Cellular vesicles derived from algae, and method for producing the cellular vesicles

Abstract

To provide cellular vesicles derived from algae having distinctive functions suitable for human health maintenance and alleviation of lifestyle diseases, and to provide a production method capable of efficiently concentrating the algae-derived cellular vesicles.SOLUTION: Provided is a bioactive cellular vesicle derived from heat treatment of algae and / or an extract thereof. Also provided is a method for producing a bioactive cellular vesicle, comprising a step of heat-treating algae and / or an extract thereof.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] The present invention relates to algae-derived cellular vesicles and a method for producing said cellular vesicles. [Background technology]

[0002] With the improvement of the standard of living in modern society and the growing interest in beauty and health, there is an increasing demand for maintaining health and improving lifestyle-related diseases through health foods, nutritional supplements, etc. On the other hand, even if existing foods are ingested, the concentration of active ingredients is very low, and the effective concentration cannot be reached unless an excessive amount is ingested.

[0003] Thus, Patent Document 1 discloses the use of extracellular vesicles derived from microcellular algae as nanocarriers for the delivery of diagnostic agents, therapeutic agents, nutritional supplements and / or cosmetic agents. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2023-520101 Summary of the Invention [Problem to be solved by the invention]

[0005] In Patent Document 1, the extracellular vesicles derived from microcellular algae are merely used as a carrier for delivering an active ingredient, and the extracellular vesicles derived from the microcellular algae are not endowed with any unique function. In addition, ultracentrifugation is used to separate and concentrate the extracellular vesicles, but there is a problem in that the operation becomes complicated when separating and concentrating a large amount of them.

[0006] The present invention has been made in consideration of the above-mentioned problems, and aims to provide algae-derived cellular vesicles that have unique functions that can be used to maintain human health and improve lifestyle-related diseases, as well as to provide a production method that can efficiently concentrate the cellular vesicles derived from such cells. [Means for solving the problem]

[0007] The present inventors conducted extensive research to solve the above problems, and discovered that cellular vesicles contained in algae such as Chlorella (Chlorellaceae), Euglenophyceae, or Laminariaceae can be used to maintain human health and improve lifestyle-related diseases, thereby completing the present invention. The cellular vesicles described in this specification include not only extracellular vesicles naturally secreted from cells, but also vesicles composed of a complex of lipids and proteins produced by artificial treatments such as heat treatment.

[0008] That is, the present invention includes the following inventions. [Invention 1] A biologically active cellular vesicle obtained by heat-treating algae and / or an extract thereof. [Invention 2] The biologically active cellular vesicle according to Invention 1, characterized in that the algae is at least one species selected from Chlorella (Chlorellaceae), Euglenophyceae, and Laminariaceae. [Invention 3] A cellular vesicle having biological activity according to Invention 1 or 2, characterized in that the biological activity activates the transcription factor NRF2. [Invention 4] An NRF2 activator according to invention 3, characterized in that it contains cellular vesicles. [Invention 5] 4. An antioxidant according to invention 3, characterized in that it contains cellular vesicles. [Invention 6] 4. An anti-inflammatory agent according to invention 3, characterized in that it contains cellular vesicles. [Invention 7] 4. A melanin synthesis inhibitor according to invention 3, characterized in that it contains cellular vesicles. [Invention 8] 3. The cell introduction agent according to invention 1 or 2, characterized in that it contains cellular vesicles. [Invention 9] 3. A food or beverage composition according to invention 1 or 2, characterized in that it contains cellular vesicles. [Invention 10] 3. A pharmaceutical composition according to claim 1 or 2, characterized in that it contains cellular vesicles. [Invention 11] A method for producing biologically active cellular vesicles, comprising a step of heat-treating algae and / or an extract thereof. [Invention 12] A method for producing cellular vesicles according to Invention 11, characterized in that the algae is at least one species selected from Chlorella (Chlorellaceae), Euglenophyceae, and Laminariaceae. [Invention 13] A method for producing biologically active cellular vesicles according to invention 11 or 12, characterized in that the heat treatment is carried out at 60°C or higher. [Invention 14] 13. A method for producing biologically active cellular vesicles according to invention 11 or 12, characterized in that it comprises a step of adding a carotenoid. [Invention 15] A method for producing biologically active cellular vesicles according to Invention 13, characterized in that it comprises a step of adding a carotenoid. Effect of the Invention

[0009] The biologically active cellular vesicles of the present invention are obtained by heat-treating algae and / or their extracts, and have biological activities such as antioxidant effect, anti-inflammatory effect, and melanin synthesis inhibition effect, and therefore can be used in food and beverage compositions or pharmaceutical compositions that are low-cost and highly safe and can be used to maintain human health and improve lifestyle-related diseases.

[0010] The method for producing biologically active cellular vesicles of the present invention can increase the yield simply by heat treatment, making it possible to easily and low-costly produce cellular vesicles that can be used to maintain human health and improve lifestyle-related diseases. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is an image of a cellular vesicle. [Diagram 2] FIG. 2 is a graph showing the components of cellular vesicles of Euglena and Chlorella in terms of fluorescent signal intensity. [Diagram 3] FIG. 3 is a graph showing the reconstitution of cellular vesicles by heating and pressurizing powdered raw materials of Euglena, Chlorella, and kelp. [Figure 4] Figure 4 is an electron microscope photograph of cellular vesicles derived from Euglena. [Diagram 5] Figure 5 is a graph showing the size of cellular vesicles derived from Euglena. [Figure 6] Figure 6 is a graph showing the difference in the amount of reconstituted cellular vesicles derived from Euglena due to differences in heat treatment conditions. [Figure 7] FIG. 7 shows the results of detection of fucoxanthin and lutein contained in cellular vesicles derived from Euglena by mass spectrometry. [Figure 8] FIG. 8 is a graph showing the accumulation effect in cellular vesicles by the addition of carotenoids. [Figure 9] FIG. 9 shows Western blotting results showing the antioxidant effect induced by heme oxygenase in cellular vesicles. [Figure 10] FIG. 10 is a graph showing that heme oxygenase induction is NRF2 sequence dependent. [Figure 11] Figure 11A) is a graph evaluating the anti-inflammatory activity of cellular vesicles from each algae based on the reduction in nitric oxide (NO) levels, and Figure 11B) is a graph evaluating the anti-inflammatory activity of cellular vesicles derived from Euglena that have accumulated carotenoids based on the reduction in nitric oxide (NO) levels. [Figure 12] FIG. 12 is a graph showing the inhibition of TNFα secretion. [Figure 13] FIG. 13A) is a photograph comparing the intestinal lengths of mice using an inflammatory bowel disease model mouse, and FIG. 13B) is a graph comparing the intestinal lengths of mice using an inflammatory bowel disease model mouse. [Figure 14] FIG. 14 is a graph showing the quantification of the mRNA level of the inflammatory cytokine TNFα extracted from the intestine of an inflammatory bowel disease model mouse. [Figure 15] Figure 15A) is a photograph evaluating the inhibition of melanin synthesis in cellular vesicles, Figure 15B) is a graph showing the inhibition of melanin synthesis in cellular vesicles, and Figure 15C) is a graph showing the inhibition of melanin synthesis in cellular vesicles derived from Euglena that have accumulated carotenoids. [Figure 16] FIG. 16 is a graph showing that NRF2 activation signaling by cellular vesicles derived from Chlorella is involved in the inhibition of melanin synthesis. [Figure 17] Figure 17 shows photographs in which the uptake of algae-derived cellular vesicles into cells was evaluated using fluorescent labels. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The biologically active cellular vesicles of the present invention are biologically active cellular vesicles extracted from algae by heat treatment, particularly biologically active cellular vesicles extracted from Chlorella (Chlorellaceae), Euglenophyceae, or Laminariaeae by heat treatment. Here, the cellular vesicles include not only extracellular vesicles naturally secreted from cells, but also vesicles composed of a lipid-protein complex produced by artificial treatment such as heat treatment.

[0013] In the present invention, "algae" is a general term for organisms that perform oxygenic photosynthesis, excluding mosses, ferns, seed plants, and the like that mainly live on land, and includes everything from cyanobacteria, which are true bacteria, to most diatoms, which are unicellular eukaryotic organisms, some green algae and zygophytes, and dinoflagellates, as well as multicellular marine algae, such as red algae, brown algae, and green algae.

[0014] In the present invention, "Chlorella" is a general term for freshwater unicellular green algae mainly belonging to the Chlorophyta division, Chlorophyceae class, and Chlorellaceae family, and is a microorganism that is almost spherical with a diameter of 2 to 10 μm, has chlorophyll in its cells, and appears green or pale green. Among them, the Chlorella genus is widely used in industries such as food, cosmetics, and animal feed, and representative species used industrially include Chlorella pyrenoidosa, Chlorella ellipsoidea, Chlorella vulgaris, and Chlorella regularis.

[0015] The type of Chlorella used in the present invention is not particularly limited and any species may be used, but it is preferable to use Chlorella that is used industrially, and Chlorella vulgaris (C. vulgaris) is particularly preferable from the viewpoint of easy availability. Since Chlorella that is used industrially is easily available, the cellular vesicles of the present invention can be easily produced.

[0016] In the present invention, "Euglena" refers to a group of unicellular eukaryotic algae in the Euglenophyceae, a group of flagellates that are motile and have chlorophyll in their cells. Among them, the genus Euglena is widely used in the pharmaceutical, cosmetic, and biofuel industries, and representative species include Euglena chadefaudii, Euglena deses, Euglena gracilis, Euglena granulata, Euglena mutabilis, Euglena proxima, Euglena spirogyra, and Euglena viridis, with Euglena gracilis being particularly used industrially.

[0017] The type of Euglena used in the present invention is not particularly limited and any species may be used, but industrially used Euglena is preferred, and Euglena gracilis is particularly preferred from the viewpoint of easy availability. Since industrially used Euglena is easily available, the cellular vesicles of the present invention can be easily produced.

[0018] In the present invention, "kelp" is a general name for seaweed belonging to the family Laminariaceae of the order Laminariales of the class Phaeophyceae. Representative species of the family Laminariaceae include the genus Laminaria, Cymathere, Saccharina, Costaria, Agarum, Arthrothamnus, Streptophyllopsis, Eisenia, Ecklonia, and Eckloniopsis, and the genera Laminaria, Cymathere, Saccharina, and Arthrothamnus are particularly used industrially as kelp products.

[0019] The type of kelp used in the present invention is not particularly limited and any type may be used, but it is preferable to use kelp that is used industrially. Since industrially used kelp is easily available, the cellular vesicles of the present invention can be easily produced.

[0020] [Algal sample preparation] (Single-celled algae such as Chlorella and Euglena) In this embodiment, the preparation of a sample for extracting cellular vesicles from unicellular algae such as Chlorella or Euglena may be performed by any method that can extract cellular vesicles from unicellular algae. For example, the culture solution of unicellular algae may be separated by centrifugation, filtration, sedimentation, or the like, and raw unicellular algae may be used as is. The culture solution of unicellular algae may be washed with water or physiological saline, or may be used in a state dispersed in a liquid such as water. In this embodiment, the unicellular algae may be freeze-dried or spray-dried to dry, and dried. Furthermore, the unicellular algae may be ultrasonicated or mechanically treated, such as by homogenization, to extract the unicellular algae. If the unicellular algae is industrially used, such as Chlorella or Euglena, fine powder of the unicellular algae or the like that is commercially available or an extract thereof may be preferably used.

[0021] (Multicellular organisms such as kelp) In this embodiment, the preparation of the sample for extracting kelp cellular vesicles may be performed by any method that can extract kelp cellular vesicles. For example, seaweed of the Laminariales family (Laminariaeae) may be dried and then crushed using a crusher. The seaweed may be dried in the sun or may be dried using a commonly used dryer. If it is an industrially used species such as Laminariaceae, fine powder of commercially available kelp or the like may be preferably used.

[0022] [Extraction of cellular vesicles from algae] The conditions for extracting and reconstituting cellular vesicles from algae are not particularly limited, but a method of extracting and reconstituting an algae culture solution or dried product by heat treatment can be adopted. When the sample for extraction is a dry powder, the extraction temperature is not particularly limited as long as an appropriate yield of the extracted and reconstituted product can be obtained, but for example, when water is used as the extraction solvent, it is 20 to 120°C, preferably 60 to 120°C, and more preferably 100 to 120°C. The extraction time is not particularly limited as long as an appropriate yield of the extracted and reconstituted product can be obtained, but for example, it is 1 to 60 minutes, preferably 10 to 20 minutes. The amount of solvent added is not particularly limited as long as an appropriate yield of the extract can be obtained, but 1 to 50 parts by mass, preferably 5 to 20 parts by mass of the extraction solvent is added to 1 part by mass of the dry powder of algae.

[0023] The obtained extract of algal cellular vesicles may be simply removed of impurities by filtering, treating with activated carbon, treating with acid, etc. The obtained extract and reconstituted product of algal cellular vesicles may be further purified to obtain a purified product. Purification may be performed using silica gel, liquid-liquid partitioning, ion exchange resin, adsorption chromatography, partition chromatography, etc.

[0024] [Application] The algal cellular vesicles of the present invention activate the NFR2 transcription factor, and thus can activate various biologically active functions associated with NFR2, such as anti-inflammatory effects, antioxidant effects, and melanin synthesis inhibition effects. Therefore, they can be used for preventing lifestyle-related diseases, preventing aging and cancer, and for whitening effects. The algal cellular vesicles of the present invention can be used not only for medicinal purposes but also for non-medicinal purposes, and can be suitably used in foods and beverages aimed at patients with these diseases and healthy individuals who are conscious of preventing these diseases.

[0025] The algal cellular vesicles of the present invention have an anti-inflammatory effect and can therefore be suitably used as an anti-inflammatory agent. It is becoming clear that chronic inflammation causes damage to biological tissues and functional impairment, which in turn leads to diseases such as arteriosclerosis, diabetes, and obesity. The algal cellular vesicles of the present invention can suppress chronic inflammation and can be used not only for medicinal purposes but also for non-medicinal purposes, and can be suitably used in foods and beverages intended for patients with these diseases and healthy individuals who are conscious of preventing these diseases.

[0026] The algal cellular vesicles of the present invention have an antioxidant effect and can be suitably used as an antioxidant. It is known that active oxygen generated in our bodies causes damage to biological tissues and is therefore related to aging, cancer, and lifestyle-related diseases. The antioxidant of the present invention can protect the body from active oxygen and can be used not only for medical purposes but also for non-medicinal purposes, and can be suitably used in foods and beverages for patients with these diseases and healthy people who are conscious of preventing these diseases.

[0027] The algal cellular vesicles of the present invention have an inhibitory effect on melanin synthesis, and therefore can be suitably used as a melanin synthesis inhibitor. The algal cellular vesicles of the present invention have an effect of inhibiting the overproduction of melanin, and can be used to prevent, improve, and treat pigmentation such as rough skin, age spots, and freckles. The melanin synthesis inhibitor of the present invention can be used not only for medical purposes but also for non-medicinal purposes, and can be suitably used in foods and beverages aimed at patients with these diseases and healthy people who are conscious of preventing these diseases.

[0028] The algal cellular vesicles of the present invention have the ability to penetrate into cells, and therefore can be suitably used as a cell introduction agent for introducing substances such as pharmaceuticals into cells. Because the algal cellular vesicles of the present invention function as a cell introduction agent, they can be used not only for pharmaceutical purposes such as drug delivery systems, but also for non-pharmaceutical purposes.

[0029] [Cosmetics] The cosmetic preparation contains the anti-inflammatory agent, antioxidant and / or melanin synthesis inhibitor of the present invention as an active ingredient, and is provided in combination with a pharma- ceutically acceptable base material or carrier. The cosmetic preparation may also contain additives such as excipients, coating agents, binders, bulking agents, disintegrants, lubricants, diluents, osmotic pressure regulators, pH regulators, emulsifiers, dispersants, stabilizers, antioxidants, surfactants, preservatives, UV absorbers, moisturizers, colorants, fragrances, thickeners, bactericides, and cell activators, within the limits of pharma- ceutically acceptable limits.

[0030] Specific examples of cosmetics include skin care cosmetics such as lotions, creams, cleansing products, packs, oil liquids, massage products, beauty serums, cleaning agents, deodorants, hand creams, and lip balms; makeup cosmetics such as makeup bases, face powders, liquid foundations, oil-based foundations, blushers, eye shadows, mascara, eyeliners, eyebrows, and lipsticks; antiperspirants, ultraviolet protection cosmetics such as sunscreen lotions and sunscreen creams, and topical skin preparations, and also include those used as quasi-drugs.

[0031] The blending ratio of the anti-inflammatory agent, antioxidant and / or melanin synthesis inhibitor in the cosmetic is appropriately set depending on the effective amount, the form of the cosmetic, and the like, and is, for example, 0.000001 to 1 mass %, preferably 0.000001 to 0.01 mass %, and more preferably 0.000003 to 0.01 mass % relative to the total amount of the cosmetic.

[0032] [Whitening cosmetics] The whitening cosmetic agent is provided by combining the melanin synthesis inhibitor of the present invention as an active ingredient with a pharma- ceutically acceptable base material or carrier. The whitening cosmetic agent may also contain additives such as excipients, coating agents, binders, bulking agents, disintegrants, lubricants, diluents, osmotic pressure regulators, pH regulators, emulsifiers, dispersants, stabilizers, antioxidants, surfactants, preservatives, UV absorbers, moisturizers, colorants, fragrances, thickeners, bactericides, cell activators, and anti-inflammatory agents, within the limits of pharma- ceutically acceptable limits.

[0033] The blending ratio of the melanin synthesis inhibitor in the whitening cosmetic agent is appropriately set depending on the effective amount and the form of the whitening cosmetic agent, and is, for example, 0.000001 to 1 mass %, preferably 0.000001 to 0.01 mass %, and more preferably 0.000003 to 0.01 mass %, relative to the total amount of the whitening cosmetic agent.

[0034] [Skin protectant] The skin protective agent contains the antioxidant and anti-inflammatory agent of the present invention as active ingredients, and is provided in combination with a pharma- ceutically acceptable base material or carrier. The skin protective agent may also contain additives such as excipients, coating agents, binders, bulking agents, disintegrants, lubricants, diluents, osmotic pressure regulators, pH regulators, emulsifiers, dispersants, stabilizers, antioxidants, surfactants, preservatives, UV absorbers, moisturizers, colorants, fragrances, thickeners, bactericides, cell activators, and anti-inflammatory agents, within the limits of pharma- ceutically acceptable limits.

[0035] The blending ratio of the antioxidant in the skin protective agent is set appropriately depending on the effective amount and the form of the skin protective agent, but is, for example, 0.000001 to 1 mass%, preferably 0.000001 to 0.01 mass%, and more preferably 0.000003 to 0.01 mass% relative to the total amount of the skin protective agent.

[0036] [Anti-wrinkle agent] The anti-wrinkle agent is provided by combining the antioxidant and anti-inflammatory agent of the present invention as active ingredients with a pharma- ceutically acceptable base material or carrier. The anti-wrinkle agent may also be appropriately mixed with additives such as excipients, coating agents, binders, bulking agents, disintegrants, lubricants, diluents, osmotic pressure regulators, pH regulators, emulsifiers, dispersants, stabilizers, antioxidants, surfactants, preservatives, UV absorbers, moisturizers, colorants, fragrances, thickeners, bactericides, cell activators, and anti-inflammatory agents within the pharma-ceutically acceptable limits.

[0037] The blending ratio of the antioxidant and anti-inflammatory agent in the anti-wrinkle agent is set appropriately depending on the effective amount and the form of the anti-wrinkle agent, and is, for example, 0.000001 to 1 mass%, preferably 0.000001 to 0.01 mass%, and more preferably 0.000003 to 0.01 mass% relative to the total amount of the anti-wrinkle agent.

[0038] [Food and beverages] The food and drink can contain the anti-inflammatory agent, antioxidant and / or melanin synthesis inhibitor of the present invention as an active ingredient. These foods and drinks can also be used for skin protection, wrinkle improvement, and whitening effects. These foods and drinks can also be provided as functional drinks or functional foods. Functional foods include foods for specified health uses, foods with nutrient functions, foods with functional claims, foods for the elderly, health supplements (balanced nutritional foods, supplements), etc.

[0039] Examples of foods include grains, potatoes, seafood, meat, eggs, oils and fats, dairy products, vegetables, beans, fruits, sugars, seaweed, confectioneries, seasonings, and processed foods.

[0040] Processed foods include, but are not limited to, seafood products such as chikuwa and kamaboko; livestock products such as ham and sausage; sweets such as cookies, biscuits, snacks, chocolate, and cake; noodles such as soba, udon, fresh noodles, Chinese noodles, and pasta; breads such as white bread and sweet bread; fermented processed foods such as natto and miso; soybean foods such as tofu and okara; pickles such as lightly pickled and bran-pickled foods; canned seafood, processed meat, vegetables, and fruit; dairy products such as butter, margarine, yogurt, cheese, and milk; and frozen desserts such as ice cream and sorbet.

[0041] Examples of beverages include, but are not limited to, fruit beverages such as roasted and dried products as substitutes for tea leaves, fruit juice beverages, vegetable juices, flavored beverages, and diluted fruit beverages; carbonated beverages; luxury beverages such as coffee, coffee beverages, coffee-containing soft drinks, cocoa beverages, black tea, green tea, matcha, oolong tea, barley tea, and roasted green tea; vinegar beverages; soft drinks such as sports drinks; milk; dairy beverages; lactic acid beverages; lactic acid bacteria beverages; soy beverages such as soy milk and prepared soy milk; alcoholic beverages such as beer, sake, shochu, liqueurs, and wine; non-alcoholic beverages; and nutritional beverages containing taurine, royal jelly, amino acids, vitamins, minerals, and iron.

[0042] When applied to food and beverages containing at least one of the anti-inflammatory agent, antioxidant and / or melanin synthesis inhibitor of the present invention as an active ingredient, the content of algae-derived cellular vesicles is not particularly limited, but is generally 0.001 to 100 mg / kg body weight per day, preferably 0.1 to 20 mg / kg body weight, and more preferably 1 to 10 mg / kg body weight, in solid content terms, for humans and animals.

[0043] [Medicines, quasi-drugs] Medicines and quasi-drugs can contain the anti-inflammatory agent, antioxidant and / or melanin synthesis inhibitor of the present invention as an active ingredient. Medicines and quasi-drugs can be formulated into an appropriate form and administered to humans or animals in any dosage form. The dosage form is not particularly limited, but examples include oral, transdermal, enteral, transmucosal, and injection.

[0044] Pharmaceuticals and quasi-drugs can be introduced into patients by compounding an active ingredient with the cell introduction agent of the present invention. Pharmaceuticals and quasi-drugs can be formulated into an appropriate form and administered to humans or animals in any dosage form. The dosage form is not particularly limited, but examples include oral, transdermal, enteral, transmucosal, and injection.

[0045] Dosage forms for oral administration include, for example, pills, powders, tablets, granules, capsules, syrups, liquids, and the like.

[0046] In the case of parenteral administration, examples include intravenous injection, intramuscular injection, transdermal absorption agent, inhalant, suppository, eye drop, nasal drop and the like.

[0047] These various preparations can be produced by conventional methods using pharma- ceutical acceptable carriers such as commonly used excipients, disintegrants, binders, lubricants, colorants, diluents and the like.

[0048] Examples of excipients include lactose, corn starch, glucose, sorbitol, calcium carbonate, kaolin, and crystalline cellulose.

[0049] Examples of disintegrants include starch, sodium alginate, carboxymethylcellulose, carboxymethylcellulose calcium, carmellose calcium, croscarmellose sodium, carboxymethylstarch sodium, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, stearate monoglyceride, lactose, and the like.

[0050] Examples of binders include dimethyl cellulose, glucose liquid, starch liquid, gelatin liquid, bound cellulose, sucrose, D-mannitol, dextrin, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl starch, ethyl cellulose, shellac, calcium phosphate, polyvinylpyrrolidone, and the like.

[0051] Lubricants include, for example, talc, magnesium stearate, calcium stearate, colloidal silica, borax, polyethylene glycol, and the like.

[0052] When the present invention is applied to pharmaceuticals or quasi-drugs containing at least one of the anti-inflammatory agent, antioxidant, and / or melanin synthesis inhibitor, the content of algae-derived cellular vesicles is not particularly limited, but is generally 0.001 to 100 mg / kg body weight per day, preferably 0.1 to 20 mg / kg body weight, and more preferably 1 to 10 mg / kg body weight, in terms of solid content, for humans and animals. EXAMPLES

[0053] The present invention will be described in more detail and specifically below with reference to examples, but the present invention is not limited to the following examples.

[0054] [Extraction of cellular vesicles from Chlorella, Euglena and Kombu] Chlorella culture solution and powder (Chlorella vulgaris) were obtained from Wakasha LLC, Euglena culture solution (Euglena gracilis) was obtained from TYD H-MEDAKA, powdered Euglena (Euglena gracilis) was obtained from Lotz, and dried kelp powder (kelp from Hidaka, Hokkaido) was obtained from Itoen. 100 mL of Chlorella culture solution was centrifuged at 8000g for 30 minutes, the supernatant was filtered through a 0.45 μm polyethersulfone (PES) filter (47 mm, Thermofisher), and the filtrate was further filtered through a 0.1 μm polyethersulfone (PES) filter (Thermofisher). A 0.22 μm filter (47 mm) can also be used. A filter made of cellulose acetate (CA) (Thermofisher) was used for the Euglena culture solution. The filtrate that passed through the filter was concentrated on the membrane using a membrane with a smaller pore size than that of a 0.03 μm or 0.05 μm polyethersulfone (PES) filter (Membrane Solution). The concentrated cellular vesicles on the membrane were washed three times with phosphate buffer (pH = 6.8) (Fuji Film Wako) and finally collected with 1 to 2 mL of phosphate buffer. The collected cellular vesicles were passed through a 0.22 μm disk filter (GVS) to remove aggregates.

[0055] [Isolation of cellular vesicles from culture medium of Chlorella and Euglena] The lipids and proteins, which are the constituents of cellular vesicles, were labeled with 1 / 50 diluted ExoSparkler Exosome Membrane Labeling Kit-Red (Dojin Co., Ltd.) and 20 μM GIF-2276 (see PCT / JP2023 / 28846). Labeling with GIF-2276 was performed at 80°C for 5 minutes. After fluorescent labeling, the vesicles were separated by gel filtration and detected by fluorescence intensity. Detection was performed using a Shimadzu high-performance liquid chromatogram system (SCL-10A vp) equipped with two tandem fluorometers (RF-10A xl). A gel filtration resin (qEV10 70 nm, Izon Science Co., Ltd.) was packed into a stainless steel column (φ8 mm × 100 mm: Senshu Science Co., Ltd.). Separation of cellular vesicles was carried out using a buffer (10 mM Tris-HCl (pH 6.8), 0.5 mM EDTA, 50 mM NaCl) at a flow rate of 1 mL / min.

[0056] Figure 1 is an image of a cellular vesicle, and Figure 2 is a graph showing the components of cellular vesicles of Euglena and Chlorella in terms of the intensity of the fluorescent signal. As shown in Figure 2, lipid and protein complexes were detected in both Euglena culture and Chlorella culture. In this gel filtration column separation, the following sizes were separated: 220 nm at 10.7 minutes, 100 nm at 12 minutes, and 30 nm at 13.3 minutes. As a result of examining the detected peaks, it was confirmed that the size distribution of cellular vesicles in Euglena culture was 300 nm-30 nm, and that in Chlorella culture was 300 nm-10 nm. The cellular vesicles are considered to have the form shown in Figure 1, and the lipids in the cell membrane of the cellular vesicles were fluorescently labeled with ExoSparkler Exosome Membrane Labeling Kit-Red, and the cell membrane proteins were fluorescently labeled with GIF-2276.

[0057] [Isolation and characterization of cellular vesicles from powdered raw materials of Chlorella, Euglena and Kelp] Figure 3 is a graph showing the reconstitution of cellular vesicles by heating and pressurizing the powdered raw materials of Euglena, Chlorella and Kombu, Figure 4 is an electron microscope photograph of cellular vesicles derived from Euglena, and Figure 5 is a graph showing the measurement of the size of cellular vesicles derived from Euglena. To reconstitute cellular vesicles from algae, 20g of the above-mentioned Chlorella powder, Euglena powder and Kombu tea were added and suspended in 100mL of phosphate buffer, and then heated and pressurized at 110°C for 20 minutes to prepare a sample for the isolation of cellular vesicles. After the heating and pressurizing treatment, the mixture was centrifuged at 8000g for 30 minutes, and the cellular vesicles of each were isolated using the same method as that for the isolation of cellular vesicles from the above-mentioned Chlorella and Euglena culture solutions.

[0058] The size of cellular vesicles was evaluated using an electron microscope. A solution of cellular vesicles from Euglena cultures was deposited on a glow-discharged carbon support film attached to copper grids U1013 (EM Japan). These grids were then stained with uranyl acetate and observed at 80 kV or 200 kV using a JEM-2100F electron microscope (JEOL). As a result, particles with a maximum diameter of 250 nm could be observed.

[0059] As shown in Figure 3, cellular vesicles were detected in all powdered raw materials, and as shown in Figure 4, cellular vesicles were confirmed by electron microscopy. When the size of the cellular vesicles derived from Euglena was measured using a Zetasizer Nano ZS (Malvern Instruments), the average size was measured to be 254 nm, as shown in Figure 5. A small number of particles were also observed in the 30-60 nm range.

[0060] [Evaluation of heat treatment of algae-derived cellular vesicles] Figure 6 is a graph showing the difference in the amount of reconstituted cellular vesicles derived from Euglena due to differences in heat treatment conditions, and the evaluation method is explained below. The effect of temperature on the reconstitution of cellular vesicles from algae was examined. 2 g of dried Euglena powder was suspended in 10 mL of phosphate buffer and heat-treated for 20 minutes. The heat treatment temperature was changed from 60°C to 120°C, and cellular vesicles were extracted. After heat treatment, the supernatant was collected by centrifugation at 8000g for 30 minutes, passed through a 0.45 μm disk filter (PES) (manufactured by GVS), and then passed through a 0.1 μm disk filter (PES) (manufactured by BM Equipment). For cellular vesicles, a 0.05 μm PES membrane was set in a filter funnel (manufactured by Shibata Chemical Co., Ltd.), and the cellular vesicles were accumulated on the filter by aspiration. After washing three times with phosphate buffer, the cells were recovered in 500 μL of phosphate buffer, and the lipids and proteins were fluorescently labeled in the same manner as described in [Isolation of cellular vesicles from culture medium of Chlorella and Euglena].

[0061] As shown in Figure 6, the reorganization of cellular vesicles was promoted by increasing the heat treatment temperature compared to samples that were not heat-treated, and it was shown that the reorganization of cellular vesicles was further promoted when the heat treatment temperature exceeded 100°C, which is the temperature of the heat and pressure treatment.

[0062] [Evaluation of carotenoids contained in cellular vesicles derived from algae] FIG. 7 shows the results of detection of fucoxanthin and lutein contained in cellular vesicles derived from Euglena by a mass spectrometer, and the analytical method is described below. The content of carotenoids in cellular vesicles was determined by adding an equal amount of chloroform (manufactured by Fujifilm Wako Co., Ltd.) to 500 μL of a suspension of cellular vesicles derived from Euglena, resuspending the suspension, centrifuging at 8000 g for 10 minutes to collect the chloroform layer, and evaporating the chloroform with an evaporator. The dried product was dissolved in 50 μL of methanol, diluted with an equal amount of distilled water, and 10 μL was analyzed by liquid chromatography. The liquid chromatography analysis conditions were a C18 column (7 mm x 150 mm: manufactured by Kanto Chemical Co., Ltd.) and separation was performed with a gradient of 10% acetonitrile (manufactured by Kanto Chemical Co., Ltd.) - 100% acetonitrile, 10 minutes (0.5 mL / min). Fucoxanthin or lutein was detected by mass spectrometry (manufactured by Waters, Xevo QTof).

[0063] As shown in FIG. 7, fucoxanthin was identified in the negative mode at 657.4166, and lutein was identified at 567.4180.

[0064] [Evaluation of carotenoid accumulation in algae-derived cytoplasmic vesicles] Figure 8 is a graph showing the accumulation effect of carotenoids in cellular vesicles, and the evaluation method is explained below. During the heat treatment to prepare cellular vesicles from algae, 1 mg of the carotenoids fucoxanthin or lutein (Fujifilm Wako Co., Ltd.) was added to prepare them. Carotenoids were extracted from cellular vesicles and separated using the method described in [Evaluation of carotenoids contained in cellular vesicles derived from algae], and fucoxanthin (449 nm) and lutein (445 nm) were detected using a spectrophotometer (ThermoFisher, NanoDropND-1000).

[0065] As shown in FIG. 8, it was confirmed that both fucoxanthin and lutein accumulated in cellular vesicles, and that fucoxanthin in particular accumulated in large amounts in cellular vesicles.

[0066] [Evaluation of the antioxidant effect of algae-derived cytoplasmic vesicles] Figure 9 shows the antioxidant effect of heme oxygenase induction in cellular vesicles by Western blotting. The evaluation method is explained below. The antioxidant effect was evaluated by induction of heme oxygenase (OH-1), a protein marker. RAW264.7 cells were cultured in 12-well plates (As One) using RPMI medium (Fuji Film Wako) supplemented with 10% fetal bovine serum (Merck) and 1x penicillin-streptomycin (Fuji Film Wako). When the cells were grown to semi-confluency, cellular vesicles derived from Euglena, adjusted to a protein concentration of 1μg / mL (measured with Protein Assay CBB solution (Nacalai)), were added to the medium at a concentration of 1-10%. To efficiently induce heme oxygenase, lipopolysaccharide (LPS: Fuji Film Wako) was added at 500ng / mL. After 12 hours of incubation, the cells were washed twice with phosphate buffer and dissolved in 150 μL of SDS sample buffer (0.125 mol / L Tris-HCl, 4 w / v% SDS, 20 w / v% Glycerol, 0.002 w / v% bromophenol blue pH 6.8, 10% mercaptoethanol) and treated at 100 °C for 10 minutes. After SDS-PAGE, the cells were blotted onto a PVDF membrane (Merck) and blocked with Blocking One. Heme oxygenase was detected with anti-heme oxygenase antibody (Gene Tex) diluted 1 / 5000 and anti-rabbit HRP antibody (Abcam) diluted 1 / 5000. A fluorescent substrate (ATTO) was used as the HRP substrate, and luminescence was detected with ChemDoc (BioRad). GAPDH-HPR (MBL) was used at a 1 / 5000 dilution as a loading control.

[0067] As shown in Figure 9, the group to which Euglena-derived cellular vesicles were added showed significant induction of heme oxygenase protein. The amount of protein expression gradually increased depending on the concentration of cellular vesicles (1%, 3%, and 10%), clearly demonstrating that the induction of heme oxygenase protein was promoted depending on the concentration of cellular vesicles.

[0068] [Evaluation of the antioxidant effect of algae-derived cytoplasmic vesicles and their relationship to NRF2 response elements] Figure 10 is a graph showing that heme oxygenase induction is dependent on the NRF2 sequence, and the evaluation method is explained below. The NRF2-dependent induction of heme oxygenase (HO-1) was evaluated by a heme oxygenase promoter assay. We prepared reporters with and without the heme oxygenase (HO-1) promoter and NRF2 response element (enhancer), which reacts with NRF2. HEK293 cells were seeded on a 24-well plate, and 12 hours later, the heme oxygenase promoter / NRF2 response enhancer was introduced. Correction was performed using a Renilla luciferase reporter plasmid (pRL-TK: Promega) and a promoterless control plasmid pTAL that was also introduced at the same time. In addition, a reporter with only the heme oxygenase promoter and no NRF2 response element was introduced. TranITX2 (Mirus) was used for introduction. 12 hours after plasmid introduction, cellular vesicles were added at a concentration of 1 μg / mL in protein equivalent, and the cells were cultured for another 12 hours. A dual luciferase assay kit (Promega) was used to detect the reporter.

[0069] As shown in Figure 10, the cellular vesicle-added group suppressed heme oxygenase promoter activity in an NRF2 response element-dependent manner. In addition, the fucocysanthin-increased group showed an enhanced suppression effect on heme oxygenase promoter activity (data not shown).

[0070] [Evaluation of the anti-inflammatory effect of algae-derived cellular vesicles] Figure 11A) is a graph evaluating the anti-inflammatory activity of cellular vesicles of each algae based on the reduction in the amount of nitric oxide (NO), and Figure 11B) is a graph evaluating the anti-inflammatory activity of cellular vesicles derived from Euglena that have accumulated carotenoids based on the reduction in the amount of nitric oxide (NO). Below, these evaluation methods are explained. Since it is known that NRF2 activation signaling is linked to anti-inflammatory signaling, we decided to evaluate anti-inflammatory activity. Anti-inflammatory activity was evaluated based on the reduction in the amount of nitric oxide (NO) induced by lipopolysaccharide (Fuji Film Wako Co., Ltd.). Mouse macrophage RAW264.7 cells were cultured in a 96-well plate in RPMI medium (Fuji Film Wako Co., Ltd.) containing 10% fetal bovine serum and 1x antibiotics. After 12 hours, 500ng / mL of lipopolysaccharide was added, and at the same time, cellular vesicles were added at a concentration of 3ng / mL in terms of protein. After 24 hours of culture, the concentration of NO accumulated in the medium was measured. The NO concentration was measured by mixing 100 μL of the supernatant with 100 μL of Griess reagent [0.1% sulfanilamide, 1% N-(1-naphthyl)ethylenediamine [Merck], 0.625% (w / v) phosphoric acid solution] and incubating at room temperature for 20 minutes.Then, the absorbance at 560 nm was measured using the Glomax Multi Detection System (Promega).

[0071] As shown in Figure 11A), the group to which cellular vesicles were added showed a significant decrease in NO accumulation in the medium. In particular, cellular vesicles derived from Chlorella showed a decrease in NO accumulation in the medium, resulting in the highest anti-inflammatory effect. Furthermore, as shown in Figure 11B), in the Euglena-derived cellular vesicles that had increased accumulation of fucoxanthin and lutein, the amount of NO accumulated in the medium was further decreased, and in particular, fucoxanthin caused a significant decrease in the amount of NO accumulated in the medium, resulting in an even greater anti-inflammatory effect.

[0072] [Evaluation of the inhibition of secretion of inflammatory cytokine TNFα by algae-derived cellular vesicles] FIG. 12 is a graph showing the inhibition of TNFα secretion, and the evaluation method is described below. It is known that NO production inhibitory activity also acts to inhibit the secretion of inflammatory cytokines. Therefore, the secretion of TNFα, an inflammatory cytokine, was measured. Mouse macrophage RAW264.7 cells were cultured in a 96-well plate in RPMI medium (manufactured by Fujifilm Wako Co., Ltd.) containing 10% fetal bovine serum and 1x antibiotics. After 12 hours, 500ng / mL of lipopolysaccharide was added, and at the same time, cellular vesicles derived from Euglena powder were also added at a protein concentration of 3 to 30ng / mL. After that, the cells were cultured for 24 hours, and the TNFα concentration accumulated in the medium was measured using an ELISA kit (manufactured by BioLegend Co., Ltd.).

[0073] As shown in Figure 12, the group with the addition of cellular vesicles significantly reduced the concentration of TNFα in the culture medium, and the amount of TNFα reduction increased depending on the concentration of cellular vesicles added, clearly demonstrating that the compound can be used effectively as an anti-inflammatory agent.

[0074] [Evaluation of the anti-inflammatory effect of algae-derived cytoplasmic vesicles at the biological level] Figure 13A) is a photograph comparing the intestinal length of mice using inflammatory bowel disease model mice, and Figure 13B) is a graph comparing the intestinal length of mice using inflammatory bowel disease model mice. The evaluation method is explained below. We examined whether algae-derived cellular vesicles have an anti-inflammatory effect at the living body level using a dextran sulfate (DSS)-induced inflammatory bowel disease model using mice (C57BL / 6J) (Nihon SLC Co., Ltd.). Extracts of cellular vesicles heated and pressurized at 120°C with 10 times the amount of phosphate buffer from Euglena powder and extracts extracted at room temperature were orally administered to model mice twice a day, morning and evening. The cellular vesicle content was adjusted to 300μg / mL in protein equivalent for both, and administered at 4mg / kg / day. The total protein content of the cellular vesicles extracted at room temperature was the same as that of the cellular vesicles adjusted at 120°C, resulting in a cellular vesicle content of approximately 1 / 5. Eight-week-old male mice were treated with 3% DSS (Biomedicals) for one week, and the colons were collected. Since colitis can be evaluated by colon shortening, the length of the colon was examined.

[0075] As shown in the photograph in Figure 13A), colon shortening was observed with 3% DSS treatment. In the group to which Euglena-derived cytoplasmic vesicles were added, the shortening effect of the colon was alleviated compared to the control, as shown in the graph in Figure 13B). In particular, the effect was particularly strong with cytoplasmic vesicles extracted at 120°C.

[0076] [Evaluation of expression of inflammatory cytokine TNFα in mouse colon] FIG. 14 is a graph showing the quantitative determination of the amount of mRNA of the inflammatory cytokine TNFα extracted from the intestine of an inflammatory bowel disease model mouse, and the evaluation method is explained below. TM RNA was extracted using an RNA purification kit (Nihon Genetic Co., Ltd.), and the amount of mRNA of the inflammatory cytokine TNFα was verified by quantitative PCR. For reverse transcription of mRNA, 500 ng of total RNA was used, and ReverTra Ace (registered trademark) (TOYOBO Corporation) was used. The following primers were used for TNFα. The following GAPDH primer set was used for correction. These primer sets were mixed with MixMyiQ using THUNDERBIRD (registered trademark) SYBR (registered trademark) qPCR Mix. TM Amplification and quantification were performed using the Single-Color Real-Time PCR Detection System (Bio-Rad).

[0077] TNFα primers Forward:5'-GCAATGGCAATTCTGATTGTAT-3' Reverse: 5'-GAAGGACTCTGGCTTTGTCTTT-3' Correction primer (GAPDH primer) Forward:5'-ACTCACGGCAAATTCAACGG-3' Reverse: 5'-GACTCCACGACATACTGAGC-3'

[0078] As shown in Figure 14, the expression level of TNFα in the mouse large intestine increased with 3% DSS treatment compared to the control, but it was confirmed that the expression level of TNFα was suppressed by treatment with Euglena cellular vesicles. In particular, the group of cellular vesicles treated at 120°C had a greater effect of suppressing TNFα expression. The above results confirmed that algae-derived cellular vesicles have the effect of suppressing inflammation in the body.

[0079] [Evaluation of inhibition of melanin synthesis in algae-derived cellular vesicles] Figure 15A) is a photograph evaluating the inhibition of melanin synthesis by cellular vesicles, and the evaluation method will be explained below. It is known that the NRF2 activation signal is linked to the anti-melanin production signal. Therefore, using mouse melanoma B16F10 cells, the amount of melanin production associated with the increase in intracellular cAMP concentration by the addition of forskolin (Fsk) was evaluated. B16F10 cells were cultured in DMEM medium in a 6-well plate for 12 hours. Then, forskolin (Fsk) (Tokyo Chemical Industry Co., Ltd.) was added to 20 μM (final concentration) and DMSO was added to 0.2% (final concentration) to induce melanin synthesis. Fsk is a substance that induces signals and promotes melanin synthesis in the same way as α-melanocyte-stimulating hormone (αMSH) in B16F10 melanoma cells. At the same time, cellular vesicles were added at a concentration of 3 ng / mL in protein equivalent. After 48 hours, the cells were washed once with phosphate buffer and collected in a 1.5 mL tube with 700 μL of phosphate buffer. To visually confirm the amount of melanin, the tube was centrifuged at 8000 g for 5 minutes to collect the cells at the bottom and photograph them.

[0080] Figure 15B) is a graph showing the inhibition of melanin synthesis in cellular vesicles, and the evaluation method is explained below. In order to quantify the amount of melanin produced in the cells, the collected cells were suspended in 400uL of distilled water, and 200μL was used for protein quantification and the remaining 200μL was used for melanin quantification. Melanin was extracted from each of the collected B16F10 melanoma cells. The method for extracting melanin is shown below. (1) 200 mL of 4N NaOH was added to each test sample and incubated at 60°C for 3 hours. (2) 100 μl of a methanol:chloroform (1:2) mixed solution was added to each test sample and stirred to extract melanin. (4) The mixture was centrifuged at 1,200 rpm for 10 minutes, and the supernatant was collected to obtain a melanin extract. (5) 100 μl of the melanin extract was dispensed into a 96-well plate, and the absorbance at 405 nm was measured. Blank treatment medium (-): The above treatment medium was not supplemented with cellular vesicles. Control treatment medium (Fsk(-)): The above treatment medium was free of cellular vesicles and FSK. Control treatment medium (Fsk(+)): The above treatment medium was added with or without cellular vesicles.

[0081] Figure 15C) is a graph showing the inhibition of melanin synthesis in cellular vesicles in which carotenoids have accumulated, and the evaluation method is explained below. An evaluation was conducted on the inhibition of melanin synthesis in euglena-derived cellular vesicles in which fucoxanthin and lutein have accumulated. The accumulation of fucoxanthin and lutein was performed using the same method as described in the section "Evaluation of the accumulation of carotenoids in algae-derived cellular vesicles," and the inhibition of melanin synthesis was performed using the method used in the evaluation of Figure 15B) above.

[0082] As shown in the results of Figures 15A) and 15B), both cellular vesicles derived from Euglena and cellular vesicles derived from Chlorella were confirmed to have an inhibitory effect on melanin synthesis. In particular, cellular vesicles derived from Chlorella were confirmed to have a high inhibitory effect on melanin synthesis. Furthermore, the results of Figure 15C) confirmed that the accumulation of carotenoids fucoxanthin and lutein in cellular vesicles derived from Euglena enhanced the inhibitory effect on melanin synthesis. In particular, the effect of fucoxanthin was high.

[0083] [Evaluation of inhibition of melanin synthesis in algae-derived cellular vesicles] Figure 16 is a graph showing that the NRF2 activation signal by cellular vesicles is involved in the inhibition of melanin synthesis, and the evaluation method will be explained below. When the NRF2 activation signal works in conjunction with the anti-melanin production signal, the induction of the melanin synthesis enzyme (tyrosinase) is involved, and it has been reported that the transcription factor MITF binding sequence on the tyrosinase promoter is involved in this induction (J Nat Med. 2022 Jan;76(1):132-143. doi: 10.1007 / s11418-021-01565-3.). Therefore, we examined the promoter inhibition effect of melanin synthesis enzyme of cellular vesicles derived from Chlorella (10 ng / mL in protein equivalent), which has a higher melanin synthesis inhibitory effect. For the evaluation, we used the normal tyrosinase promoter (mTyr) and a promoter lacking the MITF binding sequence (△MITF). The promoter assay was performed in the same manner as for heme oxygenase described in the section [Evaluation of the antioxidant effect of algae-derived cellular vesicles and the NRF2 response element].

[0084] As shown in Figure 16, Chlorella-derived cytoplasmic vesicles significantly suppressed the tyrosinase promoter activity, but did not affect the promoter lacking the MITF binding sequence. This indicates that cytoplasmic vesicles are involved in the inhibition of melanin synthesis by suppressing the tyrosinase promoter activity.

[0085] [Evaluation of uptake of algae-derived cytoplasmic vesicles into cells] Figure 17 shows a photograph of the uptake of algae-derived cellular vesicles into cells evaluated by fluorescent labeling, and the evaluation method is described below. In order to visualize the introduction of algae-derived cellular vesicles into cells, a method was examined in which cellular vesicles were labeled with a fluorescent compound such as GIF-2276, added to cultured cells, and monitored by fluorescence. Lipid labeling agents may also be used for fluorescent labeling. Labeling was performed under the labeling conditions described in the section on [Isolation of cellular vesicles from culture medium of Chlorella and Euglena], except for the condition of 12 hours at room temperature. Unreacted labeling reagents were removed using a 300K PVDF spin column (Apro Sciences), washed three times with phosphate buffer, and then recovered with 50 μL of phosphate buffer. HEK293 cells were seeded on a glass bottom dish in DMEM medium (Fuji Film Wako Co., Ltd.) containing 10% fetal bovine serum and 1x antibiotics. 24 hours after seeding, lysosomes were isolated using LysoBrite. TM The cells were stained with Red reagent (AAT Bioquest) for 20 minutes in advance, and then replaced with DMEM medium containing 1% fetal bovine serum. Labeled cellular vesicles (GIF) were then added, and after 3 hours, the cells were photographed under a microscope. The microscope used was a Keyence BZ-9000 with a 100x objective lens. The photographs are, from the left, a photograph of labeled lysosomes, a photograph of labeled cellular vesicles, and a superimposed photograph of the fluorescence of labeled lysosomes and cellular vesicles.

[0086] As shown in Figure 17, the labeled cellular vesicles were taken up into HEK293 cells and accumulated in lysosomes, demonstrating that algae-derived cellular vesicles can be suitably used as a cell introduction agent.

[0087] [Evaluation of the effects of algae-derived cellular vesicles on the skin] To consider the effect of algae-derived cellular vesicles (derived from Euglena) on the skin, 100 μL of the solution at a concentration of 1 μg / mL was applied to a band-aid and applied to the skin (fingers, wrists, and cheeks). After 24 hours, the band-aid was removed and the effect on the skin was examined. Phosphate buffer solution was used as a control. (-): No effect on the skin (+) : Slight effect (redness of the skin, etc.) (++): May affect the skin (itchiness, sores, etc.)

[0088] [Table 1]

[0089] As shown in Table 1, no significant effects were observed in the cellular vesicle-added group compared with the non-applied area and the phosphate buffer solution, and no adverse effects were observed on normal skin.

Claims

1. A biologically active cellular vesicle obtained by heat-treating algae and / or an extract thereof.

2. The biologically active cellular vesicle according to claim 1, characterized in that the algae is at least one selected from Chlorella (Chlorellaceae), Euglenophyceae, and Laminariaceae.

3. A biologically active cellular vesicle as described in claim 1 or 2, characterized in that the biological activity activates the transcription factor NRF2.

4. The NRF2 activator according to claim 3, characterized in that it contains said cellular vesicles.

5. The antioxidant according to claim 3, characterized in that it contains said cellular vesicles.

6. The anti-inflammatory agent according to claim 3, characterized in that it contains said cellular vesicles.

7. The melanin synthesis inhibitor according to claim 3, characterized in that it contains said cellular vesicles.

8. The cell introduction agent according to claim 1 or 2, characterized in that it contains the cellular vesicles.

9. The food and beverage composition according to claim 1 or 2, characterized in that it contains the cellular vesicles.

10. The pharmaceutical composition according to claim 1 or 2, characterized in that it contains said cellular vesicles.

11. A method for producing biologically active cellular vesicles, comprising a step of heat-treating algae and / or an extract thereof.

12. The method for producing cellular vesicles according to claim 11, characterized in that the algae is at least one selected from Chlorella (Chlorellaceae), Euglenophyceae, and Laminariaceae.

13. The method for producing cellular vesicles according to claim 11 or 12, characterized in that the heat treatment is performed at 60°C or higher.

14. A method for producing cellular vesicles as described in claim 11 or 12, characterized in that it includes a step of adding a carotenoid.

15. The method for producing cellular vesicles according to claim 13, further comprising the step of adding a carotenoid.

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