CN108697632B

CN108697632B - Skin improving composition containing sea cucumber extract as active ingredient

Info

Publication number: CN108697632B
Application number: CN201780011984.5A
Authority: CN
Inventors: 金熙洙; 金凡峻; 刘光镐; 朴文绪
Original assignee: Catholic Kwandong University Industry Cooperation Foundation
Current assignee: Catholic Kwandong University Industry Cooperation Foundation
Priority date: 2016-02-19
Filing date: 2017-02-16
Publication date: 2022-03-01
Anticipated expiration: 2037-02-16
Also published as: WO2017142316A1; CN108697632A; KR20170098342A; KR101826296B1

Abstract

Disclosed is a skin-improving composition comprising a sea cucumber extract as an active ingredient. The sea cucumber extract prepared by repeating freeze-drying 2 times has effects on skin regeneration activity and whitening skin.

Description

Skin improving composition containing sea cucumber extract as active ingredient

Technical Field

The present invention relates to a skin improvement composition comprising a sea cucumber extract as an active ingredient, and more particularly to an invention of a skin improvement composition comprising a sea cucumber extract prepared by repeating freeze-drying 2 times as an active ingredient.

Background

Sea cucumber is a marine invertebrate belonging to the class holothuria (holothuria) of the phylum echinodermata, and there are 14 kinds of families of 4 in korea, such as stichopus japonicus, black sea cucumber, red sea cucumber, and the like. Since ancient times, sea cucumber and ginseng have been representative tonic foods, and have been the subject of research in food nutrition. The zaishan fish score, compendium of materia medica, eastern medicine and the like describe effects of recovering primordial qi, soothing nerves and the like, and are widely used in ancient times, and the saponin component is found in recent studies, so that the effect of the saponin component on food nutrition is revealed.

In recent years, research on not only food nutrition but also the use of sea cucumber as a novel natural drug has been actively conducted, and in particular, the efficacy of sea cucumber on skin regeneration or skin whitening has been verified in recent research results. However, most of the studies on skin regeneration and skin whitening use both sea cucumber and extracts extracted from other raw materials. Therefore, it is difficult to determine the efficacy for skin regeneration and skin whitening in the case of only sea cucumber. In addition, in the case of merely studying sea cucumbers, detailed studies on what kind of difference of skin activity is present in the kind, part, extraction method, etc. of sea cucumbers have not been reported, and most of them suggest effects according to animal experiments, and do not accurately suggest effects on skin regeneration and skin whitening in actual human skin models.

Therefore, it is necessary to study whether or not sea cucumber has an effect of regenerating and whitening the skin, and it is also necessary to confirm that the skin of human beings has an effect of regenerating and whitening the skin of sea cucumber.

Disclosure of Invention

Technical problem

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 a sea cucumber extract as an active ingredient, which is prepared by repeating freeze-drying 2 times.

Means for solving the problems

As a technical means for achieving the technical problems as described above, the skin improvement composition according to an aspect of the present invention includes a sea cucumber extract prepared by repeating freeze-drying 2 times as an effective ingredient.

Here, the sea cucumber extract may be prepared by a preparation method comprising the steps of: carrying out primary freeze drying on the sea cucumber; a step of pulverizing freeze-dried sea cucumber to prepare a powder; a step of incubating the pulverized powder; evaporating and concentrating the incubated sea cucumber; filtering and condensing the evaporated and concentrated sea cucumber, and performing secondary freeze drying on the condensed sea cucumber.

The above skin improvement composition can also be prepared by a preparation method comprising the steps of: before the first freeze-drying step, ultra-low temperature freezing (deep-freezing) is carried out on the sea cucumber at the temperature of-70 ℃ to-100 ℃.

The first freeze-drying step may include: cooling the sea cucumber to a temperature of between 20 ℃ below zero and 100 ℃ below zero; and drying the cooled sea cucumber for 36 to 84 hours.

In the above incubation step, the sea cucumber powder may be incubated for 1 to 3 hours by adding 55 to 95% ethanol thereto.

In the above-mentioned step of evaporation concentration, the evaporation concentration may be carried out at a temperature of 55 ℃ to 100 ℃ for 1.5 hours to 5 hours.

The sea cucumber can be Stichopus japonicus selenka and/or Stichopus japonicus selenka.

The sea cucumber extract may be extracted from the viscera of sea cucumber.

The sea cucumber extract can be prepared by a preparation method which also comprises the following steps: the solution is prepared by dissolving the secondarily freeze-dried sea cucumber in a solvent.

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

The skin improvement may be skin regeneration activity.

The skin regenerating activity may be increased expression level of type IV collagen, increased secretion of cell activating factor (ki-67) and/or decreased secretion of matrix metalloproteinase.

The skin improvement may be skin whitening.

The skin whitening agent can inhibit melanin generation.

ADVANTAGEOUS EFFECTS OF INVENTION

The skin-improving composition of the present invention comprising a sea cucumber extract as an active ingredient has an effect of regenerating skin, for example, an increase in the expression level of type iv collagen, a decrease in the secretion level of matrix metalloproteinase, and the like, and has an effect of preventing skin blackening, for example, inhibiting melanin production, and the like.

Drawings

Fig. 1 is a view showing a preparation process for extracting a sea cucumber extract.

Fig. 2 is a view showing a process of preparing a sea cucumber extract into a solution.

FIG. 3 shows the expression levels of type IV collagen and ki-67 in comparative examples 1, 2 and 4.

FIG. 4 shows the expression levels of type IV collagen and ki-67 in examples 3 and 4.

FIG. 5 is a graph showing a comparison of expression rates of ki-67.

FIG. 6 is a standard curve diagram showing MMP-9.

FIG. 7 is a graph showing a comparison of the secretion amounts of MMP-9.

Fig. 8 is a graph showing a melanin standard curve.

Fig. 9 is a graph showing comparison of the melanin amounts in comparative example 2 and comparative example 3, and examples 1 to 6.

Detailed Description

The present invention will be described in further detail below. However, this is only given as an example, and the present invention is not limited thereto, and is defined only by the scope of the invention claimed hereinafter.

Provided is a skin cosmetic composition having effects on both skin rejuvenating activity and whitening skin.

The skin improvement composition according to an aspect of the present invention comprises a sea cucumber extract prepared by repeating freeze-drying 2 times as an effective ingredient.

The sea cucumber is a marine organism belonging to order tenodesidae (aspenodhritida), family Stichopus japonicus (stichopodae), genus Stichopus (stichopus), and 1500 species are inhabited globally. The sea cucumber that survives in korea is known as stichopus japonicus (stichopus japonica) and inhabits 14 species of 4 families.

Preferably, the sea cucumber can be Stichopus japonicus selenka or Stichopus japonicus selenka. Stichopus japonicus is sea cucumber in rock, gravel and sand areas of open sea water, and Holothuria nobilis is sea cucumber inhabiting in eutrophic water and land with strong influence.

The sea cucumber may be one or more parts selected from the group consisting of whole sea cucumber, viscera, and parts other than viscera.

The seafood drying method comprises natural drying, pressure drying, normal pressure drying and vacuum drying, and the freeze drying belongs to a vacuum drying method. The freeze drying is a drying method in which a material containing a large amount of moisture is frozen and decompressed to sublimate ice and remove moisture at the same time, thereby obtaining a dried product. Freeze-drying has the advantage that drying is performed by minimizing damage to heat-sensitive substances, and the dried substances form a structure that is easily penetrated by moisture, so that rapid rehydration of the frozen substances can be achieved.

The sea cucumber is easily deformed due to heat, and is preferably dried by freeze-drying because of its high water content. The freeze-dried sea cucumber can be dried while retaining its inherent tissue and is excellent in rehydration to water.

The effective component is sea cucumber extract as main ingredient of the skin improving composition. That is, the sea cucumber extract directly or indirectly exhibits the skin cosmetic function of the composition.

The composition is a skin improving composition. Skin improvement means whitening of the skin, improvement of the skin periphery, and protection of the skin from ultraviolet rays.

The skin-improving composition comprising the above sea cucumber extract may comprise only the sea cucumber extract as an active ingredient, and may further comprise the sea cucumber extract and other necessary ingredients as active ingredients, depending on the desired skin-improving function and formulation. Further, the above-mentioned skin cosmetic composition can be used as an external preparation for skin, and also as a food such as a health food or a medicine such as an injection preparation.

The sea cucumber extract can be prepared by a preparation method comprising the following steps: freeze-drying the sea cucumber for the first time (step S110); pulverizing the freeze-dried sea cucumber to prepare a powder (step S120); incubating the pulverized powder (step S130); evaporating and concentrating the incubated sea cucumber (step S140); filtering (step S150) and condensing (step S160) the evaporated and concentrated sea cucumber; and performing a second freeze-drying of the condensed sea cucumber (step S170).

The above skin improvement composition can be prepared by a preparation method comprising the steps of: before the first freeze-drying step, ultra-low temperature freezing (deep-freezing) is carried out on the sea cucumber at the temperature of-70 ℃ to-100 ℃. The water content of the sea cucumber can be completely removed by ultra-low temperature freezing, and the sea cucumber can be rapidly frozen at an extremely low temperature, so that the cells of the sea cucumber are not damaged and can be kept intact. The ultra-low temperature freezing may be performed by a quick freezing (quick freezing) method which rapidly passes through a temperature interval of-1 ℃ to-5 ℃ as a maximum ice crystal generation zone.

The nutrients of the sea cucumber are concentrated by the first freeze-drying (step S110). That is, moisture is removed from raw sea cucumbers, of which more than 90% by weight is formed of water, by freeze-drying, and the dried sea cucumbers contain highly concentrated nutrients under the same weight conditions as the raw sea cucumbers. Therefore, an excellent skin-improving effect can be exhibited even in a small amount.

The first freeze-drying (step S110) may include the following preparation method: freezing Stichopus japonicus to-10 deg.C to-110 deg.C; and the frozen sea cucumber is recalled for 36 hours to 84 hours.

Preferably, the freezing temperature of the above-mentioned first freeze-drying (step S110) may be-10 ℃ to-110 ℃, more preferably, -20 ℃ to-100 ℃. The freezing time of freeze-drying varies depending on the freezing temperature. The lower the freezing temperature, the shorter the freezing time, and the smaller the size of the voids and the dense tissue. This is because when this is rapidly frozen, a band develops rapidly across the ice crystals, thus forming smaller ice particles. When the freezing temperature is lower than-110 ℃, although the freeze-drying time is shortened, the ice crystal generation speed is increased and the ice crystal is reduced, so that the water permeates into the sea cucumber subjected to the first freeze-drying and the absorption speed of the water into the dried sea cucumber is reduced, and the hydration recovery capability is low. Further, when the freezing temperature is higher than-10 ℃, voids in the sea cucumber become large, and thus the hydration recovery ability becomes good, but the freeze-drying time becomes long, and the process of preparing the extract may be lengthened.

Preferably, the first frozen sea cucumber may be dried for 36 to 84 hours, and more preferably, may be dried for 48 to 72 hours. The drying refers to sublimation drying and/or secondary drying. Sublimation drying refers to drying moisture, such as water inside cells, by reducing the pressure to a value equal to or lower than the triple point of water after freezing and directly changing the pressure from solid to gas, and secondary drying refers to dehumidification by drying the moisture remaining after sublimation drying. If the drying time is shorter than 36 hours, the moisture in the sea cucumber may not be sufficiently dried, and if it is longer than 72 hours, the moisture may be excessively dried, and thus the hydration recovery ability may be deteriorated.

The first freeze-dried sea cucumber is pulverized to prepare powder (step S120). Preferably, the size of the powder may be 0.01nm to 1mm according to the purpose of use. When freeze-dried sea cucumbers are prepared into powder, the size becomes small and thus they can be easily absorbed into the body, and the surface area increases, so that they can easily react with other substances contained in the skin improvement composition.

The incubation (step S130) of the pulverized powder may include the following preparation method: adding 55% to 95% ethanol to the sea cucumber powder and incubating for 1 hour to 3 hours.

Preferably, the above ethanol may have a purity of 55% to 95%, more preferably, may have a purity of 65% to 85%. Also, preferably, the incubation may be performed for 1 hour to 3 hours, and more preferably, may be performed for 1.5 hours to 2.5 hours. The incubation (step S130) decomposes the matrix protein of the sea cucumber, and the decomposed matrix protein activates the skin cosmetic function of the sea cucumber extract. When the purity of the ethanol is less than 55%, the incubation can be performed because of the inclusion of pollutants, and when the purity of the ethanol is more than 95%, the sea cucumber protein can be coagulated because of the high purity ethanol. When the incubation time is less than 1 hour, the base protein of the sea cucumber may not be sufficiently decomposed to the extent that the extract can be prepared, and when the incubation time is more than 3 hours, the skin cosmetic function of the sea cucumber may be lost due to the decomposition of the base protein of the sea cucumber.

The above-mentioned evaporation concentration (step S140) may include a preparation method comprising the steps of: evaporating and concentrating the incubated sea cucumber at 55-100 deg.C for 1.5-5 hr.

Preferably, the above-mentioned evaporative concentration may be carried out at a temperature of 55 ℃ to 100 ℃ for 1.5 hours to 5 hours, more preferably, at a temperature of 65 ℃ to 90 ℃ for 2 hours to 4.5 hours. Through the evaporation and concentration steps, the pollution components such as ethanol, oil components, heavy metals and the like can be removed, and only pure sea cucumbers can be obtained. When the evaporation concentration temperature is less than 55 deg.C, the pollution component can not be volatilized due to low temperature, so that impurities can remain in the sea cucumber extract, and when the temperature exceeds 100 deg.C, the skin improving component of sea cucumber can be damaged due to high temperature. Further, when the evaporation concentration time is less than 1.5 hours, the time is short and ethanol, contaminated components, etc. cannot be sufficiently evaporated, and when it exceeds 5 hours, the protein of the sea cucumber component may be deformed at a low temperature by heating for a long time.

The step (step S160) of filtering (step S150) and condensing (condensing) the incubated sea cucumbers is a step of removing impurities from the evaporated and concentrated sea cucumbers and concentrating the impurities to obtain a high-purity sea cucumber extract.

The step of performing the second freeze-drying of the condensed sea cucumber (step S170) is a step of performing the second freeze-drying of the condensed sea cucumber to obtain a high-purity sea cucumber extract. Since the second freeze-drying step is to repeat the first freeze-drying step again, detailed description is omitted hereinafter.

Preferably, the sea cucumber may be a stichopus japonicus and/or a black sea cucumber, and more preferably, may be a stichopus japonicus and/or a black sea cucumber perched on a coast near a lark island on a west coast. Stichopus japonicus and Holothuria nobilis belong to gulf-shaped sea cucumbers, and Holothuria nobilis belong to open sea cucumbers. Stichopus japonicus selenka and Stichopus japonicus selenka have thick and short Boyle sac (polar vesicle) with blunt round front end, small contractility, and simple shape of tentacle bone slice (ossicle). On the contrary, the red sea cucumber, which is an open sea cucumber, has a long and thin burley sac, a pointed tip, a high contractility, and a complicated hand-touching bone piece. Compared with the red sea cucumber only eating red algae, the stichopus japonicus and the black sea cucumber ingest various baits, and thus have more nutrient components than the red sea cucumber.

The sea cucumber extract may be extracted from the viscera of sea cucumber. The viscera of sea cucumber has high oxidation resistance compared with the viscera except for the viscera due to the influence of the algae existing in the viscera. Antioxidation means the action of preventing oxidation. The antioxidant activity of the viscera of sea cucumber can prevent the aging of cells due to the oxidation of cells by removing active oxygen in the human body, thereby contributing to the beauty of skin. Therefore, the skin-improving composition comprising as an active ingredient an extract extracted from only the viscera of sea cucumber can exert excellent skin-regenerating activity and skin-whitening function.

The sea cucumber extract can be prepared by a preparation method further comprising the following steps: a step of dissolving the sea cucumber freeze-dried for the second time in a solvent to prepare a solution (step S180). The skin improvement composition comprising the sea cucumber extract as an effective ingredient can be used in various dosage forms. Therefore, the skin improvement composition may further comprise a sea cucumber extract in solution, depending on the purpose and method of use. The solvent may be one or more selected from the group consisting of polar, nonpolar, organic and inorganic solvents.

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

The butanediol, ethylhexanediol and 1, 2-hexanediol are alcohol compounds, and can dissolve various components of sea cucumber, so that a solution capable of effectively exerting skin-improving functions of a skin-improving composition containing a sea cucumber extract can be prepared.

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

The skin regeneration activity may be an increase in the expression level of type IV collagen. Preferably, the above type IV collagen may be expressed in an amount of 200% to 500% as compared with a negative control group prepared from dimethyl sulfoxide, and may be expressed in an amount of 103% to 110% as compared with ascorbic acid.

Collagen (collagen) is the major protein constituting the flesh and skin layers and connective tissues of mammals, and accounts for 25% to 35% of the proteins constituting the body. To date, 29 types of collagen, type iv collagen, have been found to be the major constituent of basement membrane and the matrix of glomerular epilepsy. Type iv collagen is composed of 4 regions such as a 7S collagen region, a non-collagen 1(NC1, non-collagenous 1) region, a non-collagen 2(NC2, non-collagenous 2) region, and a Triple Helix (TH) region. The 4 molecules of type IV collagen are linked at the terminal NH2 region, and the region with more disulfide bonds (S-S) is the 7S collagen region, which is not decomposed by various proteolytic enzymes such as bacterial collagen-decomposing enzymes. The basement membrane refers to a thin membrane existing between epidermis and dermis, and plays a role in skin regeneration. When the basement membrane is damaged, enzymes for decomposing collagen invade from the epidermal layer into the dermis, thus breaking collagen fibers, thereby generating wrinkles on the skin. The skin improvement composition may have a function of regenerating activity to the skin by a mechanism of enhancing basement membrane by increasing the expression level of type iv collagen, which is a main constituent of basement membrane.

The skin regenerating activity may be an increase in a cell activating factor (ki-67). Preferably, the expression amount of the above-mentioned cytokine (ki-67) may be 200% to 500% as compared with 0.1% dimethyl sulfoxide (dimethyl sulfoxide) as a negative control group. The cytokine (ki-67) is a protein used as a marker for cell proliferation, and is expressed in the G1, G2, S, and M phases of the cell cycle. The skin improvement composition comprising the sea cucumber extract as an active ingredient has an increased regenerative activity by affecting skin cells. Therefore, cell regeneration of the stratum corneum and the like of the skin using the skin improving composition becomes active, and the expression level of the cell activity factor (ki-67) increases.

The skin regenerating activity may be a decrease in the amount of secretion of matrix metalloprotease. Preferably, the decrease in the secretion amount of the matrix metalloproteinase is 50 to 90% of the expression amount of the matrix metalloproteinase in the negative control prepared using dimethylsulfoxide and 60 to 90% of the expression amount of the matrix metalloproteinase of ascorbic acid.

The matrix metalloproteinase refers to collagenase of type IV (type IV collagen) and stromelysin (MMP-3) for decomposing collagen of type IV. The type IV collagenase is composed of gelatinase A (gelatinase A, MMP-2) and gelatinase B (gelatinase B, MMP-9). Gelatinase A is prepared from fibroblast, endothelial cell, macrophage, etc., gelatinase B is prepared from normal cell and cancer cell, and stromelysin (MMP-3) is prepared from cancer cell. The sea cucumber extract has the function of regenerating skin activity by reducing the secretion of the matrix metalloproteinase and inhibiting the decomposition of type IV collagen.

The skin improvement may be skin whitening. Whitening skin means preventing the skin from being melanogenesis (melanogenesis) and whitening and beautifying the skin by melanin.

The skin cosmetic composition can be used for whitening skin before, during or after synthesis of melanin. The skin whitening agent has the functions of whitening skin through tyrosinase (tyrosinase) transcription regulation and saccharification regulation before melanin synthesis, tyrosinase inhibition, peroxidase inhibition, reducing agent and active oxygen elimination in the melanin synthesis, and tyrosinase decomposition, melanosome movement inhibition and stripping promotion after the melanin synthesis.

The skin whitening agent can inhibit melanin generation. Preferably, the skin improvement composition comprising the sea cucumber extract as an effective ingredient may have a decrease in melanin secretion amount of 12% to 15% as compared to that of the negative control group prepared with 1 × PBS, and may have a decrease in melanin secretion amount of 2% to 6% as compared to that of the negative control group prepared with dimethyl sulfoxide.

The inhibition of melanin production is carried out by converting tyrosine, which is an intracellular substance, from tyrosinase to dopa (dopa) and dopaquinone (dopaquinone), and then allowing melanin to be produced by the action and automation of dopachrome tautomerase (TRP-2, dopachrome tautomerase), which is a tyrosinase-related protein, and tyrosinase-related protein 1(TRP-1, 5, 6-dihydroxyindole-2-carboxylic acid oxidase (5, 6-dihydroxyindole-2-carboxylic acid oxidase)). The skin improvement composition can inhibit melanin generation by inhibiting the mechanism of formation of tyrosine into melanin by increasing tyrosinase inhibitory activity and/or decreasing tyrosinase expression.

Hereinafter, the present invention will be described in detail so that those skilled in the art to which the present invention pertains can easily carry out the present invention. However, the present invention may be realized in various forms and is not limited to the embodiments described herein.

Comparative examples 1 to 3: negative control group

Comparative examples 1 to 3 negative control groups were prepared using a maintenance medium (maintence medium) provided by korean diko high (TEGO) science and technology, 0.1% dimethyl sulfoxide (dimethyl sulfoxide) provided by korean diko high (TEGO) science and technology, and 1 × pbs (phosphate buffer saline) provided by korean diko high (TEGO) science and technology, respectively.

Comparative example 4 and comparative example 5: positive control group

In comparative example 4, ascorbic acid (L-ascorbic acid) was used for preparation, and in comparative example 5, arbutin (arbutin) was used for preparation of a positive control group.

Example 1: low concentration sea cucumber extract

The powder is prepared by washing Stichopus japonicus selenka and Holothuria nobilis collected in the sea area near the island, cooling at-30 deg.C, drying for 48 hr, and pulverizing the frozen Stichopus japonicus selenka. After incubating the sea cucumber powder in 75% ethanol for 2 hours, it was filtered through cotton (cotton) after concentrating by evaporation at 70 ℃ for 3 hours. The extract was concentrated by a vacuum evaporator (vacuum evaporator, EYELA N-12, EYEKA CA-1112, product of Tokyo physical and chemical instruments Co., Ltd., Tokyo, Japan) for 5 hours to a low concentration of 50 mg/ml. Freeze-drying the concentrated Stichopus japonicus for the second time at-30 deg.C for 48 hr.

Example 2: high concentration sea cucumber extract

The powder is prepared by washing Stichopus japonicus selenka and Holothuria nobilis collected in the sea area near the island, cooling at-30 deg.C, drying for 48 hr, and pulverizing the frozen Stichopus japonicus selenka. The sea cucumber powder was incubated in 75% ethanol for 2 hours, evaporated and concentrated at 70 ℃ for 3 hours, and then filtered through a cotton-like substance. The extract was concentrated by a vacuum evaporator (vacuum evaporator, EYELA N-12, EYEKA CA-1112, product of Tokyo physical and chemical instruments Co., Ltd., Tokyo, Japan) for 5 hours to a low concentration of 50 mg/ml. Freeze-drying the concentrated Stichopus japonicus for the second time at-30 deg.C for 48 hr.

Example 3: low concentration sea cucumber viscera extract

The viscera of Stichopus japonicus and Holothuria nobilis collected in sea area near the island are cleaned and frozen at-80 deg.C by ultra-low temperature freezer. After the first freeze-drying step of cooling and drying the ultra-low temperature frozen sea cucumber entrails at a temperature of-30 ℃ for 48 hours, the frozen sea cucumber entrails are pulverized to prepare powder. The sea cucumber viscera powder was incubated in 75% ethanol for 2 hours, then concentrated by distillation at 70 ℃ for 3 hours, and then filtered through a cotton-like material. The extract was concentrated by a vacuum evaporator (vacuum evaporator, EYELA N-12, EYEKA CA-1112, product of Tokyo physical and chemical instruments Co., Ltd., Tokyo, Japan) for 5 hours to a low concentration of 50 mg/ml. Freeze-drying the concentrated viscera of the sea cucumber at-30 deg.C for 48 hr.

Example 4: high concentration sea cucumber viscera extract

The viscera of Stichopus japonicus and Stichopus japonicus collected in the sea area near the island are cleaned and frozen at-80 deg.C by ultra-low temperature freezer. After a first freeze-drying step of cooling and drying the ultra-low temperature frozen sea cucumber entrails at a temperature of-30 ℃ for 48 hours, the frozen sea cucumber entrails are pulverized to prepare powder. The sea cucumber viscera powder was incubated in 75% ethanol for 2 hours, evaporated and concentrated at 70 ℃ for 3 hours, and then filtered through a cotton-like substance. The extract was concentrated by a vacuum evaporator (vacuum evaporator, EYELA N-12, EYEKA CA-1112, product of Tokyo physical and chemical instruments Co., Ltd., Tokyo, Japan) for 5 hours to a low concentration of 50 mg/ml. Freeze-drying the concentrated viscera of the sea cucumber at-30 deg.C for 48 hr.

Example 5: low-concentration sea cucumber extract liquid

The frozen sea cucumber is pulverized to prepare powder after a first freeze-drying step of washing stichopus japonicus and black sea cucumber collected in the sea area near the island of lark, cooling at-30 ℃, and drying for 48 hours. The sea cucumber powder was incubated in 75% ethanol for 2 hours, evaporated and concentrated at 70 ℃ for 3 hours, and then filtered through a cotton-like substance. The extract was concentrated by a vacuum evaporator (vacuum evaporator, EYELA N-12, EYEKA CA-1112, product of Tokyo physical and chemical instruments Co., Ltd., Tokyo, Japan) for 5 hours to a low concentration of 50 mg/ml. Freeze-drying the concentrated Stichopus japonicus for the second time at-30 deg.C for 48 hr. A 5% strength solution of the sea cucumber extract was prepared by mixing the second freeze-dried sea cucumber extract with butylene glycol (butylene glycol), water, ethyl hexanediol (ethyl hexanediol) and 1, 2 hexanediol (1, 2-hexanediol) solvents.

Example 6: high concentration sea cucumber extract liquid

The frozen sea cucumber is pulverized to prepare powder after a first freeze-drying step of washing stichopus japonicus and black sea cucumber collected in the sea area near the island of lark, cooling at-30 ℃, and drying for 48 hours. The sea cucumber powder was incubated in 75% ethanol for 2 hours, evaporated and concentrated at 70 ℃ for 3 hours, and then filtered through a cotton-like substance. The extract was concentrated by a vacuum evaporator (vacuum evaporator, EYELA N-12, EYEKA CA-1112, product of Tokyo physical and chemical instruments Co., Ltd., Tokyo, Japan) for 5 hours to a low concentration of 50 mg/ml. Freeze-drying the concentrated Stichopus japonicus for the second time at-30 deg.C for 48 hr. A 20% strength solution of sea cucumber extract was prepared by mixing the second freeze-dried sea cucumber extract with butylene glycol (butylene glycol), water, ethyl hexanediol (ethyl hexanediol) and 1, 2 hexanediol (1, 2-hexanediol) solvents.

Experimental example 1: wrinkle improvement test

Test substances stored at a temperature of-20 ℃ were thawed before treatment and prepared by dilution in a maintenance medium (see the following table 1). Each 3D skin culture model of control and test groups (

Korea Digao (TEGO) Tech Co.). Well wells (3.5 ml of each well was treated on the medium layer and 30. mu.l of each well was treated on the cell layer surface, and then the resulting mixture was subjected to CO treatment₂Incubator (10%, CO)₂And cultured at 37 ℃ for 24 hours. After 24 hours of incubation, the medium was changed and the control and test groups were incubated per 3D skin culture model: (

Korea Digao (TEGO) Tech Co., Ltd.) wells were repeatedly treated with 3.5ml of each medium layer and 30. mu.l of each cell layer surface, and then CO was added thereto₂Incubator (10%, CO)₂And cultured at 37 ℃ for 24 hours in a batch for 48 hours. After culturing for 48 hours, all the groups of culture liquids were collected and stored at-70 ℃ to prepare frozen blocks (frozen blocks).

TABLE 1

In the case of type IV collagen, experiments were carried out in the same manner as type IV collagen except that the process of permeabilization (permeabilization) by 0.5% Triton X-100 was omitted, and the number of ki-67 positive cells was analyzed as a percentage with respect to the total number of cells in the basal layer in the epidermal layer and is shown in Table 2 and FIG. 5 below.

TABLE 2

As shown in Table 2 above, the expression of type IV collagen was significantly increased in examples 3 and 4 as compared with that in comparative example 2, which is a negative control group. In examples 3 and 4, the expression of ki-67 was increased in N ═ 3, 12.5(± 3.6)% of comparative example 2 as a negative control group by 3.9 times, and 12.5(± 3.6)% of comparative example 2 as a negative control group by 49.1(± 7.8)% and the expression of ki-67 in example 4 was increased by 48.3(± 10.2)% of comparative example 2 as a negative control group by 3.9 times, as compared with comparative example 2 as a negative control group.

Experimental examples 1-2: MMP-9ELISA

Human MMP-9ELISA kit (R & D systems/DMP 900). For the standard curve of MMP-9, a 20ng/ml standard stock of MMP-9 was diluted according to the individual steps using 1 × calibration diluent (calibretor diluent) and a standard solution was prepared, shown in Table 3 below.

TABLE 3

100. mu.l of assay diluent (assay diluent) was put into each well of the human MMP-9 microplate, and 100. mu.l of blank solution (blank), standard solution (standard) and test solution were added to each well and reflected at normal temperature for 2 hours. After removing the reaction solution and adding 400. mu.l of a rinsing buffer (wash buffer) to each well, the total weight was repeated 3 times. After 200. mu.l of MMP-9 conjugate (conjugate) was placed in each well, it was reflected at room temperature for 1 hour in a light-shielded state. The reaction solution was removed and after adding 400. mu.l of the rinsing buffer to each well, the total weight was repeated 3 times. Before using the matrix solution (substrate solution), color reagents (color reagent) a and B contained in the kit were prepared in the same amount by mixing, and 200 μ l was added to each well, which was reflected for 30 minutes in a light-shielded state at normal temperature. The MMP-9 secretion of the test solutions was analyzed using an MMP-9 standard curve by adding 50. mu.l of stop solution (stop solution) per well and measuring the absorbance at a wavelength of 450nm for 30 minutes.

Immunofluorescent staining (immunofluorescent staining) was performed on type IV collagen using cryoclumps and according to the test method itself. MMP-9 secretion was quantitatively analyzed using the collected culture medium and ELISA kit. The absorbance results of the MMP-9 standard solution are shown in Table 4 and FIG. 6 below, and the MMP-9 secretion amount is shown in Table 5 and FIG. 7 below.

TABLE 4

TABLE 5

As shown in tables 4 and 5, the results of measuring the average secretion amount of MMP-9 showed that comparative example 1 was 14.4 (+ -3.1) ng/ml, comparative example 2 was 17.9 (+ -1.6) ng/ml, comparative example 4 as a positive control group was measured to be 14.8 (+ -2.5) ng/ml, and the secretion amount of MMP-9 was not changed as compared with comparative examples 1 and 2 as a negative control group. The average MMP-9 secretion of example 3 was 12.0 (+ -1.4) ng/ml, which was reduced by 0.7-fold compared to comparative example 2 as a negative control group.

Therefore, as shown in experimental examples 1-1 and 1-2, the skin cosmetic composition containing the sea cucumber extract as an active ingredient exhibited equal or more efficacy as compared to ascorbic acid (L-ascorin acid).

Experimental example 2: skin whitening test

Test substances stored at-20 ℃ were thawed before treatment and diluted with 1 × pbs (diluent) to prepare the following table 6.

TABLE 6

The test substances were treated to the surface of a 3D skin culture model (Neoderm, korean digo (TEGO) science) at 100 μ l per well. In CO₂Incubator (10%, CO)₂After incubation for 48 hours at 37 ℃), residual test substance remaining on the surface of the 3D skin culture model was removed by a pipette.

Then, the 3D skin culture model was washed 2 times with 200 μ l of Phosphate Buffered Saline (PBS), and scraped away from the insertion edge of the 3D skin culture model using a blade (blade). The separated 3D skin culture model tissue was placed in a 1.5ml microcentrifuge tube (e-tube), after phosphate buffered saline was completely removed, lysis buffer (soluble) was added to the 3D skin culture model^TM) Each 360. mu.l. After 5 minutes of reaction in the lysate, the 3D skin culture tissue model was removed from the membrane separation. After incubation at 95 ℃ for 45 minutes in a thermostated apparatus (heat block) and centrifugation at 13000rpm for 10 minutes, the supernatant was transferred to a new microfuge tube (eppendorf tube). Using 20mg/ml NH₄OH 100. mu.l/ml of the melanin solution was prepared, and then the prepared 100. mu.l/ml of the melanin solution was diluted stepwise as shown in Table 7 using a lysis solution to prepare a standard solution, and the results thereof are shown in Table 7. The results of transferring 200. mu.l of each of the blank solution, the standard solution and the test substance into a 96-well plate and measuring the absorbance at 450nm are shown in Table 8, and the results of analyzing the amount of melanin by quantification using a melanin standard curve are shown in the following Table 9 and FIG. 9.

TABLE 7

TABLE 8

TABLE 9

As shown in the above table, the average melanin amount of N ═ 3 of example 5 was measured to be 19.3(± 009) μ g/ml, which was reduced by 13.6% compared to comparative example 3, and the average melanin amount of N ═ 3 of example 6 was measured to be 19.2(± 0.4) μ g/ml, which was reduced by 13.8% compared to comparative example 3. The average melanin amount of N ═ 3 of example 1 was determined to be 20.6(± 1.0) μ g/ml, which was reduced by 2.2% compared to comparative example 2, and the average melanin amount of N ═ 3 of example 2 was determined to be 19.8(± 0.7) μ g/ml, which was reduced by 6.3% compared to comparative example 2. The average melanin amount of N ═ 3 of example 3 was determined to be 20.2(± 1.4) μ g/ml, which was reduced by 4.5% compared to comparative example 2, and the average melanin amount of N ═ 3 of example 4 was determined to be 21.0(± 0.6) μ g/ml, which was reduced by 0.4% compared to comparative example 2. Examples 1 to 4 exhibited whitening efficacy with a similar level to arbutin (arbutin).

The above description of the present invention is intended to be illustrative, and it will be understood by those skilled in the art that the present invention may be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. It is therefore to be understood that the various embodiments described above are illustrative in all respects, rather than restrictive. For example, each component described in a single form may be dispersed and implemented, and similarly, a plurality of components described in a dispersed form may be implemented in a combined form. The scope of the present invention is shown by the following claims, rather than the above detailed description, and it should be construed that all modifications or variations derived from the meaning and scope of the claims and the concept of equivalents thereof are included in the scope of the present invention.

Claims

1. A skin cosmetic composition comprising a sea cucumber extract prepared by repeating freeze-drying 2 times as an effective ingredient,

wherein the sea cucumber extract is prepared by a preparation method comprising the following steps:

carrying out primary freeze drying on the sea cucumber;

pulverizing the freeze-dried sea cucumber to prepare a powder;

incubating the pulverized powder in ethanol;

evaporating and concentrating the incubated sea cucumber;

filtering and condensing the evaporated and concentrated sea cucumber; and

a second freeze-drying step of the condensed sea cucumber,

wherein the sea cucumber is selected from the group consisting of Stichopus japonicus selenka, Holothuria nobilis selenka, and combinations thereof,

wherein the sea cucumber extract is extracted only by using the viscera of the sea cucumber,

the preparation method of the sea cucumber extract further comprises the following steps: preparing a solution by dissolving the sea cucumber subjected to the second freeze-drying in a solvent, and

wherein the solvent is selected from one or more of the group consisting of butanediol, water, ethylhexanediol, and 1, 2-hexanediol.

2. The skin cosmetic composition according to claim 1, wherein the skin cosmetic composition is prepared by a preparation method comprising the steps of: before the first freeze-drying step, the sea cucumber is frozen at an ultra-low temperature of-70 ℃ to-100 ℃.

3. The skin cosmetic composition according to claim 2,

the first freeze-drying step comprises:

cooling the sea cucumber to a temperature of between 20 ℃ below zero and 100 ℃ below zero; and

drying the cooled sea cucumber for 36 to 84 hours.

4. The dermocosmetic composition according to claim 1, characterized in that in said step of incubating, incubating for 1 to 3 hours by adding 55 to 95% of ethanol to said pulverized powder.

5. The skin cosmetic composition according to claim 1, wherein in the step of evaporation concentration, the evaporation concentration is carried out at a temperature of 55 ℃ to 100 ℃ for 1.5 hours to 5 hours.

6. The skin cosmetic composition according to claim 1, wherein the skin cosmetic is a skin regenerating activity.

7. The skin cosmetic composition according to claim 6, wherein the skin regenerating activity is an increase in the expression level of type IV collagen, an increase in ki-67 as a cell active factor and/or a decrease in the secretion level of matrix metalloproteinase.

8. The skin cosmetic composition according to claim 1, wherein the skin cosmetic is a whitening skin.

9. The skin cosmetic composition according to claim 8, wherein the skin whitening is melanin production inhibition.