Composition for improving skin, containing abalone viscera hydrolysate or abalone viscera extract as active ingredient

Abstract

The present invention relates to a composition containing an abalone viscera peptide hydrolysate for skin moisturization. The composition contains a peptide of the abalone viscera enzyme hydrolysate, of the present invention, inhibits skin moisturization-associated enzymes, and increases procollagen biosynthesis ability in fibroblasts so as to exhibit skin moisturization effects, and thus can be used as a natural cosmetic material. In addition, the present invention relates to a composition for improving the skin, containing, as an active ingredient, an abalone viscera extract, an abalone viscera hydrolysate or a mixture thereof. More specifically, the composition of the present invention is a natural product having the effects of skin barrier factor activation, procollagen synthesis ability, skin moisturizing factor increase and antioxidant activity, and thus can be effectively used as a cosmetic composition, a health functional food composition and a quasi-drug composition for improving skin conditions.

Description

Composition for improving skin containing abalone viscera hydrolyzate or abalone viscera extract as an effective ingredient

[0001] The present invention relates to a skin moisturizing composition comprising a peptide derived from the viscera of abalone as an active ingredient. Specifically, the present invention relates to a method for producing abalone viscera hydrolysate using the enzyme alcalase, and to a cosmetic composition and a health functional food composition utilizing the skin moisturizing effect of the hydrolysate.

[0002] In addition, the present invention relates to a composition for improving skin comprising abalone viscera extract, abalone viscera hydrolyzate, or a mixture thereof as an effective ingredient.

[0003] The skin performs a crucial role as a barrier, protecting the body from the outside world. This barrier function protects against various external stimuli, such as chemicals, air pollutants, dry environments, and ultraviolet rays, while also preventing excessive loss of body moisture through the skin. This protective function can only be maintained if the stratum corneum (horney layer), composed of keratinocytes, is formed normally. Histologically, the skin is composed of three layers: the epidermis, the dermis, and the subcutis. As the outermost layer, the epidermis plays the most crucial role in skin aging and, as such, has been the subject of intense research in the field of skin beauty.

[0004] Normally, the moisture content of the skin is about 70% in the dermis layer, but it decreases as it goes to the epidermis, reaching about 10 to 30% in the stratum corneum layer. Water supplied from the dermis layer mainly moves to the upper part of the stratum corneum through passive diffusion and is ultimately discharged to the outside. This is called transepidermal water loss (TEWL), and it is known that this transepidermal water loss is maintained at an appropriate level by sebum and epidermal lipids, which are components of the lipid membrane of the stratum corneum layer. In addition, the stratum corneum layer contains a hydrophilic substance with moisture-retaining capacity called Natural Moisturizing Factor (NMF), which is known to play an important role in moisturizing the skin. When the moisture content of the stratum corneum layer is maintained at about 10 to 30%, the skin is smooth and soft, and the body's protective function is normally performed. However, when the moisture content of the stratum corneum falls below 10%, the skin becomes rough and loses its protective function, leading to aging. For example, in the case of dry skin, the cohesion of keratinocytes weakens, and a scaling phenomenon occurs in which keratinocytes peel off from the skin surface in a whitish scale-like state. This dryness is due to the moisture content of the stratum corneum being lower than that of normal skin. In addition, even healthy skin can suffer from moisture deficiency due to harsh external environments such as wind, cold weather, sunlight, washing the face, and shaving, which can worsen the skin condition. Therefore, it is very important to maintain an appropriate moisture content in the stratum corneum of the skin.

[0005] Meanwhile, abalone viscera accounts for approximately 20% of the total weight of abalone. Abalone viscera is rich in vitamins B1 and B2, and is also high in phosphorus, calcium, iodine, and taurine, making it a health food. The general nutritional composition of abalone is 74.41% moisture, 22.32% crude protein, 0.46% crude fat, and 2.81% ash. The amino acid composition of abalone is arginine, lysine, threonine, and glutamic acid, in that order. Abalone viscera not only contains more nutrients than abalone meat, but also contains a variety of health functional ingredients. Previous research has shown that abalone viscera possesses excellent antioxidant, anticancer, and antithrombotic effects, and has been confirmed to have higher nutritional value and functionality than abalone meat.

[0006] Hyaluronidase is a general term for enzymes that hydrolyze hyaluronic acid. It is a type of carbohydrate-decomposing enzyme that cleaves the glycosidic bonds of carbohydrates, and is also called mucinase or diffusion factor. The types of hyaluronidase include endohexosaminidase type, endoglucuronidase type, endohexosamine cleavage type, and endoglucosamine cleavage type, and these hyaluronidases are present in the testis and sperm of higher animals, lysosomes in the liver, snake venom, pneumococci, and streptococci. Meanwhile, the connective tissue of the skin is composed of collagen, hyaluronic acid polysaccharides, etc. Hyaluronidase is an enzyme that breaks down hyaluronic acid. When hyaluronidase is activated, the hyaluronic acid in the skin's connective tissue breaks down, loosening the skin's connective tissue and causing a decrease in skin elasticity and wrinkles.

[0007] The skin barrier function can be destroyed by aging and various external factors, and this skin barrier damage can ultimately lead to a decrease in skin moisture and the development of wrinkles. Tight junctions are directly related to the skin barrier. Tight junctions are multiprotein complexes that link two or more cells and regulate material transport through the extracellular matrix. These complexes include occludens, claudins, and adhesion molecules. These bind to the actin cytoskeleton and cytosolic regulatory proteins through plaque proteins (zona occludens-1 and cingulin) present in the cytoplasm, and have a biological barrier function that regulates cell-to-cell adhesion and the movement of water-containing solutes and water through the pericellular space. The barrier function of these skin cells is directly involved in maintaining the slightly acidic skin surface (pH 5.5) and antibacterial peptides in the stratum corneum, and plays an important role in protecting and maintaining normal skin by blocking external invasions such as viruses and bacteria and regulating keratinocyte division and differentiation.

[0008] In this experiment, the inventors confirmed the DPPH radical scavenging activity, hydrogen peroxide scavenging activity, ABTS radical scavenging activity, and the effect of inhibiting hyalurodinase, an enzyme related to skin moisturizing, and increasing procollagen biosynthesis in fibroblasts of abalone viscera hydrolysate. In addition, while screening various natural products for the activity of skin barrier factors, the inventors also discovered that abalone viscera extract and abalone viscera hydrolysate exhibit the effect of activating skin barrier factors, thereby completing the present invention.

[0009] Based on the above, the research team aims to provide a cosmetic composition for improving skin, which includes abalone viscera extract, abalone viscera hydrolyzate, or a mixture thereof, and has the effects of activating skin barrier factors, increasing collagen synthesis, increasing skin moisturizing factors, and having antioxidant activity.

[0010] A cosmetic composition for improving skin is provided, comprising abalone entrails extract, abalone entrails hydrolyzate, or a mixture thereof as an effective ingredient.

[0011] In addition, a health functional food composition for improving skin is provided, which comprises abalone entrails extract, abalone entrails hydrolyzate, or a mixture thereof as an effective ingredient.

[0012] In addition, a pharmaceutical composition for improving skin is provided, which comprises abalone entrails extract, abalone entrails hydrolyzate, or a mixture thereof as an effective ingredient.

[0013] In addition, a cosmetic composition for moisturizing the skin is provided, which comprises abalone viscera alkalize hydrolyzate or a fraction of abalone viscera alkalize hydrolyzate.

[0014] In addition, a skin moisturizing health functional food composition comprising abalone viscera alkalize hydrolyzate or a fraction of abalone viscera alkalize hydrolyzate is provided.

[0015] The present invention relates to a functional cosmetic composition comprising abalone viscera alkalize hydrolyzate, wherein the cosmetic composition exhibits DPPH radical scavenging activity, hydrogen peroxide scavenging activity, ABTS radical scavenging activity, and inhibits skin moisturizing-related enzymes and increases procollagen biosynthesis in fibroblasts, and is therefore useful as a skin moisturizing cosmetic composition.

[0016] In addition, the abalone viscera ultrasound extract, protamex hydrolysate or mixture thereof according to the present invention exhibits excellent antioxidant activity, and increases the expression of COL1A1, COL1A2, COL3A1, Aquaporin3, HAS1, HAS2, HAS3, procollagen, filaggrin, occludin and claudin, thereby increasing the hyaluronic acid content in keratinocytes and regulating proteins related to the skin barrier and tight junctions, thereby having skin moisturizing and barrier strengthening effects. In addition, it exhibits skin moisturizing effects by increasing collagen synthesis genes in fibroblasts, thereby exhibiting excellent skin improvement effects such as skin barrier strengthening, skin moisturizing, skin elasticity strengthening and skin wrinkle improvement, and therefore, as a natural material without side effects, it can be usefully used as a composition for skin improvement.

[0017] Figure 1 is a diagram showing the hyaluronidase inhibitory activity of four types of abalone viscera enzyme hydrolysates.

[0018] Figure 2 is a diagram showing a gel filtration chromatogram of an alkalize hydrolyzate.

[0019] Figure 3 is a diagram showing the hyaluronidase inhibitory activity of the alkalize fraction.

[0020] Figure 4 is a diagram showing the cell viability of Alcalase fraction 1 (Alcalase-Fr.1).

[0021] Figure 5 is a diagram showing the pro-collagen synthesis ability of Alcalase fraction 1 (Alcalase-Fr.1).

[0022] Figure 6 is a diagram showing the base peak chromatogram of the Alcalase fraction (Alcalase-Fr.1).

[0023] Figure 7 is a diagram showing the peptide profile of the Alcalase fraction (Alcalase-Fr.1).

[0024] Figure 8 shows the antioxidant efficacy of abalone viscera ultrasonic extract, protamex hydrolysate, and ethanol extract.

[0025] Figure 9a shows the cell viability of abalone viscera ultrasonic extract, protamex hydrolysate, and ethanol extract.

[0026] Figure 9b shows the procollagen synthesis ability of abalone viscera ultrasonic extract, protamex hydrolysate, and ethanol extract.

[0027] Figure 10a shows the collagen synthesis efficacy of abalone viscera ultrasonic extract. (1) is the result showing the COL1A1 mRNA expression level of abalone viscera ultrasonic extract, (2) is the result showing the COL1A2 mRNA expression level of abalone viscera ultrasonic extract, and (3) is the result showing the COL3A1 mRNA expression level of abalone viscera ultrasonic extract.

[0028] Figure 10b shows the collagen synthesis efficacy of abalone viscera protamex hydrolysate. (1) is the result showing the COL1A1 mRNA expression level of abalone viscera protamex hydrolysate, (2) is the result showing the COL1A2 mRNA expression level of abalone viscera protamex hydrolysate, and (3) is the result showing the COL3A1 mRNA expression level of abalone viscera protamex hydrolysate.

[0029] Figure 11a shows the measurement of cell viability (1), hyaluronic acid content (2), and Aquaporin 3 expression (3) of abalone viscera ultrasonic extract.

[0030] Figure 11b shows the measurement of cell viability (1), hyaluronic acid content (2), and Aquaporin 3 expression (3) of abalone viscera protamex hydrolysate.

[0031] Figure 12a shows the mRNA expression of hyaluronic acid synthase in the ultrasonic extract of abalone intestines. (1) is the result showing the HAS1 mRNA expression level in the ultrasonic extract of abalone intestines, (2) is the result showing the HAS2 mRNA expression level in the ultrasonic extract of abalone intestines, and (3) is the result showing the HAS3 mRNA expression level in the ultrasonic extract of abalone intestines.

[0032] Figure 12b shows the mRNA expression of hyaluronic acid synthase in abalone viscera protamex hydrolysate. (1) is the result showing the HAS1 mRNA expression amount in abalone viscera protamex hydrolysate, (2) is the result showing the HAS2 mRNA expression amount in abalone viscera protamex hydrolysate, and (3) is the result showing the HAS3 mRNA expression amount in abalone viscera protamex hydrolysate.

[0033] Figure 13a shows the skin barrier function enhancement effect of abalone viscera ultrasound extract. (1) is the result showing the Filaggrin mRNA expression level of abalone viscera ultrasound extract, (2) is the result showing the Occludin mRNA expression level of abalone viscera ultrasound extract, and (3) is the result showing the Claudin mRNA expression level of abalone viscera ultrasound extract.

[0034] Figure 13b shows the skin barrier function enhancement effect of abalone viscera protamex hydrolysate. (1) is the result showing the Filaggrin mRNA expression level of abalone viscera protamex hydrolysate, (2) is the result showing the Occludin mRNA expression level of abalone viscera protamex hydrolysate, and (3) is the result showing the Claudin mRNA expression level of abalone viscera protamex hydrolysate.

[0035] Figure 14a shows the cell viability according to the mixing ratio of the mixture of abalone viscera ultrasonic extract and protamex hydrolyzate.

[0036] Figure 14b shows the procollagen synthesis ability according to the mixing ratio of a mixture of abalone viscera ultrasonic extract and protamex hydrolyzate.

[0037] The terminology used herein is for the purpose of description only and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this specification, the terms "comprises" or "has" and the like are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but should be understood to not preclude the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0038]

[0039] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0040]

[0041] Hereinafter, the present invention will be described in more detail.

[0042] The present invention provides a skin moisturizing cosmetic composition comprising abalone viscera alkalize hydrolyzate or a fraction of abalone viscera alkalize hydrolyzate.

[0043] The present invention provides a health functional food composition for moisturizing the skin, comprising abalone viscera alkalize hydrolyzate or a fraction of abalone viscera alkalize hydrolyzate.

[0044] The present invention produces a hydrolyzed abalone viscera, which is produced by a method comprising the steps of: (1) separating and drying the viscera from abalone; (2) hydrolyzing the dried viscera using an enzyme; and (3) performing centrifugation and vacuum filtration on the hydrolyzed product to separate the supernatant and then freeze-drying it.

[0045] Preferably, the drying step (1) may be far infrared radiation drying (F-IRD), but is not limited thereto.

[0046] Preferably, the enzyme may be, but is not limited to, Alcalase.

[0047] The above abalone viscera hydrolyzate is characterized in that it contains at least one of the peptides of sequence numbers 1 to 3.

[0048] The above abalone viscera hydrolyzate is characterized by exhibiting an antioxidant effect or an effect of increasing the degree of procollagen biosynthesis in the body.

[0049] The above abalone viscera hydrolyzate is characterized by exhibiting an effect of inhibiting hyaluronidase, and is preferably characterized by inhibiting hyaluronidase by 50% or more.

[0050] The above abalone viscera hydrolyzate is characterized by exhibiting antioxidant activity, and is preferably characterized by exhibiting DPPH radical removal, hydrogen peroxide removal or ABTS radical removal effects, but is not limited thereto.

[0051] In the present invention, the term "functional food" is the same as food for special health use (FoSHU), and refers to a food with high medical or healthcare effects that is processed to efficiently exhibit a bioregulatory function in addition to providing nutrition, and may be manufactured in the form of tablets, capsules, pills, granules, powders, liquids, flakes, pastes, syrups, gels, jellies, bars, or films. Here, "function" means regulating nutrients for the structure and functions of the human body or obtaining a useful effect for health purposes such as physiological actions.

[0052] When the abalone viscera hydrolysate of the present invention is used as a food additive, it can be added as is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method. The amount of the active ingredient mixed can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing food or beverage, the abalone viscera hydrolysate of the present invention can be added in an amount of 15 wt% or less, or 10 wt% or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount can be below the above range, and since there is no problem in terms of safety, the active ingredient can also be used in an amount above the above range.

[0053] In the present invention, "food" means a natural product or processed product containing one or more nutrients, preferably a product that has gone through a certain degree of processing to become directly edible, and in its usual sense, includes all health functional foods, beverages, food additives, and beverage additives.

[0054] There are no specific restrictions on the types of the above foods. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and all health functional foods in the conventional sense are included.

[0055] The present invention also provides a fraction of abalone viscera hydrolysate, characterized in that the fraction is recovered at an absorbance of 220 nm by separating the hydrolysate by protein size using a gel filtration technique, which is manufactured by a process including the steps of (1) separating and drying the viscera from abalone; (2) hydrolyzing the dried viscera using an enzyme; and (3) performing centrifugation and vacuum filtration on the hydrolysate to separate the supernatant and then freeze-drying the hydrolysate.

[0056] The above fraction is characterized in that it comprises three kinds of peptides of 1,000 Da or less and seven kinds of peptides of 1,000 Da or more, and preferably, it comprises at least one peptide selected from the group consisting of peptides of SEQ ID NOs: 1 to 3.

[0057]

[0058] In addition, the abalone entrails extract, abalone entrails hydrolyzate or a mixture thereof of the present invention exhibited effects of skin moisturizing effect, collagen synthesis ability, procollagen synthesis ability, skin barrier strengthening function, skin elasticity enhancement, skin wrinkle improvement and antioxidant activity.

[0059] Accordingly, the present invention provides a cosmetic composition for improving skin comprising abalone entrails extract, abalone entrails hydrolyzate, or a mixture thereof as an effective ingredient.

[0060] The above composition may be used for skin improvement. The skin improvement may be skin barrier strengthening, skin moisturizing function enhancement, skin wrinkle improvement, skin elasticity enhancement, or two or more of these skin improvements.

[0061] As used herein, the term “skin improvement” may mean any action that at least reduces the degree of symptoms associated with alleviating or treating a skin condition.

[0062] The term "skin barrier strengthening" as used herein may mean any action that enhances the function of the skin barrier, which is located at the outermost layer of the skin and prevents moisture and nutrient loss.

[0063] In this specification, the term “skin elasticity enhancement” may mean any action that increases skin elasticity that has decreased due to aging or other causes, or prevents the decrease in skin elasticity.

[0064] In this specification, the term “improvement of skin wrinkles” is interpreted to mean not only the action of reducing the number or depth of wrinkles on the skin, but also the inhibition of skin aging through wrinkle improvement and wrinkle suppression.

[0065] As used herein, the term "enhancing skin moisturizing function" can refer to any action that maintains skin moisture or prevents moisture loss. The moisturizing effect can help improve wrinkles and increase skin elasticity, thereby preventing and mitigating skin aging.

[0066] The composition of the present invention can increase the expression of procollagen. Collagen, which is generally associated with wrinkles in skin cells, is known to be synthesized in the form of a precursor substance called procollagen. Therefore, by measuring the amount of procollagen peptides, the degree of collagen synthesis within the cell can be determined. By increasing the expression of procollagen, the composition can exhibit effects of improving skin wrinkles and enhancing skin elasticity.

[0067] Additionally, the composition can increase the expression of collagen synthesis factors (COL1A1, COL1A2, COL3A1). By measuring the expression levels of COL1A1, COL1A2, and COL3A1, the degree of collagen synthesis within cells can be determined. By increasing the expression of collagen synthesis factors, the composition can induce collagen synthesis in the skin, thereby improving skin wrinkles and enhancing skin elasticity.

[0068] Fibroblasts are crucial for the production of collagen and other proteins in the dermal layer of the skin, which form the skin and function as a barrier. Collagen type 1 accounts for 80% of the dermal layer, while collagen type 3 accounts for over 10%, both of which play a crucial role in skin formation. Collagen type 1 contributes to skin formation and thickness, and is formed by two α1 chains and one α2 chain twisted into a triple helix. These chains are expressed from the COL1A1 and COL1A2 genes, respectively. Collagen type 3 contributes to skin elasticity and is expressed from the COL3A1 gene.

[0069] The composition can increase the expression of aquaporin 3 (AQP3). By increasing the expression of AQP3, the composition can smoothly supply moisture to skin cells and exhibit a skin moisturizing effect.

[0070] The composition can increase the expression of hyaluronic acid synthase (HAS1, HAS2, HAS3). The composition can induce hyaluronic acid synthesis by increasing the expression of mRNA of HA synthase (HAS) 1, 2, and 3, thereby exhibiting skin moisturizing and skin barrier strengthening effects.

[0071] The composition can increase the expression of filaggrin, occludin, and claudin. Filaggrin is a protein produced by keratinocytes and plays an important role in the structure and hydration of the skin. Filaggrin is also involved in the skin's immune response and the formation of the skin barrier. Furthermore, occludin and claudin are types of tight junction proteins and play a crucial role in the formation of the skin barrier and mucosal barrier. Tight junctions strengthen the connections between cells, regulating the passage of water and solutes and preventing the invasion of pathogens and foreign substances. Occludin forms the structure of tight junctions, and claudin regulates the permeability of tight junctions.

[0072] The above composition can exhibit skin moisturizing and skin barrier strengthening effects by increasing the expression of filaggrin, occludin, and claudin.

[0073] Accordingly, the composition of the present invention can increase the expression of one or more selected from the group consisting of COL1A1, COL1A2, COL3A1, Aquaporin3, HAS1, HAS2, HAS3, procollagen, filaggrin, occludin, and claudin.

[0074] In one embodiment of the present invention, the abalone viscera extract may be an ultrasonic extract. The "ultrasonic extraction" of the present invention can maximize the efficiency of extracting antioxidants and effective active substances, while also reducing energy and carbon emissions.

[0075] The above ultrasonic extract may be obtained by mixing abalone viscera and distilled water in a weight ratio of 1:20. The extract may be obtained by performing ultrasonic extraction at an ultrasonic intensity of 1000 to 1500 Hz, a stirring speed of 50 to 70 rpm, and a temperature of 4 to 8°C for 2 to 6 hours.

[0076] Additionally, in one embodiment of the present invention, the abalone viscera hydrolyzate may be hydrolyzed with Protamex. The hydrolysis may be performed at a pH of 5 to 7 and a temperature of 20 to 60°C for 12 to 36 hours.

[0077] In addition, in one embodiment of the present invention, a mixture of abalone viscera ultrasound extract and abalone viscera protamex hydrolyzate showed superior procollagen synthesis ability than a single component.

[0078] In one embodiment of the present invention, the mixture of abalone viscera ultrasonic extract and abalone viscera protamex hydrolysate may be a mixture of abalone viscera ultrasonic extract: abalone viscera protamex hydrolysate in a weight ratio of 70 to 90: 10 to 30, preferably 75 to 85: 15 to 25, and more preferably a mixture of abalone viscera ultrasonic extract: abalone viscera protamex hydrolysate in a weight ratio of 80:20.

[0079] In one embodiment of the present invention, in order to confirm the biosynthetic ability of procollagen, a precursor of collagen, the content of C-peptide separated from procollagen was measured, and as a result, it was confirmed that the procollagen biosynthetic efficacy of a mixture of abalone viscera ultrasonic extract and abalone viscera Protamex hydrolysate increased as the ratio of abalone viscera ultrasonic extract: abalone viscera Protamex hydrolysate increased to 20:80, 40:60, 60:40, and 80:20. The procollagen biosynthetic efficacy was the best in the mixed group of ultrasonic extract: Protamex hydrolysate at 80:20, compared to a single component of the extract or hydrolysate.

[0080] Since the abalone entrails extract, abalone entrails hydrolyzate, or a mixture thereof of the present invention is a natural product, it is non-toxic to cells and can be used in various ways as a cosmetic composition, a health functional food composition, or an over-the-counter drug composition.

[0081] Accordingly, the present invention provides a health functional food composition for improving skin, comprising abalone viscera extract, abalone viscera hydrolyzate, or a mixture thereof as an effective ingredient.

[0082] In addition, a pharmaceutical composition for improving skin, comprising an abalone entrails extract, abalone entrails hydrolyzate, or a mixture thereof as an effective ingredient according to one embodiment of the present invention, is provided.

[0083] The term “quasi-drug” refers to products that are used for the purpose of diagnosing, treating, improving, alleviating, managing or preventing diseases in humans or animals, and have a milder effect than pharmaceutical products. For example, according to the Pharmaceutical Affairs Act, quasi-drugs are products other than those used for pharmaceutical purposes, and include products used for treating or preventing diseases in humans or animals, and products that have a mild effect on the human body or do not act directly.

[0084] The non-medical composition may be at least one selected from the group consisting of a coating agent, a disinfectant, a detergent, a mask, a hand wash, a hair cleanser, a hair softener, a wet tissue, a body cleanser, a foam, a soap, an ointment, a cream, a lotion, an essence, and a spray, but is not limited thereto.

[0085] Quasi-drug compositions can be administered orally or parenterally in various dosage forms. When formulated, they can be prepared using commonly used diluents or excipients, such as fillers, bulking agents, binders, wetting agents, disintegrants, or surfactants.

[0086] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0087]

[0088] <Example 1. Abalone viscera alkalize hydrolyzate>

[0089] 1. Experimental contents and methods

[0090] 1.1 Preparation of abalone viscera enzyme hydrolysate

[0091] The abalone (haliotis discus) used in the experiment was provided by Cheongryong Su-san Co., Ltd. After separating the meat and viscera, only the viscera were freeze-dried (FD) and far infrared radiation dried (F-IRD). The viscera were finely ground, and 10 g each was mixed with 500 mL of distilled water. Enzymatic hydrolysis was performed for 24 h at a substrate-to-enzyme ratio of 1% (w / w) at the optimal activity conditions of Alcalase, Bromelanin, Flavorzyme, and Neutrase, as shown in Table 1. After the hydrolysis reaction, the abalone was boiled at 95–100°C for 10 min to inactivate the enzymes, centrifuged (3,200 rpm, 30 min), and filtered under reduced pressure to separate the supernatant. This was then freeze-dried and used in the experiment. The yield of each hydrolyzate was calculated according to the equation below.

[0092] Extraction yield (%) = (sample weight after extraction / original sample weight) Х 100

[0093]

[0094]

[0095] 1.2 Measurement of protein content

[0096] Protein content was determined using a modified method of Lowry et al. (1951). 1 ml of a 20:1:1 mixture of Solution A: 10% Na2CO3 dissolved in 0.5 N NaOH, Solution B: 1% CuSO4 5H2O dissolved in distilled water, and Solution C: 2% C4H4O6K2 4H2O dissolved in distilled water was mixed with 1 ml of each sample and reacted for 15 minutes at room temperature in the dark. After adding 3 ml of 1 N Folin-phenol solution and reacting for 45 minutes at room temperature in the dark, the absorbance was measured at 540 nm using a microplate reader. The protein content was quantified by comparison with the calibration curve using BSA (Bovine serum albumin) as a standard.

[0097]

[0098] 1.3 Measurement of polysaccharide content

[0099] Polysaccharide content was determined using a modified method of Dubois et al. (1956). 2 ml of each sample was mixed with 50 μl of 80% phenol solution and 5 ml of sulfuric acid, and the mixture was allowed to react at room temperature for 30 minutes. The absorbance was then measured at 480 nm using a microplate reader. Glucose was used as a standard for polysaccharide content, and quantification was performed by comparison with a calibration curve.

[0100]

[0101] 1.4 Measurement of total phenol content

[0102] Total phenolic content was determined using a modified method of Heo et al. (2006). 1 ml of 95% EtOH, 1 ml of each sample, and 5 ml of DW were mixed with 0.5 ml of 1 N folin-ciocalteu reagent and kept for 5 minutes. Then, 1 ml of 5% Na2CO3 was added, and the mixture was incubated at room temperature for 1 hour. The absorbance was measured at 700 nm using a microplate reader. Total phenolic content was quantified using gallic acid as a standard and compared with a calibration curve.

[0103]

[0104] 1.5. Measurement of DPPH radical scavenging activity

[0105] DPPH radical scavenging activity was performed using a modified method of Heo et al. (2006). 20 μl of each sample and 180 μl of 0.15 mM DPPH solution were added, mixed, and reacted at room temperature for 30 minutes. The absorbance was measured at 515 nm using a microplate reader.

[0106]

[0107] 1.6. Measurement of hydrogen peroxide scavenging activity

[0108] Hydrogen peroxide scavenging activity was performed using a modified method of Heo et al. (2006). 20 μl of each sample, 100 μl of 0.1 M phosphate buffer (pH 5.0) and 20 μl of 10 mM H2O2 were reacted at 37°C for 5 minutes, then 30 μl of 1.25 mM ABTS and 30 μl of 1 unit / ml peroxidase were added and reacted at 37°C for 10 minutes. The absorbance was measured at 405 nm using a microplate reader.

[0109]

[0110] 1.7. Measurement of ABTS radical scavenging activity

[0111] ABTS radical scavenging activity was performed using a modified method of Heo et al. (2009). 7 mM ABTS + 2.4 mM potassium persulfate was stored in a dark room at room temperature for 12 hours, and then 150 μl of ABTS solution and 50 μl of each sample were mixed and reacted for 10 minutes at room temperature in a dark room. The absorbance was measured at 414 nm using a microplate reader.

[0112]

[0113] 1.8. Measurement of hyaluronidase inhibitory activity

[0114] Hyaluronidase inhibition activity was performed using a modified method of Sahasrabudhe and Deodhar (2010). 0.1 M acetate buffer (pH 3.6) was used as the buffer. First, 10 μl of the buffer and 10 μl of the sample were mixed with 10 μl of hyaluronidase (8 mg / ml) and incubated at 37°C for 20 min. Next, 20 μl of 12.5 mM CaCl2 was added and incubated again at 37°C for 20 min. Afterwards, 50 μl of 2.4 μg / ml hyaluronic acid was added and incubated at 37°C for 40 min. Next, 2 μl of 0.4 N NaOH and 20 μl of 0.4 N potassium tetraborate were sequentially added and incubated at 100°C for 3 min to terminate the reaction. Finally, 600 μl of DMAB solution (0.4 g of DMAB dissolved in 35 ml of 100% acetic acid and 5 ml of 10 N HCl) was treated, and the color was developed at 37°C for 20 minutes, and the absorbance was measured at a wavelength of 585 nm.

[0115]

[0116] 1.9. Gel filtration chromatography of Alcalase hydrolysate

[0117] Alcalase hydrolysate was dissolved in distilled water at a concentration of 200 mg / ml and filtered through a 0.45 μm filter to prepare a sample. After injecting the prepared sample into a Sephadex G-25 column (φ2.5 X 80 cm), distilled water was passed through it at a flow rate of 1.3 ml / min to elute, and the absorbance was measured at 220 nm. Six eluted fractions (Alcalase-Fr. 1~6) were recovered according to the measured absorbance, lyophilized, and used in the experiment.

[0118]

[0119] 1.10. Evaluation of moisturizing efficacy in fibroblasts (HDF cells)

[0120] 1.10.1. Cell culture and experimental conditions

[0121] HDF used in the experiment was provided by ATCC in the United States, and HDF cells were cultured in a medium containing F-12 medium mixed with Dulbecco's Modified Eagle Medium (DMEM) and 10% fetal bovine serum (FBS) and 1% antibiotics at 37°C and 5% CO2.

[0122]

[0123] 1.10.2. Cytotoxicity evaluation

[0124] The cytotoxicity of Al-Fr.1 in HDF cells was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay. HDF cells were seeded at 4 x 104 cells / ml in 96-well plates and cultured for 16 h. Then, Al-Fr.1 was treated at various concentrations and cultured for 24 h. After that, MTT solution (10 μl, 3 mg / mL) was added and cultured for an additional 4 h at 37°C. After removing the supernatant, formazan crystals were dissolved in DMSO and the absorbance was measured at 570 nm using a microplate reader. The cytotoxicity of Al-Fr.1 was expressed as a percentage of the control.

[0125]

[0126] 1.10.3. Procollagen biosynthesis

[0127] HDF cells were seeded into 24-well plates at 4Х104 cells / ml in 96-well plates and cultured for 16 hours. After treatment with various concentrations of Alkalease fraction 1 (Al-Fr.1), the cells were cultured for 24 hours. The supernatant was then collected and the degree of peptide biosynthesis was measured using a procollagen type C-peptide assay kit.

[0128]

[0129] 1.11. Peptide profiling of Alcalase fraction 1 (Alcalase-Fr.1)

[0130] Peptide profiling was performed on Alcalase fraction 1 (Alcalase-Fr. 1) protein, which exhibited the best moisturizing effect. Alcalase fraction 1 (Alcalase-Fr. 1) was desalted using a Sepak C18 cartridge, dried using a SpeedVac, and prepared at a concentration of 10 mg / μl for analysis.

[0131] Peptide profiling was performed using a Thermo (Dionex) UHPLC Ultimate 3000 and Q-TOF mass spectrometer (ABsciexTripleTOF 5600+) to perform mass analysis of peptides, and data analysis was performed using the Protein pilot (AB SCIEX) program and the sequence DB of the Haliotidae family.

[0132] The column used was ACQUITY UPLC@ BEH 130 C18 (1.7 μm), and the column temperature was maintained at 50℃ and the flow rate was 0.3 ml / min. 8 μl of sample was injected. DW (0.1% formic acid) and ACN (0.1% formic acid) were used as the mobile phases, respectively. The analysis conditions were as follows: 0 min, 1% B; 0-5 min, 1% B; 5-95 min, 50% B; 95-100 min, 100% B; 100-105 min, 100% B; 105-106 min, 1% B; 106-120 min, 1% B. Column temperature, 50℃, flow rate, 0.3 ml / min.

[0133]

[0134] 2. Experimental results

[0135] 2.1. Yield, protein content, sugar content, and total phenol content of abalone viscera enzymatic hydrolysate

[0136] In order to extract each active ingredient from freeze-dried (FD) and far-infrared-dried (F-IFD) abalone viscera, hydrolysates were prepared using proteolytic enzyme hydrolysis techniques such as Alcalase, Bromelanin, Flavorzyme, and Neutrase, and the results of measurements of yield, protein, sugar, and total phenol content for each hydrolysate are shown in Table 2. Although the yield did not differ significantly depending on the drying method, there were differences in yield depending on the enzyme, and the enzyme with the highest yield was Alcalase hydrolysate, with 89% for freeze-drying and 88% for far-infrared-dried. The protein content measurement results showed a protein content of approximately 33-42%. The sugar content measurement results showed a small amount of 6-9%, and the total phenol content measurement results showed a small amount of 1-2%. According to these results, there was no significant difference in yield, protein, sugar, and total phenol content depending on the drying method, but there were differences depending on the enzyme.

[0137]

[0138]

[0139] 2.2. Antioxidant activity of abalone viscera enzyme hydrolysate

[0140] To confirm the antioxidant activity of the proteolytic enzyme hydrolysate, DPPH, hydrogen peroxide, and ABTS scavenging activities were measured to see the free radical scavenging ability, and the activity level was expressed as IC 50The values ​​representing the concentration required to inhibit a given biological process in a test tube by 50% are shown in Table 3. It was confirmed that the free radical scavenging activity was superior in the far-infrared-dried product than in the freeze-dried product, and by enzyme, the best scavenging activity was confirmed in Alcalase hydrolysate for DPPH, hydrogen peroxide, and ABTS. There was no significant difference in yield, protein, sugar, and total phenol content by drying method, but the antioxidant activity results confirmed that the far-infrared-dried product had better antioxidant activity than the freeze-dried product, and among the proteolytic enzyme hydrolysates, Alcalase showed the highest antioxidant activity. Therefore, the moisturizing activity was measured using the far-infrared-dried proteolytic enzyme hydrolysate.

[0141]

[0142]

[0143] 2.3. Hyaluronidase inhibitory activity of abalone viscera enzyme hydrolysate

[0144] Hyaluronic acid, one of the natural moisturizing factors, is known to be one of the direct causes of aging, such as increased wrinkles, decreased elasticity, and decreased moisture content in the skin due to a decrease in the skin moisture barrier defect, which causes epidermal atrophy. Accordingly, hyaluronidase is an enzyme that breaks down hyaluronic acid, and hyaluronidase inhibitors can be expected to have a moisturizing effect on the skin by maintaining the polymer form of hyaluronic acid. As a result of measuring the hyaluronidase inhibitory activity of proteolytic enzyme hydrolysates at concentrations of 0.5 and 1 mg / ml (Fig. 1), all hydrolysates showed high hyaluronidase inhibitory activity, with a high hyaluronidase inhibitory activity of 79-82% at a concentration of 1 mg / ml. At 0.5 mg / ml, the highest hyaluronidase inhibition efficacy of 55% was observed in alkaline hydrolysate with an inhibitory activity of 44-55%.

[0145]

[0146] 2.4. Gel filtration chromatography of alkalize hydrolysate

[0147] Alcalase hydrolysate, which exhibited the highest radical scavenging and hyaluronidase inhibitory effects, was separated by protein size using gel filtration. A total of six fractions were recovered at an absorbance of 220 nm (Fig. 2).

[0148]

[0149] 2.5. Hyaluronidase inhibitory activity of Alcalase fraction

[0150] Among the six proteins of the alkaline fraction obtained through gel filtration chromatography, Fr. 5 and 6 were not used in the experiment because the yield was too low and it would be problematic for future industrial use. The hyaluronidase inhibitory activity of the fractions Fr. 1 to 4 is shown in Fig. 3. Specifically, only Fr. 1 exhibited hyaluronidase inhibitory activity, and it was found that the inhibitory activity increased as the concentration increased, and the IC 50 The value was 180.01 μg / ml. IC of alkaline hydrolysate 50 The value was 378.42 μg / ml, confirming that the Fr.1 fraction had an approximately 3.5-fold increase in efficacy.

[0151]

[0152] 2.6. Skin moisturizing activity of Al-Fr.1 (Alcalase-Fr.1.) fraction in cells

[0153] 2.6.1 Cytotoxicity of Al-Fr.1 fraction

[0154] The survival rates of cells treated with Al-Fr.1 at concentrations of 12.5, 25, and 50 μg / ml were examined, and no significant toxicity was observed in the concentration ranges of 50, 100, 200, and 400 μg / ml. Therefore, it can be said to be a biologically safe substance in the above concentration range (Fig. 4).

[0155]

[0156] 2.6.2. Pro-collagen biosynthesis activity of Al-Fr.1 fraction

[0157] In order to confirm the biosynthetic ability of procollagen, a precursor of collagen, the content of C-peptide separated from procollagen was measured and shown in Fig. 5. As the concentration of Al-Fr.1 increased, the collagen biosynthetic ability was confirmed, and a high production amount of 427.57 ng / mL was confirmed at a high concentration of 50 μg / mL.

[0158]

[0159] 2.7. Peptide profiling of Al-Fr.1

[0160] The results of each analysis are shown in Figures 6 and 7. In Al-Fr.1, three peptides less than 1,000 Da (DTDLPAPPY (SEQ ID NO: 4), IIAPPER (SEQ ID NO: 1), TEAPLNPK (SEQ ID NO: 5)) and seven peptides more than 1,000 Da (KLPAITDPGPF (SEQ ID NO: 6), VVGTDDIELPPGIL (SEQ ID NO: 7), GGELEMPWSFDRL (SEQ ID NO: 8), SYELPDGQVITIG (SEQ ID NO: 9), EDEFLGEEVEMI (SEQ ID NO: 10), DNPLLPGPPF (SEQ ID NO: 2), SADNPLLPGPPF (SEQ ID NO: 3)) were detected (Fig. 6). In particular, three peptides, IIAPPER (SEQ ID NO: 1), DNPLLPGPPF (SEQ ID NO: 2), and SADNPLLPGPPF (SEQ ID NO: 3), were repeatedly detected, and their spectra were found to be highly reliable (Fig. 7). Therefore, three peptides, IIAPPER (SEQ ID NO: 1), DNPLLPGPPF (SEQ ID NO: 2), and SADNPLLPGPPF (SEQ ID NO: 3), were confirmed to be indicator peptides of Alcalase-Fr.1.

[0161]

[0162] <Example 2, a mixture of abalone viscera ultrasonic extract, abalone viscera Protamex hydrolyzate, or a combination thereof>

[0163] 1. Experimental contents and methods

[0164] 1.1. Preparation of Abalone viscera ultrasonic extract (AVU)

[0165] After separating the abalone meat and intestines, 1 kg of the crushed intestines was taken, mixed with 20 L of distilled water, and placed in an ultrasonic extractor (ultrasonic intensity: 1,200, stirring speed: 60 rpm, temperature: 4-8℃) for extraction for 4 hours to prepare an extract. Centrifugation (4,500 rpm, 10 min) and filtration twice (1 μm microfilter, then 0.45 μm membrane filter) were performed to separate the supernatant, which was then freeze-dried and used as a sample (AVU).

[0166]

[0167] 1.2. Preparation of abalone viscera Protamex hydrolysate (AVP; Abalone viscera Protamex hydrolystate)

[0168] The separated abalone viscera was dried by far infrared radiation drying (F-IRD) and finely ground (10 g), mixed with 500 mL of distilled water, and enzymatic hydrolysis was performed for 24 hours at a substrate-to-enzyme ratio of 1% (w / w) under the optimal activity conditions of Protamex (pH 6, 40°C). After the hydrolysis reaction, the mixture was boiled at 95-100°C for 10 minutes to inactivate the enzyme, and the supernatant was separated by centrifugation (3,200 rpm, 30 minutes) and filtration under reduced pressure, which was then lyophilized and used as a sample (AVP).

[0169]

[0170] 1.3. Preparation of a mixture of abalone viscera ultrasonic extract and abalone viscera protamex hydrolyzate (AVU+AVP)

[0171] The abalone viscera ultrasonic extract and abalone viscera protamex hydrolyzate prepared in the above 1.1. and 1.2. were mixed at a concentration of 4 mg / ml in AVU:AVP ratios of 20:80, 40:60, 60:40, and 80:20, respectively, and used as samples.

[0172]

[0173] 1.4. Preparation of Abalone viscera ethanol extract (AVE; Abalone viscera EtOH extract)

[0174] After separating the abalone meat and intestines, 10 g of the finely ground intestines were freeze-dried, and ethanol was added 20 times, extracted twice at 60℃ for 6 hours, and then centrifuged (3,200 rpm, 30 minutes) and filtered under reduced pressure to separate the supernatant, which was then concentrated and used as a sample (AVE).

[0175]

[0176] 1.5. Antioxidant activity evaluation

[0177] The antioxidant activities of abalone viscera ultrasonic extract (AVU), protamex hydrolysate (AVP), a mixture of abalone viscera ultrasonic extract and abalone viscera protamex hydrolysate (AVU+AVP), and ethanol extract (AVE) were measured using DPPH (1,1-diphenyl-2-pricrylhydrazyl) radical scavenging assay. 1.5X10 -4 180 μl of M DPPH solution and 20 μl of each sample were mixed and reacted at room temperature for 30 minutes, and then the absorbance was measured using a microplate reader at a wavelength of 517 nm.

[0178]

[0179] 1.6. Evaluation of skin barrier strengthening and moisturizing effects in keratinocytes (HaCaT cells) and fibroblasts (HDF cells)

[0180] 1.6.1. Cell culture and experimental conditions

[0181] HaCaT and HDF used in the experiment were obtained from ATCC in the United States. HaCaT cells were cultured in a medium containing 10% Fetal bovine serum (FBS) and 1% antibiotics in Dulbecco's Modified Eagle Medium (DMEM), and HDF cells were cultured in a medium containing 10% Fetal bovine serum (FBS) and 1% antibiotics in Dulbecco's Modified Eagle Medium (DMEM) mixed with F-12 medium at 37°C and 5% CO2.

[0182]

[0183] 1.6.2. Evaluation of cell viability in fibroblasts

[0184] The cell viability of each sample in HDF cells was measured using the CCK-8 (Cell Counting Kit 8) assay. 2 × 10 HDF cells were seeded in each 96-well plate. 4 After dispensing cells / well and culturing for 16 hours, the cells were treated with various concentrations of AVU, AVP, AVU+AVP, and AVE and incubated for 24 hours. Afterwards, CCK-8 solution was added and incubated for an additional 2 hours at 37°C. The absorbance was then measured using a microplate reader at a wavelength of 450 nm. The cell viability of each sample was expressed as a percentage of the control.

[0185]

[0186] 1.6.3. Procollagen biosynthesis

[0187] HDF cells were seeded at 2 × 10 in 96-well plates. 4 After dividing cells / well and culturing for 16 hours, each sample was treated at different concentrations and cultured for 24 hours. The supernatant was then collected and the degree of peptide biosynthesis was measured using a procollagen type C-peptide assay kit.

[0188]

[0189] 1.6.4. Evaluation of collagen synthesis efficacy in fibroblasts

[0190] HDF 1×10 in 6 wells 5 After dividing into cells / well and culturing for 16 hours, each sample was treated at different concentrations and incubated for 24 hours. Afterwards, total RNA was isolated using TRIzol reagent and quantified using Nanodrop. The quantified RNA was synthesized into cDNA using a PCR machine under the conditions of 65°C for 60 minutes and 95°C for 5 minutes. PCR was performed with cDNA and the following specific primers COL1A1, COL1A2, COL3A1 and SYBR green, repeating 40 cycles of 95°C for 30 seconds, 60°C for 1 minute, and 72°C for 1 minute. The constant loading amount of mRNA was confirmed with β-actin. The primer sequences used in the PCR are shown in Table 4 below.

[0191]

[0192]

[0193] 1.6.5. Evaluation of cell viability in keratinocytes

[0194] The cell viability of AVU, AVP, and AVU+AVP in HaCaT cells was measured using the CCK-8 (Cell Counting Kit 8) assay. HaCaT cells were seeded in 96-well plates at a density of 3 × 10 4 After dispensing cells / well and culturing for 16 hours, the cells were treated with various concentrations of AVU, AVP, and AVU+AVP and incubated for 24 hours. Afterwards, CCK-8 solution was added and incubated for an additional 2 hours at 37°C. The absorbance was then measured using a microplate reader at a wavelength of 450 nm. The cell viability of AVU, AVP, and AVU+AVP was expressed as a percentage of the control.

[0195]

[0196] 1.6.6. Evaluation of Hyaluronic Acid Content in Keratinocytes

[0197] HaCaT 5×10 in 24 wells 4 After dispensing cells / well and culturing for 16 hours, the medium was removed, washed twice with DPBS, and each sample was treated with medium without FBS at various concentrations and cultured for 24 hours. The culture medium was then collected and the absorbance was measured at a wavelength of 450 nm using a Hyaluronan ELISA kit (R&D system) according to the manufacturer's manual using a microplate reader.

[0198]

[0199] 1.6.7 Evaluation of skin barrier strengthening and moisturizing effects in keratinocytes

[0200] HaCaT 7×10 in 6 wells 4 After dividing cells / well and culturing for 16 hours, AVU and AVP were treated at various concentrations and cultured for 24 hours. After that, total RNA was isolated using TRIzol reagent and quantified using Nanodrop. The quantified RNA was synthesized into cDNA using a PCR machine under the conditions of 65℃ for 60 minutes and 95℃ for 5 minutes. PCR was performed with cDNA and the following specific primers: Aquaporin3, HAS1, HAS2, HAS3, Fillagrin, Occulidin, Claudin, and SYBR green, repeating 40 cycles of 95℃ for 30 seconds, 60℃ for 1 minute, and 72℃ for 1 minute. The constant loading amount of mRNA was confirmed with β-actin. The primer sequences used in the PCR are shown in Table 5 below.

[0201]

[0202]

[0203] 1.7. Free amino acid analysis

[0204] For free amino acid analysis, 70% ethanol was added to the extract, and after ultrasonic extraction for 1 hour, the extract was extracted at room temperature for 24 hours, filtered through a 0.2 um syringe filter, and analyzed by HPLC (Ultimate 3000, Thermo Fisher Scentific) and FL detector and UV detector. To quantify free amino acids, C 18 The column was analyzed at a wavelength of 338 nm.

[0205]

[0206] 2. Results

[0207] 2.1. Antioxidant activity

[0208] The antioxidant activity of AVU, AVP, and AVE was measured by DPPH radical scavenging activity, and the results are shown in Fig. 8. As the concentration of AVU, AVP, and AVE increased, the DPPH radical scavenging activity was superior, and IC 50 As a result of checking the values, AVU was 1.67 mg / ml, AVP was 0.66 mg / ml, and AVE was 1.77 mg / ml, confirming that AVP showed the best antioxidant activity.

[0209]

[0210] 2.2. Evaluation of cell viability and procollagen biosynthesis in fibroblasts

[0211] To confirm the cell viability of each sample in fibroblasts, a CCK8 assay was performed, and to evaluate the procollagen biosynthetic ability, a procollagen type C-peptide assay was performed. The results are shown in Fig. 9a and Fig. 9b. As a result of confirming the viability of cells treated with AVU and AVP at concentrations of 25, 50, 100, and 200 μg / ml, it was confirmed that the cell viability increased as the concentration increased, whereas the viability of AVE decreased at a high concentration (200 μg / ml). To confirm the biosynthetic ability of procollagen, a precursor of collagen, the content of C-peptide isolated from procollagen was measured. As a result, it was confirmed that the collagen biosynthetic efficacy of AVU and AVP increased as the concentration increased, whereas the collagen biosynthetic efficacy of AVE decreased from a concentration of 100 μg / ml. In particular, AVU showed the highest efficacy, followed by AVP in terms of excellent collagen biosynthetic efficacy. In future studies, experiments were conducted using AVU and AVP, which have excellent antioxidant efficacy and procollagen biosynthesis performance.

[0212]

[0213] 2.3. Evaluation of collagen synthesis efficacy of abalone viscera ultrasonic extract and protamex hydrolysate

[0214] Fibroblasts are crucial for the production of collagen and other proteins in the dermal layer of the skin, which form the skin and function as a barrier. Collagen type 1 accounts for 80% of the dermal layer, while collagen type 3 accounts for over 10%, both of which play a crucial role in skin formation. Collagen type 1 contributes to skin formation and thickness, and is formed by two α1 chains and one α2 chain twisted into a triple helix. These chains are expressed from the COL1A1 and COL1A2 genes, respectively. Collagen type 3 contributes to skin elasticity and is expressed from the COL3A1 gene. As the concentration of both AVU and AVP increased, the expression levels of all three genes, COL1A1, COL1A2, and COL3A1, increased. In the case of AVU, at high concentrations, the expression of COL1A1, COL1A2, and COL3A1 increased by 3.1-fold, 1.5-fold, and 2.2-fold, respectively. In the case of AVP, the expression of COL1A1, COL1A2, and COL3A1 increased by 2.2-fold, 2.4-fold, and 4.1-fold, respectively, confirming the efficacy of promoting collagen synthesis (Fig. 10a, Fig. 10b).

[0215]

[0216] 2.4. Evaluation of cell viability and skin moisturizing function enhancement effects of abalone viscera ultrasonic extract and protamex hydrolyzate

[0217] Hyaluronic acid (HA), a natural moisturizing factor, is a water-soluble polysaccharide biopolymer with moisturizing properties that prevents water evaporation, thereby maintaining skin elasticity and moisturizing function. Aquaporin 3 selectively allows water molecules to pass through, and is known to affect skin moisturizing and barrier function. Therefore, when the viability of cells treated with AVU and AVP at concentrations of 50, 100, and 200 μg / ml in HaCaT cells was examined, it was confirmed that cell viability increased as the concentration increased (Fig. 11a, Fig. 11b). Therefore, it can be said to be a biologically safe substance in the above concentration range and was used in subsequent experiments. Referring to Fig. 11a and Fig. 11b, as the concentration of AVU and AVP increased, the hyaluronic acid content increased by 125.0% and 135.4% at high concentrations, respectively, and Aquaporin 3 increased by 1.7-fold and 1.8-fold, respectively, confirming that it maintained skin elasticity, moisturized, and strengthened the barrier function. In addition, as a result of confirming the expression levels of mRNA of HA synthase (HAS) 1, 2, and 3, which play an important role in hyaluronic acid biosynthesis, the expression levels of AVU and AVP increased as the concentration increased (Fig. 12a and Fig. 12b). In the case of AVU, it was confirmed that it increased 5.0-fold, 1.4-fold, and 2.1-fold at high concentrations of HAS1, HAS2, and HAS3, respectively, and in the case of AVP, it increased 1.3-fold, 3.4-fold, and 1.7-fold, thereby increasing hyaluronic acid synthesis enzyme and helping in the production of hyaluronic acid.

[0218]

[0219]

[0220] 2.5. Evaluation of the skin barrier function enhancement effect of abalone viscera ultrasonic extract and protamex hydrolyzate.

[0221] In keratinocytes, the mRNA expression of filaggrin, a major skin barrier protein, and occludin and claudin, which are adhesive proteins that constitute the skin barrier, was confirmed (Fig. 13a, Fig. 13b). Referring to Fig. 13a and Fig. 13b, as the concentration increased, the expression of filaggrin increased by 1.3-fold, 1.5-fold, and 1.7-fold in AVU, and AVP increased by 2.1-fold at high concentrations, confirming that it can maintain skin moisture and strengthen the skin barrier function by preventing the penetration of external foreign substances. In addition, occludin increased by 2.1-fold and 3.1-fold, respectively, at high concentrations in AVU and AVP, and claudin increased by 1.3-fold, 1.7-fold, and 2.8-fold in AVP as the concentration increased. Therefore, it was confirmed that AVU and AVP exhibit efficacy by regulating gene expression involved in skin barrier function and moisture supply.

[0222]

[0223] 2.6. Amino acid content of abalone viscera ultrasonic extract and Protamex hydrolysate

[0224] The amino acid content analysis results of Avalon visera ultrasonication extract (AVU) and protamex hydrolyzate (AVP) are shown in Table 6. AVU showed a higher amino acid content at 75,666 g / kg and AVP 224,927 g / kg. In the case of AVU, Alanine accounted for 65% at 49,413 g / kg, followed by Arginine at 5,544 g / kg, Glycine at 4,595 g / kg, and Glutamic acid at 4,440 g / kg. In the case of AVP, Taurine accounted for the highest proportion at 67,152 g / kg, followed by Glutamine at 17,399 g / kg, Arginine at 16,116 g / kg, and Glutamic acid at 15,612 g / kg. When hydrolytic enzymes were used rather than the ultrasonic extraction method, a higher amino acid content was observed.

[0225]

[0226]

[0227] 2.7. Evaluation of survival rate and procollagen biosynthesis performance of a mixture of abalone viscera ultrasonic extract and Protamex hydrolysate

[0228] To confirm cell viability, CCK8 assay analysis was performed, and to evaluate procollagen biosynthesis ability, procollagen type C-peptide assay analysis was performed. The results are shown in Fig. 14a and Fig. 14b. Referring to Fig. 14a and Fig. 14b, the cell viability results showed a viability of over 100% in all groups. To confirm the biosynthesis ability of procollagen, a precursor of collagen, the content of C-peptide isolated from procollagen was measured, and the procollagen biosynthesis efficacy was confirmed to increase as the ratio increased from 20:80, 40:60, 60:40, and 80:20. In addition, it was confirmed that the procollagen biosynthesis efficacy was the best in the group where the ratio of AVU:AVP was 80:20 in the AVU+AVP mixture compared to when single extracts and hydrolysates were processed.

Claims

1. A cosmetic composition for improving skin, comprising abalone viscera extract, abalone viscera hydrolyzate or a mixture thereof as an effective ingredient.

2. In paragraph 1, A cosmetic composition wherein the above skin improvement is an antioxidant effect, strengthening of the skin barrier, strengthening of the skin moisturizing function, improvement of skin wrinkles, enhancement of skin elasticity, or two or more skin improvements thereof.

3. In paragraph 1, A cosmetic composition, wherein the hydrolyzate is an alkaline hydrolyzate or protamex hydrolyzate of abalone viscera produced by hydrolysis with alkaline or protamex.

4. In paragraph 3, A cosmetic composition wherein the hydrolysis is performed at a pH of 5 to 7 and a temperature of 20 to 60°C for 12 to 36 hours.

5. In paragraph 1, A cosmetic composition wherein the above extract is an ultrasonic extract.

6. In paragraph 1, A cosmetic composition, wherein the extract is obtained by mixing abalone entrails and distilled water in a weight ratio of 1:20 and performing ultrasonic extraction at an ultrasonic intensity of 1000 to 1,500 Hz, a stirring speed of 50 to 70 rpm, and a temperature of 4 to 8°C for 2 to 6 hours.

7. In paragraph 1, A cosmetic composition, wherein the composition increases the expression of at least one selected from the group consisting of COL1A1, COL1A2, COL3A1, Aquaporin3, HAS1, HAS2, HAS3, procollagen, filaggrin, occludin, and claudin.

8. In paragraph 1, A cosmetic composition wherein the composition improves antioxidant activity or collagen production ability.

9. In paragraph 1, A cosmetic composition wherein the above mixture is a mixture of abalone viscera ultrasound extract and abalone viscera protamex hydrolyzate in a weight ratio of 70 to 90: 10 to 30.

10. In paragraph 3, A cosmetic composition wherein the above alkaline hydrolyzate inhibits hyaluronidase.

11. In paragraph 3, A cosmetic composition comprising a fraction of the hydrolyzate recovered at an absorbance of 220 nm by separating the above alkaline hydrolyzate by protein size using a gel filtration technique.

12. A health functional food composition for improving skin, comprising abalone viscera extract, abalone viscera hydrolyzate or a mixture thereof as an active ingredient.

13. A pharmaceutical composition for improving skin, comprising abalone entrails extract, abalone entrails hydrolyzate or a mixture thereof as an active ingredient.

14. A cosmetic composition for moisturizing the skin comprising abalone viscera alkalize hydrolyzate or a fraction of abalone viscera alkalize hydrolyzate.

15. A health functional food composition for moisturizing the skin, comprising abalone viscera alkalize hydrolyzate or a fraction of abalone viscera alkalize hydrolyzate.

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