Composition for Improving Skin Wound Regeneration, Anti-Inflammation, Skin Moisturizing, and Skin Barrier Comprising Extract of Pulsatilla koreana as Active Ingredient

Abstract

The present invention relates to a composition containing a complex of two components, berberine and Pulsatilla koreana extract, as an active ingredient. The composition of the present invention is non-cytotoxic, promotes cell growth, effectively prevents wrinkles by promoting the production of type 1 procollagen, improves skin elasticity by directly promoting collagen production, promotes skin wound regeneration, exhibits anti-inflammatory effects by inhibiting nitric oxide production, exhibits excellent skin moisturizing effects by promoting hyaluronic acid production, strengthens skin barrier formation by increasing the expression of the profilaggrin gene, and promotes angiogenesis by increasing the motility of vascular endothelial cells, so it can be usefully utilized in cosmetics, food, pharmaceuticals, and quasi-pharmaceuticals for improving skin condition. Furthermore, the composition of the present invention is not only very safe for the human body, but also has excellent stability.

Description

Technology Field

[0001] The present invention relates to a composition for promoting skin wound regeneration, anti-inflammation, skin moisturization, and strengthening the skin barrier containing Pulsatilla koreana extract as an active ingredient, and more specifically, to a composition for promoting skin wound regeneration, anti-inflammation, skin moisturization, strengthening the skin barrier, anti-inflammation, and promoting angiogenesis containing a complex of berberine and Pulsatilla koreana extract as an active ingredient. Background Technology

[0003] Some of the substances that play a crucial role in maintaining homeostasis in living organisms are physiologically active substances derived from various organisms. Extensive research has been conducted on numerous physiologically active substances to date, and among them, the study of physiologically active substances isolated from microorganisms, plants, or animals is a very important area in the fields of life science and medicine.

[0004] There are substances known to be effective in improving skin wrinkles and elasticity, moisturizing, whitening, and antioxidant properties, such as vitamin C, retinoic acid, and transforming growth factor (TGF), which have been developed in the past. However, materials such as vitamin C, retinoic acid, transforming growth factor (TGF), animal placenta-derived protein (JP 1996-231370A), betulinic acid (JP 1996-208424A), and Chlorella extract (JP 2001-072567A) have had problems such as limited usage amounts due to safety issues like irritation and redness when applied to the skin, or their effects are so minimal that they cannot practically be expected to have an effect on the skin.

[0005] Furthermore, recently, there has been an active trend in the development of cosmetics that enhance functionality by introducing physiologically active substances derived from natural sources to further strengthen the skin's own inherent defense mechanisms. Consequently, there is an urgent need for the development of natural materials that are safe for the body, have stable active ingredients, and, above all, are more effective than existing substances for improving skin wrinkles, moisturizing, and whitening.

[0006] Meanwhile, cosmetic ingredients must strike a balance between safety, efficacy, and physical properties. Furthermore, as economic feasibility is a crucial factor, cosmetic materials are developed using various methods, such as natural extraction and fermentation by microorganisms, to optimize skin penetration, anti-aging properties, physiological activity, safety, and physical characteristics. Moreover, as the boundaries between cosmetics, food, and pharmaceuticals blur, a diverse range of cosmeceutical products continues to experience high growth through complex functional ingredients and biomaterials that combine the safety of cosmetics with the efficacy of pharmaceuticals and health functional foods.

[0007] Against this backdrop, the inventors have diligently researched to discover combinations of natural extracts or naturally derived compounds that exhibit excellent effects in improving skin conditions—such as improving skin elasticity, promoting skin wound regeneration, anti-inflammation, skin moisturization, and strengthening the skin barrier—and can be actively utilized in the field. As a result, the combination of berberine and Pulsatilla koreana extract exhibits the following effects: i) it is non-cytotoxic and promotes cell growth; ii) it promotes the production of Type 1 procollagen to effectively prevent wrinkles and directly promotes collagen production to improve skin elasticity; iii) it promotes skin wound regeneration; iv) it exhibits anti-inflammatory effects by inhibiting nitric oxide production; v) it exhibits excellent effects on skin moisturization by promoting hyaluronic acid production; vi) it strengthens skin barrier formation by increasing the expression of the profilaggrin gene; and vii) it promotes angiogenesis by increasing the motility of vascular endothelial cells. Consequently, it can be usefully utilized in cosmetics, foods, pharmaceuticals, and quasi-pharmaceuticals for improving skin conditions. It was confirmed that there is a [unclear], and the present invention was completed. Prior art literature

[0009] (0001) Patent Publication No. 10-2016-0093420 (2016.08.08), cosmetic composition for improving dark circles comprising berberine or a pharmaceutically acceptable salt thereof (0002) Patent Publication No. 10-2004-0078449 (2004.09.10), cosmetic composition having berberine baicalinate as an active ingredient (0003) Patent Publication No. 10-2015-0044863 (2015.04.27), anti-aging, anti-inflammatory, and antioxidant cosmetic composition containing Pulsatilla koreana plant placental cell culture extract The problem to be solved

[0010] Accordingly, the main objective of the present invention is to provide a complex of berberine and Pulsatilla extract that exhibits excellent effects in improving skin elasticity, promoting skin wound regeneration, anti-inflammatory, skin moisturizing, and strengthening the skin barrier.

[0011] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings. means of solving the problem

[0013] According to one aspect of the present invention, the present invention relates to a berberine compound and a pasque flower ( Pulsatilla koreana The present invention provides a cosmetic composition containing a complex of two components of an extract as an active ingredient.

[0014] The inventors explored the effects of various natural substances in order to find a substance that exhibits excellent effects in improving skin condition, and as a result of research efforts to further improve the skin condition improvement efficacy of ingredients previously known for their skin condition improvement effects, a berberine compound and pasque flower ( Pulsatilla koreana It was discovered that the combination of extracts dramatically increased synergistic effects for improving skin elasticity, promoting skin wound healing, anti-inflammatory effects, skin moisturizing, and strengthening the skin barrier, and it was also confirmed that there were no safety issues when applied to the human body.

[0015] The "Berberine" of the present invention is a quaternary ammonium salt derived from protoberberine of the benzylisoquinoline alkaloid group. The berberine compound is Coptis chinensis (goldthread), Berberis vulgaris (barberry), Berberis petiolaris , Berberis aristata , Berberis thunbergii , Mahonia aquifolium , Hydrastis canadensis It is extracted from the roots, stems, rhizomes, etc., of various plants and is a naturally derived component. The above-mentioned berberine compound may be chemically synthesized or extracted by methods known in the art, or commercially available substances may be used. In addition, the above-mentioned berberine compound can be extracted from natural plants, in this case Coptis chinensis (Coptis japonica), or Berberis vulgarisIt is preferable to use a berberine compound extracted from the barberry.

[0016] In addition, the above "pasqueflower ( Pulsatilla koreana )" refers to a perennial plant of the Ranunculaceae family, order Ranunculales, class Dicotyledon.

[0017] In addition, the term "active ingredient" in this specification means an ingredient that exhibits the intended activity alone, or can exhibit activity together with a carrier that is inactive itself.

[0018] The term "extract" as used in the present invention includes the extract itself and all formulations of extracts that can be formed using the extract, such as an extract obtained by extracting a plant component, a diluted or concentrated extract of said extract, a dried product obtained by drying said extract, a modified or purified product of said extract, or a mixture thereof. Preferably, said extract of the present invention may be prepared and used in the form of a dried powder after extraction.

[0019] In the extraction of the Pulsatilla koreana according to the present invention, the extraction method is not particularly limited and can be performed according to methods commonly used in the relevant technical field. Non-limiting examples of the extraction method include hot water extraction, ultrasonic extraction, filtration, and reflux extraction, and these may be performed individually or in combination with two or more extraction methods.

[0020] In the present invention, the type of extraction solvent used to extract the Pulsatilla koreana is not particularly limited, and any solvent known in the relevant technical field may be used. Non-limiting examples of the extraction solvent include water (purified water); lower alcohols having 1 to 4 carbon atoms, such as methanol, ethanol, propyl alcohol, butyl alcohol; polyhydric alcohols, such as glycerin, butylene glycol, propylene glycol; and hydrocarbon solvents, such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; or mixtures thereof. Preferably, water (purified water), lower alcohols, 1,3-butylene glycol, and ethyl acetate may be used alone or mixed in a combination of two or more types.

[0021] In the present invention, the extract obtained by hot water extraction or cold maceration extraction can be used as is or dried using freeze-drying, hot air drying, spray drying, etc., after filtering to remove suspended solid particles by filtering particles using, for example, nylon, etc.

[0022] In the cosmetic composition of the present invention, the berberine compound and the pasque flower ( Pulsatilla koreana The composition of the extract is not limited thereto, but based on the raw material components, the above-mentioned berberine compound and the above-mentioned pasque flower ( Pulsatilla koreana The extract can be used by mixing it in a weight ratio of 1:90-110.

[0023] In the cosmetic composition of the present invention, the complex of the two components is characterized by increasing the expression of type 1 procollagen protein to exhibit activity for improving skin wrinkles and skin elasticity. In a preferred embodiment of the present invention, it was confirmed that the expression of type 1 procollagen protein significantly increased by the combination of a berberine compound and Pulsatilla koreana extract, and it was confirmed that the complex of the berberine compound and Pulsatilla koreana extract of the present invention is effective in improving skin wrinkles and skin elasticity (see FIG. 2).

[0024] In the cosmetic composition of the present invention, the two-component complex is characterized by promoting the regeneration of skin wounds and strengthening the skin barrier by increasing filaggrin gene expression. In a preferred embodiment of the present invention, it has been confirmed that the complex of the berberine compound and Pulsatilla koreana extract of the present invention exhibits a synergistic effect on skin regeneration compared to their single components (see FIG. 3). Furthermore, it has been confirmed that the two-component complex of the present invention, which combines the berberine compound and Pulsatilla koreana extract, exhibits excellent effects in improving skin condition by contributing to the formation of a skin barrier through increasing the expression of the profilaggrin gene (see FIG. 6).

[0025] In the cosmetic composition of the present invention, the complex of the two components is characterized by exhibiting anti-inflammatory activity by inhibiting the production of nitric oxide, an inflammatory mediator. In a preferred embodiment of the present invention, it has been confirmed that the berberine compound or the Pulsatilla extract each significantly reduces the production of nitric oxide to a certain extent, but the effect is negligible, and that the combination of the two components exhibits an excellent synergistic effect in inhibiting the inflammatory response (see FIG. 4).

[0026] In the cosmetic composition of the present invention, the complex of the two components is characterized by promoting hyaluronic acid synthesis to exhibit skin moisturizing activity. In a preferred embodiment of the present invention, it has been confirmed that while the skin moisturizing effect of the berberine compound or Pulsatilla extract is weak, the production of hyaluronic acid increases significantly depending on the combination of these two components, thereby exhibiting a synergistic effect on skin moisturization (see FIG. 5).

[0027] In the cosmetic composition of the present invention, the two-component complex is characterized by promoting angiogenesis by promoting the migration of vascular endothelial cells. In a preferred embodiment of the present invention, it has been confirmed that the migration rate of human umbilical vein endothelial cells (HUVEC) increases through a combination of two components, a berberine compound and an angioides extract, thereby promoting angiogenesis (see FIG. 7).

[0028] The cosmetic composition of the present invention is non-toxic to skin cells and has activity that further promotes the growth of skin cells (see FIG. 1). Since its addition does not impair the quality of the cosmetic material, it is suitable for use in cosmetic compositions. In particular, since it has functions related to improving skin elasticity, promoting skin wound regeneration, anti-inflammatory effects, skin moisturization, and strengthening the skin barrier, it is desirable to use it in functional cosmetic compositions.

[0029] The cosmetic composition of the present invention may be prepared in any one formulation selected from skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, essence, nourishing essence, mask pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, and body cleanser, but is not limited thereto.

[0030] The cosmetic composition of the present invention may additionally include one or more cosmetically acceptable carriers that are incorporated into general skin cosmetics, and may appropriately incorporate, for example, oils, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, colorants, preservatives, fragrances, etc., as conventional ingredients, but is not limited thereto.

[0031] Cosmetically acceptable carriers included in the cosmetic composition of the present invention vary depending on the formulation of the cosmetic composition.

[0032] When the formulation of the present invention is an ointment, paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. may be used as a carrier component, but is not limited thereto. These may be used alone or in a mixture of two or more types.

[0033] When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be used as a carrier component, and in particular, in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether may be additionally included, but is not limited thereto. These may be used alone or in a mixture of two or more types.

[0034] When the formulation of the present invention is a solution or an emulsion, a solvent, a solubilizing agent, or an emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, etc., may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan may be used, but are not limited thereto. These may be used alone or in a mixture of two or more.

[0035] When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol, or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used as carrier components, but are not limited thereto. These may be used alone or in a mixture of two or more.

[0036] When the formulation of the present invention is a soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolysates, isethionates, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerol, sugars, etc. may be used as carrier components, but are not limited thereto. These may be used alone or in a mixture of two or more types.

[0037] In the cosmetic composition of the present invention, the berberine compound and pasque flower ( Pulsatilla koreanaThe two-component complex of the extract may specifically be included in a range of 0.0001% to 50% by weight of the total weight of the cosmetic composition in terms of dry weight, and more specifically in a range of 0.0005% to 10% by weight. Within the above range, there is an advantage of exhibiting excellent efficacy in improving skin elasticity, promoting skin wound healing, anti-inflammatory, skin moisturizing, and strengthening the skin barrier, and there is an advantage of stabilizing the formulation of the composition.

[0038] According to another aspect of the present invention, the present invention relates to a berberine compound and a pasque flower ( Pulsatilla koreana The present invention provides a food composition containing a complex of two components of ) extract as an active ingredient. The above berberine compound and Pulsatilla ( Pulsatilla koreana A complex of two components of ) extract exhibits excellent effects in improving skin elasticity, promoting skin wound regeneration, anti-inflammatory, skin moisturizing, and strengthening the skin barrier, and can be usefully utilized as a food composition for improving skin condition. The berberine compound of the present invention and Pulsatilla ( Pulsatilla koreana The two-component complex of the extract and its effects on improving skin elasticity, promoting skin wound regeneration, anti-inflammatory, skin moisturizing, and strengthening the skin barrier are as described above. The food composition may be used in the form of a health functional food, but is not limited thereto.

[0039] The food composition of the present invention may be included in the form of the above-mentioned two-component complex and a processed product thereof. In addition, the composition may include a food-grade acceptable food additive in addition to the active ingredient.

[0040] In the present invention, "food auxiliary additive" refers to a component that can be added to food as an auxiliary component, and can be appropriately selected and used by a person skilled in the art as an additive for manufacturing health functional foods of each formulation. Examples of food auxiliary additives include various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., but the types of food auxiliary additives of the present invention are not limited by the above examples.

[0041] The food composition of the present invention may include a health functional food. In the present invention, "health functional food" refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., using raw materials or ingredients having functional properties useful to the human body. Here, "functional properties" means obtaining effects useful for health purposes, such as regulating nutrients or physiological actions regarding the structure and function of the human body. The health functional food of the present invention can be manufactured using methods commonly used in the industry, and can be manufactured by adding raw materials and ingredients commonly added in the industry during such manufacturing.

[0042] In addition, the formulation of the above-mentioned health functional food may be manufactured without restriction as long as it is a formulation recognized as a health functional food. The food composition of the present invention can be manufactured in various forms of formulations. Unlike general pharmaceuticals, it uses naturally derived compounds and natural plant extracts that are non-cytotoxic as raw materials, and thus has the advantage of not causing side effects that may occur during long-term consumption of the food. It is highly portable and can be consumed as an adjuvant to enhance effects such as improving skin elasticity, promoting skin wound regeneration, anti-inflammatory effects, skin moisturization, and strengthening the skin barrier.

[0043] There are no restrictions on the form that the health functional food of the present invention may take, and it may include all foods in the conventional sense and may be used interchangeably with terms known in the art, such as functional food. Furthermore, the health functional food of the present invention may be manufactured by mixing known additives with other appropriate auxiliary ingredients that may be included in food, at the choice of a person skilled in the art. Examples of foods that may be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and the berberine compound according to the present invention and Pulsatilla ( Pulsatilla koreana It can be manufactured by adding to juices, teas, jellies, and juices made using a complex of two components of the extract as the main ingredient. In addition, it includes foods used as animal feed.

[0044] According to another aspect of the present invention, the present invention relates to a berberine compound and a pasque flower ( Pulsatilla koreana The present invention provides a quasi-drug composition containing a complex of two components of ) extract as an active ingredient. The above berberine compound and Pulsatilla ( Pulsatilla koreana The two-component complex of ) extracts exhibits excellent effects in improving skin elasticity, promoting skin wound regeneration, anti-inflammatory, skin moisturizing, and strengthening the skin barrier, and can be usefully utilized as a quasi-drug composition for improving skin condition. The berberine compound of the present invention and Pulsatilla koreana ( Pulsatilla koreana The two-component complex of the extract and its effects on improving skin elasticity, promoting skin wound regeneration, anti-inflammatory, skin moisturizing, and strengthening the skin barrier are as described above.

[0045] The term "quasi-drug" as used in the present invention refers to articles used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing diseases in humans or animals, among which the effects are milder than those of pharmaceuticals. For example, according to the Pharmaceutical Affairs Act, quasi-drugs are defined as articles excluding those used for pharmaceutical purposes, and include products used for the treatment or prevention of diseases in humans or animals, and products that have mild effects on the human body or do not act directly on it. The quasi-drug composition of the present invention is used for the purpose of improving skin conditions related to improving skin elasticity, promoting skin wound regeneration, anti-inflammation, skin moisturization, and strengthening the skin barrier. While there are no specific limitations on the formulation, it may preferably be a formulation for oral administration, skin administration, or transdermal administration.

[0046] In addition, for each formulation, the quasi-drug composition may arbitrarily select and combine other ingredients according to the formulation or intended use of other quasi-drugs. The mixing amount of the active ingredient may be appropriately determined according to the intended use (inhibition or alleviation). For example, it may include conventional adjuvants such as thickeners, stabilizers, solubilizers, vitamins, pigments, and fragrances, and carriers. Effects of the invention

[0048] The features and advantages of the present invention are summarized as follows:

[0049] (1) The present invention relates to a berberine compound and a pasque flower ( Pulsatilla koreana The present invention provides a composition containing a complex of two components of an extract as an active ingredient.

[0050] (2) The composition of the present invention is non-cytotoxic, promotes cell growth, promotes the production of type 1 procollagen to effectively prevent wrinkles, directly promotes collagen production to improve skin elasticity, promotes skin wound healing, inhibits nitric oxide production to show anti-inflammatory effects, promotes hyaluronic acid production to show excellent skin moisturization effects, increases the expression of profilaggrin gene to strengthen skin barrier formation, and increases the motility of vascular endothelial cells to promote angiogenesis, so it can be usefully utilized in cosmetics, food, pharmaceuticals and quasi-pharmaceuticals for improving skin condition.

[0051] (3) In addition, the composition of the present invention is not only very safe for the human body, but also has excellent stability. Brief explanation of the drawing

[0053] Figure 1 is a graph summarizing the changes in cell viability of human skin fibroblasts according to treatment with different concentrations of a berberine compound (Preparation Example 1), Pulsatilla extract (Preparation Example 2), and a two-component complex of a berberine compound and Pulsatilla extract (Preparation Example 3). Figure 2 is a graph summarizing the changes in type 1 procollagen protein expression according to treatment at different concentrations of a berberine compound (Preparation Example 1), Pulsatilla extract (Preparation Example 2), and a two-component complex of a berberine compound and Pulsatilla extract (Preparation Example 3). Figure 3 is a graph summarizing the changes in wound repair rates of human keratinocytes (HaCaT) according to treatment with different concentrations of a berberine compound (Preparation Example 1), Pulsatilla extract (Preparation Example 2), and a two-component complex of a berberine compound and Pulsatilla extract (Preparation Example 3). Figure 4 is a graph summarizing the change in nitric oxide production according to treatment at different concentrations of a berberine compound (Preparation Example 1), Pulsatilla extract (Preparation Example 2), and a two-component complex of a berberine compound and Pulsatilla extract (Preparation Example 3). Figure 5 is a graph summarizing the change in hyaluronic acid production according to treatment at different concentrations of a berberine compound (Preparation Example 1), Pulsatilla extract (Preparation Example 2), and a two-component complex of a berberine compound and Pulsatilla extract (Preparation Example 3). Figure 6 is a graph summarizing the changes in mRNA expression of the propyllagrin gene according to treatment at different concentrations of a berberine compound (Preparation Example 1), Pulsatilla extract (Preparation Example 2), and a two-component complex of a berberine compound and Pulsatilla extract (Preparation Example 3). Figure 7 is a graph showing the migration rate of human umbilical vein endothelial cells (HUVEC) according to treatment at different concentrations of a berberine compound (Preparation Example 1), Pulsatilla extract (Preparation Example 2), and a two-component complex of a berberine compound and Pulsatilla extract (Preparation Example 3). Specific details for implementing the invention

[0054] The present invention will be described in more detail below through examples. These examples are merely illustrative of the present invention, and therefore the scope of the present invention should not be interpreted as being limited by these examples.

[0056] Example 1. Preparation of a complex of berberine and Pulsatilla

[0057] 1-1. Berberine's Preparation

[0058] Berberine hydrochloride purchased from Sigma Aldrich (USA) was used (Preparation Example 1). The chemical formula of berberine hydrochloride is [C 20 H 18 It is ClNO4, and its molecular weight is 371.81.

[0060] 1-2. Preparation of Pulsatilla Extract

[0061] Crushed pasqueflower with a dry weight of 100g ( Pulsatilla koreanaThe whole plant of ) was heated and reflux-extracted with 10 times its weight in purified water for 12 hours, cold-macerated, and then filtered using filter paper (Glassfiber, Sartorius, USA) with a filtration size of 1.2 μm. The filtered extract was concentrated under reduced pressure at 50°C or below, and then completely dried using a vacuum dryer (Eyela Rotary evaporator, Tokyo Rikakika Co., LTD, Japan) to obtain Pulsatilla extract (Preparation Example 2).

[0063] 1-3. Preparation of a complex of berberine and pasqueflower

[0064] A complex of berberine and pasque flower (Preparation Example 3) was prepared by mixing 1 mg of the berberine sample from Example 1-1 and 100 mg of the pasque flower extract sample from Example 1-2.

[0066] Example 2. Confirmation of cytoprotective effect through cytotoxicity test (MTT Assay)

[0067] The effects of berberine compounds and / or Pulsatilla koreana extract on the viability of human dermal fibroblasts (NHDFs) were evaluated.

[0068] Cytotoxicity was measured using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay. The MTT (Methyl thiazol tetrazolium) assay is used to measure cell viability and involves the conversion of the MTT reagent into a formazan stain that produces a purple color. After 72 hours of incubation, the volume of the medium was reduced to 1 mL, and 100 µL of 1 mg / mL MTT reagent was added to each wall. Subsequently, NHDFs cells were cultured at 37°C for 2 hours in the presence of 5% CO2 and 95% O2. UVB was applied to NHDFs cells at 150 mJ / cm² 2The conditions were investigated, and the sample of Preparation Example 1 prepared in Example 1 was diluted to concentrations of 0.1 μg / ml, 0.3 μg / ml, and 1 μg / ml and treated, and the samples of Preparation Examples 2 and 3 were diluted to concentrations of 10 μg / ml, 30 μg / ml, and 100 μg / ml, respectively and treated, and then cultured for 24 hours. After 24 hours, 1 mg / ml of MTT was treated, and after 3 hours, the formazan generated by the MTT treatment in the cells was dissolved in DMSO solvent and the absorbance was measured at 570 nm.

[0069] Referring to [Figure 1], which summarizes the experimental results, it was observed that the cell viability of NHDFs cells decreased due to UVB irradiation. It was observed that this decrease in the viability of NHDFs cells due to UVB irradiation was improved by treatment with berberine compounds and / or Pulsatilla koreana extract.

[0070] Specifically, in the test group treated with the single component of the berberine compound of Preparation Example 1, the survival rate of NHDFs cells increased by 102% (0.1 μg / ml), 108% (0.3 μg / ml), and 115% (1% μg / ml), respectively, compared to the UVB irradiation treatment group; in the test group treated with the Pulsatilla extract of Preparation Example 2, the survival rate of NHDFs cells increased by 103% (10 μg / ml), 106% (30 μg / ml), and 111% (100 μg / ml), respectively, compared to the UVB irradiation treatment group; and in the test group treated with the complex of berberine and Pulsatilla of Preparation Example 3, the survival rate of NHDFs cells increased by 108% (10 μg / ml), 121% (30 μg / ml), and 128% (100 μg / ml), respectively, compared to the UVB irradiation treatment group.

[0071] Based on these results, it was confirmed that the berberine compound and / or Pulsatilla extract of the present invention are non-cytotoxic, and that their combination further promotes the growth of skin cells compared to a single component.

[0073] Example 3. Improvement of skin wrinkles and elasticity through collagen synthesis

[0074] Changes in the expression of type 1 procollagen protein following treatment with berberine compounds and / or Pulsatilla extract were analyzed.

[0075] For the cells used in the experiment, 2 ml of DMEM medium was placed in a 40 mm cell culture dish, and human dermal fibroblasts (NHDFs) were added at a rate of approximately 1.2 x 10⁶ 5 After inoculating at the cell concentration, the cells were cultured for 24 hours at 37°C in a 5% CO2 environment before use. Subsequently, UVB was irradiated at 150 mJ / cm² 2 After investigating under the conditions, the sample of Preparation Example 1 prepared in Example 1 was diluted to concentrations of 0.1 μg / ml, 0.3 μg / ml, and 1 μg / ml and treated, and the samples of Preparation Examples 2 and 3 were diluted to concentrations of 10 μg / ml, 30 μg / ml, and 100 μg / ml, respectively and treated, and cultured for 3 days. Afterward, the culture medium was harvested, centrifuged at 4°C and 7,500 rpm for 5 minutes, and the change in the expression level of Type 1 procollagen (Procollagen Type IC Peptide EIA Kit, Takara, Shiga, Japan) protein was confirmed by the ELISA method. All experiments were measured in three repetitions.

[0076] Referring to [Figure 2], which quantifies the change in type 1 procollagen protein expression, it was confirmed that the expression of type 1 procollagen protein in the complex treatment group increased in a concentration-dependent manner compared to the single-component treatment group of berberine compound or Pulsatilla extract.

[0077] Specifically, UVB irradiation (150 mJ / cm²) 2Based on the type 1 procollagen protein expression level (Con.) which was reduced to 2 / 3 of the level due to ), the test group treated with berberine compounds showed an increase in type 1 procollagen protein expression of 101% (0.1 μg / ml), 117% (0.3 μg / ml), and 121% (1 μg / ml), respectively, and an increase of 104% (10 μg / ml), 112% (30 μg / ml), and 119% (100 μg / ml), confirming that each individual component has some activity of increasing type 1 procollagen protein expression on its own.

[0078] In contrast, the test group treated with the complex composed of the berberine compound and Pulsatilla extract of the present invention showed type 1 procollagen protein expression at UVB irradiation (150 mJ / cm² 2 Based on the untreated sample group, the levels increased by 121% (10 μg / ml), 137% (30 μg / ml), and 149% (100 μg / ml), respectively.

[0079] Based on the above experimental results, it was confirmed that the expression of type 1 procollagen protein was significantly increased by the combination of the berberine compound and the Pulsatilla extract, and it was confirmed that the complex of the berberine compound and the Pulsatilla extract of the present invention is effective in improving skin wrinkles and skin elasticity. Therefore, it was confirmed that the complex of the berberine compound and the Pulsatilla extract of the present invention acts on the TGF-β / Smad signaling pathway to promote the production of type 1 procollagen, and thereby directly promotes collagen production, resulting in an excellent effect of improving skin elasticity.

[0081] Example 4. Skin wound regeneration promoting activity

[0082] The skin regeneration activity of berberine compounds and / or Pulsatilla extract was tested.

[0083] When human keratinocytes (HaCaT), which are primary cultured skin cells, proliferated to 80% in a 100 mm cell culture dish, the skin cells were wounded using the tip of a sterile tip (200 µl). The sample of Preparation Example 1 prepared in Example 1 was injected at concentrations of 0.1 µg / ml, 0.3 µg / ml, and 1 µg / ml, and the samples of Preparation Examples 2 and 3 were injected at concentrations of 10 µg / ml, 30 µg / ml, and 100 µg / ml, respectively. After 48 hours of injection, the cell regeneration effect was confirmed. Cell proliferation was measured by staining the cells using the MTT (methylthiazole-2-yl-2.5-diphenyl tetrazolium bromide) assay and the Diff-quic stain method, then examining the results under a microscope, and finally measuring the cell regeneration rate compared to the untreated group.

[0084] Referring to [Figure 3], which summarizes the measured cell regeneration rates, wound repair rates improved by 105% (0.1 μg / ml), 114% (0.3 μg / ml), and 136% (1 μg / ml) compared to the untreated sample group according to treatment with the berberine compound of Preparation Example 1, wound repair rates improved by 107% (10 μg / ml), 121% (30 μg / ml), and 129% (100 μg / ml) compared to the untreated sample group according to treatment with the Pulsatilla extract of Preparation Example 2, and wound repair rates improved by 112% (10 μg / ml), 138% (30 μg / ml), and 152% (100 μg / ml) compared to the untreated sample group according to treatment with the complex of the berberine compound and Pulsatilla extract of Preparation Example 3. Through the above results, it was confirmed that the complex of the berberine compound and Pulsatilla extract of the present invention exhibits a synergistic effect on skin regeneration compared to their single components.

[0085] In addition, when the complex of the berberine compound of Preparation Example 3 and the Pulsatilla extract was applied to damaged skin cells, it was observed that the morphological shape of the regenerated skin cells grew in the same way as normal skin cells.

[0087] Example 5. Anti-inflammatory activity

[0088] The inhibitory effect of amino acid mixtures and / or mineral complex fermented extracts on nitric oxide (NO) production was measured. Nitric oxide is a representative cytotoxic substance involved in inducing inflammation.

[0089] Human keratinocytes (HaCaT) were obtained from the Korean Cell Line Bank (KTCC, Seoul, Korea) and used. The cells were inoculated into DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS (Fetal bovine serum) supplemented with 10% FBS, 100 µg / ml penicillin, and 100 µg / ml streptomycin, and cultured at 37°C and 5% CO2.

[0090] The three samples prepared in Example 1 were each diluted to different concentrations (Preparation Example 1: 0.1 μg / ml, 0.3 μg / ml, and 1 μg / ml; Preparation Examples 2 and 3: 10 μg / ml, 30 μg / ml, and 100 μg / ml) and simultaneously treated with a new medium containing LPS (1 μg / ml), known as an endotoxin, and cultured for 24 hours. Afterward, 100 μl of the cell culture supernatant and 100 μl of Griess reagent [1% (w / v) sulfanilamide, 0.1% (w / v) naphtylethylenediamine in 2.5% (v / v) phosphoric acid] were mixed and reacted in a 96-well plate for 10 minutes.

[0091] After the Griess reagent reaction described above, the amount of nitric oxide produced was measured by measuring the absorbance at 540 nm using an ELISA reader. The concentration of the produced nitrite was calculated using a standard curve prepared by dissolving sodium nitrite in DMEM solution. For comparison, 10 μg / ml of dexamethasone was used as a positive control.

[0092] Referring to [Figure 4] which summarizes the experimental results, it was observed that the amount of nitric oxide produced increased by more than twofold with LPS treatment, and the amount of nitric oxide produced decreased in a concentration-dependent manner with treatment with berberine compounds and / or Pulsatilla extract.

[0093] Specifically, in the berberine compound treatment group, the production of nitric oxide decreased by 8% (0.1 μg / ml), 19% (0.3 μg / ml), and 33% (1 μg / ml), respectively, compared to the LPS treatment group (Con); in the Pulsatilla koreana extract treatment group, the production of nitric oxide decreased by 7% (10 μg / ml), 14% (30 μg / ml), and 38% (100 μg / ml), respectively, compared to the LPS treatment group (Con); and in the test group treated with a complex of berberine compound and Pulsatilla koreana extract, the production of nitric oxide decreased by 28% (10 μg / ml), 49% (30 μg / ml), and 62% (100 μg / ml), respectively, compared to the LPS treatment group (Con).

[0094] To summarize the above results, it was confirmed that berberine compounds or Pulsatilla extracts each significantly reduce the production of nitric oxide to a certain extent, but the effect is minimal, and that the combination of the two components exhibits an excellent synergistic effect in suppressing inflammatory responses.

[0096] Example 6. Skin moisturizing activity

[0097] Changes in hyaluronic acid (Hyaluronan) production following treatment with berberine compounds and / or Pulsatilla extract were measured.

[0098] Hyaluronic acid (HA, Hyaluronan, Hyaluronic acid) is primarily synthesized by epidermal keratinocytes and dermal fibroblasts and plays a crucial role in binding with moisture to create an environment where cells can function normally. A decrease in hyaluronic acid in the skin is one of the direct causes of reduced skin elasticity and moisture content. Therefore, this experiment investigated the production of hyaluronic acid within human fibroblasts.

[0099] Add 1 ml of culture medium to human immortalized keratinocytes (HaCaT) in a 24-well cell culture dish and approximately 1 × 10⁶ 5 After inoculating with cell concentrations, the samples were cultured for 24 hours at 37°C in a 5% CO2 environment. Subsequently, the samples of Preparation Example 1 were diluted to concentrations of 0.1 μg / ml, 0.3 μg / ml, and 1 μg / ml and treated, and the samples of Preparation Examples 2 and 3 were diluted to concentrations of 10 μg / ml, 30 μg / ml, and 100 μg / ml, respectively, and cultured for 24 hours. Afterward, the culture medium was harvested and centrifuged for 5 minutes at 4°C and 7,500 rpm, and the change in the production amount of hyaluronic acid (Hyaluronan kit, R&D Systems, Inc., Minneapolis, MN, USA) was checked using the ELISA method.

[0100] Referring to [Figure 5], which quantifies the change in the amount of hyaluronic acid produced, the test group treated with berberine compounds showed a minimal effect, with hyaluronic acid production increasing by 103% (0.1 μg / ml), 107% (0.3 μg / ml), and 109% (1 μg / ml), respectively, compared to the untreated negative control group. In addition, the test group treated with Pulsatilla koreana extract showed a hyaluronic acid production increase of 104% (10 μg / ml), 108% (30 μg / ml), and 112% (100 μg / ml), respectively, indicating that the effect of the single components on increasing hyaluronic acid production was not very significant.

[0101] In contrast, the amount of hyaluronic acid produced in the test group treated with the complex of the berberine compound and Pulsatilla extract of the present invention increased by 125% (10 μg / ml), 139% (30 μg / ml), and 149% (100 μg / ml), respectively, compared to the untreated group, and a synergistic effect on skin moisturization was observed due to the combination of these two components.

[0102] Through the above results, it was confirmed that although the skin moisturizing effect of berberine compounds or Pulsatilla extract is weak, the combination of these two ingredients significantly increases hyaluronic acid production, showing a synergistic effect on skin moisturization.

[0104] Example 7. Skin barrier strengthening activity

[0105] Changes in filaggrin (FLG, stratum corneum formation factor) gene expression following treatment with berberine compounds and / or Pulsatilla koreana extract were analyzed.

[0106] For the experiment, human keratinocytes (HaCaT) were used as the cell line, and cells were cultured at a rate of 1×10⁶ in DMEM medium supplemented with 10% FBS in 100 mm cell culture dishes. 6 The cells were inoculated at a concentration and cultured for 24 hours in a 37°C, 5% CO2 incubator. After culture, the samples from Preparation Example 1 were diluted to concentrations of 0.1 μg / ml, 0.3 μg / ml, and 1 μg / ml and treated, and the samples from Preparation Examples 2 and 3 were diluted to concentrations of 10 μg / ml, 30 μg / ml, and 100 μg / ml, respectively and treated, and additionally cultured for 24 hours under the same conditions. Subsequently, the cells were harvested using Trizol reagent (Invitrogen, USA), and RNA was isolated. RT-PCR was performed using a PCR instrument (Step One Plus, Applied Biosystems, USA), and CyberGreen (SYBR Green supermix, Applied Biosystems, USA) reagent was added along with profilaggrin, GAPDH, and cDNA to activate the polymerase at 94°C for 5 minutes, followed by 40 cycles of polymerization at 95°C for 30 seconds, 54°C for 1 minute, and 72°C for 1 minute.

[0107] The primers used are as follows:

[0108] - Propilaggrin: forward 5'-AAG CTT CAT GGT GAT GCG AC-3', reverse 5'-TCA AGC AGA AGA GGA AGG CA-3'

[0109] - GAPDH: Forward 5'-ACC ACA GTC CAT GCC ATC AC-3', Reverse 5'-CCA CCA CCC TGT TGC TGT AG-3'.

[0110] RT-PCR was performed using the above primers, and the experimental results are summarized in [Figure 6].

[0111] Filaggrin exists in the form of profilaggrin, a protein that forms keratohyalin granules in the granular layer of the epidermis, and then, after being broken down into filaggrin during the final differentiation process of keratinocytes, aggregates keratin filaments to form a hard and flat structure of keratinocytes when forming the membrane of keratinocytes, thereby performing the role of a brick in the skin barrier.

[0112] Referring to [Figure 6] which summarizes the experimental results, it was found that the mRNA expression of profilaggrin increased with treatment with berberine compounds and / or Pulsatilla extract, and the expression of the profilaggrin gene increased more distinctly in the group treated with the complex of these compounds compared to the individual single-component treatment groups.

[0113] Specifically, compared to the untreated sample group, the mRNA expression of propyllagrin increased by 105% (0.1 μg / ml), 112% (0.3 μg / ml), and 119% (1 μg / ml) in the test group treated with the berberine compound of Preparation Example 1, and by 107% (10 μg / ml), 110% (30 μg / ml), and 121% (100 μg / ml) in the test group treated with the Pulsatilla extract of Preparation Example 2, respectively; whereas, in the test group treated with the complex of Preparation Example 3, the mRNA expression of propyllagrin increased by 115% (1% v / v), 129% (3% v / v), and 143% (10% v / v) respectively.

[0114] Through the above results, it was confirmed that the complex of the two components of the present invention, which combines a berberine compound and an anemone extract, increases the expression of the profilaggrin gene and contributes to the formation of a skin barrier, thereby showing excellent effects in improving skin condition.

[0116] Example 8. Effect of promoting vascular endothelial cell migration (HUVECs migration)

[0117] We measured and analyzed the motility of vascular endothelial cells, which is utilized as a common method for assessing angiogenesis. Boyden's chamber (Transwell) was used to analyze cell motility.

[0118] 600 µl of serum-free M199 culture medium was placed in each 24-well plate. The culture medium was treated with the sample of Preparation Example 1 prepared in Example 1 at concentrations of 0.1 µg / ml, 0.3 µg / ml, and 1 µg / ml, and the samples of Preparation Examples 2 and 3 were treated with concentrations of 10 µg / ml, 30 µg / ml, and 100 µg / ml, respectively. Gelatin (1 mg / ml) was spread on the bottom surface of the transwell, and 2 x 10⁶ on the top surface 4 Human umbilical vein endothelial cells (HUVECs) were attached at cell concentrations.

[0119] After about 4 hours, the cells on the upper surface were removed using a cotton swab, and the cells that had moved to the lower surface were stained with hematoxylin and eosin, and the number of stained cells was counted and measured.

[0120] Referring to [Figure 7] which summarizes the experimental results, it was confirmed that a combination of two components could further promote the migration of human umbilical vein endothelial cells (HUVEC) compared to a single component of berberine compound or Pulsatilla extract.

[0121] Specifically, treatment with TNF-α at 20 ng / ml reduced the migration of human umbilical vein endothelial cells (HUVECs) by more than 20%, but the berberine compound (Preparation Example 1) treatment group showed migration rates of 105%, 118%, and 127% increased compared to the TNF-α treatment group at concentrations of 0.1 μg / ml, 0.3 μg / ml, and 1 μg / ml, respectively, and the Pulsatilla extract treatment group (Preparation Example 2) showed migration rates of 107%, 115%, and 121% increased compared to the TNF-α treatment group at concentrations of 10 μg / ml, 30 μg / ml, and 100 μg / ml, respectively. In contrast, the migration rate of human umbilical vein endothelial cells was observed to be increased by 109%, 124%, and 139%, respectively, compared to the TNF-α treatment group at concentrations of 10 μg / ml, 30 μg / ml, and 100 μg / ml for the two-component complex treatment group (Preparation Example 3).

[0122] Through the results above, it was confirmed that the angiogenesis-promoting effect is further improved through the combination of two components: berberine compounds and Pulsatilla extract.

[0123] To summarize the experimental results above, it was confirmed that the complex of berberine compounds and Pulsatilla extract i) has no cytotoxicity and promotes cell growth, ii) effectively prevents wrinkles by promoting the production of type 1 procollagen and improves skin elasticity by directly promoting collagen production, iii) promotes skin wound healing, iv) exhibits anti-inflammatory effects by inhibiting nitric oxide production, v) shows excellent skin moisturizing effects by promoting hyaluronic acid production, vi) strengthens skin barrier formation by increasing the expression of the profilaggrin gene, and vii) promotes angiogenesis by increasing the motility of vascular endothelial cells, thus confirming that it can be usefully utilized in cosmetics, food, pharmaceuticals, and quasi-pharmaceuticals for improving skin condition.

[0124] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 Berberine and pasqueflower Pulsatilla koreana Contains a complex of two components of ) extract as an active ingredient, wherein the above berberine and the above pasque flower ( Pulsatilla koreana A cosmetic composition for promoting skin wound regeneration, strengthening the skin barrier, and promoting the migration of vascular endothelial cells, characterized in that the extract is blended in a weight ratio of 1:90-110, the two-component complex promotes the regeneration of skin wounds, the two-component complex promotes neovascularization by promoting the migration of vascular endothelial cells, and the two-component complex strengthens the skin barrier by increasing filaggrin gene expression. Claim 2 delete Claim 3 A cosmetic composition for promoting skin wound regeneration, strengthening the skin barrier, and promoting the migration of vascular endothelial cells, characterized in that, in claim 1, the two-component complex further exhibits skin wrinkle improvement and skin elasticity improvement activities by increasing the expression of type 1 procollagen protein. Claim 4 delete Claim 5 A cosmetic composition for promoting skin wound regeneration, strengthening the skin barrier, and promoting the migration of vascular endothelial cells, characterized in that, in claim 1, the two-component complex further exhibits anti-inflammatory activity by inhibiting the production of nitric oxide, an inflammatory mediator. Claim 6 A cosmetic composition for promoting skin wound regeneration, strengthening the skin barrier, and promoting the migration of vascular endothelial cells, characterized in that, in claim 1, the two-component complex further exhibits skin moisturizing activity by promoting hyaluronic acid synthesis. Claim 7 delete Claim 8 Berberine and pasqueflower Pulsatilla koreana Contains a complex of two components of ) extract as an active ingredient, wherein the above berberine and the above pasque flower ( Pulsatilla koreana A food composition for promoting skin wound regeneration, strengthening the skin barrier, and promoting the migration of vascular endothelial cells, characterized in that the extract is blended in a weight ratio of 1:90-110, the two-component complex promotes the regeneration of skin wounds, the two-component complex promotes neovascularization by promoting the migration of vascular endothelial cells, and the two-component complex strengthens the skin barrier by increasing filaggrin gene expression. Claim 9 Berberine and pasqueflower Pulsatilla koreana Contains a complex of two components of ) extract as an active ingredient, wherein the above berberine and the above pasque flower ( Pulsatilla koreana A quasi-drug composition for promoting skin wound regeneration, strengthening the skin barrier, and promoting the migration of vascular endothelial cells, characterized in that the extract is blended in a weight ratio of 1:90-110, the two-component complex promotes the regeneration of skin wounds, the two-component complex promotes neovascularization by promoting the migration of vascular endothelial cells, and the two-component complex strengthens the skin barrier by increasing filaggrin gene expression.

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