Composition, method for producing composition, raw material, use of extracellular vesicles (EVS) derived from seaweed, agent for improving stability, method for improving stability, agent for increasing cyclic adenosine monophosphate, method for producing agent for increasing cyclic adenosine monophosphate, permeable composition, and permeation enhancer

Abstract

Provided is a composition that includes an extract from seaweed and has excellent action and efficacy. This composition is characterized by including extracellular vesicles (EVs) and carotenoids.

Description

Composition, method for producing composition, raw material, use of seaweed-derived extracellular vesicles (EVs), stability improver, method for improving stability, agent for increasing cyclic adenosine monophosphate, method for producing agent for increasing cyclic adenosine monophosphate, permeation composition, and permeation enhancer

[0001] (First Invention) The present invention relates to a composition comprising extracellular vesicles (EVs) and a carotenoid such as at least one compound selected from the group consisting of fucoxanthin and its derivatives, and a method for producing the composition; raw materials for obtaining a composition comprising extracellular vesicles (EVs) and a carotenoid such as at least one compound selected from the group consisting of fucoxanthin and its derivatives; the use of seaweed-derived extracellular vesicles (EVs) for improving the stability of carotenoids; a carotenoid stability improver comprising seaweed-derived extracellular vesicles (EVs); and a method for improving carotenoid stability by allowing seaweed-derived extracellular vesicles (EVs) to coexist with carotenoids.

[0002] (Second Invention) The present invention also relates to a composition comprising extracellular vesicles (EVs) derived from seaweed gametophytes and a specified protein and a method for producing the same, a cyclic adenosine monophosphate (cAMP) increaser containing the composition and a method for producing the same, and raw materials used for extracting extracellular vesicles (EVs) derived from seaweed gametophytes, a specified protein, and extracellular vesicles (EVs) derived from seaweed gametophytes.

[0003] (Third Invention) The present invention also relates to a composition comprising at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, and a method for producing the composition; a raw material for obtaining the composition comprising at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof; a composition capable of penetrating into cellular tissue, comprising at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof; and a penetration enhancer into cellular tissue, comprising at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof.

[0004] Fucoxanthin (C), a red pigment found in large amounts in brown algae such as wakame and kelp, ４２ H ５８ O ６ Fucoxanthin (fucoxanthin) possesses various excellent physiologically active functions, including anti-obesity, anti-diabetic, anti-inflammatory, and even skin-whitening effects, in addition to its excellent antioxidant properties, and its use as a functional food ingredient is highly anticipated. However, fucoxanthin's low stability against heat and light limits its use in foods, beverages, cosmetics, pharmaceuticals, and other applications. For this reason, various techniques for stabilizing fucoxanthin have been studied. Furthermore, various studies have been conducted on the extraction of components from seaweed and microalgae and their uses.

[0005] Patent Document 1 discloses a technique for stabilizing fucoxanthin by blending a thickener such as hyaluronic acid or xanthan gum with propolis extract and / or peanut seed coat extract.

[0006] Patent Document 2 discloses a technique for inclusion of one or more species selected from fucoxanthin and its derivatives with one or more species selected from cyclodextran and its derivatives.

[0007] Patent Document 3 discloses a method for isolating a lipophilic extract from seaweed gametophytes, in which an aqueous-alcoholic suspension of brown algal gametophyte cells is mixed with at least one fatty acid triglyceride containing 8 to 22 carbon atoms, and then water is added to isolate the lipophilic extract. Patent Document 4 discloses a cosmetic preparation for topical use, which contains a freeze-dried product of brown algal gametophyte cells as an active ingredient, and also discloses that the freeze-dried product of brown algal cells contains 1% or more of fucoxanthin.

[0008] Patent Document 4 discloses a freeze-dried product of brown algae cells that is enriched in fucoxanthin.

[0009] Patent Document 5 discloses a method for extracting water-soluble components of seaweed, which comprises adding water to fine pieces or powder of seaweed and mixing them, followed by wet grinding at room temperature to dissolve and extract the water-soluble components of the seaweed in water.

[0010] Patent Document 6 discloses a wakame protein-containing composition obtained by adding water, a salt solution, or a weak alkaline solution to the thallus or dried granules of wakame seaweed, wet grinding the thallus or dried granules, extracting soluble components, and separating the protein from the extract.

[0011] Patent Document 7 discloses a method for isolating a lipophilic extract from seaweed gametophytes, in which an aqueous-alcoholic suspension of brown algae gametophyte cells is mixed with at least one fatty acid triglyceride containing 8 to 22 carbon atoms, and then water is added to isolate the lipophilic extract.

[0012] Patent Document 8 discloses a cosmetic preparation for topical use that contains a freeze-dried product of gametophyte cells of brown algae as an active ingredient, and also discloses that the freeze-dried product of brown algae cells contains 1% or more of fucoxanthin.

[0013] Patent Document 9 discloses a process for producing fucoxanthin and / or polysaccharides from microalgae, which may include an absorption enhancer.

[0014] Furthermore, Patent Document 10 discloses a composition containing carotenoids and fatty acids extracted from microalgae, and discloses that the fucoxanthin content is 1.7 to 2.0% / DW and the linoleic acid content is 0.23% / DW.

[0015] Japanese Patent No. 7057568 Japanese Patent Application Laid-Open No. 2021-127369 Japanese Patent No. 6731425 Japanese Patent No. 4979592 Japanese Patent Application Laid-Open No. 2004-49072 Japanese Patent Application Laid-Open No. 2004-97021 Japanese Patent No. 6731425 Japanese Patent No. 4979592 Special Publication No. 2019-506133 Special Publication No. 2018-512432

[0016] (First Invention) The first invention will be described below. Fucoxanthin (C), a red pigment found in large amounts in brown algae such as wakame seaweed and kelp, is a compound of the present invention. ４２ H ５８ O ６Fucoxanthin has a variety of excellent physiologically active functions, including anti-obesity, anti-diabetic, anti-inflammatory, and even skin-whitening effects, in addition to its excellent antioxidant properties, and its use as a functional food ingredient is highly anticipated. However, fucoxanthin's low stability against heat and light limits its use in foods, beverages, cosmetics, pharmaceuticals, and other applications. For this reason, various techniques for stabilizing fucoxanthin have been studied.

[0017] For example, Patent Document 1 discloses a technique for stabilizing fucoxanthin by blending a thickener such as hyaluronic acid or xanthan gum with propolis extract and / or peanut seed coat extract. Patent Document 2 discloses a technique for encapsulating one or more compounds selected from fucoxanthin and its derivatives with one or more compounds selected from cyclodextran and its derivatives. Patent Document 3 discloses a method for isolating a lipophilic extract from seaweed gametophytes, in which an aqueous-alcoholic suspension of brown algal gametophyte cells is mixed with at least one fatty acid triglyceride containing 8 to 22 carbon atoms, followed by addition of water to isolate the lipophilic extract. Patent Document 4 discloses a cosmetic preparation for topical use containing a freeze-dried product of brown algal gametophyte cells as an active ingredient, and also discloses that the freeze-dried product of brown algal cells contains 1% or more fucoxanthin.

[0018] The method of Patent Document 1 requires the addition of a thickener, which reduces the handleability of the resulting fucoxanthin composition. Furthermore, the method of Patent Document 2 uses cyclic saccharides with inclusion function, such as cyclodextran. However, because cyclodextran can inclusion a relatively wide range of substances, when applied to cosmetics, pharmaceuticals, and food products, there is a possibility that compounds or ions that are undesirable for intracellular uptake may be included together with fucoxanthin. Therefore, care must be taken when coexisting with fucoxanthin encapsulated by cyclodextran, resulting in handling problems. Furthermore, Patent Document 3 is a technology for isolating lipophilic extracts such as fucoxanthin, but does not consider stabilizing fucoxanthin. Patent Document 4 discloses obtaining fucoxanthin by ethanol extraction of freeze-dried product of brown algae gametophyte cells, but does not consider stabilizing fucoxanthin.

[0019] The first invention has been made in view of the above-mentioned problems, and aims to provide a composition containing a carotenoid such as fucoxanthin that is easy to handle and has improved stability against heat and light, and a method for producing the same.

[0020] The present invention relates to a composition comprising extracellular vesicles (EVs) and a carotenoid. In the present invention, extracellular vesicles (EVs) are a collective term for vesicles with a lipid bilayer structure secreted from living cells, and the concept of extracellular vesicles includes exosomes, microvesicles, and apoptotic bodies. Exosomes have a diameter of 1 to 200 nm. Exosomes are formed by the inward budding of late endosomal membranes, which then fuse with the plasma membrane to form complete particles, which are then secreted extracellularly by exocytosis. Microvesicles (MVs) are generated by the outward budding and separation of the plasma membrane, and have a diameter of 100 to 1,000 nm. Apoptotic bodies are generated when cells undergo organized cell death (apoptosis), and have a diameter of 50 to 5,000 nm.

[0021] The carotenoids used in the present invention include at least one compound selected from the group consisting of fucoxanthin and its derivatives contained in brown algae, diadinoxanthin, diatoxanthin, α-carotene, β-carotene, rutin, and antheraxanthin contained in red algae, and β-carotene, zeaxanthin, rutin, and siphonaxanthin contained in green algae. These carotenoids are generally unstable to heat and light, and fucoxanthin in particular has a variety of excellent physiologically active functions, such as anti-obesity, anti-diabetic, anti-inflammatory, and even whitening effects, in addition to its excellent antioxidant effect. However, it is the most unstable to heat and light and is therefore the carotenoid for which the stabilizing effect of the present invention is most effective.

[0022] In the present invention, carotenoids, such as at least one compound selected from the group consisting of fucoxanthin and its derivatives, coexist with extracellular vesicles (EVs), thereby making carotenoids such as fucoxanthin and its derivatives less susceptible to decomposition by heat or light. The mechanism by which the coexistence of carotenoids such as fucoxanthin and its derivatives with extracellular vesicles (EVs) inhibits their decomposition is unclear, but is hypothesized as follows: For example, fucoxanthin is a xanthophyll with a unique chemical structure containing an allene bond, an epoxide structure, and a conjugated double bond. It is also hypothesized that the decomposition proceeds when the conjugated double bond is cleaved by ozone, oxygen radicals, and hydroxyl radicals generated by heat or light. It is hypothesized that extracellular vesicles trap and inactivate such ozone, oxygen radicals, and hydroxyl radicals on their lipid membrane surfaces, and that their coexistence with extracellular vesicles inhibits the decomposition of fucoxanthin by heat, light, or the like. In addition, carotenes such as beta-carotene also contain conjugated double bonds that are easily broken by ozone, oxygen radicals, and hydroxyl radicals generated by heat and light. It is thought that extracellular vesicles trap and deactivate such ozone, oxygen radicals, and hydroxyl radicals on the surface of their lipid membranes, and that by coexisting with the extracellular vesicles, they prevent the decomposition of carotenes by heat, light, etc.

[0023] Examples of fucoxanthin derivatives include fucoxanthin hydrolysates or derivatives thereof, fucoxanthin esters (for example, esters with amino acids, carboxylic acids, inorganic acids, or fatty acids), salts thereof, and fucoxanthin glycosides. More specific examples of fucoxanthin derivatives include, but are not limited to, monoesters and homogeneous or heterogeneous diesters selected from the following: fucoxanthinol, which is a hydrolysis product; amarousiaxanthin A, which is fucoxanthinol that has undergone dehydration and isomerization; esters of fucoxanthin with amino acids such as glycine and alanine; esters and salts of fucoxanthin with carboxylic acids such as acetic acid and citric acid; esters and salts of fucoxanthin with inorganic acids such as phosphoric acid and sulfuric acid; and fatty acid esters of fucoxanthin with 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; and glycosides such as glucosides.

[0024] In the present invention, the extracellular vesicles (EVs) are preferably derived from seaweed. This is because seaweed-derived extracellular vesicles encapsulate fucoxanthin or a derivative thereof in their lipid membrane or release fucoxanthin or a derivative thereof attached to the lipid membrane outside the seaweed cells, thereby contributing to the stabilization of fucoxanthin and its derivatives. Note that, in the present invention, seaweed refers to a group of marine species of multicellular algae, and does not include microalgae, which are unicellular algae.

[0025] In the present invention, the seaweed is preferably a gametophyte and / or sporophyte of seaweed. Seaweeds exhibit various morphologies in their life cycle, including sporophytes (thallus, discus, filamentous body), male and female gametophytes (thallus, discus, filamentous body), etc. Male and female gametophytes are preferred in the present invention because they contain a high amount of carotenoids, such as fucoxanthin and its derivatives.

[0026] In the present invention, the seaweed is preferably a female gametophyte of seaweed, because the female gametophyte has the highest content of fucoxanthin or a derivative thereof among the various forms in the life cycle of seaweed.

[0027] In the present invention, the seaweed is preferably brown algae. Seaweed includes green algae, red algae, brown algae, etc., and brown algae are preferred because they contain a large amount of fucoxanthin or a derivative thereof.

[0028] In the present invention, the seaweed may be a filamentous sporophyte. Extracellular vesicles (EVs) can also be extracted from the filamentous sporophyte.

[0029] In the present invention, a composition containing extracellular vesicles (EVs) and a carotenoid such as at least one compound selected from the group consisting of fucoxanthin and its derivatives preferably contains an aqueous solvent or a mixed solvent consisting of an aqueous solvent and an organic solvent, and the organic solvent is preferably an organic solvent that dissolves carotenoids such as at least one compound selected from the group consisting of fucoxanthin and its derivatives. In the present invention, a composition containing extracellular vesicles (EVs) and a carotenoid such as fucoxanthin or its derivatives preferably contains an aqueous solvent. This is because the lipid membrane of extracellular vesicles is less likely to be destroyed in an aqueous solvent, and their morphology is easily maintained. The lipid membrane of extracellular vesicles is easily destroyed in organic solvents such as alcohols that dissolve carotenoids such as fucoxanthin and its derivatives. The aqueous solvent is water or a solvent in which water-soluble salts are dissolved, and includes pure water, seawater, etc. The aqueous solvent may also contain antifoaming agents, thixotropic agents, pH adjusters, etc., as appropriate. In addition, artificial seawater, filtered seawater, deep seawater, etc. can also be used as seawater. Furthermore, in the present invention, the composition containing extracellular vesicles (EVs) and carotenoids such as fucoxanthin or its derivatives may contain a mixed solvent consisting of an aqueous solvent and an organic solvent. The organic solvent is preferably one that dissolves carotenoids such as at least one compound selected from the group consisting of fucoxanthin and its derivatives. This is because the inclusion of an aqueous solvent makes the extracellular vesicles (EVs) less likely to be destroyed, even when the composition contains an organic solvent that dissolves carotenoids such as at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0030] In the present invention, the composition containing extracellular vesicles (EVs) and at least one carotenoid, such as fucoxanthin and its derivatives, is preferably a powdered composition. Powdered compositions can reduce the volume per component compared to solvent-containing compositions, thereby reducing transportation costs. Furthermore, when used as a cosmetic, pharmaceutical, or food ingredient, they are easily mixed with other raw materials, making them useful as industrial raw materials.

[0031] In the present invention, it is desirable that at least a portion of the carotenoids, such as at least one compound selected from the group consisting of fucoxanthin and its derivatives, be attached to or encapsulated in the extracellular vesicles (EVs), because this makes the carotenoids, such as at least one compound selected from the group consisting of fucoxanthin and its derivatives, contained in the composition less susceptible to decomposition by heat or light.

[0032] In the present invention, the carotenoids, such as at least one compound selected from the group consisting of fucoxanthin and its derivatives, may be present both attached to or encapsulated in the extracellular vesicles (EVs) and free from the extracellular vesicles (EVs). It is believed that the extracellular vesicles (EVs) trap ozone and radicals generated by heat or light. Therefore, even if the carotenoids, such as at least one compound selected from the group consisting of fucoxanthin and its derivatives, are not in contact with the extracellular vesicles (EVs), the compounds are less susceptible to decomposition by heat or light, ensuring high stability.

[0033] The present invention also encompasses a method for producing a composition containing extracellular vesicles (EVs) derived from seaweed gametophytes and / or sporophytes and a carotenoid such as at least one compound selected from the group consisting of fucoxanthin and its derivatives, the method comprising the following steps (1) and (2): Step (1): Dispersing seaweed gametophytes and / or sporophytes in an aqueous solvent to obtain a dispersion of the seaweed gametophytes and / or sporophytes; Step (2): Separating and removing the seaweed gametophytes and / or sporophytes from the dispersion of the seaweed gametophytes and / or sporophytes obtained in step (1) to obtain an aqueous solvent composition containing the extracellular vesicles (EVs) and a carotenoid such as at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0034] Marine algae gametophytes and / or sporophytes release extracellular vesicles (EVs) in an aqueous medium. Marine algae gametophytes and / or sporophytes are dispersed in an aqueous solvent, and the extracellular vesicles (EVs) are extracted into the dispersion. The extracellular vesicles (EVs) encapsulate carotenoids such as fucoxanthin or its derivatives in their lipid membranes, or fucoxanthin or its derivatives are attached to the lipid membrane surface of the extracellular vesicles (EVs). Carotenoids such as fucoxanthin and its derivatives are insoluble in water, but are dispersed in an aqueous solvent together with the extracellular vesicles (EVs) by being encapsulated in the extracellular vesicles (EVs) or by being attached to the lipid membrane on the surface of the extracellular vesicles (EVs). If the aqueous solvent contains an organic solvent, such as ethanol or butylene glycol, that dissolves carotenoids such as fucoxanthin and its derivatives, the extracellular vesicles immediately after release from the gametophytes and / or sporophytes are unstable, resulting in the dissolution and destruction of the lipid membranes of the extracellular vesicles. Therefore, the solvent for dispersing the gametophytes and / or sporophytes is preferably an aqueous solvent, and preferably does not contain such an organic solvent, and preferably does not contain an organic solvent that dissolves carotenoids such as at least one compound selected from the group consisting of fucoxanthin and its derivatives. By removing the seaweed gametophytes and / or sporophytes from the dispersion, an aqueous solvent composition containing extracellular vesicles (EVs) derived from the seaweed gametophytes and / or sporophytes and carotenoids such as at least one compound selected from the group consisting of fucoxanthin and its derivatives can be obtained.

[0035] The present invention preferably relates to a method for producing a composition comprising extracellular vesicles (EVs) derived from seaweed gametophytes and / or sporophytes and carotenoids such as at least one compound selected from the group consisting of fucoxanthin and its derivatives, further comprising step (5-1). Step (5-1): The aqueous solvent composition obtained in step (2) is dried to obtain a powdery composition comprising the extracellular vesicles (EVs) and carotenoids such as at least one compound selected from the group consisting of fucoxanthin and its derivatives. The solvent is removed from the composition obtained in step (2) comprising extracellular vesicles (EVs) derived from seaweed gametophytes and / or sporophytes and carotenoids such as at least one compound selected from the group consisting of fucoxanthin and its derivatives, to obtain a powdery composition.

[0036] The present invention preferably relates to a method for producing a composition containing extracellular vesicles (EVs) derived from seaweed gametophytes and / or sporophytes and carotenoids such as at least one compound selected from the group consisting of fucoxanthin and its derivatives, further comprising steps (3) and (4). Step (3): To the seaweed gametophytes isolated in step (2), an organic solvent capable of dissolving carotenoids such as at least one compound selected from the group consisting of fucoxanthin and its derivatives is added to obtain an organic solvent composition containing carotenoids such as at least one compound selected from the group consisting of fucoxanthin and its derivatives. Step (4): To the organic solvent composition containing carotenoids such as at least one compound selected from the group consisting of fucoxanthin and its derivatives obtained in step (3), an aqueous solvent composition containing the extracellular vesicles (EVs) obtained in step (2) and carotenoids such as at least one compound selected from the group consisting of fucoxanthin and its derivatives is added to obtain a mixed solvent composition of the aqueous solvent and the organic solvent.

[0037] In step (3), an organic solvent capable of dissolving carotenoids, such as at least one compound selected from the group consisting of fucoxanthin and its derivatives, is added to the seaweed gametophytes and / or sporophytes separated in step (2). Carotenoids, such as fucoxanthin and its derivatives, in the seaweed gametophytes and / or sporophytes are eluted into the organic solvent. An aqueous solvent composition containing the extracellular vesicles (EVs) obtained in step (2) and carotenoids, such as at least one compound selected from the group consisting of fucoxanthin and its derivatives, is added to the organic solvent composition containing carotenoids, such as at least one compound selected from the group consisting of fucoxanthin and its derivatives, obtained in step (3), to obtain a mixed solvent composition of an aqueous solvent and an organic solvent containing extracellular vesicles (EVs) derived from seaweed gametophytes and / or sporophytes and carotenoids, such as at least one compound selected from the group consisting of fucoxanthin and its derivatives. It is believed that once the extracellular vesicles (EVs) are dispersed in an aqueous solvent, a protective membrane formed on their surface by hydration with water molecules is formed. Therefore, even if the EVs are subsequently mixed with an organic solvent capable of dissolving a carotenoid, such as at least one compound selected from the group consisting of fucoxanthin and its derivatives, the lipid membrane of the extracellular vesicles is unlikely to be destroyed by the organic solvent. Furthermore, the organic solvent composition containing a carotenoid, such as at least one compound selected from the group consisting of fucoxanthin and its derivatives, obtained in step (3) may also contain an aqueous solvent. This is because the gametophytes and / or sporophytes of seaweed contain an aqueous solvent, and the aqueous solvent contained in the gametophytes and / or sporophytes may be contaminated with the organic solvent. In the mixed solvent composition of an aqueous solvent and an organic solvent obtained in step (4), the weight ratio of the organic solvent to the aqueous solvent is preferably organic solvent / aqueous solvent ≦9 / 1, and preferably organic solvent / aqueous solvent = 1 / 10 to 10 / 10.

[0038] The present invention preferably provides a method for producing a composition containing extracellular vesicles (EVs) derived from seaweed gametophytes and / or sporophytes and carotenoids such as at least one compound selected from the group consisting of fucoxanthin and its derivatives, further comprising step (5-2). Step (5-2): The mixed solvent composition containing the aqueous solvent and the organic solvent obtained in step (4) is dried to obtain a powdery composition containing the extracellular vesicles (EVs) and carotenoids such as at least one compound selected from the group consisting of fucoxanthin and its derivatives. The powdery composition is obtained by removing the solvent from the mixed solvent composition containing extracellular vesicles (EVs) derived from seaweed gametophytes and / or sporophytes and carotenoids such as at least one compound selected from the group consisting of fucoxanthin and its derivatives obtained in step (4).

[0039] In the present invention, the sporophyte may be a filamentous body.

[0040] The present invention also encompasses a raw material comprising a collection of seaweed gametophytes and / or sporophytes, which can be used to extract extracellular vesicles (EVs) and carotenoids such as at least one compound selected from the group consisting of fucoxanthin and its derivatives. Seaweed gametophytes and / or sporophytes are approximately 200 μm to 2000 μm in size, making them easy to cultivate in a small space, and contain a higher amount of carotenoids such as fucoxanthin and its derivatives than other forms in the seaweed life cycle, such as sporophytes (thallus, discus, filament), making them excellent raw materials for extracting extracellular vesicles (EVs) and carotenoids such as at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0041] The seaweed is preferably brown algae, since brown algae contain the highest amount of carotenoids, such as fucoxanthin and its derivatives, among all seaweeds.

[0042] The seaweed is preferably a female gametophyte, since it contains more carotenoids, such as fucoxanthin and its derivatives, than a male gametophyte.

[0043] In the raw material of the present invention, the sporophyte may be a filamentous body.

[0044] Seaweed-derived extracellular vesicles (EVs) can be used to improve the stability of carotenoids, such as at least one compound selected from the group consisting of fucoxanthin and its derivatives. The present invention also encompasses the use of seaweed-derived extracellular vesicles (EVs) to improve the stability of carotenoids, such as at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0045] The present invention also includes a stability enhancer for carotenoids such as at least one compound selected from the group consisting of fucoxanthin and its derivatives, including extracellular vesicles (EVs) derived from seaweed.

[0046] The present invention also encompasses a method for improving the stability of carotenoids such as at least one or more compounds selected from the group consisting of fucoxanthin and its derivatives, which comprises causing seaweed-derived extracellular vesicles (EVs) to coexist with at least one or more compounds selected from the group consisting of fucoxanthin and its derivatives.

[0047] According to the present invention, there are provided a composition containing a carotenoid such as fucoxanthin that has improved stability against heat and light, and a method for producing the same. The composition of the present invention contains extracellular vesicles (EVs) and a carotenoid such as at least one compound selected from the group consisting of fucoxanthin and its derivatives, and the carotenoid such as at least one compound selected from the group consisting of fucoxanthin and its derivatives is inhibited from being decomposed by heat or light, resulting in excellent stability and storage stability. Therefore, the composition can be used as a variety of industrial raw materials. Examples of industrial raw materials include those with a wide range of applications, such as pharmaceuticals, food products, and cosmetics.

[0048] The present invention relates to a composition comprising extracellular vesicles (EVs) and a carotenoid. Hereinafter, the present invention will be specifically described using an example in which the carotenoid is at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0049] The present invention relates to a composition comprising extracellular vesicles (EVs) and at least one compound selected from the group consisting of the carotenoid fucoxanthin and its derivatives. In the composition of the present invention, at least one compound selected from the group consisting of fucoxanthin and its derivatives coexists with the extracellular vesicles (EVs), making fucoxanthin and its derivatives less susceptible to decomposition by heat or light. The mechanism by which the coexistence of fucoxanthin and its derivatives with extracellular vesicles (EVs) inhibits their decomposition is unknown, but is hypothesized as follows: Fucoxanthin is a xanthophyll with a unique chemical structure containing an allene bond, an epoxide structure, and a conjugated double bond. It is hypothesized that the decomposition proceeds when the conjugated double bond is cleaved by ozone, oxygen radicals, or hydroxyl radicals generated by heat or light. It is thought that extracellular vesicles trap and inactivate such ozone, oxygen radicals, and hydroxyl radicals on the surface of their lipid membranes, and that their coexistence with extracellular vesicles may prevent the decomposition of fucoxanthin and its derivatives by heat, light, or other factors.

[0050] Examples of fucoxanthin derivatives include fucoxanthin hydrolysates or derivatives thereof, fucoxanthin esters (eg, 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, fucoxanthinol, which is a hydrolysis product of fucoxanthin; amarousiaxanthin A, which is fucoxanthinol that has undergone dehydration and isomerization; esters of fucoxanthin with amino acids such as glycine and alanine; esters and salts of acetic acid, fucoxanthin with carboxylic acids such as citric acid; esters and salts of fucoxanthin with inorganic acids such as phosphoric acid and sulfuric acid; monoesters and homogeneous or heterogeneous diesters selected from fatty acid esters of fucoxanthin with 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; glycosides such as glucosides; and the like. The fucoxanthin derivative may be of one type or of two or more types.

[0051] 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, and may be, for example, 0.001 to 50 wt % in the composition.

[0052] Furthermore, extracellular vesicles (EVs) derived from cells capable of producing fucoxanthin or its derivatives can be used. Examples of cells capable of producing fucoxanthin or its derivatives include microalgae such as diatoms, golden algae, raphidophytes, and haptophytes. More specifically, unicellular diatoms belonging to the genus Phaeodactylum, unicellular diatoms belonging to the genus Thalassiosira, and unicellular haptophytes belonging to the genus Isochrysis are preferred. Furthermore, cells capable of producing fucoxanthin or its derivatives can be derived from seaweed, including brown algae, red algae, and green algae. In the present invention, the extracellular vesicles (EVs) are preferably derived from seaweed. The seaweed-derived extracellular vesicles (EVs) may be derived from one type of seaweed or from two or more types of seaweed. Examples of green algae include Enteromorpha japonica, Porphyra nigra, Miru, Hiramir, Kuromir, and Caulerpa lentilata. Examples of red algae include Taoyagisou, Asakusa nori, Fukurofunori, Susabinori, Tanshisai (Japanese name: Haitan Amanori), Uppuri nori, Tanegashima Amanori, Millipede nori, Tosakanori, Shikinori, Dulse, Amakusanori, Akaba, and Fujimatsumo. Examples of brown algae include Undaria pinnatifida, Mozuku (Okinawa Mozuku, Itomozuku), Habanori, Hirome, Aowakame, Sagarame, Kayamonori, Kelp, Laminaria makinobu, Narrow-leaved Laminaria, Long-leaved Laminaria, Mitsuishi Kelp, Ecklonia cava, Kurome, Sargassum serrata, Eisenia bifida, Eisenia bifida, Eisenia bifida, Sesquiolepis gracilis, and Habanori wakame. Brown algae are most desirable as seaweed because they contain a large amount of fucoxanthin or its derivatives. Among brown algae, wakame is preferred. Seaweed includes sporophytes (thallus, discus, filament), male and female gametophytes (thallus, discus, filament), etc. Male and female gametophytes are desirable in the present invention because they contain a large amount of fucoxanthin or its derivatives. Female gametophytes are particularly advantageous because they contain a larger amount of fucoxanthin or its derivatives than male gametophytes.

[0053] In the present invention, the composition containing extracellular vesicles (EVs) and fucoxanthin or its derivatives preferably contains an aqueous solvent. This is because the lipid membrane of extracellular vesicles is less likely to break down in an aqueous solvent, and the morphology of the extracellular vesicles is more likely to be maintained. The aqueous solvent is water or a solvent containing water-soluble salts dissolved in water, such as pure water or seawater. Antifoaming agents, thixotropic agents, pH adjusters, etc. may be added to the aqueous solvent as appropriate. Furthermore, artificial seawater, filtered seawater, deep seawater, etc. 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 fulvic acid, and sodium citrate. Furthermore, the aqueous solvent may be phosphate-buffered saline. The composition of the present invention may also contain an organic solvent in addition to the aqueous solvent. The organic solvent is preferably an organic solvent capable of dissolving at least one compound selected from the group consisting of fucoxanthin and its derivatives. Furthermore, in the present invention, the composition containing extracellular vesicles (EVs) and fucoxanthin or a derivative thereof may 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. This is because the inclusion of an aqueous solvent makes the extracellular vesicles (EVs) less likely to be destroyed even when the organic solvent is contained as a solvent. The weight ratio of the organic solvent to the aqueous solvent is preferably organic solvent / aqueous solvent ≦9 / 1, and preferably organic solvent / aqueous solvent = 1 / 10 to 10 / 10.

[0054] In one aspect of the present invention, the composition containing extracellular vesicles (EVs) and at least one compound selected from the group consisting of fucoxanthin and its derivatives is preferably a powdered composition. Powdered compositions can reduce the volume per component compared to compositions containing a solvent, thereby reducing transportation costs. Furthermore, when used as a cosmetic, pharmaceutical, or food ingredient, the composition can be easily mixed with other raw materials, making it useful as an industrial raw material.

[0055] In the present invention, it is desirable that at least one compound selected from the group consisting of fucoxanthin and its derivatives be attached to or encapsulated in the extracellular vesicles (EVs). This is because at least one compound selected from the group consisting of fucoxanthin and its derivatives is less susceptible to decomposition by heat or light. Extracellular vesicles (EVs) contain various physiologically active substances, including proteins, nucleic acids such as DNA and RNA, from the cells from which they originated. Carotenoids such as fucoxanthin and its derivatives are also thought to be encapsulated in the extracellular vesicles (EVs) or attached to the surface or within the lipid membrane of the extracellular vesicles.

[0056] In the present invention, the at least one compound selected from the group consisting of fucoxanthin and its derivatives may be present both attached to or encapsulated in the extracellular vesicles (EVs) and free from the extracellular vesicles (EVs). Even when not in contact with the extracellular vesicles (EVs), the at least one compound selected from the group consisting of fucoxanthin and its derivatives is presumed to trap ozone and radicals generated by heat or light, which makes the at least one compound selected from the group consisting of fucoxanthin and its derivatives less susceptible to decomposition by heat or light, ensuring high stability.

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

[0058] Next, a method for producing a composition containing the extracellular vesicles (EVs) of the present invention and at least one compound selected from the group consisting of fucoxanthin and its derivatives will be described.

[0059] The composition of the present invention can be produced, for example, by a method using seaweed gametophytes and / or sporophytes as raw materials, comprising the following steps (1) and (2): Step (1): Dispersing seaweed gametophytes and / or sporophytes in an aqueous solvent to obtain a dispersion of the seaweed gametophytes and / or sporophytes; Step (2): Separating and removing the seaweed gametophytes and / or sporophytes from the dispersion obtained in step (1) to obtain an aqueous solvent composition containing the extracellular vesicles (EVs) and at least one compound selected from the group consisting of fucoxanthin and its derivatives. The method comprising steps (1) and (2) above allows for the production of a composition containing extracellular vesicles (EVs) derived from seaweed gametophytes and / or sporophytes and at least one compound selected from the group consisting of fucoxanthin and its derivatives. The present invention also encompasses a method for producing a composition comprising extracellular vesicles (EVs) derived from seaweed gametophytes and / or sporophytes and at least one compound selected from the group consisting of fucoxanthin and its derivatives, the method comprising steps (1) and (2) above. The production method of the present invention may include steps other than steps (1) and (2). For example, it may include a drying step, as described below. The present invention also encompasses a composition comprising extracellular vesicles (EVs) derived from seaweed gametophytes and / or sporophytes and at least one compound selected from the group consisting of fucoxanthin and its derivatives, which is obtained by the production method of the present invention.

[0060] The gametophyte and sporophyte of the seaweed used in step (1) may be female gametophytes or filamentous sporophytes, respectively. Examples of seaweed include the seaweeds mentioned above, preferably brown algae, and more preferably wakame.

[0061] In the following production method, the female gametophyte of seaweed will be described as an example, but the present invention is not limited thereto. First, an example of a method for obtaining gametophytes of seaweed will be described using Undaria pinnatifida as an example.

[0062] (I) Cultivation of seaweed gametophytes Gametophytes developed from wakame zoospores are separated into one male and one female, grown, mechanically shredded, attached to threads, and fertilized on the threads. The wakame juveniles developed from these are grown in indoor cultures and in natural seawater. Sporophytes (mekabu) are obtained from the grown wakame thallus (adult).

[0063] 1. Preserving sporophytes 1) Place sporophytes in a plastic bag or container and store in a cool, dark place at 15-20°C. At temperatures below 15°C, zoospores are not easily released when returned to seawater. If sporophytes are removed from seawater and stored in a cool, dark place, zoospores can be released for 2-3 days.

[0064] 2. Collecting zoospores 1) The room temperature for releasing zoospores should be 15-20°C. High temperatures are not recommended as they shorten the swimming time of zoospores. 2) Cut the sporophyll into pieces about 3-4 cm square. The part close to the rhizoid releases spores well, but cut off the part of the sporophyll surface that is as clean as possible. Lightly wipe off any dirt from the cut leaf pieces with absorbent paper or similar.

[0065] 3) Prepare three beakers containing 100 mL of sterilized seawater, wash the leaf pieces in turn, and then place them in a petri dish containing 50 mL of sterilized seawater. 4) Place the petri dish containing the leaf pieces on the stage of a stereomicroscope and shine light from above to release the zoospores. Adjusting the stereomicroscope to a dark field setting makes it easier to observe the release of zoospores. After about 10 minutes of irradiating it with an optical fiber or similar, the zoospores will be sufficiently released.

[0066] 5) Prepare a capillary tube. Use a hematocrypt tube that has been heated and stretched, or a Pasteur pipette with the tip stretched out until it is sufficiently thin. 6) Prepare a petri dish filled with 50 mL of PESI culture medium.

[0067] 7) Under a stereomicroscope, aspirate an appropriate amount of zoospores and drop them into a petri dish. When aspirating, be careful not to let the capillary touch the bottom of the petri dish or the leaf fragment (this is often the case when aspirating diatoms). After dropping, shake the petri dish thoroughly by hand to make the zoospore density uniform. 8) Prepare about four different types of petri dishes, each with a different amount of zoospore solution aspirated. If the amount of zoospores is large, the gametophyte density will increase, and the gametophytes will be too close to each other, making them difficult to isolate.

[0068] 9) After collecting the zoospores, the petri dishes are cultured at 10-30°C with a 12-14 hour light period (1000-1500 lux). Large temperature fluctuations, high temperatures, and high light levels can cause male and female gametophytes to look similar, making them difficult to distinguish, so care must be taken to maintain consistent culture conditions. 10) After two weeks, the gametophytes will reach a size where they can be sexed. Because there is a risk of fertilization, sex the gametophytes and isolate them as soon as possible.

[0069] 11) At this stage, there is little contamination with diatoms, but if diatoms do appear, discard the petri dish. If it is not possible to discard it, germanium dioxide can be used to suppress diatom growth, and gametophytes that are not contaminated by diatoms can be isolated.

[0070] 3. Isolation of male and female gametophytes 1) Place the petri dish in which the gametophytes are being cultured on the stage of an inverted microscope and search for a female gametophyte suitable for isolation. Isolate those in which the gametophytes are sufficiently separated and clearly sexed. Attach a tube to a Pasteur pipette, separate the female gametophytes from the petri dish, and aspirate them. Place each aspirated female gametophyte into a microplate filled with PESI medium. A schematic diagram of female and male gametophytes is shown in Figure 1-1. 2) Culture for one month at 10-30°C with a 12-14 hour light period (1500-2000 lux).

[0071] 4. Preservation of female gametophytes 1) After culturing in a microplate, remove the female gametophyte. Usually, the female gametophyte is large enough to be seen with the naked eye, so it can be picked up and removed from the microplate with ophthalmic tweezers. If it has not grown to a sufficient size, it can be aspirated using a Pasteur pipette under an inverted microscope. 2) For storage, place in a screw-cap test tube and store at 10-30°C with a 14-hour light period (1000-1500 lux). After storage, the medium is changed every two months using PESI medium.

[0072] (II) Cultivation of Seaweed Gametophytes As a culture medium for female gametophytes, Provasoli's Enriched Seawater (PES) or an improved version of it, the PESI culture medium, is preferred. The PES culture medium has the following composition. (See "Phycology Experiments and Practices," edited by Ariga Hirokatsu, Inoue Isao, Tanaka Jiro, Yokohama Yasutsugu, and Yoshida Tadao, Kodansha Scientific (2000) and JP 2009-201480 A.) The PESI culture medium can be prepared with reference to Plant Tissue Culture, 6(2), 55-62 (1989), etc. The detailed composition will be described later in the Examples.

[0073] (PES culture solution) Tris hydroxymethyl aminomethane 5.0g NaNO ３ 3.5g Na ２ -glycerophophate 500mg Fe stock solution 250mL P-2 metal mix 250mL Vitamin 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

[0074] (Fe stock solution) ２ -EDTA 2H ２ O 330mg Fe(NH ４ )２ (SO ４ ) ２ ・6H ２ O 351mg Distilled water 500mL

[0075] (P-2 metal mix) Na ２ -EDTA 2H ２ O 500mg H ３ BO ３ 570 mg FeCl ３ ・6H ２ O 24.5mg MnSO ４ ・4H ２ O 82.0mg CoSO ４ ・7H ２ O (4.8mg / mL) 0.5mL ZnSO ４ ・7H ２ O 11.0mg Distilled water 500mL

[0076] A seaweed culture solution is prepared by adding a PES or PESI culture solution to seawater. The seawater used may be natural seawater or artificial seawater sterilized with ozone, ultraviolet light, an autoclave, or the like, or filtered seawater obtained by filtering natural seawater. The artificial seawater is prepared to contain primarily 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 natural seawater is used, deep seawater may also be used. Deep seawater is seawater found in the deep sea at depths of 200 m or more. Deep seawater has a high salt concentration, making it difficult for bacteria and other organisms that are harmful to seaweed cultivation to survive. Deep seawater is free from artificial contamination, has low bacteria due to its low temperature, and is free from the presence of viable phytoplankton due to its lack of sunlight. Deep seawater is also rich in nitrogen (N) from nitrates, phosphorus (P) from phosphates, and silicon (Si) from silicates, making it ideal for the growth of seaweed.

[0077] The seaweed female gametophyte is placed in the prepared seaweed culture solution and subjected to aeration culture. The aeration culture consists of a preliminary culture and a main culture. The seaweed female gametophyte is cultured in an artificial environment, such as an environment in which the water temperature, light intensity, and sunshine duration are controlled. Any environment can be used as long as the light intensity, sunshine duration, and water temperature can be strictly controlled. For example, the seaweed may be cultured in a container capable of growing seaweed in an incubator where the light intensity, sunshine duration, and internal temperature are regulated, or in an aquarium where the light intensity, sunshine duration, and water temperature can be controlled.

[0078] In the preliminary culture environment, a daylight fluorescent lamp was used as the light source, and the irradiation light intensity was 1 to 100 μmol / m ２ The water temperature is preferably 5 to 30°C, and the sunlight duration is preferably 8 to 24 hours. Cultivation is usually carried out under a photoperiod with alternating light and dark periods, but continuous light irradiation is also possible. The culture period may be between 1 and 40 days, but is preferably 5 to 10 days. A minimum of 5 days is sufficient.

[0079] In the main culture environment, a green light source (490-550 nm) is used, and the irradiation dose is 10-200 μmol / m ２ The preferred conditions are: 1 / s, water temperature 5 to 30°C, and sunshine duration 8 to 24 hours. Cultivation is usually carried out under a photoperiod with alternating light and dark periods, but continuous light irradiation is also possible. The culture period may be between 1 and 40 days, but is preferably 5 to 10 days. Seaweed growth promoters such as fatty acids can be added to the seaweed culture solution used in the present invention as appropriate. The culture tank is also equipped with an agitator, a vibrator, a temperature controller, a pH adjuster, a turbidity meter, a light controller, air, O ２ , CO ２ The seaweed may be cultured by any suitable liquid culture method, such as a batch culture method, a semi-batch culture method (fed-batch culture method), or a continuous culture method (perfusion culture method).

[0080] After the primary culture of the seaweed female gametophytes, the seaweed gametophytes are removed from the culture medium by scooping them up with a plankton net or filtering the culture medium. The aggregation of seaweed gametophytes thus obtained can be preferably used as a raw material for extracting, for example, extracellular vesicles (EVs) and at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0081] (III) Aqueous Solvent Extraction of Seaweed Gametophytes and / or Sporophytes Seaweed gametophytes and / or sporophytes are dispersed in an aqueous solvent to obtain a dispersion of the seaweed gametophytes and / or sporophytes (step (1)). By dispersing the seaweed gametophytes and / or sporophytes in the aqueous solvent, extracellular vesicles (EVs) and fucoxanthin and its derivatives are extracted from the seaweed gametophytes and / or sporophytes into the aqueous solvent. Preferably, filaments of the seaweed female gametophytes and / or sporophytes are dispersed in the aqueous solvent, and extracellular vesicles (EVs) and at least one compound selected from the group consisting of fucoxanthin and its derivatives are extracted from the filaments of the seaweed female gametophytes and / or sporophytes into the dispersion. Extracellular vesicles (EVs) are easily dissolved in aqueous solvents, and vesicles composed of lipid membranes are easily disrupted by organic solvents that dissolve at least one compound selected from the group consisting of fucoxanthin and its derivatives. Therefore, extraction with an aqueous solvent is preferable. Furthermore, in the case of seaweed gametophytes and / or sporophytes, extraction with an aqueous solvent followed by extraction with an organic solvent is more effective than direct extraction with an organic solvent because it facilitates the extraction of at least one compound selected from the group consisting of fucoxanthin and its derivatives. An aqueous solvent is water or a solvent containing water-soluble salts dissolved in water, 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, deep seawater, etc. 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 fulvic acid, and sodium citrate. The aqueous solvent may also be phosphate buffered saline (PBS). The aqueous solvent in which the seaweed gametophytes and / or sporophytes are dispersed desirably does not contain an organic solvent that dissolves at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0082] The extraction temperature is preferably 4°C to 60°C, more preferably 20°C to 30°C. The extraction time is preferably 0.5 to 5 hours. Seaweed gametophytes (preferably female gametophytes) and / or sporophytes (preferably filaments) are dispersed in an aqueous solvent, followed by stirring and infiltration. The aqueous solvent is preferably used in a ratio of 0.01 to 1 L per 1 g of seaweed gametophytes and / or sporophytes (dry weight). The seaweed gametophytes and / or sporophytes are separated and removed from the resulting seaweed gametophyte and / or sporophyte dispersion to obtain an aqueous solvent composition containing extracellular vesicles (EVs) and at least one compound selected from the group consisting of fucoxanthin and its derivatives (step (2)). In this specification, the aqueous solvent composition obtained in step (2) is also referred to as an aqueous solvent extract, or an aqueous solvent extract of seaweed gametophytes and / or sporophytes. The seaweed gametophytes and / or sporophytes, which are the extraction residue, and impurities separated from the gametophytes and / or sporophytes are removed from the aqueous solvent by methods such as filtration using a filter or column, centrifugation, etc. In this manner, a composition (aqueous solvent composition) is obtained that contains the aqueous solvent, extracellular vesicles (EVs), and at least one compound selected from the group consisting of fucoxanthin and its derivatives dissolved in the aqueous solvent. The seaweed gametophytes and / or sporophytes separated in step (2) can be used for extraction with an organic solvent, as described below.

[0083] Furthermore, by removing the aqueous solvent from the aqueous solvent composition containing the extracellular vesicles (EVs) and at least one compound selected from the group consisting of fucoxanthin and its derivatives, a powdery composition containing the extracellular vesicles (EVs) and at least one compound selected from the group consisting of fucoxanthin and its derivatives can be obtained. In one aspect, the production method of the present invention preferably includes the above steps (1) and (2), as well as step (5-1). Step (5-1): The aqueous solvent composition obtained in step (2) is dried to obtain a powdery composition containing the extracellular vesicles (EVs) and at least one compound selected from the group consisting of fucoxanthin and its derivatives. Any method capable of drying the aqueous solvent composition can be used to remove the aqueous solvent, including heating, freeze-drying, and vacuum drying. However, because extracellular vesicles are composed of proteins and lipids, freeze-drying is preferred.

[0084] (IV) Extraction with Organic Solvent After Extraction of Seaweed Gametophytes and / or Sporophytes with an Aqueous Solvent Preferably, an organic solvent is added to the seaweed gametophytes and / or sporophytes that have been extracted with an aqueous solvent (the seaweed gametophytes and / or sporophytes separated in step (2) above) to obtain an organic solvent composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives (step (3)). In this specification, the organic solvent composition obtained in step (3) may also be referred to as an "organic solvent extract." Fucoxanthin or a derivative thereof is extracted from the seaweed gametophytes and / or sporophytes by immersing the seaweed gametophytes and / or sporophytes that have 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 allows at least one compound selected from the group consisting of fucoxanthin and its derivatives present in the cells of the seaweed gametophytes and / or sporophytes to elute into the organic solvent. The organic solvent does not substantially contain extracellular vesicles (EVs). This is because extracellular vesicles (EVs) released into the organic solvent are unstable because they do not form a protective membrane hydrated with water molecules on their surface, resulting in destruction of the lipid membrane. The organic solvent used can be any organic solvent that dissolves at least one compound selected from the group consisting of fucoxanthin and its derivatives. Examples of such organic solvents 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, organic chlorinated hydrocarbons such as chloroform, aliphatic hydrocarbons such as hexane, and aromatic hydrocarbons such as benzene and toluene, either alone or in combination. Among these, ethanol and butanediol are preferred. At least one compound selected from the group consisting of fucoxanthin and its derivatives is eluted in the organic solvent.Because fucoxanthin and its derivatives are substantially insoluble in aqueous solvents, the organic solvent contains a higher amount of fucoxanthin and its derivatives than the aqueous solvent extract (II). The organic solvent may contain, but preferably does not contain, an aqueous solvent. When the organic solvent contains an aqueous solvent, the aqueous solvent is derived primarily from water contained in the seaweed gametophytes and / or sporophytes. In the extraction with an organic solvent, 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. The seaweed gametophytes and / or sporophytes are preferably dispersed in the organic solvent and stirred or infiltrated. The organic solvent is preferably used in a ratio of 0.01 to 1 L per 1 g of seaweed gametophytes. After extraction with an organic solvent, the seaweed gametophytes and / or sporophytes, which are the extraction residue, and impurities separated from the gametophytes and / or sporophytes may be removed from the organic solvent. The method for removing the seaweed gametophytes and / or sporophytes from the organic solvent is not particularly limited, and methods such as filtration using a filter or column, centrifugation, etc. can be used. In this manner, it is desirable to obtain a composition (organic solvent composition) containing the organic solvent and at least one compound selected from the group consisting of fucoxanthin and its derivatives dissolved in the organic solvent.

[0085] (V) Mixing of an aqueous solvent extract of seaweed gametophytes and / or sporophytes with an organic solvent extract In the present invention, it is preferable to add the aqueous solvent composition obtained in step (2) to the organic solvent composition obtained in step (3) to obtain a mixed solvent composition containing an aqueous solvent and an organic solvent (step (4)). The aqueous solvent extract of seaweed gametophytes and / or sporophytes prepared in (III) contains extracellular vesicles (EVs) and at least one compound selected from the group consisting of fucoxanthin and its derivatives. The organic solvent extract prepared in (IV) is usually substantially free of extracellular vesicles. By mixing the aqueous solvent extract of seaweed gametophytes and / or sporophytes prepared in (I) with the organic solvent extract prepared in (IV), a mixed solvent composition containing extracellular vesicles (EVs), at least one compound selected from the group consisting of fucoxanthin and its derivatives, as well as an aqueous solvent and an organic solvent can be obtained. The mixing ratio of the aqueous solvent composition obtained in step (2) to the organic solvent composition obtained in step (3) is preferably aqueous solvent composition / organic solvent composition = 10 / 10 to 10 / 1 by weight. In one embodiment, by mixing an aqueous solvent extract of female gametophytes and / or sporophytes with an organic solvent extract, a composition containing a high amount of at least one compound selected from the group consisting of fucoxanthin and its derivatives and extracellular vesicles (EVs) also present can be obtained. This allows for the production of a composition in which the stability of at least one compound selected from the group consisting of fucoxanthin and its derivatives is excellent. Furthermore, it is believed that once dispersed in an aqueous solvent, a protective film formed on the surface of the extracellular vesicles (EVs) by hydration with water molecules is formed. Therefore, even when the EVs are subsequently mixed with an organic solvent capable of dissolving at least one compound selected from the group consisting of fucoxanthin and its derivatives, the lipid film of the extracellular vesicles (EVs) is unlikely to be destroyed by the organic solvent.

[0086] In the present invention, an aqueous solvent extract of seaweed gametophytes and / or sporophytes or a mixed solvent composition of an aqueous solvent extract of seaweed gametophytes and / or sporophytes and an organic solvent extract may be dried to produce a powder composition containing extracellular vesicles (EVs) and at least one compound selected from the group consisting of fucoxanthin and its derivatives (steps (5-1) and (5-2)). In one aspect, the production method of the present invention preferably includes steps (1) to (4) and step (5-2). Step (5-2): The mixed solvent composition containing the aqueous solvent and the organic solvent obtained in step (4) is dried to obtain a powder composition containing the extracellular vesicles (EVs) and at least one compound selected from the group consisting of fucoxanthin and its derivatives. Various drying methods can be used, such as heat drying, freeze drying, and vacuum drying. Since extracellular vesicles (EVs) are composed of proteins and lipids, freeze drying, which does not involve heating, is preferred. The average particle size of the powder composition is preferably 0.1 to 10 μm.

[0087] In the present invention, it is preferable to mix fats and oils such as fatty acid triglycerides with the composition and not isolate fucoxanthin or its derivatives. This is because isolation would impair the stability of fucoxanthin and its derivatives. Furthermore, even if mixed with fats and oils such as fatty acid triglycerides, once extracted with water, extracellular vesicles (EVs) are hydrophilized and do not elute or disperse in the fats and oils, making it difficult to isolate fucoxanthin or its derivatives from extracellular vesicles (EVs) using the fats and oils.

[0088] The composition of the present invention, which contains the extracellular vesicles (EVs) and at least one compound selected from the group consisting of fucoxanthin and its derivatives, has excellent stability against heat and light and can be used in a variety of industrial applications, including, in particular, health foods expected to have cholesterol-lowering and antithrombosis effects, pharmaceuticals intended to have antitumor effects, neuroprotective effects, and blood sugar level suppression effects, and cosmetics intended to have a melanin production suppression effect.

[0089] As described above, seaweed gametophytes and / or sporophytes can be suitably used as raw materials for extracting extracellular vesicles (EVs) and at least one compound selected from the group consisting of fucoxanthin and its derivatives. The present invention also encompasses raw materials containing aggregates of seaweed gametophytes and / or sporophytes, which are used for extracting at least one compound selected from the group consisting of extracellular vesicles (EVs) and fucoxanthin and its derivatives. The seaweed is preferably a brown alga. The seaweed is preferably a filamentous body of a female gametophyte and / or sporophyte.

[0090] By coexisting seaweed-derived extracellular vesicles (EVs) 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. Seaweed-derived extracellular vesicles (EVs) can be used to improve the stability of at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0091] The present invention also includes the following uses: an agent for improving the stability of at least one compound selected from the group consisting of fucoxanthin and its derivatives; and a method for improving the stability of at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0092] Use of seaweed-derived extracellular vesicles (EVs) for improving the stability of at least one compound selected from the group consisting of fucoxanthin and its derivatives. A stability improver for at least one compound selected from the group consisting of fucoxanthin and its derivatives, comprising seaweed-derived extracellular vesicles (EVs). A method for improving the stability of at least one compound selected from the group consisting of fucoxanthin and its derivatives, comprising allowing seaweed-derived extracellular vesicles (EVs) to coexist with at least one compound selected from the group consisting of fucoxanthin and its derivatives. The seaweed-derived extracellular vesicles (EVs), and preferred embodiments thereof, are the same as those of the composition of the present invention described above. The following items are disclosed in this specification regarding the present invention.

[0093] The present disclosure (1) is a composition comprising extracellular vesicles (EVs) and a carotenoid.

[0094] The present disclosure (2) relates to the composition according to the present disclosure (1), wherein the extracellular vesicles (EVs) are derived from seaweed.

[0095] The present disclosure (3) is the composition according to the present disclosure (2), wherein the seaweed is a gametophyte and / or a sporophyte of the seaweed.

[0096] The present disclosure (4) is the composition according to the present disclosure (2) or (3), wherein the seaweed is a female gametophyte of seaweed.

[0097] The present disclosure (5) is the composition according to the present disclosure (2) or (3), wherein the seaweed is a filamentous sporophyte.

[0098] The present disclosure (6) is the composition according to any one of the present disclosures (2) to (5), wherein the seaweed is brown algae.

[0099] The present disclosure (7) is the composition according to any one of the present disclosures (1) to (6), wherein the composition contains an aqueous solvent or a mixed solvent of an aqueous solvent and an organic solvent, and the organic solvent dissolves a carotenoid.

[0100] The present disclosure (8) is the composition according to the present disclosure (7), wherein the aqueous solvent is water or a solvent in which a water-soluble salt is dissolved in water.

[0101] The present disclosure (9) is the composition according to the present disclosure (8), wherein the water-soluble salt is at least one selected from the group consisting of sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, ammonium sulfate, potassium phosphate, potassium dihydrogen phosphate, iron phosphate, iron fulvic acid, and sodium citrate.

[0102] The present disclosure (10) is the composition according to the present disclosure (8) or (9), wherein the solvent in which the water-soluble salt is dissolved in water is seawater or phosphate buffered saline.

[0103] The present disclosure (11) is the composition according to any one of the present disclosures (7) to (10), wherein the organic solvent is at least one selected from the group consisting of methanol, ethanol, propanol, isopropanol, n-butanol, 1,3-butanediol, 1,4-butanediol, methyl ethyl ketone, acetone, methyl acetate, ethyl acetate, chloroform, hexane, benzene, and toluene.

[0104] The present disclosure (12) is the composition according to any one of the present disclosures (7) to (11), wherein the weight ratio of the organic solvent to the aqueous solvent is organic solvent / aqueous solvent ≦9 / 1.

[0105] The present disclosure (13) is the composition according to any one of the present disclosures (1) to (12), wherein the composition is a powdery composition.

[0106] The present disclosure (14) is the composition according to any one of the present disclosures (1) to (13), wherein at least a portion of the carotenoid is attached to or encapsulated in the extracellular vesicles (EVs).

[0107] The present disclosure (15) is the composition according to any one of the present disclosures (1) to (14), wherein the carotenoid coexists with one attached to or encapsulated in the extracellular vesicles (EVs) and one released from the extracellular vesicles (EVs).

[0108] The present disclosure (16) is the composition according to any one of the present disclosures (1) to (15), wherein the carotenoid is at least one compound selected from the group consisting of fucoxanthin and derivatives thereof.

[0109] The present disclosure (17) is the composition according to the present disclosure (16), wherein the total content of at least one compound selected from fucoxanthin and its derivatives in the composition is 0.001 to 50 wt %.

[0110] The present disclosure (18) is the composition according to any one of the present disclosures (1) to (17), wherein the extracellular vesicles (EVs) are vesicles having a lipid bilayer structure secreted from living cells.

[0111] The present disclosure (19) is the composition according to any one of the present disclosures (1) to (18), wherein the extracellular vesicles (EVs) include at least one selected from the group consisting of exosomes, microvesicles, and apoptotic bodies.

[0112] The present disclosure (20) is the composition according to any one of the present disclosures (1) to (19), wherein the extracellular vesicles (EVs) contain exosomes, and the size of the exosomes is 1 to 200 nm in diameter.

[0113] The present disclosure (21) is the composition according to any one of the present disclosures (1) to (20), wherein the extracellular vesicles (EVs) include microvesicles (MVs), and the size of the microvesicles (MVs) is 100 to 1000 nm in diameter.

[0114] The present disclosure (22) is the composition according to any one of the present disclosures (1) to (21), wherein the extracellular vesicles (EVs) contain apoptotic bodies, and the apoptotic bodies have a diameter of 50 to 5,000 nm.

[0115] The present disclosure (23) is the composition according to any one of the present disclosures (1) to (22), wherein, when the composition is separated by liquid chromatography under the following conditions, a peak of the lipid membrane constituting the extracellular vesicles (EVs) and a peak of the protein appear at the same retention time on the liquid chromatography chart. Liquid chromatography conditions: Column packing: qEV10 70 nm manufactured by Izon Science Column shape: Stainless steel column manufactured by Senshu Scientific Co., Ltd., diameter 8 mm x length 100 m Developing solvent: Aqueous solution containing 10 mM tris(hydroxymethyl)aminomethane, pH = 7.6, 30 mM NaCl, and 0.5 mM EDTA Developing time gradient: Uniform Solution flow rate: 1 mL / min Injection volume: 10 μL Column temperature: 30°C Sample chamber temperature: 10°C

[0116] The present disclosure (24) provides a method for producing a composition containing extracellular vesicles (EVs) derived from seaweed gametophytes and / or sporophytes and a carotenoid, the method comprising the following steps (1) and (2): Step (1): Dispersing seaweed gametophytes and / or sporophytes in an aqueous solvent to obtain a dispersion of the seaweed gametophytes and / or sporophytes; Step (2): Separating and removing the seaweed gametophytes and / or sporophytes from the dispersion of the seaweed gametophytes and / or sporophytes obtained in step (1) to obtain an aqueous solvent composition containing the extracellular vesicles (EVs) and the carotenoid.

[0117] The present disclosure (25) is a method for producing a composition containing extracellular vesicles (EVs) derived from gametophytes and / or sporophytes of the seaweed described in the present disclosure (24), and a carotenoid, further comprising step (5-1): Step (5-1): The aqueous solvent composition obtained in step (2) is dried to obtain a powdery composition containing the extracellular vesicles (EVs) and the carotenoid.

[0118] The present disclosure (26) is a method for producing a composition containing extracellular vesicles (EVs) derived from gametophytes and / or sporophytes of the seaweed described in the present disclosure (24) and a carotenoid, further comprising steps (3) and (4). Step (3): An organic solvent capable of dissolving the carotenoid is added to the gametophytes and / or sporophytes of the seaweed separated in step (2), thereby obtaining an organic solvent composition containing the carotenoid. Step (4): The aqueous solvent composition containing the extracellular vesicles (EVs) and the carotenoid obtained in step (2) is added to the organic solvent composition containing the carotenoid obtained in step (3), thereby obtaining a mixed solvent composition containing the aqueous solvent and the organic solvent.

[0119] The present disclosure (27) is a method for producing a composition containing extracellular vesicles (EVs) derived from gametophytes and / or sporophytes of the seaweed described in the present disclosure (25), and a carotenoid, further comprising step (5-2): drying the mixed solvent composition containing the aqueous solvent and the organic solvent obtained in step (4) to obtain a powdery composition containing the extracellular vesicles (EVs) and the carotenoid.

[0120] The present disclosure (28) is a method for producing the composition according to any one of the present disclosures (24) to (27), wherein the sporophyte is a filamentous body.

[0121] The present disclosure (29) is a method for producing the composition according to any one of the present disclosures (24) to (28), wherein the carotenoid is at least one compound selected from the group consisting of fucoxanthin and derivatives thereof.

[0122] The present disclosure (30) is a raw material comprising a collection of seaweed gametophytes and / or sporophytes, which is used to extract extracellular vesicles (EVs) and carotenoids.

[0123] The present disclosure (31) is a raw material disclosed in the present disclosure (30), which is obtained by freeze-drying and powdering an aggregate of gametophytes and / or sporophytes of the seaweed.

[0124] The present disclosure (32) is the raw material according to the present disclosure (30), wherein the seaweed is a female gametophyte.

[0125] The present disclosure (33) is the raw material according to the present disclosure (30), wherein the seaweed is a filamentous body of the sporophyte.

[0126] The present disclosure (34) is the raw material according to any one of the present disclosures (30) to (33), wherein the seaweed is brown algae.

[0127] The present disclosure (35) is the raw material according to any one of the present disclosures (30) to (34), wherein the carotenoid is at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0128] The present disclosure (36) is the use of extracellular vesicles (EVs) derived from seaweed to improve the stability of carotenoids.

[0129] The present disclosure (37) is the use of seaweed-derived extracellular vesicles (EVs) according to the present disclosure (36), wherein the carotenoid is at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0130] The present disclosure (38) is a carotenoid stability enhancer containing extracellular vesicles (EVs) derived from seaweed.

[0131] The present disclosure (39) is the agent for improving the stability of a carotenoid according to the present disclosure (38), wherein the carotenoid is at least one compound selected from the group consisting of fucoxanthin and derivatives thereof.

[0132] The present disclosure (40) is a method for improving the stability of carotenoids by coexisting seaweed-derived extracellular vesicles (EVs) with carotenoids.

[0133] The present disclosure (41) is the method for improving the stability of a carotenoid according to the present disclosure (40), wherein the carotenoid is at least one compound selected from the group consisting of fucoxanthin and derivatives thereof.

[0134] (Second Invention) The second invention will be described below. Seaweeds such as wakame (Undaria pinnatifida) and kombu (kelp) are rich in nutrients such as protein, dietary fiber, vitamins, and minerals, and their extracts have been utilized in foods, cosmetics, pharmaceuticals, and the like. For example, Patent Document 5 discloses a method for extracting water-soluble components from seaweed, which comprises adding water to fine pieces or powder of seaweed, mixing the mixture, and then wet-grinding the mixture at room temperature to dissolve and extract the water-soluble components of the seaweed. Patent Document 6 also discloses a wakame protein-containing composition obtained by adding water, a salt solution, or a weak alkaline solution to thallus or dried, finely ground wakame seaweed thallus, followed by wet-grinding to extract the soluble components, and then separating the protein from the extract.

[0135] Furthermore, Patent Document 7 discloses a method for isolating a lipophilic extract from seaweed gametophytes, in which an aqueous-alcoholic suspension of brown algal gametophyte cells is mixed with at least one fatty acid triglyceride containing 8 to 22 carbon atoms, and then water is added to isolate the lipophilic extract. Patent Document 8 discloses a cosmetic preparation for topical use, which contains a freeze-dried product of brown algal gametophyte cells as an active ingredient, and also discloses that the freeze-dried product of brown algal cells contains 1% or more of fucoxanthin.

[0136] Incidentally, a substance called cAMP (cyclic adenosine monophosphate) is known as a second messenger that plays an important role in intracellular signal transmission. This substance activates an enzyme called protein kinase A (PKA) and regulates various intracellular reactions. cAMP is also known to have a wide range of effects in the body, including activating transcription factors, which are proteins that regulate gene transcription, cell growth and differentiation, regulating neurotransmission, and activating hair papilla cells, and is expected to be applied in the fields of pharmaceuticals and cosmetics. For example, Japanese Patent Application Laid-Open No. 4-124122 discloses an anti-graying hair blackening agent that combines a cAMP increaser and a protein kinase C inhibitor. In this patent application Laid-Open No. 4-124122, adenosine, forskolin, etc. are listed as cAMP increasers.

[0137] The inventors of the present application have conducted various studies to determine whether the seaweed extracts described in Patent Documents 5 to 8 described above can be used to increase the amount of cAMP in cells, since they are relatively inexpensive natural materials. However, the extracts described in Patent Documents 5 to 8 were unable to sufficiently increase the amount of cAMP in cells.

[0138] The second invention was made in consideration of the above-mentioned problems, and aims to provide a seaweed gametophyte and / or sporophyte extract composition that can effectively increase the amount of cAMP in cells, and a method for producing the same.

[0139] As a result of extensive research, the present inventors have found that it is possible to increase intracellular cAMP (cyclic adenosine monophosphate) by applying to cells a composition in which a specific protein constituting an enzyme coexists with extracellular vesicles (EVs) derived from the gametophyte and / or sporophyte of seaweed.

[0140] The present invention relates to a composition comprising at least one protein selected from the group consisting of 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.

[0141] As used herein, extracellular vesicles (EVs) are a general term for vesicles with a lipid bilayer structure secreted from living cells, and the concept of extracellular vesicles includes exosomes, microvesicles, and apoptotic bodies. Exosomes have a diameter of 1 to 200 nm. Exosomes are formed by the inward budding of late endosomal membranes, which then fuse with the cell membrane to form complete particles, which are then secreted extracellularly by exocytosis. Microvesicles (MVs) are generated by the outward budding and separation of the cell membrane, and have a diameter of 100 to 1,000 nm. Apoptotic bodies are generated when cells undergo organized cell death (apoptosis), and have a diameter of 50 to 5,000 nm.

[0142] The composition of the present invention contains, as a protein, at least one protein selected from ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit.

[0143] The ribulose-1,5-bisphosphate carboxylase large subunit is a large subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase (hereinafter also referred to as "RuBisCO"), the most important enzyme in photosynthesis, and plays a role in the catalytically active site of RuBisCO. ２ The small subunit of ribulose 1,5-bisphosphate carboxylase catalyzes the binding of ribulose 1,5-bisphosphate (RuBP) to ribulose 1,5-bisphosphate (RuBP) to produce 3-phosphoglycerate (3-PGA). This reaction is the first step in the photosynthetic Calvin cycle and is a key reaction in carbon fixation in photosynthesis. The small subunit of ribulose 1,5-bisphosphate carboxylase is a small subunit of RuBisCO, the most important enzyme in photosynthesis. Although it is not directly involved in the catalytically active site of RuBisCO, it binds to the large subunit of RuBisCO, increasing its stability and promoting its activity. RuBisCO is composed of eight large subunits (LSUs) and eight small subunits (SSUs).

[0144] As explained above, ribulose-1,5-bisphosphate carboxylase / oxygenase is an enzyme involved in photosynthesis. In the present invention, it is believed that the large subunit (LSU) and small subunit (SSU) of RuBisCO, which constitute this enzyme, act by binding to receptors in the cell membrane of mammalian cells.

[0145] When the composition of the present invention is applied to mammalian cells, the extracellular vesicles (EVs) help proteins comprising the large and small subunits of ribulose 1,5-bisphosphate carboxylase / oxygenase bind to receptors in the cell membrane of mammalian cells. When the proteins comprising the large and small subunits of ribulose 1,5-bisphosphate carboxylase / oxygenase bind to receptors in the cell membrane of mammalian cells, the receptors are activated, which activate proteins called G proteins. This G protein activates an enzyme called adenylate cyclase, which is thought to convert adenosine triphosphate (ATP) to cAMP (cyclic adenosine monophosphate). 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 follicle papilla cells.

[0146] The composition of the present invention may further contain at least one selected from ATP synthase subunit β, thiamine biosynthesis protein G, and histone H2B. ATP synthase is composed of two parts, F1 and F0, and ATP synthase subunit β is present in the F1 part and plays an important role in forming the catalytic site for ATP synthesis. ATP synthase subunit β catalyzes the reaction of synthesizing ATP by combining ADP (adenosine diphosphate) and phosphate. Histone H2B is a protein present in the nucleus of eukaryotic cells. 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 around DNA, compactly storing DNA within the nucleus and regulating gene expression by controlling DNA accessibility. Thiamine biosynthesis protein G (THP) 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), a key step in the thiamine biosynthesis pathway. The "G" in THP comes from its involvement in the seventh step of the thiamine biosynthesis pathway. The thiamine biosynthetic pathway is a complex pathway involving multiple enzymes, with each step named by an alphabet. Thiamine biosynthesis protein G plays a key role in the seventh step of this pathway, combining hydroxymethyldihydropyrimidine (HMP) with a pyrimidine dimer to produce hydroxymethyldihydropyrimidine dimer (HMP-PP).These proteins are also thought to exhibit the same action as at least one protein selected from ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit.

[0147] In the composition of the present invention, the extracellular vesicles (EVs) must be derived from seaweed. However, the term "seaweed" as used herein refers to a group of marine species of multicellular algae, and does not include microalgae, which are unicellular algae.

[0148] In the composition of the present invention, the seaweed must be a gametophyte and / or sporophyte of seaweed. Seaweeds have various morphologies in their life cycle, including sporophytes (thallus, discus, filamentous body), male and female gametophytes (thallus, discus, filamentous body), etc. Male and female gametophytes and filamentous sporophytes are preferred in the present invention because they are easy to extract extracellular vesicles (EVs) and the aforementioned proteins from.

[0149] JP 2023-520101 A discloses extracellular vesicles derived from microalgae, which are biolipid-membrane nanovesicles with particle sizes in the range of 50-300 nm (small extracellular vesicles, sEV) or 300-2 μm (large extracellular vesicles, lEV), and which are contained in a lipid bilayer membrane. The extracellular vesicles are derived from natural photosynthetic, non-fermentative microalgae and contain at least the extracellular vesicle protein marker Alix and, optionally, one or more additional protein markers selected from the group consisting of enolase, actin, and any combination thereof. However, these are extracellular vesicles derived from microalgae and contain the proteins Alix, enolase, and actin, which differ from those of the present invention. It should be noted that, although ribulose 1,5-bisphosphate carboxylase / oxygenase is an enzyme involved in photosynthesis, it is not commonly extracted from all photosynthetic algae, as can be seen from JP-A No. 2023-520101.

[0150] In the composition of the present invention, the seaweed is preferably a filamentous female gametophyte and / or a filamentous sporophyte of the seaweed, because the filamentous female gametophyte and the filamentous sporophyte are easy to extract extracellular vesicles (EVs) and proteins from and contain the highest amount of fucoxanthin or its derivatives, described below, among the various forms in the life cycle of seaweed.

[0151] In the composition of the present invention, the seaweed is preferably brown algae. Examples of seaweed include green algae, red algae, and brown algae. Brown algae are preferred because they are easy to extract extracellular vesicles (EVs) and proteins from and contain a large amount of fucoxanthin or its derivatives, as described below.

[0152] The composition of the present invention, which 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 seaweed gametophytes and / or sporophytes, preferably contains an aqueous solvent or a mixed solvent of an aqueous solvent and an organic solvent, and the organic solvent is preferably an organic solvent that dissolves at least one compound selected from the group consisting of fucoxanthin and its derivatives, as described below.

[0153] In the composition of the present invention, the composition containing at least one protein selected from the group consisting of ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit and extracellular vesicles (EVs) preferably contains an aqueous solvent. This is because the lipid membrane of extracellular vesicles is less likely to be destroyed in an aqueous solvent, and the morphology of the EVs is easily maintained. The lipid membrane of the EVs is easily destroyed and the protein is easily denatured in organic solvents such as alcohol, which dissolve fucoxanthin and its derivatives, as described below.

[0154] The aqueous solvent is water or a solvent in which a water-soluble salt is 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. Furthermore, artificial seawater, filtered seawater, deep seawater, etc. can also be used as seawater.

[0155] The composition of the present invention may also contain a mixed solvent consisting of an aqueous solvent and an organic solvent. The organic solvent is preferably one that dissolves at least one compound selected from the group consisting of fucoxanthin and its derivatives, as described below. This is because the inclusion of an aqueous solvent makes it difficult for extracellular vesicles (EVs) to be destroyed, even when the composition contains an organic solvent that dissolves at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0156] In the 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.

[0157] The composition of the present invention may contain at least one protein selected from ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, extracellular vesicles (EVs) derived from seaweed gametophytes and / or sporophytes, and at least one compound selected from the group consisting of fucoxanthin and its derivatives. Although fucoxanthin does not contribute to an increase in cAMP (cyclic adenosine monophosphate) in mammalian cells, it is known to inhibit melanin production and has antioxidant properties, making it a beneficial substance.

[0158] Examples of fucoxanthin derivatives include fucoxanthin hydrolysates or derivatives thereof, fucoxanthin esters (eg, esters with amino acids, carboxylic acids, inorganic acids, or fatty acids), salts thereof, and fucoxanthin glycosides. More specific examples of fucoxanthin derivatives include, but are not limited to, monoesters and homogeneous or heterogeneous diesters selected from the following: fucoxanthinol, which is a hydrolysis product; amarousiaxanthin A, which is fucoxanthinol that has undergone dehydration and isomerization; esters of fucoxanthin with amino acids such as glycine and alanine; esters and salts of fucoxanthin with carboxylic acids such as acetic acid and citric acid; esters and salts of fucoxanthin with inorganic acids such as phosphoric acid and sulfuric acid; and fatty acid esters of fucoxanthin with 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; and glycosides such as glucosides.

[0159] In the composition of the present invention, the 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 seaweed gametophytes and / or sporophytes is preferably in powder form. Powdered compositions can be produced with a smaller volume per component than solvent-containing compositions, thereby reducing transportation costs. Furthermore, when used as a cosmetic, pharmaceutical, food ingredient, reagent, or raw material thereof, it can be easily mixed with other raw materials, making it useful as an industrial raw material.

[0160] In the composition of the present invention, it is desirable that at least one protein selected from ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and 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) facilitate binding of the protein to receptors present on the cell membrane of mammalian cells.

[0161] The agent for increasing intracellular cyclic adenosine monophosphate (cAMP) of the present invention includes the composition of the present invention. 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).

[0162] The method for producing a composition of the present invention is a method for producing a composition 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 seaweed gametophytes and / or sporophytes, and comprises the following steps (1) and (2): Step (1): The gametophytes and / or sporophytes of the seaweed are dried to obtain a dried body, and then the dried body is pulverized to obtain a powder of the gametophytes and / or sporophytes of the seaweed, and the powder is dispersed in an aqueous solvent adjusted to a temperature of 5° C. or less. Step (2): The aqueous solvent in which the powder has been dispersed is centrifuged to separate it into a solid content and a supernatant, and the supernatant is collected.

[0163] The gametophytes and / or sporophytes of marine algae release extracellular vesicles (EVs) and proteins in an aqueous medium at a low temperature of 5° C. or less. The gametophytes and / or sporophytes of marine algae are dispersed in an aqueous solvent, and the extracellular vesicles (EVs) and proteins are extracted into the dispersion. At least one protein selected from ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit is insoluble in water, but is dispersed in an aqueous solvent together with extracellular vesicles (EVs) by being encapsulated in the EVs or by being attached to the lipid membrane on the surface of the EVs. At least one protein selected from ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit is extracted in an aqueous solvent at a low temperature of 5°C or less, so that the protein is not decomposed and can be extracted while maintaining its structure.

[0164] The extracellular vesicles (EVs) may be derived from the female gametophyte of the seaweed or from the filaments of the sporophyte of the seaweed, and the female gametophyte and the filaments of the sporophyte are preferred in the present invention because they are easy to extract the extracellular vesicles (EVs) and the aforementioned proteins from.

[0165] If the aqueous solvent contains an organic solvent such as ethanol or butylene glycol that dissolves the aforementioned fucoxanthin and its derivatives, the extracellular vesicles (EVs) immediately after release from the gametophyte and / or sporophyte will be unstable, and the lipid membranes of the extracellular vesicles (EVs) will be dissolved and destroyed. Therefore, the solvent for dispersing the gametophyte and / or sporophyte is preferably an aqueous solvent, and it is preferable that the aqueous solvent does not contain such an organic solvent, and it is preferable that the aqueous solvent does not contain an organic solvent that dissolves at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0166] A dispersion containing dried powder of seaweed gametophytes and / or sporophytes is centrifuged to separate the solid portion and the supernatant, and the solid portion is removed from the dispersion to separate the supernatant, thereby obtaining an aqueous solvent composition 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 seaweed gametophytes and / or sporophytes. Centrifugation is preferably carried out in an atmosphere adjusted to 5° C. or below.

[0167] The method for producing the composition of the present invention may include a step of adding to the supernatant at least one compound selected from the group consisting of fucoxanthin and its derivatives. The 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.

[0168] The method for producing the composition of the present invention may include a 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 seaweed gametophytes and / or sporophytes. Removal of the aqueous solvent results in a powdery composition.

[0169] The composition produced by the method for producing a composition of the present invention may also include at least one protein selected from ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and a raw material used to extract extracellular vesicles (EVs) derived from seaweed gametophytes and / or sporophytes. The gametophyte and / or sporophyte of seaweed are approximately 200 μm to 2000 μm in size and can be easily cultured in a small space. Furthermore, compared to other forms in the life cycle of seaweed, such as sporophytes (thallus, disc, filament), they contain extracellular vesicles (EVs), ribulose-1,5-bisphosphate carboxylase / oxygenase large subunits, and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunits. Since it is easy to extract at least one protein selected from extracellular vesicles (EVs) and extracellular subunits from the extract, and it also contains a large amount of fucoxanthin, it is also an excellent raw material for extracting extracellular vesicles (EVs) and proteins, or, if necessary, at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0170] The seaweed used in the method for producing the composition of the present invention is preferably brown algae, as this facilitates the extraction of at least one protein selected from the group consisting of ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, as well as extracellular vesicles (EVs). Furthermore, brown algae contain the highest amount of fucoxanthin and its derivatives among all seaweeds, making them advantageous for extracting fucoxanthin and other derivatives.

[0171] The seaweed used in the method for producing the composition of the present invention is preferably a female gametophyte and / or a filamentous sporophyte. This is because it is easy to extract at least one protein selected from the group consisting of ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, as well as extracellular vesicles (EVs). Furthermore, the content of fucoxanthin and its derivatives is higher than that of male gametophytes, allowing for efficient extraction.

[0172] 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.

[0173] The method of the present invention for producing an agent for increasing intracellular cyclic adenosine monophosphate (cAMP) may include the method of producing the composition of the present invention described above.

[0174] The raw material of the present invention comprises a collection of seaweed gametophytes and / or sporophytes, and is used to extract 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 seaweed gametophytes and / or sporophytes. The composition of the present invention can be produced using such a raw material.

[0175] In the raw material of the present invention, the seaweed is preferably a brown alga. Also, in the raw material of the present invention, the seaweed is preferably a female gametophyte or a filamentous body of a sporophyte.

[0176] 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, improve cell growth and differentiation, neurotransmission, and activate dermal papilla cells. Furthermore, in addition to the effect of increasing cAMP in mammalian cells, the composition of the present invention is also thought to have an effect of 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 variety of industrial raw materials. As an industrial raw material, a wide range of applications can be cited, including pharmaceuticals, food products, cosmetics, and reagents. The "ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit" and "ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit" used in the present invention are described in JP 2018-131414 A, JP 2023-120242 A, Japanese Patent No. 7298905 A, Production Research These terms have been established as technical terms, as described in, for example, Vol. 64, No. 3 (2012), pp. 351-357, and in Japanese Patent Application Laid-Open No. 9-252778. Histone H2B is also described in Japanese Patent Application Laid-Open No. 3705768 and Japanese Patent Application Laid-Open No. 7104689, and ATP synthase subunit β is described in Japanese Patent Application Laid-Open No. 2011-188744, and all of these terms are used by those skilled in the art as technical terms. 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 as terms meaning the same protein.Therefore, in the present invention, the term "ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit" is synonymous with the term "ribulose-bisphosphate carboxylase large chain." Furthermore, the terms "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 the present invention, the term "ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit" is synonymous with the term "ribulose-bisphosphate carboxylase small subunit."

[0177] The composition of the present invention will now be described in more detail.

[0178] Ribulose 1,5-bisphosphate carboxylase / oxygenase is an enzyme involved in photosynthesis, and in the present invention, it is believed 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 the proteins constituting the large and small subunits of ribulose 1,5-bisphosphate carboxylase / oxygenase in binding to the receptor in the cell membrane of mammalian cells. When the proteins constituting the large and small subunits of ribulose 1,5-bisphosphate carboxylase / oxygenase bind to the receptor in the cell membrane of mammalian cells, the receptor is activated, which activates a protein called a G protein. This G protein activates an enzyme called adenylate cyclase, which is believed to convert adenosine triphosphate (ATP) to cAMP (cyclic adenosine monophosphate).

[0179] By increasing cAMP (cyclic adenosine monophosphate) in mammalian cells, it is expected that the activation of transcription factors, which are proteins that regulate gene transcription, cell growth and differentiation, improvement of neurotransmission, and activation of dermal papilla cells will be achieved. Furthermore, the composition of the present invention is also thought to have an effect of inhibiting melanin production in mammalian cells, as well as anti-inflammatory and antioxidant effects on mammalian cells. For this reason, it is expected to be used as a raw material or additive for pharmaceuticals, cosmetics, foods, reagents, and the like. In other words, the composition of the present invention is expected to be used as a melanin production inhibitor, anti-inflammatory agent, and antioxidant.

[0180] The proteins contained in the compositions of the present invention are identified by the following method. Specifically, proteins are separated in a gel according to molecular weight using SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). Proteins separated in the gel are visualized as molecular weight bands using a coloring agent. The darker colored portions of the molecular weight bands are excised as protein bands to obtain gel fragments. These gel fragments are then degraded into peptides using trypsin, and the degraded peptide fragments are separated using a liquid chromatograph called nanoLC, which has been made highly sensitive by miniaturizing the packing material and column diameter. The amino acid sequences of the peptides are identified using a tandem mass spectrometer (MS / MS) following LC. The results are compared with proteins registered in a database to identify the proteins contained in the gel fragments. Even when the biological species of origin of a protein is unknown, it is possible to infer the biological species and protein by comparing it with a database of all biological species to find proteins with the same amino acid sequence. The database used was MASCOT from Matrix Science. Of the peptides obtained by enzymatic digestion with trypsin, only a portion of the peptides can be sequenced using a mass spectrometer. However, for biological species with a genome database, a protein can be identified if only a few peptide amino acids match. Such protein identification can be performed, for example, at Nippon Proteomics Co., Ltd. (6-6-3 Minamiyoshinari, Aoba-ku, Sendai, Miyagi Prefecture, 989-3204).

[0181] The composition of the present invention may further contain fucoxanthin or a derivative thereof. This is because fucoxanthin or a derivative thereof is not involved in increasing intracellular cAMP, but is known to inhibit melanin production and have antioxidant effects. Examples of fucoxanthin derivatives include fucoxanthin hydrolysates or derivatives thereof, fucoxanthin esters (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, fucoxanthinol, which is a hydrolysis product of fucoxanthin; amarousiaxanthin A, which is fucoxanthinol that has undergone dehydration and isomerization; esters of fucoxanthin with amino acids such as glycine and alanine; esters and salts thereof of acetic acid, fucoxanthin with carboxylic acids such as citric acid; esters and salts thereof of fucoxanthin with inorganic acids such as phosphoric acid and sulfuric acid; monoesters and homogeneous or heterogeneous diesters selected from fatty acid esters of fucoxanthin with 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; glycosides such as glucosides; and the like. The fucoxanthin derivative may be of one type or of two or more types.

[0182] 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, and may be, for example, 0.001 to 50 wt % in the composition.

[0183] In addition, extracellular vesicles (EVs) released from gametophytes and / or sporophytes of seaweeds such as green algae, red algae, and brown algae can be used. Examples of green algae include Enteromorpha japonica, Acanthus nigricans, Miru (sea urchin), Hirami (sea urchin), Kuromi (sea urchin), and Caulerpa lentillifera. Examples of red algae include Taoyagisou (sea urchin), Asakusa nori (sea urchin), Fukurofunori (sea urchin), Susabinori (sea urchin), Tanshisai (Japanese name: Haitan Amanori), Uppuri nori (sea urchin), Tanegashima Amanori (sea urchin), Mukadenori (sea urchin), Tosakanori (sea urchin), Shikinori (sea urchin), Dulse (sea urchin), Amakusa nori (sea urchin), Akaba (sea urchin), and Fujimatsumo (sea urchin). Examples of brown algae include Undaria pinnatifida (wakame), Mozuku (Okinawa mozuku, Itomozuku), Habanori (sea urchin), Hirome (sea urchin), Aowakame (sea urchin), Sagarame (sea urchin), Kayamonori (sea urchin), Laminaria japonica (kelp), Laminaria japonica (kombu), Narrow-leaved Laminaria, Long-leaved Laminaria, Mitsuishi-kombu (sea urchin), Ecklonia cava (kajime), Kurome (black seaweed), Sargassum serrata (sea urchin), Eisenia bifida (Eisenia bifida), and Seiyo nori (sea urchin). Of the seaweeds, brown algae are the most desirable. This is because it contains a high content of fucoxanthin or its derivatives. Among brown algae, wakame is preferred. Seaweed includes sporophytes (thallus, discus, filamentous bodies), male and female gametophytes (thallus, discus, filamentous bodies), etc. Male and female gametophytes are desirable in the present invention because they are likely to release extracellular vesicles (EVs) and at least one protein selected from ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and also because they contain a large amount of fucoxanthin or a derivative thereof. In particular, the female gametophyte and / or sporophyte filaments are advantageous because they allow easy extraction of enzyme-constituting proteins and extracellular vesicles at low temperatures below 5°C, and contain a higher amount of fucoxanthin or its derivatives than the male gametophyte.

[0184] 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, including pure water and seawater. Antifoaming agents, thixotropic agents, pH adjusters, etc. may be added to the aqueous solvent as appropriate. Furthermore, artificial seawater, filtered seawater, deep seawater, etc. 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 fulvic acid, 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. The organic solvent is preferably an organic solvent capable of dissolving at least one compound selected from the group consisting of fucoxanthin and its derivatives. 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. This is because the inclusion of an aqueous solvent makes it difficult for extracellular vesicles (EVs) to be destroyed, even when the organic solvent is included as a solvent. The weight ratio of the organic solvent to the aqueous solvent is preferably organic solvent / aqueous solvent ≦9 / 1, and preferably organic solvent / aqueous solvent = 1 / 10 to 10 / 10.

[0185] In one aspect of the present invention, a composition comprising at least one protein selected from the group consisting of ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs) derived from seaweed gametophytes, is preferably in powder form. Powdered compositions can reduce the volume per component compared to solvent-containing compositions, thereby reducing transportation costs. Furthermore, when used as a cosmetic, pharmaceutical, reagent, or food ingredient, the composition can be easily mixed with other raw materials, making it useful as an industrial raw material.

[0186] In the present invention, at least one protein selected from the group consisting of ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, ATP synthase subunit beta, thiamine biosynthesis protein G, or histone H2B is used. It is desirable that the H2B) be attached to or encapsulated in the extracellular vesicles (EVs). This is because extracellular vesicles (EVs) inherently have the property of transporting proteins between cells, and the extracellular vesicles (EVs) facilitate the binding of the aforementioned proteins to receptors on the cell membrane that constitutes mammalian cells.

[0187] The composition of the present invention may contain components other than those described above, as long as the effects of the present invention are not impaired. Examples of such components include excipients and additives that can be used in cosmetics, pharmaceuticals, reagents, foods, and raw materials thereof.

[0188] Next, a method for producing the composition of the present invention will be described.

[0189] The composition of the present invention can be produced, for example, by a method using seaweed gametophytes as a raw material, comprising the following steps (1) and (2): Step (1): The seaweed gametophytes are dried to obtain a dried product, which is then pulverized to obtain a seaweed gametophyte powder, and the powder is dispersed in an aqueous solvent adjusted to a temperature of 5° C. or less. Step (2): The aqueous solvent in which the powder has been dispersed is centrifuged to separate it into a solid content and a supernatant, and the supernatant is collected.

[0190] A method including the above steps (1) and (2) can be used to produce a composition 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 seaweed gametophytes. The production method of the present invention may include steps other than steps (1) and (2). For example, it may include a drying step, as described below.

[0191] The seaweed gametophyte used in step (1) may be a female gametophyte or a filamentous sporophyte. Examples of seaweed include the seaweeds mentioned above, preferably brown algae, and more preferably wakame.

[0192] In the following production method, the female gametophyte of seaweed will be described as an example, but the present invention is not limited thereto. First, an example of a method for obtaining gametophytes of seaweed will be described using Undaria pinnatifida as an example.

[0193] (I) Cultivation of seaweed gametophytes Gametophytes developed from wakame zoospores are separated into one male and one female, grown, mechanically shredded, attached to threads, and fertilized on the threads. The wakame juveniles developed from these are grown in indoor cultures and in natural seawater. Sporophytes (mekabu) are obtained from the grown wakame thallus (adult).

[0194] 1. Preserving sporophytes 1) Place sporophytes in a plastic bag or container and store in a cool, dark place at 15-20°C. At temperatures below 15°C, zoospores are not easily released when returned to seawater. If sporophytes are removed from seawater and stored in a cool, dark place, zoospores can be released for 2-3 days.

[0195] 2. Collecting zoospores 1) The room temperature for releasing zoospores should be 15-20°C. High temperatures are not recommended as they shorten the swimming time of the zoospores.

[0196] 2) Cut the spore leaves into pieces about 3-4 cm square. The part closest to the rhizoid is best for releasing spores, but cut off the part of the spore leaf surface that is as clean as possible. Lightly wipe off any dirt from the cut leaf pieces with absorbent paper or similar.

[0197] 3) Prepare three beakers containing 100 mL of sterilized seawater, wash the leaf pieces in turn, and then place them in a petri dish containing 50 mL of sterilized seawater.

[0198] 4) Place the dish containing the leaf fragment on the stage of a stereomicroscope and shine light from above to release the zoospores. Adjusting the stereomicroscope to a dark field setting makes it easier to observe the release of zoospores. After 10 minutes of illumination with an optical fiber or similar, sufficient zoospores will be released.

[0199] 5) Prepare a capillary tube. A capillary tube can be prepared by heating and stretching a hematocrypt tube, or by stretching the tip of a Pasteur pipette until it is sufficiently elongated.

[0200] 6) Prepare a petri dish filled with 50 mL of PESI culture medium.

[0201] 7) Under a stereomicroscope, aspirate an appropriate amount of zoospores and drop them into a petri dish. When aspirating, be careful not to let the capillary tube touch the bottom of the petri dish or the leaf fragment (this is often the case when aspirating diatoms). After dropping, shake the petri dish thoroughly by hand to ensure a uniform zoospore density.

[0202] 8) Prepare about four different types of petri dishes, each with a different amount of zoospore fluid. If the amount of zoospores is large, the gametophyte density will be high, and the gametophytes will be too close to each other, making them difficult to isolate.

[0203] 9) After collecting the zoospores, the petri dishes are cultured at 10-30°C with a 12-14 hour light period (1000-1500 lux). When there are large temperature fluctuations or when the temperature is high or the light intensity is high, the male and female gametophytes will look similar and be difficult to distinguish, so care must be taken to maintain consistent culture conditions.

[0204] 10) After two weeks, the gametophytes reach a size where they can be sexed. Because there is a risk of fertilization, sex the gametophytes as soon as possible and isolate them.

[0205] 11) At this stage, there is little contamination with diatoms, but if diatoms do appear, discard the petri dish. If it is not possible to discard it, germanium dioxide can be used to suppress diatom growth, and gametophytes that are not contaminated by diatoms can be isolated.

[0206] 3. Isolation of male and female gametophytes 1) Place the petri dish in which the gametophytes are being cultured on the stage of an inverted microscope and search for a female gametophyte suitable for isolation. Isolate those in which the gametophytes are sufficiently separated and clearly sexed. Attach a tube to a Pasteur pipette, separate the female gametophytes from the petri dish, and aspirate them. Place each aspirated female gametophyte into a microplate filled with PESI medium. A schematic diagram of female and male gametophytes is shown in Figure 2-1. 2) Culture for one month at 10-30°C with a 12-14 hour light period (1500-2000 lux).

[0207] 4. Preservation of female gametophytes 1) After culturing in a microplate, remove the female gametophyte. Usually, the female gametophyte is large enough to be seen with the naked eye, so it can be picked up and removed from the microplate with ophthalmic tweezers. If it has not grown to a sufficient size, it can be aspirated using a Pasteur pipette under an inverted microscope.

[0208] 2) For storage, place in a screw-cap test tube and store at 10-30°C with 14 hours of light (1000-1500 lux). After storage, the medium is replaced with PESI medium once every two months.

[0209] (II) Cultivation of Seaweed Gametophytes As a culture medium for female gametophytes, Provasoli's Enriched Seawater (PES) or an improved version of it, the PESI culture medium, is preferred. The PES culture medium has the following composition. (See "Phycology Experiments and Practices," edited by Ariga Hirokatsu, Inoue Isao, Tanaka Jiro, Yokohama Yasutsugu, and Yoshida Tadao, Kodansha Scientific (2000) and JP 2009-201480 A.) The PESI culture medium can be prepared with reference to Plant Tissue Culture, 6(2), 55-62 (1989), etc. The detailed composition will be described later in the Examples.

[0210] (PES culture solution) Tris hydroxymethyl aminomethane 5.0g NaNO ３ 3.5g Na ２ -glycerophophate 500mg Fe stock solution 250mL P-2 metal mix 250mL Vitamin 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

[0211] (Fe stock solution) ２ -EDTA 2H ２ O 330mg Fe(NH４ ) ２ (SO ４ ) ２ ・6H ２ O 351mg Distilled water 500mL

[0212] (P-2 metal mix) Na ２ -EDTA 2H ２ O 500mg H ３ BO ３ 570 mg FeCl ３ ・6H ２ O 24.5mg MnSO ４ ・4H ２ O 82.0mg CoSO ４ ・7H ２ O (4.8mg / mL) 0.5mL ZnSO ４ ・7H ２ O 11.0mg Distilled water 500mL

[0213] A seaweed culture solution is prepared by adding a PES or PESI culture solution to seawater. The seawater used may be natural seawater or artificial seawater sterilized with ozone, ultraviolet light, an autoclave, or the like, or filtered seawater obtained by filtering natural seawater. The artificial seawater is prepared to contain primarily 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 natural seawater is used, deep seawater may also be used. Deep seawater is seawater found in the deep sea at depths of 200 m or more. Deep seawater has a high salt concentration, making it difficult for bacteria and other organisms that are harmful to seaweed cultivation to survive. Deep seawater is free from artificial contamination, has low bacteria due to its low temperature, and is free from the presence of viable phytoplankton due to its lack of sunlight. Deep seawater is also rich in nitrogen (N) from nitrates, phosphorus (P) from phosphates, and silicon (Si) from silicates, making it ideal for the growth of seaweed.

[0214] The seaweed female gametophyte is placed in the prepared seaweed culture solution and cultured in an aerated state. The aerated culture consists of a preliminary culture and a main culture. The seaweed female gametophyte is cultured in an artificial environment, such as an environment in which the water temperature, light intensity, and sunshine duration are controlled. Any environment can be used as long as the light intensity, sunshine duration, and water temperature can be strictly controlled. For example, the seaweed may be cultured in a container capable of cultivating seaweed in an incubator where the light intensity, sunshine duration, and internal temperature are regulated, or in an aquarium where the light intensity, sunshine duration, and water temperature can be controlled.

[0215] In the preliminary culture environment, a daylight fluorescent lamp was used as the light source, and the irradiation light intensity was 1 to 100 μmol / m ２ The water temperature is preferably 5 to 30°C, and the sunlight duration is preferably 8 to 24 hours. Cultivation is usually carried out under a photoperiod with alternating light and dark periods, but continuous light irradiation is also possible. The culture period may be between 1 and 40 days, but is preferably 5 to 10 days. A minimum of 5 days is sufficient.

[0216] In the main culture environment, a green light source (490-550 nm) is used, and the irradiation dose is 10-200 μmol / m ２ The preferred conditions are: 1 / s, water temperature 5 to 30°C, and sunshine duration 8 to 24 hours. Cultivation is usually carried out under a photoperiod with alternating light and dark periods, but continuous light irradiation is also possible. The culture period may be between 1 and 40 days, but is preferably 5 to 10 days. Seaweed growth promoters such as fatty acids can be added to the seaweed culture solution used in the present invention as appropriate. The culture tank is also equipped with an agitator, a vibrator, a temperature controller, a pH adjuster, a turbidity meter, a light controller, air, O ２ , CO ２ The seaweed may be cultured by any suitable liquid culture method, such as a batch culture method, a semi-batch culture method (fed-batch culture method), or a continuous culture method (perfusion culture method).

[0217] After the primary culture of the seaweed female gametophytes, the seaweed gametophytes are removed from the culture medium by scooping them up with a plankton net or filtering the culture medium. The resulting aggregate of seaweed gametophytes can be preferably used as a raw material for extracting, for example, extracellular vesicles (EVs) and at least one protein selected from the group consisting of ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit.

[0218] (III) Aqueous solvent extraction of seaweed gametophytes Seaweed gametophytes are dried by freeze-drying or other methods to form a powder, and this powder is dispersed in an aqueous solvent cooled to below 5°C to obtain a dispersion of the seaweed gametophytes (step (1)). By dispersing dried powder of seaweed gametophytes in an aqueous solvent, extracellular vesicles (EVs) and at least one protein selected from 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, a dry powder of female gametophytes 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 ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit are extracted from the female gametophytes of seaweed into the dispersion.

[0219] At least one protein selected from extracellular vesicles (EVs), ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit, and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit is easily eluted in a cooled aqueous solvent, and vesicles made of lipid membranes are easily disrupted by organic solvents, so extraction with an aqueous solvent is preferred. In the case of an aqueous solvent whose temperature exceeds 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 it difficult to extract the protein.

[0220] 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, deep seawater, etc. 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 fulvic acid, and sodium citrate. The aqueous solvent may also be phosphate-buffered saline (PBS). It is desirable that the aqueous solvent in which seaweed gametophytes are dispersed does not contain an organic solvent.

[0221] The extraction temperature is preferably 5°C or lower. The extraction time is preferably 0.1 to 5 hours. Seaweed gametophytes (preferably female gametophytes) are preferably dispersed in an aqueous solvent and then stirred or infiltrated. The aqueous solvent is preferably used in a ratio of 0.01 to 1 L per 1 g of seaweed gametophytes (dry weight). The seaweed gametophytes are separated and removed from the resulting seaweed gametophyte dispersion to obtain an aqueous solvent composition containing extracellular vesicles (EVs) (step (2)). In this specification, the aqueous solvent composition obtained in step (2) is also referred to as an aqueous solvent extract or an aqueous solvent extract of seaweed gametophytes. The seaweed gametophytes, which are the extraction residue, and impurities separated from the gametophytes are separated into a solid residue and a supernatant by methods such as filtration using a filter or column or centrifugation, and the solids are further removed from the aqueous solvent by filtration or other methods, and the supernatant is collected to obtain a composition. Step (2) is also preferably performed at a temperature of 5°C or lower. This is to inhibit the degradation of proteins and intracellular vesicles (EVs).

[0222] In this manner, a composition (aqueous solvent composition) is obtained that contains an aqueous solvent, at least one protein selected from the group consisting of ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs). The seaweed gametophytes isolated in step (2) can be used for extraction with an organic solvent, as described below.

[0223] Furthermore, a powdery composition can be obtained by removing the aqueous solvent from an aqueous solvent composition containing at least one protein selected from the group consisting of ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit and extracellular vesicles (EVs). The method for removing the aqueous solvent can be any method capable of drying the aqueous solvent composition, including heating, freeze-drying, and vacuum drying. However, because extracellular vesicles are composed of proteins and lipids, freeze-drying is preferred.

[0224] (IV) It is also possible to add an organic solvent to the seaweed gametophyte residue (the seaweed gametophyte solid separated in step (1) above) removed after dispersing dried seaweed gametophytes in an aqueous solvent, thereby obtaining an organic solvent composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives (for convenience, referred to as step (3)). In this specification, the organic solvent composition obtained in step (3) is sometimes referred to as an organic solvent extract.

[0225] Fucoxanthin or its derivatives can be extracted from seaweed gametophytes by immersing seaweed gametophyte residues 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 present in the cells of the seaweed gametophyte residues to elute into the organic solvent. Note that extracellular vesicles (EVs) are substantially not contained in the organic solvent. This is because extracellular vesicles (EVs) released into the organic solvent are unstable because they do not form a protective membrane hydrated with water molecules on their surface, resulting in destruction of the lipid membrane.

[0226] The organic solvent to be used may be an alcohol such as methanol, ethanol, propanol, isopropanol, n-butanol, or butanediol (1,3-butanediol (1,3-butylene glycol), 1,4-butanediol (1,4-butylene glycol)), a ketone such as methyl ethyl ketone or acetone, an ester such as methyl acetate or ethyl acetate, an organic chlorine-based hydrocarbon such as chloroform, an aliphatic hydrocarbon such as hexane, or an aromatic hydrocarbon such as benzene or toluene, either alone or in combination of two or more thereof. Of these, ethanol and butanediol are preferred as the organic solvent.

[0227] At least one compound selected from the group consisting of fucoxanthin and its derivatives is eluted in the organic solvent. Because fucoxanthin and its derivatives are substantially insoluble in aqueous solvents, the organic solvent contains a higher amount of fucoxanthin and its derivatives than the aqueous solvent extract (II). The organic solvent may contain an aqueous solvent, but preferably does not. When the organic solvent contains an aqueous solvent, it is mainly derived from the water contained in the seaweed gametophyte.

[0228] In the extraction with an organic solvent, 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. The seaweed gametophytes are preferably dispersed in an organic solvent and then stirred and infiltrated. The organic solvent is preferably used at a ratio of 0.01 to 1 L per 1 g of seaweed gametophytes. After extraction with an organic solvent, the seaweed gametophytes, which are the extraction residue, and impurities separated from the gametophytes may be removed from the organic solvent. The method for removing the seaweed gametophytes from the organic solvent is not particularly limited, and methods such as filtration using a filter or column, or centrifugation can be used. In this manner, it is desirable to obtain a composition (organic solvent composition) containing an organic solvent and at least one compound selected from the group consisting of fucoxanthin and its derivatives dissolved in the organic solvent.

[0229] In the present invention, it is preferable to mix oils and fats such as fatty acid triglycerides with the composition and not isolate fucoxanthin or its derivatives. This is because isolation would impair the stability of fucoxanthin and its derivatives. Furthermore, even if mixed with oils and fats such as fatty acid triglycerides, once extracted with water, the extracellular vesicles (EVs) are hydrophilized and do not elute or disperse in the oil, making it difficult to isolate fucoxanthin or its derivatives from the extracellular vesicles (EVs) using the oil.

[0230] The composition of the present invention, which 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), can be used for various industrial applications. In particular, the composition of the present invention is expected to promote glycogenolysis in liver cells and increase blood glucose levels by increasing cAMP (cyclic adenosine monophosphate) in mammalian cells, and also promote lipolysis in adipocytes and increase energy supply, making it an effective ingredient for various health foods. Furthermore, the composition of the present invention is expected to activate hair follicle papilla cells, increase cardiac contractility, dilate airways by promoting relaxation of airway smooth muscle, promote neurotransmission in nerve cells, and control immune responses by regulating the activation and inhibition of immune cells, making it suitable for pharmaceutical and other uses. Furthermore, the composition of the present invention is also useful in cosmetics, as it promotes cell growth by activating signal transduction pathways that promote cell growth and differentiation. Thus, the composition of the present invention can be used as an agent for increasing cAMP (cyclic adenosine monophosphate) in cells. The form of use of the thickener is not particularly limited, and examples thereof include the composition itself, excipients, binders, lubricants, disintegrants, surfactants, buffers, preservatives, flavorings, colorings, oils, pigments, water, alcohols, thickeners, antiseptics, antioxidants, and chelating agents, which may be used alone or in combination of two or more, but are not limited to these.When used as an external preparation for skin, the dosage form of the present invention can be any form used in ordinary pharmaceuticals, cosmetics, quasi-drugs, etc., as long as it is suitable for obtaining this medicinal effect, and examples include external liquid preparations such as lotions, liniments, aqueous solutions, and emulsions, external solid preparations such as powders and dissolving tablets, external semi-solid preparations such as creams, film-forming agents, ointments, and jellies, soaps, etc. Note that oral preparations, injections, etc. may also be effective, and several methods may be used in combination.

[0231] As described above, seaweed gametophytes can be suitably used as a raw material for extracting 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). The raw material may also include an aggregate of seaweed gametophytes.

[0232] The composition of the present invention may include a raw material used for extracting 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). The seaweed is preferably a brown alga. The seaweed is preferably a female gametophyte or a filamentous sporophyte.

[0233] Furthermore, by allowing seaweed-derived extracellular vesicles (EVs) to coexist 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. Seaweed-derived extracellular vesicles (EVs) can be used to improve the stability of at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0234] This specification describes the following:

[0235] The present disclosure (42) relates to 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.

[0236] The present disclosure (43) is the composition according to the present disclosure (42), wherein the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit is a subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase (hereinafter referred to as "RuBisCO"), an enzyme used in photosynthesis, and is responsible for the catalytically active site of the RuBisCO.

[0237] The present disclosure (44) is the composition according to the present disclosure (42) or (43), wherein the ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit is detected in the region of 35,000 to 63,000 Da by electrophoresis.

[0238] The present disclosure (45) is the composition according to any one of the present disclosures (42) to (44), further comprising at least one selected from ATP synthase subunit beta, thiamine biosynthesis protein G, and histone H2B.

[0239] The present disclosure (46) is the composition according to any one of the present disclosures (42) to (45), wherein the seaweed is a female gametophyte of seaweed.

[0240] The present disclosure (47) is the composition according to any one of the present disclosures (42) to (46), wherein the seaweed is a filamentous sporophyte.

[0241] The present disclosure (48) is the composition according to any one of the present disclosures (42) to (47), wherein the seaweed is brown algae.

[0242] The present disclosure (49) is the composition according to any one of the present disclosures (42) to (48), wherein the composition contains an aqueous solvent or a mixed solvent consisting of an aqueous solvent and an organic solvent.

[0243] The present disclosure (50) is the composition according to the present disclosure (49), wherein the aqueous solvent is water or a solvent in which a water-soluble salt is dissolved in water.

[0244] The present disclosure (51) is the composition according to the present disclosure (50), wherein the water-soluble salt is at least one selected from the group consisting of sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, ammonium sulfate, potassium phosphate, potassium dihydrogen phosphate, iron phosphate, iron fulvic acid, and sodium citrate.

[0245] The present disclosure (52) is the composition according to the present disclosure (50) or (51), wherein the solvent in which the water-soluble salt is dissolved in water is seawater or phosphate buffered saline.

[0246] The present disclosure (53) is the composition according to any one of the present disclosures (50) to (52), wherein the organic solvent is at least one selected from the group consisting of methanol, ethanol, propanol, isopropanol, n-butanol, 1,3-butanediol, 1,4-butanediol, methyl ethyl ketone, acetone, methyl acetate, ethyl acetate, chloroform, hexane, benzene, and toluene.

[0247] The present disclosure (54) is the composition according to any one of the present disclosures (50) to (53), wherein the weight ratio of the organic solvent to the aqueous solvent is organic solvent / aqueous solvent ≦9 / 1.

[0248] The present disclosure (55) is the composition according to any one of the present disclosures (42) to (54), wherein the composition further comprises at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0249] The present disclosure (56) is the composition according to the present disclosure (55), wherein the total content of at least one compound selected from fucoxanthin and its derivatives in the composition is 0.001 to 50 wt %.

[0250] The present disclosure (57) is a composition according to any one of the present disclosures (42) to (56), in which 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 seaweed gametophytes.

[0251] The present disclosure (58) is the composition according to any one of the present disclosures (42) to (57), wherein the composition is a powdery composition.

[0252] The present disclosure (59) is an agent for increasing intracellular cyclic adenosine monophosphate (cAMP), comprising the composition according to any one of the present disclosures (42) to (58).

[0253] The present disclosure (60) is a cellular melanin production inhibitor comprising the composition according to any one of the present disclosures (42) to (59).

[0254] The present disclosure (61) is the composition according to any one of the present disclosures (42) to (60), wherein the extracellular vesicles (EVs) are vesicles having a lipid bilayer structure secreted from living cells.

[0255] The present disclosure (62) is the composition according to any one of the present disclosures (42) to (60), wherein the extracellular vesicles (EVs) include at least one selected from the group consisting of exosomes, microvesicles, and apoptotic bodies.

[0256] The present disclosure (63) is the composition according to any one of the present disclosures (42) to (62), wherein the extracellular vesicles (EVs) contain exosomes, and the size of the exosomes is 1 to 200 nm in diameter.

[0257] The present disclosure (64) is the composition according to any one of the present disclosures (42) to (63), wherein the extracellular vesicles (EVs) include microvesicles (MVs), and the size of the microvesicles (MVs) is 100 to 1000 nm in diameter.

[0258] The present disclosure (65) is the composition according to any one of the present disclosures (42) to (64), wherein the extracellular vesicles (EVs) contain apoptotic bodies, and the size of the apoptotic bodies is 50 to 5,000 nm in diameter.

[0259] The present disclosure (66) is the composition according to any one of the present disclosures (42) to (65), wherein, when the composition is separated by liquid chromatography under the following conditions, a peak of the lipid membrane constituting the extracellular vesicles (EVs) and a peak of the protein appear at the same retention time on the liquid chromatography chart. Liquid chromatography conditions: Column packing: qEV10 70 nm manufactured by Izon Science Column shape: Stainless steel column manufactured by Senshu Scientific Co., Ltd., diameter 8 mm x length 100 m Developing solvent: Aqueous solution containing 10 mM tris(hydroxymethyl)aminomethane, pH = 7.6, 30 mM NaCl, and 0.5 mM EDTA Developing time gradient: Uniform Solution flow rate: 1 mL / min Injection volume: 10 μL Column temperature: 30°C Sample chamber temperature: 10°C

[0260] The present disclosure (67) 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 seaweed gametophytes and / or sporophytes, the method comprising the following steps (1) and (2): Step (1): The gametophytes and / or sporophytes of the seaweed are dried to obtain a dried body, and then the dried body is pulverized to obtain a powder of the gametophytes and / or sporophytes of the seaweed, and the powder is dispersed in an aqueous solvent adjusted to a temperature of 5° C. or less. Step (2): The aqueous solvent in which the powder has been dispersed is centrifuged to separate it into a solid content and a supernatant, and the supernatant is collected.

[0261] The present disclosure (68) is a method for producing the composition according to the present disclosure (67), comprising the step of adding at least one compound selected from the group consisting of fucoxanthin and its derivatives to the supernatant.

[0262] The present disclosure (69) is a method for producing the composition according to the present disclosure (67) or (68), 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 seaweed gametophytes and / or sporophytes.

[0263] The present disclosure (70) is a method for producing an agent for increasing intracellular cyclic adenosine monophosphate (cAMP), comprising the method for producing the composition according to any one of the present disclosures (67) to (69).

[0264] The present disclosure (71) is a method for producing the composition according to any one of the present disclosures (67) to (70), wherein the extracellular vesicles (EVs) are derived from the female gametophyte of the seaweed.

[0265] The present disclosure (72) is a method for producing the composition according to any one of the present disclosures (67) to (71), wherein the extracellular vesicles (EVs) are derived from filaments of the sporophyte of the seaweed.

[0266] The present disclosure (73) relates to a raw material that includes a collection of seaweed gametophytes and is used to extract 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 seaweed gametophytes.

[0267] The present disclosure (74) is the raw material according to the present disclosure (73), wherein the seaweed is a female gametophyte.

[0268] The present disclosure (75) is the raw material according to the present disclosure (73) or (74), wherein the seaweed is a filamentous body of a sporophyte.

[0269] The present disclosure (76) is the raw material according to any one of the present disclosures (73) to (75), wherein the seaweed is brown algae.

[0270] (Third Invention) The third invention will be described below. Fucoxanthin (C), a red pigment found in large amounts in brown algae such as wakame seaweed and kelp, is a compound of the present invention. ４２ H ５８ O ６ Fucoxanthin has a variety of excellent physiologically active functions, including anti-obesity, anti-diabetic, anti-inflammatory, and even skin-whitening effects, in addition to its excellent antioxidant properties, and its use as a functional food ingredient is highly anticipated. For this reason, various technologies related to various compositions containing fucoxanthin are being researched.

[0271] For example, Patent Document 9 discloses a process for producing fucoxanthin and / or polysaccharides from microalgae, and discloses that an absorption enhancer may be included. Also, Patent Document 10 discloses a composition containing carotenoids and fatty acids extracted from microalgae, and discloses that the fucoxanthin content is 1.7 to 2.0% DW and the linoleic acid content is 0.23% DW.

[0272] However, Patent Document 9 does not disclose any specific fucoxanthin penetration enhancers, and Patent Document 10 does not consider the permeability of fucoxanthin.

[0273] The present invention has been made in view of the above problems, and an object of the present invention is to provide a composition in which the permeability of fucoxanthin into cell tissues is improved, and a method for producing the same.

[0274] The present invention is characterized in that it comprises at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, and the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 1.1 to 22.0.

[0275] The composition of the present invention contains at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, and the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives and the linoleic acid or a salt thereof to the linoleic acid or a salt thereof ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 1.1 to 22.0. Linoleic acid or a salt thereof easily penetrates into cellular tissues such as the skin, and it is presumed that the at least one compound selected from the group consisting of fucoxanthin and its derivatives also penetrates into cellular tissues such as the skin in a manner induced by the penetration of linoleic acid or a salt thereof. If the amount of at least one compound selected from the group consisting of fucoxanthin and its derivatives is too high, the effect of linoleic acid or its salts on penetrating into the interior of cellular tissues, which is presumed to be the effect of linoleic acid or its salts on fucoxanthin, etc., cannot be fully exerted, and conversely, if there is too much linoleic acid or its salts, the linoleic acid or its salts will preferentially penetrate into the interior of cellular tissues, making it difficult for fucoxanthin, etc. to penetrate into the interior of cellular tissues. For this reason, it is considered necessary for the weight ratio of at least one compound selected from the group consisting of fucoxanthin and its derivatives to linoleic acid or its salts to be 1.1 to 22.0. The weight ratio ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is not particularly limited as long as it is in the range of 1.1 to 22.0, and may be, for example, 1.182 to 19.5, 9.0 to 22.0, 5.0 to 15.0, 1.182 to 9.0, or 9.0 to 19.5.

[0276] Examples of fucoxanthin derivatives include fucoxanthin hydrolysates or derivatives thereof, fucoxanthin esters (for example, esters with amino acids, carboxylic acids, inorganic acids, or fatty acids), salts thereof, and fucoxanthin glycosides. More specific examples of fucoxanthin derivatives include, but are not limited to, monoesters and homogeneous or heterogeneous diesters selected from the following: fucoxanthinol, which is a hydrolysis product; amarousiaxanthin A, which is fucoxanthinol that has undergone dehydration and isomerization; esters of fucoxanthin with amino acids such as glycine and alanine; esters and salts of fucoxanthin with carboxylic acids such as acetic acid and citric acid; esters and salts of fucoxanthin with inorganic acids such as phosphoric acid and sulfuric acid; and fatty acid esters of fucoxanthin with 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; and glycosides such as glucosides.

[0277] As the salt of linoleic acid, metal salts, ammonium salts, etc. can be used. Furthermore, as the metal salt, alkali metal salts and alkaline earth metal salts can be used, and as the alkali metal, lithium, sodium, potassium, etc. can be used, and as the alkaline earth metal, calcium, magnesium, etc. can be used.

[0278] The composition of the present invention preferably does not contain microalgae extract. This is because microalgae extracts often contain uncharacterized proteins and impurities, which may cause allergic reactions or may be accompanied by an unpleasant odor, making them undesirable. Microalgae grow in either marine or freshwater systems. Microalgae are single-celled species that exist individually, in chains, or in groups, and are capable of photosynthesis. Microalgae extract refers to a composition extracted from microalgae using a solvent. Solvents used include aqueous solvents, organic solvents, supercritical fluids, subcritical fluids, and the like. For the same reason, the composition of the present invention preferably does not contain any material derived from microalgae.

[0279] In the present invention, the at least one compound selected from the group consisting of fucoxanthin and its derivatives, and linoleic acid or a salt thereof are preferably derived from seaweed, as naturally derived compositions have fewer adverse effects on living organisms. The composition of the present invention may also contain at least one compound selected from the group consisting of fucoxanthin and its derivatives, and linoleic acid or a salt thereof derived from seaweed, as well as at least one compound selected from the group consisting of industrially synthesized fucoxanthin and its derivatives, and linoleic acid or a salt thereof, with the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or a salt thereof adjusted. Furthermore, the term "seaweed" as used herein refers to a group of marine species of multicellular algae, and does not include unicellular microalgae.

[0280] In the present invention, the seaweed is preferably a seaweed gametophyte. Seaweeds exhibit various morphologies in their life cycle, including sporophytes (thallus, discus, and filament), male and female gametophytes (thallus, discus, and filament). Male and female gametophytes are preferred in the present invention because they contain a high amount of fucoxanthin and its derivatives, and linoleic acid and its salts.

[0281] In the present invention, the seaweed is preferably the female gametophyte of seaweed, because the female gametophyte has the highest content of fucoxanthin and its derivatives among the various forms in the life cycle of seaweed.

[0282] In the present invention, the seaweed is preferably brown algae. Seaweed includes green algae, red algae, brown algae, etc., but brown algae are preferred because they contain a large amount of fucoxanthin and its derivatives.

[0283] The composition of the present invention preferably does not contain an extract of Phaeodactylum tricornutum. Phaeodactylum tricornutum is a microalga, and extracts extracted from Phaeodactylum tricornutum are likely to contain allergens and substances that cause malodors. The extraction of Phaeodactylum tricornutum is carried out in accordance with Patent Document 10, JP-A-2018-512432. The solvents used are methanol, ethanol, and supercritical fluid carbon dioxide.

[0284] In the present invention, the composition may further contain a polysaccharide.

[0285] In the present invention, the composition contains an organic solvent or a mixed solvent consisting of an aqueous solvent and an organic solvent. The organic solvent preferably dissolves at least one compound selected from the group consisting of fucoxanthin and its derivatives. The organic solvent is preferably an organic solvent capable of dissolving at least one compound selected from the group consisting of fucoxanthin and its derivatives. This is because compositions containing fucoxanthin or its derivatives and linoleic acid or a salt thereof are easily soluble in organic solvents. The composition of the present invention may further contain an aqueous solvent in addition to the organic solvent. The aqueous solvent is water or a solvent in which a water-soluble salt is dissolved, such as pure water or seawater. Antifoaming agents, thixotropic agents, pH adjusters, etc. may be added to the aqueous solvent as appropriate. Furthermore, artificial seawater, filtered seawater, deep seawater, etc. can also be used as seawater. In addition, the composition of the present invention may contain a mixed solvent consisting of an aqueous solvent and an organic solvent. The organic solvent is preferably one capable of dissolving at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0286] In the present invention, the composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof is preferably a powdered composition. Powdered compositions can reduce the volume per component compared to compositions containing a solvent, thereby reducing transportation costs. Furthermore, when used as a cosmetic, pharmaceutical, or food ingredient, they can be easily mixed with other raw materials, making them useful as industrial raw materials.

[0287] The present invention provides a method for producing a composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, comprising the following steps (1) to (3): Step (1): Dispersing seaweed gametophytes in an aqueous solvent to obtain a dispersion of the seaweed gametophytes. Step (2): Separating and removing the aqueous solvent from the dispersion of the seaweed gametophytes obtained in step (1). Step (3): To the seaweed gametophytes from which the aqueous solvent has been separated and removed in step (2), an organic solvent capable of dissolving at least one compound selected from the group consisting of fucoxanthin and its derivatives is added to obtain a composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, wherein the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 1.1 to 22.0.

[0288] The present invention also provides a method for producing a composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, the method comprising the following steps (1) to (4): Step (1): Dispersing seaweed gametophytes in an aqueous solvent to obtain a dispersion of the seaweed gametophytes; Step (2): Separating and removing the aqueous solvent from the dispersion of the seaweed gametophytes obtained in step (1); Step (3): Adding an organic solvent to the seaweed gametophytes from which the aqueous solvent has been separated and removed in step (2) to obtain an organic solvent composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof. Step (4): At least one compound selected from the group consisting of fucoxanthin and its derivatives and / or linoleic acid or a salt thereof is added to the organic solvent composition obtained in step (3), to obtain a composition in which the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is adjusted to 1.1 to 22.0.

[0289] A composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof is obtained by dispersing seaweed gametophytes in an aqueous solvent, separating and removing the aqueous solvent from the seaweed gametophytes, and then adding to the seaweed gametophytes an organic solvent capable of dissolving at least one compound selected from the group consisting of fucoxanthin and its derivatives. If the weight ratio of at least one compound selected from the group consisting of fucoxanthin and its derivatives to linoleic acid or a salt thereof in this composition is within the range of 1.1 to 22.0, no adjustment of the composition is necessary. However, if the weight ratio of at least one compound selected from the group consisting of fucoxanthin and its derivatives to linoleic acid or a salt thereof is outside the range of 1.1 to 22.0, the weight ratio of at least one compound selected from the group consisting of fucoxanthin and its derivatives or linoleic acid or a salt thereof is adjusted to 1.1 to 22.0 by adding at least one compound selected from the group consisting of fucoxanthin and its derivatives or linoleic acid or a salt thereof. When at least one compound selected from the group consisting of fucoxanthin and its derivatives, or linoleic acid or a salt thereof is added, commercially available industrially synthesized products can be used. The amount of fucoxanthin and linoleic acid extracted is increased when the seaweed gametophytes are first dispersed in an aqueous solvent and then an organic solvent capable of dissolving at least one compound selected from the group consisting of fucoxanthin and its derivatives is added. The reason for this is unclear, but it is presumed that first dispersing the seaweed gametophytes in an aqueous solvent allows the organic solvent to more easily penetrate into the cells.

[0290] Examples of the organic solvent that can be used in the present invention 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, organic chlorine-based hydrocarbons such as chloroform, aliphatic hydrocarbons such as hexane, and aromatic hydrocarbons such as benzene and toluene.

[0291] The composition of the present invention may contain an aqueous solvent in addition to the organic solvent. The aqueous solvent is water or a solvent in which water-soluble salts are dissolved, and includes pure water, seawater, etc. Antifoaming agents, thixotropic agents, pH adjusters, etc. may be added to the aqueous solvent as appropriate. Furthermore, artificial seawater, filtered seawater, deep seawater, etc. can also be used as the seawater. Examples of salts include sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, ammonium sulfate, potassium phosphate, potassium dihydrogen phosphate, iron phosphate, iron fulvic acid, sodium citrate, etc. Furthermore, the aqueous solvent may be phosphate-buffered saline.

[0292] The weight ratio of the organic solvent to the aqueous solvent is preferably organic solvent / aqueous solvent=1 / 10 to 10 / 10.

[0293] The method for producing the composition of the present invention may further include a drying step of drying the composition to form a powder.

[0294] The present invention relates to a raw material used for extracting and preparing a composition that is composed of an aggregate of seaweed gametophytes, and that contains at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, wherein the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 1.1 to 22.0.

[0295] This is because seaweed gametophytes contain a large amount of at least one compound selected from the group consisting of fucoxanthin and its derivatives, and linoleic acid or a salt thereof.

[0296] The seaweed is preferably brown algae, since brown algae contain the highest amount of fucoxanthin and its derivatives among all seaweeds.

[0297] The seaweed is preferably a female gametophyte, since the content of fucoxanthin and its derivatives is higher in the female gametophyte than in the male gametophyte.

[0298] The composition of the present invention capable of penetrating into cell tissue contains at least one compound selected from the group consisting of fucoxanthin and its derivatives, and linoleic acid or a salt thereof, and is characterized in that the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 1.1 to 22.0.

[0299] The cell tissue penetration enhancer of the present invention comprises at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, and is characterized in that the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 1.1 to 22.0.

[0300] By using the permeable composition and permeation enhancer of the present invention, fucoxanthin can be suitably permeated into cell tissues.

[0301] According to the present invention, a composition can be provided in which the permeability of fucoxanthin into cellular tissues is improved. By using the composition of the present invention, at least one compound selected from the group consisting of fucoxanthin and its derivatives is permeated into cellular tissues by the action of linoleic acid or a salt thereof, thereby imparting to cellular tissues the various excellent physiologically active functions of fucoxanthin, such as its excellent antioxidant effect, anti-obesity effect, anti-diabetic effect, anti-inflammatory effect, and even whitening effect. Therefore, the composition of the present invention can be used in a wide range of applications, including pharmaceuticals, food products, and cosmetics.

[0302] The composition of the present invention will now be described in more detail. The composition of the present invention comprises at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, and the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 1.1 to 22.0. Linoleic acid or a salt thereof easily penetrates into cellular tissue, and it is presumed that the at least one compound selected from the group consisting of fucoxanthin and its derivatives also penetrates into cellular tissue in a manner that is induced by the penetration of linoleic acid or a salt thereof. However, if the amount of at least one compound selected from the group consisting of fucoxanthin and its derivatives is too high, the effect of linoleic acid or its salts on penetrating into the interior of cellular tissues, which is presumed to be relative to fucoxanthin, etc., will not be fully exerted, and conversely, if there is too much linoleic acid or its salts, the linoleic acid or its salts will preferentially penetrate into the interior of cellular tissues, making it difficult for fucoxanthin, etc. to penetrate into the interior of cellular tissues. For this reason, it is considered necessary for the weight ratio of at least one compound selected from the group consisting of fucoxanthin and its derivatives to linoleic acid or its salts ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or its salts]) to be 1.1 to 22.0.

[0303] Examples of cellular tissues include epithelial tissues such as the epidermis of the skin, the inner wall of the digestive tract, the alveoli, and the endothelium of blood vessels; connective tissues such as bone, cartilage, blood, lymph, adipose tissue, tendons, and ligaments; muscle tissues such as skeletal muscle, cardiac muscle, and smooth muscle; and nervous tissues such as the brain, spinal cord, and nerves.

[0304] The composition of the present invention preferably does not contain microalgae extract. This is because microalgae extracts often contain uncharacterized proteins and impurities, which can cause allergic reactions or have an unpleasant odor, making them undesirable. Microalgae grow in either marine or freshwater systems. Microalgae are single-celled species that exist individually, in chains, or in groups, and are photosynthetic. Although the term "micro" is used, size itself is not essential; the term "microalgae" refers to single-celled algae. Microalgae extract refers to a composition extracted from microalgae using a solvent. Solvents used include aqueous solvents, organic solvents, supercritical fluids, and subcritical fluids. For the same reason, the composition of the present invention preferably does not contain any material derived from microalgae. Examples of microalgae include Phaeodactylum tricornutum, Navicula pelliculosa, Amphora, and Isochrysis aff. Galbana, Odontella aurita, Nitzscia closterium, Cylindrotheca closterium, Chaetoseros sp. , Emiliania huxleyi, Phaeodactykum sp. , Iso chrysis sp. , Amphora sp. , Naviculla lensi, Naviculla incerta and Chaeotocerous sp. , Phaeodactylum tricornutum (P. tricornutum), OPMS30543 strain (Pavlova), and the like.

[0305] Furthermore, it is desirable that the composition of the present invention does not contain an extract of Phaeodactylum tricornutum, which is a microalga, and an extract extracted from Phaeodactylum tricornutum is likely to contain allergens and substances that cause bad odors.

[0306] Examples of fucoxanthin derivatives include fucoxanthin hydrolysates or derivatives thereof, fucoxanthin esters (eg, esters with amino acids, carboxylic acids, inorganic acids, or fatty acids), salts thereof, and fucoxanthin glycosides. Specific examples of fucoxanthin derivatives include, but are not limited to, fucoxanthinol, which is a hydrolysis product of fucoxanthin; amarousiaxanthin A, which is fucoxanthinol that has undergone dehydration and isomerization; esters of fucoxanthin with amino acids such as glycine and alanine; esters and salts of acetic acid, fucoxanthin with carboxylic acids such as citric acid; esters and salts of fucoxanthin with inorganic acids such as phosphoric acid and sulfuric acid; monoesters and homogeneous or heterogeneous diesters selected from fatty acid esters of fucoxanthin with 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; glycosides such as glucosides; and the like. The fucoxanthin derivative may be of one type or two or more types.

[0307] 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, 0.0011 to 50 wt % in the composition. The total content of linoleic acid or a salt thereof in the composition of the present invention is also not particularly limited, but may be, for example, 0.001 to 45 wt % in the composition.

[0308] Furthermore, seaweeds can be used as raw materials for producing at least one compound selected from the group consisting of fucoxanthin and its derivatives, or linoleic acid or a salt thereof, and include brown algae, red algae, green algae, etc. In the present invention, seaweeds refer to a group of marine species that are multicellular algae, and do not include microalgae that are unicellular algae. Examples of green algae include Enteromorpha japonica, Acanthus pulcherrima, Miru (mildew), Hirami (flatworm), Kuromi (blackworm), and Caulerpa lentillifera. Examples of red algae include Taoyagisou (seaweed), Asakusa nori (porphyra), Fukurofunori (porphyra), Susabinori (porphyra), Tanshisai (Japanese name: Haitan Amanori), Uppuri nori (porphyra), Tanegashima Amanori (porphyra), Millipede nori (porphyra), Tosakanori (porphyra), Shikinori (porphyra), Dulse (porphyra), Amakusa nori (porphyra), Akaba (porphyra), Fujimatsumo (porphyra), and Danshisai (porphyra). Examples of brown algae include Undaria pinnatifida, Mozuku (Okinawa mozuku, Itomozuku), Habanori (porphyra), Hirome (seaweed), Aowakame (porphyra), Sagarame (seaweed), Kayamonori (kelp), Laminaria japonica (kombu), Laminaria japonica (kombu), Narrow-leaved Laminaria, Long-leaved Laminaria, Mitsuishi-kombu (kelp), Ecklonia cava (kajime), Kurome (black seaweed), Hondawara (seaweed), Eisenia bifida (Eisenia moniliformis), Arame (Eisenia bifida), and Western Habanori wakame (wakame). Brown algae are the most desirable seaweeds because they contain a large amount of fucoxanthin or its derivatives. Among brown algae, wakame seaweed is the most desirable. Seaweeds include sporophytes (thallus, discus, filament), male and female gametophytes (thallus, discus, filament), etc. Male and female gametophytes are desirable in the present invention because they contain a large amount of fucoxanthin or its derivatives. Female gametophytes are particularly advantageous because they contain a larger amount of fucoxanthin or its derivatives and linoleic acid or its salts than male gametophytes.

[0309] The composition of the present invention may further comprise a polysaccharide.

[0310] The composition of the present invention desirably contains an organic solvent. The organic solvent can be one that dissolves at least one compound selected from the group consisting of fucoxanthin and its derivatives. Examples of such organic solvents 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, organic chlorinated hydrocarbons such as chloroform, aliphatic hydrocarbons such as hexane, and aromatic hydrocarbons such as benzene and toluene, either alone or in combination. Among these, ethanol and butanediol (butylene glycol) are preferred.

[0311] The present invention may contain an aqueous solvent in addition to an organic solvent. 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. Furthermore, artificial seawater, filtered seawater, deep seawater, etc. 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 fulvic acid, and sodium citrate. The aqueous solvent may also be phosphate-buffered saline. The composition of the present invention may contain an organic solvent in addition to the aqueous solvent. The organic solvent is preferably an organic solvent capable of dissolving at least one compound selected from the group consisting of fucoxanthin and its derivatives. 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.

[0312] In one aspect of the present invention, the composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof is preferably a powdered composition. Powdered compositions can reduce the volume per component compared to compositions containing a solvent, thereby reducing transportation costs. Furthermore, when used as a cosmetic, pharmaceutical, or food ingredient, they can be easily mixed with other raw materials, making them useful as industrial raw materials.

[0313] The composition of the present invention may contain ingredients other than those described above, as long as the ingredients do not impair the effects of the present invention. Examples of such ingredients include excipients and additives that can be used in cosmetics, pharmaceuticals, foods, and raw materials for these.

[0314] Next, a method for producing the composition of the present invention containing at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof will be described.

[0315] The method for producing a composition of the present invention is, for example, a method for producing a composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, using seaweed gametophytes as a raw material, and comprising the following steps (1) to (3):

[0316] Step (1): Dispersing seaweed gametophytes in an aqueous solvent to obtain a seaweed gametophyte dispersion. Step (2): Separating and removing the aqueous solvent from the seaweed gametophyte dispersion obtained in Step (1). Step (3): Adding an organic solvent to the seaweed gametophytes from which the aqueous solvent has been separated and removed in Step (2) to obtain a composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, wherein the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to linoleic acid or a salt thereof ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 1.1 to 22.0.

[0317] Another embodiment of the method for producing a composition of the present invention is a method for producing a composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives, and linoleic acid or a salt thereof, using, for example, seaweed gametophytes as a raw material, the method comprising the following steps (1) to (4):

[0318] Step (1): Dispersing seaweed gametophytes in an aqueous solvent to obtain a seaweed gametophyte dispersion. Step (2): Separating and removing the aqueous solvent from the seaweed gametophyte dispersion obtained in Step (1). Step (3): Adding an organic solvent to the seaweed gametophytes from which the aqueous solvent has been separated and removed in Step (2) to obtain an organic solvent composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof. Step (4): Adding at least one compound selected from the group consisting of fucoxanthin and its derivatives and / or linoleic acid or a salt thereof to the organic solvent composition obtained in Step (3) to obtain a composition in which the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is adjusted to 1.1 to 22.0.

[0319] The seaweed gametophyte used in the method for producing the composition of the present invention is preferably a female gametophyte. Examples of seaweed include the seaweeds mentioned above, preferably brown algae, and more preferably wakame.

[0320] In the following production method, the female gametophyte of seaweed will be described as an example, but the present invention is not limited thereto. First, an example of a method for obtaining gametophytes of seaweed will be described using Undaria pinnatifida as an example.

[0321] (I) Cultivation of seaweed gametophytes Gametophytes developed from wakame zoospores are separated into one male and one female, grown, mechanically shredded, attached to threads, and fertilized on the threads. The wakame juveniles developed from these are grown in indoor cultures and in natural seawater. Sporophytes (mekabu) are obtained from the grown wakame thallus (adult).

[0322] 1. Preserving sporophytes 1) Place sporophytes in a plastic bag or container and store in a cool, dark place at 15-20°C. At temperatures below 15°C, zoospores are not easily released when returned to seawater. If sporophytes are removed from seawater and stored in a cool, dark place, zoospores can be released for 2-3 days.

[0323] 2. Collecting zoospores 1) The room temperature for releasing zoospores should be 15-20°C. High temperatures are not recommended as they shorten the swimming time of the zoospores.

[0324] 2) Cut the spore leaves into pieces about 3-4 cm square. The part closest to the rhizoid is best for releasing spores, but cut off the part of the spore leaf surface that is as clean as possible. Lightly wipe off any dirt from the cut leaf pieces with absorbent paper or similar.

[0325] 3) Prepare three beakers containing 100 mL of sterilized seawater, wash the leaf pieces in turn, and then place them in a petri dish containing 50 mL of sterilized seawater.

[0326] 4) Place the dish containing the leaf fragment on the stage of a stereomicroscope and shine light from above to release the zoospores. Adjusting the stereomicroscope to a dark field setting makes it easier to observe the release of zoospores. After 10 minutes of illumination with an optical fiber or similar, sufficient zoospores will be released.

[0327] 5) Prepare a capillary tube. A capillary tube can be prepared by heating and stretching a hematocrypt tube, or by stretching the tip of a Pasteur pipette until it is sufficiently elongated.

[0328] 6) Prepare a petri dish filled with 50 mL of PESI culture medium.

[0329] 7) Under a stereomicroscope, aspirate an appropriate amount of zoospores and drop them into a petri dish. When aspirating, be careful not to let the capillary tube touch the bottom of the petri dish or the leaf fragment (this is often the case when aspirating diatoms). After dropping, shake the petri dish thoroughly by hand to ensure a uniform zoospore density.

[0330] 8) Prepare about four different types of petri dishes, each with a different amount of zoospore fluid. If the amount of zoospores is large, the gametophyte density will be high, and the gametophytes will be too close to each other, making them difficult to isolate.

[0331] 9) After collecting the zoospores, culture the petri dishes at 10-30°C with a 12-14 hour light period (1000-1500 lux). Care must be taken to maintain consistent culture conditions, as gametophytes are subject to large temperature fluctuations and males and females will look similar, making them difficult to distinguish, at high temperatures and high light levels.

[0332] 10) After two weeks, the gametophytes reach a size where they can be sexed. Because there is a risk of fertilization, sex the gametophytes as soon as possible and isolate them.

[0333] 11) At this stage, there is little contamination with diatoms, but if diatoms do appear, discard the petri dish. If it is not possible to discard it, germanium dioxide can be used to suppress diatom growth, and gametophytes that are not contaminated by diatoms can be isolated.

[0334] 3. Isolation of male and female gametophytes 1) Place the petri dish in which the gametophytes are being cultured on the stage of an inverted microscope and search for a female gametophyte suitable for isolation. Isolate those in which the gametophytes are sufficiently separated and clearly sexed. Attach a tube to a Pasteur pipette, separate the female gametophytes from the petri dish, and aspirate them. Place each aspirated female gametophyte into a microplate filled with PESI medium. A schematic diagram of female and male gametophytes is shown in Figure 3-1. 2) Culture for one month at 10-30°C with a 12-14 hour light period (1500-2000 lux).

[0335] 4. Preservation of female gametophytes 1) After culturing in a microplate, remove the female gametophyte. Usually, the female gametophyte is large enough to be seen with the naked eye, so it can be picked up and removed from the microplate with ophthalmic tweezers. If it has not grown to a sufficient size, it can be aspirated using a Pasteur pipette under an inverted microscope. 2) For storage, place in a screw-cap test tube and store at 10-30°C with a 14-hour light period (1000-1500 lux). After storage, the medium is changed every two months using PESI medium.

[0336] (II) Cultivation of Seaweed Gametophytes As a culture medium for female gametophytes, Provasoli's Enriched Seawater (PES) or an improved version of it, the PESI culture medium, is preferred. The PES culture medium has the following composition. (See "Phycology Experiments and Practices," edited by Ariga Hirokatsu, Inoue Isao, Tanaka Jiro, Yokohama Yasutsugu, and Yoshida Tadao, Kodansha Scientific (2000) and JP 2009-201480 A.) The PESI culture medium can be prepared with reference to Plant Tissue Culture, 6(2), 55-62 (1989), etc. The detailed composition will be described later in the Examples.

[0337] (PES culture solution) Tris hydroxymethyl aminomethane 5.0g NaNO ３ 3.5g Na ２ -glycerophophate 500mg Fe stock solution 250mL P-2 metal mix 250mL Vitamin 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

[0338] (Fe stock solution) ２ -EDTA 2H ２ O 330mg Fe(NH ４ ) ２ (SO ４ ) ２ ・6H ２ O 351mg Distilled water 500mL

[0339] (P-2 metal mix) Na ２ -EDTA 2H ２ O 500mg H ３ BO ３ 570 mg FeCl ３ ・6H ２O 24.5mg MnSO ４ ・4H ２ O 82.0mg CoSO ４ ・7H ２ O (4.8mg / mL) 0.5mL ZnSO ４ ・7H ２ O 11.0mg Distilled water 500mL

[0340] A seaweed culture solution is prepared by adding a PES or PESI culture solution to seawater. The seawater used may be natural seawater or artificial seawater sterilized with ozone, ultraviolet light, an autoclave, or the like, or filtered seawater obtained by filtering natural seawater. The artificial seawater is prepared to contain primarily 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 seawater may also be used. Deep seawater is seawater found in the deep sea at depths of 200 m or more. Deep seawater has a high salt concentration, making it difficult for bacteria and other organisms that are harmful to seaweed cultivation to survive. Deep seawater is free from artificial contamination, has low bacteria due to its low temperature, and is free from the presence of viable phytoplankton due to its lack of sunlight. Deep seawater is also rich in nitrogen (N) from nitrates, phosphorus (P) from phosphates, and silicon (Si) from silicates, making it ideal for the growth of seaweed.

[0341] The seaweed female gametophyte is placed in the prepared seaweed culture solution and subjected to aeration culture. The aeration culture consists of a preliminary culture and a main culture. The seaweed female gametophyte is cultured in an artificial environment, such as an environment in which the water temperature, light intensity, and sunshine duration are controlled. Any environment can be used as long as the light intensity, sunshine duration, and water temperature can be strictly controlled. For example, the seaweed may be cultured in a container capable of growing seaweed in an incubator where the light intensity, sunshine duration, and internal temperature are regulated, or in an aquarium where the light intensity, sunshine duration, and water temperature can be controlled.

[0342] In the preliminary culture environment, a daylight fluorescent lamp was used as the light source, and the irradiation light intensity was 1 to 100 μmol / m ２ The optimum conditions are: 1.5-2.5°C / s, water temperature 5-30°C, and sunlight duration 8-24 hours. Cultivation is usually carried out under a photoperiod with alternating light and dark periods, but continuous light irradiation is also possible. 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.

[0343] In the main culture environment, a green light source (490-550 nm) is used, and the irradiation dose is 10-200 μmol / m ２ The preferred conditions are: 1 / s, water temperature 5 to 30°C, and sunshine duration 8 to 24 hours. Cultivation is usually carried out under a photoperiod with alternating light and dark periods, but continuous light irradiation is also possible. The culture period may be between 1 and 40 days, but is preferably 5 to 10 days. Seaweed growth promoters such as fatty acids can be added to the seaweed culture solution used in the present invention as appropriate. The culture tank is also equipped with an agitator, a vibrator, a temperature controller, a pH adjuster, a turbidity meter, a light controller, air, O ２ , CO ２ The seaweed may be cultured by any suitable liquid culture method, such as a batch culture method, a semi-batch culture method (fed-batch culture method), or a continuous culture method (perfusion culture method).

[0344] After the primary culture of the seaweed female gametophytes, the seaweed gametophytes are removed from the culture medium by scooping them up with a plankton net or filtering the culture medium. The resulting aggregate of seaweed gametophytes can be used as a raw material for extracting, for example, at least one compound selected from the group consisting of fucoxanthin and its derivatives, and linoleic acid or a salt thereof.

[0345] (III) Dispersion and Extraction of Seaweed Gametophytes in an Aqueous Solvent Seaweed gametophytes are dispersed in an aqueous solvent to obtain a dispersion of the seaweed gametophytes (step (1)). Dispersing seaweed gametophytes in an aqueous solvent also allows for the extraction of fucoxanthin and a portion of its derivatives from the seaweed gametophytes into the aqueous solvent. If necessary, female seaweed gametophytes are dispersed in an aqueous solvent, and at least one compound selected from the group consisting of fucoxanthin and its derivatives is partially extracted from the female seaweed gametophytes into the dispersion. Furthermore, in the case of seaweed gametophytes, rather than directly extracting them with an organic solvent, dispersing the seaweed gametophytes in an aqueous solvent, separating and removing the aqueous solvent, and then subjecting the seaweed gametophytes to extraction with an organic solvent facilitates the extraction of at least one compound selected from the group consisting of fucoxanthin and its derivatives, as well as linoleic acid or a salt thereof. The aqueous solvent is water or a solvent in which water-soluble salts are dissolved, and includes pure water, seawater, etc. Antifoaming agents, thixotropic agents, pH adjusters, etc. may be added to the aqueous solvent as appropriate. Furthermore, artificial seawater, filtered seawater, deep seawater, etc. may 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 fulvic acid, and sodium citrate. Furthermore, the aqueous solvent may be phosphate-buffered saline (PBS).

[0346] The extraction temperature is preferably 0°C to 60°C. To prevent deterioration of fucoxanthin, it is more preferable to perform extraction while cooling with ice (at 0°C). The extraction time is preferably 0.5 to 5 hours. Seaweed gametophytes (preferably female gametophytes) are dispersed in an aqueous solvent and then stirred and soaked. The aqueous solvent is preferably used in a ratio of 0.01 to 1 L per 1 g of seaweed gametophytes (dry weight). The seaweed gametophytes are separated and removed from the resulting seaweed gametophyte dispersion to obtain an aqueous solvent composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives (step (2)). In this specification, the aqueous solvent composition obtained in step (2) is also referred to as an aqueous solvent extract, or an aqueous solvent extract of seaweed gametophytes or sporophytes. The seaweed gametophytes and impurities remaining after extraction are removed from the aqueous solvent by filtration using a filter or column, centrifugation, or other methods. The seaweed gametophytes separated in step (2) can be used for extraction with an organic solvent as described below.

[0347] (IV) It is preferable to disperse seaweed gametophytes in an aqueous solvent, separate the gametophytes from the aqueous solvent (the seaweed gametophytes separated in step (2) above), and then add an organic solvent to obtain an organic solvent composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives (step (3)). Herein, the organic solvent composition obtained in step (3) is sometimes referred to as an organic solvent extract. The seaweed gametophytes extracted with an aqueous solvent are immersed in an organic solvent capable of dissolving at least one compound selected from the group consisting of fucoxanthin and its derivatives, thereby extracting at least one compound selected from the group consisting of fucoxanthin and its derivatives, and linoleic acid or a salt thereof, from the seaweed gametophytes. The organic solvent allows at least one compound selected from the group consisting of fucoxanthin and its derivatives, and linoleic acid or a salt thereof, present in the cells of the seaweed gametophytes, to elute into the organic solvent.

[0348] The organic solvent used can be one that dissolves at least one compound selected from the group consisting of fucoxanthin and its derivatives. Examples of such organic solvents 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, organic chlorine-based hydrocarbons such as chloroform, aliphatic hydrocarbons such as hexane, and aromatic hydrocarbons such as benzene and toluene. These organic solvents can be used alone or in combination. Among these, ethanol and butanediol are preferred. At least one compound selected from the group consisting of fucoxanthin and its derivatives is eluted in the organic solvent. Because fucoxanthin and its derivatives are substantially insoluble in aqueous solvents, the content of fucoxanthin and its derivatives in the organic solvent is higher than in the aqueous solvent extract of (II). The organic solvent may contain an aqueous solvent, but preferably does not. When an aqueous solvent is contained in the organic solvent, it is primarily derived from the water contained in the seaweed gametophytes. In extraction with an organic solvent, the extraction temperature is preferably 0°C to 60°C. To prevent deterioration of fucoxanthin and increase the amount of fucoxanthin extracted, extraction while cooling with ice (cooling at 0°C) is more preferable. Furthermore, the extraction time is preferably 0.5 to 5 hours. The seaweed gametophytes are preferably dispersed in the organic solvent and stirred or infiltrated. It is preferable to use 0.01 to 1 L of organic solvent per 1 g of seaweed gametophytes. After extraction with an organic solvent, the seaweed gametophytes and impurities remaining as extraction residues may be removed from the organic solvent. The method for removing the seaweed gametophytes and other impurities from the organic solvent is not particularly limited, and methods such as filtration using a filter or column, or centrifugation, can be used. In this way, a composition (organic solvent composition) can be obtained that contains an organic solvent, at least one compound selected from the group consisting of fucoxanthin and its derivatives dissolved in the organic solvent, and linoleic acid or a salt thereof.

[0349] (V) A composition is obtained by mixing an aqueous solvent extract of seaweed gametophytes, an organic solvent extract, at least one compound selected from the group consisting of fucoxanthin and its derivatives, and linoleic acid or a salt thereof. If the weight ratio of at least one compound selected from the group consisting of fucoxanthin and its derivatives to linoleic acid or a salt thereof in this composition is within the range of 1.1 to 22.0, no adjustment of the composition is necessary. However, if the weight ratio of at least one compound selected from the group consisting of fucoxanthin and its derivatives to linoleic acid or a salt thereof is outside the range of 1.1 to 22.0, the weight ratio of at least one compound selected from the group consisting of fucoxanthin and its derivatives or linoleic acid or a salt thereof to linoleic acid or a salt thereof is adjusted to 1.1 to 22.0 by adding at least one compound selected from the group consisting of fucoxanthin and its derivatives or linoleic acid or a salt thereof. When at least one compound selected from the group consisting of fucoxanthin and its derivatives or linoleic acid or a salt thereof is added, commercially available industrially synthesized products can be used.

[0350] In the method for producing the composition of the present invention, the composition may be dried to form a powder. Various methods can be used to dry the composition, such as heat drying, freeze drying, and vacuum drying. In this case, the average particle size of the powder contained in the composition is preferably 0.1 to 10 μm.

[0351] The composition of the present invention, which contains at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, and is characterized in that the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt is 1.1 to 22.0, has excellent permeability to cell tissue and can be used in a variety of industrial applications, including, in particular, various health foods expected to have cholesterol-lowering and antithrombosis effects, pharmaceuticals intended to have antitumor effects, neuroprotective effects, and blood sugar level elevation suppression effects, and cosmetics intended to have a melanin production-suppressing effect.

[0352] As described above, seaweed gametophytes contain at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, and can be suitably used as a raw material for preparing a composition characterized in that the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to linoleic acid or a salt thereof is 1.1 to 22.0.

[0353] The present invention also encompasses a raw material used to extract and prepare a composition comprising a collection of seaweed gametophytes, at least one compound selected from the group consisting of fucoxanthin and its derivatives, and linoleic acid or a salt thereof, wherein the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt is 1.1 to 22.0. The seaweed is preferably a brown alga. The seaweed is preferably a female gametophyte.

[0354] The present invention also relates to a composition capable of penetrating cell tissue, which comprises at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, and the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt is 1.1 to 22.0.

[0355] The present invention also relates to a cell tissue penetration enhancer comprising at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, wherein the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt is 1.1 to 22.0.

[0356] By using the permeable composition and permeation enhancer of the present invention, fucoxanthin can be suitably permeated into cell tissues.

[0357] The present specification describes the following:

[0358] The present disclosure (77) is a composition comprising at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, wherein the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 1.1 to 22.0.

[0359] Disclosure (78) is the composition according to Disclosure (77), in which the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or a salt thereof ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 1.182 to 19.5.

[0360] The present disclosure (79) is the composition according to the present disclosure (77), wherein the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or a salt thereof ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 9.0 to 22.0.

[0361] The present disclosure (80) is the composition according to the present disclosure (77), wherein the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or a salt thereof ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 5.0 to 15.0.

[0362] The present disclosure (81) is the composition according to the present disclosure (77), wherein the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or a salt thereof ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 1.182 to 9.0.

[0363] The present disclosure (82) is the composition according to the present disclosure (77), wherein the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or a salt thereof ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 9.0 to 19.5.

[0364] The present disclosure (83) is the composition according to any one of the present disclosures (77) to (82), wherein the salt of linoleic acid is at least one selected from the group consisting of alkali metal salts, alkaline earth metal salts, and ammonium salts.

[0365] The present disclosure (84) is the composition according to any one of the present disclosures (77) to (83), wherein the at least one compound selected from the group consisting of fucoxanthin and its derivatives, and the linoleic acid or a salt thereof are derived from seaweed.

[0366] The present disclosure (85) is the composition according to the present disclosure (84), wherein the seaweed is a gametophyte of seaweed.

[0367] The present disclosure (86) is the composition according to the present disclosure (84) or (85), wherein the seaweed is a female gametophyte of seaweed.

[0368] The present disclosure (87) is the composition according to any one of the present disclosures (84) to (86), wherein the seaweed is brown algae.

[0369] The present disclosure (88) is the composition according to any one of the present disclosures (77) to (87), wherein the composition does not contain an extract of Phaeodactylum tricornutum.

[0370] The present disclosure (89) is the composition according to any one of the present disclosures (77) to (88), further comprising a polysaccharide.

[0371] The present disclosure (90) is the composition according to any one of the present disclosures (77) to (89), wherein the composition contains an organic solvent or a mixed solvent of an aqueous solvent and an organic solvent, and the organic solvent dissolves at least one compound selected from the group consisting of fucoxanthin and its derivatives.

[0372] The present disclosure (91) is the composition according to the present disclosure (90), wherein the aqueous solvent is water or a solvent in which a water-soluble salt is dissolved in water.

[0373] The present disclosure (92) is the composition according to the present disclosure (91), wherein the water-soluble salt is at least one selected from the group consisting of sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, ammonium sulfate, potassium phosphate, potassium dihydrogen phosphate, iron phosphate, iron fulvic acid, and sodium citrate.

[0374] The present disclosure (93) is the composition according to the present disclosure (91), wherein the solvent in which the water-soluble salt is dissolved in water is seawater or phosphate buffered saline.

[0375] The present disclosure (94) is the composition according to any one of the present disclosures (90) to (93), wherein the organic solvent is at least one selected from the group consisting of methanol, ethanol, propanol, isopropanol, n-butanol, 1,3-butanediol, 1,4-butanediol, methyl ethyl ketone, acetone, methyl acetate, ethyl acetate, chloroform, hexane, benzene, and toluene.

[0376] The present disclosure (95) is the composition according to any one of the present disclosures (90) to (94), wherein the weight ratio of the organic solvent to the aqueous solvent is organic solvent / aqueous solvent ≦9 / 1.

[0377] The present disclosure (96) is the composition according to any one of the present disclosures (77) to (95), wherein the total content of at least one compound selected from fucoxanthin and its derivatives in the composition is 0.001 to 50 wt %.

[0378] The present disclosure (97) is the composition according to any one of the present disclosures (77) to (96), wherein the composition is a powdery composition.

[0379] The present disclosure (98) provides a method for producing a composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, the method comprising the following steps (1) to (3): Step (1): Dispersing seaweed gametophytes in an aqueous solvent to obtain a dispersion of the seaweed gametophytes. Step (2): Separating and removing the aqueous solvent from the dispersion of the seaweed gametophytes obtained in step (1). Step (3): Adding an organic solvent to the seaweed gametophytes from which the aqueous solvent has been separated and removed in step (2) to obtain a composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives and the linoleic acid or a salt thereof, wherein the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or a salt thereof ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 1.1 to 22.0.

[0380] The present disclosure (99) is a method for producing a composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, the method comprising the following steps (1) to (4): Step (1): Dispersing seaweed gametophytes in an aqueous solvent to obtain a dispersion of the seaweed gametophytes; Step (2): Separating and removing the aqueous solvent from the dispersion of the seaweed gametophytes obtained in step (1); Step (3): Adding an organic solvent to the seaweed gametophytes from which the aqueous solvent has been separated and removed in step (2), to obtain an organic solvent composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof. Step (4): At least one compound selected from the group consisting of fucoxanthin and its derivatives and / or linoleic acid or a salt thereof is added to the organic solvent composition obtained in step (3), to obtain a composition in which the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is adjusted to 1.1 to 22.0.

[0381] The present disclosure (100) is a method for producing the composition according to the present disclosure (98) or (99), further comprising a drying step of drying the composition to form a powder.

[0382] The present disclosure (101) relates to a raw material used for extracting and preparing a composition that is composed of an aggregate of seaweed gametophytes, and that contains at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, wherein the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 1.1 to 22.0.

[0383] The present disclosure (102) is the raw material according to the present disclosure (101), wherein the seaweed is brown algae.

[0384] The present disclosure (103) is the raw material according to the present disclosure (101) or (102), wherein the seaweed is a female gametophyte.

[0385] The present disclosure (104) is a composition capable of penetrating cell tissue, comprising at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, wherein the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 1.1 to 22.0.

[0386] The present disclosure (105) is a penetration enhancer for cell tissue, comprising at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, wherein the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 1.1 to 22.0.

[0387] Figure 1-1 is a schematic diagram showing male and female gametophytes of Undaria pinnatifida. Figure 1-2 is a liquid chromatography chart of proteins and lipids in extracellular vesicles (EVs) in an aqueous extract of Undaria pinnatifida female gametophyte (solid line: protein, dotted line: lipid). Figure 1-3 is an electron microscope photograph of a dried product (powder composition) obtained by drying an aqueous extract of Undaria pinnatifida female gametophyte. Figure 1-4 is a liquid chromatography chart of proteins and lipids in extracellular vesicles (EVs) in an aqueous extract of filaments of the sporophyte of Porphyra gracilis (solid line: protein, dotted line: lipid). Figure 1-5 is a liquid chromatography chart of proteins and lipids in extracellular vesicles (EVs) in an aqueous extract of male gametophyte of Sagarame seaweed (solid line: protein, dotted line: lipid). Figure 1-6 is a liquid chromatography chart of proteins and lipids in extracellular vesicles (EVs) in an aqueous extract of filaments of Asakusanori sporophytes (solid line: protein, dotted line: lipid). Figure 1-7A is a liquid chromatography chart of fucoxanthin in an aqueous extract of Undaria pinnatifida female gametophytes. Figure 1-7B is a liquid chromatography calibration curve for fucoxanthin. In Figure 1-7A, the peak at approximately 13 minutes corresponds to fucoxanthin. Also, in Figure 1-7A, the peak at approximately 11.3 minutes corresponds to chlorophyll. Figure 1-8 is a liquid chromatography chart of proteins and lipids in extracellular vesicles in an extract of Undaria pinnatifida female gametophytes extracted with water and then further extracted with 100% butylene glycol (solid line: protein, dotted line: lipid). Figure 1-9 is a liquid chromatography chart of fucoxanthin in an extract obtained by extracting wakame female gametophytes with water and then further extracting them with 100% butylene glycol. In Figure 1-9, the peak at around 13 minutes corresponds to fucoxanthin. Also, in Figure 1-9, the peak at around 11.3 minutes corresponds to chlorophyll. Figure 1-10 is a chart of liquid chromatography analysis of proteins and lipids in extracellular vesicles in an ethanol extract of wakame female gametophytes (solid line: protein, dotted line: lipid).Figure 1-11 is a chart showing the analysis of proteins and lipids in extracellular vesicles (EVs) in an extract of water extracted from wakame thallus by liquid chromatography (solid line: protein, dotted line: lipid). Figure 1-12 is a chart showing the analysis of proteins and lipid membranes in extracellular vesicles in a dilution of a water extract of wakame female gametophyte diluted with a mixed solvent of phosphate-buffered saline and butylene glycol by liquid chromatography (solid line: protein, dotted line: lipid). Figure 2-1 is a schematic diagram showing wakame male and female gametophytes. Figure 2-2 is a chart showing the analysis of proteins and lipids in extracellular vesicles (EVs) in an aqueous extract of wakame female gametophyte by liquid chromatography (solid line: protein, dotted line: lipid). Figure 2-3A is a TEM image (50,000x magnification) of extracellular vesicles (EVs) extracted from wakame female gametophyte. Figure 2-3B is a TEM image (100,000x magnification) of extracellular vesicles (EVs) extracted from the female gametophyte of Undaria pinnatifida. Figure 2-3C is a TEM image (250,000x magnification) of extracellular vesicles (EVs) extracted from the female gametophyte of Undaria pinnatifida. Figure 2-3D is a TEM image (300,000x magnification) of extracellular vesicles (EVs) extracted from the female gametophyte of Undaria pinnatifida. Figure 2-4 is a liquid chromatography chart of the proteins and lipids of extracellular vesicles (EVs) in an aqueous extract of Porphyra gracilis (uppuri-nori) (solid line: protein, dotted line: lipid). Figure 2-5 is a liquid chromatography chart of the proteins and lipids of extracellular vesicles (EVs) in an aqueous extract of the male gametophyte of Sagarame (sagarame) (solid line: protein, dotted line: lipid). Figure 2-6 is a liquid chromatography chart of proteins and lipids in extracellular vesicles (EVs) in an aqueous extract of filaments of Asakusanori sporophytes (solid line: protein, dotted line: lipid). Figure 2-7 is a liquid chromatography chart of proteins and lipids in extracellular vesicles in an extract of Undaria pinnatifida female gametophytes extracted with water and then further extracted with 100% butylene glycol (solid line: protein, dotted line: lipid). Figure 2-8A is a liquid chromatography chart of fucoxanthin in a BG extract of Undaria pinnatifida female gametophytes. In Figure 2-8A, the peak at around 13 minutes corresponds to fucoxanthin, and the peak at around 11.3 minutes corresponds to chlorophyll.Figure 2-8B is a liquid chromatography chart of fucoxanthin in a BG extract of Undaria pinnatifida thallus. In Figure 2-8B, the peak at around 13 minutes corresponds to fucoxanthin, and the peak at around 11.3 minutes corresponds to chlorophyll. Figure 2-9 is a diagram showing the separation of proteins in the composition of the present invention based on molecular weight by SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). Figure 2-10 is a graph comparing the increase in cAMP in PSVK1 (human keratinocyte) cells using the aqueous extracts of Example 2-1-1, Example 2-1-2, Comparative Example 2-1-1, Comparative Example 2-1-2, Comparative Example 2-2, and Comparative Example 2-3. The vertical axis shows the amount of luminescence produced by the luciferin reaction; the lower the luminescence, the higher the amount of cAMP in the cells. Figure 2-11 is a graph comparing the increase in cAMP in PSVK1 (human keratinocyte) cells using the aqueous extracts of Examples 2-2, 2-3, and 2-4. The vertical axis shows the amount of luminescence produced by the luciferin reaction; the lower the luminescence, the higher the amount of cAMP in the cells. Figure 2-12 is a chart showing the analysis of proteins and lipids in extracellular vesicles of an extract obtained by ethanol extraction of the female gametophyte of Undaria pinnatifida (Wakame) by liquid chromatography (solid line: protein, dotted line: lipid). Figure 2-13 is a chart showing the analysis of proteins and lipids in extracellular vesicles of an extract obtained by water extraction of Undaria pinnatifida (Wakame) thallus (solid line: protein, dotted line: lipid). Figure 2-14 is a chart showing the analysis of proteins and lipids in extracellular vesicles of an extract obtained by water extraction of the thallus of Cladosiphon glabra (Mozuku seaweed) by liquid chromatography (solid line: protein, dotted line: lipid). Figure 2-15 is a chart showing the analysis of proteins and lipid membranes of extracellular vesicles by liquid chromatography in a diluted solution prepared by diluting a water extract of wakame female gametophyte with a mixed solvent of phosphate-buffered saline and butylene glycol (solid line: protein, dotted line: lipid).Figure 2-16 shows the separation of proteins in an aqueous extract of wakame thallus (adult) by SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) based on molecular weight. Figure 2-17 shows a comparison of the melanin production per viable cell in aqueous extracts of the filamentous sporophytes of Porphyra japonica, Porphyra asakusanori, Wakame female gametophytes, and Wakame thallus (adult). Figure 3-1 is a schematic diagram showing female and male gametophytes. Figure 3-2 is a liquid chromatography chart of fucoxanthin in an extract obtained by water extraction of wakame female gametophytes followed by further extraction with 100% butylene glycol. In Figure 3-2, the peak around 13 minutes corresponds to fucoxanthin. The peak around 11 minutes in Figure 3-2 corresponds to chlorophyll a. Figure 3-3A is a chart showing the results of LC / MS analysis of linoleic acid, with time (min) on the horizontal axis. The peak around 4.2 minutes is the linoleic acid peak. Although Figure 3-3A appears to have four peaks from 4.17 to 4.31 minutes, this represents the measurement of a single linoleic acid, and a calibration curve can be created using the area of ​​the peak around 4.2 minutes. The detector used is a Q Exactive Focus (manufactured by Thermo Fisher Scientific Inc.), which has the ability to alternately measure negatively and positively charged molecular ions (positive-negative alternating scan). Linoleic acid molecular ions have a negative charge, and when negatively charged molecular ions are measured for a specified time, a portion of the linoleic acid peak appears on the chart. After a specified time has elapsed, the instrument begins measuring positively charged molecular ions, causing the rise of the linoleic acid molecular ion peak to stop, resulting in a shoulder on the peak. After measuring positive charges for a specified time, the instrument resumes measuring negatively charged molecular ions, causing the linoleic acid peak to rise again. Thus, because positively and negatively charged molecular ions are alternately measured, a shoulder appears on the negatively charged linoleic acid peak.However, what is being measured is the negatively charged molecular ion of linoleic acid. Figure 3-3B is a chart showing the results of an LC / MS analysis of linoleic acid, with the mass-to-charge ratio (m / z) on the horizontal axis. The peak at m / z = 279.23 in Figure 3-3B is the peak of the molecular ion of linoleic acid. Figure 3-4 shows the results of a tape stripping test. The vertical axis represents permeability, and the horizontal axis represents the weight ratio of fucoxanthin to linoleic acid.

[0388] The first, second and third inventions described above are disclosed in Japanese Patent Application Nos. 2024-158354, 2024-193728, 2024-108281, 2024-193729, 2025-21698, Japanese Patent No. 7057568, JP 2021-127369, JP 6731425, JP 4979592, JP 2004-49072, JP 2004-97021, JP 2019-506133, JP 2018-512432, WO2024 / 053316, Biochemical and Biophysical Research Communications Volume 696, February 12, 2024, 149505 "Simple methods for measuring milk exosomes using fluorescent compound GIF-2250 / 2276", Hirokatsu Ariga, Isao Inoue, Jiro Tanaka, Yasutsugu Yokohama, Tadao Yoshida (eds.), "Phycology Experiments and Practice", Kodansha Scientific (2000), JP 2009-201480 A, Plant Tissue Culture, 6 (2), 55-62 (1989), JP 2023-520101 A, JP 4-124122 A, JP 2018-131414 A, JP 2023-120242 A, Japanese Patent No. 7298905, Production Research Vol. 64 No. 3 (2012) pp. 351-357, JP 9-252778 A, Japanese Patent No. 3705768 A, Japanese Patent No. 7104689 A, and the entire disclosures of JP 2011-188744 are incorporated (incorporated by reference).

[0389] (Examples of the First Invention) The first invention will be described based on examples, but the first invention is not limited to only the following examples.

[0390] (Preparation of PESI culture solution) Tris, NaNO3, and PEG-1000 were added to approximately 300 mL of distilled water according to the following composition. ３ The following ingredients were added in the specified amounts in that order and thoroughly dissolved: β-glycerophosphate disodium (Na2-glycerophosphate), Fe stock solution, P-II metal mix, and KI. The pH of the solution was then adjusted to 7.8, and the solution was diluted to 1000 mL with distilled water. The resulting PESI culture solution was dispensed into containers and sterilized in an autoclave (121°C, 20 minutes), after which the sterilized PESI culture solution was stored refrigerated at 4°C.

[0391] The composition of the PESI culture medium was: 2-amino-2-hydroxymethyl-1,3-propanediol (Tris(hydroxymethyl)aminomethane; Tris), 5.0 g; NaNO3, 3.5 g; Na ２ -glycerophosphate, 500 mg; Fe stock solution (see composition below), 250 mL; P-II metal mix (see composition below), 250 mL; KI stock solution (0.1 mg / mL), 10 mL. The above was made up to 1000 mL (pH 7.8) with distilled water.

[0392] The composition of Fe stock solution is Na ２ -EDTA 2H ２ O, 330mg; Fe(NH ４ ) ２ (SO ４ ) ２ ・6H ２ The above was dissolved in distilled water to make a 500 mL solution (Fe:EDTA molar ratio = 1:1) and stored in a refrigerator at 4°C.

[0393] The composition of P-II metal mix is ​​Na ２ -EDTA 2H ２ O, 500mg; H ３ BO ３ , 570mg; FeCl ３ ・6H ２ O, 24.5 mg; MnSO ４・4H ２ O, 82.0 mg; CoSO ４ ・7H ２ O stock solution (4.8mg / mL), 0.5mL; ZnSO ４ ・7H ２ The above was added to distilled water in order, and finally distilled water was added to make a 500 mL solution. The solution was stored refrigerated at 4°C. KI stock solution was prepared by adding 20 mg of KI to 200 mL of distilled water, and this was stored refrigerated at 4°C.

[0394] (A-1. Preparation of a sample of wakame female gametophyte) (1) Natural seawater was filtered through a cartridge filter with 1 μm mesh size, and the filtered seawater was heated and pressurized in an autoclave at 121°C, 2 atmospheres, and for 50 minutes to obtain 10 L of sterilized seawater. Next, 20 mL of the previously prepared PESI culture solution was added per 1 L of sterilized seawater to obtain culture seawater.

[0395] (2) Naruto wakame female gametophytes were cultured and grown from Naruto wakame collected near the mouth of the Yoshino River according to the procedure described above under "(I) Cultivation of Seaweed Gametophytes." 300 mg of wakame female gametophytes and culture seawater were placed in a 5 L glass Erlenmeyer flask, and preliminary aerated culture (hereinafter referred to as "preliminary culture") was performed until the wakame female gametophyte reached 10 g wet weight (g wet weight). The aerated culture was performed as follows: 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, the tip of the Pasteur pipette was immersed in the culture seawater, and then air was pumped from an air pump through a silicone tube and the Pasteur pipette into the culture seawater to perform aerated culture. This aerated culture was performed in the same manner for the preliminary culture and the main culture. The culture conditions for the preliminary culture were a temperature of 20°C, a daylight fluorescent light source, and a light intensity of 50 μmol m －２ s －１ The photoperiod was set to 12L (light) and 12D (dark).

[0396] (3) 10 gw.w of pre-cultured wakame female gametophytes and culture seawater were placed in a 20 L cylindrical polycarbonate container (manufactured by Nikko Hansen Co., Ltd.) and cultured under aeration (main culture) until the total volume reached 50 gw.w. The main culture conditions were a temperature of 20°C, a green LED (wavelength 518 nm) (manufactured by Nippon Medical Instruments Manufacturing Co., Ltd., 3LH-64) as the light source, and a light intensity of 50 to 100 μmol m. －２ s －１ The photoperiod was set to 12L (light), 12D (dark).

[0397] (4) After the main culture was completed, the female gametophytes were collected by filtering through a 50 μm plankton net (Sefar Inc, DIN110). The collected female gametophytes were lightly sandwiched between commercially available paper towels along with the plankton net to absorb moisture and dehydrate, and then transferred to a plastic bag with a zipper. The gametophytes were crushed thinly to facilitate freeze-drying, and then frozen and stored in a freezer set at -60°C. The fully frozen gametophytes were freeze-dried overnight in a freeze dryer (Tokyo Rikakikai Co., Ltd., FD-1) to prepare a female gametophyte sample.

[0398] (A-2. Sample preparation of filamentous sporophytes of Porphyra gracilis) (1) Mature Porphyra gracilis thallus was collected in December 2023 from a rocky area near the low tide line in Aizaki City, Niigata Prefecture. Zygospores, which are sporophytes released from the mature algae, were washed with sterilized seawater using a Hibette to obtain a zygospore mixture. This mixture was diluted to a concentration of approximately 1,000 spores / ml. (2) This diluted solution was cultured at a temperature of 20°C and a light intensity of 20 μmol / m ２ The spores were cultured for 15 days under the conditions of 12L:12D photoperiod, aeration, and PES medium. The zygospores germinated and grew into filaments. The filaments were entangled and spherical. The average spherical size was 5 mm. The filaments were frozen and stored in a freezer set at -60°C. The fully frozen filaments were freeze-dried overnight in a freeze dryer (Tokyo Rikakikai Co., Ltd., FD-1) to prepare a sporophyte filament sample.

[0399] (A-3. Preparation of Sagarame Gametophyte Samples) (1) In October 2023, growing Sagarame was collected from Mochimune Beach, Shizuoka City, Shizuoka Prefecture, and leaves bearing ascophytes were cut off. These leaf pieces were stored in a refrigerator for approximately one day, after which zoospores were released in sterilized seawater and allowed to attach to glass slides. (2) These zoospore-infested slides were transferred to a glass tank containing 3 L of culture solution and cultured for one month. The water temperature was 20°C, and illumination was provided using white fluorescent lamps with an illuminance of 20,000 ux and a light / dark cycle of 10 L:14 D. PESI medium was used as the culture solution, and half of the water was replaced every two days. To measure gametophyte growth, 20 individuals with the most advanced growth were selected every two days, and only male gametophytes were extracted from these and continued to be cultured under the same conditions to obtain Sagarame male gametophytes. These were then stored in a refrigerator at 4°C. This male gametophyte was frozen and stored in a freezer set at −60° C. The fully frozen gametophyte was freeze-dried overnight in a freeze dryer (FD-1, manufactured by Tokyo Rikakikai Co., Ltd.) to prepare a male gametophyte sample.

[0400] (A-4. Preparation of a sample of the filamentous sporophyte of Asakusa-nori) (1) Asakusa-nori cultivated near the mouth of the Ibi River was collected, and the tip of the filamentous sporophyte was cut into 3 cm pieces. ２ The leaf pieces were cut off to a length of about 100 mm. The leaf pieces were spread on a glass plate and the surface was washed with sterilized seawater. (2) A sterilized petri dish was prepared, a slide glass was placed on the bottom, PES culture medium was poured into it, and the leaf pieces from (1) were floated on it. The petri dish was left to stand for 14 days under a 2000 Lux fluorescent light. Carpospores fell to the bottom of the dish and accumulated, forming colonies. (3) The carpospores were sucked up with a dropper and placed in PES culture medium. They were cultured at a temperature of 16°C under a 2000 Lux fluorescent light with a light / dark cycle of 12L:12D, and filaments formed from germinated carpospores were obtained. The filaments were entangled with each other to form a spherical shape, and the average length of the spheres was 3 mm. The filaments were frozen and stored in a freezer set at -60°C. The thoroughly frozen gametophytes were freeze-dried overnight in a freeze dryer (Tokyo Rikakikai Co., Ltd., FD-1) to prepare samples of sporophyte filaments.

[0401] (B-1. Water extraction treatment of wakame female gametophyte, sagarame male gametophyte, and filamentous sporophyte, and analysis of the water extract) Each sample was subjected to water extraction treatment by the following method.

[0402] (B-1-1. Water extraction treatment of wakame female gametophytes) (1) 10 mg of the female gametophyte sample prepared in (A-1) above was suspended in 1 mL of distilled water and stirred at 200 rpm for 1 hour at 25°C using a rotator (RT-5N, manufactured by TAITEC). (2) The solution alone was removed from the 2 mL tube containing the stirred female gametophyte sample solution in (1) using a micropipette, and separated into a solid precipitate and a supernatant. (3) The supernatant from (2) was filtered through a 0.45 μm PES filter (manufactured by GVS Japan Co., Ltd.), and the filtrate was further filtered through a 0.1 μm PES filter (manufactured by Membrane Solutions LLC). (4) 0.5 mL of this filtrate was concentrated (6000 rpm for 10 minutes) using a 300K spin column (manufactured by Nippon Pall Co., Ltd.), and washed and filtered twice using 200 to 300 μL of PBS (phosphate buffered saline), and 100 μL of the filtrate was recovered.

[0403] (B-1-2. Water Extract of Filaments of Sporophytes of Porphyra japonica) The freeze-dried filaments of the sporophytes of Porphyra japonica obtained in (A-2) above were pulverized into powder. Using this powder, a water extract of filaments of the sporophytes of Porphyra japonica was obtained in the same manner as in (B-1-1) above.

[0404] (B-1-3. Water extract of Sagarame gametophyte) The freeze-dried male gametophyte of Sagarame obtained in (A-3) above was pulverized into powder. Using this powder, a water extract of Sagarame male gametophyte was obtained by the same method as in (B-1-1) above.

[0405] (B-1-4. Water Extract of Asakusanori Sporophyte Filaments) The freeze-dried Asakusanori sporophyte filaments obtained in (A-4) above were pulverized into powder. Using this powder, a water extract of Asakusanori sporophyte filaments was obtained in the same manner as in (B-1-1) above.

[0406] (B-2. Analysis of Each Water Extract) Each water extract was analyzed by the following method.

[0407] (B-2-1. Analysis of extracellular vesicles in aqueous extracts of wakame female gametophytes) (1) 1 μL of a 1 mM solution of GIF-2276 (manufactured by GIFU EXOSOME Co., Ltd.: a labeling reagent for detecting protein in EVs. It is a fluorescent substance with Ex / Em of 475 / 530 nm) and 2 μL of ExoSparkler Exosome Membrane Labeling Kit-Red (manufactured by Dojindo Laboratories, Ltd.: a labeling reagent for detecting lipid membranes. It is a fluorescent substance with Ex / Em of 560 / 600 nm) were mixed with the concentrate prepared in (4) of (B-1-1) above, and the mixture was labeled by heating in 80°C hot water for 5 minutes.

[0408] (2) The following liquid chromatography apparatus A was prepared for the detection of extracellular vesicles. Column packing: qEV10 70 nm manufactured by Izon Science; Column shape: Stainless steel column manufactured by Senshu Scientific Co., Ltd., diameter 8 mm x length 100 m; Detector: RF-10AxL manufactured by Shimadzu Corporation; Developing solvent: 10 mM tris(hydroxymethyl)aminomethane pH = 7.6, aqueous solution containing 30 mM NaCl and 0.5 mM EDTA; Development time gradient: uniform; Solution flow rate: 1 mL / min; Injection volume: 10 μL; Column temperature: 30°C; Sample chamber temperature: 10°C; Delivery pump: LC10ADVP manufactured by Shimadzu Corporation.

[0409] (3) The concentrate labeled in (1) was analyzed using the liquid chromatography system A in (2). The chart of the results is shown in Figure 1-2 (solid line: protein, dotted line: lipid). The horizontal axis of the chart represents time (minutes), and the vertical axis represents the absorbance of fluorescence (protein: 530 nm, lipid membrane: 600 nm) and is unitless.

[0410] As described in WO2024 / 053316 and Biochemical and Biophysical Research Communications Volume 696, February 12, 2024, 149505, "Simple methods for measuring milk exosomes using fluorescent compound GIF-2250 / 2276," GIF-2276 binds to Lys residues of proteins that constitute extracellular vesicles (EVs) to modify the extracellular vesicles (EVs). Furthermore, the aforementioned literature indicates that 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 protein peak appear at the same retention time on a liquid chromatography chart, it is believed that extracellular vesicles (EVs) are present in the sample. Since the lipid membrane peak and protein peak appear at the same retention time in Figure 1-2, it can be concluded that extracellular vesicles (EVs) are present in the water extract of wakame female gametophytes.

[0411] As a precaution, a water extract of wakame female gametophytes was frozen at -50°C using liquid nitrogen, and then freeze-dried at a reduced pressure of 4 Pa ​​to prepare a powder composition. A transmission electron micrograph of the powder composition obtained by drying is shown in Figure 1-3 (scale: 100 nm). The electron microscope used was a JEOL JEM-2100, and the photographing conditions were an accelerating voltage of 200 kV, negative staining with uranyl acetate, and a magnification of 40,000 times. The spherical object in the center of the photograph in Figure 1-3 and the white granular objects seen around it are extracellular vesicles (EVs).

[0412] (B-2-2. Analysis of extracellular vesicles in the aqueous extract of filaments of the sporophyte of Porphyra uppuri) The aqueous extract of filaments of the sporophyte of Porphyra uppuri obtained in (B-1-2) above was analyzed using liquid chromatography apparatus A in the same manner as in (B-2-1) above. The chart of the results is shown in Figure 1-4.

[0413] (B-2-3. Analysis of extracellular vesicles in the aqueous extract of male gametophytes of Sagarame) The aqueous extract of male gametophytes of Sagarame obtained in (B-1-3) above was analyzed using liquid chromatography apparatus A in the same manner as in (B-2-1) above. The chart of the results is shown in Figure 1-5. Compared to female gametophytes, the amount of extracellular vesicles (EVs) appears to be relatively small.

[0414] (B-2-4. Analysis of extracellular vesicles in the aqueous extract of filaments of Asakusanori sporophytes) The aqueous extract of filaments of Asakusanori sporophytes obtained in (B-1-4) above was analyzed using liquid chromatography apparatus A in the same manner as in (B-2-1) above. The chart of the results is shown in Figure 1-6.

[0415] (B-3. Analysis of fucoxanthin in aqueous extract) (1) The following liquid chromatography apparatus B was prepared for detecting fucoxanthin. Column: diameter 3.0 mm x length 150 mm, packing material was 25437-96 RP-18GP 150-3 manufactured by Kanto Chemical Co., Inc., a packing material for high-performance liquid chromatography with a particle size of 5.0 μm. Detector: absorbance was measured using a spectrophotometer (UV-1800 manufactured by Shimadzu Corporation). Wavelength: 450 nm Developing solvent A: 10% acetonitrile prepared by mixing LC / MS grade acetonitrile (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 LC / MS grade acetonitrile (Kanto Chemical Co., Ltd., 01033-76) and 0.05 wt % formic acid aqueous solution in an 8:2 ratio Developing solvent C: 100% acetonitrile Development time gradient: 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: 5 μL Column temperature: 40°C Sample chamber temperature: 15°C Delivery pump: Shimadzu Corporation, LC10ADVP

[0416] 0.6589 mg of fucoxanthin standard (Fujifilm Wako Pure Chemical Industries, Ltd.) was weighed out, 1 L of ethanol was added, and the solution was thoroughly stirred to prepare a 1 mM fucoxanthin solution. 1 mL of this solution was further diluted 10-fold with ethanol to prepare a 0.1 mM (100 μM) standard solution. This standard solution was used to confirm the retention time of fucoxanthin in liquid chromatography apparatus B (1). This 100 μM fucoxanthin standard solution was also diluted 2-fold (50 μM), 3.33-fold (30 μM), 10-fold (10 μM), and 33.3-fold (3 μM) with ethanol. Next, a calibration curve for fucoxanthin concentration in liquid chromatography apparatus B (1) was prepared using the standard solution and the diluted solution. The calibration curve is shown in Figure 1-7B. The calibration curve is expressed as y = 110489x + 22725 (x is the fucoxanthin concentration, and y is the peak area). Fucoxanthin at a concentration of 1 M was analyzed using the liquid chromatography apparatus B in (1), and it was confirmed that the peak area and fucoxanthin concentration appearing on the chart conformed to the calibration curve.

[0417] (2) The concentrate prepared in (B-1-1.)(4) above was analyzed using liquid chromatography apparatus B described in (1). The chart of the results is shown in Figure 1-7A. The horizontal axis of the chart represents time (minutes), and the vertical axis represents absorbance at 450 nm (unitless). The peak around 13 minutes in Figure 1-7A is the fucoxanthin peak, and the concentrate prepared in (B-1-1.)(4) contained fucoxanthin. The calibration curve indicated a concentration of 3.44 mg / mL (= 0.00523 mol / L). Since fucoxanthin is a hydrophobic substance, it does not dissolve in water and is therefore not detected in aqueous extracts. However, in this Example, fucoxanthin was detected in the water extract, and therefore it is believed that fucoxanthin is attached to or encapsulated in extracellular vesicles (EVs) and released together with the EVs into the water solvent.

[0418] (C. 100% butylene glycol (BG) extraction of wakame female gametophytes after aqueous extraction and analysis of BG extract) (C-1. BG extraction of aqueous extract of wakame female gametophytes) After aqueous extraction of wakame female gametophytes using the same procedures as in (1) and (2) above in (B-1-1), the wakame female gametophyte residue was thoroughly drained, and 10 mg of the solid matter was suspended in 1 mL of 100% butylene glycol (1,3-butylene glycol, hereinafter referred to as "BG") and stirred at 8000 rpm for 1 hour using a rotator (RT-5N, manufactured by TAITEC) to prepare a BG extract.

[0419] (C-2. Analysis of Extracellular Vesicles in BG Extract) The BG extract prepared in (C-1) above was analyzed using the liquid chromatography apparatus A described in (B-2-1) above. The chart of the results is shown in Figure 1-8 (solid line: protein, dotted line: lipid). The horizontal axis of the chart is time (minutes), and the vertical axis is the absorbance of fluorescence (protein: 530 nm, lipid membrane: 600 nm), which is unitless. The protein and lipid peaks observed in Figure 1-2 around 10 to 12 minutes are not observed in Figure 1-8. Therefore, extracellular vesicles (EVs) are not observed in the BG extract prepared in (C-1) above.

[0420] (C-3. ​​Analysis of fucoxanthin in BG extract) The BG extract prepared in (C-1) above was analyzed using the liquid chromatography apparatus B described in (B-3) above. The liquid chromatography chart is shown in Figure 1-9. The horizontal axis of the chart represents time (minutes), and the vertical axis represents absorbance at 450 nm (unitless). The fucoxanthin concentration in the BG extract was 146.64 mg / mL (=0.223 mol / L).

[0421] (D. Fucoxanthin Stability Evaluation Test) (D-1-1. Preparation of Samples for Evaluation Test)

[0422] (1) The aqueous extract of female gametophytes prepared in (B-1-1) above was placed in a 2 mL tube and designated Sample 1-1. The BG extract of female gametophytes prepared in (C-1) above was placed in a 2 mL tube and designated Sample 1-2. (2) Sample 1-2 and Sample 1-1 were mixed at a volume ratio of 1:3 (Sample 1-2:Sample 1-1 = 1:3), and the mixture was placed in a 2 mL tube and designated Sample 1-3. As can be seen from Figures 1-8 and 1-9, extracellular vesicles (EVs) were not present in the BG extract of female gametophytes prepared in (C-1) above (Sample 1-2), and only fucoxanthin was detected at a concentration of 146.64 mg / mL. On the other hand, Sample 1-1 was found to contain fucoxanthin at a concentration of 3.44 mg / mL, and Sample 1-3, a mixture of Samples 1-1 and 1-2, can be said to be a mixed composition of fucoxanthin and extracellular vesicles (EVs). Sample 1-3 is also a mixed solvent composition containing an aqueous solvent and an organic solvent. (3) Sample 1-2 and water were mixed at a volume ratio of 1:3 (Sample 1-2:water = 1:3) to obtain Sample 1-4.

[0423] (D-2-1. Evaluation Test of Fucoxanthin Stability) (1) For Samples 1-3 and 1-4, the initial concentrations of fucoxanthin were measured using the liquid chromatography apparatus B described in (B-3) above and the calibration curve prepared in (B-3). The respective concentrations were 39.24 mg / mL for Sample 1-3 and 36.66 mg / mL for Sample 1-4. (2) Next, Samples 1-3 and 1-4 were placed in an incubator and subjected to a stress test under the following conditions: temperature: 60°C, humidity: 100%, time: 3 days. (3) For Samples 1-3 and 1-4 after the stress test, the concentrations of fucoxanthin in the samples were measured using the liquid chromatography apparatus B and the calibration curve prepared in (B-3). The residual fucoxanthin was calculated, assuming the initial concentration to be 100%. The residual rates (%) of fucoxanthin in Samples 1-3 and 1-4 are shown in Table 1-1.

[0424]

[0425] As can be seen from Table 1-1, Sample 1-3, a mixed composition of fucoxanthin and extracellular vesicles (EVs), had an extremely high fucoxanthin residual rate of 4.249%, which was 9.6 times higher than the 0.444% residual rate of Sample 1-4, which contained only fucoxanthin. Thus, the mixed composition of fucoxanthin and extracellular vesicles (EVs) can be said to be a composition with excellent stability. For the purpose of taking the electron micrograph in Figure 1-3, a powder composition of fucoxanthin and extracellular vesicles (EVs) was prepared by freeze-drying an aqueous extract of wakame (Undaria pinnatifida) female gametophyte corresponding to Sample 1-1. When this powder composition was dispersed in a 25% BG aqueous solution and subjected to the above-mentioned stress test, the residual fucoxanthin rate was 4% on the third day, an extremely high fucoxanthin residual rate comparable to that of Sample 1-3. Furthermore, a mixed solvent composition of an aqueous solvent and an organic solvent containing fucoxanthin and extracellular vesicles (EVs), corresponding to Sample 1-3, can be frozen to -50°C using liquid nitrogen and then lyophilized by reducing the pressure to 4 Pa ​​to prepare a powder composition consisting of fucoxanthin and extracellular vesicles (EVs). In this way, the composition of the present invention can be freely prepared in either a solution or powder form, and the viscosity can also be freely adjusted. Furthermore, because the extracellular vesicles (EVs) contain only components contained in the cells from which they were derived, there is no need to worry about contamination with components that may harm the living body, and they are easy to handle.

[0426] (D-2-2. Confirmation of the fucoxanthin-stabilizing effect of the aqueous extract of the filaments of the sporophyte of Porphyra uppuri) A sample was prepared from the aqueous extract of the filaments of the sporophyte of Porphyra uppuri obtained in (B-1-2) above using the same procedure as in (D-1-1) above, and a fucoxanthin stability evaluation test was carried out using the same procedure as in (D-2-1). The residual rate of fucoxanthin on the third day was 2.959%.

[0427] (D-2-3. Confirmation of the fucoxanthin-stabilizing effect of the aqueous extract of Sagara seaweed male gametophyte) A sample was prepared using the aqueous extract of Sagara seaweed male gametophyte obtained in (B-1-3) above in the same manner as in (D-1-1) above, and a fucoxanthin stability evaluation test was carried out in the same manner as in (D-2-1). The residual rate of fucoxanthin on the third day was 3.241%. The extract of female gametophyte had superior fucoxanthin stability.

[0428] (D-2-4. Confirmation of the stabilizing effect of fucoxanthin in the aqueous extract of filaments of Asakusanori sporophytes) A ​​sample was prepared using the aqueous extract of filaments of Asakusanori sporophytes obtained in (B-1-4) above in the same manner as in (D-1-1) above, and a fucoxanthin stability evaluation test was carried out in the same manner as in (D-2-1). The residual rate of fucoxanthin on the third day was 2.945%.

[0429] (Test Example 1-1) 10 mg of a sample of female gametophytes obtained in the same manner as in (A-1) above was suspended in 1 mL of 70 wt % ethanol (the remaining 30% was water) and stirred with a rotator (TAITEC, RT-5N) at 200 rpm for 1 hour at 25° C. Next, the solution alone was removed from the 2 mL tube containing the stirred female gametophyte sample solution with a micropipette, and separated into a solid precipitate and a supernatant.

[0430] The supernatant was filtered through a PES filter and labeled in the same manner as described above (B-2-1), and the extract was analyzed. The liquid chromatography chart is shown in Figure 1-10 (solid line: protein, dotted line: lipid). As can be seen from Figure 1-10, the protein and lipid peaks observed in Figure 1-2 around 10 to 12 minutes are not observed in Figure 1-10. Therefore, it can be seen that extracellular vesicles (EVs) were not observed in the 70% ethanol extract of female gametophytes.

[0431] (Test Example 1-2) (1) 10 g of dried wakame (Marukome Co., Ltd.) consisting of thallus (adult) was crushed in a mixer mill to obtain a powder, and 10 mg of the powder was suspended in 1 mL of distilled water and stirred at 200 rpm for 1 hour at 25°C using a rotator (TAITEC, RT-5N). (2) The solution was removed from the 2 mL tube containing the stirred wakame sample solution from (1) using a micropipette, and the solution was separated into a solid precipitate and a supernatant. (3) The supernatant from (2) was filtered through a 0.45 μm PES filter (GVS Japan Co., Ltd.), and the filtrate was further filtered through a 0.1 μm PES filter (Membrane Solutions LLC). (4) 0.5 mL of this filtrate was concentrated (6000 rpm, 10 min) using a 300K spin column (manufactured by Nippon Pall Co., Ltd.), washed twice with 200-300 μL of PBS (phosphate-buffered saline), and 100 μL of filtrate (PBS solution) was recovered. This filtrate was labeled and the extract was analyzed using the same method as described above (B-2-1). The liquid chromatography chart is shown in Figure 1-11 (solid line: protein, dotted line: lipid).

[0432] As can be seen from Figure 1-11, the protein and lipid peaks observed around 10 to 12 minutes in Figure 1-2 are not observed in Figure 1-11. Therefore, it can be seen that extracellular vesicles (EVs) are not observed in the water extract of the thallus. Generally, when wakame is used commercially or industrially, the thallus is used, but extracellular vesicles (EVs) cannot be obtained from the thallus.

[0433] (Test Example 1-3) A 10-fold dilution of the water extract of wakame female gametophytes obtained by the process described above in (B-1-1) was prepared by adding a mixed solvent of phosphate-buffered saline (PBS) and BG (weight ratio: BG / PBS = 9 / 1) to 20 μL of the water extract of wakame female gametophytes. The presence or absence of extracellular vesicles (EVs) in this 10-fold diluted water extract of wakame female gametophytes was confirmed using liquid chromatography system A. The results are shown in Figure 1-12 (solid line: protein, dotted line: lipid). As seen in Figure 1-2, Figure 1-12 confirms peaks of protein and lipid membranes around 10 to 12 minutes. This indicates that extracellular vesicles (EVs) once extracted with water can be present even when transferred to a solvent with a high organic solvent concentration, such as BG / PBS = 9 / 1.

[0434] (Test Example 1-4) 100 μL of caprylic triglyceride was added to 100 μL of the water extract of wakame female gametophyte obtained by the process in (B-1-1) above, and the mixture was shaken for 5 minutes and then allowed to stand to separate the water extract of wakame female gametophyte and caprylic triglyceride into two phases. The caprylic triglyceride phase was then drawn up with a pipette, and the presence or absence of extracellular vesicles (EVs) in this caprylic triglyceride was confirmed using liquid chromatography apparatus A. The chart of the results is the same as that in Figure 1-11, and no peak corresponding to extracellular vesicles (EVs) was confirmed.

[0435] (Examples of the Second Invention) The second invention will be described based on examples, but the second invention is not limited to the following examples. Note that the term "thallus" used in the following description refers to the thallus (adult) of the sporophyte.

[0436] (Preparation of Solution) (Preparation of PESI Culture Solution) Add Tris, NaNO3, and PEG-1000 to approximately 300 mL of distilled water according to the following composition. ３ , β-glycerophosphate disodium (Na ２Prescribed amounts of PEG-1000-glycerophosphate, Fe stock solution, P-II metal mix, and KI were added in this order and thoroughly dissolved. The pH of the solution was then adjusted to 7.8, and the solution was diluted to 1000 mL with distilled water. The resulting PESI culture solution was dispensed into containers and sterilized in an autoclave (121°C, 20 minutes), after which the sterilized PESI culture solution was stored refrigerated at 4°C.

[0437] The PESI culture solution contained 2-Amino-2-hydroxymethyl-1,3-propanediol (Tris (hydroxymethyl) aminomethane; Tris), 5.0 g; NaNO ３ , 3.5g; Na ２ -glycerophosphate, 500 mg; Fe stock solution (see composition below), 250 mL; P-II metal mix (see composition below), 250 mL; KI stock solution (0.1 mg / mL), 10 mL; were added to distilled water and dissolved, and finally distilled water was added to make a 1000 mL solution (pH 7.8).

[0438] Fe stock solution is Na ２ -EDTA 2H ２ O, 330mg; Fe(NH ４ ) ２ (SO ４ ) ２ ・6H ２ 351 mg of O was dissolved in distilled water, and finally the solution was made up to 500 mL with distilled water (Fe:EDTA molar ratio = 1:1). The solution was stored in a refrigerator at 4°C.

[0439] P-II metal mix is ​​Na ２ -EDTA 2H ２ O, 500 mg; H ３ BO ３ ,570mg; FeCl ３ ・6H ２ O, 24.5 mg; MnSO ４ ・4H ２ O, 82.0 mg; CoSO ４ ・7H ２O stock solution (4.8 mg / mL), 0.5 mL; ZnSO ４ ・7H ２ 11.0 mg of KI was added to distilled water in this order, and finally the solution was made up to 500 mL with distilled water. The solution was stored in a refrigerator at 4°C. KI stock solution was prepared by adding 200 mL of distilled water to 20 mg of KI, and this was stored in a refrigerator at 4°C.

[0440] (A-1. Preparation of a sample of wakame female gametophyte) (1) Natural seawater was filtered through a cartridge filter with 1 μm mesh size, and the filtered seawater was heated and pressurized in an autoclave at 121°C, 2 atmospheres, and for 50 minutes to obtain 10 L of sterilized seawater. Next, 20 mL of the previously prepared PESI culture solution was added per 1 L of sterilized seawater to obtain culture seawater.

[0441] (2) Naruto wakame female gametophytes were cultured and grown from Naruto wakame collected near the mouth of the Yoshino River according to the procedure described above under "(I) Cultivation of Seaweed Gametophytes." 300 mg of wakame female gametophytes and culture seawater were placed in a 5 L glass Erlenmeyer flask, and preliminary aerated culture (hereinafter referred to as "preliminary culture") was performed until the wakame female gametophyte reached 10 g wet weight (g wet weight). The aerated culture was performed as follows: 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, the tip of the Pasteur pipette was immersed in the culture seawater, and then air was pumped from an air pump through a silicone tube and the Pasteur pipette into the culture seawater to perform aerated culture. This aerated culture was performed in the same manner for the preliminary culture and the main culture. The culture conditions for the preliminary culture were a temperature of 20°C, a daylight fluorescent light source, and a light intensity of 50 μmol m －２ s －１ The photoperiod was set to 12L (light) and 12D (dark).

[0442] (3) 10 gw.w of pre-cultured wakame female gametophytes and culture seawater were placed in a 20 L cylindrical polycarbonate container (manufactured by Nikko Hansen Co., Ltd.) and cultured under aeration (main culture) until the total volume reached 50 gw.w. The main culture conditions were a temperature of 20°C, a green LED (wavelength 518 nm) (manufactured by Nippon Medical Instruments Manufacturing Co., Ltd., 3LH-64) as the light source, and a light intensity of 50 to 100 μmol m. －２ s －１ The photoperiod was set to 12L (light), 12D (dark).

[0443] (4) After the main culture was completed, the female gametophytes were collected by filtering through a 50 μm plankton net (Sefar Inc, DIN110). The collected female gametophytes were lightly sandwiched between commercially available paper towels along with the plankton net to absorb moisture and dehydrate, and then transferred to a plastic bag with a zipper. The gametophytes were crushed thinly to facilitate freeze-drying, and then frozen and stored in a freezer set at -60°C. The fully frozen gametophytes were freeze-dried overnight in a freeze dryer (Tokyo Rikakikai Co., Ltd., FD-1) to prepare a female gametophyte sample.

[0444] (A-2. Sample preparation of filaments of sporophytes of Porphyra gracilis) (1) Mature Porphyra gracilis thallus was collected from a rocky area near the low tide line in Aizaki City, Niigata Prefecture. Zygospores, which are sporophytes released from the mature algae, were washed with sterilized seawater using a Hibette to obtain a zygospore mixture. This mixture was diluted to a concentration of approximately 1,000 spores / ml. (2) The diluted solution was cultured at a temperature of 20°C and a light intensity of 20 μmol / m ２ The spores were cultured for 15 days under the conditions of 12L:12D photoperiod, aeration, and PES medium. The zygospores germinated and grew into filaments. The filaments were entangled and spherical. The average spherical size was 5 mm. The filaments were frozen and stored in a freezer set at -60°C. The fully frozen filaments were freeze-dried overnight in a freeze dryer (Tokyo Rikakikai Co., Ltd., FD-1) to prepare a sporophyte filament sample.

[0445] (A-3. Preparation of Sagarame Gametophyte Samples) (1) Sagarame growing on Mochimune Beach, Shizuoka City, Shizuoka Prefecture, was collected, and leaf sections bearing ascophytes were cut off. These leaf sections were stored in a refrigerator for approximately one day, after which zoospores were released in sterilized seawater and allowed to attach to glass slides. (2) These zoospore-bearing slides 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 provided using white fluorescent lamps with an illuminance of 20,000 ux and a light / dark cycle of 10 L:14 D. PESI medium was used as the culture medium, and half of the water was replaced every two days. To measure gametophyte growth, 20 individuals with the most advanced growth were selected every two days, and only male gametophytes were extracted from among them and continued to be cultured under the same conditions to obtain male gametophytes of Sagarame. These were then stored in a refrigerator at 4°C. This male gametophyte was frozen and stored in a freezer set at −60° C. The fully frozen gametophyte was freeze-dried overnight in a freeze dryer (FD-1, manufactured by Tokyo Rikakikai Co., Ltd.) to prepare a male gametophyte sample.

[0446] (A-4. Preparation of a sample of the filamentous sporophyte of Asakusa-nori) (1) Asakusa-nori cultivated near the mouth of the Ibi River was collected, and the tip of the filamentous sporophyte was cut into 3 cm pieces. ２ The leaf pieces were cut off to a length of about 100 mm. The leaf pieces were spread on a glass plate and the surface was washed with sterilized seawater. (2) A sterilized petri dish was prepared, a slide glass was placed on the bottom, PES culture medium was poured into it, and the leaf pieces from (1) were floated on it. The petri dish was left to stand for 14 days under a 2000 Lux fluorescent light. Carpospores fell to the bottom of the dish and accumulated, forming colonies. (3) The carpospores were sucked up with a dropper and placed in PES culture medium. They were cultured at a temperature of 16°C under a 2000 Lux fluorescent light with a light / dark cycle of 12L:12D, and filaments formed from germinated carpospores were obtained. The filaments were entangled with each other to form a spherical shape, and the average length of the spheres was 3 mm. The filaments were frozen and stored in a freezer set at -60°C. The thoroughly frozen gametophytes were freeze-dried overnight in a freeze dryer (Tokyo Rikakikai Co., Ltd., FD-1) to prepare samples of filamentous sporophytes.

[0447] (B-1. Water extraction treatment of wakame female gametophyte, sagarame male gametophyte, filamentous sporophyte and wakame thallus (adult)) Each sample was subjected to water extraction treatment by the following method.

[0448] (B-1-1. Water extraction treatment of wakame female gametophytes and wakame thallus (adult) (Example 2-1 and Comparative Example 2-1)) (1) 1 g of powder sample obtained by crushing the female gametophytes prepared in (A-1) above was suspended in 40 mL of distilled water and stirred at 200 rpm for 0.5 hours at 4°C using a rotator (RT-5N, manufactured by TAITEC).

[0449] (2) The stirred female gametophyte sample solution from (1) was centrifuged at 4°C, 8000 rpm, and 20 minutes using a centrifuge to separate the solid precipitate from the supernatant.

[0450] (3) The supernatant of (2) was filtered through a coffee filter, and the filtrate was filtered through a 0.1 μm PES filter (manufactured by Membrane Solutions LLC).

[0451] (4) 20 mL of this filtrate was concentrated (6000 rpm for 10 min) using a 300K spin column (manufactured by Nippon Pall Co., Ltd.), washed and filtered three times using 2 mL of PBS (phosphate buffered saline), and 0.5 mL of the filtrate was recovered (40-fold concentrated). This was used as the water extract of the wakame female gametophyte of Example 2-1.

[0452] (5) 10 g of dried wakame (Marukome Co., Ltd.) consisting of thallus (adult) was crushed in a mixer mill to obtain powder, and 1 g of the 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).

[0453] (6) The stirred thallus sample solution from (5) was centrifuged at 4°C, 8000 rpm, and 20 minutes using a centrifuge to separate the solid precipitate from the supernatant.

[0454] (7) The supernatant of (6) was filtered through a coffee filter, and the filtrate was filtered through a 0.1 μm PES filter (manufactured by Membrane Solutions LLC).

[0455] (8) 20 mL of this filtrate was concentrated (6000 rpm for 10 min) using a 300K spin column (manufactured by Nippon Pall Co., Ltd.), washed and filtered three times using 2 mL of PBS (phosphate buffered saline), and 0.5 mL of the filtrate was recovered (40-fold concentrated). This was used as the water extract of the wakame thallus (adult) according to Comparative Example 2-1.

[0456] (B-1-2. Aqueous Extract of Filaments of Sporophytes of Porphyra Uppuri) The freeze-dried filaments of the sporophytes of Porphyra Uppuri obtained in (A-2) above were pulverized into powder. This powder was used to obtain a water extract of filaments of the sporophytes of Porphyra Uppuri in the same manner as in (B-1-1) above. This was designated as the water extract of filaments of the sporophytes of Porphyra Uppuri according to Example 2-2.

[0457] (B-1-3. Water extract of Sagarame gametophyte) The freeze-dried male gametophyte of Sagarame obtained in (A-3) above was pulverized into powder. This powder was used to obtain a water extract of Sagarame male gametophyte in the same manner as in (B-1-1) above. This was designated as the water extract of Sagarame male gametophyte according to Example 2-3.

[0458] (B-1-4. Water Extract of Asakusanori Sporophyte Filaments) The freeze-dried Asakusanori sporophyte filaments obtained in (A-4) above were pulverized into powder. This powder was used to obtain a water extract of Asakusanori sporophyte filaments in the same manner as in (B-1-1) above. This was used as the water extract of Asakusanori sporophyte filaments according to Example 2-4.

[0459] (B-2. Analysis of Each Water Extract) Each water extract was analyzed by the following method.

[0460] (B-2-1. Analysis of extracellular vesicles in aqueous extracts of wakame female gametophytes) (1) 1 μL of a 1 mM solution of GIF-2276 (manufactured by GIFU EXOSOME Co., Ltd.: a labeling reagent for detecting protein in EVs. It is a fluorescent substance with an Ex / Em of 475 / 530 nm) and 2 μL of ExoSparkler Exosome Membrane Labeling Kit-Red (manufactured by Dojindo Laboratories, Inc.: a labeling reagent for detecting lipid membranes. It is a fluorescent substance with an Ex / Em of 560 / 600 nm) were mixed with 100 μL of the aqueous extract of the female gametophyte according to Example 2-1 prepared in (4) of (B-1-1) above, and the mixture was labeled by heating in 80°C hot water for 5 minutes.

[0461] (2) The following liquid chromatography apparatus A was prepared for the detection of extracellular vesicles. Column packing: qEV10 70 nm manufactured by Izon Science; Column shape: Stainless steel column manufactured by Senshu Scientific Co., Ltd., diameter 8 mm x length 100 m; Detector: RF-10AxL manufactured by Shimadzu Corporation; Developing solvent: 10 mM tris(hydroxymethyl)aminomethane pH = 7.6, aqueous solution containing 30 mM NaCl and 0.5 mM EDTA; Development time gradient: uniform; Solution flow rate: 1 mL / min; Injection volume: 10 μL; Column temperature: 30°C; Sample chamber temperature: 10°C; Delivery pump: LC10ADVP manufactured by Shimadzu Corporation.

[0462] (3) The concentrate labeled in (1) was analyzed using the liquid chromatography system A in (2). The chart of the results is shown in Figure 2-2 (solid line: protein, dotted line: lipid). The horizontal axis of the chart represents time (minutes), and the vertical axis represents the absorbance of fluorescence (530 nm for protein, 600 nm for lipid membrane) and is unitless.

[0463] As described in WO2024 / 053316 and Biochemical and Biophysical Research Communications Volume 696, February 12, 2024, 149505, "Simple methods for measuring milk exosomes using fluorescent compound GIF-2250 / 2276," GIF-2276 binds to Lys residues of proteins that constitute extracellular vesicles (EVs) to modify the extracellular vesicles (EVs). Furthermore, the aforementioned literature indicates that ExoSparkler Exosome Membrane Labeling Kit-Red (abbreviated as Exo-SP or EXoSP in the aforementioned literature) modifies the lipid membrane of extracellular vesicles (EVs). When the lipid membrane peak and protein peak appear at the same retention time on a liquid chromatography chart, it is believed that extracellular vesicles (EVs) are present in the sample. In Figure 2-2, since the lipid membrane peak and protein peak appear at the same retention time, it can be concluded that extracellular vesicles (EVs) are present in the aqueous extract of wakame female gametophytes, etc. To be sure, the aqueous extract of wakame female gametophytes was frozen at -50°C using liquid nitrogen, and then lyophilized under reduced pressure of 4 Pa ​​to prepare a powdered composition. Transmission electron micrographs of the powdered composition obtained by drying are shown in Figures 2-3A, 2-3B, 2-3C, and 2-3D (scale: 100 nm, 50 nm, 20 nm, 20 nm). The electron microscope used was a JEOL JEM-2100, and the imaging conditions were an accelerating voltage of 200 kV and negative staining with uranyl acetate. The magnifications for Figures 2-3A, 2-3B, 2-3C, and 2-3D were 50,000x, 100,000x, 250,000x, and 300,000x, respectively. The spherical object in the center of the photographs in Figures 2-3A and 2-3B and the white granular objects around it are extracellular vesicles (EVs). Figures 2-3C and 2-3D are enlarged images of the white granular EVs.

[0464] (B-2-2. Analysis of extracellular vesicles in the aqueous extract of filaments of the sporophyte of Porphyra uppuri) The aqueous extract of filaments of the sporophyte of Porphyra uppuri in Example 2-2 was analyzed using liquid chromatography apparatus A in the same manner as in (B-2-1) above. The chart of the results is shown in Figure 2-4. Protein and lipid peaks were observed at the same positions, indicating the presence of EVs. Note that proteins other than those constituting EVs were also observed.

[0465] (B-2-3. Analysis of extracellular vesicles in the aqueous extract of male gametophytes of Sagara seaweed) The aqueous extract of male gametophytes of Sagara seaweed according to Example 2-3 was analyzed using liquid chromatography apparatus A in the same manner as in (B-2-1) above. The chart of the results is shown in Figure 2-5. Compared to female gametophytes, the amount of extracellular vesicles (EVs) appears to be relatively small.

[0466] (B-2-4. Analysis of extracellular vesicles in the aqueous extract of filaments of Asakusanori sporophytes) The aqueous extract of filaments of Asakusanori sporophytes in Example 2-4 was analyzed using liquid chromatography apparatus A in the same manner as in (B-2-1) above. The chart of the results is shown in Figure 2-6. Protein and lipid peaks were observed at the same positions, indicating the presence of EVs. Note that proteins and lipids different from the proteins and lipids that constitute EVs were also observed.

[0467] (B-3. Analysis of Fucoxanthin) (1) The following liquid chromatography apparatus B was prepared for the detection of fucoxanthin. Column: diameter 3.0 mm x length 150 mm, packing material was 25437-96 RP-18GP 150-3 manufactured by Kanto Chemical Co., Inc., a packing material for high-performance liquid chromatography with a particle size of 5.0 μm. Detector: absorbance was measured using a spectrophotometer (UV-1800 manufactured by Shimadzu Corporation). Wavelength: 450 nm Developing solvent A: 10% acetonitrile prepared by mixing LC / MS grade acetonitrile (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 LC / MS grade acetonitrile (Kanto Chemical Co., Ltd., 01033-76) and 0.05 wt % formic acid aqueous solution in an 8:2 ratio Developing solvent C: 100% acetonitrile Development time gradient: 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: 5 μL Column temperature: 40°C Sample chamber temperature: 15°C Delivery pump: Shimadzu Corporation, LC10ADVP

[0468] 0.6589 mg of fucoxanthin standard (Fujifilm Wako Pure Chemical Industries, Ltd.) was weighed out, 1 L of ethanol was added, and the solution was stirred thoroughly to prepare a 1 mM fucoxanthin solution. 1 mL of this solution was further diluted 10-fold with ethanol to prepare a 0.1 mM (100 μM) standard solution. This standard solution was used to confirm the retention time of fucoxanthin in liquid chromatography apparatus B (1). This 100 μM fucoxanthin standard solution was also diluted 2-fold (50 μM), 3.33-fold (30 μM), 10-fold (10 μM), and 33.3-fold (3 μM) with ethanol. Next, a calibration curve of fucoxanthin concentration in liquid chromatography apparatus B (1) was created using the standard solution and the diluted solution.

[0469] The calibration curve is expressed as y = 110489x + 22725 (x is the fucoxanthin concentration, and y is the peak area). Fucoxanthin at a concentration of 1 M was analyzed using the liquid chromatography apparatus B in (1), and it was confirmed that the peak area and fucoxanthin concentration appearing on the chart conformed to the calibration curve.

[0470] (C. 100% butylene glycol (BG) extraction treatment of wakame female gametophytes and wakame thallus after water extraction treatment and analysis of BG extract) BG extraction treatment and analysis of BG extract were carried out using wakame female gametophytes and wakame thallus using the following method.

[0471] (C-1. BG extraction treatment of aqueous extracts of wakame female gametophytes and wakame thallus (adult) (Comparative Example 2-2 and Comparative Example 2-3)) (1) After performing aqueous extraction treatment of wakame female gametophytes using the same procedures as in (1) and (2) of (B-1-1) above, the wakame female gametophytes, which are the extraction residue (solid content), were thoroughly drained, and 1 g of the solid matter 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. Subsequently, the supernatant was removed, and the precipitate was suspended in 20 mL of 100% BG, 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 sterilized filter to prepare a BG extract, which was used as the BG extract of the wakame female gametophyte according to Comparative Example 2-2.

[0472] (2) 10 g of dried wakame (Marukome Co., Ltd.) consisting of thallus (adult) was crushed in a mixer mill to obtain a powder, and 1 g of the powder was suspended in 0.5 mL of 100% butylene glycol (BG) and stirred at 4°C and 8000 rpm for 0.3 hours using a rotator (TAITEC, RT-5N). The supernatant was then removed, and the resulting precipitate was suspended in 20 mL of 100% BG. The suspension was then stirred at 200 rpm for 0.5 hours using a rotator (TAITEC, RT-5N) at 4°C. 1 mL of the supernatant was filtered through a 0.2 μm sterilized filter to prepare a BG extract. This was designated the wakame thallus BG extract of Comparative Example 2-2.

[0473] (C-2. Analysis of Extracellular Vesicles in BG Extract) The BG extract of wakame female gametophyte according to Comparative Example 2-2 prepared in (C-1) above was analyzed using the liquid chromatography apparatus A in (B-2-1). The chart of the results is shown in Figure 2-7 (solid line: protein, dotted line: lipid). The horizontal axis of the chart is time (minutes), and the vertical axis is the absorbance of fluorescence (protein: 530 nm, lipid membrane: 600 nm), which is unitless. The protein and lipid peaks around 10 to 12 minutes seen in Figure 2-2 are not observed in Figure 2-7. Therefore, extracellular vesicles (EVs) are not observed in the BG extract prepared in (C-1).

[0474] (C-3. ​​Analysis of fucoxanthin in BG extract) The BG extracts of wakame female gametophytes and wakame thallus prepared in (C-1.) (1) and (2) above (Comparative Example 2-2 and Comparative Example 2-3, respectively) were analyzed using the liquid chromatography apparatus B described in (1). Liquid chromatography charts are shown in Figures 2-8A and 2-8B. The horizontal axis of the charts represents time (minutes). The vertical axis represents absorbance at 450 nm (unitless). In Figures 2-8A and 2-8B, the peak at around 13 minutes corresponds to fucoxanthin, and the peak at around 11.3 minutes corresponds to chlorophyll.

[0475] D. Identification of Proteins in Water Extracts Proteins in each water extract were identified in the following manner.

[0476] (D-1. Identification of proteins in the water extract of wakame female gametophyte) (1) 100 mg of a powder sample obtained by grinding 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 4°C using a rotator (TAITEC, RT-5N).

[0477] (2) The stirred female gametophyte sample solution from (1) was centrifuged at 4°C, 8000 rpm, and 20 minutes using a centrifuge to separate the solid precipitate from the supernatant.

[0478] (3) The supernatant of (2) was filtered through a coffee filter, and the filtrate was filtered through a 0.1 μm PES filter (manufactured by Membrane Solutions LLC).

[0479] (4) 10 mL of this filtrate was concentrated (6000 rpm for 10 minutes) using a 300K spin column (manufactured by Nippon Pall Co., Ltd.), washed and filtered twice using 250 μL of PBS (phosphate buffered saline), and 100 μL of the filtrate was recovered.

[0480] (5) The following samples were prepared: Sample A: PM007-0500 (standard sample), manufactured by BIO-HELIX; Sample B: Water extract sample prepared in (1) to (4) of D above; Sample C: Liquid passed through a 300K spin column in (1) to (4) of D above; Sample D: Filtrate after treatment in (1) to (3) of D above.

[0481] (6) 5 μL of each of Samples A to D in (1) was mixed with 3 times the amount of SDS-Sample buffer (TAKARA Bio: 786-701) containing 10% mercaptoethanol, and the mixture was heated at 100° C. for 10 minutes.

[0482] (7) Samples A to D treated in (6) were electrophoresed using an acrylamide gel (ATTO E-T520L) containing a running buffer (for SDS-PAGE, Tris-glycine system, Nakarai 30329-74).

[0483] (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 2-9.

[0484] (9) In the acrylamide gel, two darkly colored bands were cut out with a scalpel from the electrophoresis bands of sample B. Based on the bands of sample A, the cut-out portions corresponded to the molecular weight region of 35,000 to 63,000 Da and the molecular weight region of 10,000 to 17,000 Da.

[0485] (10) The excised acrylamide gel fragments were sent to Nippon Proteomics Co., Ltd. (6-6-3 Minamiyoshinari, Aoba-ku, Sendai, Miyagi Prefecture, 989-3204) for protein identification. Protein identification was performed using the following procedure. First, the gel fragments were decomposed into peptides using trypsin, and the decomposed peptide fragments were separated using a liquid chromatograph called nanoLC, which has high sensitivity due to the miniaturization of the packing material and column diameter. The amino acid sequence of the peptides was identified using a tandem mass spectrometer MS / MS following LC, and the results were compared with proteins registered in a database (Matrix Science Co., Ltd.) to identify the proteins contained in the gel fragments.

[0486] As a result of the identification, in the molecular weight region of 35,000 to 63,000 Da, a ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit with a molecular weight (MW) of 52,369 and an ATP synthase subunit beta with a molecular weight (MW) of 44,542 were confirmed. In addition, in the molecular weight region of 10,000 to 17,000 Da, ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit with a molecular weight (MW) of 15,249 and histone H2B with a molecular weight (MW) of 12,789 were confirmed. The dark bands are the 35 x 10 bands shown by the bands of the standard sample. ３ ~63 x 10 ３ Da range, and 10 x 10 ３ ~17 x 10 ３ Since the protein was confirmed to be in the range of 1,5 Da, it was determined that the main components of the protein were the ribulose 1,5-bisphosphate carboxylase / oxygenase large subunit and a small subunit that could bind to it.

[0487] (D-2. Identification of proteins in a water extract of filaments of Uppuri Porphyra sporophytes) Using a powder sample obtained by pulverizing the filaments of Uppuri Porphyra sporophytes prepared in (A-2) above, the filtrate obtained in steps (1) to (4) of (D-1) above was subjected to electrophoresis in steps (6) to (10) of (D-1) above to separate the protein bands and identify the proteins. A ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit with a molecular weight (MW) of 52,440 and an ATP synthase subunit beta with a molecular weight (MW) of 51,165 were identified. Thiamine biosynthesis protein G with a molecular weight (MW) of 26,838 was also identified.

[0488] (D-3. Identification of proteins in aqueous extracts of male gametophytes of Sagarame) Using a powder sample obtained by pulverizing the male gametophytes of Sagarame prepared in (A-3) above, the filtrate obtained in steps (1) to (4) of (D-1) above was subjected to electrophoresis in steps (6) to (10) of (D-1) above to separate the protein bands and identify the proteins. A ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit with a molecular weight (MW) of 52,400 was identified. Other bands likely to be proteins were present, but no corresponding proteins were found in the database.

[0489] (D-4. Identification of proteins in a water extract of filaments of Asakusanori sporophytes) Using a powder sample obtained by pulverizing the filaments of Asakusanori sporophytes prepared in (A-4) above, the filtrate obtained in steps (1) to (4) of (D-1) above was subjected to electrophoresis in steps (6) to (10) of (D-1) above to separate the protein bands and identify the proteins. A ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit with a molecular weight (MW) of 52,400 was identified. Other bands likely to be proteins were present, but no corresponding proteins were found in the database.

[0490] (E. Measurement of cAMP-increasing effect of aqueous extract and BG extract) (1) The aqueous extract of the wakame female gametophyte according to Example 2-1 and the aqueous extract of the wakame thallus according to Comparative Example 2-1 were diluted 300-fold and 100-fold using a medium (Keratinocyte Growth Medium 2 (available from TakaraBio Co., Ltd. as C-20011)), to prepare a total of four types of samples. The 300-fold and 100-fold diluted versions of the aqueous extract of the wakame female gametophyte were designated Example 2-1-1 and Example 2-1-2, respectively, and the 300-fold and 100-fold diluted versions of the aqueous extract of the wakame thallus were designated Comparative Example 2-1-1 and Comparative Example 2-1-2, respectively.

[0491] (2) The BG extract of the wakame female gametophyte according to Comparative Example 2-2 and the BG extract of the wakame thallus according to Comparative Example 2-3 were diluted 100-fold with the above-mentioned medium.

[0492] (3) 5 x 10 cells in a 96-well plate ４ PSVK1 cells (human keratinocytes: JCRB1093, JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition) at the time of passage (concentration) of 1 / well were placed, and 150 μL of the above medium was added, followed by culturing for 48 hours.

[0493] (4) Next, 150 μL of the medium was aspirated, and 100 μL of the same medium was added again. 100 μL of the medium from Example 2-1-1, Example 2-1-2, Comparative Example 2-1-1, Comparative Example 2-1-2, Comparative Example 2-2, and Comparative Example 2-3, as well as the (negative) control, was added to each of three wells of each well plate. The well holes were 7 samples x 3 wells, for a total of 21 wells. Furthermore, this well plate was incubated at 25°C for 90 minutes. After incubation, Promega cAMP Glo ＴＭ The increase in cAMP was measured using a measurement kit called "Promega V1501 Assay" (manufactured by Promega Corporation). The attached V1501 reagent group was used for the measurement. First, the sample and medium were removed from the well plate, and 20 μL of Lysis Buffer was added to each well. The wells were left 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 Glu were added. ＴＭ cAMP Glo consisting of Reacton Buffer 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® Reagent was added to each well and left for 10 minutes to stop the PKA reaction, and the remaining ATP was used to generate light using the luciferin-luciferase reaction. ＴＭ The cAMP measurement kit, the "CAMP Assay," utilizes the fact that cAMP stimulates protein kinase A (PKA) holoenzyme activity, and the activated protein kinase A (PKA) reacts with its substrate protein, consuming ATP during the reaction. The measurement principle is based on the fact that, if there is a lot of cAMP, there is less ATP available for the luciferin-luciferase reaction, and luminescence is suppressed; the higher the cAMP concentration, the less luminescence there is.

[0494] (5) Using a Promega Glo MAX Multi Detection System (Promega Corporation), the luminescence intensity of each well of the well plate was measured for 2 seconds. The results are shown in Figure 2-10. The luminescence intensity was the average value of three wells for each sample. As can be seen from Figure 2-10, the aqueous extract from wakame female gametophytes, which contains extracellular vesicles (EVs), ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit, and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, produced less luminescence than in the control test, confirming its effect in increasing cAMP levels. Furthermore, it was found that the lower the dilution rate, the lower the luminescence and the greater the effect in increasing cAMP levels. On the other hand, the aqueous extract from wakame thallus, which does not contain proteins or EVs (see Test Examples 2-1 and 2-4 described below), showed no difference in luminescence compared to the comparative test in which only medium was added, indicating that it had no effect on increasing cAMP.Furthermore, when compared with the comparative test, the BG extract from wakame female gametophyte or wakame thallus, which contains fucoxanthin but does not contain EVs, could not be confirmed to have any effect on increasing cAMP.

[0495] Incidentally, it is also possible to prepare a powdery composition comprising extracellular vesicles (EVs), ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit, and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit by freezing the water extract from Undaria pinnatifida female gametophytes corresponding to Examples 2-1-1 and 2-1-2 to -50°C using liquid nitrogen and then lyophilizing by reducing the pressure to 4 Pa. In this way, the composition of the present invention can be freely prepared in either a solution or powder form, and the viscosity can also be freely adjusted. Furthermore, since extracellular vesicles (EVs) contain only components contained in the cells from which they were derived, there is no need to worry about contamination with components that are harmful to the living body, and they are easy to handle.

[0496] The water extract of the filaments of the sporophytes of Porphyra gracilis according to Example 2-2 was diluted 300-fold with a medium (Keratinocyte Growth Medium 2: Keratinocyte Growth Medium 2 (available from TakaraBio Co., Ltd. as C-20011)). The water extract of the male gametophyte of Sagarame according to Example 2-3 was diluted 300-fold with a medium (Keratinocyte Growth Medium 2: Keratinocyte Growth Medium 2 (available from TakaraBio Co., Ltd. as C-20011)). The water extract of the filaments of the Asakusanori sporophyte of Example 2-4 was diluted 300-fold using a medium (Keratinocyte Growth Medium 2 (available from TakaraBio Co., Ltd. as C-20011)). The cAMP-increasing effect was then measured using the same method as in (E) above. The results are shown in Figure 2-11. As shown in Figure 2-11, the water extract of the male gametophyte and the water extract of the filaments of the sporophyte were found to have a cAMP-increasing effect. Furthermore, Figures 2-10 and 2-11 reveal that the water extract of the gametophyte had a greater cAMP-increasing effect than the water extract of the filaments of the sporophyte.

[0497] (Comparative Example 2-4) A female gametophyte sample was obtained in the same manner as in (A-1) above, and then 10 mg of the female gametophyte sample was suspended in 1 mL of 70 wt % ethanol (the remaining 30% was water) and stirred with a rotator (TAITEC, RT-5N) at 200 rpm for 1 hour at 25° C. Next, the solution alone was removed from the 2 mL tube containing the stirred female gametophyte sample solution with a micropipette, and the solution was separated into a solid precipitate and a supernatant.

[0498] The supernatant was filtered through a PES filter and labeled in the same manner as described above (B-2-1), and the extract was analyzed. The liquid chromatography chart is shown in Figure 2-12 (solid line: protein, dotted line: lipid). As can be seen from Figure 2-12, the protein and lipid peaks observed in Figure 2-2 around 10 to 12 minutes were not observed in Figure 2-12. Therefore, it can be seen that extracellular vesicles (EVs) were not observed in the 70% ethanol extract of female gametophytes.

[0499] (Test Example 2-1) (1) 10 g of dried wakame seaweed (manufactured by Marukome Co., Ltd.) consisting of thallus (adult) and 10 g of dried mozuku seaweed (manufactured by JF Okinawa Gyoren) were crushed into powder using a mixer mill, and 1 g of the powder was suspended in 40 mL of distilled water and stirred at 200 rpm for 0.5 hours at 4°C using a rotator (manufactured by TAITEC, RT-5N).

[0500] (2) The stirred thallus (adult) sample solution from (1) was centrifuged at 4°C, 8000 rpm, and 20 minutes using a centrifuge to separate the solid precipitate from the supernatant.

[0501] (3) The supernatant of (2) was filtered through a coffee filter, and the filtrate was filtered through a 0.1 μm PES filter (manufactured by Membrane Solutions LLC).

[0502] (4) 20 mL of this filtrate was concentrated (6000 rpm for 10 minutes) using a 300K spin column (manufactured by Nippon Pall Co., Ltd.), washed and filtered three times using 2 mL of PBS (phosphate buffered saline), and 0.5 mL of the filtrate was recovered (40-fold concentrated).

[0503] (5) The water extracts of these wakame and mozuku thallus were labeled with GIF-2276 and ExoSparkler Exosome Membrane Labeling Kit-Red in the same manner as in (B-2-1) above, and the presence or absence of extracellular vesicles (EVs) was confirmed using liquid chromatography apparatus A. Figure 2-13 shows the chromatography chart for the water extract of wakame thallus, and Figure 2-14 shows the chromatography chart for the water extract of mozuku thallus.

[0504] As can be seen from Figures 2-13 and 2-14, the protein and lipid peaks observed around 10 to 12 minutes in Figure 2-2 are not observed in Figures 2-13 and 2-14. Therefore, it can be seen that extracellular vesicles (EVs) are not detected in the water extract of the thallus. Generally, when wakame and mozuku are used commercially or industrially, the thallus is used, but extracellular vesicles (EVs) cannot be obtained from the thallus. The reason for this is unclear, but it is presumed that the EVs cannot be released in the first place, or that they are destroyed when released. When the water extract of the wakame thallus obtained in (4) above was used to analyze proteins in the same manner as in (D-1) above, the electrophoretic bands were pale in color, but the presence of ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit was confirmed.

[0505] (Test Example 2-2) A 10-fold dilution of the water extract of the wakame female gametophyte from Example 2-1 was prepared by adding a mixed solvent of phosphate-buffered saline (PBS) and BG (weight ratio: BG / PBS = 9 / 1) to 20 μL of the water extract of the wakame female gametophyte. The presence or absence of extracellular vesicles (EVs) in this 10-fold diluted water extract of the wakame female gametophyte was confirmed using liquid chromatography system A, with labeling performed in the same manner as described above (B-2-1). The resulting chart is shown in Figure 2-15 (solid line: protein, dotted line: lipid). As seen in Figure 2-2, Figure 2-15 confirms peaks of protein and lipid membranes around 10 to 12 minutes. This indicates that extracellular vesicles (EVs) once extracted with water can be present even when transferred to a solvent with a high organic solvent concentration, such as BG / PBS = 9 / 1.

[0506] (Test Example 2-3) 0.5 mL of caprylic triglyceride was added to 0.5 mL of the water extract of wakame female gametophyte from Example 2-1, and the mixture was shaken for 5 minutes and then allowed to stand to separate the water extract of wakame female gametophyte and caprylic triglyceride into two phases. The caprylic triglyceride phase was then drawn up with a pipette, and the presence or absence of extracellular vesicles (EVs) in this caprylic triglyceride was confirmed using liquid chromatography apparatus A, after labeling in the same manner as in (B-2-1) above. The resulting chart is the same as in Figure 2-13, and no peaks corresponding to extracellular vesicles (EVs) were observed.

[0507] (Test Example 2-4) (1) 0.1 g of a powder sample obtained by crushing dried wakame (manufactured by Marukome Co., Ltd.) consisti...

Claims

A composition comprising extracellular vesicles (EVs) and a carotenoid. The composition of claim 1 , wherein the extracellular vesicles (EVs) are derived from seaweed. The composition according to claim 2 , wherein the seaweed is a gametophyte and / or a sporophyte of the seaweed. The composition according to claim 2 or 3, wherein the seaweed is a female gametophyte of seaweed. The composition according to claim 2 or 3, wherein the seaweed is a filamentous sporophyte. The composition according to claim 2 or 3, wherein the seaweed is a brown algae. The composition contains an aqueous solvent or a mixed solvent consisting of an aqueous solvent and an organic solvent, The composition of claim 1 or 2, wherein the organic solvent dissolves the carotenoid. The composition according to claim 1 or 2, wherein the composition is a powder composition. The composition according to claim 1 or 2, wherein at least a portion of the carotenoid is attached to or encapsulated in the extracellular vesicles (EVs). The composition according to claim 1 or 2, wherein the carotenoid coexists with the carotenoid attached to or encapsulated in the extracellular vesicles (EVs) and the carotenoid released from the extracellular vesicles (EVs).

3. The composition according to claim 1, wherein the carotenoid is at least one compound selected from the group consisting of fucoxanthin and derivatives thereof. A method for producing a composition containing extracellular vesicles (EVs) derived from seaweed gametophytes and / or sporophytes and carotenoids, comprising the following steps (1) and (2): Step (1): Dispersing seaweed gametophytes and / or sporophytes in an aqueous solvent to obtain a dispersion of the seaweed gametophytes and / or sporophytes. Step (2): The seaweed gametophytes and / or sporophytes are separated and removed from the dispersion of the seaweed gametophytes and / or sporophytes obtained in step (1) to obtain an aqueous solvent composition containing the extracellular vesicles (EVs) and the carotenoid. A method for producing a composition comprising extracellular vesicles (EVs) derived from gametophytes and / or sporophytes of the seaweed according to claim 12 and carotenoids, further comprising step (5-1). Step (5-1): The aqueous solvent composition obtained in step (2) is dried to obtain a powdery composition containing the extracellular vesicles (EVs) and the carotenoid. A method for producing a composition comprising extracellular vesicles (EVs) derived from gametophytes and / or sporophytes of the seaweed described in claim 12 and carotenoids, further comprising steps (3) and (4). Step (3): An organic solvent capable of dissolving the carotenoid is added to the gametophyte and / or sporophyte of the seaweed separated in step (2), to obtain an organic solvent composition containing the carotenoid. Step (4): The aqueous solvent composition containing the extracellular vesicles (EVs) and the carotenoid obtained in step (2) is added to the organic solvent composition containing the carotenoid obtained in step (3) to obtain a mixed solvent composition containing the aqueous solvent and the organic solvent. A method for producing a composition comprising extracellular vesicles (EVs) derived from gametophytes and / or sporophytes of the seaweed according to claim 14 and carotenoids, further comprising step (5-2). Step (5-2): The mixed solvent composition containing the aqueous solvent and the organic solvent obtained in step (4) is dried to obtain a powdery composition containing the extracellular vesicles (EVs) and the carotenoid. The method for producing a composition according to any one of claims 12 to 15, wherein the sporophyte is a filamentous body. The method for producing a composition according to any one of claims 12 to 15, wherein the carotenoid is at least one compound selected from the group consisting of fucoxanthin and derivatives thereof. A raw material comprising a collection of seaweed gametophytes and / or sporophytes, which is used to extract extracellular vesicles (EVs) and carotenoids.

19. The raw material according to claim 18, wherein the seaweed is a female gametophyte. The raw material according to claim 18, wherein the seaweed is a filamentous sporophyte. The raw material according to any one of claims 18 to 20, wherein the seaweed is brown algae. The raw material according to any one of claims 18 to 20, wherein the carotenoid is at least one compound selected from the group consisting of fucoxanthin and derivatives thereof. Use of seaweed-derived extracellular vesicles (EVs) to improve the stability of carotenoids. The use of seaweed-derived extracellular vesicles (EVs) according to claim 23, wherein the carotenoid is at least one compound selected from the group consisting of fucoxanthin and its derivatives. A carotenoid stability enhancer comprising extracellular vesicles (EVs) derived from seaweed. The carotenoid stability enhancer according to claim 25, wherein the carotenoid is at least one compound selected from the group consisting of fucoxanthin and derivatives thereof. A method for improving the stability of carotenoids by coexisting seaweed-derived extracellular vesicles (EVs) with carotenoids.

28. The method for improving the stability of a carotenoid according to claim 27, wherein the carotenoid is at least one compound selected from the group consisting of fucoxanthin and its derivatives. A composition comprising at least one protein selected from the group consisting of 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. The composition according to claim 29, further comprising at least one selected from the group consisting of ATP synthase subunit beta, thiamine biosynthesis protein G, and histone H2B.

30. The composition of claim 29, wherein the seaweed is a female gametophyte of seaweed.

30. The composition of claim 29, wherein the seaweed is a filamentous sporophyte.

30. The composition of claim 29, wherein the seaweed is a brown algae.

30. The composition of claim 29, wherein the composition comprises an aqueous solvent or a mixture of an aqueous solvent and an organic solvent.

30. The composition according to claim 29, further comprising at least one compound selected from the group consisting of fucoxanthin and derivatives thereof. The composition according to claim 29, wherein the at least one protein selected from the group consisting of ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit is encapsulated in or attached to extracellular vesicles (EVs) derived from seaweed gametophytes.

30. The composition of claim 29, wherein the composition is a powdered composition. An agent for increasing intracellular cyclic adenosine monophosphate (cAMP), comprising the composition according to any one of claims 29 to 37. A method for producing a composition comprising at least one protein selected from the group consisting of ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs) derived from seaweed gametophytes and / or sporophytes, the method comprising the following steps (1) and (2): Step (1): The gametophytes and / or sporophytes of the seaweed are dried to obtain a dried body, and then the dried body is pulverized to obtain a powder of the gametophytes and / or sporophytes of the seaweed, and the powder is dispersed in an aqueous solvent adjusted to a temperature of 5°C or below. Step (2): The aqueous solvent in which the powder is dispersed is separated into a solid matter and a supernatant by centrifugation, and the supernatant is collected.

40. A method for producing the composition according to claim 39, comprising the step of adding to the supernatant at least one compound selected from the group consisting of fucoxanthin and its derivatives. The method for producing the composition according to claim 39, further comprising the step of removing the aqueous solvent from the supernatant to obtain a powder containing at least one protein selected from the group consisting of ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit and ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit, and extracellular vesicles (EVs) derived from seaweed gametophytes and / or sporophytes. A method for producing an agent for increasing intracellular cyclic adenosine monophosphate (cAMP), comprising the method for producing the composition according to any one of claims 39 to 41.

40. The method for producing a composition according to claim 39, wherein the extracellular vesicles (EVs) are derived from the female gametophyte of the seaweed.

40. The method for producing a composition according to claim 39, wherein the extracellular vesicles (EVs) are derived from the filaments of the sporophyte of the seaweed. The raw material contains an aggregate of seaweed gametophytes and / or sporophytes, and is used to extract 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 seaweed gametophytes.

46. ​​The raw material according to claim 45, wherein the seaweed is a female gametophyte.

46. ​​The raw material according to claim 45, wherein the seaweed is a filamentous sporophyte. The raw material according to any one of claims 45 to 47, wherein the seaweed is brown algae. A composition comprising at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, wherein the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 1.1 to 22.

0.

50. The composition according to claim 49, wherein the at least one compound selected from the group consisting of fucoxanthin and its derivatives, and the linoleic acid or a salt thereof are derived from seaweed.

51. The composition of claim 50, wherein the seaweed is a seaweed gametophyte.

51. The composition of claim 50, wherein the seaweed is a female gametophyte of seaweed.

51. The composition of claim 50, wherein the seaweed is a brown algae.

50. The composition of claim 49, wherein the composition does not include an extract of Phaeodactylum tricornutum. The composition according to any one of claims 49 to 54, further comprising a polysaccharide. The composition according to any one of claims 49 to 54, comprising an organic solvent or a mixed solvent of an aqueous solvent and an organic solvent, wherein the organic solvent dissolves at least one compound selected from the group consisting of fucoxanthin and derivatives thereof. The composition according to any one of claims 49 to 54, wherein the composition is a powder composition. A method for producing a composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives, and linoleic acid or a salt thereof, comprising the following steps (1) to (3): Step (1): Dispersing seaweed gametophytes in an aqueous solvent to obtain a dispersion of the seaweed gametophytes. Step (2): The aqueous solvent is separated and removed from the dispersion of the seaweed gametophytes obtained in step (1). Step (3): An organic solvent is added to the seaweed gametophytes from which the aqueous solvent has been separated and removed in step (2), to obtain a composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, wherein the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 1.1 to 22.

0. A method for producing a composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives, and linoleic acid or a salt thereof, comprising the following steps (1) to (4): Step (1): Dispersing seaweed gametophytes in an aqueous solvent to obtain a dispersion of the seaweed gametophytes. Step (2): The aqueous solvent is separated and removed from the dispersion of the seaweed gametophytes obtained in step (1). Step (3): An organic solvent is added to the seaweed gametophytes from which the aqueous solvent has been separated and removed in step (2), to obtain an organic solvent composition containing at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof. Step (4): At least one compound selected from the group consisting of fucoxanthin and its derivatives and / or linoleic acid or a salt thereof is added to the organic solvent composition obtained in step (3), to obtain a composition in which the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is adjusted to 1.1 to 22.

0.

60. A method for producing a composition according to claim 58 or 59, further comprising a drying step of drying the composition to form a powder. A raw material used to extract and prepare a composition composed of an aggregate of seaweed gametophytes, which contains at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, and in which the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or its salt]) is 1.1 to 22.

0.

62. The raw material according to claim 61, wherein the seaweed is brown algae.

62. The raw material of claim 61, wherein the seaweed is a female gametophyte. A composition capable of penetrating into cell tissue, comprising at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, wherein the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 1.1 to 22.

0. A penetration enhancer for cell tissue, comprising at least one compound selected from the group consisting of fucoxanthin and its derivatives and linoleic acid or a salt thereof, wherein the weight ratio of the at least one compound selected from the group consisting of fucoxanthin and its derivatives to the linoleic acid or its salt ([at least one compound selected from the group consisting of fucoxanthin and its derivatives] / [linoleic acid or a salt thereof]) is 1.1 to 22.0.

Images