Antioxidant and fibroblast promoter

Abstract

The purpose of the present invention is to provide a novel component for ameliorating biological symptoms such as sagging of the skin.　An antioxidant according to the present invention is characterized by containing dragon fruit-derived extracellular vesicles. A fibroblast promoter according to the present invention is characterized by containing dragon fruit-derived extracellular vesicles. The dragon fruit is, for example, a red dragon fruit.

Description

Antioxidant and fibroblast promoter

[0001] The present invention relates to an antioxidant, a fibroblast promoting agent, and uses thereof.

[0002] Various ingredients have been developed to improve symptoms in the body, such as on the skin, and active ingredients derived from natural sources are particularly desired from the standpoint of safety.

[0003] Therefore, an object of the present invention is to provide a new component for improving various biological symptoms such as sagging skin.

[0004] To achieve the above object, the antioxidant of the present invention is characterized by containing extracellular vesicles derived from dragon fruit.

[0005] The fibroblast promoting agent of the present invention is characterized by containing extracellular vesicles derived from dragon fruit.

[0006] The present inventors have newly discovered that dragon fruit-derived extracellular vesicles have antioxidant properties and can promote the expression of genes involved in the production of collagen and elastin in fibroblasts that produce these substances. Therefore, according to the present invention, for example, oxidation can be prevented and the production of collagen, elastin, etc. can be promoted, thereby making it possible to improve symptoms in the body related to these substances.

[0007] Fig. 1 is a graph showing the particle size distribution of extracellular vesicles derived from red dragon fruit in Example 1. Fig. 2 is a graph showing the antioxidant activity of extracellular vesicles derived from red dragon fruit in Example 2. Fig. 3 is a graph showing the effect of extracellular vesicles derived from red dragon fruit on fibroblasts in Example 3. Fig. 4 is a graph showing the particle size distribution of extracellular vesicles derived from red dragon fruit in Example 4. Fig. 5 shows the results of Western blotting for detecting markers in a red dragon fruit-derived EVs sample in Example 5.

[0008] [1] An antioxidant comprising extracellular vesicles derived from dragon fruit. [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 particle size distribution of the extracellular vesicles is a mean of 50 to 500 nm. [4] The antioxidant according to any one of [1] to [3], wherein the antioxidant is for parenteral administration. [5] The antioxidant according to [4], wherein the antioxidant is for transdermal administration. [6] The antioxidant according to any one of [1] to [3], wherein the antioxidant is for oral administration. [7] The antioxidant according to any one of [1] to [6], wherein the dragon fruit is red dragon fruit. [8] A fibroblast-promoting agent comprising extracellular vesicles derived from dragon fruit. [9] The fibroblast stimulating agent 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 stimulating agent according to [9], wherein the particle size distribution of the extracellular vesicles is a mean of 50 to 500 nm.

[11] The fibroblast stimulating agent according to any one of [8] to

[10] , wherein the extracellular vesicles are administered parenterally.

[12] The fibroblast stimulating agent according to

[11] , wherein the extracellular vesicles are administered transdermally.

[13] The fibroblast stimulating agent according to any one of [8] to

[10] , wherein the fibroblast stimulating agent is administered orally.

[14] The fibroblast stimulating agent according to any one of [8] to

[13] , wherein the fibroblast stimulating agent promotes at least one of collagen production and elastin production by fibroblasts.

[15] The fibroblast stimulating agent according to any one of [8] to

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

[16] An antioxidant method, comprising administering dragon fruit-derived extracellular vesicles to a subject.

[17] A method for promoting collagen production in fibroblasts, comprising contacting fibroblasts with dragon fruit-derived extracellular vesicles.

[18] A method for promoting elastin production in fibroblasts, comprising contacting fibroblasts with dragon fruit-derived extracellular vesicles.

[19] A method for promoting elastin production in fibroblasts, comprising contacting fibroblasts with dragon fruit-derived extracellular vesicles.

[0009] Unless otherwise specified, terms used in this specification can be used in the sense commonly used in the art.

[0010] As used herein, "extracellular vesicles" refer to membrane vesicles secreted from cells, hereinafter also referred to as EVs. Examples of extracellular vesicles include exosomes derived from endosomes secreted via the endocytosis pathway, and microvesicles derived from plasma membranes.

[0011] (1) Dragon Fruit-Derived Extracellular Vesicles As described above, the antioxidant and fibroblast promoting agent of the present invention are characterized by containing dragon fruit-derived extracellular vesicles. First, dragon fruit-derived extracellular vesicles will be described below.

[0012] In the present invention, the extracellular vesicles may be derived from dragon fruit. Dragon fruit is a plant of the genus Cactus in the family Cactaceae. In the present invention, unless otherwise specified, dragon fruit may refer to the fruit, the entire plant including the fruit, or any part of the plant, and may include any of these meanings. Dragon fruit is also known as pitaya. The type of dragon fruit is not particularly limited, and examples include red-fleshed red dragon fruit (Hylocereus costaricensis, Hylocereus polyrhizus), white-fleshed white dragon fruit (Hylocereus undatus), yellow-skinned white-fleshed golden dragon fruit (Hylocereus polyhizus), and peach-fleshed pink dragon fruit (hybrid: Hylocereus undatus x Hylocereus ocanponis), with red dragon fruit being preferred.

[0013] For example, the dragon fruit material used to prepare the extracellular vesicles may be the entire dragon fruit plant, or any part of the dragon fruit. Examples of the parts include the fruit, fruit skin, pulp, seeds, pulp containing seeds, and leaves, or a mixture of two or more parts. The material is preferably the pulp or pulp containing seeds. The fruit may be, for example, fully ripe or unripe, with fully ripe being preferred.

[0014] The dragon fruit-derived extracellular vesicles can be prepared, for example, from a liquid fraction derived from the dragon fruit material. The liquid fraction can be, for example, a juice obtained by squeezing the dragon fruit material, a mixture of crushed dragon fruit material and a solvent, or an extract from the dragon fruit material. The juicing method is not particularly limited, and examples include squeezing, rotation, and rotary squeezing. Specifically, a preferred juice is obtained by squeezing the fruit, pulp, or pulp containing seeds as the dragon fruit material. The juice is preferably a pulp juice or a pulp juice containing seeds, and more preferably a pulp juice from which seeds have been removed. The pulp juice from which seeds have been removed can be prepared, for example, by squeezing pulp containing seeds and removing the seeds. The pulp juice from which seeds have been removed can be obtained by centrifuging the pulp juice containing seeds to remove the precipitate containing the seeds. The extraction method is not particularly limited, and examples thereof include a method of adding a solvent to the crushed material and recovering a liquid fraction as an extract. The liquid fraction may be, for example, a concentrated liquid such as the squeezed juice, the mixed liquid, or the extract. The crushed dragon fruit material may also be, for example, a paste. The crushed material may be, for example, crushed pulp including seeds. The solvent is not particularly limited, and examples thereof include an aqueous solvent such as water, a buffer solution such as phosphate buffer solution (PBS), or physiological saline.

[0015] The dragon fruit material and the crushed material may be, for example, immediately after preparation or after storage. In the case of storage after storage, for example, they may be stored at room temperature, refrigerated, frozen, or returned to room temperature after refrigerated or frozen storage. The liquid fraction may be, for example, a freshly prepared liquid, a liquid stored at room temperature, a liquid stored in a refrigerator, or a liquid stored frozen.

[0016] The liquid fraction may be, for example, the squeezed juice, the mixed liquid, or the extract, which may be used as is, or a concentrated liquid may be used. Examples of the concentrated liquid include a concentrated liquid obtained by concentrating the squeezed juice, the mixed liquid, or the extract. The concentrated squeezed juice is also called, for example, dragon fruit extract. The liquid fraction may also be, for example, a dried form of the squeezed juice or a mixed liquid (also called a concentrated reconstituted liquid) obtained by mixing the dried form of the extract with a solvent. The dried form can be prepared, for example, by drying the squeezed juice or the extract, and a powder form is preferred from the viewpoint of handling. Examples of the drying process include freeze-drying.

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

[0018] Prior to the preparation of extracellular vesicles, for example, contaminants such as fibers and debris may be removed from the liquid fraction. The removal of contaminants may be achieved, for example, by separation by standing, centrifugation, or filtration. In the case of standing, the precipitated contaminants can be removed, for example, by stirring the liquid fraction, standing, and then recovering the supernatant. In the case of centrifugation, the contaminants can be removed by coarse centrifugation of the liquid fraction and recovering the supernatant. After removing contaminants from the liquid fraction by standing, for example, the contaminants may be further removed by coarse centrifugation. The conditions for standing are not particularly limited, and are, for example, 5 to 60 minutes (e.g., 20 minutes). The conditions for coarse centrifugation are not particularly limited, and are, for example, 400 to 3000 × g (e.g., 2000 × g) for 5 to 30 minutes (e.g., 10 minutes). After the impurity removal treatment, the liquid fraction is preferably subjected to particle observation using, for example, a nanoparticle analysis system to confirm whether or not any impurities remain.

[0019] The filtration may be, for example, filtration using a filter medium. When removing the impurities from the liquid fraction using the filter medium, for example, the remaining material on the filter medium is removed as impurities, and a filtrate fraction is recovered. The filter medium may be, for example, a filter. The size of the pores in the filter medium is not particularly limited, and may be, for example, pores that allow the extracellular vesicles to pass through. The liquid fraction after the standing or coarse centrifugation may be further filtered using a filter medium.

[0020] The method for preparing the extracellular vesicles from the liquid fraction is not particularly limited, and for example, ultracentrifugation can be used. Specifically, the liquid fraction is subjected to ultracentrifugation, and a precipitate fraction containing extracellular vesicles can be collected as the extracellular vesicle fraction. The conditions for the ultracentrifugation are not particularly limited, and for example, are 50,000 to 150,000 × g (100,000 × g) and 50 to 140 minutes (e.g., 70 minutes). The precipitate fraction may be suspended in the solvent and stored in a refrigerator or freezer, for example.

[0021] As a specific example, when the squeezed juice is used, the juice is stirred and allowed to stand at 4°C to room temperature, and the liquid is recovered so as to remove precipitated impurities. The recovered liquid is then subjected to the coarse centrifugation to recover a supernatant fraction, which is then further subjected to ultracentrifugation to recover a precipitated fraction as an extracellular vesicle fraction.

[0022] Furthermore, when using a dried form of the squeezed juice (e.g., powder), the dried form can be suspended in a solvent, impurities can be removed by coarse centrifugation, and the recovered liquid fraction can be subjected to ultracentrifugation to obtain the extracellular vesicle fraction.

[0023] Furthermore, when using an extract obtained by concentrating the squeezed juice, for example, the solvent is added as necessary, the mixture is stirred, the coarse centrifugation is performed, the supernatant fraction is recovered, and this supernatant fraction is further subjected to ultracentrifugation, and the precipitate fraction is recovered as an extracellular vesicle fraction.

[0024] The method for preparing the extracellular vesicles from the liquid fraction may be, for example, filtration using a filter (also known as ultrafiltration). In this case, for example, a filter having pores large enough to allow the extracellular vesicles of dragon fruit to pass through, a filter having pores large enough to prevent the extracellular vesicles from passing through, or a combination of both, may be used.

[0025] The dragon fruit-derived extracellular vesicles have a mean particle size distribution of, for example, 50 to 500 nm.

[0026] The extracellular vesicles used in the present invention may have an average value of, for example, 320±120 nm, 320±100 nm, 320±80 nm, 320±50 nm, 320±30 nm, 320±20 nm, 320±10 nm, or 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, or 290±5 nm. The mode can also be referred to as, for example, the peak in the particle size distribution. 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, or 80±5 nm.

[0027] Furthermore, the extracellular vesicles used in the present invention may have an average particle size of, for example, 83±21 nm, 83±30 nm, 83±20 nm, 83±10 nm, 83±5 nm, 83±30 nm, 83±20 nm, 83±10 nm, or 83±5 nm. When the average particle size of the extracellular vesicles is 83±21 nm, the mode in the particle size distribution is, for example, 67±20 nm, 67±10 nm, or 67±5 nm.

[0028] The method for measuring the particle size distribution of the extracellular vesicles is not particularly limited, and examples thereof include laser diffraction, light scattering methods such as dynamic light scattering, particle trajectory analysis, nanotracking analysis, etc. For the measurement, a commercially available device such as NanoSight (trade name, Quantum Design) can be used.

[0029] (2) Antioxidant As described above, the antioxidant of the present invention is characterized by containing extracellular vesicles derived from dragon fruit. The extracellular vesicles contained in the antioxidant are, for example, those isolated from dragon fruit. The antioxidant of the present invention is characterized by containing the extracellular vesicles, and other configurations and conditions are not particularly limited. The description of (1) above can be used for the dragon fruit-derived extracellular vesicles.

[0030] The antioxidant of the present invention may contain, for example, only the dragon fruit-derived extracellular vesicles, or may contain other components. The other components may be, for example, other components contained in the liquid fraction derived from dragon fruit, or may further include other additive components. The other additive components are not particularly limited and can be selected appropriately depending on the target site and method of use of the antioxidant of the present invention.

[0031] The antioxidant of the present invention may be, for example, for pharmaceutical use, for specified health uses, or for cosmetic use. The antioxidant of the present invention may be, for example, a composition containing the dragon fruit-derived extracellular vesicles.

[0032] When the antioxidant of the present invention is a pharmaceutical composition, its administration route is not particularly limited and may be, for example, parenteral or oral. Examples of parenteral administration include transdermal, subcutaneous, intravenous, intraarterial, intraperitoneal, intranasal, topical, and intestinal administration. When the antioxidant of the present invention is a pharmaceutical composition, its form is not particularly limited and can be appropriately selected depending on the administration route. Specific examples of such forms include liquids such as solutions and suspensions, emulsions, gels, sols, ointments, granules, tablets, and capsules. When the antioxidant of the present invention is for oral administration, the composition may be, for example, a so-called supplement.

[0033] When the antioxidant of the present invention is a cosmetic composition, the route of administration is, for example, transdermal, and specific application sites include, for example, the skin of the face, neck, upper body, lower body, etc. When the antioxidant of the present invention is a cosmetic composition, the form thereof is not particularly limited, and examples thereof include lotions, gels, emulsions, creams, oils, ointments, etc.

[0034] The other additive components are not particularly limited and can be appropriately selected depending on the route of administration and the dosage form. Specific examples include excipients, diluents, antioxidants, preservatives, bulking agents, humectants, thickeners, viscosity stabilizers, UV filters, binders, vitamins, fragrances, and colorants. Examples of the other additive components include pharmaceutically acceptable ingredients.

[0035] In the antioxidant of the present invention, the content of the dragon fruit-derived extracellular vesicles is not particularly limited and can be appropriately determined depending on, for example, the type, age, sex, symptoms, etc. of the living body to be administered. Specifically, in the case of a transdermal composition, the content of the extracellular vesicles is, for example, 0.01 to 5% (w / w). In addition, in the case of an oral composition, the daily dose of the extracellular vesicles is, for example, 5 to 1000 mg, 25 mg, etc., and the number of administrations per day is not particularly limited and can be, for example, 1 to 3 times, 1 to 2 times, or 1 time.

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

[0037] (3) Fibroblast Stimulator The fibroblast stimulator of the present invention is characterized by containing the dragon fruit-derived extracellular vesicles. The fibroblast stimulator of the present invention is characterized by containing the dragon fruit-derived extracellular vesicles, and other configurations and conditions are not particularly limited. The description of (1) above can be applied to the dragon fruit-derived extracellular vesicles. Furthermore, the description of the antioxidant of the present invention can be applied to the fibroblast stimulator of the present invention. In the present invention, promotion of fibroblasts means, for example, promotion of elastin production or promotion of collagen production.

[0038] (4) Symptom Improvement As described above, the dragon fruit-derived extracellular vesicles exhibit, for example, antioxidant activity and fibroblast-promoting activity. The fibroblast-promoting activity, for example, promotes collagen production and / or elastin production by the fibroblasts. Therefore, the antioxidant and fibroblast-promoting agent of the present invention can improve skin conditions, such as wrinkle reduction, sagging, anti-aging, moisturizing, reduction / elimination of nasolabial folds, skin quality improvement, tightening of enlarged pores, improvement of sagging pores, pore cleansing, keratin plug removal, acne scar reduction, exfoliation, removal of dark spots, improvement of skin texture, increased transparency, improved barrier function, normalization of stratum corneum turnover, and increase in skin-beautifying bacteria. In the present invention, "improvement" refers to, for example, suppression of deterioration, alleviation of symptoms, or cure of symptoms. The antioxidant and fibroblast-promoting agent of the present invention can also be referred to as, for example, symptom-improving compositions.

[0039] (5) Antioxidation Method The antioxidant method of the present invention is characterized by comprising a step of administering the antioxidant of the present invention. The present invention is characterized by using the antioxidant of the present invention, and other steps, conditions, etc. are not limited in any way. The description of the antioxidant of the present invention can be used in the present invention.

[0040] The administration method is not particularly limited, and as described above, examples thereof include oral administration and parenteral administration. In the case of parenteral administration, examples thereof include transdermal, subcutaneous, intravenous, intraarterial, intraperitoneal, intranasal, topical, and intraintestinal administration. In the case of transdermal administration, specific application sites include, for example, the skin of the face, neck, upper body, lower body, etc.

[0041] The administration form is not particularly limited and may be, for example, in vivo or in vitro. In the case of in vitro administration, the administration target (also referred to as the subject) may be, for example, a cell, tissue, or organ. In the case of in vivo administration, the administration target may be, for example, a human or the non-human animal. In addition, in the case of in vivo administration, for example, either oral administration or parenteral administration may be used.

[0042] The conditions for administration are not particularly limited and can be appropriately set depending on, for example, the administration method, administration site, type of subject, age, sex, symptoms, etc. of the subject.

[0043] (6) Fibroblast Promotion Method The fibroblast promotion method of the present invention is characterized by comprising a step of administering the fibroblast promotion agent of the present invention. The present invention is characterized by using the fibroblast promotion agent of the present invention, and other steps and conditions, etc. are not limited in any way. The description of the fibroblast promotion agent of the present invention can be used in the present invention. The fibroblast promotion method of the present invention refers to, for example, the promotion of elastin production or collagen production in fibroblasts.

[0044] The administration method is not particularly limited, and as described above, examples thereof include oral administration and parenteral administration. In the case of parenteral administration, examples thereof include transdermal, subcutaneous, intravenous, intraarterial, intraperitoneal, intranasal, topical, and intraintestinal administration. In the case of transdermal administration, specific application sites include, for example, the skin of the face, neck, upper body, lower body, etc.

[0045] The administration form is not particularly limited and may be, for example, in vivo or in vitro. When the administration is in vitro, the administration target may be, for example, a cell, tissue, or organ. When the administration is in vivo, the administration target may be, for example, a human or the non-human animal. When the administration is in vivo, the administration may be, for example, oral or parenteral.

[0046] The conditions for administration are not particularly limited and can be appropriately set depending on, for example, the administration method, administration site, type of subject, age, sex, symptoms, etc. of the subject.

[0047] (7) Uses The present invention relates to extracellular vesicles derived from dragon fruit for use in antioxidants. The present invention relates to extracellular vesicles derived from dragon fruit for producing antioxidants.

[0048] The present invention relates to extracellular vesicles derived from dragon fruit for use in promoting fibroblasts. The present invention relates to extracellular vesicles derived from dragon fruit for use in producing a fibroblast promoting agent.

[0049] The above-mentioned description can be applied to the dragon fruit-derived extracellular vesicles of the present invention.

[0050] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these.

[0051] Example 1 Extracellular vesicles were collected from red dragon fruit material.

[0052] (1) Powder: 1 g of commercially available powder of red dragon fruit juice (juice extracted from the pulp without seeds) (product name: Red Dragon Fruit Powder, manufactured by Bioactives Japan) was mixed with 1 ml of sterile distilled water. The mixture was thoroughly stirred and allowed to stand at room temperature for 20 minutes, after which precipitated impurities were removed and the supernatant was collected. The supernatant was filtered through a 0.22 μm pore size filter to collect the filtrate. The filtrate was then subjected to ultracentrifugation (100,000 g, 70 minutes, 4°C) to collect the precipitated fraction (pellet). 1 g of the pellet was suspended in 10 ml of distilled water and collected as an EVs sample. The EVs sample was subjected to a nanoparticle analysis system (product name: NanoSight, LM10, laser 405 nm, Malvern) to confirm the particle size distribution of extracellular vesicles contained in the EVs sample. The analysis software used was NTA3.4 (Malvern Panalytical).

[0053] The particle size distribution results are shown in the graph in Figure 1. In Figure 1, the vertical axis represents particle concentration (particles / ml) and the horizontal axis represents particle diameter (nm). The EVs sample (1 g pellet / 10 ml) had an extracellular vesicle concentration of 8.92 x 10 １０ particles / ml, and the extracellular vesicle concentration of the diluted sample diluted 200 times was 4.46 × 10 ８ The mean was 319.9 nm, the mode was 589.6 nm, and the SD was 80.8 nm.

[0054] Example 2 The antioxidant capacity of extracellular vesicles derived from red dragon fruit was evaluated.

[0055] The EVs sample prepared in Example 1 was diluted with distilled water to give diluted sample 1 (8 × 10 １０A concentration of 1000 particles / ml was prepared. Furthermore, several diluted samples were prepared by diluting the diluted sample 1 with distilled water. The antioxidant activity of these diluted samples was then evaluated using a commercially available kit (product name: DPPH Antioxidant Assay Kit, Dojindo, D678) according to the manufacturer's instructions. This kit allows the evaluation of antioxidant activity based on the ability to scavenge artificially generated radicals (2,2-diphenyl-1-picrylhydrazyl: DPPH).

[0056] The results are shown in the graph in Figure 2. In Figure 2, the vertical axis represents the DPPH radical scavenging rate (%), and the horizontal axis represents the dilution ratio of the EVs sample. As shown in Figure 2, when the extracellular vesicle concentration was 8 x 10 １０ It was found that the use of diluted sample 1 (1.0E+00) at particles / ml effectively eliminated 90% of radicals.

[0057] [Example 3] The effect of extracellular vesicles derived from red dragon fruit on fibroblasts was evaluated.

[0058] The sample was diluted sample 1 (8 × 10 １０ Human dermal fibroblasts (Human Dermal Fibroblast, Normal, Cryopreserved particles / ml) were used. <nhdf-c>The expression levels of target genes involved in type I collagen production (Collagen I (COL1A1) Human) and elastin production (Elastin (ELN) Human) in exosomes (Funakoshi) were measured (n = 2). The expression level of the actin gene was also measured as a control for expression levels. Cells were cultured in a dedicated culture medium (Fibroblast Basal Medium, Funakoshi) and the EVs were added to the subconfluent cells. The addition amount was set to a concentration at which 100 EV particles were incorporated per cell. Seventy-two hours after addition, the cells were harvested, and mRNA was purified using RNAeasy reagent (Giagen). Meanwhile, as a reference example, the expression levels of the genes were similarly measured using exosomes (mean particle size distribution: 110 nm) derived from adipose tissue-derived mesenchymal stem cells (ADMSCs), which are known to promote collagen and elastin production in fibroblasts (n = 2).

[0059] The results are shown in the graph in Figure 3. In Figure 3, the vertical axis represents the relative gene expression level, specifically, "expression level of target gene / expression level of actin gene." As shown in Figure 3, the red dragon fruit-derived EVs sample was found to be able to further promote the expression of both target genes involved in type I collagen production and target genes involved in elastin production, compared to the ADMSC-derived exosomes used as a reference example.

[0060] Example 4 Extracellular vesicles were collected from red dragon fruit material.

[0061] (1) Extract: 1 g of commercially available red dragon fruit juice (squeezed pulp from the seeds removed) extract (Shirakawa Farm Co., Ltd.) was dissolved in 1 ml of sterile distilled water and centrifuged (2,000 g, 10 minutes, 4°C) to recover the supernatant. This supernatant was then subjected to ultracentrifugation (100,000 g, 70 minutes, 4°C) to recover the precipitated fraction (pellet). 1 g of the pellet was suspended in 10 ml of distilled water and recovered as an EVs sample. The EVs sample was subjected to a nanoparticle analysis system (trade name NanoSight, LM10, laser 405 nm, Malvern) to confirm the particle size distribution of the extracellular vesicles contained in the EVs sample. Analysis software used was NTA3.4 (Malvern Panalytical Co., Ltd.).

[0062] The particle size distribution results are shown in the graph of Figure 4. In Figure 4, the vertical axis represents particle concentration (particles / ml) and the horizontal axis represents particle diameter (nm). The EVs sample (1 g pellet / 10 ml) had an extracellular vesicle concentration of 2 x 10 １０ particles / ml. Mean: 82.9 nm Mode: 67.1 nm

[0063] [Example 5] Plant-derived extracellular vesicle markers were detected in EVs samples collected from red dragon fruit material.

[0064] HSC70 (molecular weight 70 kDa) is generally known as a marker for plant-derived extracellular vesicles. Therefore, HSC70 was detected by Western blotting under reducing conditions using an anti-HSC70 antibody for the EV samples isolated by ultracentrifugation in Example 4. The antibody used was an antibody against plant HSC70 (Anti-HSC70 (Plant) Antibody, Catalog # SPC-302D, StoresMarq Biosciences Inc.). Furthermore, spinach leaf-derived extracellular vesicles, which are known to be HSC70-positive, were used as a positive control for plant-derived extracellular vesicles. Spinach leaf-derived extracellular vesicles were prepared in the same manner as in Example 4 using commercially available spinach. The amount of sample added to each lane was 10 μg protein per lane.

[0065] These results are shown in Figure 5. Figure 5 is an image showing the results of Western blotting for a red dragon fruit-derived EV sample. Marker indicates a marker, lane 1 is no sample, lane 2 is the EV sample, and lane 3 is a positive control (extracellular vesicles derived from spinach leaves). As shown in Figure 5, an HSC70 band was detected in the red dragon fruit-derived EV sample, as in the positive control. This confirmed that the EV sample isolated in Example 1 was extracellular vesicles derived from a plant (red dragon fruit).

[0066] Although the present invention has been described above with reference to the embodiments, the present invention is not limited to the above-described embodiments. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

[0067] This application claims priority based on Japanese Patent Application No. 2022-135848, filed on August 29, 2022, the disclosure of which is incorporated herein in its entirety.

[0068] According to the present invention, for example, oxidation can be prevented and the production of collagen, elastin, etc. can be promoted, thereby making it possible to improve symptoms of the body in which these substances are involved.

Claims

1. An antioxidant characterized by containing extracellular vesicles derived from dragon fruit.

2. The antioxidant according to claim 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 claim 1 or 2, wherein the particle size distribution of the extracellular vesicles has an average value (mean) of 50 to 500 nm.

4. The antioxidant according to claim 1 or 2, which is for parenteral use.

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

6. An antioxidant according to claim 1 or 2, for oral use.

7. The antioxidant according to claim 1 or 2, wherein the dragon fruit is red dragon fruit.

8. A fibroblast stimulant characterized by containing extracellular vesicles derived from dragon fruit.

9. The fibroblast stimulant according to claim 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 stimulant according to claim 9, wherein the particle size distribution of the extracellular vesicles has an average value (mean) of 50 to 500 nm.

11. A fibroblast stimulant according to claim 8 or 9, which is for parenteral use.

12. A fibroblast stimulant according to claim 11, for transdermal use.

13. A fibroblast stimulant according to claim 8 or 9, for oral use.

14. A fibroblast promoter according to claim 8 or 9, which promotes at least one of collagen production and elastin production by fibroblasts.

15. The fibroblast stimulant according to claim 8 or 9, wherein the dragon fruit is red dragon fruit.

16. Extracellular vesicles derived from dragon fruit were added to the subject. An antioxidant method characterized in that the addition to the target is performed in vitro or in vivo to a non-human animal.

17. Extracellular vesicles derived from dragon fruit were brought into contact with fibroblasts. A method for promoting collagen production in fibroblasts, characterized in that the contact is in vitro or in vivo contact with a non-human animal.

18. Extracellular vesicles derived from dragon fruit were brought into contact with fibroblasts. A method for promoting elastin production in fibroblasts, characterized in that the contact is in vitro or in vivo contact with a non-human animal.