Plant exosome composition and application thereof

Abstract

The invention provides a plant exosome composition and application thereof. The plant exosome composition comprises a ganoderma lucidum exosome, a gingko exosome and a glycyrrhiza glabra exosome in a mass ratio of (1-10): (1.5-10): (2-6). According to the plant exosome, the forms of the lucid ganoderma, the gingko and the glycyrrhiza glabra exosome are combined, the respective advantages of the lucid ganoderma, the gingko and the glycyrrhiza glabra can be better played, the content of active matters is increased, and then the plant exosome has the excellent effects of resisting oxidation, relieving and resisting inflammation, whitening and removing freckles and brightening the skin color.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant exosomes, and specifically relates to a plant exosome composition and applications thereof. Background Art

[0002] Ganoderma lucidum is a filamentous fungus that belongs to the class Hymenomycetes, order Polyporaceae, family Ganodermaceae, and genus Ganoderma. Wild Ganoderma lucidum is found in Lijiang, Baoshan, Tengchong, and Qujing, Yunnan Province, while cultivated Ganoderma lucidum is found in South my country, Southwest China, and Northeast China. Ganoderma lucidum has important medicinal properties and contains active substances such as extracellular and intracellular polysaccharides, triterpenes (such as ganoderic acid), proteases, and alkaloids. In traditional Chinese medicine, Ganoderma lucidum is sweet and neutral in nature, and enters the heart, lung, liver, and kidney meridians. It has the functions of replenishing qi and calming the mind, relieving cough and relieving asthma, and can be used to treat restlessness, insomnia, palpitations, cough and asthma due to lung deficiency, shortness of breath due to consumptive disease, and loss of appetite.

[0003] Ginkgo biloba L., commonly known as the duck's foot tree, maidenhair tree, and ginkgo nuts, is an endangered plant endemic to my country. Its leaves, resembling fans and duck's feet, are tender green in spring and summer, turning yellow in autumn, making them very beautiful and beloved by the public. Ginkgo is a tall tree, reaching up to 40 meters in height and a diameter at breast height of 4 meters. Its cultivation range in my country is widespread, ranging from northeastern China to southern China, and from eastern China to southwest China. Ginkgo belongs to the Ginkgoales, Ginkgoaceae, and Ginkgo genus, with over ten cultivated species. Ginkgo contains substances such as ginkgolic acid, ginkgol, and ginkgol, and possesses medicinal value, with the leaves being the primary medicinal part. Compounds in ginkgo leaves include flavonoids, ginkgolides, bilobalide, organic acids, phenols, polyprenols, and steroids. Ginkgo leaves are sweet, bitter, astringent, and neutral in nature, and enter the heart and lung meridians. They have the functions of promoting blood circulation and removing blood stasis, dredge meridians and relieve pain, astringe the lungs and relieve asthma, and clear turbidity and reduce lipids.

[0004] Glycyrrhiza glabra L., also known as licorice, European licorice, and fallen licorice, is a perennial herb found primarily in Xinjiang and Gansu in northwest my country. It belongs to the genus Glycyrrhiza, family Leguminosae, and family Caprifoliaceae. Flowering occurs from May to June, and fruiting occurs from July to September. The roots and rhizomes of Glycyrrhiza glabra are sturdy, 0.5 to 3 cm in diameter, and contain glycyrrhizin, liquiritin, and glycyrrhizic acid. Its nature and flavor are sweet and neutral, and it enters the heart, lung, spleen, and stomach meridians. In Traditional Chinese Medicine, it tonifies the spleen and replenishes Qi, clears away heat and toxins, eliminates phlegm and relieves cough, relieves pain, and harmonizes various medicinal herbs. In addition to their pharmaceutical applications, Ganoderma lucidum, Ginkgo biloba, and Glycyrrhiza glabra are also used in cosmetics. They are typically added in the form of extracts, such as White Crane Ganoderma extract, Chinese Ganoderma extract, Ginkgo biloba leaf extract, Ginkgo biloba root extract, and Glycyrrhiza glabra extract.

[0005] Currently, extracts of Ganoderma lucidum, Ginkgo biloba, or Glycyrrhiza glabra are typically extracted using water or ethanol as solvents through methods such as leaching, heating, evaporation, and concentration, or spray drying. These methods fail to produce vesicles, and the heating involved in the process can also affect the biological activity of the active ingredients, leading to loss of activity. Summary of the Invention

[0006] In response to the deficiencies of the prior art, the present invention provides a plant exosome composition and its application. When used in cosmetics, the plant exosome composition can have the effects of anti-oxidation, soothing and anti-inflammatory, whitening and freckle removal, and brightening skin tone.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a plant exosome composition, comprising Ganoderma lucidum exosomes, Ginkgo biloba exosomes and Glycyrrhiza glabra exosomes in a mass ratio of (1-10):(1.5-10):(2-6).

[0009] Wherein "1-10" can be 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 or 9.5, etc.;

[0010] “1.5-10” can mean 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 or 9.5, etc.;

[0011] “2-6” can be 2.2, 2.5, 2.8, 3, 3.2, 3.5, 3.8, 4, 4.2, 4.5, 4.8, 5, 5.2, 5.5 or 5.8, etc.

[0012] Exosomes are lipid bilayer structures containing proteins, lipids, nucleic acids, and polysaccharides. Plant exosomes can play a role in signaling, defense responses, and immune regulation. They facilitate the transport of substances across membranes. In addition to containing flavonoids, lactones, polysaccharides, and triterpenes, they also contain substances involved in cellular defense and immune regulation. Incorporating intact plant exosomes into cosmetics can enhance the efficacy of Ganoderma lucidum, Ginkgo biloba, and Glycyrrhiza glabra.

[0013] In the present invention, Ganoderma lucidum, Ginkgo biloba and Glycyrrhiza glabra are combined in the form of exosomes, which can better bring out the respective advantages of Ganoderma lucidum, Ginkgo biloba and Glycyrrhiza glabra, increase the content of active ingredients, and thus make the plant-containing exosomes have excellent antioxidant, soothing and anti-inflammatory, whitening and freckle removal, brightening skin tone, repairing skin barrier and improving skin tolerance.

[0014] Preferably, the Ganoderma lucidum exosomes are isolated from Ganoderma lucidum mycelial culture cells.

[0015] Preferably, the Ginkgo exosomes are isolated from Ginkgo callus cells.

[0016] Preferably, the Glycyrrhiza glabra exosomes are isolated from the rhizomes of Glycyrrhiza glabra.

[0017] Preferably, the Ganoderma lucidum exosomes are prepared by the following steps:

[0018] (1) taking a Ganoderma lucidum block and placing it in a culture medium to culture and obtain mycelium;

[0019] (2) washing the mycelium with liquid culture medium, and then pouring the liquid culture medium into a container with glass beads for cultivation to obtain mycelium cells of Ganoderma lucidum fermentation;

[0020] (3) washing the fermented mycelial cells of Ganoderma lucidum with water, then stirring and crushing them to obtain a slurry, centrifuging the slurry and collecting the supernatant to obtain a Ganoderma lucidum cell fermentation liquid;

[0021] (4) adding Ganoderma lucidum cell fermentation liquid, conjugated fatty acids, and bisamino polyethylene glycol to water, mixing, and then dialysis filtering to obtain a dialysate;

[0022] (5) The dialysate was centrifuged to obtain Ganoderma lucidum exosomes.

[0023] Preferably, the components of the culture medium in step (1) are glucose, magnesium sulfate, potassium dihydrogen phosphate, vitamin B1, peeled potatoes and water.

[0024] Preferably, the culture temperature in step (1) is 25-35°C (for example, 26°C, 28°C, 30°C, 32°C or 34°C), and the culture time is 5-15 days (for example, 6 days, 8 days, 10 days, 12 days or 14 days).

[0025] Preferably, the combination of the liquid culture medium in step (2) is glucose, peptone, yeast extract, potassium dihydrogen phosphate, magnesium sulfate, vitamin B1 and water.

[0026] Preferably, the culture temperature in step (2) is 25-35°C (for example, 26°C, 28°C, 30°C, 32°C or 34°C), and the culture time is 5-10 days (for example, 6 days, 7 days, 8 days or 9 days).

[0027] Preferably, the specific steps of the centrifugation in step (3) include: centrifuging the slurry at 3000-4000G (for example, 3100G, 3300G, 3500G, 3700G or 3900G, etc.) for 40-80min (for example, 45min, 50min, 55min, 60min, 65min, 70min or 75min, etc.), taking a supernatant and centrifuging it at 8000-15000G (for example, 9000G, 10000G, 11000G, 12000G, etc.), and centrifuging it at 15000G. The fermentation liquid is centrifuged at 13000G or 14000G for 40-90 min (for example, 45 min, 50 min, 60 min, 70 min, 80 min or 85 min), the secondary supernatant is centrifuged at 80000-120000G (for example, 9000G, 10000G or 11000G) for 90-120 min (for example, 95 min, 100 min, 105 min, 110 min or 115 min), and the tertiary supernatant is obtained to obtain the Ganoderma lucidum fermentation liquid.

[0028] Preferably, the mass ratio of the Ganoderma lucidum fermentation broth, conjugated fatty acids and bisamino polyethylene glycol in step (4) is (1-3):(0.001-0.1):(0.0005-0.05).

[0029] Wherein "1-3" can be 1.1, 1.3, 1.5, 1.8, 2, 2.2, 2.5, 2.7 or 2.9, etc.;

[0030] “0.001-0.1” can mean 0.003, 0.005, 0.008, 0.01, 0.03, 0.05, 0.07 or 0.09, etc.;

[0031] “0.0005-0.05” can mean 0.0007, 0.0009, 0.001, 0.003, 0.005, 0.008, 0.01 or 0.03, etc.

[0032] Preferably, the mass ratio of the Ganoderma lucidum fermentation liquid to water in step (4) is 1:(1.5-25), for example, it can be 1:2, 1:5, 1:7, 1:10, 1:12, 1:15, 1:18, 1:20, 1:22 or 1:24, etc.

[0033] Preferably, the conjugated fatty acid in step (4) comprises any one of conjugated linoleic acid, α-linolenic acid or arachidonic acid, or a combination of at least two thereof.

[0034] Preferably, the bisamino polyethylene glycol in step (4) comprises any one of bisamino-PEG4-NHS ester, bisamino-PEG6 or bisamino-PEG12, or a combination of at least two thereof.

[0035] Preferably, the centrifugation step in step (5) is 80,000-120,000G (for example, 90,000G, 100,000G, or 110,000G, etc.) and centrifuged for 60-90min (for example, 65min, 70min, 75min, 80min, or 85min, etc.).

[0036] Preferably, the ginkgo exosomes are prepared by the following steps:

[0037] (1) Ginkgo biloba leaves were sterilized and crushed, and then inoculated into a sterile liquid culture medium for cultivation;

[0038] (II) inoculating the surviving callus into a culture medium in a triangular flask and culturing it to obtain a Ginkgo callus suspension cell solution;

[0039] (III) filtering the suspended cell solution of the ginkgo callus tissue, then stirring and crushing the solution to obtain a slurry, centrifuging the slurry and collecting the supernatant to obtain the ginkgo cell solution;

[0040] (IV) adding the ginkgo cell solution, conjugated fatty acids, and bisamino polyethylene glycol to water, mixing, and then diafiltration to obtain a dialysate;

[0041] (V) Ginkgo exosomes were obtained by centrifuging the dialysate.

[0042] Preferably, the disinfection in step (I) is performed using a 75% ethanol aqueous solution.

[0043] Preferably, the sterile liquid culture medium in step (I) comprises potassium nitrate, ammonium nitrate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride dihydrate, potassium iodide, boric acid, manganese sulfate tetrahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, copper sulfate pentahydrate, cobalt chloride hexahydrate, ferrous sulfate heptahydrate, disodium EDTA, inositol, glycine, vitamin B1, vitamin B5, vitamin B6, sucrose, agar and water.

[0044] Preferably, the culture temperature in step (I) is 25-35°C (for example, 26°C, 28°C, 30°C, 32°C, or 34°C), and the culture time is 1-3 days (for example, 1 day, 2 days, or 3 days).

[0045] Preferably, the flask culture medium in step (II) comprises potassium dihydrogen phosphate, boric acid, calcium chloride, zinc sulfate heptahydrate, manganese sulfate tetrahydrate, potassium iodide, sodium molybdate dihydrate, magnesium sulfate, ferrous sulfate heptahydrate, copper sulfate pentahydrate, nickel chloride, cobalt chloride, disodium EDTA, vitamin B1, glycine, vitamin B6, inositol, niacin, sucrose, agar, naphthaleneacetic acid, 6-furfuryladenine and water.

[0046] Preferably, the culture temperature in step (II) is 20-30°C (for example, 21°C, 23°C, 25°C, 27°C or 29°C), and the culture time is 15-20 days (for example, 16 days, 17 days, 18 days or 19 days).

[0047] Preferably, the culture in step (II) is first irradiated with light for 10-15 h (for example, 11 h, 12 h, 13 h or 14 h, etc.), and then carried out in the dark.

[0048] Preferably, the specific steps of the centrifugation in step (III) include: centrifuging the slurry at 3000-4000G (for example, 3100G, 3300G, 3500G, 3700G or 3900G, etc.) for 40-80min (for example, 45min, 50min, 55min, 60min, 65min, 70min or 75min, etc.), taking a supernatant at 8000-15000G (for example, 9000G, 10000G, 11000G, 12000G, etc.), and centrifuging the supernatant at 15000G. , 13000G or 14000G, etc.) for 40-90 min (for example, 45 min, 50 min, 60 min, 70 min, 80 min or 85 min, etc.), the secondary supernatant was centrifuged at 80000-120000G (for example, 9000G, 10000G or 11000G, etc.) for 90-120 min (for example, 95 min, 100 min, 105 min, 110 min or 115 min, etc.), and the tertiary supernatant was collected to obtain the ginkgo cell fluid.

[0049] Preferably, the mass ratio of the ginkgo cell fluid, conjugated fatty acid and bisamino polyethylene glycol in step (4) is (1-3):(0.001-0.1):(0.0005-0.05).

[0050] Wherein "1-3" can be 1.1, 1.3, 1.5, 1.8, 2, 2.2, 2.5, 2.7 or 2.9, etc.;

[0051] “0.001-0.1” can mean 0.003, 0.005, 0.008, 0.01, 0.03, 0.05, 0.07 or 0.09, etc.;

[0052] “0.0005-0.05” can mean 0.0007, 0.0009, 0.001, 0.003, 0.005, 0.008, 0.01 or 0.03, etc.

[0053] Preferably, the mass ratio of the ginkgo cell solution to water in step (4) is 1:(1.5-25), for example, it can be 1:2, 1:5, 1:7, 1:10, 1:12, 1:15, 1:18, 1:20, 1:22 or 1:24.

[0054] Preferably, the conjugated fatty acid in step (IV) comprises any one of conjugated linoleic acid, α-linolenic acid or arachidonic acid, or a combination of at least two thereof.

[0055] Preferably, the bisamino polyethylene glycol in step (IV) comprises any one of bisamino-PEG4-NHS ester, bisamino-PEG6 or bisamino-PEG12, or a combination of at least two thereof.

[0056] Preferably, the centrifugation step in step (V) is 80,000-120,000G (for example, 90,000G, 100,000G, or 110,000G, etc.) and centrifuged for 60-90min (for example, 65min, 70min, 75min, 80min, or 85min, etc.).

[0057] Preferably, the Glycyrrhiza glabra exosomes are prepared by the following steps:

[0058] (a) sterilizing the rhizome of Glycyrrhiza glabra and then slicing it, and then breaking the cell wall to obtain a cell slurry;

[0059] (b) centrifuging the cell slurry and collecting the supernatant to obtain Glycyrrhiza glabra cell slurry;

[0060] (c) adding the Glycyrrhiza glabra cell fluid, conjugated fatty acids, and bisaminopolyethylene glycol to water, mixing, and then diafiltration to obtain a dialysate;

[0061] (d) The dialysate was centrifuged to obtain Glycyrrhiza glabra exosomes.

[0062] Preferably, the disinfection in step (a) is performed using a 75% ethanol aqueous solution.

[0063] Preferably, the centrifugation step in step (b) comprises: centrifuging the cell slurry at 3000-4000G (for example, 3100G, 3300G, 3500G, 3700G or 3900G, etc.) for 40-80min (for example, 45min, 50min, 55min, 60min, 65min, 70min or 75min, etc.), taking a supernatant and centrifuging it at 8000-15000G (for example, 9000G, 10000G, 11000G, 12000G, etc.), and centrifuging it at 15000G. The supernatant is centrifuged at 80000-120000G (for example, 9000G, 10000G or 11000G) for 90-120 min (for example, 95 min, 100 min, 105 min, 110 min or 115 min), and the supernatant is collected for 3 times to obtain the cell fluid of Glycyrrhiza glabra.

[0064] Preferably, the mass ratio of the Glycyrrhiza glabra cell sap, conjugated fatty acids and bisamino polyethylene glycol in step (4) is (1-3):(0.001-0.1):(0.0005-0.05).

[0065] Wherein "1-3" can be 1.1, 1.3, 1.5, 1.8, 2, 2.2, 2.5, 2.7 or 2.9, etc.;

[0066] “0.001-0.1” can mean 0.003, 0.005, 0.008, 0.01, 0.03, 0.05, 0.07 or 0.09, etc.;

[0067] “0.0005-0.05” can mean 0.0007, 0.0009, 0.001, 0.003, 0.005, 0.008, 0.01 or 0.03, etc.

[0068] Preferably, the mass ratio of the Glycyrrhiza glabra cell sap to water in step (4) is 1:(1.5-25), for example, it can be 1:2, 1:5, 1:7, 1:10, 1:12, 1:15, 1:18, 1:20, 1:22 or 1:24, etc.

[0069] Preferably, the conjugated fatty acid in step (c) comprises any one of conjugated linoleic acid, α-linolenic acid or arachidonic acid, or a combination of at least two thereof.

[0070] Preferably, the bisamino polyethylene glycol in step (c) comprises any one of bisamino-PEG4-NHS ester, bisamino-PEG6 or bisamino-PEG12, or a combination of at least two thereof.

[0071] Preferably, the centrifugation step in step (d) is 80,000-120,000G (for example, 90,000G, 100,000G, or 110,000G, etc.) for 60-90min (for example, 65min, 70min, 75min, 80min, or 85min, etc.).

[0072] Any specific point value within the above numerical range can be selected and will not be described in detail here.

[0073] In a second aspect, the present invention provides a use of the plant exosome composition as described in the first aspect in the preparation of cosmetics or cosmetic raw materials.

[0074] In a third aspect, the present invention provides a freeze-dried powder containing plant exosomes. The raw materials for preparing the freeze-dried powder include, in parts by weight, 0.01-2 parts of the plant exosome composition described in the first aspect, 0.5-2.5 parts of mannitol, 0.1-1.5 parts of trehalose, 0.05-1 part of soluble collagen and 0.01-0.5 part of sodium hyaluronate.

[0075] The amount of the plant exosome composition added to the raw materials for preparing the freeze-dried powder of the present invention is 0.02 parts, 0.05 parts, 0.07 parts, 0.09 parts, 0.1 parts, 0.2 parts, 0.3 parts, 0.4 parts, 0.5 parts, 0.6 parts, 0.7 parts, 0.8 parts, 0.9 parts, 1 parts, 1.1 parts, 1.2 parts, 1.3 parts, 1.4 parts, 1.5 parts, 1.6 parts, 1.7 parts, 1.8 parts or 1.9 parts, etc.;

[0076] The amount of mannitol added can be 0.6 parts, 0.7 parts, 0.8 parts, 0.9 parts, 1 part, 1.1 parts, 1.2 parts, 1.3 parts, 1.4 parts, 1.5 parts, 1.6 parts, 1.7 parts, 1.8 parts, 1.9 parts, 2 parts, 2.1 parts, 2.2 parts, 2.3 parts or 2.4 parts, etc.;

[0077] The added amount of trehalose can be 0.2 parts, 0.3 parts, 0.4 parts, 0.5 parts, 0.6 parts, 0.7 parts, 0.8 parts, 0.9 parts, 1 parts, 1.1 parts, 1.2 parts, 1.3 parts or 1.4 parts, etc.;

[0078] The amount of the soluble collagen added can be 0.06 parts, 0.07 parts, 0.08 parts, 0.09 parts, 0.1 parts, 0.2 parts, 0.3 parts, 0.4 parts, 0.5 parts, 0.6 parts, 0.7 parts, 0.8 parts or 0.9 parts, etc.;

[0079] The added amount of the sodium hyaluronate can be 0.02 parts, 0.03 parts, 0.04 parts, 0.05 parts, 0.06 parts, 0.07 parts, 0.08 parts, 0.09 parts, 0.1 parts, 0.2 parts, 0.3 parts or 0.4 parts, etc.

[0080] The Ganoderma lucidum exosomes, Ginkgo biloba exosomes or Glycyrrhiza glabra exosomes used in the freeze-dried powder of the present invention can better retain the activity of the beneficial ingredients of Ganoderma lucidum, Ginkgo biloba or Glycyrrhiza glabra. The addition of freeze-dried excipients and skin conditioners, such as mannitol, trehalose, soluble collagen, sodium hyaluronate and other substances, after dissolution and mixing, can work synergistically to jointly exert the effects of skin anti-oxidation, soothing and anti-inflammatory, whitening and freckle removal, and brightening skin tone.

[0081] Any specific point value within the above numerical range can be selected and will not be described in detail here.

[0082] In a fourth aspect, the present invention provides a method for preparing the freeze-dried powder containing plant exosomes as described in the third aspect, the preparation method comprising:

[0083] The plant exosome composition, mannitol, trehalose, soluble collagen, sodium hyaluronate and water are evenly mixed, and then vacuum freeze-dried to obtain the freeze-dried powder containing the plant exosomes.

[0084] Preferably, the vacuum freeze-drying conditions are as follows: pre-freezing temperature -50°C to -45°C (for example, -46°C, -47°C, -48°C or -49°C), pre-freezing time 0.5-2h (for example, 0.6h, 0.8h, 1h, 1.2h, 1.5h or 1.8h); primary sublimation temperature -15°C to -5°C (for example, -6°C, -8°C, -10°C, -12°C or -14°C), primary sublimation time 3-10h (for example, 4h, 5h, 6h, 7h, 8h or 9h, etc.), vacuum degree 15-25Pa (for example, it can be 16Pa, 17Pa, 18Pa or 19Pa, etc.); secondary sublimation temperature 0℃-10℃ (for example, it can be 1℃, 3℃, 5℃, 7℃ or 9℃, etc.), secondary sublimation time 2-8h (for example, it can be 3h, 4h, 5h, 6h or 7h, etc.), vacuum degree 20-40Pa (for example, it can be 22Pa, 25Pa, 28Pa, 30Pa, 32Pa, 35Pa or 38Pa, etc.).

[0085] Any specific point value within the above numerical range can be selected and will not be described in detail here.

[0086] In a fifth aspect, the present invention provides a use of the freeze-dried powder containing plant exosomes as described in the third aspect in the preparation of cosmetics.

[0087] Preferably, the cosmetic has the effects of any one or a combination of at least two of anti-oxidation, soothing and anti-inflammatory, whitening and freckle removal, or brightening skin tone.

[0088] Compared with the prior art, the present invention has the following beneficial effects:

[0089] In the present invention, Ganoderma lucidum, Ginkgo biloba and Glycyrrhiza glabra are combined in the form of exosomes, which can better bring out the respective advantages of Ganoderma lucidum, Ginkgo biloba and Glycyrrhiza glabra, increase the content of active ingredients, and thus make the plant-containing exosomes have excellent antioxidant, soothing and anti-inflammatory, whitening and freckle removal, and skin brightening effects. DETAILED DESCRIPTION

[0090] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0091] The sources of some raw materials in the Examples or Comparative Examples of the present invention are as follows:

[0092] Ganoderma lucidum was collected from Tengchong City, Yunnan Province;

[0093] Ginkgo leaves were collected from Nanxiong City, Guangdong Province;

[0094] The rhizomes of Glycyrrhiza glabra (Licorice) were collected from the Altay region of Xinjiang Uygur Autonomous Region;

[0095] Ganoderma lucidum extract was from Guangzhou Baoyi Biotechnology Co., Ltd., specification 6%;

[0096] Ginkgo biloba leaf extract was from Guangzhou Baoyi Biotechnology Co., Ltd., with a specification of 6%;

[0097] Glycyrrhiza glabra extract was obtained from Guangzhou Baoyi Biotechnology Co., Ltd., with a specification of 6%;

[0098] Soluble collagen was obtained from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd. with a specification of >90%;

[0099] Sodium hyaluronate was obtained from Bloomage Biotechnology Co., Ltd., with a specification of 200-400 kDa;

[0100] The rest of the raw materials can be used as long as they are purchased from regular dealers.

[0101] Preparation Example 1

[0102] Preparation of Ganoderma lucidum exosomes

[0103] (1) Take about 2cm of low temperature storage 3The Ganoderma lucidum clumps were placed on potato culture medium plates (20 g glucose, 1.5 g magnesium sulfate, 3.0 g potassium dihydrogen phosphate, 0.05 g vitamin B1, 200 g peeled potatoes cut into small pieces, added with 1 L ultrapure water, boiled for 30 min, filtered through gauze, the filtrate was re-adjusted to 1 L, the pH was adjusted to 6.0, sterilized at 115° C. for 30 min, and poured into a petri dish to cool) and cultured at 30° C. for 5 days. The peripheral Ganoderma lucidum clumps were cut and placed on potato culture medium test tube slant plates, and activated and cultured at 30° C. for 7 days.

[0104] (2) The mycelia on the slant were washed with 5 ml of liquid culture medium (35 g glucose, 5 g peptone, 2.5 g yeast extract, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 0.05 g vitamin B1, fixed to 1 L, adjusted to pH = 5.5, and sterilized at 115°C for 30 min), poured into a 100 ml Erlenmeyer flask culture medium with glass beads and cultured at 30°C for 5 days. 20 ml of the culture solution was added to a 500 ml Erlenmeyer flask culture medium with glass beads and cultured at 30°C for 2 days. 50 ml of the culture solution was then added to 500 ml of liquid culture medium (35 g glucose, 5 g peptone, 5 g yeast extract, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 0.05 g vitamin B1, fixed to 1 L, adjusted to pH = 5.5, and sterilized at 115°C for 30 min) and fermented at 30°C with shaking at 150 rpm for 96 h.

[0105] (3) The fermented Ganoderma mycelial cells were rinsed with water and disrupted by low-speed stirring (ultrasonic cell disruptor SCIENTZ-IID, Ningbo Xinzhi). The resulting slurry was centrifuged at 4000 G (low-temperature desktop centrifuge CL55, Hunan Xiangyi) for 60 min, and the supernatant was centrifuged at 15000 G (high-speed centrifuge Avanti JE, Beckman Coulter, USA) for 60 min. The supernatant was centrifuged at 120000 G (ultrahigh-speed centrifuge Optima XE, Beckman Coulter, USA) for 110 min. The supernatant was obtained as the Ganoderma cell fermentation broth.

[0106] (4) 30 parts of Ganoderma lucidum cell fermentation broth, 0.5 parts of conjugated linoleic acid, 0.3 parts of α-linolenic acid, 0.5 parts of bisamino-PEG4-NHS ester, and 65 parts of water were mixed evenly, dialyzed with a 5KDa dialysis membrane for 18 hours, and centrifuged at 100000G for 75 minutes to obtain Ganoderma lucidum exosomes.

[0107] Preparation Example 2

[0108] Preparation of Ganoderma lucidum exosomes

[0109] (1) Take about 2cm of low temperature storage 3The Ganoderma lucidum clumps were placed on potato culture medium plates (20 g glucose, 1.5 g magnesium sulfate, 3.0 g potassium dihydrogen phosphate, 0.05 g vitamin B1, 200 g peeled potatoes cut into small pieces, added with 1 L ultrapure water, boiled for 30 min, filtered through gauze, the filtrate was re-adjusted to 1 L, the pH was adjusted to 6.0, sterilized at 115° C. for 30 min, and poured into a petri dish to cool) and cultured at 35° C. for 8 days. The peripheral Ganoderma lucidum clumps were cut and placed on potato culture medium test tube slant plates, and activated and cultured at 30° C. for 7 days.

[0110] (2) The mycelia on the slant were washed with 5 ml of liquid culture medium (35 g glucose, 5 g peptone, 2.5 g yeast extract, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 0.05 g vitamin B1, fixed to 1 L, adjusted to pH = 5.5, and sterilized at 115°C for 30 min), poured into a 100 ml Erlenmeyer flask culture medium with glass beads and cultured at 30°C for 5 days. 20 ml of the culture solution was added to a 500 ml Erlenmeyer flask culture medium with glass beads and cultured at 30°C for 2 days. 50 ml of the culture solution was then added to 500 ml of liquid culture medium (35 g glucose, 5 g peptone, 5 g yeast extract, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 0.05 g vitamin B1, fixed to 1 L, adjusted to pH = 5.5, and sterilized at 115°C for 30 min) and fermented at 30°C with shaking at 150 rpm for 96 h.

[0111] (3) The fermented Ganoderma mycelium cells were rinsed with water and disrupted by low-speed stirring (ultrasonic cell disruptor SCIENTZ-IID, Ningbo Xinzhi). The resulting slurry was centrifuged at 3800 G (low-temperature desktop centrifuge CL55, Hunan Xiangyi) for 60 min, and the supernatant was centrifuged at 12000 G (high-speed centrifuge Avanti JE, Beckman Coulter, USA) for 90 min. The supernatant was centrifuged at 80000 G (ultrahigh-speed centrifuge Optima XE, Beckman Coulter, USA) for 120 min. The supernatant was obtained as the Ganoderma cell fermentation broth.

[0112] (4) 30 parts of Ganoderma lucidum cell fermentation broth, 0.1 part of α-linolenic acid, 0.15 parts of arachidonic acid, 0.2 parts of bisamino-PEG 6, 0.1 parts of bisamino-PEG 12, and 69 parts of water were mixed uniformly. After 18 hours of dialysis using a 5 kDa dialysis membrane, the mixture was centrifuged at 90,000 g for 90 minutes to obtain Ganoderma lucidum exosomes.

[0113] Preparation Example 3

[0114] Preparation of Ginkgo Exosomes:

[0115] (1) Take a few young leaves of ginkgo biloba newly grown at the end of March, rinse them with running water, disinfect the surface with 75% ethanol solution, rinse with sterile water, cut into small pieces the size of rice grains, and inoculate them in 5 ml of sterile liquid culture medium (potassium nitrate 0.19%, ammonium nitrate 0.165%, potassium dihydrogen phosphate 0.017%, magnesium sulfate 0.037%, calcium chloride dihydrate 0.044%, potassium iodide 0.000083%, boric acid 0.00062%, manganese sulfate tetrahydrate 0.00223%, zinc sulfate heptahydrate 0.00086%, sodium molybdate dihydrate 0.000025%, copper sulfate pentahydrate 0.0000025%, cobalt chloride hexahydrate 0.0000025%, ferrous sulfate heptahydrate 0.00278%, disodium EDTA 0.00372%, inositol 0.01%, glycine 0.0002%, vitamin B1 0.00001%, vitamin B5 0.00005%, vitamin B6 0.00005%, sucrose 3%, agar 0.6%, autoclaved at 121°C for 20 min) in test tubes, inoculating one small piece per test tube;

[0116] (2) The surviving callus tissue was inoculated into 500 ml Erlenmeyer flask culture medium (potassium nitrate 0.19%, ammonium nitrate 0.165%, potassium dihydrogen phosphate 0.017%, magnesium sulfate 0.037%, calcium chloride dihydrate 0.044%, potassium iodide 0.000083%, boric acid 0.00062%, manganese sulfate tetrahydrate 0.00223%, zinc sulfate heptahydrate 0.00086%, sodium molybdate dihydrate 0.000025%, copper sulfate pentahydrate 0.0000025%, cobalt chloride hexahydrate 0.0000025%, ferrous sulfate heptahydrate 0.00278%, disodium EDTA 0.00372%, inositol 0.01%, glycine 0.0002%, vitamin B1 0.00001%, vitamin B5 0.00005%, vitamin B6 0.00005%, sucrose 3%, agar 0.6%, after high-pressure sterilization at 121℃ for 20min, add sterile-filtered 1mg / ml naphthaleneacetic acid 1ml / L, 1mg / ml 6-benzylpurine 0.5ml / L), inoculate 5-8 pieces per bottle, light period 14h, dark period 10h, 25±2℃, shaking at 120rpm for 16 days;

[0117] (3) Ginkgo biloba cell suspension was collected by filtration, rinsed with water, and centrifuged at 3600 G (low-temperature desktop centrifuge CL55, Hunan Xiangyi) for 75 min. The supernatant was centrifuged at 12000 G (high-speed centrifuge Avanti JE, Beckman Coulter, USA) for 80 min. The supernatant was centrifuged at 100000 G (ultrahigh-speed centrifuge Optima XE, Beckman Coulter, USA) for 100 min to obtain the Ginkgo biloba cell suspension.

[0118] (4) Mix 30 parts of ginkgo cell fluid, 0.2 parts of α-linolenic acid, 0.25 parts of bisamino-PEG12, and 65 parts of water. After dialysis with a 3 kDa dialysis membrane for 15 h, centrifuge at 100,000 G for 75 min to obtain ginkgo exosomes.

[0119] Preparation Example 4

[0120] Preparation of Ginkgo Exosomes:

[0121] (1) Take a few young leaves of ginkgo biloba newly grown at the end of March, rinse them with running water, disinfect the surface with 75% ethanol solution, rinse with sterile water, cut into small pieces the size of rice grains, and inoculate them in 5 ml of sterile liquid culture medium (potassium nitrate 0.19%, ammonium nitrate 0.165%, potassium dihydrogen phosphate 0.017%, magnesium sulfate 0.037%, calcium chloride dihydrate 0.044%, potassium iodide 0.000083%, boric acid 0.00062%, manganese sulfate tetrahydrate 0.00223%, zinc sulfate heptahydrate 0.00086%, sodium molybdate dihydrate 0.000025%, copper sulfate pentahydrate 0.0000025%, cobalt chloride hexahydrate 0.0000025%, ferrous sulfate heptahydrate 0.00278%, disodium EDTA 0.00372%, inositol 0.01%, glycine 0.0002%, vitamin B1 0.00001%, vitamin B5 0.00005%, vitamin B6 0.00005%, sucrose 3%, agar 0.6%, autoclaved at 121°C for 20 min) in test tubes, inoculating one small piece per test tube;

[0122] (2) The surviving callus tissue was inoculated into 500 ml Erlenmeyer flask culture medium (0.017% potassium dihydrogen phosphate, 0.00062% boric acid, 0.0085g% calcium chloride, 0.00086% zinc sulfate heptahydrate, 0.00223% manganese sulfate tetrahydrate, 0.000083% potassium iodide, 0.000025% sodium molybdate dihydrate, 0.037% magnesium sulfate, 0.00278% ferrous sulfate heptahydrate, 0.0000025% copper sulfate pentahydrate, 0.0000025% nickel chloride, 0.0000025% cobalt chloride, 0.0000025% disodium EDTA, 1.0mg vitamin B1, 0.0002% glycine, 0.000002% vitamin B6) 0.00005%, inositol 0.02%, niacin 0.00005%, B6 0.00005%, sucrose 3%, agar 0.6%, autoclave at 121°C for 20 min, add sterile-filtered 1 mg / ml naphthaleneacetic acid 1 ml / L, 1 mg / ml 6-furfuryladenine 0.5 ml / L), inoculate 5-8 pieces per bottle, light period 12 h, dark period 10 h, 25±2°C, shaking at 120 rpm for 16 days;

[0123] (3) Ginkgo biloba cell suspensions were collected by filtration, rinsed with water, and disrupted with low-speed stirring (ultrasonic cell disruptor SCIENTZ-IID, Ningbo Xinzhi). The resulting cell slurry was centrifuged at 3800 G (low-temperature desktop centrifuge CL55, Hunan Xiangyi) for 80 min. The supernatant was centrifuged at 14000 G (high-speed centrifuge Avanti JE, Beckman Coulter, USA) for 80 min. The supernatant was then centrifuged at 110000 G (ultrahigh-speed centrifuge Optima XE, Beckman Coulter, USA) for 110 min to obtain the Ginkgo biloba cell suspension.

[0124] (4) Mix 30 parts of Ginkgo biloba cell fluid, 0.22 parts of arachidonic acid, 0.2 parts of bisamino-PEG12, and 60 parts of water. After dialysis using a 3 kDa dialysis membrane for 18 hours, centrifuge at 120,000 G for 65 minutes to obtain Ginkgo biloba exosomes.

[0125] Preparation Example 5

[0126] Preparation of Glycyrrhiza glabra exosomes:

[0127] (1) 1200 g of 5-10 cm long and 0.5-1.5 cm in diameter Glycyrrhiza glabra rhizomes were collected and washed with water to remove dirt. The surface was then disinfected with 75% ethanol solution. The ethanol was then rinsed with water to remove the ethanol. The rhizomes were cut into 1-2 mm thick slices and the cells were disrupted using a low-speed blender (BL1036A, Midea Electric).

[0128] (2) The resulting cell slurry was centrifuged at 4000G (low-temperature desktop centrifuge CL55, Hunan Xiangyi) for 60 min, and the supernatant was centrifuged at 15000G (high-speed centrifuge Avanti JE, Beckman Coulter, USA) for 45 min. The supernatant was centrifuged at 100000G (ultrahigh-speed centrifuge Optima XE, Beckman Coulter, USA) for 90 min to obtain Glycyrrhiza glabra cell slurry.

[0129] (3) 35 parts of Glycyrrhiza glabra cell fluid, 0.05 parts of conjugated linoleic acid, 0.08 parts of α-linolenic acid, 0.05 parts of arachidonic acid, 0.1 parts of bisamino-PEG4-NHS ester, 0.08 parts of amino-PEG12, and 60 parts of water were mixed uniformly. After 15 hours of dialysis using a 5 kDa dialysis membrane, the mixture was centrifuged at 120,000 g for 70 minutes to obtain Glycyrrhiza glabra exosomes.

[0130] Preparation Example 6

[0131] Preparation of Glycyrrhiza glabra exosomes:

[0132] (1) 1200 g of 5-10 cm long and 0.5-1.5 cm in diameter Glycyrrhiza glabra rhizomes were collected and washed with water to remove dirt. The surface was then disinfected with 75% ethanol solution. The ethanol was then rinsed with water to remove the ethanol. The rhizomes were cut into 1-2 mm thick slices and the cells were disrupted using a low-speed blender (BL1036A, Midea Electric).

[0133] (2) The obtained cell slurry was centrifuged at 4000G (low-temperature desktop centrifuge CL55, Hunan Xiangyi) for 80 minutes. The supernatant was centrifuged at 15000G (high-speed centrifuge Avanti JE, Beckman Coulter, USA) for 65 minutes. The supernatant was centrifuged at 100000G (ultrahigh-speed centrifuge Optima XE, Beckman Coulter, USA) for 90 minutes to obtain Glycyrrhiza glabra exosomes;

[0134] (3) 35 parts of Glycyrrhiza glabra cell fluid, 0.1 parts of conjugated linoleic acid, 0.05 parts of α-linolenic acid, 0.05 parts of arachidonic acid, 0.05 parts of bisamino-PEG4-NHS ester, 0.05 parts of amino-PEG12, and 65 parts of water were mixed uniformly. After 18 hours of dialysis using a 5 kDa dialysis membrane, the mixture was centrifuged at 120,000 g for 60 minutes to obtain Glycyrrhiza glabra exosomes.

[0135] Examples 1-3

[0136] The embodiment provides three types of freeze-dried powders, the raw materials of which are shown in the following table:

[0137]

[0138] The preparation method of the freeze-dried powder comprises: uniformly mixing Ganoderma lucidum exosomes, Ginkgo biloba exosomes, Glycyrrhiza glabra exosomes, mannitol, trehalose, soluble collagen, sodium hyaluronate and water, and then vacuum freeze-drying to obtain the freeze-dried powder containing plant exosomes.

[0139] Comparative Example 1

[0140] This comparative example provides a freeze-dried powder. The only difference between the freeze-dried powder and Example 1 is that Ganoderma lucidum extract, Ginkgo biloba extract and Glycyrrhiza glabra extract are used to replace Ganoderma lucidum exosomes, Ginkgo biloba exosomes and Glycyrrhiza glabra exosomes in equal amounts, respectively. The remaining components and proportions are consistent with those in Example 1. The preparation method refers to Example 1.

[0141] Comparative Example 2

[0142] This comparative example provides a freeze-dried powder containing plant exosomes. The freeze-dried powder differs from Example 1 only in that an equal amount of Ganoderma lucidum extract is used to replace Ganoderma lucidum exosomes. The remaining components and proportions are consistent with those in Example 1. The preparation method refers to Example 1.

[0143] Comparative Example 3

[0144] This comparative example provides a freeze-dried powder containing plant exosomes. The freeze-dried powder differs from Example 1 only in that an equal amount of ginkgo extract is used to replace ginkgo exosomes. The remaining components and proportions are consistent with those in Example 1. The preparation method refers to Example 1.

[0145] Comparative Example 4

[0146] This comparative example provides a freeze-dried powder containing plant exosomes. The freeze-dried powder differs from Example 1 only in that an equal amount of Glycyrrhiza glabra extract is used to replace Glycyrrhiza glabra exosomes. The remaining components and proportions are consistent with those in Example 1. The preparation method refers to Example 1.

[0147] Comparative Example 5

[0148] This comparative example provides a freeze-dried powder containing plant exosomes. The freeze-dried powder differs from Example 1 only in that it does not include Ganoderma lucidum exosomes, and the reduced amount thereof is distributed to Ginkgo biloba exosomes and Glycyrrhiza glabra exosomes in proportion to the number of parts. The remaining components and proportions are consistent with those in Example 1, and the preparation method is referred to Example 1.

[0149] Comparative Example 6

[0150] This comparative example provides a freeze-dried powder containing plant exosomes. The freeze-dried powder differs from Example 1 only in that it does not include Ginkgo biloba exosomes, and the reduced amount thereof is distributed to Ganoderma lucidum exosomes and Glycyrrhiza glabra exosomes in proportion to the number of parts. The remaining components and proportions are consistent with those in Example 1, and the preparation method is referred to Example 1.

[0151] Comparative Example 7

[0152] This comparative example provides a freeze-dried powder containing plant exosomes. The freeze-dried powder differs from Example 1 only in that it does not include Glycyrrhiza glabra exosomes, and the reduced amount thereof is distributed to Ganoderma lucidum exosomes and Ginkgo biloba exosomes in proportion to the number of parts. The remaining components and proportions are consistent with those in Example 1, and the preparation method is referred to Example 1.

[0153] Comparative Example 8

[0154] This comparative example provides a freeze-dried powder containing plant exosomes. The freeze-dried powder differs from Example 1 only in that it does not include Ginkgo biloba exosomes and Glycyrrhiza glabra exosomes, and the reduced amount is supplemented by Ganoderma lucidum exosomes. The remaining components and proportions are consistent with those in Example 1, and the preparation method refers to Example 1.

[0155] Comparative Example 9

[0156] This comparative example provides a freeze-dried powder containing plant exosomes. The freeze-dried powder differs from Example 1 only in that it does not include Ganoderma lucidum exosomes and Glycyrrhiza glabra exosomes, and the reduced amount is supplemented by Ginkgo exosomes. The remaining components and proportions are consistent with those in Example 1, and the preparation method refers to Example 1.

[0157] Comparative Example 10

[0158] This comparative example provides a freeze-dried powder containing plant exosomes. The freeze-dried powder differs from Example 1 only in that it does not include Ganoderma lucidum exosomes and Ginkgo biloba exosomes, and the reduced amount is supplemented by Glycyrrhiza glabra exosomes. The remaining components and proportions are consistent with those in Example 1, and the preparation method refers to Example 1.

[0159] Test Example 1: DPPH Antioxidant Test

[0160] DPPH free radicals have a strong absorption peak at 517nm wavelength, resulting in a purple color. When DPPH free radicals combine with electrons provided by antioxidant peptides, the purple color disappears and becomes colorless or pale yellow. The color change is reflected by measuring the absorbance before and after the reaction using a UV-visible spectrophotometer. The antioxidant capacity of the lyophilized powder sample is determined by comparing it with the scavenging capacity of glutathione and using the relative half-scavenged amount.

[0161] The lyophilized powders of Examples 1-3 and Comparative Examples 1-10 were dissolved in a solvent to obtain lyophilized powder reconstituted solutions, which were used as test samples. The preparation method of the lyophilized powder reconstituted solutions was as follows:

[0162] Weigh 1 part of glycerol, 0.5 part of 1,2-hexanediol, 0.2 part of ethylhexylglycerol, 0.002 part of disodium EDTA, 0.002 part of disodium hydrogen phosphate, 0.015 part of sodium dihydrogen phosphate, and 98.281 parts of water, dissolve and mix uniformly, filter through 300 mesh to obtain a solvent solution, add the lyophilized powder to the solvent solution (solid-liquid ratio is 0.3 g / mL), and mix uniformly to obtain the lyophilized powder reconstitution solution.

[0163] Separately, dissolve L-reduced glutathione to a volume of 10.0 mg / mL as the control stock solution. Weigh 5.0 mg of DPPH and dissolve it in an appropriate amount of anhydrous ethanol. Ultrasound-proof the solution in the dark to fully dissolve it. Then, dilute the volume to 100 ml with anhydrous ethanol to prepare a 50 μg / ml DPPH solution.

[0164] The glutathione control group was prepared according to Table 1. The freeze-dried powder sample test group was prepared according to Table 2.

[0165] Table 1

[0166]

[0167] Table 2

[0168]

[0169] After the six sample solutions were mixed evenly, they were reacted in the dark at room temperature for 30 minutes, and the absorbance was measured at a wavelength of 517 nm.

[0170] The free radical scavenging rate was calculated according to the following formula:

[0171] P=[1-(As-Ac) / Ab]×100%

[0172] P is the clearance rate, and As, Ac, and Ab correspond to the absorbance values ​​of samples No. 1, No. 2, and No. 3 in the table, respectively.

[0173] The test results are shown in Table 3.

[0174] Table 3

[0175]

[0176] The results show that compared with Comparative Examples 1-4, the antioxidant capacity of the freeze-dried powders of the three plant exosomes of the present invention is superior to that of the comparative example using ordinary extracts. Comparative Examples 5-10 show that the three have a synergistic effect in antioxidant activity.

[0177] Test Example 2: Anti-inflammatory and soothing test

[0178] Mouse mononuclear macrophage leukemia cells RAW264.7 were cultured with DMEM complete medium (from Thermo Fisher Scientific). When the cells adhered to 80% to 90%, the original medium was discarded and the cells were washed twice with pH 7.0 phosphate buffer. The cells were passaged at a ratio of 1:3 and cultured in a 37°C, 5% CO2 incubator. RAW264.7 cells in the logarithmic growth phase were taken and the cell density was adjusted to 2×10 5 / ml, inoculated into a 12-well culture plate, 1ml per well, and discarded the supernatant after culturing for 24 hours. A blank control group, a model group (bacterial lipopolysaccharide LPS 1μg / ml) and a freeze-dried powder sample group (the reconstituted solution of Examples 1-9 and Comparative Examples 1-4, the preparation method refers to Test Example 1) were set up, and each group was given the corresponding concentration of test sample and 1μg / ml LPS to jointly intervene in the cells. After the sample intervened in the cells for 24 hours, the cell supernatant was collected. The IL-1β content was detected according to the operating instructions of the interleukin 1β (IL-1β) kit (Shanghai Biyuntian) (results are shown in Table 5). The nitric oxide (NO) content was determined according to the Griess method. After the cell treatment was completed, the cell supernatant was taken into a 1.5ml centrifuge tube and centrifuged at 600×g and 4°C for 5 minutes. The standard was diluted with complete culture medium to 0, 1, 2, 5, 10, 20, 40, 60, and 100μmol / L. Add 50 μl of sample supernatant and standard to each well of a 96-well plate. Add 50 μl each of Griess Reagent I and II, which had been brought to room temperature, to each well. Measure absorbance at 540 nm and calculate the NO content of the sample using the standard curve (see Table 4 for results).

[0179] Table 4

[0180]

[0181]

[0182] NO is an important biomarker in the inflammatory process, and its excessive release can exacerbate the inflammatory response. By testing the effect of lyophilized powder on NO content in the supernatant of RAW264.7 cells, it was found that compared with the blank control group, the amount of NO released by RAW264.7 cells after LPS induction was significantly increased. Treatment with lyophilized powder and solution effectively inhibited NO release.

[0183] Table 5

[0184]

[0185]

[0186] IL-1β plays a key role in the development of inflammation and autoimmune diseases and is produced in large quantities in LPS-induced inflammation models.

[0187] By detecting the effect of the lyophilized powder reconstitution solution on the expression of IL-1β in RAW264.7 cells, it was found that compared with the control group, the mRNA expression level of IL-1β in RAW264.7 cells was significantly increased after LPS stimulation. This indicates that the lyophilized powder reconstitution solution of the present invention can significantly inhibit the expression of IL-1β in RAW264.7 inflammatory cells induced by LPS after pretreatment.

[0188] Test Example 3: Whitening and Spot Removal - Human Trial Trichromatic Stimulation Value Scoring

[0189] Test principle: The skin color is quantified using the three primary color stimulus values ​​determined by the International Commission on Illumination, and the individual type angle ITA is calculated according to the formula o (Individual Typology Angle) value, which represents the whiteness of the skin.

[0190] ITA o =arctan[(L*-50) / b*]×180×π -1

[0191] L* represents the brightness coordinate, b* represents the chromaticity coordinate

[0192] ITA o Larger values ​​indicate lighter skin. ITA° is an objective skin color classification method used in dermatology and cosmetic research. It can be used to categorize skin color as follows: very light (>55°), light (55°–41°), medium (41°–28°), tan (28°–10°), tan (10°––30°), and black (<–30°). ITA° correlates with total melanin content, as well as eumelanin and pheomelanin content. This objective skin color classification method can address the reliability issues of self-report and subjective evaluation using the FST.

[0193] The trial recruited 78 subjects aged 36 to 45 years, including 39 females and 39 males, who were randomly divided into 13 groups, with 6 subjects in each group. o The value ranged from 10° to 41°. Participants with the following conditions were excluded from the trial: those with birthmarks, congenital or hereditary pigmentary abnormalities; those who had used preparations that affected skin color in the past week; those who had used antihistamines in the past week or immunosuppressants in the past month; those who had used anti-inflammatory drugs on the test site in the past two months; those with unresolved inflammatory skin diseases; those who were breastfeeding or pregnant; those with sensitive constitutions; those who were allergic to the test product ingredients; and other individuals deemed by the researchers to be ineligible for the trial requirements.

[0194] The subjects cleaned their faces before using the samples. According to the groups, the freeze-dried powders of Examples 1-3 and Comparative Examples 1-10 were reconstituted with the solvent solution (refer to Test Example 1) and applied to the test area of ​​the face. The subjects sat or lay on their backs with their faces facing up for 20 minutes to allow the samples to be absorbed. ITA was tested at the beginning of the test, on the 14th day, on the 28th ± 1st day, and on the 56th ± 2nd day. o Before the test, the subjects cleaned the test area, dried it with a lint-free paper towel, and sat quietly in the test waiting area for 20 minutes without drinking water or other beverages. The instrument (tristimulus colorimeter LS-150, Konica Minolta, Japan) was calibrated first, and the ITA of the test area applied by the subjects was measured.o The values ​​were measured at different locations in the test area and the average value was taken. The results are shown in Table 6.

[0195] Table 6

[0196]

[0197]

[0198] By testing the effect of freeze-dried powder solution on skin ITA o It was found that compared with the comparative example 1 in which conventional extracts were added, the freeze-dried powder solution of the present invention can more significantly increase the ITA of the skin by adding plant exosomes. o value.

[0199] Test Example 4: Safety Evaluation Experiment

[0200] The specific method is as follows: 40 females aged 18-35 years old with no history of allergies were selected, and each subject used the freeze-dried powder solution of Examples 1-3 (refer to Test Example 1). After cleaning the subject's back, the spot tester with the sample was applied to the selected position on the back with a non-irritating adhesive tape. After affixing, lightly press it with your fingers to evenly stick it on the skin for 48 hours. The subjects kept the patch area dry within 48 hours, and avoided strenuous exercise, scratching the patch area, and prolonged sunlight exposure. After 48 hours, the tester was removed and marked. After 30 minutes, when the indentation disappeared, the test was made under sufficient light.

[0201] The grading standards for adverse skin reactions are shown in Table 7:

[0202] Table 7

[0203]

[0204] The results are shown in Table 8:

[0205] Table 8

[0206]

[0207] From the data in Table 8, it can be seen that after removing the spot tester, no adverse reactions were observed on the skin of the volunteers. It can be concluded that the raw materials used in the product of the present invention are safe and non-irritating, have no adverse reactions to the human body, and are highly safe.

[0208] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the plant exosome composition and its application, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacement of various raw materials in the product of the present invention, addition of auxiliary ingredients, selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

[0209] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0210] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A plant exosome composition, characterized in that The plant exosome composition comprises Ganoderma lucidum exosomes, Ginkgo biloba exosomes and Glycyrrhiza glabra exosomes in a mass ratio of (1-10):(1.5-10):(2-6).

2. The plant exosome composition according to claim 1, wherein The Ganoderma lucidum exosomes are prepared by the following steps: (1) taking a Ganoderma lucidum block and placing it in a culture medium to culture and obtain mycelium; (2) washing the mycelium with liquid culture medium, and then pouring the culture medium into a triangular flask with glass beads for cultivation to obtain mycelium cells of Ganoderma lucidum fermentation; (3) washing the fermented mycelial cells of Ganoderma lucidum with water, then stirring and crushing them to obtain a slurry, centrifuging the slurry and collecting the supernatant to obtain Ganoderma lucidum fermentation liquid; (4) adding Ganoderma lucidum fermentation liquid, conjugated fatty acids, and bisamino polyethylene glycol to water, mixing, and then dialysis filtering to obtain a dialysate; (5) The dialysate was centrifuged to obtain Ganoderma lucidum exosomes.

3. The plant exosome composition according to claim 2, wherein The culture temperature in step (1) is 25-35° C. and the culture time is 5-15 days; Preferably, the culture temperature in step (2) is 25-35°C and the culture time is 5-10 days; Preferably, the centrifugation step in step (3) comprises: centrifuging the slurry at 3000-4000G for 40-80min, taking the first supernatant and centrifuging it at 8000-15000G for 40-90min, taking the second supernatant and centrifuging it at 80000-120000G for 90-120min, and taking the third supernatant to obtain the Ganoderma lucidum fermentation liquid; Preferably, the mass ratio of the Ganoderma lucidum fermentation broth, conjugated fatty acids, and bisamino polyethylene glycol in step (4) is (1-3):(0.001-0.1):(0.0005-0.05); Preferably, the mass ratio of the Ganoderma lucidum fermentation liquid to water in step (4) is 1:(1.5-25); Preferably, the conjugated fatty acid in step (4) comprises any one or a combination of at least two of conjugated linoleic acid, α-linolenic acid or arachidonic acid; Preferably, the bisamino polyethylene glycol in step (4) comprises any one of bisamino-PEG4-NHS ester, bisamino-PEG6 or bisamino-PEG12, or a combination of at least two thereof; Preferably, the centrifugation step in step (5) is performed at 80,000-120,000 G for 60-90 min.

4. The plant exosome composition according to any one of claims 1 to 3, wherein The ginkgo exosomes are prepared by the following steps: (1) Ginkgo biloba leaves were sterilized and crushed, and then inoculated into a sterile liquid culture medium for cultivation; (II) inoculating the surviving callus into a culture medium in a triangular flask and culturing it to obtain a Ginkgo callus suspension cell solution; (III) filtering the suspended cell solution of the ginkgo callus tissue, then stirring and crushing the solution to obtain a slurry, centrifuging the slurry and collecting the supernatant to obtain the ginkgo cell solution; (IV) adding the ginkgo cell solution, conjugated fatty acids, and bisamino polyethylene glycol to water, mixing, and then diafiltration to obtain a dialysate; (V) Ginkgo exosomes were obtained by centrifuging the dialysate.

5. The plant exosome composition according to claim 4, wherein Step (I) disinfection is performed using 75% ethanol aqueous solution; Preferably, the culture temperature in step (I) is 25-35° C. and the culture time is 1-3 days; Preferably, the culture temperature in step (II) is 20-30°C and the culture time is 15-20 days; Preferably, the culture in step (II) is first irradiated with light for 10-15 hours and then carried out in the dark; Preferably, the centrifugation step in step (III) comprises: centrifuging the slurry at 3000-4000G for 40-80 minutes, taking the first supernatant and centrifuging it at 8000-15000G for 40-90 minutes, taking the second supernatant and centrifuging it at 80000-120000G for 90-120 minutes, and taking the third supernatant to obtain the ginkgo cell fluid; Preferably, the conjugated fatty acid in step (IV) comprises any one or a combination of at least two of conjugated linoleic acid, α-linolenic acid or arachidonic acid; Preferably, the bisamino polyethylene glycol in step (IV) comprises any one or a combination of at least two of bisamino-PEG4-NHS ester, bisamino-PEG6 or bisamino-PEG12; Preferably, the centrifugation step in step (V) is performed at 80,000-120,000 G for 60-90 min.

6. The plant exosome composition according to any one of claims 1 to 5, wherein The exosomes of Glycyrrhiza glabra are prepared by the following steps: (a) sterilizing the rhizome of Glycyrrhiza glabra and then slicing it, and then breaking the cell wall to obtain a cell slurry; (b) centrifuging the cell slurry and collecting the supernatant to obtain Glycyrrhiza glabra cell slurry; (c) adding the Glycyrrhiza glabra cell fluid, conjugated fatty acids, and bisaminopolyethylene glycol to water, mixing, and then diafiltration to obtain a dialysate; (d) The dialysate was centrifuged to obtain Glycyrrhiza glabra exosomes.

7. The plant exosome composition according to claim 6, wherein Step (a) disinfection is performed using a 75% ethanol aqueous solution; Preferably, the centrifugation step in step (b) comprises: centrifuging the cell slurry at 3000-4000G for 40-80min, taking the first supernatant and centrifuging it at 8000-15000G for 40-90min, taking the second supernatant and centrifuging it at 80000-120000G for 90-120min, and taking the third supernatant to obtain the Glycyrrhiza glabra cell slurry; Preferably, the conjugated fatty acid in step (c) comprises any one or a combination of at least two of conjugated linoleic acid, α-linolenic acid or arachidonic acid; Preferably, the bisamino polyethylene glycol in step (c) comprises any one or a combination of at least two of bisamino-PEG4-NHS ester, bisamino-PEG6 or bisamino-PEG12; Preferably, the centrifugation step in step (d) is performed at 80,000-120,000 G for 60-90 min.

8. Use of the plant exosome composition according to any one of claims 1 to 7 in the preparation of cosmetics or cosmetic raw materials.

9. A freeze-dried powder containing plant exosomes, characterized in that: In parts by weight, the raw materials for preparing the lyophilized powder include 0.01-2 parts of the plant exosome composition according to any one of claims 1 to 7, 0.5-2.5 parts of mannitol, 0.1-1.5 parts of trehalose, 0.05-1 part of soluble collagen, and 0.01-0.5 parts of sodium hyaluronate.

10. A method for preparing the freeze-dried powder containing plant exosomes according to claim 9, characterized in that: The preparation method comprises: The plant exosome composition, mannitol, trehalose, soluble collagen, sodium hyaluronate and water are evenly mixed, and then vacuum freeze-dried to obtain the freeze-dried powder containing the plant exosomes.

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