Composition for Anti-inflammation, antioxidation, skin whitening, wrinkle reduction, or skin moisturization, comprising ultrasonic extract of undaria pinnatifida sporophyll as active ingredient

Abstract

The present invention relates to a composition for anti-inflammation, antioxidation, skin whitening, wrinkle reduction, or skin moisturization, comprising an ultrasonic extract of Undaria pinnatifida sporophyll, as an active ingredient. The ultrasonic extract obtained from Undaria pinnatifida sporophyll, according to the present invention, has scavenging activity against free radicals that cause skin inflammation and inhibitory activity against reactive oxygen species production, can regulate skin inflammation mechanisms through not only inhibitory activity against the inflammatory factor COX-2 but also regulation of HO-1 / NRF2 and ERK signaling pathways, and has inhibitory activity against collagenase, MMP-1 protein expression, hyaluronidase, tyrosinase, or melanin production. Thus, the ultrasonic extract of Undaria pinnatifida sporophyll, of the present invention, can be effectively used for the preparation of functional cosmetics and health functional foods, having anti-inflammatory, antioxidant, skin whitening, wrinkle reduction or skin moisturization activity.

Description

Composition for anti-inflammation, anti-oxidation, skin whitening, wrinkle improvement or skin moisturizing containing ultrasonic extract of seaweed sporophyll as an active ingredient

[0001] The present invention relates to a composition for anti-inflammation, anti-oxidation, skin whitening, wrinkle improvement or skin moisturizing, which contains an ultrasonic extract of seaweed sporophyll as an active ingredient.

[0002] The skin serves as the body's first line of defense. It protects against changes in temperature and humidity, UV rays, bacteria, and various environmental stimuli, and plays a crucial role in maintaining homeostasis. However, skin exposed to environmental stimuli like UV rays and bacterial infections cannot perform its normal functions, accelerating skin aging processes such as wrinkles and loss of elasticity. To prevent this aging and maintain healthy skin, cosmetics containing bioactive substances derived from various plants are used to maintain skin function and health.

[0003] Meanwhile, the inflammatory response in the skin begins as a defense mechanism against skin damage caused by physical stimuli, chemicals, or bacteria, and involves various immune cells and inflammatory cytokines. Representative inflammatory cytokines include tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8). In addition, activated macrophages not only produce inflammatory cytokines but also excessive amounts of nitric oxide (NO) and prostaglandin E2 (PGE2), further activating the inflammatory process.

[0004] The inflammatory response of the skin is regulated by several complex genes, and the major genes known are cyclooxygenase-2 (COX-2) and inducible nitric oxide synthases (iNOS). Among them, COX-2 has been found to induce the production of PGE2 by inflammatory cytokines, cell growth factors, cancer cell promoting factors, and reactive oxygen species (ROS), and to cause inflammatory skin diseases such as psoriasis, vitiligo, and lupus erythematosus.

[0005] In addition, it is known that when exposed to ultraviolet rays (UV), reactive oxygen species (ROS) are generated in the skin, and the excessively generated reactive oxygen species cause oxidation and react with cell membranes, DNA, and major components of the skin, such as lipids, proteins, polysaccharides, and nucleic acids, damaging cells and causing an inflammatory response in the skin.

[0006] Meanwhile, anti-oxidation refers to preventing various oxidation reactions that occur in the body. Lipids that exist as biomembranes or lipoproteins are attacked by free radicals generated in the body and form various types of peroxides. Peroxides and their decomposition products are highly reactive and change the structure and function of surrounding biomolecules, causing aging and various chronic diseases.

[0007] There are several antioxidant defense mechanisms in the body that can neutralize these free radicals and protect the body. Human diseases and aging are caused by O generated during metabolic processes in the body. 2-It can be caused by oxidation reactions such as (superoxide), NO(nitric oxide), NO2(nitrogen dioxide), OH(hydroxyl), ROO(proxyl), RO(alkoxyl), HO2(hydroperoxyl) radicals, and as a means of defending the body from these harmful radicals, superoxide dismutase (SOD), an antioxidant enzyme, reduces superoxide radicals and protects the body from oxidative damage.

[0008] In addition, active oxygen (O2) generated during human physiological processes - , H2O2, OH and O2) are highly reactive and the free radical reactions caused by them have a destructive effect on major macromolecules including lipids. Their production can be excessively increased by various factors that cause abnormalities in normal metabolic processes, photosensitivity reactions, drug metabolism processes and other cellular metabolism, and exposure to harmful effects due to uncontrolled high levels of free radicals can occur.

[0009] Therefore, it is expected that it will be useful in treating various diseases caused by oxidative damage by suppressing free radical reactions in the body through reactive oxygen metabolites or removing harmful active oxygen accumulated in the human body, and research is continuing to discover candidate substances.

[0010] Therefore, substances that can suppress the expression or activity of these skin inflammation-inducing factors can be used as agents for improving and treating skin inflammation.

[0011] In particular, consumer interest in skin beauty and health has been increasing significantly across various age groups recently, and many beauty-related cosmetics and foods are being developed with the goal of preventing skin aging, suppressing skin stress, improving skin inflammation, and maintaining healthy skin through antioxidant activity.

[0012] Meanwhile, the sporophyll is the reproductive organ of the seaweed, which belongs to the brown algae family, the Goniaceae. As the seaweed grows in spring, wrinkles form at the lower edge of the stem, transforming into sporophylls where zoospore sacs develop. When the water temperature exceeds 14℃, the zoospores are released and the mother plant thaws. This phenomenon continues until 22℃. In autumn, eggs and sperm are released from the male and female gametophytes, respectively, which fertilize and soon germinate to form spores. These grow into the thallus, which is the main body of the seaweed.

[0013] The spore leaves of seaweed contain about 20% sulfated polysaccharides with a high sulfate content, which are called sulfated fucose polysaccharides or fucoidan. They are known to have physiologically active functions such as anticancer, antiviral, antitumor, and immune-enhancing functions.

[0014] However, most of these seaweed sporophylls are discarded during the seaweed processing process, and thus, technology to recycle them into functional products is required. However, there has been no research on the development of functional cosmetics and foods using seaweed sporophylls.

[0015] Accordingly, the inventors of the present invention obtained an ultrasonic extract having various excellent physiological activities from the spore leaves of Undaria pinnatifida, and confirmed that the ultrasonic extract of the spore leaves obtained has anti-inflammatory, antioxidant, skin whitening, wrinkle improvement and skin moisturizing activities, thereby completing the present invention.

[0016] Accordingly, the purpose of the present invention is to provide a method for producing an ethanol precipitate fraction of an ultrasonic extract of Undaria pinnatifida having an activity of inhibiting or improving skin inflammation, the method comprising the steps of (1) obtaining an ultrasonic extract of Undaria pinnatifida; and (2) adding ethanol to the ultrasonic extract of Undaria pinnatifida obtained in step (1) to obtain an ethanol precipitate fraction.

[0017] Another object of the present invention is to provide an ethanol precipitate fraction of an ultrasonic extract of Undaria pinnatifida having an activity of inhibiting or improving skin inflammation, produced by the method of the present invention.

[0018] Another object of the present invention is to provide a cosmetic composition, pharmaceutical composition and health functional food for preventing, improving or treating skin inflammation, which contain an ethanol precipitate fraction of an ultrasonic extract of seaweed sporophyll as an active ingredient.

[0019] Another object of the present invention is to provide an antioxidant composition comprising an ultrasonic extract of seaweed sporophyll or an ethanol precipitate fraction of the ultrasonic extract as an active ingredient.

[0020] Another object of the present invention is to provide an antioxidant health functional food or antioxidant cosmetic composition comprising the antioxidant composition of the present invention.

[0021] Another object of the present invention is to provide a cosmetic composition for skin whitening, wrinkle improvement and skin moisturizing, which contains an ultrasonic extract of seaweed sporophyll as an active ingredient.

[0022] Furthermore, another object of the present invention is to provide a health food composition for skin whitening, wrinkle improvement and skin moisturizing, which contains an ultrasonic extract of seaweed sporophyll as an effective ingredient.

[0023] Therefore, the present invention provides a method for producing an ethanol precipitate fraction of an ultrasonic extract of Undaria pinnatifida having skin inflammation suppression or improvement activity, comprising the steps of (1) obtaining an ultrasonic extract of Undaria pinnatifida; and (2) adding ethanol to the ultrasonic extract of Undaria pinnatifida obtained in step (1) to obtain an ethanol precipitate fraction.

[0024] In one embodiment of the present invention, the ultrasonic extract of step (1) may be obtained by adding purified water to the powder of seaweed sporophyll and ultrasonicating it at a temperature of 28 to 32°C and 1000 to 1100 W for 7 to 9 hours.

[0025] In one embodiment of the present invention, the ethanol precipitate fraction may be obtained by adding ethanol to an ultrasonic extract of the kelp sporophyll, performing ethanol precipitation at a temperature of 4 to 6°C for 20 to 28 hours, and then centrifuging to obtain a precipitate.

[0026] In one embodiment of the present invention, the ethanol precipitated fraction may contain a polysaccharide derived from an ultrasonic extract of seaweed sporophyll.

[0027] In addition, the present invention provides an ethanol precipitate fraction of an ultrasonic extract of Undaria pinnatifida having skin inflammation suppression or improvement activity, prepared by the method of the present invention.

[0028] In addition, the present invention provides a cosmetic composition for preventing or improving skin inflammation, which comprises an ethanol precipitate fraction of the ultrasonic extract of the seaweed sporophyll of the present invention as an active ingredient.

[0029] In addition, the present invention provides a pharmaceutical composition for preventing or treating skin inflammation, which comprises an ethanol precipitate fraction of the ultrasonic extract of the seaweed sporophyll of the present invention as an active ingredient.

[0030] In addition, the present invention provides a health functional food for preventing or improving skin inflammation, which comprises an ethanol precipitate fraction of the ultrasonic extract of the seaweed sporophyll of the present invention as an effective ingredient.

[0031] In addition, the present invention provides an antioxidant composition comprising an ultrasonic extract of seaweed sporophyll or an ethanol precipitate fraction of the ultrasonic extract as an active ingredient.

[0032] In one embodiment of the present invention, the ultrasonic extract may be an ultrasonic extract obtained by adding purified water to a powder of seaweed sporophyll and ultrasonicating it at a temperature of 28 to 32°C for 7 to 9 hours.

[0033] In one embodiment of the present invention, the ethanol precipitate fraction may be a precipitate obtained by adding ethanol to an ultrasonic extract of seaweed sporophyll obtained by ultrasonic treatment for 8 hours, performing ethanol precipitation overnight at 4°C, and then centrifuging.

[0034] In one embodiment of the present invention, the ultrasonic extract and the ethanol precipitation fraction of the above-mentioned seaweed sporophyll may contain polysaccharides derived from the seaweed sporophyll.

[0035] The present invention also provides an antioxidant health functional food comprising the antioxidant composition of the present invention.

[0036] The present invention also provides an antioxidant cosmetic composition comprising the antioxidant composition of the present invention.

[0037] In addition, the present invention provides a method for producing an ethanol precipitate fraction of an ultrasonic extract of Undaria pinnatifida having antioxidant activity, comprising the steps of (1) obtaining an ultrasonic extract of Undaria pinnatifida; and (2) adding ethanol to the ultrasonic extract of Undaria pinnatifida obtained in step (1) to obtain an ethanol precipitate fraction.

[0038] In one embodiment of the present invention, the ultrasonic extract may be obtained by adding purified water to the powder of the seaweed sporophyll and ultrasonicating it at 1,000 to 1,100 W and a temperature of 28 to 32°C for 7 to 9 hours, and the ethanol precipitate fraction may be obtained by adding ethanol to the ultrasonic extract of the seaweed sporophyll and performing ethanol precipitation overnight at 4°C, followed by centrifugation.

[0039] In addition, the present invention provides a cosmetic composition for skin whitening, wrinkle improvement, and skin moisturizing, which contains an ultrasonic extract of seaweed sporophyll as an active ingredient.

[0040] In one embodiment of the present invention, the ultrasonic extract may be an ultrasonic extract obtained by adding purified water to a powder of seaweed sporophyll and ultrasonicating it at a temperature of 28 to 32°C for 7 to 9 hours.

[0041] In one embodiment of the present invention, the ultrasonic extract may be obtained by performing filter cloth and housing filtration on the ultrasonic extract obtained after the ultrasonic treatment, performing ion exchange, and then sterilizing and freeze-drying.

[0042] In one embodiment of the present invention, the ultrasonic extract of the seaweed sporophyll may have antioxidant activity, collagenase inhibitory activity, MMP-1 protein expression inhibition, hyaluronic acid decomposition enzyme inhibition, tyrosinase activity inhibition, or melanin production inhibition activity.

[0043] Furthermore, the present invention provides a health food composition for skin whitening, wrinkle improvement and skin moisturizing, which contains an ultrasonic extract of seaweed sporophyll as an effective ingredient.

[0044] In one embodiment of the present invention, the ultrasonic extract may be an ultrasonic extract obtained by adding purified water to a powder of seaweed sporophyll and ultrasonicating it at a temperature of 28 to 32°C for 7 to 9 hours.

[0045] In one embodiment of the present invention, the ultrasonic extract may be obtained by performing filter cloth and housing filtration on the ultrasonic extract obtained after the ultrasonic treatment, performing ion exchange, and then sterilizing and freeze-drying.

[0046] In one embodiment of the present invention, the ultrasonic extract of the seaweed sporophyll may have antioxidant activity, collagenase inhibitory activity, MMP-1 protein expression inhibition, hyaluronic acid decomposition enzyme inhibition, tyrosinase activity inhibition, or melanin production inhibition activity.

[0047] The ultrasonic extract obtained from the seaweed sporophyll according to the present invention has scavenging activity against free radicals, which are skin inflammation-inducing factors, and suppressing activity against the production of reactive oxygen species, and can regulate the skin inflammation mechanism through the regulation of the signal transduction pathway of HO-1 / NRF2 and ERK as well as the inhibitory activity of the inflammatory factor COX-2, and has collagenase inhibitory activity, MMP-1 protein expression inhibition, hyaluronic acid decomposition enzyme inhibition, tyrosinase activity inhibition, or melanin production inhibition activity, so that the seaweed sporophyll ultrasonic extract of the present invention can be usefully used in the manufacture of functional cosmetics and health functional foods having anti-inflammation, antioxidant, skin whitening, wrinkle improvement, or skin moisturizing activities.

[0048] Figure 1 is a schematic diagram showing a process for obtaining an ultrasonic extract from seaweed sporophyll and an ethanol precipitate fraction from the ultrasonic extract.

[0049] Figure 2 shows the results of MTT assay analysis to determine whether the ethanol precipitated fractions (UEFs) of the ultrasonic extract of the seaweed sporophyll of the present invention have cytotoxicity on human keratinocyte cells (HaCaT cells) (A), RAW 264.7 cells (B), and BEAS-2B cells (C).

[0050] Figure 3 shows the results of analyzing the effect on the production of ROS induced by LPS through regulation of the HO-1 / NRF2 signaling pathway in RAW 264.7 cells by confirming the anti-inflammatory activity of the ethanol precipitated fraction (UEF3) of the ultrasonic extract of Undaria pinnatifida of the present invention, (A-B) the level of ROS production confirmed by DCF-DA and observed under a fluorescence microscope, (C) the expression level of HO-1 confirmed by Western blot, (D) the expression level of HO-1 represented as a graph, and (E) the results of analyzing the expression level of KEAP1 and the translocation of NRF2 by Western blot.

[0051] Figure 4 shows the analysis of the effect of the ethanol precipitated fraction of the ultrasonic extract of Undaria pinnatifida on the expression of COX-2 induced by ultraviolet (UVB) radiation through regulation of the MAPK signaling pathway in HaCaT cells, and (AB) COX-2 and MAPK phosphorylation levels were analyzed by Western blot.

[0052] Figure 5 is a schematic diagram showing the process of obtaining an ultrasonic extract from seaweed sporophyll and an ethanol fraction from the ultrasonic extract.

[0053] Figure 6 shows the yield (black line) and polysaccharide content (gray bar) of ultrasonic extracts obtained from seaweed sporophyll according to the ultrasonic treatment time. UPE_2, UPE_4, UPE_6, and UPE_8 represent extracts obtained by ultrasonic treatment for 2 hours, 4 hours, 6 hours, and 8 hours, respectively.

[0054] Figure 7 shows the results of analyzing the effect of ultrasonic extract of Undaria pinnatifida (UPE) on cell viability of Vero cells (A), protection of Vero cells from hydrogen peroxide (B), and effect on ROS production (C).

[0055] Figure 8 shows the results of analyzing the effects on Vero cell protection (A) and ROS production (B) from hydrogen peroxide for the ethanol precipitated fraction (UPE_8P) and supernatant (UPE_8S) obtained after adding ethanol to the ultrasonic extract of Undaria pinnatifida (treated for 8 hours) and performing ethanol precipitation.

[0056] Figure 9 shows the results of analyzing the effects of the ethanol precipitated fraction (UPE_8P) of the ultrasonic extract of Undaria pinnatifida sporophyll on the expression of Bax and Bcl-2 proteins (A, B, C) and the Bax / Bcl-2 expression ratio (D) in Vero cells treated with hydrogen peroxide.

[0057] Figure 10 is a schematic diagram showing the process of obtaining an ultrasonic extract from seaweed sporophyll.

[0058] Figure 11 shows the results of DPPH radical scavenging activity analysis according to treatment concentration of the ultrasonic extract of seaweed sporophyll according to the present invention.

[0059] Figure 12 shows the results of MTS analysis to determine whether there is cytotoxicity after treating HaCaT cells and B16F10 cell lines with the ultrasonic extract of seaweed sporophyll according to the present invention at different concentrations.

[0060] Figure 13 shows the results of analyzing the collagenase inhibitory activity according to the treatment concentration of the ultrasonic extract of seaweed sporophyll according to the present invention. NC represents the negative control group treated with only the substrate, PC represents the positive control group treated with the substrate and enzyme solution, and Vit.C represents the group treated with vitamin C.

[0061] Figure 14 shows the results of confirming the inhibitory activity against hyaluronidase and the protein expression inhibitory activity of hyaluronidase according to the treatment concentration of the ultrasonic extract of seaweed sporophyll according to the present invention.

[0062] Figure 15 shows the results of confirming the whitening activity of the ultrasonic extract of the seaweed sporophyll according to the present invention. A shows the mushroom tyrosinase inhibitory activity of the ultrasonic extract of the seaweed sporophyll according to the treatment concentration, B shows the activity of inhibiting malanine production increased by α-MSH in the B16F10 cell line, and C shows the results of L-DOPA staining.

[0063] The present invention is characterized by providing an anti-inflammatory, antioxidant, skin whitening, wrinkle improvement or skin moisturizing composition containing an ultrasonic extract of seaweed sporophyll as an active ingredient.

[0064] The inventors of the present invention, while conducting research to develop a functional material using the seaweed sporophyll discarded during the processing process, first discovered that the ultrasonic extract obtained from the seaweed sporophyll has excellent skin whitening, wrinkle improvement, and skin moisturizing activities.

[0065] In the present invention, the ultrasonic extract of the above-mentioned seaweed sporophyll may be an extract obtained by ultrasonic treatment of seaweed sporophyll, preferably, an ultrasonic extract obtained by adding purified water to seaweed sporophyll powder and ultrasonic treatment at 1,000 to 1,100 W and 28 to 32°C for 7 to 9 hours, and more preferably, an extract obtained by ultrasonic treatment at 1,080 W and 30°C for 8 hours.

[0066] If the above ultrasonic extraction is performed under conditions outside the range of the ultrasonic treatment conditions described above, there is a problem in that the active ingredient having the above activity is obtained from the seaweed sporophyll at a very low yield, and furthermore, a problem in that the activity of the active ingredient may be lost or altered may occur.

[0067] Therefore, it is important to obtain the ultrasonic extract of the seaweed sporophyll of the present invention under the ultrasonic treatment conditions described above.

[0068] In addition, the ultrasonic extract of the seaweed sporophyll of the present invention may be obtained by ultrasonic treatment, filtering through a filter cloth and a housing, performing ion exchange, and then sterilizing and freeze-drying.

[0069] The inventors of the present invention analyzed the antioxidant activity of the ultrasonic extract of the seaweed sporophyll prepared by the method of the present invention, and as a result, it was found to have DPPH radical scavenging activity, and it was confirmed that the extract also contained a large amount of total polyphenols and total flavonoids having antioxidant activity.

[0070] In addition, in another embodiment of the present invention, the wrinkle formation inhibition effect of the ultrasonic extract of the seaweed sporophyll of the present invention was analyzed, and as a result, it was confirmed that when treated with the extract of the present invention, it has an activity of inhibiting collagenase, which is known to induce wrinkle formation as a collagen fiber decomposing enzyme, and it was also shown that the expression of MMP-1 protein in human skin epidermal cells was inhibited by treatment with the extract of the present invention.

[0071] Dermal cells in the skin secrete matrix metalloproteinases (MMPs), enzymes that break down extracellular matrix proteins such as collagen and elastin fibers. Collagen, in particular, makes up the majority of the skin's matrix structure, and when it is reduced by these enzymes, wrinkles and elasticity develop. Specifically, MMP-1, produced by dermal fibroblasts, acts to break down types I, II, and III collagen. Therefore, substances that inhibit MMP-1 can improve and suppress skin wrinkles.

[0072] In another embodiment of the present invention, the moisturizing function of the ultrasonic extract of the seaweed sporophyll of the present invention was analyzed, and as a result, it was found that the extract of the present invention has an activity of inhibiting hyaluronidase (hyaluronic acid decomposition enzyme), which is an enzyme that decomposes hyaluronic acid, which plays an important role in skin moisturizing, and also inhibits the expression of hyaluronidase protein.

[0073] Therefore, the ultrasonic extract of the seaweed sporophyll of the present invention has the effect of maintaining or enhancing skin moisture by inhibiting hyaluronidase activity.

[0074] In addition, the ultrasonic extract of the seaweed sporophyll of the present invention has the characteristic of having skin whitening activity.

[0075] According to one embodiment of the present invention, it was shown that tyrosinase activity was inhibited by treatment with an ultrasonic extract of seaweed sporophyll, and melanin production was also inhibited.

[0076] It is known that the chemical action for melanin production, which determines skin color, is that melanocytes, which are melanin-producing cells, synthesize melanin, which has the function of protecting cells from ultraviolet rays, within intracellular organelles called melanosomes, and then the melanin moves from the melanocytes to keratinocytes. It is reported that the melanin moved to keratinocytes in this way determines skin color.

[0077] It is known that the precursor required in the above melanin biosynthesis process is an amino acid called tyrosine, which is converted into dihydroxyphenylalanine (DOPA), and then through a substance called dopaquinone, and ultimately converted into melanin.

[0078] In this regard, tyrosinase (TYR), tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP*?*2 / DOPA, chrome tautomerase) are known as melanin-forming enzymes, and it is known that the above melanin-forming enzymes are regulated by a transcription factor called MITF (microphthalmia-associated transcription factor). Tyrosinase, which plays an important role in the melanin synthesis pathway, converts L-tyrosine into L-DOPA (L-3,4-dihydroxyphenylalanine), rapidly converts the produced L-DOPA into L-dopaquinone, and ultimately forms melanin through various subsequent reactions. Melanin synthesized by tyrosinase darkens skin color, and suppressing melanin synthesis for health or cosmetic reasons can lighten skin color and induce a skin whitening effect.

[0079] In this respect, the ultrasonic extract of the seaweed sporophyll of the present invention can effectively inhibit tyrosinase activity and has the activity of inhibiting melanin production, thereby inducing a skin whitening effect.

[0080] Therefore, the ultrasonic extract of the seaweed sporophyll of the present invention has antioxidant activity, collagenase inhibitory activity, MMP-1 protein expression inhibition, hyaluronic acid decomposition enzyme inhibition, tyrosinase activity inhibition, or melanin production inhibition activity, and can be usefully used for skin whitening, wrinkle improvement, and skin moisturizing.

[0081] Therefore, the present invention can provide a cosmetic composition for skin whitening, wrinkle improvement, and skin moisturizing, which contains an ultrasonic extract of seaweed sporophyll as an effective ingredient.

[0082] In addition to the above-mentioned effective ingredients, the cosmetic composition of the present invention may contain other ingredients commonly contained in cosmetics, as needed. Examples of such components include fat components, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, pH adjusters, alcohols, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, purified water, water-soluble vitamins, fat-soluble vitamins, high molecular weight peptides, high molecular weight polysaccharides, sphingolipids, and seaweed extracts.

[0083] The cosmetic composition of the present invention can be prepared in the form of an emulsified formulation and a solubilized formulation commonly used in the art.

[0084] In addition, the components included in the cosmetic composition of the present invention may include components commonly used in cosmetic compositions in addition to the above-mentioned components as effective ingredients, and may further include conventional auxiliary agents and carriers such as stabilizers, pigments, and natural fragrances.

[0085] Products to which the composition of the present invention can be added include, for example, cosmetics such as mist, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, UV protection cream, moisture cream, hand cream, foundation, essence, nutrition essence, mask pack, pressed powder, loose powder, eye shadow, and soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, and body cleanser.

[0086] When the formulation of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0087] When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane, butane or dimethyl ether may be additionally included.

[0088] In the case where the formulation of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0089] When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used as carrier components.

[0090] In addition, the present invention can provide a health food composition for skin whitening, wrinkle improvement, and skin moisturizing, which contains an ultrasonic extract of the seaweed sporophyll of the present invention as an effective ingredient.

[0091] In one embodiment of the present invention, the cytotoxicity of the ultrasonic extract of the seaweed sporophyll prepared by the method of the present invention was analyzed, and it was found that it was safe as it did not induce cytotoxicity.

[0092] The term “food” herein refers to a natural or processed product containing one or more nutrients, preferably one that has gone through a certain degree of processing to become directly edible, and in its general sense includes all foods, food additives, functional foods, and beverages.

[0093] Foods to which the composition according to the present invention can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, functional foods, etc. In addition, foods in the present invention include, but are not limited to, special nutritional foods (e.g., formulated milk, infant and toddler food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g., ramen, noodles, etc.), bread, health supplements, seasoned foods (e.g., soy sauce, soybean paste, red pepper paste, mixed paste, etc.), sauces, confectionery (e.g., snacks), candies, chocolates, gum, ice cream, processed dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi, pickled vegetables, etc.), beverages (e.g., fruit drinks, vegetable drinks, soy milk, fermented drinks, etc.), and natural seasonings (e.g., ramen soup, etc.). The above food, beverage or food additive can be manufactured by a conventional manufacturing method.

[0094] The above “functional food” refers to a food group or food composition that has been designed and processed to sufficiently exert its internal regulatory functions, such as regulating biological defense rhythms, disease prevention, and recovery, by using physical, biochemical, or bioengineering techniques to provide added value to the food so that the food’s function can be performed and expressed for a specific purpose. Specifically, it may be a health functional food. The above functional food may include food additives that are acceptable from a food science perspective, and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of functional foods.

[0095] Additionally, in the present invention, the term "beverage" refers to a general term for anything consumed to quench thirst or enjoy a flavor, and includes functional beverages. The beverage, other than including the antioxidant composition as an essential ingredient in the indicated proportions, has no particular limitations on other ingredients, and, like conventional beverages, may contain various flavorings or natural carbohydrates as additional ingredients.

[0096] Furthermore, in addition to those described above, a food containing the composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., and the above components may be used independently or in combination.

[0097] In a food containing a composition according to the present invention, the amount of the composition according to the present invention may be comprised in an amount of 0.001 wt% to 90 wt% of the total food weight, preferably 0.1 wt% to 40 wt%, and in the case of a beverage, it may be comprised in an amount of 0.001 g to 2 g, preferably 0.01 g to 0.1 g, based on 100 ml. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since the active ingredient does not have any problems in terms of safety, it may be used in an amount greater than the above range, and thus is not limited to the above range.

[0098] Furthermore, the present invention can provide a pharmaceutical composition for skin whitening or wrinkle improvement, which comprises an ultrasonic extract of seaweed sporophyll as an active ingredient.

[0099] As described above, the ultrasonic extract of the seaweed sporophyll of the present invention has tyrosinase inhibitory activity and melanin production inhibitory activity at the same time, and thus has an excellent whitening effect and can be used as a pharmaceutical composition for skin whitening. In addition, it can inhibit collagenase activity involved in wrinkle formation and has MMP-1 protein expression inhibitory activity, and thus can be used as a pharmaceutical composition for improving skin wrinkles.

[0100] In the present invention, the term 'whitening effect' refers to not only brightening the skin tone by inhibiting the synthesis of melanin pigment, but also improving skin hyperpigmentation such as freckles or blemishes caused by ultraviolet rays, hormones, or genetics.

[0101] Additionally, 'wrinkle improvement' refers to suppressing or inhibiting the formation of wrinkles on the skin, or alleviating wrinkles that have already formed.

[0102] Additionally, "moisturizing" or "skin hydration" refers to increasing moisture in the skin and maintaining its hydration. Skin moisturizing can help improve wrinkles and increase elasticity.

[0103] In addition, the pharmaceutical composition for skin whitening of the present invention can be used as a pharmaceutical composition for preventing or treating hyperpigmentation disease, and the present invention can provide a pharmaceutical composition for preventing or treating hyperpigmentation disease, which comprises the ultrasonic extract of the seaweed sporophyll of the present invention as an active ingredient.

[0104] The above hyperpigmentation disease may be selected from the group consisting of, but is not limited to, pigmentation arising from blemishes, freckles, lentigines, nevi, drug-induced pigmentation, post-inflammatory pigmentation, and dermatitis.

[0105] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is one commonly used in the preparation of formulations and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0106] The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc.

[0107] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. A skilled physician can easily determine and prescribe a dosage effective for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10,000 mg / kg.

[0108] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0109] The present invention is characterized by providing an antioxidant composition comprising an ultrasonic extract of seaweed sporophyll or an ethanol precipitate fraction of the ultrasonic extract as an effective ingredient.

[0110] The present inventors, while conducting research to discover a material with excellent antioxidant activity from natural materials, established an optimal manufacturing process capable of obtaining an extract of Undaria pinnatifida and its fractions with excellent antioxidant activity from Undaria pinnatifida, and found that an ultrasonic extract of Undaria pinnatifida and an ethanol precipitate fraction of the ultrasonic extract had excellent antioxidant activity.

[0111] In the present invention, the ultrasonic extract of the above-mentioned seaweed sporophyll may be an extract obtained by ultrasonic treatment of seaweed sporophyll, preferably, an ultrasonic extract obtained by adding purified water to seaweed sporophyll powder and ultrasonic treatment at 1,000 to 1,100 W and 28 to 32°C for 7 to 9 hours, and more preferably, an extract obtained by ultrasonic treatment at 1,080 W and 30°C for 8 hours.

[0112] If the above ultrasonic extraction is performed under conditions outside the range of the ultrasonic treatment conditions described above, there is a problem in that the effective ingredient having antioxidant activity from the seaweed sporophyll is obtained in a very low yield, and furthermore, the problem in that the antioxidant activity of the effective ingredient may be lost or altered may occur.

[0113] Therefore, it is important to obtain the ultrasonic extract of the seaweed sporophyll of the present invention under the ultrasonic treatment conditions described above.

[0114] In addition, in another embodiment of the present invention, the extraction yield, polysaccharide content, and antioxidant activity of ultrasonic extracts obtained according to ultrasonic treatment time were analyzed. As a result of the analysis, it was shown that the extract obtained by ultrasonic treatment for 8 hours had the highest extraction yield and contained the most polysaccharides compared to the extracts obtained by ultrasonic treatment for 2 hours, 4 hours, and 6 hours, and it was also shown to be able to most effectively suppress cellular oxidative stress caused by H2O2.

[0115] Therefore, through these results, the inventors of the present invention were able to find out that the ultrasonic extract of the seaweed sporophyll of the present invention has antioxidant activity, and in particular, it was found that the extract obtained by ultrasonic treatment for 8 hours has excellent antioxidant activity.

[0116] Next, the inventors of the present invention added ethanol to the ultrasonic extract of the seaweed sporophyll obtained by ultrasonic treatment for 8 hours, performed ethanol precipitation, performed centrifugation, and then obtained the precipitate and supernatant, respectively. The major components and antioxidant activity of each of the precipitate and supernatant were analyzed.

[0117] As a result, it was confirmed that the ethanol precipitate fraction (precipitate) contained the most polysaccharides compared to the ultrasonic extract of the seaweed sporophyll and the supernatant after ethanol precipitation.

[0118] Through these results, the inventors were able to determine that the ethanol precipitate fraction of the ultrasonic extract of the seaweed sporophyll is a polysaccharide fraction.

[0119] In addition, the results of the antioxidant activity analysis showed that the group in which oxidative stress was induced by H2O2 treatment showed a significantly lower cell viability due to increased cell death caused by oxidative stress compared to the normal group, whereas the group treated with the ultrasonic extract of Undaria sporophyll of the present invention showed an increase in cell viability as the cell viability decreased by H2O2 treatment was recovered, whereas the group treated with the supernatant obtained after ethanol precipitation of the ultrasonic extract of Undaria sporophyll showed a low cell viability similar to the H2O2 treatment group. These results indicate that the supernatant obtained after ethanol precipitation of the ultrasonic extract of Undaria sporophyll contains almost no active ingredient with antioxidant activity.

[0120] However, the group treated with the ethanol precipitate fraction of the ultrasonic extract of the seaweed sporophyll showed a greater increase in cell viability than the group treated with the ultrasonic extract of the seaweed sporophyll, demonstrating the best antioxidant activity.

[0121] In addition, in another embodiment of the present invention, the mechanism of antioxidant activity of the ethanol precipitate fraction of the ultrasonic extract of Undaria sporophyll according to the present invention was analyzed, and as a result, when the ethanol precipitate fraction of the ultrasonic extract of Undaria sporophyll was treated in cells induced with oxidative stress, the expression of the Bax protein, which induces apoptosis, was suppressed, while the expression of the Bcl-2 protein, which inhibits apoptosis, was increased.

[0122] These results show that the ethanol precipitate fraction of the ultrasonic extract of the seaweed sporophyll of the present invention has antioxidant activity by inhibiting cell death caused by oxidative stress and protecting cells from oxidative stress.

[0123] Accordingly, the inventors of the present invention were able to determine that the ethanol precipitate fraction of the ultrasonic extract of the seaweed sporophyll according to the present invention contains active ingredients having antioxidant activity, and that the ethanol precipitate fraction can be used in a useful industrial product having antioxidant activity.

[0124] Therefore, the present invention can provide an antioxidant composition comprising an ultrasonic extract of seaweed sporophyll or an ethanol precipitate fraction of the ultrasonic extract as an effective ingredient.

[0125] Preferably, the above-mentioned ultrasonic extract of the seaweed sporophyll may be an extract obtained by ultrasonic treatment at 1,080 W and 30°C for 8 hours, and the above-mentioned ethanol precipitate fraction may be a precipitate obtained by adding ethanol to the ultrasonic extract of the seaweed sporophyll obtained by ultrasonic treatment for 8 hours, performing ethanol precipitation overnight at 4°C, and then centrifuging.

[0126] In addition, the present invention can provide an antioxidant health functional food comprising the antioxidant composition of the present invention.

[0127] In one embodiment of the present invention, the cytotoxicity of the ultrasonic extract of the seaweed sporophyll prepared by the method of the present invention and the ethanol precipitate fraction of the ultrasonic extract were analyzed, and it was found that neither of them induced cytotoxicity, indicating that they were stable.

[0128] Furthermore, the present invention can provide a method for producing an ethanol precipitate fraction of an ultrasonic extract of Undaria pinnatifida having antioxidant activity, wherein the method preferably comprises the steps of: (1) obtaining an ultrasonic extract of Undaria pinnatifida; and (2) adding ethanol to the ultrasonic extract of Undaria pinnatifida obtained in step (1) to obtain an ethanol precipitate fraction.

[0129] The sporophyll is the reproductive organ of the brown algae, Gonophyceae. As the seaweed grows in spring, wrinkles form along the lower edge of the stem, transforming into sporophylls where zoospore sacs develop. When the water temperature exceeds 14℃, the zoospores are released and the mother plant thaws. This phenomenon continues until 22℃. In autumn, eggs and sperm are released from the male and female gametophytes, respectively, which fertilize and soon germinate to form spores. These grow into the thallus, which is the main body of the seaweed.

[0130] The spore leaves of seaweed contain about 20% of sulfated polysaccharides with a high sulfate content, which are called sulfated fucose polysaccharides or fucoidan. They are known to have physiological activities such as anticancer, antiviral, antitumor, and immune enhancement.

[0131] Meanwhile, most of these seaweed sporophylls are discarded during the seaweed processing process, and therefore, research is not being conducted to establish a manufacturing process for functional products using seaweed sporophylls.

[0132] Accordingly, the inventors of the present invention established an optimal manufacturing process capable of obtaining a fraction having antioxidant activity from seaweed sporophyll discarded as waste.

[0133] The method for producing an ethanol precipitate fraction of the ultrasonic extract of Undaria pinnatifida having the antioxidant activity according to the present invention first obtains an ultrasonic extract of Undaria pinnatifida.

[0134] The above ultrasonic extract can be obtained by adding purified water to the powder of seaweed sporophyll and treating it with ultrasonic waves at 1,000 to 1,100 W and a temperature of 28 to 32°C for 7 to 9 hours.

[0135] The above seaweed sporophyll can be crushed to a size of 2 to 3 mm, and in one embodiment of the present invention, it was crushed to a size of 2.5 mm and used.

[0136] The above ultrasonic treatment can be performed under the conditions described above, 1,000 to 1,100 W, 28 to 32°C, and 7 to 9 hours. However, if the treatment is performed under conditions outside the above conditions, it may be difficult to obtain active ingredients having antioxidant activity from the seaweed sporophyll, and the activity of the effective active ingredients contained in the seaweed sporophyll may be destroyed or lost. Therefore, it is preferable to perform the ultrasonic treatment under the above conditions, and in one embodiment of the present invention, the extract was obtained by ultrasonic treatment at 1,080 W and 30°C for 8 hours.

[0137] The above ultrasonic treatment performed in the present invention is an industrial-scale ultrasonic treatment condition, and through this process, a large amount of ultrasonic extract of the seaweed sporophyll can be easily obtained.

[0138] When the ultrasonic extract of the seaweed sporophyll is obtained, ethanol is then added to the ultrasonic extract to obtain an ethanol precipitate fraction.

[0139] At this time, before adding ethanol, the obtained ultrasonic extract of the seaweed sporophyll is subjected to filtration and filter treatment, and an additional sterilization process is performed to obtain a sterilized ultrasonic extract of the seaweed sporophyll.

[0140] The above ethanol precipitate fraction can be obtained in the form of an ethanol precipitate settled at the bottom by adding ethanol to an ultrasonic extract of the sporophyll of Undaria pinnatifida, performing ethanol precipitation at a temperature of 4 to 6°C for 20 to 28 hours, and then centrifuging. In one embodiment of the present invention, ethanol precipitation was performed overnight at 4°C, and centrifugation was performed at 4°C for 10 minutes at a speed of 12,000 g to obtain a precipitate.

[0141] The present invention is characterized by providing a method for obtaining a fraction having excellent effects of suppressing, improving and treating skin inflammation from the spore leaves of seaweed.

[0142] Specifically, the present invention is characterized in that it provides a method for producing an ethanol precipitate fraction of an ultrasonic extract of Undaria pinnatifida having an activity of inhibiting or improving skin inflammation, the method comprising the steps of (1) obtaining an ultrasonic extract of Undaria pinnatifida; and (2) adding ethanol to the ultrasonic extract of Undaria pinnatifida obtained in step (1) to obtain an ethanol precipitate fraction.

[0143] The sporophyll is the reproductive organ of the brown algae, Gonophyceae. As the seaweed grows in spring, wrinkles form along the lower edge of the stem, transforming into sporophylls where zoospore sacs develop. When the water temperature exceeds 14℃, the zoospores are released and the mother plant thaws. This phenomenon continues until 22℃. In autumn, eggs and sperm are released from the male and female gametophytes, respectively, which fertilize and soon germinate to form spores. These grow into the thallus, which is the main body of the seaweed.

[0144] The spore leaves of seaweed contain about 20% of sulfated polysaccharides with a high sulfate content, which are called sulfated fucose polysaccharides or fucoidan. They are known to have physiological activities such as anticancer, antiviral, antitumor, and immune enhancement.

[0145] Meanwhile, most of these seaweed sporophylls are discarded during the seaweed processing process, and therefore, research is not being conducted to establish a manufacturing process for functional products using seaweed sporophylls.

[0146] Accordingly, the inventors of the present invention have established an optimal manufacturing process capable of obtaining a fraction with excellent anti-inflammatory and improvement activity from seaweed sporophyll discarded as waste.

[0147] The method for producing a fraction having excellent anti-inflammatory and improving activity from the sporophyll of the present invention preferably comprises the steps of (1) obtaining an ultrasonic extract of the sporophyll of the present invention; and (2) adding ethanol to the ultrasonic extract of the sporophyll of the present invention obtained in step (1) to obtain an ethanol precipitated fraction.

[0148] The above method is explained in more detail as follows.

[0149] First, an ultrasonic extract of the seaweed sporophyll is obtained.

[0150] The above ultrasonic extract can be obtained by adding purified water to the powder of seaweed sporophyll and treating it with ultrasonic waves at 1,000 to 1,100 W and a temperature of 28 to 32°C for 7 to 9 hours.

[0151] Here, the above seaweed sporophyll can be crushed to a size of 2 to 3 mm, and in one embodiment of the present invention, it was crushed to a size of 2.5 mm and used.

[0152] In addition, the above ultrasonic treatment can be performed under the conditions described above, 1,000 to 1,100 W, 28 to 32°C, and 7 to 9 hours. However, if the treatment is performed under conditions outside the above conditions, it may be difficult to obtain an extract with excellent anti-inflammatory activity from the seaweed sporophyll, and the activity of the effective active ingredients contained in the seaweed sporophyll may be destroyed or lost. Therefore, it is preferable to perform the ultrasonic treatment under the above conditions, and in one embodiment of the present invention, the extract was obtained by ultrasonic treatment at 1,080 W, 20 kHz, and 30°C for 8 hours.

[0153] The above ultrasonic treatment performed in the present invention is an industrial-scale ultrasonic treatment condition, and through this process, a large amount of ultrasonic extract of the seaweed sporophyll can be easily obtained.

[0154] When the ultrasonic extract of the seaweed sporophyll is obtained, ethanol is then added to the ultrasonic extract to obtain an ethanol precipitate fraction.

[0155] At this time, before adding ethanol, the obtained ultrasonic extract of the seaweed sporophyll is subjected to filtration and filter treatment, and an additional sterilization process is performed to obtain a sterilized ultrasonic extract of the seaweed sporophyll.

[0156] The above ethanol precipitate fraction can be obtained in the form of an ethanol precipitate settled at the bottom by adding ethanol to an ultrasonic extract of the sporophyll of Undaria pinnatifida, performing ethanol precipitation at a temperature of 4 to 6°C for 20 to 28 hours, and then centrifuging. In one embodiment of the present invention, ethanol precipitation was performed at 4°C for 24 hours, and centrifugation was performed at a speed of 5,000 g for 15 minutes to obtain a precipitate.

[0157] In addition, the ethanol precipitation fraction according to the present invention can be obtained in the form of a plurality of ethanol precipitation fractions by repeatedly performing the process of obtaining a precipitate using ethanol several times. In one embodiment of the present invention, ethanol was added to the ultrasonic extract of the seaweed sporophyll obtained in the process of step (1) to perform ethanol precipitation, and then centrifuged to obtain a first ethanol precipitation fraction (UEF1). After separating the supernatant after the centrifugation, ethanol was added thereto again, and ethanol precipitation and centrifugation were performed to obtain a second ethanol precipitation fraction (UEF2). After separating the supernatant, ethanol was added thereto again, and ethanol precipitation and centrifugation were performed to obtain a third ethanol precipitation fraction (UEF3). After separating the supernatant, ethanol was added thereto again, and ethanol precipitation and centrifugation were performed to obtain a fourth ethanol precipitation fraction (UEF4). In addition, the fourth ethanol precipitation fraction The supernatant of the fraction was taken and used as the supernatant experimental group in the examples.

[0158] Furthermore, the inventors of the present invention conducted an analysis of the major components contained in the ethanol precipitate fraction of each of the ultrasonic extracts of the seaweed sporophyll produced by the method of the present invention and the supernatant obtained in the process of obtaining the ultrasonic extract and the final ethanol precipitate fraction.

[0159] As a result, as shown in Tables 1 to 4 of the examples below, it was confirmed that there were differences in the types and contents of the main components contained in each fraction, ultrasonic extract, and supernatant.

[0160] Through this, it was found that even if the process of obtaining a solvent precipitate using the same solvent is performed repeatedly, there are differences in the types or contents of effective ingredients in the obtained fractions depending on each processing step, i.e., the number of repetitions.

[0161] The present inventors conducted an experiment to confirm whether the ethanol precipitated fractions of the ultrasonic extract of the seaweed sporophyll of the present invention obtained through the above process have anti-inflammatory, improving, and therapeutic effects.

[0162] As a result, the ethanol precipitated fractions of the ultrasonic extract of the seaweed sporophyll of the present invention were found to have the activity of scavenging free radicals known as inflammation-inducing factors, and were also found to have the effect of inhibiting the production of reactive oxygen species.

[0163] In addition, it was confirmed that this anti-inflammatory activity is due to the activation of the HO-1 / NRF2 signaling pathway. When the ethanol precipitated fraction of the present invention was treated, the expression of HO-1, known as an antioxidant factor, was found to increase, and it was confirmed that this increase in HO-1 expression was due to the movement of NRF2, known as a factor that induces the expression of anti-inflammatory factors, from the cytosol to the nucleus.

[0164] In another embodiment of the present invention, it was analyzed whether the ethanol precipitate fraction of the present invention could suppress the expression of COX-2 due to ultraviolet irradiation, which acts as a cause of skin inflammation. As a result, it was shown that the group treated with the ethanol precipitate fraction of the ultrasonic extract of the seaweed sporophyll of the present invention had the expression of COX-2, an inflammatory factor, suppressed by ultraviolet irradiation.

[0165] In addition, in the results of the anti-inflammatory activity analysis, it was shown that the ethanol precipitated fractions of the ultrasonic extract of the seaweed sporophyll obtained by the method of the present invention have anti-inflammatory activity, and in particular, the 3rd ethanol precipitated fraction (UEF3) was shown to have the best anti-inflammatory activity compared to other precipitated fractions, and in the case of the UEF3 fraction, unlike other fractions, it was shown to have an activity of inhibiting the phosphorylation of ERK1 / 2 related to the inflammatory mechanism.

[0166] Through these results, the inventors of the present invention were able to determine that the ethanol precipitate fraction of the ultrasonic extract of the seaweed sporophyll obtained by the method of the present invention has anti-inflammatory activity and can be usefully used for the prevention, improvement, and treatment of skin inflammation.

[0167] Therefore, the present invention can provide an ethanol precipitate fraction of an ultrasonic extract of seaweed sporophyll having skin inflammation suppression or improvement activity, prepared by the method of the present invention.

[0168] Preferably, the ethanol precipitation fraction may be a third ethanol precipitation fraction (UEF3).

[0169] The ethanol precipitate fraction of the ultrasonic extract of the seaweed sporophyll according to the present invention contains carbohydrates, proteins, polyphenols and fucoxanthin, and various types of monosaccharides.

[0170] In the case of the above 3rd ethanol precipitation fraction (UEF3), which was confirmed to have the best anti-inflammatory activity, it contains 32.72% Fucose, 0.5% Rhamnose, 62.63% Galactose, 2.23% Glucose, 1.84% Xylose, and 0.08% Fructose as monosaccharides.

[0171] In addition, the 3rd ethanol precipitation fraction (UEF3) was found to contain the most sulfate polysaccharide with a high sulfate group content compared to other ethanol precipitation fractions, and was found to contain 33.89±0.05% of sulfate polysaccharide.

[0172] In addition, it was confirmed that the ethanol precipitate fraction of the ultrasonic extract of the seaweed sporophyll according to the present invention is stable because it does not cause cytotoxicity. In one embodiment of the present invention, it was confirmed that the ethanol precipitate fraction of the present invention does not cause cytotoxicity for all of the human keratinocyte cell line (HaCaT), the mouse macrophage cell line (Raw264.7), and the human normal bronchial epithelial cell line (BEAS-2B).

[0173] Therefore, the present invention can provide a cosmetic composition for preventing or improving skin inflammation, which comprises an ethanol precipitate fraction of an ultrasonic extract of seaweed sporophyll prepared by the method of the present invention as an effective ingredient.

[0174] In the present invention, the skin inflammation may include all skin inflammatory diseases that involve an inflammatory reaction occurring in the skin, and may be any one selected from the group consisting of atopic dermatitis, contact dermatitis, seborrheic dermatitis, and acne, but is not limited thereto.

[0175] The cosmetic composition and food composition described in the present invention are as described above.

[0176] Hereinafter, the present invention will be described in more detail through examples. These examples are intended to more specifically illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0177]

[0178] 1. Preparation of a fraction with excellent anti-inflammatory activity from seaweed sporophyll extract and experimental method for confirming anti-inflammatory activity

[0179] (1) Materials and reagents

[0180] DMEM (Dulbecco's Modified Eagle's Medium) / high glucose containing L-glutamine supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (antibiotics) for cell culture was purchased from Hyclone (Logan, UT, USA). DMEM / high glucose containing L-glutamine or without phenol red (serum-free medium) was purchased from Hyclone (Logan, UT, USA) and used to induce starvation of HaCaT cells. LPS derived from E. coli O111:B4, supplied by Sigma-Aldrich (St. Louis, MO, USA), was diluted with sterile phosphate-buffered saline (PBS) to a concentration of 1 mg / mL. NAC (N-acetyl-L-cysteine), EGCG ((-)-Epigallocatechin gallate), and fucoidan derived from seaweed were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used after dilution in DMSO. In addition, primary antibodies against KEAP-1 (1:1000), HO-1 (1:1000), COX-2 (1:1000), p-ERK1 / 2 (1:1000), ERK1 / 2 (1:1000), p-JNK1 / 2 (1:1000), JNK1 / 2 (1:1000), p-p38 (1:1000), and p38 (1:1000) were purchased from Cell signaling (Beverly, MA, USA) and used. β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All antibodies were diluted using 1X Tris-Buffered Saline, 0.1% Tween® 20 Detergent (TBST) buffer. HRP (horseradish peroxidase)-conjugated pierce goat anti rabbit IgG (H+L) was used as the secondary antibody.

[0181]

[0182] (2) Preparation of seaweed sporophyll extract using ultrasound and preparation of fractions using ethanol precipitation

[0183] Dried U. pinnatifidasporophyll (Wando-gun, South Korea) was purchased and stored at 4℃ until use. The U. pinnatifidasporophyll was pulverized, and 6 kg of the powder was extracted with 600 L of water for 8 h at 20 kHz, 1,080 W, and 30℃ using an industrial-scale ultrasonic extraction system (Mirae Ultrasonic, Bucheon, South Korea). After housing filtration and filter treatment, the solution was sterilized (121℃, 15 min), and then freeze-dried to obtain the ultrasonic extract of U. pinnatifidasporophyll (UPE). The extract was then subjected to ethanol precipitation. 1 L of ethanol was added to 0.5 L of a solution containing 30 g of the ultrasonic extract of U. pinnatifidasporophyll, and the solution was allowed to settle at 4℃ for 1 day. Afterwards, centrifugation (5,000 x g for 15 minutes) was performed to collect the precipitate and named 'UEF1'. After the 'UEF1' precipitate was separated, 1 L of ethanol was added to the supernatant and precipitated for 1 day, then centrifuged (5,000 x g, 15 minutes) to obtain the precipitate 'UEF2' again. By repeating the same process as above, ethanol precipitate fractions 'UEF3' and 'UEF4' were obtained, respectively. The four ethanol precipitate fractions (UEF1 to UEF4) obtained in this way were freeze-dried after removing ethanol. This process is shown in Fig. 1.

[0184]

[0185] (3) Yield and component analysis

[0186] The yield of the fractions obtained in the above process (2) was measured from the dry weight ratio of each fraction to the extract. Total carbohydrate content was measured by the phenol-sulfuric acid method using glucose as a standard and a total carbohydrate analysis kit (Sigma-Aldrich, St. Louis, MO, USA). Protein content was quantified using a DC protein assay kit (Bio-rad, Hercules, CA, USA), and bovine serum albumin (Sigma-Aldrich) was used as a standard. Total polyphenol content was measured according to the method of Zhang et al. Twenty microliters of sample solution and sequential standard solutions were added to a 96-well microplate, and 100 μL of Folin-Ciocalteu reagent was added to each well with thorough mixing and left to stand for 5 minutes. Then, 80 μL of 7.5% sodium carbonate solution was added and mixed well again. The microplate was covered and left to stand in a dark environment at room temperature for 2 hours. Finally, the absorbance was measured at a wavelength of 750 nm using a spectrophotometer. The content of fucoxanthin was analyzed using a high-performance liquid chromatography (HPLC) system. For fucoxanthin extraction, 0.5 g of the sample was dissolved in 10 mL of methanol at 25°C for 2 hours. The precipitate obtained by centrifugation (3,000 rpm, 15 minutes) was dissolved again in 10 mL of methanol at 25°C for 1 hour and then centrifuged. The supernatant was evaporated and dissolved in a solvent (deionized water-ethyl acetate = 1:1, v / v) to collect the ethyl acetate layer. The solvent layer was then evaporated, and the sample was finally dissolved in 75% acetonitrile. Then, quantitative analysis was performed at 30°C using a high-performance liquid chromatography (HPLC) system (Dionex, Sunnyvale, CA, USA) equipped with an Inertsil ODS-3 column (4.6 × 250 mm, 5 μm) (GL Science, Tokyo, Japan).The sample was eluted using 75% acetonitrile solvent at a flow rate of 1 mL / min, detected using a UV detector at 450 nm, and the concentration of eluted fucoxanthin was calculated using the peak area.

[0187]

[0188] (4) Analysis of molecular weight and sugar composition

[0189] Molecular weight was analyzed by size exclusion chromatography using an HPLC system (Agilent, Palo Alto, CA, USA). Specifically, the sample was dissolved in distilled water, filtered through a 0.45 μm syringe filter, and then injected into the HPLC system. Analysis was performed using Shodex SB-804HQ and SB-802.5HQ OHPak columns (Showa Denko, Tokyo, Japan) at 55°C and eluted with water at a flow rate of 0.6 mL / min. Peaks were detected with a refractive index (RI) detector, and the molecular weight of each peak was measured using maltooligosaccharides from glucose to maltohexaose, and the Pullulan series (Shodex Standard Pullulan kit P-82, Showa Denko) was used as a molecular weight marker.

[0190] In addition, monosaccharide composition analysis was performed using a HPAEC-PAD (High-Performance Anion-Exchange Chromatography coupled with Pulsed Amperometric Detection, Dionex, Sunnyvale, CA, USA) system. The sample (2 mg / mL) was hydrolyzed with trifluoroacetic acid (TFA), then CarboPac ™ It was loaded onto a PA10 column (2 x 250 mm, 10 μm particle size), and the solvent was eluted using a gradient system of 18 mM NaOH / 200 mM NaOH at a flow rate of 1.0 mL / min.

[0191]

[0192] (5) Analysis of sulfate content

[0193] Analysis of the sulfate polysaccharide content was performed according to the BaCl2 gelation method (Dodgson, KS; Price, RG A note on the determination of the ester sulfate content of sulfated polysaccharides. Biochem. J. 1962, 84, 106-110).

[0194]

[0195] (6) Free radical scavenging activity analysis

[0196] The antioxidant activity of phlorotannins purified from Ecklonia cava on free radical scavenging using ESR and H2O2-mediated DNA damage was investigated using the ultrasonic extract of Undaria pinnatifida and the precipitate (UEF) obtained from the ethanol extract, according to the method reported by Ahn et al. (G.-N. Ahn, K.-N. Kim, S.-H. Cha, C.-B. Song, J. Lee, M.-S. Heo, …, J.-S. Kim). Antioxidant activities of phlorotannins purified from Ecklonia cava on free radical scavenging using ESR and H2O2-mediated DNA damage. European Food Research and Technology, 226 (1) (2007), pp. 71-79).

[0197]

[0198] (7) Cell culture

[0199] Human immortalized keratinocytes (HaCaT), human bronchial epithelial cells (BEAS-2B), and mouse macrophage RAW 264.7 cells were cultured in DMEM medium containing 10% FBS and 1% antibiotics. Cells were cultured at 37°C in a CO2 incubator manufactured by Eppendorf (Hamburg, Germany) under 5% CO2 and humidified conditions. When the cells reached 70–80% confluency, they were subcultured into new culture flasks and cultured for 2 days.

[0200]

[0201] (8) Cytotoxicity test

[0202] 1x10 HaCaT cells 5 Cells were grown in 96-well plates at a density of 10 cells / mL. When the cells reached 70–80% confluency, the medium was replaced with serum-free medium. In addition, the UPE (25, 50, and 100 μg / mL) obtained in the above process was pretreated for 12 h to induce cell starvation. Then, 10 μL / well of MTT solution purchased from Sigma-Aldrich (St. Louis, MO, USA) was treated and reacted for 24 h. 80 μL / well of the mixture was removed, and 100 μL / well of DMSO was added 2 h later. The absorbance was measured at a wavelength of 595 nm, and the cell viability was evaluated using statistical analysis. For cytotoxicity tests on RAW 264.7 cells and BEAS-2B cells, the cells were each seeded at a density of 3 × 10 5 10 cells / mL and 3 x 10 4 The cells were grown in a 96-well plate at a density of 10 cells / mL, and when the cells reached 70-80% confluency, they were treated with UPE (25, 50, 100 μg / mL) obtained in the above process for 24 hours, and the analysis of cell viability was performed using the same method described above.

[0203]

[0204] (9) Measurement of intracellular ROS

[0205] RAW 264.7 cells were seeded at 3 x 10 in a 96-well plate. 5 Cells were seeded at a density of 10 cells / mL, and when the cells reached 60–70% confluency, they were pretreated with UPE (100 μg / mL) and NAC (25 μM) for 1 hour. Then, LPS (1 μg / mL) was treated, and the cells were cultured for 24 hours. Then, 2',7'-dichlorofluorescein diacetate (DCFH-DA) was diluted in serum-free medium to a concentration of 20 μM and added to the cells, which were then washed twice with sterile PBS. Fluorescence was detected at an absorbance between 485 nm and 538 nm using a fluorescence plate reader purchased from Molecular Devices (San Jose, CA, USA). The localization of ROS within the cells was analyzed using a fluorescence microscope from Leica-Microsystems (Wetzlar, Germany), and the images were edited using LAS X (Leica Microsystems, Wetzlar, Hessen Germany).

[0206]

[0207] (10) Western blotting

[0208] RAW 264.7 cells were seeded at 3 x 10 in 6-well plates. 5 The cells were cultured at a density of 10 cells / mL. The fractionated UPE was pretreated, and when the cells reached 70-80% confluency, LPS was treated. HaCaT cells were seeded at 1 x 10 in 60 mm and 100 mm cell culture dishes at a dose of 3 mL or 10 mL per dish. 5 The cells were grown at a density of 10 cells / mL. When the cells reached 60-70% confluency, the medium was replaced with serum-free medium. Fractionated UPE and EGCG were pretreated, and then cell starvation was performed for 12 h, followed by UVB (0.03 J / cm 2) was investigated 1 hour after TCE treatment.

[0209] Afterwards, cells were cultured for an appropriate period of time and harvested with lysis buffer from Cell signaling (Beverly, MA, USA) together with protease and phosphatase inhibitors from Thermo Fisher Scientific (Waltham, Massachusetts, USA). The cells were centrifuged at 12,000 × g for 15 min at 4°C, and the cell supernatant was transferred to a new prechilled tube. The cell lysate was subjected to protein quantification using a DC protein assay kit from Bio-Rad Inc. (Hercules, California, USA).

[0210] Protein separation was performed by electrophoresis on 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel), and the separated proteins were transferred to a PVDF membrane with a pore size of 0.45 μm from Millipore (Burlington, Massachusetts, USA). Blocking was then performed with TBST solution containing 5% skim milk, and the primary antibody was reacted overnight at 4°C and 8 rpm. Afterwards, the secondary antibody was dissolved in 5% skim milk at a ratio of 1:5000 and reacted at room temperature for 1 hour at 8 rpm. Protein bands were measured using a chemiluminescence detection kit from ATTO (Amherst, New York, USA), and imaged using Gene Gnome XRQ NPC from Syngene (India). Protein band quantification was analyzed using Image J.

[0211]

[0212] (11) Statistical processing

[0213] The results were presented by calculating the mean ± standard deviation (SD) of the three independent variables using GraphPad Prism (Boston, MA, USA). Statistical analysis was performed using Student's t-test and two-sample equal variances, and a p<0.05 was considered statistically significant.

[0214]

[0215] <Example 1>

[0216] Composition analysis of each fraction obtained from the ultrasonic extract of seaweed sporophyll

[0217] The inventors of the present invention obtained an extract by ultrasonic treatment of the sporophyll of Undaria pinnatifida for 8 hours using the method described in the above experimental method, and performed a compositional analysis on each of the four ethanol precipitate fractions (UEF1 to UEF4) sequentially obtained by adding ethanol to the ultrasonic extract. Table 1 below shows the yield and composition of the ethanol precipitate fractions of the ultrasonic extract of Undaria pinnatifida.

[0218]

[0219] As a result, as shown in Table 1 above, among the precipitate fractions, the UEF1 fraction was found to have the highest yield compared to other fractions, and also had the highest contents of total carbohydrates, proteins, polyphenols, and fucoxanthin.

[0220]

[0221] In addition, the present inventors performed molecular weight analysis of polysaccharides contained in each of the ethanol precipitate fractions (UEF1 to UEF4). As a result, as shown in Table 2 below, it was confirmed that the ultrasonic extract (UPE) of Undaria pinnatifida sporophyll was composed of two main peaks with peak molecular weights of 1,486 and 0.186 kDa, and the area ratios of each peak were 24.62% and 75.38%. In addition, while the molecular weight of the large peak (peak 1) tended to decrease while the area ratio of peak 1 gradually increased while the precipitate fractionation using ethanol was performed. In addition, a small amount of peaks corresponding to several hundred kDa were detected in the UEF1, UEF2, and UEF3 fractions. Table 2 below shows the results of molecular weight analysis of polysaccharides.

[0222]

[0223] Next, the sugar composition analysis of polysaccharides contained in the ultrasonic extract (UPE) and ethanol precipitate fractions (UEFs) of Undaria pinnatifida sporophyll was performed. As a result, as shown in Table 3 below, galactose and fucose were identified as the main components of polysaccharides in UPE and UEF, and were found to be contained in more than 80% of the content in all samples except the supernatant. Meanwhile, the main components of the supernatant were found to be glucose and xylose. Table 3 below shows the sugar composition analysis of polysaccharides, and ND indicates that they were not detected.

[0224]

[0225] Additionally, the content of sulfated polysaccharides in the ultrasonic extract (UPE) and ethanol precipitate fractions (UEFs) of Undaria pinnatifida sporophyll was analyzed. Table 4 below shows the results of the analysis of sulfated polysaccharide content.

[0226]

[0227] As a result, as shown in Table 4 above, among the ethanol precipitated fractions, the UEF3 fraction was found to contain the most sulfate (33.89±0.05%), followed by UEF4 (26.95±0.10%), UEF2 (25.65±0.15%), and UEF1 (13.02±0.15%). In addition, the ultrasonic extract and supernatant were found to have significantly lower sulfate contents than the other precipitated fractions.

[0228]

[0229] <Example 2>

[0230] Determination of cytotoxicity of ethanol precipitated fractions obtained from ultrasonic extracts of seaweed sporophyll

[0231] The cytotoxicity of ethanol precipitated fractions (UPEs) of the ultrasonic extract of Undaria pinnatifida obtained by the method of the present invention was analyzed. To this end, HaCaT, RAW 264.7, and BEAS-2B cells were treated with the ethanol precipitated fractions at various concentrations, and cell viability was analyzed.

[0232]

[0233] As a result, as shown in Fig. 2, it was found that no cytotoxicity was induced in HaCaT, RAW 264.7, and BEAS-2B cells at all treatment concentrations. Through this, the inventors of the present invention were able to determine that the ethanol precipitated fraction of the ultrasonic extract of the Undaria pinnatifida of the present invention does not cause cytotoxicity and is therefore safe.

[0234]

[0235] <Example 3>

[0236] Free radical scavenging and reactive oxygen species inhibition activities of ethanol precipitated fractions obtained from ultrasonic extracts of seaweed sporophyll

[0237] The free radical scavenging activity and reactive oxygen species inhibition activity of the ethanol precipitated fractions (UPEs) of the ultrasonic extract of seaweed sporophyll obtained by the method of the present invention were analyzed, and the results of the free radical scavenging activity analysis are shown in Table 5 below.

[0238]

[0239] As a result, as shown in Table 5 above, all samples were found to have free radical scavenging activity, but in particular, the ethanol precipitated fraction, UEF3, was found to have the highest DPPH and alkyl radical scavenging activity.

[0240] In addition, the inventors of the present invention confirmed whether the ethanol precipitated fraction of the ultrasonic extract of the seaweed sporophyll has an inhibitory effect on reactive oxygen species. As a result, as shown in Fig. 3, it was found to have an activity of inhibiting the production of reactive oxygen species induced by LPS, and in particular, the UEF3 and supernatant treatment groups were found to have excellent effects (Figs. 3A, 3B).

[0241] Furthermore, it was confirmed whether the above-mentioned reactive oxygen species inhibition activity is achieved through HO-1 regulation. HO-1 is known as an antioxidant-related gene that regulates various biological processes such as inflammation, cell proliferation, and apoptosis, and HO-1 can be activated by NRF2 translocation from the cytoplasm to the nucleus and is known to have a protective effect against external stimuli.

[0242] Accordingly, the inventors of the present invention analyzed whether the UEF3 precipitate fraction, which has excellent radical scavenging and reactive oxygen species inhibition activities, can regulate HO-1 expression. As a result, UEF3 was found to downregulate KEAP1 in the cytoplasm, while upregulating HO-1 expression (Fig. 3C, 3D), and to induce the translocation of NRF2 to the nucleus (Fig. 3E).

[0243]

[0244] <Example 4>

[0245] Analysis of the anti-inflammatory effect of the ethanol-precipitated fraction obtained from the ultrasonic extract of Undaria pinnatifida sporophyll in skin keratinocytes through inhibition of COX-2 expression and activation of the MAPK signaling pathway.

[0246] Ultraviolet (UV) rays, UVB, are a crucial inducer of skin inflammation, activating inflammatory pathways such as MAPKs and abnormally inducing the expression of the inflammatory marker COX-2. Therefore, the inventors of the present invention investigated whether the ethanol-precipitated fraction of the ultrasonic extract of Undaria pinnatifida (Undaria pinnatifida) sporophyll of the present invention could inhibit the expression of COX-2, an inflammatory factor induced by UVB irradiation, in HaCaT cells, which are keratinocytes.

[0247]

[0248] As a result, as shown in Fig. 4, COX-2 expression was significantly reduced in the UEF2, UEF3, UEF4, and supernatant treatment groups compared to the UEF1 fraction, and it was also confirmed that there was phosphorylation inhibitory activity of MAPK. Furthermore, it was confirmed that only the UEF3 fraction had phosphorylation inhibitory activity of ERK1 / 2 compared to the other fractions.

[0249] Through these results, the inventors of the present invention were able to find out that the ethanol precipitate fraction of the ultrasonic extract of Undaria sporophyll has excellent radical scavenging ability that causes skin inflammation, has activity to inhibit the production of reactive oxygen species, and can suppress the induction of expression of COX-2, an inflammatory factor caused by ultraviolet irradiation, and can be usefully used as a material for preventing, improving, and treating skin inflammation. In particular, it was confirmed that the UEF3 fraction, which is the third ethanol precipitate fraction obtained using ethanol from the ultrasonic extract of Undaria sporophyll, has excellent anti-inflammatory activity compared to other fractions, and thus it was found that the UEF3 precipitate fraction contains effective ingredients with excellent anti-inflammatory activity.

[0250]

[0251] 2. Preparation of extracts and fractions with excellent antioxidant activity from seaweed sporophyll extract and experimental method for confirming antioxidant activity

[0252] (1) Materials

[0253] Undaria pinnatifida sporophylls were purchased from a local market in Wando (Jeollanam-do, Korea). The undaria pinnatifida sporophylls were dried, ground to powder using a blender (Hanil, Korea), and used in the experiments. All reagents for cell culture, including Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and phosphate-buffered saline (PBS), were purchased from Gibco (Grand Island, NY, USA). In addition, primary antibodies (Bax, Bcl-2, β-actin) and secondary antibodies (HRP-conjugated antibodies) for Western blotting were purchased from Cell Signaling Technology (Danvers, MA, USA). Other reagents required for Western blotting were from Bio-Rad (Hercules, CA, USA).

[0254]

[0255] (2) Ultrasonic extraction and ethanol precipitation

[0256] Ultrasonic extraction of Undaria pinnatifida sporophyll was performed using MX sonic (MX-12S2, Mirae Ultasonic Tech., Korea), and the extraction process is as shown in Figure 1. 40 L of water was added to 400 g of crushed Undaria pinnatifida powder, and ultrasonic extracts were obtained using MX sonic at 1,080 W, 80% amplitude, 20 kHz, and 30°C for different extraction times (2, 4, 6, and 8 hours). After extraction, the extract was centrifuged (4,000 rpm, 15 min, 4°C) to obtain the supernatant, which was then freeze-dried. The obtained extracts were named UPE_2, UPE_4, UPE_6, and UPE_8 according to the extraction time. Subsequently, ethanol precipitation was performed using the following method. After 8 hours of ultrasonic extraction, ethanol was added to the UPE_8 ultrasonic extract, and it was allowed to precipitate overnight at 4℃. Then, centrifugation (12,000xg, 10 min, 4℃) was performed to recover the supernatant and precipitate, respectively, and these were lyophilized. The supernatant and precipitate obtained through ethanol precipitation were named UPE_8S (supernatant) and UPE_8P (precipitate), respectively.

[0257]

[0258] (3) Composition analysis

[0259] The composition analysis of the ultrasonic extract and ethanol fraction obtained in the process (2) above was performed using the following methods. The total protein content was analyzed using the BCA analysis method, the total polyphenol content was analyzed using the Folin-Ciocalteu method, the total polysaccharide content was analyzed using the phenol-sulfuric acid method, and the total sulfate content was analyzed using the barium-gelatin method.

[0260]

[0261] (4) Analysis of molecular weight and monosaccharide composition using HPLC

[0262] Molecular weight analysis of the ultrasonic extract and ethanol fraction of the present invention was performed using an HPLC system (Agilent 1100, CA, USA) coupled with an ELSD detector (Evaporative Light Scattering Detector, Agilent). The columns used were Shodex OHPak SB-804HQ (8 × 300 mm, 10 μm particle size, Showa Denko, Tokyo, Japan) and SB-802.5HQ (8 × 300 mm, 6 μm particle size). The sample (2 mg / mL in deionized water) was filtered using a 0.45 μm syringe filter, and the filtered sample was loaded onto the column. The injection volume was 100 μL and the flow rate was 0.6 mL / min. The peak was detected with an RI detector, and the molecular weight was calculated using the pullulan standard Shodex standard P-82.

[0263] The composition analysis of monosaccharides was performed using a HPAEC-PAD (High-Performance Anion-Exchange Chromatography coupled with Pulsed Amperometric Detection, Dionex, Sunnyvale, CA, USA) system, and CarboPac ™ A PA10 column (2 × 250 mm, particle size 10 μm) was used for separation. All samples (2 mg / mL) were hydrolyzed with trifluoroacetic acid (TFA) and then injected into the column at an injection volume of 20 μL. Column analysis was performed at a flow rate (1.0 mL / min) using a solvent of 18 mM NaOH / 200 mM NaOH.

[0264]

[0265] (5) Cell culture and cell viability analysis

[0266] Vero cells, monkey kidney cells, were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified incubator with 5% CO2. To investigate the effect of the ultrasonic extract or ethanol fraction of the present invention on cell viability, an MTT assay was performed, in which Vero cells were seeded at 1 x 10 in a 96-well plate. 4 Cells were seeded at a density of 10 cells / well and cultured for 24 h. Afterwards, each well was treated with the concentration-specific ultrasonic extract or ethanol fraction together with hydrogen peroxide (H2O2). After 24 h of culture, MTT solution (Sigma-Aldrich Co., St. Louis, MO, USA) was added to each well and reacted for 3 h. Afterwards, the medium was removed, and DMSO was added to each well to dissolve the colored formazan crystals. The absorbance was measured at 570 nm using a microplate reader, and the cell viability was calculated based on the absorbance of the medium-treated group as a control.

[0267]

[0268] (6) Measurement of intracellular reactive oxygen species

[0269] To analyze the effect of the ultrasonic extract or ethanol fraction of the present invention on the production of intracellular ROS in Vero cells, DCFH-DA fluorescence analysis was used. Vero cells were seeded in 96-well plates at a density of 1 x 10 4Cells were seeded at a density of 10 cells / well and cultured for 24 hours. Then, each well was treated with ultrasonic extracts or ethanol fractions at various concentrations and cultured for an additional 1 hour. Afterwards, 10 μM DCFH-DA fluorescent solution (Sigma-Aldrich Co.) was treated to each well for 30 minutes, and after replacing the medium with fresh medium, 1 mM hydrogen peroxide (H2O2) was added to each well and reacted for 30 minutes. The fluorescence intensity was measured at a wavelength of 485 nm (excitation) / 535 nm (emission) using a microplate reader.

[0270]

[0271] (7) Western blotting

[0272] Vero cells were seeded at 1 x 10 in 60mm cell culture dishes. 5The cells were seeded at a density of 10 cells / dish and cultured for 24 hours. The samples obtained in the present invention were then treated with 1 mM H2O2 to the cells. After 2 hours, total protein was extracted from the cells with RIPA buffer (protease and phosphatase inhibitor cocktail, Thermo Fisher Scientific, Waltman, MA, USA), and the protein content was measured using BCA assay (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific). Thirty micrograms of protein corresponding to each sample was electrophoresed on a 12% SDS-PAGE gel (Bio-Rad) and transferred to a PVDF membrane (Bio-Rad). The membrane was blocked with blocking buffer (EveryBlot Blocking Buffer, Bio-Rad) and then incubated with specific primary antibodies (Bax, anti-rabbit, 1:1000 dilution; Bcl-2, anti-rabbit, 1:1000 dilution; β-actin, anti-rabbit, 1:2000 dilution) overnight at 4°C. The membrane was then washed with TBS-T (Tris-buffered saline containing Tween) and incubated again with anti-rabbit secondary antibody (1:5000) for 2 h at room temperature. After a second wash with TBS-T, protein bands were detected using ECL reagent and observed with ChemiDoc™ XRS+ (with Image Lab™ Software, Bio-Rad).

[0273]

[0274] (8) Statistical processing

[0275] Results are expressed as the mean ± standard deviation of experiments performed in triplicate, and statistical analysis was performed using SPSS Statistics 20 (SPSS Inc., Chicago, IL, USA). Significant differences between samples were analyzed using one-way analysis of variance (ANOVA) and Duncan's test (P < 0.05).

[0276]

[0277] <Example 5>

[0278] Yield and total polysaccharide content of ultrasonic extracts from seaweed sporophyll according to ultrasonic treatment time

[0279] As shown in Fig. 5, the extraction yield and total polysaccharide content of the ultrasonic extracts obtained from the seaweed sporophyll were compared and analyzed by varying the ultrasonic treatment time (2, 4, 6, and 8 hours).

[0280] As a result, the longer the sonication time, the higher the extraction yield (Fig. 6). The extraction yield of the group sonicated for 2 hours was 27.55% (UPE_2), the 4-hour treatment group showed 30.22% (UPE_4), the 6-hour treatment group showed 30.67% (UPE_6), and the 8-hour treatment group showed 31.91% (UPE_8).

[0281] In addition, the total polysaccharide content of each extract was analyzed using the phenol-sulfuric acid method, and the results showed that, similar to the extraction yield results, the total polysaccharide content increased as the ultrasonic treatment time increased (P < 0.05). The UPE_2 group extracted for 2 hours showed a total polysaccharide content of 53.33%, and the UPE_8 group extracted with ultrasonic treatment for 8 hours showed a significantly highest total polysaccharide content of 69.22% (P < 0.05) (Fig. 6).

[0282] Based on these results, the inventors were able to determine that when ultrasonic treatment is performed for 8 hours, not only can a high extraction yield be secured, but total polysaccharides can also be obtained in the highest content.

[0283]

[0284] <Example 6>

[0285] Analysis of the effect of ultrasonic extracts of seaweed sporophyll on cell viability and on the production of reactive oxygen species by hydrogen peroxide.

[0286] The cytotoxicity and antioxidant activity of the ultrasonic extract of the seaweed sporophyll obtained by the method of the present invention were investigated.

[0287] First, in order to analyze whether there is cytotoxicity, the ultrasonic extract of the seaweed sporophyll of the present invention was treated to Vero cells at various concentrations, and then the cell viability was analyzed.

[0288] As a result, as shown in Fig. 7A, the ultrasonic extract of the present invention from the spore leaves of Undaria pinnatifida did not induce cell toxicity. Furthermore, regardless of the extraction time by ultrasonic treatment, all ultrasonic extracts were found to have no effect on the cell viability of Vero cells at all treatment concentrations.

[0289]

[0290] To confirm the presence of antioxidant activity, Vero cells were first treated with 1 mM H2O2 to induce oxidative stress in the cells, and at the same time, the ultrasonic extract of the seaweed sporophyll of the present invention was treated, and then the cell viability and the level of reactive oxygen species production were analyzed.

[0291] As a result, it was found that the cell viability of Vero cells in the group treated with H2O2 significantly decreased from 100% to 59.60% compared to the group not treated with H2O2. On the other hand, the group treated with the ultrasonic extract of the seaweed sporophyll of the present invention showed a higher cell viability than the group treated with only H2O2, and the cell viability was found to increase in a treatment concentration-dependent manner. In particular, the group treated with UPE_8 400 μg / mL obtained by performing an 8-hour ultrasonic treatment showed the highest cell viability of 83.15% (Fig. 7B).

[0292] In addition, as a result of analyzing the level of ROS production, it was shown that the amount of ROS significantly increased from 100% to 393.17% (P < 0.05) in the group treated with H2O2 compared to the group that was not treated with anything. On the other hand, it was shown that the increased amount of ROS decreased according to the treatment with the ultrasonic extract of the seaweed sporophyll of the present invention, and the UPE_2, UPE_4, UPE_6, and UPE_8 extract treatment groups (400 μg / m) showed ROS production of 148.71, 149.98, 150.92, and 149.89%, respectively, confirming that ROS production of 393.17% could be significantly suppressed (Fig. 7C).

[0293] It is known that excessive production of reactive oxygen species (ROS) caused by oxidative stress causes oxidative damage to cellular macromolecules, induces cell death, and affects cell viability.

[0294] In this respect, it was found that the ultrasonic extract of the seaweed sporophyll of the present invention has antioxidant activity that can effectively suppress the production of reactive oxygen species due to oxidative stress, and thus can protect cells from oxidative stress.

[0295] In addition, considering the extraction yield, polysaccharide content, and cell protection effect according to the sonication time, it was found that the UPE_8 extract obtained by sonication for 8 hours had excellent antioxidant activity.

[0296]

[0297] <Example 7>

[0298] Analysis of sugar content, monosaccharide composition, and molecular weight of ethanol fractions (supernatant and precipitate) obtained from ultrasonic extract of Undaria pinnatifida (UPE_8)

[0299] Through the experiments of the above examples, it was found that among the ultrasonic extracts of the seaweed sporophyll of the present invention, the UPE_8 extract obtained by ultrasonic treatment for 8 hours had the best antioxidant activity. Accordingly, the inventors of the present invention recovered the ethanol precipitate (UPE_8P) and supernatant (UPE_8S) obtained by adding ethanol to the UPE_8 ultrasonic extract and precipitating it with ethanol, and performed analyses of the sugar content, monosaccharide composition, and molecular weight contained in UPE_8P and UPE_8S. Table 6 below shows the results of the sugar content analysis, Table 7 shows the results of the molecular weight analysis, and Table 8 shows the results of the monosaccharide composition analysis.

[0300]

[0301]

[0302]

[0303] As a result, as shown in Table 6 above, the UPE_8 extract extracted by ultrasonic treatment for 8 hours was found to contain 67.52±1.94% of sugar content, and the supernatant (UPE_8S) and precipitate (UPE_8P) obtained after ethanol precipitation of the ultrasonic extract were found to contain 53.08±3.72% and 80.29±5.14% of sugar content, respectively.

[0304] Through this, it was found that the ethanol precipitate (ethanol precipitate fraction; UPE_8P) recovered by performing ethanol precipitation contained a large amount of polysaccharides derived from seaweed sporophyll, and thus was a polysaccharide fraction.

[0305]

[0306] In addition, as a result of molecular weight analysis, as shown in Table 7 above, two main peaks were detected for each sample, and in the case of UPE_8, which is an ultrasonic extract, the molecular weight of the peak with the highest molecular weight was confirmed to be 1,062 kDa, and the content was confirmed to be 30.27%. In addition, it was confirmed that the 110 Da peak occupied 69.73% of the total area. On the other hand, when ethanol precipitation was performed, high molecular weight polysaccharides were found to be precipitated by ethanol and contained in the precipitate. The analysis result of UPE_8P confirmed that the ratio of the peak with a molecular weight of 745 kDa was 94.89%. In addition, in the case of UPE_8S, which is the supernatant recovered after ethanol precipitation, most of the components (96.33%) showed a molecular weight of 175 Da, indicating that the polysaccharides were well separated by ethanol.

[0307]

[0308] Meanwhile, it is known that the biofunctional activity of polysaccharides is greatly affected by the type of monosaccharide that constitutes the polysaccharide. Accordingly, the present inventors performed an analysis of the monosaccharide composition contained in UPE_8, UPE_8S, and UPE_8P. As a result, as shown in Table 8, the UPE_8 ultrasonic extract was composed of four monosaccharides (fucose, galactose, glucose, and xylose), and galactose was analyzed to be the main monosaccharide at 22 μg / mg (55.64%), followed by fucose at 18 μg / mg, accounting for 31.90%.

[0309] In addition, in the fractions obtained by ethanol precipitation by adding ethanol to the ultrasonic extract, the monosaccharide content of UPE_8S (supernatant) was significantly reduced, whereas the monosaccharide content of UPE_8P, which is an ethanol precipitate, was increased. Through these results, the inventors were able to determine that ethanol precipitation performed by adding ethanol to the ultrasonic extract is a useful method for isolating polysaccharides present in the UPE_8 extract.

[0310]

[0311] <Example 8>

[0312] Analysis of cell protective activity against oxidative stress of ultrasonic extract of Undaria pinnatifida (UPE_8) and its ethanol fraction (supernatant and precipitate)

[0313] In order to analyze the antioxidant activity of the ultrasonic extract of the spore leaves of the present invention (UPE_8) and its ethanol fractions (UPE_8S and UPE_8P), the cell viability and the level of production of reactive oxygen species were analyzed in Vero cells treated with the ultrasonic extract and ethanol fraction of the present invention in which oxidative stress was induced with hydrogen peroxide.

[0314] As a result, it was confirmed that the cell viability of cells in which oxidative stress was induced by 1 mM H2O2 was approximately 50% lower than that of the untreated group. On the other hand, the groups treated with the ultrasonic extract of Undaria pinnatifida (UPE_8) and the ethanol precipitate of the ultrasonic extract (UPE_8P) showed that cells were protected from oxidative stress and cell viability was restored. In particular, UPE_8P showed a cell viability of 97.17% at a concentration of 400 μg / mL, demonstrating a superior cytoprotective effect compared to the UPE_8 ultrasonic extract-treated group. On the other hand, UPE_8S showed very little effect in protecting cells from cytotoxicity caused by oxidative stress at all treatment concentrations (Fig. 8A).

[0315] In addition, in the analysis results of reactive oxygen species production, the group treated with hydrogen peroxide showed an increase in reactive oxygen species production of approximately 1.6 times compared to the control group (164.18% increase), whereas in the group treated with UPE_8P of the present invention, the production of reactive oxygen species was effectively suppressed in a treatment concentration-dependent manner, and the group treated with 400 ug / ml showed a level almost similar to the normal group (Fig. 8B).

[0316] Through these results, the inventors of the present invention were able to predict that the ethanol precipitate of the ultrasonic extract of the UPE_8P, which has the best antioxidant activity, contains a large amount of polysaccharide components, and that this antioxidant activity may be due to the polysaccharide components present in the UPE_8P.

[0317]

[0318] <Example 9>

[0319] Analysis of the effect of ethanol precipitate of ultrasonic extract of Undaria pinnatifida (UPE_8P) on the expression of apoptosis-related proteins.

[0320] Through the results of the above <Example 8>, it was confirmed that the UPE_8P ethanol precipitate of the present invention has the best antioxidant activity, and further, it was investigated whether the UPE_8P ethanol precipitate also affects the expression of apoptosis-related proteins in cells.

[0321] To this end, the protein expression levels of the Bcl-2 protein family (pro-apoptosis; Bax, Bok, and Bad and anti-apoptosis; Bcl-2, Bcl-XL, and Mcl-1), which are apoptosis-related proteins, were analyzed using Western blot for groups treated with UPE_8P and groups not treated with oxidative stress-induced Vero cells.

[0322]

[0323] As a result, as shown in Fig. 9, compared to the untreated control group, the group treated with H2O2 to induce oxidative stress showed an increase in the expression of the Bax protein belonging to the pro-apoptosis family, whereas the expression of the Bcl-2 protein belonging to the anti-apoptosis family was decreased. On the other hand, when the UPE_8P ethanol precipitate of the present invention was treated, the expression level of the Bax protein, which induces apoptosis, was effectively suppressed, and the expression level of the Bcl-2 protein, which inhibits apoptosis, was effectively increased.

[0324] Through these results, it was found that the ethanol precipitate fraction (i.e., polysaccharide fraction) of the ultrasonic extract of the seaweed sporophyll obtained by the method of the present invention can effectively protect cells from cell death due to oxidative stress, and thus has excellent antioxidant activity.

[0325]

[0326] 3. Preparation of extracts and fractions with excellent skin whitening, wrinkle improvement, and skin moisturizing activities from seaweed sporophyll extracts and experimental methods for confirming their activities.

[0327] (1) Materials

[0328] Undaria pinnatifida sporophylls were purchased from a local market in Wando (Jeollanam-do, Korea). The undaria pinnatifida sporophylls were dried, ground to powder using a blender (Hanil, Korea), and used in the experiments. All reagents for cell culture, including Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and phosphate-buffered saline (PBS), were purchased from Gibco (Grand Island, NY, USA). In addition, primary antibodies (Bax, Bcl-2, β-actin) and secondary antibodies (HRP-conjugated antibodies) for Western blotting were purchased from Cell Signaling Technology (Danvers, MA, USA). Other reagents required for Western blotting were from Bio-Rad (Hercules, CA, USA).

[0329]

[0330] (2) Antioxidant activity evaluation

[0331] ① Measurement of total polyphenol content (Total phenolic contents, TPC assay)

[0332] In a 96-well plate, 20 μL of 1N Folin-Ciocalteu reagent and 60 μL of 20% NaCO were added to each well of the extracts prepared by concentration and 40 μL of gallic acid for the standard curve. Afterwards, the reaction was performed on a shaker for 30 minutes under light protection with foil, and the absorbance was measured at 700 nm. The total polyphenol content in the extract was expressed as mg gallic acid equivalents per g of sample (mg GAE / g) by substituting the absorbance value according to the gallic acid standard curve.

[0333]

[0334] ② Measurement of total flavonoid contents (TFC assay)

[0335] 25 μL of extracts prepared by concentration and quercetin for standard curve in a 96-well plate, 125 μL of distilled water, 5% NaNO28 μL was added to each well and reacted. After 5 minutes, 15 μL of 10% AlCl3 was added and reacted for 6 minutes. After that, 50 μL of 1 M NaOH and 27 μL of distilled water were dispensed, mixed well with a pipette, and the absorbance was measured at 510 nm. The total flavonoid content in the extract was expressed as mg Quercetin equivalents per g of sample (mg QE / g) by substituting the absorbance value according to the Quercetin standard curve.

[0336]

[0337] ③ Measurement of DPPH radical scavenging activity

[0338] 80 μL of 0.2 mM DPPH (2,2-diphenyl-1-picrylhydrazyl) radical solution was added to 80 μL of extracts prepared by concentration and distilled water in a 96-well plate, and the mixture was allowed to react for 30 minutes under light-shielding conditions. The absorbance was measured at 517 nm, and the DPPH radical scavenging activity was calculated using the following formula. Vitamin C was used as a positive control.

[0339] DPPH radical scavenging activity (%) = [(OD DPPH -OD sample ) / OD DPPH ]× 100

[0340]

[0341] (3) Cytotoxicity evaluation

[0342] Cell viability was measured using the MTS assay, a technique known in the art. Human epidermal cells (HaCaT, keratinocytes) were seeded at 1 × 10 in a 96-well plate. 4Cells were seeded at a concentration of 10 cells / well and cultured (37°C, 5% CO2, overnight), then the medium was replaced with DMEM medium without FBS and cultured (overnight). After that, the medium was replaced with medium containing extracts at different concentrations and cultured for 24 hours. For melanin-producing cells (B16F10, melanocytes), 3.5 × 10 3 Dispense at a concentration of 10 cells / well and culture overnight, then replace with a medium containing extracts at different concentrations and culture for 72 hours.

[0343] After removing the culture medium and replacing it with fresh medium, 20 μL of a solution containing MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and PMS (phenazine methyl sulfate) in a 20:1 ratio was added and reacted at 37°C for 30 minutes. The absorbance was measured at 490 nm to evaluate the toxicity of the extract to cells.

[0344]

[0345] (4) Measurement of enzyme activity inhibition effect

[0346] ① Measurement of collagenase inhibitory activity

[0347] Collagenase inhibitory activity was measured by mixing 20 μL of the present invention's seaweed sporophyll extract prepared at various concentrations in a 96-well plate, 50 μL of 0.5 mg / mL substrate (4-henylazo-benzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg), and 30 μL of 0.5 mg / mL Clostridium histolyticum collagenase, and incubating at 37°C for 45 minutes. After stopping the reaction by adding 100 μL of 6% citric acid, the absorbance was measured at 324 nm. Vitamin C was used as a positive control.

[0348]

[0349] ② Measurement of hyaluronidase inhibitory activity

[0350] The activity of hyaluronic acid degrading enzyme (Hyalruonidase) was measured using the Hyaluronidase Inhibitor Screening Assay Kit (BioAssay Systems). 20 μL of 100 U / mL human hyaluronidase was added to 10 μL of the present invention's seaweed sporophyll extract prepared at various concentrations in a 96-well plate, and the mixture was incubated for 15 minutes. 20 μL of the substrate solution was then added and incubated for 20 minutes. 80 μL of the stop reagent was then added, and the absorbance was measured at 600 nm after 10 minutes. L-Ascorbyl palmitate was used as a positive control.

[0351]

[0352] ③ Analysis of mushroom tyrosinase inhibitory activity

[0353] Tyrosinase inhibitory activity assay was performed by adding 20 μL of 0.02 mg / mL mushroom tyrosinase to 40 μL of the present invention's seaweed sporophyll extract prepared at various concentrations in a 96-well plate and reacting for 10 minutes. After that, 40 μL of 1 mM L-DOPA (L-3,4-dihydroxyphenylalanine) was added and reacted for 30 minutes, and then measuring the absorbance at 475 nm. Vitamin C was used as a positive control.

[0354]

[0355] (5) Melanin secretion assay and L-DOPA staining

[0356] B16F10 cells were seeded at 1×10 in a 96-well Ultra-Low Attachment (ULA) plate, a 3D cell culture plate. 4 After dividing the cells into a concentration of 10 cells / well and culturing overnight, 100 nM α-MSH and the extract of the present invention were treated and cultured for 72 hours. Afterwards, the secreted melanin content was measured at an absorbance of 490 nm using the cultured medium.

[0357] B16F10 cells were seeded at 2×10 in a 6cm dish. 5 Cells / dish were divided into 100 μg / dish and cultured overnight, and then treated with the extract. After 1 hour, 100 nM α-MSH and the extract were treated together and cultured for 72 hours. After removing the medium, proteins were extracted from the cells and quantitative protein analysis was performed using BCA (Bicinchoninic acid) analysis, followed by 8% SDS-PAGE using zymogram sample buffer. The gel was then washed twice with 0.1 M sodium phosphate monobasic buffer for 30 minutes each. After that, 10 mM L-DOPA solution was added and reacted (37°C, 1 hour), and when a dark band was observed, a photograph of the gel was taken.

[0358]

[0359] (6) Confirmation of MMP-1 protein expression induced by UVB irradiation

[0360] HaCaT cells were seeded at 3×10 in a 6 cm dish. 5 After dividing the cells into a concentration of 10 cells / dish and culturing overnight, the medium was replaced with DMEM medium without FBS and cultured overnight. After that, the medium was replaced with medium containing extracts at different concentrations and cultured for 1 hour. After 1 hour, UVB 0.01 J / cm 2 After irradiation, the cells were cultured for 24 hours. After removing the cultured medium, protein extraction and quantitative protein analysis were performed through BCA analysis, and protein expression was confirmed through Western blotting.

[0361]

[0362] (7) Confirmation of HYAL2 protein expression

[0363] HaCaT cells were seeded at 3×10 in a 6 cm dish. 5 The cells were divided into 100 cells / dish and cultured overnight. The medium was replaced with DMEM medium without FBS and cultured overnight. Afterwards, the medium was replaced with medium containing the extracts at each concentration and cultured for 24 hours. After removing the cultured medium, protein extraction and BCA analysis were performed for protein quantification, and protein expression was confirmed through Western blotting.

[0364]

[0365] <Example 10>

[0366] Preparation of ultrasonic extract of seaweed sporophyll

[0367] An ultrasonic extract was obtained from the Undaria pinnatifida sporophyll using the following method. The ultrasonic extraction was performed using an MX sonic (MX-12S2, Mirae Ultasonic Tech., Korea), and the extraction process is as shown in Fig. 10. Specifically, 40 L of water was added to 400 g of the powder of the crushed Undaria pinnatifida sporophyll, and an ultrasonic extract was obtained using an MX sonic at 1,080 W, 80% amplitude, 20 kHz, and 30°C for 8 hours. The obtained extract was then filtered through a filter cloth, further filtered through a 20-inch housing, and ion-exchanged using an ion-exchange filter for 24 hours. Then, sterilization was performed at 121°C for 15 minutes, and then freeze-dried to obtain the final ultrasonic extract of Undaria pinnatifida according to the present invention.

[0368]

[0369] <Example 11>

[0370] Confirmation of antioxidant activity of ultrasonic extract of seaweed sporophyll of the present invention

[0371] The antioxidant activity of the ultrasonic extract of the seaweed sporophyll of the present invention obtained by the method of <Example 10> above was confirmed by measuring the total polyphenol content and total flavonoid content and analyzing DPPH radical scavenging activity.

[0372]

[0373] As a result, as shown in Table 9 and Fig. 11, the total polyphenol content of the ultrasonic extract of the seaweed sporophyll of the present invention was found to be 7.02±0.60 mg GAE / g, and the total flavonoid content was found to be 0.85±0.05 mg QE / g. As a result of the DPPH radical scavenging activity analysis, the DPPH radical scavenging activity of the ultrasonic extract of the seaweed sporophyll was found to increase in a concentration-dependent manner in the 100-800 μg / mL concentration treatment range. On the other hand, the DPPH radical scavenging activity was found to be lower in the 1600 μg / mL concentration treatment group compared to the 800 μg / mL treatment group.

[0374] Through these results, the inventors of the present invention were able to determine that the ultrasonic extract of the seaweed sporophyll produced by the method of the present invention has antioxidant activity and can be usefully utilized in the production of antioxidant materials.

[0375]

[0376] <Example 12>

[0377] Cytotoxicity assay

[0378] The cytotoxicity of the ultrasonic extract of the seaweed sporophyll obtained by the method of the present invention was investigated using the MTS assay described above on human skin epidermal cells (HaCaT) and melanin-producing cells (B16F10).

[0379]

[0380] As a result, as shown in Fig. 12, in the case of human skin epidermal cells (HaCaT), cell viability of more than 90% was observed in the extract treatment concentration range of 6.25 to 800 μg / mL, and in the case of melanin-producing cells (B16F10), cell viability of more than 90% was observed in the extract treatment concentration range of 6.25 to 200 μg / mL.

[0381]

[0382] <Example 13>

[0383] Confirmation of the wrinkle formation inhibition effect according to the ultrasonic extract treatment of the seaweed sporophyll of the present invention

[0384] In order to confirm whether the ultrasonic extract of Undaria pinnatifida prepared by the method of the present invention has the activity of inhibiting skin wrinkle formation, the inventors of the present invention evaluated collagenase inhibitory activity and analyzed the degree of inhibition of the expression of Matrix metalloproteinase-1 (MMP-1) protein involved in wrinkle formation. Collagenase is an enzyme that degrades collagen fibers in the extracellular matrix (ECM) and is known to play an important role in wrinkle formation, and MMP-1 protein is known to play an important role in skin wrinkle formation by destroying collagen.

[0385]

[0386] As a result, as shown in Fig. 13, it was confirmed that when treated with the ultrasonic extract of the seaweed sporophyll, the collagenase inhibitory activity increased in a treatment concentration-dependent manner compared to the untreated group, and the expression level of MMP-1 protein increased by ultraviolet irradiation in human skin epidermal cells was shown to be reduced by the treatment with the ultrasonic extract of the seaweed sporophyll.

[0387]

[0388] <Example 14>

[0389] Confirmation of moisturizing effect according to ultrasonic extract treatment of seaweed sporophyll of the present invention

[0390] An experiment was conducted to determine whether the ultrasonic extract of Undaria pinnatifida (Undaria pinnatifida) of the present invention can enhance skin hydration. To this end, the hyaluronidase inhibitory activity was evaluated and the effect on the expression of hyaluronic acid decomposition enzyme (HYAL2) protein in human epidermal cells (HaCaT) was analyzed. Hyaluronic acid (HA), which exists in the skin, is known to contain a large amount of moisture and plays an important role in skin hydration, elasticity, and wrinkle prevention. Hyaluronidase (HYAL) is an enzyme that has the activity to decompose hyaluronic acid. Therefore, inhibiting hyaluronidase enzyme activity can enhance skin hydration.

[0391]

[0392] As a result, as shown in Fig. 14, when treated with the ultrasonic extract of the seaweed sporophyll of the present invention, it was shown that the inhibition of the enzymatic activity of hyaluronic acid was increased in a treatment concentration-dependent manner, and this inhibition of the enzymatic activity was shown to be superior to the group treated with L-ascorbyl palmitate, which is known as a hyaluronidase inhibitor. In addition, when human skin epidermal cells were treated with the ultrasonic extract of the seaweed sporophyll of the present invention, it was shown that the protein expression of hyaluronic acid decomposition enzyme (HYAL2) was inhibited in a treatment concentration-dependent manner.

[0393] These results indicate that the ultrasonic extract of the seaweed sporophyll of the present invention has excellent skin moisturizing promoting effects.

[0394]

[0395] <Example 15>

[0396] Confirmation of the whitening promotion effect according to the ultrasonic extract treatment of the seaweed sporophyll of the present invention

[0397] Furthermore, the inventors of the present invention analyzed the whitening activity of the ultrasonic extract of Undaria pinnatifida sporophyll. To this end, tyrosinase inhibition activity, melanin secretion activity, and L-DOPA staining were performed. Tyrosinase is an enzyme that plays a key role in melanin biosynthesis. In addition, a melanin secretion assay was performed to confirm whether the melanin secretion activity and L-DOPA staining inhibited the increased melanin secretion induced by α-melanocyte stimulating hormone (α-MSH) in a melanogenic cell line (B16F10). In addition, B16F10 cells were cultured in Ultra-Low Attachment (ULA) 96-well plates for 3D cell culture, which exhibits structural characteristics similar to tissues and organs rather than 2D cells, and the inhibition of tyrosinase activity increased by α-MSH was confirmed through L-DOPA staining. The analysis was performed at the level where L-DOPA is oxidized and a dark band appears on the gel depending on intracellular tyrosinase activity.

[0398]

[0399] As a result of the analysis, it was shown that the ultrasonic extract of the seaweed sporophyll of the present invention has tyrosinase inhibitory activity (Fig. 15A), and the amount of melanin secretion increased by α-MSH was also shown to be inhibited by treatment with the ultrasonic extract of the seaweed sporophyll (Fig. 15B), and the activity of tyrosinase in cells increased by α-MSH was also shown to be inhibited (Fig. 15C).

[0400] Through these results, the inventors of the present invention were able to find out that the ultrasonic extract of the seaweed sporophyll of the present invention can promote and improve skin whitening through inhibition of tyrosinase activity.

[0401]

[0402] The present invention corresponds to the results of the following national research project.

[0403] Project ID: 1525013773, Project Number: 202205052, Ministry of Oceans and Fisheries, Project Management (Specialist) Agency: Korea Institute of Ocean Science and Technology Promotion, Research Project Name: Marine and Fisheries New Industry Technology Commercialization Support, Research Project Name: Standardization of Industrial Seaweed Sporophyll Extraction Process Using Ultrasound and Development of Anti-Pollution Functional Cosmetic Food Materials, Project Implementing Agency: 3FC Co., Ltd., Research Period: April 1, 2022 - December 31, 2023

[0404]

[0405] The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

Claims

1. (1) A step of obtaining an ultrasonic extract of the kelp sporophyll; and (2) a step of adding ethanol to the ultrasonic extract of the seaweed sporophyll obtained in step (1) to obtain an ethanol precipitate fraction; A method for producing an ethanol precipitate fraction of an ultrasonic extract of Undaria pinnatifida having an activity for suppressing or improving skin inflammation.

2. In paragraph 1, A method for producing an ethanol precipitate fraction of an ultrasonic extract of Undaria pinnatifida having skin inflammation suppression or improvement activity, characterized in that the ultrasonic extract of step (1) above is obtained by adding purified water to Undaria pinnatifida powder and ultrasonicating it at a temperature of 28 to 32°C and 1000 to 1100 W for 7 to 9 hours.

3. In paragraph 1, A method for producing an ethanol precipitate fraction of an ultrasonic extract of Undaria pinnatifida having skin inflammation-suppressing or -improving activity, characterized in that the ethanol precipitate fraction is obtained by adding ethanol to an ultrasonic extract of Undaria pinnatifida, performing ethanol precipitation at a temperature of 4 to 6°C for 20 to 28 hours, and then centrifuging to obtain a precipitate.

4. In paragraph 3, A method for producing an ethanol precipitate fraction of an ultrasonic extract of Undaria pinnatifida having an activity of inhibiting or improving skin inflammation, characterized in that the ethanol precipitate fraction contains a polysaccharide derived from an ultrasonic extract of Undaria pinnatifida.

5. An ethanol precipitate fraction of an ultrasonic extract of Undaria pinnatifida having skin inflammation suppression or improvement activity, prepared by the method of any one of claims 1 to 4.

6. A cosmetic composition for preventing or improving skin inflammation, comprising an ethanol precipitate fraction of the ultrasonic extract of the seaweed spore leaves of Article 5 as an effective ingredient.

7. A pharmaceutical composition for preventing or treating skin inflammation, comprising an ethanol precipitate fraction of the ultrasonic extract of the seaweed spore leaves of Article 5 as an effective ingredient.

8. A health functional food for preventing or improving skin inflammation, containing the ethanol precipitate fraction of the ultrasonic extract of the seaweed spore leaves of Article 5 as an effective ingredient.

9. An antioxidant composition comprising an ultrasonic extract of seaweed sporophyll or an ethanol precipitate fraction of the ultrasonic extract as an effective ingredient.

10. In paragraph 9, An antioxidant composition characterized in that the above ultrasonic extract is an ultrasonic extract obtained by adding purified water to the powder of seaweed sporophyll and ultrasonicating it at a temperature of 28 to 32°C and 1,000 to 1,100 W for 7 to 9 hours.

11. In paragraph 9, An antioxidant composition characterized in that the above ethanol precipitate fraction is a precipitate obtained by adding ethanol to an ultrasonic extract of Undaria pinnatifida obtained by ultrasonic treatment for 8 hours, performing ethanol precipitation overnight at 4°C, and then centrifuging.

12. In paragraph 9, An antioxidant composition, characterized in that the ultrasonic extract of the above-mentioned Undaria pinnatifida and the above-mentioned ethanol precipitate fraction contain polysaccharides derived from Undaria pinnatifida.

13. An antioxidant health functional food comprising an antioxidant composition according to any one of claims 9 to 12.

14. An antioxidant cosmetic composition comprising an antioxidant composition according to any one of claims 9 to 12. 15.(1) A step for obtaining an ultrasonic extract of the kelp sporophyll; and (2) a step of adding ethanol to the ultrasonic extract of the seaweed sporophyll obtained in step (1) to obtain an ethanol precipitate fraction; A method for preparing an ethanol precipitate fraction of an ultrasonic extract of Undaria pinnatifida having antioxidant activity.

16. In paragraph 15, The above ultrasonic extract is obtained by adding purified water to the powder of seaweed sporophyll and sonicating it at 1,000 to 1,100 W and a temperature of 28 to 32°C for 7 to 9 hours. A method for producing an ethanol precipitate fraction of an ultrasonic extract of Undaria pinnatifida having antioxidant activity, characterized in that the ethanol precipitate fraction is a precipitate obtained by adding ethanol to an ultrasonic extract of Undaria pinnatifida, performing ethanol precipitation at 4°C overnight, and then centrifuging.

17. A cosmetic composition for skin whitening, wrinkle improvement and skin moisturizing, containing an ultrasonic extract of seaweed sporophyll as an effective ingredient.

18. In paragraph 17, A cosmetic composition for skin whitening, wrinkle improvement and skin moisturizing, characterized in that the above ultrasonic extract is an ultrasonic extract obtained by adding purified water to the powder of seaweed sporophyll and ultrasonicating it at a temperature of 28 to 32°C and 1,000 to 1,100 W for 7 to 9 hours.

19. In paragraph 18, A cosmetic composition for skin whitening, wrinkle improvement and skin moisturizing, characterized in that the ultrasonic extract is obtained by performing filter cloth and housing filtration on the ultrasonic extract obtained after the ultrasonic treatment, performing ion exchange, and then sterilizing and freeze-drying.

20. In paragraph 17, The ultrasonic extract of the above seaweed sporophyll is A cosmetic composition for skin whitening, wrinkle improvement and skin moisturizing, characterized by having antioxidant activity, collagenase inhibitory activity, MMP-1 protein expression inhibition, hyaluronic acid decomposition enzyme inhibition, tyrosinase activity inhibition or melanin production inhibition activity.

21. A health food composition for skin whitening, wrinkle improvement and skin moisturizing, containing an ultrasonic extract of seaweed sporophyll as an effective ingredient.

22. In paragraph 21, A health food composition for skin whitening, wrinkle improvement and skin moisturizing, characterized in that the above ultrasonic extract is an ultrasonic extract obtained by adding purified water to the powder of seaweed sporophyll and ultrasonicating it at a temperature of 28 to 32°C and 1,000 to 1,100 W for 7 to 9 hours.

23. In paragraph 22, A health food composition for skin whitening, wrinkle improvement and skin moisturizing, characterized in that the ultrasonic extract is obtained by performing filter cloth and housing filtration on the ultrasonic extract obtained after the ultrasonic treatment, performing ion exchange, and then sterilizing and freeze-drying.

24. In paragraph 21, The ultrasonic extract of the above seaweed sporophyll is A health food composition for skin whitening, wrinkle improvement and skin moisturizing, characterized by having antioxidant activity, collagenase inhibitory activity, MMP-1 protein expression inhibition, hyaluronic acid decomposition enzyme inhibition, tyrosinase activity inhibition or melanin production inhibition activity.

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