KR101826296B1 - Skin regenerating and whitening composition containing sea cucumber extract - Google Patents

Abstract

A skin improving composition containing a sea cucumber extract as an active ingredient is disclosed. The sea cucumber extract prepared by repeating lyophilization twice is effective for skin regeneration activity and skin whitening.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a skin improving composition containing a sea cucumber extract as an active ingredient,

The present invention relates to a skin improving composition containing an extract of sea cucumber as an active ingredient, and more particularly, to a skin improving composition containing an active ingredient of a sea cucumber extract prepared by repeating lyophilization twice.

Sea cucumber is a marine invertebrate belonging to the Holothuroidea. There are 4 species and 14 species in Korea such as blue sea cucumber, black sea cucumber and red sea ginseng. Sea cucumbers, together with ginseng, have been the subject of food and nutrition research since ancient times as a typical aquatic food. It is known that it has been widely used since ancient times because of its effects on restorative and nervous stability, and recent studies have found that saponin is found in food and nutrition.

In recent years, not only research on food and nutrition of sea cucumber, but also research on natural products have been actively conducted. Especially, recent studies have proved the effect of sea cucumber on skin regeneration or skin whitening. However, most of the studies on skin regeneration and skin whitening were carried out simultaneously with extracts from sea cucumber and other raw materials. Therefore, it was difficult to confirm the effect of sea cucumber alone on skin regeneration and skin whitening. In addition, there has been no detailed study on the difference in skin activity depending on the type, region, and extraction method of sea cucumbers when studying only sea cucumber. In most cases, And skin whitening effects on the skin.

Therefore, it has been necessary to study whether sea cucumbers have effects on skin regeneration and skin whitening, and it has also been necessary to confirm that human skin has an effect on skin regeneration and whitening of sea cucumber.

Patent Registration No. 10-1141929 (Apr. 25, 2012) Published Japanese Patent Application No. 10-2015-0111699 (Oct. 10, 2015)

The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide a skin improving composition comprising an extract of sea cucumber produced by repeated freeze-drying twice as an active ingredient.

As a technical means to achieve the above technical object, the skin improving composition according to one aspect of the present invention contains the sea cucumber extract prepared by repeating lyophilization twice as an active ingredient.

The sea cucumber extract may be prepared by first lyophilizing sea cucumber, preparing a powder by grinding lyophilized sea cucumber, preparing a powder by pulverizing the lyophilized sea cucumber, incubating the pulverized powder, Evaporating concentrated sea cucumbers, filtering and condensing evaporated concentrated sea cucumbers, and lyophilizing the condensed sea cucumbers.

The skin improving composition may further include deep-freezing the sea cucumber from -70 ° C to -100 ° C before the first lyophilization step.

The first lyophilization step may include a step of cooling the sea cucumber to -20 ° C to -100 ° C and a step of drying the cooled sea cucumber for 36 hours to 84 hours.

The incubation step may be carried out by adding 55% to 95% ethanol to the sea cucumber powder and incubating the mixture for 1 hour to 3 hours.

The evaporation step may be carried out at a temperature ranging from 55 ° C to 100 ° C for 1.5 hours to 5 hours by evaporation.

The sea cucumber may be a blue sea cucumber and / or a black sea cucumber.

The above-mentioned sea cucumber extract may be extracted using only sea cucumber.

The sea cucumber extract may be prepared by preparing a solution by dissolving the second lyophilized sea cucumber in a solvent.

The solvent may be one or more selected from the group consisting of butylene glycol, water, ethyl hexanediol, and 1,2-hexanediol.

The skin improvement may be a skin regeneration activity.

The skin regeneration activity may be an increase in expression level of collagen type 4, an increase in cellular activity factor (ki-67), and / or a decrease in substrate decomposition enzyme secretion amount.

The skin improvement may be a skin whitening.

The skin whitening may be a melanin formation inhibition.

The skin improving composition containing the sea cucumber extract of the present invention as an active ingredient has an effect of regenerating the skin such as an increase in the expression amount of collagen type 4 and a decrease in the amount of substrate decomposition enzyme and a skin whitening effect for preventing blackening of the skin such as inhibition of melanin formation .

Fig. 1 shows a production process for extracting sea cucumber extract.
2 shows a process for preparing a sea cucumber extract as a solution.
Fig. 3 shows the expression levels of collagen type 4 and ki-67 of Comparative Examples 1, 2 and 4. Fig.
Fig. 4 shows the expression levels of collagen type 4 and ki-67 of Examples 3 and 4. Fig.
Fig. 5 shows a comparison graph of the expression ratio of ki-67.
6 shows a standard curve graph of MMP-9.
Fig. 7 shows a comparative graph of the secretion amount of MMP-9.
Figure 8 shows a melanin standard curve.
9 shows comparative graphs of the amounts of melanin in Comparative Examples 2 and 3 and Examples 1 to 6. Fig.

Hereinafter, the present invention will be described in more detail. However, it should be understood that the present invention is not limited thereto, and the present invention is only defined by the scope of the following claims.

The present invention provides a skin cosmetic composition having an effect on skin regeneration activity and skin whitening.

The skin improving composition according to one aspect of the present invention contains the sea cucumber extract prepared by repeating lyophilization twice as an active ingredient.

The sea cucumber is a marine life belonging to aspiodchirotida, stichopodidae, and stichopus, and 1,500 species live in the world. Sea cucumbers in the Republic of Korea have the scientific name stichopus japonicus, and 4 and 14 species are inhabited.

The sea cucumber can be preferably a blue sea cucumber and a black sea cucumber. Chunghae is a sea cucumber which is inhabited by rocks, pebbles, and sandy areas of aquatic waters.

The sea cucumber may be at least one site selected from the group consisting of the whole of sea cucumbers, the parts excluding the viscera and the viscera.

Drying methods of seafood include natural drying, pressure drying, atmospheric drying, and vacuum drying. The above freeze drying is a drying method belonging to vacuum drying. The lyophilization is a drying method in which a material containing a large amount of water is frozen and decompressed to sublimate ice to remove moisture to obtain a dried material. The lyophilization method has advantages of minimizing the damage of the heat-sensitive material and drying it, and the dried material has a structure that allows easy penetration of moisture, thereby allowing rapid rehydration of the frozen material.

Since the sea cucumber is susceptible to deformation of the tissue by heat and has a high moisture content, it is suitable to dry the sea cucumber by freeze-drying. The lyophilized sea cucumber can be dried while maintaining its original structure, and the rehydration of water is excellent.

The effective ingredient means that the main component of the skin-improving composition is a sea cucumber extract. That is, the sea cucumber extract directly or indirectly expresses the skin cosmetic function of the composition.

The composition is a skin improving composition. Skin improvement means skin whitening, skin wrinkle improvement, and functions that help protect skin from ultraviolet rays.

The skin improving composition containing the sea cucumber extract may contain only the sea cucumber extract as an active ingredient or may contain other necessary ingredients as well as the sea cucumber extract according to the desired skin improving function and formulation. The skin-care composition may be used as an external preparation for skin, or may be used as a food such as a health food or a drug such as an injection.

The above-mentioned sea cucumber extract may be prepared by first lyophilizing sea cucumber (S110), grinding the freeze-dried sea cucumber to produce powder (S120), incubating the pulverized powder (S130), concentrating the incubated sea cucumber (S140), filtration (S150) and condensation (S160) of the evaporated concentrated sea cucumber, and second lyophilization (S170) of the condensed sea cucumber.

The skin-improving composition may be prepared by a manufacturing method comprising deep-freezing the sea cucumber from -70 ° C to -100 ° C before the first lyophilization step. Deep-freezing can completely remove water from the sea cucumber, and because the cryo-freezing rapidly freezes, the cells of the sea cucumber can be kept intact without destruction. The deep-freezing can be performed in a quick freezing mode in which the temperature range of -1 ° C to -5 ° C, which is the maximum ice crystal generation temperature, is rapidly passed.

The sea cucumber dried in the primary freeze-drying (S110) is concentrated in nutrients. That is, when the water is released from the sea cucumber made of water by 90% or more of free water, the dried sea cucumber contains highly concentrated nutrients compared with the sea cucumber. Therefore, it is possible to exhibit an excellent skin improving effect even in a small amount.

The first lyophilization step (S110) may include a step of freezing the sea cucumber to -10 ° C to -110 ° C and a step of drying the frozen sea cucumber for 36 hours to 84 hours.

The freezing temperature of the first lyophilization step (S110) may preferably be from -10 ° C to -110 ° C, more preferably from -20 ° C to -100 ° C. The freeze-drying time varies depending on the freezing temperature. The lower the freezing temperature, the shorter the freezing time and the smaller the pore size and the dense texture. This is because rapid freezing causes ice particles to be formed because ice crystals pass quickly. When the freezing temperature is lower than -110 ° C., the freeze-drying time is shortened. However, since the rate of formation of ice crystals increases and the ice crystal becomes smaller, the rate at which the water penetrates into the first freeze-dried sea cucumber and is absorbed by the dried sea cucumber And the hydration restoring force may be low. When the freezing temperature is higher than -10 ° C, the voids inside the sea cucumber become larger and the hydration restoring force improves. However, the freeze-drying time becomes longer and the process of producing the extract may be longer.

The primary frozen sea cucumber may preferably be dried for 36 to 84 hours, more preferably for 48 to 72 hours. The drying means sublimation drying and / or secondary drying. Sublimation drying means that the pressure is lowered to below the triple point of water after freezing and the water is dried by changing the solid to direct gas. Secondary drying means dehumidifying to dry remaining water even after sublimation drying, it means. If the drying time is shorter than 36 hours, the moisture in the sea cucumber may not be sufficiently dried. If the drying time is longer than 72 hours, the water may be excessively dried to deteriorate the hydration restoring force.

The first lyophilized sea cucumber is crushed to prepare a powder (S120). The size of the powder may be preferably from 0.01 nm to 1 mm, depending on the purpose of use. When the lyophilized sea cucumber is made into a powder, the size of the powder becomes small, so it can be easily absorbed into the body, and the surface area can be increased to easily react with other substances contained in the skin improving composition.

The step (S130) of incubating the pulverized powder may be performed by preparing a method of adding 55% to 95% ethanol to the sea cucumber powder and incubating the powder for 1 hour to 3 hours.

The ethanol may preferably be 55% to 95% pure, more preferably 65% to 85% pure. Incubation is preferably carried out for 1 to 3 hours, more preferably for 1.5 to 2.5 hours. The incubation step (S130) may degrade the substrate protein of sea cucumber, and the degraded substrate protein may activate the skin-beauty function of the sea cucumber extract. When the purity of the ethanol is less than 55%, contaminants may be contained and incubated. When the purity of the ethanol is more than 95%, the sea cucumber protein may be coagulated due to high purity ethanol. If the incubation time is less than 1 hour, the substrate protein of the sea cucumber may not be decomposed sufficiently to make the extract. If the incubation time is more than 3 hours, the skin cosmetic function of the sea cucumber may be lost due to decomposition of the substrate protein of the sea cucumber have.

The evaporation concentration step (S140) may be performed by a manufacturing method comprising evaporating the incubated sea cucumber at 55 to 100 DEG C for 1.5 to 5 hours.

The evaporation concentration can be preferably conducted at 55 to 100 DEG C for 1.5 to 5 hours, more preferably 65 to 90 DEG C for 2 to 4.5 hours. Through the above evaporation and concentration step, only the pure sea cucumbers can be obtained by removing contaminants such as ethanol, oil components, and heavy metals. If the evaporation concentration temperature is lower than 55 ° C, the contamination component is not volatilized due to the low temperature, so that impurities may remain in the sea cucumber extract. If the temperature exceeds 100 ° C, the skin improvement component of the sea cucumber may be destroyed due to high temperature. When the evaporation time is less than 1.5 hours, ethanol, pollutants and the like may not evaporate due to a short time. If the evaporation time is less than 1.5 hours, the protein may be deformed even at a low temperature due to heating for a long time .

The step of filtering (S150) and condensing (S160) the incubated sea cucumber is a step for obtaining high purity sea cucumber extract by filtering and concentrating the remaining impurities from evaporated concentrated sea cucumber.

The step of performing the second lyophilization of the condensed sea cucumber (S170) is a step for obtaining a high purity sea cucumber extract by lyophilizing the condensed sea cucumber. In the second lyophilization step, the first lyophilization step is repeated, and a detailed description thereof will be omitted.

The sea cucumbers can be preferably blue sea cucumber and / or black sea cucumber, more preferably blue sea cucumber and / or black sea cucumber inhabiting the coast near Baekryeong in the west coast. Blue sea cucumber and black sea cucumber belong to my chronic sea cucumber, and red sea cucumber belongs to outer sea cucumber. Chronic sea cucumber and black sea hens are thick and short in the polian vesicle, the tip is formed in a round shape, the shrinkage is low, and the tentacle ossicle is simple in shape. On the other hand, red sea ginseng, a foreign sea cucumber, is thin and long, has a pointed shape at its tip, is shrunken, and has a complex shape. The blue sea cucumber and black sea shrimp have various nutritional components compared with red sea ginseng because they eat various foods compared to red sea cucumbers which mainly eat red algae.

The above-mentioned sea cucumber extract may be extracted using only sea cucumber. Sea cucumber intestine has high antioxidant ability compared to the area except intestine due to the effect of seaweed present in the intestine. Antioxidant means an action to prevent oxidation. The antioxidant power of the sea cucumber can prevent the aging of the cells due to oxidization of the cells by removing the active oxygen in the human body, which can help skin beauty. Accordingly, the skin improving composition containing the extract extracted from the sea cucumber alone as an effective ingredient can exert excellent functions for skin regeneration activity and skin whitening.

The above-mentioned sea-cucumber extract may be a skin-improving composition which is prepared by further dissolving the second lyophilized sea cucumber in a solvent to prepare a solution (S180). The skin improving composition containing the sea cucumber extract as an active ingredient can be used in various formulations. Therefore, depending on the purpose and method of use, the skin-improving composition may contain a sea cucumber extract of the solution. The solvent may be one or more solvents selected from the group consisting of polar, non-polar, organic and inorganic solvents.

The solvent may preferably be one or more selected from the group consisting of butylene glycol, water, ethyl hexanediol, and 1,2-hexanediol.

Butyleneglycol, ethylhexanediol, and hexanediol are alcoholic compounds that can dissolve various components of sea cucumber and can produce a solution that can effectively exert the skin improving function of the skin care composition containing the sea cucumber extract.

The skin improvement may be a skin regeneration activity. The skin regeneration activity may preferably be an increase in the expression level of collagen type IV (collagen type IV), an increase in cellular activity factor (ki-67) and / or a decrease in the secretion amount of matrix metalloproteinases.

The skin regeneration activity may be increased expression of collagen type 4. The expression level of the collagen type 4 may be 200% to 500%, preferably 103% to 110%, relative to the ascorbic acid, as compared to the negative control prepared with dimethylsulfoxide.

Collagen is a major protein that constitutes the flesh and connective tissues of mammals, accounting for 25% to 35% of the proteins that make up the body. To date, 29 types of collagen have been identified. Collagen type 4 is a major component of basement membrane and glomerular epileptic matrix. Collagen type 4 is composed of four regions such as 7S collagen region, NC1 (non collagenous 1) region, NC2 (non collagenous 2) region and TH (triple helix) region. The four molecules of collagen type 4 are connected to each other at the NH2 terminal site. The site where the disulfide bond (SS) is abundant is the 7S collagen region, which is not degraded by various proteases such as bacterial collagenase . The basement membrane is a thin membrane between the epidermis and the epidermis and is responsible for skin regeneration. When the basement membrane is damaged, an enzyme that breaks down the collagen into the dermis in the epidermal layer breaks and collagen fibers are destroyed. The skin improving composition may have a function for skin regeneration activity through a mechanism of enhancing the basement membrane by increasing the expression amount of collagen type 4, which is a main component of the basement membrane.

The skin regeneration activity may be an increase in the cellular activity factor (ki-67). The expression amount of the cell activation factor (ki-67) may be 200% to 500%, as compared to 0.1% dimethyl sulfoxide, which is a negative control. Cell activator (ki-67) is a protein used as a marker of cell proliferation and is expressed in the G1, G2, S, and M phases of the cell cycle. A skin improving composition containing an extract of sea cucumber as an active ingredient has an effect on skin cells to increase regeneration activity. Therefore, cell regeneration such as the stratum corneum of the skin in which the skin improving composition is used becomes active and the expression amount of the cell activation factor (ki-67) is increased.

The skin regeneration activity may be a decrease in substrate decomposition enzyme secretion amount. The reduction of the substratease is preferably 50% to 90% of the amount of substratease expression of the negative control prepared with dimethylsulfoxide, and 60% to 90% of the amount of substratease expression of ascorbic acid .

The proteolytic enzyme refers to type IV collagenase, stromelysin (MMP-3), which degrades collagen type 4. Type 4 collagenase is composed of gelatinase A (MMP-2) and gelatinase B (gelatinase B, MMP-9). MMP-2 is produced in fibroblasts, endothelial cells and macrophages. MMP-9 is produced in both normal cells and cancer cells. Stromelysin (MMP-3) is produced in cancer cells. The sea cucumber extract may have a function for skin regeneration activity through a mechanism of reducing collagen type 4 degradation by decreasing secretion amount of the substratease.

The skin improvement may be a skin whitening. Skin whitening means to prevent the skin from becoming melanogenesis by the melanin pigment, making the skin whiter and beautiful.

The skin-care composition may have a skin-whitening function before, during or after synthesis of melanin. Before melanin synthesis, through tyrosinase transcriptional regulation and glycation control, through tyrosinase inhibition, peroxidase inhibition, reducing agent and reactive oxygen scavenging during melanin synthesis, tyrosinase degradation, melanin body movement inhibition, Whitening function can be done.

The skin whitening may be a melanin formation inhibition. The decrease in the amount of melanin secretion in the skin-improving composition containing the sea cucumber extract as an active ingredient may preferably be 12% to 15% lower than that of the negative control prepared with 1X PBS, and the negative control The melanin secretion amount of the melanin is decreased by 2% to 6%.

The melanin inhibition is caused by tyrosine tyrosinase, which converts melanin into dopa and dopaquinone, and then the tyrosinase-related proteins TRP-2 (dopacrome tautomerase) and TRP-1 (5,6- dihydroxyindole-2-carboxylic acid oxidase. The skin improving composition may inhibit the formation of melanin by inhibiting the mechanism by which tyrosine is formed into melanin by increasing tyrosinase inhibitory activity and / or decreasing tyrosinase expression.

Hereinafter, embodiments of the present invention will be described in detail 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.

Comparative Example  1 to 3: negative control group

Comparative Examples 1 to 3 were prepared using a maintenance medium provided by Tegoscience, 0.1% dimethyl sulfoxide provided by Tegoscience, 1X PBS (phosphate buffered saline) provided by Tegoscience, As a negative control.

Comparative Example  4 and 5: positive control group

Comparative Example 4 was made with L-Ascorbin acid and Comparative Example 5 was made with arbutin as a positive control.

Example  1: low concentration sea cucumber extract

The frozen sea cucumber was crushed and prepared as a powder after the first freeze - drying step in which the blue sea cucumber and the black sea cucumbers collected from the sea near Baekryeong Island were washed, cooled at -30 ° C and dried for 48 hours. The sea cucumber powder was incubated in 75% ethanol for 2 hours, then evaporated at 70 ° C for 3 hours, filtered through cotton. and concentrated at a low concentration of 50 mg / ml for 5 hours using a vacuum evaporator (EYELA N-12, EYEKACE-1112, Tokyo, Japan). The concentrated sea cucumber was secondly lyophilized under the conditions of -30 占 폚 for 48 hours.

Example  2: High concentration of sea cucumber extract

The frozen sea cucumber was crushed and prepared as a powder after the first freeze - drying step in which the blue sea cucumber and the black sea cucumbers collected from the sea near Baekryeong Island were washed, cooled at -30 ° C and dried for 48 hours. The sea cucumber powder was incubated in 75% ethanol for 2 hours, then evaporated at 70 ° C for 3 hours, filtered through cotton. and concentrated at a low concentration of 50 mg / ml for 5 hours using a vacuum evaporator (EYELA N-12, EYEKACE-1112, Tokyo, Japan). The concentrated sea cucumber was secondly lyophilized under the conditions of -30 占 폚 for 48 hours.

Example  3: Low-concentration sea cucumber extract

The inside of the blue sea cucumber and the black sea cucumber collected in the sea area near Baekryeong Island were washed and then subjected to deep-freezing at -80 캜 using a dip-freezer. After the first freeze-drying step in which the deep-frozen sea cucumber was cooled at -30 ° C for 48 hours, the frozen sea cucumber was pulverized to prepare powder. The ginseng embedded powder was incubated in 75% ethanol for 2 hours, then evaporated at 70 ° C for 3 hours, concentrated by evaporation, and then filtered through cotton. and concentrated at a low concentration of 50 mg / ml for 5 hours using a vacuum evaporator (EYELA N-12, EYEKACE-1112, Tokyo, Japan). The concentrated sea cucumber was lyophilized at -30 ° C for 48 hours.

Example  4: Extract of high concentration sea cucumber

The inside of the blue sea cucumber and the black sea cucumber collected in the sea area near Baekryeong Island were washed and then subjected to deep-freezing at -80 캜 using a dip-freezer. After the first freeze-drying step in which the deep-frozen sea cucumber was cooled at -30 ° C for 48 hours, the frozen sea cucumber was pulverized to prepare powder. The sea cucumber powder was incubated in 75% ethanol for 2 hours, then concentrated at 70 ° C for 3 hours, then filtered through cotton. and concentrated at a low concentration of 50 mg / ml for 5 hours using a vacuum evaporator (EYELA N-12, EYEKACE-1112, Tokyo, Japan). The concentrated sea cucumber was lyophilized at -30 ° C for 48 hours.

Example  5: Low concentration sea cucumber extract liquid

The frozen sea cucumber was crushed and prepared as a powder after the first freeze - drying step in which the blue sea cucumber and the black sea cucumbers collected from the sea near Baekryeong Island were washed, cooled at -30 ° C and dried for 48 hours. The sea cucumber powder was incubated in 75% ethanol for 2 hours, then evaporated at 70 ° C for 3 hours, filtered through cotton. and concentrated at a low concentration of 50 mg / ml for 5 hours using a vacuum evaporator (EYELA N-12, EYEKACE-1112, Tokyo, Japan). The concentrated sea cucumber was secondly lyophilized under the conditions of -30 占 폚 for 48 hours. The second lyophilized sea cucumber extract was mixed with butylene glycol, water, ethyl hexanediol and 1,2-hexanediol so as to prepare 5% concentration of sea cucumber extract solution.

Example  6: High concentration sea cucumber extract liquid

The frozen sea cucumber was crushed and prepared as a powder after the first freeze - drying step in which the blue sea cucumber and the black sea cucumbers collected from the sea near Baekryeong Island were washed, cooled at -30 ° C and dried for 48 hours. The sea cucumber powder was incubated in 75% ethanol for 2 hours, then evaporated at 70 ° C for 3 hours, filtered through cotton. and concentrated at a low concentration of 50 mg / ml for 5 hours using a vacuum evaporator (EYELA N-12, EYEKACE-1112, Tokyo, Japan). The concentrated sea cucumber was secondly lyophilized under the conditions of -30 占 폚 for 48 hours. The second lyophilized sea cucumber extract was mixed with butylene glycol, water, ethyl hexanediol and 1,2-hexanediol to prepare a 20% solution of sea cucumber extract.

Experimental Example  1: Wrinkle improvement test

The test substances stored at -20 ℃ were thawed immediately before treatment and diluted in maintenance medium (see Table 1 below). The control and test groups were treated with 3.5 ml of the medium layer per well of a 3D skin culture model (Neoderm ^® , Tegoscience) in an amount of 30 μl each on the surface of the cell layer, followed by incubation in a CO ₂ incubator (10% CO ₂ , 37 ° C.) Time. After cultivation for 24 hours, the replacement of the medium, and control and test groups 3D cultured skin model (Neoderm ^®, Tego Science ㈜) After each 3.5ml per well in culture medium layer, by iterating over the cell layer surface 30㎕ CO ₂ incubator (10 % CO ₂ , 37 ° C) for 24 hours and cultured for a total of 48 hours. Forty-eight hours after the culture, the culture media of all groups were collected, stored at -70 ° C, and frozen blocks were prepared.

Group Stock
Conc. Final
Conc. Stock
Volume Diluent
volume Final
Volume Final DMSO
Conc. (V / v) Comparative Example 1 - - - - 10ml - Comparative Example 2 100% 0.1% 10 μl 9,990 l 10ml 0.1% Comparative Example 4 20 mg 200 쨉 l 100 μl 9,900 l 10ml - Example 3 50 mg 50 μl 15 μl 14,985 l 15ml 0.1% Example 4 500 mg 500 μl 15 μl 14,985 l 15ml 0.1%

Experimental Example  1-1: Collagen Type 4  And ki -67

Frozen blocks were separated from the cryostat at a temperature of -20 ℃ in a total thickness of 5 μm and fixed at -20 ℃ in acetone: MeOH (1: 1). After washing 3 times with 1X PBS, permeabilization was performed with 0.5% triton X-100. After washing three times with 1 × PBS, the cells were blocked with 1% BSA, washed three times with 1 × PBS, and reacted with 1 ^st antibody (mouse anti-human collagen type IV). After repeated washing 3 times with 1X PBS, 2 ^nd antibody (anti-mouse IgG FITC) was reacted for 1 hour. After repeated washing 3 times with 1X PBS, the cells were sealed with fluorescent mounting solution and observed under a fluorescence microscope.

In case of collagen type 4, the same procedure as in collagen type 4 was carried out except that permeabilization using 0.5% triton X-100 was omitted. The number of Ki-67 positive cells relative to the total number of cells located in the basal layer in the epidermal layer as a percentage The results are shown in Table 2 and FIG.

Percentage (%) of Ki-67 positive cells Fold
Change
(NC-2) Fold
Change
(NC-2) Image 1 Image 2 Ave. SD Comparative Example 1 One 16.1 13.8 14.0 1.5 1.0 - 2 12.5 13.6 Comparative Example 4 One 39.1 50.0 45.7 9.7 3.3 - 2 57.1 36.4 Comparative Example 2 One 10.3 16.7 12.5 3.6 - 1.0 2 14.3 8.8 Example 3 One 40.0 48.0 49.1 7.8 - 3.9 2 56.3 44.7 3 60.7 44.8 Example 4 One 58.1 51.9 48.3 10.2 - 3.9 2 34.8 58.3 3 37.0 50.0

As shown in Table 2, in Examples 3 and 4, the expression of collagen type 4 was markedly increased as compared with the negative control, The expression of ki-67 at N = 3 in Examples 3 and 4 was increased in comparison with that in Comparative Example 2 in negative control, and the expression ratio of ki-67 according to Example 3 was 12.5 (+/- 3.6)% in Comparative Example 2, And the expression level of Ki-67 in Example 4 was increased to 3.9-fold by 48.3 (± 10.2)% from 12.5 (± 3.6)% of the negative control, which was 49.1 (± 7.8)%.

Experimental Example  1-2: MMP -9 ELISA

human MMP-9 ELISA kit (R & D systems / DMP900). For the standard curve of MMP-9, 20 ng / ml MMP-9 standard stock solution was diluted stepwise with 1X calibrator diluent, and standard solution was prepared and shown in Table 3 below.

Standard MMP-9 Standard
Conc. (Ng / ml) Standard stock (ng / ml) Diluent Final Volume One 20 1,000 μl - 500 μl 2 10 1) 500 μl 500 μl 3 5 2) 500 μl 500 μl 4 2.5 3) 500 μl 500 μl 5 1.25 4) 500 μl 1,000 μl

100 μl of assay diluent was added to each well of human MMP-9 microplate, and 100 μl of each blank, standard and test solution was added thereto, followed by reaction at room temperature for 2 hours. The reaction solution was discarded, and 400 μl of wash buffer was added per well. 200 μl of MMP-9 conjugate per well was added and incubated at room temperature for 1 hour in the absence of light. The reaction solution was discarded, and 400 μl of the solution was added to wash buffer and removed. The substrate solution was prepared by mixing equal amounts of the color reagents A and B contained in the kit immediately before use, and 200 μl of the solution was added to each well. The absorbance was measured at 450 nm wavelength within 30 min by adding 50 μl of stop solution per well, and the amount of MMP-9 secretion in the test solution was analyzed using MMP-9 standard curve.

Immunofluorescence staining was performed on collagen type 4 using a frozen block according to its own test method. The amount of MMP-9 secretion was quantitatively analyzed using an ELISA kit using the collected culture. The absorbance results of MMP-9 standard solution are shown in Table 4 and FIG. 6, and the amount of MMP-9 secretion is shown in Table 5 and FIG.

Standard MMP-9 Final Conc. (Ng / ml) Values
(ng / ml) Absorbance
(450 nm) Ave. SD One 20 19.6 0.907 0.912 0.007 19.8 0.917 2 10 10.6 0.504 0.912 0.007 10.4 0.494 3 5 5.6 0.281 0.499 0.026 4.8 0.245 4 2.5 2.3 0.130 0.263 0.002 2.3 0.133 5 1.25 1.0 0.071 0.066 0.007 0.7 0.062

Absorbance
(450 nm) MMP-9 content (ng / ml) Fold
Change
(NC-2) Fold
Change
(NC-2) Result Ave. SD Comparative Example 1 One 0.768 16.5 14.4 3.1 1.0 - 2 0.574 12.2 Comparative Example 4 One 0.613 13.1 14.8 2.5 1.0 - 2 0.772 16.6 Comparative Example 2 One 0.882 19.0 17.9 1.6 - 1.0 2 0.781 16.8 Example 3 One 0.493 10.4 12.0 1.4 - 0.7 2 0.609 13.0 3 0.599 12.8 Example 4 One 0.886 19.1 17.3 2.3 - 1.0 2 0.831 17.9 3 0.688 14.7

As shown in Tables 4 and 5, the average amount of secretion of MMP-9 was 14.4 (± 3.1) ng / ml in Comparative Example 1 and 17.9 (± 1.6) ng / ml in Comparative Example 2, Example 4 was measured at 14.8 (± 2.5) ng / ml, and the secretion level of MMP-9 was not changed compared to the negative control samples 1 and 2. The mean MMP-9 secretion amount in Example 3 was 12.0 (占 1.4) ng / ml, which was 0.7 times smaller than that in Comparative Example 2 which was the negative control.

Therefore, as shown in Experimental Examples 1-1 and 1-2, a skin-care composition containing an extract of sea cucumber as an active ingredient has an efficacy equal to or higher than that of L-ascorbic acid.

Experimental Example  2: skin whitening test

The test substances stored at -20 ° C were thawed just before treatment, diluted with 1X PBS (Diluent), and prepared as shown in Table 6 below.

sample Stock
Conc. Final
Conc. Stock
Volume Diluent
volume Final
Volume Final DMSO
Conc. (V / v) Example 1 50 mg 50 μl 5μl 4,955 μl 5ml 0.1% Example 2 500 mg 500 μl 5μl 4,955 μl 5ml 0.1% Example 3 50 mg 50 μl 5μl 4,955 μl 5ml 0.1% Example 4 500 mg 500 μl 5μl 4,955 μl 5ml 0.1%

The test material was treated with 100 μl per well on the surface of a 3D skin culture model (Neoderm ^® , Tegoscience). After 48 hours incubation in a CO ₂ incubator (10% CO ₂ , 37 ° C), the remaining test material remaining on the surface of the 3D skin culture model was removed using a pipet.

The 3D skin culture model was then washed twice with 200 [mu] l PBS and detached from the insert edge of the 3D skin culture model using a blade. After separating the 3D skin culture model tissue into a 1.5 ml e-tube, PBS was completely removed and 360 μl of lysis buffer (Solvable ^™ ) was added to the 3D skin culture model. After reacting for 5 minutes in lysis buffer, 3D skin culture tissue model tissue was removed from the membrane. After incubation at 95 ° C for 45 min in a heat block and centrifugation at 13,000 rpm for 10 min, the supernatant was transferred to a new eppendorf tube. 100 μl / ml of melanin solution was prepared using 20 mg / ml NH ₄ OH, and 100 μl / ml melanin solution was diluted with lysis buffer as shown in Table 7 to prepare a standard solution. The results are shown in Table 7 below . The standard solutions were blanked on a 96-well plate, 200 μl of the standard solution and the test substance were transferred, and the absorbance at 450 nm was measured. The results are shown in Table 8. The amounts of melanin were quantified using the melanin standard curve, Is shown in Fig.

Standard Melanin Final Conc.
(μg / ml) Volume of Melanin stock
(100 [mu] g / ml) Lysis buffer Final volume Example 1 100 1,000 μl - 500 μl Example 2 50 1) 500 μl 500 μl Example 3 25 2) 500 μl 500 μl Example 4 12.5 3) 500 μl 500 μl

Standard Melanin Final Conc.
(μg / ml) Values
(μg / ml) Absorbance
(405 nm) Ave. Std.Dev. One 100 99.5 0.832 0.827 0.006 98.4 0.823 2 50 51.8 0.438 0.439 0.001 52.0 0.440 3 25 25.5 0.221 0.222 0.001 25.7 0.222 4 12.5 12.4 0.113 0.113 0.001 12.5 0.114 5 6.25 5.7 0.058 0.057 0.000 5.7 0.057 6 3.125 2.2 0.029 0.029 0.000 2.3 0.030

Sample N Absorbance
(405 nm) Melanin content
(μg / ml) Melanin content
(%) Comparative Example 3 (%) Comparative Example 2 Contrast reduction amount (%) result Ave. SD Result Ave. SD Comparative Example 3 One 0.192 21.9 22.3 0.5 98.2 100.0 2.5 - - 2 0.198 22.7 101.8 Comparative Example 5 One 0.172 19.6 18.9 1.1 87.9 84.7 4.7 15.3 - 2 0.160 18.1 81.2 Example 5 One 0.178 20.3 19.3 0.9 91.0 86.4 4.7 13.6 - 2 0.166 18.8 84.3 3 0.164 18.6 93.4 Example 6 One 0.172 19.6 19.2 0.4 87.9 86.2 2.0 13.8 - 2 0.170 19.3 86.5 3 0.166 18.7 83.9 Comparative Example 2 One 0.195 22.3 21.1 1.7 105.7 100.0 8.1 - - 2 0.175 19.9 94.3 Example 1 One 0.191 21.8 20.6 1.0 103.1 97.8 4.7 - 2.2 2 0.178 20.3 96.1 3 0.175 19.9 94.3 Example 2 One 0.177 20.1 19.8 0.7 95.5 93.7 3.1 - 6.3 2 0.177 20.1 95.5 3 0.168 19.0 90.0 Example 3 One 0.190 21.7 20.2 1.4 102.8 95.5 6.8 - 4.5 2 0.175 19.9 94.4 3 0.166 18.8 89.3 Example 4 One 0.189 21.6 21.0 0.6 102.3 99.6 2.6 - 0.4 2 0.184 21.0 99.4 3 0.180 20.5 97.1

As shown in the above table, the average amount of melanin of N = 3 in Example 5 was measured as 19.3 (± 009) μg / ml and decreased by 13.6% compared with Comparative Example 3, and the average melanin amount of N = 3 in Example 6 was 19.2 (± 0.4) μg / ml, which was 13.8% lower than that of Comparative Example 3. The mean melanin level of N = 3 of Example 1 was measured to be 20.6 (± 1.0) μg / ml, which was 2.2% lower than that of Comparative Example 2 and the mean melanin amount of N = 3 of Example 2 was 19.8 (± 0.7) And was 6.3% lower than that of Comparative Example 2. The average melanin level of N = 3 of Example 3 was measured as 20.2 (占 1.4) 占 퐂 / ml, which was 4.5% lower than that of Comparative Example 2. The average melanin amount of N = 3 of Example 4 was 21.0 (占 0.6) ml, and was decreased by 0.4% compared with Comparative Example 2. Examples 1 to 4 were found to have a similar level of whitening efficacy to arbutin.

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. It will be possible. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims rather than the detailed description and all changes or modifications derived from the meaning and scope of the claims and their equivalents are to be construed as being included within the scope of the present invention do.

Claims (14)

A skin cosmetic composition comprising as an active ingredient an extract of sea cucumber, which is prepared by repeating lyophilization twice,
The sea-
Lyophilization of sea cucumber;
Pulverizing the lyophilized sea cucumber to prepare a powder;
Incubating the pulverized powder in ethanol;
Evaporating and concentrating the incubated sea cucumber;
Filtering and condensing the evaporated concentrated sea cucumber;
Secondly lyophilizing the condensed sea cucumber; And
The second lyophilized sea cucumber is dissolved in at least one alcohol-based solvent selected from the group consisting of butylene glycol, ethyl hexanediol and 1,2-hexanediol, ;
, Wherein the method comprises the steps of:
Wherein the sea cucumber is any one selected from the group consisting of blue sea cucumber, black sea cucumber, and combinations thereof,
The above sea cucumber extracts were extracted using sea cucumber viscera only
≪ / RTI > delete The method according to claim 1,
The skin-
Deep-freezing the sea cucumber from -70 ° C to -100 ° C before the first lyophilization step
≪ / RTI > by weight of the composition. The method according to claim 1,
In the first lyophilization step,
Cooling the sea cucumber to -20 캜 to -100 캜; And
Drying the cooled sea cucumber for 36 to 84 hours
≪ / RTI > by weight of the composition. The method according to claim 1,
Wherein the incubation step comprises:
And 55% to 95% ethanol is added to the pulverized powder, followed by incubation for 1 hour to 3 hours. The method according to claim 1,
The step of evaporating and concentrating comprises:
And concentrated by evaporation at 55 ° C to 100 ° C for 1.5 hours to 5 hours. delete delete delete delete The method according to claim 1,
The skin-
Skin regeneration activity. 12. The method of claim 11,
The skin regeneration activity,
Wherein the composition is any one selected from the group consisting of an increase in the expression level of collagen type 4, an increase in cell activity factor (ki-67), a decrease in secretion amount of matrix metalloproteinase, and combinations thereof. The method according to claim 1,
The skin-
Wherein the composition is skin whitening. 14. The method of claim 13,
The skin whitening,
Melanin production inhibitor.

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