WO2023008768A1 - Cosmetic composition for skin improvement, comprising recoflavone or salt thereof - Google Patents

Abstract

The present invention relates to a cosmetic composition and a pharmaceutical composition, which comprise, as an active ingredient, Recoflavone or a salt thereof, and thus have an antioxidant effect and the excellent effects of skin whitening, elasticity, wrinkle reduction, moisturizing, anti-inflammation or the like, particularly, excellent skin-related effects.

Description

Cosmetic composition for skin improvement containing lecoflavone or a salt thereof

The present invention relates to a cosmetic composition having excellent effects such as anti-oxidation, skin whitening, elasticity, wrinkle improvement, moisturizing and anti-inflammatory, and particularly excellent in anti-aging of the skin.

The skin is the largest organ of the human body, and has a great influence on appearance and image. It plays an important role in maintaining biological homeostasis such as function, excretion function, respiratory function, body temperature regulation, and immune function. However, in recent years, various factors that threaten skin health, such as worsening air pollution such as yellow dust or fine dust, strong ultraviolet rays, and overwork and stress in daily life, are gradually increasing. The accumulation of these skin harmful factors causes skin inflammation and skin barrier damage due to the generation of active oxygen, and accelerates skin aging.

Skin aging can be largely divided into intrinsic aging and extrinsic aging (photoaging). The clinical features of endogenous aging are relatively mild, and include fine wrinkles, skin dryness, and loss of elasticity. In the case of extrinsic aging (photoaging), it is characterized by the occurrence of thick and deep wrinkles and fine wrinkles compared to intrinsic aging, and in severe cases, it causes skin dryness, loss of elasticity, and pigment diseases, or increases the probability of occurrence of benign tumors and skin cancer.

The elasticity or moisturizing of the skin is maintained by moisture present in the stratum corneum of the epidermis, which is the outermost layer of the skin, and collagen present in the dermis. The moisture content of the stratum corneum is determined by the sebaceous membrane, which is a mixture of lipids produced and secreted from the epidermis, and natural moisturizing factor (NMF), a water-soluble component present in the stratum corneum. The stratum corneum of healthy epidermis contains 15-20% water, and when the water content falls below 10%, the skin becomes dry, loses luster and elasticity, and wrinkles increase.

Collagen is a major matrix protein produced by fibroblasts of the skin and is present in the extracellular matrix. Its important functions include mechanical robustness of the skin, resistance and cohesiveness of connective tissue, support of cell adhesion, cell division and differentiation. (in the growth of organisms or wound healing), etc. are known. Collagen increases the enzymatic activity of collagenase that decomposes it, so that collagen reduction is accelerated. In particular, the reduction is promoted by an increase in age and photoaging by ultraviolet irradiation. It is known that it is closely related to the formation of wrinkles in the skin. In addition, elastin fibers form a cross-linked bond with collagen and are an important skin component for wrinkle formation, which is involved in skin elasticity. Deficiency and aggregation of elastin fibers and significant increase in the activity of elastase, an elastin degrading enzyme, have been found to be one of the causes of skin wrinkles. Currently, retinol, retinyl palmitate, adenosine, TGF, animal placenta-derived protein, chlorella extract, and the like are known as wrinkle-improving cosmetics. Retinol, the most well-known, is a substance that promotes collagen synthesis and inhibits the elastase enzyme, but is unstable and has limitations in usage due to safety issues such as irritation and redness when applied to the skin. It is known that it is difficult to expect wrinkle improvement effects.

The color or brightness of the skin is genetically determined according to the concentration and distribution of melanin in the human skin, but it is also influenced by environmental or physiological conditions such as solar ultraviolet rays, fatigue or stress. Melanin is sequentially converted into DOPA and Dopaquinone by enzymes such as Tyrosinase, TRP-1 (Tyrosinase related protein-1), and TRP-2 from Tyrosine, which is a kind of amino acid. , it is produced through a non-enzymatic oxidation reaction. Although the pathway by which melanin is produced is known, the cause of triggering tyrosinase in the mechanism inducing melanin synthesis has not yet been clarified in detail. On the other hand, as commonly known whitening ingredients, substances that inhibit tyrosinase enzyme activity such as kojic acid or arbutin, paper mulberry extract, oil-soluble licorice extract, niacinamide, alpha-B There are Alpha-Bisabolol, Hydroquinone, Vitamin C (L-Ascorbic acid) or their derivatives, and various plant extracts. However, when these substances are applied to the skin, there are limitations in usage due to safety issues such as skin irritation and redness, or they affect the stability of formulations such as precipitation and browning, or have insignificant effects. There are unexpected problems.

Inflammation is an immune response of the human body in response to wounds or diseases, and oxidative stress such as ultraviolet rays, active oxygen, and free radicals activate inflammatory factors to cause various diseases and aging of the skin. In general, substances used for the purpose of anti-inflammatory include non-steroidal anti-inflammatory drugs (anti-inflammatory drugs) or steroid-based anti-inflammatory drugs (anti-inflammatory drugs). Among these, steroids are the most frequently used drugs in dermatological diseases due to their strong anti-inflammatory effects. However, the biggest problem is that its use is limited due to side effects such as skin atrophy, stretch marks, skin ulcers, infection, acne, etc., or resistance to drugs when used for a long time. As an alternative, a non-steroidal system is used, but there are side effects different from those of steroid ointments, such as skin weakness at the site of use of the ointment or skin sensitivity to sunlight, which limits its use. Therefore, it is urgently needed to develop active ingredients that are safe without side effects and have excellent material stability when applied to the human body, while having better anti-aging effects on the skin than previously known substances with antioxidant, skin whitening, elasticity, wrinkle improvement, moisturizing and anti-inflammatory effects. is being demanded

Accordingly, the inventors of the present invention completed the present invention by discovering that lecoflavone can improve various skin problems while looking for a material that can solve various problems of the skin at the same time. Accordingly, an object of the present invention is to provide a substance for skin improvement or a composition of the substance containing lecoflavone or a salt thereof as an active ingredient.

In order to achieve the above object, the present invention provides a substance for skin improvement or a composition of the substance containing lecoflavone or a salt thereof as an active ingredient.

The composition may be a pharmaceutical or cosmetic composition.

The skin improvement may be selected from the group consisting of antioxidant, skin inflammation improvement, skin aging prevention, skin protection, skin moisturizing, skin whitening, skin wrinkle improvement, and combinations thereof.

The present invention also provides a composition for the prevention or treatment of hyperpigmentation disease comprising lecoflavone or a salt thereof as an active ingredient.

The present invention also provides a composition for preventing or treating xeroderma, comprising lecoflavone or a salt thereof as an active ingredient.

The present invention also provides a skin improvement method comprising the step of treating the skin of a subject with lecoflavone or a salt thereof.

The composition for improving skin according to the present invention contains lecoflavone or a salt thereof as an active ingredient, thereby providing antioxidation, improvement of skin inflammation, prevention of skin aging, skin moisturizing, skin whitening, improvement of skin wrinkles, prevention or treatment of hyperpigmentation diseases, etc. Having a variety of effects, it is possible to significantly improve the skin.

The composition for skin improvement according to the present invention contains lecoflavone or a salt thereof as an active ingredient, thereby exhibiting anti-aging effects, specifically anti-oxidation, skin inflammation improvement, skin moisturizing, skin whitening, skin elasticity enhancement, and skin wrinkle prevention effects. , Ultimately, it has the advantage of excellent skin anti-aging effect. In addition, skin improvement can be expected through various effects such as prevention or treatment of hyperpigmentation disease or xeroderma.

1 is a graph showing the collagen production ability of the group treated with lecoflavone according to the present invention.

Figure 2 is a graph showing the melanin production ability of the group treated with lecoflavone according to the present invention.

Figure 3 is a graph showing the aquaporin 3 mRNA expression level in the group treated with recoflavone according to the present invention.

Figure 4 is a graph showing the amount of TNF-α mRNA expression in the group treated with recoflavone according to the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention provides a composition for skin improvement comprising lecoflavone or a salt thereof as an active ingredient. Specifically, the lecoflavone is a compound having the structure of Formula 1 below, which is also called the chemical name of 7-carboxymethyloxy-3',4',5-trimethoxy flavone:

[Formula 1]

As one aspect, the present invention may be a pharmaceutical composition or a cosmetic composition.

In one aspect, the skin improvement may be selected from the group consisting of antioxidant, skin inflammation improvement, skin aging prevention, skin moisturizing, skin whitening, skin wrinkle improvement, and combinations thereof.

In one aspect, the skin inflammation improving composition may exhibit an anti-inflammatory effect on various inflammation-related diseases of the human body as well as skin inflammation, and in particular, may exhibit an anti-inflammatory effect on inflammation and related diseases caused by fine dust.

In addition, the composition according to the present invention may have synergistic effects in antioxidation, skin inflammation improvement, skin aging prevention, skin moisturizing, skin whitening, or skin wrinkle improvement by including lecoflavone or a salt thereof as an active ingredient.

The composition according to the present invention may improve skin, and the skin improvement may be selected from the group consisting of antioxidant, skin inflammation improvement, skin aging prevention, skin moisturizing, skin whitening, skin wrinkle improvement, and combinations thereof.

Recoflavone inhibits ROS and exhibits antioxidant activity. Aerobic organisms use oxygen for energy metabolism. When oxygen in a living body is subjected to various physical, chemical and biological stresses, O ₂ -(superoxide), NO·(nitric oxide), NO ₂ ·(nitrogen dioxide), OH Harmful active oxygen species such as radicals such as ·(hydroxyl), ROO·(proxyl), RO·(alkoxyl), NO ₃ ·(peroxynitrite), and ABTS+ are generated. These reactive oxygen species are unstable and have high oxidizing power, so they easily react with biomaterials and cause stress in the skin. It oxidizes proteins and lipids in cell membranes in the body to cause inflammation, damages DNA to cause mutations or cancer, and attacks collagen and skin fibers that give skin elasticity, causing skin aging and wrinkle formation. It is important to discover antioxidant substances or materials that remove active oxygen species, such as reactive oxygen species. Specifically, it was confirmed that lecoflavone exhibits antioxidant activity.

Recoflavone inhibits MAPK and NF-Kb, and inhibits the expression of cytokines involved in inflammatory responses. Inflammatory cytokines are secreted from immune cells to kill and neutralize foreign antigens when they are introduced. Inflammatory cytokines involved in this case typically include tumor necrosis factor (TNF-α), interleukin-1 (IL-1), and interleukin-8 (IL-8).

Therefore, inhibition of the expression of these inflammatory cytokines can alleviate the inflammatory response. Specifically, recoflavone exhibits an anti-inflammatory effect by significantly reducing the expression level of TNF-α mRNA induced by fine dust.

Recoflavone inhibits MMP production through inhibition of MAPK and NF-kB, and enhances the production of procollagen, thereby promoting collagen production, and these actions can improve or prevent skin aging or wrinkles. .

Also, in the present invention, the “prevention of skin aging” means suppression or delay of skin aging by using the lecoflavone of the present invention.

In addition, in the present invention, the "improvement of skin wrinkles" means maintaining or strengthening the ability related to wrinkles and elasticity of the skin. Skin wrinkles are known to be caused by a decrease in collagen, a major component of the extracellular matrix. Collagen is a major matrix protein produced by dermal fibroblasts, accounting for about 30% of the total weight of biological proteins.The main functions of collagen are mechanical firmness of skin, resistance and cohesion of connective tissue, and cell adhesion. Support, induction of cell division and differentiation, etc. In addition, collagen is a matrix protein that plays an important role in retaining water in the dermal layer, and these matrix proteins adsorb water and increase the water retention capacity within the structure they form, making the skin more beautiful. It is known to be involved in maintaining the elasticity of the skin, allowing it to maintain a state containing appropriate moisture.On the other hand, in the dermis, as human skin fibroblasts age, the productivity of fibers and matrix decreases, and cell My peroxide level increases rapidly, and I am affected by various contaminants and stress, so that the function of the cell cannot be performed smoothly, and the metabolic activity of the cell is lowered. As a result, the formation of wrinkles is promoted due to functional and structural damage.

Recoflavones increase the expression of aquaporins. Aquaporin is a type of membrane protein and is known to play a role as a water channel. Several types of aquaporin family are known, but among them, aquaporin-3 is known to play the most important role in water passage, so research on substances activating aquaporin-3 is active. This is because the substance that activates aquaporin-3 is related to the moisturizing function of the skin.

Lecoflavones inhibit the production of melanin. The melanin production mechanism uses intracellular tyrosine as a substrate and an enzyme called tyrosinase produces DOPAquinone, and melanin is produced from dopaquinone through an autoxidation reaction and an enzymatic reaction. In addition, inflammatory substances expressed by stress such as ultraviolet rays and harmful environments are expressed in keratinocytes, and the expressed inflammatory substances activate melanocytes, which are melanin-producing cells, to form melanosomes. Stimulates the synthesis of melanin. In addition, expression of enzymes such as tyrosinase, TRP-1 (Tyrosinase related protein-1) and TRP-2, transport of melanin in melanocytes, transfer of melanin from melanocytes to keratinocytes ( pigmentation occurs by accelerating the transparent process. When such a series of processes occur excessively, the skin tone is darkened (pigmentation), and melasma, freckles, melanoma, and the like occur. Therefore, in order to prevent this pigmentation phenomenon, it can be suppressed by controlling some processes in the melanin production process.

In addition, the present invention can be used for preventing, improving, or treating diseases related to hyperpigmentation according to the melanin production inhibitory effect of lecoflavone. Accordingly, another aspect of the present invention provides a composition for preventing or treating a disease related to hyperpigmentation comprising lecoflavone. Specifically, the hyperpigmentation disease is at least one disease selected from the group consisting of melasma, freckles, spots, solar lentigo, drug-induced pigmentation, inflammatory pigmentation, senile pigmentation, and pregnancy-induced pigmentation.

When the composition according to the present invention is a cosmetic composition, it contains lecoflavone as an active ingredient, and can be used as a cosmetic composition, such as a pH adjuster, a sweetener, a binder, a surfactant, a sequestering agent, a preservative, a bactericidal preservative, an anti-discoloration agent, a moisturizer, Opacifier, antioxidant, detergent, astringent, solvent, emulsion stabilizer, emulsifier, sunscreen, viscosity reducing agent, viscosity increasing agent, antistatic agent, colorant, flavoring agent, film forming agent, skin protectant, skin softener, skin conditioning agent , It can be prepared in the form of a basic cosmetic composition, a color cosmetic composition, a hair or scalp product composition, and soap, together with a hair conditioning agent, a suspending agent, and an absorbent. For example, the formulation of the cosmetic composition of the present invention is not limited thereto, but skin lotion, skin softener, skin toner, astringent lotion, softening lotion, nutrient lotion, astringent, lotion, milk lotion, moisture lotion, nutrient lotion, body cream , Massage Cream, Nutrition Cream, Moisture Cream, Hand Cream, Essence, Nutrition Essence, Pack, Soap, Shampoo, Face Wash (Cleansing Foam, Cleansing Lotion, Cleansing Cream, Cleansing Water, Soap), Mask Pack, Body Oil, Body Lotion, It may be a formulation such as body cleanser, treatment, beauty essence, lotion, foundation, lipstick, makeup base, press powder, loose powder, concealer, eye shadow, shampoo, rinse, hair conditioner, hair gel, and hair vinegar.

Examples of the excipients include, but are not limited to, pH adjusters, sweeteners, binders, surfactants, sequestering agents, preservatives, bactericidal preservatives, anti-discoloration agents, moisturizers, opacifiers, antioxidants, detergents, astringents, solvents, Emulsion stabilizer, emulsifier, sunscreen, viscosity reducing agent, viscosity increasing agent, antistatic agent, coloring agent, flavoring agent, film forming agent, skin protectant, skin softener, skin conditioning agent, hair conditioning agent, suspending agent, absorbent, etc. can be included with For example, when preparing a face wash and soap with the cosmetic composition of the present invention, it can be easily prepared by adding the lecoflavone of the present invention to a conventional face wash and soap base. In the case of preparing a cosmetic composition, it can be prepared by adding the lecoflavone of the present invention to a conventional cosmetic composition base. Herein, synthetic or natural materials such as flavors, chelating agents, pigments, antioxidants, preservatives, and proteins, minerals, and vitamins for the purpose of improving physical properties may be additionally added.

The content of lecoflavone contained in the cosmetic composition of the present invention is not limited thereto, but may be 0.00001 to 10 weight (w/v)% or 0.0001 to 5 weight (w/v)% based on the total weight of the composition. but is not limited thereto.

In another aspect, the present invention provides a pharmaceutical composition for improving excessive pigmentation, atopic dermatitis, scarring, or dry skin, containing lecoflavone of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The composition containing lecoflavone of the present invention may be in various oral or parenteral formulations, but preferably may be a parenteral formulation. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose or lactose ( lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, and emulsions. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.

The present invention also relates to the improvement of hyperpigmentation such as melasma, freckles, spots, sunburn, drug-induced pigmentation, inflammatory pigmentation, senile pigmentation, pregnancy-related pigmentation, etc., including lecoflavone as an active ingredient, atopic dermatitis, It can be provided in the form of an external skin preparation for improving dry skin including xeroderma, such as scar improvement and senile dryness, and dermatitis.

When lecoflavone is used as an external skin preparation, additional buffers, solvents, solubilizers, softeners, emulsifiers, thickeners, colorants, flavoring agents, penetrating agents, coating agents, chelating agents, antioxidants, reducing agents, lubricants, suspending agents, pH adjuster, plasticizer, solubilizer, sweetener, gelling agent, binder, curing agent, surfactant, corrigent, base, foaming agent, preservative, release control agent, denaturant, excipient, dispersant, opacifying agent, disintegrant, oxidizing agent, sustained release agent, antifoaming agent , stabilizers, alkalizing agents, emollients, warming agents, etc., and any other ingredients commonly used in external preparations for the skin, and adjuvants commonly used in the field of dermatology. In addition, the components may be introduced in an amount generally used in the field of skin science.

When the pharmaceutical composition is provided in a formulation for external application to the skin, it may have a formulation such as, but not limited to, an ointment, patch, gel, cream or spray.

The pharmaceutical composition of the present invention may be particularly preferably used as a parenteral preparation, and for example, the external skin preparation may include a suitable pharmaceutically acceptable base such as petrolatum and stearyl alcohol; suitable pharmaceutically acceptable surfactants such as polysorbate and sorbitan sesquioleate; suitable pharmaceutically acceptable moisturizers such as glycerin; suitable pharmaceutically acceptable solvents; and a flavoring agent, a colorant, a stabilizer, a viscosifying agent, and the like can be prepared by a conventional method for preparing an external preparation for skin.

In addition, when the composition of the present invention is a pharmaceutical composition, it may contain at least one active ingredient exhibiting the same or similar function in addition to the active ingredient lecoflavone. For example, it may include known skin whitening, elasticity enhancement, wrinkle improvement, moisturizing effects, and anti-inflammatory components. When additional skin whitening, elasticity enhancement, wrinkle improvement, moisturizing effects and anti-inflammatory components are included, the skin whitening, elasticity enhancement, wrinkle improvement and moisturizing effects of the composition of the present invention can be further enhanced. When adding the above components, skin safety according to combined use, ease of formulation, and stability of active ingredients may be considered. In one embodiment of the present invention, the composition is a hyperpigmentation improving component known in the art, hydroquinone, vitamin-C, nicotinic acid amide, glutathione, tranexamic acid; As anti-inflammatory ingredients known in the art, COX-2 inhibitors such as celecoxib or pyrocoxib, naproxen, ibuprofen, flubiprofen, ketoprofen, loxoprofen, diclofenac, etodolac, indomethacin , NSAID anti-inflammatory agents such as mefenamic acid, piroxicam, and clonixin ricinate, steroid-based anti-inflammatory agents such as prednisone, prednisolone, methylprednisolone, triamcinolone, desonide, corticosterone, betamethasone, and dexamethasone, and allantoin; As the dry skin improving component, D-panthenol, tocopherol acetate, allantoin, heparinoid, diphenhydramine, urea; And it may further include one or two or more components selected from the group consisting of derivatives thereof and various plant extracts. Additional components may be included in an amount of 0.0001 wt (w / v)% to 10 wt (w / v)% based on the total weight of the composition, and the content range is skin safety, ease of formulation of the compound of Formula 1 etc. can be adjusted according to the requirements.

When the pharmaceutical composition of the present invention contains an effective amount of the compound of Formula 1, it is preferable to improve hyperpigmentation such as liver spots, melasma, freckles, and senile dark spots, improve dry skin, and improve dermatitis. effect can be provided.

In the present invention, the term 'effective amount' means an agent capable of improving hyperpigmentation such as liver spots, melasma, freckles, and senile dark spots, atopic dermatitis, dry skin, and dermatitis. amount of the compound.

The effective amount of the lecoflavone contained in the composition of the present invention will vary depending on the form in which the composition is commercialized, how the compound is applied to the skin, and how long the compound stays on the skin. For example, when the composition is commercialized as a pharmaceutical product, it may contain the lecoflavone at a higher concentration than when the composition is commercialized as a cosmetic product that is routinely applied to the skin.

As another aspect, the present invention may be a skin improvement method comprising the step of treating a subject with lecoflavone of Chemical Formula 1 or a salt thereof.

In a specific aspect, the subject may be a human or mammal.

The method of treating the subject may be a method of orally or parenterally administering an effective amount of lecoflavone or a salt thereof to the subject. Methods of such oral or parenteral administration are well known in the art and an appropriate method can be selected.

The skin improvement may be one selected from the group consisting of excessive pigmentation improvement, atopic dermatitis, scar improvement, dry skin improvement, and combinations thereof.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it is to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. It will be self-evident.

[Example]

In order to confirm the skin improvement effect of lecoflavone, an active ingredient of the composition according to the present invention, cytotoxicity, collagen production ability, melanin production inhibition ability, Aqauporin 3 mRNA expression, TNF-α mRNA expression, and ORAC index were analyzed through the following tests. did

Recoflavone used in this experiment was prepared according to the preparation method of WO 98/04541.

1) All tests were repeated three times, and the results were expressed as mean ± standard deviation.

2) The values measured as a result of cytotoxicity, collagen production ability, melanin production inhibition ability, Aquaporin 3 mRNA expression and TNF-α mRNA expression test were analyzed using the MannWhitney U test after each Kruskal-Wallis test, and Bonferroni correction was performed (p <0.0125).

3) ORAC index test results The measured values were analyzed using the Mann-whitney U test (p<0.05).

4) Each value measured as a result of the test was checked for significance using IBM SPSS statistics version 21.0 with a hypothesized mean difference of 5% (p<0.05).

Reference Example 1: Cell line selection and cell culture

Human dermal fibroblast (HDF) cell line (Lonza), B16F10 cell line (Korea Cell Line Bank) and HaCaT cell line to evaluate the cytotoxicity, collagen production ability, melanin production inhibition ability, Aquaporin3 mRNA expression and TNF-α mRNA expression of lecoflavone (tentative name) (Addexbio) was used. All cells were inoculated at 1 × 10 ⁶ cells/dish in a 100 mm ² culture dish, and then Dulbecco's Modified containing penicillin (100 IU/ml), streptomycin (100 μg/mL), and 10% Fetal Bovine Serum (FBS). Eagle's Medium (DMEM) was added and cultured in an incubator (Sanyo, Japan) containing 37°C and 5% carbon dioxide.

Example 1: Confirmation of cytotoxicity

Cytotoxicity was conducted through a well-known MTT assay, and did not show cytotoxicity in all cell types and all test concentrations used in the test.

As described above, it was confirmed that lecoflavone, an active ingredient of the composition of the present invention, does not affect the toxicity of various cells such as fibroblast, melanocyte, and keratinocyte at all test concentrations.

Example 2: Confirmation of collagen production ability

Collagen is produced intracellularly as procollagen and then secreted extracellularly to polymerize into collagen fibers. At this time, the N-terminal and C-terminal propeptides of procollagen are released by endopeptidase. Therefore, the ELISA test method for detecting type I collagen in cells with the Procollagen Type I C-Peptide PIP EIA kit, which quantifies the C-terminal peptide (PIP) of human procollagen type I using a monoclonal antibody, is used to measure the amount of collagen in cells. This is a useful test method.

In this Example 2, HDF cells subcultured according to the intracellular collagen production test method of the Guidelines for Efficacy Evaluation of Functional Cosmetics of the Ministry of Food and Drug Safety were used, and the final treatment concentration was 0.001 (w / v)%, 0.005 ( w/v)%, 0.01(w/v)% Recoflavone was treated to be 0.1(w/v)%, and the positive control group (Transforming Growth Factor beta, TGF-β) had a final treatment concentration of 2.5 ng /mL, 5 ng/mL, 10 ng/mL, 20 ng/mL were treated in each well and cultured for 24 hours under cell culture conditions, and then Procollagen Type I C-Peptide PIP EIA kit (Takara, Japan) was used. The amount of collagen production was measured and the amount of protein was obtained from the cells.

The results are shown in Figure 1. As can be seen in the figure, the active ingredient of the composition according to the present invention, lecoflavone, is 1.50% at 0.001 (w / v)% concentration, 3.76% at 0.005 (w / v)% concentration, 0.01 (w / v) )% concentration and 8.95% at 0.1 (w / v)% concentration, respectively, it was confirmed that the collagen production ability increased compared to the untreated group and could help improve skin wrinkles.

Example 3: Melanin production inhibition test

Melanin, which determines the color of the skin, is a reddish brown or dark brown polymer compound produced by the oxidation reaction of tyrosinase enzyme in the melanosomes of melanin-forming cells. It is known that excessively produced and accumulated melanin can cause skin diseases. As a method for evaluating the degree of inhibition of cell melanin production, mouse melanoma B16F10 cells can be used to evaluate the melanin production inhibition ability of a sample by measuring the amount of melanin produced intracellularly and melanin secreted extracellularly.

In this Example 3, B16F10 cells subcultured according to the intracellular melanin production inhibition test method of the Guidelines for Effectiveness Evaluation of Functional Cosmetics were used, and the final treatment concentration was 0.001 (w / v)%, 0.005 (w / v) %, 0.01 (w / v)%, 0.1 (w / v)% Recoflavone was treated, respectively, and the final treatment concentration was 0.00125 (w / v)%, 0.0025 (w / v)%, 0.005 (w / v)%, 0.01 (w / v)% diluted positive control (arbutin) was treated, respectively, and cultured for 72 hours in cell culture conditions, and then the amount of extracellular melanin production was measured using a microplate reader, The amount of intracellular melanin and protein were determined from the cells. IBMX (3-Isobutyl-1-methylxanthine) was used as a melanin production inducing substance.

The results are shown in FIG. 2 . As can be seen from the figure, the active ingredient of the composition according to the present invention, lecoflavone, is 2.72% at 0.001 (w / v)% concentration, 6.49% at 0.005 (w / v)% concentration, 0.01 (w / v) 7.31% at % concentration and 10.14% at 0.1 (w/v)% concentration showed melanin production inhibitory ability, respectively.

Example 4. Aquaporin 3 and TNF-α mRNA expression test

RT-qPCR is a test method that can quantitatively analyze the expression of a specific mRNA in cells by monitoring the amount of amplification product in real time by measuring the fluorescence generated in the process of amplifying the DNA product. In this experiment, changes in the expression levels of Aquaporin 3 and TNF-α mRNA according to sample treatment were measured using RT-qPCR. The change in Aquaporin 3 mRNA expression level was 0.0005 (w/v)%, 0.001 (w/v)%, 0.005 (w/v)%, 0.01 (w/v) at the final treatment concentration using subcultured HaCaT cells. Cells were treated with Recoflavone to be 0.5 µM, 1 µM, 2 µM, and 4 µM for the positive control group (retinoic acid) and cultured in cell culture conditions for 24 hours. After culturing, RNA was extracted from the cells and cDNA was generated and measured by performing RT-qPCR. Changes in the expression level of TNF-α mRNA were observed at final treatment concentrations of 0.0005 (w/v)%, 0.001 (w/v)%, 0.005 (w/v)%, and 0.01 (w/v)% using subcultured HaCaT cells. )% of Recoflavone and 1 μg/mL of fine dust (Sigma, USA) were treated on the cells together, and the positive control (Dexamethasone ) and fine dust (Sigma, USA) 1 μg/mL were treated with cells and cultured for 24 hours under cell culture conditions, RNA was extracted from the cells, cDNA was generated, and RT-qPCR was measured.

The results are shown in Figures 3 to 4. As can be seen from the figure, the active ingredient of the composition according to the present invention, lecoflavone, is 1.18 at 0.0005 (w / v)% concentration, 1.34 at 0.001 (w / v)% concentration, and 0.005 (w / v)% concentration It was confirmed that the Aquaporin 3 mRNA expression level of 1.83 was shown at 1.57 and 0.01 (w / v)% concentrations, respectively, indicating a moisturizing effect. In addition, as can be seen in Figure 4, lecoflavones are 1.58 at 0.0005 (w / v)% concentration, 1.46 at 0.001 (w / v)% concentration, 1.39 at 0.005 (w / v)% concentration, 1.31 at 0.01% concentration It was confirmed that the anti-inflammatory effect was shown by showing the expression level of TNF-α mRNA.

Example 5. ORAC index confirmation test

Active oxygen is naturally produced by metabolism and enzymes of intracellular organs and plays a role in controlling substance reactions. However, when excessive active oxygen is generated, it may cause skin aging through skin cell and tissue damage. It is known that cell damage can be prevented by scavenging free radicals using antioxidants to protect skin cells and delay or prevent aging.

ORAC (Oxygen Radical Absorption Capacity) assay is a test method that measures the ORAC index of a sample by using the decrease in fluorescence by peroxyl radical, a type of free radical. It is a test method using the ORAC Antioxidant Assay Kit (Zen-Bio, USA). . Fluorescein, a fluorescent dye used in ORAC assay, loses its fluorescence due to oxidation. Due to the anti-oxidation ability of the sample, the oxidation of fluorescein is suppressed and the fluorescence lasts for a long time. In other words, by using the principle that the fluorescence destruction rate is reduced by the antioxidant when the fluorescence is oxidized and the fluorescence is reduced, the net area under the fluorescence reduction curve depicting the change in fluorescence value is calculated, and it is calculated. The ORAC index of a sample can be calculated by substituting it into a calibration curve prepared with trolox, a standard antioxidant.

In this example, in each well of a 96-well plate, 150 μL of fluorescein sodium salt solution was mixed with 25 μL of sample lecoflavone (tentative name) (5% sol) diluted as follows or 25 μL of positive control (5 ppm ascorbic acid solution), respectively. did

After taking 10 μL of lecoflavone (5% sol), it was diluted to a final 1 mL by adding assay buffer (×100).

After reacting at 37 ° C for 10 minutes, 25 μL of AAPH was added and the fluorescence value of the mixed reaction solution was measured at excitation 485 nm / emission 530 nm at 1 minute intervals for 45 minutes.

AUC (Area Under Curve) was calculated by Equation 1 using the measured fluorescence value. The Net AUC value was calculated by Equation 2 using the difference between the AUC value of the sample treatment group and the AUC value of the untreated group. A standard calibration curve was prepared through the Trolox content and Net AUC values, and the ORAC index was calculated by substituting the Net AUC values of the sample treatment group into the standard calibration curve. The test was repeated three times, and the result value was expressed as an ORAC index and expressed as the average ± standard deviation of the three experiments.

[Equation 1]

AUC (Area Under Curve) = 0.5+(F1/F0)+(F2/F0)+(F3/F0)+...+0.5×(F45/F0)

[Equation 2]

Net AUC = AUC sample treatment group - AUC untreated group

As a result, as a result of evaluating the ORAC index for the recoflavone according to the present invention, an ORAC index (μmole TE/L) of 4478.24 was calculated. This index indicates that lecoflavone, an active ingredient of the composition according to the present invention, exhibits an excellent antioxidant effect.

Claims (21)

1. A cosmetic composition for skin improvement comprising lecoflavone or a salt thereof as an active ingredient.
2. The cosmetic composition for skin improvement according to claim 1, wherein the skin improvement is skin aging prevention.
3. The cosmetic composition for skin improvement according to claim 2, wherein the anti-aging of the skin is one selected from the group consisting of skin moisturizing, skin whitening, skin wrinkle improvement, antioxidant, and combinations thereof.
4. The cosmetic composition for skin improvement according to claim 1, which inhibits TNF-α and inhibits the expression of inflammatory cytokines.
5. The cosmetic composition for skin improvement according to claim 1, characterized in that it improves collagen production ability.
6. The cosmetic composition for skin improvement according to claim 1, characterized in that it activates aquaporin-3.
7. The cosmetic composition for skin improvement according to claim 1, characterized in that it inhibits the production of melanin.
8. According to claim 1, skin lotion, skin softener, skin toner, astringent lotion, softening lotion, nutrient lotion, astringent, lotion, milk lotion, moisture lotion, nutrient lotion, body cream, massage cream, nutrient cream, moisture cream, hand Cream, Essence, Nutrition Essence, Pack, Soap, Shampoo, Cleansing Foam, Cleansing Lotion, Cleansing Cream, Cleansing Water, Soap, Mask Pack, Body Oil, Body Lotion, Body Cleanser, Treatment, Essence, Emulsion, Foundation, Lipstick, A cosmetic composition for skin improvement having a formulation selected from the group consisting of makeup base, press powder, loose powder, concealer, eye shadow, shampoo, rinse, hair conditioner, hair gel and hair vinegar.
9. A pharmaceutical composition for skin improvement comprising lecoflavone or a salt thereof as an active ingredient.
10. The pharmaceutical composition for skin improvement according to claim 9, wherein the skin improvement is one selected from the group consisting of excessive pigmentation improvement, atopic dermatitis, scar improvement, dry skin improvement, and combinations thereof.
11. A pharmaceutical composition for preventing or improving hyperpigmentation disease, comprising lecoflavone or a salt thereof as an active ingredient.
12. The hyperpigmentation disease according to claim 11, characterized in that the hyperpigmentation disease is selected from the group consisting of melasma, freckles, spots, solar lentigo, drug-induced pigmentation, inflammatory pigmentation, senile pigmentation, and pregnancy-induced pigmentation. A pharmaceutical composition for the prevention or treatment of
13. Pharmaceutical composition for preventing or improving xeroderma containing lecoflavone or a salt thereof as an active ingredient
14. According to claim 13, wherein the xeroskin disease is characterized in that selected from the group consisting of atopic dermatitis, scars, senile dryness and xeroderma due to topical application of drugs, a pharmaceutical composition for preventing or treating dry skin.
15. The pharmaceutical composition according to claim 9, 11 or 13, further comprising a pharmaceutically acceptable diluent or excipient.
16. The pharmaceutical composition according to claim 9, 11 or 13, which is an oral or parenteral formulation.
17. The pharmaceutical composition according to claim 16, wherein the parenteral preparation is an external preparation for the skin.
18. According to claim 1, Retinol, retinyl palmitate, adenosine, TGF, animal placenta-derived protein, betulinic acid, and skin elasticity or wrinkle improvement component selected from the group consisting of chlorella extract;

    A whitening component selected from the group consisting of Kojic acid, Arbutin, Hydroquinone, Niacinamide, Vitamin-C (L-Ascorbic acid) and derivatives thereof;

    COX-2 inhibitor, prednisolone and anti-inflammatory components selected from the group consisting of allantoin further comprising one or two or more components selected from the group consisting of, a cosmetic composition for improving skin.
19. The hyperpigmentation improving component according to claim 9, claim 11 or claim 13, hydroquinone, vitamin-C, nicotinic acid amide, glutathione, tranexamic acid; As anti-inflammatory components, COX-2 inhibitors, NSAID-based anti-inflammatory drugs, steroid-based anti-inflammatory drugs and allantoin; As the dry skin improving component, D-panthenol, tocopherol acetate, allantoin, heparinoid, diphenhydramine, urea; And those further comprising one or two or more components selected from the group consisting of derivatives thereof, a pharmaceutical composition for improving skin.
20. A method of improving skin comprising the step of treating the skin of a subject with lecoflavone or a salt thereof.
21. The method of claim 20, wherein the skin improvement is characterized in that one selected from the group consisting of excessive pigmentation improvement, atopic dermatitis, scar improvement, dry skin improvement, and combinations thereof.

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