Composition containing extracellular vesicles derived from microalgae

Abstract

The purpose of the present invention is to provide a composition containing extracellular vesicles derived from microalgae. In one embodiment of the present invention, the composition contains (A) extracellular vesicles derived from microalgae, and, optionally, one or more chemicals selected from the group consisting of (B-1) to (B-3), where: (B-1) is one or more chemicals selected from the group consisting of primary alcohols and polyhydric alcohols, (B-2) is one or more chemicals selected from the group consisting of organic acids and salts thereof, and (B-3) is a chelating agent.

Description

Composition containing extracellular vesicles derived from microalgae

[0001] The present invention relates to a composition containing extracellular vesicles derived from microalgae.

[0002] Many cell types secrete lipid bilayer membrane-containing vesicles. These vesicles are called extracellular vesicles (ECVs), and typically contain functional molecules such as proteins and nucleic acids within the lipid bilayer membrane. ECVs are involved in the delivery of functional molecules within the body, and in recent years, they have been shown to be involved in various biological phenomena and diseases.

[0003] For example, it is known that extracellular vesicles produced by specific species of lactic acid bacteria are used to prevent hair loss and promote hair growth (Patent Document 1).

[0004] Special table 2018-529720 publication

[0005] However, much remains unknown about extracellular vesicles, and there have been few reports, particularly about microalgae-derived extracellular vesicles themselves and their applications.

[0006] Therefore, an object of the present invention is to provide a composition containing extracellular vesicles derived from microalgae, in which the extracellular vesicles have excellent stability.

[0007] In order to solve the above problems, the inventors conducted extensive research and found that compositions containing extracellular vesicles derived from microalgae are more stable than extracellular vesicles derived from other sources, and also have good properties when combined with a base, leading to the completion of the present invention.

[0008] That is, the present invention provides a composition comprising (A) extracellular vesicles derived from microalgae.

[0009] One aspect of the present invention is as follows: [1] (A) A composition containing extracellular vesicles derived from microalgae.

[0010] According to the present invention, a composition containing extracellular vesicles derived from microalgae and having excellent stability can be obtained.

[0011] [Composition Comprising Extracellular Vesicles] The composition of the present invention comprises (A) extracellular vesicles derived from microalgae. It may further comprise one or more species selected from the group consisting of (B-1) to (B-3): (B-1) one or more species selected from the group consisting of primary alcohols and polyhydric alcohols, (B-2) one or more species selected from the group consisting of organic acids and salts thereof, and (B-3) a chelating agent.

[0012] (Extracellular vesicles) The extracellular vesicles derived from microalgae in this specification are typically contained in the culture supernatant of the microalgae, and when preparing a composition containing extracellular vesicles, the culture supernatant can be used as is, or can be crudely or purified from the culture supernatant before use. The composition is a preparation obtained from the culture supernatant and may contain secretions obtained by culture in addition to the extracellular vesicles.

[0013] (Microalgae Culture Supernatant) In this specification, microalgae refer to organisms that perform oxygen-producing photosynthesis, excluding mosses, ferns, and spermatophytes, and have a cell size of 0.1 μm to 1000 μm in diameter. Labyrinthula, which are protists closely related to microalgae, are also included.

[0014] Microalgae include, for example, prokaryotic Cyanobacteria, as well as eukaryotic Glaucophyta, Rhodophyta (red algae), Chlorophyta, Cryptophyta (cryptophytes), Haptophyta (haptophytes), Heterokontophyta, Dinophyta (dinoflagellates), Euglenida, and Chlorarachniophyta.

[0015] Examples of microalgae include microalgae belonging to the genus Pavlova, the genus Euglena (Japanese name: Midorimushi, protozoa / green algae), the genus Spirulina (cyanophyceae), the genus Chlorella (green algae), the genus Dunaliella (green algae), the genus Nannochloropsis (true eyespot algae), the genus Licoricerum (green algae), the genus Botryococcus (green algae), and the Labyrinthulomycete Thraustochytrids. The microalgae exemplified above may be used singly or in combination of two or more.

[0016] 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 genus Pavlova, the genus Euglena, the genus Spirulina, and the genus Chlorella are preferred, microalgae belonging to at least one species selected from the group consisting of the genus Pavlova, the genus Euglena, and the genus Spirulina are more preferred, microalgae belonging to at least one species selected from the group consisting of the genus Pavlova and the genus Euglena are even more preferred, and microalgae belonging to the genus Pavlova are particularly preferred.

[0017] Pavlova is included in the haptophyte division, and examples of microalgae of the genus Pavlova include P. calceolate, P. granifera, P. gyrans, P. lutheri, P. pinguis, and P. salina, with P. granifera and / or P. gyrans being preferred.

[0018] Examples of microalgae belonging to the genus Euglena include E. gracilis, E. longa, E. caudata, E. oxyuris, E. tripteris, E. proxima, E. Viridis, E. socialis, E. ehrenbergii, E. Deses, E. pisciformis, E. spirogyra, E. acus, E. geneculata, E. intermedia, E. mutabilis, E. sanguinea, E. stellata, E. terricola, E. Klebsi, E. rubra, E. Examples of suitable strains include E. cyclopicola, and E. gracilis and / or E. longa are preferred.

[0019] Examples of microalgae of the genus Spirulina include the genus Altrospira, which has a scientific name change from Spirulina, and include S. platensis (A. platensis) and S. maxima (A. maxima), with S. platensis being preferred.

[0020] Examples of microalgae of the genus Chlorella include C. vulgaris, C. saccharophila, C. ellipsoidea, C. pyrenoidosa, C. sorokiniana, and C. lobophora, with C. vulgaris being preferred.

[0021] The microalgae exemplified in the present invention are classified as lower organisms and exhibit common characteristics in the lipids contained within their cells. Examples of lipids commonly contained in these microalgae include so-called betaine lipids. Betaine lipids have a unique structure different from phospholipids and the like commonly found in higher organisms. Furthermore, the betaine lipids commonly contained in microalgae are thought to contribute to the lipids that constitute the membrane structure of the extracellular vesicles secreted by these microalgae. Therefore, it is presumed that the membranes of these extracellular vesicles have common membrane properties that reflect the properties unique to microalgae. Therefore, the effects of the present invention are expected when using extracellular vesicles of any microalgae.

[0022] The microalgae exemplified above are widely distributed in seawater, freshwater such as ponds and marshes, and brackish water, and may be separated from these and used, or any microalgae that has already been isolated may be used.

[0023] The microalgae exemplified above include closely related species and mutant strains thereof, as long as they exhibit the effects of the present invention. Mutant strains also include those obtained by genetic methods such as gene recombination, transduction, and transformation.

[0024] As used herein, the term "microalgae culture supernatant" refers to the supernatant of a culture solution obtained after culturing microalgae. For example, the term "microalgae culture supernatant" refers to the supernatant of a culture solution obtained by culturing microalgae using a known culture method and removing the microalgae using a known separation means such as centrifugation.

[0025] 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, including the following examples.

[0026] (Cultivation Conditions) Microalgae can be cultured using a culture medium. A carbon source is added to the culture medium as a nutrient source. The carbon source can be an inorganic carbon source (CO ２ , NaHCO ３ , Na ２ CO ３ The culture medium is not limited, but it is preferable to use an autotrophic medium that does not contain an organic carbon source such as glucose as a nutrient source. Examples of such a medium include culture media supplemented with nutrient salts such as a nitrogen source, a phosphorus source, and minerals, such as Cramer-Myers medium and modified Cramer-Myers medium.

[0027] 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.

[0028] The pH of the culture medium is not particularly limited as long as the effects of the present invention are achieved, and examples 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.

[0029] The culture temperature is not particularly limited as long as the effects of the present invention are achieved, but examples include 15 to 40°C, 20 to 34°C, and 23 to 28°C.

[0030] The culture period is not particularly limited as long as the effects of the present invention are achieved, but examples include 4 to 30 days, 4 to 20 days, and 5 to 15 days.

[0031] Depending on the type of microalgae, the type of light source, the presence or absence of light and dark, aerobic conditions, anaerobic conditions, etc. can be appropriately selected.

[0032] In one embodiment, when culturing marine microalgae such as pavlova, any known method can be used as long as the effects of the present invention are achieved. For example, the culture can be carried out under conditions that satisfy at least one of the following: Seawater concentration: 50% seawater Culture medium concentration: 2x IMK medium Culture solution volume: 800 mL Light source: Side: fluorescent light (100 to 150 μmol) Light / dark cycle: 12 hours of light and 12 hours of dark pH: pH of about 7.4 at the start of each culture Aeration: performed Mechanical stirring: none Culture period: approximately 10 days Culture temperature: 25°C to 28°C

[0033] (Recovery of Culture Supernatant) After culturing microalgae under the culture conditions exemplified above, the culture supernatant is recovered using a known separation method such as centrifugation. The method for recovering the culture supernatant is not particularly limited, as long as it can remove or substantially remove the microalgae and appropriately recover the culture supernatant. In an exemplary embodiment, the centrifugation performed to obtain the culture supernatant may be performed for 30 to 60 minutes at a centrifugal force of, for example, 1,000 to 20,000 x g, 1,500 to 20,000 x g, 1,500 to 15,000 x g, or 1,500 to 10,000 x 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 x g, and then centrifuged at a high speed of 10,000 to 20,000 x g to further remove cells or cell-related debris and residues. The supernatant thus obtained is collected and subjected to the next step.

[0034] (A) Extracellular Vesicles) As used herein, extracellular vesicles refer to vesicles secreted by microalgae, typically having an average diameter of 10 nm to 1 μm. Such an average diameter is also referred to herein as the average particle size. Although not limited thereto, it is preferred that the composition containing the extracellular vesicles of the present invention does not contain microalgae cells themselves. Here, the term diameter or particle size when specifying the size of extracellular vesicles, as used herein, refers to the maximum dimension of the extracellular vesicles, since extracellular vesicles are not necessarily spherical. Diameter or particle size can be measured using microscopy techniques, particularly techniques for measuring nanoparticle size using transmission electron microscopy. Furthermore, nanoparticle tracking analysis (NTA), which is based on the analysis of both light scattering and Brownian motion, can also be used. In the present invention, the diameter or particle size of extracellular vesicles is measured using NanoSight (Malvern Panalytical, NanoSight L10) for extracellular vesicles with a particle size of 50 nm or more, and using a transmission electron microscope for extracellular vesicles with a particle size of less than 50 nm.

[0035] The cumulative 10% value (D10) of the particle diameter of pavlova-derived extracellular vesicles is not particularly limited as long as the effects of the present invention are achieved, and examples thereof include 50 to 120 nm, 60 to 110 nm, and 65 to 100 nm. Furthermore, the cumulative 50% value (D50) of the particle diameter is, for example, 100 to 180 nm, 110 to 170 nm, and 115 to 160 nm. Furthermore, the cumulative 90% value (D90) of the particle diameter is, for example, 180 to 300 nm, 190 to 290 nm, and 200 to 280 nm. As used herein, the cumulative 10% value (D10) of the particle diameter of extracellular vesicles refers to the diameter below which 10% of all extracellular vesicles are located. The cumulative 50% value (D50) of the particle diameter of extracellular vesicles refers to the diameter below which 50% of all extracellular vesicles are located, 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.

[0036] The cumulative 10% value (D10) of particle diameter of 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.

[0037] The cumulative 10% value (D10) of particle diameter of the Chlorella-derived extracellular vesicles is not particularly limited as long as the effects of the present invention are achieved, and examples thereof include 90 to 150 nm, 100 to 140 nm, 110 to 130 nm, etc. Furthermore, the cumulative 50% value (D50) of the particle diameter is, for example, 180 to 240 nm, 190 to 230 nm, 200 to 220 nm, etc. Furthermore, the cumulative 90% value (D90) of the particle diameter is, for example, 320 to 380 nm, 330 to 370 nm, 340 to 360 nm, etc.

[0038] The average particle number of extracellular vesicles of microalgae is, for example, 1.0 × 10 per ml when the composition is in a liquid state. 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 or more, and 1.0 × 10 １５ Less than or equal to 1.0×10 １４ Less than or equal to 1.0×10 １３ Less than or equal to 1.0×10 １２ Less than or equal to 1.0×10 １１ The average number of particles in the extracellular vesicles of microalgae is, for example, 1.0 × 10 ４ ~1.0 x 10 １５ pieces, 1.0×10 ４ ~1.0 x 10 １４ pieces, 1.0×10 ４ ~1.0 x 10 １３ pieces, 1.0×10 ４~1.0×10 １２ 1.0×10 ４ ~1.0×10 １１ 1.0×10 ５ ~1.0×10 １５ 1.0×10 ５ ~1.0×10 １４ 1.0×10 ５ ~1.0×10 １３ 1.0×10 ５ ~1.0×10 １２ 1.0×10 ５ ~1.0×10 １１ 1.0×10 ６ ~1.0×10 １５ 1.0×10 ６ ~1.0×10 １４ 1.0×10 ６ ~1.0×10 １３ 1.0×10 ６ ~1.0×10 １２ 1.0×10 ６ ~1.0×10 １１ 1.0×10 ７ ~1.0×10 １５ 1.0×10 ７ ~1.0×10 １４ 1.0×10 ７ ~1.0×10 １３ 1.0×10 ７ ~1.0×10 １２ 1.0×10 ７ ~1.0×10 １１ 1.0×10 ８ ~1.0×10 １５ 1.0×10 ８ ~1.0×10 １４ 1.0×10 ８ ~1.0×10 １３ 1.0×10 ８ ~1.0×10 １２ 1.0×10 ８ ~1.0×10 １１ 1.0×10 ９ ~1.0×10 １５ 1.0×10 ９ ~1.0×10 １４ 1.0×10 ９ ~1.0×10１３ pieces,, 1.0×10 ９ ~1.0 x 10 １２ pieces, 1.0×10 ９ ~1.0 x 10 １１ The number of extracellular vesicles can be determined using NanoSight (Malvern Panalytical, Nanosite L10) or the like, which uses nanoparticle tracking analysis (NTA) based on the analysis of both light scattering and Brownian motion.

[0039] (Method for Obtaining Extracellular Vesicles) Methods for obtaining the extracellular vesicles of the present invention include a method for preparing them from the culture supernatant of algae.

[0040] In one embodiment, the extracellular vesicles of the present invention are obtained by heating a culture or culture supernatant of microalgae.

[0041] The extracellular vesicles of the present invention can also be obtained by heating a microalgae culture or culture supernatant at a temperature, for example 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, or 100°C or higher and 200°C or lower, 150°C or lower, or 120°C or lower, for a time period of 30 seconds or longer, 1 minute or longer, 2 minutes or longer, 3 minutes or longer, or 4 minutes or longer, 30 minutes or shorter, 20 minutes or shorter, or 10 minutes or shorter.

[0042] Alternatively, in another embodiment, the extracellular vesicles of the present invention can be obtained by filtering the culture supernatant of microalgae.

[0043] The membrane pore size of the filter used for filtration is preferably 0.001 to 0.5 μm, more preferably 0.005 μm to 0.3 μm, and even more preferably 0.01 μm to 0.25 μm.

[0044] In yet another embodiment, the method for obtaining extracellular vesicles of the present invention can also include a purification step. Specific embodiments of the purification step include purification by ultrafiltration (e.g., tangential flow filtration), ultracentrifugation, and affinity purification using antibodies. Of these, purification by ultrafiltration is preferred, although not limited thereto.

[0045] When purification by ultrafiltration is used, it may be performed using a hollow fiber membrane to filter from the inside to the outside of the fiber, using a spiral membrane including a filtration membrane and a support membrane, using a tubular membrane on a hollow cylinder, or using a flat membrane. Of these, but not limited to, it is preferable to perform filtration using a hollow fiber membrane to obtain a concentrated liquid containing extracellular vesicles on the inside of the hollow fiber membrane.

[0046] The molecular weight cutoff of a cutoff membrane is preferably 10 kDa to 1,000 kDa, 50 kDa to 1,000 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 2,000 kDa or less, 1,500 kDa or less, 1,000 kDa or less, 750 Dka or less, or 500 Dka or less are also preferred.

[0047] The extracellular vesicles of the present invention may be prepared by one or a combination of two or more of these preparation methods, for example, but not limited to, a combination of a heat treatment step and a purification step, a combination of a purification step and a filtration step, or a combination of a heat treatment step, a purification step, and a filtration step.

[0048] The extracellular vesicles may be in the form of a purified liquid of the culture supernatant, or may be in the form of a solid or semi-solid obtained by drying the purified liquid.

[0049] The extracellular vesicles can optionally be further purified to provide a pure population of extracellular vesicles.

[0050] The extracellular vesicles may be in the form of a purified liquid of the culture supernatant itself, or may be a solid or semi-solid obtained by drying the purified liquid.

[0051] (Compositions containing extracellular vesicles) In one aspect of the present invention, it is possible to prepare compositions containing any of the extracellular vesicles obtained in this manner.

[0052] In such a composition, the average particle number of extracellular vesicles of the microalgae is, for example, 1.0 × 10 per 100 g. ３ pcs or more, 1.0×10 ４ pcs or more, 1.0×10 ５ pcs or more, 1.0×10 ６ or more, and 1.0 × 10 １６ Less than or equal to 1.0×10 １５ Less than or equal to 1.0×10 １４ Less than or equal to 1.0×10 １３ Less than or equal to 1.0×10 １２ Less than or equal to 1.0×10 １１ Less than or equal to 1.0×10 １０ Less than or equal to 1.0×10 ９ Less than or equal to 1.0×10 ８ Less than or equal to 1.0×10 ７ Less than or equal to 1 x 10 ６ The average particle number of extracellular vesicles of microalgae is, for example, 1.0 × 10 ３ ~1.0 x 10 １４ pieces, 1.0×10 ３ ~1.0 x 10 １３ pieces, 1.0×10 ３ ~1.0 x 10 １２ pieces, 1.0×10 ３ ~1.0 x 10 １１ pieces, 1.0×10 ３ ~1.0 x 10 １０ pieces, 1.0×10 ３ ~1.0 x 10 ９ pieces, 1.0×10 ３ ~1.0 x 10 ８ pieces, 1.0×10 ３ ~1.0 x 10 ７ pieces, 1.0×10 ４ ~1.0 x 10 １４ pieces, 1.0×10 ４ ~1.0 x 10 １３ pieces, 1.0×10 ４ ~1.0 x 10 １２ pieces, 1.0×10 ４ ~1.0 x 10 １１ pieces, 1.0×10 ４ ~1.0 x 10 １０ pieces, 1.0×10 ４ ~1.0 x 10 ９1.0×10 ４ ~1.0×10 ８ 1.0×10 ４ ~1.0×10 ７ 1.0×10 ５ ~1.0×10 １４ 1.0×10 ５ ~1.0×10 １３ 1.0×10 ５ ~1.0×10 １２ 1.0×10 ５ ~1.0×10 １１ 1.0×10 ５ ~1.0×10 １０ 1.0×10 ５ ~1.0×10 ９ 1.0×10 ５ ~1.0×10 ８ 1.0×10 ５ ~1.0×10 ７ 1.0×10 ６ ~1.0×10 １４ 1.0×10 ６ ~1.0×10 １３ 1.0×10 ６ ~1.0×10 １２ 1.0×10 ６ ~1.0×10 １１ 1.0×10 ６ ~1.0×10 １０ 1.0×10 ６ ~1.0×10 ９ 1.0×10 ６ ~1.0×10 ８ 1.0×10 ６ ~1.0×10 ７ 1.0×10 ７ ~1.0×10 １４ 1.0×10 ７ ~1.0×10 １３ 1.0×10 ７ ~1.0×10 １２ 1.0×10 ７ ~1.0×10 １１ 1.0×10 ７ ~1.0×10 １０ 1.0×10 ８ ~1.0×10 １４ 1.0×10８ ~1.0 x 10 １３ pieces, 1.0×10 ８ ~1.0 x 10 １２ pieces, 1.0×10 ８ ~1.0 x 10 １１ pieces, 1.0×10 ８ ~1.0 x 10 １０ pieces, 1.0×10 ９ ~1.0 x 10 １４ pieces, 1.0×10 ９ ~1.0 x 10 １３ pieces, 1.0×10 ９ ~1.0 x 10 １２ pieces, 1.0×10 ９ ~1.0 x 10 １１ pieces, 1.0×10 ９ ~1.0 x 10 １０ Examples include:

[0053] 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, but 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 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 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.

[0054] The content of microalgal extracellular vesicles in the composition is adjusted appropriately 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 in the state of a purified liquid 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 microalgal extracellular vesicles can be, for example, 50% by mass or less relative to the total amount of the composition, and examples thereof include 40% by mass or less, 30% by mass or less, 20% by mass or less, and 15% by mass or less. The content of the extracellular vesicles of microalgae may be, for example, 0.001 to 50% by mass, 0.001 to 40% by mass, 0.001 to 30% by mass, 0.001 to 20% by mass, 0.001 to 15% by mass, 0.01 to 50% by mass, 0.01 to 40% by mass, 0.01 to 30% by mass, 0.01 to 20% by mass, 0.01 to 15% by mass, 0.1 to 50% by mass, 0.1 to 40% by mass, 0.1 to 30% by mass, 0.1 to 20% by mass, 0.1 to 15% by mass, 1 to 50% by mass, 1 to 40% by mass, 1 to 30% by mass, 1 to 20% by mass, or 1 to 15% by mass, relative to the total amount of the composition.

[0055] The content of microalgal extracellular vesicles is adjusted appropriately depending on the type and amount of other components, dosage form, etc., and is not limited, and can be, for example, 0.00001 μg / mL or more, 0.0001 μg / mL or more, 0.001 μg / mL or more, 0.01 μg / mL or more, 0.1 μg / mL or more, or 1 μg / mL or more in terms of protein amount relative to the total amount of the composition. Other examples include 100 μg / mL or less, 10 μg / mL or less, 1 μg / mL, 0.1 μg / mL or less, and 0.01 μg / mL or less.

[0056] ((B-1) One or more alcohols selected from the group consisting of primary alcohols and polyhydric alcohols) The component (B-1) of the present invention is at least one alcohol selected from the group consisting of primary alcohols and polyhydric alcohols.

[0057] The primary alcohol that can be used in the present invention is not limited as long as it is used in the pharmaceutical or cosmetic fields, and in some cases in the food field. From the viewpoint of significantly achieving the effects of the present invention, the primary alcohol preferably has 1 to 3 carbon atoms. Examples of such primary alcohols include methanol, ethanol, n-propanol, and isopropanol. Furthermore, the primary alcohol used in the present invention preferably contains at least ethanol, and more preferably is ethanol. In the present invention, one type of primary alcohol can be used, or two or more types can be used in combination.

[0058] The polyhydric alcohols that can be used in the present invention are not limited as long as they are used in the pharmaceutical or cosmetic fields, and in some cases in the food field. In this specification, the term "polyhydric alcohol" refers to an alcohol containing two or more hydroxy (—OH) groups per molecule. From the perspective of achieving the effects of the invention, dihydric to hexahydric alcohols are preferably used, with dihydric to pentahydric alcohols being more preferred, and dihydric to trihydric alcohols being even more preferred. Examples of dihydric alcohols include dipropylene glycol, 1,3-butylene glycol, 1,3-propanediol, 3-methyl-1,3-butanediol, propylene glycol, pentanediol, hexanediol, octanediol, and polyethylene glycol; trihydric alcohols include glycerin; tetrahydric alcohols include pentaerythritol; pentahydric alcohols include diglycerin and xylitol; and hexahydric alcohols include sorbitol and inositol. Polyhydric alcohols can be used singly or in combination of two or more. The polyhydric alcohol is preferably one or more selected from the group consisting of glycerin, dipropylene glycol, 1,3-butylene glycol, 1,3-propanediol, pentanediol, and polyethylene glycol, and particularly preferably glycerin, 1,3-butylene glycol, 1,3-propanediol, and polyethylene glycol.

[0059] Examples of polyethylene glycol include Macrogol 200 (liquid), Macrogol 300 (liquid), and Macrogol 1540 (paste) as specified in the Pharmaceutical Excipients Standards; Macrogol 400 (liquid), Macrogol 1500 (paste), and Macrogol 4000 (solid) as specified in the Japanese Pharmacopoeia; and polyethylene glycol 200 (liquid), polyethylene glycol 300 (liquid), polyethylene glycol 400 (liquid), polyethylene glycol 600 (liquid), polyethylene glycol 1000 (wax), polyethylene glycol 1500 (paste), polyethylene glycol 1540 (wax), polyethylene glycol 2000 (wax), and polyethylene glycol 4000 (solid) as specified in the Quasi-drug Ingredients Standards 2006. As described above, the properties of polyethylene glycol vary depending on the molecular weight. In the present invention, liquid, paste, or wax polyethylene glycol is preferably used, more preferably liquid or paste, and even more preferably liquid.

[0060] The total content of the component (B-1) relative to the total amount of the composition of the present invention is preferably 1 to 60 mass %, more preferably 3 to 50 mass %, and even more preferably about 5 to 40 mass %.

[0061] The content of the primary alcohol relative to the total amount of the composition of the present invention is preferably from 0.01 to 30% by mass, more preferably from 1 to 25% by mass, and even more preferably from about 3 to 20% by mass.

[0062] The total content of the polyhydric alcohol relative to the total amount of the composition of the present invention is preferably from 0.5 to 50% by mass, more preferably from 2 to 40% by mass, and even more preferably from about 3 to 30% by mass.

[0063] In the composition of the present invention, the number of the component (A) per 1% by mass of the primary alcohol is 1×10 to 1×10 １０ 1×10 is preferred, ２ ~1 x 10 ９ 1×10 is more preferable. ３ ~1 x 10 ８ 1×10 is more preferable,４ ~1 x 10 ７ is even more preferred.

[0064] In the composition of the present invention, the number of the component (A) per 1% by mass of the polyhydric alcohol is 1×10 to 1×10 １０ 1×10 is preferred, ２ ~1 x 10 ９ 1×10 is more preferable. ３ ~1 x 10 ８ 1×10 is more preferable, ４ ~1 x 10 ７ is even more preferred.

[0065] ((B-2) One or more selected from the group consisting of organic acids and salts thereof) The component (B-2) of the present invention is an organic acid or a salt thereof. In the present invention, the organic acid is an acid of an organic compound having an organic group and a molecular weight of 1000 or less, preferably 700 or less, more preferably 500 or less, even more preferably 350 or less, and particularly preferably 300 or less. The organic acid is preferably a water-soluble organic acid. The organic acid may be a carboxylic acid having a carboxyl group, a sulfonic acid having a sulfo group, or a phosphoric acid ester having a phosphoric acid group. More specifically, examples of organic acids include, but are not limited to, ascorbic acid, tranexamic acid, citric acid, salicylic acid, lactic acid, tartaric acid, malic acid, succinic acid, oxalic acid, gluconic acid, fumaric acid, aspartic acid, pyrrolidonecarboxylic acid, ε-aminocaproic acid, glutamic acid, aminoethylsulfonic acid, ellagic acid, kojic acid, glycyrrhizic acid, phytic acid, ferulic acid, glycolic acid, and azelaic acid. Among these, at least one selected from the group consisting of ascorbic acid, tranexamic acid, citric acid, salicylic acid, lactic acid, tartaric acid, malic acid, succinic acid, gluconic acid, aspartic acid, pyrrolidonecarboxylic acid, ε-aminocaproic acid, glutamic acid, aminoethylsulfonic acid, ellagic acid, kojic acid, glycyrrhizic acid, phytic acid, ferulic acid, glycolic acid, and azelaic acid can be exemplified. Of these, at least one selected from the group consisting of ascorbic acid, tranexamic acid, citric acid, salicylic acid, lactic acid, tartaric acid, malic acid, succinic acid, gluconic acid, pyrrolidonecarboxylic acid, ε-aminocaproic acid, glutamic acid, kojic acid, glycyrrhizic acid, phytic acid, ferulic acid, glycolic acid, and azelaic acid is preferred, at least one selected from the group consisting of ascorbic acid, tranexamic acid, citric acid, salicylic acid, lactic acid, succinic acid, tartaric acid, gluconic acid, kojic acid, phytic acid, glycolic acid, and glycyrrhizic acid is more preferred, and at least one selected from citric acid and succinic acid is even more preferred.

[0066] In the present invention, the organic acid salt refers to a salt of such an organic acid. The salt formed by the organic acid salt is a pharmaceutically acceptable salt. Examples of the organic acid salt include, but are not limited to, salts with organic bases (e.g., salts with tertiary amines such as trimethylamine salt, triethylamine salt, monoethanolamine salt, triethanolamine salt, and pyridine salt; basic ammonium salts such as arginine or lysine); and salts with inorganic bases (e.g., inorganic acid salts such as hydrochloride, sulfate, and phosphate; ammonium salt; alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; or zinc salt and aluminum salt). Among these, preferred organic acid salts are triethanolamine salt, monoammonium salt, sodium salt, potassium salt, dipotassium salt, magnesium salt, and zinc salt, and particularly preferred organic acid salts are sodium salt, dipotassium salt, and magnesium salt.

[0067] In the present invention, preferred organic acid salts include sodium ascorbate, tranexamic acid hydrochloride, calcium tranexamate, sodium salicylate, calcium salicylate, magnesium salicylate, potassium salicylate, sodium lactate, sodium tartrate, sodium citrate, disodium citrate, trisodium citrate, potassium citrate, sodium succinate, disodium succinate, sodium oxalate, calcium gluconate, zinc gluconate, sodium pyrrolidone carboxylate, zinc pyrrolidone carboxylate, sodium glutamate, dipotassium glycyrrhizinate, monoammonium glycyrrhizinate, sodium phytate, sodium glycolate, and ammonium glycolate. Among these, at least one selected from the group consisting of sodium ascorbate, tranexamic acid hydrochloride, calcium tranexamate, sodium salicylate, sodium lactate, sodium tartrate, sodium citrate, disodium citrate, trisodium citrate, sodium succinate, disodium succinate, calcium gluconate, zinc gluconate, zinc pyrrolidonecarboxylate, sodium pyrrolidonecarboxylate, sodium glutamate, dipotassium glycyrrhizinate, monoammonium glycyrrhizinate, sodium phytate, sodium glycolate, ammonium glycolate, and sodium succinate and disodium succinate is preferred, and at least one selected from the group consisting of sodium ascorbate, sodium salicylate, sodium lactate, sodium citrate, disodium citrate, trisodium citrate, zinc gluconate, sodium pyrrolidonecarboxylate, sodium glutamate, sodium succinate, and disodium succinate is more preferred.

[0068] The total content of the organic acid or salt thereof relative to the total amount of the composition of the present invention is preferably from 0.0001 to 30% by mass, more preferably from 0.001 to 10% by mass, and even more preferably from about 0.01 to 5% by mass.

[0069] In the composition of the present invention, the number of molecules per 1% by mass of the organic acid or salt thereof of component (A) is 1×10 to 1×10 １６ 1×10 is preferred,２ ~1 x 10 １０ 1×10 is more preferable. ３ ~1 x 10 ８ 1×10 is more preferable, ４ ~1 x 10 ７ is even more preferred.

[0070] ((B-3) Chelating Agent) The component (B-3) of the present invention is a chelating agent. Examples of the chelating agent used in the present invention include ethylenediaminetetraacetic acid (edetic acid), ethylenediaminetetraacetic acid salts (sodium salts (sodium edetate: Japanese Pharmacopoeia, EDTA-2Na, etc.), potassium salts, etc.), phytic acid, gluconic acid, polyphosphoric acid, metaphosphoric acid, etc. Among these, disodium edetate is preferred.

[0071] The total content of the chelating agent relative to the total amount of the composition of the present invention is preferably 0.0001 to 2% by mass, more preferably 0.001 to 1% by mass, and even more preferably about 0.01 to 0.5% by mass.

[0072] In the composition of the present invention, the number of the chelating agent of the component (A) per 1% by mass is 1×10 to 1×10 １６ 1×10 is preferred, ２ ~1 x 10 １０ 1×10 is more preferable. ３ ~1 x 10 ８ 1×10 is more preferable, ４ ~1 x 10 ７ is even more preferred.

[0073] (C) Water) The composition of the present invention may also contain water. The total content of water relative to the total amount of the composition of the present invention is preferably 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, 80% by mass or more, or 90% by mass or more, and is preferably 99% by mass or less, 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, or 40% by mass or less.

[0074] In the composition of the present invention, the number of components (A) per 1% by mass of water is 1×10 to 1×10 １６1×10 is preferred, ２ ~1 x 10 １４ 1×10 is more preferable. ３ ~1 x 10 １２ 1×10 is more preferable, ４ ~1 x 10 １１ In one embodiment, the number of the component (A) per 1% by mass of water is 1×10 to 1×10 １０ 1×10 is preferred, ２ ~1 x 10 ９ 1×10 is more preferable. ３ ~1 x 10 ８ 1×10 is more preferable, ４ ~1 x 10 ７ is even more preferred.

[0075] The pH of the composition of the present invention may be 2.5 or more, 3.5 or more, 4.0 or more, 5.0 or more, or 6.0 or more, and may be 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, or 8 or less. The pH of the composition may be pH 2.5 to 12, pH 3.5 to 10, or pH 5 to 8.

[0076] (Nonionic Surfactant) The composition of the present invention may contain a nonionic surfactant in addition to the above-described components (A) and (B), as long as the effects of the present invention are not impaired. The nonionic surfactant that can be used in the present invention is not limited, but is preferably a nonionic surfactant with an HLB of less than 15, more preferably a surfactant with an HLB of 14.5 or less, and even more preferably a surfactant with an HLB of 14 or less. The HLB of the nonionic surfactant may be 6 to 14.5, preferably 8 to 14.5, and more preferably 10 to 14.5.

[0077] The hydrophilic-lipophilic balance (HLB) value in the present invention is a value calculated by Kawakami's formula (the following (Formula 1)) using the hydrophilic group molecular weight (Mw) and lipophilic group molecular weight (MO) of the surfactant molecule: HLB value = 7 + 11.7 log Mw / Mo (Formula 1)

[0078] Examples of nonionic surfactants include polyglycerin fatty acid esters such as hexaglyceryl monolaurate (HLB value 14.5), hexaglyceryl monomyristate (HLB value 11), hexaglyceryl monostearate (HLB value 9.0), hexaglyceryl monooleate (HLB value 9.0), decaglyceryl monomyristate (HLB value 14.0), decaglyceryl monostearate (HLB value 12.0), decaglyceryl monoisostearate (HLB value 12.0), decaglyceryl monooleate (HLB value 12.0), decaglyceryl distearate (HLB value 9.5), and decaglyceryl diisostearate (HLB value 10.0); Polyoxyethylene glycerin fatty acid esters such as polyoxyethylene 5-mol-added (POE(5)), hereinafter the same) glyceryl monostearate (HLB value 9.5), POE(15) glyceryl monostearate (HLB value 13.5), POE(5) glyceryl monooleate (HLB value 9.5), POE(15) glyceryl monooleate (HLB value 14.5), etc.; POE(20) sorbitan monostearate (HLB value 14.9), POE(6) sorbitan monostearate (HLB value 9.5), POE(20) sorbitan tristearate (HLB value 10.5), POE(20) sorbitan monoisostearate (HLB value 15.0), POE(6) sorbitan monooleate (HLB value 10.0), POE(20) sorbitan trioleate (HLB value 11.0), and other polyoxyethylene sorbitan fatty acid esters; Polyoxyethylene sorbitol fatty acid esters such as POE (60) tetrastearate sorbitol (HLB value 13.0), POE (30) tetraoleate sorbitol (HLB value 11.5), POE (40) tetraoleate sorbitol (HLB value 12.5), and POE (60) tetraoleate sorbitol (HLB value 14.0); polyoxyethylene lanolin, lanolin alcohol, and beeswax derivatives such as POE (10) lanolin (HLB value 12.0), POE (20) lanolin (HLB value 13.0), POE (5) lanolin alcohol (HLB value 12.5), and POE (20) sorbitol beeswax (HLB value 9.5);Polyoxyethylene castor oils and hydrogenated castor oils such as POE (20) castor oil (HLB value 10.5), POE (40) castor oil (HLB value 12.5), POE (50) castor oil (HLB value 14.0), POE (60) castor oil (HLB value 14.0), POE (20) hydrogenated castor oil (HLB value 10.5), POE (30) hydrogenated castor oil (HLB value 11.0), POE (40) hydrogenated castor oil (HLB value 13.5), and POE (60) hydrogenated castor oil (HLB value 14.0); Polyoxyethylene sterols / hydrogenated sterols such as POE (5) phytosterol (HLB value 9.5), POE (10) phytosterol (HLB value 12.5), and POE (25) phytostanol (HLB value 14.5); POE (2) lauryl ether (HLB value 9.5), POE (4.2) lauryl ether (HLB value 11.5), POE (9) lauryl ether (HLB value 14.5), POE (5.5) cetyl ether (HLB value 10.5), POE (7) cetyl ether (HLB value 11.5), POE (10) cetyl ether (HLB value 13.5), POE (4) stearyl ether (HLB value 9.0), POE (7) oleyl ether (HLB value 10.5), POE (10) oleyl ether (HLB value 14.5), POE (10) behenyl ether (HLB value 10.0), POE (2) (C12-15) alkyl ether (HLB value 9.0), POE (4) (C12-15) alkyl ether (HLB value 10.5), POE (5) secondary alkyl ether (HLB value 10.5), POE (7) secondary alkyl ether (HLB value 12.0), POE (9) alkyl ether (HLB value 13.5), POE (12) alkyl ether (HLB value 14.5) and other polyoxyethylene alkyl ethers; polyoxyethylene polyoxypropylene alkyl ethers such as 1-mol-added polyoxypropylene (referred to as "POP(1)"; the same applies hereinafter), POP(4) cetyl ether (HLB value 9.5), POE(10)POP(4) cetyl ether (HLB value 10.5), POE(20)POP(8) cetyl ether (HLB value 12.5), POE(20)POP(6) decyltetradecyl ether (HLB value 11.0), and POE(30)POP(6) decyltetradecyl ether (HLB value 12.0);Examples include polyethylene glycol fatty acid esters such as 10-mol polyethylene glycol monolaurate (referred to as "PEG (10)"; the same applies hereinafter) (HLB value 12.5), PEG (10) monostearate (HLB value 11.0), PEG (10) monooleate (HLB value 11.0), and PEG diisostearate (HLB value 9.5); and polyoxyethylene glyceryl isostearates such as PEG (8) glyceryl isostearate (HLB value 10.0), PEG (10) glyceryl isostearate (HLB value 10.0), PEG (15) glyceryl isostearate (HLB value 12.0), PEG (20) glyceryl isostearate (HLB value 13.0), PEG (25) glyceryl isostearate (HLB value 14.0), and PEG glyceryl isostearate (30).

[0079] The total content of nonionic surfactants having an HLB of less than 15 relative to the total amount of the composition of the present invention is preferably from 0.01 to 5 mass%, more preferably from 0.1 to 4 mass%, and even more preferably from about 0.5 to 3 mass%.

[0080] (Additives) The composition of the present invention may contain various additives other than those described above, provided that the effects of the present invention are not impaired. Examples of additives include antioxidants, thickeners, pH adjusters, inorganic salts, stabilizers, UV absorbers or UV scattering agents, irritation reducers, colorants, and fragrances. One or more additives may be used.

[0081] Here, the inorganic salt is preferably sodium chloride, disodium hydrogen phosphate, potassium chloride, or potassium dihydrogen phosphate.

[0082] [Use] In one embodiment, the composition containing the extracellular vesicles of the present invention is preferably used as an oral or external preparation.

[0083] The composition of the present invention can be added to or mixed with pharmaceuticals, quasi-drugs, cosmetics, foods, beverages, feed, or pet foods. Alternatively, it can be used as is as pharmaceuticals, quasi-drugs, cosmetics, foods, beverages, feed, or pet foods. Furthermore, it can also be used as a raw material for preparing such products or as a so-called premix product.

[0084] When used in so-called external preparations such as pharmaceuticals, quasi-drugs, and cosmetics, the composition of the present invention is preferably used in skin care cosmetics (e.g., lotions, creams, facial creams, facial lotions, emulsions, packs, liquid facial cleansers, soaps, and serums), makeup cosmetics (e.g., eyeliners and eyebrow makeup), and scalp and hair cosmetics (e.g., scalp lotions, scalp creams, shampoos, hair conditioners, hair treatments, hair essences, hair mists, and hair tonics), more preferably in lotions, creams, facial creams, facial lotions, emulsions, packs, facial cleansers, soaps, and serums, and particularly preferably in lotions, facial lotions, and serums, but is not limited to these.

[0085] When used in so-called external preparations such as pharmaceuticals, quasi-drugs, and cosmetics, the dosage form of the composition of the present invention is not particularly limited, and examples thereof include solutions, suspensions, emulsions, creams, gels, liniments, lotions, ointments, and aerosols. Among these, solutions, suspensions, emulsions, lotions, and aerosols are preferred, and solutions, lotions, and aerosols are more preferred. Furthermore, premixed products may be liquids containing, for example, extracellular vesicles, water, glycol, preservatives, excipients, solubilizers, pH adjusters, and the like.

[0086] When used in so-called external preparations such as pharmaceuticals, quasi-drugs, and cosmetics, the daily dose 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, ingestion or administration route, ingestion or administration schedule, formulation form, and other factors. Examples of the daily dose 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 dosage of microalgal extracellular vesicles 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. The daily dose of microalgal 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 dosage is 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 in divided doses one to three times a day, and the dosage does not limit the scope of the present specification in any way.

[0087] When used in so-called external preparations such as pharmaceuticals, quasi-drugs, and cosmetics, the daily adult dosage of microalgal extracellular vesicles 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, and other factors. Examples of the daily adult dosage of microalgal extracellular vesicles in terms of protein amount 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 microalgal extracellular vesicles 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. The daily dose of microalgal 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, in 1 to 3 divided doses per day, and the dosage does not limit the scope of the present specification in any way.

[0088] In another embodiment, the daily dose of microalgal extracellular vesicles for an adult is, in terms of number, for example, 1 x 10 or more, 1 x 10 ２ pcs or more, 1×10 ３ pcs or more, 1×10 ４ The daily dose of extracellular vesicles of microalgae for an adult is, in terms of the number of particles, for example, 1 x 10 to 1 x 10 １０ pieces, 1×10 to 1×10 ９ pieces, 1×10 to 1×10 ８ pieces, 1×10 to 1×10 ７ pieces, 1×10 ２ ~1 x 10 １０ pieces, 1×10 ２ ~1 x 10 ９ pieces, 1×10 ２ ~1 x 10 ８ pieces, 1×10 ２ ~1 x 10 ７ pieces, 1×10 ３ ~1 x 10 １０ pieces, 1×10 ３ ~1 x 10 ９ pieces, 1×10 ３ ~1 x 10 ８ pieces, 1×10 ３ ~1 x 10 ７ pieces, 1×10 ４ ~1 x 10 １０ pieces, 1×10 ４ ~1 x 10 ９ pieces, 1×10 ４ ~1 x 10 ８ pieces, 1×10 ４ ~1 x 10 ７ Examples include:

[0089] The composition of the present invention can be taken or administered once to 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 twice a day is preferred.

[0090] 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, i.e., a health food, a functional food, a food for patients, or a food for specified health uses.It can also be used as a so-called doctor's supplement recommended or presented by a doctor in an internal medicine department, an orthopedic surgery department, an animal hospital, or the like in a hospital and / or clinic.

[0091] Health foods, functional foods, foods for patients, and foods for specified health uses can be used in various dosage forms, such as solid preparations (tablets, orally disintegrating tablets, granules, fine granules, powders, capsules, chewable tablets, candy, etc.), liquid preparations (syrups, suspensions), and liquid foods. Food preparations can be manufactured in the same manner as known pharmaceutical preparations, by mixing the active ingredient with a food-acceptable carrier, such as a suitable excipient, and then manufacturing the preparation using conventional means. The dosage form is not limited, but is preferably an orally disintegrating tablet, chewable tablet, candy, granules, powder, or liquid preparation, from the viewpoint of significantly achieving the effects of the present invention.

[0092] 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.

[0093] 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.

[0094] The daily oral intake or administration amount of microalgal extracellular vesicles for an adult can be determined appropriately 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 microalgal extracellular vesicles 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. The daily oral intake or administration amount of microalgal 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 500 μg, 5 to 600 μg, 5 to 200 μg, 5 to 500 μg, 5 to 500 μg, 5 to 400 μg, 5 to 300 μg, 5 to 200 μg, 5 to 500 μg, 5 to 500 μg, 5 to 500 μg, 5 to 500 μg, 5 to 500 μg, 5 to 500 μg, 5 to 500 μg, 5 to 6 ... Examples include up to 100 μg, 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.

[0095] In another embodiment, the daily oral intake or administration amount of microalgal extracellular vesicles for an adult is, in terms of the number of particles, for example, 1 x 10 or more, 1 x 10 or more, ２ pcs or more, 1×10 ３ pcs or more, 1×10 ４ The daily oral intake or administration amount of extracellular vesicles of microalgae for an adult is, in terms of the number of particles, for example, 1 × 10 ９Less than or equal to 1 x 10 ８ Less than or equal to 1 x 10 ７ Less than or equal to 1 x 10 ６ Less than or equal to 1 x 10 ５ The daily oral intake or administration amount of extracellular vesicles of microalgae for an adult is, in terms of the number of particles, for example, 1 x 10 to 1 x 10 ９ pieces, 1×10 to 1×10 ８ pieces, 1×10 to 1×10 ７ pieces, 1×10 to 1×10 ６ pieces, 1×10 ２ ~1 x 10 ９ pieces, 1×10 ２ ~1 x 10 ８ pieces, 1×10 ２ ~1 x 10 ７ pieces, 1×10 ２ ~1 x 10 ６ pieces, 1×10 ３ ~1 x 10 ９ pieces, 1×10 ３ ~1 x 10 ８ pieces, 1×10 ３ ~1 x 10 ７ pieces, 1×10 ３ ~1 x 10 ６ pieces, 1×10 ４ ~1 x 10 ９ pieces, 1×10 ４ ~1 x 10 ８ pieces, 1×10 ４ ~1 x 10 ７ pieces, 1×10 ４ ~1 x 10 ６ Examples include pieces, etc.

[0096] The oral intake or dosage per day 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.

[0097] The composition of the present invention can be ingested or administered once to 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.

[0098] When the present invention is used as feed, pet food, etc., the target organisms are not particularly limited, but are preferably mammals, reptiles, amphibians, birds, and fish, and more preferably mammals other than humans.

[0099] When the present invention is used as a feed or pet food, it may be given to a pet in divided doses several times a day by adding it to the staple food, or it may be given to a pet as a snack at any time.

[0100] [Method for Improving Stability] The present invention relates to a method for improving the stability of microalgae-derived extracellular vesicles by adding one or more selected from the group consisting of (B-1) to (B-3) to (A) microalgae-derived extracellular vesicles. The method comprises: (B-1) one or more selected from the group consisting of primary alcohols and polyhydric alcohols; (B-2) one or more selected from the group consisting of organic acids and salts thereof; and (B-3) a chelating agent. The present invention also relates to a composition comprising (A) microalgae-derived extracellular vesicles and having improved stability. The present invention also relates to a composition comprising (A) microalgae-derived extracellular vesicles and one or more selected from the group consisting of (B-1) to (B-3), having improved stability of the microalgae-derived extracellular vesicles. (B-1) one or more selected from the group consisting of primary alcohols and polyhydric alcohols, (B-2) one or more selected from the group consisting of organic acids and salts thereof, and (B-3) a chelating agent. Here, improved stability refers to, for example, little change in the particle number and / or size of extracellular vesicles before and after storage of a composition containing extracellular vesicles. Extracellular vesicles are expected to have various physiologically active functions, and maintaining their number and physical properties will lead to the maintenance of the properties of a composition containing them.

[0101] The present invention includes the following aspects. [1] A composition containing (A) extracellular vesicles derived from microalgae. [2] A composition containing (A) extracellular vesicles derived from microalgae; and one or more selected from the group consisting of (B-1) to (B-3): (B-1) one or more selected from the group consisting of primary alcohols and polyhydric alcohols, (B-2) one or more selected from the group consisting of organic acids and salts thereof, and (B-3) a chelating agent. [3] The composition according to [2], wherein the composition contains component (B-1), and as component (B-1), one or more selected from the group consisting of primary alcohols having 1 to 3 carbon atoms, dihydric to trihydric polyhydric alcohols, and polyethylene glycol. [4] The composition according to [3], wherein the primary alcohol is one or more selected from the group consisting of methanol, ethanol, n-propanol, and isopropanol. [5] The composition according to [3], wherein the dihydric to trihydric polyhydric alcohol is one or more selected from the group consisting of dipropylene glycol, 1,3-butylene glycol, 1,3-propanediol, 3-methyl-1,3-butanediol, propylene glycol, pentanediol, hexanediol, octanediol, polyethylene glycol, and glycerin. [6] The composition according to any one of [2] to [5], wherein the composition contains a component (B-2), and as the component (B-2), one or more selected from the group consisting of organic acids having a molecular weight of 300 or less and salts thereof. [7] The composition according to [6], wherein the organic acid having a molecular weight of 300 or less and salts thereof are one or more selected from the group consisting of ascorbic acid, tranexamic acid, citric acid, salicylic acid, lactic acid, succinic acid, tartaric acid, gluconic acid, kojic acid, phytic acid, glycolic acid, and glycyrrhizic acid. [8] The composition according to any one of [2] to [7], wherein the composition contains component (B-3), and component (B-3) contains ethylenediaminetetraacetate. [9] A composition (excluding anti-aging compositions) containing (A) extracellular vesicles derived from microalgae.

[10] The composition according to any one of [1] to [9], further containing water.

[11] The composition according to any one of [1] to

[10] , wherein the microalgae belong to the genus Pavlova, Euglena, Spirulina, or Chlorella.

[12] The composition according to any one of [1] to

[11] , which is for oral or topical use.

[13] A composition for topical use comprising (A) pavlova-derived extracellular vesicles; and (B-1) 1,3-butylene glycol.

[14] A composition for topical use comprising (A) pavlova-derived extracellular vesicles; and (B-1) 1,3-propanediol.

[15] A composition for topical use comprising (A) pavlova-derived extracellular vesicles; and (B-1) dipropylene glycol.

[16] A composition for topical use comprising (A) pavlova-derived extracellular vesicles; and (B-1) glycerin.

[17] A topical composition comprising (A) pavlova-derived extracellular vesicles and (B-1) 1,2-pentanediol.

[18] A topical composition comprising (A) pavlova-derived extracellular vesicles and (B-1) polyethylene glycol.

[19] A topical composition comprising (A) pavlova-derived extracellular vesicles and (B-1) ethanol.

[20] A topical composition comprising (A) pavlova-derived extracellular vesicles and (B-2) citric acid and / or a salt thereof.

[21] A topical composition comprising (A) pavlova-derived extracellular vesicles and (B-3) ethylenediaminetetraacetate.

[22] A method for improving the stability of microalgae-derived extracellular vesicles by adding one or more selected from the group consisting of (B-1) to (B-3) to the microalgae-derived extracellular vesicles: (B-1) one or more selected from the group consisting of primary alcohols and polyhydric alcohols, (B-2) one or more selected from the group consisting of organic acids and salts thereof, and (B-3) a chelating agent.

[23] A method for improving the stability of microalgae-derived extracellular vesicles in a composition by adding one or more selected from the group consisting of (A) microalgae-derived extracellular vesicles and (B-1) one or more selected from the group consisting of primary alcohols having 1 to 3 carbon atoms, dihydric to trihydric polyhydric alcohols, and polyethylene glycols.

[24] (A) A method for improving the stability of microalgae-derived extracellular vesicles in an externally applied composition or an internally applied composition by adding one or more selected from the group consisting of (B-1) to (B-3) to the microalgae-derived extracellular vesicles: (B-1) one or more selected from the group consisting of primary alcohols having 1 to 3 carbon atoms, dihydric to trihydric polyhydric alcohols, and polyethylene glycols; (B-2) one or more selected from the group consisting of organic acids with a molecular weight of 300 or less and salts thereof; and (B-3) ethylenediaminetetraacetate.

[25] The method according to any one of

[22] to

[24] , wherein the composition further contains water.

[26] The method according to any one of

[22] to

[25] , wherein the microalgae belong to the genus Pavlova, Euglena, Spirulina, or Chlorella.

[0102] Next, the present invention will be specifically explained by way of examples, but the present invention is not limited to the following examples.

[0103] (Preparation Example) Pavlova (Pavlova sp.), Spirulina (Arthrospira platensis), or Chlorella (Chlorella vulgaris) was cultured using commercially available algae medium by conventional methods to obtain a culture supernatant. Each culture supernatant was heat-treated at 105 ° C. for 4 minutes. After heating, 3000 mL of the supernatant was centrifuged at 3000 × g and room temperature for 60 minutes, and then filtered through a 0.2 μm filter. Then, using a 300 kDa hollow fiber membrane filter (MiniKros sampler S02-E300-05-N: manufactured by Repligen), the supernatant was concentrated to a volume of 150 mL or less by tangential flow. Then, the mixture was concentrated and washed using 10,000 mL of PBS, and finally 100 mL was recovered with PBS. Thereafter, each concentrated sample was sterilized using a 0.22 μm filter, and the particle count and other parameters were measured using NanoSight.

[0104] Each sample was diluted appropriately 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 (manufactured by Malvern Panalytical) in accordance with the accompanying software. Particle diameter and particle size measurements were performed three times, and the average values ​​were used as the average particle diameter and average particle size of each sample.

[0105] As a result of the above, the following samples were obtained: Pavlova culture supernatant ultrafiltration sample (protein concentration 574 μg / mL) Spirulina culture supernatant ultrafiltration sample (protein concentration 29.6 μg / mL) Chlorella culture supernatant ultrafiltration sample (protein concentration 10.4 μg / mL)

[0106] In the ultrafiltrated sample of pavlova culture supernatant, pavlova-derived extracellular vesicles had a particle number of 1.79 × 10 １２ The particle size was 146 nm, the cumulative 10% value of particle size (D10) was 75 nm, the cumulative 50% value of particle size (D50) was 135.3 nm, and the cumulative 90% value of particle size (D90) was 229.3 nm.

[0107] In the ultrafiltrated sample of Spirulina culture supernatant, Spirulina-derived extracellular vesicles had a particle number of 2.88 × 10 １１ The 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.

[0108] In the ultrafiltration sample of Chlorella culture supernatant, Chlorella-derived extracellular vesicles had a particle number of 8.18 x 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.

[0109] (Examples and Comparative Examples) Compositions were prepared according to the formulations shown in Tables 1 to 3. The raw material components were mixed to prepare the compositions. The pH of the prepared compositions was 3.8 to 6.9.

[0110] The main raw materials used in the examples are as follows. Pavlova-derived extracellular vesicle-containing solution The sample prepared in the production example was used as is. Human stem cell-derived extracellular vesicle-containing solution (product name: RemyStem-JP, manufacturer: Anti-Aging Co., Ltd.) (7.28 x 10 ８ Particles / mL) Extracellular Vesicles derived from Lactobacillus paracasei 180913-R1 strain (manufacturer: Cosmo Bio Inc.) (3.9x10 １０ particles / mL)

[0111] [Evaluation of Particle Size and Particle Number Stability] The state of each composition was evaluated immediately after preparation and after standing at 40°C for one week. That is, the particle number and particle diameter of extracellular vesicles in the composition were measured. NanoSight was used to measure particle diameter, and each composition was appropriately diluted with purified water. From the obtained particle number and particle diameter, the particle number reduction rate and particle size variation rate were calculated using the following formula: Particle number reduction rate = (particle number immediately after preparation - particle number after storage at 40°C) / (particle number immediately after preparation) x 100 Particle size variation rate = [(particle size immediately after preparation - particle size after storage at 40°C) / (particle size immediately after preparation) x 100] Note that [ ] indicates an absolute value.

[0112] The evaluation results of the compositions of the Examples and Comparative Examples are shown in Tables 1 to 3. The numerical values ​​in the tables represent mass %. The evaluation results are shown as follows:

[0113] <Particle count reduction rate> 20% or more × 14% or more, less than 20% △ 5% or more, less than 14% ○ Less than 5% ◎

[0114] <Particle size fluctuation rate> Absolute value 20% or more × Absolute value 10% or more but less than 20% △ Absolute value less than 10% ○

[0115] As shown in the table, there was little change in the particle number and particle size of pavlova-derived extracellular vesicles before and after storage. Pavlova-derived extracellular vesicles remained stable even when base ingredients were added. On the other hand, human-derived and lactic acid bacteria-derived extracellular vesicles showed changes in particle number and particle size before and after storage.

[0116] Furthermore, all of the compositions of the examples had a smooth feel on the skin, were excellent in moisturizing feeling, and had a good feel when used.

Claims

1. A composition containing extracellular vesicles derived from microalgae.

2. A composition containing: (A) extracellular vesicles derived from microalgae; and one or more selected from the group consisting of (B-1) to (B-3): (B-1) one or more selected from the group consisting of primary alcohols and polyhydric alcohols, (B-2) one or more selected from the group consisting of organic acids and their salts, and (B-3) a chelating agent.

3. A composition (excluding the anti-aging composition) containing extracellular vesicles derived from microalgae.

4. The composition according to claim 1 or 2, further containing water.

5. The composition according to claim 1 or 2, wherein the microalgae belong to microalgae of the genus Pavlova, Euglena, Spirulina, or Chlorella.

6. The composition according to claim 1 or 2, for oral or external use.

7. A method for improving the stability of extracellular vesicles derived from microalgae by co-existing one or more selected from the group consisting of (B-1) to (B-3) with the extracellular vesicles derived from microalgae: (B-1) one or more selected from the group consisting of primary alcohols and polyhydric alcohols, (B-2) one or more selected from the group consisting of organic acids and their salts, and (B-3) a chelating agent.

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