Anti-aging composition

Abstract

To provide a composition that improves diseases or conditions associated with aging.SOLUTION: Provided is an anti-aging composition, a mitochondrial activity-enhancing composition, or a collagen production-enhancing composition, comprising a culture supernatant and / or extracellular vesicles of microalgae.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an anti-aging composition. The present invention also relates to a composition for promoting mitochondrial activity and collagen production. [Background technology]

[0002] The average life expectancy of Japanese people continues to increase, and both men and women are now over 80 years old. However, while life expectancy is increasing, the decline in quality of life (QOL) due to age-related diseases and conditions, such as decreased cognitive ability and muscle strength, locomotive syndrome, osteoporosis, and cataracts, has become a social issue.

[0003] Various functional ingredients are being explored for treating diseases and conditions associated with aging, not only in the fields of pharmaceuticals but also in the fields of food, beverages, and supplements.

[0004] For example, in Patent Document 1, the mitochondrial activity and collagen production ability of Arthrospira platensis, an alga of the genus Arthrospira, are evaluated, and its use as an anti-aging agent is proposed. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2022-184634 A Summary of the Invention [Problem to be solved by the invention]

[0006] However, the search for materials to solve the social issues related to aging is still insufficient, and the discovery of new functional materials is required. In particular, there are not yet enough reports on the use of extracellular vesicles produced by various organisms in the field of anti-aging.

[0007] Therefore, the present invention aims to evaluate the effect of extracellular vesicles when used in the anti-aging field and to provide a composition for improving diseases and conditions associated with aging. [Means for solving the problem]

[0008] In order to solve the above problems, the inventors conducted intensive research and discovered that culture supernatant and / or extracellular vesicles of microalgae have the function of improving diseases and conditions associated with aging, thereby completing the present invention.

[0009] That is, the present invention provides the compositions described below.

[0010] [1] An anti-aging composition comprising culture supernatant and / or extracellular vesicles of microalgae.

[0011] [2] A composition for promoting mitochondrial activity, comprising culture supernatant and / or extracellular vesicles of microalgae.

[0012] [3] A composition for promoting collagen production, comprising culture supernatant and / or extracellular vesicles of microalgae.

[0013] [4] The composition according to any one of [1] to [3], wherein the microalgae culture supernatant is obtained by a method comprising purifying the culture supernatant by filtration through a filter having a membrane pore size of 0.001 μm to 0.2 μm.

[0014] [5] The extracellular vesicles or extracellular vesicle-containing material described in [4], wherein the molecular weight cut-off of the filter is 10 KDa to 1000 KDa.

[0015] [6] The composition according to any one of [1] to [5], characterized in that it is obtained by heating the culture supernatant of the microalgae at 40°C or higher.

[0016] [7] The composition according to any one of [1] to [6], wherein the particle diameter of the extracellular vesicles is 50 nm to 700 nm.

[0017] [8] The composition according to any one of [1] to [7], wherein the microalgae is microalgae belonging to the genus Pavlova, Euglena, Spirulina, or Chlorella.

[0018] [9] The composition according to any one of [1] to [8], which is a food or drink, a cosmetic, a medicine, or a quasi-drug. Effect of the Invention

[0019] According to the present invention, mitochondrial activity and collagen production can be promoted by using culture supernatant and / or extracellular vesicles of microalgae, and it is possible to provide a composition suitable for improving diseases and conditions associated with aging. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 is a graph showing the results of nanoparticle analysis of an ultrafiltration sample of Pavlova culture supernatant in Test Example 1. [Diagram 2] FIG. 2 is a graph showing the results of nanoparticle analysis of the Spirulina culture supernatant ultrafiltration sample in Test Example 1. [Diagram 3] FIG. 3 is a graph showing the results of nanoparticle analysis of the ultrafiltration sample of Chlorella culture supernatant in Test Example 1. [Figure 4] FIG. 4 is a graph showing the results of nanoparticle analysis of the heated ultrafiltration sample of Pavlova culture supernatant in Test Example 1. [Diagram 5] FIG. 5 is a graph showing the results of the evaluation test of mitochondrial membrane potential in Test Example 2. [Figure 6] FIG. 6 is a graph showing the results of an evaluation test of collagen production using senescent fibroblasts in Test Example 3. [Figure 7]FIG. 7 is a graph showing the results of a test for evaluating skin-related gene expression using fibroblasts in Test Example 4. [Figure 8] FIG. 8 is a graph showing the results of a test for evaluating skin-related gene expression using fibroblasts in Test Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] [Anti-aging composition] In one embodiment, the anti-aging composition of the present invention contains culture supernatant and / or extracellular vesicles of microalgae.

[0022] (Microalgae culture supernatant and / or extracellular vesicles) In this specification, microalgae refers to organisms that perform photosynthesis and generate oxygen, excluding mosses, ferns, and seed plants, and have a cell size of 0.1 μm to 1000 μm in diameter. Labyrinthules, which are protists closely related to microalgae, are also included.

[0023] Microalgae include, for example, prokaryotic cyanobacteria, as well as eukaryotic organisms of the Glaucophyta, Rhodophyta (red algae), Chlorophyta, Cryptophyta (cryptophyta), Haptophyta (haptophyta), Heterokontophyta, Dinophyta (dinoflagellates), Euglenida, and Chlorarachniophyta.

[0024] Examples of microalgae include microalgae belonging to the genus Pavlova, the genus Euglena (Japanese name: Midorimushi, protozoa / green algae), the genus Spirulina (cyanophyte), the genus Chlorella (green algae), the genus Dunaliella (green algae), the genus Nannochloropsis (true eyespot algae), the genus Ligamentum (green algae), the genus Botryococcus (green algae), and the Labyrinthula Thraustochytrid. The above-listed microalgae may be used alone or in combination of two or more.

[0025] Among microalgae, from the viewpoint of significantly achieving the effects of the present invention, microalgae belonging to at least one species selected from the group consisting of the genera Pavlova, Euglena, Spirulina, and Chlorella are preferred, microalgae belonging to at least one species selected from the group consisting of the genera Pavlova, Euglena, and Spirulina are more preferred, microalgae belonging to at least one species selected from the group consisting of the genera Pavlova and Euglena are even more preferred, and microalgae belonging to the genus Pavlova are particularly preferred.

[0026] Pavlova is included in the haptophyte division, and examples of microalgae in the genus Pavlova include P. calceolate, P. granifera, P. gyrans, P. lutheri, P. pinguis, P. salina, etc., with P. granifera and / or P. gyrans being preferred.

[0027] Examples of microalgae of the genus Euglena include E. gracilis, E. longa, E. caudata, E. oxyuris, E. tripteris, E. proxima, E. viridis, E. sociabilis, E. ehrenbergii, E. deses, E. pisciformis, E. spirogyra, E. acus, E. geniculata, E. intermedia, E. mutabilis, E. sanguinea, E. stellata, E. terricola, E. klebsi, E. rubra, and E. cyclopicola, with E. gracilis and / or E. longa being preferred.

[0028] Examples of microalgae of the genus Spirulina include the genus Arthrospira, whose scientific name has been changed from Spirulina, and examples of such microalgae include S. platensis (A. platensis) and S. maxima (A. maxima), with S. platensis being preferred.

[0029] Examples of microalgae belonging to the genus Chlorella include C. vulgaris, C. saccharophila, C. Ellipsoidea, C. pyrenoidosa, C. sorokiniana, and C. lobophora, with C. vulgaris being preferred.

[0030] The microalgae exemplified above are widely distributed in seawater, fresh water such as ponds and swamps, and brackish water, and may be separated from these and used, or any microalgae that has already been isolated may be used.

[0031] The microalgae exemplified above include closely related species and mutants thereof, as long as they provide the effects of the present invention. Mutants include those obtained by genetic methods such as gene recombination, transduction, and transformation.

[0032] In this specification, the culture supernatant of microalgae refers to the supernatant of a culture solution after culturing microalgae. For example, the culture supernatant of a culture solution obtained by culturing microalgae by a known culture method and removing the microalgae by a known separation means such as centrifugation can be mentioned.

[0033] The culture conditions for preparing the culture supernatant of microalgae are not particularly limited as long as the effects of the present invention are achieved, and known methods can be used. Examples of the culture conditions include the following.

[0034] (Culture conditions) Microalgae can be cultured using a culture medium. A carbon source is added to the culture medium as a nutrient source, and the carbon source is an inorganic carbon source (CO 2 , NaHCO 3 , Na 2 CO 3The culture medium is preferably an autotrophic medium that does not contain an organic carbon source such as glucose as a nutrient source, although there is no limitation thereon. For example, a culture medium containing a nitrogen source, a phosphorus source, a mineral, or other nutrient salts, such as Cramer-Myers medium or modified Cramer-Myers medium, may be used.

[0035] Depending on the type of microalgae, when seawater is used, it is possible to use a culture medium for marine microalgae or artificial seawater, or it is also possible to use commercially available products such as IMK medium.

[0036] The pH of the culture medium is not particularly limited as long as the effects of the present invention are achieved, and examples thereof include 2 to 8, 3 to 8, 4 to 8, 5 to 8, 2 to 7.5, 3 to 7.5, 4 to 7.5, and 5 to 7.5.

[0037] The culture temperature is not particularly limited as long as the effects of the present invention are achieved, and examples thereof include 15 to 40°C, 20 to 34°C, and 23 to 28°C.

[0038] The culture period is not particularly limited as long as the effects of the present invention are achieved, and examples include 4 to 30 days, 4 to 20 days, and 5 to 15 days.

[0039] Depending on the type of microalgae, the type of light source, the presence or absence of light and darkness, aerobic or anaerobic conditions, etc. can be appropriately selected.

[0040] In one embodiment, when culturing marine microalgae such as pavlova, a known method can be used as long as the effects of the present invention are achieved. For example, Seawater concentration: 50% seawater Medium concentration: IMK medium 2x concentration Culture solution volume: 800mL Light source: Side: Fluorescent lamp (100~150μmol) Light / dark cycle: 12 hours light and 12 hours dark pH: pH approximately 7.4 at the start of each culture Aeration: Implementation Mechanical agitation: None Cultivation period: Approximately 10 days Culture temperature: 25℃~28℃ The cells can be cultured under conditions that satisfy at least one of the following:

[0041] (Collection of culture supernatant) After culturing the microalgae under the culture conditions exemplified above, the culture supernatant is recovered using a known separation means such as centrifugation, but there is no particular limitation on the method as long as it can appropriately recover the culture supernatant of the microalgae.

[0042] In the present invention, extracellular vesicles of microalgae can also be used. In this specification, extracellular vesicles refer to vesicles secreted from microalgae, which usually have an average diameter of 10 nm to 1 μm. Although not limited thereto, when using extracellular vesicles of microalgae in the composition of the present invention, it is preferable that the composition does not contain the cells of the microalgae themselves.

[0043] The method for separating extracellular vesicles can be a known separation means and is not particularly limited. For example, the method for separating extracellular vesicles can be, but is not limited to, a technique such as centrifugation.

[0044] In one embodiment, the method for producing extracellular vesicles includes, but is not limited to, (1) a step of centrifuging a culture supernatant of a microalgae to obtain a supernatant; and (2) a step of further centrifuging the obtained material, if necessary, to obtain a precipitate. This method can produce microalgae-derived extracellular vesicles exhibiting effective activity.

[0045] In an exemplary embodiment, the centrifugation in steps (1) and (2) may be performed for 30 to 60 minutes at a centrifugal force of, for example, 1,000 to 20,000×g, 1,500 to 20,000×g, 1,500 to 15,000×g, or 1,500 to 10,000×g. In this case, the centrifugation may be performed stepwise by changing the speed or time. For example, the culture supernatant may be separated by centrifugation at a low speed of 1,500 to 2,000×g, and then the culture supernatant may be separated by centrifugation at a high speed of 10,000 to 20,000×g to further remove cells or cell-related debris and residual materials.

[0046] As a method for producing extracellular vesicles, a method of filtering a culture supernatant of microalgae can also be used. The membrane pore size of the filter used for filtering is, for example, preferably 0.001 μm to 0.2 μm, more preferably 0.005 μm to 0.1 μm, and further preferably 0.01 μm to 0.05 μm.

[0047] Here, the filter may be a hollow fiber membrane that filters from the outside to the inside of the fiber. Alternatively, the filter may be a spiral membrane including a filtration membrane and a support membrane, a tubular membrane on a hollow cylinder, or a flat membrane. Among these, it is preferable to use a hollow fiber membrane.

[0048] A hollow fiber membrane is a type of permeable membrane, and refers to a membrane body formed into a thin straw-like capillary. A hollow fiber membrane module is a hollow fiber membrane bundle in which a number of hollow fiber membranes are bundled together and stored in a sealed state in a housing container formed of a vinyl chloride synthetic resin or a metal such as aluminum. In general, the diameter (inner diameter) of each hollow fiber membrane is about 100 μm to several mm, but in order to efficiently circulate liquid, it is preferably 100 μm to 500 μm, and more preferably 100 μm to 300 μm. In addition, the length of each hollow fiber membrane can be adjusted depending on the application, but if it is too long, the pressure for flowing the liquid increases, so that in the form of a hollow fiber membrane bundle, the length excluding the fixing parts at both ends, that is, the so-called effective length, is preferably formed to be 120 mm to 450 mm, more preferably 100 mm to 300 mm, and even more preferably 150 mm to 250 mm. The membrane thickness of the hollow fiber membrane is generally about 10 μm to 100 μm, preferably 20 μm to 60 μm, more preferably 20 μm to 50 μm, and further preferably 30 μm to 50 μm. In the present invention, from the viewpoint of purification efficiency, it is preferable to use a hollow fiber membrane module (the hollow fiber membrane constitutes the hollow fiber membrane module).

[0049] The membrane area of ​​the hollow fiber membrane in the hollow fiber membrane module used in the present invention can be the converted value at the middle part of the thickness, i.e., the average value of the outer surface area and the inner surface area, taking into account the difference in diameter between the inside and outside of the hollow fiber membrane. Since the membrane area of ​​the hollow fiber membrane and the processing speed are proportional to each other, using a hollow fiber membrane module with a larger membrane area will increase the processing speed, shorten the processing time, and reduce damage to extracellular vesicles. On the other hand, since the membrane area of ​​the hollow fiber module and the dead volume (amount remaining in the flow path) during purification are also proportional to each other, using a hollow fiber module with a larger membrane area will increase the loss accordingly. Therefore, it is preferable to select a hollow fiber membrane module with an appropriate membrane area according to the purpose. The membrane area of ​​the hollow fiber membrane in the hollow fiber membrane module used in the present invention is 1 cm 2 ~3,000cm 2For example, if the purpose is to concentrate the solution to a small volume, a 20cm diameter is selected. 2 It is preferable to use a module with a small membrane area of ​​about 115 cm. In addition, when the purpose is concentration, filtration, removal of impurities, etc. of cell culture supernatant, a module with a membrane area of ​​about 115 cm is used according to the amount of processing. 2 Approximately 1,000cm 2 Approximately 1,600cm 2 It is preferable to use a hollow fiber membrane module of about

[0050] The pore size of the hollow fiber membrane used in the present invention (including the hollow fiber membrane provided in the hollow fiber membrane module) can be selected according to the purpose, but for example, when removing impurities larger than the desired extracellular vesicles, a pore size that does not allow only the impurities to pass through is selected. Such a pore size is preferably 0.001 to 0.2 μm, more preferably 0.005 μm to 0.1 μm, and even more preferably 0.01 μm to 0.05 μm. It may also be 0.0001 μm or more, 0.0005 μm or more, 0.001 μm or more, 0.005 μm or more, 0.01 μm or more, 0.015 μm or more, 0.2 μm or less, 0.15 μm or less, 0.1 μm or less, 0.05 μm or less, 0.04 μm or less, or 0.03 μm or less.

[0051] The cutoff molecular weight is preferably 10 kDa to 1,000 kDa, 50 kDa to 1000 kDa, or 100 kDa to 1,000 kDa, and more preferably 100 kDa to 500 kDa. Cutoff membranes of 10 kDa or more, 50 kDa or more, 100 kDa or more, or 150 kDa or more are preferred, and cutoff membranes of 2000 kDa or less, 1500 kDa or less, 1000 kDa or less, 750 Dka or less, or 500 DKa or less are also preferred.

[0052] After culturing microalgae, the culture supernatant after removing the cells can be successively diluted and concentrated to remove unnecessary substances and at the same time exchange the solvent to obtain the required extracellular vesicles.

[0053] The material of the filter, particularly the hollow fiber membrane provided in the hollow fiber membrane module, is not particularly limited as long as it can be used for the purpose of appropriately separating extracellular vesicles derived from cells, and examples thereof include polyethersulfone (PES), modified polyethersulfone (mPES), polyacrylonitrile (PAN), etc. Among these, from the viewpoints of high hydrophilicity and low protein adsorption, polyethersulfone (PES) and modified polyethersulfone (mPES) are preferred, and modified polyethersulfone (mPES) is more preferred.

[0054] The extracellular vesicles of the present invention can also be obtained by heating the culture supernatant of a microalgae under temperature conditions, including but not limited to, 40°C or higher, 50°C or higher, 60°C or higher, 70°C or higher, 80°C or higher, 90°C or higher, 100°C or higher, 200°C or lower, 150°C or lower, or 120°C or lower, for a time period of 30 seconds or more, 1 minute or more, 2 minutes or more, 3 minutes or more, 4 minutes or more, 30 minutes or less, 20 minutes or less, or 10 minutes or less.

[0055] The extracellular vesicles of the present invention can also be obtained by a method comprising heating a culture supernatant of a microalga and concentrating or purifying the heated culture supernatant by filtering.

[0056] The present invention includes extracellular vesicles obtained from the culture supernatant of microalgae, and can also be obtained as a concentrate or purified liquid (extracellular vesicle-containing material) obtained by filtering the culture supernatant. The extracellular vesicle-containing material can be in the form of a concentrate or purified liquid of the culture supernatant itself, or it can be a solid or semi-solid obtained by drying the concentrate or purified liquid.

[0057] The extracellular vesicles of the present invention can optionally be further purified to provide a highly pure population of extracellular vesicles.

[0058] The extracellular vesicles of the present invention may be in the form of a concentrated or purified liquid of the culture supernatant itself, but may also be in the form of a solid or semi-solid obtained by drying the concentrated or purified liquid.

[0059] In the composition of the present invention, the average particle number of the extracellular vesicles of the microalgae is, for example, 1.0 × 10 3 pcs or more, 1.0×10 4 pcs or more, 1.0×10 5 pcs or more, 1.0×10 6 pcs or more, 1.0×10 7 pcs or more, 1.0×10 8 pcs or more, 1.0×10 9 1.0×10 14 pcs or less, 1.0×10 13 pcs or less, 1.0×10 12 pcs or less, 1.0×10 11 pcs or less, 1.0×10 10 The average particle number of extracellular vesicles of microalgae is, for example, 1.0 × 10 4 ~1.0×10 14 pieces, 1.0×10 4 ~1.0×10 13 pieces, 1.0×10 4 ~1.0×10 12 pieces, 1.0×10 4 ~1.0×10 11 pieces, 1.0×10 4 ~1.0×10 10 pieces, 1.0×10 5 ~1.0×10 14 pieces, 1.0×10 5 ~1.0×10 13 pieces, 1.0×10 5 ~1.0×10 12 pieces, 1.0×10 5 ~1.0×10 11 pieces, 1.0×10 5 ~1.0×10 10 pieces, 1.0×10 6 ~1.0×10 14 pieces, 1.0×10 6 ~1.0×10 13 pieces, 1.0×10 6 ~1.0×10 12 pieces, 1.0×10 6 ~1.0×10 11 pieces, 1.0×10 6 ~1.0×10 10 pieces, 1.0×10 7 ~1.0×10 14pieces, 1.0×10 7 ~1.0×10 13 pieces, 1.0×10 7 ~1.0×10 12 pieces, 1.0×10 7 ~1.0×10 11 pieces, 1.0×10 7 ~1.0×10 10 pieces, 1.0×10 8 ~1.0×10 14 pieces, 1.0×10 8 ~1.0×10 13 pieces, 1.0×10 8 ~1.0×10 12 pieces, 1.0×10 8 ~1.0×10 11 pieces, 1.0×10 8 ~1.0×10 10 pieces, 1.0×10 9 ~1.0×10 14 pieces, 1.0×10 9 ~1.0×10 13 pieces, 1.0×10 9 ~1.0×10 12 pieces, 1.0×10 9 ~1.0×10 11 pieces, 1.0×10 9 ~1.0×10 10 The number of extracellular vesicles can be measured using an exosome measurement system (ExoCounter, JVC Kenwood Corporation) that uses a method of sandwich detection specific to surface antigens using antibodies on a disk and nanobeads.

[0060] The average particle size of the extracellular vesicles of microalgae is not particularly limited as long as the effects of the present invention are achieved, and examples include 10 nm or more, 20 nm or more, 30 nm or more, 40 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, and the like; and 700 nm or less, 600 nm or less, 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, and the like. The average particle size of the extracellular vesicles of microalgae is, for example, 10 to 700 nm, 10 to 600 nm, 10 to 500 nm, 10 to 400 nm, 10 to 300 nm, 10 to 250 nm, 30 to 700 nm, 30 to 600 nm, 30 to 500 nm, 30 to 400 nm, 30 to 300 nm, 30 to 250 nm, 50 to 700 nm, 50 to 600 nm, 50 to Examples of the thickness include 500 nm, 50 to 400 nm, 50 to 300 nm, 50 to 250 nm, 70 to 700 nm, 70 to 600 nm, 70 to 500 nm, 70 to 400 nm, 70 to 300 nm, 70 to 250 nm, 100 to 700 nm, 100 to 600 nm, 100 to 500 nm, 100 to 400 nm, 100 to 300 nm, and 100 to 250 nm.

[0061] The cumulative 10% value (D10) of the particle diameter of the Pavlova-derived extracellular vesicles is not particularly limited as long as the effects of the present invention are exhibited, and examples thereof include 80 to 140 nm, 90 to 130 nm, and 100 to 120 nm. In addition, the cumulative 50% value (D50) of the particle diameter is, for example, 120 to 210 nm, 130 to 200 nm, and 140 to 190 nm. In addition, the cumulative 90% value (D90) of the particle diameter is, for example, 200 to 320 nm, 210 to 310 nm, and 220 to 300 nm. In this specification, the cumulative 10% value (D10) of the particle diameter of the extracellular vesicles means a diameter below which 10% of the whole extracellular vesicles are. The cumulative 50% value (D50) of the particle diameter of the extracellular vesicles means a diameter below which 50% of the whole extracellular vesicles are, and is synonymous with the median diameter. The cumulative 90% particle diameter (D90) of extracellular vesicles means the diameter below which 90% of all extracellular vesicles fall.

[0062] The cumulative 10% value (D10) of the particle diameter of the Spirulina-derived extracellular vesicles is not particularly limited as long as the effects of the present invention are achieved, and examples thereof include 50 to 110 nm, 60 to 100 nm, 70 to 90 nm, etc. Furthermore, the cumulative 50% value (D50) of the particle diameter is, for example, 100 to 160 nm, 110 to 150 nm, 120 to 140 nm, etc. Furthermore, the cumulative 90% value (D90) of the particle diameter is, for example, 190 to 250 nm, 200 to 240 nm, 210 to 230 nm, etc.

[0063] The particle diameter of the Chlorella-derived extracellular vesicles is not particularly limited as long as it exhibits the effects of the present invention, and may be, for example, 90 to 150 nm, 100 to 140 nm, 110 to 130 nm, etc. The particle diameter is, for example, 180 to 240 nm, 190 to 230 nm, 200 to 220 nm, etc. The particle diameter is, for example, 320 to 380 nm, 330 to 370 nm, 340 to 360 nm, etc. The particle diameter is, for example, 320 to 380 nm, 330 to 370 nm, 340 to 360 nm, etc.

[0064] The content of the culture supernatant of the microalgae is appropriately adjusted depending on the type and amount of other components, the formulation, etc., and is not limited, and may be, for example, 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.5% by mass or more, 1% by mass or more, 10% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, etc. In addition, the content of the culture supernatant of the microalgae may be, for example, 100% by mass or less, and may be 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 10% by mass or less, 1% by mass or less, etc., based on the total amount of the composition. The content of the microalgae culture supernatant may be, for example, 0.01 to 100% by mass, 0.01 to 90% by mass, 0.01 to 50% by mass, 0.01 to 30% by mass, 0.01 to 10% by mass, 0.01 to 1% by mass, 0.1 to 100% by mass, 0.1 to 90% by mass, 0.1 to 50% by mass, 0.1 to 30% by mass, 0.1 to 10% by mass, 0.1 to 1% by mass, 0.5 to 100% by mass, 0.5 to 90% by mass, 0.5 to 50% by mass, 0.5 to 30% by mass, 0.5 to 10% by mass, 0.5 to 1% by mass, 1 to 100% by mass, 1 to 90% by mass, 1 to 50% by mass, 1 to 30% by mass, 1 to 10% by mass, and the like, relative to the total amount of the composition.

[0065] The content of microalgae extracellular vesicles is appropriately adjusted depending on the type and amount of other components, dosage form, etc., and is not limited, but can be, for example, 0.001% by mass or more relative to the total amount of the composition, and may be 0.005% by mass or more, 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 1% by mass or more, 5% by mass or more, 10% by mass or more, etc. Furthermore, the content of microalgae extracellular vesicles can be, for example, 50% by mass or less relative to the total amount of the composition, such as 40% by mass or less, 30% by mass or less, 20% by mass or less, 15% by mass or less ... 0.001~50 mass%, 0.001~40 mass%, 0.001~30 mass%, 0.001~20 mass%, 0.001~15 mass%, 0.01~50 mass%, 0.01~40 mass%, 0.01~30 mass%, 0.01~20 mass%, 0.01~15 mass%, 0.1~50 mass%, 0 Examples include 1-40% by mass, 0.1-30% by mass, 0.1-20% by mass, 0.1-15% by mass, 1-50% by mass, 1-40% by mass, 1-30% by mass, 1-20% by mass, 1-15% by mass, 5-50% by mass, 5-40% by mass, 5-30% by mass, 5-20% by mass, 5-15% by mass, etc.

[0066] The content of microalgal extracellular vesicles is appropriately adjusted depending on the type and amount of other components, dosage form, etc., and is not limited, and can be, for example, 0.01 μg / mL or more, 0.1 μg / mL or more, 1 μg / mL or more, 10 μg / mL or more, or 100 μg / mL or more in terms of protein amount relative to the total amount of the composition. Examples of the content include 100,000 μg / mL or less, 10,000 μg / mL or less, 1000 μg / mL, 100 μg / mL or less, and 10 μg / mL or less.

[0067] [Application] In one embodiment, the composition of the present invention is used for anti-aging purposes. In this specification, anti-aging refers to improving diseases and conditions associated with aging. Diseases associated with aging include, but are not limited to, myocardial infarction, cerebral infarction, osteoporosis, dementia, mild cognitive impairment, rheumatoid arthritis, cataracts, etc. In addition, conditions associated with aging include, but are not limited to, skin conditions such as spots, wrinkles, and dullness, decreased motor function due to aging, decreased energy metabolism due to aging, decreased vascular flexibility due to aging, decreased vascular dilation due to aging, decreased bone density due to aging, decreased cognitive function (memory, judgment, attention, spatial recognition, etc.) due to aging, decreased muscle mass due to aging, decreased macular pigment density of the eye due to aging, blurred or hazy vision, dry skin due to aging, increased gray hair, decreased hair volume, etc.

[0068] In the examples (test examples) described later, it has been confirmed that the culture supernatant and / or extracellular vesicles of microalgae promote collagen production in aged fibroblasts. Fibroblasts and collagen are present in the dermis, with type I collagen accounting for 80% of the total. In addition to type I collagen, the existence of types III, V, XII and XIV collagen is known. A decrease in type I collagen is considered to be one of the causes of wrinkles and sagging. It is considered important to keep the production and decomposition cycle of collagen, which constitutes the extracellular matrix, normal in order to keep the skin in normal condition. Therefore, by using the composition of the present invention, it is possible to promote collagen production, maintain the normality of the skin, and prevent or improve wrinkles and / or sagging.

[0069] In addition, about 90% of bone matrix is ​​made up of collagen. Osteoblasts promote the formation of collagen networks outside the cells by producing and releasing collagen fibrils, thereby promoting bone formation. Therefore, by using the composition of the present invention, it is possible to promote collagen production in bones and promote bone formation.

[0070] In addition, in the Examples (Test Examples) described later, it has been confirmed that the culture supernatant and / or extracellular vesicles of microalgae activate mitochondria. Aging can be caused by a decrease in the number and / or activity of mitochondria that produce energy in cells with age.

[0071] The target mitochondria are not particularly limited, and examples thereof include skin mitochondria and muscle mitochondria. Improvement of skin mitochondrial function is involved in, for example, suppression or improvement of skin wrinkle formation. Improvement of muscle mitochondrial function is involved in, for example, improvement of motor function decline and suppression of muscle weakness. In addition to the above, activation of mitochondria is known to be involved in improvement of metabolic function, extension of life span, recovery of nervous system abnormality, improvement of cognitive function, etc. Therefore, by using the composition of the present invention, it is possible to promote mitochondrial activity and / or energy production and improve the above functions.

[0072] Furthermore, in the Examples (Test Examples) described later, it has been confirmed that the culture supernatant and / or extracellular vesicles of microalgae enhance the gene expression of HSP47 (Heat shock protein 47). HSP47 is known to respond to external stress and improve and restore the condition of the skin. HSP47 is known to respond to external stress and improve and restore the condition of the skin. Furthermore, HSP47 is important for maintaining the quality of collagen. In addition, since HSP47 decreases with aging, enhancing the gene expression of HSP47 and increasing HSP47 makes it possible to produce collagen with the correct structure and maintain the elasticity of the skin.

[0073] The composition of the present invention can be used by adding to or mixing with medicines, quasi-drugs, cosmetics, foods, beverages, feed, or pet foods, or can be used as it is as medicines, quasi-drugs, cosmetics, foods, beverages, feed, or pet foods.

[0074] When the composition of the present invention is used by adding to or mixing with so-called external preparations such as medicines, quasi-drugs, and cosmetics, the composition of the present invention can be used as a medicine, quasi-drug, or cosmetic that explicitly or implicitly has efficacy, effects, or functions such as anti-aging, skin elasticity, skin firmness, improvement of wrinkles, texture, moisture, dryness, roughness, and the like.

[0075] When the above functionality is not clearly stated, it is also possible to use the composition of the present invention by indicating, for example, that it is suitable for those seeking anti-aging, those seeking skin elasticity, those seeking skin firmness, those who are concerned about wrinkles, those who are concerned about fine lines, those who want to smooth skin texture, those seeking moisturized skin, those who are concerned about dry skin, those who are concerned about rough skin, those who are concerned about pores, those who are concerned about skin fluctuations, those who are concerned about pores, etc., or a similar indication.

[0076] When used in so-called external preparations such as medicines, quasi-drugs, and cosmetics, the composition of the present invention is preferably prepared as an external preparation for skin beautification, an external preparation for anti-aging, etc. Among these, particularly preferred applications of the composition of the present invention include skin care cosmetics (e.g., lotions, creams, facial creams, facial lotions, milky lotions, packs, liquid facial cleansers, soaps, beauty essences), makeup cosmetics (e.g., eyeliners, eyebrows), and scalp and hair cosmetics (e.g., scalp lotions, scalp creams, shampoos, hair conditioners, hair treatments, hair essences, hair mist, and hair tonics), and most preferably, may be lotions, creams, facial creams, facial lotions, milky lotions, packs, facial cleansers, soaps, and beauty essences, but are not limited thereto.

[0077] Thus, when used in so-called external preparations such as medicines, quasi-drugs, and cosmetics, the dosage form of the composition of the present invention is not particularly limited, and examples thereof include liquids, suspensions, emulsions, creams, gels, liniments, lotions, ointments, and aerosols, etc. Among these, liquids, suspensions, emulsions, lotions, and aerosols are preferred, and liquids, lotions, and aerosols are more preferred.

[0078] When used in so-called external preparations such as medicines, quasi-drugs, and cosmetics, the daily dose of microalgal extracellular vesicles for adults can be appropriately determined depending on the individual's condition, body weight, sex, age, activity of the material, ingestion or administration route, ingestion or administration schedule, formulation form, or other factors. Examples of the daily dose of microalgal extracellular vesicles for adults include 1 μg or more, 5 μg or more, 10 μg or more, 15 μg or more, 20 μg or more, 30 μg or more, 40 μg or more, 50 μg or more, 70 μg or more, 100 μg or more, 150 μg or more, and 200 μg or more. In addition, the daily dosage of extracellular vesicles of microalgae for an adult can be, for example, 5 mg or less, 3 mg or less, 1 mg or less, 900 μg or less, 800 μg or less, 700 μg or less, 600 μg or less, 500 μg or less, 400 μg or less, 300 μg or less, 200 μg or less, or 100 μg or less. In addition, the daily dosage of microalgae extracellular vesicles for adults is, for example, 1 to 1000 μg, 1 to 900 μg, 1 to 800 μg, 1 to 700 μg, 1 to 600 μg, 1 to 500 μg, 1 to 400 μg, 1 to 300 μg, 1 to 200 μg, 1 to 100 μg, 5 to 1000 μg, 5 to 900 μg, 5 to 800 μg, 5 to 700 μg, 5 to 600 μg, 5 to 500 μg, 5 to 400 μg, 5 to 300 μg, 5 to 200 μg, 5 to 100 μg. g, 10 to 1000 μg, 10 to 900 μg, 10 to 800 μg, 10 to 700 μg, 10 to 600 μg, 10 to 500 μg, 10 to 400 μg, 10 to 300 μg, 10 to 200 μg, 10 to 100 μg, 20 to 1000 μg, 20 to 900 μg, 20 to 800 μg, 20 to 700 μg, 20 to 600 μg, 20 to 500 μg, 20 to 400 μg, 20 to 300 μg, 20 to 200 μg, and 20 to 100 μg. The dosage of microalgal extracellular vesicles varies depending on various factors such as the condition and age of the subject, but based on an adult, generally, 1 μg / kg to 200 mg / kg of the composition, and in another aspect, 50 μg / kg to 50 mg / kg, may be administered 1 to 3 times a day, and the dosage is not intended to limit the scope of the present specification in any way.

[0079] When used in so-called external preparations such as medicines, quasi-drugs, and cosmetics, the daily dose of microalgal extracellular vesicles for adults can be appropriately determined depending on the individual's condition, body weight, sex, age, activity of the material, ingestion or administration route, ingestion or administration schedule, formulation form, or other factors. The daily dose of microalgal extracellular vesicles for adults can be, for example, 1 μg or more, 5 μg or more, 10 μg or more, 15 μg or more, 20 μg or more, 30 μg or more, 40 μg or more, 50 μg or more, 70 μg or more, 100 μg or more, 150 μg or more, or 200 μg or more in terms of protein amount. In addition, the daily dosage of extracellular vesicles of microalgae for an adult can be, for example, 5 mg or less, 3 mg or less, 1 mg or less, 900 μg or less, 800 μg or less, 700 μg or less, 600 μg or less, 500 μg or less, 400 μg or less, 300 μg or less, 200 μg or less, or 100 μg or less. In addition, the daily dosage of microalgae extracellular vesicles for adults is, for example, 1 to 1000 μg, 1 to 900 μg, 1 to 800 μg, 1 to 700 μg, 1 to 600 μg, 1 to 500 μg, 1 to 400 μg, 1 to 300 μg, 1 to 200 μg, 1 to 100 μg, 5 to 1000 μg, 5 to 900 μg, 5 to 800 μg, 5 to 700 μg, 5 to 600 μg, 5 to 500 μg, 5 to 400 μg, 5 to 300 μg, 5 to 200 μg, 5 to 100 μg. g, 10 to 1000 μg, 10 to 900 μg, 10 to 800 μg, 10 to 700 μg, 10 to 600 μg, 10 to 500 μg, 10 to 400 μg, 10 to 300 μg, 10 to 200 μg, 10 to 100 μg, 20 to 1000 μg, 20 to 900 μg, 20 to 800 μg, 20 to 700 μg, 20 to 600 μg, 20 to 500 μg, 20 to 400 μg, 20 to 300 μg, 20 to 200 μg, and 20 to 100 μg. The dosage of microalgal extracellular vesicles varies depending on various factors such as the condition and age of the subject, but based on an adult, the composition may be administered at 1 μg / kg to 200 mg / kg, or in another aspect, 50 μg / kg to 50 mg / kg, 1 to 3 times a day, and the dosage is not intended to limit the scope of the present specification in any way.

[0080] In another embodiment, the daily dose of microalgal extracellular vesicles for an adult is, for example, 1 × 10 7 pcs or more, 1×10 8 pcs or more, 1×10 9 The daily dose of microalgae extracellular vesicles for adults is, for example, 1 × 10 14 pcs or less, 1×10 13 pcs or less, 1×10 12 pcs or less, 1×10 11 pcs or less, 1×10 10 The daily dose of microalgae extracellular vesicles for adults is, for example, 1 × 10 7 ~1×10 14 pieces, 1×10 7 ~1×10 13 pieces, 1×10 7 ~1×10 12 pieces, 1×10 7 ~1×10 11 pieces, 1×10 8 ~1×10 14 pieces, 1×10 8 ~1×10 13 pieces, 1×10 8 ~1×10 12 pieces, 1×10 8 ~1×10 11 pieces, 1×10 9 ~1×10 14 pieces, 1×10 9 ~1×10 13 pieces, 1×10 9 ~1×10 12 pieces, 1×10 9 ~1×10 11 Examples include:

[0081] The daily dose of the microalgae culture supernatant for an adult can be appropriately determined depending on the individual's condition, body weight, sex, age, activity of the material, administration schedule, formulation form, or other factors. The daily dose of the microalgae culture supernatant for an adult can be, for example, the amount of microalgae extracellular vesicles contained in the above-mentioned intake or administration amount.

[0082] The composition of the present invention can be ingested or administered once or several times a day, typically 1 to 6 times a day, 1 to 3 times a day, 1 to 2 times a day, or at any period and interval, but once a day is preferred.

[0083] When the composition of the present invention is added to or mixed with food, beverage, feed, or pet food, the composition of the present invention can be used as a food or beverage that expressly or implies that it has the functions of anti-aging, skin elasticity, skin firmness, wrinkles, texture, moisture, dryness, improvement of roughness, etc., i.e., health food, functional food, food for sick people, and food for specified health uses. In addition, even if the above-mentioned functions are not expressly or implied, it can be used as a so-called doctor's supplement recommended or presented by a doctor in the internal medicine, orthopedic surgery, veterinary hospital, etc. of a hospital and / or clinic.

[0084] When the above functionality is not clearly stated, it is also possible to use the composition of the present invention by indicating, for example, that it is suitable for those seeking anti-aging, those seeking skin elasticity, those seeking skin firmness, those who are concerned about wrinkles, those who are concerned about fine lines, those who want to smooth skin texture, those seeking moisturized skin, those who are concerned about dry skin, those who are concerned about rough skin, those who are concerned about pores, those who are concerned about skin fluctuations, those who are concerned about pores, etc., or a similar indication.

[0085] Health foods, functional foods, foods for the sick, and foods for specified health uses can be used in various formulations such as solid preparations (tablets, orally disintegrating tablets, granules, fine granules, powders, capsules, chewable tablets, candies, etc.), liquid preparations (syrups, suspensions), and liquid foods. Foods in the form of formulations can be produced in the same manner as known pharmaceutical preparations, and can be produced by mixing an active ingredient with a carrier acceptable for food, such as a suitable excipient, and then using conventional means. The formulation form is not limited, but is preferably an orally disintegrating tablet, a chewable tablet, a candy, a granule, a powder, or a liquid, from the viewpoint of significantly exhibiting the effects of the present invention.

[0086] The composition of the present invention can also be manufactured as liquid beverages such as soups, juices, fruit juice drinks, milk, dairy drinks, whey drinks, lactic acid bacteria drinks, tea drinks, alcoholic drinks, coffee drinks, carbonated drinks, soft drinks, water drinks, cocoa drinks, jelly drinks, sports drinks, and diet drinks; semi-solid foods such as pudding and yogurt; noodles such as pasta, ramen, udon, and soba; confectioneries; and spreads.

[0087] The composition of the present invention can be applied in the form of oral administration, internal administration, etc. When used as a pharmaceutical composition, it may be used therapeutically or non-therapeutically.

[0088] The daily oral intake or administration amount of microalgal extracellular vesicles for an adult can be appropriately determined depending on the individual's condition, body weight, sex, age, activity of the material, intake or administration route, intake or administration schedule, formulation form, or other factors. Examples of the daily oral intake or administration amount of microalgal extracellular vesicles for an adult include 1 μg or more, 5 μg or more, 10 μg or more, 15 μg or more, 20 μg or more, 30 μg or more, 40 μg or more, 50 μg or more, 70 μg or more, 100 μg or more, 150 μg or more, and 200 μg or more. In addition, the daily oral intake or administration amount of extracellular vesicles of microalgae for an adult can be, for example, 5 mg or less, 3 mg or less, 1 mg or less, 900 μg or less, 800 μg or less, 700 μg or less, 600 μg or less, 500 μg or less, 400 μg or less, 300 μg or less, 200 μg or less, 100 μg or less, etc. The daily oral intake or administration amount of microalgal extracellular vesicles for an adult is, for example, 1 to 1000 μg, 1 to 900 μg, 1 to 800 μg, 1 to 700 μg, 1 to 600 μg, 1 to 500 μg, 1 to 400 μg, 1 to 300 μg, 1 to 200 μg, 1 to 100 μg, 5 to 1000 μg, 5 to 900 μg, 5 to 800 μg, 5 to 700 μg, 5 to 600 μg, 5 to 500 μg, 5 to 400 μg, 5 to 300 μg, 5 to 200 μg, 5 to 5 ... ~100μg, 10~1000μg, 10~900μg, 10~800μg, 10~700μg, 10~600μg, 10~500μg, 10~400μg, 10~300μg, 10~200μg, 10~100μg, 20~1000μg, 20~900μg, 20~800μg, 20~700μg, 20~600μg, 20~500μg, 20~400μg, 20~300μg, 20~200μg, 20~100μg, etc.

[0089] In another embodiment, the daily oral intake or administration amount of microalgal extracellular vesicles for an adult is, for example, 1 × 10 7 pcs or more, 1×10 8 pcs or more, 1×10 9 The daily oral intake or administration amount of microalgae extracellular vesicles for an adult is, for example, 1×10 14pcs or less, 1×10 13 pcs or less, 1×10 12 pcs or less, 1×10 11 pcs or less, 1×10 10 The daily oral intake or administration amount of microalgae extracellular vesicles for an adult is, for example, 1 × 10 7 ~1×10 14 pieces, 1×10 7 ~1×10 13 pieces, 1×10 7 ~1×10 12 pieces, 1×10 7 ~1×10 11 pieces, 1×10 8 ~1×10 14 pieces, 1×10 8 ~1×10 13 pieces, 1×10 8 ~1×10 12 pieces, 1×10 8 ~1×10 11 pieces, 1×10 9 ~1×10 14 pieces, 1×10 9 ~1×10 13 pieces, 1×10 9 ~1×10 12 pieces, 1×10 9 ~1×10 11 Examples include:

[0090] The daily oral intake or administration amount of the microalgal culture supernatant for an adult can be appropriately determined depending on the individual's condition, body weight, sex, age, activity of the material, intake or administration route, intake or administration schedule, formulation form, or other factors. The daily oral intake or administration amount of the microalgal culture supernatant for an adult can be, for example, the amount of microalgal extracellular vesicles contained in the above-mentioned intake or administration amount.

[0091] The daily oral intake or dosage for an adult may be divided into 1 to 6 capsules, 1 to 4 capsules, 1 to 3 capsules, or 1 to 2 capsules depending on the dosage form.

[0092] The composition of the present invention can be ingested or administered once or several times a day, typically 1 to 6 times a day, 1 to 3 times a day, 1 to 2 times a day, or at any period and interval, but once a day is preferred.

[0093] When the present invention is used as feed, pet food, or the like, the target organism is not particularly limited, but is preferably a mammal, reptile, amphibian, bird, or fish, and more preferably a mammal other than humans, such as a platypus, echidna, opossum, quoll, kangaroo, aardvark, rock badger, elephant, armadillo, sloth, anteater, tree shrew, flying lemur, chimpanzee, rabbit, degu, dormouse, squirrel, raccoon, mouse, hedgehog, chinchilla, ferret, camel, wild boar, giraffe, deer, cow, goat, hippopotamus, whale, dolphin, horse, rhinoceros, tapir, bat, monkey, tiger, wolf, weasel, bear, seal, dog, cat, parakeet, parrot, finch, owl, owl, etc., and more preferably a dog or cat.

[0094] When the present invention is used as feed, pet food, or the like, it may be given to the pet in divided portions several times a day by adding it to the staple food, or it may be given to the pet as a snack at any time. EXAMPLES

[0095] Next, the present invention will be specifically described with reference to examples, but the present invention is not limited to the following examples.

[0096] [Test Example 1. Preparation and Evaluation of Microalgae Culture Supernatant and / or Extracellular Vesicles 1] (Preparation Example 1) Pavlova sp., Spirulina (Arthrospira platensis), or Chlorella (Chlorella vulgaris) was cultured in a commercially available algae medium by a conventional method, and a culture supernatant was obtained. 500 mL of each culture supernatant was used and concentrated to a volume of 5 mL or less using a 200K ultrafiltration membrane (Advantec). Then, concentration washing was performed three times using 100 mL of PBS, and finally, the sample was collected with 50 mL of PBS. Then, each concentrated sample was sterilized using a 0.22 μm filter, and the particle number, etc. were measured using NanoSight.

[0097] Each of the above samples was appropriately diluted with PBS(-), and a number distribution graph (horizontal axis: particle diameter, vertical axis: particle number concentration) was created using a nanoparticle analysis system NanoSight LM10 (Malvern Panalytical) in accordance with the attached software.

[0098] The following samples were obtained as described above. These samples were diluted 50-fold and added to cells in the following test examples. Pavlova culture supernatant ultrafiltration sample (protein concentration 11.9 μg / mL) Spirulina culture supernatant ultrafiltration sample (protein concentration 29.6 μg / mL) · Chlorella culture supernatant ultrafiltration sample (protein concentration 10.4 μg / mL)

[0099] The results of the Pavlova culture supernatant ultrafiltered sample are shown in Figure 1, the results of the Spirulina culture supernatant ultrafiltered sample are shown in Figure 2, and the results of the Chlorella culture supernatant ultrafiltered sample are shown in Figure 3.

[0100] As shown in Figure 1, in the ultrafiltrated sample of Pavlova culture supernatant, Pavlova-derived extracellular vesicles had a particle number of 3.32 × 10 11 The particle size was 162.8 nm, the cumulative 10% value of particle size (D10) was 105.4 nm, the cumulative 50% value of particle size (D50) was 151.9 nm, and the cumulative 90% value of particle size (D90) was 232.3 nm.

[0101] As shown in Figure 2, in the Spirulina culture supernatant ultrafiltration sample, Spirulina-derived extracellular vesicles had a particle number of 2.88 × 10 11The particle size was 138.7 nm, the cumulative 10% value of particle size (D10) was 72.2 nm, the cumulative 50% value of particle size (D50) was 125.9 nm, and the cumulative 90% value of particle size (D90) was 217.8 nm.

[0102] As shown in Figure 3, in the ultrafiltrated sample of Chlorella culture supernatant, Chlorella-derived extracellular vesicles had a particle number of 8.18 × 10 9 The particle size was 227.7 nm, the cumulative 10% value of particle size (D10) was 121.4 nm, the cumulative 50% value of particle size (D50) was 215.8 nm, and the cumulative 90% value of particle size (D90) was 348.5 nm.

[0103] (Preparation Example 2) Pavlova sp. and Spirulina (Arthrospira platensis) were cultured in a commercially available algae medium by a conventional method, and culture supernatants were obtained. The culture supernatant was heated at 105°C for 4 minutes, and after heating, 500mL was used and concentrated to a volume of 5mL or less using a 200K ultrafiltration membrane (manufactured by Advantec). Then, concentration washing was performed three times using 100mL of PBS, and finally, the sample was collected with 50mL of PBS. Then, each concentrated sample was sterilized using a 0.22μm filter, and the particle number, etc. were measured using NanoSight.

[0104] Each of the above samples was appropriately diluted with PBS(-), and a number distribution graph (horizontal axis: particle diameter, vertical axis: particle number concentration) was created using a nanoparticle analysis system NanoSight LM10 (Malvern Panalytical) in accordance with the attached software.

[0105] The following samples were obtained as described above. The samples were diluted 50-fold and added to cells in the following test examples. Pavlova culture supernatant heated ultrafiltration sample (protein concentration 201.5 μg / mL)

[0106] The results for the heated ultrafiltration sample of Pavlova culture supernatant (referred to as Pavlova EVs2 in the figure, and the same applies below) are shown in FIG.

[0107] As shown in Figure 4, in the sample obtained by ultrafiltration and heat treatment of the Pavlova culture supernatant, the Pavlova-derived extracellular vesicles had a particle number of 8.34 × 10 11 The particle size was 191.9 nm, the cumulative 10% value of particle size (D10) was 107.6 nm, the cumulative 50% value of particle size (D50) was 180.5 nm, and the cumulative 90% value of particle size (D90) was 288.7 nm.

[0108] [Test Example 2. Evaluation test of mitochondrial membrane potential] Mitochondria have a double membrane structure consisting of an outer membrane and an inner membrane, and produce energy (ATP synthesis) by utilizing the potential difference between the outer and inner membranes. Healthy mitochondria have a high membrane potential and produce energy. On the other hand, mitochondria with impaired function have a low membrane potential and produce less energy.

[0109] In the following, the effects of microalgal culture supernatant and / or extracellular vesicles on mitochondrial membrane potential were evaluated. Human skin fibroblasts (NB1RGB cells, RIKEN BioResource Research Center, same as in the following test examples) were cultured for 70 to 89 days in a medium (same as in the following test examples) containing 10% FBS, 100 μg / mL penicillin, and 100 μg / mL streptomycin in MEMα medium to prepare senescent cells. Each ultrafiltration sample obtained in the above test example 1 was added, and the human skin fibroblast senescent cells were further cultured at 37°C for 72 hours.

[0110] Mitochondrial activity was then measured by JC-1 (Dojindo Molecular Technologies) staining using a mitochondrial membrane potential detection kit ("JC-1 MitoMP Detection kit" Dojindo) according to the manufacturer's protocol. Fluorescence was measured with a fluorescence microplate reader ("Infinite M200" TECAN) using a 535 nm / 590 nm filter pair.

[0111] When mitochondrial activity is high, the membrane potential is maintained, the small molecule fluorescent dye JC-1 aggregates, and longer wavelength fluorescence is emitted. Therefore, mitochondrial activity can be measured as the longer wavelength measured at 535nm / 590nm.

[0112] The results are shown in Figure 5. "**" indicates a significant difference compared to the control group (0 µg / mL) at p<0.01 by two-way analysis of variance followed by Tukey's test.

[0113] As shown in Figure 5, it was confirmed that the culture supernatant and / or extracellular vesicles of microalgae significantly increased JC-1 activity, which is an indicator of mitochondrial membrane potential.

[0114] [Test Example 3. Evaluation test of collagen production in aged fibroblasts] Next, the effect of microalgal culture supernatant and / or extracellular vesicles on collagen production in aged fibroblasts was evaluated. Using a medium containing each sample obtained in the above Test Example 1 (a sample obtained by heating and ultrafiltrating the culture supernatant of Pavlova, and a sample obtained by ultrafiltrating the culture supernatant of Chlorella), human dermal fibroblast senescent cells were further cultured at 37°C for 72 hours to obtain samples. A cell lysate was prepared from the obtained sample, and the amount of collagen in the cell layer per 25μL of the cell lysate containing the same protein concentration 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" manufactured by TECAN Co., Ltd.) equipped with a 380nm / 485nm filter pair.

[0115] The results are shown in Figure 6. "**" indicates a significant difference compared to the control group at p<0.01 according to two-way analysis of variance followed by Tukey's test.

[0116] As shown in FIG. 6, it was confirmed that the culture supernatant and / or extracellular vesicles of microalgae significantly increased collagen production in senescent fibroblasts.

[0117] [Test Example 4. Evaluation of skin-related gene expression in human fibroblasts] Each sample obtained in the above Test Example 1 (a sample obtained by heat-treating and ultrafiltrating the culture supernatant of Pavlova, and a sample obtained by ultrafiltrating the culture supernatant of Spirulina) was diluted 50-fold and allowed to act on human skin fibroblasts. Then, the samples were incubated at 37°C and 5% CO 2 The cells were cultured in an incubator for 48 hours.

[0118] After treating each sample for 48 hours with human fibroblasts, the medium was removed and the cells were washed with PBS at 1 mL / well. QIAzol (QIAGEN) was added at 350 mL / well. The cells were collected into 1.5 mL tubes using a cell scraper and stored at -80°C. RNA was then extracted using the miRNeasy mini Kit (QIAGEN).

[0119] The RNA concentration of the RNA samples was measured using a NanoDrop ND-1000 (Thermo Fisher Scientific), and then the samples were converted to cDNA by reverse transcription. After the reverse transcription, the cDNA sample was diluted 5-fold with RNase-free water and stored at -30°C.

[0120] Gene expression analysis of COL1A1 (collagen type I alpha 1 chain) and HSP47 (Heat shock protein 47) was performed by real-time PCR using Syber green with StepOne PLUS (Life Technologies). The control was a sample without any addition of each sample (a sample with only PBS added), and the internal standard was β-actin (also referred to as ACTB). Data analysis was performed using the ΔΔCT method. The gene expression results of COL1A1 are shown in Figure 7, and the gene expression results of HSP47 are shown in Figure 8.

[0121] 7 to 8, it was confirmed that the act of the material containing extracellular vesicles and / or extracellular vesicles of microalgae on human fibroblasts enhanced the expression of COL1A1 gene and HSP47 gene in human fibroblasts. Therefore, the material containing extracellular vesicles and / or extracellular vesicles of microalgae can be suitably used for maintaining or increasing fibrous structural proteins, and can be suitably used for anti-aging of the skin.

Claims

1. An anti-aging composition comprising culture supernatant and / or extracellular vesicles of microalgae.

2. A composition for promoting mitochondrial activity, comprising culture supernatant and / or extracellular vesicles of microalgae.

3. A composition for promoting collagen production, comprising culture supernatant and / or extracellular vesicles of microalgae.

4. The composition according to any one of claims 1 to 3, characterized in that the culture supernatant of the microalgae is obtained by a method comprising purifying the culture supernatant by filtration through a filter having a membrane pore size of 0.001 μm to 0.2 μm.

5. The composition according to any one of claims 1 to 3, wherein the molecular weight cut-off of the filter is 10 KDa to 1000 KDa.

6. The composition according to any one of claims 1 to 3, which is obtained by heating the culture supernatant of the microalgae at 40°C or higher.

7. The composition according to any one of claims 1 to 3, wherein the particle diameter of the extracellular vesicles is 50 nm to 700 nm.

8. The composition according to any one of claims 1 to 3, wherein the microalgae is microalgae belonging to the genus Pavlova, Euglena, Spirulina, or Chlorella.

9. The composition according to any one of claims 1 to 3, which is a food or drink, a cosmetic, a medicine, or a quasi-drug.

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