Composition for improving swell of skin comprising nypa fruticans wurmb extract and fucoidan extract

Abstract

The present invention relates to a composition for improving skin swelling comprising nipa palm extract and fucoidan extract. Specifically, the composition has excellent efficacy in improving skin swelling by increasing nitric oxide (NO) in the vascular endothelium to an appropriate level to maintain homeostatic function, inhibiting the activation of epithelial sodium channels (ENaC) to lower the sodium reabsorption rate, and expelling water along with sodium from the body.

Description

Technology Field

[0001] The present invention relates to a composition for improving skin swelling comprising nipa palm extract and fucoidan extract. Specifically, by including nipa palm extract and fucoidan extract as active ingredients, the invention relates to a composition having excellent efficacy in improving skin swelling by inhibiting the activation of epithelial sodium channels (ENaC), thereby lowering the reabsorption rate of sodium and expelling water along with sodium from the body. Background Technology

[0002] Skin swelling is a phenomenon caused by the excessive accumulation of water or salt in the skin, which reduces skin elasticity and hydration and is a cause of wrinkles and aging. If skin swelling becomes severe, it can cause the skin to crack, reduce elasticity and radiance, and increase wrinkles.

[0003] This skin swelling is known to be caused by various factors, including dehydration, excessive salt intake, stress, increased free radicals, lack of sleep, allergic reactions, and hormonal imbalances.

[0004] In particular, when salt accumulates excessively in the body, fluid accumulates abnormally outside the blood vessels, leading to edema, a condition in which water accumulates in the interstitial tissue. Specifically, when there is an excess of salt in the body, sodium channels (ENaC) present in the epithelial cells of the kidney's collecting ducts promote the reabsorption of sodium; consequently, to maintain homeostasis, the reabsorption of water is accelerated, causing the skin to swell due to the excess fluid.

[0005] In addition, normal vascular endothelial cells continuously secrete small amounts of nitric oxide (NO), an endothelial relaxant, to maintain vascular tone at a baseline. Chronic reduction in NO secretion leads to venous endothelium damage and inflammation, which in turn results in valve damage and can lead to varicose veins or chronic venous insufficiency. Therefore, nitric oxide secreted by vascular endothelial cells in response to a baseline reaction or stimulation maintains vascular tone appropriately and plays an important role in maintaining vascular homeostasis through mechanisms such as preventing leukocyte adhesion and inhibiting platelet aggregation and adhesion.

[0006] Although various products, such as functional foods and cosmetics, are being released to alleviate this skin swelling, most of them do not show significant effects or cause skin irritation due to physical pressure or chemical ingredients.

[0007] Meanwhile, as the demand for products utilizing natural ingredients that are non-irritating to the human body and do not cause skin problems grows, extensive research is being conducted to discover natural materials and enhance their efficacy. Furthermore, research on mixtures combining two or more types of natural extracts by utilizing their various functions has recently been actively underway. However, mixing extracts can lead to issues such as precipitation, formulation instability, and reduced usability, and there are problems where the desired efficacy is not fully realized.

[0008] Accordingly, the inventors continued research to develop a composition effective in improving skin swelling using a complex of natural extracts, and confirmed that when nipa palm extract and fucoidan extract are optimally combined, the function of α-EnaC is inhibited to lower the reabsorption rate of sodium, thereby expelling water along with sodium from the body and improving blood circulation problems caused by the abnormal reduction in NO production, resulting in an excellent effect in improving skin swelling, and thus completed the present invention. The problem to be solved

[0009] The present invention aims to provide a composition having excellent effects in improving skin swelling by including nipa palm extract and fucoidan extract as active ingredients.

[0010] The technical problems of the present invention are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0011] According to an embodiment of the present invention, a composition for improving skin swelling is provided, comprising fucoidan extract and nipa palm extract as active ingredients, wherein the fucoidan extract and nipa palm extract are included in a weight ratio of 1:0.05 to 20.

[0012] In addition, the total content of fucoidan extract and nipa palm extract may be included in an amount of 40 to 70 weight% based on the total weight of the composition.

[0013] In addition, fucoidan extract and nipa palm extract may be hot water extracted.

[0014] In addition, the fucoidan extract may be obtained by hot water extraction, and the nipa palm extract may be obtained by alcohol extraction.

[0015] In addition, nipa palm extract and fucoidan extract may be in the form of a dry powder.

[0016] In addition, the above composition can inhibit the activation of epithelial sodium channels (ENaC).

[0017] In addition, the above composition can increase the production of nitric oxide (NO) for maintaining the function of vascular endothelial cells.

[0018] In addition, a food composition comprising the above composition is provided.

[0019] In addition, a cosmetic composition comprising the above composition is provided. Effects of the invention

[0020] The composition according to the present invention can reduce the reabsorption rate of sodium by inhibiting the activation of epithelial sodium channels (ENaC) and increase the secretion of urine containing water along with sodium outside the body, thereby alleviating skin swelling caused by excess water.

[0021] The composition according to the present invention can provide excellent skin improvement effects, such as improved skin hydration, anti-inflammatory effects, improved skin elasticity, and improved skin density, in addition to the effect of improving skin swelling.

[0022] The composition according to the present invention utilizes natural extracts, is non-irritating to the human body, and can be used in various products such as food and cosmetics. Brief explanation of the drawing

[0023] A brief description of each drawing is provided to help to better understand the drawings cited in the detailed description of the invention. Figure 1 shows a schematic diagram of the structure of a sodium channel protein (ENaC). Figure 2 shows the results of measuring the α-ENaC inhibitory efficacy of Comparative Examples 1 to 3 and Examples 1 to 6, and the results of Western blot analysis of α-EnaC. Figure 3 shows the results of the HUVEC toxicity evaluation of fucoidan hot water extract and nipa palm ethanol extract at different concentrations. Figure 4 is a figure showing the increase rate of NO production relative to the steady state in Examples 9 to 21. Specific details for implementing the invention

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art. Furthermore, in describing embodiments of the present invention, detailed descriptions of related known components or functions are omitted if it is determined that such detailed descriptions would hinder understanding of the embodiments of the present invention. Additionally, while embodiments of the present invention will be described below, the technical concept of the present invention is not limited or restricted thereto and can be modified and implemented in various ways by those skilled in the art.

[0025] In this specification, when a part is described as including a certain component, it means that, unless specifically stated otherwise, it does not exclude other components but may include additional components. In this specification, the term "and / or" includes a combination of a plurality of related items or any one of a plurality of related items.

[0027] According to an embodiment of the present invention, a composition for improving skin swelling is provided, comprising nipa palm extract and fucoidan extract.

[0028] Skin swelling is a phenomenon caused by the accumulation of water in the skin due to the body's inability to properly drain fluids. Excessive sodium intake activates sodium channel proteins (ENaC), which promotes the reabsorption of sodium and water, thereby increasing body fluid levels. This increase in fluid raises the osmotic pressure in the blood, increasing pressure within blood vessels. Consequently, fluid leaks out of the blood vessels and accumulates between tissues, which can lead to skin swelling.

[0029] Epithelial sodium channels (ENaC) are essential membrane proteins that facilitate sodium ion transport and play an important role in regulating electrolyte ion balance, body fluid volume, and blood pressure. As shown in Fig. 1, epithelial sodium channels (ENaC) are structurally composed of α-EnaC, which is an essential factor for sodium channel activation, and β-EnaC and γ, which function to activate the cell surface and enhance the activity of EnaC.

[0030] To inhibit the sodium reabsorption phenomenon of these sodium channel proteins (ENaC), α-ENaC inhibitors such as amiloride or triamterene can be used, and β-ENaC and γ activation can be inhibited to inhibit cell surface activation and the enhancement of ENaC activity. In addition, sodium reabsorption can be inhibited by controlling SGK-1, which regulates the activity of sodium channel proteins (ENaC), or Nedd4-2, which promotes the degradation of sodium channel proteins.

[0031] In addition, when excessive sodium intake leads to the excessive production of reactive oxygen species (ROS) or the activation of sodium channel proteins (ENaC) causes an increase in inflammatory factors (such as cytokines like COX-2 and PGE2, cell adhesion molecules like VCAM-1, interleukin, and hs-CRP), the action of antioxidant or anti-inflammatory substances can reduce vascular permeability and alleviate swelling of the body.

[0032] The inventors have confirmed that when a nipa palm extract and a fucoidan extract are appropriately combined and applied, the reabsorption of sodium is inhibited by inhibiting the function of one of α-EnaC, β-ENaC, and γ of the sodium channel protein (EnaC), or by controlling the regulatory factor (SGK-1 or Nedd4-2) of the sodium channel protein (EnaC), thereby lowering the sodium reabsorption rate and improving skin swelling by expelling water along with sodium from the body.

[0033] Furthermore, the composition of the present invention can inhibit cell contraction and moisture loss by improving the function of tight junctions, which act as a key element in maintaining skin cell volume. By enhancing the skin cell barrier and moisture retention capabilities, skin swelling can be alleviated.

[0034] In addition, the composition of the present invention exhibits an antioxidant or anti-inflammatory effect that inhibits the reactivity of excessively generated reactive oxygen species (ROS) or inflammatory factors (cytokines such as COX-2 and PGE2, cell adhesion molecules such as VCAM-1, interleukin, and hs-CRP), thereby reducing intravascular pressure and improving swelling of the body.

[0035] Specifically, the composition of the present invention can inhibit α-EnaC activation to inhibit sodium reabsorption by the epithelial sodium channel (ENaC). Through this inhibition of α-EnaC function, the sodium reabsorption rate is reduced, and consequently, the reabsorption of water for maintaining homeostasis in the body is inhibited, thereby increasing the amount of urine containing sodium and water excreted from the body. In this way, the composition of the present invention can alleviate edema or swelling of the skin by excreting excess water accumulated in the body along with urine through a mechanism that inhibits α-EnaC activation.

[0036] Nipa palm ( Nypa fruticans Nipa palm is the only species in the Nipa subfamily, a monotypic subfamily of the palm family, and is distributed in South Asia, Southeast Asia, and Oceania. Nipa palms are young shoots of the palm tree that are very high in polyphenols, providing benefits such as antioxidant effects, improved blood circulation, anti-inflammatory effects, and cancer prevention. Additionally, they are rich in Vitamin E and saponins, which help with cardiovascular diseases, cataracts, and arthritis, and are also known for their efficacy in relieving constipation, eliminating toxins, and curing hangovers.

[0037] Fucoidan is a type of viscous, sulfated polymeric polysaccharide found in trace amounts in brown algae such as wakame and kelp. It acts as a protective barrier to defend against marine microorganisms, serving as a component that shields seaweed from ocean currents and external stimuli.

[0038] The composition of the present invention comprises both nipa palm extract and fucoidan extract. The composition exhibits excellent α-EnaC inhibitory efficacy in the kidneys, thereby lowering the sodium reabsorption rate. Consequently, it excretes sodium and water together from the body, which can improve skin swelling caused by excess water. Additionally, it exhibits excellent efficacy in regulating appropriate NO production in vascular endothelial cells, thereby maintaining normal vascular function and alleviating skin swelling through vasodilation and the alleviation of blood flow stagnation. These effects are even more superior when nipa palm extract and fucoidan extract are combined compared to when nipa palm extract and fucoidan extract are used alone.

[0039] In the present invention, "extract" refers to a substance obtained by extracting components from a natural product; it is a broad concept that includes, for example, components soluble in a solvent extracted from a natural product using water or an organic solvent, or substances obtained by extracting only specific components of a natural product. The extract may include all extracts, fractions, their dilutions, concentrates, or dried products obtained at each stage, such as extraction, fractionation, or purification.

[0040] In the present invention, the extraction method of the extract may utilize extraction methods commonly used in the art, such as hot water extraction, alcohol extraction, immersion extraction, cold maceration extraction, reflux extraction, supercritical extraction, subcritical extraction, elution method, pressing method, high temperature extraction, high pressure extraction, or ultrasonic extraction, but specifically, it is preferable to use hot water extraction or alcohol extraction.

[0041] In the examples, the fucoidan extract and nipa palm extract of the present invention may be obtained by hot water extraction. The active ingredients that influence the skin swelling alleviation effect of the fucoidan extract and nipa palm extract of the present invention are water-soluble substances that dissolve well in pure water, and the active ingredients can be effectively extracted by hot water extraction of the fucoidan extract and nipa palm extract. When the nipa palm extract is obtained by hot water extraction, the extraction amount of polyphenols, which have antioxidant, blood circulation-promoting, anti-inflammatory, and cancer-preventing effects, as well as vitamin E and saponins, which improve the therapeutic effects of cardiovascular diseases, cataracts, and arthritis, and the efficacy of constipation relief, detoxification, and hangover relief, can be effectively increased.

[0042] In another embodiment, the fucoidan extract of the present invention may be obtained by hot water extraction, and the nipa palm extract may be obtained by ethanol extraction. When the composition of the present invention is prepared by ethanol extraction of the nipa palm extract, active ingredients such as polyphenols, flavonoids, and tannins can be efficiently eluted, and preservative stability can be ensured by reducing the risk of spoilage and contamination during the extraction process through the inhibition of microbial growth. Additionally, since ethanol can be easily evaporated and removed even at low temperatures, thermal denaturation of active ingredients can be minimized, and drying, powdering, and concentration processes can be facilitated.

[0043] According to the examples, the solvent used for the extract may be water, ethanol, or a mixture thereof, and specifically, it may be purified water, ethanol, or ethanol. However, it is not limited thereto, and various solvents applicable in the relevant technical field may be used alone or in combination. For example, as an extraction solvent, water (or distilled water); lower alcohols having 1 to 4 carbon atoms, such as methanol, ethanol, propyl alcohol, butyl alcohol; polyhydric alcohols, such as propanediol, butanediol, hexanediol, methylpropanediol, glycerin, butylene glycol, propylene glycol, pentylene glycol, dipropylene glycol; and hydrocarbon solvents, such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, dichloromethane; or a mixture thereof may be used.

[0044] According to the example, hot water extraction may use hot water at 60 to 140°C. Since hot water extraction uses pure water as the extraction medium, it has the advantage of not causing problems caused by the toxicity of organic solvents.

[0045] According to the examples, the alcohol extraction may be performed using an aqueous ethanol solution of 30 to 70%, but is not limited thereto. When ethanol of the above concentration is used, the skin swelling improvement effect of the composition according to the present invention may be even better.

[0046] According to the embodiments, the extract may be in a liquid formulation or in the form of a dried powder. In this case, the drying method may include not only freeze-drying but also various methods commonly used in the art, such as vacuum drying, hot air drying, and spray drying.

[0047] According to the examples, the extract may be prepared or processed in various ways, such as by vacuum distillation or concentration.

[0048] The composition of the present invention may include nipa palm extract and fucoidan extract in a weight ratio of 1:0.05 to 20, specifically in a weight ratio of 1:0.1 to 18, and more specifically in a weight ratio of 1:0.1 to 15. When nipa palm extract and fucoidan extract are included in the above mixing ratios, the sodium reabsorption rate due to inhibition of α-EnaC function in the kidney can be sufficiently lowered, and the amount of NO expression in vascular endothelial cells increases, so the effect of alleviating skin swelling can be excellent.

[0049] In the examples, the nipa palm extract and fucoidan extract may be included in an amount of 40 to 70 weight% based on the total weight of the composition. If the nipa palm extract and fucoidan extract are included in an amount of less than 40 weight% based on the total weight of the composition, the α-Enac function inhibitory efficacy and / or NO production activation efficacy may not be sufficient, and thus the effect of improving skin swelling may be insufficient. Even if the amount is included in an amount exceeding 70 weight%, the α-EnaC inhibitory efficacy and / or NO production activation efficacy may be reduced, and thus the effect of improving skin swelling may not be sufficient. However, this is not limited thereto.

[0050] In the examples, the composition lowers the reabsorption rate of sodium and water in the kidneys by inhibiting the activation of epithelial sodium channels (EnaC), thereby providing an excellent effect in alleviating skin swelling. In addition to the effect of alleviating swelling, at least one of the following effects may be excellent: improved hydration, improved elasticity, and improved density. Specifically, the composition of the present invention can alleviate skin swelling by improving hydration within the skin and increasing elasticity and density, thereby suppressing the factors that cause skin swelling.

[0051] In another embodiment, the composition can alleviate skin swelling by increasing the production of appropriate nitric oxide (NO) in vascular endothelial cells. NO acts as a vasodilator to promote microcirculation and can reduce excessive fluid accumulation in tissues by relieving stagnant blood flow in capillaries, and in this process, vascular permeability can be normalized and lymphatic circulation can be improved.

[0053] In the examples, the composition may be a food composition.

[0054] In this specification, the term "food" refers to a food manufactured and processed using raw materials or ingredients that have functional properties useful to the human body, and is a food with high medical or therapeutic effects that is processed to efficiently exhibit biological regulatory functions in addition to providing nutrition; it may be used interchangeably with terms known in the art, such as functional food. For example, it may be manufactured as a health functional food, a health supplement, a food for special nutritional supplementation, a functional beverage, etc., or the composition according to the present invention may be added to natural foods, processed foods, etc. The food may be manufactured as a powder, granule, tablet, capsule, syrup, or beverage, etc., and there are no restrictions on the form in which the food may take, and it may include all foods in the conventional sense.

[0055] For example, beverages and various drinks, fruits and their processed foods (canned fruit, jam, etc.), fish, meat and its processed foods (ham, bacon, etc.), breads and noodles, cookies and snacks, dairy products (butter, cheese, etc.), etc. are possible, and may include all functional foods in the conventional sense. In addition, foods used as animal feed may also be included.

[0056] The food composition according to the present invention may be prepared by further including food-grade acceptable food additives and suitable other auxiliary ingredients commonly used in the art. For example, it may additionally contain flavoring agents, natural carbohydrates, sweeteners, vitamins, electrolytes, coloring agents, pectic acid, alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents, etc.

[0057] For example, natural flavoring agents including taumatin and stevia extract, or synthetic flavoring agents including saccharin and aspartame may be used as flavoring agents; natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides including maltose and sucrose, polysaccharides including dextrin and cyclotensin, and sugar alcohols including xylitol, sorbitol, and erythritol; and sweeteners may include natural sweeteners such as taumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame. It is preferable that the above food additives and other auxiliary components be added and used in minute amounts as long as they achieve their intended use. Numerically, minute amounts may be in the range of 0.0005% by weight to about 0.5% by weight based on the total weight of the product composition. The amount of the above components can be easily selected by a person skilled in the art within a range that does not impair the purpose and effects of the present invention.

[0059] In the examples, the composition may be a cosmetic composition. In the present invention, "skin" refers to tissue covering the body surface of an animal, and is a broad concept that includes not only tissue covering the body surface such as the face or body, but also the scalp.

[0060] According to the examples, when the composition of the present invention is utilized as a cosmetic composition, it can be manufactured into various products effective for improving swelling, improving moisture within the skin, increasing skin elasticity, and improving skin density.

[0061] According to the embodiments, the composition of the present invention may be manufactured into functional cosmetics such as nourishing creams, essences, and ampoules, or into basic cosmetics such as toners and lotions. In addition, it may be manufactured into soaps, cleansers, cleansing creams, cleansing waters, etc., or into color cosmetics such as lipsticks, lip balms, mascaras, blushes, shading, highlighters, makeup bases, foundations, compacts, concealers, skin covers, etc. Furthermore, it may be manufactured into products that are attached to the skin, such as face masks and hydrogel slimming patches, or into products that are sprayed onto the skin, such as mists and aerosols.

[0062] According to the embodiments, the cosmetic composition of the present invention may be provided in formulations commonly manufactured in the art. For example, it may be provided in formulations such as a solution, an emulsion obtained by dispersing an oil phase in an aqueous phase, an emulsion obtained by dispersing an aqueous phase in an oil phase, a suspension, an emulsion, a solid, a gel, an oil, a powder, a paste, or a foam aerosol.

[0063] According to the embodiments, other ingredients commonly used in the art may be further included within a range that does not impair the effects of the present invention, such as thickeners, pH adjusters, isotonic agents, surfactants, stabilizers, preservatives, antimicrobial agents, UV absorbers, disinfectants, moisturizers, blockers, antioxidants, organic pigments, inorganic pigments, fragrances, vitamins, etc. The amount of the above ingredients can be easily selected by a person skilled in the art within a range that does not impair the purpose and effects of the present invention.

[0065] In the following, examples and experimental examples are presented to further explain the present invention in more detail, but the present invention is not limited thereto.

[0067] Preparation Example. Preparation of extracts and mixtures

[0069] (1) Preparation of hot water extract of Nipa palm

[0070] Ten times the amount of distilled water was added per g of nipa palm, and hot water extraction was performed at 95±2℃ for 6 hours. The solids of the extract obtained by hot water extraction were removed, and the extract was concentrated under reduced pressure. After concentration, the extract was mixed with dextrin in a 7:3 ratio and spray-dried to obtain the nipa palm hot water extract.

[0072] (2) Preparation of nipa palm ethanol extract

[0073] Ten times the amount of 50% ethanol was added per g of nipa palm, and the extraction temperature was set to 70℃. For the first extraction, the solvent was extracted for 4 hours at 10 times the amount of the sample (v / w), and for the second extraction, the solvent was extracted for 2 hours at 10 times the amount of the sample (v / w). After the extraction was completed, the extract from which solids were removed was concentrated using a vacuum concentrator, and then freeze-dried to obtain the nipa palm ethanol extract.

[0075] (3) Preparation of hot water extract of fucoidan

[0076] Dried sea mustard was extracted with purified water, and after solid-liquid separation, the extract was concentrated to prepare a first concentrate. After removing alginic acid from the concentrate, a second concentration was performed. Then, as a detailed process for polysaccharide separation, alcohol was added and the mixture was immersed, after which the precipitate was purified. After purification was completed, the mixture was dissolved in water and freeze-dried to prepare a hot water extract of fucoidan.

[0078] (4) Preparation of a composition for improving skin swelling

[0079] The above-prepared fucoidan hot water extract and nipa palm hot water extract were mixed in the ratios shown in Table 1 below to prepare the example composition.

[0080]

[0082] The compositions of Examples 1 to 8 in Table 1 above had the concentration of the Nipa palm hot water extract adjusted based on 0.5 µg / mL of the fucoidan hot water extract.

[0084] In addition, the above-prepared fucoidan hot water extract and nipa palm ethanol extract were mixed in the ratios shown in Table 2 below to prepare the example composition.

[0085]

[0087] The compositions of Examples 9 to 21 in Table 2 above had the concentration of the fucoidan hot water extract adjusted based on 2.5 µg / mL of the nipa palm ethanol extract.

[0089] Experimental Example 1. Evaluation of Toxicity to Epidermal Cells (HaCaT)

[0090] To evaluate the toxicity of the compositions of Examples 1 to 8 on epidermal cells (HaCaT), the survival rate of epidermal cells was measured by treating epidermal cells (HaCaT) treated with NaCl at a concentration of 145 mM with the compositions of Examples 1 to 8.

[0091] Specifically, HaCaT cell lines were dispensed into a 96-well plate at an appropriate concentration of 200 µL per well and cultured for 24 hours under incubator conditions of 37 ℃ and 5% CO2. The cultured cells were treated with the compositions of Examples 1 to 8 at different concentrations and cultured for 22 hours under incubator conditions of 37 ℃ and 5% CO2. Subsequently, 10 µL of CCK-8 solution was added per well and reacted for 2 hours under the same culture conditions. Thirty minutes before the reaction was completed, 145 mM NaCl was added to re-react the cells, and the absorbance was measured at 450 nm using a microplate reader (Thermo, UV / Vis). Cell viability was calculated using Equation (1). The results are shown in Table 3 below.

[0092]

[0093]

[0095] Referring to Table 3, the compositions of Examples 1 to 8, prepared by including fucoidan hot water extract and nipa palm hot water extract in appropriate content ratios, were confirmed to have no cytotoxicity in the epidermal cell (HaCaT) toxicity evaluation.

[0097] Experimental Example 2. Evaluation of α-ENaC Inhibitory Efficacy

[0098] To evaluate the α-ENaC inhibitory efficacy of the compositions of Examples 1 to 6 above, the α-ENaC activity of the compositions of Examples 1 to 6 was measured in epithelial cells treated with NaCl at a concentration of 145 mM. In addition, for comparison, Comparative Example 1 measured the α-ENaC activity of epithelial cells without added NaCl, Comparative Example 2 measured the α-ENaC activity of epithelial cells treated with NaCl at a concentration of 145 mM, and Comparative Example 3 measured the α-ENaC activity when NaCl and an Amiloride 10 μM solution exhibiting α-ENaC inhibitory efficacy were added together to epithelial cells.

[0099] Specifically, HaCaT cell lines were dispensed at appropriate concentrations of 4 mL per well into 60 mm dishes and cultured for 24 hours in an incubator at 37 ℃ and 5% CO2. The cultured cells were treated with the compositions of Comparative Examples 1 to 3 and Examples 1 to 6 diluted to various concentrations and cultured for an additional 24 hours. Thirty minutes prior to the end of the culture, the cells were treated with NaCl at a concentration of 145 mM to induce an additional reaction. Subsequently, PRO-PREP™ Protein Extract Solution (Intron, Inc.) was added and reacted at -20 ℃ for 20 minutes, followed by centrifugation. The supernatant of the isolated proteins was transferred to a tube and stored at -20 ℃. The isolated proteins were subjected to electrophoresis using 8% SDS-PAGE and transferred to a PVDF membrane. The samples were subjected to a blocking process for 1 hour in Tris buffer containing 5% BSA, followed by treatment with the primary antibody and subsequent reaction. After treatment with the secondary antibody and reaction for a certain period using Immobilon™ Western Chemiluminescent HRP Substrate (Millipore), the samples were developed. Through this process, the degree of activity inhibition of α-ENAC, whose expression was enhanced under 145 mM NaCl (high sodium) conditions, was measured. The results are shown in Figure 2.

[0100] In addition, the improvement rate of the α-ENaC inhibitory efficacy of Comparative Example 1, Comparative Example 3, and Examples 1 to 6 was calculated based on the α-ENaC activity of Comparative Example 2, in which epithelial cells were treated with NaCl at a concentration of 145 mM and no active ingredient was included, and is shown in Table 4 below.

[0101]

[0103] Referring to Table 4 and Figure 2, it was confirmed that Examples 1 to 6, containing fucoidan hot water extract and nipa palm hot water extract in appropriate content ratios, exhibited α-ENaC inhibitory efficacy. In particular, it was confirmed that the extract mixture composition having the composition ratios of Examples 5 and 6 exhibited α-ENaC inhibitory efficacy similar to or superior to Comparative Example 3, which contained amyloride, which is generally used as a preparation for α-ENaC inhibition.

[0104] Therefore, the composition of the present invention can be expected to have the effect of lowering the sodium reabsorption rate by inhibiting the α-ENaC mechanism within the epithelial cells of the kidney that causes swelling.

[0106] Experimental Example 3. Evaluation of vascular endothelial cell (HUVEC) toxicity

[0107] To determine whether the compositions of Examples 9 to 21 above were toxic to vascular endothelial cells (HUVEC), the viability of vascular endothelial cells (HUVEC) treated with 145 mM NaCl was measured by treating them with a hot water extract of fucoidan and an alcohol extract of nipa palm.

[0108] Specifically, HUVEC cell lines were dispensed into a 96-well plate at an appropriate concentration of 100 µL per well and cultured for 24 hours under incubator conditions of 37 ℃ and 5% CO2. The cultured cells were treated with the above-mentioned fucoidan hot water extract and nipa palm ethanol extract at different concentrations and cultured for 23 hours under incubator conditions of 37 ℃ and 5% CO2. Subsequently, 10 µL of CCK-8 solution was added per well and reacted for 1 hour under the same culture conditions. Afterward, absorbance was measured at 450 nm using a microplate reader (Thermo, UV / Vis). Cell viability was calculated in the same manner as Equation (1) of Experimental Example 1, and the maximum concentration at which cytotoxicity did not occur is shown in Table 5 and Figure 3 below.

[0109]

[0111] Referring to Table 5 and Figure 3 above, in the toxicity evaluation of vascular endothelial cells (HUVEC), it was confirmed that the hot water extract of fucoidan and the ethanol extract of nipa palm did not exhibit cytotoxicity at concentrations of 100 μg / mL or less and 25 μg / mL or less, respectively. This suggests that the compositions of Examples 9 to 21, which mixed the hot water extract of fucoidan and the ethanol extract of nipa palm at concentrations below the above levels, also do not show toxicity in cells.

[0113] Experimental Example 4. Evaluation of Efficacy in Increasing NO Production

[0114] To evaluate the efficacy of the compositions of Examples 9 to 21 in increasing NO production in vascular endothelial cells, HUVEC cells were treated with each of Examples 9 to 21, and the amount of Nitric Oxide (NO) produced in the cells was measured.

[0115] Specifically, HUVEC cell lines were dispensed at an appropriate concentration of 100 μL per well into a 96-well plate and cultured for 24 hours in an incubator at 37°C and 5% CO₂. The cultured cells were pretreated for 1 hour by adding each example. Subsequently, LPS (1 μg / mL) was added to each well, and the cells were cultured for an additional 24 hours under the same incubator conditions (37°C, 5% CO₂). After the culture was completed, the supernatant from each well was collected, and the increased amount of Nitric Oxide (NO) compared to normal cells was measured. The absorbance of the reaction solution was measured at 570 nm using a Microplate Reader (Thermo, UV / Vis). LPS 1 μg / mL was used as the negative control (NC) and Celecoxib 5 μg / mL was used as the positive control (PC). All results were calculated as a percentage (%) relative to the blank controls and are shown in Table 6 and Figure 4 below. Here, the blank controls represent the NO production status of normal vascular endothelial cells that have not been treated with any composition. Even if the NO production rate is lower compared to the blank controls, if the NO production rate increases compared to the negative control (NC), it can be interpreted as having an appropriate level of increased NO production effect.

[0116]

[0118] According to Table 6 and Figure 4 above, it was confirmed that the amount of NO produced in the examples increased significantly compared to the control group (Blank Controls), and it was confirmed that NO production increased by up to 50% compared to the control group (Example 21). In particular, in Examples 9 to 12 and Examples 18 to 21, NO production increased significantly by more than about 30% to an appropriate level compared to the control group, and in Examples 13 and 17, a similar or higher rate of increase in NO production was observed compared to the positive control group (Celecoxib).

[0119] Therefore, the composition of the present invention can be expected to improve skin swelling by regulating vasodilation and increased blood flow through increasing NO production, which is an important factor in maintaining vascular homeostasis in vascular endothelial cells, to an appropriate level.

[0121] Foregoing, specific parts of the content of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Accordingly, the actual scope of the present invention is defined by the appended claims and their equivalents.

Claims

Claim 1 A composition having excellent skin swelling improvement effect, comprising fucoidan extract and nipa palm extract as active ingredients, wherein the fucoidan extract and nipa palm extract are included in a weight ratio of 1:0.05 to 20, and the composition improves skin swelling caused by fluid accumulation or reduced NO secretion by inhibiting the activation of epithelial sodium channels (ENaC) or increasing the production of nitric oxide (NO) to maintain the function of vascular endothelial cells. Claim 2 A composition for improving skin swelling according to claim 1, wherein the total content of the fucoidan extract and nipa palm extract is 40 to 70 weight% based on the total weight of the composition. Claim 3 A composition for improving skin swelling according to claim 1, wherein the fucoidan extract and nipa palm extract are obtained by hot water extraction. Claim 4 A composition for improving skin swelling according to claim 1, wherein the fucoidan extract is obtained by hot water extraction and the nipa palm extract is obtained by ethanol extraction. Claim 5 A composition for improving skin swelling according to claim 1, wherein the fucoidan extract and nipa palm extract are in the form of a dry powder. Claim 6 delete Claim 7 delete Claim 8 In claim 1, the composition is a food composition for improving skin swelling. Claim 9 In claim 1, the composition is a cosmetic composition for improving skin swelling.

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