Anti-inflammatory or aging-suppressing composition containing extracellular vesicles derived from plant, mushroom or microalga

Abstract

This anti-inflammatory or aging-suppressing composition contains extracellular vesicles derived from a plant of the family Apiaceae, Lamiaceae, Theaceae, Selaginellaceae, Brassicaceae, Asteraceae, Amaranthaceae, Amaryllidaceae, Fabaceae, Cucurbitaceae, Zingiberaceae, Polygonaceae, Dioscoraceae, Vitaceae, Rosaceae, Solanaceae, Ericaceae, Lythraceae or Rutaceae, a mushroom or a microalga.

Description

Anti-inflammatory or anti-aging composition comprising extracellular vesicles derived from plants, mushrooms, or microalgae

[0001] The present invention relates to an anti-inflammatory or anti-aging composition comprising extracellular vesicles derived from plants, mushrooms, or microalgae.

[0002] Extracellular vesicles (EVs) are a collective term for lipid bilayer-enclosed vesicles secreted from cells of various organisms, including animals and plants, and include exosomes derived from endosomal membranes. Proteins, lipids, nucleic acids, and other substances are stored inside EVs.

[0003] In addition, EVs are known to have various physiologically active actions (see Patent Documents 1 to 5).

[0004] Special Publication No. 2018-531932 Special Publication No. 2020-535189 Japanese Patent Application Laid-Open No. 2021-193070 Special Publication No. 2022-525131 Special Publication No. 2022-543061

[0005] The problem to be solved by the present invention is to provide a new composition capable of suppressing inflammation or aging.

[0006] As a result of intensive research conducted by the present inventors to solve the above-mentioned problems, they discovered that extracellular vesicles derived from specific plants, mushrooms, or microalgae have a strong inhibitory effect on inflammation or aging, and thus completed the present invention.

[0007] That is, the present invention is as follows: [1] An anti-inflammatory or anti-aging composition comprising extracellular vesicles derived from a plant, mushroom, or microalgae of the Apiaceae, Lamiaceae, Theaceae, Selaginaceae, Brassicaceae, Asteraceae, Amaranthaceae, Amaryllidaceae, Fabaceae, Cucurbitaceae, Zingiberaceae, Polygonaceae, Dioscoreaceae, Vitaceae, Rosaceae, Solanaceae, Ericaceae, Lythraceae, or Rutaceae family. [2] Parsley, angelica tree, mitsuba, violet harmony, carrot, leaf carrot, fruit carrot, coriander, dill, fennel, rosemary, spearmint, lemon palm, shiso, marjuram, sweet basil, thyme, oregano, peppermint, sage, tea plant, camellia, temaki katahiba, Brussels sprouts, cauliflower, romanesco, arugula, wasabi radish, komatsuna, wasabi, cauliflower, red heart radish, selvatico, bok choy, broccoli, mizuna, radish, radish, watercress, turnip, tatsoi, artichoke, golden kidney leaf, chrysanthemum, lettuce, burdock [3] An anti-inflammatory or anti-aging composition comprising extracellular vesicles derived from Euglena, endive, Jerusalem artichoke, romaine lettuce, radicchio, epazote, amaranth, spinach, Swiss chard, beet, okahijiki, chive, leek, onion, shallot, Morus alba, edamame, urizumame, kinuko pea, snap pea, pea sprout, watermelon, zucchini, loofah, ginger, mioga, rhubarb, yam, muscat, mulberry, paprika, chili pepper, potato, bell pepper, blueberry, pomegranate, kaffir lime, kumquat, mushroom, shiitake mushroom, Bunashimeji mushroom, or Euglena. [4] The composition according to [1] or [2], which suppresses the expression of inflammatory cytokines or aging markers. [4] The composition according to [3], wherein the inflammatory cytokine or aging marker is IL-4, IL-6, IL-13, IL-33, IL-1β, CXCL1, CXCL2, TNF-α, p15, or p16. [5] The composition according to any one of [1] to [4], for use in food, cosmetics, or pharmaceuticals.

[0008] The present invention includes the following embodiments: [A1] A method for suppressing inflammation or aging using extracellular vesicles derived from a plant, mushroom, or microalgae of the Apiaceae, Lamiaceae, Theaceae, Selaginaceae, Brassicaceae, Asteraceae, Amaranthaceae, Amaryllidaceae, Fabaceae, Cucurbitaceae, Zingiberaceae, Polygonaceae, Dioscoreaceae, Vitaceae, Rosaceae, Solanaceae, Ericaceae, Lythraceae, or Rutaceae family. [A2] Parsley, angelica tree, mitsuba, violet harmony, carrot, leaf carrot, fruit carrot, coriander, dill, fennel, rosemary, spearmint, lemon palm, shiso, marjuram, sweet basil, thyme, oregano, peppermint, sage, tea plant, camellia, temaki katahiba, Brussels sprouts, cauliflower, romanesco, arugula, wasabi radish, komatsuna, wasabi, cauliflower, red heart radish, selvatico, bok choy, broccoli, mizuna, radish, radish, watercress, turnip, tatsoi, artichoke, golden kidney leaf, chrysanthemum, lettuce, burdock A method for suppressing inflammation or aging using extracellular vesicles derived from endive, Hakata Jerusalem artichoke, romaine lettuce, radicchio, epazote, amaranth, spinach, Swiss chard, beets, okahijiki, chives, leeks, onions, shallots, Morus alba, edamame, urizumame, kinukotoba, snap peas, pea sprouts, watermelon, zucchini, loofah, ginger, myoga, rhubarb, yam, muscat, mulberry, paprika, chili pepper, potato, bell pepper, blueberry, pomegranate, kaffir lime, kumquat, mushroom, shiitake mushroom, buna-shimeji mushroom, or Euglena.

[0009] [B1] Use of extracellular vesicles derived from plants, mushrooms, or microalgae belonging to the Apiaceae, Lamiaceae, Theaceae, Selaginaceae, Brassicaceae, Asteraceae, Amaranthaceae, Amaryllidaceae, Fabaceae, Cucurbitaceae, Zingiberaceae, Polygonaceae, Dioscoreaceae, Vitaceae, Rosaceae, Solanaceae, Ericaceae, Lythraceae, or Rutaceae families, for suppressing inflammation or aging. [B2] To suppress inflammation or aging, parsley, angelica tree, mitsuba, violet harmony, carrot, leaf carrot, fruit carrot, coriander, dill, fennel, rosemary, spearmint, lemon palm, shiso, marjuram, sweet basil, thyme, oregano, peppermint, sage, tea plant, camellia, temae katahiba, Brussels sprouts, cauliflower, romanesco, arugula, wasabi radish, komatsuna, wasabi, cauliflower, red heart radish, selvatico, bok choy, broccoli, mizuna, radish, radish, watercress, turnip, tatsoi, artichoke, Use of extracellular vesicles derived from red kidney beans, chrysanthemum, lettuce, burdock, endive, Jerusalem artichoke, romaine lettuce, radicchio, epazote, amaranth, spinach, Swiss chard, beets, okahijiki, chives, leeks, onions, shallots, Morus alba (coconut) beans, edamame beans, urizun beans, snow peas, snap peas, pea sprouts, watermelon, zucchini, loofah, ginger, myoga (Japanese ginger), rhubarb, nagaimo (Japanese yam), muscat grapes, mulberry, paprika, chili peppers, potatoes, bell peppers, blueberries, pomegranates, kaffir limes, kumquats, mushrooms, shiitake mushrooms, buna-shimeji mushrooms, or Euglena.

[0010] [C1] Use of extracellular vesicles derived from plants of the Apiaceae, Lamiaceae, Theaceae, Selaginaceae, Brassicaceae, Asteraceae, Amaranthaceae, Amaryllidaceae, Fabaceae, Cucurbitaceae, Zingiberaceae, Polygonaceae, Dioscoreaceae, Vitaceae, Rosaceae, Solanaceae, Ericaceae, Lythraceae, or Rutaceae, mushrooms, or microalgae for the manufacture of a composition that inhibits inflammation or aging. [C2] For the manufacture of a composition that inhibits inflammation or aging, parsley, angelica tree, mitsuba, Violet Harmony, carrot, leaf carrot, fruit carrot, coriander, dill, fennel, rosemary, spearmint, lemon palm, shiso, majuram, sweet basil, thyme, oregano, peppermint, sage, tea plant, camellia, temarikatahiba, Brussels sprouts, cauliflower, Romanesco, arugula, wasabi radish, komatsuna, wasabi, cauliflower, red heart radish, Selvatica, bok choy, broccoli, mizuna, radish, radish, watercress, turnip, tatsoi, artichoke Use of extracellular vesicles derived from corn, red kidney beans, chrysanthemum, lettuce, burdock, endive, Jerusalem artichoke, romaine lettuce, radicchio, epazote, amaranth, spinach, Swiss chard, beetroot, okahijiki, chives, leeks, onions, shallots, Morus codon, edamame, urizun beans, snow peas, snap peas, pea sprouts, watermelon, zucchini, loofah, ginger, myoga, rhubarb, nagaimo, muscat grapes, mulberry, paprika, chili peppers, potatoes, bell peppers, blueberries, pomegranates, kaffir limes, kumquats, mushrooms, shiitake mushrooms, buna-shimeji mushrooms, or Euglena.

[0011] [D1] Extracellular vesicles derived from plants, mushrooms, or microalgae belonging to the Apiaceae, Lamiaceae, Theaceae, Selaginaceae, Brassicaceae, Asteraceae, Amaranthaceae, Amaryllidaceae, Fabaceae, Cucurbitaceae, Zingiberaceae, Polygonaceae, Dioscoreaceae, Vitaceae, Rosaceae, Solanaceae, Ericaceae, Lythraceae, or Rutaceae families, for use in inhibiting inflammation or aging. [D2] Parsley, angelica tree, mitsuba, violet harmony, carrot, leaf carrot, fruit carrot, coriander, dill, fennel, rosemary, spearmint, lemon palm, shiso, marjuram, sweet basil, thyme, oregano, peppermint, sage, tea plant, camellia, temae katahiba, Brussels sprouts, cauliflower, romanesco, arugula, wasabi radish, komatsuna, wasabi, cauliflower, red radish, selvatica, bok choy, broccoli, mizuna, radish, radish, watercress, turnip, tatsoi, artichoke Extracellular vesicles derived from yoke, red kidney beans, chrysanthemum, lettuce, burdock, endive, Jerusalem artichoke, romaine lettuce, radicchio, epazote, amaranth, spinach, Swiss chard, beet, okahijiki, chives, leeks, onions, shallots, coriander beans, edamame beans, urizun beans, snow peas, snap peas, pea sprouts, watermelon, zucchini, loofah, ginger, myoga, rhubarb, nagaimo, muscat grapes, mulberry, paprika, chili peppers, potatoes, bell peppers, blueberries, pomegranates, kaffir limes, kumquats, mushrooms, shiitake mushrooms, buna-shimeji mushrooms, or Euglena.

[0012] [E1] An anti-inflammatory or anti-aging agent comprising extracellular vesicles derived from a plant, mushroom, or microalgae belonging to the Apiaceae, Lamiaceae, Theaceae, Selaginaceae, Brassicaceae, Asteraceae, Amaranthaceae, Amaryllidaceae, Fabaceae, Cucurbitaceae, Zingiberaceae, Polygonaceae, Dioscoreaceae, Vitaceae, Rosaceae, Solanaceae, Ericaceae, Lythraceae, or Rutaceae families. [E2] Parsley, angelica tree, mitsuba, violet harmony, carrot, leaf carrot, fruit carrot, coriander, dill, fennel, rosemary, spearmint, lemon palm, shiso, marjuram, sweet basil, thyme, oregano, peppermint, sage, tea plant, camellia, temaki katahiba, Brussels sprouts, cauliflower, romanesco, arugula, wasabi radish, komatsuna, wasabi, cauliflower, red heart radish, selvatico, bok choy, broccoli, mizuna, radish, radish, watercress, turnip, tatsoi, artichoke, golden kidney leaf, chrysanthemum, lettuce, and burdock An anti-inflammatory or anti-aging agent comprising extracellular vesicles derived from coriander, endive, Hakata Jerusalem artichoke, romaine lettuce, radicchio, epazote, amaranth, spinach, Swiss chard, beet, okahijiki, chives, leeks, onions, shallots, Moroccan beans, edamame, urizumame, kinukotoba, snap peas, pea sprouts, watermelon, zucchini, loofah, ginger, myoga, rhubarb, nagaimo, muscat, mulberry, paprika, chili pepper, potato, bell pepper, blueberry, pomegranate, kaffir lime, kumquat, mushroom, shiitake mushroom, buna-shimeji mushroom, or Euglena.

[0013] In each of the aspects [A1] to [E2], the composition may suppress the expression of an inflammatory cytokine or an aging marker, the inflammatory cytokine or the aging marker may be IL-4, IL-6, IL-13, IL-33, IL-1β, CXCL1, CXCL2, TNF-α, p15, or p16, and the composition may be for use in food, cosmetics, or pharmaceuticals.

[0014] The present invention includes the following embodiments. [6] An anti-inflammatory or anti-aging composition comprising extracellular vesicles derived from parsley or Euglena. [7] The composition according to [6], whose anti-inflammatory or anti-aging effect is not weakened by heat history. [8] The composition according to [7], wherein the heat history is a heat treatment at 40°C or higher. In each of the aspects [6] to [8], the composition may be stable to heat, may exhibit anti-inflammatory and / or anti-aging effects equivalent to those of a non-heated composition, and may suppress the expression of aging markers or cytokines equivalent to those of a non-heated composition.

[0015] The present invention makes it possible to provide a composition containing novel extracellular vesicles that can suppress inflammation or aging.

[0016] The following figures show the fractions obtained by ultracentrifugation of parsley- and ginger-derived EVs, followed by sucrose density gradient ultracentrifugation. The arrow indicates the Band 2 fraction. The plant-, mushroom-, or microalgae-derived EV fraction solutions listed in Table 1 were applied to human IMR90 cells in which cellular senescence was induced by overexpressing activated Ras, and the expression levels of cellular senescence markers (IL6, p15, and p16) were measured by qRT-PCR. The following figures also show the expression levels of inflammatory cytokines (IL-6 or IL-1β) when applied to mouse macrophage cells (Raw-Blue cells) in which inflammation was induced by LPS stimulation. In Figures 2-1 to 2-14, 2-19, and 2-21, the upper panels show the results of an experiment in which senescence was induced by overexpression of activated Ras, and the lower panels show the results of an experiment in which inflammation was induced by LPS stimulation. Mouse macrophage cells (Raw-Blue cells) were sensitized with an EV fraction solution or EV sample derived from urizun bean obtained by sucrose density gradient ultracentrifugation or ultrafiltration, or PBS, and then stimulated with LPS. The results of inflammatory cytokine (IL-6 or IL-1β) expression were shown. Mouse macrophage cells (Raw-Blue cells) were sensitized with an EV sample derived from parsley or Yanaka ginger obtained by ultrafiltration, or PBS, and then stimulated with LPS. The results of nanoparticle tracking analysis of an EV fraction solution derived from urizun bean obtained by sucrose density gradient ultracentrifugation were shown. The results of nanoparticle tracking analysis of an EV sample derived from urizun bean obtained by ultrafiltration were shown. The results of nanoparticle tracking analysis of an EV fraction solution derived from parsley obtained by sucrose density gradient ultracentrifugation were shown. The results of nanoparticle tracking analysis of an EV sample derived from parsley obtained by ultrafiltration were shown. The results of nanoparticle tracking analysis of a Yanaka ginger-derived EV fraction solution obtained by sucrose density gradient ultracentrifugation are shown. The results of nanoparticle tracking analysis of a Yanaka ginger-derived EV sample obtained by ultrafiltration are shown. The results of nanoparticle tracking analysis of a Euglena-derived EV fraction solution obtained by sucrose density gradient ultracentrifugation are shown.The results of analyzing the condition of human facial skin (wrinkles, age spots, pores, and skin texture) after oral ingestion of parsley EVs using the VISIA skin imaging diagnostic device are shown. Melanin accumulated deep in human skin after oral ingestion of parsley EVs was analyzed using the VISIA skin imaging diagnostic device. Mouse macrophage cells (Raw-Blue cells) were sensitized with parsley-derived EV samples obtained by unheated or heat-treated ultrafiltration, and the results of the expression of inflammatory cytokines (IL-6) after stimulation with LPS are shown. Euglena-derived EV fraction solutions obtained by unheated or heat-treated ultracentrifugation were sensitized with mouse macrophage cells (Raw-Blue cells), and the results of the expression of inflammatory cytokines (IL-6) after stimulation with LPS are shown. After oral administration of 1.0E+11 particles / day of parsley EVs (PcELNs) for one week, mice were given 3% DSS in their drinking water to induce colitis. The weight change of the mice during this period was measured. PBS was used as a control. The phenotype of the mice after DSS ingestion is shown (Category 0: no phenotype, 1: diarrhea / bloody stool, 2: bleeding+, 3: bleeding++, 4: bleeding+++, 5: anal bleeding). The length of the large intestine (from just below the cecum to the anus) in the PBS group and the PcELNs group after DSS ingestion is shown (Day 0: N=6, Day 3: N=3, Day 5: N=3, Day 7: N=6). The large intestine from the cecum to the anus was divided into three sections, with the section closest to the cecum designated as the anterior section and the section closest to the anus designated as the distal section. RT-qPCR analysis was performed on these sections (N=3). Immunostained images of HE, Alcian blue, and β-catenin / zo-1 (scale bar: 100 μm) on DSS day 0 and day 7 were shown. The ear thickness of mice in which inflammation was induced by TNCB stimulation was measured after infiltration of Brussels sprout-derived EVs, parsley-derived EVs, or PBS was measured. The changes in the expression of inflammatory cytokines in the ears infiltrated with a solution containing EVs after inflammation induction were measured by RT-qPCR. Grip strength was measured using a grip strength test on mice that had been administered parsley- and Brussels sprout-derived EVs simultaneously for 13 weeks in a high-fat diet. Average grip strength was shown.The average muscle endurance of each mouse that had been administered parsley- and Brussels sprout-derived EVs simultaneously with a high-fat diet for 13 weeks was measured using a 4-limb hanging test. Hanging time (the time the mouse endured before falling from the wire mesh) and hanging score (the value obtained by multiplying the hanging time by the body weight of each mouse) are shown. The average weight of the soleus muscle of the hind limb of each mouse that had been administered parsley- and Brussels sprout-derived EVs simultaneously with a high-fat diet for 13 weeks is shown.

[0017] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, an embodiment of the present invention (hereinafter referred to as "the present embodiment") will be described in detail. However, the present invention is not limited to the following embodiment.

[0018] The anti-inflammatory or anti-aging composition of this embodiment contains extracellular vesicles derived from plants, mushrooms, or microalgae of the Umbelliferae, Lamiaceae, Theaceae, Selaginaceae, Brassicaceae, Asteraceae, Amaranthaceae, Amaryllidaceae, Fabaceae, Cucurbitaceae, Zingiberaceae, Polygonaceae, Dioscoreaceae, Vitaceae, Rosaceae, Solanaceae, Ericaceae, Lythraceae, or Rutaceae family. The extracellular vesicles contained in the anti-inflammatory or anti-aging composition may contain extracellular vesicles derived from two or more different plants from the same family, or may contain extracellular vesicles derived from two or more different mushrooms or microalgae. Furthermore, the extracellular vesicles may contain extracellular vesicles derived from one or more different plants selected from the same family and one or more plants selected from a different family from the family. The composition may also contain extracellular vesicles derived from plants of two or more different families. The microalgae may include extracellular vesicles derived from plants and mushrooms, may include extracellular vesicles derived from plants and microalgae, may include extracellular vesicles derived from mushrooms and microalgae, or may include extracellular vesicles derived from plants, mushrooms, and microalgae. The plants may be plants grown outdoors or may be plants grown hydroponically. The mushrooms may be wild mushrooms or mushrooms planted in artificial media or on felled logs. The microalgae may be cultured.

[0019] The extracellular vesicles of this embodiment may be extracellular vesicles extracted by a conventionally known method using plants, mushrooms, or microalgae as raw materials. Furthermore, the extracellular vesicles derived from plants, mushrooms, or microalgae may be those obtained by extracting extracellular vesicles present in plants, mushrooms, or microalgae by a conventionally known method. Since plants, mushrooms, or microalgae are used as raw materials, the solvents and reagents used for extraction may contain components derived from the plants, mushrooms, or microalgae. The Umbelliferae plant is not particularly limited, but may be, for example, parsley, angelica tree, mitsuba, Violet Harmony, carrot, leaf carrot, fruit carrot, coriander, dill, or fennel. The Lamiaceae plant is not particularly limited, but may be, for example, rosemary, spearmint, lemon palm, shiso, marjuram, sweet basil, thyme, oregano, peppermint, or sage. The shiso may be red shiso. The plant of the Theaceae family is not particularly limited, but may be, for example, tea plant or camellia. The plant of the Selaginellaceae family is not particularly limited, but may be, for example, Temarikatahiba. Temarikatahiba is also called resurrection plant or Rose of Jericho. The plant of the Brassicaceae family is not particularly limited, but may be, for example, Brussels sprouts, cauliflower, Romanesco, arugula, wasabi radish, komatsuna, wasabi, cauliflower, red heart radish, Selvatica, bok choy, broccoli, mizuna, radish, daikon radish, watercress, turnip, or Chinese cabbage. The plant of the Asteraceae family is not particularly limited, but may be, for example, artichoke, golden kidney leaf, chrysanthemum, lettuce, burdock, endive, Jerusalem artichoke, romaine lettuce, or radicchio. The Amaranthaceae plant is not particularly limited, but may be, for example, epazote, amaranth, spinach, Swiss chard, beet, or okahijiki. The Amaryllidaceae plant is not particularly limited, but may be, for example, chive, leek, onion, or shallot. The Leguminosae plant is not particularly limited, but may be, for example, moringa, edamame, urizumame, yukino pea, snap pea, or pea sprout.The Cucurbitaceae plant is not particularly limited, but may be, for example, watermelon, zucchini, or loofah. The Zingiberaceae plant is not particularly limited, but may be, for example, ginger or ginger. The Polygonaceae plant is not particularly limited, but may be, for example, rhubarb. The Dioscoreaceae plant is not particularly limited, but may be, for example, Chinese yam. The Vitaceae plant is not particularly limited, but may be, for example, Muscat. The Rosaceae plant is not particularly limited, but may be, for example, mulberry. The Solanaceae plant is not particularly limited, but may be, for example, paprika, chili pepper, potato, or bell pepper. The Ericaceae plant is not particularly limited, but may be, for example, blueberry. The Menthaceae plant is not particularly limited, but may be, for example, pomegranate. The Rutaceae plant is not particularly limited, but may be, for example, a kaffir lime or kumquat. The mushroom is not particularly limited, but may be, for example, a mushroom, a shiitake mushroom, or a Bunashimeji mushroom. Mushrooms are also called fungal plants. The microalgae is not particularly limited, but may be, for example, a Euglena.

[0020] In this embodiment, parsley, angelica tree, mitsuba, violet harmony, carrot, leaf carrot, fruit carrot, coriander, dill, fennel, rosemary, spearmint, lemon palm, shiso, majuram, sweet basil, thyme, oregano, peppermint, sage, tea plant, camellia, temarikatahiba, Brussels sprouts, cauliflower, romanesco, arugula, wasabi radish, komatsuna, wasabi, cauliflower, red heart radish, selvatico, bok choy, broccoli, mizuna, radish, radish, watercress, turnip, tatsoi, artichoke, golden kidney leaf, chrysanthemum, lettuce, goat's lettuce, Also provided is an anti-inflammatory or anti-aging composition containing extracellular vesicles derived from bow, endive, Hakata Jerusalem artichoke, romaine lettuce, radicchio, epazote, amaranth, spinach, Swiss chard, beet, okahijiki, chive, leek, onion, shallot, Morus johns, edamame, urizumame, kinukotoba, snap peas, pea sprouts, watermelon, zucchini, loofah, ginger, mioga, rhubarb, yam, muscat, mulberry, paprika, chili pepper, potato, bell pepper, blueberry, pomegranate, kaffir lime, kumquat, mushroom, shiitake mushroom, bunashimeji mushroom, or Euglena.

[0021] The extracellular vesicles used in this embodiment may be extracellular vesicles derived from parsley, Euglena, Angelica keiskei, Japanese trefoil, Violet Harmony, carrot leaves, rosemary, spearmint, lemon palm, shiso, marjuram, sweet basil, thyme, oregano, peppermint, tea plant, camellia, Temarikatahiba, Brussels sprouts, cauliflower, Romanesco, arugula, wasabi radish, komatsuna, wasabi, cauliflower, red radish, Selvatica, artichoke, golden kidney leaf, garland chrysanthemum, lettuce, burdock, epazote, amaranth, Swiss chard, Chinese chive, leek, onion, shallot, Morus alba, edamame, urizumame, snow peas, watermelon, ginger, rhubarb, Nagaimo, Muscat, mulberry, paprika, pomegranate, Kaffir lime, or mushroom. The extracellular vesicles used in this embodiment may be any combination selected from the extracellular vesicles listed above.

[0022] The anti-inflammatory or anti-aging composition of this embodiment has anti-inflammatory or anti-aging effects by containing the extracellular vesicles of this embodiment as an active ingredient. The composition may contain extracellular vesicles derived from parsley, euglena, angelica tree, mitsuba, violet harmony, ginseng leaves, rosemary, spearmint, lemon palm, shiso, majuram, sweet basil, thyme, oregano, peppermint, tea plant, camellia, temari katahiba, Brussels sprouts, cauliflower, romanesco, arugula, wasabi radish, komatsuna, wasabi, cauliflower, red radish, selvatica, artichoke, golden kidney leaf, chrysanthemum, lettuce, burdock, epazote, amaranth, Swiss chard, chives, leeks, onions, shallots, Morus alba, edamame, urizumame, snow peas, watermelon, ginger, rhubarb, Nagaimo, Muscat grapes, mulberry, paprika, pomegranate, kaffir lime, or mushrooms as an active ingredient, thereby exhibiting anti-inflammatory or anti-aging effects. By including any combination of active ingredients selected from the extracellular vesicles listed above, the composition may have anti-inflammatory or anti-aging effects.

[0023] The concentration of extracellular vesicles in the anti-inflammatory or anti-aging composition may be any amount as long as they can exert their function as an active ingredient. For example, ５ , 1.0x10 ６ , 1.0x10 ７ , 1.0x10 ８ , 1.0x10 ９ , or 1.0 x 10 １０ It may be particles / ml (or particles / g) or more.

[0024] In this embodiment, extracellular vesicles (EVs) are a general term for vesicles surrounded by a lipid bilayer secreted from cells of various organisms, such as animals and plants, and include exosomes derived from endosomal membranes. In this specification, extracellular vesicles are also referred to as EVs, and EVs has the same meaning as extracellular vesicles. In this embodiment, extracellular vesicles derived from plants, mushrooms, or microalgae can be obtained by methods known to those skilled in the art, including, but not limited to, ultracentrifugation, density gradient ultracentrifugation, ultrafiltration, isolation by chromatography, isolation using beads or antibodies, polymer precipitation, microfluidic separation technology, and the like. Specific examples of density gradient ultracentrifugation and ultrafiltration are provided below.

[0025] Density Gradient Ultracentrifugation Plants or mushrooms are juiced using a household juicer or similar device. This juice is centrifuged at 1,000 to 10,000 g for 10 to 60 minutes to remove larger debris. The resulting supernatant is then ultracentrifuged at 120,000 to 150,000 g for 60 minutes to overnight to obtain a precipitate. This precipitate is suspended in a buffer solution such as PBS, then added to a sucrose density gradient solution, and further ultracentrifuged at 120,000 to 150,000 g for 60 minutes to overnight. After ultracentrifugation, the target extracellular vesicles can be obtained by collecting the Band 1 or Band 2 fraction that accumulates at the 8-30% or 30-45% boundary. However, the collected fraction may also be mixed with a buffer solution such as PBS and further ultracentrifuged to obtain a precipitate that can be used as the final extracellular vesicle sample. Microalgae are centrifuged at 1,000 to 3,000 g for 10 to 60 minutes, and the precipitate is suspended in a buffer solution such as PBS. The suspension is disrupted using an ultrasonic disrupter, either directly or after freeze-thawing, and then centrifuged at 1,000 to 10,000 g for 10 to 60 minutes to remove larger fragments. The resulting supernatant is then ultracentrifuged at 120,000 to 150,000 g for 60 minutes to overnight to obtain a precipitate. This precipitate may be suspended in a buffer solution such as PBS and used as a sample of extracellular vesicles, or it may be added to a sucrose density gradient solution and further ultracentrifuged at 120,000 to 150,000 g for 60 minutes to overnight. After ultracentrifugation, the target extracellular vesicles can be obtained by collecting the Band 1 or Band 2 fraction accumulated at the boundary of 8 to 30% or 30 to 45%. However, if desired, the collected fraction may be mixed with a buffer solution such as PBS, and then further ultracentrifuged to obtain a precipitate, which may be used as the final sample of extracellular vesicles.

[0026] Ultrafiltration First, plants, mushrooms, or microalgae are juiced using a household juicer or the like. This juice is centrifuged at 1,000 to 10,000 g for 10 to 60 minutes to remove larger debris. The resulting supernatant is ultrafiltered using an ultrafiltration membrane appropriate for the size of the target extracellular vesicles to obtain the target extracellular vesicles. Here, filtration may be performed using two or more types of ultrafiltration membranes.

[0027] The parsley- or Euglena-derived extracellular vesicles contained in the composition of this embodiment are extracellular vesicles obtained using parsley or Euglena as a raw material, and may be obtained by density gradient ultracentrifugation or ultrafiltration. They may also be extracellular vesicles obtained by a combination of both techniques. The parsley- or Euglena-derived extracellular vesicles may be extracellular vesicles obtained by the above-mentioned treatment. As shown in the examples, the parsley- or Euglena-derived extracellular vesicles may be heat-resistant and may have the property that their anti-inflammatory or anti-aging effects are not weakened by thermal history. The thermal history may be 35°C or higher, 40°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, 75°C or higher, 80°C or higher, 85°C or higher, 90°C or higher, or 95°C or higher, or 140°C or lower, 130°C or lower, 120°C or lower, 110°C or lower, or 100°C or lower. That is, the parsley- or Euglena-derived extracellular vesicles of this embodiment may be heat-stable, for example, at temperatures of 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, or 95°C or higher. The temperature referred to here generally refers to external temperature, but may also be internal temperature. In this embodiment, the parsley- or Euglena-derived extracellular vesicles are heat-resistant, which means that they are resistant to sterilization treatments and the like, and that the anti-inflammatory or anti-aging effects of the extracellular vesicles are not attenuated. "The effects are not attenuated" means, for example, that measurement data obtained under appropriate temperature conditions for measuring each effect (e.g., room temperature, a temperature described in the Examples, or a temperature described in the action measurement protocol) are not attenuated under predetermined heating conditions; for example, the difference may be within a range of ±20% or within a range of ±10% of the original measurement data. Furthermore, the heated parsley- or Euglena-derived extracellular vesicles may be heat-stable, may exhibit anti-inflammatory and / or anti-aging effects equivalent to those of the non-heated parsley- or Euglena-derived extracellular vesicles, and may suppress the expression of aging markers or cytokines equivalent to those of the non-heated parsley- or Euglena-derived extracellular vesicles.Alternatively, parsley- or Euglena-derived extracellular vesicles may exhibit a smaller expression level of aging markers or cytokines when heated extracellular vesicles are administered to cells divided by the expression level of aging markers or cytokines when unheated extracellular vesicles are administered to cells, compared to extracellular vesicles not derived from parsley or Euglena.When the anti-inflammatory composition or anti-aging composition of this embodiment contains heat-stable parsley- or Euglena-derived extracellular vesicles, it becomes possible to easily sterilize the composition by heat without losing activity, for example, before commercialization.

[0028] The size of extracellular vesicles obtained from plants, mushrooms, or microalgae can be analyzed by methods known to those skilled in the art. For example, the particle size, particle size distribution, and concentration of extracellular vesicles can be examined by nanoparticle tracking analysis (NTA). Extracellular vesicles obtained using density gradient ultracentrifugation may have a particle size of, for example, 10 to 500 nm, and the concentration of extracellular vesicles may be high at particle sizes of 50 to 450 nm. Furthermore, extracellular vesicles obtained using ultrafiltration may have a particle size of, for example, 10 to 300 nm, and the concentration of extracellular vesicles may be high at particle sizes of 50 to 250 nm. Extracellular vesicles having a more limited particle size range may be obtained using ultrafiltration than when density gradient ultracentrifugation is used.

[0029] In this embodiment, "anti-inflammatory" refers to, for example, an effect of suppressing inflammation, an effect of preventing further inflammation, an effect of preventing inflammation, etc. The "anti-inflammatory effect" exerted by the composition of this embodiment may be exerted, for example, by suppressing the expression in cells of inflammation-inducing cytokines (IL-4, IL-6, IL-13, IL-33, IL-1β, CXCL1, CXCL2, TNF-α, etc.), thereby suppressing cytokine release. Furthermore, for example, the anti-inflammatory effect can relieve swelling, pain, fever, etc. caused by inflammation, or can even suppress inflammation not accompanied by symptoms such as swelling. The composition of this embodiment can be applied to the treatment or prevention of various types of inflammation. The inflammation may be, for example, acute inflammation or chronic inflammation, or may be, for example, inflammation caused by an allergic disease or an autoimmune disease. The composition of the present embodiment can be used for the treatment or prevention of inflammatory diseases such as dermatitis, bronchitis, arthritis, gastritis, hepatitis, nephritis, encephalitis, systemic lupus erythematosus, ulcerative colitis, Crohn's disease, and dry eye; inflammation-related diseases such as diabetes, decreased renal function, decreased liver function, cardiovascular disease, arteriosclerosis, cerebral infarction, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, narcolepsy, and cancer; and conditions such as weakened immunity, decreased muscle strength and endurance, sarcopenia (including sarcopenia due to aging), frailty, osteoporosis, osteoarthritis (e.g., knee osteoarthritis), spinal deformity, tendon degeneration, weight loss, skin blemishes, wrinkles, and presbyopia. Additionally, the composition of the present embodiment can be used for the treatment of COVID-19 infection and its sequelae, as well as other infectious diseases. The composition of the present embodiment can exert an anti-inflammatory effect when administered directly or indirectly to the affected area.

[0030] Here, the composition of this embodiment can suppress inflammation, thereby suppressing inflammation-related aging. That is, the composition of this embodiment has an anti-aging effect, and in this embodiment, if the composition has an anti-inflammatory effect, it can also be said to have an anti-aging effect. Furthermore, anti-aging refers to suppressing symptoms associated with aging or aging. The relationship between inflammation and aging will be described below.

[0031] Cellular senescence, which plays an important role in individual aging (Baker et al., Nature 479:232-236 2011), is considered a chronic inflammatory state. Senescent cells also exhibit increased expression of cellular senescence markers such as p16, p15, and p21, and secrete inflammatory cytokines (e.g., IL-6, IL-8, etc.), suggesting that inflammation is associated with the development of aging-related diseases (Birch et al., Genes Dev. 34:1565-1576 2020; Sharpless et al., Nat. Rev. Cancer 15:397-408 2015; and van Deursen, Nature 509:439-446 2014).

[0032] In this embodiment, "anti-aging" refers to an effect of inhibiting aging, an effect of preventing aging, or the like. "Anti-aging" can be used interchangeably with "anti-aging." The "anti-aging effect" exhibited by the composition of this embodiment can inhibit, for example, symptoms associated with cellular aging. For example, symptoms associated with aging of epithelial cells, bone cells, chondrocytes, nerve cells, muscle cells, adipocytes, hepatocytes, lymphocytes, and the like can be inhibited, such as wrinkles, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, narcolepsy, decline in muscle strength and endurance, sarcopenia, frailty, osteoporosis, osteoarthritis and other arthropathy, spinal deformities, tendon degeneration, weight loss, diabetes, decreased immunity, decreased kidney function, decreased liver function, cardiovascular disease, arteriosclerosis, cerebral infarction, and cancer. Furthermore, symptoms of COVID-19 infection and its sequelae, as well as those caused by other infectious diseases, can also be inhibited. As a result, effects such as an extension of lifespan can also be obtained. With regard to the "anti-aging effect," the anti-aging effect may be exerted, for example, by suppressing the expression of senescence markers (p15, p16, IL-6) or cytokines (IL-4, IL-6, IL-13, IL-33, IL-1β, CXCL1, CXCL2, TNF-α, etc.) in cells, thereby suppressing cytokine release. That is, the composition of this embodiment suppresses the expression of senescence markers (IL-6, p15, p16, etc.) or cytokines (IL-4, IL-6, IL-13, IL-33, IL-1β, CXCL1, CXCL2, TNF-α, etc.) in cells, thereby suppressing cytokine release. The composition of this embodiment can exert an anti-inflammatory effect or anti-aging effect by being administered directly or indirectly to an affected area.

[0033] For example, when the expression level of cytokines can be suppressed to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% or less compared to when the composition of this embodiment is not added or administered, etc., it can be determined that the composition has an anti-inflammatory effect or an anti-aging effect. Preferably, when the expression level of cytokines can be suppressed to 70% or less in the aging induction experiment (p15, p16, IL-6) and to 40% or less in the inflammation induction experiment (IL-6, IL-1β), compared to when the composition of this embodiment is not added or administered, etc., it can be determined that the composition has an anti-aging effect or an anti-inflammatory effect.

[0034] The anti-inflammatory composition of this embodiment may have both an anti-inflammatory effect and an anti-aging effect. Furthermore, the anti-aging composition of this embodiment may have both an anti-inflammatory effect and an anti-aging effect. That is, the anti-inflammatory composition may be an anti-aging composition, and the anti-aging composition may be an anti-inflammatory composition.

[0035] The anti-inflammatory composition or anti-aging composition of this embodiment may have a skin-improving effect, and for example, administration of the anti-inflammatory composition or anti-aging composition may promote whitening and skin turnover, and may also improve pores, age spots (senile lentigo, melasma, inflammatory pigmentation), latent spots (potential invisible spots), redness, dullness, skin texture, firmness, sagging, etc. These effects can be evaluated by methods known to those skilled in the art, for example, using an imaging diagnostic device, etc.

[0036] The composition of this embodiment may be for use in foods, cosmetics, or medicines. That is, this embodiment also provides an anti-inflammatory or anti-aging composition for use in foods, cosmetics, or medicines.

[0037] The composition can be in a form suitable for use in, for example, food, cosmetics, or medicine, and can be in a form suitable for use in, for example, but not limited to, solid, liquid, granular, particulate, powder, capsule, cream, paste, or jelly. For example, when encapsulated, it may be coated with one or more layers as needed. To achieve these forms, the composition can be produced using conventionally known additives, and, as needed, may be used, for example, but not limited to, excipients, binders, diluents, disintegrants, buffers, thickeners, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, etc., known to those skilled in the art.

[0038] The food composition is not particularly limited, but examples thereof include foods and beverages, health foods, functional foods, foods for specified health uses, functional nutritional foods, health supplements, and dietary supplements.

[0039] The cosmetic compositions are not particularly limited, but examples thereof include scalp cosmetics, skin cosmetics, finishing cosmetics, perfumes, nail cosmetics, and bath cosmetics.

[0040] Dosage forms of pharmaceutical compositions include tablets, capsules, granules, fine granules, powders, liquids, syrups, chewable tablets, oral preparations such as troches, ointments, gels, creams, injections, sublingual preparations, inhalants, suppositories, liniments, patches, eye drops, nasal drops, etc. The above dosage forms may be applied to compositions for food or cosmetic use.

[0041] When provided, the composition may be labeled with claims such as "suppresses inflammation," "for those concerned about aging," "prevents muscle loss," "suppresses muscle weakness," "increases muscle mass," "increases muscle strength," "improves muscle endurance," or "promotes skin turnover."

[0042] The composition may contain any amount of extracellular vesicles as long as they can exert an anti-inflammatory or anti-aging effect. In addition, other active ingredients may be contained as long as they do not impair the effect.

[0043] The aspects of the anti-inflammatory or anti-aging composition in this embodiment also apply to other embodiments described below.

[0044] In another embodiment, there is provided a method for suppressing inflammation or aging using extracellular vesicles derived from plants, mushrooms, or microalgae of the Apiaceae, Lamiaceae, Theaceae, Selaginaceae, Brassicaceae, Asteraceae, Amaranthaceae, Amaryllidaceae, Fabaceae, Cucurbitaceae, Zingiberaceae, Polygonaceae, Dioscoreaceae, Vitaceae, Rosaceae, Solanaceae, Ericaceae, Lythraceae, or Rutaceae family. In other embodiments, parsley, angelica tree, mitsuba, Violet Harmony, carrot, leaf carrot, fruit carrot, coriander, dill, fennel, rosemary, spearmint, lemon palm, shiso, majuram, sweet basil, thyme, oregano, peppermint, sage, tea plant, camellia, temarikatahiba, Brussels sprouts, cauliflower, Romanesco, arugula, wasabi radish, komatsuna, wasabi, cauliflower, red heart radish, Selvatico, bok choy, broccoli, mizuna, radish, radish, watercress, turnip, tatsoi, artichoke, golden kidney leaf, chrysanthemum, lettuce, Also provided is a method for suppressing inflammation or aging using extracellular vesicles derived from burdock, endive, Hakata Jerusalem artichoke, romaine lettuce, radicchio, epazote, amaranth, spinach, Swiss chard, beetroot, okahijiki, chives, leeks, onions, shallots, Morus alba (coconut) beans, edamame beans, urizumame (sweet beans), kinuko peas, snap peas, pea sprouts, watermelon, zucchini, loofah, ginger, mioga (Japanese ginger), rhubarb, yam, muscat, mulberry, paprika, chili pepper, potato, bell pepper, blueberry, pomegranate, kaffir lime, kumquat, mushroom, shiitake mushroom, bunashimeji mushroom, or Euglena. The method for suppressing inflammation or aging is carried out, for example, in order to suppress inflammation or aging inside or outside the human body, and can suppress inflammation or aging inside or outside the human body by being carried out when it is desired to suppress inflammation or aging or when treating a disease caused by inflammation or aging.

[0045] In another embodiment, there is provided an anti-inflammatory agent or an anti-aging agent comprising extracellular vesicles derived from a plant, mushroom, or microalgae of the Apiaceae, Lamiaceae, Theaceae, Selaginaceae, Brassicaceae, Asteraceae, Amaranthaceae, Amaryllidaceae, Fabaceae, Cucurbitaceae, Zingiberaceae, Polygonaceae, Dioscoreaceae, Vitaceae, Rosaceae, Solanaceae, Ericaceae, Lythraceae, or Rutaceae family. In other embodiments, parsley, angelica tree, mitsuba, Violet Harmony, carrot, leaf carrot, fruit carrot, coriander, dill, fennel, rosemary, spearmint, lemon palm, shiso, majuram, sweet basil, thyme, oregano, peppermint, sage, tea plant, camellia, temae katahiba, Brussels sprouts, cauliflower, romanesco, arugula, wasabi radish, komatsuna, wasabi, cauliflower, red heart radish, selvatico, bok choy, broccoli, mizuna, radish, radish, watercress, turnip, tatsoi, artichoke, golden kidney leaf, chrysanthemum, sanchi Also provided is an anti-inflammatory agent or an anti-aging agent comprising extracellular vesicles derived from ginger, burdock, endive, Hakata Jerusalem artichoke, romaine lettuce, radicchio, epazote, amaranth, spinach, Swiss chard, beet, okahijiki, chives, leeks, onions, shallots, Morus junos, edamame, urizumame, kinuko peas, snap peas, pea sprouts, watermelon, zucchini, loofah, ginger, myoga, rhubarb, yam, muscat, mulberry, paprika, chili pepper, potato, bell pepper, blueberry, pomegranate, kaffir lime, kumquat, mushroom, shiitake mushroom, Bunashimeji mushroom, or Euglena.

[0046] The anti-inflammatory agent or anti-aging agent may be in the form of, but is not limited to, oral preparations such as tablets, capsules, granules, fine granules, powders, liquids, syrups, chewable tablets, and lozenges, as well as ointments, gels, creams, injections, sublingual preparations, inhalants, and suppositories. To obtain these dosage forms, the agent can be produced using conventionally known additives. Industry-known excipients, binders, diluents, disintegrants, buffers, thickeners, stabilizers, emulsifiers, dispersants, suspending agents, and preservatives may be used as needed. The anti-inflammatory agent or anti-aging agent may also be incorporated into foods, cosmetics, pharmaceuticals, or the like. A composition containing an anti-inflammatory agent may be an anti-inflammatory composition, and a composition containing an anti-aging agent may be an anti-aging composition. An anti-inflammatory composition or anti-aging composition containing an anti-inflammatory agent or anti-aging agent may be for food, cosmetics, or pharmaceutical use.

[0047] The anti-inflammatory or anti-aging composition, or anti-inflammatory agent or anti-aging agent of this embodiment is administered to animals in general, including humans. The dosage and frequency of administration may be appropriately determined depending on various conditions, such as the administration route, age, weight, and symptoms of the recipient. The recipient is preferably a mammal, more preferably a human, monkey, cat, pig, horse, cow, mouse, rat, guinea pig, dog, or rabbit, and even more preferably a human.

[0048] A method for inhibiting inflammation or aging may include administering to a subject an anti-inflammatory or anti-aging composition, or an anti-inflammatory or anti-aging agent. As used herein, "method" includes therapeutic and non-therapeutic methods, and "use" includes therapeutic and non-therapeutic uses.

[0049] The present invention will be described in more detail below with reference to specific examples, although the present invention is not limited thereto.

[0050] Example 1 (1) Evaluation of the Anti-aging and Anti-inflammatory Effects of EVs in Human IMR90 Cells and Mouse Macrophage Cells (1-1) Culturing of Human Normal Fibroblast IMR90 Cells Human normal fibroblast IMR90 cells were cultured in E-MEM medium (WAKO) supplemented with 10% FBS, MEM NEAA (Gibco), and Sodium Pyruvate (Gibco) at 37°C and 3% O. ２ , 5% CO ２ The cells were cultured in an incubator under the conditions of ５ The cells were subcultured at a ratio of 100 cells / 100 mm dish. (1-2) Establishment of IMR90 Cells Capable of Inducing Oncogenic Ras. Plasmid pLNCX2 ER:ras (addgene) was introduced into retrovirus packaging cells Plat-A using polyethyleneimine and cultured for one week. This resulted in the production of retrovirus containing the ER:ras gene in the Plat-A culture supernatant. Hexadimethrine bromide (Merck) was then added to the culture supernatant containing this retrovirus, and the ER:ras gene was introduced into IMR90 cells by adding the medium to the IMR90 cells. Furthermore, G418 (Gibco) was added to the IMR90 cell medium to select only IMR90 cells into which the ER:ras gene had been introduced, establishing an IMR90 cell line capable of inducing cellular senescence via the Ras gene. (1-3) Culture of Mouse Macrophage Cells Mouse macrophage cells (Raw-Blue cells, InvivoGen) were cultured in Dulbecco's Modified Eagle Medium (Thermo Fisher) supplemented with 10% FBS, 100 μg / ml Normocin (InvivoGen), and Pen-Strep (100 U / ml) at 37°C and 5% CO ２ The cells were cultured in an incubator under the conditions of ６The cells were subcultured at a ratio of 0.1 cells / 100 mm dish. (1-4) Preparation of EVs Using Sucrose Density Gradient Ultracentrifugation EV fraction solutions were prepared from the plants and mushrooms listed in Table 1. First, the plants and mushrooms were washed with water in a plastic bucket, drained, and then juiced using a household juicer (HUROM). This juice was centrifuged at 3,000 g for 20 minutes and then at 10,000 g for 40 minutes twice using an Avati J-E (JA-14 rotor, Bechman Courlter) to remove large debris. The resulting supernatant was then ultracentrifuged at 150,000 g for 90 minutes using an Optima XE-90 (SW32 Ti rotor, Bechman Courlter) to obtain a precipitate. Because this precipitate contained EVs, the precipitate was suspended in PBS, then added to a sucrose density gradient solution (8, 30, 45, or 60%) and further ultracentrifuged at 150,000 g for 90 minutes. After ultracentrifugation, the Band 2 fraction, which accumulated at the 30-45% boundary, was collected using a 20 ml syringe (Terumo), mixed with PBS, and then ultracentrifuged at 150,000 g for 90 minutes. The precipitate was suspended in PBS to obtain an EV fraction solution. The parsley and ginger fractions after ultracentrifugation with the sucrose density gradient solution are shown in Figure 1. The arrows indicate the Band 2 fraction. The Band 2 fractions of parsley and ginger had densities of 1.18 g / ml or less. The culture solution for the microalgae was centrifuged at 3,000 g for 20 minutes, the microalgae were collected, suspended in PBS, freeze-thawed, and then disrupted using an ultrasonic disrupter (manufactured by Sonyfire Branson). The disrupted solution was centrifuged twice, at 3,000 g for 20 minutes and then at 10,000 g for 40 minutes, to remove larger fragments. The resulting supernatant was then ultracentrifuged at 150,000 g for 90 minutes to obtain a precipitate. Since this precipitate contains EVs, the precipitate was suspended in PBS for use, or added to a sucrose density gradient solution (8, 30, 45, 60%) and further ultracentrifuged at 150,000 g for 90 minutes. After ultracentrifugation, the Band 2 fraction accumulated at the 30-45% boundary was collected using a 20 ml syringe (Terumo), mixed with PBS, and then ultracentrifuged at 150,000 g for 90 minutes. The precipitate was suspended in PBS to obtain an EVs fraction solution.

[0051]

[0052] (1-5) Preparation of EVs Sample Using Ultrafiltration Membrane. Cucumber beans, parsley, and Yanaka ginger were washed with water in a plastic bucket, drained, and then juiced using a household juicer (HUROM). The juice was centrifuged at 3,000 g for 20 minutes and then at 20,000 g for 40 minutes using an Avati J-E (JA-14 rotor, Bechman Courlter) to remove large debris. The resulting supernatant was passed through a Supracap depth capsule filter (Cytiva) and then ultrafiltered using a TFF Omega membrane (Cytiva) to obtain an EVs sample.

[0053] (1-6) LPS stimulation treatment EVs prepared using sucrose density gradient ultracentrifugation or ultrafiltration membrane at a concentration of 1.0 x 10 １０ The EV fraction solution or EV sample containing 0.8 x 10 particles / ml was ５ Mouse macrophage cells seeded at 12-well cells / well were sensitized for 16 hours, washed with PBS, and stimulated with 1 μg / ml LPS (derived from Salmonella minnesota R595, manufactured by WAKO) for 24 hours to induce inflammation. After stimulation, the cells were washed with PBS, and RNA was extracted from the mouse macrophage cells using TRIsure (Nippon Genetics).

[0054] (1-7) Evaluation of the effect of EVs on the expression of senescence markers in Ras-induced human IMR90 cells and on the expression of inflammatory cytokines in mouse macrophage cells after LPS stimulation. 24 hours before the addition of the EV fraction solution or EV sample, IMR90 cells were cultured at 4-6 x 10 ４ The EV fraction solution or EV sample prepared by sucrose density gradient ultracentrifugation or ultrafiltration was seeded on IMR90 cells at 1 x 10 cells / well. １０EVs were added at a concentration of 1000particles / ml. Simultaneously, 4-hydroxytamoxifen was added to IMR90 cells to induce overexpression of activated Ras and cellular senescence. RNA was then extracted from the IMR90 cells, and the expression levels of cellular senescence markers (p15, p16, and Il6) were measured by RT-qPCR to evaluate the anti-aging effect of EVs. The results are shown in Figure 2. The effect of EVs on the expression of inflammatory cytokines (IL-6 and IL-1β) in LPS-stimulated mouse macrophage cells was evaluated using the expression of these cytokines as an indicator. IL-6 and IL-1β expression levels were measured by RT-qPCR using RNA extracted from LPS-stimulated mouse macrophage cells. The expression of senescence markers when human IMR90 cells in which Ras senescence was induced were sensitized with EV fraction solutions derived from plants, mushrooms, or microalgae listed in Table 1, which were prepared by sucrose density gradient ultracentrifugation, or PBS, and the expression of IL-6 and IL-1β when mouse macrophage cells were sensitized and stimulated with LPS are shown in Figures 2-1 to 2-21. Furthermore, the expression of IL-6 or IL-1β when mouse macrophage cells were sensitized with various concentrations of EV fraction solutions or EV samples obtained by sucrose density gradient ultracentrifugation or ultrafiltration, or PBS (control), and stimulated with LPS are shown in Figures 3-1 and 3-2. The results for the EV fraction solution or EV sample derived from cucumber bean at various concentrations obtained by sucrose density gradient ultracentrifugation or ultrafiltration are shown in Figure 3-1, and the results for the EV samples derived from parsley and Yanaka ginger at various concentrations obtained by ultrafiltration (TFF) are shown in Figure 3-2. The expression results (vertical axis) in Figures 2-1 to 2-21 and Figures 3-1 and 3-2 are shown as relative values ​​(fold change) when the expression level in cells stimulated with Ras or LPS, in which PBS was added instead of the EV fraction solution or EV sample, was set to 1, using Gapdh as a correction gene. The horizontal axis in Figure 3-2 shows the EV concentration (particles / mL).

[0055] The results in Figures 2-1 to 2-21 revealed that mouse macrophage cells exposed to EV fraction solutions derived from many of the plants, mushrooms, or microalgae listed in Table 1 showed significantly reduced expression of inflammatory cytokines compared to the PBS-containing solution. Furthermore, the results in Figures 3-1 and 3-2 show that EVs derived from cucumber beans, parsley, and Yanaka ginger dose-dependently suppressed the expression of inflammatory cytokines in mouse macrophage cells. The cucumber bean-derived EV sample prepared by ultrafiltration had a more effective inhibitory effect than the same amount of EV fraction solution prepared by sucrose density gradient ultracentrifugation.

[0056] (1-8) Nanoparticle Tracking Analysis (NTA) The particle size, particle size distribution, and concentration of EVs in EV fraction solutions derived from urizum beans, parsley, Yanaka ginger, and Euglena obtained by sucrose density gradient ultracentrifugation, and in EV samples derived from urizum beans, parsley, and Yanaka ginger obtained by ultrafiltration, were measured using a NanoSight NS300 or LM10 (manufactured by Malvern Instrument) equipped with a 405 nm blue laser. The results for urizum beans are shown in Figure 4-1, those for parsley in Figure 4-2, those for Yanaka ginger in Figure 4-3, and those for Euglena in Figure 4-4. Figures 4-1A, 4-2A, 4-3A, and 4-4 show the results for EV fraction solutions prepared by sucrose density gradient ultracentrifugation (UC), while Figures 4-1B, 4-2B, and 4-3B show the results for EV samples prepared by ultrafiltration (TFF). The results in Figures 4-1 to 4-4 indicate that the EV fraction solutions derived from cucumber beans, parsley, Yanaka ginger, and Euglena prepared by sucrose density gradient ultracentrifugation contained EVs with an average size of 128 nm (50-500 nm), 216 nm (50-500 nm), 215 nm (50-500 nm), and 211 nm (50-500 nm), respectively. Furthermore, EV samples derived from cucumber beans, parsley, and Yanaka ginger prepared by ultrafiltration contained EVs with average sizes of 125 nm (50-300 nm), 148 nm (50-300 nm), and 135 nm (50-300 nm), respectively. These results demonstrate that ultrafiltration can produce EVs with a more specific size range than sucrose density gradient ultracentrifugation.

[0057] Example 2 (2) Improvement effect on human skin Freeze-dried parsley EVs were suspended in water and administered daily before bedtime for 5 weeks at 8 x 10 ml. １２The subjects orally ingested parsley EVs (particles / day). Subsequently, facial skin conditions were analyzed using the VISIA skin imaging diagnostic device (Figures 5-1 and 5-2). The results in Figure 5-1 indicated that parsley EVs significantly improved skin whitening, skin turnover, pores, uneven skin tone, age spots (senile lentigo, melasma, inflammatory pigmentation), pigmentation, redness, dullness, skin texture, firmness, and sagging. Furthermore, the results of measuring melanin deep in the skin (Figure 5-2) showed that melanin, the source of age spots, was metabolized and significantly reduced (Figure 5-2, left and center). Furthermore, the percentile values, which indicate the percentage of the group counting from the bottom, increased compared to people of the same age who underwent VISIA analysis (Figure 5-2, right), indicating that parsley EVs significantly improved latent age spots (potential for invisible age spots). Therefore, parsley EVs are believed to have a skin-improving effect.

[0058] Example 3 (3) Stability of EVs when Heat-treated The prepared EVs solution was subjected to heat treatment under conditions commonly used for food (65°C for 10 minutes or 85°C for 30 minutes), and the EVs were dissolved in 1 x 10 １０ The solution was added to the medium at a concentration of 1 μg / mL. Mouse macrophage cells (Raw-Blue cells) were sensitized for 16 hours, then stimulated with 1 μg / mL LPS for 24 hours and washed with PBS. RNA was extracted using TRIsure, cDNA was synthesized by reverse transcription, and IL-6 expression was confirmed by qPCR. As a control, cells were cultured in a medium containing an equal volume of PBS instead of the EV solution. The relative values ​​are shown with the IL-6 expression level in the control cells set at 1 (Figure 6). The results in Figure 6-A show that the effects of plant-derived (parsley-derived) EVs, even those heat-treated at 65°C and 85°C, were not attenuated compared to unheated EVs. Furthermore, the results in Figure 6-B show that the effects of microalgae-derived (Euglena-derived) EVs and heat-treated EVs were not attenuated compared to unheated EVs. Therefore, parsley- and microalgae-derived EVs are thought to have heat resistance, and therefore can be easily sterilized to produce products without losing activity through heat treatment.

[0059] Example 4 (4) Anti-inflammatory effect in mouse colitis model mice. １１Parsley-derived EVs were administered to 8-week-old wild-type male mice (C57BL / 6J) for 1 week, and then 3% dextran sulfate sodium (DSS), which induces colitis, was added to their water supply. Subsequently, the same amount of parsley-derived EVs was administered. As shown in Figure 7A, mice administered parsley-derived EVs without DSS treatment showed similar weight gain as the PBS-fed control group, indicating no acute toxicity of parsley-derived EVs to the animals. Furthermore, as shown in Figure 7B, fecal blood was observed in both the PBS-fed and parsley-derived EVs-fed groups from day 3 of DSS administration, and increased with the number of days of administration. Anal bleeding was observed in 50% of mice in the PBS-fed group on day 6 and 100% on day 7, whereas it was observed in only 30% of mice in the parsley-derived EVs-fed group on day 6 and 50% on day 7. As shown in Figure 7C, the length of the colon from the cecum to the anus significantly decreased from day 3 onwards in the PBS-fed group, whereas significant differences were observed from day 5 onwards in the parsley-derived EVs-fed group. Next, RNA was extracted from the anterior, middle, and posterior colonic regions, starting from the region closest to the cecum, and changes in the expression of the inflammatory cytokine Il6 were examined by qPCR. As shown in Figure 7D, expression was significantly suppressed in the parsley-derived EVs-fed group on day 7 of DSS-fed compared to the PBS-fed group. Furthermore, mice were perfusion-fixed on days 0 and 7 after DSS ingestion. The colonic region from the cecum to the anus was cut into paraffin sections, stained with HE and Alcian blue (pH 2.5), and the morphology of the colonic sections was observed. As shown in Fig. 7E, on day 0 of DSS ingestion, in both the PBS-ingested group and the parsley-derived EVs-ingested group, Alcian blue-stained areas, which bind to the carboxyl and sulfate groups of acidic mucus polysaccharides, were observed throughout almost the entire colon, including the distal colon (the boxed area in the left panel of Fig. 7E). In contrast, on day 7 of DSS ingestion, in the PBS-ingested group, the Alcian blue-stained areas in the distal colon had almost disappeared, whereas in the parsley-derived EVs-ingested group, the Alcian blue-stained areas were present even in the distal colon.Furthermore, immunostaining images using epithelial cell markers β-catenin and ZO-1 revealed a significant loss of simple columnar epithelial structure in the PBS-fed group on day 7 of DSS ingestion, whereas the epithelial structure was maintained in the parsley-derived EVs-fed group.

[0060] Example 5 (5) Therapeutic Effect on Dermatitis The abdominal hair of 8-week-old C57BL / 6J male mice was shaved, and 5% TNCB (2,4,6-trinitrochlorobenzene: Tokyo Chemical Industry Co., Ltd.) dissolved in acetone:ethanol (1:9) solution was applied at 150 μl / mouse for sensitization. Six days later, 1% TNCB dissolved in acetone was applied to the front and back of both ears at 10 μl each to induce inflammation. Acetone was applied to the non-inflammation group. Six hours after inflammation induction, PBS or 1.8 × 10 １０ A 10 μl drop of a solution containing particle / μl Brussels sprout-derived EVs or parsley-derived EVs was placed on the front and back of each ear and massaged with the fingers to allow penetration. After 18 hours, the thickness of the ear was measured using a micrometer. The results are shown in Figure 8A. Measurements were taken for both ears, and the average was used to represent the ear thickness of that individual. The results in Figure 8A demonstrate that ear swelling associated with inflammation was significantly suppressed in the groups administered Brussels sprout-derived EVs or parsley-derived EVs compared to the control PBS group. Next, after measuring ear thickness, RNA was collected from the left ear, and the expression levels of inflammatory cytokines were measured by RT-qPCR. The results are shown in Figure 8B.

[0061] Example 6 (6) Evaluation of the Effects of Plant EVs on Obese Mice To quantitatively evaluate the effects of parsley- and Brussels sprout-derived EVs on aging and muscle loss associated with obesity in individual mice, mice were fed a high-fat diet for 13 weeks to induce obesity, and parsley- and Brussels sprout-derived EVs were administered to the water supply over the same period. A grip strength test to measure muscle strength (grip strength) and a 4-limb hanging test were performed on a non-obese mouse group (HFD-), an obese mouse group (HFD+), and a group of obese mice given parsley or Brussels sprout-derived EVs. The weight of the soleus muscle of each mouse was also measured. Specifically, 5-week-old C57B6J mice were obtained from Charles River and, after one week of acclimation, were divided into three groups so that the average weight of each group was equal. Each mouse was fed a high-fat diet and 3 × 10 EVs derived from parsley and Brussels sprouts for 13 weeks. ９The mice were given pure water containing EVs at a concentration of 1000 / mL. The normal diet was D12450B (Research Diet), and the high-fat diet was D12492 (Research Diet) ad libitum. The high-fat diet and the pure water containing each EV were replaced with fresh water twice a week. Grip strength tests were performed on obese mice administered each EV using a smart rat / mouse grip strength measuring device MK-380Si (Muromachi Kikai Co., Ltd.), with three measurements per mouse, and the average value was used as the grip strength value for each mouse. The results are shown in Figure 9. The 4-limb hanging test was performed using a wire hanging experiment box (Ohara Medical Industry Co., Ltd.). The wire mesh was turned upside down along with the mouse while the mouse was grasping the mesh with all limbs. The time the mouse could withstand before falling was recorded as the hanging time, and the hanging score was calculated by multiplying this endurance time by the body weight of each mouse. The results are shown in Figure 10. The soleus muscle was collected from the hind limb of each mouse that had been administered a high-fat diet and each EV for 13 weeks, and its weight was measured and evaluated. The results are shown in Figure 11. In the grip strength test, a decrease in grip strength was confirmed in the high-fat diet group due to obesity associated with the high-fat diet load, but both the mouse groups administered EVs derived from parsley and Brussels sprouts showed higher grip strength values ​​than the control obese mouse group (Figure 9). In the 4-limb Hanging Test, obesity reduced the hanging time (Fig. 10A) and the hanging score (Fig. 10C), which is the product of the hanging time and the mouse body weight. However, the obese mice administered Brussels sprout-derived EVs had significantly greater hanging time (Fig. 10B) and hanging score (Fig. 10D) than the control mice, demonstrating that the obese mice administered Brussels sprout-derived EVs have higher muscle endurance. The obese mice administered parsley-derived EVs showed a tendency to suppress the decline in muscle endurance, although no significant difference was observed in the 4-limb Hanging Test (Fig. 10B, D).Furthermore, when the soleus muscle was removed from the hind limbs of each mouse and its weight was measured, it was revealed that the soleus muscle weight was significantly increased in the obese mice that received EVs derived from parsley and Brussels sprouts compared to the control group (Figure 11). As described above, EVs derived from parsley and Brussels sprouts are thought to have the effect of suppressing the decline in muscle strength and muscle endurance, i.e., anti-aging effects.

Claims

1. An anti-inflammatory or anti-aging composition comprising extracellular vesicles derived from a plant, mushroom, or microalgae of the Umbelliferae, Lamiaceae, Theaceae, Selaginellaceae, Brassicaceae, Asteraceae, Amaranthaceae, Amaryllidaceae, Fabaceae, Cucurbitaceae, Zingiberaceae, Polygonaceae, Dioscoreaceae, Vitaceae, Rosaceae, Solanaceae, Ericaceae, Lythraceae, or Rutaceae.

2. Parsley, angelica, mitsuba, violet harmony, carrot, leaf carrot, fruit carrot, coriander, dill, fennel, rosemary, spearmint, lemon palm, shiso, marjuram, sweet basil, thyme, oregano, peppermint, sage, tea plant, camellia, temari katahiba, Brussels sprouts, cauliflower, romanesco, arugula, wasabi radish, komatsuna, wasabi, cauliflower, red heart radish, selvatico, bok choy, broccoli, mizuna, radish, radish, watercress, turnip, tatsoi, artichoke, golden chrysanthemum, chrysanthemum, lettuce, burdock An anti-inflammatory or anti-aging composition comprising extracellular vesicles derived from endive, Hakata Jerusalem artichoke, romaine lettuce, radicchio, epazote, amaranth, spinach, Swiss chard, beet, okahijiki, chives, leeks, onions, shallots, Moroccan beans, edamame, urizumame, snow peas, snap peas, pea sprouts, watermelon, zucchini, loofah, ginger, myoga, rhubarb, yam, muscat, mulberry, paprika, chili pepper, potato, bell pepper, blueberry, pomegranate, kaffir lime, kumquat, mushroom, shiitake mushroom, bunashimeji, or Euglena.

3. The composition described in claim 1 or 2, which suppresses the expression of inflammatory cytokines or aging markers.

4. The composition according to claim 3, wherein the inflammatory cytokine or aging marker is IL-4, IL-6, IL-13, IL-33, IL-1β, CXCL1, CXCL2, TNF-α, p15, or p16.

5. The composition according to claim 1 or 2 for use in food, cosmetics or medicines.

6. An anti-inflammatory or anti-aging composition comprising extracellular vesicles derived from parsley or Euglena.

7. The composition according to claim 6, wherein the anti-inflammatory or anti-aging effect is not weakened by heat history.

8. The composition according to claim 7, wherein the thermal history is a heat treatment at 40°C or higher.

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