decline, and as we age, cell proliferation and immune activity weaken, preventing the rapid recovery of damaged skin cells and causing an overall deterioration of skin components, resulting in wrinkles, loss of elasticity, and keratinization.

[0003] Skin aging can be broadly divided into intrinsic and extrinsic aging. Intrinsic aging is a natural aging phenomenon characterized clinically by a loss of elasticity, rough skin texture, deep wrinkles, and pigmentation. Extrinsic aging is caused by external environmental factors such as ultraviolet rays, reactive oxygen species, and stress. Recently, rapid industrialization and the resulting air pollution and ozone layer destruction have increased UV levels, generating powerful oxidants such as reactive oxygen species, which can disrupt connective tissue formation such as skin collagen, inhibit cell membrane function, promote DNA mutations, alter protein function, and alter intracellular signaling molecules, resulting in wrinkles, pigmentation, and skin aging. To prevent this skin damage, cosmetics and skin protectants containing a variety of skin-protecting ingredients, such as retinol, retinoids, vitamin C, flavonoids, tocopherol, and coenzyme Q10, have been developed.

[0004] In particular, ultraviolet rays (UV) from sunlight are a well-known cause of aging. Prolonged exposure to UV rays causes the stratum corneum to thicken, resulting in the denaturation of collagen and elastin, the main components of skin, and a loss of skin elasticity. Collagen and elastin are regulated by various factors, but the expression of matrix metalloproteases, such as collagenase and elastase, causes the breakdown of collagen and elastin produced, resulting in a decrease in collagen content in the skin. Various substances have been developed and used to prevent this loss of collagen and elastin, which causes elasticity loss. Among these, retinol and retinoic acid have been shown to improve elasticity, and protein fractions extracted from leguminous seeds have also been used to increase elasticity. However, these retinoids have the disadvantage of causing skin irritation and allergies, even when applied in very small amounts.

[0005] Recently, in order to develop relatively less toxic and eco-friendly cosmetics, bioactive substances such as whitening, anti-aging, anti-wrinkle, antioxidant, and anti-inflammatory substances have been discovered through extraction processes and fermentation techniques using natural products, and these substances are being used as cosmetic ingredients.

[0006] Quercus acuta THUNB. is an evergreen broad-leaved tree of the Fagaceae family, commonly found in the sunny foothills and valleys of mountains at altitudes of 170-500m in Jeollanam-do, North and South Gyeongsang-do, and Jeju Island. It grows to a height of 20m and a diameter of over 60cm, with young branches densely covered in brown hairs. The leaves are alternate, oblong or oblong-ovate, 7-13cm long, and smooth-edged. The monoecious flowers bloom in May, and the 2cm-long fruit ripens in October of the following year. The name "Aka-oak" (red oak) is said to have come from the reddish color of its wood. Its heavy wood is not only durable and resistant to cracking, but also preserves well and has excellent leaf texture, making it useful for construction, furniture, and tools (especially for planes). Acorns grow in autumn and are edible. The tannins in these fruits have an astringent effect, stopping diarrhea and providing medicinal benefits for dyspnea, colitis, diarrhoea, breast pain, gingivitis, and stomatitis. However, there has not yet been much research into the physiological activities of red oak.

[0007] Therefore, the present inventors sought to find a natural substance that has excellent skin wrinkle-reducing effects without causing side effects to the body, and focused on red oak to test its pharmacological effects. As a result, they confirmed that red oak branch extract contains large amounts of catechin and tannic acid, which are effective in inhibiting wrinkle formation, as well as potent sulfated substances as useful components. In particular, they confirmed that a 100% ethanol extract of red oak branch effectively inhibits enzymes such as collagenase, elastase, and hyaluronidase, and suppresses the expression of MMP-3 and MMP-9 genes, which are related to wrinkle formation, thereby demonstrating excellent wrinkle-reducing effects, leading to the completion of the present invention. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Korean Patent Publication No. 10-2009-0118154 Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, an object of the present invention is to provide a cosmetic composition that is safe for the human body and yet effective in improving skin wrinkles. [Means for solving the problem]

[0010] In order to achieve the above object of the present invention,

[0011] The present invention provides a cosmetic composition for reducing wrinkles, which contains an extract of red oak branches as an active ingredient.

[0012] In one embodiment of the present invention, the extract may be extracted with one or more solvents selected from the group consisting of lower alcohols having 1 to 4 carbon atoms, ethyl acetate, acetone, water, and hexane.

[0013] In one embodiment of the present invention, the extract may be a 100% ethanol extract.

[0014] In one embodiment of the present invention, the extract may contain catechin, tannic acid, genistein, and quercetin.

[0015] In one embodiment of the present invention, the extract can inhibit wrinkle formation through its inhibitory activity against collagenase, elastase and hyaluronidase.

[0016] In one embodiment of the present invention, the extract can inhibit wrinkle formation by inhibiting the expression of MMP-3 and MMP-9 genes.

[0017] In one embodiment of the present invention, the extract may be contained in the composition at a concentration of 0.0001 to 2000 μg / ml.

[0018] In one embodiment of the present invention, the cosmetic composition may be in one form selected from the group consisting of softening lotion, gel, water-soluble liquid, milk lotion, nourishing cream, massage cream, essence, oil-in-water emulsion, water-in-oil emulsion, anhydrous paste product, anhydrous solid product, oil dispersion in an aqueous phase using microspheres, ionic lipid vesicles, non-ionic lipid vesicles, ointment, cleansing foam, cleansing water, pack, body oil, oil-in-water makeup base, water-in-oil makeup base, foundation, skin cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, mascara, blush, and eyebrow pencils. [Effects of the Invention]

[0019] The red oak branch extract of the present invention contains, as useful ingredients, potent sulfated substances as well as large amounts of catechin and tannic acid, which are effective in inhibiting wrinkle formation. Furthermore, the red oak branch extract of the present invention effectively inhibits collagenase, elastase, and hyaluronidase, and can derive excellent wrinkle-improving effects by suppressing the expression of MMP-3 and MMP-9 genes, which are involved in wrinkle formation. Therefore, the composition of the present invention containing the extract as an active ingredient can be useful as a functional cosmetic composition for wrinkle improvement. Furthermore, since the extract of the present invention is a natural material derived from red oak branches, the cosmetic composition of the present invention containing the extract has the advantage of being safe for long-term use. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 is a simplified production flow chart showing the extraction process of the red oak branch extract of the present invention (QAB1: hot water extract of red oak branch, QAB2: 70% ethanol extract of red oak branch, QAB3: 100% ethanol extract of red oak branch). [Figure 2]Figure 2 shows the DPPH free radical scavenging activity of extracts from various red oak species using different extraction solvents (QAB1: hot water extract of red oak branches, QAB2: 70% ethanol extract of red oak branches, QAB3: 100% ethanol extract of red oak branches). [Figure 3] Figure 3 shows the ABTS radical scavenging activity of extracts from various red oak species extracted with different extraction solvents (QAB1: hot water extract of red oak branches, QAB2: 70% ethanol extract of red oak branches, QAB3: 100% ethanol extract of red oak branches). [Figure 4] FIG. 4 is a graph showing the results of an MTT assay showing the cytotoxicity of various concentrations of a 100% ethanol extract of red oak branches. [Figure 5] FIG. 5 shows the results of RT-PCR measurement of MMP-3 and MMP-9 mRNA expression levels in HaCaT cells treated with various concentrations of 100% ethanol extract of red oak branches. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention is characterized by providing a cosmetic composition for reducing wrinkles, which contains an extract of red oak branches as an active ingredient.

[0022] The "red oak" (Quercus acuta Thunb.) mentioned in this specification belongs to the Fagaceae family and is also known as "Kasenam, Garangnip (Jeolla Province), Kasinang, Bukkasinang, Hongkasinang, and Borenang (Jeju Island)." It is found in sunny mountain foothills and valleys, reaching a height of 20 meters and a diameter of 60 cm. Its straight trunk, numerous branches, and lush foliage create a magnificent tree shape. As a temperate evergreen broad-leaved tree, it is a common species in the temperate zones of Korea and Japan. Red oak is the dominant tree in the temperate forests of the southern islands, including Jeju Island, and the southern coastal mountains. One theory is that the name "red oak" comes from the reddish color of its wood. Red oak fruit contains tannins that act as an astringent and help stop diarrhea. It is known to be effective in treating diarrhea, colitis, breast pain, gingivitis, and stomatitis.

[0023] However, the wrinkle-reducing activity of the extract from red oak branches has not been known until now.

[0024] In the present invention, it was confirmed for the first time that the extracts of red oak branches using different extraction solvents have excellent anti-oxidant and anti-wrinkle activity, and therefore can be used as ingredients for functional cosmetics.

[0025] The extract of red oak branches according to the present invention may be obtained by extraction and separation from natural sources using extraction and separation methods known in the art. The "extract" defined in the present invention is extracted from red oak branches using an appropriate solvent, and includes, for example, crude extracts, polar solvent-soluble extracts, and non-polar solvent-soluble extracts of red oak branches.

[0026] The solvent used to extract the extract from the red oak branches may be any pharmaceutically acceptable organic solvent, including, but not limited to, water or an organic solvent, such as purified water, C1-C4 alcohols including methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, and cyclohexane, which may be used alone or in combination. In consideration of safety and yield, ethanol is preferred, and 100% ethanol is more preferred.

[0027] The extraction method can be selected from hot water extraction, cold maceration extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, squeezing, etc. The desired extract may be further subjected to a conventional fractionation step or purified using a conventional purification method. There are no limitations on the method for producing the red oak extract of the present invention, and any known method can be used.

[0028] The extract of red oak branches according to the present invention can be obtained as a liquid by maceration at room temperature, heating and filtering in a conventional manner known in the art, or by further evaporating the solvent, spray drying or freeze drying.

[0029] For example, the extract of red oak branches contained in the composition of the present invention can be prepared in a powder form by subjecting the extract obtained as described above to additional processes such as vacuum distillation and freeze-drying or spray-drying, etc. Furthermore, the extract can be further purified to obtain fractions using various types of chromatography such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, etc.

[0030] Therefore, in the present invention, the extract of red oak branches is a concept that includes all extracts, fractions and purified products obtained at each stage of extraction, fractionation or purification, as well as their diluted solutions, concentrated solutions or dried products.

[0031] The composition of the present invention corresponds to a cosmetic composition for reducing wrinkles, which contains an extract of red oak branches as an active ingredient.

[0032] The cosmetic composition of the present invention can have anti-wrinkle activity in addition to antioxidant activity.

[0033] The extract of red oak branches of the present invention contains, as useful ingredients, the powerful sulfated substances genistein and quercetin, as well as large amounts of catechin and tannic acid, which are effective in inhibiting wrinkle formation.

[0034] The extract of red oak branches of the present invention can suppress wrinkle formation by inhibiting collagenase, elastase, and hyaluronidase, and by suppressing MMP-3 and MMP-9 gene expression, so the composition of the present invention containing this as an active ingredient can be useful as a functional cosmetic composition for improving wrinkles.

[0035] In one embodiment of the present invention, the extract of red oak branches may be contained in the cosmetic composition at a concentration of 0.0001 to 2000 μg / ml.

[0036] Examples of products to which the cosmetic composition of the present invention can be added include cosmetics such as astringent lotions, softening lotions, nourishing lotions, various creams, essences, packs, and foundations, as well as cleansers, facial washes, soaps, treatments, and beauty serums.

[0037] Specific dosage forms of the cosmetic composition of the present invention include one dosage form selected from the group consisting of softening lotion, gel, water-soluble liquid, milk lotion, nourishing cream, massage cream, essence, oil-in-water emulsion, water-in-oil emulsion, anhydrous paste product, anhydrous solid product, oil dispersion in an aqueous phase using microspheres, ionic lipid vesicles, non-ionic lipid vesicles, ointment, cleansing foam, cleansing water, pack, body oil, oil-in-water makeup base, water-in-oil makeup base, foundation, skin cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, mascara, cheek color, and eyebrow pencils.

[0038] According to a preferred embodiment of the present invention, the content of the active ingredient (oak branch extract) of the present invention is 0.00001 to 40 wt %, preferably 0.0005 to 40%, and more preferably 0.0005 to 20 wt %, based on the total weight of the composition. If the content of the active ingredient (oak branch extract) is less than 0.00001 wt %, the antioxidant effect and anti-wrinkle effect will be significantly reduced, and if it exceeds 20 wt %, it may cause skin irritation and problems with the dosage form.

[0039] Meanwhile, the cosmetic composition according to the present invention can be formulated by incorporating the active ingredient (extract of red oak branches) into nanoliposomes for stabilization. By incorporating the active ingredient (extract of red oak branches) into nanoliposomes, the active ingredient is stabilized, which can solve problems such as precipitation, discoloration, and odor during formulation, and can also increase the solubility and transdermal absorption rate of the ingredient, thereby maximizing the efficacy expected from the extract.

[0040] In the present invention, nanoliposomes refer to liposomes having the form of conventional liposomes and having an average particle size of 10 to 500 nm. According to a preferred embodiment of the present invention, the average particle size of nanoliposomes is 50 to 300 nm, and more preferably 100 to 200 nm. If the average particle size of nanoliposomes exceeds 500 nm, the technical effects of the present invention, such as improved skin penetration and improved formulation stability, are very weak.

[0041] The nanoliposomes used to stabilize the active ingredient (oak branch extract) according to the present invention can be prepared from a mixture containing polyol, oily component, surfactant, phospholipid, fatty acid and water.

[0042] The polyol used in the nanoliposome of the present invention is not particularly limited, and is preferably one or more selected from the group consisting of propylene glycol, dipropylene glycol, 1,3-butylene glycol, glycerin, methylpropanediol, isopropylene glycol, pentylene glycol, erythritol, xylitol, sorbitol, and mixtures thereof. The amount used is 10 to 80% by weight, preferably 30 to 70% by weight, of the total weight of the nanoliposome.

[0043] The oil component used in the preparation of the nanoliposomes of the present invention may be any of various oils known in the art, and preferably includes hydrocarbon oils such as hexadecane and paraffin oil, synthetic ester oils, silicone oils such as dimethicone and cyclomethicone, animal and vegetable oils such as sunflower oil, corn oil, soybean oil, avocado oil, sesame oil and fish oil, ethoxylated alkyl ether oils, propoxylated alkyl ether oils, sphingoid lipids such as phytosphingosine, sphingosine and sphinganine, cerebroside cholesterol, sitosterol cholesterol sulfate, sitosterol sulfate, C 10‐40 The fatty alcohol and its mixture may be used in an amount of 1.0 to 30.0% by weight, preferably 3.0 to 20.0% by weight, based on the total weight of the nanoliposome.

[0044] Any surfactant known in the art can be used in the preparation of the nanoliposomes of the present invention. For example, anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants can be used. Anionic surfactants and nonionic surfactants are preferred. Specific examples of anionic surfactants include alkyl acyl glutamates, alkyl phosphates, alkyl lactylates, dialkyl phosphates, and trialkyl phosphates. Specific examples of nonionic surfactants include alkoxylated alkyl ethers, alkoxylated alkyl esters, alkyl polyglycosides, polyglyceryl esters, and sugar esters. Polysorbates, which belong to the nonionic surfactant category, are particularly preferred. The amount of the surfactant used may be 0.1 to 10 wt %, preferably 0.5 to 5.0 wt %, based on the total weight of the nanoliposomes.

[0045] The phospholipid, an additional component used in producing the nanoliposomes of the present invention, is an amphipathic lipid, and includes natural phospholipids (e.g., egg yolk lecithin, soybean lecithin, sphingomyelin) and synthetic phospholipids (e.g., dipalmitoylphosphatidylcholine or hydrogenated lecithin), with lecithin being preferred. Naturally derived unsaturated lecithin or saturated lecithin extracted from soybean or egg yolk is particularly preferred. Typically, naturally derived lecithin contains 23 to 95% phosphatidylcholine and 20% or less phosphatidylethanolamine. In producing the nanoliposomes of the present invention, the amount of phospholipid used is 0.5 to 20.0 wt %, preferably 2.0 to 8.0 wt %, based on the total weight of the nanoliposomes.

[0046] The fatty acid used in the production of the nanoliposome of the present invention is a higher fatty acid, preferably C 12‐22 Examples of saturated or unsaturated alkyl chain fatty acids include lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, and linoleic acid, and the amount used may be 0.05 to 3.0% by weight, preferably 0.1 to 1.0% by weight, based on the total weight of the nanoliposome.

[0047] The water used in producing the nanoliposomes of the present invention is generally deionized distilled water, and the amount used may be 5.0 to 40% by weight based on the total weight of the nanoliposomes.

[0048] Nanoliposomes can be produced by various methods known in the art, but most preferably by passing a mixture containing the above ingredients through a high-pressure homogenizer. Nanoliposomes can be produced using a high-pressure homogenizer under various conditions (e.g., pressure, number of passes, etc.) depending on the desired particle size. Preferably, nanoliposomes are produced by passing the mixture through the high-pressure homogenizer 1 to 5 times under a pressure of 600 to 1200 bar.

[0049] The cosmetic composition of the present invention can be used alone or in combination with other cosmetic compositions other than the present invention. The cosmetic composition of the present invention, which has an excellent wrinkle-reducing effect, can be used according to a normal method of use, and the frequency of use can be varied depending on the skin condition or preference of the user.

[0050] The present invention will be described in more detail below with reference to examples. These examples are intended to explain the present invention in more detail, but the scope of the present invention is not limited to these examples.

[0051] Example 1

[0052] Manufacturing of oak branch extract

[0053] The red oak (Quercus acuta Thunb.) branches used in this experiment were collected at the Jeollanam-do Forest Resources Research Institute, washed, dried, and crushed before being used as a powder. Dried red oak branches were crushed and extracts were prepared using distilled water, 70% ethanol, and 100% ethanol.

[0054] <1-1> Hot water extract of red oak branches

[0055] 100g of branch powder was soaked in 1,000mL of distilled water and extracted in an autoclave at 100℃ for 10 minutes. The extracted extract was filtered through a Whatman No. 2 filter and concentrated in a rotary vacuum evaporator before being used as a sample. The yield was 13.69g, calculated using the following formula 1.

[0056] [Formula 1]

[0057] Yield = (weight of sample after drying (g) / weight of sample before extraction (g)) x 100

[0058] <1-2> 70% ethanol extract of red oak branches

[0059] 100g of red oak branch powder was placed in 1000mL of 70% ethanol and steeped for 7 days at room temperature. After steeping, the extract was filtered through a Whatman No. 2 filter and concentrated in a rotary vacuum evaporator before use. The yield was 14.57g, calculated using Equation 1.

[0060] <1-3> 100% ethanol extract of red oak branches

[0061] 100g of red oak branch powder was placed in 1000mL of 100% ethanol and steeped for 7 days at room temperature. After steeping, the extract was filtered through a Whatman No. 2 filter and concentrated in a rotary vacuum evaporator before use. The yield was 11.66g, calculated using Equation 1.

[0062] The process for preparing the oak twig extract of the present invention is shown in detail in Figure 1, and the yield of each extract is shown in detail in Table 1 below. The 70% ethanol extract QAB2 had the highest yield at 14.57%.

[0063] [Table 1]

[0064] <Experimental Example 1>

[0065] Evaluation of antioxidant activity of red oak branch extracts

[0066] In order to evaluate the antioxidant activity of the red oak extract of the present invention prepared in Example 1, the DPPH free radical scavenging ability and ABTS radical scavenging ability were examined.

[0067] <1-1> DPPH free radical scavenging ability

[0068] DPPH free radical scavenging is a widely used method for measuring the antioxidant activity of natural extracts. In this experiment, the radical scavenging ability of samples was measured using a modified version of Blois's method, which is one of the methods for measuring antioxidant activity. Specifically, 800 μL of 0.5 mM DPPH (2,2-diphenyl-1-picrylhydrazyl, Sigma, USA) dissolved in methanol and 200 μL of each sample (50–750 μg / mL) were placed in an EP tube, vortexed, and incubated in a dark room for 15 minutes. The absorbance was then measured at 517 nm using an HT multi-detection microplate reader. Gallic acid (Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control to measure the DPPH scavenging ability (IC) of red oak extracts. 50 ) is expressed as the concentration required to reduce the absorbance of the solvent-only control by 50%.

[0069] TIFF2025116806000003.tif28161

[0070] As a result, as shown in Table 2 below and Figure 2, the DPPH IC of the extract of red oak branches was 50The range was confirmed to be 250.34–340.70 μg / mL, and the 100% ethanol extract, QAB3, was IC 50 The value was 250.34 μg / mL, showing the best DPPH scavenging ability.

[0071] <1-2> ABTS radical scavenging ability

[0072] The ABTS radical scavenging activity has the advantage of being versatile, being able to measure the radical scavenging activity of both hydrophilic and hydrophobic samples. In this experiment, the method by Jeong et al. was modified. Specifically, a 7 mM ABTS solution dissolved in distilled water was mixed with 2.45 mM potassium persulfate in a 1:1 ratio and left in the dark for 12–16 hours. The radical stock solution was diluted with PBS (pH 7.4) to an absorbance value of 0.70 ± 0.02. 200 μL of each sample (50–750 μg / mL) was added to 800 μL of the diluted solution and incubated in the dark for 15 minutes. The absorbance was then measured at 517 nm using an HT multi-detection microplate reader (Synergy HT, BIO-TEX, Winooski, VT, USA). Quercetin (Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control, and the scavenging ability of the extract against ABTS (IC 50 ) is expressed as the concentration required to reduce the absorbance of the solvent-only control by 50%.

[0073] TIFF2025116806000004.tif27161

[0074] As a result, as shown in Table 2 below and Figure 2, the IC value of ABTS in the extract of red oak branches was 50 The range was confirmed to be 117.54–199.31 μg / mL, and the 100% ethanol extract, QAB3, was IC 50 The radical scavenging activity of each extract increased in a concentration-dependent manner.

[0075] [Table 2]

[0076] <Experimental Example 2>

[0077] Total polyphenol and total flavonoid contents of red oak branch extracts

[0078] <2-1> Total polyphenol content analysis

[0079] Polyphenols are aromatic alcohol compounds found in plants, with two or more phenolic groups per molecule. They have a variety of structures and molecular weights, and their hydroxyl groups allow them to easily bind to a variety of compounds, including water-soluble proteins. Polyphenols have a variety of physiological functions, such as anti-cancer, anti-inflammatory, and antioxidant properties, and their functions vary depending on the content and components. In this experiment, the total polyphenol content of red oak branch extracts was measured according to the extraction site and extraction solvent.

[0080] Total polyphenol content was analyzed using the Folin-Ciocalteu method (Ainsworth, EA, & Gillespie, KM, Nat. Protoc., 2(4), 875-877 (2007)). Briefly, a 500μg / mL concentration of red oak branch extract was prepared. 500μL of the prepared sample was mixed with 500μL of 0.2M Folin-Ciocalteu's phenol reagent and 500μL of 2% sodium carbonate aqueous solution (w / v), sequentially, and incubated in a dark room at room temperature for 30 minutes. Absorbance was then measured at 750nm using an HT multi-detection microplate reader. A calibration curve was prepared using various diluted gallic acid standards, and the mg / g equivalent of gallic acid (GAE) was calculated using the calibration curve.

[0081] As a result, as shown in Table 3, the 100% ethanol extract of red oak branches showed the highest content of 159.99±0.97 GAE mg / g.

[0082] <2-2> Total flavonoid content analysis

[0083] Flavonoids, which exist in various forms in plants, belong to the polyphenol group and have significant anti-inflammatory, anti-cancer, anti-obesity, and antioxidant effects. In this experiment, the flavonoid content of red oak extracts was measured according to the extraction site and extraction solvent.

[0084] For total flavonoid content analysis, a red oak extract was prepared at a concentration of 500 μg / mL. 500 μL of the prepared sample was mixed with 1.5 mL of methanol, 100 μL of 1M potassium acetate, and 1.4 mL of distilled water, in that order. After incubation at room temperature for 40 minutes, the absorbance was measured at 415 nm using an HT multi-detection microplate reader. A calibration curve was created using diluted quercetin at various concentrations as a standard, and the quercetin (QUE) mg / g equivalent was calculated.

[0085] As a result, as shown in Table 3, the 100% ethanol extract of red oak branches showed the highest content of 56.87±0.60 QUE mg / g.

[0086] [Table 3]

[0087] <Experimental Example 3>

[0088] Component analysis of red oak branch extracts using LC-MS / MS analysis

[0089] In order to quantitatively analyze the active ingredients contained in the extract of red oak twigs of the present invention prepared in Example 1, 57 active ingredients (allopurinol, apigenin, caffeic acid, catechin, p-coumaric acid, epicatechin, epicatechin gallate, ethyl gallate, ferulic acid, fumaric acid, gallic acid, genistein, gentisic acid, 4-hydroxybenzoic acid, ... Polyphenols, including catechuic acid, hyperoside, luteolin, naringenin, protocatechuic acid, pyrogallol, quercetin, riboflavin, salicylic acid, shikimic acid, syringic acid, tannic acid, taxifolin, ursolic acid, and vanillic acid, were analyzed. A 10 μL sample was injected and analyzed using a C18 column (Gemini 3 μm, C18 110A 50 mm x 2.0 mm) in a column oven at 40°C and an autosampler at 15°C. Analysis was performed in anion and cation modes using Turbo Ion Spray, and the mobile phases used were water (0.1% formic acid) (A) and acetonitrile (0.1% formic acid) (B).To improve analytical performance, mobile phase B was used as the reference and the mixing ratios of each mobile phase according to the analysis time were analyzed as follows: 0–2.0 min: B (5–40%), 2.0–3.0 min: B (40–80%), 3.0–5.0 min: B (80–80%), 5.0–5.1 min: B (80–5%), 5.1–8.0 min: B (5–5%).

[0090] As a result, as shown in Table 4, the type and content of polyphenols varied depending on the extraction solvent, with the 100% ethanol extract showing the highest content at 15,392 μg / g.

[0091] In particular, the 100% ethanol extract of red oak branches was found to have significantly higher catechin and tannic acid content among the polyphenol components than other extracts. Catechin is known to affect human skin cells, which are involved in the cause of aging, and to inhibit wrinkle formation while providing moisture and elasticity to the skin. Tannic acid has been reported to inhibit elastase activity in a concentration-dependent manner, and is known to be a substance that inhibits wrinkle formation.

[0092] Meanwhile, among the polyphenols, genistein and quercetin were found to be active ingredients only in the 100% ethanol extract of red oak branches. Genistein and quercetin are powerful sulfated substances known to protect the skin from oxidative stress caused by free radicals.

[0093] Therefore, it was determined that the 100% ethanol extract of the red oak species of the present invention, which not only contains high amounts of catechin and tannic acid, which are effective in inhibiting wrinkle formation, but also contains the powerful sulfated substances genistein and quercetin as active ingredients, can be useful as a material with functions such as wrinkle improvement, skin moisturizing, and whitening.

[0094] [Table 4]

[0095] <Experimental Example 4>

[0096] Cytotoxicity evaluation of red oak branch extracts

[0097] To evaluate the cytotoxicity of the extract of red oak branches of the present invention prepared according to Example 1, an MTT assay was carried out.

[0098] In the MTT assay, the reagent MTT [3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide] is absorbed into cells and then forms formazan by mitochondrial succinate dehydrogenase. The intracellular accumulation of this substance is used as an indicator of mitochondrial activity, or more broadly, cellular activity, and is a typical method for measuring cell growth rate.

[0099] HaCaT cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 1% penicillin (Sigma-Aldrich, St. Louis, MO, USA) and 10% fetal bovine serum (FBS, Gibco BRL, USA) in an animal cell culture vessel (Sanyo CO2 Incubator, MCO-18AC, SANYO Electric Co., Ltd., OSA, JP) at 5% CO2 and 37°C. Experiments were performed after the cells had grown to approximately 90% of the floor area.

[0100] Cell suspension was added to 12 wells at 1×10 5The cells were cultured in 1 mL aliquots at a concentration of 100 cells / mL and allowed to stabilize in an incubator for 24 hours. The cells were then treated with various concentrations of red oak branch extract and cultured for 24 hours. According to Choi's method, 1 mL of 0.2 mg / mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, MO, USA) solution was added to each well and incubated for 30 minutes in an incubator. The supernatant was then removed, and 300 μL of dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) was added to each well. The cells were then shaken for 1 minute to completely dissolve the formazan. Cell viability was calculated by measuring absorbance at 540 nm using an ELISA Bio-Tek instrument Inc. (Winooski, VT, USA).

[0101] As a result, as shown in FIG. 4, it was confirmed that the 100% ethanol extract of red oak branches of the present invention did not exhibit cytotoxicity over the entire concentration range of 25 to 100 μg / mL.

[0102] <Experimental Example 5>

[0103] Wrinkle-improving effect of red oak branch extract

[0104] To evaluate the wrinkle-reducing effect of the 100% ethanol extract of red oak branches prepared in Example 1, collagenase inhibitory activity, elastase inhibitory activity, and hyaluronidase inhibitory activity were measured. In addition, the effect on the expression of wrinkle-related genes (MMP-3, mMP-9) was confirmed.

[0105] For reference, the wrinkle improvement effect is based on the actions of collagenase, which breaks down collagen, gelatinase, which breaks down gelatin, and elastase, which breaks down elastic fibers. Collagen is synthesized by fibroblasts in the skin and is known to provide mechanical strength to the skin, the resistance and cohesion of connective tissue, support cell adhesion, and induce cell division and differentiation. In addition, hyaluronidase is an enzyme that breaks down hyaluronic acid, and it functions to protect cells in the skin, moisturize them, and maintain their elasticity. Therefore, a decrease in hyaluronic acid content causes the skin to become dry and rough.

[0106] <5-1> Collagenase inhibitory activity

[0107] 0.5 mL of substrate solution (4-phenylazobenzyloxycarbinyl-Pro-Leu-Gly-Pro-DArg (0.3 mg / mL) in 0.1 M Tris-HCl buffer (pH 7.5) supplemented with 4 mM CaCl2) and 0.2 mL of 100% ethanol extracts of red oak branches prepared at different concentrations were mixed with 0.3 mL of collagenase type 1 enzyme solution (prepared at 200 units / mL). The solution was incubated at room temperature for 20 minutes, then 0.5 mL of 5% citric acid was added to terminate the reaction. 1 mL of ethyl acetate was added, and the absorbance was measured at 320 nm. Collagenase inhibitory activity was expressed as the percentage decrease in absorbance between the sample solution-treated and untreated groups.

[0108] TIFF2025116806000008.tif16161

[0109] As a result, as shown in Table 5, the collagenase inhibitory activity of the 100% ethanol extract of red oak branches was IC 50 The IC value was 390.40±7.41μg / mL, indicating excellent collagenase inhibitory activity. On the other hand, the IC value of oleic acid used as a positive control was 390.40±7.41μg / mL. 50 The value was measured as 89.52 ± 2.85 μg / mL.

[0110] <5-2> Elastase inhibitory activity

[0111] To 500 μL of 100% ethanol extract of red oak branches, 250 μL of pancreatic solution (Type I, 0.6 unit / mL) was added, followed by 500 μL of N-succinyl-(LAla)3-p-nitroanilide (1 mg / mL) dissolved in 50 mM Tris-HCl buffer (pH 8.6) as the substrate. The solution was incubated at 37°C for 30 minutes, and the absorbance was measured at 410 nm. The elastase inhibitory activity was expressed as the rate of decrease in absorbance between the group with and without the sample solution.

[0112] TIFF2025116806000009.tif15161

[0113] As a result, as shown in Table 5, the elastase inhibitory activity of the 100% ethanol extract of red oak branches was IC 50 The IC value was 309.44±11.91μg / mL, indicating excellent elastase inhibitory activity. On the other hand, the IC value of oleic acid used as a positive control was 309.44±11.91μg / mL. 50 The value was measured as 89.56±3.07 μg / mL.

[0114] <5-3> Hyaluronidase inhibitory activity

[0115] Six microliters of hyaluronidase (10 mg / mL) dissolved in 0.1 M acetate buffer (pH 3.5) was mixed with 6 μL of 100% ethanol extract of red oak twigs and incubated in a 37°C water bath for 20 minutes. To activate the hyaluronidase, 26 μL of 12.5 mM CaCl was added to the mixture, which was then incubated in a 37°C water bath for 20 minutes. After the reaction was complete, 12 μL of a substrate solution (6 mg / mL hyaluronate) dissolved in 0.1 M acetate buffer (pH 3.5) was added to the reaction mixture, which was then incubated in a 37°C water bath for 40 minutes. To terminate the substrate-enzyme reaction, 6 μL of 0.4 N NaOH and 6 μL of 0.4 M potassium tetraborate were added to the reaction mixture, which was then incubated at 100°C for 3 minutes and then cooled completely to room temperature. After adding 180 μL of DMAB solution (a coloring agent) to the substrate-enzyme mixture, the absorbance was measured at 540 nm using an ELISA reader. Hyaluronidase inhibitory activity was expressed as the percentage decrease in absorbance between the sample solution-added group and the control group.

[0116] TIFF2025116806000010.tif16161

[0117] As a result, as shown in Table 5, the hyaluronidase inhibitory activity of the 100% ethanol extract of red oak branches was IC 50 The IC value was 307.98±3.16μg / mL, indicating excellent elastase inhibitory activity. On the other hand, the IC value of oleic acid used as a positive control was 307.98±3.16μg / mL. 50 The value was measured as 70.21±0.43 μg / mL.

[0118] [Table 5]

[0119] <5-4>Effects on the expression of wrinkle formation-related genes (MMP-3, mMP-9)

[0120] In this experiment, HaCaT cells were simultaneously treated with 100% ethanol extract of red oak branches at different concentrations (25, 50, 100 μg / mL) and 1% H2O2, and the induced mRNA expression levels of MMP-3 and MMP-9 were analyzed by RT-PCR.

[0121] For reference, matrix metalloproteinases (MMPs) are broadly classified into MMP-1, mMP-2, MMP-3, and mMP-9 based on their substrate specificity. MMP-3, as stromelysin-1, breaks down proteoglycans, elastin, fibronectin, laminin, etc. in mouse skin, activating various MMPs and playing an important role in wrinkle formation, while MMP-9, as a gelatinase, has been reported to break down type 1 collagen, a major component of the basement membrane.

[0122] For RT-PCR analysis, total RNA extracts were first obtained using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the company's protocol. To synthesize cDNA, 2 μg of total RNA was reverse transcribed using oligo-dT18 primers. The synthesized cDNA was amplified using a High-Capacity cDNA Synthesis Kit (Bioneer, Daejeon, Korea) equipped with a Thermal Cycler (Bio-Rad, Hercules, CA, USA). PCR amplification products were separated by 2% agarose gel electrophoresis and stained with ethidium bromide (Sigma, St. Louis) and imaged using a gel documentation system (Fujifilm). The PCR primer sequences used were as follows: Human MMP3 sense 5'-AACCTGTCCCTCCAGAACCT-3' and antisense 5'-GGAAGAGATGGCCAAAATGA-3'; human MMP9 sense 5'-CTCGAACTTTGACAGCGACA-3' and antisense 5'-GCCATTCACGTCGTCCTTAT-3'; human GAPDH sense 5'-GAAGGTGAAGGTCGGAGTC-3' and antisense 5'-GAAGATGGTGATGGGATTTC-3'. GAPDH was used as a reference gene for normalization.

[0123] As a result, as shown in FIG. 5, it was confirmed that the mRNA expression of MMP-3 and MMP-9 was inhibited in a treatment concentration-dependent manner with the extract of red oak branches of the present invention.

[0124] From the above results, it was confirmed that the 100% ethanol extract of red oak branches of the present invention is an effective substance for improving wrinkles.

[0125] Dosage example 1: Softening lotion (skin lotion)

[0126] As shown in the table below, softening lotions were prepared in a conventional manner.

[0127] [Table 6]

[0128] Dosage form example 2: Nutritious lotion (milk lotion)

[0129] Nutritious lotions were prepared in the usual manner as shown in the table below.

[0130] [Table 7]

[0131] Dosage form example 3: Nutritious cream

[0132] Nutritious creams were prepared in the usual manner as shown in the table below.

[0133] [Table 8]

[0134] Dosage form example 4: Massage cream

[0135] Massage creams were prepared in the usual manner as shown in the table below.

[0136] [Table 9]

[0137] Dosage form example 5: Pack

[0138] Packs were prepared in the usual manner as shown in the table below.

[0139] [Table 10]

[0140] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will recognize that the present invention can be embodied in various modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims, rather than the foregoing description, and all variations within the scope of the claims are deemed to be within the scope of the present invention.

Claims

1. A cosmetic composition for improving wrinkles, comprising an extract of red oak branches as an active ingredient.

2. 2. The wrinkle-improving cosmetic composition according to claim 1, wherein the extract is extracted with one or more solvents selected from the group consisting of lower alcohols having 1 to 4 carbon atoms, ethyl acetate, acetone, water, and hexane.

3. The wrinkle-improving cosmetic composition according to claim 1, wherein the extract is a 100% ethanol extract.

4. 2. The wrinkle-improving cosmetic composition according to claim 1, wherein the extract comprises catechin, tannic acid, genistein, and quercetin.

5. The wrinkle-improving cosmetic composition according to claim 1, wherein the extract inhibits wrinkle formation by inhibiting collagenase, elastase, and hyaluronidase.

6. The wrinkle-improving cosmetic composition according to claim 1, wherein the extract inhibits wrinkle formation by suppressing MMP-3 and MMP-9 gene expression.

7. 2. The wrinkle-improving cosmetic composition according to claim 1, wherein the extract is contained in the composition at a concentration of 0.0001 to 2000 μg / ml.

8. 8. The wrinkle-improving cosmetic composition according to claim 1, wherein the cosmetic composition is in one dosage form selected from the group consisting of an emollient lotion, a gel, a water-soluble liquid, a milk lotion, a nourishing cream, a massage cream, an essence, an oil-in-water emulsion, a water-in-oil emulsion, an anhydrous paste product, an anhydrous solid product, an oil dispersion in an aqueous phase using microspheres, an ionic lipid vesicle, a non-ionic lipid vesicle, an ointment, a cleansing foam, a cleansing water, a pack, a body oil, an oil-in-water makeup base, a water-in-oil makeup base, a foundation, a skin cover, a lipstick, a lip gloss, a face powder, a two-way cake, an eye shadow, a mascara, a blush, and an eyebrow pencil.

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