KR20190035444A - An anti-aging functional cosmetic composition comprising an Undaria pinnatifida extract, an Ecklonia cava extract and glycosaminoglycan - Google Patents

Abstract

The present invention relates to an anti-aging functional cosmetic composition comprising an active ingredient of glycosaminoglycans (GAGs), an Ecklonia cava ethanol-extract (ECE), and an Undaria pinnatifida enzymatic-extract fucoidan (UPEF).

Description

An anti-aging functional cosmetic composition comprising an extract of Ganoderma lucidum, an extract of Ganoderma lucidum, and a glycosaminoglycan, and an anti-aging functional cosmetic composition comprising an extract of Ecklonia cava extract and glycosaminoglycan,

The present invention relates to a skin whitening, wrinkle-improving, antioxidative, ultraviolet (UV) and anti-inflammatory effect including Undaria pinnatifida enzymatic-extracts fucoidan (UPEF), Ecklonia cava Ethanol-extracts (ECE) and Glycosaminoglycans The present invention relates to an anti-aging functional cosmetic composition having a skin protection effect or a skin regeneration effect.

Skin is the largest organ in the human body and its area is about 1.6m2 in size. It is a large organ which weighs 16% of its weight. Skin acts as the most basic skin covering the body to protect the internal organs. It is also a very important institution that performs many functions.

However, a variety of biochemical changes within the cell can be induced by cellular damage stimuli that damage cells such as excessive physical and chemical stimuli and stresses, nutritional deficiencies, ultraviolet rays, environmental contaminants, rapid temperature changes, Induction promotes aging of the skin such as stain, freckles, dullness, loss of elasticity, keratinization, and wrinkling in the appearance of the skin.

In skin aging, pigmentation such as pigmentation or staining of the skin can be caused by internal factors such as metabolic disturbances in the living body and external factors such as ultraviolet rays. Generally, the external factors When the ultraviolet rays are irradiated to the skin, the melanocytes are stimulated by the ultraviolet ray. When the melanocyte is activated, the tyrosinase enzyme acts and the skin becomes brown, which is melanin Is generated and absorbs excess light rays to preserve the living body. Therefore, in the cosmetics industry, in order to prevent such phenomenon, it has been necessary to develop a whitening agent which inhibits the activity of melanocyte and inhibits the formation of tyrosinase enzyme and melanin pigment, thereby preventing pigmentation such as skin coloring and unevenness .

The cause of wrinkles in skin aging is largely divided into the generation of wrinkles due to natural aging and the generation of wrinkles due to exposure to ultraviolet rays (photoaging). In addition, common wrinkles are found in association with skin aging caused by natural aging and photoaging Studies on the phosphorus phenomenon have shown that the reduction and modification of collagen and elastin, the main proteins in the dermis, are directly involved in the production of wrinkles and the reduction of elasticity of the skin. In particular, collagen is a major substrate protein produced in the fibroblasts of the skin, and a decrease in collagen is closely related to the formation of wrinkles in the skin. In other words, free radicals that affect collagen reduction are known to cause destruction of connective tissues such as skin collagen, inhibition of cell membrane function, promotion of DNA mutation, modification of protein action, intercellular energy transfer, and transformation of molecules involved in metabolism , The fact that free radicals are involved in such skin aging means that antioxidants that inactivate free radicals can be used to delay the aging process. On the other hand, even in normal cells, some free radicals and other active oxygen and peroxides are produced in the metabolic process, resulting in abnormality in bio-defense mechanisms against them, or generation of active oxygen by various physical and chemical factors, The oxidative stress is caused. Therefore, inhibiting active oxygen is an important factor in cosmetic composition to prevent skin aging.

Korean Patent Publication No. 10-13038070000

An object of the present invention is miyeokgwi (Undaria pinnatifida enzymatic-extracts fucoidan, UPEF), Ecklonia cava extract, skin whitening, anti-wrinkle mutually synergistic effect of (Ecklonia cava Ethanol-extracts, ECE) and glycosaminoglycans (Glycosaminoglycan, GAGs), Antioxidant, protection against ultraviolet rays, or skin regeneration.

In one aspect of the present invention, the present invention provides an anti-aging cosmetic composition comprising, as an active ingredient, an irregular extract, a gentian extract, and a glycosaminoglycan.

The cosmetic composition of the present invention contains, as an active ingredient, a mugwort extract, a moss extract, and a glycosaminoglycan.

The cosmetic composition comprising the asparagine extract, phlegm extract and glycosaminoglycan as an active ingredient has an anti-aging effect.

As used herein, the term " gypsum " is a gingival germ of the seaweed located at the root of undaria pinnatifida , a type of brown algae inhabiting the sea. It is known to be abundant in alginic acid and fucoidan components. Virus function, and immunity enhancement function.

The term " extract " of the present invention refers to a substance extracted from a natural product, which can be obtained by an extraction process using water, an organic solvent, or a mixed solvent thereof, and a diluent or concentrate or extract of the extract is dried , Or any of these regulated or refined products.

The term " mugwort extract " of the present invention means an enzyme extract of mugwort. The soybean extract contains fucoidan as an active ingredient, and the enzyme may be Cellucalast.

In the case of the non-dialyzed extract, the undilated solution was dissolved in water and then hydrolyzed by adding cellulose. After centrifugation, the supernatant was filtered, concentrated, and the concentrated supernatant was precipitated with ethanol and calcium chloride It can be extracted by removing alginic acid, but it can be used without limitation as long as it is an extraction method using an enzyme and containing fucoidan and removing alginic acid.

The fucoidan is a sulfated polysaccharide having a sticky, viscous structure and is contained in brown algae such as seaweed and kelp, and is known to have anti-inflammatory, antiviral and antiallergic functions.

The term " Ecklonia cava " of the present invention is a perennial seaweed belonging to the seaweed family, which is distributed in the southern part of Korea including the East Sea and Jeju Island.

The term " phlegm extract " of the present invention is obtained by extracting the phloem with an ethanol solvent, and may include polyphenols and diacols.

In the examples of the present invention, the ginger extract has a total polyphenol content of 50% or more and contains 5% or more of dioecol.

The term Glycosaminoglycans (GAGs) of the present invention is one of the main components constituting the skin, and includes hyaluronic acid (HA), dermatan sulfate, chondroitin sulfate, keratan sulfate, heparan sulfate, It belongs to this. Glycosaminoglycan can be extracted mainly from animal cartilage, fish cartilage, thyroid gland, urchin skin, and the like.

The glycosaminoglycan of the present invention may be one extracted from the bark of Halocynthia roretzi .

The term " aging " of the present invention refers to changes in the function and condition of the body that accompany the passage of time, and there are symptoms such as uneven skin caused by pigmentation such as spots, freckles and blurred spots, And oxidative stress are known to be the main causes.

The term " anti-aging " of the present invention means alleviating, reducing, preventing, delaying, improving or eliminating the aging symptom through whitening, wrinkle improvement, antioxidation, protective effect against ultraviolet rays or skin regeneration effect.

The cosmetic composition of the present invention may include, without limitation, commonly accepted ingredients other than the above-mentioned effective ingredients, and may contain conventional additives such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavoring agents, have.

In addition, the cosmetic composition according to the present invention can be used as a cosmetic composition in the form of a solution, an external ointment, a cream, a foam, a nutritional lotion, a softening water, a pack, It may be formulated into one or more formulations selected from the group consisting of suspensions, emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, patches and sprays , But is not limited thereto.

In addition, the cosmetic composition of the present invention may further comprise at least one cosmetically acceptable carrier to be incorporated in a general skin cosmetic composition, and examples thereof include oil, water, a surfactant, a moisturizer, A thickening agent, a chelating agent, a coloring matter, an antiseptic, a perfume, and the like may be appropriately compounded, but the present invention is not limited thereto.

The cosmetically acceptable carrier to be contained in the cosmetic composition of the present invention varies depending on the formulations.

When the formulation of the present invention is an ointment, a paste, a cream or a gel, the carrier component may be an animal oil, a vegetable oil, a wax, a paraffin, a starch, a tracer, a cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide Mixtures of these may be used.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or a mixture thereof may be used as the carrier component, Propellants such as fluorohydrocarbons, propane / butane or dimethyl ether.

When the formulation of the invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil may be used, and fatty acid esters of cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or sorbitan may be used .

When the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is a soap, an alkali metal salt of a fatty acid, a fatty acid hemiester salt, a fatty acid protein hydrolizate, isethionate, a lanolin derivative, an aliphatic alcohol, a vegetable oil, glycerol, .

When the formulation of the present invention is an interface-active agent-containing cleansing, the carrier component is selected from the group consisting of aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

The term " whitening " of the present invention refers to a method of increasing the brightness of skin with reduced brightness due to excessive pigment such as melanin or keeping the brightness of skin at a certain level, This means specifically skin whitening.

The melanin is a pigment component widely distributed in organisms such as skin, hair, eyes and the like, melanosomes in the melanocytes of the skin of the human body are synthesized. Tyrosinase inhibits tyrosine Melanin is biosynthesized by oxidation and polymerization reactions which are converted to DOPA (3,4-dihydroxy-phenylalanine) or DOPA quinone (dopaquinone) as a starting material. Biosynthesized melanin increases resistance to skin irritation such as ultraviolet rays. However, since excessive melanin synthesis causes pigmentation such as spots, freckles, and black spots, tyrosinase activity measurement is an important indicator of skin whitening effect It is accepted.

In one embodiment of the present invention, the whitening effect of the mixed sample according to the weight ratio (w / v) of the nematode extract, milt extract and glycosaminoglycan was examined to confirm tyrosinase inhibitory activity and melanin formation inhibitory activity Respectively.

As a result of the measurement of tyrosinase inhibitory activity, a mixed sample showing ascorbic acid effect was confirmed when a single sample was treated. When the weight ratio (w / v) was expressed as UPEF: ECE: GAGs, the increase in tyrosinase inhibitory activity A mixed sample in the ratio of 2: 6: 2, 2: 7: 1, 3: 5: 2, 3: 6: 1, 4: 3: 3, 4: 5: 1, 7: 2: Melanogenesis inhibition was measured by treatment with melanoma cells (B16F10), and melanogenesis was inhibited in all the mixed samples. The melanogenesis inhibitory activity of zebrafish model was confirmed by mixing samples with weight ratios of 3: 5: 2, 3: 6: 1 and 4: 5: 1 with significant differences. As a result, a ratio of 4: 5: 1 Was found to be the most effective for inhibiting melanin.

The whitening effect was confirmed by confirming that a mixed sample of the non-mitochondrial extract, phlegm extract and glycosaminoglycan inhibited tyrosinase activity and reduced melanin production in melanoma cells and zebrafish models. w / v), and it was confirmed that the whitening effect was the best in a weight ratio of 4: 5: 1.

The term " wrinkle improvement " of the present invention means to inhibit collagen or elastin degrading enzyme, or to promote the synthesis of collagen to improve wrinkles. The collagen and elastin constitute the dermal layer of the skin, and the reduction or deformation of collagen and elastin is known to be involved in wrinkle formation and skin elasticity reduction.

In order to confirm the wrinkle-improving effect of the mixed sample according to the weight ratio (w / v) of the nematode extract, moxibustion extract and glycosaminoglycan in an embodiment of the present invention, the inhibitory activity of collagenase and elastase was evaluated Respectively.

As a result of measurement of collagenase and elastase inhibiting activity, mixed samples having elevated activity than single samples were identified, and the content of pro-collagen was measured in human fibroblasts treated with a synergistic mixture sample It was confirmed that the content of procollagen was significantly increased in the mixed sample of cells in the weight ratio of 3: 5: 2, 3: 6: 1, 4: 5: 1, and 7: 2:

It was confirmed that a mixed sample of the nonylpiperidine extract, menthaea extract and glycosaminoglycan inhibited collagenase and elastase and increased the amount of procollagen, confirming the effect of improving wrinkles. It was confirmed that there was a difference in efficacy. Among them, a mixed sample having weight ratios of 3: 5: 2, 3: 6: 1, 4: 5: 1, and 7: 2: 1 showed excellent wrinkle improving effect.

The term "antioxidant" of the present invention means an antioxidant action. The human body has a balance of prooxidant and antioxidant. However, due to various factors, And leaning toward oxidation promotes oxidative stress leading to potential cellular damage and pathological disease. Reactive oxygen species (ROS), which is a direct cause of oxidative stress, is unstable and highly reactive. It reacts easily with various bio-materials, attacks irreversible damage to cells and tissues, Cytotoxicity and carcinogenesis. Such active oxygen oxidizes and destroys cells in the body, thereby exposing them to various diseases. In addition, the antioxidant refers to a function of inhibiting cell oxidation by highly reactive free radicals or reactive oxygen species depending on the oxidative stress due to intracellular metabolism or ultraviolet rays, thereby eliminating free radicals or reactive oxygen species Lt; RTI ID = 0.0 > damage. ≪ / RTI >

In one embodiment of the present invention, in order to examine the antioxidative effect of the mixed sample according to the weight ratio of the nonylphenol extract, phytonutrient extract and glycosaminoglycan, the zebrafish model was treated with hydrogen peroxide and the survival rate and active oxygen inhibition Activity was confirmed. The survival rate was increased in a concentration ratio of 3: 5: 2 and 4: 5: 1 in a concentration-dependent manner. When a mixed sample having a weight ratio of 4: 5: 1 was treated with hydrogen peroxide, .

It was confirmed that the mixed sample of specific fractions of the non-mitochondrial extract, manganese extract and glycosaminoglycan increased the survival rate and decreased the active oxygen of the zephyrate treated with hydrogen peroxide, 4: 5: 1 mixed sample showed excellent antioxidative effect.

The term " protection against ultraviolet rays " of the present invention is intended to protect cells against stress induced by ultraviolet rays. Ultraviolet rays may be ultraviolet rays B.

In one embodiment of the present invention, in order to examine the protective effect of the ultraviolet B of the mixed sample according to the weight ratio of the nematode extract, the moxibustion extract and the glycosaminoglycan, the survival rate after irradiation with the ultraviolet B in the zebrafish model, The elimination effect on nitrogen (NO) was confirmed.

It was confirmed that the survival rate was increased in the zebrafish model treated with the mixed sample having weight ratios of 3: 5: 2, 3: 6: 1, and 4: 5: 1 by weight of the nematode extract, menthol extract and glycosaminoglycan , The nitrogen monoxide scavenging activity was measured in a mixed sample treated zebrafish model with increased survival rate. As a result, it was confirmed that nitrogen monoxide was reduced in zebrafish treated with a mixed sample having a weight ratio of 4: 5: 1.

It was confirmed that the mixture of the non-mitochondrial extract, mesophyll extract and glycosaminoglycan increased the survival rate of the zebrafish treated with ultraviolet rays and decreased the nitrogen monoxide. Thus, the nematode extract, the moth extract and the glycosaminoglycan were irradiated with ultraviolet And it was confirmed that a mixed sample having a weight ratio of 4: 5: 1 has excellent UV protection effect.

The term " skin regeneration " of the present invention means improving the proliferation of skin cells or promoting the recovery of damaged skin.

In an embodiment of the present invention, in order to examine the skin regeneration efficiency of the mixed sample according to the weight ratio of the nematode extract, the moxibustion extract and the glycosaminoglycan, cell proliferation and cell migration in the skin fibroblast (NIH3T3) cell migration, and confirmed the regeneration after wounding in the zebrafish model.

As a result of measuring the cell proliferation ability of the mixed sample, it was confirmed that the cell proliferating ability was increased as the ratio of glucosaminoglycan was increased, and the samples having synergistic effect were confirmed in the mixed sample than in the single sample treatment. Seven of the samples with synergistic effect were selected and their cell proliferative capacity was checked with the epidermal growth factor as a positive control. As a result, the weight ratio of 3: 5: 2, 3: 6: 1, 4: 3: 3, 4: 5: 1, 7 : 2: 1, the cell proliferation was significantly improved. Especially, in the mixed sample of 4: 5: 1, it was confirmed that the cell proliferation was the best, and the whitening and wrinkle improving effect (Skin regeneration) in the zebrafish model was confirmed as a result of mixing samples in the weight ratios of 3: 5: 2, 3: 6: 1 and 4: 5: 1 in all the groups except the control group The wound was recovered after work. In addition, cell migration was observed in a mixed sample having a weight ratio of 4: 5: 1, indicating that the cell migration was increased as compared with the control group.

 It was confirmed that the mixture of the non-wild type extract, the gentian extract and the glycosaminoglycan enhanced the proliferation of the skin fibroblast and promoted wound regeneration (skin regeneration) of the zebrafish model. Thus, the non-wild type extract, Minoglycan showed skin regeneration efficacy. In particular, it was confirmed that a mixed sample having a weight ratio of 4: 5: 1 has excellent skin regeneration effect.

In one embodiment of the present invention, whitening, wrinkle improvement, antioxidation, antioxidation, anti-oxidative effect and skin regeneration effect of whitening, wrinkle reduction, antioxidation, As a result of confirming the anti-aging effect such as protecting effect against ultraviolet rays and skin regeneration, the non-ginseng extract, ginseng extract and glycosaminoglycan can be used as an anti-aging cosmetic composition.

(W / v) of 2 to 7: 3 to 7: 1 to 3, or the ratio of the unmodified extract (UPEF), the bitter extract (ECE) and the glycosaminoglycan Wrinkles, wrinkles, antioxidant activity, and antioxidative activity were observed in a sample mixed with a weight ratio (w / v) of 1: 0.1 to 4: 0.1 to 1 of the above Nameless Extract (UPEF), Phytophthora exudate (ECE), and Glycosaminoglycan The effect of protection against ultraviolet rays and the synergy of skin regeneration were confirmed, and whitening, wrinkle reduction, antioxidation, ultraviolet protection effect and skin regeneration effect were observed in a sample mixed at a weight ratio (w / v) of 4: 5: 1 . Therefore, the above-mentioned non-mitotic extract (UPEF), ginger extract (ECE) and glycosaminoglycan (GAGs) can be mixed at a weight ratio (w / v) of 1: 0.1 to 4: 0.1 to 1, , Ganoderma lucidum extract and glycosaminoglycan may be mixed at a weight ratio (w / v) of 4: 5: 1.

Further, as a result of confirming the synergistic effects of whitening, wrinkle reduction, antioxidation, protection against ultraviolet rays, and skin regeneration of the above-mentioned non-ginseng extract (UPEF), ginseng extract (ECE) and glycosaminoglycans (GAGs) It can be used for whitening, wrinkle improvement, antioxidation, skin protection against ultraviolet rays, or skin regeneration.

The cosmetic composition according to the present invention may contain, in an amount of 0.01 to 20% by weight based on the total weight of the composition, the apical extract (UPEF), the ginger extract (ECE) and the glycosaminoglycan (GAGs).

As another aspect of the present invention, there is provided an anti-aging quasi-product composition comprising, as an active ingredient, a ginseng extract (UPEF), a ginger extract (ECE) and a glycosaminoglycan (GAGs).

In the present invention, the quasi-drug composition may be a quasi-drug composition for skin whitening, wrinkle reduction, antioxidation, ultraviolet screening or skin regeneration.

As described above, the above-mentioned Japanese apricot extract (UPEF), ginger extract (ECE), glycosaminoglycan (GAGs), anti-aging, skin whitening, wrinkle improvement, antioxidation, ultraviolet screening or skin regeneration are as described above.

The term "quasi-drug product" used in the present invention means products that are less active than drugs, among the products used for diagnosing, treating, improving, alleviating, treating or preventing diseases of human or animal. For example, The quasi-quasi-drug means a product that is used for the treatment of diseases of humans and animals, except for the products used for medicines, which are slightly or not directly acting on the human body, , And sterilization and insecticides to prevent infectious diseases.

The type and formulations of the quasi-drug composition of the present invention are not particularly limited, but may be disinfectant cleaner, shower foam, gagrin, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment or filter filler.

When the composition of the present invention is contained in a quasi-drug for anti-aging purposes, the composition may be used as it is or may be used together with other quasi-drugs, and may be suitably used according to a conventional method. The amount of the active ingredient in the quasi-drug composition may be suitably determined according to the purpose of use. The quasi-drug extract (UPEF), ginger extract (ECE) and glycosaminoglycan (GAGs) By weight to 20% by weight.

As another aspect of the present invention, there is provided an anti-aging dermatologic external composition comprising an ungulate extract (UPEF), a gangue extract (ECE) and glycosaminoglycan (GAGs) as an active ingredient.

In the present invention, the composition for external application for skin may be a skin external composition for skin whitening, wrinkle reduction, antioxidation, skin protection against ultraviolet rays, or skin regeneration.

As described above, the above-mentioned Japanese apricot extract (UPEF), ginger extract (ECE), glycosaminoglycan (GAGs), anti-aging, skin whitening, wrinkle improvement, antioxidation, ultraviolet screening or skin regeneration are as described above.

The composition for external application for skin according to the present invention includes, for example, ointments, lotions, soluble images, suspensions, emulsions, creams, gels, sprays, papermaking agents, And may be incorporated into any of the bases known in the art. The composition for external application for skin according to the present invention may contain 0.01 to 20% by weight, based on the total weight of the composition, of the prepuce extract (UPEF), ginger extract (ECE) and glycosaminoglycan (GAGs).

As described above, the cosmetic composition containing Undiaria pinnatifida extract, extract of Ecklonia cava and glycosaminoglycans (GAGs) according to the present invention can be used for antioxidant, whitening, wrinkle improvement, There is protective effect or skin regeneration effect.

FIG. 1 is a graph showing the activity of tyrosinase inhibitory activity after treatment of single samples of Japanese apricot extract (UPEF), ginseng extract (ECE) and glycosaminoglycan (GAGs) at 25, 50 and 100 μg / As a control group, 350 μM arbutin was used.
FIG. 2 is a graph showing the activity of inhibiting the production of melanin by treating single samples of Japanese apricot extract (UPEF), ginger extract (ECE) and glycosaminoglycan (GAGs) at 25, 50 and 100 μg / As a control, arbutin (350 μM) was used (UPE, APE, GAGS from left to right).
FIG. 3 is a graph showing the survival rates of zebrafish embryos treated with single 25 μg / ml concentrations of a single sample of Japanese apricot extract (UPEF), ginger extract (ECE) and glycosaminoglycan (GAGs).
FIG. 4 is a graph showing the whitening activity after treatment of single samples of Japanese apricot extract (UPEF), gentle extract (ECE) and glycosaminoglycan (GAGs) at 25 and 50 μg / ml concentrations in a zebrafish model.
FIG. 5 is a graph showing the results of collagenase inhibition and elastase inhibition activity after treatment with a single sample of Umbelliferae Extract (UPEF), Gingko Extract (ECE) and Glycosaminoglycan (GAGs) at a concentration of 50 μg / ml.
FIG. 6 is a graph showing the fibroblast proliferative activity after treatment of single samples of Umbelliferae extract (UPEF), menthol extract (ECE) and glycosaminoglycan (GAGs) at a concentration of 25 and 50 μg / ml, respectively.
7 is a schematic representation of an experimental model of wound regeneration (skin regeneration) in a zebra fish model.
8 is a graph showing the effect of wound regeneration (skin regeneration) on a single zebrafish model treated with a single sample of a non-digested extract (UPEF), a gangrene extract (ECE) and a glycosaminoglycan (GAGs) I checked
FIG. 9 is a graph showing the tyrosinase inhibitory activity of the mixed sample according to the mixing ratio of the unmodified extract (UPEF), the ginger extract (ECE) and the glycosaminoglycan (GAGs).
10 shows the melanogenesis inhibitory activities of seven samples selected from 262, 271, 352, 361, 433, 451 and 721 selected from the mixed samples of the unmodified extract (UPEF), menthol extract (ECE) and glycosaminoglycan Respectively.
FIG. 11 shows the survival rates of zebrafish embryos treated with 25, 50, and 50 μg / ml of each of 352, 361, and 451 of a mixture of the unmodified extract (UPEF), the menthol extract (ECE), and the glycosaminoglycan Respectively.
FIG. 12 shows the result of treating the zebrafish embryo with UPE, UPE, ECE, and GGS mixed samples 352, 361, and 451 at a concentration of 25 and 50 μg / ml, respectively, Respectively.
FIG. 13 is a graph showing a collagenase inhibitory activity of a mixed sample according to the mixing ratio of a supernatant extract (UPEF), a gentian extract (ECE), and a glycosaminoglycan (GAGs).
FIG. 14 is a graph showing the elastase inhibitory activity of a mixed sample according to the mixing ratio of a sumum extract (UPEF), a ginger extract (ECE), and a glycosaminoglycan (GAGs).
FIG. 15 shows the results of collagenase production of 7 samples selected from 262, 271, 352, 361, 433, 451, and 721 selected from among the mixed samples of Japanese apricot extract (UPEF), ginseng extract (ECE) and glycosaminoglycan (GAGs) Inhibitory activity.
FIG. 16 shows the results of ELASA production of 7 samples selected from 262, 271, 352, 361, 433, 451, and 721 selected from among the mixed samples of the nematode extract (UPEF), the ginger extract (ECE), and the glycosaminoglycan (GAGs) Inhibitory activity.
17 shows the results of promoting the synthesis of procollagen from 7 samples selected from 262, 271, 352, 361, 433, 451, and 721 selected from among the mixed samples of the umami extract (UPEF), the ginger extract (ECE), and the glycosaminoglycan Activity.
FIG. 18 is a graph showing the fibroblast proliferative capacity of a mixed sample according to the mixing ratio of the supernatant extract (UPEF), the ginger extract (ECE), and the glycosaminoglycan (GAGs).
19 shows the fibroblast proliferative activity of seven samples selected from 262, 271, 352, 361, 433, 451, and 721 selected from the group consisting of the sumate extract (UPEF), the ginger extract (ECE) and the glycosaminoglycan (GAGs) Respectively.
FIG. 20 shows the scratch and skin regeneration efficacy in the zebrafish model of the mixture of the supernatant extract (UPEF), the menthol extract (ECE) and the glycosaminoglycan (GAGs) mixed samples 352, 361 and 451.
FIG. 21 shows the cell migration effect of 451 of a mixture of a supernatant extract (UPEF), a phlegm extract (ECE), and a glycosaminoglycan (GAGs) sample.
FIG. 22 shows the cell regeneration mechanism of the sample 451 mixed with the unmodified extract (UPEF), the moxibustion extract (ECE), and the glycosaminoglycan (GAGs).
FIG. 23 shows the survival rate of zebrafish of the mixed samples 352, 361 and 451 of the unmodified extract (UPEF), the ginger extract (ECE) and the glycosaminoglycan (GAGs) (when 5 mM of hydrogen peroxide was treated).
FIG. 24 is a graph showing the activity of the active oxygen scavenging activity of a mixture of the supernatant extract (UPEF), menthol extract (ECE) and glycosaminoglycan (GAGs) 352, 361 and 451 (when 5 mM of hydrogen peroxide was treated).
FIG. 25 shows the zebrafish survival rate (UV 50 mJ / cm 2 irradiation) of a mixture of the supernatant extract (UPEF), the ginger extract (ECE) and the glycosaminoglycan (GAGs) mixed samples 352, 361 and 451.
FIG. 26 is a graph showing the activity (ROS) scavenging activity (UV 50 mJ / cm 2) of 352, 361 and 451 of a mixture of a supernatant extract (UPEF), a sensory extract (ECE) and a glycosaminoglycan (GAGs) .
Fig. 27 shows a method for producing an unsalted extract (UPEF).

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

A. Experimental Method

<Example 1> In vitro whitening efficacy evaluation

1) Measurement of inhibitory activity of tyrosinase

The tyrosinase inhibition assay uses the method reported by Jones et al. (2002) Pigment. Cell Res. 15: 335), and to tyrosinase substrate L-Dopa dopachrome Conversion was carried out by absorption monitoring at 450 nm.

Tyrosinase was prepared with 200 U / ml of pH 6.8 (assay buffer), 50 mM potassium phosphate buffer. A 2 mM working solution of substrate L-DOPA was prepared with assay buffer for each assay, samples were dissolved in 0.5 ml of 10% dimethyl sulfoxide (DMSO) and diluted with 5 ml of assay buffer . The reaction mixture consisted of 0.050 ml of 2 mM L-DOPA, 0.050 ml of 200 U / ml of mushroom tyrosinase and 0.050 ml of inhibitor. The reaction volume was adjusted with 200 [mu] L of assay buffer and assay was performed in a 96 well plate. The percentage of tyrosinase inhibition by the test sample was determined by comparing the absorbance of the sample with respect to the control.

(Negative control absorbance - sample absorbance) / negative control absorbance X 100

2) Melanin formation inhibitory activity

Inhibition of melanin production was measured under the induction of α-melanocyte stimulating hormone (α-MSH). Melanoma cell line (B16F10) is fully chilled (confluency) and incubated (incubation) overnight at 37 ℃ in a humid environment containing 5% carbon dioxide (CO ₂₎ divided by 40,000 per well cells, so the cells can grow and then grown. The next day, the inhibitor was added to the cells at a concentration of 50 nM, treated with the sample, and cultured for a total of 4 days. Color development was observed with the naked eye every morning. After color development, 200 μL of cell supernatant was removed from each well and absorbance was measured at 450 nm.

&Lt; Example 2 > Evaluation of efficacy in wrinkle in vitro

1) Measurement of collagenase inhibitory activity

In order to confirm the skin aging inhibitory effect, the collagenase inhibitory activity was measured as follows. 0.25 ml of a substrate solution obtained by adding 4 mM calcium chloride (CaCl ₂ ) to 0.1 M Tris-HCI buffer (pH 7.5) and dissolving 4-phenyl azobenzyl loxycarbonyl-Pro-Leu-Gly-Pro-Arg (0.3 mg / 0.15 ml of collagenase at a concentration of 0.2 mg / ml was added to the mixed solution, and the mixture was allowed to stand at room temperature for 20 minutes. Then, 0.5 ml of 6% citric acid was added to quench the reaction and 1.5 ml of ethylacetate Was added and the supernatant was measured for absorbance at 320 nm. The collagenase inhibitory activity was shown by the absorbance reduction ratio of the sample solution and the non-additive solution.

2) Measurement of elastase inhibitory activity

To evaluate the effect of p-nitroanilide production at 37 ° C for 30 minutes, N-succinyl- (L-Ala) 3-p-nitroanilide was used as a substrate to confirm the effect of pancreatic elastase Activity. Each test solution was prepared to have a constant concentration, and 0.1 ml of each test solution was taken in a test tube, and 0.05 ml of an elastase solution (Type I: derived from porcine pancreas, 0.6 units / ml) dissolved in 50 mM Tris-HCl buffer (pH 8.6) (L-Ala) 3-p-nitroanilide (1 mg / ml) was dissolved in 50 mM Tris-HCl buffer (pH 8.6) as a substrate and reacted for 30 minutes. A microplate reader absorbance at 410 nm was measured using a reader. The elastase inhibitory activity was expressed as the absorbance reduction rate of the sample solution added and the non-added sample.

3) Cell culture

HDFn cells as human fibroblast cells and HaCaT cells as human keratinocyte cell lines were cultured in DMEM supplemented with 5% CO 2 (CO 2) at 37 ° C using medium containing 100 U / ml penicillin and 100 μg / ml streptomycin ₂ ) conditioner incubator. The cell culture medium was changed into a new medium every 2 days. Then, 4 × 10 ⁴ cells / 300 μl cells were inoculated into a ^6- well plate at 48 wells and 2 × 10 ⁶ cells / Were stably attached and used in the experiment.

4) Collagen aggregation performance test

HDFn cells were inoculated into 48 well plates at 5 × 10 ⁴ cells and cultured for 24 hours at 37 ° C in 5% CO ₂ . Subsequently, the medium was replaced with a DMEM medium containing no fetal bovine serum (FBS), and the sample was added thereto. After culturing for 24 hours, the culture broth was collected and procollagen type protein synthesis kit Was used to measure the amount of C-terminus of the collagen precursor.

&Lt; Example 3 > Evaluation of ultraviolet protection effect

1) UV irradiation

Ultraviolet B was treated under the following conditions in order to confirm the protective effect of the sample against cytotoxicity and oxidative apoptosis caused by ultraviolet B irradiation. HDFn cells were cultured to a density of 80%, the medium was removed, washed once with phosphate buffer solution, and irradiated with ultraviolet B of 50 mJ / cm 2 using an ultraviolet irradiation device. After irradiating ultraviolet rays, various concentrations of samples were treated and cultured for 24 hours.

2) Evaluation of cell survival rate

HDFn cells were plated on a 48-well plate at 4 × 10 ⁴ cells / well. After 24 hours, the medium was replaced with a medium containing no serum and cultured for 24 hours. After 12 hours, the medium was removed and washed with PBS. 200 μl of phosphate buffer saline (PBS) was dispensed into wells, and ultraviolet B was irradiated at 50 mJ / cm 2. After UV irradiation, the PBS was removed, and the medium was replaced with the medium. After 24 hours, the medium was removed, the MTT solution was removed, and the formazan crystal was dissolved in DMSO to measure the absorbance at 450 nm.

3) Measurement of anti-wrinkle activity by ultraviolet irradiation

HDFn cells were seeded at 4 × 10 ⁴ cells / well in a 48-well plate, and then cultured for 24 hours in a medium containing no serum after 24 hours. After 12 hours, the medium was removed and washed with PBS. 200 μl of PBS was dispensed into wells, and ultraviolet B was irradiated at 50 mJ / cm 2. After irradiation with ultraviolet light B, PBS was removed and replaced with the medium. After 24 hours, the medium was removed and cells were obtained. After that, western blotting was performed to examine the effect of collagenase (Matrix metalloproteinase-1, MMP-1) expressed on irradiated ultraviolet B in HDFn cells when the sample was treated, Respectively.

Example 4 Evaluation of Cell Regeneration Efficiency

1) Cell proliferation measurement

NIH3T3 cells were plated at 5 × 10 ⁴ cells / well in a 96-well plate, and then cultured for 24 hours after 24 hours in a medium containing no serum. After the sample was treated with bromodeoxyuridine (BrdU) reagent 2 hours before the end of 24 hours, the fixed reagent was reacted at room temperature for 30 minutes. After washing the plate, BrdU antibody was attached to confirm the degree of proliferation.

2) Cell migration measurement

After the busy NIH3T3 cells to 1 × 10 ⁵ cells / well in 6well plate and then replaced with a culture medium that contains no serum 24 hours after the culture for 24 hours, and to process the samples to embellish the scratch on the surface of the whole cell sample And 24 hours later, the extent of scratching was measured to confirm the regeneration of the wound.

<Example 5> Evaluation of whitening efficacy using zebrafish

1) Measurement of whitening activity of zebrafish embryos

Add 15-20 eggs per well to a 24-well plate, add zebrafish embryo that has passed the hour post fertilization (hpf), fill the embryo media with 0.95 ml, Respectively. The degree of whitening activity was measured through a microscope in the state of awakened zebrafish larva in 3dpf, and the data was repeated three times in triplicate and expressed as an average percentage.

2) Melanin content of zebrafish embryos

In order to measure the melanin content, 100 melanogenic inhibitor-treated groups and untreated groups were treated in a pro-prep protein extraction solution (Intron) with ultrasonic treatment in an intron of about 100 mg of zebrafish embryo up to 9-48 hpf Respectively. After centrifugation, the pellet was dissolved in 1 mL of 1N sodium hydroxide (NaOH), reacted at 100 DEG C for 30 minutes, and the mixture vortexed vigorously to solubilize the melanin pigment. Absorbance was measured at 490 nm in the supernatant and 100% in the untreated group. The data were repeated three times in triplicate and expressed as an average percentage.

3) Tyrosinase inhibitory activity in zebrafish embryo

Tyrosinase inhibitory activity was performed according to the tyrosinase method. 100 embryos of approximately zebrafish embryos up to 9-48 hpf were divided into groups treated with melanogenic inhibitor (samples) and untreated groups. Ultrasonic treatment was performed in the Pro-Prep protein extract solution (Intron) And centrifuged at 10,000 g for purification. 250 μg of total protein in 100 μl of lysis buffer was transferred to a 96-well plate and 100 μl of 1.5 mM L-tyrosinase was added. 100 μl of lysis buffer and 100 μl of 1.5 mM L-tyrosinase were present in untreated group wells. After incubation at 28 ° C for 60 minutes, the absorbance was measured at 475 nm using a micro-reader (Packard Spectrocount). Phenylthiourea (PTU) and arbutin were used as controls, and the untreated group was expressed as 100%. The data were repeated three times in triplicate and expressed as an average percentage.

<Example 6> Evaluation of antioxidant efficacy using zebrafish

1) Measurement of reactive oxygen species (ROS) in zebrafish

Hydrogen peroxide, a stimulant, was treated in zebrafish larvae to determine if it had antioxidant efficacy. The active oxygen was measured 96 hours after the exposure, and the zebrafish larvae of each well were transferred to a 96-well plate for fluorescence measurement and filled with new zebrafish embryo medium. To measure the amount of reactive oxygen species, 2,7-dichlorofluorescein diacetate (DCFH-DA, Sigma), which reacts with active oxygen to produce a fluorescent substance, was used. After incubation at 28.5 ± 1 ° C for 1 hour, the zebrafish larva was washed twice with zebrafish embryo medium and anesthetized with 0.01% MS-222 to obtain CoolSNAP -Pro dolor digital camera (Olympus, Japan) was used to measure the amount of active oxygen generated, and the fluorescence intensity of each zebrafish larva was quantified using an image J program.

2) Nitric oxide (NO) measurement in zebrafish

Ultraviolet irradiation was performed after water was removed from the zebrafish larva as much as possible in order to investigate how much the sample was reduced by the stress caused by ultraviolet irradiation. Nitrogen monoxide (NO) was measured 96 hours after exposure. For the fluorescence measurement, zebrafish larva of each well was transferred to a 96-well plate and filled with new zebrafish embryo medium. Nitric oxide was used as a fluorescent reagent 2 ', 7'-difluorofluorescein diacetate (DAF-FM DA, Sigma) Respectively. 5 mM DAF-FMDA solution was added to each well and incubated at 28.5 ± 1 ° C for 2 hours. The reacted zebrafish larva was washed twice with zebrafish embryo medium and anesthetized with 0.01% MS-222. The amount of nitrogen monoxide was measured using a CoolSNAP-Pro dolor digital camera (Olympus, Japan). Fluorescence intensity of each zebrafish larva was quantified using image J program.

<Example 7> Evaluation of skin regeneration efficiency using zebrafish

In the zebrafish larva state, the tail fin was cut uniformly and the recovery of the tail portion was measured for 3 days after the sample treatment.

&Lt; Example 8 > Preparation of mixed sample

In this example, massively-produced Undaria pinnatifida enzymatic-extracts fucoidan (UPEF) and Glycosaminoglycans (GAGs) isolated from the shell of Halocynthia roretzi were used. In addition, Ecklonia cava Ethanol-extracts (ECE) were purchased from AquaGreenTec Co., Ltd., and products having a total polyphenol content of 50% or more and a diecor content of 5% or more were used (Product name: Kantai diecol).

1) Preparation method of non-gingival extract

To 1 L of distilled water was added 50 g of a groundnut powder and 500 Cell of Celluclast. The mixture was stirred at 50 캜 for 24 hours for hydrolysis. At the end of the hydrolysis reaction, the mixture was incubated at 100 캜 for 10 minutes I boiled it. After centrifugation at 3,000 rpm for 20 minutes, only the supernatant was filtered and concentrated to 1/10, followed by the addition of 3-fold volume of ethanol to precipitate the sugar at 5 ° C for 24 hours. To recover the precipitated sugar, the sugar was centrifuged at 10,000 rpm for 20 minutes to obtain a saccharide. To remove the alginic acid contained in the saccharide, 3-fold amount of 4M calcium chloride was added and reacted at 5 캜 for 6 hours to precipitate alginic acid , And alginic acid was removed by centrifugation (10,000 rpm, 20 minutes).

To the supernatant was added 3-fold ethanol again and reacted at 5 ° C for 24 hours to obtain a saccharide. The saccharide obtained by centrifugation (10,000 rpm, 20 minutes) was lyophilized and used in the experiment.

2) Mixing of Nasiridium extract, Ganoderma lucidum extract and Glycosaminoglycan

And the glycosaminoglycan were mixed at a weight ratio (w / v) shown in Table 1 to prepare a mixed sample of Table 1. A total of 36 mixed samples were made to confirm the synergistic effect of antiaging function according to mixing ratio.

Mixed sample mixing ratios (weight ratio w / v) of primate extract (UPEF), menthol extract (ECE) and glycosaminoglycan (GAGs) Mixed sample designation Exudate extract
(UPEF) Moth extract
(ECE) Glycosaminoglycan
(GAGs) 118 One One 8 127 One 2 7 136 One 3 6 145 One 4 5 154 One 5 4 163 One 6 3 172 One 7 2 181 One 8 One 217 2 One 7 226 2 2 6 235 2 3 5 244 2 4 4 253 2 5 3 262 2 6 2 271 2 7 One 316 3 One 6 325 3 2 5 334 3 3 4 343 3 4 3 352 3 5 2 361 3 6 One 415 4 One 5 424 4 2 4 433 4 3 3 442 4 4 2 451 4 5 One 514 5 One 4 523 5 2 3 532 5 3 2 541 5 4 One 613 6 One 3 622 6 2 2 631 6 3 One 712 7 One 2 721 7 2 One 811 8 One One

B. Experimental Results

&Lt; Comparative Experimental Example 1 > Evaluation of whitening efficacy of a single sample

FIG. 1 shows the activity of inhibiting the tyrosinase activity of a single sample in order to examine the whitening activity of the whitening activity of the whitening activity of the whitish extracts (UPEF), phytotoxic extract (ECE) and glycosaminoglycans (GAGs) Inhibitory activity was measured by colorimetric assay of DOPA chrome produced by tyrosinase. Each sample was treated with concentrations of 25, 50 and 100 μg / ml to determine tyrosinase inhibitory activity. As a result, positive control (positive control) concentrations of 50 and 100 μg / It was confirmed that the inhibitory activity was better than that of arbutin.

FIG. 2 is a graph showing the effect of inhibiting the activity of inhibiting the production of melanin in a single sample of the primate extract (UPEF), the ginger extract (ECE) and the glycosaminoglycan (GAGs). The melanin content of each sample was examined by using 25, 50, and 100 μg / ㎖ concentration to confirm the melanogenesis inhibitory activity. As a result, it was confirmed that the content of melanin was decreased in the ECE sample. In addition, it was confirmed that the content of melanin was significantly reduced at a concentration of 100 μg / ml of the ginseng extract (UPEF) and glycosaminoglycan (GAGs).

In addition, the whitening effect of zebra extract (UPEF), ginger extract (ECE), and glycosaminoglycan (GAGs) in the zebrafish model was confirmed. Zebrafish is a recent animal model that has very similar genes to humans. Compared with conventional mouse models, it is possible to breed in a narrow space, it is relatively easy to manage, cheaper than mice, and life cycle is short, so it is very easy to experiment, so we measured whitening activity using zebrafish.

FIG. 3 shows the toxicity of a single sample of Umbelliferae Extract (UPEF), Gentian Extract (ECE), and Glycosaminoglycan (GAGs) in order to measure the whitening activity in a zebrafish model. The zebrafish embryos, which had been spawning for 8 hours, were continuously treated with each concentration (25, 50 ㎍ / ㎖) until hatching and the survival rate was measured by counting the number of hatching. As a result, it was confirmed that the survival rate was slightly lowered to 90% at the concentration of 50 ㎍ / ㎖ of the nematode extract (UPEF), but there was no significant difference, and the whitening activity experiment was carried out at the same concentration.

FIG. 4 is a graph comparing the whitening activity of a single sample of Japanese apricot extract (UPEF), ginger extract (ECE), and glycosaminoglycan (GAGs) at 25 and 50 μg / ml concentrations in a zebrafish model. To determine the whitening activity of the whitening activity of the whitening extracts (UPEF), the ginger extract (ECE) and the glycosaminoglycans (GAGs), each sample was added to the zebrafish embryos which had been scattered for 8 hours at 25, 50 μg / ) For up to 35 hours and then melanin content was confirmed when hatching occurred. The results showed that the melanin content of the zebrafish larva was decreased and the melanin content of the zebrafish larva image was decreased when the samples were treated as compared to the control group without any treatment. The whitening effect was excellent at a concentration of 50 / / ml of glycan (GAGs).

&Lt; Comparative Experimental Example 2 > Evaluation of wrinkle improving effect of single sample

FIG. 5 is a graph showing the effect of collagenase and elastase inhibitory activity of a single sample of Umbelliferae extract (UPEF), menthol extract (ECE) and glycosaminoglycan (GAGs). Epigallocatechin gallate (EGCG), known to be a positive control for improving wrinkles, was used. In the case of collagenase inhibitory activity, epigallocatechin gallate inhibited And the activity was 80%. Elastase inhibitory activity was also significantly inhibited by the non-sperm extract (UPEF) and the mite extract (ECE).

&Lt; Comparative Experimental Example 3 > Evaluation of Cell Regeneration Efficiency of Single Samples

Fig. 6 shows the fibroblast proliferative potency of the non-digested extract (UPEF), the gangue extract (ECE) and the glycosaminoglycan (GAGs). Mouse fibroblast NIH3T3 cells were seeded at a density of 5 × 10 ⁴ cells / well on a 96-well plate. After 24 hours, the cells were cultured for 24 hours in a medium containing no serum. After 24 hours, The BrdU reagent was previously treated 2 hours before. After the reaction, the immobilized reagent was reacted at room temperature for 30 minutes. After washing the plate, BrdU antibody was attached to confirm the degree of proliferation.

Epidermal growth factor (EGF) was used as a positive control and UPE and ECE were superior to positive control in cell proliferation.

FIG. 7 is a graphical representation of an experiment to confirm wound and skin regeneration efficacy in a zebrafish model.

FIG. 8 shows the scratching and skin regeneration efficacy in the zebra fish model of Japanese apricot extract (UPEF), ginger extract (ECE) and glycosaminoglycan (GAGs). In the zebrafish larva state, the tail part was cut uniformly, and then the tail part was restored for 4 days at the interval of 2 days after the sample treatment.

(5 days) after cutting off the tail, it was confirmed that it was fully recovered at a concentration of 50 μg / ml of the non-digested extract (UPEF), the ginger extract (ECE) and the glycosaminoglycan (GAGs) . On the fourth day (7 days), it was confirmed that the cells recovered even at a concentration of 25 μg / ml. However, in the control group that did not process anything, it was confirmed that the recovery was not completed even on the fourth day.

<Experimental Example 1> Evaluation of whitening efficacy of a mixed sample

FIG. 9 is a graph showing the effect of tyrosinase inhibitory activity on the whitening effect of the mixed samples according to the mixing ratio of the whitish extract (UPEF), the gentle extract (ECE) and the glycosaminoglycan (GAGs). In order to ascertain whether there is a rising effect according to the mixing ratio of UPG, UPE, ECE and GAGS, tyrosinase inhibitory activity was measured by making a total of 36 mixing ratios See Table 1 in Example 8).

As a result of measuring the tyrosinase inhibitory activity of the sample according to the mixing ratio, it was confirmed that the whitening activity was increased with the increase of the proportion of gangrene extract (ECE) or the ratio of glycosaminoglycan (GAGs) The samples with ascending effect were confirmed by mixing ratio. Therefore, seven melanin inhibitory activities of 262, 271, 352, 361, 433, 451 and 721 were investigated.

FIG. 10 compares the melanin inhibitory activities of seven samples, 262, 271, 352, 361, 433, 451 and 721, which have an elevating effect on tyrosinase inhibitory activity. Melanin content of melanin was lower than that of α-MSH, and 352, 361, and 451 were significantly different from other blend ratios. Therefore, the whitening activity in the zebrafish model was confirmed using 352, 361, and 451 mixed ratio samples, which were significantly different from each other.

In order to measure the whitening activity of 352, 361, and 451 mixed samples with the highest activity, it was first confirmed to be toxic in the zebrafish model. The procedure was the same as the single sample test method. FIG. 11 shows the result of confirming the toxicity of the mixed sample, and it was confirmed that all the samples were free of toxicity.

352, 361, and 451 mixed ratios were tested in the same manner as the single sample test method. FIG. 12 shows that the melanin content of the zebrafish larva and melanin content were decreased when the samples were treated as compared to the control group without any treatment, It was confirmed that the activity was most excellent at a concentration of 50 / / ml.

&Lt; Experimental Example 2 > Evaluation of wrinkle improving effect of mixed samples

1) Collagenase and elastase inhibitory activity according to the mixing ratio of UPG, UPE, ECE, and GAGS

Figures 13 and 14 show the effect of increasing the amount of collagenase (collaganase) in 36 total mixed samples in order to ascertain whether there is a rising effect according to the mixing ratio of UPG, UPE, ECE, and GAGS. ) And elastase inhibitory activity were measured. As a result of measurement of the collaganase inhibitory activity of the sample according to the mixing ratio, it was confirmed that the inhibitory activity was increased as the ratio of glycosaminoglycan (GAGs) was increased. In the elastase inhibitory activity, It was found that as the content of extracts (UPEF) and gangrene extract (ECE) increases, the inhibitory activity increases as the content of glycosaminoglycan (GAGs) decreases. In addition, samples with ascending efficacy were identified by mixing ratio than when single samples were treated.

15 and 16, seven of the mixing ratios 262, 271, 352, 361, 433, 451 and 721 having ascending effect were selected and the collagenase and elastase inhibitory activity Were again measured with a positive control. Epigallocatechin gallate (EGCG) and urosolic acid (UA) were used as positive controls. The inhibitory activity of the enzyme was significantly higher than that of the positive control at all mixing ratios.

FIG. 17 shows the effect of promoting pro-collagen synthesis in cells treated with the above seven mixed samples

Collagen, a major constituent of extracellular matrix, is a major substrate protein produced by the fibroblasts of the skin. It is composed of the mechanical rigidity of the skin, the connective tissue resistance and the binding force of tissue, the support of cell adhesion, the induction of cell division and differentiation Function. It is known that collagen (type I, II, III, IV, V) is synthesized in the form of a pro-collagen precursor and has a function of cleaving and separating from collagen molecules when collagen polymerization occurs . Therefore, by measuring the amount of procollagen, the degree of collagen biosynthesis in the cell can be grasped.

The pro-collagen content of seven samples with elevated efficacy was confirmed, and the pro-collagen content was significantly increased at a mixing ratio of 352, 361, 451, and 721.

&Lt; Experimental Example 3 > Evaluation of Cell Regeneration Efficiency of Mixed Samples

In FIG. 18, cell regeneration efficacy was confirmed by the mixing ratio of the unmodified extract (UPEF), the ginger extract (ECE) and the glycosaminoglycan (GAGs). Fibroblast proliferative activity of 36 mixed ratio samples was confirmed in order to ascertain whether there was a rising effect according to the mixing ratio of UPG, UPE, ECE, and GAGS. As a result of the measurement of the fibroblast proliferative capacity of the sample according to the mixing ratio, it was confirmed that the cell proliferative capacity was increased as the ratio of glycosaminoglycans (GAGs) was increased, and the cell proliferating ability was also increased Samples with efficacy were identified and a total of seven mixing ratios with elevated efficacy were selected.

Seven samples with elevated efficacy were selected from the results of the measurement of fibroblast proliferative activity. In FIG. 19, the cell regeneration efficiency of the seven samples was confirmed again with positive control, and as a positive control, Epidermal Growth Factor, EGF) was used. As a result of the cell regeneration activity, the activity was significantly higher at 352, 361, 433, 451, and 721 than the positive control, and the highest activity was observed at the 451 mixture ratio.

Figure 20 shows that the 352, 361, 433, 451, and 721 mixed ratios of 352, 361, and 451, which were commonly used for whitening and wrinkle improvement, had a significant effect on fibroblast proliferative activity. The wound regeneration efficiency was confirmed. The experimental method was the same as that of the single-sample method. As a result of examining the wound regeneration efficiency of the sample, it was observed that all the groups except the control group completely recovered on the second day (5days) after the sample treatment there was.

21 shows the cell migration effect of the 451 mixture ratio sample. As a result, it was confirmed that the cell migration was increased in the 451 mixture ratio compared to the control group.

22 shows the results of western blot analysis of the degree of protein expression of p-EGFR and p-AKT in order to confirm the pathway through which cell regeneration occurs. The quantified protein (40 μg) was sampled using a 4 × sample buffer and boiled at 95 ° C. for 10 minutes. The boiled samples were electrophoresed on a 10% SDS-PAGE gel at 130 V for 90 minutes. After electrophoresis, proteins were transferred to 300 mA for 70 minutes using a 0.45 mu m nitrocellulose membrane. 5% skim milk was prepared in 1 x TBST and then blocked for 1 hour at room temperature and incubated for 24 hours at 4 ° C using a primary anti-body . All primary anti-bodies were diluted in 1% skim milk. After incubation at 4 ° C, the cells were washed 3 times for 10 minutes at room temperature with 1 × TBST for 30 minutes, and then incubated at room temperature with a secondary antibody diluted 1: 3000 in 1% skim milk. Lt; / RTI &gt; After attaching the secondary anti-body at room temperature, the plate was washed with 1 × TBST for 3 minutes for 30 minutes, and the ECL solution was reacted to the membrane (membarane) and analyzed with the Vilber Fusion solo system.

As a result of the analysis, it was confirmed that a positive control Epidermal Growth Factor (EGF) binds to Epidermal Growth Factor Receptor (EGFR) and cell proliferation occurs through phosphor-AKT , 451 in the mixed ratio sample, the cell proliferation was observed through phosphor-AKT, which is a sub-mechanism after binding to other receptors other than EGFR binding.

<Experimental Example 4> Evaluation of Antioxidant Efficacy

When the whitening, wrinkle improvement, and cell regeneration activities of the 7 samples selected above were evaluated comprehensively, the 352, 361 and 451 mixed ratio samples showed the highest activity.

FIG. 23 shows the survival rate and the ROS inhibitory activity of the 352, 361, and 451 samples when 5 mM H ₂ O ₂ was treated to evaluate the antioxidant activity of the 352, 361, and 451 samples in the zebrafish. For the 8-hour egg laying, 352, 361 and 451 mixed ratios were treated from 25 ㎍ / ㎖ to 50 ㎍ / ㎖ and treated with 5 mM H ₂ O ₂ after 1 hour. After incubation at 37 ° C for 3 days, the survival rate was determined by counting the number.

The survival rate of the control group was about 96% and the survival rate of the group treated with 5 mM hydrogen peroxide (H ₂ O ₂ ) was less than 60%. The survival rate of the group treated with the 352 and 451 mixture ratios increased with increasing concentration and increased to 70% at 50 ㎍ / ㎖. Therefore, it has been confirmed that there is a protective effect against oxidative stress caused by hydrogen peroxide (H ₂ O ₂ ).

FIG. 24 shows the reactive oxygen species (ROS) scavenging activity in the zebrafish of the mixing ratio samples. The procedure for treating the mixed ratio sample and H ₂ O ₂ was the same as the above-mentioned survival rate measurement, and the scavenging activity was measured after 3 days by staining the ROS generated with the DCF-DA reagent.

Fluorescence photographs of zebrafish showed that the group treated with H ₂ O ₂ had a brighter fluorescence than the control group that did nothing. Since DCF-DA reagent is a reagent that expresses green fluorescence in combination with ROS, it can be confirmed that ROS was generated in zebrafish by treating H ₂ O ₂ . The amount of ROS was increased by treatment with H ₂ O ₂ , but it decreased by 37% when treated with 50 μg / ml of 451 mixing ratio sample. As a result, it was confirmed that the 451 mixture ratio sample had scavenging activity for ROS caused by H ₂ O ₂ .

Experimental Example 5 UV Protection Effect in Zebrafish Model of 352, 361 and 451 according to Mixing Ratios of Sweet Potato Extract (UPEF), Ginseng Extract (ECE) and Glycosaminoglycan (GAGs)

1) Measurement of zebrafish survival rate of mixed ratio samples

FIG. 25 is a graph showing the survival rate and the NO inhibitory activity when irradiated with 50 mJ / cm 2 of ultraviolet ray B to evaluate ultraviolet (UV) protection efficacy of 352, 361, and 451 mixed ratio samples in zebrafish. The zebrafish larva was treated with 352, 361, and 451 mixed ratios from 25 ㎍ / ㎖ to 50 ㎍ / ㎖ and irradiated with ultraviolet B 50mJ / ㎠ after 1 hour. After incubation at 37 ° C for 3 days, the survival rate was determined by counting the number.

The survival rate of the control group was about 98% and the survival rate of the UVB group was 85% or less. In the groups treated with 352, 361, and 451 mixed ratios, it was confirmed that the concentration increased to about 95% at the concentration of 50 μg / ml, and it was confirmed that the protective effect against the stress due to ultraviolet B was confirmed.

2) NO scavenging activity in zebrafish of mixed ratio samples

FIG. 26 shows the results of measuring the scavenging activity by staining the NO generated with the DAF-FM-DA reagent three days after irradiating the mixed ratio sample and ultraviolet ray B at 50 mJ / cm 2.

Fluorescence photographs of the zebrafish showed that the group irradiated with ultraviolet ray B 50 mJ / cm 2 had brighter fluorescence than the control group which did nothing. Since DAF-FM-DA reagent is a reagent that binds to NO and expresses green fluorescence, it can be confirmed that NO is generated in zebrafish by irradiating ultraviolet ray B. The amount of NO was increased by irradiating ultraviolet ray B, but when it was treated with 50 μg / ml of 451 mixing ratio sample, it was confirmed that the amount of NO decreased by 26%, and the 451 mixing ratio sample showed an elimination activity against NO generated due to ultraviolet ray B I could confirm.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, Of the right.

Claims (12)

An anti-aging cosmetic composition comprising, as an active ingredient, an irregular extract, a gentian extract, and a glycosaminoglycan. 2. The cosmetic composition according to claim 1, wherein the apricot extract is a Celluclast enzyme extract. 2. The cosmetic composition according to claim 1, wherein the apricot extract comprises fucoidan. 2. The cosmetic composition according to claim 1, wherein the glycosaminoglycan is separated from the skins. 2. The cosmetic composition according to claim 1, wherein the phlegm extract is an extract of phytotoxic ethanol containing polyphenol and diegol. [Claim 2] The cosmetic composition according to claim 1, wherein the apricot extract, gentian extract and glycosaminoglycan are mixed at a weight ratio (w / v) of 1: 0.1-4: 0.1-1. 2. The cosmetic composition according to claim 1, wherein the non-ginseng extract, ginseng extract and glycosaminoglycan are mixed at a weight ratio (w / v) of 4: 5: 1. The cosmetic composition according to claim 1, wherein the cosmetic composition is for skin whitening, wrinkle reduction, antioxidation, ultraviolet screening or skin regeneration. An anti-aging quasi-drug composition comprising, as an active ingredient, an irregular extract, a phlegm extract and a glycosaminoglycan. The quasi-drug composition according to claim 9, wherein the quasi-drug composition is for skin whitening, wrinkle reduction, antioxidation, ultraviolet screening or skin regeneration. An anti-aging dermatological external preparation composition comprising, as an active ingredient, an irregular extract, a gentian extract, and a glycosaminoglycan. 12. The composition for external application for skin according to claim 11, wherein the composition for external application for skin is for skin whitening, wrinkle reduction, antioxidation, ultraviolet screening or skin regeneration.

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