KR102297395B1 - Cosmetic composition comprising extract of capelin egg or sailfin sandfish egg and method for preparing the extract of the same - Google Patents

Abstract

The present application relates to a cosmetic composition comprising an extract of hot smelt roe or Dorumuk roe as an active ingredient, for antioxidant, skin whitening, wrinkle improvement, or skin cell regeneration. The composition of the present application is safe for the skin, and effectively inhibits melanin production, inhibits collagenase activity, reduces elastase activity, and has excellent skin cell regeneration effect.

Description

COSMETIC COMPOSITION COMPRISING EXTRACT OF CAPELIN EGG OR SAILFIN SANDFISH EGG AND METHOD FOR PREPARING THE EXTRACT OF THE SAME

The present application relates to a cosmetic composition comprising a hot smelt roe or an oyster roe extract as an active ingredient, and a method for preparing the extract.

It is known that about 8,000 kinds of raw materials are used in cosmetics, and the domestic market for cosmetics raw materials is estimated to be 300 billion won, but more than 80% of expensive functional raw materials are imported.

Korea's major importers of cosmetic raw materials are 24,546,000 dollars from Japan, 10,641 thousand dollars from Germany, 9,723,000 dollars from the United States, 9,075 thousand dollars from France, and 3,036,000 dollars from China.

Recently, the consumption of high-functional cosmetics in Southeast Asia including China has rapidly increased, so it is urgent to develop functional raw materials and cosmetics. Localization of raw materials and development of new materials in the functional cosmetic raw materials and cosmetics market is an industrial field that can create enormous high added value.

In the meantime, the development of cosmetic raw materials has been mainly extracted and isolated from terrestrial vegetable resources. The development of raw materials using terrestrial plants has reached the limit of research on new materials as many studies have been conducted for a long time.

In order to solve this problem, 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 development technology for resources using marine animals is very weak.

Recently, functional cosmetics using marine resources, such as seaweeds, which have excellent antioxidant ability, have been developed. In order to supplement this problem, it is necessary to develop a marine resource material market that has a function as a material derived from marine resources. The placenta cosmetics market started gaining popularity in Korea after 2000, and most placental cosmetics are made up of sheep placenta from overseas (New Zealand, Australia, etc.). After 2006, the use of human placenta as a cosmetic ingredient in Korea became impossible, so the commercialization of animal placenta increased.

However, they are reluctant to use animal-derived placenta due to mad cow disease, etc. In Korea, placenta is mainly used, but the price is very high and it is difficult to use it, so there is a very high interest in developing cosmetic raw materials to replace it.

The Thai cosmetics market is the largest market among ASEAN countries and the level of consumption is very high. In addition, as the ASEAN Economic Community comes into effect, the market is expected to expand further in the future. The most popular cosmetic in Thailand is placental cosmetics.

For the past three years, cosmetics using marine organisms have gained great popularity, and it has continued to this day. In particular, cosmetics using marine placenta are rapidly increasing. However, marine placental cosmetics currently sold in the market are products that use plankton, a source of food for marine organisms with a similar placenta origin, or use nutrient-rich raw materials like placenta. The placenta is a raw material that can be obtained from mammals, and it is impossible to obtain it from the sea.

Therefore, the present inventor intends to develop as a cosmetic raw material for Marine placenta using fish eggs, which are rich in nutrients like the placenta of mammals and are a source material for the birth of a life.

The problem to be solved by the present application is to provide a cosmetic composition having antioxidant, skin whitening, skin wrinkle improvement or skin regeneration effects by including an extract of hot smelt roe or Dorumuk roe as an active ingredient.

In order to solve the above problems, one embodiment of the present application includes an extract obtained by hydrolyzing hot smelt eggs or smelt eggs with a proteolytic enzyme as an active ingredient, and the proteolytic enzyme includes Neutrase, , It provides a cosmetic composition, characterized in that for antioxidant, skin wrinkle improvement, or skin elasticity improvement.

In one embodiment of the present application, the extract may be hydrolyzed by a proteolytic enzyme of the hot smelt roe or black smelt roe.

In one embodiment of the present application, the extract may be a product obtained by extracting the hot smelt roe or smelt roe with water, saline, or an organic solvent.

In one embodiment of the present application, the proteolytic enzyme is, papain (Papain), pepsin (Pepsin), trypsin (Trypsin), alpha-chymotrypsin (α-Chymotrypsin), sumizyme (Sumizyme), kojizyme (Kojizyme) ), pancreatin, kallikrein, cathepsin, thermolisin, subtilisin, bromelain, ficin, actinidin ), peptidase (Peptidase) and transglutaminase (Transglutaminase) may be selected from the group consisting of.

In one embodiment of the present application, the active ingredient may be included in an amount of 0.00001 wt% to 10 wt% based on the total weight of the total composition.

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 wrinkles or improving skin elasticity.

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

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

One embodiment of the present application comprises the steps of grinding after mixing hot smelt eggs or smelt eggs with water or saline; After the grinding step, hydrolyzing at 45° C. to 60° C. by adding Neutrase as a proteolytic enzyme; inactivating the proteolytic enzyme by heating; and obtaining an extract through centrifugation or filtration; It provides a method for producing a hot smelt roe or a roe extract containing a smelt.

In one embodiment of the present application, after the grinding step, hydrolyzing by adding a proteolytic enzyme; and heating to inactivate the enzyme; may further include.

In one embodiment of the present application, the protease may be added in an amount of 0.1 wt % to 10 wt % based on the total weight of the grinding material.

In one embodiment of the present application, the hydrolyzing may be performed by stirring at a temperature of 30°C to 60°C for 1 hour to 10 hours.

In one embodiment of the present application, before the grinding step, the steps of removing the organic solvent after removing the fat by immersing the hot smelt eggs or smelt eggs in an organic solvent; may further include.

The extract of hot smelt roe or Dorumuk roe of the present application has the advantage of excellent antioxidant effect due to high DPPH radical and ABTS radical scavenging activity.

In addition, since it effectively inhibits melanin production, it can be usefully used as a functional cosmetic composition for skin whitening, or a composition for external application for skin that can prevent or improve skin pigmentation diseases.

And, it can be usefully used as a functional cosmetic composition for improving wrinkles by effectively promoting collagen synthesis and inhibiting elastase activity, or as a composition for external application for skin that can prevent or improve skin wrinkles.

In addition, since it has excellent skin cell regeneration activity, it can be usefully used as a functional cosmetic for skin cell regeneration or a composition for external application for skin.

1 and 2 show the DPPH radical scavenging ability of the extract according to the embodiment of the present application.
3 and 4 show the ABTS radical scavenging ability of the extract according to an embodiment of the present application.
Figure 5 shows the tyrosinase inhibitory activity of the extract according to the embodiment of the present application.
Figure 6 shows the elastase inhibitory activity of the extract according to the embodiment of the present application.
Figure 7 shows the collagenase inhibitory activity of the extract according to the embodiment of the present application.
Figure 8 shows 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.

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

Throughout the specification, when a part "includes" a certain element, it means that other elements may be further included, rather than excluding other elements, unless otherwise stated.

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 a commonly used dictionary 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 provides a cosmetic composition comprising an extract of hot smelt roe or Dorumuk roe as an active ingredient.

One embodiment of the present application provides a cosmetic composition comprising, as an active ingredient, an extract obtained after grinding a hot smelt roe or smelt roe by mixing water, distilled water, and saline.

One embodiment of the present application provides a cosmetic composition comprising, as an active ingredient, an extract obtained by immersing hot smelt roe or smelt roe in ethanol and then filtration.

One embodiment of the present application provides a cosmetic composition comprising, as an active ingredient, an extract obtained by hydrolyzing hot smelt eggs or smelt eggs with a proteolytic enzyme.

In one embodiment of the present application, the method for preparing the extract comprises the steps of mixing hot smelt eggs or smelt eggs with water or saline, followed by grinding; and obtaining an extract through centrifugation or filtration; may include.

In one embodiment of the present application, the method for preparing the extract comprises the steps of mixing hot smelt eggs or smelt eggs with water or saline, followed by grinding; After the grinding step, hydrolyzing at 45° C. to 60° C. by adding Neutrase as a proteolytic enzyme; inactivating the proteolytic enzyme by heating; and obtaining an extract through centrifugation or filtration; It provides a method for producing a hot smelt roe or a roe extract containing a smelt.

In one embodiment of the present application, the method for preparing the extract comprises: immersing a hot smelt egg or roe deermuk in an organic solvent to remove fat and then removing the organic solvent; grinding after mixing hot smelt eggs or smelt eggs with water or saline; and obtaining an extract through centrifugation or filtration; may include.

The immersion time in the organic solvent may be 3 hours to 10 hours, but is not limited thereto.

The organic solvent may be at least one selected from the group consisting of ethanol, methanol, butanol, ether, ethyl acetate and hexane, but is not limited thereto.

In one embodiment of the present application, the proteolytic enzyme 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 one, more specifically flavozyme (Flavourzyme), pro tamax (Protamex), may be neutrase (Neutrase) or papain (Papain), but is not limited thereto.

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 addition, the hydrolysis temperature may be a temperature at which the proteolytic enzyme has high activity, and may be appropriately adjusted according to the type of the enzyme. For example, the hydrolysis temperature ranges from 45°C to 60°C for Flavozyme, Protamex or Neutrase, 35°C to 40°C for trypsin, and 35°C to 40°C for papain. can be

In the case of hydrolysis time, the reaction may proceed while stirring for 1 hour to 10 hours, more specifically 2 hours to 9 hours.

After performing the hydrolysis, the enzyme may be inactivated by heating at 70° C. to 120° C., specifically, at 80° C. to 100° C. for 1 minute to 20 minutes.

In one embodiment of the present application, the protease may be added in an amount of 0.1 wt % to 10 wt % based on the total weight of the grinding material. When the amount of the proteolytic enzyme is less than 0.1% by weight, the proteolytic effect is insignificant, and when it exceeds 10% by weight, there may be no difference in the effect according to the increase in the content.

In the step of obtaining the extract, impurities may be removed using a conventional filtration method or apparatus, for example, a centrifugation method or filtration using a micro filter to obtain an extract from which impurities are removed. For example, as a usable micro filter, a filter having pores having a size of 0.1 μm to 5 μm may be used. Specifically, the filtration may be performed using a filter having pores having a size of 3 μm. As another example, centrifugation may be performed at 1000 rpm to 5000 rpm for 5 to 60 minutes. Specifically, the precipitate is discarded by centrifugation at 2000 rpm to 4000 rpm for 10 to 20 minutes, and only the supernatant can be collected and used.

In one embodiment of the present application, the active ingredient may be included in an amount of 0.00001 wt% to 10 wt% based on the total weight of the total composition.

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 ingredient stays on the skin for a long period of time, it is okay to include the extract at a lower concentration than that of the wash-off type cosmetics. something to do. 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 1 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 10% by weight, unwanted reactions such as allergies may occur. This is to prevent this from occurring or there may be problems with skin safety.

In one embodiment of the present application, the cosmetic composition may be for antioxidant. In the present application, the term 'antioxidant effect' refers to the oxidation of cells by highly reactive free radicals or reactive oxygen species (ROS) depending on intracellular metabolism or oxidative stress caused by the effect 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 wrinkles or improving skin elasticity. In the present application, the term 'skin wrinkle improvement or skin elasticity improvement effect' refers to increasing skin elasticity, inhibiting or inhibiting loss of skin elasticity, or alleviating already reduced elasticity

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

In the cosmetic composition according to an embodiment of the present application, in addition to the extract as an active ingredient, components 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, astringent lotion, softening lotion, lotion, essence, nutrition gel or 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, methyltaurate, 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 provided only to help the understanding of the present application, and do not limit the scope of the present application.

<Example 1 and Example 2> Preparation of ethanol soaked extract

After adding the same amount of 95% ethanol to the eggs of hot smelt fish ( Mallotus villosus ), immersion for 5 hours, and filtering with Whatman filter No. 1 to prepare an extract (Example 1).

Drummook ( Arctoscopus) japonicus ), an extract (Example 2) was prepared in the same manner as in Example 1.

<Examples 3 and 4> Preparation of ethanol-immersed saline extract

After adding 95% ethanol in the same amount to the eggs of hot-smelt fish, the ethanol was removed after immersion for 5 hours. Thereafter, the same amount of sterilized saline was added, crushed, and filtered through Whatman filter No. 1 to prepare an extract (Example 3).

An extract (Example 4) was prepared from the egg of Dorumuk in the same manner as in Example 3.

<Example 5 and Example 6> Preparation of salt extract

After washing the eggs of the hot-smelt fish with sterile saline, the same amount of sterilized saline was added to the eggs, followed by pulverization, and then filtered through Whatman filter No. 1 to prepare an extract (Example 5).

An extract (Example 6) was prepared from the egg of Dorumuk in the same manner as in Example 5.

<Examples 7 and 8> Preparation of distilled water extract

After washing the eggs of hot smelt with distilled water, distilled water was added in the same amount as the eggs, followed by pulverization, and then filtered through Whatman filter No. 1 to prepare an extract (Example 7).

An extract (Example 8) was prepared from the egg of Dorumuk in the same manner as in Example 7.

<Examples 9 to 12> Preparation of hydrolyzed protein extract of hot smelt roe

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. After that, 1% of the weight of the triturated material was added to flavozyme and hydrolyzed at 55° C. for 3 hours. After that, the enzyme was inactivated by heating for 10 minutes, and then filtered through a Whatman filter to prepare an extract (Example 9).

In Example 9, an extract (Example 10) was prepared by adding Protamex instead of Flavozyme.

In Example 9, an extract (Example 11) was prepared by adding Neutrase instead of Flavozyme.

In Example 9, an extract (Example 12) was prepared by adding papain instead of Flavozyme and setting the hydrolysis temperature to 35°C.

<Example 13 to Example 16> Preparation of hydrolyzed protein extract of smelt roe

After washing the eggs of the hot-smelt fish with sterile distilled water, 95% ethanol was added in the same amount, immersed for 5 hours, and then the ethanol was removed. Thereafter, distilled water was added in the same amount to triturate. After that, 1% of the weight of the triturated material was added to flavozyme and hydrolyzed at 55° C. for 3 hours. After that, the enzyme was inactivated by heating for 10 minutes, and then filtered through a Whatman filter to prepare an extract (Example 13).

In Example 13, an extract (Example 14) was prepared by adding Protamex instead of Flavozyme.

In Example 13, an extract (Example 15) was prepared by adding Neutrase instead of Flavozyme.

In Example 13, an extract (Example 16) was prepared by adding papain instead of Flavozyme and setting the hydrolysis temperature to 35°C.

<Comparative Examples 1 to 3>

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

<Experimental Example 1> Measurement of DPPH radical scavenging ability

The DPPH assay was performed by Yoshida et al. (1989) were measured according to the method used. In a 96-well plate, 100 μl of the solvent was added to the control, and 100 μl of the diluted solution of the extract of Example was added to the test group. Put 100 μl into every well, add 50 μl of DPPH (0.5 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH)/ethanol) solution, mix, and react at room temperature at 25° C. for 30 minutes. Absorbance at 517 nm was measured. Each radical scavenging ability for the solvent control was calculated as a percentage according to Equation 1 below and shown in Table 1 and FIGS. 1 and 2 below. The DPPH radical scavenging ability of Examples 1 to 8 and Example 12 is shown in FIGS. 1 and 2, and Examples 9 to 12 are shown in Table 1.

Referring to Table 1 below, Example 11 using neutrase showed the best DPPH radical scavenging activity, and Example 11 using neutrase was tested at different concentrations. The IC50 of Example 11 was 28.6%. It was confirmed that the placenta extract of Comparative Example 1 had almost no DPPH radical scavenging activity.

Sample name (%) DPPH Radical Scavenging Activity (%)
unprocessed menstruum 81.4 ±2.4 Example 9
(Flavourzyme) 100 20.7 ±11.4 Example 10
(Protamex) 100 79.1 ±0.8 Example 11
(Neutrace) 100 90.0 ±8.7 Example 12
(Papain) 100 89.7 ±1.5 Example 11
(Neutrace) 0.025 9.7 ±0.2 0.05 9.7 ±0.4 0.25 15.4 ±0.4 0.5 18.8 ±0.2 2.5 18.6 ±1.1 5 22.5 ±0.7 Comparative Example 1
(placenta extract) 0.025 4.4 ±1.1 0.05 4.9 ±0.6 0.25 2.2 ±1.2 0.5 4.0 ±1.1 2.5 7.6 ±0.5 5 11.7 ±1.0

<Experimental Example 2> Measurement of ABTS radical scavenging ability

ABTS assay was measured by modifying the method of Re et al. (1999). That is, after mixing 5 mL of 7 mM ABTS and 88 μL of 140 mM potassium persulfate, blocking light at room temperature for 16 hours to form ABTS cations. Thereafter, 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. As a solvent control group, the radical scavenging ability of the control group was calculated as a percentage according to Equation 2 below and shown in Table 2 and FIGS. 3 and 4 below. The ABTS radical scavenging ability of Examples 1 to 8 and Example 12 is shown in FIGS. 3 and 4, and Examples 9 to 12 are shown in Table 2. Referring to FIG. 3, the extracts prepared by different extraction processes all showed high ABTS radical scavenging activity, confirming the applicability as a cosmetic raw material. It was confirmed that this showed a better effect.

In Table 2 below, Example 11 using neutrase exhibited the most excellent ABTS radical scavenging activity, and the concentration of Example 11 using neutrase was varied. As a result, the IC50 for the ABTS radical scavenging activity of the placenta extract of Comparative Example 1 was 3.8%, but the IC50 of Example 11 was 0.2%.

Sample name (%) ABTS Radical Scavenging Activity (%) unprocessed menstruum 41.3 ±0.7 Example 9
(Flavourzyme) 100 77.4 ±1.1 Example 10
(Protamex) 100 84.1 ±0.1 Example 11
(Neutrace) 100 87.5 ±0.7 Example 12
(Papain) 100 73.8 ±0.7 Example 11
(Neutrace) 0.025 25.0 ±0.4 0.05 33.2 ±0.7 0.25 66.9 ±1.0 0.5 81.1 ±0.7 2.5 97.7 ±0.6 5 97.7 ±0.3 Comparative Example 1
(placenta extract) 0.025 8.6 ±1.2 0.05 12.8 ±6.7 0.25 11.2 ±2.7 0.5 16.7 ±1.9 2.5 38.0 ±2.3 5 61.1 ±8.0

<Experimental Example 3> Measurement of B16F0 cytotoxicity

To see the melanin production inhibitory ability of the sample, melanocytes, B16F0, were purchased from KCTC and used. The B16F0 cell line in a cell culture flask was cultured in _{a CO 2} incubator _{controlled at 37 ° C, humidity 95%, and CO 2} 5% with a medium containing 10% FBS in DMEM. (Penicillin streptomycin, Gibco, CA, USA) was used. When about 80% of the cells covered the dish, it was washed with phosphate-buffered saline-EDTA (PBS-EDTA), treated with 0.05% Trypsin EDTA in 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.

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.} Then, samples were treated by concentration (Table 1) and cultured for 72 hours in an incubator controlled at _{95% humidity, 5% CO 2 , and 37°C.} After removing the medium, 9 ml of the medium (DMEM (21063-029, fee phenol red, GIBCO, Grand Island, NY), free FBS) was added to 1 ml of reagent and diluted, and 100 μl of each well was treated. After reaction at 37° C. for 60 minutes, absorbance was measured at 490 nm with a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA). Cell viability is shown in Table 3 by expressing the absorbance of the sample-treated group compared to the non-treated control group.

In Table 3 below, it was confirmed that the extract of Example 11 did not exhibit B16F0 cytotoxicity at a concentration of 0.05 to 1%, but in the case of arbutin, Comparative Example 2, cytotoxicity was confirmed at a concentration of 1%, resulting in a concentration of 0.05 to 0.5%. Arbutin was used for whitening cell experiments.

Sample name (%) Toxicity to B16F0 cells (%) Example 11
(Neutrace) Control 100.0 ±5.7 0.05 109.3 ±11.4 0.1 110.6 ±7.5 0.5 105.7 ±10.5 0.1 102.3 ±4.3 0.2 98.6 ±2.1 0.5 96.3 ±4.4 One 99.8 ±7.8 Comparative Example 2
(Arbutin) Control 100.0 ±4.2 0.05 99.2 ±3.5 0.1 97.7 ±9.1 0.5 103.5 ±8.8 0.1 102.7 ±8.7 0.5 101.8 ±6.9 One 88.3 ±5.9 ^*

*P <0.05, compared with Control group

<Experimental Example 4> Measurement of extracellular melanin content

^{After inoculating 1×10 5} cells per well in a 6-well plate, the cells were cultured for 24 hours under cell culture conditions. The medium was removed, and a new medium containing the test solution and 2% FBS was added and cultured. At this time, the melanin production process was promoted by treating the medium with 100 nM α-MSH. The cell medium was transferred to another container, and the remaining cells were washed with PBS, and then the amount of protein was obtained using cell lysis buffer. The absorbance of the medium was measured at 490 nm, and the amount of melanin was calculated from a calibration curve using synthetic melanin. 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 Table 4 below. In Table 4 below, the melanin production amount similar to that of Comparative Example 2 at the 1% concentration of Example 11 was shown.

Sample name (%) Extracellular melanin production (mg/g protein) Extracellular melanogenesis inhibitory activity (%) Example 11
(Neutrace) (-)Control 15.5 ± 0.8 ^* - (+)Control 104.6 ±9.4 0.0 ±8.9 0.1 68.3 ±7.4 ^* 34.7 ±7.0 0.2 44.1 ±3.1 ^* 57.8 ±3.0 0.5 17.2 ±1.5 ^* 83.6 ±1.4 One 16.3 ±2.3 ^* 84.4 ±2.2 Comparative Example 2
(Arbutin) 0.01 50.0 ±0.5 ^* 52.7 ±0.4 0.1 16.3 ±4.7 ^* 84.6 ±4.4

*P <0.05, compared with (+)Control group

<Experimental Example 5> Measurement of intracellular melanin content

6-well plate to 1 × 10 ⁵ per ^{well (1 × 10 5 cells /} 1.2ml / well) was inoculated to and cultured for 24 hours in cell culture conditions. The medium was removed, and a new medium containing the test solution and 2% FBS was added and incubated. At this time, the melanin production process was promoted by treating the medium with 100 nM α-MSH. After removing the medium, 100 μl of 1N sodium hydroxide solution was added to the pellet, dissolved in a thermostat at 60° C. for 1 hour, and then centrifuged. The absorbance was measured at 490 nm with a microplate reader with the supernatant, and the amount of melanin was obtained from a calibration curve using synthetic melanin. The amount of melanin was converted to 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 Table 5 below. In Table 5 below, the 1% concentration of Example 11 showed a 1.2-fold higher melanin inhibitory activity compared to the 0.1% concentration of Comparative Example 2.

Sample name (%) Intracellular melanin production (mg/g protein) Inhibitory activity of intracellular melanin production (%) Example 11
(Neutrace) (-)Control 92.0 ±2.6 ^* - (+)Control 179.6 ±3.2 0.0 ±1.1 0.1 148.2 ±9.0 ^* 17.5 ±3.3 0.2 118.6 ±11.7 ^* 34.0 ±4.3 0.5 105.5 ±8.6 ^* 41.2 ±3.1 One 70.2 ±7.6 ^* 60.9 ±2.8 Comparative Example 2
(Arbutin) 0.01 151.2 ±4.2 ^* 15.8 ±1.5 0.1 88.5 ±4.3 ^* 50.7 ±1.6

*P <0.05, compared with (+)Control group

< Experimental example 6> Tyrosinase ( Tyrosinase ) measurement of inhibitory activity

Dissolve the sample in ethanol or a suitable solvent, dilute it by setting the concentration range that can confirm the inhibition of tyrosinase activity, but process it so that it becomes at least 5 concentrations, and put 220 μL of 0.1 M phosphate buffer (pH 6.5) in a test tube, sample solution 20 µL and 20 µL of mushroom tyrosinase solution (1500 U/mL to 2000 U/mL) (or human tyrosinase) were added in this order. 40 μL of 1.5 mM tyrosine solution was added to this solution, reacted at 37 °C for 10 to 15 minutes, and absorbance was measured at 490 nm. The sample concentration (IC50) when the activity inhibition rate was 50% was calculated using an appropriate program, and the sample solution was corrected by using the solvent as the blank sample solution, and the results are shown in FIG. 5 . In FIG. 5, it was confirmed that Examples 5 and 8 showed superior tyrosinase inhibitory activity than the 5% concentration of arbutin of Comparative Example.

<Experimental Example 7> Elastase production inhibitory activity measurement

Elastase inhibitory activity was used by purchasing Invitrogen's EnzChek® Elastase Assay Kit (600 assays) reagent. First, 0.5 ml 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, 0.5 ml of 1 x reaction buffer was added to the elastase vial to prepare 100 U/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 processed in triplicate by 50 μl in a 96-well plate. After adding the sample, 100 μl of the elastase enzyme was treated with control and test samples, and DW was treated with the remaining control blank and sample blank. After mixing well, 50 μl of DQ elastin was added to all wells. After reacting for 1 hour at room temperature with the light blocked, measure the fluorescence intensity with an ELISA plate reader (VICTOR X3™, PerkinElmer, US) at the intensity excitation wavelength 485 nm and emission wavelength 535 nm, and calculate according to Equation 3 below. The results are shown in FIG. 6 and Table 6 below.

In FIG. 6 , in the case of Examples 5 and 8, it was confirmed that the elastase inhibitory activity increased as the concentration increased.

In Table 6 below, the IC50 in Example 11 was 18.6%, and the IC50 in Comparative Example 1 was 41.3%. Comparative Example 1 was about 1.9 times less effective than Example 11 at the same concentration.

Sample name (%) Elastase Inhibitory (%) Example 11
(Neutrace) 1.25 10.3 ±1.1 2.5 18.8 ±0.9 12.5 41.9 ±0.5 25 60.8 ±0.9 Comparative Example 1
(placenta extract) 1.25 13.7 ±7.7 2.5 15.6 ±5.4 12.5 28.7 ±6.2 25 32.6 ±6.5

< Experimental example 8> fibroblasts (CCD- 986sk ) to measure the cytotoxicity of

DMEM (Dulbecco's Modified Eagle's Medium) containing penicillin (100 IU/mL), streptomycin (100 μg/mL), and 10% FBS (fetal bovine serum) after inoculation of fibroblasts (CCD-986sk) on the bottom of a culture dish ) medium or a medium having the same or higher growth potential, maintained at 37°C, and cultured in an incubator containing 5% carbon dioxide.

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.} After that, samples were treated by concentration and incubated for 24 hours in an incubator controlled at _{95% humidity, 5% CO 2 , and 37°C.} After removing the medium, 9 ml of medium (DMEM, free FBS) was added to 1 ml of reagent, diluted, and 100 μl of each well was treated. After reaction at 37° C. for 60 minutes, absorbance was measured at 490 nm with a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA), and procollagen-producing activity was confirmed at a concentration without cytotoxicity.

<Experimental Example 9> Measurement of procollagen production activity in fibroblasts

Procollagen type I peptide EIA kit (Takara Biomedical Co.) reagent was used. 100 μl of the antibody-PoD conjugate solution was put into the well, then 20 μl of a 1/5 diluted culture solution and standard solution were added, incubated at 37° C. for 3 hours, the culture solution was removed from the well, and then washed 4 times with 400 μl of phosphate buffer (PBS). . 100 μl of the color reagent was added, incubated at room temperature for 15 minutes, 100 μl of 1 N sulfuric acid was added, and then measured with an ELISA reader at 450 nm. Collagen standards were dissolved in water, diluted to 0,10, 20, 40, 80, 160, 320, and 640 ng/mL, respectively, and a calibration curve was prepared, and the results are shown in Table 7 below. The 1% concentration of Example 11 exhibited a procollagen generating activity 2.5 times higher than 1% of Comparative Example 1, and the 1% concentration of Example 11 was 2 times higher than 1% of Comparative Example 1 procollagen production activity.

Sample name (%) Procollagen Contents (pg/g protein) Procollagen Contents
(%) Control 26.2 ±3.1 100.0 ±11.8 Example 11
(Neutrace) 0.05 96.0 ±3.6 ^* 366.2 ±13.8 ^* 0.1 147.7 ±5.2 ^* 563.2 ±19.8 ^* 0.5 153.1 ±0.8 ^* 584.0 ±3.1 ^* One 192.7 ±1.5 ^* 734.7 ±5.6 ^* Comparative Example 1
(placenta extract) 0.1 65.5 ±7.0 ^* 249.7 ±26.5 ^* One 75.6 ±4.5 ^* 288.4 ±17.1 ^*

*P <0.05, compared with (+)Control group

< Experimental example 10> collagenase Inhibitory activity measurement

Collagenase inhibitory activity was used by purchasing Invitrogen's EnzChek® Gelatinase/Collagenase Assay Kit (250-2000 Assays) reagent. First, 1.0 ml of water was added to a 1 mg DQ collagen vial to prepare a DQ collagen stock solution (1 mg/ml). Reaction buffer was diluted by adding 18 ml of water to 2 ml of 10xreaction buffer. Next, a collagenase enzyme reagent was prepared. The working solution was diluted with reaction buffer to a final concentration of 0.2 U/ml. Prepare the sample in triplicate by 50 μl in 96 well plate. Add 20 μl of DQ collagen and 30 μl of reaction buffer to the sample blank, and 30 μl of working solution to the sample, and leave it at room temperature for 1 to 2 hours in a state of blocking light. Then, the fluorescence intensity excitation wavelength 485 nm and emission wavelength 535 The fluorescence intensity was measured at nm with an ELISA plate reader (VICTOR X3™, PerkinElmer, US), and is shown in FIG. 7 and Table 8 below. 7, it was confirmed that the collagenase inhibitory activity was higher than that of adenosine at the same concentration in Examples 5 and 8. In Table 8 below, it was confirmed that Example 15 was the highest.

Sample name (%) Collagenase inhibitory activity (%) unprocessed menstruum 0.6 ±1.0 Example 13
(Flavourzyme) 100 15.0 ±0.9 Example 14
(Protamex) 100 29.5 ±0.8 Example 15
(Neutrace) 100 31.0 ±1.0 Example 16
(Papain) 100 18.1 ±1.6

< Experimental example 11> Human origin of dermal keratinocytes (HaCaT) Cytotoxicity measurement

Cell toxicity was measured using HaCaT cells, which are human-derived epidermal cells. That is, the HaCaT cell line cultured in a cell culture flask was passaged by removing the cells from the flask with a 10 ml pipette, and the cells were cultured in DMEM/LOW medium containing 10% FBS. 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.} After that, samples were treated by concentration (Table 1) and cultured for 24 hours in an incubator controlled at _{95% humidity, 5% CO 2 , and 37°C.} After removing the medium, 9 ml of medium (DMEM, free FBS) was added to 1 ml of reagent, diluted, and 100 μl of each well was treated. After reaction at 37° C. for 60 minutes, absorbance was measured at 490 nm with a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA), and regeneration activity was confirmed at a concentration without cytotoxicity.

<Experimental Example 12> Measurement of regenerative activity of human-derived keratinocytes (HaCaT)

The HaCaT cell line was dispensed in a 96 well plate at a concentration of ^{2×10 3} cells/ml, and cultured in a CO _{2 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. In order to measure the viability of the cell line, the cell counting kit-8 (CCK-8, Dojindo Mol. Tech., Rockville., MD, USA) was used at 450 nm with a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA). Absorbance was measured. The regenerative efficacy of cells was expressed as the absorbance of the sample-treated group compared to the non-treated control group, and is shown in FIG. 8 and Tables 9 and 10 below. In FIG. 8, it was confirmed that Examples 5 and 8 showed similar or higher regenerative activity compared to allantoin of Comparative Example 3.

In Table 9 below, it was confirmed that the 0.5% concentration of Example 11 on the 3rd day of proliferative activity showed about 1.2 times higher regenerative activity compared to 1 μg/ml allantoin.

sample Skin cell (HaCaT) regeneration activity (%) Day 1 Day 2 Day 3 Control 100.0 ±4.5 Example 11
(Neutrace)
density(%) 0.01 99.5 ±7.7 99.2 ±4.4 119.0 ±4.7 ^* 0.05 98.6 ±7.3 104.4 ±5.2 121.9 ±4.9 ^* 0.1 92.7 ±2.8 102.6 ±5.1 126.7 ±3.6 ^* 0.2 94.0 ±3.2 106.2 ±9.9 131.1 ±6.6 ^* 0.5 98.7 ±5.1 111.1 ±9.7 144.5 ±11.1 ^* Comparative Example 3
(Allantoin)
(μg/ml) 0.1 97.1 ±5.8 104.2 ±8.1 119.9 ±6.8 ^* One 98.4 ±7.9 115.1 ±7.1 ^* 121.1 ±7.6 ^*

* p <0.05, compared with Control group

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

< Formulation example 1> Soft lotion ( skin lotion )

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

ingredient content (wt%) Extract of Example 11 0.25 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%) Extract of Example 11 0.25 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%) Extract of Example 11 0.25 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

Above, the present application has been described in detail with reference to Examples and Experimental Examples, but the present application is not limited by the Examples and Experimental Examples, and those of ordinary skill in the art within the technical spirit and scope of the present application Various modifications and changes are possible by

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (13)

Contains as an active ingredient an extract obtained by hydrolyzing hot smelt eggs or smelt eggs with proteolytic enzymes,
The proteolytic enzyme includes Neutrase,
A cosmetic composition, characterized in that it is used for antioxidant, skin wrinkle improvement, or skin elasticity improvement. delete The method according to claim 1,
The extract is
A cosmetic composition, characterized in that the hot smelt eggs or smelt eggs are extracted with water, saline or an organic solvent. The method according to claim 1,
The proteolytic enzyme is
Papain, Pepsin, Trypsin, Alpha-Chymotrypsin, Sumizyme, Kojizyme, Pancreatin, Kallikrein, Kallikrein Cathepsin, Thermolisin, Subtilisin, Bromelain, Ficin, Actinidin, Peptidase and Transglutaminase ( Transglutaminase) cosmetic composition further comprising one selected from the group consisting of. The method according to claim 1,
The extract is a cosmetic composition, characterized in that it is included in an amount of 0.00001 wt% to 10 wt% based on the total weight of the composition. delete delete The method according to claim 1,
The cosmetic composition is a cosmetic composition, characterized in that for skin whitening. The method according to claim 1,
The cosmetic composition is a cosmetic composition, characterized in that for skin cell regeneration. grinding after mixing hot smelt eggs or smelt eggs with water or saline;
After the grinding step, hydrolyzing at 45° C. to 60° C. by adding Neutrase as a proteolytic enzyme;
inactivating the proteolytic enzyme by heating; and
obtaining an extract through centrifugation or filtration;
A method for producing a hot smelt roe or roe extract containing a smelt. delete 11. The method of claim 10,
The method for producing a hot smelt roe or Dorumuk roe extract, characterized in that the proteolytic enzyme is added in an amount of 0.1% to 10% by weight based on the total weight of the crushed product. 11. The method of claim 10,
Prior to the grinding step,
Removing the organic solvent after removing the fat by immersing the hot smelt roe or the roe deermuk roe in an organic solvent; A method for producing a hot smelt roe or roe extract of Dorumuk further comprising a.

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