KR102845814B1 - Cosmetic composition for improving wrinkles containing branch extract of Quercus acuta Thunb as an active ingredient - Google Patents

Abstract

The present invention relates to a cosmetic composition, and more particularly, to a cosmetic composition having an anti-wrinkle activity, comprising an extract of a branch of Quercus amurense as an active ingredient. The extract of the branch of Quercus amurense of the present invention contains a large amount of catechin and tannic acid, which are effective in inhibiting wrinkle formation, in addition to a powerful antioxidant as useful ingredients. In addition, the extract of the branch of Quercus amurense of the present invention can effectively inhibit collagenase, elastase, and hyaluronidase, and can induce an excellent anti-wrinkle effect by inhibiting the expression of MMP-3 and MMP-9 genes related to wrinkle formation. Therefore, the composition of the present invention comprising the extract as an active ingredient can be usefully used as a functional cosmetic composition for improving wrinkles. In addition, the extract of the present invention is a natural material derived from Quercus amurense of the branch, and the cosmetic composition of the present invention comprising the extract has the advantage of being safe even for long-term use.

Description

Cosmetic composition for improving wrinkles containing branch extract of Quercus acuta Thunb as an active ingredient

The present invention relates to a cosmetic composition, and more particularly, to a cosmetic composition having wrinkle-improving activity, which comprises an extract of a red oak branch as an active ingredient.

The skin, in direct contact with the external environment, functions to protect against temperature, humidity, antigens, and UV rays. However, physical and chemical stimuli such as external pollutants, UV rays, and stress can deteriorate skin functions. As we age, cell proliferation and immune activity weaken, preventing skin cells from quickly repairing damage and leading to an overall deterioration of skin composition, resulting in wrinkles, loss of elasticity, and keratinization.

Skin aging is broadly divided into intrinsic and extrinsic aging. Intrinsic aging is a natural phenomenon that is clinically characterized by decreased 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 led to increased UV exposure. This increases the generation of powerful oxidizing substances such as reactive oxygen species. These substances can destroy connective tissues such as collagen, impair cell membrane function, promote DNA mutations, alter protein function, and alter intracellular signaling molecules. This can lead to wrinkles, pigmentation, and other skin aging effects. To prevent this damage, cosmetics and skin protectants containing various skin-protecting ingredients, such as retinol, retinoids, vitamin C, flavonoids, tocopherols, and coenzyme Q10, are being developed.

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, and collagen and elastin, the main components of the skin, to degenerate, resulting in a loss of elasticity. Collagen and elastin are regulated by various factors. The expression of matrix metalloproteinases, such as collagenase and elastase, degrades the collagen and elastin produced, ultimately resulting in a decrease in collagen content within the skin. Various substances have been developed and used to suppress the decline in collagen and elastin, which are the cause of this loss of elasticity. Among these, retinol and retinoic acid have been shown to improve elasticity, and protein fractions derived from Leguminosae seeds have also been used to enhance elasticity. However, these retinoids have the disadvantage of causing skin irritation and allergies even when applied in very small amounts.

Recently, in order to develop cosmetics that are relatively less toxic and eco-friendly, natural substances have been used for whitening, anti-aging, anti-wrinkle, anti-oxidation, and anti-inflammatory properties through extraction processes and fermentation technologies, and these have been used as cosmetic materials.

Red oak ( Quercus acuta THUNB.) is an evergreen broad-leaved tree of the genus Quercus in the oak family, growing abundantly in sunny mountain slopes and valleys at elevations of 170–500 m in South Jeolla Province, South Gyeongsang Province, and Jeju Island. It grows to a height of 20 m and a diameter of over 60 cm, and young branches are covered with dense brown hairs. The leaves are alternate, oblong or oblong-ovate, 7–13 cm long, and have smooth margins. The flowers are monoecious and bloom in May, and the fruit is 2 cm long and ripens in October of the following year. The name "red oak" comes from the red color of its wood. The wood is heavy, does not split easily, and preserves well. The texture of the leaves is superior to that of other woods, so it is used as a building material, furniture material, and tool material (especially as a material for planes). In autumn, acorns ripen and are edible. Their tannins have astringent and anti-diarrheal properties, making them effective in treating dysentery, colitis, diarrhea, milk sores, gingivitis, and stomatitis. However, little research has been conducted on the physiological activities of the red acorn tree.

Accordingly, the inventors of the present invention, while searching for a natural substance that has excellent wrinkle improvement effect without side effects in the body, focused on the red twig tree and tested its pharmacological effect. As a result, it was confirmed that the red twig branch extract contains a large amount of catechin and tannic acid, which are effective in inhibiting wrinkle formation, as well as a powerful antioxidant as a useful ingredient. In particular, the 100% ethanol extract of the red twig tree effectively inhibits enzymes such as collagenase, elastase, and hyaluronidase, and has an excellent wrinkle improvement effect by suppressing the expression of MMP-3 and MMP-9 genes related to wrinkle formation, thereby completing the present invention.

Korean Patent Publication No. 10-2009-0118154

Therefore, the purpose of the present invention is to provide a cosmetic composition that is safe for the human body and effective in improving skin wrinkles.

In order to achieve the above purpose of the present invention,

The present invention provides a cosmetic composition for improving wrinkles containing an extract of a red oak branch as an active ingredient.

In one embodiment of the present invention, the extract can 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.

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

In one embodiment of the present invention, the extract may include catechin, tannic acid, genistein, and quercetin.

In one embodiment of the present invention, the extract can suppress wrinkle formation through the inhibitory activity of collagenase, elastase, and hyaluronidase.

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

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

In one embodiment of the present invention, the cosmetic composition may be one formulation selected from the group consisting of a flexible toner, a gel, a water-soluble liquid, a milk lotion, a nourishing cream, a massage cream, an essence, an oil-in-water type emulsion, a water-in-oil type emulsion, an anhydrous product for face, an anhydrous solid product, an oil dispersion in an aqueous phase using spherules, 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 type makeup base, a water-in-oil type 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 cheek color, and an eyebrow pencil.

The red oak branch extract of the present invention contains a large amount of catechin and tannic acid, which are effective in inhibiting wrinkle formation, as well as powerful antioxidants as useful ingredients. In addition, the red oak branch extract of the present invention can effectively inhibit collagenase, elastase, and hyaluronidase, and can induce an excellent wrinkle improvement effect by inhibiting the expression of MMP-3 and MMP-9 genes related to wrinkle formation, and therefore, the composition of the present invention containing the extract as an effective ingredient can be usefully used as a functional cosmetic composition for improving wrinkles. In addition, the extract of the present invention is a natural material derived from red oak branches, and the cosmetic composition of the present invention containing it has the advantage of being safe even for long-term use.

Figure 1 is a manufacturing process diagram briefly showing the extraction process of the red thorn branch extract of the present invention (QAB1: hot water extract of red thorn branch, QAB2: 70% ethanol extract of red thorn branch, QAB3: 100% ethanol extract of red thorn branch).
Figure 2 is a graph showing the DPPH free radical scavenging activity of red thorn branch extracts using different extraction solvents (QAB1: hot water extract of red thorn branch, QAB2: 70% ethanol extract of red thorn branch, QAB3: 100% ethanol extract of red thorn branch).
Figure 3 is a graph showing the ABTS radical scavenging activity of red thorn branch extracts using different extraction solvents (QAB1: hot water extract of red thorn branch, QAB2: 70% ethanol extract of red thorn branch, QAB3: 100% ethanol extract of red thorn branch).
Figure 4 is a graph showing the results of an MTT assay showing the cytotoxicity of 100% ethanol extracts of red oak branches at different concentrations.
Figure 5 shows the results of RT-PCR measurement of MMP-3 and MMP-9 mRNA expression levels according to concentration-specific treatment with 100% ethanol extract of red oak branches in HaCaT cells.

The present invention is characterized in that it provides a cosmetic composition for improving wrinkles containing an extract of a red oak branch as an active ingredient.

The red thorn tree ( Quercus acuta Thunb.) mentioned in this specification belongs to the oak family and is also called gasaenamu, garangnip (Jeolla-do), gasinang, bukgasinang, honggasinang, and beorenanang (Jeju-do). It grows on sunny mountainsides and valleys, reaching a height of 20 m and a diameter of 60 cm. The trunk grows straight, with many branches and lush leaves, forming a magnificent tree. It is a temperate evergreen broad-leaved tree and is a common species in the temperate zones of Korea and Japan. Quercus acuta is a major tree that formed temperate forests in southern islands, including Jeju Island, and in the southern coastal mountains. The name "Quercus acuta" (Quercus acuta) comes from the red color of its wood. The tannin in the fruit of Quercus acuta has an astringent effect and is known to have a diarrhea-stopping effect, and is known to be effective in treating dysentery, colitis, diarrhea, milk fever, gingivitis, and stomatitis.

However, the wrinkle improvement activity of red thorn tree branch extract is currently unknown.

In the present invention, it was confirmed for the first time that red oak branch extracts obtained by using different extraction solvents have excellent antioxidant functionality and wrinkle improvement activity, and thus can be used as a material for functional cosmetics.

The red oak branch extract according to the present invention can be obtained by extracting and separating from nature using extraction and separation methods known in the art, and the ‘extract’ defined in the present invention is extracted from the branch of the red oak using an appropriate solvent, and includes, for example, a crude extract, a polar solvent-soluble extract, or a non-polar solvent-soluble extract of the branch of the red oak.

Any pharmaceutically acceptable organic solvent may be used as a suitable solvent for extracting the extract from the branches of the above-mentioned red thorn tree, and water or an organic solvent may be used, and is not limited thereto, for example, purified water, alcohols having 1 to 4 carbon atoms including methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, and cyclohexane, and various solvents may be used alone or in mixtures. Considering safety and yield, etc., ethanol (alcohol) is preferably used, and more preferably, 100% ethanol is good to use.

Any one of the following extraction methods may be selected and used: hot water extraction, cold immersion extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, dissolution, and pressing. Furthermore, the desired extract may be further subjected to a conventional fractionation process, or purified using a conventional purification method. There are no limitations on the method for producing the red oak branch extract of the present invention, and any known method may be used.

The red oak branch extract according to the present invention can be obtained in liquid form by cooling, heating and filtering at room temperature using conventional methods known in the art, or the solvent can be further evaporated, spray-dried or freeze-dried.

For example, the red oak branch extract included in the composition of the present invention can be manufactured into a powder form by additional processes such as reduced pressure distillation and freeze drying or spray drying of the extracted extract as described above. In addition, the extract can be further purified to obtain a fraction using various chromatography methods such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, etc.

Therefore, in the present invention, the red oak branch extract is a concept that includes all extracts, fractions and purified products obtained at each stage of extraction, fractionation or purification, as well as their dilutions, concentrates or dried products.

The composition of the present invention corresponds to a cosmetic composition for improving wrinkles containing an extract of red oak branches as an active ingredient.

The cosmetic composition of the present invention may have an activity of inhibiting wrinkle formation in addition to antioxidant activity.

The red oak branch extract of the present invention contains a large amount of catechin and tannic acid, which are effective in inhibiting wrinkle formation, as well as genistein and quercetin, which are powerful antioxidants.

The red oak branch extract of the present invention can suppress wrinkle formation through the inhibitory activity of collagenase, elastase and hyaluronidase and the suppression of MMP-3 and MMP-9 gene expression, and therefore, the composition of the present invention containing the extract as an active ingredient can be usefully used as a functional cosmetic composition for improving wrinkles.

In one specific example of the present invention, the red oak branch extract may be included in the cosmetic composition at a concentration of 0.0001 to 2000 μg/ml.

Products to which the cosmetic composition of the present invention can be added include, for example, cosmetics such as astringent toners, emollient toners, nourishing toners, various creams, essences, packs, foundations, and cleansing products, facial cleansers, soaps, treatments, and beauty solutions.

Specific formulations of the cosmetic composition of the present invention include one formulation selected from the group consisting of a flexible toner, 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 product for face, an anhydrous solid product, an oil dispersion in an aqueous phase using spherules, an ionic lipid vesicle, a nonionic 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 cheek color, and an eyebrow pencil.

According to a preferred embodiment of the present invention, the content of the active ingredient (extract of red thorn branch) of the present invention is 0.00001-40 wt%, preferably 0.0005-40 wt%, and more preferably 0.0005-20 wt%, based on the total weight of the composition. If the content of the active ingredient (extract of red thorn branch) is less than 0.00001 wt%, the antioxidant effect and wrinkle improvement effect are significantly reduced, and if it exceeds 20 wt%, it may cause skin irritation and problems in the formulation may arise.

Meanwhile, the cosmetic composition according to the present invention can be formulated by stabilizing the active ingredient (extract of red thorn tree branches) by including it within nanoliposomes. By including the active ingredient (extract of red thorn tree branches) within nanoliposomes, the active ingredient is stabilized, thereby resolving problems such as precipitation, discoloration, and odor change during formulation. In addition, the solubility and percutaneous absorption rate of the ingredient can be increased, thereby maximizing the efficacy expected from the extract.

In the present invention, "nanoliposome" refers to a liposome having the form of a conventional liposome and having an average particle diameter of 10 to 500 nm. According to a preferred embodiment of the present invention, the average particle diameter of the nanoliposome is 50 to 300 nm, and more preferably 100 to 200 nm. When the average particle diameter of the nanoliposome exceeds 500 nm, the technical effects sought by the present invention, such as improved skin penetration and improved formulation stability, are very minimal.

Nanoliposomes used to stabilize the active ingredient (red oak branch extract) according to the present invention can be prepared by a mixture containing a polyol, an oily component, a surfactant, a phospholipid, a fatty acid, and water.

The polyol used in the nanoliposome of the present invention is not particularly limited, and is preferably at least one 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 wt%, preferably 30 to 70 wt%, based on the total weight of the nanoliposome.

The oil component used in the production of the nanoliposome of the present invention may be 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, sphingonoid lipids such as phytosphingosine, sphingosine and sphinganine, cerebroside cholesterol, sitosterol cholesteryl sulfate, sitosteryl sulfate, C _10-40 fatty alcohols and mixtures thereof. The amount used may be 1.0 to _30.0 wt% based on the total weight of the nanoliposome, and preferably 3.0 to 20.0 wt%.

Any surfactant known in the art may be used in the preparation of the nanoliposomes of the present invention. For example, anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants may 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. Particularly preferred surfactants are polysorbates, which belong to the nonionic surfactants. The amount used may be 0.1 to 10 wt%, and preferably 0.5 to 5.0 wt%, based on the total weight of the nanoliposomes.

Another component used in the preparation of the nanoliposome of the present invention is a phospholipid, which is an amphiphilic lipid, and includes natural phospholipids (e.g., egg yolk lecithin or soy lecithin, sphingomyelin) and synthetic phospholipids (e.g., dipalmitoylphosphatidylcholine or hydrogenated lecithin), and is preferably lecithin. In particular, natural unsaturated lecithin or saturated lecithin extracted from soybean or egg yolk is preferable. Typically, natural lecithin contains 23 to 95% phosphatidylcholine and 20% or less phosphatidylethanolamine. In the preparation of the nanoliposome of the present invention, the amount of phospholipid used is 0.5 to 20.0 wt%, and preferably 2.0 to 8.0 wt%, based on the total weight of the nanoliposome.

The fatty acid used in the preparation of the nanoliposome of the present invention is a higher fatty acid, preferably a saturated or unsaturated fatty acid with a _{C 12-22 alkyl} chain, such as lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, and linoleic acid. The amount used may be 0.05 to 3.0 wt% based on the total weight of the nanoliposome, and preferably 0.1 to 1.0 wt%.

The water used in the preparation of the nanoliposome of the present invention is generally deionized distilled water, and the amount used may be 5.0 to 40 wt% based on the total weight of the nanoliposome.

Nanoliposomes can be manufactured using various methods known in the art, but most preferably, they are manufactured by applying a mixture containing the above components to a high-pressure homogenizer. The manufacture of nanoliposomes using a high-pressure homogenizer can be performed under various conditions (e.g., pressure, number of cycles, etc.) depending on the desired particle size. Preferably, nanoliposomes are manufactured by passing the mixture through a high-pressure homogenizer 1 to 5 times under a pressure of 600 to 1200 bar.

The cosmetic composition of the present invention can be used alone or in combination, or in combination with other cosmetic compositions. Furthermore, the cosmetic composition of the present invention, with its excellent wrinkle-improving effect, can be used according to conventional methods, and the frequency of use can vary depending on the user's skin condition or preference.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to more specifically illustrate the present invention, and the scope of the present invention is not limited to these examples.

<Example 1>

Preparation of red thorn tree branch extract

The Quercus acuta Thunb. branches used in this experiment were collected from the Jeollanam-do Forest Resources Research Institute, washed, dried, and ground into powder. The dried Quercus acuta branches were ground, and extracts were prepared using distilled water, 70% ethanol, and 100% ethanol.

<1-1> Hot water extract of red oak branches

100 g of eggplant powder was soaked in 1,000 mL of distilled water, and extracted using an autoclave at 100°C for 10 minutes. The extracted extract was filtered through filter paper (Whatman No. 2) and concentrated using a rotary vacuum evaporator to be used as a sample. The yield was 13.69 g, calculated using the following equation 1.

[Formula 1]

Yield = (Weight of sample after drying (g) / Weight of sample before extraction (g)) × 100

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

100 g of red oak branch powder was added to 1000 mL of 70% ethanol and extracted by steeping at room temperature for 7 days. The steeped extract was filtered through filter paper (Whatman No. 2) and concentrated using a rotary vacuum evaporator to be used as a sample. The yield was 14.57 g, calculated using Equation 1 above.

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

100 g of red oak branch powder was added to 1000 mL of 100% ethanol and extracted by steeping at room temperature for 7 days. The steeped extract was filtered through filter paper (Whatman No. 2) and concentrated using a rotary vacuum evaporator to be used as a sample. The yield was 11.66 g, calculated using Equation 1 above.

The manufacturing process of the red oak branch extract of the present invention is described in detail in Fig. 1, and the yield of each extract is described in detail in Table 1 below. The yield of QAB2, a 70% ethanol extract, was found to be the highest at 14.57%.

Yield of the red oak branch extract of the present invention sample Yield (g) QAB1 13.69 QAB2 14.57 QAB3 11.66 QAB1: Hot water extract of red oak branches
QAB2: 70% ethanol extract of red oak branches
QAB3: 100% ethanol extract of red oak branches

Experimental Example 1

Evaluation of antioxidant activity of red oak branch extracts

In order to evaluate the antioxidant activity of the red oak branch extract of the present invention prepared in the above <Example 1>, DPPH free radical scavenging activity and ABTS radical scavenging activity were investigated.

<1-1> DPPH free radical scavenging activity

DPPH free radical assay is a widely used method for measuring the antioxidant activity of natural extracts. In this experiment, the radical scavenging activity of the samples was measured using a modified version of Blois' method among antioxidant activity measurement methods. 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 reacted in a darkroom 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. The DPPH scavenging activity (IC ₅₀ ) of the Quercus amurense branch extract was expressed as the concentration required to reduce the absorbance by 50% of the solvent-only control.

As a result, as shown in Table 2 and Figure 2 below, the DPPH IC ₅₀ range of the red oak branch extract was confirmed to be 250.34 to 340.70 μg/mL, and QAB3, a 100% ethanol extract, showed the best DPPH scavenging ability with an IC ₅₀ value of 250.34 μg/mL.

<1-2> ABTS radical scavenging ability

ABTS radical scavenging activity has the advantage of being widely applicable because it can measure the radical scavenging activity of both hydrophilic and hydrophobic samples. In this experiment, the method of Jeong et al. was modified and measured. Specifically, a 7 mM ABTS solution dissolved in distilled water and 2.45 mM potassium persulfate were mixed in a 1:1 ratio and left in the dark for 12–16 h. The radical stock solution was diluted with PBS (pH 7.4) to an absorbance value of 0.70 ± 0.02. 200 μL of samples prepared at various concentrations (50–750 μg/mL) were added to 800 μL of the diluted solution and reacted in a darkroom for 15 min. Afterwards, the absorbance was 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 ABTS scavenging activity (IC ₅₀ ) of the extract was expressed as the concentration required to reduce the absorbance by 50% of the control using only the solvent.

As a result, as shown in Table 2 and Figure 2 below, the IC ₅₀ range of ABTS of the red oak branch extract was confirmed to be 117.54 ~ 199.31 μg/mL, and QAB3, a 100% ethanol extract, showed the best DPPH scavenging activity with an IC ₅₀ value of 117.54 μg/mL. The radical scavenging activity of each extract increased in a concentration-dependent manner.

Antioxidant activity of red oak branch extracts using different extraction solvents sample DPPH IC ₅₀ (μg/mL) ABTS IC ₅₀ (μg/mL) QAB1 340.70 ± 2.57 199.31 ± 0.81 QAB2 296.59 ± 3.54 150.60 ± 1.89 QAB3 250.34 ± 2.64 117.54 ± 5.72 Positive control (gallic acid) 30.83 ± 0.45 - Positive control (quercetin) - 26.24 ± 0.95 QAB1: Hot water extract of red oak branches
QAB2: 70% ethanol extract of red oak branches
QAB3: 100% ethanol extract of red oak branches

Experimental Example 2

Total polyphenol and total flavonoid contents of red oak branch extracts

<2-1> Analysis of total polyphenol content

Polyphenols are aromatic alcohol compounds found in plants, each molecule containing two or more phenol groups. They come in a variety of structures and molecular weights, and their hydroxyl groups facilitate binding to various compounds, including water-soluble proteins. Polyphenols possess various physiological functions, including anticancer, anti-inflammatory, and antioxidant properties, and their functions vary depending on their content and composition. In this experiment, the total polyphenol content of red oak branch extracts was measured according to the extraction site and solvent.

Total polyphenol content analysis was measured using the Folin-Ciocalteu method (Ainsworth, E. A., & Gillespie, K. M., Nat. Protoc., 2(4), 875-877(2007)). Briefly, the concentration of the red oak branch extract was prepared at 500 μg/mL, and 500 μL of the prepared sample was sequentially mixed with 500 μL of 0.2 M Folin-Ciocalteu’s phenol reagent and 500 μL of 2% sodium carbonate aqueous solution (w/v), and the mixture was reacted in a dark room at room temperature for 30 min. Afterwards, the absorbance was measured at 750 nm using an HT multi-detection microplate reader. A calibration curve was created using gallic acid diluted at various concentrations as a standard substance, and then the mg/g gallic acid (GAE) equivalent was converted using the calibration curve.

As a result, as shown in Table 3, the 100% ethanol extract of the red twig showed the highest content at 159.99 ± 0.97 GAE mg/g.

<2-2> Analysis of total flavonoid content

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 branch extracts was measured according to the extraction site and solvent.

For the analysis of total flavonoid content, the concentration of the red thorn branch extract was prepared at 500 μg/mL, and 500 μL of the prepared sample was sequentially mixed with 1.5 mL of methanol, 100 μL of 1 M potassium acetate, and 1.4 mL of distilled water. After reacting at room temperature for 40 min, the absorbance was measured at 415 nm using an HT multi-detection microplate reader. A calibration curve was created using quercetin diluted at various concentrations as a standard substance, and then the quercetin (QUE) mg/g equivalent was converted.

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

Total polyphenol and total flavonoid contents of red oak branch extracts using different extraction solvents sample TPC (GAE mg/g) TFC (QUE mg/g) QAB1 120.93 ± 0.76 22.11 ± 0.30 QAB2 122.49 ± 0.56 35.88 ± 0.30 QAB3 159.99 ± 0.97 56.87 ± 0.60 QAB1: Hot water extract of red oak branches
QAB2: 70% ethanol extract of red oak branches
QAB3: 100% ethanol extract of red oak branches

Experimental Example 3

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

In order to quantitatively analyze the effective ingredients contained in the red oak branch extract of the present invention manufactured through the above <Example 1>, 57 kinds (Allopurinol; Apigenin; Caffeic acid; Catechin; p-Coumaric acid; Epicatechin; Epicatechin gallate; Ethyl gallate; t-Ferulic acid; Fumaric acid; Gallic acid; Genistein; Gentisic acid; 4-Hydroxybenzoic acid; Hyperoside; Polyphenols including luteolin, naringenin, protocatechuic acid, pyrogallol, quercetin, riboflavin, salicylic acid, shikimic acid, syringic acid, tannic acid, taxifolin, ursolic acid, and vanillic acid were analyzed. 10 μL of sample was injected into a C18 column (Gemini 3 μm, C18 110A 50 mm × 2.0 mm) and analyzed using an autosampler (15°C) in a column oven (40°C). Turbo Ion Spray was used for analysis in negative and positive ion modes, and water (0.1% formic acid) (A) and acetonitrile (0.1% formic acid) (B) were used as mobile phases. To improve analytical performance, the mixing ratio of each mobile phase according to the analysis time was analyzed as follows based on mobile phase B: 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%).

As a result, as shown in Table 4, there were differences in the types and contents of polyphenols depending on the extraction solvent, and the 100% ethanol extract showed the highest content at 15,392 μg/g.

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

Meanwhile, among the polyphenol components, genistein and quercetin were found to be effective only in the 100% ethanol extract of red oak branches. Genistein and quercetin are powerful antioxidants known to protect the skin from oxidative stress caused by free radicals.

Therefore, it was determined that the 100% ethanol extract of the red oak branches of the present invention, which not only contains a high content of catechin and tannic acid, which are effective in inhibiting wrinkle formation, but also contains genistein and quercetin, which are powerful antioxidants, as active ingredients, can be usefully used as a material having functions such as wrinkle improvement, skin moisturizing, and whitening.

LC-MS/MS analysis results of red oak branch extracts using different extraction solvents No. standard material Concentration (μg/g) QAB1 QAB2 QAB3 1 Allopurinol 24.40 10.00 9.92 2 Apigenin - 4.96 7.37 3 Caffeic acid 0.56 1.23 1.57 4 Catechin 7,320.00 9400.00 11300.00 5 p-Coumaric acid 4.48 8.34 0.77 6 Epicatechin 234.00 271.00 335.00 7 Epicatechin gallate 4.51 35.30 26:30 8 Ethyl gallate - 39.30 154.00 9 t-Ferulic acid 13:40 5.38 8.51 10 Fumaric acid 20.50 15.60 2.31 11 Gallic acid 91.80 23.80 10:40 12 Genistein - - 1.31 13 Gentisic acid 8.01 4.91 29.40 14 4-Hydroxybenzoic acid 7.80 4.48 3.87 15 Hyperoside 133.00 279.00 332.00 16 Luteolin 10:40 23.50 152.00 17 Naringenin 0.52 1.60 2.88 18 Nicotinic acid - 5.15 11.80 19 Protocatechuic acid 41.30 23.90 48.40 20 Pyrogallol 11.70 - - 21 Quercetin - - 76.60 22 Riboflavin 0.50 - 0.44 23 Salicylic acid 138.00 91.50 170.00 24 Shikimic acid 133.00 184.00 159.00 25 Syringic acid 58.90 26:30 61.00 26 Tannic acid 250.00 724.00 1520.00 27 Taxifolin 9.63 68.90 101.00 28 Ursolicacid - 821.00 830.00 29 Vanillic acid 59.80 35.80 37.00 Total (μg/g) 8,576.21 12,108.95 15,392.85 QAB1: Hot water extract of red oak branches
QAB2: 70% ethanol extract of red oak branches
QAB3: 100% ethanol extract of red oak branches

Experimental Example 4

Cytotoxicity evaluation of red thorn branch extract

In order to evaluate the cytotoxicity of the red oak branch extract of the present invention manufactured through the above <Example 1>, an MTT assay was performed.

The MTT assay is a representative method for measuring cell growth rate, as the MTT [3-(4,5-dimethylthizol-2-yl)-2,5-diphenyl tetrazolium bromide] reagent is absorbed into cells and then forms formazan by succinate dehydrogenase in the mitochondria. The accumulation of this substance within the cells indicates mitochondrial activity, and more broadly, cell activity.

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). Cultures were performed in an animal cell culture incubator (Sanyo CO2 Incubator, MCO-18AC, SANYO Electric Co., Ltd., OSA, JP) at 37°C and 5% _CO2 . Experiments were performed by subculturing cells when they reached approximately 90% of the confluence.

Cell suspension was dispensed into 12 wells at a concentration of 1 × 10 ⁵ cells/mL (1 mL each) and stabilized in an incubator for 24 h. Red oak branch extract was treated at various concentrations and incubated for 24 h. According to Choi's method, 0.2 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, MO, USA) solution was added 1 mL per well and left in an incubator for 30 min. Afterwards, the supernatant was removed, and 300 μL of dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) was added per well and shaken for 1 min to completely dissolve the formazan. Cell viability was calculated by measuring absorbance at 540 nm using ELISA Bio-Tek instrument Inc (Winooski, VT, USA).

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

Experimental Example 5

Wrinkle-improving effect of red thorn tree branch extract

In order to evaluate the wrinkle improvement effect of the 100% ethanol extract of the red oak branches of the present invention prepared through the above <Example 1>, collagenase inhibitory activity, elastase inhibitory activity, and hyaluronidase inhibitory activity were measured. In addition, the effect on the expression of wrinkle formation-related genes (MMP-3, MMP-9) was confirmed.

For reference, the wrinkle-improving 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 have functions such as providing mechanical strength to the skin, resistance to connective tissue, tissue cohesion, supporting cell adhesion, and inducing cell division and differentiation. Furthermore, hyaluronidase is an enzyme that breaks down hyaluronic acid, which helps protect cells in the skin, maintain moisture, and maintain elasticity. Therefore, when hyaluronic acid content decreases, the skin becomes dry and rough.

<5-1> Collagenase inhibitory activity

0.3 mL of collagenase type 1 enzyme bath prepared at a concentration of 200 unit/mL was added to a mixture of 0.5 _mL of substrate solution containing 4-phenylazobenzyloxycarbinyl-Pro-Leu-Gly-Pro-DArg (0.3 mg/mL) dissolved in 0.1 M Tris-HCl buffer (pH 7.5) and 0.2 mL of 100% ethanol extracts of red oak branches prepared at various concentrations. The added solution was reacted at room temperature for 20 minutes, then 0.5 mL of 5% citric acid was added to stop the reaction, and 1 mL of ethyl acetate was added to measure the absorbance at 320 nm. The collagenase inhibitory activity was expressed as the rate of decrease in absorbance between the sample solution addition group and the non-added group.

As a result, as shown in Table 5, the collagenase inhibitory activity of the 100% ethanol extract of the red oak branches was confirmed to have an IC ₅₀ value of 390.40 ± 7.41 μg/mL, demonstrating excellent collagenase inhibitory activity. Meanwhile, the IC ₅₀ value of oleic acid used as a positive control was measured to be 89.52 ± 2.85 μg/mL.

<5-2> Elastase inhibitory activity

To 500 μL of 100% ethanol extract of the branches of Quercus amurense, 250 μL of pancreatic solution (Type Ⅰ, 0.6 unit/mL) was added, followed by the addition of 500 μL of N-succinyl-(LAla)3-p-nitroanilide (1 mg/mL) dissolved in 50 mM Tris-HCl buffer (pH 8.6) as a substrate. The solution was reacted at 37°C for 30 min, and the absorbance was measured at 410 nm. Elastase inhibitory activity was expressed as the decrease in absorbance between the sample solution-added group and the non-added group.

As a result, as shown in Table 5, the elastase inhibitory activity of the 100% ethanol extract of the red oak branches was confirmed to have an IC ₅₀ value of 309.44 ± 11.91 μg/mL, demonstrating excellent elastase inhibitory activity. Meanwhile, the IC ₅₀ value of oleic acid used as a positive control was measured to be 89.56 ± 3.07 μg/mL.

<5-3> Hyaluronidase inhibitory activity

6 μL 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 branches, and the mixture was reacted in a 37°C water bath for 20 minutes. To activate hyaluronidase, 6 μL of 12.5 mM CaCl2 was added to the mixture, and the mixture was reacted in a 37°C water bath for 20 minutes. After the reaction was completed, 12 μL of substrate solution hyaluronate (6 mg/mL) dissolved in 0.1 M acetate buffer (pH 3.5) was added to the reaction mixture, and the mixture was reacted in a 37°C water bath for 40 minutes. To stop 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 solution, and the reaction was incubated at 100°C for 3 minutes, after which it was completely cooled to room temperature. 180 μL of DMAB solution, a chromogenic agent, was added to the substrate-enzyme mixture, and the absorbance value was measured at 540 nm using an ELISA reader. Hyaluronidase inhibition activity was expressed as the rate of decrease in absorbance between the sample solution-added group and the non-added group.

As a result, as shown in Table 5, the hyaluronidase inhibitory activity of the 100% ethanol extract of the red oak branches was confirmed to have an IC ₅₀ value of 307.98 ± 3.16 μg/mL, demonstrating excellent elastase inhibitory activity. Meanwhile, the IC ₅₀ value of oleic acid used as a positive control was measured to be 70.21 ± 0.43 μg/mL.

Collagenase, elastase, and hyaluronidase inhibitory activities of red oak branch extracts sample collagenase
IC ₅₀ value (μg/mL) Elastase
IC ₅₀ value (μg/mL) hyaluronidase
IC ₅₀ value (μg/mL) QAB3 390.40 ± 7.41 309.44 ± 11.91 307.98 ± 3.16 oleic acid 89.52 ± 2.85 89.56±3.07 70.21±0.43 QAB3: 100% ethanol extract of red oak branches

<5-4> Effect on the expression of wrinkle formation-related genes (MMP-3, MMP-9)

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% concentration of _H2O2 , and the mRNA expression levels of MMP-3 and MMP-9 induced were analyzed by RT-PCR.

For reference, matrix metalloproteinases (MMPs) are broadly classified into MMP-1, MMP-2, MMP-3, and MMP-9 according to their substrate specificity. MMP-3, as stromelysin-1, degrades proteoglycan, elastin, fibronectin, and laminin in mouse skin and activates various MMPs, playing an important role in wrinkle formation. MMP-9 is a gelatinase that has been reported to degrade type collagen, a major component of the basement membrane.

For RT-PCR analysis, total RNA was first extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Total RNA (2 g) was reverse transcribed using an oligo-dT18 primer to synthesize cDNA. 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 following PCR primer sequences were used: 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.

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 concentration-dependent manner depending on the treatment concentration of the red oak branch extract of the present invention.

Through the above results, it was confirmed that the 100% ethanol extract of the red oak branch of the present invention is an effective substance for improving wrinkles.

Formulation Example 1: Softening Toner (Skin Lotion)

As shown in the table below, a flexible toner was manufactured according to a conventional method.

Flexible toner formulation example of the present invention Mixing ingredients Content (weight%) Red twig extract 1~5 1,3-butylene glycol 6.0 glycerin 4.0 DL-Panthenol 0.3 Allantoin 0.1 Polysorbate 20 0.5 ethanol 15.0 Benzophenone-9 0.05 fragrance, preservatives a very small amount purified water to 100

Formulation Example 2: Nourishing Toner (Milk Lotion)

As shown in the table below, a nutritious toner was prepared according to the usual method.

Nutritional toner formulation example of the present invention Mixing ingredients Content (weight%) Red twig extract 1~5 Glyceryl Stearate SE 1.5 Cetearyl alcohol 1.5 lanolin 1.5 Polysorbate 60 1.3 Sorbitan stearate 0.5 hydrogenated vegetable oils 4.0 mineral oil 5.0 Trioctanoin 2.0 Dimethicone 0.8 Tocopherol acetate 0.5 carboxyvinyl polymer 0.12 glycerin 5.0 1,3-butylene glycol 3.0 Sodium hyaluronate 5.0 triethanolamine 0.12 Uniside-U 13 0.02 incense a very small amount distilled water to 100

Formulation Example 3: Nourishing Cream

As shown in the table below, a nourishing cream was prepared according to a conventional method.

Nutritional cream formulation example of the present invention Mixing ingredients Content (weight%) Red twig extract 1~5 Glycerin monostearate 1.5 Cetearyl alcohol 1.5 Stearic acid 1.0 Polysorbate 60 1.5 Sorbitan stearate 0.6 isostearyl isosterate 5.0 Squalane 5.0 mineral oil 35.0 Dimethicone 0.5 Hydroxyethylcellulose 0.12 glycerin 6.0 triethanolamine 0.7 Uniside-U13 0.02 incense a very small amount distilled water to 100

Formulation Example 4: Massage Cream

Massage cream was manufactured according to the conventional method as shown in the table below.

Massage cream formulation example of the present invention Mixing ingredients Content (weight%) Red twig extract 1~5 Propylene glycol 6 glycerin 4.0 triethanolamine 0.5 beeswax 2.0 Tocopheryl acetate 0.1 Polysorbate 60 3.0 Sorbitan sesquioleate 2.5 Cetearyl alcohol 2.0 liquid paraffin 30.0 Carboxyvinyl polymer 0.5 incense a very small amount distilled water to 100

Formulation Example 5: Pack

The pack was manufactured according to the conventional method as shown in the table below.

Pack formulation example of the present invention Mixing ingredients Content (weight%) Red twig extract 1~5 glycerin 10.0 Betaine 5.0 REG 1500 2.0 Allantoin 0.1 DL-Panthenol 0.3 EDTA-2Na 0.02 Benzophenone-9 0.04 Hydroxyethyl cellulose 0.1 Sodium hyaluronate 80. Carboxyvinyl polymer 0.2 triethanolamine 0.18 octyldodecanol 0.3 Octyldodeces-16 0.4 ethanol 6.0 Uniside-U13 0.01 incense a very small amount distilled water to 100

The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

Claims (8)

A cosmetic composition for improving wrinkles containing 100% ethanol extract of red oak branches as an active ingredient. delete delete In the first paragraph,
A cosmetic composition for improving wrinkles, characterized in that the extract comprises catechin, tannic acid, genistein, and quercetin. In the first paragraph,
A cosmetic composition for improving wrinkles, characterized in that the extract inhibits wrinkle formation through the inhibitory activity of collagenase, elastase, and hyaluronidase. In the first paragraph,
A cosmetic composition for improving wrinkles, characterized in that the extract inhibits wrinkle formation by suppressing the expression of MMP-3 and MMP-9 genes. In the first paragraph,
A cosmetic composition for improving wrinkles, characterized in that the extract is included in the composition at a concentration of 0.0001 to 2000 μg/ml. In any one of the first and fourth to seventh paragraphs,
The cosmetic composition for improving wrinkles is characterized in that the cosmetic composition is one formulation selected from the group consisting of a flexible toner, a gel, a water-soluble liquid, a milk lotion, a nourishing cream, a massage cream, an essence, an oil-in-water type emulsion, a water-in-oil type emulsion, an anhydrous product for face makeup, an anhydrous solid product, an oil dispersion in an aqueous phase using spherules, an ionic lipid vesicle, a nonionic lipid vesicle, an ointment, a cleansing foam, a cleansing water, a pack, a body oil, an oil-in-water type makeup base, a water-in-oil type 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 cheek color, and an eyebrow pencil.