Antioxidant and fibroblast promoter

Abstract

The purpose of the present invention is to provide a novel component for improving various biological symptoms such as cutis laxa. The antioxidant of the present invention is characterized by containing pitaya-derived extracellular vesicles. The fibroblast promoter according to the present invention is characterized by containing pitaya-derived extracellular vesicles. The pitaya is, for example, red pitaya.

Description

Technical Field

[0001] The present invention relates to an antioxidant, a fibroblast promoter, and their uses. Background Art

[0002] In order to improve the symptoms of organisms such as the skin, various components have been developed. Moreover, especially from the viewpoints of safety and the like, active ingredients derived from natural products are required. Summary of the Invention Problems to be Solved by the Invention

[0003] Therefore, an object of the present invention is to provide a new ingredient for improving various organism symptoms such as skin laxity. Means for Solving the Problems

[0004] In order to achieve the above object, the antioxidant of the present invention is characterized by containing extracellular vesicles derived from pitaya.

[0005] The fibroblast promoter of the present invention is characterized by containing extracellular vesicles derived from pitaya. Effects of the Invention

[0006] The present inventors newly found that extracellular vesicles derived from pitaya have antioxidant ability, and for fibroblasts that produce collagen and elastin, they can promote the expression of genes involved in their production. Therefore, according to the present invention, for example, oxidation can be prevented, and in addition, the production of collagen, elastin, etc. can be promoted, so that the symptoms of organisms in which they are involved can be improved. Brief Description of the Drawings

[0007] Figure 1 It is a chart showing the particle size distribution of extracellular vesicles derived from red-fleshed pitaya in Example 1. Figure 2 It is a chart showing the antioxidant ability of extracellular vesicles derived from red-fleshed pitaya in Example 2. Figure 3 It is a chart showing the effect of extracellular vesicles derived from red-fleshed pitaya on fibroblasts in Example 3. Figure 4 It is a chart showing the particle size distribution of extracellular vesicles derived from red-fleshed pitaya in Example 4. Figure 5 It is a figure showing the result of a Western blot for detecting markers of EVs samples derived from red-fleshed pitaya in Test Example 5. Detailed Description of the Invention

[0008] [1] An antioxidant, characterized by containing extracellular vesicles derived from pitaya. [2] The antioxidant according to [1], wherein the extracellular vesicles are at least one of extracellular vesicles derived from dragon fruit juice and extracellular vesicles derived from dragon fruit leaves. [3] The antioxidant according to [1] or [2], wherein the average value (mean) of the particle size distribution of the extracellular vesicles is 50 to 500 nm. [4] The antioxidant according to any one of [1] to [3] is for non-oral use. [5] The antioxidant according to [4] is for percutaneous use. [6] The antioxidant according to any one of [1] to [3] is for oral use. [7] The antioxidant according to any one of [1] to [6], wherein the dragon fruit is red-fleshed dragon fruit. [8] A fibroblast promoter, characterized by containing extracellular vesicles derived from dragon fruit. [9] The fibroblast promoter according to [8], wherein the extracellular vesicles are at least one of extracellular vesicles derived from dragon fruit juice and extracellular vesicles derived from dragon fruit leaves.

[10] The fibroblast promoter according to [9], wherein the average value (mean) of the particle size distribution of the extracellular vesicles is 50 to 500 nm.

[11] The fibroblast promoter according to any one of [8] to

[10] is for non-oral use.

[12] The fibroblast promoter according to

[11] is for percutaneous use.

[13] The fibroblast promoter according to any one of [8] to

[10] is for oral use.

[14] The fibroblast promoter according to any one of [8] to

[13] promotes the production of at least one of collagen and elastin by fibroblasts.

[15] The fibroblast promoter according to any one of [8] to

[14] , wherein the dragon fruit is red-fleshed dragon fruit.

[16] An antioxidant method, characterized by adding extracellular vesicles derived from dragon fruit to a subject.

[17] A method for promoting the production of collagen by fibroblasts, characterized by contacting fibroblasts with extracellular vesicles derived from dragon fruit.

[18] A method for promoting the production of elastin by fibroblasts, characterized by contacting fibroblasts with extracellular vesicles derived from dragon fruit.

[19] A method for promoting the production of elastin by fibroblasts, characterized by contacting extracellular vesicles derived from dragon fruit.

[0009] Unless otherwise specified, the terms used in this specification can be used with the meanings commonly used in the relevant technical field.

[0010] In this specification, "extracellular vesicle" refers to membrane vesicles secreted from cells, hereinafter also referred to as EVs. Examples of extracellular vesicles include exosomes derived from endosomes secreted by the endocytic pathway and microvesicles (microparticles) derived from the plasma membrane.

[0011] (1) Extracellular vesicles derived from pitaya As described above, the antioxidant and the fibroblast promoter of the present invention are characterized by containing extracellular vesicles derived from pitaya. Below, the extracellular vesicles derived from pitaya will be described first.

[0012] In the present invention, the extracellular vesicles only need to be derived from pitaya. Pitaya is a plant of the genus Hylocereus in the family Cactaceae. In the present invention, unless otherwise specified, pitaya can be a fruit, an entire plant including the fruit, or any part thereof, and can have any meaning. Pitaya is also known as pitaya. There is no particular limitation on the variety of pitaya. For example, it can include red-fleshed pitayas such as Hylocereus costaricensis and Hylocereus polyrhizus, white-fleshed pitayas such as Hylocereus undatus, yellow-skinned and white-fleshed pitayas such as Hylocereus polyrhizus, and pink pitayas of the peach flesh variety (hybrid: Hylocereus undatus × Hylocereus ocanponis). Preferably, it is a red-fleshed pitaya.

[0013] In the preparation of the extracellular vesicles, for example, as the pitaya material, the entire pitaya plant can be used, or any part of the pitaya can be used. Examples of the part include fruits, fruit peels, pulp, seeds, pulp containing seeds, leaves, etc., or a mixture of any two or more parts can also be used. As the material, pulp or pulp containing seeds is preferably used. The fruit can be fully ripe or not fully ripe, and preferably fully ripe.

[0014] The extracellular vesicles derived from pitaya can be prepared, for example, from the liquid fraction sourced from the pitaya material. The liquid fraction can be, for example, the juice obtained by juicing the pitaya material, or a mixture of the crushed pitaya material and a solvent, or an extract sourced from the pitaya material. There are no particular limitations on the juicing method, and examples include pressing, rotation, rotary pressing, etc. As a specific example, as the pitaya material, it is preferably a fruit, pulp, or pulp containing seeds, and the juice (fruit juice) obtained by juicing it. The juice is preferably, for example, the juice of the pulp or the juice of the pulp containing seeds, and more preferably the juice of the pulp from which the seeds have been removed. The juice of the pulp from which the seeds have been removed can be prepared, for example, by removing the seeds from the juice obtained by juicing the pulp containing seeds. The removal of the seeds can be carried out by centrifuging the juice of the pulp containing seeds to remove the precipitate containing seeds. There are no particular limitations on the extraction method, and examples include adding a solvent to the crushed material and recovering the liquid fraction as an extract. The liquid fraction can be, for example, a concentrated solution such as the juice, the mixture, or the extract. In addition, the crushed material of the pitaya material can also be, for example, a paste. Examples of the crushed material include the crushed material of the pulp containing seeds. The solvent is not particularly limited and can be, for example, water, a buffer solution such as phosphate buffer (PBS), or an aqueous solvent such as physiological saline.

[0015] The pitaya material and the crushed material can be, for example, freshly prepared substances or stored substances. In the case of stored substances, they can be, for example, substances stored at room temperature, substances stored in a refrigerator, substances stored frozen, or substances restored to room temperature after being stored in a refrigerator or frozen. The liquid fraction can be, for example, freshly prepared liquid, liquid stored at room temperature, liquid stored in a refrigerator, or liquid stored frozen.

[0016] The liquid fraction can directly use the juice, the mixture, or the extract, or a concentrated solution can be used. Examples of the concentrated solution include concentrated solutions obtained by concentrating the juice, the mixture, or the extract. The concentrate of the juice is also referred to as pitaya extract. In addition, the liquid fraction can be a mixture (also referred to as a concentrated and reconstituted solution) formed by mixing the dried body of the juice or the dried body of the extract with a solvent. The dried body can be prepared, for example, by drying the juice or the extract. From the perspective of processing, it is preferably in powder form. Examples of the drying treatment include freeze-drying, etc.

[0017] Preparation of extracellular vesicles from the liquid fraction can be carried out under, for example, cooling conditions to room temperature conditions (e.g., 4°C to 37°C), and centrifugation and the like are preferably carried out at, for example, 4°C ± 2°C.

[0018] The liquid fraction can, for example, remove inclusions such as fibers and debris before preparing extracellular vesicles. Removal of the inclusions can be, for example, separation by standing, centrifugation, or filtration. In the case of standing, for example, the precipitated inclusions can be removed by stirring the liquid fraction, allowing it to stand, and then recovering the supernatant. In the case of centrifugation, the inclusions can be removed by centrifuging the liquid fraction and recovering the supernatant. The liquid fraction can, for example, be further subjected to centrifugation to remove inclusions after removing inclusions by standing. The conditions for standing are not particularly limited, for example, 5 to 60 minutes (e.g., 20 minutes). The conditions for centrifugation are not particularly limited, for example, 400 to 3000 × g (e.g., 2000 × g), 5 to 30 minutes (e.g., 10 minutes). For example, it is preferable to observe the particles of the liquid fraction that has been subjected to the inclusion removal treatment using a nanoparticle analysis system or the like to confirm the residual inclusions.

[0019] In addition, examples of the filtration include filtration using a filter material. When removing the inclusions from the liquid fraction using the filter material, for example, the residue on the filter material is removed as inclusions, and the filtrate fraction is recovered. The filter material can, for example, be a filter or the like. The pore size of the filter material is not particularly limited, for example, as long as the pores allow the extracellular vesicles to pass through. In addition, the liquid fraction after standing or centrifugation can be further filtered using a filter material.

[0020] The method for preparing the extracellular vesicles from the liquid fraction is not particularly limited, for example, ultracentrifugation can be employed. Specifically, the liquid fraction can be ultracentrifuged, and the precipitated fraction containing extracellular vesicles is recovered as the extracellular vesicle fraction. The conditions for ultracentrifugation are not particularly limited, for example, 50,000 to 150,000 × g (100,000 × g), 50 to 140 minutes (e.g., 70 minutes). The precipitated fraction can, for example, be suspended in the solvent and stored refrigerated or frozen.

[0021] As a specific example, when using the juice extract (juice), for example, the juice is stirred, allowed to stand at 4°C to room temperature, and the liquid is recovered to remove the precipitated inclusions. Then, the recovered liquid is centrifuged, the supernatant fraction is recovered, and this supernatant fraction is further ultracentrifuged to recover the precipitated fraction as the extracellular vesicle fraction.

[0022] In addition, when using the dried product (e.g., powder) of the juice extract, for example, the dried product can be suspended in a solvent, impurities can be removed by centrifugation, and the recovered liquid fraction can be subjected to the ultracentrifugation, thereby obtaining the extracellular vesicle fraction.

[0023] In addition, when using the extract obtained by concentrating the juice extract, for example, the solvent is added as needed and stirred, centrifugation is performed, the supernatant fraction is recovered, and the supernatant fraction is further subjected to ultracentrifugation to recover the precipitate fraction as the extracellular vesicle fraction.

[0024] In addition, the method for preparing the extracellular vesicles from the liquid fraction can be, for example, filtration using a filter material (also referred to as ultrafiltration). In this case, for example, a filter material having pores with a size through which the extracellular vesicles of pitaya pass, a filter material having pores with a size through which the extracellular vesicles do not pass, or a combination of both can be used.

[0025] The average value (mean) of the particle size distribution of the extracellular vesicles derived from pitaya is, for example, 50 to 500 nm.

[0026] The average value of the extracellular vesicles used in the present invention can be, for example, 320 ± 120 nm, 320 ± 100 nm, 320 ± 80 nm, 320 ± 50 nm, 320 ± 30 nm, 320 ± 20 nm, 320 ± 10 nm, 320 ± 5 nm. When the average value of the extracellular vesicles is 320 ± 120 nm, the mode in the particle size distribution is, for example, 290 ± 90 nm, 290 ± 87 nm, 290 ± 80 nm, 290 ± 50 nm, 290 ± 30 nm, 290 ± 20 nm, 290 ± 10 nm, 290 ± 5 nm. The mode can also be referred to as the peak in the particle size distribution. In addition, in this case, the SD in the particle size distribution of the extracellular vesicles is, for example, 80 ± 40 nm, 80 ± 30 nm, 80 ± 20 nm, 80 ± 10 nm, 80 ± 5 nm.

[0027] In addition, the average value of the extracellular vesicles used in the present invention can be, for example, 83 ± 21 nm, 83 ± 30 nm, 83 ± 20 nm, 83 ± 10 nm, 83 ± 5 nm. When the average value of the extracellular vesicles is 83 ± 21 nm, the mode in the particle size distribution is, for example, 67 ± 20 nm, 67 ± 10 nm, 67 ± 5 nm.

[0028] The method for measuring the particle size distribution of the extracellular vesicles is not particularly limited. For example, light scattering methods such as laser diffraction method and dynamic light scattering method, particle trajectory analysis method, nano tracking analysis method, etc. can be used. The measurement can be carried out using commercially available devices such as NanoSight (trade name, QuantumDesigh Corporation), etc.

[0029] (2) Antioxidant As described above, the antioxidant of the present invention is characterized by containing extracellular vesicles derived from pitaya. The extracellular vesicles contained in the antioxidant are, for example, extracellular vesicles isolated from the pitaya. The antioxidant of the present invention is characterized by containing the extracellular vesicles, and there are no particular restrictions on other structures and conditions. The extracellular vesicles derived from pitaya can refer to the description in (1) above.

[0030] The antioxidant of the present invention can, for example, contain only the extracellular vesicles derived from pitaya, or can also contain other components. The other components can be, for example, other components contained in the liquid fraction derived from the pitaya, or can also be other added components. The other added components are not particularly limited and can be appropriately selected according to the target site, usage method, etc. of using the antioxidant of the present invention.

[0031] The antioxidant of the present invention can be used, for example, in medicine, can also be used in specific health care, and can also be used in cosmetics. The antioxidant of the present invention can be, for example, a composition containing the extracellular vesicles derived from pitaya.

[0032] When the antioxidant of the present invention is a pharmaceutical composition, its administration route is not particularly limited. For example, it can be non-oral or oral. In the case of non-oral administration, for example, transdermal, subcutaneous, intravenous, intra-arterial, intraperitoneal, intranasal, topical, enteral, etc. can be cited. When the antioxidant of the present invention is a pharmaceutical composition, its form is not particularly limited and can be appropriately selected according to the administration route. As specific examples of the form, for example, liquid agents such as solutions and suspensions, emulsions, gels, sols, ointments, granules, tablets, capsules, etc. can be cited. When the antioxidant of the present invention is for oral use, the composition can be, for example, a so-called supplement.

[0033] When the antioxidant of the present invention is a cosmetic composition, its administration route is, for example, transdermal, and specific application sites can be cited, such as the skin of the face, neck, upper body, lower body, etc. When the antioxidant of the present invention is a cosmetic composition, its form is not particularly limited. For example, forms such as lotions, gels, emulsions, creams, oils, ointments, etc. can be cited.

[0034] The other additive components are not particularly limited and can be appropriately selected according to the administration route and form. As specific examples, excipients, diluents, antioxidants, preservatives, extenders, wetting agents, thickening agents, viscosity stabilizers, UV blockers, binders, vitamins, fragrances, pigments, etc. can be cited. The other additive components can be, for example, pharmaceutically acceptable components, etc.

[0035] In the antioxidant of the present invention, the content of the extracellular vesicles derived from pitaya is not particularly limited. For example, it can be appropriately set according to the type, age, sex, symptoms, etc. of the organism of the administration subject. As a specific example, in the case of a percutaneous composition, the content of the extracellular vesicles is, for example, 0.01 to 5% (w / w), etc. In addition, in the case of an oral composition, the daily dosage of the extracellular vesicles is, for example, 5 to 1000 mg, 25 mg, etc., and the number of daily administrations is not particularly limited, for example, 1 to 3 times, 1 to 2 times, 1 time.

[0036] The administration subject is, for example, a human or a non-human animal. Examples of non-human animals include mice, rats, rabbits, pigs, cows, camels, dogs, cats, etc.

[0037] (3) Fibroblast promoter The fibroblast promoter of the present invention is characterized by containing the extracellular vesicles derived from pitaya. The fibroblast promoter of the present invention is characterized by containing the extracellular vesicles, and other structures and conditions are not particularly limited. The extracellular vesicles derived from pitaya can refer to the description of (1) above. In addition, the fibroblast promoter of the present invention can refer to the description of the antioxidant of the present invention. In the present invention, promoting fibroblasts means, for example, promoting the production of elastin or collagen.

[0038] (4) Symptom improvement As described above, the extracellular vesicles derived from pitaya exhibit, for example, antioxidant ability and fibroblast promoting ability. The fibroblast promoting ability is, for example, promoting the fibroblasts to produce collagen and / or elastin. Therefore, according to the antioxidant of the present invention and the fibroblast promoter of the present invention, skin symptoms can be improved, such as improving wrinkles, improving sagging, anti-aging, moisturizing, reducing and removing nasolabial folds, improving skin texture, tightening open pores, improving sagging pores, cleaning pores, removing horny plugs, improving acne scars, keratin care, removing dark spots, improving skin texture, increasing transparency, improving barrier function, normalizing stratum corneum renewal, increasing skin-friendly bacteria, etc. In the present invention, improvement means, for example, inhibiting deterioration, alleviating symptoms, curing symptoms, etc. The antioxidant of the present invention and the fibroblast promoter of the present invention can also be referred to as symptom-improving compositions, for example.

[0039] (5) Antioxidant method The antioxidant method of the present invention is characterized by including the step of administering the antioxidant of the present invention. The present invention is characterized by using the antioxidant of the present invention, and there are no restrictions on other steps and conditions, etc. The present invention can refer to the description of the antioxidant of the present invention.

[0040] The administration method is not particularly limited. As described above, oral administration or parenteral administration can be cited. In the case of parenteral administration, for example, transdermal, subcutaneous, intravenous, intra-arterial, intraperitoneal, intranasal, topical, enteral, etc. can be cited. In the case of transdermal administration, specific application sites can be cited, for example, the skin of the face, neck, upper body, lower body, etc.

[0041] The administration mode is not particularly limited. For example, it can be in vivo or in vitro. In the case of in vitro, the administration object (also called the subject) can be cited, for example, cells, tissues or organs, etc. In the case where the administration is in vivo, the administration object can be cited, for example, humans or the non-human animals. In addition, in the case of in vivo, for example, it can be any one of oral administration and parenteral administration.

[0042] The administration conditions are not particularly limited. For example, they can be appropriately set according to the administration method, administration site, type of administration object, age, gender, symptoms, etc. of the administration object.

[0043] (6) Fibroblast promotion method The fibroblast promotion method of the present invention is characterized by including the step of administering the fibroblast promoter of the present invention. The present invention is characterized by using the fibroblast promoter of the present invention, and there are no restrictions on other steps and conditions, etc. The present invention can refer to the description of the fibroblast promoter of the present invention. The fibroblast promotion method of the present invention means, for example, promoting fibroblasts to produce elastin or collagen.

[0044] The administration method is not particularly limited. As described above, oral administration or parenteral administration can be cited. In the case of parenteral administration, for example, transdermal, subcutaneous, intravenous, intra-arterial, intraperitoneal, intranasal, topical, enteral, etc. can be cited. In the case of transdermal administration, specific application sites can be cited, for example, the skin of the face, neck, upper body, lower body, etc.

[0045] The administration mode is not particularly limited. For example, it can be in vivo or in vitro. In the case where the administration is in vitro, the administration object can be cited, for example, cells, tissues or organs, etc. In the case where the administration is in vivo, the administration object can be cited, for example, humans or the non-human animals. In addition, in the case of in vivo, for example, it can be any one of oral administration and parenteral administration.

[0046] There are no particular restrictions on the administration conditions, and they can be appropriately set according to the administration method, administration site, type of administration subject, age, gender, symptoms, etc. of the administration subject.

[0047] (7) Use The present invention is an extracellular vesicle derived from pitaya, which is used for antioxidant. The present invention is an extracellular vesicle derived from pitaya, which is used for manufacturing an antioxidant.

[0048] The present invention is an extracellular vesicle derived from pitaya, which is used for promoting fibroblasts. The present invention is an extracellular vesicle derived from pitaya, which is used for manufacturing a fibroblast promoter.

[0049] The extracellular vesicle derived from pitaya in the present invention can refer to the above description.

[0050] Hereinafter, the present invention will be described in detail by way of examples, etc., but the present invention is not limited thereto. Examples

[0051] [Example 1] Recover extracellular vesicles from red-fleshed pitaya materials.

[0052] (1) Powder Mix 1 g of a commercially available powder of red-fleshed pitaya juice (juice of the pulp with seeds removed, trade name: red-fleshed pitaya powder, manufactured by Bioactives Japan Co., Ltd.) with 1 ml of sterile distilled water. Stir the mixture well, let it stand at room temperature for 20 minutes, then remove the precipitated impurities and recover the supernatant. Filter the supernatant through a filter with a pore size of 0.22 μm to recover the filtrate. Then, subject the filtrate to ultracentrifugation (100,000×g, 70 minutes, 4°C) to recover the precipitate fraction (particles). Suspend 1 g of the particles in 10 ml of distilled water and recover it as an EVs sample. Send the EVs sample into a nanoparticle analysis system (trade name NanoSight, LM10, laser 405 nm, Malvern Co., Ltd.) to confirm the particle size distribution of the extracellular vesicles contained in the EVs sample. Use NTA3.4 (Malvernpanalytical Co., Ltd.) as the analysis software.

[0053] The results of the particle size distribution are shown in Figure 1 the chart of. In Figure 1 , the vertical axis represents the particle concentration (particles / ml), and the horizontal axis represents the particle size (nm). The concentration of the extracellular vesicles in the EVs sample (1 g of particles / 10 ml) is 8.92×10 10 particles / ml, and the concentration of the extracellular vesicles in the diluted sample further diluted 200 times is 4.46×10 8particles / ml. Mean: 319.9 nm Mode: 589.6 nm SD: 80.8 nm

[0054] [Example 2] Evaluate the antioxidant capacity of extracellular vesicles derived from red pitaya.

[0055] Dilute the EVs sample prepared in Example 1 with distilled water to obtain Diluted Sample 1 (8×10 10 particles / ml). Furthermore, dilute Diluted Sample 1 with distilled water to obtain multiple diluted samples. Then, for these diluted samples, use a commercially available kit (product name: DPPH Antioxidant Assay Kit, Dojindo, D678) to evaluate the antioxidant capacity according to its instruction manual. According to this kit, the scavenging ability of artificially generated free radicals (2,2-diphenyl-1-picrylhydrazyl: DPPH) can be evaluated as the antioxidant capacity.

[0056] Show the result in the Figure 2 chart. In the Figure 2 , the vertical axis represents the DPPH free radical scavenging rate (%), and the horizontal axis represents the dilution factor of the EVs sample. As Figure 2 shown, it can be seen that by using Diluted Sample 1 (1.0E+00) with an extracellular vesicle concentration of 8×10 10 particles / ml, 90% of the free radicals can be effectively scavenged.

[0057] [Example 3] Evaluate the effect of extracellular vesicles derived from red pitaya on fibroblasts.

[0058] Use Diluted Sample 1 (8×10 10 particles / ml) of the EVs prepared in Example 1 as the sample. Then, by the following method, measure human dermal fibroblasts (Human Dermal Fibroblast, Normal, Cryopreserved <nhdf-c>(Funakoshi)) in the expression levels (n = 2) of the target genes involved in type I collagen production (Collagen I (COL1A1) Human) and the target genes involved in elastin production (Elastin (ELN) Human). It should be noted that as a control for the expression level, the expression level of the actin gene was also measured. The cells were cultured using a dedicated medium (Fibroblast Basal Medium, Funakoshi), and the EVs were added in a sub-confluent state. The addition amount was set to a concentration of 100 EV particles per cell intake. The cells were harvested 72 hours after addition, and the mRNA was purified using the RNAeasy reagent (Qiagen). On the other hand, as a reference example, exosomes derived from adipose tissue-derived mesenchymal stem cells (ADMSC) (average particle size distribution: 110 nm) were used, and the expression levels of the above genes were measured in the same manner (n = 2). It is known that exosomes can promote the production of collagen and elastin by fibroblasts.)

[0059] The results are shown in Figure 3 the chart of. In Figure 3 , the vertical axis represents the relative gene expression level, specifically expressed as "expression level of the target gene / expression level of the actin gene". As Figure 3 shown, according to the EVs sample from pitaya, it can be seen that compared with the exosomes derived from ADMSC in the reference example, it can further promote the expression of both the target gene involved in type I collagen production and the target gene involved in elastin production.)

[0060] [Example 4] Extracellular vesicles were recovered from pitaya materials.)

[0061] (1) Extract 1 g of a commercially available extract (manufactured by Shirakawa Farm) of pitaya juice (juice of the pulp with seeds removed) was dissolved in 1 ml of sterile distilled water, and centrifuged (2,000 × g, 10 minutes, 4 °C), and the supernatant was recovered. The supernatant was further ultracentrifuged (100,000 × g, 70 minutes, 4 °C), and the pellet fraction (particles) was recovered. 1 g of the particles was suspended in 10 ml of distilled water and recovered as an EVs sample. The EVs sample was fed into a nanoparticle analysis system (trade name NanoSight, LM10, laser 405 nm, Malvern), and the particle size distribution of the extracellular vesicles contained in the EVs sample was confirmed. The analysis software used was NTA3.4 (Malvern panalytical).)

[0062] The results of the particle size distribution are shown in Figure 4 the chart of. In Figure 4 Among them, the vertical axis represents the particle concentration (particles / ml), and the horizontal axis represents the particle size (nm). The concentration of extracellular vesicles in the EVs sample (1 g particles / 10 ml) is 2×10 10 particles / ml. Mean: 82.9 nm Mode: 67.1 nm

[0063] [Example 5] The EVs sample recovered from the red pitaya material was tested for markers of extracellular vesicles of plant origin.

[0064] HSC70 (molecular weight 70 kDa) is generally considered a marker of extracellular vesicles of plant origin. Therefore, for the EVs sample separated by the ultracentrifugation method of Example 4, an anti-HSC70 antibody was used to detect HSC70 by Western blotting under reducing conditions. The antibody used was an antibody against plant HSC70 (Anti-HSC70(Plant) Antibody, Catalog #SPC-302D, StressMarq Biosciences Inc.). In addition, as a positive control for extracellular vesicles of plant origin, extracellular vesicles derived from spinach leaves known to be HSC70-positive were used. Commercially available spinach was used to prepare extracellular vesicles derived from spinach leaves in the same manner as in Example 4. The amount of sample added to each lane was 10 μg / lane in terms of protein mass.

[0065] These results are shown in Figure 5 . Figure 5 is an image showing the Western blotting results of the EVs sample derived from red pitaya. The Marker is the marker, lane 1 is without sample, lane 2 is the EVs sample, and lane 3 is the positive control (extracellular vesicles derived from spinach leaves). As Figure 5 shown, a band of HSC70 was detected in the EVs sample derived from red pitaya in the same manner as in the positive control. From this, it can be confirmed that the EVs sample isolated in Example 1 is an extracellular vesicle of plant (red pitaya) origin.

[0066] As described above, the present invention has been described with reference to the embodiments, but the present invention is not limited to the above embodiments. Within the scope of the present invention, various changes that can be understood by those skilled in the art can be made to the structure and details of the present invention.

[0067] This application claims priority based on Japanese Patent Application 2022-135848 filed on August 29, 2022, the entire disclosure of which is incorporated herein.

Industrial Applicability

[0068] According to the present invention, for example, oxidation can be prevented, and in addition, the production of collagen, elastin, etc. can be promoted, so that the symptoms of the organism in which they are involved can be improved.

Claims

1. An antioxidant, characterized in that it contains extracellular vesicles derived from pitaya.

2. The antioxidant according to claim 1, wherein the extracellular vesicles are at least one of extracellular vesicles derived from pitaya juice and extracellular vesicles derived from pitaya leaves.

3. The antioxidant according to claim 1 or 2, wherein the average value mean of the particle size distribution of the extracellular vesicles is 50 - 500 nm.

4. The antioxidant according to any one of claims 1 to 3, which is for non - oral use.

5. The antioxidant according to claim 4, which is for percutaneous use.

6. The antioxidant according to any one of claims 1 to 3, which is for oral use.

7. The antioxidant according to any one of claims 1 to 6, wherein the pitaya is red - flesh pitaya.

8. A fibroblast promoter, characterized in that it contains extracellular vesicles derived from pitaya.

9. The fibroblast promoter according to claim 8, wherein the extracellular vesicles are at least one of extracellular vesicles derived from pitaya juice and extracellular vesicles derived from pitaya leaves.

10. The fibroblast promoter according to claim 9, wherein the average value mean of the particle size distribution of the extracellular vesicles is 50 - 500 nm.

11. The fibroblast promoter according to any one of claims 8 to 10, which is for non - oral use.

12. The fibroblast promoter according to claim 11, which is for percutaneous use.

13. The fibroblast promoter according to any one of claims 8 to 10, which is for oral use.

14. The fibroblast promoter according to any one of claims 8 to 13, which promotes the production of at least one of collagen and elastin by fibroblasts.

15. The fibroblast promoter according to any one of claims 8 to 14, wherein the pitaya is red - flesh pitaya.

16. An antioxidant method, characterized in that extracellular vesicles derived from pitaya are added to a subject.

17. A method for promoting the production of collagen by fibroblasts, characterized in that fibroblasts are contacted with extracellular vesicles derived from pitaya.

18. A method for promoting the production of elastin by fibroblasts, characterized in that fibroblasts are contacted with extracellular vesicles derived from pitaya.

19. A method for promoting the production of elastin by fibroblasts, characterized in that it contacts extracellular vesicles derived from pitaya.

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