KR102433235B1 - Extract of fish egg using magma seawater, cosmetic composition comprising the same and method for preparing the same - Google Patents

Abstract

The present application includes the steps of pre-treatment of fish roe; preparing a mixture by mixing pretreated fish roe and lava seawater in a weight ratio of 1:5 to 1:1; hydrolyzing the mixture with a hydrolase; centrifuging the hydrolyzed mixture; And filtering with a mesh (Mesh) filter; It provides a method for preparing a fish roe extract comprising a. The fish roe extract prepared by the manufacturing method of the present application is rich in mineral content, has an excellent antioxidant effect, and has advantages in skin whitening, skin wrinkle improvement and skin cell regeneration activity.

Description

Fish roe extract extracted using lava seawater, a cosmetic composition containing the same, and a method for manufacturing the extract

The present application relates to a fish roe extract extracted using lava seawater and a cosmetic composition comprising the same.

Due to the development of modern medicine and science, people's life expectancy is increasing and the income level is increasing, so interest in cosmeceuticals is rapidly increasing. Recently, in the cosmeceutical market, cosmetics made from marine organisms are attracting attention because they are natural cosmetics and have therapeutic and functional effects.

The development of raw materials using marine resources is very urgent, and resource research using marine organisms is being conducted in Korea, which has a clean area of the southern coast, but the technology for developing resources using marine animals is very weak.

In addition, although functional cosmetics using marine resources such as seaweeds with excellent antioxidant ability are being developed, they are applied only to a few products such as pack products, so there is a problem that industrialization as a wide range of cosmetic materials is still insignificant.

Therefore, it is necessary to develop a market for marine resource materials that have functionality as materials derived from marine resources.

Korean Patent Registration No. 10-140446 (published on May 30, 2014)

One embodiment of the present application includes the steps of pre-processing the fish eggs to solve the conventional problems as described above; preparing a mixture by mixing pretreated fish roe and lava seawater in a weight ratio of 1:5 to 1:1; hydrolyzing the mixture with a hydrolase; centrifuging the hydrolyzed mixture; And filtering with a mesh (Mesh) filter; It provides a method for preparing a fish roe extract comprising a.

Another embodiment of the present application provides a fish roe extract prepared by the above preparation method.

Another embodiment of the present application provides a cosmetic composition comprising the fish roe extract and lava seawater as active ingredients.

In order to solve the above problems, one embodiment of the present application includes the steps of pre-processing the fish; preparing a mixture by mixing pretreated fish roe and lava seawater in a weight ratio of 1:5 to 1:1; hydrolyzing the mixture with a hydrolase; centrifuging the hydrolyzed mixture; And filtering with a mesh (Mesh) filter; It provides a method for preparing a fish roe extract comprising a.

In one embodiment of the present application, the filtering may include: a first filtering step of filtering the mixture with a mesh filter of 1 μm to 5 μm; and a second filtration step of filtering the mixture through a mesh filter of 0.1 μm to 0.5 μm after the first filtration step; may include.

In one embodiment of the present application, the hydrolyzing may include heating the mixture at a temperature of 80°C to 100°C for 3 minutes to 10 minutes; cooling at room temperature and grinding with a homogenizer; and after heating to 40°C to 60°C, hydrolyzing by adding a proteolytic enzyme; may include.

In one embodiment of the present application, the fish roe may be an egg of the family Osmeridae selected from the group consisting of smelt, raw smelt, and hot smelt.

In one embodiment of the present application, the hydrolase is, flavozyme (Flavourzyme), protamex (Protamex), neutrase (Neutrase), papain (Papain), pepsin (Pepsin), trypsin (Trypsin), Alpha-Chymotrypsin, Alcalase, Sumizyme, Kojizyme, Pancreatin, Kallikrein, Cathepsin, Thermolysin ( thermolisin), subtilisin, bromelain, ficin, actinidin, peptidase and transglutaminase selected from the group consisting of It may be any one or two or more.

In one embodiment of the present application, the hydrolase may be a liquid proteolytic enzyme.

In one embodiment of the present application, the pre-treatment of the fish eggs may include: removing salt by immersing the fish eggs in fresh water for 1 hour to 10 hours; may further include.

In one embodiment of the present application, the fish roe extract recovered through the centrifugation step may be coated with mineral-vanadium obtained from lava seawater through salting-out.

One embodiment of the present application provides a fish roe extract prepared by the above preparation method.

One embodiment of the present application provides a cosmetic composition comprising the fish roe extract and lava seawater as active ingredients.

In one embodiment of the present application, the cosmetic composition may include a liposome or a niosome having a fish roe extract inside some.

In one embodiment of the present application, the some (some) may be dispersed in a hydrocolloid state in lava seawater.

In one embodiment of the present application, the average particle diameter of the some may be in the range of 5 nm to 200 nm.

In one embodiment of the present application, the cosmetic composition may be for antioxidant.

In one embodiment of the present application, the cosmetic composition may be for improving skin whitening.

In one embodiment of the present application, the cosmetic composition may be for skin wrinkle improvement.

In one embodiment of the present application, the cosmetic composition may be for skin regeneration.

The method for preparing a fish roe extract according to an embodiment of the present application has an advantage in that the content of minerals such as minerals and vanadium is abundant.

The fish roe extract prepared by the method for manufacturing a fish roe extract according to an embodiment of the present application is harmless to the human body and has the advantage of providing a cosmetic composition with excellent safety.

The fish roe extract and the cosmetic composition comprising the same according to an embodiment of the present application have an advantage in that the DPPH radical and ABTS radical scavenging activity are high, and thus the antioxidant effect is excellent.

Since the fish roe extract and the cosmetic composition comprising the same according to an embodiment of the present application effectively inhibit melanin production, there is an advantage in that it is possible to prevent or improve a functional cosmetic composition for skin whitening or a skin pigmentation disease.

The fish roe extract and the cosmetic composition comprising the same according to an embodiment of the present application effectively promote collagen synthesis and inhibit elastase activity to prevent or improve functional cosmetic composition for wrinkle improvement or skin wrinkles There is this.

The fish roe extract and the cosmetic composition comprising the same according to an embodiment of the present application have excellent skin cell regeneration activity and thus can be usefully used as a functional cosmetic for skin cell regeneration.

1 shows the DPPH radical scavenging ability of an extract according to an embodiment of the present application.
Figure 2 shows the ABTS radical scavenging ability of the extract according to the embodiment of the present application.
Figure 3 shows the skin whitening inhibitory activity of the extract according to the embodiment of the present application.
4 is a view showing the measurement of the amount of procollagen (procollagen) produced in Example 1 and Comparative Example 1 according to the Examples of the present application.
5 is a view showing the measurement of the elastase production inhibitory activity of Example 1 and Comparative Example 1 according to the Examples of the present application.
6 is a view showing the measurement of the skin cell regeneration activity of the extract according to the embodiment of the present application.

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

The following specific structural or functional descriptions are only exemplified to describe embodiments according to the concept of the present application, and the embodiments according to the concept of the present application may be implemented in various forms, and It should not be construed as limiting.

Since the embodiment according to the concept of the present application may have various changes and may have various forms, specific embodiments will be described in detail herein. However, this is not intended to limit the embodiments according to the concept of the present application to a specific disclosed form, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present application.

The terminology used herein is used only to describe specific embodiments and is not intended to limit the present application. The singular expression includes the plural expression unless the context clearly dictates otherwise.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and are not to be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present specification. .

One embodiment of the present application, pre-processing the fish eggs; preparing a mixture by mixing pretreated fish roe and lava seawater in a weight ratio of 1:5 to 1:1; hydrolyzing the mixture with a hydrolase; centrifuging the hydrolyzed mixture; And filtering with a mesh (Mesh) filter; It provides a method for preparing a fish roe extract comprising a.

In the step of pre-treating the fish eggs, the fish eggs may be immersed in fresh water for 1 hour to 10 hours to remove salt. Specifically, it may be immersed for 1 hour to 6 hours. Alternatively, the fish eggs may be washed in fresh water for pretreatment.

When preparing a mixture of pretreated fish roe and lava seawater, the fish roe and lava seawater are mixed in a weight ratio of 1:5 to 1:1, so that the weight of the fish roe is 1/6 to 1/2 based on the mixed weight of the fish roe and lava seawater. It is preferable to be If the weight of the fish roe is less than 1/6, the fish roe may not be uniformly mixed in the lava seawater, and if the weight of the fish roe exceeds 1/2, the hydrolysis efficiency may decrease.

The lava seawater may be desalted lava seawater that has undergone a desalting treatment step using a cationic or anionic ion exchange resin, or desalted lava seawater that has undergone a desalting treatment step using an electrolysis method that decomposes sodium chloride components through an electrolysis device. . There is an advantage that minerals and inorganic components can be increased by going through the above step, and there is an advantage that a uniform mixture can be prepared when preparing a mixture with the fish eggs.

The centrifugation step may be stirred at 300 rpm to 400 rpm. When stirring at less than 300 rpm, there is a problem that the hydrolysis does not proceed and the mixture may agglomerate, and if stirred at more than 400 rpm, there is a problem that the aqueous colloid may be broken.

In one embodiment of the present application, the filtering may include: a first filtering step of filtering the mixture with a mesh filter of 1 μm to 5 μm; and a second filtration step of filtering the mixture through a mesh filter of 0.1 μm to 0.5 μm after the first filtration step; may include.

In the first filtering step, if the filter is less than 1 μm, there is a problem that a large amount of extracts are agglomerated with each other and are not filtered, and when it exceeds 5 μm, there is a problem in that extracts with too large a size are filtered and do not dissolve in the solvent.

In the second filtering step, if the filter is less than 0.1 μm, extracts of too small size may not be filtered and some may not be formed. have.

If the filtering step is performed sequentially, even if a large amount of extract is filtered, there is an advantage that the extract of a uniform size can be filtered without clumping.

In one embodiment of the present application, the hydrolyzing may include heating the mixture at a temperature of 80°C to 100°C for 3 minutes to 10 minutes; cooling at room temperature and grinding with a homogenizer; and after heating to 40°C to 60°C, hydrolyzing by adding a proteolytic enzyme; may include.

In the hydrolyzing step, if the mixture is heated at a temperature of less than 80 ° C., the activity of the enzyme may not proceed, and when heated at a temperature exceeding 100 ° C., there is a problem that the enzyme may be inactivated.

If the temperature at which the mixture is heated is less than 3 minutes, there is a problem that hydrolysis is not completely accomplished, and if it exceeds 10 minutes, there is a problem that the enzyme may be inactivated.

In addition, if heated at a temperature of less than 40 ℃ after grinding with a homogenizer, there is a problem that the aqueous colloid may not be uniformly dispersed, and when heated at a temperature exceeding 60 ℃, there is a problem that the colloid is decomposed.

In one embodiment of the present application, the fish roe may be an egg of the family Osmeridae selected from the group consisting of smelt, raw smelt, and hot smelt. More specifically, the roe may be hot smelt fish.

In this application, “lava seawater” is obtained from “salt sewage contained in volcanic lava in the eastern part of Jeju Island”.

Lava seawater is naturally filtered by the volcanic lava layer formed in the eastern part of Jeju due to volcanic activity 400,000 years ago, and is Jeju's unique underground water containing various rare minerals. Lava seawater is very clean because it is naturally filtered in the process of breaking through basalt, and contains various kinds of potassium and rare minerals that are essential for the human body. In addition, various functions and efficacy of lava seawater have already been proven through experiments and epidemiological investigations. This lava seawater contains sodium (Na) component 10800/, chlorine (Cl ^- ) 19422/, potassium (K) 416/, calcium (Ca) 407/, magnesium (Mg) 1329/, iron (Fe) 0.01/, lithium ( Li) 0.37/, Zn 0.016/, copper (Cu) 0.014/, manganese (Mn) 0.003/, molybdenum (Mo) 0.011/, vanadium (V) 0.022/, germanium (Ge) 0.001/, strontium (Sr) It contains 8.36/, silicon (Si) 10.8/, selenium (Se) 0.008/, boton (B) 4.97/.

Specifically, 'Jeju-do eastern region' means Gujwa-eup, Seongsan-eup, and Pyoseon-myeon of Jeju-do in terms of administrative district, and 'salt groundwater' means groundwater containing a certain amount of salt or more. It can be understood as meaning including both (electrical conductivity of 1,700 μs/cm to 17,350 μs/cm) and saltwater underground water (electrical conductivity of 17,350 μs/cm or more), but preferably, electrical conductivity is 17,350 μs/cm or more Alternatively, it may be understood to mean saltwater underground water having a salt concentration of 30% (permilliage) or higher (usually, the salt concentration of seawater is 32 to 35%).

In one embodiment of the present application, the hydrolase is, flavozyme (Flavourzyme), protamex (Protamex), neutrase (Neutrase), papain (Papain), pepsin (Pepsin), trypsin (Trypsin), Alpha-Chymotrypsin, Alcalase, Sumizyme, Kojizyme, Pancreatin, Kallikrein, Cathepsin, Thermolysin ( thermolisin), subtilisin, bromelain, ficin, actinidin, peptidase and transglutaminase selected from the group consisting of It may be any one or two or more, but is not limited thereto.

In one embodiment of the present application, the hydrolase may be a liquid proteolytic enzyme. More specifically, the hydrolase may be Neutrase.

When hydrolytic enzymes are used in liquid form, there is an advantage that lava seawater and fish eggs, which are the subject of hydrolysis, can be hydrolyzed uniformly.

When hydrolyzing with the proteolytic enzyme, pH and temperature can be adjusted according to the activity range of the enzyme. For example, in the case of pepsin, pH 2.0 to 4.0, trypsin, pH 7.0 to 9.0, protamax, pH 5.5 to 7.5, and flavozyme, pH 5.0 to 7.0. In addition, an organic acid may be used to adjust the pH. The organic acid may be any organic acid capable of adjusting the pH, and may include, for example, any one or two or more selected from citric acid, lactic acid, acetic acid, formic acid, oxalic acid, uric acid, and the like.

In one embodiment of the present application, the fish roe extract recovered through the centrifugation step may be coated with mineral-vanadium obtained from lava seawater through salting-out.

The fish roe extract prepared according to the method for preparing a fish roe extract using lava seawater has an advantage in that the content of minerals and vanadium is rich, and the content of free and constituent amino acids is excellent. This is because lava seawater has a richer content of minerals and vanadium than general seawater or freshwater, and has excellent free and constituent amino acid content. In particular, lysine is 180~185mg/100g, leucine is 155~160mg/100g, proline is 130~135mg/100g, arginine is 105~110mg/100g, glycine is 90~95mg/100g, histidine is 50~55mg/100g and Methionine has the advantage that it contains a content of 40 to 45 mg/100 g.

One embodiment of the present application provides a fish roe extract prepared by the above preparation method.

The fish roe extract prepared by the above manufacturing method has an advantage in that it uses lava seawater, not general seawater or freshwater, to have a rich content of minerals or vanadium, and excellent content of free and constituent amino acids. In particular, lysine 180~185mg/100g, leucine 155~160mg/100g, proline 130~135mg/100g, arginine 105~110mg/100g, glycine 90~95mg/100g, histidine 50~55mg/100g and methionine 40~ It has the advantage that it contains a content of 45mg/100g.

One embodiment of the present application provides a cosmetic composition comprising the fish roe extract and lava seawater as active ingredients.

One embodiment of the present application provides a cosmetic composition comprising the fish roe extract, lava seawater and vitamin C as active ingredients.

The cosmetic composition may further include a vitamin C stabilizer.

The vitamin C stabilizer may be selected from the group consisting of ethylated ascorbic acid (Ethyl Ascorbic Acid), L-Ascorbic Acid 2-Glucoside (AA2G), Magnesium ascorbyl phosphate (MAP), and caffeic acid derivatives.

When the vitamin C is further included, the whitening activity and wrinkle improvement of the mixture can be improved.

The vitamin C stabilizer may be extracted from kakadu plum, and the yield may be 5000 to 6000 mg/100g. The kakadu plum has an advantage in that it contains abundant vitamin C compared to the conventional species containing vitamin C.

And, the vitamin C stabilizer may be a caffeic acid derivative represented by the following formula (1).

The compound has the advantage of helping the stabilization of pure vitamin C, and has the advantage of preventing oxidation.

In one embodiment of the present application, the cosmetic composition may include liposomes or niosomes with fish roe extract inside some.

The inside of the some (some) may contain active ingredients such as minerals in addition to the fish roe extract.

As used herein, the term 'active ingredient' refers to an amount of a compound capable of promoting the regeneration of damaged skin, improving wrinkles, enhancing elasticity, or exhibiting an antioxidant effect, a whitening effect, or a skin regeneration effect. . The content of the extract included in the composition of the present application will vary depending on the form in which the composition is commercialized, the method in which the compound is applied to the skin, and the time it stays on the skin.

In one embodiment of the present application, the active ingredient may be included in an amount of 0.00001 to 20% by weight based on the total weight of the total composition. Although not limited thereto, specifically, the composition may include the extract in an amount of 0.00001 wt% to 10 wt%, more specifically 0.0001 wt% to 3 wt%, based on the total weight of the composition. When the composition of the present application contains less than 0.00001% by weight of the extract, sufficient skin regeneration, wrinkle improvement, elasticity enhancement, and whitening effects cannot be expected, and when it contains more than 20% by weight, unwanted reactions such as allergies This is to prevent this from occurring or there may be problems with skin safety.

In one embodiment of the present application, the fish roe extract may be included in an amount of 0.001 to 1% by weight based on the total weight of the composition. If it is less than 0.001% by weight, the desired effect is insignificant, and if it is more than 1% by weight, there may be a problem in formulation stability.

In one embodiment of the present application, the lava seawater may be included in an amount of 0.001 to 2% by weight based on the total weight of the composition. If it is less than 0.001% by weight, the desired effect is insignificant, and if it is more than 2% by weight, there may be a problem in formulation stability.

In one embodiment of the present application, the vitamin C may be included in an amount of 0.1 to 17% by weight based on the total weight of the composition. If it is less than 0.1% by weight, the desired effect is insignificant, and if it is more than 17% by weight, there may be a problem in formulation stability.

In one embodiment of the present application, the vitamin C stabilizer may be included in an amount of 0.01 to 5% by weight based on the total weight of the composition. If it is less than 0.01% by weight, the desired effect is insignificant, and if it is more than 5% by weight, there may be a problem in formulation stability.

In the present application, the term 'active ingredient' refers to an amount of a compound capable of promoting the regeneration of damaged skin, improving wrinkles, enhancing elasticity, or exhibiting an antioxidant effect, a whitening effect, or a skin regeneration effect. . The content of the extract included in the composition of the present application will vary depending on the form in which the composition is commercialized, the method in which the compound is applied to the skin, and the time it stays on the skin.

In the case of wash-off type cosmetics such as makeup removers and detergents, in which active ingredients stay on the skin within a short period of time when commercialized as cosmetics, a relatively high concentration of the extract may be included. On the other hand, in the case of leave-on type cosmetics, such as lotions, emulsions, creams, and essences, in which the active ingredients stay on the skin for a long period of time, it is okay to include the extract at a lower concentration than the wash-off type cosmetics. something to do.

In addition, in order to maintain the shape of the some, a small amount of surfactant may be further included during hydrolysis.

In one embodiment of the present application, the some may be dispersed in a hydrocolloidal state in lava seawater.

The hydrophilic colloidal state may be a gelatinous colloid in unstable equilibrium with the water it contains, and a dispersing agent may be additionally included in the dispersed solution.

Surfactants are used as the dispersing agent included in the dispersion solution of the present invention. Particularly preferred in the present invention are excellent emulsifying, dispersing and penetrating effects, extremely stable in acid and alkali solutions, and even in aqueous solutions in which acids and alkalis coexist. It exhibits excellent interfacial properties and may be nonionic as a homogeneous liquid phase.

In addition, the penetrating agent included in the dispersion solution of the present invention is a kind of surfactant to facilitate the penetration of colloidal particles, and is characterized by particularly strong wet permeability. Particularly preferred in the present invention is a homogeneous liquid, anionic, pH (in 1% aqueous solution) of 7.0±1.0, specific gravity (at 25°C) of 1.05±0.05, easily soluble in cold water, anionic, nonionic It can be used in combination with and has the advantage of exhibiting a stable effect even in acid and alkali, stable even in hard water, and having low foaming properties. Since the physical property is low-foaming, there is an advantage that it can be uniformly mixed in the cosmetic composition.

In one embodiment of the present application, the average particle diameter of the some may be in the range of 5 nm to 200 nm.

If the average particle diameter of the some is less than 5 nm, the shape of the liposome or niosome may not be maintained, and if the average particle diameter exceeds 200 nm, the liposome or niosome may not exist in an aqueous colloidal state in lava seawater. there is

In one embodiment of the present application, the cosmetic composition may be for antioxidant.

In the present application, the term 'antioxidation' refers to oxidation of cells by highly reactive free radicals or reactive oxygen species (ROS) depending on intracellular metabolism or oxidative stress caused by the influence of ultraviolet rays. It refers to inhibiting, and includes reducing damage to cells by removing free radicals or reactive oxygen species.

In one embodiment of the present application, the cosmetic composition may be for improving skin whitening.

In the present application, the term 'improving whitening' refers to not only brightening skin tone by inhibiting the synthesis of melanin pigment, but also improving skin hyperpigmentation such as spots or freckles caused by ultraviolet rays, hormones, or heredity.

In one embodiment of the present application, the cosmetic composition may be for skin wrinkle improvement.

As used herein, the term 'skin wrinkle improvement' refers to an increase in skin elasticity, which can improve skin wrinkles caused by aging by inhibiting or inhibiting loss of skin elasticity or alleviating already reduced elasticity. say that

In one embodiment of the present application, the cosmetic composition may be for skin regeneration.

In the cosmetic composition according to an embodiment of the present application, in addition to the extract as an active ingredient, ingredients commonly added to the cosmetic composition, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and conventional adjuvants, and carriers. Additional can be added.

The cosmetic composition of the present application may be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , oil, powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto. More specifically, it may be prepared in the form of a nourishing cream, an astringent lotion, a softening lotion, a lotion, an essence, a nourishing gel or a massage cream.

When the formulation of the present application is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, gum tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, etc. can be used

When the formulation of the present application is a powder or a spray, toss, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane /may contain propellants such as butane or dimethyl ether.

When the formulation of the present application is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol fatty ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present application is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Adult cellulose, aluminum metahydroxide, bentonite, agar or gum tracanth may be used.

When the formulation of the present application is surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid as carrier components Amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester may be used.

Hereinafter, the present application will be described in more detail through Examples, Comparative Examples, and Experimental Examples, but the following examples are only provided to aid understanding of the present application, and do not limit the scope of the present application.

< Example 1>

In Example 1, after washing the eggs of hot smelt with sterilized distilled water, the same amount of distilled water was added thereto, followed by grinding in a bath for 10 minutes. Thereafter, 1% of the weight of the triturated material was added with Neutrase and hydrolyzed at 55° C. for 3 hours. After that, the enzyme was inactivated by heating for 10 minutes, followed by first filtration with a 1 μm mesh filter and second filtration with a 0.1 μm mesh filter. prepared.

< comparative example 1 to comparative example 3>

Placenta extract as Comparative Example 1, arbutin as Comparative Example 2, and allantoin as Comparative Example 3 were used.

< Experimental example 1> DPPH radical extinction ability measurement

The DPPH assay was performed by Yoshida et al. (1989) were measured according to the method used. In a 96-well plate, 12 μl of the solvent was added to the control and 12 μl of the diluted solution of the extract of Example was added to the test group, respectively. After adding 6 μl of ethanol to all wells, 222 μl of DPPH (0.03 mM 1,1-diphenyl-2-picrylhy-drazyl(DPPH)/ethanol) solution was added and mixed. After reacting at room temperature of 25° C. for 30 minutes, absorbance was measured at 517 nm. In order to find out the radical scavenging activity of each of Example 1 and Comparative Example 1, it was calculated as a percentage according to Equation 1 and is shown in Table 1 and FIG. 1 below.

Sample concentration (%) DPPH Radical Scavenging (%) Example 1 Comparative Example 1 control 0.0 ± 0.2 0.025 8.6 ± 0.3 4.4 ± 1.1 0.05 7.9 ± 0.2 4.9 ± 0.6 0.25 10.8 ± 0.1 2.2 ± 1.2 0.5 13.1 ± 0.3 4.0 ± 1.1 2.5 20.5 ± 0.2 7.6 ± 0.5 5 20.9 ± 1.0 11.7 ± 1.0 IC ₅₀ (%) 0.664 3.165

In Table 1, the half maximal inhibitory concentration (IC50) of Example 1 was 0.664%, and the IC50 of Comparative Example 1 used as a positive control was 3.165%. In Experimental Example 1 compared to Comparative Example 1, Example 1 had about 4.8 times higher DPPH radical scavenging activity.

That is, it could be confirmed that one embodiment of the present application had excellent DPPH radical scavenging ability.

< Experimental example 2> ABTS radical extinction ability measurement

ABTS radical scavenging activity was determined by Re et al. (1999) was modified and measured. That is, after mixing 5 ml of 7mM ABTS and 88 μl of 140mM potassium persulfate, light was blocked at room temperature for 16 hours to form ABTS cations. Then, this solution was diluted with PBS so that the absorbance value at 414 nm was 1.5. After mixing 190 μl of the prepared diluted solution and 10 μl of the sample, the mixture was reacted at room temperature for 6 minutes, and absorbance was measured at 734 nm.

In order to find out the radical scavenging activity of each of Example 1 and Comparative Example 1, it was calculated as a percentage according to Equation 2 and is shown in Table 2 and FIG. 2 below.

Sample concentration (%) ABTS Radical Scavenging (%) Example 1 Comparative Example 1 Control 0.0 ± 0.8 0.025 22.3 ±0.9 8.6 ± 1.2 0.05 29.5 ± 0.1 12.8 ± 6.7 0.25 61.9 ± 2.0 11.2 ± 2.7 0.5 77.5 ± 2.5 16.7 ± 1.9 2.5 98.1 ± 0.2 38.0 ± 2.3 5 97.8 ± 1.2 61.1 ± 8.0 IC ₅₀ 0.221 22.594

The half maximal inhibitory concentration (IC50) of Example 1 was 0.221%, and the IC50 of Comparative Example 1 used as a positive control was 22.594%. In Experimental Example 1, compared to Comparative Example 1, Example 1 had about 102.2 times higher ABTS radical scavenging activity. That is, it was confirmed that one Example in the present application had excellent ABTS radical scavenging ability.

< Experimental example 3> Skin whitening inhibitory activity, B16F0 Cytotoxicity measurement

B16F0, a melanocyte, was purchased from the Korea Research Institute of Biotechnology and Biotechnology (KCTC) and used to test the melanin production inhibitory ability of the sample. In a cell culture flask, the B16F0 cell line was cultured in DMEM medium containing 10% Fetal Bovine Serum (FBS, Lonza, Valais, Switzerland) medium. The cell line was cultured in an incubator controlled at 95% humidity and 5% CO ₂ ,37°C, and antibiotics for the medium (Penicillin streptomycin, Gibco) were used to suppress contamination or proliferation of microorganisms. When about 80% of the cells covered the dish, it was washed with phosphated-buffered saline-EDTA (PBS-EDTA) and treated with 1 ml of 0.05% Trypsin EDTA to remove the cells attached to the bottom of the dish and subcultured. The medium was changed every 48 hours to culture the cells.

Cell line viability was measured using MTS (CellTiter 96®AQueous One Solution Cell Proliferation Assay, Promega, USA) reagent. To check the toxicity to B16F0 cells, the B16F0 cell line was aliquoted in a 96-well plate at 1x10 ⁴ cells/well and then stabilized for 24 hours. Thereafter, the sample was diluted in the medium for each concentration, dispensed by 100 μl, and then cultured for 24 hours in an incubator controlled at 95% humidity, 5% CO ₂ ,37°C. After removing the medium, 9 ml of the medium was added to 1 ml of MTS (CellTiter 96®AQueous One Solution Cell Proliferation Assay) reagent and diluted, and then treated at 100 μl per well. Humidity 95%, 5% CO ₂ After 60 minutes of reaction in an incubator controlled at 37 °C, absorbance was measured at 490 nm with a microplate reader (Molecular Devices, Versa Max ELISA Microplate Reader, USA). The cell viability is shown in Table 3 by expressing the absorbance in comparison with Example 1 without treating Comparative Example 2, which is albutin, a raw material notified by the Ministry of Food and Drug Safety, as a control.

Sample concentration (%) Example 1 (%) Comparative Example 2 (%) Control 100.00 ± 5.74 25 100.26 ± 5.38 99.16 ± 3.47 50 112.26 ± 2.39* 97.67 ± 9.10 250 109.65 ± 9.65 103.49 ± 8.80 500 94.73 ± 3.65 102.74 ± 8.72 2,500 99.90 ± 3.28 101.77 ± 6.88 5,000 107.10 ± 7.13 88.32 ± 5.86*

* p <0.05, VS Control

In Table 3, Example 1 confirmed that B16F0 did not show cytotoxicity at all concentrations, and a concentration of 25 to 25,000 ppm was used for the whitening inhibitory activity experiment, but in Comparative Example 2, arbutin was cytotoxic at a concentration of 5,000 ppm. Thus, 25 to 25,000 ppm arbutin was used in the whitening inhibitory activity experiment.

< Experimental example 4> Skin whitening inhibitory activity test

The melanin production inhibition experiment was conducted according to the guidelines for functional cosmetics of the Ministry of Food and Drug Safety. B16F0 cells per well were seeded at 1×10 ⁵ cells/1.2ml/well in a 6-well plate and stabilized in cell culture conditions for 24 hours. The medium was removed, and 100 nM α-MSH, FBS, a test solution and a fresh medium were added and cultured for 72 hours. After removing the culture medium, the adhered cells were placed in 120 μl of 1N sodium hydroxide solution, dissolved in a thermostat at 60° C. for 1 hour, and centrifuged (2,000 rpm, 4° C., 10 minutes). 100 μl of the supernatant was taken in a 96 well plate, and the absorbance was measured at 490 nm to calculate the melanin content. The amount of melanin was converted into the amount of melanin per a certain number of cells or the amount of melanin per certain protein, and compared with the results of the blank sample solution treated with the solvent in which the sample was dissolved, it is shown in Tables 4 and 3 below.

Sample name 100 nM α-MSH Amount of melanin production in cells
(mg/g protein) Inhibitory activity of intracellular melanin production (%) (-)Control - - 170.26 ± 19.06** - (+)Control - + 362.19 ± 11.24 - Example 1 (ppm) 500 + 279.69 ± 12.52** 48.52 ± 3.46 1,000 + 180.45 ± 5.46** 66.78 ± 1.51 2,500 + 99.06 ± 8.03** 72.65 ± 2.22 5,000 + 62.37 ± 8.36** 88.52 ± 10.08 Comparative Example 2 (ppm) 500 + 230.93 ± 7.55** 36.24 ± 2.08 1,000 + 138.73 ± 17.77** 61.70 ± 4.91

*p<0.05, VS (+)Control, **p<0.01, VS (+)Control

In Table 4, the melanin content of (+)Control, the untreated group treated with α-MSH, was 362.2 mg/g protein, and the melanin content of 1% Example 1 was 62.4 mg/g protein, which was treated with α-MSH. It showed melanin inhibitory activity of 88.5% compared to the untreated group. This is about 1.4 times higher melanin inhibitory activity than that of Comparative Example 2, 0.1% arbutin (61.7%).

That is, it could be confirmed that one example in the present application had excellent melanin inhibitory activity.

< Experimental example 5> Measurement of anti-wrinkle activity, HS68 cell culture and cytotoxicity

It was carried out according to the guidelines for functional cosmetics of the Ministry of Food and Drug Safety.

First, the cell line used in the experiment was HS68. As the medium, DMEM (HyClone, SH30243.01, Thermo-Fisher Scientific, Waltham, MA) medium containing 10% Fetal Bovine Serum (FBS, Lonza, Valais, Switzerland) medium was used. The cell line was cultured in an incubator controlled at 95% humidity, 5% CO ₂ ,37°C, and antibiotics for the medium (Penicillin streptomycin, Gibco, CA, USA) were used to suppress contamination or proliferation of microorganisms. When about 80% of the cells covered the dish, they were washed with phosphate-buffered saline-EDTA (PBS-EDTA), treated with 0.05% Trypsin EDTA for 1 ml, and the cells attached to the bottom of the dish were removed and subcultured. The medium was changed every 48 hours to culture the cells.

In order to confirm the toxicity to HS68 cells, HS68 cells were aliquoted in a 96-well plate at 1x104 cells/well and then stabilized for 24 hours. After removing the medium and diluting the sample in a new medium to an appropriate concentration, 100 μl each was dispensed, and cultured for 24 hours in an incubator controlled at 95% humidity, 5% CO ₂ , 37°C.

After removing the medium, 9 ml of the medium was added to 1 ml of MTS (CellTiter 96®AQueous One Solution Cell Proliferation Assay) reagent and diluted, and then treated at 100 μl per well. Humidity 95%, 5% CO ₂ After 60 minutes of reaction in an incubator controlled at 37 °C, absorbance was measured at 490 nm with a microplate reader (Molecular Devices, Versa Max ELISA Microplate Reader, USA). The cell viability is shown in Table 5 by expressing the placenta extract of Comparative Example 1 as a control and not treating the sample as compared to Example 1 as absorbance.

Sample concentration (%) Example 1 (%) Comparative Example 1 (%) Control 100.00 ± 3.06 0.005 96.64 ± 3.96 106.72 ± 10.41 0.01 96.62 ± 4.61 107.92 ± 11.76 0.05 98.63 ± 6.02 121.67 ± 10.3** 0.1 105.44 ± 7.12 114.17 ± 9.21* 0.5 134.34 ±13.94** 112.44 ± 7.41* One 161.57 ± 6.22** 112.63 ± 8.35*

*p<0.05, VS Control, **p<0.01, VS Control

In Table 5, Example 1 and Comparative Example 1 confirmed that no cytotoxicity was shown at all concentrations, and the concentration of the samples of Example 1 and Comparative Example 1 was used at the same concentration of 0.005 to 1% during the wrinkle improvement activity test. .

< Experimental example 6> measurement of wrinkle improvement activity, procollagen ( procollagen ) production

For the measurement of C-peptide, the C-peptide EIA kit was obtained from Takara Bio Inc. was purchased from and used. 100 μl of antibody-POD conjugate solution was dispensed into 96 wells, and 20 μl of sample or standard was dispensed, mixed, and reacted at 37° C. for 3 hours. After completion of the reaction, 96 wells were washed repeatedly 4 times with PBS (Phosphate Buffer Saline solution, ph 7.4). After washing, 100 μl of substrate solution was dispensed into each well, left at room temperature for 15 minutes, and 100 μl of stop solution was added and stirred. The result was obtained by absorption at 450 nm. Results were calculated as procollagen content per cell protein. Example 1 and Comparative Example 1 are shown in Table 6 and Figure 4 below to find out the respective procollagen generating activity.

Sample name (%) Procollagen
(pg/g protein) Procollagen (%) Control 26.23 ± 3.19 100.00 ± 12.17 Example 1 (%) 0.05 59.92 ± 3.80 228.44 ± 14.48 0.1 102.43 ± 8.56 390.51 ± 32.65 0.5 124.91 ± 4.29 476.23 ± 16.37 One 145.48 ± 2.33 554.64 ± 8.88 Comparative Example 1 (%) 0.1 65.35 ± 3.40 249.15 ± 12.96 One 75.75 ± 6.79 288.78 ± 25.88

In Table 6, the production amount of procollagen (pg) of the untreated group (control) was 26.23 pg/g protein, and the amount of procollagen production of 1% Example 1 was 145.48 pg/g protein, and the amount of procollagen production compared to the control group was 145.48 pg/g protein. increased by about 5.5 times. In comparison with 1%, the amount of procollagen production of 1 was 75.75 pg/g protein.

That is, Example 1 showed that the amount of procollagen production was about 1.9 times higher than that of Comparative Example 1.

< Experimental example 7> measurement of wrinkle improvement activity, elastase Measurement of production inhibitory activity

Elastase inhibitory activity was used by purchasing Invitrogen's EnzChek® Elastase Assay Kit (600 assays) reagent. First, 0.5ml of DW was added to a 1 mg DQ elastin vial to prepare a DQ elastin stock solution (1 mg/ml). 18 ml of DDW was added to 2 ml of 10 x reaction buffer to dilute the reaction buffer. Next, 0.5ml of 1 x reaction buffer was added to the elastase vial to prepare 100U/ml. The enzyme working solution was diluted with 1 x reaction buffer so that the final concentration was 0.1-0.2 U/ml. Samples were carried out in triples of 50 μl each in a 96 well plate. After adding the sample, 100 μl of the elastase enzyme was treated to the control and test samples, and DW was treated to the remaining control blank and sample blank. After mixing well, 50 μl of DQ elastin was added to all wells. After 1 hour reaction at room temperature with light blocked, measure the fluorescence intensity with an ELISA plate reader (VICTOR X3™, PerkinElmer, US) at the intensity excitation wavelength 485nm and emission wavelength 535nm. Calculate the result according to Equation 3 below. It is shown in Table 7 and FIG. 5 below.

a: absorbance after reaction of blank sample solution

b: absorbance after reaction of the sample solution

a',b': absorbance measured by substituting a buffer for elastase

Sample concentration (ppm) Elastase inhibitory activity (%) Example 1 (%) Comparative Example 1 (%) Control 0.00 ± 0.86 6.25 15.52 ± 3.56 18.05 ± 1.76 12.5 23.78 ± 1.62 15.65 ± 5.40 62.5 52.65 ± 2.25 28.67 ± 6.22 125 68.73 ± 0.10 32.57 ± 6.54 IC ₅₀ (%) 16.62 20.43

In Table 7, the IC50 (the half maximal inhibitory concentration) of Example 1 was 16.62%, and the IC50 of Comparative Example 1 was 20.43%.

That is, in this experiment, compared to Comparative Example 1, Example 1 was confirmed that about 1.2 times the elastase (elastase) inhibitory activity is excellent.

< Experimental example 8> skin cell regeneration activity, HaCaT Cytotoxicity measurement

Cell toxicity was measured using HaCaT cells, which are human-derived epidermal cells. Cell line viability was measured using MTS (CellTiter 96®AQueous One Solution Cell Proliferation Assay, Promega, USA) reagent. After dispensing the cells at a concentration of 1×10 ⁴ cells/well in a 96 well plate, the cells were stabilized for 24 hours in an incubator controlled at 95% humidity, 5% CO ₂ , and 37°C. Thereafter, samples were treated by concentration and incubated for 24 hours in an incubator controlled at 95% humidity, 5% CO ₂ ,37°C. After removing the medium, 9 ml of a medium (DMEM low glucose, free FBS) was added to 1 ml of the reagent and diluted, and then treated at 100 μl per well. After reaction at 37°C for 60 minutes, absorbance was measured at 490 nm with a microplate reader (Molecular Devices, Versa Max ELISA Microplate Reader, USA), and cell viability was compared with allantoin, which is known to be effective for skin soothing and skin cell proliferation. Example 3 is shown in Table 8 as a control, expressed as absorbance compared to Example 1.

Sample name Concentration (%, μg/ml) HaCaT Cell Viability (%) Control - 100.00 ± 2.37 Example 1 (%) 0.01 98.38 ± 7.71 0.05 97.02 ± 2.90 0.1 95.91 ± 4.19 0.5 96.98 ± 8.31 One 95.25 ± 4.50 Comparative Example 3 (μg/ml) 0.1 121.16 ± 4.35** 0.5 118.37 ± 0.95** One 125.56 ± 5.70** 5 115.95 ± 6.80** 10 115.14 ± 2.34**

**p<0.01, VS Control

In Table 8, it was confirmed that Example 1 and Comparative Example 3 did not show cytotoxicity at all concentrations, so that Example 1 was used at a concentration of 0.01 to 1%, and Comparative Example 3 was 0.1 to 10. % was used.

< Experimental example 9> Measurement of skin cell regeneration activity

The HaCaT cell line was dispensed in a 96-well plate at a concentration of 2×10 ³ cells/ml, and cultured in a CO2 incubator controlled at 37°C, humidity 95%, and CO ₂ 5% for 24 hours. Each sample was treated in a new medium in an appropriate concentration range that does not show cytotoxicity, and then cultured for 1, 2, or 3 days. To measure the viability of cell lines, absorbance at 450 nm with a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA) using Cell Counting Kit-8 (CCK-8, Dojindo Mol. Tech., Rockville., MD, USA) was measured. Cell regeneration efficacy is shown in Table 9 and Figure 6 by expressing the absorbance of Example 1 compared to Comparative Example 3, which is a control group that is not treated with the sample.

Sample name sample concentration Day 1 Day 2 Day 3 Control - 100.00 ±8.55 100.00 ± 5.74 100.00 ± 8.38 Example 1 (%) 0.01 101.04 ± 15.01 114.38 ± 4.78* 122.78 ± 11.45* 0.05 107.6 ± 13.48 129.37 ± 4.83** 125.48 ± 3.88** 0.1 111.37 ± 13.84 133.46 ± 5.88** 140.12 ± 5.40** 0.5 98.61 ± 12.74 137.54 ± 7.02** 139.18 ±7.23** One 101.53 ± 10.29 140.5 ± 10.02** 145.73 ± 12.03** Comparative Example 3 (μg/ml) 0.1 100.93 ± 14.03 111.01 ± 4.75* 119.94 ± 6.75** One 94.59 ± 2.66 112.14 ± 6.27* 121.08 ± 7.62**

*p<0.05, VS Control, **p<0.01, VS Control.

In Table 9, it was confirmed that the results of the proliferation activity on the 2nd and 3rd days of Example 1 showed similar or high regenerative activity compared to Comparative Example 3. In particular, the 3rd day proliferative activity of 1% Example 1 was about 146%, and the 3rd day proliferative activity of 1% Comparative Example 3 as a positive control was about 121%.

That is, when 1% of Example 1 was used, it was confirmed that about 1.2 times superior human skin cell proliferation and proliferation effect compared to Comparative Example 3 at 1 μg/ml was used.

Formulation examples of the cosmetic composition according to the present invention will be described below, but various formulations other than the following examples are applicable, which is not intended to limit the present invention, but merely to describe in detail.

[Formulation Example 1] Softening lotion (skin lotion)

A softening lotion was prepared in a conventional manner according to the composition shown in Table 10 below.

ingredient Content (wt%) Fish roe extract of Example 1 0.1 lava seawater 0.1 glycerin 3.5 oleyl alcohol 1.5 ethanol 5.5 Polysorbate 80 3.2 carboxyl vinyl polymer 1.0 butylene glycol 2.0 propylene glycol 2.0 preservatives, fragrances appropriate amount Purified water remaining amount total 100

[Formulation Example 2] Nourishing lotion (milk lotion)

Nutrient lotion was prepared in a conventional manner according to the composition shown in Table 11 below.

ingredient Content (wt%) Fish roe extract of Example 1 0.1 lava seawater 0.1 glycerin 3.0 butylene glycol 3.0 propylene glycol 3.0 Carboxyvinyl Polymer 0.1 beeswax 4.0 Polysorbate 60 1.5 Caprylic/Capric Liglycerides 5.0 squalane 5.0 Sorbitasequioleate 1.5 cetearyl alcohol 1.0 triethanolamine 0.2 preservatives, fragrances appropriate amount Purified water remaining amount total 100

[Formulation Example 3] Nourishing Cream

Nutrient creams were prepared in a conventional manner according to the composition shown in Table 12 below.

ingredient Content (wt%) Fish roe extract of Example 1 0.1 lava seawater 0.1 glycerin 3.5 butylene glycol 3.0 liquid paraffin 7.0 beta glucan 7.0 Carbomer 0.1 Caprylic/Capric Liglycerides 3.0 squalane 5.0 cetearyl glucoside 1.5 Sorbitan Stearate 0.4 Polysorbate 60 1.2 triethanolamine 0.1 preservatives, fragrances appropriate amount Purified water remaining amount total 100

As above, a specific part of the present application has been described in detail, and for those of ordinary skill in the art, this specific technique is only a preferred embodiment, and it is clear that the scope of the present application is not limited thereto.

Accordingly, the substantial scope of the present application will be defined by the appended claims and their equivalents.

Claims (15)

pre-treating the fish eggs;
preparing a mixture by mixing pretreated fish roe and lava seawater in a weight ratio of 1:5 to 1:1;
hydrolyzing the mixture with a hydrolase;
centrifuging the hydrolyzed mixture; and
filtering with a mesh filter;
A method for preparing a fish roe extract comprising a.
The method according to claim 1,
The filtering step is
a first filtration step of filtering the mixture through a mesh filter of 1 μm to 5 μm; and
a second filtration step of filtering the mixture through a mesh filter of 0.1 μm to 0.5 μm after the first filtration step;
A method for preparing a fish roe extract comprising a.
The method according to claim 1,
The hydrolyzing step is
heating the mixture at a temperature of 80° C. to 100° C. for 3 minutes to 10 minutes;
cooling at room temperature and grinding with a homogenizer; and
After heating to 40°C to 60°C, hydrolyzing by adding a proteolytic enzyme;
A method for preparing a fish roe extract comprising a.
The method according to claim 1,
The fish roe is
A method for producing a fish roe extract, characterized in that it is an egg of the family Osmeridae selected from the group consisting of smelt, raw smelt and hot smelt.
The method according to claim 1,
The hydrolase is
Flavozyme, Protamex, Neutrase, Papain, Pepsin, Trypsin, Alpha-Chymotrypsin, Alcalase ), Sumizyme, Kojizyme, pancreatin, kallikrein, cathepsin, thermolisin, subtilisin, bromelain ), ficin (Ficin), actinidin (Actinidin), peptidase (Peptidase) and transglutaminase (Transglutaminase) A method for producing a fish roe extract, characterized in that any one or two or more selected from the group consisting of.
6. The method of claim 5,
The hydrolase is a method for producing a fish roe extract, characterized in that the liquid proteolytic enzyme.
The method according to claim 1,
The step of pre-processing the fish eggs,
removing salt by immersing the fish eggs in fresh water for 1 hour to 10 hours;
Fish roe extract manufacturing method, characterized in that it further comprises.
The method according to claim 1,
The fish roe extract recovered through the centrifugation step is a mineral-vanadium obtained from lava seawater through a salting-out process, characterized in that it is coated.
A fish roe extract prepared by the method of any one of claims 1 to 8.
A cosmetic composition comprising the fish roe extract of claim 9 and lava seawater as active ingredients.
11. The method of claim 10,
The cosmetic composition,
A cosmetic composition comprising a liposome or a niosome provided with a fish roe extract inside some.
12. The method of claim 11,
The cosmetic composition, characterized in that the some (some) is dispersed in a hydrophilic colloidal state in lava seawater.
12. The method of claim 11,
A cosmetic composition, characterized in that the average particle diameter of the some is in the range of 5 nm to 200 nm.
11. The method of claim 10,
The cosmetic composition is a cosmetic composition, characterized in that for antioxidant or skin regeneration.
11. The method of claim 10,
The cosmetic composition is a cosmetic composition, characterized in that for improving skin whitening or skin wrinkle improvement.

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