KR102484277B1 - Manufacturing Method Of Raw Material Composition For Improving Skin Beauty - Google Patents

Abstract

The present invention relates to a manufacturing method of a raw material composition for skin care and improvement, and more specifically, to a manufacturing method of a raw material composition which contains functional peptides improving skin elasticity and skin whitening, reducing skin wrinkles, and the like, and which can be ingested or externally used by adding the composition to food or cosmetics. The manufacturing method of a raw material composition for skin care and improvement uses animal raw materials containing elastin.

Description

Manufacturing method of raw material composition for improving skin beauty {Manufacturing Method Of Raw Material Composition For Improving Skin Beauty}

The present invention relates to a method for producing a raw material composition for improving skin beauty, and more particularly, as a raw material composition containing a functional peptide that improves elasticity, whitening, wrinkles, etc., which can be ingested or externally applied by adding to food, cosmetics, etc. It relates to a method for producing a raw material composition for improving skin beauty.

Skin is composed of epidermis, dermis, and subcutaneous tissue, of which the dermis is composed of elastin, collagen, and hyaluronic acid. The dermis forms the elasticity of the skin with collagen, elastin connecting collagen, and hyaluronic acid filling the gap. Wrinkles and sagging of the skin occur because elastin and collagen fibers are damaged. When elastin fibers are cut due to external factors such as active oxygen caused by stress or smoking or ultraviolet rays, or when defective elastin that is too thick or too thin is stretched, the balance of the fiber structure is disturbed. As a result, wrinkles are formed due to loss of elasticity, and the ability of skin and fat to adhere to muscles and bones is also reduced, resulting in skin and fat sagging.

Elastin is produced in fibroblasts of the dermis and has the property of elasticity, and is present in large amounts in tissues that require elasticity and elasticity in the body, such as the dermis of the skin, blood vessels, and ligaments. In the case of human tissues, about 80% of ligaments and about 50% of arteries %, about 20% of the lungs, and about 5% of the dermis. Due to the elasticity and fibrous structure of elastin, elastin is known to have effects such as elasticity of skin and ligaments, prevention of arteriosclerosis (improvement of blood flow), and inhibition of platelet aggregation, and the concentration of soluble elastin in the body is also used as an indicator of arteriosclerosis. . In particular, since collagen is a substance involved in the strength of tissues, its elasticity is insignificant, and glycoproteins such as hyaluronic acid do not have properties such as elasticity. , the elasticity of ligaments, and the contractility of lungs and blood vessels cannot be made without elastin.

Elastin is not much at the age of 0, peaks around the age of 25, gradually decreases with age, and rapidly decreases after the age of 40. On the other hand, hyaluronic acid is the highest at the age of 0, gradually decreases with age, and at the age of 20, it is about half of what it was at birth. In fact, it is known that the skin is soft around the age of 0, which is rich in hyaluronic acid instead of low in elastin, and the skin is most elastic in the late 20s. Therefore, the existence of elastin as well as hyaluronic acid is very important for skin elasticity and its maintenance.

Elastin is contained in a lot of bovine aorta, tendon, cartilage, chicken skin, fish, etc. Even if you frequently consume foods using these materials, in the case of crosslinked elastin, it does not dissolve well in solvents such as water, so the absorption rate in the body decreases. Since it is difficult, such as processing at 120 ° C or higher for more than 16 hours, it is efficient to supplement elastin synthesis in the body by ingesting elastin decomposition products through supplements. According to a study conducted by Nippon Ham Central Research Institute, 13 healthy adult males and females were given a bottle of elastin-containing health drink daily for 8 weeks, and then 4 weeks after and 8 weeks after drinking the drink, respectively. , When the elasticity of the cheek area (elasticity rate at which the skin returns to its original state) was measured using a measuring instrument over a total of 3 times, when eating foods with elastin added, cells that synthesize elastin (fibroblasts) are activated or , it was confirmed that the production of collagen increased.

Elastin supplements appeared around 2003, and were made from arterioles of tuna, skipjack tuna, yellowtail, etc. or blood vessels of pigs. Since the amount of elastin that can be extracted from these materials is very small, most of the nutrients are collagen and hyaluronic acid. , Plagenta (placenta), and chondroitin sulfate were often mixed together.

Therefore, there is a need for an elastin degrading composition that increases the content of elastin by maximally extracting elastin from raw materials such as bonito, while appropriately decomposing the extracted elastin to increase the absorption rate in the body and activate the synthesis of elastin in the body so that various benefits of elastin can be easily obtained. .

The present invention is to solve these problems, and proposes a method for producing a raw material composition containing a peptide capable of increasing the extraction amount of the elastin-derived raw material composition from animal raw materials and improving skin health such as skin elasticity, moisture content and whitening. want to do

According to one aspect of the present invention for solving the above problems, the first step of cutting the animal raw material containing elastin;

A second step of removing proteins other than elastin by adding an alkaline solution to the cut animal raw material;

a third step of neutralizing the alkaline solution by adding an acidic solution and washing with water to obtain insoluble materials including elastin;

A fourth step of obtaining an elastin degradation product by decomposing the insoluble matter for each enzyme using two or more proteolytic enzymes (proteases); and

A fifth step of degreasing and deodorizing by adding and stirring activated carbon dissolved in water to the elastin degradation product;

In the fourth step, 0.5 to 0.7% of the weight of the insoluble matter obtained in the third step is added with water to decompose at 65 to 80 ° C. and pH 6.5 to 8.5 for 120 to 180 minutes. Step 4-1;

1.3 to 1.7% of papain by weight of the insoluble matter obtained in the third step is added to the decomposition product obtained in the step 4-1 to decompose at 75 to 85 ° C. and pH 6.5 to 7.5 for 120 to 180 minutes Step 4-2; and

A method for producing a raw material composition for improving skin beauty is provided, comprising a 4-3 step of inactivating the thermoase and the papain by heating at 90 to 95 ° C. for 25 to 35 minutes.

Preferably, the second step includes adding 0.1 mol/L NaOH of 3 times the weight of the animal raw material and stirring at 80 to 90 ° C. for 40 to 60 minutes,

The third step is provided with a method for producing a raw material composition for improving skin care, including neutralizing by stirring at 80 to 90 ° C. for 25 to 35 minutes using citric acid.

Preferably, the fifth step is provided with a method for producing a raw material composition for improving skin care, characterized in that stirring at 90 to 95 ° C. for 25 to 35 minutes using two or more activated carbons.

Preferably, the animal raw material is provided with a method for producing a raw material composition for improving skin care, characterized in that the arterioles of bonito, tuna, barfish, cod, yellowtail or salmon.

Preferably, a sixth step of first filtering using diatomaceous earth, second filtering using a mesh, and then concentrating 4 to 10 times using an evaporator; and

A seventh step of pasteurizing at 80 to 85 ° C. for 25 to 35 minutes and drying at 80 to 150 ° C.

Preferably, the raw material composition prepared according to the above manufacturing method contains the peptide of SEQ ID NO: 1 as an essential component,

There is provided a method for producing a raw material composition for improving skin care, characterized in that it further comprises one or more peptides selected from the group of peptides of SEQ ID NOS: 2 to 10.

SEQ ID NO: 1 - (valine-glycine-proline-glycine)

SEQ ID NO: 2 - (valine-glycine-proline-glycine-glycine)

SEQ ID NO: 3-(Phenylalanine-glycine-proline-glycine-glycine-alanine-glycine)

SEQ ID NO: 4-(Threonine-glycine-glycine-proline-glycine)

SEQ ID NO: 5-(glycine-valine-glycine-glycine-proline-glycine)

SEQ ID NO: 6-(Phenylalanine-glycine-glycine-proline)

SEQ ID NO: 7-(valine-glycine-proline-glycine-leucine)

SEQ ID NO: 8- (valine-glycine-proline-glycine-phenylalanine)

SEQ ID NO: 9-(Phenylalanine-glycine-proline-glycine-leucine)

SEQ ID NO: 10-(Phenylalanine-glycine-proline-glycine-phenylalanine)

Preferably, the peptide of SEQ ID NO: 1 is provided in a method for producing a raw material composition for improving skin care, characterized in that it is included in 0.5 to 5% by weight.

Preferably, the raw material composition comprises the peptide of SEQ ID NO: 2,

The SEQ ID NO: 2 peptide is provided with a method for producing a raw material composition for improving skin care, characterized in that it is included in 0.1 to 1.5% by weight.

Preferably, the raw material composition comprises the peptide of SEQ ID NO: 3,

The content of the SEQ ID NO: 3 peptide is provided with a method for producing a raw material composition for improving skin care, characterized in that it is included in 0.01 to 1.2% by weight.

The manufacturing method of the raw material composition according to the present invention decomposes elastin obtained from animal raw materials to a level below the oligopeptide to increase absorption in the body, thereby activating the synthesis of elastin in the body.

In addition, when elastin obtained from animal raw materials is treated by the method for preparing a raw material composition according to the present invention, the content of elastin-derived peptides in the raw material composition is high. In particular, the raw material composition prepared according to the present invention has a high content of peptides having an effect of improving skin elasticity, moisture, and dullness.

According to the preparation method of the raw material composition according to the present invention, 7.5% to 13% by weight of animal raw materials, preferably 8% to 11% by weight of elastin-derived peptides can be obtained.

In addition, the raw material composition prepared according to the present invention contains peptides of a specific sequence in a constant composition ratio, and thus has an effect of improving skin elasticity, moisture, and dullness.

Figure 1a shows the cell viability of each concentration of the raw material composition for HaCaT cells, which are keratinocytes.
Figure 1b shows the cell viability of HS27 human fibroblasts at different concentrations of the raw material composition.
Figure 1c shows the cell viability by concentration of the raw material composition for B16F10 melanocytes.
Figure 2 shows the results of measuring the gene expression of UGTrel7, GlcNAc, and Elastin, which are skin moisturizing related factors, in HaCaT keratinocytes irradiated with ultraviolet rays.
Figure 3 is the result of measuring the gene expression of LCB1 (SPT) and DEGS1, which are skin moisturizing related factors, in HaCaT keratinocytes irradiated with ultraviolet rays.
4 shows the results of measuring the protein expression of HAS2 and CerS4 (LASS4), which are skin moisturizing related factors, in HaCaT keratinocytes irradiated with ultraviolet rays.
Figure 5 is the result of measuring the amount of hyaluronic acid and sphingomyelin secretion in HaCaT keratinocytes irradiated with ultraviolet rays.
6 is a result of measuring the transepidermal water content measured in the back tissue of a mouse whose skin moisture was reduced by irradiating UVB.
7 is a result of measuring the gene expression of UGTrel8, GlcNAc, and Fibrillin-1, which are skin moisturizing related factors, in the skin tissues of the experimental animals irradiated with ultraviolet rays.
8 is a result of measuring the gene expression of LCB1 (SPT) and DEGS1, which are skin moisturizing related factors, in the skin tissues of the experimental animals irradiated with ultraviolet rays.
9 is a result of measuring the protein expression of HAS2 and CerS4 (LASS4), which are skin moisturizing related factors, in the skin tissues of the experimental animals irradiated with ultraviolet rays.
10 shows the results of measuring gene expression of wrinkle formation-related factors TGFβR1, procollagen type I, and collagen type I in HS27 human fibroblasts irradiated with ultraviolet rays.
11 and 12 show the results of measuring the protein expression of wrinkle formation-related factors MAPK8 (JNK), c-Fos, c-Jun, MMP's (1, 3, 9), and Smad3 in HS27 human fibroblasts irradiated with ultraviolet rays.
13 is a result of measuring the secretion of inflammatory cytokines (IL-1β, IL-6, TNF-α) in HS27 human fibroblasts irradiated with ultraviolet rays.
14 shows the results of morphological observation of back skin tissues after irradiating ultraviolet rays to the skin of experimental animals.
15 shows histopathological changes observed through H&E staining in the back skin tissue after irradiating the skin of the experimental animal with ultraviolet rays.
16 is a result of measuring the gene expression of TGFβR1, procollagen type I, and collagen type I, which are wrinkle formation-related factors, in skin tissue of the back of experimental animals irradiated with ultraviolet rays.
17 and 18 are the results of measuring the protein expression of wrinkle formation-related factors MAPK8 (JNK), c-Fos, c-Jun, MMP's (1, 3, 9), and Smad3 in the skin tissue of the back of experimental animals irradiated with ultraviolet rays. .
19 shows the results of measuring the activities of SOD, Catalase, and GPx, which are antioxidant enzymes, in the back skin tissues of experimental animals irradiated with ultraviolet rays.
20 is a result of measuring melanin production inhibitory ability in B16F10 melanocytes differentiated into IBMX.
21 and 22 show the results of measuring the protein expression of whitening-related factors PKA, CREB, MITF, TRP-1 and TRP-2 in B16F10 melanocytes differentiated into IBMX.
23 is a result of measuring tyrosinase activity in B16F10 melanocytes differentiated with IBMX.
24 shows the measurement results of Nitric oxide (NO), Glutathione (GSH), and cAMP levels in B16F10 melanocytes differentiated with IBMX.
25 and 26 show the results of measuring the protein expression of PKA, CREB, MITF, TRP-1, and TRP-2, which are whitening-related factors, in skin tissues of the backs of experimental animals irradiated with ultraviolet rays.
27 is a result of measuring tyrosinase activity in the back skin tissue of an experimental animal irradiated with ultraviolet rays.
28 is a measurement result of nitric oxide (NO) level and cAMP level in the skin tissue of the back of the experimental animal irradiated with ultraviolet rays.
29 shows the synthesis process of the SEQ ID NO: 1 peptide.
30 shows the process of purifying and drying the synthesized SEQ ID NO: 1 peptide.

Hereinafter, the present invention will be described in more detail. Those skilled in the art of the present invention or similar fields are omitted from the description of the present specification that can be sufficiently recognized and inferred.

<Raw material composition containing peptide>

In one aspect of the present invention, a raw material composition for improving skin beauty is provided, comprising the peptide of SEQ ID NO: 1 as an essential component and further comprising one or more peptides selected from the group of peptides of SEQ ID NOS: 2 to 10.

SEQ ID NO: 1 - (valine-glycine-proline-glycine)

SEQ ID NO: 2 - (valine-glycine-proline-glycine-glycine)

SEQ ID NO: 3-(Phenylalanine-glycine-proline-glycine-glycine-alanine-glycine)

SEQ ID NO: 4-(Threonine-glycine-glycine-proline-glycine)

SEQ ID NO: 5-(glycine-valine-glycine-glycine-proline-glycine)

SEQ ID NO: 6-(Phenylalanine-glycine-glycine-proline)

SEQ ID NO: 7-(valine-glycine-proline-glycine-leucine)

SEQ ID NO: 8- (valine-glycine-proline-glycine-phenylalanine)

SEQ ID NO: 9-(Phenylalanine-glycine-proline-glycine-leucine)

SEQ ID NO: 10-(Phenylalanine-glycine-proline-glycine-phenylalanine)

Preferably, the raw material composition for improving skin beauty may include the peptides of SEQ ID NO: 2 and SEQ ID NO: 3.

Preferably, the raw material composition for improving skin beauty may include 0.5 to 5% by weight of the peptide of SEQ ID NO: 1, more preferably, 1 to 3% by weight. In addition, when the peptide of SEQ ID NO: 2 is further included, the peptide of SEQ ID NO: 2 may be included in an amount of 0.1 to 1.5% by weight, and when the peptide of SEQ ID NO: 3 is further included, the peptide of SEQ ID NO: 3 is 0.01 to 1.2 wt%. It can be included in an amount of %.

The raw material composition of the present invention can preserve, restore, and strengthen skin or tissue, and specifically, improve skin or tissue moisturization, suppress wrinkles to improve elasticity or wrinkles, and alleviate pigmentation caused by ultraviolet light or the like. This can help with whitening. Containing 1 to 3% by weight of the peptide of SEQ ID NO: 1 plays a large role in reducing skin elasticity, improving dryness and whitening, and preferably further includes the peptide of SEQ ID NO: 3 in an amount of 0.01 to 1.2% by weight. Doing so further enhances the effect, and more preferably, further including the SEQ ID NO: 2 peptide in an amount of 0.1 to 1.5% by weight can further enhance the effect.

The raw material composition of the present invention may be in the form of a liquid solution, suspension, solid material or powder.

The raw material composition of the present invention may be included in food or cosmetics. Being included in foods or cosmetics includes all substances included as active ingredients or additives in foods or cosmetics. When included in cosmetics, it includes those added together with various main agents, auxiliary agents, and other ingredients commonly used in cosmetic raw materials, as long as the purpose and effect of the present invention are not impaired, if necessary. Other components are not particularly limited as long as they do not interfere with the skin's elasticity, moisture content, and whitening-improving action of the present invention. oils, fatty acids, alcohols, esters, surfactants, fragrances and the like. When these components are used in combination with the raw material composition, they act synergistically, and sometimes bring about an action and effect that is superior to what is usually expected.

When the raw material composition of the present invention is included in food in the form of a solid or powder, the amount of the raw material composition may be 25 mg/day to 500 mg/day. Preferably it is 25 mg/day to 300 mg/day, more preferably 25 mg/day to 170 mg/day. In the above numerical range, the reduction in skin elasticity, dryness, and whitening improvement effect are excellent, and the preparation of the food is easy and the intake amount is appropriate.

When the raw material composition of the present invention is used as food or cosmetic, containing 0.5 to 5% by weight of the SEQ ID NO: 1 peptide with respect to the total weight of the raw material composition plays a large role in reducing skin elasticity, improving dryness and whitening, , preferably 1 to 3% by weight, more preferably, further comprising the peptide of SEQ ID NO: 3 in an amount of 0.01 to 1.2% by weight further enhances the effect, and more preferably, SEQ ID NO: 2 The effect may be further enhanced by further including the peptide in an amount of 0.1 to 1.5% by weight.

When the raw material composition for improving skin beauty of the present invention is added to food and used, it is preferable to add it in an ingestible concentration as described above.

The food refers to food that is less likely to harm human health and is consumed by oral or digestive tract administration in normal social life, and is not limited to food, pharmaceuticals, quasi-drugs, etc., for example , orally ingested general foods, health foods, health functional foods, quasi-drugs, and pharmaceuticals. In addition, the food may be formulated so as not to interfere with the activity of the raw material composition, or may be a nutritional supplement food containing the raw material composition as a main component.

When the raw material composition of the present invention is contained in food, examples of the food include beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, and acidic drinks; Frozen confectionery such as ice cream and shaved ice; noodles such as udon, somen, Chinese noodles, and instant noodles; pudding, candy, candy, gum, chocolate, frozen confectionery, snack confectionery, biscuits, jelly, jam, cream, baked goods, and bread Confectionery products such as crab, salmon, clams, tuna, sardine, shrimp, bonito, mackerel, whale, saury, squid, scallops, abalone, sea urchin; processed fish cakes, ham, sausage, etc.; processed oils , Dairy products such as fermented milk; Salad oil, frying oil, margarine, mayonnaise, shortening, whipped cream, dressing, etc. , beef rice bowl, meat sauce, egg soup, omurice, dumplings, steamed dumplings, hamburgers, meatballs, and other retort pouch foods; various types of health foods or nutritional supplements such as vitamin complexes; tablets, caps There are medicines, quasi-drugs, etc., such as a pill, a drink, and a troche, but are not limited thereto. In addition, it may include auxiliary raw materials, additives, etc. commonly used when manufacturing the food, and is not particularly limited and may be appropriately selected according to the purpose, but, for example, glucose, fructose, sucrose, maltose ), sorbitol, stevioside, rubusoside, corn syrup, lactose, citric acid, tartaric acid, lingoic acid, succinic acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin Fatty acid esters, polyglycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, gum arabic, carrageenan, casein, gelatin, pectin, agar, B vitamins, nicotinic acid amide, calcium pantothenate, amino acids, calcium salts, pigments, flavors, A storage agent etc. are mentioned. For example, when the raw material composition for improving skin beauty is added to beverages, it is prepared by a general manufacturing method of the beverage, but the raw material composition for improving skin beauty is in the form of a liquid solution, suspension, solid or powder, and other additives. etc. may be added in the same way.

The addition amount of the raw material composition of the present invention in the above food varies depending on the type of target food and cannot be generally defined, but it is good to add within a range that does not impair the original taste of the food, and for various foods, 0.001 to 50% by mass is preferred, and 0.01 to 20% by mass is more preferred. Further, in the case of food in the form of granules, tablets or capsules, it is preferably 0.01% by mass to 100% by mass, and more preferably 5% by mass to 100% by mass.

When the raw material composition of the present invention is included in cosmetics, examples of the cosmetics include liquid solutions, suspensions, powders, emulsions, lotions, sprays, aerosols, ointments, creams and foams, packs, jellies, lip creams, lipsticks, bath additives, astringents, and the like, but are not limited thereto. For example, when the raw material composition for improving skin beauty is added to a cream, it is prepared by a general manufacturing method of the cream, but the amount of the cream in the entire cream can be appropriately adjusted depending on the type of cosmetic raw material or the physiological activity of the extract, , Preferably it can be blended at 0.0001 to 10% by mass, more preferably at 0.001 to 1% by mass. The raw material composition for improving skin beauty may be added in the same manner as other additives in the form of a liquid solution, suspension, solid material or powder.

In addition to the above peptide group, the raw material composition of the present invention is in the group containing vitamin A, vitamin K, vitamin E, minoxidil, estradiol, collagen, hyaluronic acid, antioxidants, food additives, excipients and additives. One or more selected ones may be further included.

Vitamin A, vitamin K, minoxidil and estradiol help to increase the synthesis of elastin in the body, and collagen and hyaluronic acid are substances that make up the skin dermis, so synergistic effects are expected when taken together. In order to reduce active oxygen, which is known to have a great effect on the reduction of elastin in the body, the efficacy is increased when used with antioxidants, especially vitamin E, known as a natural antioxidant.

The peptide used in the raw material composition of the present invention may be chemically synthesized or extracted from the arterioles of animals or fish. In the present invention, the extraction method and the method for synthesizing the peptide of SEQ ID NO: 1 are described.

<Method for producing raw material composition>

The raw material composition of the present invention described above can be obtained by screening and processing animal tissues (animal raw materials), specifically, arteries of fish such as bonito, tuna, barlin, cod, yellowtail, salmon, as well as mammals such as pigs and cattle. It can be obtained from the bulb, preferably from the artery bulb of skipjack tuna. Considering the large amount of water available, the weight of arterioles, and the content of elastin contained in arterioles, it is preferable to isolate arterioles from the heart of skipjack tuna (Katsuwonus pelamis) and enzymatically treat them. In addition, the raw material composition of the present invention may be prepared by synthesizing and processing each peptide included in the raw material composition.

1. Method of extraction from animal raw materials

Hereinafter, a method for preparing the raw material composition of the present invention using an animal raw material (eg, pig skin tissue or skipjack tuna artery bulb) will be described. The present invention is not limited to the following production methods, and conventionally known methods can be used.

(1) Animal raw material preparation and mincing

An animal raw material containing elastin is prepared and finely cut using a meat chopper or the like at room temperature.

(2) Alkaline treatment, neutralization and cleaning

1) Add an alkaline solution to the cut animal raw materials, and stir at 80 to 90 ° C for 40 to 60 minutes. Specifically, 0.1 mol/L NaOH of 3 times the weight of the animal raw material may be added. Through this alkaline treatment, unnecessary proteins such as collagen included in animal raw materials, except for elastin, can be removed.

2) Neutralize by adding an acidic solution in the same mole number as the alkaline solution. If strong acid is used, there is a concern that elastin included in animal raw materials may be degraded. For example, citric acid can be used, and since it can be consumed, it is safe when manufactured as food or cosmetics. It may vary depending on the weak acid added, but in the case of citric acid, it is neutralized by stirring at 80 to 90 ° C. for 25 to 35 minutes.

3) After neutralization, about 2 times the weight of the raw artery bulb is added and washed for about 25 to 35 minutes at room temperature. Washing is preferably carried out twice or more.

After washing, insoluble matter including elastin is obtained, and has a weight of 50 to 70% of the weight of the raw artery bulb.

(3) enzyme treatment

By using two or more proteolytic enzymes (proteases), the insoluble matter is decomposed by each enzyme to obtain elastin degradation products including various polypeptides. Specifically, as shown below, when Thermoase (eg, Thermoase C160) and papain (eg, Papain W-40) are added according to the weight of the insoluble matter, and the enzymes are separately treated and subjected to a two-step decomposition reaction, the conventional More elastin-derived raw material compositions can be obtained than the method of

In the case of using Thermoase C160 and Papain W-40, the pH range in which decomposition reactions are possible overlaps at 6.5 to 7.5, so there is no need to undergo a separate pH adjustment or pH confirmation step during each enzyme treatment. In addition, since the pH range is close to neutral, a separate buffer solution or the like is not required, and the pH range can be easily adjusted with only water.

1) Treatment with Thermoase

After washing, water and Thermoase are added to the remaining insoluble matter. At this time, water is about 1.5 to 2.5 times the weight of the insoluble matter, preferably 2 times the weight, and about 0.5 to 0.7% of the weight of the insoluble matter is added to Thermoase. about 65 to 80 ° C, preferably about 65 to 70 ° C, more preferably 70 ° C, the pH is 6.5 to 8.5, preferably pH 7.0 to 8.5, more preferably pH 7.0 to 7.5, and 2 to 3 time, preferably about 2 to 2.5 hours to react while stirring.

2) Treatment with Papain

Papain is added in an amount of 1.3 to 1.7% by weight based on the weight of the insoluble matter remaining after washing, at about 75 to 80° C., preferably at about 80° C., at a pH of 6.5 to 7.5, preferably at pH 7.0, and at 2 to 80° C. The reaction is stirred for 3 hours, preferably about 2 to 2.5 hours.

3) Enzyme inactivation

After the reaction is complete, the enzymes are inactivated by heating at 90 to 95°C, preferably about 90°C for 25 to 35 minutes.

(4) Degreasing and deodorizing

Activated carbon is dissolved in a small amount of water and used, but the activated carbon is used in an amount of about 3% of the weight of the insoluble material before enzyme treatment. Activated carbon dissolved in water can be degreased and deodorized by stirring at 90 to 95 ° C. for 25 to 35 minutes together with the elastin decomposition product that has been treated with the enzyme.

Activated carbon generally has uses such as deodorization and decolorization, but some of them may filter out peptides included in elastin decomposition products. Activated carbon may use two or more activated carbons, preferably two or more activated carbons that do not filter out peptides included in the elastin decomposition product, and more preferably, SEQ ID NOS: 1 to 10 included in the elastin decomposition product. Two or more activated carbons that do not filter out the peptides of The total amount of activated carbon added is about 3% by weight of the weight of the insoluble matter before enzyme treatment. Specifically, it is possible to use F-WY and F-GZW60, which are less likely to filter out the peptides of SEQ ID NOS: 1 to 10 and have deodorizing, degreasing and decolorizing effects, and each can be used alone in two steps or mixed. can

(5) Filtration and concentration

After primary filtration using diatomaceous earth, secondary filtration is performed through a mesh (eg, 200 mesh). Filtration can be carried out at about 60 ° C for 50 to 90 minutes.

It can be concentrated 4 to 10 times using an evaporator for 5 to 6 hours at about 50 to 65 ° C and a vacuum degree of -0.08 to -0.1 MPa.

(6) Sterilization and drying

After pasteurization at 80 to 85 ° C. for 25 to 35 minutes using a pasteurizer, impurities are finally removed by passing through 200 mesh at 60 ° C. In the case of high-temperature sterilization, there is a risk of destruction of the peptides included in the raw material composition, so pasteurization is recommended.

It can be dried at 80 to 150 ° C., specifically, considering the destruction prevention and drying efficiency of peptides, about 6 It can dry for hours. The raw material composition can be obtained in powder form.

The raw material composition prepared according to the manufacturing method according to the present invention includes the peptide of SEQ ID NO: 1 as an essential component, and further includes one or more peptides selected from the peptide group of SEQ ID NOS: 2 to 10.

SEQ ID NO: 1 - (valine-glycine-proline-glycine)

SEQ ID NO: 2 - (valine-glycine-proline-glycine-glycine)

SEQ ID NO: 3-(Phenylalanine-glycine-proline-glycine-glycine-alanine-glycine)

SEQ ID NO: 4-(Threonine-glycine-glycine-proline-glycine)

SEQ ID NO: 5-(glycine-valine-glycine-glycine-proline-glycine)

SEQ ID NO: 6-(Phenylalanine-glycine-glycine-proline)

SEQ ID NO: 7-(valine-glycine-proline-glycine-leucine)

SEQ ID NO: 8- (valine-glycine-proline-glycine-phenylalanine)

SEQ ID NO: 9-(Phenylalanine-glycine-proline-glycine-leucine)

SEQ ID NO: 10-(Phenylalanine-glycine-proline-glycine-phenylalanine)

The raw material composition prepared according to the manufacturing method according to the present invention may include 0.5 to 5% by weight of the peptide of SEQ ID NO: 1.

Preferably, the raw material composition may further include the SEQ ID NO: 2 peptide in an amount of 0.1 to 1.5% by weight.

Preferably, the raw material composition may further include the SEQ ID NO: 3 peptide in an amount of 0.01 to 1.2% by weight.

[Example of manufacturing method using animal raw materials]

Hereinafter, a method for preparing the raw material composition of the present invention using heart artery bulbs of skipjack tuna (Katsuwonus pelamis) will be described.

(1) Preparation and mincing of bonito arterioles

Prepare 500 kg of bonito artery bulbs, and chop them finely at room temperature using a meat chopper.

(2) Alkaline treatment, neutralization and cleaning

1) Add 0.1 mol/L NaOH of about 3 times the weight (1500 kg) of the arterial sphere as a raw material to the arterial sphere, and stir at about 85°C for 40 to 60 minutes.

2) Citric acid (aqueous solution) in the same mole number as the NaOH was added, and neutralized by stirring at about 85° C. for about 30 minutes.

3) After neutralization, water of about twice the weight (1000 kg) of the raw arterial sphere was added and washed for about 30 minutes at room temperature, followed by washing twice.

After washing, about 250 kg of insoluble matter including elastin was obtained.

(3) enzyme treatment

Enzymatic (protease) treatment was performed in two stages, and elastin in arterioles was degraded into various polypeptides. As follows, Thermoase C160 (Amano Enzyme INC.) and papain (Papain W-40, Amano Enzyme INC.) were added according to the weight of the insoluble matter, and each decomposition reaction was carried out in two stages, resulting in more elastin-derived raw materials than conventional methods. composition was obtained.

1) Treatment with Thermoase C160

After washing, water and Thermoase are added to the remaining insoluble matter. At this time, water is about twice the weight of insolubles (500kg), and about 0.6% (1.5kg) of Thermoase is added. The mixture was stirred and reacted at about 70° C. for 2 hours.

2) Treatment with papain (Papain W-40)

Papain was added in an amount of about 1.5% by weight (3.75 kg) based on the weight of the insoluble matter remaining after washing, and reacted by stirring at about 80° C. for 2 hours.

3) Enzyme inactivation

After the reaction was complete, the enzymes were inactivated by heating at about 90° C. for 30 minutes.

(4) Degreasing and deodorizing

Activated carbons F-GZW60 and F-WY were dissolved in a small amount of water and used, but the activated carbons were used in a mixture of about 1.5% of the weight of the insoluble material before enzyme treatment (3.75 kg each). Degreased and deodorized by stirring at about 90 ° C. for 30 minutes together with the elastin decomposition product that had been treated with the enzyme.

(5) Filtration and concentration

After primary filtration using diatomaceous earth, secondary filtration was performed through 200 mesh. Filtration was performed at about 60° C. for about 60 minutes.

The total weight of elastin degradation product and water after filtration was about 625 kg.

It was concentrated about 5 times using an evaporator for 5 hours at about 50 ~ 65 ℃ and vacuum degree -0.08 ~ -0.1MPa.

(6) Sterilization and drying

After pasteurization at about 85 ° C. for 30 minutes using a pasteurizer, impurities were finally removed by passing through 200 mesh at 60 ° C.

It was dried for about 6 hours using a spray dryer (Spray dryer, in-let temperature: 150° C., outlet temperature: 88° C.) to obtain 45 kg of the raw material composition in powder form.

The results of analyzing the peptide content of SEQ ID NOs: 1 to 3 in the raw material composition (total N = 6) obtained by performing the same method five more times with Agilent 6470 Triple Quad LC/MS/MS are as follows.

In the raw material composition prepared by the above production method, the peptide of SEQ ID NO: 1 was found in an average of 1.55% by weight in the range of 1.279 to 1.771% by weight, and the peptide of SEQ ID NO: 2 was found in an average of 1.03% by weight in the range of 0.708 to 1.302% by weight. %, and the peptide of SEQ ID NO: 3 showed an average of 0.87% by weight in the range of 0.622 to 1.022% by weight.

Looking at Example 1 (paragraphs [0030] to [0033]) of Japanese Patent Registration No. 5087042, which is a conventional manufacturing method, the solution in which the soluble solid content (Brix) was measured was 10 g of purified elastin, 0.05 g of thermolysin, and papain. It is 30 ml of 0.05 g and 1/15 N phosphate buffer (pH 7.0). Soluble solid content (Brix) represents the amount of solute (g) in 100 g of solution. The mass of the phosphate buffer solution is not described, but considering only elastin and enzyme, the soluble solid content by enzyme is 0.1/10.1 * 100 = 0.99%. , the effect of the phosphate buffer on the soluble solids is considered to be very small. Therefore, since the amount of the purified elastin-derived peptide is about 5.0%, it can be seen that about 0.5g was extracted. Therefore, according to the conventional manufacturing method, about 5% of the peptide can be extracted from the purified elastin, and about 77% (206g/266g*100) of the elastin purified from the raw material arteriole can be obtained. ), about 3.87% of the peptides can be extracted.

In comparison, in the case of using the production method of the present invention, about 250 to 325 kg of purified elastin can be obtained after enzymatic treatment using 500 kg of raw material arterioles, and finally about 45 kg of a raw material composition (powder) containing peptides can be obtained. can Therefore, according to the manufacturing method of the present invention, about 13.8 to 18% of peptides can be extracted from purified elastin, and about 9% of peptides can be extracted from raw arterial cells. Therefore, compared to the conventional manufacturing method, it is possible to extract about 176-206% more peptides from purified elastin, and about 133% more peptides can be extracted from raw arterioles.

According to the production method of the present invention, 7.5% to 13% by weight of animal raw materials, preferably 8% to 11% by weight of elastin-derived peptides can be obtained.

2. Chemical Synthesis of SEQ ID NO: 1 Peptide

Hereinafter, a method for preparing the peptide of SEQ ID NO: 1 included in the raw material composition of the present invention through chemical synthesis will be described. The present invention is not limited to the following manufacturing methods. The following chemical synthesis process may refer to FIGS. 29 and 30.

The peptide of SEQ ID NO: 1 is a peptide composed of four amino acids of Val-Gly-Pro-Gly, and the chemical formula is represented by C14H24N4O5, and the structural formula is as follows.

(1) Crude peptide synthesis process

1) Fmoc-AA-wang resin is swollen using DMF as a solid phase, and 20% (v/v) piperidine is added.

2) Drain off residual piperidine using DMF and wash.

3) A mixed solution containing an amino acid (valine) and a coupling reagent (HBTU) is added to the resin.

4) The residual amino acid mixture was washed with DMF and treated with 20% piperidine to remove the Fmoc group protecting the amine group of amino acids.

5) Drain off residual piperidine using DMF and wash.

6) Prepare a mixed solution containing amino acid (glycine) and coupling reagent (HBTU) and repeat steps 3) to 5).

7) Prepare a mixed solution containing amino acid (proline) and coupling reagent (HBTU) and repeat steps 3) to 5).

8) Prepare a mixed solution containing amino acid (glycine) and coupling reagent (HBTU) and repeat steps 3) to 5) above.

9) A peptide is synthesized on the resin.

10) Recover the peptide by adding trifluoroacetic acid (TFA).

11) Filter and dry.

(2) Process of purifying SEQ ID NO: 1 peptide from crude peptide

1) Dissolve the crude peptide obtained in the above process in H ₂ O/Acetonitrile.

2) Pass the lysate, 0.05% TFA/Purified water and 0.05% TFA/Acetonitrile through HPLC to separate the peptide of SEQ ID NO: 1.

3) Lyophilization to finally obtain the peptide of SEQ ID NO: 1.

Peptides of SEQ ID NOs: 2 to 10 can also be synthesized in the same manner as above.

[Example 1] Raw material composition

One embodiment of the raw material composition according to the present invention is an elastin degradation product obtained from skipjack tuna arterioles by the above-described preparation method, comprising about 1.55% by weight of the peptide of SEQ ID NO: 1, about 1.03% by weight of the peptide of SEQ ID NO: 2, and the above sequence 3 at about 0.87% by weight.

[Example 2] SEQ ID NO: 1 Peptide

An example of the peptide according to the present invention is the peptide of SEQ ID NO: 1 obtained by the chemical synthesis method among the above production methods, with a purity of 98.1% (Shimadzu HPLC LabSolution) and a molecular weight of 328.3 Da (AXIMA Assurance, MALDI-TOF). , Shimadzu).

[Example 3] SEQ ID NO: 2 Peptide

The peptide of SEQ ID NO: 2 was synthesized in the same manner as in Example 2, but added in the order of valine, glycine, proline, glycine, and glycine according to the constituting amino acid sequence.

The purity was 95.2% (Shimadzu HPLC LabSolution), and the molecular weight was measured as 386.2 Da (AXIMA Assurance, MALDI-TOF, Shimadzu).

[Example 4] SEQ ID NO: 3 Peptide

The SEQ ID NO: 3 peptide was synthesized in the same manner as in Example 2, but added in the order of phenylalanine, glycine, proline, glycine, glycine, alanine, and glycine according to the constituting amino acid sequence.

The purity was 99.3% (Shimadzu HPLC LabSolution), and the molecular weight was measured as 562.2 Da (AXIMA Assurance, MALDI-TOF, Shimadzu).

<Confirmation of functionality of peptides and raw material compositions>

1. SEQ ID NOS: 1 to 3 Peptides and Functional Research Methods of Raw Material Compositions

1) Research purpose

This study is to evaluate the efficacy of improving skin health (whitening, wrinkles, moisturizing) of the raw material composition of Example 1 and the peptides of Examples 2 to 4 in an in vitro system.

2) Positive control substance

① Evaluation of melanin production inhibitory ability: arbutin

② Evaluation of collagen production: retinol

③ Evaluation of moisturizing activity: N-acetylglucosamine (GlcNAc)

Each of the above positive control substances were purchased from Sigma-Aldrich and used.

3) Tyrosinase activity inhibition measurement method

The ability of tyrosinase, which oxidizes DOPA to DOPA quinone, to inhibit DOPA oxidase activity was evaluated. Test materials (SEQ ID NO: 1 to 3 peptides and raw material compositions) were dissolved in distilled water and diluted to various concentrations, and test solutions were prepared for each test material.

170μL of 0.1M phosphate buffer (pH 7.0), 10μL of each diluted test solution, 10μL of 10mM L-DOPA, and 10μL of 2000 unit/mL mushroom tyrosinase were sequentially added, mixed, and reacted at 37℃ for 15 minutes. was measured.

The absorbance of the blank test solution was measured by adding distilled water instead of the test solution and reacting in the same way, and the enzyme activity inhibition ability was calculated by the following formula.

Enzyme activity inhibition (%) = {(A-B)/A}x100

A: absorbance after reaction of blank test solution, B: absorbance after reaction of test solution

4) Method for measuring collagenase activity inhibition

Similarly, a test solution diluted in various concentrations by dissolving the test substance in distilled water is prepared, and the substrate solution is 4-phenylazobenzyloxy carbonyl-Pro-Leu-Gly-Pro-D-Arg in 0.1M tris-HCl buffer (containing 4mM CaCl ₂ ). pH 7.5) at a concentration of 0.3 mg/mL.

0.1 mL of each diluted test solution, 0.25 mL of substrate solution, and 0.15 mL of 0.2mg/mL collagenase were sequentially added, mixed, and reacted at room temperature for 20 minutes.

Thereafter, 0.5 mL of 6% Citric acid was added to stop the reaction, and 2 mL of ethyl acetate was added to measure absorbance at a wavelength of 320 nm, and enzyme activity inhibition was calculated by the following formula.

The absorbance of the blank test solution was measured by adding distilled water instead of the test solution and reacting in the same way.

Enzyme activity inhibition (%) = {(A-B)/A}x100

A: absorbance after reaction of blank test solution, B: absorbance after reaction of test solution

5) Evaluation method for inhibiting melanin production in B16F10 cells

① Measurement of cell viability

The viability of B16F10 cells was measured by MTT Assay (Denizot F and Lang R. J Immunologic Method 89: 271-277, 1986).

B16F10 cells are dispensed into a 24-well plate to be 5x10 ⁴ cells/well, cultured for 24 hours, and then cultured for 72 hours after exchanging the cell culture medium with a culture medium containing diluted test substances at various concentrations. Thereafter, the culture medium was removed, and 1 mg/mL MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) solution was added thereto, followed by further incubation for 2 hours. After removing the MTT solution and adding isopropanol (dissolving blue formazan), the absorbance was measured at 570 nm.

② Measurement of melanin content produced by B16F10 cells

The B16F10 cells were dispensed into a 12-well plate to be 5x10 ⁴ cells/well, cultured for 24 hours, and the cell culture medium was cultured with a culture medium containing 100 nM α-melanocyte-stimulating hormone (α-MSH) and diluted test substances at various concentrations. After exchange, incubate for 72 hours. After washing the cell monolayer with PBS, the cells were collected by treatment with 0.25% Trypsin-2.65mM EDTA, and centrifuged at 12,000 rpm for 10 minutes to obtain a pellet. 1N NaOH containing 10% dimethylsulfoxide (DMSO) was added to the pellet, dissolved at 60 ° C, and absorbance was measured at a wavelength of 490 nm.

After measuring the protein using the BCA protein assay kit and standardizing the amount of melanin production by the amount of protein, the value of the group treated with only α-MSH was set to 100 and expressed as a relative value.

6) Evaluation method for skin wrinkle improvement in HS68 cells

① Measurement of cell viability

HS68 cells are dispensed into a 48-well plate to be 1x10 ⁴ cells/well, cultured for 24 hours, and then cultured for 24 hours after exchanging the cell culture medium with a medium containing a test substance diluted in various concentrations. And MTT assay was performed in the same way as in B16F10 cells.

② Measurement of collagen and MMP-1 content produced and secreted from HS68 cells

HS68 cells are dispensed into a 48-well plate to be 5x10 ⁴ cells/well, cultured for 24 hours, and then cultured for 24 hours after exchanging the cell culture medium with a serum-free medium containing test substances diluted in various concentrations. The cell culture medium was collected, centrifuged at 5,000 rpm for 10 minutes, and the supernatant was stored at -70°C.

Collagen and MMP-1 contents in the cell culture medium were measured using the procollagen type I C-peptide (PIP) EIA kit (Takara) and the Human MMP-1 ELISA kit (Sigma-Aldrich) according to the method suggested by the manufacturer.

7) Evaluation method for skin moisturizing improvement in HaCaT cells

① Measurement of cell viability

HaCaT cells are distributed in a 24-well plate to be 5x10 ⁴ cells/well, cultured for 24 hours, and then cultured for 24 hours after exchanging the cell culture medium with a medium containing a test substance diluted in various concentrations. And MTT assay was performed in the same way as in B16F10 cells.

② Measurement of hyaluronic acid (HA) content produced and secreted by HaCaT cells

HaCaT cells are distributed in a 24-well plate to be 5x10 ⁴ cells/well, cultured for 24 hours, and then cultured for 24 hours after exchanging the cell culture medium with a medium containing a test substance diluted in various concentrations. The cell culture medium was collected, centrifuged at 5,000 rpm for 10 minutes, and the supernatant was stored at -70°C.

HA content in the cell culture medium was measured using a Hyaluronan ELISA kit (R&D Systems) according to the method suggested by the manufacturer.

8) Statistical processing

All analytical values are presented as mean±SEM. The collected results were analyzed using the GraphPad Prism 5.0 program, and Student's t-test and one-way analysis variance were used to compare the difference between the test substance treatment group and the control group. Only when p < 0.05 or more was judged statistically significant.

2. Tyrosinase activity inhibition evaluation

In order to investigate the effect of the four test substances on the activity of Tyrosinase, a major restriction enzyme in the melanin production process, the ability of Tyrosinase to inhibit DOPA oxidase activity was evaluated. The measurement results are shown in Table 1 below (Tyrosinase activity inhibitory ability (%)).

E represents the raw material composition according to Example 1, P1 represents the peptide of SEQ ID NO: 1 according to Example 2, P2 represents the peptide of SEQ ID NO: 2 according to Example 3, and P3 represents the peptide of SEQ ID NO: 3 according to Example 4.

Looking at Table 1, overall, P1 showed a relatively high Tyrosinase activity inhibitory ability with respect to P2 and P3.

As the concentration of P1 increased, the inhibitory ability gradually increased, and therefore, the highest value was measured at 1,000 μg/mL at 31.9±1.2%.

E and P2 showed the highest values of 13.6±1.9% and 3.6±1.4%, respectively, at 50 μg/mL, E showed values ranging from 1.7±0.6% to 13.6±1.9%, and P2 showed values of 2.0±0.3% Values in the range of ~ 3.6 ± 1.4% were found.

The inhibition of P3 gradually increased as the concentration increased, and the highest was measured at 6.8±1.7% at 1,000 μg/mL.

In conclusion, P1 to 3 and E showed Tyrosinase activity inhibitory ability.

3. Collagenase activity inhibition evaluation

In order to investigate the effect of the four test substances on the activity of collagenase, an enzyme responsible for collagen degradation, the ability to inhibit collagenase activity was evaluated. The measurement results are shown in Table 2 below (collagenase activity inhibition ability (%)).

E showed the inhibitory ability in the range of 3.0±0.4% to 17.2±3.8%, and the highest value appeared at 400μg/mL.

P1 exhibited an inhibitory capacity ranging from 5.2±1.0% to 16.9±1.7%, with the highest value at 200 μg/mL.

P2 exhibited inhibition in the range of 5.2±1.4% to 17.4±2.6%, with values of 17.4±2.0%, 17.4±1.6%, and 17.4±2.6% at 100, 200, and 400 μg/mL, respectively.

P3 exhibited an inhibitory capacity ranging from 3.5±2.0% to 17.4±2.5%, with values of 17.4±2.5% and 17.4±1.3% at 100 and 200 μg/mL, respectively.

In conclusion, P1 to 3 and E are collagenase activity inhibition was observed.

4. Evaluation of melanin production inhibition in B16F10 cells

1) Cytotoxicity evaluation

The cell viability of B16F10 cells was expressed as a percentage of the control group (0 μg/mL) not treated with the test substance. The measurement results are shown in Table 3 (survival rate (% of control) of B16F10 cells) below.

Treatment with 4 types of test materials significantly increased cell viability compared to the control group. This suggests that the test substance does not exhibit cytotoxicity to B16F10 cells, and melanin production inhibitory activity was studied for all concentrations of the test substance.

2) Evaluation of melanin production inhibition in B16F10 cells

In order to investigate the effect of the four test substances on melanin production, B16F10 cells induced to produce melanin with α-MSH were treated with the four test substances, and then the intracellular melanin content was measured. The measurement results are shown in Table 4 below (melanin production (% of control) of B16F10 cells).

Looking at Table 4, melanin production was significantly increased in the group treated with α-MSH compared to the group not treated with α-MSH.

When 200 μg/mL of arbutin, a positive control, was treated, melanin production was significantly reduced compared to the group treated with α-MSH.

As the concentration increased in all of P1 to P3 and E, melanin production decreased.

Compared to the group treated with α-MSH, E was significantly reduced to 89.3±2.2% and 82.9±4.4% when treated with 200 and 400 μg/mL, respectively.

Compared to the group treated with α-MSH in P1 and P2, the melanin content decreased significantly from the 5μg/mL treatment, and was 85.8±3.1% and 81.9±3.8%, respectively, at the highest concentration (40μg/mL). significantly decreased.

Compared to the group treated with α-MSH in P3, the melanin content was significantly decreased from the treatment of 1 μg/mL, and significantly decreased to 82.2±1.3% at the highest concentration (40 μg/mL), respectively.

In conclusion, P1 to 3 and E significantly inhibited melanin production in B16F10 cells induced by α-MSH.

5. Evaluation of skin elasticity (wrinkle) improvement efficacy in HS68 cells

1) Cytotoxicity evaluation

The cell viability of HS68 cells was expressed as a percentage of the control group (0 μg/mL) not treated with the test substance. The measurement results are shown in Table 5 below (survival rate (% of control) of HS68 cells).

Treatment with 4 types of test materials significantly increased cell viability compared to the control group. This suggests that the test substance does not exhibit cytotoxicity to HS68 cells, and collagen production and secretion studies were performed for all concentrations of the test substances.

2) HS68 Evaluating collagen production and secretion efficiency in cells

In order to investigate the effect of the four test substances on collagen production and secretion of HS68 cells, which are skin fibroblasts, the cell culture medium in which the cells were conditioned was collected and the collagen content was measured. The measurement results are shown in Table 6 (collagen production (% of control) of HS68 cells).

Looking at Table 6, when 0.5 μM of retinol, a positive control, was treated, collagen was significantly increased by 153.3±16.0% compared to the control group.

In all of P1 to P3 and E, collagen production generally increased as the concentration increased.

Compared to the control group, E significantly increased the collagen content from the treatment of 50 μg / mL, and significantly increased to 199.5 ± 7.4% at the highest concentration (400 μg / mL).

In P1, compared to the control group, the collagen content significantly increased from the treatment of 5 μg/mL, and significantly increased to 188.8±11.7% at the highest concentration (40 μg/mL).

In P2 and P3, compared to the control group, the collagen content significantly increased from the treatment with 1 μg/mL, and significantly increased to 189.8±15.2% and 228.8±9.4%, respectively, at the highest concentration (40 μg/mL).

3) HS68 Evaluation of MMP-1 production and secretion efficacy in cells

In order to investigate the effect of the four test substances on the production and secretion of MMP-1 in HS68 cells, which are skin fibroblasts, the cell culture medium in which the cells were conditioned was collected and the MMP-1 content was measured. The measurement results are shown in Table 7 (MMP-1 production (% of control) of HS68 cells).

Looking at Table 7, when the positive control substance retinol was treated with 0.5 μM, MMP-1 was significantly reduced by 83.2±2.2% compared to the control group (0 μg/mL).

E showed a tendency to decrease MMP-1 production compared to the control group, and at 200 μg/mL, MMP-1 production decreased to 88.4±6.8%.

In P1 to P3, compared to the control group, MMP-1 production was significantly reduced from the case of 10 μg/mL treatment.

P1 and P3 were significantly decreased to 73.0±7.7% and 70.2±3.0%, respectively, at the highest concentration (40 μg/mL), and P2 was significantly decreased to 76.6±5.0% at 20 μg/mL.

In conclusion, P1 to 3 and E significantly increased collagen production and significantly decreased MMP-1 production in HS68 cells.

6. Evaluation of skin moisturizing improvement efficacy in HaCaT cells

1) Cytotoxicity evaluation

The cell viability of HaCaT cells was expressed as a percentage of the control group (0 μg/mL) not treated with the test substance. The measurement results are shown in Table 8 (Viability of HaCaT cells (% of control)).

All four types of test substance treatment had no significant effect on cell viability or significantly increased it compared to the control group. This suggests that the test substance does not exhibit cytotoxicity to HaCaT cells, and HA production and secretion studies were performed for all concentrations of the test substances.

2) Evaluation of HA production and secretion efficacy in HaCaT cells

In order to investigate the effect of the four test materials on the HA production secretion of HaCaT cells, the cell culture medium in which the cells were conditioned was collected and the HA content was measured. The measurement results are shown in Table 9 (Hyaluronic acid production (% of control) of HaCaT cells).

Looking at Table 9, when 1μM of GlcNAc, a positive control, was treated, HA was significantly increased by 161.8±12.6% compared to the control (Oμg/mL).

In all of P1 to P3 and E, the production of HA generally increased as the concentration increased.

Compared to the control group, E significantly increased the HA content from the treatment of 10 μg / mL, and significantly increased to the maximum value of 169.5 ± 4.4% at 200 μg / mL.

In P1, compared to the control group, the HA content significantly increased from the treatment of 10 μg/mL and significantly increased to 191.5±13.7% at the highest concentration (40 μg/mL).

Compared to the control group, P2 and P3 significantly increased the HA content from the treatment with 1 μg / mL, and significantly increased to 170.7 ± 9.9% and 180.4 ± 16.3%, respectively, at the highest concentration (40 μg / mL).

In conclusion, P1 to 3 and E significantly increased HA production in HaCaT cells.

The above results indicate that P1 to 3 and E have skin whitening, moisturizing and anti-wrinkle effects in an in vitro system.

<Evaluation of efficacy of raw material composition in skin damage model by UVB irradiation>

In the skin damage model by UVB irradiation, the efficacy of skin moisturizing, anti-wrinkle and whitening activity was confirmed in vitro and in vivo.

The following bonito elastin powder refers to a powdered product of the raw material composition of Example 1.

1. Materials and Methods

1) In vitro experiments

As shown in the table below, cells related to each function were cultured and subjected to a cytotoxicity test (MTT assay), followed by irradiation with ultraviolet rays (UVB irradiation amount: 50 mJ/cm ² ), and an acceptable concentration according to the cytotoxicity test results. Using the raw material composition and the following measurement method, each item was measured and evaluated.

The test groups were NC (Normal Control), C (Control), PC 1 (Positive Control 1, L-ascorbic acid 100 μg/mL), PC 2 (Positive Control 2, Arbutin 100 μg/mL), KE 50 (Skipjack Tuna [ Katsuwonus pelamis ], KE 50 μg/mL), KE 100 (KE 100 μg/mL), KE 200 (KE 200 μg/mL), and KE 400 (KE 400 μg/mL).

① Cell culture

Each cell was supplemented with 10% fetal bovine serum (Hyclone laboratories, Logan, Utah, USA), 100 mg/L penicillin-streptomycin (Hyclone Laboratories, Logan, Utah, USA) in high-glucose Dulbecco's modified Eagle's medium (DMEM) (Hyclone Laboratories, Logan, Utah, USA). , Logan, Utah, USA), 2 mmol/L L-glutamine (Hyclone laboratories, Logan, Utah, USA), sodium pyruvate (Hyclone laboratories, Logan, Utah, USA), 100 mg/L penicillin-streptomycin (Hyclone Laboratories, Logan, Utah, USA) were used, including incubation at 37°C and 5% CO ₂ .

② Cytotoxicity test

Cytotoxicity test (MTT assay) was performed by referring to the method of Berridge et al. Each cell was dispensed in a 96 well plate at 1×10 ⁴ cells/well, stabilized overnight, and samples were treated in each well by concentration. After 24 hours, 20 μL of 5 mg/mL MTT solution was added and incubated for 4 hours. After removing the medium, 100 μL/well of DMSO was added and the absorbance was measured at 560 nm.

③ RT-PCR (RNA extraction and real-time quantitative PCR)

Each cell was dispensed in a 6-well plate at 5×10 ⁵ cells/well, stabilized overnight, washed with DPBS, and 50 mJ/well using a UVB lamp (5 Sankyo Denki G5T5 lamps, Sankyo Denki Co., Yokohama, Japan). cm ² was irradiated, and L-ascorbic acid 100 μg/mL and bonito elastin powder as positive controls were treated at concentrations of 50, 100, 200, and 400 μg/mL. After 24 hours, cells were collected with 0.25% trypsin-EDTA, and RNA was extracted using RNasy Mini Kit (QIAGEN, Hilden, Germany) and synthesized into cDNA using iScript™ cDNA Synthesis Kit (Bio-Rad). To measure the expression of genes, real-time quantitative PCR was performed using SYBR Green (iQ SYBR Green Supermix, Bio-Rad Bio-Rad Laboratories Inc.), and the device was CFX ConnectTM real-time PCR detection system (Bio-Rad Laboratories Headquarters , Hercules, CA, USA) was used. Each primer sequence to be used in the gene amplification reaction is shown in Tables 1 and 2. After hot start at 95 ° C for 10 min, PCR analysis was performed with 40 cycling at 95 ° C for 15 s, 57 ° C for 15 s, and 72 ° C for 30 s. After all cycles were completed, melting curve analysis was performed to confirm the specificity of the primer. Analysis of the results was performed with CFX Maestro™ Analysis Software (Bio-Rad Laboratories Headquarters, Hercules, CA, USA) provided by BioRad.

④ Western blot

Each cell was dispensed in a 6-well plate at 5×10 ⁵ cells/well, stabilized overnight, washed with DPBS, and 50 mJ using a UVB lamp (5 Sankyo Denki G5T5 lamps, Sankyo Denki Co., Yokohama, Japan). /cm ² was irradiated, and L-ascorbic acid 100 μg/mL and skipjack elastin powder as positive controls were treated at concentrations of 50, 100, 200, and 400 μg/mL. After 24 hours, the medium was removed, homogenized by adding lysis buffer containing protease inhibitors, kept on ice for a certain period of time, and then centrifuged (12,000 rpm, 20 minutes, 4°C) to separate proteins. Protein quantification was quantified by the Bradford method using BSA and bio-rad protein assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA USA). Using sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE, 10%), 20 μL of each sample was loaded and separated by electrophoresis, and after transferring the protein to a nitrocellulose membrane, 5% skim milk (dissolved in TBST buffer) After blocking for 1 hour with TBS containing 0.5% Tween 20), primary antibodies HAS2, CerS4 (LASS4), beta-actin, MAPK8 (JNK), c-Fos, c-Jun, MMP's (1, 3, 9) , Smad3, PKA, CREB, MITF, TRP-1, TRP-2 (Cell signaling Technology, Beverly, MA, USA) After reacting at room temperature for 3 hours, the membrane was separated with HRP-polymerized secondary antibody (Cell signaling Technology, Beverly, MA, USA). MA, USA) for 60 minutes, developed using enhanced chemiluminescent (ECL, Amershampharmacia Biotech, UK), and then photographed using Easy photo. The Western blot band images taken were measured for band density using Image J software (NIH, Bethesda, MD).

⑤ Hyaluronan (hyaluronic acid) secretion measurement

HaCaT cells were cultured and dispensed at 1×10 ⁴ cells/well in a 96 well plate, stabilized overnight, washed with DPBS, and UVB lamp (5 Sankyo Denki G5T5 lamps, Sankyo Denki Co., Yokohama, Japan) at 50 °C. After mJ/cm2 irradiation, 100 μg/mL of L-ascorbic acid and 50, 100, 200, and 400 μg/mL concentrations of skipjack elastin powder were treated as positive controls. After 24 hours, the supernatant was separated and absorbance was measured at 540 nm with an ELISA reader (Bio-Rad Laboratories Headquarters, Hercules, CA, USA) using a Hyaluronic acid (HA) ELISA Kit (Biovision Inc., CA, USA). did

⑥ Measurement of Sphingomyelin content

Samples are prepared in the same way as in the measurement of hyaluronan secretion, but 24 hours after sample preparation, the cells are homogenized (1% Triton X-100 in chloroform), centrifuged (13,000 rpm, 10min), and the supernatant is collected and stored at 50 ° C. chloroform was removed, and the remaining pellet was dissolved in assay buffer, and absorbance was measured at 595 nm with an ELISA reader (Bio-Rad Laboratories Headquarters, Hercules, CA, USA) using a Sphingomyelin Assay Kit (abcam, cambridge, UK).

⑦ Measurement of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) secretion

HS27 cells were cultured, and samples were prepared in the same way as for measuring the amount of hyaluronan secretion, but 24 hours after sample preparation, the secretion of IL-1β, IL-6, and TNF-α was measured using the supernatant using the Duoset ELISA kit (R&D system , Minneapolis, MN, USA) was measured at 655 nm using an ELISA reader (Bio-Rad Laboratories Headquarters, Hercules, CA, USA).

⑧ Measurement of Tyrosinase activity

B16F10 cells were cultured and dispensed into 2×10 ⁵ cells/well in a 6-well plate, stabilized overnight, and then positive control Arbutin 100 μg/mL and skipjack elastin powder 50, 100, 200, 400 μg/mL were used as differentiation reagents Treatment with 250 μM of phosphorus IBMX induced the differentiation of melanin pigment for 3 days, and the sample and medium were replaced every 24 hours. After 72 hours, after homogenizing using assay buffer, the supernatant obtained by centrifugation (10,000 × g, 15 min, 4 ℃) was used as an ELISA reader ( Bio-Rad Laboratories Headquarters, Hercules, CA, USA).

⑨ Melanin production inhibition test

B16F10 cells were cultured and dispensed into 5×10 ⁴ cells/well in a 24 well plate and stabilized overnight. Then, positive control Arbutin 100 μg/mL and skipjack elastin powder 50, 100, 200, 400 μg/mL were used as differentiation reagents Treatment with 250 μM of phosphorus IBMX induced the differentiation of melanin pigment for 3 days, and the sample and medium were replaced every 24 hours. After 72 hours, each well was washed with DPBS, cells were separated by dispensing 125 μL of Cell lysis reagent buffer, and proteins were quantified in the supernatant with Bradford Dye Reagent. 300 μL was added and dissolved at 100° C. for 10 minutes, and absorbance was measured at 450 nm with an ELISA reader (Bio-Rad Laboratories Headquarters, Hercules, CA, USA).

⑩ Nitric oxide (NO) production measurement

B16F10 cells were cultured and dispensed into 2×10 ⁵ cells/well in a 6-well plate, stabilized overnight, and then positive control Arbutin 100 μg/mL and skipjack elastin powder 50, 100, 200, 400 μg/mL were used as differentiation reagents Treatment with 250 μM of phosphorus IBMX induced the differentiation of melanin pigment for 3 days, and the sample and medium were replaced every 24 hours. After 72 hours, after homogenizing using assay buffer, the supernatant obtained by centrifugation (13,000 rpm, 5 min, 4 ℃) was used as a Nitric Oxide Assay Kit (abcam, cambridge, UK) at 540 nm using an ELISA reader (Bio -Rad Laboratories Headquarters, Hercules, CA, USA).

⑪ Measurement of Glutathione (GSH) production

B16F10 cells were cultured and dispensed into 2×10 ⁵ cells/well in a 6-well plate, stabilized overnight, and then positive control Arbutin 100 μg/mL and skipjack elastin powder 50, 100, 200, 400 μg/mL were used as differentiation reagents Treatment with 250 μM of phosphorus IBMX induced the differentiation of melanin pigment for 3 days, and the sample and medium were replaced every 24 hours. After 72 hours, the cells were lysed using assay buffer, and the supernatant obtained was analyzed with an ELISA reader (Bio-Rad Laboratories Headquarters, Hercules, CA) at 405 nm using Glutathione Assay Kit (BioVison Inc., Mountain View, CA, USA). , USA).

⑫ cAMP level measurement

B16F10 cells were cultured and dispensed into 2×10 ⁵ cells/well in a 6-well plate, stabilized overnight, and then positive control Arbutin 100 μg/mL and skipjack elastin powder 50, 100, 200, 400 μg/mL were used as differentiation reagents Treatment with 250 μM of phosphorus IBMX induced the differentiation of melanin pigment for 3 days, and the sample and medium were replaced every 24 hours. After 72 hours, the cells were lysed with 0.1M HCl, and the supernatant obtained was analyzed with an ELISA reader (Bio-Rad Laboratories Headquarters, Hercules, CA, USA) at 540 nm using the cAMP ELISA Kit (Enzo Life Sciences, PA, USA). ) was measured using.

2) In vivo experiment

① Treatment of laboratory animals

Experimental animals used in this study were supplied with 5-week-old male SKH-Ⅰ hairless mice weighing around 20 g from Saeron Bio Co., Ltd. and kept in an animal breeding room at a temperature of 23±2℃, a light/dark cycle of 12 hr (light/dark cycle), and a humidity of 50 °C. After stabilizing for one week under constant conditions at ± 5 ° C, it was used in the experiment. During the adaptation period, the weight was measured and randomly divided into groups of 8 animals with a weight close to the average weight. They were allowed to freely consume a prescribed diet and drinking water. This study was conducted with the approval of the Animal Research Ethics Committee of Kyung Hee University. (KHGASP-20-347) Experimental design animals (n=8/group) are shown in Table 10 below.

Groups UVB Dietary administration Normal Control - AIN 93G diet Control + AIN 93G diet PC1 + AIN 93G diet + L-ascorbic acid 200 mg/kg·b.w. PC2 + AIN 93G diet + Arbutin 200 mg/kg·b.w. KE10 + AIN 93G diet + Katsuwonus pelamis Elastin 10 mg/kg bw KE20 + AIN 93G diet + Katsuwonus pelamis Elastin 20 mg/kg bw KE30 + AIN 93G diet + Katsuwonus pelamis Elastin 30 mg/kg bw

PC1: Positive Control 1, L-ascorbic acid 200 mg/kg b.w.

PC2: Positive Control 2, Arbutin 200 mg/kg b.w.

KE 10: Skipjack Tuna [ Katsuwonus pelamis ], KE 10 mg/kg bw

KE 20: Skipjack Tuna [ Katsuwonus pelamis ], KE 20 mg/kg bw

KE 30: Skipjack Tuna [ Katsuwonus pelamis ], KE 30 mg/kg bw

② Administration method

The concentration of bonito elastin powder used in this experiment was calculated using the mouse conversion factor (12.33) in order to match the human test concentration of 100 mg / day to the experimental animal standard. was added and set to 10, 20, 30 mg/kg·b.w., and L-ascorbic acid and arbutin as positive controls were set to 200 mg/kg·b.w. AIN 93G was provided as a basic diet, and samples per group were administered orally.

As described in the table below, each item related to each function was measured and evaluated using an acceptable concentration of the raw material composition and the following measurement method.

UGTrel8; UDP-glucuronic acid, GlcNAc; UDP-N-acetylglucosamine, LCB1 (SPT); Long chain base 1 (Serine palmitoyltransferase), DEGS1; Dihydroceramide desaturase 1, GAPDH; glyceraldehyde-3-phosphate dehydrogenase

③ Induction of moisture reduction by UV irradiation

UV irradiation was performed using a UVB lamp (5 Sankyo Denky G5T5 lamps, Sankyo Denki Co., Yokohama, Japan) with a minimum erythromal dose (MED) of 150 mJ/cm ² , three times a week on the back of the mouse. The site was irradiated, 1MED (150 mJ/cm ² ) for 1 week, 2MED (300 mJ/cm ² ) for 2 weeks, 3MED (450 mJ/cm ² ) for 3 weeks, and 4MED (600 mJ/cm for 4 to 8 weeks) ² ) to induce a decrease in moisture retention. After 8 weeks of testing, the experimental animals in each group were sacrificed to obtain blood and skin tissue.

④ Measurement of transepidermal water loss (TEWL)

For the measurement of transepidermal water content, the research method of Lee et al. was referred to. In order to determine the moisturizing ability of the skin, the electronic terminal of a moisture meter (howskin) was bonded to the back of the mouse, and the amount of skin transepidermal water loss was measured at the 8th week of this experiment.

⑤ RNA extraction from mouse skin tissue and real-time quantitative PCR

After sacrificing the experimental animal, the separated skin tissue was collected and stored in 1 mL Trizol (QIAZEN) for homogenization, then 200 μL Chloroform was added to collect the supernatant, and RNA was extracted using the RNasy Mini Kit (QIAGEN, Hilden, Germany). extract The subsequent process is the same as the RT-PCR process in the in vitro system. Each primer sequence to be used in the gene amplification reaction is shown in Tables 3 and 4.

⑥ Western blot

After sacrificing the experimental animals, the separated skin tissues were collected, homogenized in lysis buffer with protease inhibitor added, kept on ice for a certain period of time, and then centrifuged (12,000 rpm, 20 minutes, 4℃) to separate proteins. The subsequent process is the same as the Western blot process in the in vitro system.

⑦ Skin histopathological observation

For skin histopathological observation, the H&E staining method in the research method of Kim et al. was referred. For tissue preparation, after sacrificing the mouse, the back skin was removed, attached flat to filter paper, fixed in 10% neutral formalin, paraffin-embedded through normal tissue processing, and sectioned with a tissue section (RM2125, Leica, Wetzlar, Germany) to 4 μm thickness and attached to a slide coated with polylysine. The tissue sections were paraffin-removed using xylene, hydrated with alcohol and distilled water for 10 minutes, washed with distilled water, and then stained with H&E (Hemaoxylin & Eosin) to observe the changes in skin tissue.

⑧ Morphological observation of the skin

For morphological observation, the wrinkle photographing method in Wu et al.'s research method was referred to. To observe morphological changes, close-up images were taken of the mouse's back with a digital camera.

⑨ Measurement of antioxidant enzyme activity

After sacrificing the experimental animals, the separated skin tissue was collected and the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were tested using the SOD Assay kit, Catalase Assay kit, and Glutathione Peroxidase Activity Assay Kit (BioVison Inc., Mt. Absorbance was measured at 655 nm with an ELISA reader (Bio-Rad Laboratories Headquarters, Hercules, CA, USA) using View, CA, USA.

⑩ Tyrosinase activity measurement

After sacrificing the experimental animals, the separated skin tissues were collected, homogenized using assay buffer, and the supernatant obtained by centrifugation (10,000g, 15min, 4℃) was used as Tyrosinase activity Assay Kit (abcam, cambridge, UK). and measured at 490 nm using an ELISA reader (Bio-Rad Laboratories Headquarters, Hercules, CA, USA).

⑪ Nitric oxide (NO) production measurement

After sacrificing the experimental animals, the separated skin tissue was collected, homogenized using assay buffer, and then centrifuged (13,000 rpm, 5 min, 4℃) to obtain the supernatant using Nitric Oxide Assay Kit (abcam, cambridge, UK). was measured at 540 nm using an ELISA reader (Bio-Rad Laboratories Headquarters, Hercules, CA, USA).

⑫ cAMP level measurement

After sacrificing the experimental animals, the separated skin tissue was collected, homogenized using 0.1M HCl, and centrifuged (13,000 rpm, 5min, 4℃), and the resulting supernatant was used as a cAMP ELISA Kit (Enzo Life Sciences, PA, USA) was measured at 540 nm using an ELISA reader (Bio-Rad Laboratories Headquarters, Hercules, CA, USA).

3) Statistical processing

The statistical program SPSS (Statistical package for social science version 23.0, SPSS Inc., Chicago, IL, USA) was used to analyze the results of this experiment, and the measurement results of each experimental item were expressed as mean ± standard deviation (mean ± SD). did The average difference between groups was confirmed by one-way ANOVA, and statistical significance was tested at the P<0.05 level by Duncan's test.

2. Results and discussion

1) Moisturizing the skin

go) in vitro result

① Cytotoxicity test of skipjack elastin powder in HaCaT cells

To confirm the cytotoxicity of skipjack elastin powder, MTT assay was performed. The cell viability of HaCaT cells, which are keratinocytes, according to the concentration of skipjack elastin is shown in FIG. 1A. As a result of treating skipjack elastin powder by concentration, all concentration groups showed no significant difference from the 0 μg/mL group without sample treatment, and it was judged that skipjack elastin powder did not affect cytotoxicity. After setting, the study was conducted.

② Effect of bonito elastin powder on skin moisturizing related gene expression in UV-irradiated HaCaT cells

Photoaging by ultraviolet rays (UV) is extrinsic aging characterized by a decrease in the number and function of dermal and epidermal cells, pigmentation, dryness, thick skin, loss of elasticity, and the like. The stratum corneum, located at the outermost layer of the skin, plays a role in protecting the skin from external stimuli and maintaining moisture and lipid composition. As a component of the skin's extracellular matrix (ECM), hyaluronic acid plays an essential role in water balance. , GlcNAc) and can contain a significant amount of water, thereby regulating the skin barrier function and hydrating the extracellular matrix to maintain moisture homeostasis in the tissue. In the stratum corneum, ceramide constitutes about 35-40% of lipids and generally follows the de novo pathway, one of which is initiated by serine palmitoyl transferase (SPT; LCB1), followed by ceramide synthase (CerS4) and dihydroceramide desaturase 1 (DEGS1). synthesized by the action of enzymes in In this study, the gene expression of skin moisturizing factors UGTrel7, GlcNAc, Elastin, LCB1 (SPT), and DEGS1 was measured in HaCaT keratinocytes irradiated with UV rays, and the results are shown in FIGS. 2 and 3.

As a result of measuring the gene expression level of UGTrel7 (FIG. 2 (A)), the UV irradiation group (C) significantly decreased compared to the normal control group (NC). Among the positive control group (PC) and skipjack elastin powder treatment group, KE100, KE200, and KE400 showed significant increases by 89.8, 31.5, 56.8, and 102.4%, respectively, compared to the ultraviolet irradiation group (C), and the KE400 group showed positive control ( PC) did not show a significant difference. The KE50 group showed no significant difference from the UV irradiation group (C).

As a result of measuring the gene expression level of GlcNAc (((B) in FIG. 2), the ultraviolet irradiation group (C) significantly decreased compared to the normal control group (NC). Among the positive control group (PC) and skipjack elastin powder treatment group, KE100 , KE200 and KE400 showed significant increases by 72.9, 33.2, 39.5, and 86.5%, respectively, compared to the UV irradiation group (C), and the KE400 group showed no significant difference from the positive control group (PC). There was no significant difference from the UV irradiation group (C).

As a result of measuring the gene expression level of Elastin (FIG. 2(C)), the ultraviolet irradiation group (C) significantly decreased compared to the normal control group (NC). The positive control group (PC) and the bonito elastin powder treatment group (KE50, KE100, KE200, KE400) showed a significant increase of 57.5, 32.6, 64.1, 69.3, and 99.7%, respectively, compared to the UV irradiation group (C), and the KE400 The group showed no significant difference from the normal control group (NC).

As a result of measuring the gene expression level of LCB1 (SPT) (Fig. 3 (A)), the UV irradiation group (C) significantly decreased compared to the normal control group (NC). The positive control group (PC) and the bonito elastin powder treatment group (KE50, KE100, KE200, KE400) showed a significant increase of 90.4, 45.9, 62.6, 75.3, and 73.4%, respectively, compared to the UV irradiation group (C), and the KE200 The group showed no significant difference from the positive control group (PC).

As a result of measuring the gene expression level of DEGS1 (FIG. 3(B)), the UV irradiation group (C) significantly decreased compared to the normal control group (NC). The positive control group (PC) and skipjack elastin powder treatment group (KE50, KE100, KE200, KE400) showed a significant increase of 95.9, 59.9, 60.8, 86.0, and 115.1%, respectively, compared to the UV irradiation group (C), and KE400 The group showed no significant difference from the normal control group (NC) and the positive control group (PC).

③ Effect of bonito elastin powder on skin moisturizing related protein expression in UV-irradiated HaCaT cells

Hyaluronic acid plays a role in tissue moisture, elasticity and structure control, tissue repair, and free radical removal, and is synthesized by three isoform enzymes, HAS-1, HAS-2, and HAS-3. It has been reported that HAS is downregulated by UV irradiation, which leads to loss of hyaluronic acid. Ceramide is a central metabolite of sphingolipid metabolism and a key intermediate in the synthesis of glycosphingolipids and sphingomyelin, accounting for about half of all lipid species in the stratum corneum and playing a key role in maintaining epidermal barrier homeostasis. In this study, the protein expression of HAS2 and CerS4 (LASS4), which are skin moisturizing-related factors, was measured in HaCaT keratinocytes irradiated with UV rays, and the results are shown in FIG.

As a result of measuring the protein expression level of HAS2 (top and bottom left of FIG. 4), the UV irradiation group (C) was significantly reduced compared to the normal control group (NC). The positive control group (PC) and skipjack elastin powder treatment group (KE50, KE100, KE200, KE400) showed a significant increase of 61.9, 45.9, 121.2, 119.2, and 99.9%, respectively, compared to the UV irradiation group (C), and KE100 , KE200 and KE400 groups showed no significant difference from the normal control group (NC).

As a result of measuring the protein expression level of CerS4 (LASS4) (top and bottom right of FIG. 4), the UV irradiation group (C) significantly decreased compared to the normal control group (NC). The positive control group (PC) and skipjack elastin powder treatment groups (KE50, KE100, KE200, KE400) showed significant increases of 22.4, 19.8, 18.5, 31.9, and 42.6%, respectively, compared to the UV irradiation group (C).

④ Effect of skipjack elastin powder on hyaluronic acid secretion in UV-irradiated HaCaT cells

The effect on hyaluronic acid secretion was measured in HaCaT keratinocytes irradiated with UV light, and the results are shown in FIG. 5 .

As a result of measuring the amount of hyaluronic acid secretion (left side of FIG. 5), the ultraviolet irradiation group (C) significantly decreased compared to the normal control group (NC). Among the positive control group (PC) and the bonito elastin powder treatment group, KE100, KE200, and KE400 showed significant increases by 23.8, 37.3, 42.3, and 47.5%, respectively, compared to the UV irradiation group (C), and the KE400 group showed the normal control group ( NC) showed a significantly increased result. The KE50 group showed no significant difference from the UV irradiation group (C).

⑤ Effect of skipjack elastin powder on sphingomyelin secretion in UV-irradiated HaCaT cells

The effect on the amount of sphingomyelin secretion was measured in HaCaT keratinocytes irradiated with UV light, and the results are shown in FIG. 5 .

As a result of measuring the amount of sphingomyelin secretion (right side of FIG. 5), the UV irradiation group (C) significantly decreased compared to the normal control group (NC). The positive control group (PC) and skipjack elastin powder treatment group (KE50, KE100, KE200, KE400) showed significant increases by 46.4, 18.8, 36.8, 40.1, and 63.2%, respectively, compared to the UV irradiation group (C), and KE400 The group showed no significant difference from the normal control group (NC).

me) in vivo result

① Effect on epidermal moisture content of experimental animals

The stratum corneum of healthy skin epidermis contains 30-40% of water, and when the water content falls below 10%, the skin becomes dry and loses luster and elasticity, leading to wrinkles. The transepidermal water content measured in the back tissue of mice whose skin moisture was reduced by irradiation with UVB was measured, and the results are shown in FIG. 6.

As a result of measuring the transepidermal water content in the back tissues of experimental animals whose skin moisture was reduced by irradiating UVB, the UV irradiation group (C) significantly decreased compared to the normal control group (NC). Positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and bonito elastin powder treatment groups (KE10, KE20, KE30) were 26.7, 13.8, 13.0, 25.6, respectively, compared to the UV irradiation group (C). , showed a significant increase by 33.3%, and the KE30 group showed no significant difference from the normal control group (NC).

② Effect of bonito elastin powder on skin moisturizing-related gene expression in the skin tissue of experimental animals

Gene expression of UGTrel8, GlcNAc, Fibrillin-1, LCB1 (SPT), and DEGS1, which are skin moisturizing-related factors, was measured in the skin tissues of the experimental animals irradiated with ultraviolet rays, and the results are shown in FIGS. 7 and 8.

As a result of measuring the gene expression level of UGTrel8 (left side of FIG. 7), the UV-irradiated group (C) significantly decreased compared to the normal control group (NC). Among the positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and skipjack elastin powder treatment group, KE20 and KE30 were 146.2, 80.7, 37.2, and 99.3% respectively compared to the UV irradiation group (C). showed an increase in The KE10 group showed no significant difference from the ultraviolet irradiation group (C).

As a result of measuring the gene expression level of GlcNAc (middle of FIG. 7), the UV-irradiated group (C) significantly decreased compared to the normal control group (NC). Among the positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and bonito elastin powder treatment group, KE30 significantly increased by 76.1, 37.9, and 49.6%, respectively, compared to the ultraviolet irradiation group (C). seemed The KE10 and KE20 groups showed no significant difference from the UV irradiation group (C).

As a result of measuring the gene expression level of fibrillin-1 (right side of FIG. 7), the ultraviolet irradiation group (C) significantly decreased compared to the normal control group (NC). Among the positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and skipjack elastin powder treatment group, KE20 and KE30 were 105.1, 69.4, 72.2, and 78.4% respectively compared to the UV irradiation group (C). , and KE20 and KE30 showed no significant difference from positive control group 1 (PC1; L-ascorbic acid). The KE10 group showed no significant difference from the ultraviolet irradiation group (C).

As a result of measuring the gene expression level of LCB1 (SPT) (left side of FIG. 8), the UV irradiation group (C) significantly decreased compared to the normal control group (NC). Among the positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and skipjack elastin powder treatment group, KE20 and KE30 were 74.2, 47.5, 62.7, and 70.7% respectively compared to the UV irradiation group (C). , and KE20 and KE30 showed no significant difference from positive control group 1 (PC1; L-ascorbic acid). The KE10 group showed no significant difference from the ultraviolet irradiation group (C).

As a result of measuring the gene expression level of DEGS1 (right side of FIG. 8), the UV-irradiated group (C) significantly decreased compared to the normal control group (NC). Among the positive control group 1 (PC1; L-ascorbic acid) and the bonito elastin powder treatment group, KE20 and KE30 showed significant increases by 77.9, 48.3, and 66.8%, respectively, compared to the ultraviolet irradiation group (C), and KE30 was the positive control group. 1 (PC1; L-ascorbic acid) did not show a significant difference. Positive control group 2 (PC2; Arbutin) and KE10 group showed no significant difference from UV irradiation group (C).

③ Effect of bonito elastin powder on the expression of proteins related to skin moisturizing in the back skin tissue of experimental animals

The protein expression of HAS2 and CerS4 (LASS4), which are skin moisturizing related factors, was measured in the skin tissues of the experimental animals irradiated with ultraviolet rays, and the results are shown in FIG. 9 .

As a result of measuring the protein expression level of HAS2 (top and bottom left of FIG. 9), the UV irradiation group (C) was significantly reduced compared to the normal control group (NC). Among the positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and skipjack elastin powder treatment group, KE30 significantly increased by 64.8, 46.6, and 42.4%, respectively, compared to the UV irradiation group (C). KE30 showed no significant difference from positive control group 1 (PC1; L-ascorbic acid). The KE10 and KE20 groups showed no significant difference from the UV irradiation group (C).

As a result of measuring the protein expression level of CerS4 (LASS4) (top and bottom right of FIG. 9), the UV irradiation group (C) significantly decreased compared to the normal control group (NC). Among the positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and bonito elastin powder treatment group, KE30 significantly increased by 28.6, 26.5, and 24.1%, respectively, compared to the ultraviolet irradiation group (C). KE30 was not significantly different from the normal control group (NC) and positive control group 1 (PC1; L-ascorbic acid). The KE10 and KE20 groups showed no significant difference from the UV irradiation group (C).

2) Anti-wrinkle

go) in vitro result

① Cytotoxicity test of bonito elastin powder in HS27 cells

To confirm the cytotoxicity of skipjack elastin powder, MTT assay was performed. Figure 1b shows the cell viability of HS27 human fibroblasts at different concentrations of skipjack elastin. As a result of treating skipjack elastin powder by concentration, all concentration groups showed a significant increase compared to the 0 μg/mL group without sample treatment, and it was judged that skipjack elastin powder did not affect cytotoxicity, based on this After setting the concentration as , the study was conducted.

② Effect of bonito elastin powder on expression of genes related to wrinkle formation in HS27 cells irradiated with UV rays

UVB irradiation affects dermal fibroblasts and causes collagen loss by reducing procollagen type I production through TGF-β and Smad pathways, which are major regulators of procollagen synthesis. Collagen type Ⅰ occupies the largest part in the skin tissue and gives elasticity to the skin. It is decomposed and suppressed by MMP's known as collagenase. 10 shows the results of gene expression measurement of TGFβR1, procollagen type I, and collagen type I, which are wrinkle formation-related factors, in HS27 human fibroblasts irradiated with ultraviolet rays in this study.

As a result of measuring the gene expression level of TGFβR1 (left side of FIG. 10), the UV irradiation group (C) significantly decreased compared to the normal control group (NC). The positive control group (PC) and skipjack elastin powder treatment group (KE50, KE100, KE200, KE400) showed a significant increase of 126.4, 79.9, 104.5, 112.6, and 172.1%, respectively, compared to the UV irradiation group (C), and KE400 The group showed no significant difference from the normal control group (NC).

As a result of measuring the gene expression level of procollagen type I (middle of FIG. 10), the UV irradiation group (C) significantly decreased compared to the normal control group (NC). The positive control group (PC) and the bonito elastin powder treatment group (KE50, KE100, KE200, KE400) showed a significant increase of 74.5, 40.1, 60.2, 66.2, and 100.7%, respectively, compared to the UV irradiation group (C), and the KE400 The group showed no significant difference from the normal control group (NC).

As a result of measuring the gene expression level of collagen type I (right side of FIG. 10), the UV irradiation group (C) significantly decreased compared to the normal control group (NC). Among the positive control group (PC) and the bonito elastin powder treatment group, P200 and P400 showed significant increases by 66.3, 42.1, and 71.2%, respectively, compared to the ultraviolet irradiation group (C), and the KE200 and KE400 groups were the positive control group (PC) did not show any significant difference with The KE50 and KE100 groups showed no significant difference from the UV irradiation group (C).

③ Effect of bonito elastin powder on the expression of proteins related to wrinkle generation in HS27 cells irradiated with UV rays

UVB irradiation activates the MAPK pathway by generating reactive oxygen species (ROS). Among the MAPK pathways, phosphorylation of JNK further increases MMP expression through phosphorylation of c-Fos and c-Jun. In particular, it induces the expression of MMPs 1,3,9 to degrade collagen and other connective tissue components. MMP-1 is a collagenase that decomposes collagen type I, a major component of the skin dermis, MMP-3 activates inactivated pro-MMP-1, and MMP-9 additionally hydrolyzes collagen fragments. In this study, the protein expression of MAPK8 (JNK), c-Fos, c-Jun, MMP's (1, 3, 9), and Smad3, which are wrinkle formation-related factors, were measured in HS27 human fibroblasts irradiated with ultraviolet rays, and the results are shown in FIGS. shown in 12.

As a result of measuring the protein expression level of MAPK8 (JNK), there was a significant increase in the UV-irradiated group (C) compared to the normal control group (NC). The positive control group (PC) and skipjack elastin powder treatment groups (KE50, KE100, KE200, KE400) showed significant reductions of 40.6, 33.1, 38.4, 54.3, and 56.1%, respectively, compared to the UV irradiation group (C).

As a result of measuring the protein expression level of c-Fos, the UV-irradiated group (C) significantly increased compared to the normal control group (NC). The positive control group (PC) and skipjack elastin powder treatment groups (KE50, KE100, KE200, KE400) showed significant reductions of 23.7, 29.0, 57.3, 62.2, and 55.9%, respectively, compared to the UV irradiation group (C). In particular, the KE100 and KE400 groups did not show a significant difference from the normal control group (NC), and the KE200 group showed a decrease compared to the normal control group (NC).

As a result of measuring the protein expression level of c-Jun, the UV-irradiated group (C) significantly increased compared to the normal control group (NC). The positive control group (PC) and the bonito elastin powder treatment group (KE50, KE100, KE200, KE400) showed significant decreases by 40.0, 14.2, 47.7, 47.4, and 46.7%, respectively, compared to the UV irradiation group (C), and the KE100 , KE200 and KE400 groups showed no significant difference from the normal control group (NC).

As a result of measuring the protein expression level of MMP-1, the ultraviolet irradiation group (C) significantly increased compared to the normal control group (NC). The positive control group (PC) and the bonito elastin powder treatment group (KE50, KE100, KE200, KE400) showed a significant decrease by 29.2, 25.0, 22.6, 26.6, and 32.3%, respectively, compared to the UV irradiation group (C), and the KE50 , KE200 and KE400 groups showed no significant difference from the positive control group (PC).

As a result of measuring the protein expression level of MMP-3, the UV irradiation group (C) significantly increased compared to the normal control group (NC). Skipjack elastin powder treatment groups (KE50, KE100, KE200, KE400) showed significant reductions by 14.8, 28.9, 26.4, and 23.9%, respectively, compared to the UV irradiation group (C), and the KE100, KE200, and KE400 groups were the normal control group. (NC) did not show a significant difference. The positive control group (PC) did not show a significant difference from the UV irradiation group (C).

As a result of measuring the protein expression level of MMP-9, the UV irradiation group (C) significantly increased compared to the normal control group (NC). The positive control group (PC) and skipjack elastin powder treatment groups (KE50, KE100, KE200, KE400) showed significant reductions of 44.4, 29.3, 39.1, 37.5, and 50.1%, respectively, compared to the UV irradiation group (C).

As a result of measuring the protein expression level of Smad3, the UV-irradiated group (C) significantly decreased compared to the normal control group (NC). Among the positive control group (PC) and skipjack elastin powder treatment group, KE400 showed a significant increase of 64.0 and 26.8%, respectively, compared to the ultraviolet irradiation group (C). KE50, KE100, and KE200 showed no significant difference from the UV irradiation group (C).

④ Effect of bonito elastin powder on secretion of inflammatory cytokines (IL-1β, IL-6, TNF-α) in HS27 cells irradiated with UV rays

When exposed to UVB, epidermal hyperplasia and erythema appear, and inflammatory cytokines are released during this process. Excessively increased reactive oxygen species (ROS) activate various receptors and growth factors on the cell surface and increase the activation of transcription factors such as NF-kB, AP-1, and MAPK. Then, it causes overexpression of inflammatory cytokines and MMPs and reduces the synthesis of procollagen. IL-1, IL-6, IL-8, TNF-α, and IL-10 are the most expressed cytokines in the skin, and these factors cause inflammation, immunosuppression, and wrinkle formation through degradation of the extracellular matrix. do. In this study, the effect on the secretion of inflammatory cytokines (IL-1β, IL-6, TNF-α) in HS27 human fibroblasts irradiated with ultraviolet rays was measured, and the results are shown in FIG. 13 .

As a result of measuring the amount of IL-1β secretion (left side of FIG. 13), there was a significant increase in the UV irradiation group (C) compared to the normal control group (NC). Among the positive control group (PC) and bonito elastin powder treatment group, KE100, KE200, and KE400 showed significant reductions by 24.1, 19.8, 19.5, and 29.8%, respectively, compared to the UV irradiation group (C), and KE400 showed a significant decrease in the normal control group (NC ) did not show a significant difference. The KE50 group showed no significant difference from the UV irradiation group (C).

As a result of measuring the amount of IL-6 secretion (middle of FIG. 13), the UV irradiation group (C) significantly increased compared to the normal control group (NC). The positive control group (PC) and the bonito elastin powder treatment group (KE50, KE100, KE200, KE400) showed a significant decrease by 37.0, 17.0, 33.7, 37.5, and 40.2%, respectively, compared to the UV irradiation group (C), and the KE100 , KE200 and KE400 groups showed no significant difference from the positive control group (PC).

As a result of measuring the amount of TNF-α secretion (right side of FIG. 13), there was a significant increase in the UV irradiation group (C) compared to the normal control group (NC). The positive control group (PC) and the bonito elastin powder treatment group (KE50, KE100, KE200, KE400) showed a significant decrease by 36.4, 19.5, 24.3, 32.9, and 35.9%, respectively, compared to the UV irradiation group (C), and the KE100 , KE200 and KE400 groups showed no significant difference from the positive control group (PC).

me) in vivo result

① Morphological observation of experimental animal skin

14 shows the morphological observation results of the back skin tissue after irradiating the skin of the experimental animal with ultraviolet rays. After UVB irradiation, wrinkle formation began to noticeably increase from the 4th week, and keratin formation and elasticity loss also increased due to dryness due to photoaging. As a result of checking the degree of wrinkle formation before sacrificing the experimental animals after 8 weeks, it was confirmed that deep wrinkles and dead skin cells were formed in the ultraviolet irradiation group (C) compared to the normal control group (NC). Positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and bonito elastin powder treatment groups (KE10, KE20, KE30) showed that the degree of wrinkle formation was suppressed compared to the ultraviolet irradiation group (C). It was confirmed, and in particular, it was confirmed that wrinkle formation and keratin formation were significantly suppressed in the KE30 group to the extent that they could be distinguished with the naked eye.

② Histopathological observation of the skin of experimental animals (H&E staining)

When the skin barrier function is damaged, abnormal symptoms such as a decrease in the water content of the stratum corneum and an increase in transepidermal water loss are observed. In order to keep it normal, abnormal metabolism and excessive decomposition of ceramides in moisture-preserving substances occur, and the skin surface changes to a rough state due to epidermal thickening and abnormal thickness increase of the stratum corneum. In order to confirm this, after irradiating the skin of the experimental animal with ultraviolet rays, histopathological changes were observed through H&E staining in the back skin tissue, and the results are shown in FIG. 15.

As a result of observation through H&E staining, it was confirmed that the thickness of the epidermis significantly increased in the ultraviolet irradiation group (C) compared to the normal control group (NC). Positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and bonito elastin powder treatment groups (KE10, KE20, KE30) showed a decrease in epidermal thickness compared to the ultraviolet irradiation group (C). Significantly decreased results were shown in the KE20 and KE30 groups.

③ Effect of bonito elastin powder on the expression of genes related to wrinkle formation in the skin tissue of the experimental animals

FIG. 16 shows the gene expression measurement results of TGFβR1, procollagen type I, and collagen type I, which are wrinkle formation-related factors, in the skin tissues of the experimental animals irradiated with ultraviolet rays.

As a result of measuring the gene expression level of TGFβR1 (left side of FIG. 16), the UV irradiation group (C) significantly decreased compared to the normal control group (NC). Positive control group 1 (PC1; L-ascorbic acid) and bonito elastin powder treatment groups (KE10, KE20, KE30) showed significant increases by 63.4, 37.6, 47.0, and 89.9%, respectively, compared to the UV irradiation group (C). , KE30 did not show a significant difference from the normal control group (NC). The positive control group 2 (PC2; Arbutin) group did not show a significant difference from the UV irradiation group (C).

As a result of measuring the gene expression level of procollagen type I (middle of FIG. 16), the UV irradiation group (C) significantly decreased compared to the normal control group (NC). Among the positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and skipjack elastin powder treatment group, KE20 and KE30 were 129.5, 51.0, 61.8, and 83.7% respectively compared to the UV irradiation group (C). showed an increase in KE10 did not show a significant difference from the ultraviolet irradiation group (C).

As a result of measuring the gene expression level of collagen type I (right side of FIG. 16), the UV irradiation group (C) significantly decreased compared to the normal control group (NC). Positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and bonito elastin powder treatment groups (KE10, KE20, KE30) were 93.7, 42.5, 26.8, 35.2, respectively, compared to the UV irradiation group (C). , showed a significant increase of 94.9%, and KE30 did not show a significant difference from the positive control group 1 (PC1; L-ascorbic acid).

④ Effect of bonito elastin powder on the expression of proteins related to wrinkle formation in the skin tissue of the experimental animals

17 and 18 show the results of measuring the protein expression of MAPK8 (JNK), c-Fos, c-Jun, MMP's (1, 3, 9), and Smad3, which are wrinkle formation-related factors, in the back skin tissues of experimental animals irradiated with ultraviolet rays.

As a result of measuring the protein expression level of MAPK8 (JNK), there was a significant increase in the UV-irradiated group (C) compared to the normal control group (NC). Positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin) and bonito elastin powder treatment groups (KE10, KE20, KE30) were 19.0, 29.6, 30.5, 32.7 compared to the UV irradiation group (C), respectively. , showed a significant decrease by 36.0%.

As a result of measuring the protein expression level of c-Fos, the UV-irradiated group (C) significantly increased compared to the normal control group (NC). Positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and bonito elastin powder treatment groups (KE10, KE20, KE30) were 39.4, 44.1, 37.3, 47.9 compared to the UV irradiation group (C), respectively. , 49.0% showed a significant decrease. In particular, the KE10 group showed no significant difference from the normal control group (NC), and the KE20 and KE30 groups showed a decrease compared to the normal control group (NC).

As a result of measuring the protein expression level of c-Jun, the UV-irradiated group (C) significantly increased compared to the normal control group (NC). Positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and bonito elastin powder treatment groups (KE10, KE20, KE30) were 18.9, 36.2, 30.3, 31.9, respectively, compared to the UV irradiation group (C). , showed a significant decrease by 37.8%. In particular, the KE10 and KE20 groups did not show a significant difference from the normal control group (NC), and the KE30 showed a decrease compared to the normal control group (NC).

As a result of measuring the protein expression level of MMP-1, the ultraviolet irradiation group (C) significantly increased compared to the normal control group (NC). Positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and bonito elastin powder treatment group (KE10, KE20, KE30) were 30.1, 17.0, 11.8, 17.1, respectively, compared to the UV irradiation group (C). , showed a significant decrease by 29.5%, and the KE30 group showed no significant difference from the positive control group 1 (PC1; L-ascorbic acid).

As a result of measuring the protein expression level of MMP-3, the UV irradiation group (C) significantly increased compared to the normal control group (NC). Among the positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and skipjack elastin powder treatment group, KE20 and KE30 were 16.7, 7.9, 23.1, and 30.6% respectively compared to the UV irradiation group (C). resulted in a decrease in The KE10 group showed no significant difference from the ultraviolet irradiation group (C).

As a result of measuring the protein expression level of MMP-9, the UV irradiation group (C) significantly increased compared to the normal control group (NC). Positive control group 2 (PC2; Arbutin) and bonito elastin powder treatment groups (KE10, KE20, KE30) showed significant reductions by 37.6, 30.2, 36.0, and 30.8%, respectively, compared to the UV irradiation group (C), and the KE20 group showed no significant difference from the normal control group (NC). Positive control group 1 (PC1; L-ascorbic acid) showed no significant difference from UV irradiation group (C).

As a result of measuring the protein expression level of Smad3, the UV-irradiated group (C) significantly decreased compared to the normal control group (NC). Positive control group 1 (PC1; L-ascorbic acid) and bonito elastin powder treatment groups (KE10, KE20, KE30) showed significant increases of 122.5, 47.2, 20.8, 21.0, and 78.2%, respectively, compared to the UV irradiation group (C). KE30 showed no significant difference from the normal control group (NC).

⑤ Effect of skipjack elastin powder on the activity of antioxidant enzymes (SOD, Catalase, GPx) in the back skin tissue of experimental animals

Reactive oxygen species (ROS) generated when excessive exposure to UVB causes cell damage and cell death and promotes photoaging. In addition, it induces oxidative damage by causing damage to lipids, proteins, collagen, DNA, etc., depletion of intercellular antioxidants, deterioration of cell function due to increased intracellular lipid peroxidation concentration, skin inflammation, and the like. Free radicals generated by UVB irradiation are converted into H ₂ O ₂ by SOD, and catalase and GPx act as antioxidants to remove hydrogen peroxide through detoxification. In this study, antioxidant enzymes such as SOD, Catalase, and GPx activity were measured in the back skin tissues of experimental animals irradiated with ultraviolet rays, and the results are shown in FIG. 19.

As a result of measuring SOD activity (left side of FIG. 19), the UV irradiation group (C) significantly decreased compared to the normal control group (NC). Among the positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and skipjack elastin powder treatment group, KE20 and KE30 were significantly higher than those of the UV irradiation group (C) by 34.8, 25.0, 18.9, and 21.6%, respectively. , and the KE30 group showed no significant difference from the positive control group 1 (PC1; L-ascorbic acid). The KE10 group showed no significant difference from the ultraviolet irradiation group (C).

As a result of measuring catalase activity (middle of FIG. 19), the ultraviolet irradiation group (C) significantly decreased compared to the normal control group (NC). Positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and bonito elastin powder treatment group (KE10, KE20, KE30) were 90.0, 53.4, 36.3, 69.4 compared to the UV irradiation group (C), respectively. , 72.9% showed a significant increase.

As a result of GPx activity measurement (right side of FIG. 19), the UV irradiation group (C) significantly decreased compared to the normal control group (NC). Positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and bonito elastin powder treatment groups (KE10, KE20, KE30) were 46.7, 31.0, 14.7, 31.5 compared to the UV irradiation group (C), respectively. , showed a significant increase of 37.3%, and the KE30 group showed no significant difference from the positive control group 1 (PC1; L-ascorbic acid).

3) whitening

go) in vitro result

① Cytotoxicity test of skipjack elastin powder in B16F10 cells

To confirm the cytotoxicity of skipjack elastin powder, MTT assay was performed. Figure 1c shows the cell viability of B16F10 melanocytes at different concentrations of skipjack elastin. As a result of treating skipjack elastin powder by concentration, all concentration groups showed a gradual increase compared to the 0 μg/mL group without sample treatment, and it was judged that skipjack elastin powder did not affect cytotoxicity. After setting the concentration based on the background, the study was conducted.

② Effect of skipjack elastin powder on melanin production inhibition in B16F10 cells

Melanin synthesis occurs in melanocytes and melanoma cells in an enzymatic process catalyzed by tyrosinase, TRP-1 and TRP-2, and excessive pigmentation occurs in the skin due to the increased number of melanocytes caused by the activity of melanin-producing enzymes. IBMX increases the expression of tyrosinase, TRP-1 and TRP-2 through the PKA pathway by regulating the activation of transcription factors such as MITF and CREB. Involved in pigmentation. In this study, the ability to inhibit melanin production was measured in B16F10 melanocytes differentiated into IBMX, and the results are shown in FIG. 20 .

As a result of measuring the melanin production inhibitory ability, it was confirmed that melanin synthesis was induced by IBMX as the differentiated control group (C) significantly increased compared to the normal control group (NC). The positive control group (PC) and skipjack elastin powder treatment group (KE50, KE100, KE200, KE400) showed significant reductions of 63.1, 28.6, 30.7, 32.6, and 43.2%, respectively, compared to the differentiated control group (C).

③ Effect of bonito elastin powder on whitening-related protein expression in B16F10 cells

Melanin is produced by various factors such as α-melanocyte stimulating hormone, cAMP, nitric oxide (NO), and estrogen. Increased intracellular cAMP activates PKA to increase phosphorylation of CREB, which leads to activation of MITF. MITF regulates the growth, differentiation and function of melanocytes by activating enzymes involved in melanin production, such as tyrosinase, the rate-limiting enzyme of melanin synthesis, and TRP-1 and TRP-2, which regulate subsequent synthesis steps in melanocytes. In this study, the protein expression of PKA, CREB, MITF, TRP-1, and TRP-2, which are whitening-related factors, were measured in B16F10 melanocytes differentiated into IBMX, and the results are shown in FIGS. 21 and 22 .

As a result of measuring the protein expression level of PKA, the differentiated control group (C) significantly increased compared to the normal control group (NC). Among the positive control group (PC) and skipjack elastin powder treatment group, KE100, KE200, and KE400 showed a significant decrease by 39.0, 24.5, 31.2, and 35.9%, respectively, compared to the differentiated control group (C), and the positive control group (PC) and KE200 , The KE400 group did not show a significant difference from the normal control group (NC). The KE50 group did not show a significant difference from the differentiation control group (C).

As a result of measuring the protein expression level of CREB, the differentiated control group (C) significantly increased compared to the normal control group (NC). The positive control group (PC) and skipjack elastin powder treatment group (KE50, KE100, KE200, KE400) showed significant reductions by 13.3, 19.9, 35.2, 30.9, and 56.0%, respectively, compared to the differentiated control group (C), and the KE400 group showed no significant difference from the normal control group (NC).

As a result of measuring the protein expression level of MITF, the differentiated control group (C) significantly increased compared to the normal control group (NC). The positive control group (PC) and the bonito elastin powder treatment group (KE50, KE100, KE200, KE400) showed significant reductions by 45.9, 16.3, 33.8, 72.5, and 76.0%, respectively, compared to the differentiated control group (C). The KE400 group showed no significant difference from the normal control group (NC).

As a result of measuring the protein expression level of TRP-1, the differentiated control group (C) significantly increased compared to the normal control group (NC). The positive control group (PC) and skipjack elastin powder treatment group (KE50, KE100, KE200, KE400) showed significant reductions by 44.8, 45.7, 48.6, 53.8, and 46.4%, respectively, compared to the differentiated control group (C), and the KE200 group showed no significant difference from the normal control group (NC).

As a result of measuring the protein expression level of TRP-2, the differentiated control group (C) significantly increased compared to the normal control group (NC). The positive control group (PC) and the bonito elastin powder treatment group (KE50, KE100, KE200, KE400) showed significant reductions of 16.8, 24.6, 27.5, 28.9, and 49.4%, respectively, compared to the differentiated control group (C).

④ Effect of bonito elastin powder on tyrosinase activity in B16F10 cells

23 shows the results of measuring tyrosinase activity in B16F10 melanocytes differentiated with IBMX.

As a result of measuring Tyrosinase activity, the differentiated control group (C) significantly increased compared to the normal control group (NC). The positive control group (PC) and the bonito elastin powder treatment group (KE50, KE100, KE200, KE400) showed significant reductions of 43.4, 15.3, 18.3, 24.4, and 34.8%, respectively, compared to the differentiated control group (C).

⑤ Effects of bonito elastin powder on Nitric oxide (NO), Glutathione (GSH), and cAMP levels in B16F10 cells

Nitric oxide (NO), a free radical gas, is considered as an intracellular and intercellular messenger molecule and causes melanin synthesis by increasing cGMP content and MITF through activation of guanylate cyclase. Glutathione (GSH) is an antioxidant that can prevent cell damage caused by reactive oxygen species. When is increased, the amount of reduced form of glutathione (GSH) is reduced or its synthesis is inhibited. An increase in cAMP increases melanin synthesis through activation of the PKA pathway. In this study, Nitric oxide (NO), Glutathione (GSH), and cAMP levels were measured in B16F10 melanocytes differentiated into IBMX, and the results are shown in FIG. 24 .

As a result of Nitric oxide (NO) measurement (left side of FIG. 24), the differentiated control group (C) significantly increased compared to the normal control group (NC). The positive control group (PC) and the bonito elastin powder treatment group (KE50, KE100, KE200, KE400) showed significant reductions of 54.3, 28.9, 29.5, 33.2, and 40.8%, respectively, compared to the differentiated control group (C).

Glutathione (GSH) measurement results (middle of FIG. 24) showed a significant decrease in the differentiated control group (C) compared to the normal control group (NC). The positive control group (PC) and skipjack elastin powder treatment group (KE50, KE100, KE200, KE400) showed a significant increase of 105.2, 21.7, 46.2, 46.0, and 97.6%, respectively, compared to the differentiated control group (C), and the KE400 group showed no significant difference from the positive control group (PC).

As a result of cAMP measurement (right side of FIG. 24 ), the differentiated control group (C) significantly increased compared to the normal control group (NC). The positive control group (PC) and skipjack elastin powder treatment group (KE50, KE100, KE200, KE400) showed significant reductions by 51.2, 25.1, 27.5, 35.9, and 43.2%, respectively, compared to the differentiated control group (C), and the KE400 group showed no significant difference from the positive control group (PC).

me) in vivo result

① Effect of bonito elastin powder on the expression of whitening-related proteins in the skin tissue of the experimental animals

25 and 26 show the results of measuring the protein expression of PKA, CREB, MITF, TRP-1, and TRP-2, which are whitening-related factors, in the back skin tissues of experimental animals irradiated with ultraviolet rays.

As a result of measuring the protein expression level of PKA, the ultraviolet irradiation group (C) significantly increased compared to the normal control group (NC). Among the positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and skipjack elastin powder treatment group, KE20 and KE30 were 27.4, 27.9, 18.6, and 17.7% respectively compared to the UV irradiation group (C). , and the KE20 and KE30 groups showed no significant difference from the positive control group 2 (PC2; Arbutin). The KE10 group showed no significant difference from the ultraviolet irradiation group (C).

As a result of measuring the protein expression level of CREB, the ultraviolet irradiation group (C) significantly increased compared to the normal control group (NC). Positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and bonito elastin powder treatment groups (KE10, KE20, KE30) were 24.0, 26.0, 19.2, 20.8, respectively, compared to the UV irradiation group (C). , showed a significant decrease by 29.5%, and the KE30 group showed no significant difference from the normal control group (NC) and the positive control group 2 (PC2; Arbutin).

As a result of measuring the protein expression level of MITF, the ultraviolet irradiation group (C) significantly increased compared to the normal control group (NC). Positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and bonito elastin powder treatment groups (KE10, KE20, KE30) were 39.2, 55.9, 23.4, 29.3 compared to the UV irradiation group (C), respectively. , showed a significant decrease by 40.5%, and the KE30 group showed no significant difference from the normal control group (NC).

As a result of measuring the protein expression level of TRP-1, there was a significant increase in the UV irradiation group (C) compared to the normal control group (NC). Positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and bonito elastin powder treatment groups (KE10, KE20, KE30) were 24.7, 32.1, 15.3, 20.4 compared to the UV irradiation group (C), respectively. , showed a significant decrease by 33.1%, and the KE30 group showed no significant difference from the normal control group (NC) and the positive control group 2 (PC2; Arbutin).

As a result of measuring the protein expression level of TRP-2, the UV irradiation group (C) significantly increased compared to the normal control group (NC). Positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and bonito elastin powder treatment groups (KE10, KE20, KE30) were 43.3, 64.0, 54.1, 66.8, respectively, compared to the UV irradiation group (C). , showed a significant decrease by 65.8%, and the KE20 and KE30 groups showed no significant difference from the positive control group 2 (PC2; Arbutin).

② Effect of bonito elastin powder on tyrosinase activity in the back skin tissue of experimental animals

27 shows the results of measuring tyrosinase activity in the back skin tissues of experimental animals irradiated with ultraviolet rays.

As a result of measuring Tyrosinase activity, the ultraviolet irradiation group (C) significantly increased compared to the normal control group (NC). Positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and bonito elastin powder treatment groups (KE10, KE20, KE30) were 21.6, 37.5, 14.6, 24.2, respectively, compared to the UV irradiation group (C). , significantly decreased by 28.6%.

③ Effect of bonito elastin powder on nitric oxide (NO) levels in the skin tissues of the experimental animals

The nitric oxide (NO) level measurement results in the back skin tissues of the experimental animals irradiated with ultraviolet rays are shown on the left side of FIG. 28 .

As a result of Nitric oxide (NO) measurement, the ultraviolet irradiation group (C) significantly increased compared to the normal control group (NC). Positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and bonito elastin powder treatment groups (KE10, KE20, KE30) were 29.4, 34.4, 11.3, 22.5 compared to the UV irradiation group (C), respectively. , decreased significantly by 28.1%, and the KE30 group showed no significant difference from the positive control group 2 (PC2; Arbutin).

④ Effect of bonito elastin powder on cAMP levels in the back skin tissue of experimental animals

The cAMP level measurement results in the back skin tissues of the experimental animals irradiated with ultraviolet rays are shown on the right side of FIG. 28 .

As a result of cAMP measurement, the UV irradiation group (C) significantly increased compared to the normal control group (NC). Among the positive control group 1 (PC1; L-ascorbic acid), positive control group 2 (PC2; Arbutin), and skipjack elastin powder treatment group, KE20 and KE30 were 23.2, 39.0, 23.8, and 24.4% respectively compared to the UV irradiation group (C). decreased to The KE10 group showed no significant difference from the ultraviolet irradiation group (C).

4. Conclusion

1) moisturizing

- As a result of checking the cell viability to confirm the experimental appropriate concentration of bonito elastin powder, there was no significant difference from 0 μg/mL without sample treatment up to the concentration of 100-1000 μg/mL, and the safety range up to 1000 μg/mL concentration Considering the concentration, 50, 100, 200, and 400 μg/mL were set as the experimental concentration.

- As a result of measuring the expression of skin moisturizing related genes in UV-irradiated HaCaT keratinocytes, UGTrel7, GlcNAc, Elastin, and DEGS1 showed the greatest increase at the concentration of 400 μg/mL among the skipjack elastin powder-treated group compared to the UV-irradiated group (C). and the 400 μg/mL group of Elastin and DEGS1 showed no significant difference from the normal control group (NC). LCB1 (SPT) showed the greatest increase in the 200 μg/mL group of the skipjack elastin powder treatment group compared to the UV irradiation group (C).

- As a result of measuring the expression of skin moisturizing related proteins in UV-irradiated HaCaT keratinocytes, HAS2 was higher than the normal control group (NC) in all groups except for the concentration of 50 μg/mL among the skipjack elastin powder-treated group compared to the UV-irradiated group (C). There was no significant difference, and CerS4 (LASS4) showed the greatest increase in the 400 μg/mL group among the bonito elastin powder treatment group compared to the UV irradiation group (C).

- As a result of measuring hyaluronic acid and sphingomyelin secretion in UV-irradiated HaCaT keratinocytes, the highest increase was shown at the concentration of 400 μg/mL among the skipjack elastin powder-treated group compared to the UV-irradiated group (C), and hyaluronic acid at 400 μg /mL group increased more than the normal control group (NC), and the 400 μg/mL group of sphingomyelin showed no significant difference from the normal control group (NC).

- Animal test concentrations of skipjack elastin powder were set at 10, 20, and 30 mg/kg·b.w. calculated using the mouse conversion factor (12.33) based on the human test concentration of 100 mg/day.

- As a result of confirming the effect of bonito elastin powder on the body weight change, food intake, diet efficiency and organ weight of the experimental animals during the animal experiment, there was no significant difference from the normal control group (NC), so the intake of bonito elastin powder It was confirmed that it did not affect the experiment.

- As a result of confirming the effect of intake of bonito elastin powder on the epidermal moisture content of experimental animals, the group ingesting bonito elastin powder significantly increased compared to the UV irradiation group (C), in particular, the 30 mg/kg b.w. showed no significant difference from the normal control group (NC).

- As a result of confirming the effect on the expression of moisturizing-related genes in the skin tissues of experimental animals that ingested skipjack tuna elastin powder, UGTrel8, GlcNAc, Fibfillin-1, LCB1 (SPT), and DEGS1 all consumed skipjack tuna elastin powder compared to the UV irradiation group (C) Among the groups, the increase was the highest in the 30 mg/kg b.w. group, and the 30 mg/kg b.w. group of Fbrillin-1, LCB1 (SPT), and DEGS1 showed a significant difference from the positive control group 1 (PC1; L-ascorbic acid). Didn't see.

- As a result of confirming the effect on the expression of moisturizing-related proteins in the skin tissues of experimental animals that consumed bonito elastin powder, both HAS2 and CerS4 (LASS4) were 30 mg/kg·b.w. , and the 30 mg/kg·b.w. group of CerS4 (LASS4) showed no significant difference from the normal control group (NC).

2) Wrinkles

- As a result of checking the cell viability to confirm the experimental appropriate concentration of skipjack elastin powder, it was significantly increased compared to the 0 μg/mL group without sample treatment up to 100-1000 μg/mL concentration, and it is safe up to 1000 μg/mL concentration Considering the range concentration, 50, 100, 200, 400 μg/mL was set as the experimental concentration.

- As a result of measuring the expression of wrinkle formation-related genes in HS27 human fibroblasts irradiated with ultraviolet rays, TGFβR1, procollagen type Ⅰ, and collagen type Ⅰ increased the most in the 400 μg/mL group among the bonito elastin powder treatment group compared to the ultraviolet irradiation group (C) In particular, the 400 μg/mL group of TGFβR1 and procollagen type I showed no significant difference from the normal control group (NC).

- As a result of measuring the expression of wrinkle formation-related proteins in HS27 human fibroblasts irradiated with ultraviolet light, MAPK (JNK) showed the greatest decrease in the 200 and 400 μg/mL group among the skipjack elastin powder treated groups compared to the ultraviolet irradiated group (C), c-Fos showed the greatest decrease in the 200 μg/mL group among the bonito elastin powder treated groups compared to the ultraviolet irradiation group (C), and showed a decrease compared to the normal control group (NC). c-Jun showed no significant difference from the normal control group (NC) in the 100, 200, and 400 μg/mL groups among the skipjack elastin powder treated groups compared to the ultraviolet irradiation group (C), and MMP-1 and MMP-9 Compared to the irradiation group (C), the most decreased in the 400 μg/mL group among the bonito elastin powder treatment groups. MMP-3 showed no significant difference from the normal control group (NC) in the 100, 200, and 400 μg/mL groups among the bonito powder treated groups compared to the ultraviolet irradiation group (C). Smad3 showed the greatest increase in the 400 μg/mL group among the bonito powder treated groups compared to the UV irradiation group (C).

- As a result of measuring the amount of inflammatory cytokine secretion in HS27 human fibroblasts irradiated with ultraviolet rays, IL-1β, IL-6, and TNF-α were the highest in the 400 μg/mL group among the skipjack elastin powder-treated groups compared to the ultraviolet irradiation group (C). decreased, and the IL-1β 400 μg/mL group showed no significant difference from the normal control group (NC).

- Animal test concentrations of bonito elastin powder were set at 10, 20, and 30 mg/kg·b.w. calculated using the mouse conversion factor (12.33) based on the human test concentration of 100 mg/day.

- As a result of confirming the effect of intake of bonito elastin powder on skin wrinkle formation of experimental animals, wrinkle formation and keratin formation were most inhibited in the 30 mg/kg b.w. group of the 30 mg/kg b.w. .

- As a result of histopathological observation of the skin tissue of experimental animals that ingested skipjack tuna elastin powder, the thickness of the epidermis decreased the most in the 20 and 30 mg/kg b.w. showed results.

- As a result of confirming the effect on the expression of genes related to wrinkle formation in the skin tissues of experimental animals that ingested skipjack tuna elastin powder, TGFβR1, procollagen type Ⅰ, and collagen type Ⅰ were all 30 mg of the group consuming skipjack tuna elastin powder compared to the ultraviolet irradiation group (C) /kg·b.w. group showed the greatest increase, and in particular, the 30 mg/kg·b.w. group of TGFβR1 showed no significant difference from the normal control group (NC).

- As a result of measuring the expression of wrinkle formation-related proteins in the skin tissues of experimental animals that ingested skipjack tuna elastin powder, MAPK (JNK) was the highest in the 30 mg/kg b.w. decreased a lot. c-Fos decreased the most in the 20 and 30 mg/kg·b.w. groups among the skipjack elastin powder intake groups compared to the ultraviolet irradiation group (C), and showed a decrease compared to the normal control group (NC). c-Jun decreased the most in the 30 mg/kg·b.w. group among the bonito elastin powder intake group compared to the ultraviolet irradiation group (C) and decreased more than the normal control group (NC). MMP-1 and MMP-3 decreased the most in the 30 mg/kg b.w. group among the bonito elastin powder intake group compared to the ultraviolet irradiation group (C), and MMP-9 decreased the most in the skipjack tuna elastin powder intake group compared to the ultraviolet irradiation group (C) The most decreased in the 20 mg/kg·b.w. group and showed no significant difference from the normal control group (NC). Smad3 increased the most in the 30 mg/kg·b.w. group among the skipjack elastin powder intake groups compared to the ultraviolet irradiation group (C), and showed no significant difference from the normal control group (NC).

- As a result of measuring the activity of antioxidant enzymes in the skin tissues of experimental animals that ingested bonito elastin powder, SOD and GPx increased the most in the 30 mg/kg b.w. group among the 30 mg/kg b.w. Catalase increased the most in the 20, 30 mg/kg b.w.

3) whitening

- As a result of checking the cell viability to confirm the experimental appropriate concentration of skipjack elastin powder, it showed a tendency to increase in a concentration-dependent manner compared to the 0 μg/mL group without sample treatment up to 100-1000 μg/mL concentration. Considering the concentration up to mL as a safe range concentration, 50, 100, 200, and 400 μg/mL were set as concentrations for the experiment.

- As a result of measuring the ability to inhibit melanin production in B16F10 melanin-producing cells differentiated with IBMX, the 400 μg/mL group showed the greatest decrease among the bonito elastin powder-treated group compared to the differentiation control group (C).

- As a result of measuring the expression of whitening-related proteins in B16F10 melanin-producing cells differentiated with IBMX, PKA, CREB, and MITF decreased the most in the 400 μg/mL group among the bonito elastin powder-treated group compared to the differentiation control group (C), and the normal control group ( NC) did not show a significant difference. TRP-1 decreased the most in the 200 μg/mL group among the bonito powder treated groups compared to the differentiated control group (C), and showed no significant difference from the normal control group (NC). TRP-2 decreased the most in the 400 μg/mL group among the bonito powder treated groups compared to the differentiation control group (C).

- As a result of measuring tyrosinase activity in B16F10 melanin-producing cells differentiated with IBMX, it was most decreased in the 400 μg/mL group among the skipjack elastin powder-treated group compared to the differentiation control group (C).

- As a result of measuring nitric oxide (NO), glutathione (GSH), and cAMP levels in B16F10 melanin-producing cells differentiated with IBMX, nitric oxide (NO) and cAMP were 400 μg in the bonito elastin powder-treated group compared to the differentiation control group (C). /mL concentration showed the most decrease, and glutathione (GSH) showed the greatest increase in the 400 μg/mL group among the bonito elastin powder treatment group compared to the differentiation control group (C).

- Animal test concentrations of bonito elastin powder were set at 10, 20, and 30 mg/kg·b.w. calculated using the mouse conversion factor (12.33) based on the human test concentration of 100 mg/day.

- As a result of measuring the expression of whitening-related proteins in the skin tissues of experimental animals that ingested skipjack tuna elastin powder, PKA decreased the most in the 20 mg/kg·b.w. group among the 20 mg/kg b.w. CREB, MITF, and TRP-1 decreased the most in the 30 mg/kg·b.w. group among the bonito elastin powder intake group compared to the ultraviolet irradiation group (C), and showed no significant difference from the normal control group (NC). TRP-2 decreased the most in the 20 and 30 mg/kg·b.w. groups among the skipjack elastin powder intake groups compared to the ultraviolet irradiation group (C).

- As a result of measuring the activity of tyrosinase in the skin tissues of experimental animals that ingested skipjack tuna elastin powder, the 30 mg/kg·b.w. group decreased the most among the 30 mg/kg·b.w.

- As a result of measuring the nitric oxide (NO) level in the skin tissues of experimental animals that ingested skipjack tuna elastin powder, the 30 mg/kg·b.w. group decreased the most among the 30 mg/kg·b.w.

- As a result of measuring the cAMP level in the skin tissues of experimental animals that ingested skipjack tuna elastin powder, the most decreased in the 20, 30 mg/kg·b.w.

Judging from the above results, skipjack elastin powder had a concentration of 400 μg/mL in cell experiments and 30 mg/kg·b.w. in animal experiments. concentration appears to be the most effective concentration in moisturizing, wrinkle and whitening related mechanisms. It is judged that skipjack elastin powder can help increase the moisture content of the skin by increasing the expression of moisturizing factors, and help suppress the expression of wrinkle formation-related factors to affect skin elasticity, and the expression of melanin production-related factors It is thought to be able to help with whitening by adjusting.

<Clinical trial study to confirm the functionality of the raw material composition>

1. A 12-week, randomized, double-blind, placebo-controlled human application study to evaluate the efficacy and safety of skipjack elastin on skin wrinkle improvement and moisturization

1) Test process

The human application test was conducted on men and women with wrinkles and dry skin between the ages of 35 and 60.

The number of test subjects is as follows.

For the test food, the raw material composition (bonito elastin) according to Example 1 was used as the test food and a control food (placebo) was used, and the test subjects were given the test food or the control food once a day for 12 weeks, once a day. Capsules were ingested (test food was 100mg/day). Subjects were randomly assigned to test groups or control groups according to gender and age (less than 50 years old, over 50 years old). The test process is as follows.

2) Efficacy and safety evaluation variables

The primary efficacy evaluation variables are skin wrinkles (visual evaluation, Mark-Vu, PRIMOS ^lite ) and the amount of skin moisture, and the secondary efficacy evaluation variables are transepidermal water loss, skin elasticity, whitening, eye wrinkle volume, and the degree of improvement of the human body tester Evaluation, human application test The improvement of the subject is also evaluated.

Safety evaluation variables are adverse events, clinical pathology tests (hematology/hematochemical tests, urine tests), vital signs (blood pressure, pulse), and body measurements (weight).

2. Efficacy evaluation results

1) Primary efficacy evaluation

The primary efficacy evaluation of this human application test was the change in skin wrinkles (visual evaluation, Mark-Vu, PRIMOS ^lite ) and skin moisture, and the degree of improvement between the test group and the control group was analyzed and compared to evaluate whether there was a statistically significant difference. .

As a result of analyzing the change in the skin wrinkle grade of the left and right outer corner of the eye measured by visual evaluation and Mark-Vu with PP Set, the test group decreased by 0.05±0.34 (p=0.3047) and the control group decreased by 0.14±0.41 after 12 weeks of intake. increased (p=0.0325), indicating a statistically significant difference between intake groups (p=0.0365 ^(W) ).

In the results analyzed with the FA set, after 12 weeks of intake, the test group decreased by 0.04±0.31 (p=0.3047) and the control group increased by 0.14±0.40 (p=0.0325), showing a statistically significant difference between the intake groups (p=0.0278). ^(W) ).

As a result of analyzing the change in Ra (average roughness) of the left and right outer corner of the eye measured with PRIMOS ^lite with the PP set, after 12 weeks of intake, the test group decreased by 1.48±0.92μm (p<0.0001), and the control group decreased by 0.09±1.22μm. increased (p=0.6090), indicating a statistically significant difference between intake groups (p<0.0001 ^(W) ).

In the results analyzed with the FA set, after 12 weeks of intake, the test group decreased by 1.58±1.01 μm (p<0.0001), and the control group increased by 0.09±1.21 μm (p=0.6288), showing a statistically significant difference between the intake groups (p <0.0001 ^(W) ).

As a result of analyzing the change in Rmax (maximum height: sum of mountain height and bone depth) of the left and right outer corner of the eye measured with PRIMOS ^lite with PP Set, the test group decreased by 7.45 ± 8.53 μm after 12 weeks of intake (p<0.0001 ), and the control group increased by 1.70±10.40μm (p=0.2782), showing a significant difference between intake groups (p<0.0001 ^[3] ).

In the results analyzed with the FA set, after 12 weeks of intake, the test group decreased by 8.69±8.85 μm (p<0.0001), and the control group increased by 1.42±10.26 μm (p=0.3480), showing a statistically significant difference between the intake groups (p <0.0001 ^[3] ).

As a result of analyzing the change in Rp (maximum cross-sectional mountain height) of the left and right outer corner of the eye measured with PRIMOS ^lite with PP Set, after 12 weeks of intake, the test group decreased by 4.20±9.03μm (p=0.0061) and the control group decreased by 0.08± 8.43μm increased (p=0.9510), showing a statistically significant difference between intake groups (p=0.0420 ^[3] ).

In the results analyzed with the FA set, after 12 weeks of intake, the test group decreased by 4.95±9.25 μm (p=0.0006) and the control group decreased by 0.33±8.55 μm (p=0.7897), showing a statistically significant difference between the intake groups (p=0.7897). =0.0141 ^[3] ).

As a result of analyzing the change in Rv (maximum cross-sectional bone height) of the left and right eyebrows measured with PRIMOS ^lite with PP Set, after 12 weeks of intake, the test group decreased by 4.72±7.28μm (p=0.0004) and the control group decreased by 1.12± 6.90μm increased (p=0.2820), showing a statistically significant difference between intake groups (p=0.0003 ^(W) ).

In the results analyzed with the FA set, after 12 weeks of intake, the test group decreased by 5.34±7.20 μm (p<0.0001), and the control group increased by 1.20±6.93 μm (p=0.2401), showing a statistically significant difference between the intake groups (p <0.0001 ^(W) ).

As a result of analyzing the change in Rz (10-point average roughness) of the left and right outer corner of the eye measured with PRIMOS ^lite with PP Set, after 12 weeks of intake, the test group decreased by 6.87±4.15μm (p<0.0001), and the control group decreased by 0.69± 6.10μm increased (p=0.4500), showing a statistically significant difference between intake groups (p<0.0001 ^[3] ).

In the results analyzed with the FA set, after 12 weeks of intake, the test group decreased by 7.55±4.69μm (p<0.0001), and the control group increased by 0.67±6.05μm (p=0.4485), showing a statistically significant difference between the intake groups (p <0.0001 ^[3] ).

As a result of analyzing the amount of change in skin moisture measured by Corneometer® CM825 with PP Set, the test group increased by 4.24±2.66 AU (p<0.0001) and the control group by 3.06± An increase of 2.15 AU (p<0.0001) showed a statistically significant difference (p=0.0160 ^(W) ).

In the results analyzed with the FA set, after 12 weeks of ingestion, the test group increased by 4.20± 2.97 AU (p<0.0001), and the control group increased by 3.02± 2.12 AU (p<0.0001), showing a statistically significant difference between the intake groups (p<0.0001). =0.0327 ^(W) ).

2) Secondary efficacy evaluation

The secondary efficacy evaluation was to evaluate whether there was a statistically significant difference by analyzing and comparing the degree of improvement between the test group and the control group as the amount of change in whitening and eye wrinkle volume evaluation.

As a result of analyzing the change in the whitening melanin index of the left and right eye corners and the tip of the nose at the right angle intersection measured by Mexameter® MX 18 with PP Set, after 12 weeks of intake, the test group decreased by 2.86± 7.24 (p=0.0181), and the control group decreased by 0.13 A decrease of ± 7.43 (p = 0.9047), indicating a statistically significant difference between intake groups (p = 0.0303 ^[3] ).

In the results analyzed with the FA set, there was no statistically significant difference between intake groups.

As a result of analyzing the change in left and right Eye Wrinkle Volume measured by PRIMOS ^lite with PP Set, after 12 weeks of intake, the test group increased by 4.10± 2.56 mm ³ (p<0.0001), and the control group decreased by 0.05± 2.93 mm ³ ( p=0.6131) There was a statistically significant difference between intake groups (p<0.0001 ^(W) ).

In the results analyzed with the FA set, after 12 weeks of intake, the test group increased by 4.00± 2.54 mm ³ (p<0.0001) and the control group decreased by 0.13± 2.89 mm ³ (p=0.7946), showing a statistically significant difference between the intake groups. (p<0.0001 ^(W) ).

3) Safety evaluation

In the safety evaluation, after being randomly assigned to the human application test, the human application test subject who ingested the food for the human application test at least once was set as the analysis target (Safety Set), and a total of 99 people (50 in the test group and 49 in the control group) applied to the human body Test subjects were included in the Safety Set.

There were 3 adverse reactions from a total of 2 (4.00%) subjects in the test group, and 3 adverse reactions from a total of 3 (6.12%) subjects in the control group, but statistically significant between intake groups. No significant difference was noted (p=0.6777 ^(F) ).

A total of 3 adverse reactions occurred in the test group, and the adverse reactions that occurred were chronic complex periodontitis (Gastro-intestinal system disorders, 2.00%), temporary anxiety (Psychiatric disorders, 2.00%), and Chilblains ([UNKNOWN], 2.00%). . A total of 3 adverse reactions occurred in the control group, and the adverse reactions that occurred were shoulder pain (Musculo-skeletal system disorders, 2.04%), acute vaginitis (Reproductive disorders, female, 2.04%), and contact dermatitis (Skin and appendages disorders, 2.04%). %) was.

There were no serious adverse reactions, and there were no dropouts due to adverse reactions.

In the investigation of symptom severity of adverse reactions, the test group had 3 mild cases and the control group had 3 mild cases.

Regarding the relationship with food for human application test, the test group had 3 cases of 'clearly not related' and the control group had 3 cases of 'clearly not related', which was judged by the tester, and there was no statistically significant difference between the intake groups. .

Clinical pathology tests for safety evaluation in this human application test were conducted at Visit 1 and Visit 5. Test items were evaluated by dividing into hematology, blood chemistry, and urine tests.

In the WBC category of hematological examination, after 12 weeks of ingestion, the test group increased by 0.53±1.14 x10 ³ /μL (p=0.0010) and the control group decreased by 0.06±1.37 x 10 ³ /μL (p=0.8796), showing statistical significance between intake groups. A significant difference was found (p=0.0089 ^(W) ), but there were no subjects judged to be clinically meaningful by the tester. After 12 weeks of ingestion, the mean and standard deviation of the test group were 5.81±1.51x10 ³ /μL, and the mean and standard deviation of the control group were 4.96±1.22x10 ³ /μL, all within the normal range of 4.0 x 10 ³ /μL to 10.0 x 10 Results of around ³ /μL were shown.

In other hematological tests, there was no statistically significant difference between the intake groups after 12 weeks of intake.

In all items of the blood chemistry test, there was no statistically significant difference between the intake groups after 12 weeks of intake.

In all urine test items, there was no statistically significant difference between the test group and the control group before and after intake.

There was no statistically significant difference between the intake groups after 12 weeks of intake in vital signs (blood pressure, pulse) and anthropometric measurements (weight).

It is obvious to those skilled in the art that the present invention is not limited to the above embodiments and can be variously modified or modified without departing from the technical gist of the present invention. it did

<110> DAEHAN CHEMTECH CO., LTD <120> Manufacturing Method Of Raw Material Composition For Improving Skin Beauty <130> P21-0399 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 4 <212> PRT <213> artificial sequence <220> <223> A peptide derived from an elastin extracted from Katsuwonus pelamis arterae occipitalis <400> 1 Val Gly Pro Gly One <210> 2 <211> 5 <212> PRT <213> artificial sequence <220> <223> A peptide derived from an elastin extracted from Katsuwonus pelamis arterae occipitalis <400> 2 Val Gly Pro Gly Gly 1 5 <210> 3 <211> 7 <212> PRT <213> artificial sequence <220> <223> A peptide derived from an elastin extracted from Katsuwonus pelamis arterae occipitalis <400> 3 Phe Gly Pro Gly Gly Ala Gly 1 5 <210> 4 <211> 5 <212> PRT <213> artificial sequence <220> <223> A peptide derived from an elastin extracted from Katsuwonus pelamis arterae occipitalis <400> 4 Thr Gly Gly Pro Gly 1 5 <210> 5 <211> 6 <212> PRT <213> artificial sequence <220> <223> A peptide derived from an elastin extracted from Katsuwonus pelamis arterae occipitalis <400> 5 Gly Val Gly Gly Pro Gly 1 5 <210> 6 <211> 4 <212> PRT <213> artificial sequence <220> <223> A peptide derived from an elastin extracted from Katsuwonus pelamis arterae occipitalis <400> 6 Phe Gly Gly Pro One <210> 7 <211> 5 <212> PRT <213> artificial sequence <220> <223> A peptide derived from an elastin extracted from Katsuwonus pelamis arterae occipitalis <400> 7 Val Gly Pro Gly Leu 1 5 <210> 8 <211> 5 <212> PRT <213> artificial sequence <220> <223> A peptide derived from an elastin extracted from Katsuwonus pelamis arterae occipitalis <400> 8 Val Gly Pro Gly Phe 1 5 <210> 9 <211> 5 <212> PRT <213> artificial sequence <220> <223> A peptide derived from an elastin extracted from Katsuwonus pelamis arterae occipitalis <400> 9 Phe Gly Pro Gly Leu 1 5 <210> 10 <211> 5 <212> PRT <213> artificial sequence <220> <223> A peptide derived from an elastin extracted from Katsuwonus pelamis arterae occipitalis <400> 10 Phe Gly Pro Gly Phe 1 5

Claims (9)

A first step of cutting an animal raw material containing elastin;
A second step of removing proteins other than elastin by adding an alkaline solution to the cut animal raw material;
a third step of neutralizing the alkaline solution by adding an acidic solution and washing with water to obtain insoluble materials including elastin;
A fourth step of obtaining an elastin degradation product by decomposing the insoluble matter for each enzyme using two or more proteolytic enzymes (proteases); and
A fifth step of degreasing and deodorizing by adding and stirring activated carbon dissolved in water to the elastin degradation product;
The animal is excluding humans,
In the fourth step,
Step 4-1 of decomposing at 65 to 80° C. and pH 6.5 to 8.5 for 120 to 180 minutes by adding 0.5 to 0.7% of thermoase by weight of the insoluble matter obtained in the third step together with water;
1.3 to 1.7% of papain by weight of the insoluble matter obtained in the third step is added to the decomposition product obtained in the step 4-1 to decompose at 75 to 85 ° C. and pH 6.5 to 7.5 for 120 to 180 minutes Step 4-2; and
4-3 step of inactivating the thermoase and the papain by heating at 90 to 95 ° C. for 25 to 35 minutes. A method for producing a raw material composition for improving skin beauty, comprising:
The raw material composition prepared according to the above production method contains the peptide of SEQ ID NO: 1 as an essential component,
A method for producing a raw material composition for improving skin care, characterized in that it further comprises one or more peptides selected from the group of peptides of SEQ ID NOS: 2 to 10.
SEQ ID NO: 1 - (valine-glycine-proline-glycine)
SEQ ID NO: 2 - (valine-glycine-proline-glycine-glycine)
SEQ ID NO: 3-(Phenylalanine-glycine-proline-glycine-glycine-alanine-glycine)
SEQ ID NO: 4-(Threonine-glycine-glycine-proline-glycine)
SEQ ID NO: 5-(glycine-valine-glycine-glycine-proline-glycine)
SEQ ID NO: 6-(Phenylalanine-glycine-glycine-proline)
SEQ ID NO: 7-(valine-glycine-proline-glycine-leucine)
SEQ ID NO: 8- (valine-glycine-proline-glycine-phenylalanine)
SEQ ID NO: 9-(Phenylalanine-glycine-proline-glycine-leucine)
SEQ ID NO: 10-(Phenylalanine-glycine-proline-glycine-phenylalanine) The method of claim 1,
The second step is
Adding 0.1 mol/L NaOH of 3 times the weight of the animal raw material and stirring at 80 to 90 ° C. for 40 to 60 minutes;
The third step is
A method for producing a raw material composition for improving skin care, comprising neutralizing by stirring at 80 to 90 ° C. for 25 to 35 minutes using citric acid. The method of claim 1,
The fifth step is
A method for producing a raw material composition for improving skin care, characterized by stirring at 90 to 95 ° C. for 25 to 35 minutes using two or more activated carbons. The method of claim 1,
The animal raw material is a method for producing a raw material composition for improving skin beauty, characterized in that the arterioles of bonito, tuna, barfish, cod, yellowtail or salmon. The method of claim 1,
A sixth step of first filtering using diatomaceous earth, second filtering using a mesh, and then concentrating 4 to 10 times using an evaporator; and
A seventh step of pasteurizing at 80 to 85 ° C for 25 to 35 minutes and drying at 80 to 150 ° C; delete The method of claim 1,
The peptide of SEQ ID NO: 1 is characterized in that it is contained in 0.5 to 5% by weight, a method for producing a raw material composition for improving skin care. The method of claim 1,
The raw material composition comprises the peptide of SEQ ID NO: 2,
The SEQ ID NO: 2 peptide is characterized in that it is contained in 0.1 to 1.5% by weight, a method for producing a raw material composition for improving skin care. The method of claim 1,
The raw material composition comprises the peptide of SEQ ID NO: 3,
The content of the SEQ ID NO: 3 peptide is characterized in that it is included in 0.01 to 1.2% by weight, a method for producing a raw material composition for improving skin care.

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