Composition, cyclic adenosine monophosphate increase agent, method for producing the composition, method for producing the cyclic adenosine monophosphate increase agent, and raw material

Abstract

The present invention provides a seaweed gametophyte and / or sporophyte extract composition and a method for producing the same that can effectively increase the amount of cAMP in cells. [Solution] A composition comprising at least one protein selected from ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs) derived from the gametophyte and / or sporophyte of seaweed.

Description

[Technical Field]

[0001] The present invention relates to a composition comprising extracellular vesicles (EVs) derived from the gametophyte of seaweed and a predetermined protein. The present invention also relates to a cyclic adenosine monophosphate (cAMP) enhancer containing the composition. The present invention also relates to a method for producing the composition and a method for producing the increasing agent of the cyclic adenosine monophosphate. Furthermore, this invention relates to raw materials. [Background technology]

[0002] Seaweed such as wakame and kelp are rich in nutrients such as protein, dietary fiber, vitamins, and minerals, and their extracts have been used in food, cosmetics, pharmaceuticals, and other products. For example, Patent Document 1 discloses a method for extracting water-soluble components of seaweed, characterized by adding water to finely chopped or powdered seaweed and mixing it, then wet grinding it at room temperature to dissolve and extract the water-soluble components of the seaweed in water. Furthermore, Patent Document 2 discloses a wakame protein-containing composition obtained by adding water, a saline solution, or a weakly alkaline aqueous solution to the thallus or dried granules of a wakame seaweed, wet grinding to extract soluble components, and then separating the protein from this extract.

[0003] Furthermore, Patent Document 3 discloses a method for isolating a lipophilic extract from the gametophyte of seaweed, which involves mixing an aqueous-alcoholic suspension of brown algae gametophyte cells with at least one fatty acid triglyceride containing 8 to 22 carbon atoms, and then adding water to isolate the lipophilic extract. Patent Document 4 discloses a cosmetic preparation for topical use characterized by containing a freeze-dried product of brown algae gametophyte cells as an active ingredient, and also discloses that the freeze-dried product of brown algae cells contains 1% or more fucoxanthin.

[0004] Incidentally, a substance called cAMP (cyclic adenosine monophosphate) is known as a second messenger that plays an important role in intracellular signal transduction. This substance activates an enzyme called protein kinase A (PKA) and regulates various intracellular reactions. Furthermore, cAMP is known to play a wide range of roles in the body, including activating transcription factors (proteins that regulate gene transcription), cell growth and differentiation, regulating neurotransmission, and activating dermal papilla cells, and is expected to have applications in the pharmaceutical and cosmetic fields. For example, Patent Document 6 discloses a gray hair prevention and blackening agent that contains a cAMP increasing agent and a protein kinase C inhibitor. In Patent Document 6, adenosine and forskolin are listed as examples of cAMP increasing agents. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2004-49072 [Patent Document 2] Japanese Patent Publication No. 2004-97021 [Patent Document 3] Patent No. 6731425 [Patent Document 4] Patent No. 4979592 [Patent Document 5] Special Publication No. 2023-520101 [Patent Document 6] Japanese Patent Application Publication No. 4-124122 [Overview of the project] [Problems that the invention aims to solve]

[0006] The inventors of this application investigated whether the amount of cAMP in cells could be increased using the seaweed extracts described in Patent Documents 1 to 4, which are relatively inexpensive natural materials. However, the extracts from Patent Documents 1 to 4 were not able to sufficiently increase the amount of cAMP in cells.

[0007] The present invention has been made in view of the above-mentioned problems, and aims to provide a seaweed gametophyte and / or sporophyte extraction composition and a method for producing the same that can effectively increase the amount of cAMP in cells. [Means for solving the problem]

[0008] As a result of diligent research, the inventors have discovered that by applying a composition containing a predetermined protein constituting an enzyme and extracellular vesicles (EVs) derived from the gametophyte and / or sporophyte of seaweed to cells, it is possible to increase cAMP (cyclic adenosine monophosphate) in cells.

[0009] The present invention relates to a composition comprising at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs) derived from the gametophyte and / or sporophyte of seaweed.

[0010] In this invention, extracellular vesicles (EVs) refer to a general term for vesicles having a lipid bilayer structure secreted from living cells, and the concept of extracellular vesicles includes exosomes, microvesicles, and apoptotic bodies. Exosomes are 1 to 200 nm in diameter. Exosomes are formed by inward budding of the late endosomal membrane, and then the endosome fuses with the cell membrane to form a complete particle which is secreted extracellularly by exocytosis. Microvesicles (MVs) are generated by the outward budding and separation of the cell membrane and are 100 to 1000 nm in diameter. Apoptotic bodies are produced when cells undergo systematic cell death (apoptosis) and are 50 to 5000 nm in diameter.

[0011] The composition of the present invention comprises, as a protein, at least one selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit.

[0012] The ribulose-1,5-bisphosphate carboxylase large subunit is a major subunit of Ribulose-1,5-bisphosphate carboxylase / oxygenase (hereinafter also referred to as "RuBisCO"), the most important enzyme in photosynthesis, and is responsible for the catalytic active site of RuBisCO. In other words, RuBisCO catalyzes the reaction in which carbon dioxide (CO2) is bound to ribulose-1,5-bisphosphate (RuBP) to produce 3-phosphoglycerate (3-PGA). This reaction is the first step in the Calvin cycle of photosynthesis and constitutes a major reaction in carbon fixation in photosynthesis. The ribulose 1,5-bisphosphate carboxylase small subunit is the small subunit of RuBisCO, the most important enzyme in photosynthesis. Although it is not directly involved in the catalytic active site of RuBisCO, it binds to the large subunit of RuBisCO to enhance the stability of RuBisCO and promote its activation. RuBisCO is composed of 8 large subunits (large subunit, LSU) and 8 small subunits (small subunit, SSU).

[0013] As described above, ribulose 1,5-bisphosphate carboxylase / oxygenase is an enzyme involved in photosynthesis. In the present invention, it is considered that the large subunit (large subunit, LSU) and small subunit (small subunit, SSU) of RuBisCO that make up this enzyme bind to and act on receptors in the cell membrane of mammalian cells.

[0014] When the composition of the present invention acts on mammalian cells, extracellular vesicles (EVs) assist the proteins constituting the ribulose 1,5-bisphosphate carboxylase / oxygenase large subunit and small subunit to bind to the receptors of the cell membrane constituting mammalian cells. When the proteins constituting the ribulose 1,5-bisphosphate carboxylase / oxygenase large subunit and small subunit bind to the receptors of the cell membrane constituting mammalian cells, the receptors are activated, and a protein called G protein is activated. This G protein is presumed to activate an enzyme called adenylate cyclase, and further, this enzyme converts adenosine triphosphate (ATP) into cAMP (cyclic adenosine monophosphate). By increasing cAMP (cyclic adenosine monophosphate) in mammalian cells, activation of transcription factors, which are proteins that regulate gene transcription, improvement of cell growth and differentiation, improvement of neurotransmission, and activation of dermal papilla cells are expected.

[0015] In the composition of the present invention, it may further contain at least one selected from ATP synthase subunit beta, Thiamine biosynthesis protein G, and Histone H2B. ATP synthase is composed of two parts, F1 and F0. The subunit beta of ATP synthase is present in the F1 part and plays an important role in forming the catalytic site of ATP synthesis. The subunit beta of ATP synthase catalyzes the reaction of binding ADP (adenosine diphosphate) and phosphate to synthesize ATP. Histone H2B is a protein present in the nucleus of eukaryotes. It is a type of histone protein that binds to DNA to form a structure called a nucleosome. It is one of the core histone proteins that wrap DNA, plays a role in compactly storing DNA in the nucleus, and regulates gene expression by controlling the accessibility of DNA. Furthermore, Thiamine biosynthesis protein G is an enzyme involved in the biosynthesis of vitamin B1 (thiamine). It is said to catalyze the phosphorylation of hydroxymethyldihydropyrimidine (HMP) to hydroxymethyldihydropyrimidine diphosphate (HMP-PP), which is an important step in the thiamine biosynthesis pathway. The "G" in Thiamine biosynthesis protein G is derived from the fact that this protein is involved in the 7th stage of the thiamine biosynthesis pathway. The thiamine biosynthesis pathway is a complex pathway involving multiple enzymes, and each stage is named with an alphabet. Thiamine biosynthesis protein G plays a role in the 7th stage of this pathway by binding hydroxymethyldihydropyrimidine (HMP) and a pyrimidine dimer to generate hydroxymethyldihydropyrimidine diphosphate (HMP-PP). These proteins are thought to exert similar effects to at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit.

[0016] In the composition of the present invention, the extracellular vesicles (EVs) must be derived from seaweed, but the term "seaweed" in this invention refers to a group of marine species of multicellular algae and does not include microalgae, which are unicellular algae.

[0017] In the composition of the present invention, the seaweed must be the gametophyte and / or sporophyte of the seaweed. Seaweed exhibits various forms throughout its life cycle, including sporophytes (thallus, discus, filamentous) and gynandrophytes (thallus, discus, filamentous). Gynandrophytes and the filamentous sporophytes are desirable in this invention because they facilitate the extraction of extracellular vesicles (EVs) and the aforementioned proteins.

[0018] Furthermore, Patent Document 5 discloses extracellular vesicles derived from microalgae, wherein the extracellular vesicles are bio-lipid membrane nanovesicles having particle sizes in the range of 50-300 nm (small extracellular vesicles, sEVs) or 300-2 μm (large extracellular vesicles, lEVs), contained within a lipid bilayer membrane, and the extracellular vesicles are derived from natural photosynthetic non-fermenting microalgae and contain at least the extracellular vesicle protein marker Alix and optionally one or more further protein markers selected from the group consisting of enolase, actin, and any combination thereof. However, these are extracellular vesicles derived from microalgae, and the proteins they contain are Alix, enolase, and actin, which are different from those of the present invention. Furthermore, while ribulose-1,5-bisphosphate carboxylase / oxygenase is an enzyme involved in photosynthesis, it can be understood from Patent Document 5 that it is not commonly extracted from all photosynthetic algae.

[0019] In the composition of the present invention, it is preferable that the seaweed be the filamentous form of the female gametophyte and / or sporophyte of the seaweed. This is because the filamentous form of the female gametophyte and sporophyte is easily used to extract extracellular vesicles (EVs) and proteins, and it contains the highest amount of fucoxanthin or its derivatives, as described later, among the various forms in the life cycle of seaweed.

[0020] In the composition of the present invention, the seaweed is preferably a brown alga. Examples of seaweed include green algae, red algae, and brown algae, but brown algae are preferred because they allow for easy extraction of extracellular vesicles (EVs) and proteins, and have a high content of fucoxanthin or its derivatives, as described later.

[0021] In the composition of the present invention, the composition comprising at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit and extracellular vesicles (EVs) derived from the gametophyte and / or sporophyte of seaweed preferably contains an aqueous solvent or a mixed solvent consisting of an aqueous solvent and an organic solvent, wherein the organic solvent preferably dissolves at least one compound selected from the group consisting of fucoxanthin and its derivatives, as described later.

[0022] In the compositions of the present invention, compositions comprising at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs), preferably contain an aqueous solvent. This is because the lipid membrane of extracellular vesicles is less likely to break down in an aqueous solvent, and their morphology is more easily maintained. In organic solvents such as alcohols used to dissolve fucoxanthin and its derivatives, as described later, the lipid membrane of extracellular vesicles is easily destroyed, and the proteins are also easily denatured.

[0023] The aqueous solvent mentioned above is water or a solvent in which water-soluble salts are dissolved, and includes pure water, seawater, phosphate-buffered saline (PBS), etc. Antifoaming agents, thixotropic agents, pH adjusters, etc., may be added to the aqueous solvent as appropriate. Artificial seawater, filtered seawater, or deep-sea water can also be used as seawater.

[0024] Furthermore, the composition of the present invention may also contain a mixed solvent comprising an aqueous solvent and an organic solvent. Preferably, the organic solvent is capable of dissolving at least one compound selected from the group consisting of fucoxanthin and its derivatives, as described later. The presence of an aqueous solvent makes it less likely for extracellular vesicles (EVs) to be destroyed, even when an organic solvent that dissolves at least one compound selected from the group consisting of fucoxanthin and its derivatives is used as the solvent.

[0025] In a mixed solvent composition of an aqueous solvent and an organic solvent, the weight ratio of the organic solvent to the aqueous solvent is preferably organic solvent / aqueous solvent ≤ 9 / 1, and more preferably organic solvent / aqueous solvent = 1 / 10 to 10 / 10.

[0026] The composition of the present invention may further contain at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, extracellular vesicles (EVs) derived from the gametophyte and / or sporophyte of seaweed, and at least one compound selected from the group consisting of fucoxanthin and its derivatives. Fucoxanthin does not contribute to the increase of cAMP (cyclic adenosine monophosphate) in mammalian cells, but it is a beneficial substance known for its ability to suppress melanin production and its antioxidant effects.

[0027] Examples of fucoxanthin derivatives include hydrolysates or derivatives of fucoxanthin, esters of fucoxanthin (e.g., esters with amino acids, carboxylic acids, inorganic acids, or fatty acids), salts thereof, and fucoxanthin glycosides. More specifically, derivatives of fucoxanthin include, but are not limited to, fucoxanthinol, a hydrolysis product; amarauciaxanthin A, obtained by dehydrating and isomerizing fucoxanthinol; esters of fucoxanthin with amino acids such as glycine and alanine; esters of fucoxanthin with carboxylic acids such as acetic acid and citric acid and their salts; esters of fucoxanthin with inorganic acids such as phosphoric acid and sulfuric acid and their salts; or monoesters and identical or different diesters selected from fucoxanthin with fatty acid esters of highly unsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid, unsaturated fatty acids such as oleic acid and linoleic acid, and saturated fatty acids such as palmitic acid and stearic acid, as well as glycosides such as glucosides.

[0028] In the composition of the present invention, the composition comprising at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs) derived from the gametophyte and / or sporophyte of seaweed, is preferably in powder form. Powdered compositions have a smaller volume per component compared to compositions containing solvents, thus reducing transportation costs. Furthermore, they are easily mixed with other raw materials when used in cosmetics, pharmaceuticals, food ingredients, reagents, or their raw materials, making them useful as industrial raw materials.

[0029] In the composition of the present invention, it is desirable that at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit is encapsulated in or attached to the extracellular vesicles (EVs). This is because the extracellular vesicles (EVs) readily bind the protein to receptors located on the cell membrane of mammalian cells.

[0030] The intracellular cyclic adenosine monophosphate (cAMP) increasing agent of the present invention includes the composition of the present invention described above. As described above, the composition of the present invention has the effect of increasing intracellular cyclic adenosine monophosphate (cAMP), and is therefore useful as an agent for increasing intracellular cyclic adenosine monophosphate (cAMP).

[0031] The present invention provides a method for producing a composition comprising at least one protein selected from ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs) derived from the gametophyte and / or sporophyte of seaweed, comprising the following steps (1) and (2). Step (1): The gametophyte and / or sporophyte of the seaweed are dried to obtain a dried product, and then the dried product is pulverized to obtain a powder of the gametophyte and / or sporophyte of the seaweed, and the powder is dispersed in an aqueous solvent adjusted to a temperature of 5°C or lower. Step (2): The aqueous solvent in which the powder is dispersed is separated into solids and supernatant by centrifugation, and the supernatant is collected.

[0032] The gametophyte and / or sporophyte of seaweed release extracellular vesicles (EVs) and proteins in an aqueous medium at temperatures below 5°C. The gametophyte and / or sporophyte of seaweed are dispersed in an aqueous solvent, and the extracellular vesicles (EVs) and proteins are extracted into the dispersion. At least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit is insoluble in water, but it is dispersed in the aqueous solvent together with the extracellular vesicles (EVs) by being encapsulated within the EVs or by attaching to the lipid membrane on the surface of the EVs. By extracting at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit in an aqueous solvent at a low temperature of 5°C or below, the protein can be extracted without degradation and while maintaining its structure.

[0033] Extracellular vesicles (EVs) may originate from the female gametophyte of the seaweed, or from the filamentous sporophyte of the seaweed. The filamentous structures of the female gametophyte and sporophyte are desirable in this invention because they facilitate the extraction of extracellular vesicles (EVs) and the aforementioned proteins.

[0034] If the aqueous solvent contains organic solvents that dissolve fucoxanthin and its derivatives, such as ethanol or butylene glycol, the extracellular vesicles (EVs) immediately after release from the gametophyte and / or sporophyte are unstable, and the lipid membranes of the extracellular vesicles (EVs) are dissolved and destroyed. For this reason, it is desirable that the solvent used to disperse the gametophyte and / or sporophyte be aqueous, and that such organic solvents are not included in the aqueous solvent. It is also preferable that the solvent does not contain organic solvents that dissolve at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0035] A dispersion of dried powder of the gametophyte and / or sporophyte of seaweed is separated into a solid portion and its supernatant by centrifugation. The solid portion is removed from the dispersion to obtain the supernatant, which contains at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs) derived from the gametophyte and / or sporophyte of seaweed. It is desirable to perform the centrifugation in an atmosphere adjusted to 5°C or below.

[0036] The method for producing the composition of the present invention may include the step of adding at least one compound selected from the group consisting of fucoxanthin and its derivatives to the supernatant liquid. At least one compound selected from the group consisting of fucoxanthin and its derivatives may be chemically synthesized or extracted from the gametophyte and / or sporophyte of seaweed.

[0037] The method for producing the composition of the present invention may include the step of removing the aqueous solvent from the supernatant to obtain a powder containing at least one protein selected from ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs) derived from the gametophyte and / or sporophyte of seaweed. By removing the aqueous solvent, a powdered composition is obtained.

[0038] The composition produced by the method for producing the composition of the present invention may include at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, as well as raw materials used to extract extracellular vesicles (EVs) derived from the gametophyte and / or sporophyte of seaweed. The gametophytes and / or sporophytes of seaweed are approximately 200 μm to 2000 μm in size, making them easy to culture in small spaces. Furthermore, compared to other forms in the life cycle of seaweed, such as the sporophyte (thallus, discus, filamentous), it is easier to extract extracellular vesicles (EVs) and at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit. Additionally, they have a high fucoxanthin content, making them excellent raw materials for extracting extracellular vesicles (EVs), proteins, or, if necessary, at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0039] The seaweed used in the method for producing the composition of the present invention is preferably brown algae. This is because it is easier to extract at least one protein or extracellular vesicles (EVs) selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit from brown algae. Furthermore, brown algae have the highest content of fucoxanthin and its derivatives among all types of seaweed, which is advantageous when extracting fucoxanthin and the like.

[0040] The seaweed used in the method for producing the composition of the present invention is preferably the filamentous form of the female gametophyte and / or sporophyte. This is because it is easier to extract at least one protein or extracellular vesicles (EVs) selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit. Furthermore, the content of fucoxanthin and its derivatives is higher than that of the male gametophyte, allowing for more efficient extraction.

[0041] Extracellular vesicles (EVs) derived from seaweed can be used to improve the stability of at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0042] The method for producing the intracellular cyclic adenosine monophosphate (cAMP) increasing agent of the present invention may include the method for producing the composition of the present invention described above.

[0043] The raw material of the present invention comprises an aggregate of gametophytes and / or sporophytes of seaweed and is used to extract at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, as well as extracellular vesicles (EVs) derived from the gametophytes and / or sporophytes of seaweed. By using such raw materials, the composition of the present invention described above can be manufactured.

[0044] In the raw materials of the present invention, the seaweed is preferably brown algae. Furthermore, in the raw material of the present invention, the seaweed is preferably in the form of a female gametophyte or a filamentous sporophyte. [Effects of the Invention]

[0045] According to the present invention, it is possible to increase cAMP (cyclic adenosine monophosphate) in mammalian cells, which is expected to activate transcription factors, which are proteins that regulate gene transcription, promote cell growth and differentiation, improve neurotransmission, and activate dermal papilla cells. Furthermore, the composition of the present invention is thought to have effects other than increasing cAMP in mammalian cells, such as inhibiting melanin production in mammalian cells, as well as anti-inflammatory and antioxidant effects on mammalian cells. As a result, the composition of the present invention can be used as a raw material for various industrial applications. These applications include a wide range of uses such as pharmaceuticals, food products, cosmetics, and reagents. Furthermore, the terms "ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit" and "ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit" used in this invention are established technical terms, as they can be found in publications such as Japanese Patent Publication No. 2018-131414, Japanese Patent Publication No. 2023-120242, Japanese Patent No. 7298905, Production Research Vol. 64 No. 3 (2012) pp. 351-357, and Japanese Patent Publication No. Hei 9-252778. Furthermore, histone H2B is described in Japanese Patent Publication No. 3705768 and Japanese Patent Publication No. 7104689, and ATP synthase subunit β is described in Japanese Patent Publication No. 2011-188744, among others, and all of these are used as technical terms by those skilled in the art. Furthermore, the terms "ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit" and "ribulose bisphosphate carboxylase large chain" are sometimes used by those skilled in the art to refer to the same protein. Therefore, in this invention, "ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit" is synonymous with "ribulose bisphosphate carboxylase large chain." Furthermore, "Ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit" and "Ribulose bisphosphate carboxylase small subunit" are sometimes used by those skilled in the art as terms meaning the same protein. Therefore, in this invention, "Ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit" is synonymous with "Ribulose bisphosphate carboxylase small subunit". [Brief explanation of the drawing]

[0046] [Figure 1] Figure 1 is a schematic diagram showing the male and female gametophytes of wakame seaweed. [Figure 2]Figure 2 is a liquid chromatography chart of proteins and lipids in extracellular vesicles (EVs) in an aqueous extract of the female gametophyte of wakame seaweed (solid lines: proteins, dotted lines: lipids). [Figure 3A] Figure 3A is a TEM image of extracellular vesicles (EVs) extracted from the female gametophyte of wakame seaweed (50,000x magnification). [Figure 3B] Figure 3B is a TEM image (100,000x magnification) of extracellular vesicles (EVs) extracted from the female gametophyte of wakame seaweed. [Figure 3C] Figure 3C is a TEM image (250,000x magnification) of extracellular vesicles (EVs) extracted from the female gametophyte of wakame seaweed. [Figure 3D] Figure 3D is a TEM image of extracellular vesicles (EVs) extracted from the female gametophyte of wakame seaweed (300,000x magnification). [Figure 4] Figure 4 is a liquid chromatography chart of proteins and lipids in extracellular vesicles (EVs) in an aqueous extract of Uppuru Inori (solid lines: proteins, dotted lines: lipids). [Figure 5] Figure 5 is a liquid chromatography chart of proteins and lipids in extracellular vesicles (EVs) in aqueous extracts of the male gametophyte of Sargassum fuscipes (solid lines: proteins, dotted lines: lipids). [Figure 6] Figure 6 is a liquid chromatography chart of proteins and lipids in extracellular vesicles (EVs) in an aqueous extract of the filamentous sporophytes of *Lysimachia japonica* (solid lines: proteins, dotted lines: lipids). [Figure 7] Figure 7 is a liquid chromatography chart of proteins and lipids in extracellular vesicles in an extract obtained by water extraction of the female gametophyte of wakame seaweed, followed by extraction with 100% butylene glycol (solid lines: proteins, dotted lines: lipids). [Figure 8A] Figure 8A is a liquid chromatography chart of fucoxanthin in the BG extract of the female gametophyte of wakame seaweed. In Figure 8A, the peak around 13 minutes corresponds to fucoxanthin, and the peak around 11.3 minutes corresponds to chlorophyll. [Figure 8B]Figure 8B is a liquid chromatography chart of fucoxanthin in a BG extract of wakame seaweed thallus. In Figure 8B, the peak around 13 minutes corresponds to fucoxanthin, and the peak around 11.3 minutes corresponds to chlorophyll. [Figure 9] Figure 9 shows the proteins in the composition of the present invention separated based on molecular weight by SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). [Figure 10] Figure 10 is a graph comparing the increase in cAMP in PSVK1 (human keratinocyte) cells using water extracts from Examples 1-1, 1-2, Comparative Examples 1-1, 1-2, 2, and 3. The vertical axis shows the amount of luminescence due to the luciferin reaction; a lower amount of luminescence indicates a higher amount of cAMP in the cells. [Figure 11] Figure 11 is a graph comparing the increase in cAMP in PSVK1 (human keratinocyte) cells using the aqueous extracts from Examples 2, 3, and 4. The vertical axis shows the amount of luminescence due to the luciferin reaction; a lower amount of luminescence indicates a higher amount of cAMP in the cells. [Figure 12] Figure 12 is a chart showing the analysis of extracellular vesicle proteins and lipids in an extract obtained by ethanol extraction of the female gametophyte of wakame seaweed, using liquid chromatography (solid lines: proteins, dotted lines: lipids). [Figure 13] Figure 13 is a chart showing the analysis of extracellular vesicle proteins and lipids in water-extracted wakame seaweed thallus using liquid chromatography (solid lines: proteins, dotted lines: lipids). [Figure 14] Figure 14 is a chart showing the analysis of extracellular vesicle proteins and lipids in an extract obtained by water extraction of the thallus of *Mozuku* seaweed using liquid chromatography (solid lines: proteins, dotted lines: lipids). [Figure 15]Figure 15 is a chart showing the results of liquid chromatography analysis of extracellular vesicle proteins and lipid membranes in diluted solutions of aqueous extracts of the female gametophyte of wakame seaweed, prepared by diluting with a mixture of phosphate-buffered saline and butylene glycol (solid lines: proteins, dotted lines: lipids). [Figure 16] Figure 16 shows the proteins in an aqueous extract of adult wakame seaweed thallus, separated by molecular weight using SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). [Figure 17] Figure 17 compares the amount of melanin produced per living cell for aqueous extracts of the filamentous sporophytes of *Uriaria japonica*, *Uriaria serrata*, *Wakame seaweed* female gametophytes, and *Wakame seaweed* thallus (adult). [Modes for carrying out the invention]

[0047] The composition of the present invention comprises at least one protein selected from ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs) derived from the gametophyte and / or sporophyte of seaweed.

[0048] Ribulose 1,5-bisphosphate carboxylase / oxygenase is an enzyme involved in photosynthesis, but in this invention, it is thought to act by binding to a receptor in the cell membrane of mammalian cells. When the composition of the present invention is applied to mammalian cells, extracellular vesicles (EVs) assist in the binding of proteins constituting the ribulose 1,5-bisphosphate carboxylase / oxygenase large and small subunits to receptors on the cell membrane of mammalian cells. When the proteins constituting the ribulose 1,5-bisphosphate carboxylase / oxygenase large and small subunits bind to receptors on the cell membrane of mammalian cells, the receptors are activated, which in turn activates a protein called a G protein. This G protein is thought to activate an enzyme called adenylate cyclase, which further converts adenosine triphosphate (ATP) to cAMP (cyclic adenosine monophosphate).

[0049] Increasing cAMP (cyclic adenosine monophosphate) in mammalian cells is expected to activate transcription factors, which are proteins that regulate gene transcription, promote cell growth and differentiation, improve neurotransmission, and activate hair papilla cells. Furthermore, the composition of the present invention is believed to have effects such as inhibiting melanin production in mammalian cells, as well as anti-inflammatory and antioxidant effects on mammalian cells. Therefore, it is expected to have uses as a raw material or additive in pharmaceuticals, cosmetics, food products, reagents, and other products.

[0050] The identification of proteins contained in the composition of the present invention is carried out by the following method. Specifically, proteins are separated in a gel according to their molecular weight using SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). The molecular weight bands of the separated proteins are visualized using a chromogenic agent. The darker areas of the molecular weight bands are excised as protein bands to obtain gel fragments. These gel fragments are then degraded into peptides using trypsin. The resulting peptide fragments are separated using a highly sensitive liquid chromatography called nanoLC, achieved by miniaturizing the packing material and column diameter. The amino acid sequence of the peptides is then identified using a tandem mass spectrometer (MS / MS) following the LC. The results are then compared with proteins registered in a database to identify the proteins contained in the gel fragments. Even if the species of origin of the protein is unknown, the species and protein can be estimated by comparing the results with a database of all species to find proteins with the same amino acid sequence. The database used was MASCOT from Matrix Science. Of the peptides obtained by enzymatic degradation with trypsin, only a portion can have their amino acid sequences determined using a mass spectrometer. However, in species with genome databases, a protein can be identified if even a small number of peptide amino acids match. The identification of such proteins can be carried out, for example, by Nippon Proteomics Co., Ltd. (6-6-3 Minamiyoshinari, Aoba-ku, Sendai City, Miyagi Prefecture 989-3204, Japan).

[0051] The composition of the present invention may further contain fucoxanthin or its derivatives. This is because fucoxanthin or its derivatives do not contribute to the increase of cAMP in cells, but they have been shown to inhibit melanin production and have antioxidant effects. Examples of fucoxanthin derivatives include hydrolysates or derivatives of fucoxanthin, esters of fucoxanthin (e.g., esters with amino acids, carboxylic acids, inorganic acids, or fatty acids), salts thereof, and fucoxanthin glycosides. Examples of fucoxanthin derivatives include, but are not limited to, monoesters and diesters of the same or different types, glycosides, etc., selected from fucoxanthin derivatives such as fucoxanthin, fucoxanthin, fucoxanthin, fucoxanthin, ferro

[0052] The total content of at least one compound selected from the group consisting of fucoxanthin and its derivatives in the composition of the present invention is not particularly limited, but may be, for example, 0.001 to 50% by weight in the composition.

[0053] Furthermore, extracellular vesicles (EVs) released from the gametophyte and / or sporophyte of seaweed can be used. Examples of seaweed include green algae, red algae, and brown algae. Examples of green algae include *Ulva lactuca*, *Monostroma*, *Euphyllia*, *Euphyllia glabrescens*, *Euphyllia erythrosora*, and *Caulerpa lentillifera*. Examples of red algae include *Ulva perforata*, *Ulva serrata*, *Ulva perforata*, *Porphyra japonica*, *Porphyra japonica* (Japanese name: *Haitan Amanori*), *Ulva perforata*, *Porphyra japonica*, *Ulva perforata*, *Ulva perforata*, *Ulva perforata*, *Ulva perforata*, and *Ulva perforata*. Examples of brown algae include *Wakame*, *Mozuku* (Okinawa mozuku, Itomozuku), *Haba nori*, *Hirome*, *Ao wakame*, *Sagara*, *Kayamonori*, *Kelp*, *Makonbu*, *Hosome konbu*, *Naga konbu*, *Mitsui konbu*, *Kajime*, *Kurome*, *Hondawara*, *Arame*, and *Euphyllia glabrescens*. Among seaweeds, brown algae are the most desirable because they contain a high amount of fucoxanthin or its derivatives. Among brown algae, wakame is particularly preferred. Seaweed can take various forms, including sporophytes (thallus, discus, filamentous) and gynandrophytes (thallus, discus, filamentous). Gynandrophytes are desirable in this invention because they readily release at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, as well as extracellular vesicles (EVs), and they contain a high amount of fucoxanthin or its derivatives. In particular, the filamentous forms of the female gametophyte and / or sporophyte are advantageous because they readily release enzyme-constituting proteins and extracellular vesicles at low temperatures of 5°C or below, and they contain a higher amount of fucoxanthin or its derivatives compared to the male gametophyte.

[0054] The composition of the present invention preferably contains an aqueous solvent. This is because the lipid membrane of extracellular vesicles (EVs) is less likely to break down in an aqueous solvent, and their morphology is more easily maintained. The aqueous solvent is water or a solvent in which water-soluble salts are dissolved, and includes pure water and seawater. Antifoaming agents, thixotropic agents, pH adjusters, etc., may be added to the aqueous solvent as appropriate. Artificial seawater, filtered seawater, or deep-sea water can also be used as seawater. Examples of salts include sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, ammonium sulfate, potassium phosphate, potassium dihydrogen phosphate, iron phosphate, iron fulvicate, and sodium citrate. The aqueous solvent may also be phosphate-buffered saline (PBS). The composition of the present invention may also contain an organic solvent in addition to the aqueous solvent. As the organic solvent, an organic solvent that dissolves at least one compound selected from the group consisting of fucoxanthin and its derivatives is preferred. Furthermore, the composition of the present invention may also contain a mixed solvent consisting of an aqueous solvent and an organic solvent. In the present invention, the organic solvent is preferably an organic solvent that dissolves at least one compound selected from the group consisting of fucoxanthin and its derivatives. The organic solvent may be one type or two or more types. The presence of an aqueous solvent prevents the destruction of extracellular vesicles (EVs) even when an organic solvent is present. The weight ratio of the organic solvent to the aqueous solvent is preferably organic solvent / aqueous solvent ≤ 9 / 1, and organic solvent / aqueous solvent = 1 / 10 to 10 / 10.

[0055] In one embodiment of the present invention, the composition comprising at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs) derived from the gametophyte of seaweed, is preferably a powder composition. Since powder compositions have a smaller volume per component compared to compositions containing solvents, transportation costs can be reduced. Furthermore, when used as a cosmetic, pharmaceutical, reagent, or food ingredient, they are easily mixed with other raw materials and are useful as industrial raw materials.

[0056] In the present invention, it is desirable that at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, as well as ATP synthase subunit beta, thiamine biosynthesis protein G, or histone H2B, be attached to or encapsulated within the extracellular vesicles (EVs). This is because extracellular vesicles (EVs) inherently have the property of transporting proteins between cells, and therefore the extracellular vesicles (EVs) can more easily assist the aforementioned proteins in binding to receptors on the cell membranes that constitute mammalian cells.

[0057] The composition of the present invention may contain components other than those described above, as long as they do not impair the effects of the present invention. Examples of such components include excipients and additives that can be used in cosmetics, pharmaceuticals, reagents, foods, and their raw materials.

[0058] Next, the method for producing the composition of the present invention will be described.

[0059] The composition of the present invention can be produced, for example, using the gametophyte of seaweed as a raw material by a method comprising the following steps (1) and (2). Step (1): The gametophyte of the seaweed is dried to obtain a dried product, and then the dried product is crushed to obtain a powder of the seaweed gametophyte. This powder is then dispersed in an aqueous solvent adjusted to a temperature of 5°C or lower. Step (2): The aqueous solvent in which the powder is dispersed is separated into solids and supernatant by centrifugation, and the supernatant is collected.

[0060] A composition can be produced by a method including steps (1) and (2) above, comprising at least one protein selected from ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs) derived from the gametophyte of seaweed. The manufacturing method of the present invention may include steps other than steps (1) and (2). For example, it may include a drying step, which will be described later.

[0061] The gametophyte of the seaweed used in step (1) above may be a female gametophyte or a filamentous sporophyte. Examples of seaweed include those mentioned above, preferably brown algae, and preferably wakame.

[0062] The following method will be explained using the female gametophyte of seaweed as an example, but it is not limited to this. First, we will explain one example of how to obtain the gametophyte of seaweed, using wakame as an example.

[0063] (I) Development of the gametophyte of seaweed The gametophytes that develop from the zoospores of wakame seaweed are separated into one male and one female, propagated, mechanically shredded, attached to or adhering to threads, and fertilized on the threads. The young wakame leaves that emerge from this stage will be grown in indoor culture and in natural seawater. From the mature wakame thallus (adult), spore-bearing leaves (mekabu) will be obtained.

[0064] 1. Preservation of spore-bearing fronds 1) Place the spore-bearing fronds in a plastic bag or container and store them in a cool, dark place at 15-20°C. Below 15°C, zoospores will not be easily released when returned to seawater. If the spore-bearing fronds are removed from seawater and stored in a cool, dark place, zoospore release is possible for 2-3 days.

[0065] 2. Collection of zoospores 1) The ideal room temperature for releasing zoospores is 15-20°C. Higher temperatures are undesirable because they shorten the zoospore's swimming time.

[0066] 2) Cut the spore-bearing fronds into pieces about 3-4 cm square. The part closest to the rhizoids releases spores well, but cut off the part of the spore-bearing frond surface that is as clean as possible. Gently wipe off any dirt from the cut leaf pieces with absorbent paper or similar.

[0067] 3) Prepare three beakers containing 100 mL of sterile seawater, wash the leaf fragments one by one, and then place them in petri dishes containing 50 mL of sterile seawater.

[0068] 4) Place the petri dish containing the leaf fragments on the stereomicroscope stage and shine light from above the petri dish to induce zoospore release. Adjusting the stereomicroscope to a dark-field setting makes it easier to observe zoospore release. After about 10 minutes of illumination using an optical fiber or similar device, sufficient zoospores will be released.

[0069] 5) Prepare a capillary tube. Use either a heated and stretched hematocrit tube, or a Pasteur pipette tip that has been sufficiently stretched to a long, thin shape.

[0070] 6) Prepare a petri dish filled with 50 mL of PESI culture medium.

[0071] 7) Aspirate an appropriate amount of zoospores under a stereomicroscope and drop them into a petri dish. Take care to prevent the capillary tube from touching the bottom of the petri dish or leaf fragments during aspiration (diatoms are often aspirated). After dropping, shake the petri dish thoroughly by hand to equalize the zoospore density.

[0072] 8) Prepare about four different petri dishes, each with a different amount of zoospore fluid aspirated. A large amount of zoospores will increase the density of the gametophytes, causing them to grow too close together and making isolation difficult.

[0073] 9) After collecting zoospores, the petri dishes should be cultured at 10°C to 30°C with 14 to 12 hours of light (1000 to 1500 lux). Since the gametophytes can become similar in shape and difficult to distinguish between males and females when there are large temperature fluctuations, high temperatures, or high light levels, care should be taken to maintain homeostasis in the culture conditions.

[0074] 10) The gametophytes will reach a size where their sex can be determined in two weeks. Due to the risk of fertilization, the sex of the gametophytes should be determined and isolated as soon as possible.

[0075] 11) At this stage, there is little to no contamination by diatoms, but if diatoms appear, discard the petri dish. If discarding is not possible, germanium dioxide can be used to suppress diatom growth, and the gametophyte that is not contaminated by diatoms can be isolated.

[0076] 3. Isolation of male and female gametophytes 1) Place the petri dish containing the cultured gametophytes on the stage of the inverted microscope and search for female gametophytes suitable for isolation. Isolate those gametophytes that are sufficiently separated from each other and whose sex is clearly defined. Attach a tube to a Pasteur pipette and separate the female gametophytes from the petri dish and aspirate them. Place the aspirated female gametophytes one by one into microplates filled with PESI medium. Schematic diagrams of female and male gametophytes are shown in Figure 1. 2) Cultivate for one month at 10-30°C with 14-12 hours of light (1500-2000 lux).

[0077] 4. Preservation of the female gametophyte 1) Remove the female gametophytes from the microplate after culturing. Normally, the female gametophytes are large enough to be seen with the naked eye, so pick them up from the microplate with ophthalmic forceps. If they are not large enough, you can use a Pasteur pipette to aspirate them under an inverted microscope.

[0078] 2) When storing, place in a screw-cap test tube and store at 10°C to 30°C with 14 hours of light (1000 to 1500 lux). After storage, the PESI medium is replaced once every two months.

[0079] (II) Cultivation of seaweed gametophytes For the culture medium of the female gametophyte, Provasoli's Enriched Seawater (PES) or its improved form, PESI culture medium, is preferable. The PES culture medium has the following composition. (See [Hirokazu Ariga, Isao Inoue, Jiro Tanaka, Yasutsugu Yokohama, and Tadao Yoshida, eds., "Phycology: Experiments and Practical Training," Kodansha Scientific (2000)] and Japanese Patent Publication No. 2009-201480.) Furthermore, PESI culture media can be prepared by referring to Plant Tissue Culture, 6(2), 55-62 (1989), etc. The detailed composition will be described later in the examples.

[0080] (PES culture solution) Tris hydroxymethyl aminomethane 5.0g NaNO33.5g Na2-glycerophosphate 500mg Fe stock solution 250mL P-2 metal mix 250mL Vitamine B12 stock solution (0.1mg / mL) 1.0mL Thiamine-HCl stock solution (1.0mg / mL) 5.0mL Biotine stock solution (0.1mg / mL) 0.5mL Distilled water 1000mL

[0081] (Fe stock solution) Na2-EDTA·2H2O 330mg Fe(NH4)2(SO4)2·6H2O 351mg Distilled water 500mL

[0082] (P-2 metal mix) Na2-EDTA·2H2O 500mg H3BO 3570mg FeCl3·6H2O 24.5mg MnSO4·4H2O 82.0mg CoSO4·7H2O (4.8 mg / mL) 0.5 mL ZnSO4·7H2O 11.0mg Distilled water 500mL

[0083] A culture solution for seaweed is prepared by adding PES or PESI culture solution to seawater. The seawater used may be natural or artificial seawater that has been sterilized by ozone, ultraviolet light, or autoclave, or filtered seawater obtained by filtering natural seawater may be used. Artificial seawater is prepared to mainly contain cations such as sodium ions, magnesium ions, potassium ions, and calcium ions, and anions such as chloride ions and sulfate ions. The salinity of the artificial seawater is preferably 1.0% by mass or more and 3.5% by mass or less. When using natural seawater, deep-sea water may also be used. Deep-sea water is seawater distributed in the deep sea at depths of 200m or more. Because deep-sea water has a high salt concentration, bacteria and other organisms that hinder seaweed cultivation are less likely to survive. In addition, deep-sea water is free from artificial pollution, has low temperatures resulting in fewer bacteria, and is free from the presence of phytoplankton due to the absence of sunlight. Furthermore, deep-sea water is rich in nitrogen (N) in the form of nitrates, phosphorus (P) in the form of phosphates, and silicon (Si) in the form of silicates, making it suitable for seaweed growth.

[0084] Female gametophytes, which are seaweed, are placed in the prepared seaweed culture medium, and aeration culture is performed. Aeration culture consists of a preliminary culture and a main culture. The female gametophytes of seaweed are cultured in an artificial environment, which includes an environment in which water temperature, light intensity, and daylight hours are controlled. Any environment is acceptable as long as light intensity, daylight hours, and water temperature can be strictly controlled. For example, seaweed may be cultured in a container that allows cultivation within an incubator where light intensity, daylight hours, and internal temperature are controlled, or it may be cultured in a tank in an environment where light intensity, daylight hours, and water temperature can be controlled.

[0085] In the preliminary culture environment, a daylight-white fluorescent lamp is used as the light source, with an irradiation intensity of 1-100 μmol / m². 2 The water temperature should be between 5 and 30°C, and the daylight hours should preferably be between 8 and 24 hours. Cultivation is usually carried out under alternating light and dark periods, but continuous light irradiation is also permitted. The cultivation period may be between 1 and 40 days, but is preferably between 5 and 10 days. A minimum of 5 days is also sufficient.

[0086] In this culture environment, a green light source (490-550 nm) is used, with an irradiation intensity of 10-200 μmol / m². 2 The water temperature should be between 5 and 30°C, and the daylight hours should preferably be between 8 and 24 hours. Cultivation is usually carried out under alternating light and dark periods, but continuous light irradiation is also permitted. The cultivation period may be between 1 and 40 days, but is preferably between 5 and 10 days. The culture solution for seaweed used in this invention may contain appropriate seaweed growth promoters such as fatty acids. The culture tank may also be equipped with one or more devices selected from a stirring device, a vibrating device, a temperature control device, a pH adjustment device, a turbidity measuring device, a light control device, a device for measuring the concentration of specific gases such as air, O2, and CO2, and a pressure measuring device. Seaweed cultivation can be carried out using any suitable liquid culture method, such as batch culture, semi-batch culture (fed-batch culture), or continuous culture (perfusion culture).

[0087] After culturing the female gametophytes of seaweed, they are either scooped up with a plankton net or filtered to extract the gametophytes from the culture medium. The aggregate of seaweed gametophytes obtained in this way can be preferably used as a raw material for extracting, for example, extracellular vesicles (EVs) and at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit.

[0088] (III) Aqueous solvent extraction of gametophytes of seaweed The gametophyte of the seaweed is dried by freeze-drying or other means to obtain a powder, and this powder is dispersed in an aqueous solvent cooled to 5°C or below to obtain a dispersion of the gametophyte of the seaweed (step (1)). By dispersing dried powder of seaweed gametophytes in an aqueous solvent, extracellular vesicles (EVs) and at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit are extracted from the seaweed gametophytes into the aqueous solvent. Preferably, the dried powder of the female gametophyte of seaweed is dispersed in an aqueous solvent cooled to 5°C or below, and extracellular vesicles (EVs) and at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit are extracted from the female gametophyte of seaweed into the dispersion.

[0089] Extracellular vesicles (EVs) and at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit readily elute in cooled aqueous solvents, and since vesicles composed of lipid membranes are easily destroyed by organic solvents, extraction with aqueous solvents is preferable. Furthermore, in aqueous solvents above 5°C, at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit will be destroyed, making protein extraction difficult.

[0090] An aqueous solvent is water or a solvent in which water-soluble salts are dissolved, and includes pure water and seawater. Antifoaming agents, thixotropic agents, pH adjusters, etc., may be added to the aqueous solvent as appropriate. Artificial seawater, filtered seawater, or deep-sea water can also be used as seawater. Examples of salts include sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, ammonium sulfate, potassium phosphate, potassium dihydrogen phosphate, iron phosphate, iron fulvicate, and sodium citrate. Phosphate-buffered saline (PBS) may also be used as the aqueous solvent. The aqueous solvent used to disperse the gametophyte of seaweed should preferably be free of organic solvents.

[0091] The extraction temperature is preferably 5°C or lower. The extraction time is preferably 0.1 to 5 hours. It is desirable to disperse the gametophyte of the seaweed (preferably the female gametophyte) in an aqueous solvent and stir or permeate it. It is desirable to use the aqueous solvent at a ratio of 0.01 to 1 L per 1 g of dry weight of the seaweed gametophyte. The gametophytes of the seaweed are separated and removed from the resulting dispersion of gametophytes of the seaweed to obtain an aqueous solvent composition containing extracellular vesicles (EVs) (step (2)). In this specification, the aqueous solvent composition obtained in step (2) may also be referred to as an aqueous solvent extract, or as an aqueous solvent extract of the gametophyte of seaweed. The extraction residue, which consists of the gametophyte of seaweed and impurities separated from the gametophyte, is separated into solid residue and supernatant by methods such as filtration using filters or columns, or centrifugation. The solids are then removed from the aqueous solvent by further filtration, and the supernatant is collected to obtain the composition. It is also desirable to carry out step (2) under conditions of 5°C or below, in order to suppress the degradation of proteins and intracellular vesicles (EVs).

[0092] In this way, a composition (aqueous solvent composition) is obtained that contains an aqueous solvent, at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs). The gametophyte of the seaweed separated in step (2) can be used for extraction with the organic solvent described later.

[0093] Furthermore, a powdered composition is obtained by removing the aqueous solvent from an aqueous solvent composition containing at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, along with extracellular vesicles (EVs). To remove the aqueous solvent, it is sufficient to dry the aqueous solvent composition, and methods such as heating, freeze-drying, and vacuum drying are available. However, since extracellular vesicles are composed of proteins and lipids, freeze-drying is preferable.

[0094] (IV) After dispersing the dried gametophytes of seaweed in an aqueous solvent, the gametophyte residue of the seaweed (the solid portion of the gametophytes of seaweed separated in step (1) above) is removed, and an organic solvent is added to this residue to obtain an organic solvent composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives (for convenience, this is referred to as step (3)). In this specification, the organic solvent composition obtained in step (3) may also be referred to as an organic solvent extract.

[0095] Fucoxanthin or its derivatives can be extracted from the gametophyte of seaweed by immersing the gametophyte residue of seaweed, which has been extracted with an aqueous solvent, in an organic solvent that dissolves at least one compound selected from the group consisting of fucoxanthin and its derivatives. The organic solvent causes at least one compound selected from the group consisting of fucoxanthin and its derivatives, which are present in the cells of the gametophyte residue of seaweed, to elute into the organic solvent. Extracellular vesicles (EVs) are substantially not present in the organic solvent. This is because extracellular vesicles (EVs) released into the organic solvent are unstable because they do not have a protective membrane formed by hydration with water molecules on their surface, and their lipid membranes are destroyed.

[0096] Organic solvents that can be used include alcohols such as methanol, ethanol, propanol, isopropanol, n-butanol, and butanediol (1,3-butanediol (1,3-butylene glycol), 1,4-butanediol (1,4-butylene glycol)), ketones such as methyl ethyl ketone and acetone, esters such as methyl acetate and ethyl acetate, organochlorine hydrocarbons such as chloroform, aliphatic hydrocarbons such as hexane, and aromatic hydrocarbons such as benzene and toluene. These organic solvents can be used individually or in combination of two or more. Among these, ethanol and butanediol are preferred as organic solvents.

[0097] At least one compound selected from the group consisting of fucoxanthin and its derivatives is eluted into the organic solvent. Since fucoxanthin and its derivatives are substantially insoluble in aqueous solvents, the content of fucoxanthin and its derivatives in the organic solvent is higher than that of the aqueous solvent extract in (II). The organic solvent may contain an aqueous solvent, but it is preferable that it does not. If an aqueous solvent is present in the organic solvent, it mainly originates from water contained in the gametophyte of the seaweed.

[0098] In extraction with organic solvents, the extraction temperature is preferably 4°C to 60°C, and more preferably 20°C to 30°C. The extraction time is preferably 0.5 to 5 hours. It is desirable to disperse the seaweed gametophyte in the organic solvent and allow it to be stirred or permeated. The organic solvent should be used at a ratio of 0.01 to 1 L per 1 g of seaweed gametophyte. After extraction with an organic solvent, the extraction residue, which includes the gametophyte of the seaweed and impurities separated from the gametophyte, may be removed from the organic solvent. The method for removing the gametophyte of the seaweed from the organic solvent is not particularly limited, and methods such as filtration using filters or columns, or centrifugation can be used. In this way, it is desirable to obtain a composition (organic solvent composition) that contains an organic solvent and at least one compound selected from the group consisting of fucoxanthin and its derivatives eluted in the organic solvent.

[0099] Incidentally, in the present invention, it is preferable not to isolate fucoxanthin and its derivatives by mixing oils and fats such as fatty acid triglycerides into the composition. This is because isolation would impair the stability of fucoxanthin and its derivatives. Furthermore, even when mixed with oils and fats such as fatty acid triglycerides, extracellular vesicles (EVs) that have been extracted with water become hydrophilic and do not dissolve or disperse in the oils and fats. Therefore, it is difficult to isolate fucoxanthin and its derivatives containing extracellular vesicles (EVs) using oils and fats.

[0100] The composition comprising at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit of the present invention, along with extracellular vesicles (EVs), can be used in various industrial applications. In particular, the composition of the present invention is expected to increase cAMP (cyclic adenosine monophosphate) in mammalian cells, thereby promoting glycogenolysis in liver cells and raising blood glucose levels, and also to promote lipolysis in adipocytes and increase energy supply, making it effective as a raw material for various health foods. Furthermore, it can activate hair papilla cells, increase cardiac contractility, dilate airways by promoting relaxation of airway smooth muscle, facilitate neurotransmission in nerve cells, and regulate immune responses by modulating the activation or suppression of immune cells, making it promising for pharmaceutical applications. In addition, it is beneficial for cosmetic applications as it promotes cell growth by activating signaling pathways that promote cell growth and differentiation. Thus, the composition of the present invention can be used as an increaser for cAMP (cyclic adenosine monophosphate) in cells. The form of use as an increaser is not particularly limited, and examples include using the composition as is, or using it as an excipient, binder, lubricant, disintegrant, surfactant, buffer, preservative, fragrance, colorant, oil, pigment, water, alcohol, thickener, antiseptic, antioxidant, or chelating agent, one or more of these can be used together. However, it is not limited to these. When used as a topical preparation for the skin, the dosage form of the present invention can be any form commonly used in pharmaceuticals, cosmetics, quasi-drugs, etc., as long as it is suitable for obtaining the therapeutic effect. Examples include topical liquid preparations such as lotions, liniments, aqueous solutions, and emulsions; topical solid preparations such as powders and dissolvable tablets; and topical semi-solid preparations such as creams, film preparations, ointments, and jellies, as well as soaps. Oral preparations and injections may also be effective, and several methods may be used in combination.

[0101] As described above, the gametophyte of seaweed can be suitably used as a raw material for extracting at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, as well as extracellular vesicles (EVs). Furthermore, the raw material may contain aggregates of seaweed gametophytes.

[0102] The composition of the present invention may include at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, as well as raw materials used to extract extracellular vesicles (EVs). The seaweed is preferably a brown alga. The seaweed is preferably a female gametophyte or a filamentous sporophyte.

[0103] Furthermore, by co-administering extracellular vesicles (EVs) derived from seaweed with at least one compound selected from the group consisting of fucoxanthin and its derivatives, the stability of at least one compound selected from the group consisting of fucoxanthin and its derivatives can be improved. Extracellular vesicles (EVs) derived from seaweed can be used to improve the stability of at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0104] This specification contains the following information:

[0105] The present invention (1) is a composition comprising at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs) derived from the gametophyte and / or sporophyte of seaweed.

[0106] The present invention (2) is the composition according to the present invention (1), wherein the composition further comprises at least one selected from ATP synthase subunit beta, thiamine biosynthesis protein G, and histone H2B.

[0107] The present invention (3) is the composition according to the present invention (1) or (2), wherein the seaweed is the female gametophyte of the seaweed.

[0108] The present invention (4) is a composition according to any one of the present inventions (1) to (3), wherein the seaweed is a filamentous sporophyte.

[0109] The present invention (5) is a composition according to any one of the present inventions (1) to (4), wherein the seaweed is a brown alga.

[0110] The present invention (6) is a composition according to any one of the present inventions (1) to (5), wherein the composition comprises an aqueous solvent or a mixed solvent consisting of an aqueous solvent and an organic solvent.

[0111] The present invention (7) is a composition according to any one of the present inventions (1) to (6), wherein the composition further comprises at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0112] The present invention (8) is a composition according to any one of the present inventions (1) to (7), wherein at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit is encapsulated in or attached to extracellular vesicles (EVs) derived from the gametophyte of seaweed.

[0113] The present invention (9) is a composition according to any one of the present inventions (1) to (8), wherein the composition is a powdered composition.

[0114] The present invention (10) is an agent for increasing intracellular cyclic adenosine monophosphate (cAMP) comprising the composition described in any one of the above claims (1) to (9).

[0115] The present invention (11) is a method for producing a composition comprising at least one protein selected from ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit and extracellular vesicles (EVs) derived from the gametophyte and / or sporophyte of seaweed, comprising the following steps (1) and (2). Step (1): The gametophyte and / or sporophyte of the seaweed are dried to obtain a dried product, and then the dried product is pulverized to obtain a powder of the gametophyte and / or sporophyte of the seaweed, and the powder is dispersed in an aqueous solvent adjusted to a temperature of 5°C or lower. Step (2): The aqueous solvent in which the powder is dispersed is separated into solids and supernatant by centrifugation, and the supernatant is collected.

[0116] The present invention (12) is a method for producing the composition described in the present invention (11), comprising the step of adding at least one compound selected from the group consisting of fucoxanthin and its derivatives to the supernatant liquid.

[0117] The present invention (13) is a method for producing the composition described in the present invention (11) or (12), comprising the step of removing the aqueous solvent from the supernatant to obtain a powder containing at least one protein selected from ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs) derived from the gametophyte and / or sporophyte of seaweed.

[0118] The present invention (14) is a method for producing an intracellular cyclic adenosine monophosphate (cAMP) increasing agent, comprising a method for producing the composition described in any one of the above claims (11) to (13).

[0119] The present invention (15) is a method for producing the composition according to any one of the present inventions (11) to (14), wherein the extracellular vesicles (EVs) are derived from the female gametophyte of the seaweed.

[0120] The present invention (16) is a method for producing the composition according to any one of the present inventions (11) to (15), wherein the extracellular vesicles (EVs) are derived from the filamentous sporophytes of the seaweed.

[0121] The present invention (17) is a raw material comprising an aggregate of gametophytes of seaweed, used for extracting at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, as well as extracellular vesicles (EVs) derived from the gametophytes of seaweed.

[0122] The present invention (18) is a raw material described in the present invention (17), wherein the seaweed is a female gametophyte.

[0123] The present invention (19) is a raw material according to the present invention (17) or (18), wherein the seaweed is a filamentous sporophyte.

[0124] The present invention (20) is a raw material according to any of the present inventions (17) to (19), wherein the seaweed is a brown algae. [Examples]

[0125] The present invention will be described based on examples, but the present invention is not limited to these examples. The term "thallus" used in the following description refers to the thallus (adult) of a sporophyte.

[0126] (Preparation of the solution) (Preparation of PESI culture medium) To approximately 300 mL of distilled water, Tris, NaNO3, β-glycerophosphate disodium (Na2-glycerophosphate), Fe stock solution, P-II metal mix, and KI were added in the specified amounts in the order shown below, and thoroughly dissolved. Then the pH of the solution was adjusted to 7.8, and the volume was increased to 1000 mL with distilled water. The resulting PESI culture medium was dispensed into containers, sterilized by autoclaving (121°C, 20 minutes), and then the sterile PESI culture medium was stored refrigerated at 4°C.

[0127] PESI culture medium is 2-Amino-2-hydroxymethyl-1,3-propanediol(Tris(hydroxymethyl)aminomethane;Tris),5.0g; NaNO3, 3.5g; Na2-glycerophosphate, 500 mg; Fe stock solution (see composition below), 250 mL; P-II metal mix (see composition below), 250 mL; KI stock solution(0.1mg / mL),10mL; It was dissolved in distilled water, and finally diluted with distilled water to make a 1000 mL solution (pH 7.8).

[0128] Fe stock file is Na2-EDTA·2H2O, 330mg; Fe(NH4)2(SO4)2·6H2O, 351mg The mixture was dissolved in distilled water, and finally diluted with distilled water to make a 500 mL solution (Fe:EDTA molar ratio = 1:1). It was stored refrigerated at 4°C.

[0129] P-II metal mix is Na2-EDTA·2H2O, 500mg; H3BO3, 570mg; FeCl3·6H2O, 24.5 mg; MnSO4·4H2O, 82.0 mg; CoSO4·7H2O stock solution(4.8mg / mL),0.5mL; ZnSO4·7H2O, 11.0 mg; The ingredients were added to distilled water in order, and finally, 500 mL of distilled water was added to make a solution. It was stored in the refrigerator at 4°C. Additionally, the KI stock solution was prepared by adding 200 mL of distilled water to 20 mg of KI and storing it refrigerated at 4°C.

[0130] (A-1. Sample preparation of the female gametophyte of wakame seaweed) (1) Natural seawater was filtered through a cartridge filter with a 1 μm mesh, and the filtered seawater was heated and pressurized in an autoclave at 121°C, 2 atmospheres, and for 50 minutes to obtain 10 liters of sterile seawater. Then, 20 mL of the previously prepared PESI culture solution was added to 1 L of sterile seawater to obtain the culture seawater.

[0131] (2) Naruto wakame seaweed female gametophytes were cultured and grown from wakame collected near the mouth of the Yoshino River, following the procedure described in "(I) Cultivation of seaweed gametophytes" above. 300 mg of wakame female gametophytes and culture seawater were placed in a 5L glass Erlenmeyer flask, and preliminary aeration culture (hereinafter referred to as "preliminary culture") was performed until the wakame female gametophytes reached 10 gw.w (wet weight g). The aeration culture was carried out using the following method. First, a Pasteur pipette (IK-PAS-9P manufactured by Iwaki Glass Co., Ltd.) was attached to the tip of a silicone tube (SR-1554 manufactured by Tigers Polymer Co., Ltd.), and an air pump (APN-057R manufactured by Iwaki Corporation) was attached to the other end of the silicone tube to assemble an aeration device. Next, after immersing the tip of the Pasteur pipette in the culture seawater, aeration culture was performed by supplying air to the culture seawater from an air pump via a silicone tube and the Pasteur pipette. This aeration culture was carried out in the same manner for both the preliminary culture and the main culture. The culture conditions for the preliminary culture were a temperature of 20°C, using a daylight-white fluorescent lamp as the light source, with a light intensity of 50 μmol m³. -2 s -1 The photoperiod was set to 12L (light) and 12D (dark).

[0132] (3) 10 gw.w of pre-cultured female gametophytes of wakame and culture seawater were placed in a 20 L cylindrical polycarbonate container (manufactured by Nikko Hansen Co., Ltd.) and cultured until the volume reached 50 gw.w (main culture). The culture conditions for this culture were: temperature 20°C, green LED (wavelength 518nm) (manufactured by Nippon Medical Equipment Co., Ltd., 3LH-64) as the light source, light intensity: 50-100 μmol m -2 s -1 The photoperiod was set to 12L (light) and 12D (dark).

[0133] (4) After the main culture was completed, the female gametophytes were poured into a 50 μm mesh plankton net (Sefar Inc, DIN110) and filtered to collect the algae. The collected female gametophytes, still in the plankton net, were lightly sandwiched between commercially available paper towels to absorb and dehydrate them, and then transferred to a resealable plastic bag. The gametophytes were flattened to facilitate freeze-drying and frozen in a freezer set to -60°C. The fully frozen gametophytes were freeze-dried overnight in a freeze-dryer (Tokyo Rika Kikai Co., Ltd., FD-1) to obtain female gametophyte samples.

[0134] (A-2. Sample preparation of the filamentous sporophyte of *Uppluinori*) (1) Mature Uppuruinori thalli were collected from rocky areas near the low tide line in Aizaki City, Niigata Prefecture. The zygospores, which are sporophytes released from these mature thalli, were hivet-washed with sterile seawater to obtain a zygospore mixture. This mixture was diluted to a concentration of approximately 1000 spores / ml. (2) This diluted solution was cultured for 15 days under the following conditions: culture temperature 20°C, light intensity 20 μmol / m2 / S, photoperiod 12 L:12 D, aeration, and PES medium. The zygotes germinated and grew into filamentous structures. The filamentous structures were intertwined with each other and formed spheres. The average diameter of the spheres was 5 mm. These filamentous tissues were frozen and stored in a freezer set to -60°C. The fully frozen filamentous tissues were freeze-dried overnight in a freeze-dryer (FD-1, manufactured by Tokyo Rika Kikai Co., Ltd.) to obtain samples of sporophyte filaments.

[0135] (A-3. Sample preparation of the gametophyte of the Japanese butterfish) (1) At Mochimune Beach in Shizuoka City, Shizuoka Prefecture, we collected Sargassum fuscoguttatum from the growing area and cut off the leaf portions that had ascospores. After storing these leaf portions in a refrigerator for about a day, we allowed them to release zoospores in sterilized seawater and attached them to a microscope slide. (2) The slides on which these zoospores were attached were transferred to a glass tank containing 3 L of culture medium and cultured for one month. The water temperature was 20°C, and illumination was performed using a white fluorescent lamp with an illuminance of 2000 1ux and a light-dark cycle of 10 L:14 D. PESI medium was used as the culture medium, and half of the water was changed every two days. To measure the growth of the gametophytes, the 20 individuals with the most advanced growth were selected every two days, and only the male gametophytes were taken from these and cultured under the same conditions to obtain male gametophytes of Sargassum. These were stored in a refrigerator at 4°C. The male gametophytes were frozen and stored in a freezer set to -60°C. The fully frozen gametophytes were freeze-dried overnight in a freeze-dryer (FD-1, manufactured by Tokyo Rika Kikai Co., Ltd.) to obtain male gametophyte samples.

[0136] (A-4. Sample preparation of the filamentous sporophyte of *Lysimachia japonica*) (1) Collect Asakusa seaweed cultivated near the mouth of the Ibi River, and cut the tip of the thallus to 3 cm. 2 A portion was cut off. These leaf fragments were spread on a glass plate and the surface was washed with sterile seawater. (2) A sterilized petri dish was prepared, a microscope slide was placed at the bottom, and the PES culture solution was poured in. The leaf fragments from (1) were then floated in the dish. This petri dish was left undisturbed for 14 days under a 2000 Lux fluorescent lamp. Carbospores had fallen to the bottom of the petri dish and accumulated, forming colonies. (3) The carpospores were drawn up with a dropper and placed in the PES culture medium. The culture was performed at a temperature of 16°C under 2000 Lux fluorescent lighting with a light-dark cycle of 12 L:12 D, and filamentous bodies were obtained from the germinated carpospores. The filamentous bodies were intertwined with each other and formed a spherical shape, with an average spherical length of 3 mm. These filamentous bodies were frozen and stored in a freezer set to -60°C. The fully frozen gametophytes were freeze-dried overnight in a freeze-dryer (FD-1, manufactured by Tokyo Rika Kikai Co., Ltd.) to obtain filamentous samples of the sporophytes.

[0137] (B-1. Water extraction treatment of the female gametophyte of wakame seaweed, the male gametophyte of sagara seaweed, the filamentous sporophyte of sporophyte, and the thallus (adult) of wakame seaweed) Each sample was subjected to water extraction using the following method.

[0138] (B-1-1. Water extraction treatment of female gametophytes and thallus (adults) of wakame seaweed (Example 1 and Comparative Example 1)) (1) 1 g of the powder sample obtained by crushing the female gametophyte prepared in (A-1) above was suspended in 40 mL of distilled water and stirred at 200 rpm for 0.5 hours at a temperature of 4°C using a rotator (TAITEC, RT-5N).

[0139] (2) The stirred female gametophyte sample solution from (1) was centrifuged using a centrifuge at 4°C, 8000 rpm for 20 minutes to separate it into a solid precipitate and a supernatant.

[0140] (3) The supernatant from (2) was filtered through a coffee filter, and this filtrate was filtered through a 0.1 μm PES filter (manufactured by Membrane Solutions LLC).

[0141] (4) 20 mL of this filtrate was concentrated using a 300 K spin column (manufactured by Nippon Pall Co., Ltd.) at 6000 rpm for 10 min, washed three times with 2 mL of PBS (phosphate-buffered saline), and filtered. 0.5 mL of the filtrate was collected (40-fold concentration). This was used as the aqueous extract of the female gametophyte of wakame according to Example 1.

[0142] (5) 10 g of dried wakame seaweed (manufactured by Marukome Co., Ltd.), consisting of thallus (adult), was crushed into a powder using a mixer mill. Then, 1 g of this powder was suspended in 40 mL of distilled water and stirred at 200 rpm for 0.5 hours at 4°C using a rotator (TAITEC, RT-5N).

[0143] (6) The stirred thallus sample solution from (5) was centrifuged using a centrifuge at 4°C, 8000 rpm, and 20 minutes to separate it into a solid precipitate and a supernatant.

[0144] (7) The supernatant from (6) was filtered through a coffee filter, and this filtrate was filtered through a 0.1 μm PES filter (manufactured by Membrane Solutions LLC).

[0145] (8) 20 mL of this filtrate was concentrated using a 300 K spin column (manufactured by Nippon Pall Co., Ltd.) at 6000 rpm for 10 min, washed three times with 2 mL of PBS (phosphate-buffered saline), and filtered. 0.5 mL of the filtrate was collected (40-fold concentration). This was used as the aqueous extract of the thallus (adult) of wakame seaweed according to Comparative Example 1.

[0146] (B-1-2. Water extraction of the filamentous sporophyte of *Uppluinori*) The freeze-dried filamentous material of the Uppuruinori sporophyte obtained in (A-2.) above was pulverized and powdered. Using this powder, an aqueous extract of the filamentous material of the Uppuruinori sporophyte was obtained by the same method as in (B-1-1.) above. This was used as the aqueous extract of the filamentous material of the Uppuruinori sporophyte according to Example 2.

[0147] (B-1-3. Water extraction of the gametophyte of the Japanese sardine) The freeze-dried male gametophytes of Sargassum obtained in (A-3.) above were pulverized and powdered. Using this powder, an aqueous extract of the male gametophytes of Sargassum was obtained by the same method as in (B-1-1.) above. This was used as the aqueous extract of the male gametophytes of Sargassum according to Example 3.

[0148] (B-1-4. Water extraction of the filamentous sporophyte of *Lysimachia japonica*) The freeze-dried filamentous material of the Asakusa-no-ri sporophyte obtained in (A-4.) above was pulverized and powdered. Using this powder, an aqueous extract of the Asakusa-no-ri sporophyte filamentous material was obtained by the same method as in (B-1-1.) above. This was used as the aqueous extract of the Asakusa-no-ri sporophyte filamentous material for Example 4.

[0149] (B-2. Analysis of each aqueous extract) Each aqueous extract was analyzed using the following method.

[0150] (B-2-1. Analysis of extracellular vesicles in aqueous extract of the female gametophyte of wakame seaweed) (1) 1 μL of a 1 mM solution of GIF-2276 (manufactured by GIFU EXOSOME Co., Ltd.: a labeling reagent for protein detection of EVs. It is a fluorescent substance with Ex / Em: 475 / 530 nm) and 2 μL of ExoSparkler Exosome Membrane Labeling Kit-Red (manufactured by Dojin Chemical Laboratories Co., Ltd.: a labeling reagent for lipid membrane detection. It is a fluorescent substance with Ex / Em: 560 / 600 nm) were mixed with 100 μL of an aqueous extract of the female gametophyte from Example 1 prepared in (4) of (B-1-1.) above, and labeled by heating with 80°C hot water for 5 minutes.

[0151] (2) The following liquid chromatography apparatus A was prepared for the detection of extracellular vesicles. Column packing material: Izon Science qEV10 70nm Column shape: Stainless steel column manufactured by Senshu Kagaku Co., Ltd., 8mm diameter x 100m length Detector: RF-10AxL manufactured by Shimadzu Corporation Developing solvent: Aqueous solution containing 10 mM tris(hydroxymethyl)aminomethane (pH=7.6), 30 mM NaCl, and 0.5 mM EDTA. Gradient in development time: uniform Solution flow rate: 1 mL / min Injection volume: 10μL Column temperature: 30℃ Sample room temperature: 10℃ Liquid transfer pump: Shimadzu Corporation LC10ADVP

[0152] (3) The concentrated samples labeled in (1) were analyzed using liquid chromatography apparatus A in (2). The resulting chart is shown in Figure 2 (solid line: protein, dotted line: lipid). The horizontal axis of the chart is in units of time (minutes), and the vertical axis shows the absorbance of fluorescence (530 nm for protein, 600 nm for lipid membrane), and is dimensionless.

[0153] As described in WO2024 / 053316 and Biochemical and Biophysical Research Communications Volume 696, 12 February 2024, 149505, "Simple methods for measuring milk exosomes using fluorescent compound GIF-2250 / 2276," GIF-2276 modifies extracellular vesicles (EVs) by binding to Lys residues of proteins that constitute extracellular vesicles (EVs). Furthermore, from the aforementioned literature, it is known that the ExoSparkler Exosome Membrane Labeling Kit-Red (abbreviated as Exo-SP or EXoSP in the aforementioned literature) modifies the lipid membrane of extracellular vesicles (EVs). If the lipid membrane peak and the protein peak appear at the same retention time on a liquid chromatography chart, it is considered that extracellular vesicles (EVs) are present in the sample. In Figure 2, since the lipid membrane peak and the protein peak appear at the same retention time, it can be concluded that extracellular vesicles (EVs) are present in the aqueous extract of the female gametophyte of wakame seaweed, etc. As a precaution, an aqueous extract of the female gametophyte of wakame seaweed was frozen at -50°C using liquid nitrogen, and freeze-dried under reduced pressure to 4 Pa ​​to prepare a powdered composition. Transmission electron microscope images of the powdered composition obtained by drying are shown in Figures 3A, 3B, 3C, and 3D (scale: 100nm, 50nm, 20nm, 20nm). The electron microscope used was a JEOL JEM-2100, and the imaging conditions were an acceleration voltage of 200kV and negative staining with uranyl acetate. The magnifications for Figures 3A, 3B, 3C, and 3D were 50,000x, 100,000x, 250,000x, and 300,000x, respectively. The spherical object in the center of the photographs in Figures 3A and 3B, and the white, granular objects surrounding it, are extracellular vesicles (EVs). Figures 3C and 3D are magnified views of the white, granular extracellular vesicles (EVs).

[0154] (B-2-2. Analysis of extracellular vesicles in aqueous extract of the filamentous sporophyte of *Uppluinori*) The aqueous extract of the filamentous sporophytes of *Inophyllum uliginosum* from Example 2 was analyzed using liquid chromatography apparatus A in the same procedure as described in (B-2-1) above. The resulting chart is shown in Figure 4. Since the protein and lipid peaks are observed at the same position, EVs are present. In addition, proteins other than those constituting the EVs were also observed.

[0155] (B-2-3. Analysis of extracellular vesicles in aqueous extract of male gametophytes of Sargassum fuscipes) The aqueous extract of the male gametophyte of Sargassum fuscipes according to Example 3 was analyzed using liquid chromatography apparatus A in the same procedure as described in (B-2-1) above. The results are shown in Figure 5. The amount of extracellular vesicles (EVs) appears to be relatively smaller compared to the female gametophyte.

[0156] (B-2-4. Analysis of extracellular vesicles in aqueous extract of filamentous sporophytes of *Lysimachia japonica*) The aqueous extract of the filamentous sporophytes of *Lysimachia japonica* according to Example 4 was analyzed using liquid chromatography apparatus A in the same procedure as described in (B-2-1) above. The resulting chart is shown in Figure 6. Since the protein and lipid peaks are observed at the same position, EVs are present. In addition, proteins and lipids other than those constituting the EVs are also observed.

[0157] (B-3. Analysis of Fucoxanthin) (1) The following liquid chromatography apparatus B was prepared for the detection of fucoxanthin. Column: 3.0 mm in diameter x 150 mm in length. The packing material used is 25437-96 RP-18GP 150-3, manufactured by Kanto Chemical Co., Ltd., a high-performance liquid chromatography packing material with a particle size of 5.0 μm. Detector: Absorbance was measured using a spectrophotometer (Shimadzu Corporation UV-1800). Wavelength: 450 nm Developing solvent A: 10% acetonitrile prepared by mixing acetonitrile for LC / MS (manufactured by Kanto Chemical Co., Ltd., 01033-76) and 0.05 wt% formic acid aqueous solution in a 1:9 ratio. Developing solvent B: 80% acetonitrile prepared by mixing acetonitrile for LC / MS (manufactured by Kanto Chemical Co., Ltd., 01033-76) and 0.05 wt% formic acid aqueous solution in an 8:2 ratio. Developing solvent C: 100% acetonitrile Gradient of development time: Solvent A (0 min) → Solvent B (1 min) → Solvent C (10 min) → Solvent C (15 min) → Solvent A (15.1 min) → Solvent A (20 min) Solution flow rate: 0.5 mL / min Injection volume: 5μL Column temperature: 40℃ Sample room temperature: 15℃ Liquid transfer pump: Shimadzu Corporation LC10ADVP

[0158] Furthermore, 0.6589 mg of fucoxanthin standard (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was weighed, and 1 L of ethanol was added and the mixture was thoroughly stirred to prepare a 1 mM fucoxanthin solution. This solution was then diluted 10-fold with ethanol to prepare a 0.1 mM (100 μM) standard solution. The retention time of fucoxanthin in liquid chromatography apparatus B of (1) was confirmed using this standard solution. In addition, this 100 μM fucoxanthin standard solution was diluted with ethanol to 2-fold (50 μM), 3.33-fold (30 μM), 10-fold (10 μM), and 33.3-fold (3 μM). Then, a calibration curve for fucoxanthin concentration in liquid chromatography apparatus B of (1) was created using the standard solution and the diluted solutions.

[0159] The calibration curve is represented by y = 110489x + 22725 (where x is the concentration of fucoxanthin and y is the peak area). It was also confirmed that the peak area and fucoxanthin concentration appearing on the chart matched the above calibration curve when 1M fucoxanthin was analyzed using liquid chromatography apparatus B (1).

[0160] (C. 100% butylene glycol (BG) extraction of the female gametophyte and thallus of wakame seaweed after water extraction, and analysis of the BG extract) The following methods were used to perform BG extraction and analyze the BG extracts using the female gametophytes and thalli of wakame seaweed.

[0161] (C-1. BG extraction treatment of aqueous extracts of wakame female gametophyte and wakame thallus (adult) (Comparative Examples 2 and 3)) (1) After performing water extraction of the female gametophyte of wakame seaweed using the same procedure as in (1) and (2) above (B-1-1.), 1 g of the solid matter of the female gametophyte of wakame seaweed, which is the extraction residue (solid content), after draining the water thoroughly, was suspended in 0.5 mL of 100% butylene glycol (1,3-butylene glycol, hereinafter referred to as "BG") and stirred in a rotator (TAITEC, RT-5N) at 4°C, 8000 rpm, for 0.3 hours. Then, 20 mL of 100% BG was added to the precipitate after removing the supernatant and suspended, and stirred in a rotator (TAITEC, RT-5N) at 4°C, 8000 rpm, for 0.3 hours. 1 mL of the supernatant was filtered through a 0.2 μm sterile filter to prepare the BG extract. This was used as the BG extract of the female gametophyte of wakame seaweed according to Comparative Example 2.

[0162] (2) 10 g of dried wakame seaweed (manufactured by Marukome Co., Ltd.), consisting of thallus (adult), was crushed into a powder using a mixer mill. 1 g of this powder was suspended in 0.5 mL of 100% butylene glycol (BG), and stirred in a rotator (TAITEC, RT-5N) at 4°C, 8000 rpm, for 0.3 hours. The supernatant was then removed, and 20 mL of 100% BG was added to the precipitate and suspended. The mixture was stirred in a rotator (TAITEC, RT-5N) at 200 rpm, for 0.5 hours, at 4°C. 1 mL of the supernatant was filtered through a 0.2 μm sterile filter to prepare the BG extract. This was used as the BG extract of the wakame thallus according to Comparative Example 2.

[0163] (C-2. Analysis of extracellular vesicles in BG extract) The BG extract of the female gametophyte of wakame seaweed related to Comparative Example 2, prepared in (C-1.) above, was analyzed using liquid chromatography apparatus A in (B-2-1.). The results are shown in Figure 7 (solid line: protein, dotted line: lipid). The horizontal axis of the chart is in units of time (minutes), and the vertical axis is the absorbance of fluorescence (530 nm for protein, 600 nm for lipid membrane), and is dimensionless. The protein and lipid peaks around 10-12 minutes, which can be seen in Figure 2, are not observed in Figure 7. Therefore, extracellular vesicles (EVs) are not observed in the BG extract prepared in (C-1.).

[0164] (C-3. ​​Analysis of fucoxanthin in BG extract) The BG extracts of the female gametophyte and thallus of wakame seaweed prepared in (1) and (2) of (C-1.) above (Comparative Example 2 and Comparative Example 3, respectively) were analyzed using the liquid chromatography apparatus B of (1). The liquid chromatography charts are shown as Figures 8A and 8B. The horizontal axis of the chart is in units of time (minutes), and the vertical axis is the absorbance at 450 nm (unitless). In Figures 8A and 8B, the peak around 13 minutes corresponds to fucoxanthin, and the peak around 11.3 minutes corresponds to chlorophyll.

[0165] (D. Identification of proteins in aqueous extracts) The proteins in each aqueous extract were identified using the following method.

[0166] (D-1. Identification of proteins in aqueous extracts of the female gametophyte of wakame seaweed) (1) 100 mg of the powder sample obtained by crushing the female gametophyte prepared in (A-1) above was suspended in 4 mL of distilled water and stirred at 200 rpm for 0.5 hours at a temperature of 4°C using a rotator (TAITEC, RT-5N).

[0167] (2) The stirred female gametophyte sample solution from (1) was centrifuged using a centrifuge at 4°C, 8000 rpm for 20 minutes to separate it into a solid precipitate and a supernatant.

[0168] (3) The supernatant from (2) was filtered through a coffee filter, and this filtrate was filtered through a 0.1 μm PES filter (manufactured by Membrane Solutions LLC).

[0169] (4) 10 mL of this filtrate was concentrated using a 300 K spin column (manufactured by Nippon Pall Co., Ltd.) at 6000 rpm for 10 min, washed twice with 250 μL of PBS (phosphate-buffered saline), filtered, and 100 μL of the filtrate was collected.

[0170] (5) The following samples were prepared. Sample A: BIO-HELIX product, product name PM007-0500 (standard sample) Sample B: Water-extracted sample prepared in (1) to (4) of D above. Sample C: Liquid permeated through a 300K spin column in steps (1) to (4) of D above. Sample D: Filtrate after processing (1) to (3) of D above

[0171] (6)5 μL of samples A to D from (1) were mixed with three times the amount of 10% mercaptoethanol-containing SDS-Sample buffer (TAKARA Bio: 786-701), and the mixture was heated at 100°C for 10 minutes.

[0172] (7) Samples A to D treated in (6) were subjected to electrophoresis using an acrylamide gel (ATTO E-T520L) containing electrophoresis buffer (for SDS-PAGE, Tris-glycine system, Nakarai 30329-74).

[0173] (8) The acrylamide gel surface was washed with distilled water, and the protein bands were stained with CBB (Coomassie Brilliant Blue) stain (CBB staining solution for protein detection: Kanto Chemical Rapid CBB KANTO 3S 36533-79). The results are shown in Figure 9.

[0174] (9) In the electrophoresis bands of Sample B from the acrylamide gel, two areas with intense coloration were cut out with a scalpel. Based on the bands of Sample A, the cut-out areas correspond to the molecular weight regions of 35,000 to 63,000 Da and 10,000 to 17,000 Da.

[0175] (10) The excised acrylamide gel fragments were sent to Japan Proteomics Co., Ltd. (6-6-3 Minamiyoshinari, Aoba-ku, Sendai, Miyagi Prefecture 989-3204) to identify the proteins. Protein identification was performed using the following procedure. First, the gel fragments were broken down into peptides using trypsin. The broken-down peptide fragments were then separated using a liquid chromatography called nanoLC, which has been made highly sensitive by miniaturizing the packing material and column diameter. Following the LC, the amino acid sequence of the peptides was identified using a tandem mass spectrometer (MS / MS). The results were then compared with proteins registered in a database (Matrix Science, Inc.) to identify the proteins contained in the gel fragments.

[0176] As a result of the identification, in the molecular weight range of 35,000 to 63,000 Da, the large subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase with a molecular weight (MW) of 52,369 and the beta subunit of ATP synthase with a molecular weight (MW) of 44,542 were confirmed. Also, in the molecular weight range of 10,000 to 17,000 Da, the small subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase with a molecular weight (MW) of 15,249 and histone H2B with a molecular weight (MW) of 12,789 were confirmed. Note that the dark bands were within the range indicated by the bands of the standard sample of 35×10 3 ~63×10 3 Da, and within the range between 10×10 3 ~17×10 3 Da. Therefore, it was determined that the main components of the protein were the large subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase and the small subunit that could bind to it.

[0177] (Identification of proteins in the aqueous extract of the filamentous bodies of Upleinori spores) Using the filtrate obtained by the procedures (1) to (4) in (D-1.) above with the powder sample obtained by pulverizing the filamentous bodies of Upleinori spores prepared in (A-2) above, the protein bands were separated by electrophoresis according to the procedures (6) to (10) in (D-1.), and the proteins were identified. The ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit with a molecular weight (MW) of 52440 and the ATP synthase subunit beta with a molecular weight (MW) of 51165 were identified. Thiamine biosynthesis protein G with a molecular weight (MW) of 26838 was also identified.

[0178] (D-3. Identification of proteins in aqueous extracts of the male gametophyte of Sargassum fuscipes) Using the powder sample obtained by crushing the male gametophyte of the Sargassum species prepared in (A-3) above, the filtrate obtained by procedures (1) to (4) in (D-1) above was subjected to electrophoresis to separate the protein bands and identify the protein. A ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit with a molecular weight (MW) of 52400 was identified. Other bands resembling proteins were also present, but none matched in the database.

[0179] (D-4. Identification of proteins in aqueous extracts of the filamentous sporophytes of *Lysimachia japonica*) Using the powder sample obtained by grinding the filamentous sporophytes of *Lysimachia japonica* prepared in (A-4) above, the filtrate obtained by procedures (1) to (4) in (D-1) above was subjected to electrophoresis to separate the protein bands and identify the protein. A large subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase with a molecular weight (MW) of 52400 was identified. Other bands resembling proteins were also present, but none matched in the database.

[0180] (E. Measurement of the cAMP-increasing effect of water extract and BG extract) (1) The aqueous extracts of the female gametophyte of wakame seaweed according to Example 1 and the aqueous extract of the thallus of wakame seaweed according to Comparative Example 1 were diluted 300-fold and 100-fold respectively using a culture medium (Keratinocyte Growth Medium 2 (available from TakaraBio Co., Ltd. as C-20011)) to prepare a total of four samples. The 300-fold and 100-fold dilutions of the aqueous extract of the female gametophyte of wakame seaweed were designated as Examples 1-1 and Example 1-2, respectively, and the 300-fold and 100-fold dilutions of the aqueous extract of the thallus of wakame seaweed were designated as Comparative Examples 1-1 and Comparative Example 1-2, respectively.

[0181] (2) The BG extract of the female gametophyte of wakame seaweed according to Comparative Example 2 and the BG extract of the thallus of wakame seaweed according to Comparative Example 3 were diluted 100-fold in the above culture medium.

[0182] (3) 96 well plate, 5 x 10 4 PSVK1 cells (human keratinocytes: JCRB1093, National Institute of Biomedical Innovation, Health and Nutrition, JCRB Cell Bank) at a cell / well cell count (concentration) during passage were placed in a well, 150 μL of the above culture medium was added, and the cells were cultured for 48 hours.

[0183] (4) Next, after aspirating 150 μL of the culture medium, 100 μL of the same medium was added again, and 100 μL each of the culture medium for Example 1-1, Example 1-2, Comparative Example 1-1, Comparative Example 1-2, Comparative Example 2 and Comparative Example 3, as well as the (negative) control, were added to three wells of each well plate. There were 7 samples × 3 wells, so 21 wells were used. Furthermore, these well plates were incubated at 25°C for 90 minutes. After the culture is complete, Promega cAMP Glo TMThe increase in cAMP was measured using a measurement kit called Assay (manufactured by Promega Corporation). The included V1501 reagent set was used for the measurement. First, the sample and culture medium were drained from the well plate, and 20 μL of Lysis Buffer was added to each well. The cells were left to stand for 15 minutes to lyse the PSV1 cells and release cAMP. Next, 2.5 μL of Protein kinase A and 1.0 mL of cAMP Glo were added. TM cAMP Glo, which consists of Reacton Buffers TM 40 μL of detection solution was added to each well, and the PKA reaction was allowed to proceed for 20 minutes. Next, Kinase-Glo(R) Reagent was added to each well and left for 10 minutes to stop the PKA reaction, and the remaining ATP was used to induce luminescence using the luciferin-luciferase reaction. Furthermore, Promega cAMP Glo TM The Assay cAMP measurement kit utilizes the fact that cAMP stimulates the holoenzyme activity of protein kinase A (PKA), and the activated protein kinase A (PKA) reacts with its substrate protein, consuming ATP during this reaction. This measurement principle is based on the fact that if there is a large amount of cAMP, the amount of ATP available for the luciferin-luciferase reaction decreases, and luminescence is suppressed; therefore, the higher the concentration of cAMP, the lower the amount of luminescence.

[0184] (5) The luminescence of each well plate was measured for 2 seconds using the Promega Glo MAX Multi Detection System (manufactured by Promega Corporation). The results are shown in Figure 10. The luminescence was calculated using the average value of three wells for each sample. As can be seen from Figure 10, the aqueous extract from the female gametophyte of wakame seaweed, which contains extracellular vesicles (EVs) and the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, showed lower luminescence and a confirmed increase in cAMP compared to the comparative test. Furthermore, it was found that lower dilution ratios resulted in lower luminescence and a greater increase in cAMP. On the other hand, for water extracts from wakame thallus that do not contain proteins and EVs (see Test Examples 1 and 4 below), there was no difference in luminescence compared to the comparative test with only the culture medium added, indicating no effect on increasing cAMP. Furthermore, for BG extracts from wakame female gametophytes and wakame thallus that contain fucoxanthin but do not contain EVs, no effect on increasing cAMP was confirmed compared to the comparative test.

[0185] Incidentally, it is also possible to prepare a powdered composition consisting of extracellular vesicles (EVs) and ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit by cooling and freezing the aqueous extract from the female gametophyte of wakame corresponding to Examples 1-1 and 1-2 to -50°C using liquid nitrogen, and then freeze-drying it under reduced pressure to 4 Pa. Thus, the composition of the present invention can be freely prepared in both solution and powder form, and its viscosity can be freely adjusted. Furthermore, since the extracellular vesicles (EVs) only contain components present in the source cells, there is no concern about contamination with harmful components, and the composition is easy to handle.

[0186] The aqueous extract of the filamentous sporophytes of *Inophyllum uppurum* according to Example 2 was diluted 300-fold using a culture medium (Keratinocyte Growth Medium 2 (available as TakaraBio Co., Ltd. C-20011)). The aqueous extract of the male gametophyte of Sargassum fuscoguttatum according to Example 3 was diluted 300-fold using a culture medium (Keratinocyte Growth Medium 2 (available as TakaraBio Co., Ltd. C-20011)). The aqueous extract of the filamentous sporophytes of *Liriope muscaria* according to Example 4 was diluted 300-fold using a culture medium (Keratinocyte Growth Medium 2 (available from TakaraBio Co., Ltd. as C-20011)). Subsequently, the effect of increasing cAMP was measured using the same method as described in (E) above. The results are shown in Figure 11. As shown in Figure 11, aqueous extracts of the male gametophyte and the filamentous sporophyte were found to have a cAMP-increasing effect. Furthermore, Figures 10 and 11 reveal that the aqueous extract of the gametophyte has a greater cAMP-increasing effect than the aqueous extract of the filamentous sporophyte.

[0187] (Comparative Example 4) A female gametophyte sample was obtained in the same manner as described above (A-1). Then, 10 mg of the female gametophyte sample was suspended in 1 mL of 70 wt% ethanol (the remaining 30% being water) and stirred at 200 rpm for 1 hour at 25°C using a rotator (TAITEC, RT-5N). Next, the solution was removed from the 2 mL tube containing the stirred female gametophyte sample solution using a micropipette and separated into solid precipitate and supernatant.

[0188] The supernatant was filtered using a PES filter, labeled, and analyzed using the same method as described in (B-2-1) above. The liquid chromatography chart is shown in Figure 12 (solid line: protein, dotted line: lipid). As can be seen from Figure 12, the protein and lipid peaks around 10-12 minutes, which are observed in Figure 2, are not observed in Figure 12. Therefore, it can be understood that extracellular vesicles (EVs) are not observed in the 70% ethanol extract of the female gametophyte.

[0189] (Test Example 1) (1) 10 g of dried wakame seaweed (manufactured by Marukome Co., Ltd.) and 10 g of dried mozuku seaweed (manufactured by JF Okinawa Fisheries Cooperative Association), consisting of thallus (adult) parts, were crushed into powder using a mixer mill. Then, 1 g of this powder was suspended in 40 mL of distilled water and stirred in a rotator (TAITEC, RT-5N) at 200 rpm for 0.5 hours at a temperature of 4°C.

[0190] (2) The stirred thallus (adult) sample solution from (1) was centrifuged at 4°C, 8000 rpm for 20 minutes using a centrifuge to separate it into a solid precipitate and a supernatant.

[0191] (3) The supernatant from (2) was filtered through a coffee filter, and this filtrate was filtered through a 0.1 μm PES filter (manufactured by Membrane Solutions LLC).

[0192] (4) 20 mL of this filtrate was concentrated using a 300 K spin column (manufactured by Nippon Pall Co., Ltd.) at 6000 rpm for 10 min, washed three times with 2 mL of PBS (phosphate-buffered saline), filtered, and 0.5 mL of the filtrate was collected (40-fold concentration).

[0193] (5) The aqueous extracts of the thallus of these wakame and mozuku seaweeds were labeled with GIF-2276 and ExoSparkler Exosome Membrane Labeling Kit-Red, as described in (B-2-1) above, and the presence or absence of extracellular vesicles (EVs) was confirmed using liquid chromatography apparatus A. Figure 13 is a chromatography chart of the aqueous extract of the wakame thallus, and Figure 14 is a chromatography chart of the aqueous extract of the mozuku thallus.

[0194] As can be seen from Figures 13 and 14, the protein and lipid peaks around 10-12 minutes, which are observed in Figure 2, are not observed in Figures 13 and 14. Therefore, it can be understood that extracellular vesicles (EVs) are not observed in aqueous extracts of the thallus. Generally, when wakame and mozuku are used commercially and industrially, the thallus is used, but extracellular vesicles (EVs) cannot be obtained from the thallus. The reason for this is not clear, but it is hypothesized that either the extracellular vesicles (EVs) cannot be released at all, or they are destroyed when they are released. Furthermore, when the aqueous extract of the wakame thallus obtained in (4) above was analyzed for proteins using the same method as in (D-1) above, although the electrophoretic band color was faint, the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit were identified.

[0195] (Test Example 2) To 20 μL of the aqueous extract of the female gametophyte of wakame seaweed according to Example 1, a mixed solvent of PBS (phosphate-buffered saline) and BG (weight ratio BG / PBS = 9 / 1) was added to prepare a 10-fold dilution of the aqueous extract of the female gametophyte of wakame seaweed. The presence or absence of extracellular vesicles (EVs) in the 10-fold diluted water extract of the female gametophyte of wakame seaweed was confirmed using liquid chromatography apparatus A after labeling in the same manner as described above (B-2-1). The resulting chart is shown in Figure 15 (solid line: protein, dotted line: lipid). In Figure 15, as can be seen in Figure 2, peaks for protein and lipid membranes can be observed around 10 to 12 minutes. This indicates that extracellular vesicles (EVs) extracted with water can remain even when transferred to a solvent with a high concentration of organic solvent, such as BG / PBS=9 / 1.

[0196] (Test Example 3) To 0.5 mL of the aqueous extract of the female gametophyte of wakame seaweed according to Example 1, 0.5 mL of caprylic acid triglyceride was added and shaken for 5 minutes, then allowed to stand to separate the aqueous extract of the female gametophyte of wakame seaweed and the caprylic acid triglyceride into two phases. Next, the caprylic acid triglyceride phase was pipetted, and the presence or absence of extracellular vesicles (EVs) in this caprylic acid triglyceride was confirmed using liquid chromatography apparatus A after labeling in the same manner as described in (B-2-1.) above. The resulting chart was the same as in Figure 13, and no peaks corresponding to extracellular vesicles (EVs) were observed.

[0197] (Test Example 4) (1) 0.1 g of a powder sample obtained by crushing dried wakame seaweed (manufactured by Marukome Co., Ltd.), consisting of thallus (adult) parts, was suspended in 10 mL of distilled water and stirred at 200 rpm for 0.5 hours at a temperature of 4°C using a rotator (TAITEC, RT-5N). (2) The stirred female gametophyte sample solution from (1) was centrifuged using a centrifuge at 4°C, 8000 rpm for 30 minutes to separate it into a solid precipitate and a supernatant. (3) The supernatant from (2) was filtered through a coffee filter, and this filtrate was filtered through a 0.45 μm PES filter (manufactured by Membrane Solutions LLC) and a 0.1 μm PES filter (manufactured by Membrane Solutions LLC). (4) 20 mL of this filtrate was concentrated (6000 rpm for 10 min) using a spin column (manufactured by Nippon Pole Co., Ltd.) at 300 K, washed and filtered twice using 1 mL of PBS (phosphate-buffered saline), and 200 μL of the filtrate was collected. This was used as the water extract of the wakame thallus according to Example 1. (5) The following samples were prepared Sample A: Product name PM007-0500 (standard sample) manufactured by BIO-HELIX Sample E: The water extraction sample prepared in (1) to (4) above. Sample F: The liquid passed through a 300 K spin column in (1) to (4) above Sample G: The filtrate after performing the treatments in (1) to (3) above (6) In the same manner as in (D-1.) above, protein identification was attempted by electrophoresis. The results are shown in Fig. 16. As understood from Fig. 16, the protein band did not develop color, and no protein could be confirmed in the water extract of the wakame thallus. As understood from Fig. 13, no extracellular vesicles (EVs) could be confirmed in the water extract of the wakame thallus. Therefore, it is presumed that the protein is attached to the surface of the extracellular vesicles (EVs) or exists inside them.

[0198] (F. Measurement of the melanin inhibitory effect of the water extract) (1) Phosphate-buffered saline (PBS) containing 1 × 10 5 cells / 0.05 ml was placed in a 24-well plate.

[0199] (2) A culture solution was prepared by adding 10% fetal bovine serum (manufactured by Merck, Fetal Calf Serum (FCS)) and penicillin-streptomycin solution (manufactured by FUJIFILM Wako Pure Chemical Corporation, product name: WAKO 168-2319) to DMEM high glucose medium (manufactured by FUJIFILM Wako Pure Chemical Corporation, product name: WAKO 043-30085). This was poured into a 24-well plate and cultured overnight.

[0200] (3) The culture solution was aspirated with a Pasteur pipette.

[0201] (4) To a new DMEM high glucose medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product name: WAKO 043-30085), Forskolin (manufactured by Tokyo Chemical Industry Co., Ltd., product name: F0855), an activator of melanoma cells, was added to a concentration of 20 μM. Further, a water extract of the filamentous form of the sporophyte of Upleinori according to Example 2 prepared in (B-1-2.), a water extract of the filamentous form of the sporophyte of Asaxanolide according to Example 4 prepared in (B-1-4.), a water extract of the female gametophyte of Undaria pinnatifida according to Example 1 prepared in (B-1-1.), and a water extract of the frond of Undaria pinnatifida according to Test Example 1 were added at 2 μL (corresponding to 0.1 vol% of each extract), 6 μL (corresponding to 0.3 vol% of each extract), and 20 μL (corresponding to 1 vol% of each extract), respectively, to prepare culture solutions (three types with the water extract according to Example 2 added, three types with the water extract according to Example 4 added, three types with the water extract according to Example 1 added, and three types with the water extract according to Test Example 1 added). Also, as control culture solutions, two types of culture solutions were prepared: a culture solution containing only DMEM high glucose medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product name: WAKO 043-30085), and a culture solution in which Forskolin (manufactured by Tokyo Chemical Industry Co., Ltd., product name: F0855), an activator of melanoma cells, was added to a concentration of 20 μM to DMEM high glucose medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product name: WAKO 043-30085).

[0202] (5) The above 14 types of culture solutions were each poured into the 24-well plate of (3) (10 wells were left empty) and cultured at 25°C for 48 hours.

[0203] (6) Discard the culture medium, aspirate the melanoma cells from each of the 14 wells and transfer them to a sample tube, wash each cell with 1 mL of phosphate-buffered saline (PBS), add more phosphate-buffered saline (PBS) and mix, then aspirate 700 μL to collect the melanoma cells, and centrifuge them using a centrifugal separator at 10,000 rpm for 3 minutes. Discard the supernatant after centrifugation, add 50 μL of pure water to the remaining solids and suspend them, then add more pure water to make a total volume of 100 μL.

[0204] (7) 100 μL of each of the 14 types of suspensions prepared in (6) was divided into two 50 μL portions.

[0205] (8) A 4N sodium hydroxide aqueous solution was added to one of the 50 μL suspensions and heated at 50°C for 3 hours to disrupt the cell membranes and disperse the intracellular melanin into the suspension.

[0206] (9) The absorbance of 450 nm visible light (OD450) of a suspension obtained by disrupting the cell membrane and dispersing the melanin inside the cell was measured using an ultra-trace ultraviolet-visible spectrophotometer (manufactured by NanoDrop1000, product name: ND-1000).

[0207] (10) For the other 50 μL suspensions, Coomassie Brilliant Blue was conjugated to living cells in the suspension using Protein Assay CBB (manufactured by Nacalai Tesque Co., Ltd.), and the absorbance of visible light at 600 nm (OD600) was measured using a micro-UV-Vis spectrophotometer.

[0208] (11) The OD450 / OD600 value was calculated as the amount of melanin produced per living cell. The results for each culture medium are shown in Figure 17.

[0209] As can be seen from Figure 17, it was found that aqueous extracts of the filamentous sporophytes of *Uriaria japonica* (Example 2), the filamentous sporophytes of *Uriaria saccharifolia* (Example 4), and the female gametophytes of *Wakame seaweed* (Example 1) all exhibited high melanin production inhibitory effects even in the presence of Forskolin, a melanoma cell activator. On the other hand, in the case of an aqueous extract of the thallus (adult) of wakame seaweed (Test Example 1), it was found that there was almost no melanin production inhibitory effect in the presence of Forskolin.

Claims

1. A composition comprising at least one protein selected from the ribose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs) derived from the gametophyte and / or sporophyte of seaweed.

2. The composition according to claim 1, further comprising at least one selected from ATP synthase subunit β, thiamine biosynthesis protein G, and histone H2B.

3. The composition according to claim 1, wherein the seaweed is the female gametophyte of the seaweed.

4. The composition according to claim 1, wherein the seaweed is a filamentous sporophyte.

5. The composition according to claim 1, wherein the seaweed is brown algae.

6. The composition according to claim 1, wherein the composition comprises an aqueous solvent or a mixed solvent consisting of an aqueous solvent and an organic solvent.

7. The composition according to claim 1, further comprising at least one compound selected from the group consisting of fucoxanthin and its derivatives.

8. The composition according to claim 1, wherein at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit is encapsulated in or attached to extracellular vesicles (EVs) derived from the gametophyte of seaweed.

9. The composition according to claim 1, wherein the composition is a powdered composition.

10. An agent for increasing intracellular cyclic adenosine monophosphate (cAMP) comprising the composition according to any one of claims 1 to 9.

11. A method for producing a composition comprising at least one protein selected from the ribose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs) derived from the gametophyte and / or sporophyte of seaweed, comprising the following steps (1) and (2). Step (1): The gametophyte and / or sporophyte of the seaweed are dried to obtain a dried product, and then the dried product is pulverized to obtain a powder of the gametophyte and / or sporophyte of the seaweed, and the powder is dispersed in an aqueous solvent adjusted to a temperature of 5°C or lower. Step (2): The aqueous solvent in which the powder is dispersed is separated into solids and supernatant by centrifugation, and the supernatant is collected.

12. A method for producing the composition according to claim 11, comprising the step of adding at least one compound selected from the group consisting of fucoxanthin and its derivatives to the supernatant liquid.

13. A method for producing the composition according to claim 11, comprising the step of removing the aqueous solvent from the supernatant to obtain a powder containing at least one protein selected from ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs) derived from the gametophyte and / or sporophyte of seaweed.

14. A method for producing an intracellular cyclic adenosine monophosphate (cAMP) increase agent, comprising a method for producing the composition according to any one of claims 11 to 13.

15. A method for producing the composition according to claim 11, wherein the extracellular vesicles (EVs) are derived from the female gametophyte of the seaweed.

16. The method for producing the composition according to claim 11, wherein the extracellular vesicles (EVs) are derived from the filamentous sporophytes of the seaweed.

17. A raw material comprising an aggregate of gametophytes and / or sporophytes of seaweed, used for extracting at least one protein selected from the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and the ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs) derived from the gametophytes of seaweed.

18. The raw material according to claim 17, wherein the seaweed is a female gametophyte.

19. The raw material according to claim 17, wherein the seaweed is a filamentous sporophyte.

20. The raw material according to any one of claims 17 to 19, wherein the seaweed is a brown algae.

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