Anti-aging agent

Abstract

The purpose of the present invention is to provide an anti-aging agent that contains an active ingredient capable of being efficiently produced, and exhibits excellent anti-aging action and skin protective action. An anti-aging agent according to the present invention is characterized by: containing extracellular vesicles derived from seaweed as an active ingredient; and exhibiting anti-aging action.

Description

Anti-aging agent

[0001] The present invention relates to an anti-aging agent that contains an active ingredient that can be efficiently produced and that exhibits excellent anti-aging and skin protective effects, the use of the active ingredient to inhibit aging, and a method for inhibiting aging using the active ingredient.

[0002] Exosomes are a type of extracellular vesicle. They are membrane vesicles with a diameter of approximately 30 to 200 nm secreted by various cells. They are surrounded by a lipid bilayer membrane derived from secretory cells and contain various proteins, RNA, etc. Extracellular vesicles have long been thought to be involved in the release of unnecessary cellular contents. However, it has been shown that nucleic acid substances such as mRNA and miRNA may be transferred to other cells via extracellular vesicles, and they have attracted attention as a new intercellular communication medium. In particular, cancer cells have been shown to release extracellular vesicles as an intercellular communication medium for cell proliferation, immunosuppression, metastasis, etc. Therefore, the use of extracellular vesicles in cancer diagnosis is also being considered.

[0003] In addition, the use of extracellular vesicles for the treatment of skin diseases and anti-aging has also been investigated. For example, Patent Document 1 discloses a composition for treating skin diseases containing stem cell-derived exosomes. Patent Document 2 discloses a therapeutic and preventive agent for aging diseases, etc., containing exosomes derived from mesenchymal stem cells. Patent Document 3 discloses the use of vesicles produced by inducing stem cell apoptosis in the preparation of skin products, etc. Patent Document 4 discloses an anti-aging cosmetic composition containing a protein component prepared from a conditioned medium of adipose-derived stem cells. Furthermore, Patent Document 5 discloses an external skin preparation containing exosome-like vesicles derived from mammalian milk.

[0004] As mentioned above, extracellular vesicles used as active ingredients in therapeutic compositions and the like are mainly isolated from stem cells. However, the amount of extracellular vesicles obtained from stem cells and the like is extremely small, posing practical problems. In contrast, Patent Document 6 discloses a collagen production promoter containing kale-derived exosomes as an active ingredient. Patent Document 7 discloses a cosmetic composition containing broccoli exosomes.

[0005] International Publication No. 2019 / 146612 Pamphlet Special Publication No. 2022-542953 Special Publication No. 2023-513394 Special Publication No. 2023-511352 Japanese Patent Application Laid-Open No. 2022-160101 Japanese Patent Application Laid-Open No. 2022-92330 Japanese Patent Application Laid-Open No. 2023-549283

[0006] As mentioned above, the use of extracellular vesicles for the treatment of skin diseases and anti-aging has also been investigated. However, extracellular vesicles have traditionally been isolated primarily from stem cells, but stem cell culture efficiency is low. The use of exosomes derived from terrestrial plants such as kale and broccoli has also been investigated, but the growth rate of terrestrial plants is not particularly fast. Therefore, the present invention aims to provide an anti-aging agent that contains an active ingredient that can be efficiently produced and exhibits excellent anti-aging and skin-protecting effects, the use of the active ingredient to inhibit aging, and a method for inhibiting aging using the active ingredient.

[0007] The present inventors have conducted extensive research to solve the above problems. As a result, they have discovered that extracellular vesicles derived from seaweed exhibit excellent anti-aging effects, and have completed the present invention. The present invention is described below.

[0008] [1] An anti-aging agent characterized by containing extracellular vesicles derived from seaweed as an active ingredient and exhibiting anti-aging activity. [2] The anti-aging agent according to [1] above, wherein the seaweed is a seaweed of the Ulvales order and / or a seaweed of the Hibuliales order. [3] The anti-aging agent according to [2] above, wherein the seaweed of the Ulvales order is Enteromorpha sieboldiana and / or Enteromorpha sieboldiana. [4] The anti-aging agent according to [2] above, wherein the seaweed of the Hibuliales order is Artemisia nigra. [5] The anti-aging agent according to any of [1] to [4] above, wherein the anti-aging activity is a skin protective activity. [6] The anti-aging agent according to [5] above, wherein the skin protective activity is one or more skin protective activities selected from the group consisting of collagen production promotion, cell adhesion structure formation promotion, antioxidant protein production promotion, hyaluronic acid production promotion, and skin protective factor production promotion.

[0009] [7] Use of extracellular vesicles derived from seaweed to inhibit aging. [8] The use according to [7] above, wherein the seaweed is a seaweed of the Ulvales order and / or a seaweed of the Hibuliales order. [9] The use according to [8] above, wherein the seaweed of the Ulvales order is Enteromorpha spp. and / or Enteromorpha natans.

[10] The use according to [8] above, wherein the seaweed of the Hibuliales order is Artemisia nigra.

[11] The use according to any of [7] to

[10] above, wherein skin aging is inhibited.

[12] The use according to

[11] above, wherein skin aging is inhibited by one or more skin protective effects selected from the group consisting of collagen production promotion, cell adhesion structure formation promotion, antioxidant protein production promotion, hyaluronic acid production promotion, and skin protective factor production promotion.

[0010]

[13] A method for inhibiting aging, comprising a step of administering extracellular vesicles derived from seaweed to a patient.

[14] The method according to

[13] , wherein the seaweed is a seaweed of the Ulvales order and / or a seaweed of the Hibuliales order.

[15] The method according to

[14] , wherein the seaweed of the Ulvales order is Enteromorpha spp. and / or Enteromorpha natans.

[16] The method according to

[14] , wherein the seaweed of the Hibuliales order is Artemisia nigra.

[17] The method according to any one of

[13] to

[16] , wherein skin aging is inhibited.

[18] The method according to

[17] , wherein skin aging is inhibited by one or more skin protective effects selected from the group consisting of collagen production promotion, cell adhesion structure formation promotion, antioxidant protein production promotion, hyaluronic acid production promotion, and skin protective factor production promotion.

[0011] The active ingredient of the anti-aging agent of the present invention is derived from seaweed, which is said to grow 30 to 60 times faster than terrestrial plants, making it an overwhelmingly fast growth source. Furthermore, seaweed can grow with only seawater and sunlight, and does not require soil, fertilizer, pesticides, etc., as do terrestrial plants. Therefore, the active ingredient of the anti-aging agent of the present invention can be efficiently produced. Furthermore, the active ingredient of the anti-aging agent of the present invention exhibits excellent anti-aging and skin-protecting effects. Therefore, the anti-aging agent of the present invention is industrially extremely advantageous as a technology to combat the coming aging society.

[0012] FIG. 1 is a graph showing the excellent antioxidant enzyme gene expression promoting effect of the EVs of the present invention on a control example and human-derived extracellular vesicles (EVs). FIG. 2 is a graph showing the excellent hyaluronic acid synthase gene expression promoting effect of the EVs of the present invention on a control example and human EVs. FIG. 3 is a graph showing the excellent filaggrin gene expression promoting effect of the EVs of the present invention on a control example and human EVs. FIG. 4 is a graph showing the results of an experiment confirming the effect of a control example, human EVs, and the EVs of the present invention on the expression of the inflammatory cytokine IL-1β gene. FIG. 5 is a graph showing the excellent collagen production promoting effect of the EVs of the present invention on a control example. FIG. 6 is a graph showing the excellent skin protection effect of the EVs of the present invention on a control example. Figure 7 (1) is a graph showing the excellent antioxidant enzyme gene expression promoting effect of the EVs of the present invention compared to the control example and seaweed extract, Figure 7 (2) is a graph showing the hyaluronic acid synthase gene expression promoting effect, and Figure 7 (3) is a graph showing the filaggrin gene expression promoting effect.

[0013] The anti-aging agent according to the present invention contains extracellular vesicles derived from seaweed as an active ingredient and exhibits anti-aging effects.

[0014] Seaweed refers to algae that grow in the ocean, and unlike terrestrial plants, many of them do not have distinct roots, stems, or leaves, and their roots are not used to absorb nutrients from the ground, but simply to attach to rocks and other surfaces. While terrestrial plants reproduce through sexual or asexual reproduction, seaweed reproduces through branching roots, which act as appressoria, or through spores and eggs.

[0015] The seaweed used in the present invention is not particularly limited as long as it produces extracellular vesicles, but for example, seaweeds of the Ulvales order and / or Hibuliales order can be used. According to the inventor's experimental findings, extracellular vesicles can be efficiently obtained from these seaweeds, and the extracellular vesicles derived from these seaweeds exhibit excellent anti-aging and skin protective effects.

[0016] Examples of the Ulvales family include the family Bolbocoleaceae, the family Kornmanniaceae, the family Phaeophilacea, the family Ulvaceae, and the family Ulvelaceae, with the family Ulvalaceae being preferred. Examples of the Ulvales family include the genera Blidingia, Percursaria, Ulva, Ulvaria, and Umbraulva, with the genus Ulva being preferred. Examples of seaweeds of the genus Ulva include Ulva adhaerens, Ulva arasakii, Ulva clathrata, Ulva clathratioides, Ulva compressa, Ulva conglobata, Ulva fasciata, Ulva fenestrata, Ulva flexuosa, Ulva flexuosa, and Ulva flexuosa. intestinalis), Ulva lactuca, Ulva linza, Ulva meridionalis, Ulva ohnoi, Ulva pertusa, Ulva prolifera, Ulva reticulata, Ulva rigida, Ulva spinulosa, Ulva sublittoralis, Ulva tanneri, Ulva tepida), and Enteromorpha sieboldii and / or Enteromorpha sieboldii are preferred.

[0017] Examples of the Ulotrichales include the Capsosiphonaceae, Collinsiellaceae, Gayraliaceae, Gomontiaceae, and Ulotrichalaceae, with the Ulotrichalaceae being preferred. Examples of the Ulotrichalaceae include the genus Gomontia Bornet and the genus Monostroma Thuret, with the genus Monostroma being preferred. Examples of the Monostroma genus include Monostroma alittoralis, Monostroma angicava, Monostroma arcticum, Monostroma crassidermum, Monostroma crassissimum, Monostroma grevillei, and Monostroma nitidum, with Monostroma being preferred.

[0018] Extracellular vesicles are particles released from cells that are surrounded by a lipid bilayer membrane and cannot replicate because they lack a nucleus. They are primarily classified as exosomes, microvesicles, and apoptotic vesicles. Exosomes are endosomal membrane-derived vesicles approximately 30-200 nm in size formed during endocytosis and are composed primarily of lipids, proteins, and nucleic acids. Microvesicles range in size from 100-1,000 nm and differ from exosomes in that they bud directly from the cell membrane and are secreted extracellularly, although it is difficult to completely distinguish them from exosomes. Apoptotic vesicles are particles budded from the membrane of cells undergoing apoptosis and are on the order of micrometers in size. Extracellular vesicles have a similar structure to the parent cell membrane and may retain the membrane proteins present in the parent cell membrane.

[0019] Extracellular vesicles can be obtained by a general method other than using seaweed as a raw material. For example, the raw seaweed is immersed in seawater or water such as purified water. By immersing the seaweed in seawater or water, extracellular vesicles are released into the seawater or water. The seaweed may be cut into pieces of approximately 5 mm or more and 10 mm or less. The temperature during immersion can be, for example, approximately 5°C or more and 60°C or less, or room temperature. Heating may increase the amount of extracellular vesicles released. However, room temperature is preferred because heating may cause the growth of unwanted bacteria. The immersion time may also be adjusted appropriately within a range that allows extracellular vesicles to be efficiently obtained, and can be, for example, approximately 10 hours or more and 5 days or less. However, the longer the immersion time, the higher the possibility of bacterial growth unless measures to inhibit bacterial growth are taken, and there is also the possibility of sporulation. Therefore, the immersion time is preferably 3 days or less or 2 days or less.

[0020] After soaking the seaweed in seawater or water, extracellular vesicles can be purified from the seawater or water. For example, common methods such as ultracentrifugation, density gradient centrifugation, immunoprecipitation, chromatography, precipitation, and ultrafiltration can be used. Specifically, after soaking, the liquid is separated from the seaweed by filtration or other methods. If the raw seaweed is cut, filtration or other methods may not be necessary. Next, in the ultracentrifugation method, the mixture is centrifuged at 200 g or more and 20,000 g or less for approximately 1 minute to 60 minutes to recover the supernatant containing the extracellular vesicles, and then ultracentrifuged at 50,000 g or more and 200,000 g or less for approximately 30 minutes to 120 minutes to precipitate the extracellular vesicles, thereby isolating the extracellular vesicles. Centrifugation and ultracentrifugation may be performed multiple times, removing the precipitated residue or replacing the liquid with physiological saline or the like, and increasing the rotation speed or time.

[0021] Other isolation methods also involve roughly purifying extracellular vesicles by performing the same centrifugation process as described above. The vesicles can then be purified using the appropriate isolation method. For example, in the case of immunoprecipitation, it may be possible to isolate extracellular vesicles using beads bound to antibodies against marker proteins for seaweed-derived extracellular vesicles.

[0022] After isolating the extracellular vesicles, they may be confirmed by standard methods, such as by checking the expression of a marker protein for seaweed-derived extracellular vesicles, measuring the zeta potential, or observing them under an electron microscope.

[0023] Furthermore, after isolating extracellular vesicles, they may be subjected to a sterilization treatment or a sterilization treatment. For example, although some extracellular vesicles are large, the size of exosomes, which are the main extracellular vesicles, is about 30 to 200 nm, whereas the size of general bacteria is about 1 to 5 μm, and many fungi are larger than bacteria. Therefore, the extracellular vesicles can be sterilized by filtering the dispersion of extracellular vesicles through a sterilization filter with a pore size of less than 1 μm.

[0024] The extracellular vesicles (EVs) of the present invention can be used as an active ingredient in an anti-aging agent and can inhibit aging. According to the inventor's experimental findings, as shown in the examples below, the extracellular vesicles of the present invention exhibit skin protective effects such as promoting collagen production, promoting the formation of cell adhesion structures, promoting antioxidant protein production, promoting hyaluronic acid production, and promoting the production of skin protective factors. Therefore, they can be used to inhibit skin aging, specifically to reduce wrinkles, improve skin firmness, and maintain skin freshness. Therefore, the anti-aging agent of the present invention can be used as a skin protective agent, particularly a topical skin protective agent. Furthermore, in the present disclosure, "inhibition" of aging includes not only the treatment of aging and the halting or delay of the progression of aging, but also the prevention of aging.

[0025] Furthermore, because the extracellular vesicles that are the active ingredients of the anti-aging agent of the present invention are derived from seaweed, they are believed to be harmless or minimally harmful, and in fact, according to the inventor's experimental findings, they are not believed to increase the production of extracellular cytokines or induce inflammatory reactions. Therefore, the anti-aging agent of the present invention can be used not only to improve or treat symptoms such as wrinkles and loss of skin elasticity, but also as a preventative measure before symptoms appear.

[0026] The dosage form of the anti-aging agent of the present invention is not particularly limited, and examples thereof include creams, ointments, lotions, gels, sprays, packs, patches, plasters, liniments, etc. as external preparations. It is also possible to use it as a bath additive or to add it to seawater during thalassotherapy. In addition to the specific extracellular vesicles that are the active ingredient, the anti-aging agent of the present invention may contain pharmaceutically acceptable additives, such as excipients, depending on the dosage form.

[0027] In addition, since there are reports that extracellular vesicles are absorbed by oral ingestion, the anti-aging agent according to the present invention may be an oral preparation, such as a tablet, capsule, granule, fine granule, powder, liquid, syrup, suspension, emulsion, or elixir.

[0028] The proportion of extracellular vesicles, which are the active ingredient in the anti-aging agent of the present invention, may be adjusted appropriately within a range in which the anti-aging effect is effectively exerted. For example, in the case of an external preparation, the proportion of extracellular vesicles is adjusted to 1 x 10 8 pcs / mL or more, 1×10 14 The concentration can be 5 × 10 8 pcs / mL or more, 1×10 9 pcs / mL or more, 1×10 10 pcs / mL or more, 1×10 11 cells / mL or 1 x 10 12 The ratio of extracellular vesicles in the topical preparation is preferably 5 μg / mL or more, and 2.5 × 10 13 The concentration of extracellular vesicles can be adjusted to, for example, 1% by mass or more and 50% by mass or less. The concentration is preferably 25 μg / mL or more, 50 μg / mL or more, 500 μg / mL or more, or 1 g / mL or more. In the case of oral preparations, the concentration of extracellular vesicles can be adjusted to, for example, 1% by mass or more and 50% by mass or less.

[0029] The method for inhibiting aging according to the present invention comprises administering extracellular vesicles derived from seaweed to a patient. Patients to whom the extracellular vesicles should be administered include not only humans but also non-human animals. Examples of non-human animals include pets such as dogs, cats, rabbits, and guinea pigs; livestock such as cows, pigs, chickens, horses, sheep, and goats; animals kept at zoos and safari parks, such as lions, tigers, elephants, and giraffes; and wild animals such as monkeys, wild boars, deer, and bears.

[0030] The dosage of the extracellular vesicles contained in the anti-aging agent according to the present invention may be adjusted appropriately depending on the age, sex, symptoms, etc. of the patient. For example, in the case of an external preparation, 0.1 μg / cm of the extracellular vesicles may be applied to the skin per application. 2 Above, 0.1g / cm 2 The following can be applied. In the case of oral preparations, the dosage per administration can be 0.0001 mg / kg body weight or more and 100.0 mg / kg body weight or less. The number of administrations per day can be, for example, 1 time or more and 5 times or less.

[0031] This application claims the benefit of priority based on Japanese Patent Application No. 2024-24007, filed on February 20, 2024. The entire contents of the specification of Japanese Patent Application No. 2024-24007, filed on February 20, 2024, are incorporated herein by reference.

[0032] The present invention will be explained in more detail below with reference to examples. However, the present invention is not limited to the following examples, and it is of course possible to carry out the invention by making appropriate modifications within the scope of the above and below-described aims, and all such modifications are included in the technical scope of the present invention.

[0033] Example 1: Preparation of Green Streamer EVs Green Streamer collected in Okinawa Prefecture was cut into pieces approximately 5-10 mm long and immersed in seawater at room temperature for 1 day. The seawater was filtered and centrifuged at 8,000 g for 30 minutes, and the resulting supernatant was further centrifuged at 11,000 g for 80 minutes to obtain EVs. The obtained EVs were dispersed in physiological saline and sterilized by filtering through a 0.22 μm filter ("Millex" manufactured by Merck Millipore). The protein concentration in the obtained EV dispersion was measured by the Bradford method using a protein quantification reagent ("Protein Assay Bradford Reagent" manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and was found to be 0.4 μg / mL. In this example, the concentration of the EV dispersion was set to 0.4 μg / mL based on the amount of protein contained in the EVs (Reference: Scientific Committee Report "Extracellular Vesicles Including Exosomes (EVs) * Regarding the publication of the "Report on Therapeutic Formulations Using Immunoglobulins (IgE)," please see: https: / / immunology.w3.kanazawa-u.ac.jp / 000249829.pdf.

[0034] Example 2: Preparation of Southern Aonori EVs A 0.6 μg / mL Southern Aonori EVs dispersion was prepared in the same manner as in Example 1, except that Southern Aonori collected in Tokushima Prefecture was used instead of Green Aonori.

[0035] Example 3: Preparation of Human Egus EVs A 0.4 μg / mL Human Egus EVs dispersion was prepared in the same manner as in Example 1, except that Human Egus collected in Kochi Prefecture was used instead of Enteromorpha spp.

[0036] Test Example 1: Measurement of particle size distribution The particle sizes of the EVs contained in each of the EVs dispersions obtained in Examples 1 to 3 were measured by the scattering intensity distribution method. The results are shown in Table 1.

[0037]

[0038] Test Example 2: Gene Expression Test Normal human epidermal keratinocytes (NHEK) derived from a 55-year-old woman were seeded at a concentration of 30,000 cells / mL into 2 mL of a serum-free liquid medium for epidermal keratinocyte proliferation ("HuMedia-KG2" manufactured by KURABO Corporation) and cultured at 37°C for 1 day. Next, each of the EV dispersions from Examples 1 to 3 was added so that the concentration of each EV in the medium was 0.02 μg / mL, and the medium was cultured at 37°C. For comparison, no EVs were added to the medium, and human EVs ("Purified Exosome, HEK293, Human Embryonic Kidney Cell Line" manufactured by System Biosciences) were added to the medium in the same manner and cultured. Three days after the addition of each EV, a medium sample was collected and mixed with a high-efficiency real-time PCR master mix (THUNDERBIRD (R) Next SYBR (R) Gene expression of antioxidant enzymes superoxide dismutase 2 (SOD2), hyaluronan synthase (HAS-1), filaggrin, and interleukin-1β (IL-1β) was quantified using "qPCR Mix" (Toyobo Co., Ltd.). Total RNA was normalized for each reaction using β-actin complementary DNA as an internal standard. The results for SOD2 are shown in Figure 1, those for hyaluronan synthase in Figure 2, those for filaggrin in Figure 3, and those for IL-1β in Figure 4. In the figures, "**" indicates a significant difference at p<0.01 based on two-way analysis of variance followed by Tukey's test.

[0039] As shown in Figure 1, the EVs of the present invention significantly promoted the expression of the antioxidant enzyme SOD2 gene compared to when no EVs were used or when human EVs were used, suggesting that they can effectively protect the skin from active oxygen.

[0040] As shown in Figure 2, the EVs of the present invention significantly promoted the expression of hyaluronic acid synthase compared to when no EVs were used or when human EVs were used, suggesting that the amount of hyaluronic acid produced, which exists between skin cells and has protective and moisturizing effects on skin tissue, increased.

[0041] As shown in Figure 3, it was revealed that the EVs derived from Enteromorpha spp. and Rhizome nigricans according to the present invention significantly promoted the expression of filaggrin compared to the case where no EVs were used or the case where human EVs were used. Filaggrin is a type of basic protein produced in epidermal granular cells. As the granular cells migrate into stratum corneum cells and further migrate from the lower to the upper layers of the stratum corneum, it is broken down by proteases into amino acids, and these amino acids function as natural moisturizing factors. It is also known that if filaggrin is not produced, abnormalities occur in the stratum corneum, reducing the skin's barrier function and causing dermatitis. Therefore, it is believed that the EVs according to the present invention promote the expression of filaggrin, maintaining the health and function of the skin.

[0042] As shown in Figure 4, it was demonstrated that the EVs according to the present invention do not significantly promote the expression of IL-1β. Since IL-1β is a typical inflammatory cytokine, it was suggested that the EVs according to the present invention do not cause an inflammatory response.

[0043] Test Example 3: Collagen Production Test EVs according to the present invention were added to MEM-α medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (2 mL) at a concentration of 0.02 μg / mL. Human dermal fibroblast senescent cells were then seeded and cultured at 37°C for 24 hours. For comparison, culture was also performed in a medium without EVs. After culture, a medium sample was collected, and the cells were lysed by sonication. The total protein content in the resulting cell lysate was measured by the Bradford method using a protein quantification reagent (Protein Assay Bradford Reagent, Fujifilm Wako Pure Chemical Industries, Ltd.). Depending on the results, PBS was added to dilute the cell lysate to prepare samples with a uniform protein concentration. The amount of collagen per 20 μL of the sample was evaluated using a collagen quantification kit (manufactured by Cosmo Bio Co., Ltd.) according to the manufacturer's protocol. Fluorescence was measured using a fluorescence microplate reader (Infinite M200, TECAN) equipped with a 380 nm / 485 nm filter pair. The results are shown in Figure 5. In Figure 5, "**" indicates a significant difference at p<0.01 in two-way analysis of variance followed by Tukey's test. As shown in the results in Figure 5, collagen production was significantly increased in senescent cells cultured in a culture medium containing the EVs of the present invention compared to control senescent cells cultured without the addition of EVs. Collagen accounts for approximately 70% of the dermis layer inside the skin and is a component that provides firmness and elasticity to the skin, maintaining it fresh and healthy. Therefore, it is believed that the EVs of the present invention have a skin-beautifying effect.

[0044] Test Example 4: Measurement of intercellular electrical resistance of epithelial cells Normal human epidermal keratinocytes (NHEK) derived from a 55-year-old woman were suspended at 100,000 cells / mL in 100 μL of serum-free liquid medium for epidermal keratinocyte proliferation ("HuMedia-KG2" manufactured by KURABO Corporation), seeded in a culture plate ("Culture Insert for 24 wells, 0.4 μm PET" manufactured by Corning), placed in a 24-well plate containing 500 μL of serum-free liquid medium for epidermal keratinocyte proliferation, and cultured at 37° C. for 1 day. Next, each of the EV dispersions from Examples 1 to 3 was added so that the concentration of each EV in the medium was 0.02 μg / mL, and the medium was cultured at 37° C. for 3 days. Thereafter, the intercellular electrical resistance (TEER) was measured using an integrity meter (Millicell ERS-2 Resistance Measurement System, manufactured by Merck). The results are shown in Figure 6. As the results shown in Figure 6 show, the use of EVs derived from Enteromorpha oryzae according to the present invention significantly increased the intercellular electrical resistance (TEER) compared to when no EVs were used. Since the stronger the adhesion between epithelial cells, the greater the electrical resistance between the upper and lower culture media for cultured cells. It is therefore believed that the EVs according to the present invention enhance adhesion between skin keratinocytes, thereby improving the skin's barrier function.

[0045] Comparative Example 1: Preparation of Green Aonori Extract Dried powder of Green Aonori (10 g) was soaked in twice the amount of water at room temperature for 24 hours. The solids were separated by filtration, and the filtrate was freeze-dried to obtain an extract. The amount of protein contained in the obtained extract was measured by the Bradford method as in Example 1. Note that since dried powder of Green Aonori was used as the raw material, it is believed that the obtained extract does not contain EVs.

[0046] Test Example 5: Comparison of EVs and Extract Normal human epidermal keratinocytes (NHEK) derived from a 55-year-old woman were suspended at 100,000 cells / mL in 100 μL of serum-free liquid medium for epidermal keratinocyte proliferation ("HuMedia-KG2" manufactured by KURABO Corporation), and seeded at 500 μL / well in a culture plate ("Culture Insert 24-well 0.4 μm PET" manufactured by Corning) and cultured at 37°C for 1 day. Next, the EVs derived from Enteromorpha spp. of Example 1 and the Enteromorpha spp. extract of Comparative Example 1 were added to the culture plate so that the protein amount was 0.02 μg / well, and the plate was cultured at 37°C for 24 hours. For comparison, human epidermal keratinocytes were cultured in the same manner except that the EVs and extract were not added. A medium sample was collected from each well, and the gene expression of antioxidant enzymes superoxide dismutase 2 (SOD2), hyaluronic acid synthase (HAS-1), and filaggrin was quantified in the same manner as in Test Example 2. Each measurement was performed three times, and the average value was calculated. The results are shown in Figure 7. In Figure 7, "**" indicates a significant difference of p<0.01 according to the Tukey's test following two-way analysis of variance.

[0047] As shown in Figure 7, the EVs of the present invention significantly promoted the expression of the antioxidant enzyme SOD2, hyaluronic acid synthase, and filaggrin genes compared to when no EVs were used or when seaweed extract was used, suggesting that they effectively exert a protective effect on the skin, etc.

Claims

1. An anti-aging agent that contains extracellular vesicles derived from seaweed as an active ingredient and exhibits anti-aging effects.

2. The anti-aging agent according to claim 1, wherein the seaweed is a seaweed of the Ulvales order and / or a seaweed of the Hibuliales order.

3. The anti-aging agent according to claim 2, wherein the seaweed of the Ulvales order is Enteromorpha spp. and / or Enteromorpha spp.

4. The anti-aging agent according to claim 2, wherein the seaweed of the order Hibiscales is Astragalus.

5. The anti-aging agent according to claim 1, wherein the anti-aging effect is a skin protecting effect.

6. The anti-aging agent according to claim 5, wherein the skin protective effect is one or more skin protective effects selected from the group consisting of collagen production promotion, cell adhesion structure formation promotion, antioxidant protein production promotion, hyaluronic acid production promotion, and skin protective factor production promotion.

7. Use of extracellular vesicles derived from seaweed to inhibit aging.

8. The use according to claim 7, wherein the seaweed is of the Ulvales order and / or the Hibuloplast order.

9. The use according to claim 8, wherein the seaweed of the order Ulva is Enteromorpha sieboldii and / or Enteromorpha minamiana.

10. The use according to claim 8, wherein the algae of the order Hypocales is Astragalus.

11. The use according to any one of claims 7 to 10 for inhibiting skin aging.

12. The use described in claim 11, which inhibits skin aging through one or more skin protective effects selected from the group consisting of collagen production promotion, cell adhesion structure formation promotion, antioxidant protein production promotion, hyaluronic acid production promotion, and skin protective factor production promotion.

13. A method for inhibiting aging, comprising the step of administering extracellular vesicles derived from seaweed to a patient.

14. The method according to claim 13, wherein the seaweed is a seaweed of the Ulvales order and / or a seaweed of the Hydrobium order.

15. The method according to claim 14, wherein the seaweed of the order Ulva is Enteromorpha japonica and / or Enteromorpha minamiana.

16. The method according to claim 14, wherein the algae of the order Hypocales is Astragalus.

17. The method according to claim 13, which inhibits skin aging.

18. The method described in claim 17, which inhibits skin aging by one or more skin protective effects selected from the group consisting of collagen production promotion, cell adhesion structure formation promotion, antioxidant protein production promotion, hyaluronic acid production promotion, and skin protective factor production promotion.

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